recent advances in bioenergy research (sssnire 3rd conference )

8/10/2019 Recent Advances in Bioenergy Research (SSSNIRE 3rd Conference )

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Recent Advances in Bioenergy Research

Volume III

Edited by

SACHIN KUMAR, A.K. SARMA, S.K. TYAGI, Y.K. YADAV

Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala, India



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ISBN 978-81-927097-2-7

© Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala-2014

Electronic version published by SSS-NIRE

ALL RIGHTS RESERVED



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CONTENTS

Preface xiv

Contributors xvi

Part-I: Biomass and Energy Management 1

1 Thermogravimetric characterization of wood stalks as gasification 2

and pyrolysis feedstock

Rakesh Punia, Sachin Kumar

Abstract 2

1.1 Introduction 2

1.2 Materials and Methodology 4

1.3 Results & Discussion 6

1.4 Conclusion 10 References 11

2 Assessment of Solid Waste Management and Energy Recovery 13

from Waste Materials in Lucknow Zoo: A Case Study

Vinayak V. Pathak, Richa Kothari, A.K. Chopra, Lhaihoichong Singson

Abstract 13

2.1 Introduction 13

2.2 Materials and Methods 15

2.3 Results and Discussion 16

2.4 Conclusion 21

References 21

3 A bioprospection of euphorbia cotinifolia for biofuel: 23

chromatography study

Punam Puri, Amita Mahajan, Anjana Bhatia, Navjot Kaur

Abstract 23

3.1 Introduction 23

3.2 Objectives 24

3.3 Methodology 253.4 Results and discussion 26

3.5 Discussion 29

3.6 Conclusion 29

References 29

4 Cost effective electrical power generation in punjab using 31

agricutural biomass

Suman

Abstract 31

4.1 Introduction 314.2 Status of Bio-energy Resources in Punjab 32



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4.3 Power Consumption in Punjab 34

4.4 Existing Technologies for Biomass Conversion 36

4.5 Biomass as a Coal Substitute 37

4.6 Environmental Criteria 37

4.7 Conclusion 38

References 39

5 Development of quality testing methodologies of fuel briquettes 40

Madhurjya Saikia, Bichitra Bikash

Abstract 40

5.1 Introduction 40

5.2 Parameters of Quality Assessment 41

5.3 Parameters of Combustion Characteristics of Briquettes 43

5.4 Conclusion 45References 45

Part-II: Thermochemical Conversion 48

6 Thermal and catalytic cracking of non-edible oil seeds to liquid fuel 49

Krushna Prasad Shadangi, Kaustubha Mohanty

Abstract 49

6.1 Introduction 49

6.2 Pyrolysis and its Types 51

6.3 Process parameters that affect the yield 51

6.4 Fuel properties of seed pyrolytic oil 53

6.5 Catalytic pyrolysis of non-edible seeds 55

6.6 Conclusion 56

References 56

7 Evaluation of micro gasifier cookstove performance with 58

handmade biomass pellets using region-specific fuels and

assessment of deployment potential

Debkumar Mandal, Vikas Dohare, Vijay H. Honkalaskar, Anurag Garg,

Upendra V. Bhandarkar, Virendra Sethi

Abstract 58

7.1 Introduction 59



7.4 Conclusions 67

References 67

8 Production of hydrocarbon liquid by pyrolysis of Camellia sinensis (tea) 68

seed deoiled cake and characterization of products Nabajit Dev Choudhury, Priyanko Protim Gohai,

Bichitra Bikash,



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Sashi Dhar Baruah, Rupam Kataki

Abstract 68

8.1 Introduction 68


8.3 Results and discussion 72

8.4 Conclusions 77

References 77

9 Comparative study of different biomass cookstove model: 79

An experimental study

K. Pal, A.K. Pandey, P Gera, S.K. Tyagi

Abstract 79

9.1 Introduction 79

9.2 Mathematical Modeling 819.3 Materials and Methods 86

9.4 Analysis of Cookstove 91


9.6 Conclusions 95

References 96

Part-III: Biochemical Conversion 98

10 Bioprospecting halotolerant cellulase from saline environment of 99

Bhitarkanika National Park, Odisha

Dash Indira, Sahoo Moumita, Dethose Ajay, C.S. Kar, R. Jayabalan

Abstract 99

10.1 Introduction 100

10.2 Materials and methods 101


10.5 Conclusions 106

References 107

11 Isolation and molecular characterization of cellulolytic fungi used 110for conversion of sugarcane biomass for bioethanol production

A.M. Chetan, K.M. Harinikumar, P. Bhavani, H.B. Manoj Kumar,

T. Madhu, Ningaraj Dalawai

Abstract 110


11.2 Material and Methods 111

11.3 Result and discussion 114

References 120

12 Application of thermostable cellulase in bioethanol production from 121lignocellulosic waste

Neha Srivatsava, Rekha Rawat, Harinder Singh Oberoi



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Abstract 121


12.2 Bioethanol production: Current production status and Challenges 123

12.3 Microorganism for thermostable cellulase production 124

12.4 Thermostable enzymes 125

12.5 Application of thermostable cellulases 128

12.6 Concluding remarks 129

References 129

13 Endoglucanases: characterization and its role in bioconversion of 135

cellulosic biomass

Rekha Rawat, Neha Srivastava, Harinder Singh Oberoi

Abstract 135


13.2 Mechanism of cellulolysis 136

13.3 Effect Classification of Endoglucanases 137

13.4 Structure of endoglucanases 137

13.5 Mechanism of cellulose hydrolysis by endoglucanases 139

13.6 Microbial sources of endoglucanase enzyme 139

13.7 Application of endoglucanases 140

13.8 Significance of thermostable endoglucanases 142

13.9 Factors responsible for thermal stability 142

13.10Conclusion 144

References 144

14 Comparative study of fermentation efficiency for bioethanol 149

production by isolates

Richa Arora, Shuvashish Behera, Sachin Kumar

Abstract 149



14.3 Result and Discussion 151

14.4 Conclusion 154

References 154

15 Sweet Sorghum - An ideal feedstock for bioethanol production 156

Reetika Sharma, Gurvinder Singh Kocher, Harinder Singh Oberoi

Abstract 156


15.2 Origin and biology of sweet sorghum 158

15.3 Cultivation and harvesting of sweet sorghum 159

15.4 Inherent advantages of sweet sorghum 160

15.5 Technical hurdles 16315.6 Bioethanol production from sweet sorghum 163

15.7 Energy ratio and environmental sustainability 165



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15.8 Small-medium scale bioethanol production plant from sweet sorghum 166


References 168

16 Fermentation of glucose and xylose sugar for the production of 175ethanol and xylitol by the newly isolated NIRE-GX1 yeast

Shuvashish Behera, Richa Arora, Nilesh Kumar Sharma, Sachin Kumar

Abstract 175


16.2 Material and method 177


16.4 Conclusion 180

References 181

17 Comparative bioethanol production by S. cerevisiae and Z. mobilis 183from saccharified Sweet Potato Root Flour ( Ipomoea batata L)

using immobilized α- amylases and glucoamylase

Preeti Krishna Dash, Sonali Mohapatra, Manas Ranjan Swain,

Hrudaya Nath Thatoi

Abstract 183




17.4 Importance of enzyme Immobilization 18917.5 Conclusion 191

References 191

18 Genetic modifications in yeast for simultaneous utilization of 194

glucose and xylose

Nilesh Kumar Sharma, Shuvashish Behera, Sachin Kumar

Abstract 194


18.2 Necessity of pentose (C5) sugar fermenting organisms 196

18.3 Problems with pentose (C5) sugar fermenting yeast 19718.4 Need of Genetic Engineering for xylose fermentation 198

18.5 Conclusion and future prospects 199

References 202

19 To optimize the process of alcohol production from banana peel 208

Mohit Jain, Anand Kumar Gupta, Sayan Chatterjee

Abstract 208


19.2 Materials used 210

19.3 Methodology 211

19.4 Results & Discussion 212



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References 216

20 Commercial production of bio-CNG & organic manure from 218

pressmud biomethanationPreetam Holkar, A.V. Mohan Rao, K.K. Meher

Abstract 218


20.2 Feed preparation 220

20.3 CSTR Digesters 220

20.4 Biogas cleaning process 222

20.5 BIO-CNG production & composition 223

20.6 Biogas utilization / storage devices cascades 224

20.7 Biogas based power 22420.8 Digestate (Organic Manure) 224


References 226

21 Feasibility of filling biogas in cylinders 228

S.S. Sooch, Jasdeep Singh Saini

Abstract 228



21.3 Discussion 23121.4 Conclusions 231

References 231

22 Effect of pretreatment on bioconversion of wheat straw for 232

the production of biogas

Nishshesh Singh, Vivek Saini, Pranshu Gupta, Rajan Sharma,

G Sanjay Kumar, Avanish K. Tiwari

Abstract 232


22.2 Material and methods 234



References 240

23 Ultrasonic pretreatment to enhance biohydrogen production 242

from food waste

Abhijit Gadhe, Shriram Sonawane, Mahesh Varma

Abstract 242

23.1 Introduction 24223.2 Material and methods 245




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23.4 Conclusion 250

References 250

24 Biological hydrogen production by facultative anaerobic bacteria 253

Enterobacter aerogens (MTCC 8100) Virendra Kumar, Richa Kothari, S.K.Tyagi

Abstract 253





References 261

25 Enhanced biohydrogen production from glycerol using pretreated 263

mixed culture

Anbalagan Krishnasamy, Mohanraj Sundaresan, Kodhaiyolii Shanmugam,

Pugalenthi Velan

Abstract 263





References 269

Part-IV: Chemical Conversion 272

26 Isolation and characterization of freshwater microalgae Scenedesmus 273

from contaminated field samples for bioenergy generation

Mayur M. Phukan, B.K. Konwar

Abstract 273



26.3 Discussion 278


References 284

27 Prospects of biodiesel production from non-edible oil seeds of 286

North East India: a review

Debashis Sut, Rupam Kataki

Abstract 286


27.2 Non-edible vegetable oils resources 289

27.3 R Fatty acid profiles of the biodiesel 292

27.4 Properties of the biodiesel 293

27.5 Conclusion 294



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References 294

28 A critical review of enzymatic transesterification: 298

a sustainable technology for biodiesel production

Neetu Singh, M.K. Jha, A.K. Sarma

Abstract 298


28.2 Biodiesel 299

28.3 Lipases as biocatalysts in biodiesel synthesis 303

28.4 Lipase immobilization 303

28.5 Variables affecting the enzymatic transesterification 305

28.6 Conclusion 307

References 307

29 Single step reaction for biodiesel production of Jatropha curcus seeds 313

Sanjaykumar N. Dalvi, Swati R. Sonawane

Abstract 313





References 318

30 Production of biodiesel from edible and non-edible oils: 319a comparative study

A.D. Singh, R. Rao, L.B. Reddy, H.K. Raghuvanshi, A.I. Kankia, H. Sharma,

S. Srivastava

Abstract 319


30.2 Scope for the Study 321

30.3 Research Methodology 321


References 325

31 Production of biodiesel from neem oil using synthesized iron nanocatalyst 327

Mookan Rengasamy, Sundaresan Mohanraj, Krishnasamy Anbalagan,

Shanmugam Kodhaiyolii, Velan Pugalenthi

Abstract 327





References 336

32 Influence of free fatty acids content in catalytic activity of [BSMIM] Cl 339



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ionic liquid for biodiesel production from non edible acidic oils

Subrata Das, Ashim Jyoti Thakur, Dhanapati Deka

Abstract 339




32.4 Conclusion 344

References 345

33 Analysis of physical properties and biodiesel production from 347

different accessions of Jatropha curcas

Dheeraj Singh, Chiranjib Banerjee, Animesh Sinha, Diwaker Prasad Nirala,

Santosh Prasad, Rajib Bandopadhyay

Abstract 347


33.2 Source: Jatropha Curcus 349

33.3 Different criteria which effect the biodiesel production 351


33.5 Production of Biodiesel 353


33.7 Conclusion 356

References 357

34 Analysis of exhaust emission from a diesel engine fueled with 359transesertified vegetable oils

Hemanandh J., Narayanan K.V.

Abstract 359


34.2 Background 360

34.3 Methodology 361

34.4 Results & Discussions 364

34.5 Conclusion 367

References 368

35 Genetic enhancement of Pongamia pinnata for bio-energy 370

M.V.R. Prasad

Abstract 370


35.2 Carbon Sequestration by Pongamia 372

35.3 Sybiotic Nitrogen Fixation and Soil Amelioration by Pongamia 373

35.4 Selection of Elite Trees 373

35.5 Vegetative Propagation 374

35.6 Vayusap Plantations 374References 378



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Part-V: Electrochemical Processes 380

36 Evaluation of electrical properties under different operating conditions 381

of bio-electrochemical system treating thin stillage

S. Ghosh Ray, M.M. Ghangrekar

Abstract 381



36.3 Experimental results and discussion 387

36.4 Conclusion 390

References 391

37 Effect of salinity, acetate addition and alteration of sediment on 394

performance of benthic microbial fuel cells D.A. Jadhav, M.M. Ghangrekar

Abstract 394




37.4 Future perspectives 405


References 406

38 Biohydrogen production using single-chamber membrane-free 409microbial electrolysis cell with a stainless steel cathode

Sundaresan Mohanraj, Krishnasamy Anbalagan, Kodhaiyolii Shanmugam,

Velan Pugalenthi

Abstract 409





References 416

39 Feasibility of interlinking two technologies for simultaneously 418

two bioenergies generation

Prashant Pandey, Vikas Shinde, S.P. Kale, R.L. Deopurkar

Abstract 418



39.3 Results 423

39.4 Discussion 428

39.4 Conclusions 428References 429



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Part-VI: Hybrid Systems 432

40 Development of nano based thermic fluid: rheological aspects of 433

new energy system

Vijay Juwar, Shriram Sonawane

Abstract 433


40.2 Materials and method 434



References 442

41 Conversion of plastic wastes into liquid fuels – a review 444

Arun Joshi, Rambir, Rakesh Punia

Abstract 444


41.2 Target of waste plastics into liquid fuel 447

41.3 Plastics recycling technologies 448

41.4 Process technology 448

41.5 Advantages of process of fuel production 450

41.4 Conclusion and recommendation 452

References 453

42 Kinetics of NOx reduction in BioDeNOx process water: effect of 455temperature and iron chelate

B. Chandrashekhar, Heena Tabassum, Nidhi Sahu, Padmaraj Pai, R.A. Pandey

Abstract 455



42.3 Analytical methods 459


42.4 Conclusion 465

References 465

43 Status of waste treatment, utilization and management in agro processing 467

Yogender Singh, Y.K. Yadav

Abstract 467


43.2 Agro processing industrial wastes treatment/utilization 470

43.3 Waste water in agriculture and food processing 472

43.4 Importance of waste management 474

43.5 Challenges in Waste management 474

43.6 Conclusions 474References 474



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Preface

Due to increasing prices of petroleum products, shortage of electricity supply, degradation of

environment and availability of millions of tons of surplus biomass, R&D activities in the

area of bio-energy including biodiesel, bioethanol, biomethanation, biomass gasification,

biomass cookstove, etc. have received the tremendous attention all over the world. Keeping

this trend in mind, the Govt. of India has already initiated the blending of 5% ethanol in the

gasoline, which is likely to increase up to 10% in the coming years. While, the Jatropha

Mission has been initiated for the promotion of Biodiesel in transportation and agriculture

sectors and it is expected that by 2020 at least 10% of the liquid fuel used in transportation

sector can be replaced by biodiesel. Similarly, MNRE has already initiated the dissemination

of 10 millions of improved biomass cookstoves in the 12th

five year plan through carbon

revenue. The renewable energy currently has made remarkable share (12.5%) of total primary

commercial energy supply of 228 GW, while the major share of around 70% of the total

generation capacity is from thermal (coal, gas, oil).

Since, energy security and diversification of the energy mix is a major policy driver for

renewables. Growth of renewables generally contributes to energy diversification, in terms of

the technology portfolio and geographical sources. Use of renewables can also reduce fuel

imports and insulate the economy to some extent from fossil fuel price rises and swings. This

not only increases the certainty for energy security but also symbolize the steady economic

growth of a country. However, the concentrated growth of variable renewables can make it

harder to balance power systems, which must be duly addressed. The electricity sector in

India had an installed capacity of around 228 GW, the world's fifth largest. Non-renewable

Power Plants constitute 87.55% of the installed capacity, and Renewable Power Plants

constitute of around 12.45% of total installed capacity while the major share is of biomass

based energy generation.

After receiving the great response of first two volumes on ‘Recent Advances in Bioenergy

Research’, we are introducing the third volume in the form of a book. The book is divided in

six parts viz. Part-I: Biomass and Energy Management; Part-II: Thermo-chemical Conversion;

Part-III: Chemical Conversion; Part-IV: Biochemical Conversion; Part-V: Electrochemical

Processes; and Part-VI: Hybrid Systems. Each section includes respective chapters from



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Eminent Academician, Scientists and Researchers in the field. We are grateful for their

commendable contribution for this book.

Emphasis is given in such a way that the current trends of research and investigation

in the bioenergy sector can easily be worked out from the in-depth study of this book. Our

efforts will be successful if the readers dig up the expected gain out of these articles.

Sachin Kumar



xvi

Contributors

Arora Richa, Biochemical Conversion Division, Sardar Swarn Singh National Institute of

Renewable Energy, Kapurthala

Bandopadhyay Rajib, Birla Institute of Technology, Mesra, Ranchi

Banerjee Chiranjib, Birla Institute of Technology, Mesra, Ranchi

Baruah Sashi Dhar, Regional Research Laboratory, Jorhat, Assam

Behera Shuvashish, Biochemical Conversion Division, Sardar Swarn Singh National

Institute of Renewable Energy, Kapurthala

Bhandarkar Upendra V., Department of Mechanical Engineering, Indian Institute of

Technology Bombay, Mumbai

Bhatia Anjana, Department of Botany, HMV College, Jalandhar

Bhavani P., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru

Bikash Bichitra, Assam Down Town University, Guwahati, Assam

Chandrashekhar B., Environmental Biotechnology Division, CSIR-National Environmental

Engineering Research Institute (CSIR-NEERI), Nagpur, Maharashtra

Chatterjee Sayan, University School of Biotechnology, Guru Gobind Singh Indraprastha

University, New Delhi

Chetan A.M., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru Chopra A.K., Department of Zoology and Environmental Sciences, Gurukula Kangri

Vishwavidyalya , Haridwar

Choudhury Nabajit Dev, Assam Down Town University, Guwahati, Assam

Dalawai Ningaraj, Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru

Dalvi Sanjaykumar N., Department of Physics, S. N. Arts, D. J. M. Commerce & B. N. S.

Science College, Sangamner, Dist. Ahmednagar, Maharashtra

Das Subrata, Department of Energy, Tezpur University, Tezpur, Assam

Dash Indira, Food and Bioprocess Technology Laboratory, National Institute of Technology,

Rourkela, Odisha

Dash Preeti Krishna, Department of Biotechnology, College of Engineering and

Technology, Biju Pattnaik University of Technology, Bhubaneswar

Deka Dhanapati, Department of Energy, Biomass Conversion Laboratory, Tezpur

University, Tezpur, Assam

Deopurkar R.L., Department of Microbiology, University of Pune, Ganeshkhind, Pune,

Maharastra

Dethose Ajay, Food and Bioprocess Technology Laboratory, National Institute of

Technology, Rourkela, Odisha



xvii

Dohare Vikas, Centre for Environmental Science and Engineering, Indian Institute of


Gadhe Abhijit, Department of Chemical Engineering, Visvesvaraya National Institute of

Technology (VNIT), Nagpur

Garg Anurag, Centre for Environmental Science and Engineering, Indian Institute of


Gera P., Dr. B. R. A. National Institute of Technology, Jalandhar

Ghangrekar M.M., Department of Civil Engineering, Indian Institute of Technology,

Kharagpur

Gohai Priyanko Protim, Department of Energy, Tezpur University, Napaam, Tezpur, Assam

Gupta Anand Kumar, University School of Biotechnology, Guru Gobind Singh

Indraprastha University, New Delhi

Gupta Pranshu, Chemical Engineering Department, University of Petroleum & Energy

Studies, Dehradun

Harinikumar K.M., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru

Hemanandh J., Department of Mechanical Engineering, Sathyabama University, Chennai

Holkar Preetam, Spectrum Renewable Energy Pvt Ltd, Kodoli, Warnanagar, Maharastra

Honkalaskar Vijay H., Centre for Technology Alternatives for Rural Areas, Indian Institute

of Technology Bombay, Mumbai

Jadhav D.A., School of Water Resources, Indian Institute of Technology, Kharagpur

Jain Mohit, University School of Biotechnology, Guru Gobind Singh Indraprastha

University, New Delhi

Jayabalan R., Food and Bioprocess Technology Laboratory, National Institute of


Jha M.K., Department of Chemical Engineering, Dr B R Ambedkar NIT, Jalandhar

Joshi Arun, Department of Chemical Engineering, Doon College of Engineering and

Technology, Dehradun

Juwar Vijay, Department of Chemical Engineering Visvesvaraya National Institute of


Kale S.P., Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research

Centre, Navi Mumbai, Maharastra

Kankia A.I., School of Biotechnology, Lovely Professional University, Phagwara, Punjab

Kar C.S., Office of the Principal CCF (Wildlife) & Chief Wildlife Warden, Bhubaneswar,

Odisha

Kataki Rupam, Department of Energy, Tezpur University, Napaam, Tezpur, Assam



xviii

Kaur Navjot, Department of Botany, HMV College, Jalandhar

Kocher Gurvinder Singh, Department of Microbiology, Punjab Agricultural University,

Ludhiana, Punjab

Konwar B.K., Department of Molecular Biology & Biotechnology, School of Science,

Tezpur University, Assam

Kothari Richa, Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar

University, Vidya Vihar, Lucknow

Krishnasamy Anbalagan, Department of Biotechnology, Bharathidhasan Institute of

Technology, Anna University, Tiruchirappalli, Tamil Nadu

Kumar G Sanjay, Chemical Engineering Department, University of Petroleum & Energy

Studies, Dehradun

Kumar H.B. Manoj, Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru

Kumar Sachin, Biochemical Conversion Division, Sardar Swarn Singh National Institute of


Kumar Virendra, Department of Environmental Science, Babasaheb Bhimrao Ambedkar

University, Lucknow

Madhu T., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru

Mahajan Amita, Department of Bio-chemistry, RBIEBT, Kharar

Mandal Debkumar, Centre for Environmental Science and Engineering, Indian Institute ofTechnology Bombay, Mumbai

Meher K.K., Spectrum Renewable Energy Pvt Ltd, Kodoli, Warnanagar, Maharastra

Mohanty Kaustubha, Department of Chemical Engineering, Indian Institute of Technology

Guwahati, Guwahati

Mohapatra Sonali, Department of Biotechnology, College of Engineering and Technology,

Biju Pattnaik University of Technology, Bhubaneswar

Narayanan K.V., Department of Mechanical Engineering, Sathyabama University, Chennai

Nirala Diwaker Prasad, Biotechnology, Genetics and Tree Improvement Division, Institute

of Forest Productivity, Lalgutwa, Ranchi

Oberoi Harinder Singh, Central Institute of Post Harvest Engineering and Technology,

Ludhiana, Punjab

Pai Padmaraj, Environmental Biotechnology Division, CSIR-National Environmental


Pal K., Thermochemical Conversion Division, Sardar Swarn Singh National Institute of


Pandey A.K., Thermochemical Conversion Division, Sardar Swarn Singh National Institute

of Renewable Energy, Kapurthala



xix

Pandey Prashant, School of Studies in Biotechnology, Jiwaji University, Gwalior- 474011,

Madhya Pradesh

Pandey R.A., Environmental Biotechnology Division, CSIR-National Environmental


Pathak Vinayak V., Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar

University, Vidya Vihar, Lucknow

Phukan Mayur M., Department of Molecular Biology & Biotechnology, School of Science,

Tezpur University, Assam

Prasad M.V.R., VAYUGRID, Bangalore

Prasad Santosh, Biotechnology, Genetics and Tree Improvement Division, Institute of Forest

Productivity, Lalgutwa, Ranchi

Punia Rakesh, Doon College of Engineering and Technology, Dehradun

Puri Punam, Department of Life Sciences–Biotechnology, Punjab Technical University,

Kapurthala

Raghuvanshi H.K., School of Biotechnology, Lovely Professional University, Phagwara,

Punjab

Rambir, Department of Chemical Engineering, Doon College of Engineering and

Technology, Dehradun

Rao A.V. Mohan, Spectrum Renewable Energy Pvt Ltd, Kodoli, Warnanagar, Maharastra

Rao R., School of Biotechnology, Lovely Professional University, Phagwara, Punjab

Rawat Rekha, Central Institute of Post Harvest Engineering and Technology, Ludhiana,

Punjab

Ray S. Ghosh, Advanced Technology Development Centre, Indian Institute of Technology,

Kharagpur

Reddy L.B., School of Biotechnology, Lovely Professional University, Phagwara, Punjab

Rengasamy Mookan, Department of Petrochemical Technology, Bharathidhasan Institute of


Sahoo Moumita, Food and Bioprocess Technology Laboratory, National Institute of


Sahu Nidhi, Environmental Biotechnology Division, CSIR-National Environmental


Saikia Madhurjya, Dibrugarh University Institute of Engineering and Technology,

Dibrugarh, Assam

Saini Jasdeep Singh, Department of Civil Engineering, Punjab Agricultural University,

Ludhiana, Punjab



xx

Saini Vivek, Chemical Engineering Department, University of Petroleum & Energy Studies,

Dehradun

Sarma A.K., Chemical Conversion Division, Sardar Swarn Singh National Institute of


Sethi Virendra, Centre for Environmental Science and Engineering, Indian Institute of


Shadangi Krushna Prasad, Department of Chemical Engineering, Indian Institute of

Technology Guwahati, Guwahati

Shanmugam Kodhaiyolii, Department of Biotechnology, Bharathidhasan Institute of


Sharma H., School of Biotechnology, Lovely Professional University, Phagwara, Punjab

Sharma Nilesh Kumar, Biochemical Conversion Division, Sardar Swarn Singh National

Institute of Renewable Energy, Kapurthala

Sharma Rajan, Chemical Engineering Department, University of Petroleum & Energy

Studies, Dehradun

Sharma Reetika, Department of Microbiology, Punjab Agricultural University, Ludhiana,

Punjab

Shinde Vikas, Department of Microbiology, University of Pune, Ganeshkhind, Pune,

Maharastra

Singh A.D., School of Biotechnology, Lovely Professional University, Phagwara, Punjab

Singh Dheeraj, Birla Institute of Technology, Mesra, Ranchi

Singh Neetu, Department of Chemical Engineering, Dr B R Ambedkar NIT, Jalandhar

Singh Nishshesh, Chemical Engineering Department, University of Petroleum & Energy

Studies, Dehradun

Singh Yogender, Department of Food Engineering and Technology, S.L.I.E.T., Longowal,

Punjab

Singson Lhaihoichong, Department of Environmental Sciences, Babasaheb BhimraoAmbedkar University, Vidya Vihar, Lucknow

Sinha Animesh, Biotechnology, Genetics and Tree Improvement Division, Institute of Forest

Productivity, Lalgutwa, Ranchi

Sonawane Shriram, Department of Chemical Engineering Visvesvaraya National Institute of


Sonawane Shriram, Department of Chemical Engineering, Visvesvaraya National Institute

of Technology (VNIT), Nagpur

Sonawane Swati R., Department of Chemistry, S. N. Arts, D. J. M. Commerce & B. N. S.

Science College, Sangamner, Dist. Ahmednagar, Maharashtra



xxi

Sooch S.S., School of Energy Studies for Agriculture, Punjab Agricultural University,

Ludhiana, Punjab

Srivastava S., School of Biotechnology, Lovely Professional University, Phagwara, Punjab

Srivatsava Neha, Central Institute of Post Harvest Engineering and Technology, Ludhiana,

Punjab

Suman, Punjab University, SSGRC, Hoshiarpur

Sundaresan Mohanraj, Department of Biotechnology, Bharathidhasan Institute of


Sut Debashis, Department of Energy, Tezpur University, Napaam, Tezpur, Assam

Swain Manas Ranjan, Department of Biotechnology, IIT Madras, Chennai

Tabassum Heena, Environmental Biotechnology Division, CSIR-National EnvironmentalEngineering Research Institute (CSIR-NEERI), Nagpur, Maharashtra

Thakur Ashim Jyoti, Department of Chemical Sciences, Tezpur University, Tezpur, Assam

Thatoi Hrudaya Nath, Department of Biotechnology, College of Engineering and

Technology, Biju Pattnaik University of Technology, Bhubaneswar

Tiwari Avanish K., Centre for alternate energy research, University of petroleum & energy

Studies, Dehradun

Tyagi S.K., Thermochemical Conversion Division, Sardar Swarn Singh National Institute of


Varma Mahesh, Department of Chemical Engineering, Visvesvaraya National Institute of


Velan Pugalenthi, Department of Biotechnology, Bharathidhasan Institute of Technology,

Anna University, Tiruchirappalli, Tamil Nadu

Yadav Y.K., Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala



Recent Advances in Bioenergy Research Vol. III 2014

1

Part I

Biomass and Energy Management




2

CHAPTER 1

THERMOGRAVIMETRIC CHARACTERIZATION OF WOOD

STALKS AS GASIFICATION AND PYROLYSIS FEEDSTOCK

Rakesh Punia, Sachin Kumar

Abstract

Energy is an integral part of a society and plays an important role in its socio-economic

development. A nation economic development can be assessed by the pattern of consumptionand quality of energy availability. Energy and chemicals from agriculture and regional

available wood residue can be efficiently produced by gasification and pyrolysis methods.

Indian has abundance of biomass sources. Selected physical and chemical properties of

biomass are related to thermochemical conversion, which has been observed to determine the

thermochemical conversion. The main objective of the present work is to investigate the

comparison of use of different biomass fuels and their parameters on the performance of small

scale downdraft gasifier, biomass fuels selected are locally available plant stalks such as

Prosopis juliflora (Kikkar), Eucalyptus (Sefeda), Pigeon pea (Arhar Dal), Albizia procera

(Surash), Melia sp. (Bakain) and Mulberry sp. (Sahatoot). Fuel feedstock characterisation of

selected biomass has been carried out at macroscopic as well as microscopic levels such as

determination of dry density, calorific value, proximate & ultimate analysis, and

thermogravimetric analysis (TGA). On the basis of characteristics, it is found that Melia sp.,

and Eucalyptus could be energy efficient feedstock for small scale downdraft gasifier possess

high fixed carbon content and calorific value as compared to other selected wood stalks.

Key Words: Wood stalks , Gasification, Thermogravimeter analyser, Reaction kinetics.

1.1 Introduction

To maintain the ecology, sustainable and equitable development become the critical

issues in most parts of world. The alarming population coupled with developmental activities

based on decisions for resource scarcity in many parts of India. A judicious choice of energy

utilization is required to achieve growth in a sustainable manner (Keyhani et al., 2010).

Gasification and pyrolysis can convert lignocellulosic materials to synthesis gas (syngas)

without the need for delignification. For different applications, the syngas from gasification




3

can be further converted and separated to other chemicals (by various reforming processes) or

fuel gas or hydrogen for fuel cell (Anjireddy et al., 2011).

Gasification is a thermochemical process by which any carbonaceous feed can be

converted to gaseous products with useable heating value (primarily carbon monoxide and

hydrogen in a controlled oxidizing atmosphere). Pyrolysis in particular converts biomass into

high energy content biofuels provided that the adequate temperature and heating rate are

reached and may be used to fuel internal combustion engines and gas turbines after an

intermediate process that converts the feedstock into a liquid or gaseous biofuel (Goyal et al.,

2008; Fantozzi et al., 2003) Pyrolysis is one of the first step of all thermochemical processes

occurring in an inert atmosphere (Fantozzi et al., 2010). Energy generation from biomass is

environmental friendly and does not increase the CO2 in the atmosphere. Biomass energy can

be generated locally and can make any country energy self-sustainable and less dependent on

foreign petroleum resources (McKendry, 2002). Interest in bioenergy has been enhanced

because it also manages the biomass wastes.

The advantage of gasification has ability to utilize a wide range of feedstocks ranging

from any plant residue, organic by-product (with protein, lignin or oil) of industry or even

municipal wastes , as compared with other bioenergy generation techniques. Gasification and

pyrolysis are efficiently viable options for processing biomass feed stocks, which cannot be

fermented to ethanol technically or economically (Kumar et al., 2008). Mathematical

modeling to predict the product gas qualities during gasification and pyrolysis requires the

reaction kinetics knowledge of biomass volatilization and its subsequent reactions.

Thermogravimetric analysis (TGA) is very useful in determining the reaction kinetics of

gasification and pyrolysis. It has been used extensively for the characterization of various

feedstocks. This method have been used by many researchers to determine the kinetics

parameter for bagasse in a nitrogen atmosphere (Nassar et al., 1996), rice husk in an oxygen

atmosphere (Mansaray and Ghaly, 1999), rapeseed straw and stalks in a nitrogen atmosphere

(Karaosmanoglu et al, 2001), forestry wastes in a nitrogen atmosphere (Lapuerta et al., 2004)

and poplar wood in a nitrogen atmosphere (Katarzyna et a.l, 2012). However, there is a lack

of kinetics information on gasification or pyrolysis of different wood stalks. This research

study surrounds to the thermochemical conversions properties of biomass and determine its

reaction kinetics in inert and oxidizing atmospheres using a thermogravimetric technique.




4

1.2 Materials and Methodology

Variety of biomass samples were collected from various accessible locations. Prosopis

juliflora, Eucalyptus, Albizia procera, Melia sp. and Mulberry sp. wood stalks were

purchased from Kapurthala timber market. Pigeon pea (Arhar Dal) stalks were collected from

SSS-NIRE (Kapurthala) campus. Biomass samples were collected from gasifier feed stock in

such a manner to represent homogeneity of biomass characteristics. Collected biomass

samples were sun dried for five days. Wood stalk dust/flakes were prepared on saw mill and

ground in grinder for analysis purpose. Ground wood samples were screened using vibratory

screen for attaining particle size of about 425 µm. Moisture content was removed from

screened samples by drying in oven Broadly, biomass characterization are categorized in two

types ; macroscopic and microscopic property. Macroscopic analysis of biomass fuel

properties are proximate analysis, ultimate analysis, particle size, bulk density, calorific value,

ash fusion temperature, etc. Biomass fuel properties for microscopic analysis includes thermal

properties, chemical kinetics, and mineral data, etc.

1.2.1 Proximate analysis

The proximate analysis of the sample was done as per ASTM standards. The

parameters namely moisture content (MC) (ASTM D3172-73), volatile matter (VM) (ASTMD3175-73) and ash content (ASTM D3174-73).

1.2.2 Ultimate analysis

The ultimate analysis of the biofuel is done with CHNS Analyzer. The modern

elemental analyzers of Elementor (Vario MICRO Cube), analysis samples from 1 mg to 800

mg solid or liquid samples.

1.2.3 Calorific value

This important characteristics of a fuel were determined by bomb calorimeter

(Toshniwal -CC01-M3). The crushed sample was compressed from the ground sample was

compacted from an original average density of 460 to 1200 kg/m3. The pellet was then

combusted to determine energy content.

1.2.4 Biomass density

The size and density affects the burning characteristics of biomass fuel by heating and

drying rate during combustion. It also dictates how the material is likely to behave during

subsequent thermo-chemical or biological processing as a fuel or feed stocks. Average mass




5

of is measured by Micro-balance and volume of dry biomass samples is determined by water

displacement method.

1.2.5 Kinetic study (activation energy)

In general, the microscopic characterisation of gasifier feedstock is done by

Thermogravimetric analysis (TGA), which is used to determine the kinetics parameter in

presence of oxygen or inert atmosphere. Pyrolysis is a sub-category of gasification, the

difference being this process takes place in an inert atmosphere (generally nitrogen)

Thermogravimetric analysis experiment is performed using on Perkin-Elmer (STA 6000)

equipment. The temperature of furnace and weighting system of TGA were calibrated

according to the manufacturer’s recommendation. Temperature calibration was performed by

measuring Curie points of alumel, nickel, perkalloy and iron.

Depending on the density of biomass, samples weight is placed in the pan of the TGA

microbalance. Approximately 20-42 mg of the wood powder were taken in crucible pan for

TGA experiment. Air is used as purge gases and all TGA experiments were conducted at a

constant purge flow rate of 20 ml/min. Residual weight of the sample and the derivative of

weight, with respect to time and temperature (differential thermogravimetry analysis, DTG),

were recorded. A program is prepared and saved in which samples were held at 30

o

C for 1min and heated to 850oC at the rate of 10oC/min and then held at 850oC for 1min.

1.2.5.1 Procedure to determine parameters of reaction kinetics

To calculate chemical kinetics of reaction using the procedure of Duvvuri et al. (1975)

as applied by Mansaray et al. (1999) and Karaosmanoglu et al. (2001) which is explained as

bellow :

Global kinetics of the vitalization reaction can be written as

−

= k xn (1)

Where, x is the sample weight, k the reaction constant and n the order of the reaction.

Applying the Arrhenius equation,

k = Ae-E/RT (2)

The combined form of the above two equations (1) and (2) can be written in linear form as

ln [

] = () − (

) + ln (

) , Put x =

(3)




6

where, wo is the initial weight at the starting stage, wf is the final weight at the end of TGA

and w is the weight at any temperature, dw/dt the ratio of change in weight to change in time,

A the pre-exponential factor and R the universal gas constant (gas value).

Eq. (3) is of the form

y = B + C x + D z, (4)

where

y = ln[

], x =

, z = ln (

) ,

B = ln(A); C = (-

), D = n

The constants B, C, D were estimated by multi-linear regression. The determination of

activation energy form selected data is done by regression calculation with Analysis Tool of

MS-Excel.

1.3 Results & Discussion

Analysis results for selected biomass samples which includes Proximate Analysis,

Ultimate Analysis, Density Measurement, Calorific Value, Activation Energy are contained in

this section.

1.3.1 Proximate Analysis

Proximate analysis data (Table-1) shows that the moisture contents of analyzed

biomasses were in between 5 to 8% (by weight) which is under the range of Downdraft

Gasifier biomass feedstock. Volatile Matter and Fixed Carbon has major role in heating value

of biomass fuels. Maximum VM of Melia was found (83.79%). Maximum Fixed carbon

content was of Eucalyptus and Pigeon pea has lowest (7.44%) FC value. Ash in the biomass

was in the range of 0.7 to 4% which plays a catalyst role during biomass gasification.

Table-1: Proximate analysis data of biomass feed stocks.

Proximate analysis (Wt.%)

Biomass Moisture Content Volatile Matter Fixed Carbon Ash

Prosopis juliflora 6.32 82.46 9.02 2.52

Eucalyptus 5.93 80.53 10.82 2.67

Pigeon pea 8.27 82.53 7.44 1.53

Albizia procera 6.72 83.58 8.07 0.79

Melia (Bakain) 5.03 83.79 8.73 1.37

Mulberry 6.24 81.93 10.04 1.69




7

Table-2: Results of biomass calorific value and Dry Density

Biomass Samples Calorific value (HHV)

MJ/ kg.

Dry Density

(Kg/m3)

Prosopis juliflora (Kikkar) 17.8038 496.711

Eucalyptus 17.4344 443.109

Pigeon pea (Arhar) 16.0138 272.509

Albizia procera 16.8509 326.300

Melia (Bakain) 17.8643 378.561

Mulberry (Sahatoot) 16.4012 251.785

On observing dry density data, it is evident that P. juliflora has substantially highest

density than other biomasses. Pigeon pea and Mulberry wood stalks have comparatively

lower dry density than other biomass samples.

1.3.2 Ultimate Analysis

Among all the biomass samples, Melia and Eucalyptus have comparatively higher

fixed carbon wt. % which indicates that they could produce syngas of high calorific value.

Nitrogen for analyzed biomass was less than 1% (by weight) of biomass. As evident from

data (Table-3), sulfur content is missing in all the samples.

Table-3: Ultimate Analysis of Biomass Samples

1.3.3 Thermogravimeter analysis (TGA)

Wood samples were characterized by thermogravimeter analyser under Nitrogen

(Inert) and Air atmosphere. The reduction in weight of biomass with steady state rise in the

reactor temperature were analysed.

Ultimate analysis (wt.%)

Biomass Samples Carbon Hydrogen Nitrogen Sulphur Oxygen

Prosopis juliflora 47.391 6.117 0.400 0.000 45.92

Eucalyptus 48.193 5.958 0.335 0.000 45.457Pigeon pea 47.314 5.837 0.450 0.000 46.073

Albizia procera 46.431 6.851 0.470 0.000 47.044

Melia (Bakain) 48.738 6.463 0.550 0.000 44.247

Mulberry(Sahatoot) 45.285 5.963 0.095 0.000 48.221




8

1.3.3.1 TGA in Nitrogen (inert) atmosphere

TGA plots of selected biomass in nitrogen atmosphere are similar to (Fig. 1). The first

stage of weight loss ranged from 30oC to around 125oC was clearly distinct from the other

stages of weight loss. It may correspond to the loss of water and light volatile compounds in

the biomass sample (Mansaray and Ghaly, 1999). Following the first stage, there was

negligible weight loss (< 0.5%) in the temperature range of 160–250oC. The second phase of

weight loss started around 250oC. The derivative plot of the region between 250 and 850oC

showed only one observable peak (Fig. 1).

Fig. 1: Typical TGA and DTG diagram of biomass in nitrogen atmosphere

When the data between 250 and 850oC were used for determining parameters of

reaction kinetics, the r2 values for the multiple-regression were less than 0.80, and the

predicted values deviated from the experimental data. This suggested that there may have

been two different reaction stages of weight loss occurring in this region (250–850oC). Total

of three distinct stages (Fig. 1) represented the global kinetics of weight loss occurring during

TGA of biomass in inert atmosphere.

1.3.3.2 TGA in Air (Oxidizing) Atmosphere

In an air atmosphere, the TGA plots similar to (Fig. 2) clearly suggested that there

were three stages of weight loss. The first stage in the oxidizing atmosphere ranged from 25 to

115–140 oC. It was very similar to the first stage in the inert atmosphere. The weight loss




9

between the end of the first stage (130oC) and the start of the next stage (240oC) was much

less (<5%). The second stage of weight loss ranged from 240 to 350–400oC, a very large

weight loss (~70%) at a very high rate (>10%/ oC) as compared with the inert atmosphere.

Separation between the second and third stages in the oxidizing atmosphere was very clear.

During the third stage, which ranged from 400 to 560oC, there was a small amount of

weight loss (~10%) at a slower rate. The weight loss in the third stage was very much lower

as compared with the second stage and also as compared with weight loss during the third

stage in an inert atmosphere. Also, the third stage in the oxidizing atmosphere had a very

narrow temperature range as compared with the third stage in the inert atmosphere. This

suggests that the third stage in the inert and oxidizing atmospheres were different. The amount

of oxygen in the air atmosphere in our experiment was sufficient for oxidation of a small

amount of biomass particles (~15–20 mg).

Fig. 2 -Typical TGA and DTG diagram of biomass in air atmosphere

1.3.3.3 Parameters of Reaction Kinetics

Kinetics of weight loss in air atmosphere at a heating rate of 10 oC/min was similar to

that in nitrogen atmosphere . But, at higher rates of 30 and 50 oC/min the reaction during the

second stage occurred very rapidly and activation energies were higher than activation

energies in nitrogen atmosphere.




10

Collected data is studied according stages from I to III and ash point of biomass. The

first stage of weight loss corresponds to the loss of water and light volatile compounds in

analysed biomass. The low moisture content in biomass samples resulted in low weight loss

during this stage of weight loss. The second stage may correspond to the major loss (65 to

80% wt.), due to the decomposition of cellulose and hemicellulose components and partial

loss of the lignin component of biomass. Following this stage, there was a continuous and

slow weight loss from 450–550 to 850oC, due to the thermal degradation of lignin or complex

high-molecular weight components of biomass. TGA stages shows that the performance of

eucalyptus and melia as gasifier feedstock are better than other selected biomasses. The data

between 250 and 450oC were used for determining parameters of reaction kinetics. Biomass

weight percent at the interval of 25oC are collected from main TGA data (Table 4).

Table-4 Activation Energy during second stage in an air atmosphere and N2 Atmosphere

Activation Energy

Biomass

Air Atmosphere N2 Atmosphere

E (kJ mol-1

) E (kJ mol-1

)

Prosopis juliflora 86.34 64.31

Eucalyptus 88.62 75.34

Pigeon pea 85.34 68.29

Albizia procera 86.74 66.74

Melia 78.53 68.53

Mulberry 76.35 59.57

1.4 Conclusions

TGA of wood stalks in nitrogen atmosphere indicates distinct three stages of weight

loss. A comparative study of different regionally available gasifier feed stocks i.e. Prosopis juliflora, Eucalyptus, Albizia procera, Melia sp. and Mulberry sp.was done on the basis of

determination of dry density, proximate analysis, ultimate analysis and thermogravimetric

analysis. Proximate analysis data shows highest fixed carbon content in Eucalyptus as

compared to Mulberry, which indicates high heating value. Ultimate analysis of all the

selected biomasses was found that all the samples were free from sulfur. The carbon

percentage was higher in Melia, and Eucalyptus as compared to other biomasses. Dry density

of Prosopis juliflora and Eucalyptus were found to be highest among all the selected

biomasses. On the compilation of TGA data, it was found that activation energy of Eucalyptus




11

is highest in both the cases (inert / air) which indicates that more external energy is required to

initiate the combustion process.

Acknowledgments

One of the authors (Rakesh Punia) would like to thank to Dr. A.K. Jain, the Director

of SSS-NIRE for their permission to perform experiments at SSS-NIRE, Kapurthala.

References

1. Bhavanam A. and Sastry R.C. (2011) Biomass Gasification Processes in Downdraft

Fixed Bed Reactors: A Review. Int. J. Chem. Eng. App., 2:425-443.

2. Duvvuri M.S., Muhlenkamp S.P., Iqbal K.Z. and Welker J.R. (1975) The pyrolysis of

natural fuels. J. Fire Flamm., 6:468-477.

3. Fantozzi F., D’Alessandro B. and Bidini G. (2003) IPRP – Integrated pyrolysis

regenerated plant – gas turbine and externally heated rotary-kiln pyrolysis as a biomass

waste energy conversion system. Influence of thermodynamic parameters. Proc. Inst.

Mech. Eng. A- J. Pow., 217:519-527.

4.

Fantozzi F., Laranci P. and Bidini G. (2010) CFD simulation of biomass pyrolysissyngas vs. natural gas in a microturbine annular combustor. Proc. ASME Turbo Expo:

Power for Land, Sea and Air, 14-18, Glasgow, UK.

5. Goyal H., Seal D. and Saxena R. (2008) Bio-fuels from thermochemical conversion of

renewable resources: a review. Renew. Sust. Energy Rev., 12:504517.

6. Karaosmanoglu F., Cift B.D. and Ergudenler A.I. (2001) Determination of reaction

kinetics of straw and stalk of rapeseed using thermogravimetric analysis. Energy

Sources, 23:767-774.

7. Keyhani A., Ghasemi-Varnamkhasti M., Khanali M. and Abbaszadeh R. (2010) An

assessment of wind energy potential as a power generation source in the capital of Iran,

Tehran. Energy, 35:188-201.

8. Kumar A., Lijun W., Dzenis Y.A., Jones D.D. and Hanna M.A. (2008)

Thermogravimetric characterization of corn stover as gasification and pyrolysis

feedstock. Biomass Bioenergy, 32:460- 467.

9. Lapuerta M., Hernandez J.J. and Rodriguez J. (2004) Kinetics of devolatilisation of

forestry wastes from thermogravimetric analysis. Biomass Bioenergy, 27:385-391.




12

10. Mansaray G.K. and Ghaly A.E. (1999) Determination of kinetic parameters of rice

husks in oxygen using thermogravimetric analysis. Biomass Bioenergy, 17:19-31.

11. McKendry P. (2002) Energy production from biomass (Part I): overview of biomass.

Bioresour. Technol., 83:37-46.

12. Nassar M.M., Ashour E.A. and Wahid S.S. (1996) Thermal characteristics of bagasse. J.

App. Polymer Sci., 61: 885-890.

13.

Slopiecka K., Bartocci P. and Fantozzi F. (2012) Thermogravimetric analysis and

kinetic study of poplar wood pyrolysis. App. Energy, 97: 491-497




13

CHAPTER 2

ASSESSMENT OF SOLID WASTE MANAGEMENT AND

ENERGY RECOVERY FROM WASTE MATERIALS IN

LUCKNOW ZOO: A CASE STUDY

Vinayak V. Pathak, Richa Kothari, A.K. Chopra, Lhaihoichong Singson

Abstract

Zoo is the facility in which animals are captured within enclosures, displayed to the publicand they may breed also. Thus zoo is the centers which provide entertainment, education and

protecting endangered species. The animals and the visitors inside the zoo generate large

amount of solid waste. The present work is a case study of Lucknow zoo in order to identify

the sources of solid waste generation and their sustainable management with an approach of

energy recovery. According to findings number of mammals (468), birds (100) and reptiles

(378) were present which generated 482 kg of fresh animal waste per day. On the other hand

various plant species such as Madhuca longifolia, Aegle marmelos, Poltalthia longifolia,

Cycas circinnalis, Ficus benhalensis etc were also observed which were produce 6.5 to 7 kg

of biomass per day. The selected zoo attracts more than 900,000 to 1000,000 of tourists and

visitors annually which contribute in solid waste generation. Various suggestive measures for

treatment of animal waste, plant waste and anthropogenic waste were identified for

conversion of this high organic waste in to energy rich biofuel.

Key words: Solid waste, Sustainable, Energy recovery, Biomass, Biofuel

2.1

Introduction

Wastes have been recognized as valuable sources whether it is organic or inorganic.

Various sources have been identified for the generation of waste like industrial, domestic,

agricultural or commercial etc. Animal waste generated from agricultural and commercial

practices primarily used for plant nutrient (feedstuff) and feedstock for energy production

(methane) (Morse, 1995; Williams, 1995; Gunaseelan, 1997). Animal waste are mainly

produced either in operations like dairy farming, poultry farming or in Zoo areas. Zoos are

the areas in which animals are confined within a definite boundary, where they breed and




14

displayed for the tourists, hence Zoo provide education and entertainment with protection of

endangered species (Gibson, 1980).

All animals and tourists produce large amount of waste inside the zoo premises which

consist of both organic as well as inorganic waste. Zoos having access of millions of people

have the ability to educate and communicate a number of people for sustainable lifestyles. A

serious contribution towards sustainable future can be made by the Zoo by following the

sustainability in their policies, strategies and management. A number of criteria such as

expenses, environmental impact, profitability, complexity and sponsorship are considered in

order to develop zoo as eco-development.

In India Central Zoo Authority (CZA) is the organizing body of Central government for

all zoos which is also associate member of world Association of Zoo and Aquarium (WAZA).

CZA classify zoos depending on area, number of animals and annual attendance of visitors.

CZA provide financial support to zoo and evaluate it time to time in order to provide

recognition.

Lucknow zoo at present has about 468 mammals, 378 reptiles, and many different

species of wild animals. The main attraction of this zoo is Royal Bengal tiger, White Tiger,

Lion, Wolf, Barking deer, Hog Deer, Asiatic Elephant, Giraffe, Zebra, Hill Mynahs, Giantsquirrels, Great pied hornbills, Golden pheasant, Silver Pheasant etc. Zoo not only protect

these animals but also achieve successful breeding of Swamp deer, Black buck, Hog deer,

barking deer, White Tiger, Indian wolf, and several pheasants..

A number of plants are also protected inside the zoo such as Madhuca longifolia,Aegle

marmelos, Poltalthia longifolia, Cycas circinnalis, Ficus benghalensis, tectona grandis,

Eucalyptus hybrid, Santalum album, Acacia nilotica, mangifer indica, Tamaridus indica,

cassia siamia, Delonix regia etc. Apart from being a conservation centre of various animalsit also provide various facilities such as an aquarium , nocturnal house, Botanical garden,

Museum, Jurassic Park, Solar power house etc. The Lucknow zoo attracts 9,00,000 to

1,00,00,000 of tourists due to the above-mentioned facilities which resulted in large amount

of solid waste generation.

This consists of complex waste such as polythene bags, wrappers, papers, plastic bottles

and waste from cafeteria. On the other hand animal present inside the zoo produces large

amount of organic waste. This is why the main objective of the present study is based on how



Recent Adva

to manage solid waste pro

for waste to energy on theo

Fig.1 Month wise total vi

2.2 Materials and Meth

2.2.1

Preparation of quest

In order to obtain tec

waste generation, its utili

categorized mainly in three

i. Animal waste

ii. Biomass waste

iii.

Anthropogenic was

2.2.2 Identification of diff

Various routes of co

vary depending on the type

it can be reduced or ma

predication is done on the

nces in Bioenergy Research

15

lems in zoo premises with identification of

retical assessment of data collected. .

itors in Lucknow Zoo (Data collected from t

Lucknow Zoo).

ods

onnaire

hnical information regarding management o

zation questionnaire method is adapted.

parts:

e

rent routes for conversion of waste to ener

version for waste to energy have been iden

of waste. According to the waste collected

age by using various advanced and tech

asis of data collected by the questionnaire m

Vol. III 2014

conversion techniques

he official website of

Zoo, amount of solid

he questionnaire was

gy

tified. The routes may

rom the zoo premises,

ical processes. The

ethod.



Recent Adva

Fi

2.3

Results and Discuss

2.3.1 Data collection by q

According to the dat

mammals, 378 reptiles and

waste which is valuable


16

ig. 2 Questionnaire for the animal waste

g. 3 Questionnaire for the Biomass waste

on

estionnaire method

a collected from the questionnaire it was f

100 birds. These animals are the source of l

or the bio-energy production. Apart from

Vol. III 2014

und that Zoo has 468

rge amount of organic

the animal waste big



Recent Adva

amount of plant derived w

and big trees were present.

Anthropogenic wast

plastic wrappers, bottles, p

the data collected from the

from the different type of

way without harming the e

Fig. 4

Table-1. Quantitati

Name of the animals

Himalayan black bear

Sloth bear

Giraffe

Rhinoceros

Swamp dear

Barking dear

Hog deer

Samber deer

Spotted deerRabbit

Total


17

aste is also collected in the Zoo as there is

was mainly consisting of non-biodegrada

olythene and food waste as a biodegradable

questionnaire different alternative of energ

aste materials. Thus the solid waste can be

vironment.

Questionnaire for the anthropogenic s wa

ve measurement of the Fresh animal waste in

No. of animals Waste

3 1 Ibs×3

3 1Ibs ×3

2 5 Ibs ×

1 1000 Ib

57 3500 g

23 3500 g

30 3500 g

15 3500 g

198 3500 g10 2800 g

342 482 kg

Vol. III 2014

more than 1000 small

ble materials such as

waste. On the basis of

sources can be opted

utilized in sustainable

te

the Zoo premises

enerated per day

(1 Kg. =2.2 Ibs )

s×1

day




18

Table-2.Quantitative measurement of plant derived waste inside the zoo premises

Sources Composition

Twigs Approx. 2 Kg/day

Dry leaves Approx. 3.5-4.0 Kg/day

Total Approximate 6.5-7.0 Kg/day

2.3.2 Potential alternative approaches for utilization of waste material

The suggestive measures based on data collected from the questionnaire can be taken

for the best use of solid waste generated inside the Zoo premises. These alternatives routes of

conversion may vary depending upon the type and composition of organic waste. Following

conversion routes are identified for the energy recovery from the waste collected from the

Zoo.

2.3.3 Animal and biomass waste

Animal and biomass waste having organic content are utilized as a feedstock in process

of biological conversion (Li et al., 2010) and thermo chemical conversion. Both waste vary

in chemical composition, physical form and quantity produced. The main factor involve the

variation of animal waste are

i.

Digestive physiology of various species

ii. Composition and form of the diet

iii.

Stage of growth and productivity of animal

On the other hand plant derived waste mainly differs in respect to their composition and

nature of degradability.

Fig. 4 Conversion platform for animal and biomass waste in to energy




19

a) Biological conversion

This process mainly involves biological treatment of animal and biomass waste by (i)

anaerobic digestion with full scale production of combustible biogas by using phototrophicmicroorganism such as Algae, (ii) fermentative process for production of Bio-hydrogen and

(iii) Plant nutrient to support crop production.

Anaerobic digestion involves breakdown of complex organic materials and the biogas is

formed by following hydrolysis, acidogenesis, acetogenesis and methanogenesis (Van

Haandel and Van der Lubbe, 2007). The solid compound gets liquefy in acidogenesis and

converted in to acid, alcohol and volatile fatty acids.

Bio-hydrogen production is carried out by following three steps such as

photosynthetically by algae in two stage photosynthesis and H2 production, photobiologically

by photofermentataive bacteria and by anaerobic fermentative bacteria.

Fig.5 Flow Diagram of Anaerobic Digestion Process and End Points of products

b) Thermo-chemical conversion

This is a high temperature chemical reforming process that breaks apart the bond of

organic matter and converts these intermediated in to char, syngas and highly oxygenated bio-

oil. The advantages of thermo-chemical conversion involve small footprints, efficient

nutrients recovery, no fugitive gas emission, short processing time on the order of minutes




20

and high temperature elimination of pathogens and pharmaceutically active compound

(Cantrell et al., 2007).

Pyrolysis, gasification and direct liquefaction these three processes are mainly identified

for the thermo-chemical conversion. Pyrolysis process mainly drive out in absence of oxygen

and it converts organic waste in to a mixture of char and volatile gases. Slow pyrolysis has

benefits of energy production and carbon credit generated from carbon sequestration (Tri et

al., 1996; Budenheim et al., 1994).

Direct liquefaction process includes hydrolysis of organic waste (lignocellulosic

materials) in to bio-oil. It proceeds in a pressurized environment (5-20 Mpa) and typically

occurs at low temperature.

This process convert organic portion of dry weight or biomass in to the minor by-

product and primarily non condensable permanent gases, CO, CO2, H2 and low molecular

hydrocarbon gases with the help of air, oxygen or stem as a reaction medium (McKendry,

2002).

2.3.4 Anthropogenic waste

These wastes are produced due to human activities and mainly consist of fraction of

degradable and non-degradable wastes. These wastes may create serious hygienic problem as

it contains high amount of organic matter and pathogens. Vermicompsting can be applied for

the degradable waste, which enhance the soil fertility and increases crop yield. Non

biodegradable waste can be managed by incineration. This process involves burning of solid

waste in a properly designed furnace under suitable temperature and operating conditions.

Incineration provide reduction of waste to one tenth of their volume without producing

offensive gases this process not only help in the elimination of odor but also protect the wall

of incinerator.

2.3.5 Evaluation of the Biogas and Electricity production

The total amount of organic waste collected inside the zoo premises was found to be

490 kg/day (approx.). This amount of organic waste is capable to produce 25.48 m3 /day

biogas and 25 kWh of electricity.




21

Fig. 6 Main thermo Chemical conversion Processes, their Intermediate products andsuggested End use

2.4

Conclusion

Waste management is highly important issue for Zoos as it poses as a good source of

various types of waste like animal waste, biomass and anthropogenic waste. These can be

managed using the 3 R’s (Recycling study guide, 1998) (Reduce, Reuse and Recycle of the

reduction principal of sustainability). The visitors of the zoo should be strictly prohibited from

throwing unwanted materials within the zoo premises in order to reduce the volume of waste

to great extent. From the present study area it has been found that of all the three wastes

(animal waste, biomass and anthropogenic waste) can be managed properly as per the

suggestive measures identified in the present study.

References

1.

Budenheim D.L. and Wydeven T., 1994. Advances in Space Research (ISSN 0273-1

177), 14:113-123.

2. http://www.lucknowzoo.com/list_of_visitors1.html

3.

Keri B. Cantrell, Thomas ucey, Kyoung S., Ro, Patrick G. hunt- Livestock waste to energy

generation opportunities, United states Department of Agriculture, ARS, coastal Plains soil,

Water and plant research center, 2611 W, lucas St. Florance, Sc29501, USA.

4.

Li, R., Chen, S., Li, X., 2010. Biogas production from anaerobic co-digestion of food

waste with dairy manure in a two-phase digestion system. Appl. Biochem. Biotechnol.

160, 643–654.




22

5. McKendry, P., 2002. Energy production from biomass (part 1): overview of biomass.

Bioresource Technol., 83: 37–46

6. Morse, D. 1995. Environmental considerations of livestock producers. J. Anim. Sci.

73:2733−2740.

7. P. W. Gibson (Bienergy Organizers, Inc., Baltimore, MD), Baltimore Zoo Digester

Project: Final

8.

Recycling Study Guide, Wisconsin Department of Natura Resources, January, 1988.

9. Report, DOE/R3/06058–T1, U.S. Department of Energy, 1980.

10. Tri, T.O., Edeen, MA., and Henninger, D.L., SAE 26th International Conference on

Environmental Systems, Monterey, CA. Paper #961592, 8p. July 8-1 1, 1996.

11.

V. N. Gunaseelan, “Anaerobic Digestion of Biomass for Methane Production: A

Review,” Biomass and Bioenergy 13, 83–114 (1997).

12. Van Haandel, A.C., van der Lubbe, J., 2007. Handbook biological waste water treatment:

design and optimisation of activated sludge systems, first ed. Quist, Leidschendam.

13. Williams, P.E.V. 1995. Animal production and European pollution problems. Anim. Feed

Sci. Technol. 32:106−115.




23

CHAPTER 3

BIOPROSPECTION OF EUPHORBIA COTINIFOLIA FOR

BIOFUEL: CHROMATOGRAPHY STUDY

Punam Puri, Amita Mahajan, Anjana Bhatia & Navjot Kaur

Abstract

Biodiesel have been receiving attention in recent years to overcome energy crisis. India is rich

in biodiversity and known for vast treasure of knowledge about use of plants for various

purposes. Many Euphorbiaceae species have been proposed as potential biofuel crops.

E.cotinifolia as one of the potential biofuel crop in future. In this research paper the focus will

be on the chemical constitutions that have been identified from Euphorbia cotinifolia species.

With different modes of chromatography, different lipids were determined. TLC in the

adsorption mode (silica gel), the principle application in lipid analysis is for the separation of

different lipid classes from plant tissues. It is a relatively easy matter to resolve each of the

main simple lipids from a tissue in one step, i.e. cholesterol esters, triglycerides, free fattyacids, cholesterol and diacylglycerols, using mobile phases consisting of a mixture of hexane

and diethyl ether, with a little formic acid to ensure that the free acids migrate successfully.

The aim of the study was to investigate the biochemical constituents and thin layer

chromatography (TLC) of the ethanol and acetone extracts of Euphorbia cotinifolia.

Keywords: - Euphorbia cotinifolia, chromatography, lipids analysis, complex lipids.

3.1 Introduction

Renewable energy is an alternative solution for fossil fuel because it is clean and

environmentally safe. Professor Melvin Calvin (Calvin, 1977) revived the idea that

hydrocarbon-producing plants could be used as future oil and other chemical sources. He also

gave the energy farming concept. The plant families mainly Euphorbiaceae and

Asclepiadaceae were screened for assessing their suitability as a source of low molecular

weight (mw) and non-polar petroleum-like hydrocarbons. Air-dried plant materials were

successively extracted with acetone and benzene, and the extracts were analyzed

spectroscopically for yield of rubber, wax, glycerides, isoprenoides and other terpenoides. In

the Euphorbiaceae family, the genus Euphorbia comprises 2000 species ranging from annuals




24

to trees and is subdivided into many subgenera and sections. This family is one of the largest

families in the plant world. All contain latex and have unique flower structures (Barla et al.,

2006; Chaudhry et al., 2001; Jassbi et al., 2006). In the search for new plants with a high

potential for the production of chemicals and liquid fuels as alternative energy sources,

several Euphorbia species have been previously examined for possible economic utilisation

(Zarrouk and Cherif, 1983; Hemmers and Gülz, 1986; Villalobos and Correal, 1992). This

genus has been investigated in view of different specialties, like more energy content, as

alternative source of hydrocarbons, laticifers, phytochemicals and systematic (4-10). Plants of

the genus Euphorbia are well known for their chemical diversity of their isoprenoid

constituents. An analytical screening program has been conducted by the USDA [11–15] to

evaluate and identify plant species as source of high energy, easily extractable compounds

suitable for fuel, chemicals and petroleum-sparing chemical feedstock. Plant families that

yielded more than one promising species were Anacardiaceae, Asclepiadaceae,

Caprifoliaceae, Compositae, Eupforbiaceae and Labiaceae. This research emphasized on

phytochemical screening of latex extract of E.Cotinifolia by chromatography techniques.

Chromatography is a method for separating the components of a mixture by differential

adsorption between stationary phases and a mobile (moving) phase. Thin-layer

chromatography and column chromatography are different types of liquid chromatography. Acolumn (or other support for TLC, see below) holds the stationary phase and the mobile phase

carries the sample through it. Sample components that partition strongly into the stationary

phase spend a greater amount of time in the column and are separated from components that

stay predominantly in the mobile phase and pass through the column faster. As the

components elute from the column they can be quantified by a detector and/or collected for

further analysis. An analytical instrument can be combined with a separation method for on-

line analysis (16-18).

3.2

Objectives

The general project objective is to evaluate the potential of integrating biofuel raw

material production in wasteland, adapted technology and their implications on energy access,

ecological sustainability, food security, economic and social efforts for wellbeing for future

generation.The present proposal deals with a plant of Euphorbia cotinifolia which will be

taken up for fatty acid and hydrocarbon estimation. In this research paper main objectives

are:-




25

i) Extraction of oil and different lipids polar as well as non polar lipids with respective

solvents by column chromatography technique.

ii) To study phyto-chemical screening of plant extracts of E.cotinifolia.

iii)

To find out the same compound in the known and unknown sample.

iv) Further analysis of biocrude oil (viscosity, density, cetane number, distillation range

water content, discussion of fuel properties of triglycerides etc.).

3.3 Methodology

3.3.1 Materials and methods

Biocrude of E. cotinifolia of acetone extract had determined through soxhlet extraction

method.

3.3.2 Phytochemical screening of plant extracts E.Cotinifolia

The latex acetone extract of E.Cotinifolia was analyzed by thin layer chromatography

(TLC) and column chromatography.

3.3.3 Method for Thin layer chromatography

TLC on silica gel G plates, on Silica Gel G plates (Loba chemie Pvt.Ltd.107,

Wodehouse road, Mumbai, India).Prepared silica gel slurry .Silica gel plate of layer thickness

0.25 mm kept in oven activation for 2-3 hrs at 700 C. Sample of acetone extract of

E.Cotinifolia and standard oleic acid were loaded and allowed to run in developing solvent

Petroleum Ether: Diethyl Ether: Acetone (7:3:0.1), spray reagent concentrated sulphuric acid,

p-anisaldehyde and glacial acetic acid (2:1:100). After development, the phytocompounds

plates were transferred into iodine chamber (resublimed iodine, Avarice laboratories Pvt. Ltd.,

India).

3.3.4 Method for Column chromatography

Weighed 5gm silica gel 60-120 (Avarice laboratories Pvt. Ltd., India) and made slurry

in water. Poured the gel into the column tightly so that no air spaces were left. This left a

space of 4-5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column

securely to a ring stand .5ml sample of 3 mg acetone extract was loaded in the silica gel filled

column. Once the sample was in the column, fresh eluting solvent was added to the top and

we were ready to begin the elution process. Only force the solvent to the very top of the silica:




26

do not let the silica go dry. Add fresh solvent as necessary. The following solvent system ran

in the column chromatography were:-

1)

Hexane: petroleum ether (90:10) respectively.

2) Hexane: petroleum ether (50:50) resp.

3) Petroleum ether: diethyl ether (50:50) resp.

4) Diethyl ether: acetone (50:50) resp.

5) Acetone: methanol (50:50) resp.

6)

Methanol 100%

The first solvent running in the column was hexane and petroleum ether in (90:10

ratios) respectively and the total quantity was 100 ml. There was appearance of 3 colored

bands in the column. The colored bands were travel down the column as the compound was

eluted. As soon as the colored compound began to elute, the collection beaker was changed.

The colored fractions were collected separately. Similarly all the above solvents ran in the

column and collected different fractions from the running column.

3.4

Results

Results of TLC

Fig. 1 Biocrude of plant extract spp. E.Cotinifolia

Fig. 2 Performing TLC




27

Fig 3. Results of TLC Fig 4. Results of TLC

Results of column chromatography

Fig. 5. Shows loaded sample in coloumnchromatography

Fig 6. Shows band formation by using

different solvents running in the column

chromatography.




28

Fig 7. Shows different colored band

formation by using solvent hexane:

petroleum ether (90:10) respectively.

Fig 8. Shows band is travelling down in the

column.

Fig 9. Shows oil sample was obtained by

running first solvent i.e. hexane: petroleum

ether (90:10)

Fig 10. Shows five fractions in

eppendorf vials were obtained from

respective solvents.




29

3.5 Discussion

The results obtained from TLC and column chromatographies were good. In TLC

standard sample was oleic acid travel with acetone extract of biocrude of E. Cotinifolia.in the

results arrows shows that there was a same compound in E. Cotinifolia which were present in

the known sample. In fig. 4, TLC results shows that the standard oleic ran with oil sample

extracted during column chromatography. The good results were obtained.

In column chromatography the results were also good and surprising we got oil in very

small fractions from 3 mg sample during running the solvent hexane and petroleum ether

(90:10) and other five fractions were collected from respective solvents. In future we will do

the triglycerides analyses (viscosity, density, cetane number, distillation range water content

discussion of fuel properties of triglycerides etc.). The sample will be forward to SAIF Centre

Chandigarh for IR, GC/MS AND NMR data.

3.6 Conclusion

The bio-energy system makes a significant contribution to the world’s growing energy

needs. The renewable sources would only be able to compete with the fossil fuel resources, if

special plant crops containing energy-producing, hydrocarbon-like material are breed and

cultivated. A great advantage of utilization of such plants is by replacing the current use of the

traditional food crops for fuel production and providing the biodiesel industry with a more

consistent "green" supply. Large scale experiments would be required to analyses of the

different classes of secondary metabolites isolated from this plant. In this paper more

emphasize on phyto-chemical analysis of compounds and extraction of components from

different running solvents system and all these extractions further analysed by different

techniques. Hope this will be the one of the future petrocrop in the world.

References

1. Calvin M. Chem Eng News 1978;20:31–6.

2.

Barla A, Biraman H, Kultur S, et al. Secondary metabolites from Euphorbia

helioscopia and their Vasodepressor activity.Turk J Chem, 30, 2006, 325- 332.

3. Zarrouk M. and Cherif A. (1983), Lipid contents of Biol. Plant. 42, 417Ð422.

4. Kalita D, Saikia CN (2004). Chemical constituents and energy content of some latex

bearing plants, Bioresource Technology 92 (3), 219-227.




30

5. Monacelli B, Valletta A, Rascio N, Moro I, Pasqua G (2005). Laticifers in

Camptotheca acuminata Decne: distribution and structure. Protoplasma 226 (3-4),

155-161.

6.

Bruni, R, Muzzoli, M, Ballero, M, Loi, MC, Fantin,0G, Poli, F, Sacchetti, G. (2004).

Tocopherols,Fatty Acid acids and sterols in seeds of four Sardinian wild

7. Mallavadhani UV, Satyanarayana KVS, Mahapatra A, et al. (2006). Development of

diagnostic, microscopic and chemical markers of some Euphorbia latexes Journal of

Integratıve Plant Biology 48 (9), 1115-1121.

8. Shi HM, Williams ID, Sung HHY, et al. (2005). Cytotoxic diterpenoids from the roots

of Euphorbia ebracteolata, Planta Medıca 71 (4), 349-354.

9.

Jiao, W, Mao, ZH; Dong, WW, et al. (). Euphorbia factor L-8: a diterpenoid from the

seeds of Euphorbia lathyris. Acta Crystallographica Section E-Structure Reports

Online, 64, (03).

10.

Suarez-Cervera M, Gillespie L, Arcalis E, L Thomas A., Lobraeau –Callen D.,

Seoane– Camba JA., (2001). Taxonomic significance of sporoderm structure in pollen

of Euphorbiaceae: Tribes Plukenetieae and Euphorbiceae. Grana 40 (1-2), 78-104.

11.

Abbott TP, Patterson RE, Tjark LW, Palmer DM, Bogby MO. Econ Bot 1990;44:278–

84.12.

Bagby MO, Buchanan RA, Otey FH. In: Klass DL, editor. Biomass as a non fossil fuel

source. ACS Symposium. Series, vol. 144. 1981. p. 125–36.

13. Campbell TA. Econ Bot 1983; 37:174–80.

14. Carr ME, Bagby MO, Roth WB. J Amer Oil Chem Soc 1986;63:1460–4.

15. Seiler GJ, Carr ME, Bagby MO. Econ Bot 1991;45:4–14.

16. Archer J. P. Martin (1952). "The development of partition chromatography". Nobel

Lecture, December 12, 1952. Nobel Lectures, Chemistry 1942-1962, ElsevierPublishing Company, Amsterdam, 1964.

17.

Ettre, L. S. (2001). "The Predawn of Paper Chromatography". Chromatographia, vol.

54, pp. 409-414.

18. Frederick Sanger (1988). "Sequences, Sequences, and Sequences". Annual Review of

Biochemistry, vol. 57 (1988), pp. 1-28




31

CHAPTER 4

COST EFFECTIVE ELECTRICAL POWER GENERATION IN

PUNJAB USING AGRICUTURAL BIOMASS

Suman

Abstract

Energy is the key input to drive and improve the life cycle. The impact of the traditional fossil

fuels on our environment and the fact that these are fast depleting sources of energy, have

encouraged the need to find alternative energy sources to fossil fuels. Biomass can be burned

for fuel by itself or co-fired with other fuels. But in recent years Biomass and Coal co-fired

based systems are receiving more attention due to high Power Generation Efficiency and

reduced Green House Gas (GHG) emissions. This paper critically analyzes the scope,

potential and implementation of agricultural -Biomass conversion to Energy in Punjab

context. Brief descriptions of potential conversion routes have been included, with their

possible and existing scope of implementation. As far as possible, the most recent statistical

data have been reported from various sources. The discussion reveals that a large potential

exists for the Biomass feed-stocks from the various kinds of waste Biomass. The analysis to

identify irreversibility and the ways to improve the performance of Power Generation systems

is discussed. The Energy generated from various kinds of Biomass products is analyzed and

its role to improve the Power Generation systems is also presented.

Keywords: Biomass, Electrical energy, Efficiency, Greenhouse gas (GHG), Green Economy,

Power Generation

4.1

Introduction

In recent years, the World is facing Energy crisis, Economic, Green & clean

Environmental problems. A lot of research efforts are put to find economically viable and

sustainable energy resources to reduce this energy crisis with green and clean environment.

With growing population, improvement in the living standard of the humanity,

industrialization of the developing countries, the Global demand for energy is expected to

increase. India rank fifth in the world in total energy consumption (with installed capacity

228.722 GW up to September 2013). Coming to Power production in the country, India ranks




32

sixth in the world with increased installed power capacity from 1362MWh to 855.3 billion

kWh (up to 2012) since independence [6]. This achievement is impressive but not sufficient.

The country still encounters peak and energy shortage of 9 % & -8.7 % respectively (up to

2013). The major sources which meet the energy requirement of India are coal and oil. The

use of these fuels is a problem because of the reasons: a) The natural formation of Coal and

Oil is a very slow process which takes long time .b) Emission of Green house gases. c) These

are fast depleting sources of Energy. Moreover, India is dependent on the imports for Oil

requirements. In 2004–05, 72% of India’s total oil consumption was dependent on the imports

[2]. This figure reached to 76.5% during 2009–10, 78% for 20010–11, and the tentative figure

for 2011–12 is 80.5% [3]. These imports are increasing year after year with the growing

economy of the country and contribute in continuous increase of the import bills. By 2025, it

will be importing 90% of its crude oil from OPEC countries. Therefore, Utilization of

renewable energy sources is one of the best ways to meet the objectives as : a) These are the

energy sources that will never run out. b) These sources are Environmental friendly means

reduce Green house effect and provide clean Environment. c) Social –cost benefits. The

major Renewable sources of Energy available freely are Solar energy, Wind energy, Small

Hydropower, Biomass, Biogas, and Energy recovery from Municipal and Industrial wastes.

4.2 Status of Bio-energy Resources in Punjab

India’s energy basket has a mix of all the resources available including renewable.

Biomass contributes as the world’s fourth largest energy source up to 14% and in developing

countries it can be as high as 35% of the primary energy. Punjab the “Grain Bowl” of India is

the major agriculture state of the country. Agricultural biomass has immense potential for

power production in Punjab. Punjab has made tremendous progress not only in the agriculture

sector but in the industrial, transport and household sectors. This has increased energy

demand significantly. This state does not have its own resources of conventional fuels such as

coal, petroleum products for electricity energy. The state has to depend on neighboring states

for petroleum products and on the far-off states for coal. But the state has plenty of renewable

energy sources, such as biomass, wind and solar energy, which can be exploited to provide

sustainable energy base for socio-economic development. Table 1: shows the various type of

biomass available in Punjab [3].

Punjab "Granary of India” is historically considered to be one of the most fertile areason Earth. The region is ideal for growing rice, wheat, cotton, sugarcane, maize and




33

vegetables covering nearly 1.5 percent of India's land. Today the state produces nearly India’s

11% rice, 22% wheat, 18 % cotton and 3 % sugarcane .In worldwide terms; this represents

1/30th or 3% of the world's production of these crops, so Punjab produces world’s 1% of

cotton, 2% of its wheat, 2% rice and 0.3% of sugarcane. Table2: Shows production of major

crops during the recent years with area in Punjab & it is clear that from the last few years’

production of major crops increases which result in increase in the bio waste product [5].

Table 1: Type of Biomass

S.No. Type of biomass Name of crop

1 Straw Wheat ,Paddy, Barley, Pulses

2 Stalk Cotton, Maize, Rapeseed &mustard

3 Bagasse Tops & leaves Sugarcane Sugarcane

4 Cobs Maize

5 Husk Paddy

Table-2: Production of Major Crops during the Recent Years

Year

Type of

Bio-

Mass

2009- 10 2010-11 2011-12 2012-13 2013-14(E)

Area

(lac

ha.)

Production

(lac MT)

Area

(lac

ha.)

Production

(lac MT)

Area

(lac

ha.)

Production

(lac MT)

Area

(lac

ha.)

Production

(lac MT)

Area

(lac

ha.)

Production

(lac MT)

Rice 28.02 112.36 28.31 108.37 28.18 105.42 28.45 113.69 27.50 110.00

Sugar

cane

0.60 40.56 0.70 49.04 0.80 56.53 0.82 56.73 0.95 66.50

Wheat 34.02 151.69 35.10 164.72 32.03 125 34.52 140.60 35 161.02

cotton 5.11 20.06 4.83 18.22 5.15 16.21 4.81 16.44 5.20 19.58

Maize 1.39 4.75 1.33 4.91 1.26 5.02 1.29 4.71 1.50 5.40




34

On the basis of survey of ratios of various major crop residues for the year 2012-2013,

the net production of residue could be around 481 Lac MT ,as described in greater detail in

Table 3.

Table -3: Estimation of Biomass Production in Punjab (Crop Wise Data)

Crop Main Crop Production

(Lac MT)

Type of

Residue

Crop to

Residue

ratio

Residue

Quantity

(Lac MT)

Rice 113.69 Husk 0.3 34.107

Straw 1.3 147.797

Wheat 140.60 Straw 1.5 210.9

Sugarcane 56.73 Bagasse 0.3 17.019

Tops & leaves 0.09 5.105

Cotton 16.44 Stalk 3.5 57.54

Gin Waste 0.1 1.644

Maize 4.71 Stalk+Cobs 1.5 7.065

Grand Total 481.177

Further studies indicate that about 15-20% of the agriculture residue is available for

power generation rest is used for other purposes such as cooking & cattle feed. So we have

nearly 100 Lac MT crop residue is available for Power Generation [1].

4.3 Power Consumption in Punjab

The Total Demand of Electricity in Punjab is 48724 MU. The availability of Energy in

Punjab is approximately 46119 MU, facing energy shortage of 5.2%. Total energy in Punjabstate is provided by the PSPCL with its own Thermal Plants and Hydro Plants. Electricity

demand of Punjab will vary with changes in weather. On an average, the demand of power in

Punjab will vary between 1,039LU to 2,072LU while the availability of power will also vary

between 873LU to 1,584LU during different months of the year [4].

The variation in annual demand and energy requirement for the year April 2012 to

March 2013 are given in Table 4. The common pool projects are the Bhakra Nangal Complex,

the Dehar Power Plant & the Pong Power Plant. Punjab shares about 51% of the Power

generated from the Bhakra Nangal Complex. 48% from the Power generated at the Pong




35

Project. By including this share of generation Punjab is still deficit with 2600 MU Power.

According to PSPCL estimates, the power-supply gap will vary between 4% to 31% the entire

year.

Table -4: Month Wise Power Supply Position of Punjab in 2012-13

Table -5: Anticipated Power Supply Position in the Punjab during 2013-14

Energy Peak

Requirement

(MU)

Availability

(MU)

Surplus(+)/Deficit(-)

(MU)

Requirement

(MU)

Availability

(MU)

Surplus(+)/Deficit

(-)

(MU)

50850 40819 -10031 -19.7 12200 9075 -3125 -25.6

Year Demand Energy

Peak

Demand

Demand

Met

Surplus

(+) /

Deficit (-)

(%)

Surplus

/Deficit

Energy

requirement

Availability Surplus(

+)

/Deficit

(-)

(%)

Surplus/

Deficit

April -12 6391 5246 -1145 -17.9 3031 2948 -83 -2.7

May -12 7236 6091 -1145 -15.8 3763 3651 -112 -3.0

June -12 10474 8452 -2022 -19.3 5437 5053 -384 -7.1

July -12 11520 8073 -3447 -29.9 6611 5867 -744 -11.3

Aug-12 9114 8751 -363 -4.0 5923 5374 -549 -9.3

Sep-12 8147 8147 0 0.0 4745 4622 -123 -2.6

Oct-12 8441 6860 -1581 -18.7 3813 3671 -142 -3.7

Nov-12 5676 4502 -1174 -20.7 3040 2941 -99 -3.3

Dec-12 5336 5336 0 0.0 2517 2362 -155 -6.2

Jan-13 5797 5197 -600 -10.4 3055 2938 -117 -3.8

Feb-13 5197 5018 -179 -3.4 3917 3844 -73 -1.9

Mar -13 5264 5264 0 0.0 2872 2848 -24 -0.8




36

The studies carried out for formulating the anticipated power supply in Punjab for the

next year 2013-14, as shown in Table 5. indicate that there would be Energy shortage of

19.7% and Peak shortage Of 25.6% in Punjab.

4.4 Existing Technologies for Biomass Conversion

Due to technological developments and cost reductions, renewable solar, hydro, wind

and biomass energy are gaining momentum across the globe. There are a variety of processes

and technologies that convert biomass into heat, steam, electricity, and other types of fuel &

products. Some of them are depicted in Table 6 [2].

Table 6: Waste Agricultural Biomass to Energy – Technology

S.No Type of

Technology

Examples of Types of

Waste Handled

Byproducts Applications

1 Direct

Combustion

Crop residues such as

wheat straw, rice straw,

rice husk, Bagasse

Carbon Dioxide, Water

& Heat

Power Generation ,

Heating , Cooking

2 Gasification Crop residues such as

wheat straw, rice straw,rice husk

Syngas, Heat, Some

CO2 and H2O

Power Generation ,

Heating , Cooking ,Transportation

3 Pyrolysis Crop residues such as

wheat straw, rice straw,

rice husk

Bio- Ethanol Power Generation ,

Transportation

4 Fermentation Sugarcane & starch

substrates like wheat,

maize, sugar beet

Solids (charcoal),

Liquids (Pyrolysis oils)

and a mix of

Combustible gases

Power Generation ,

Transportation ,

Heating

5 Esterification Rape-seed Glycerine and

RME(RapeMethyl

Ester)

Power Generation ,

Transportation

Above mentioned technologies which are already installed must be upgraded keeping

requirements in mind while those which are presently running Global like Fermentation,

Esterification. Brazil recovered from oil crisis because of development of Cars powered by




37

100% Ethanol or Petrol or combination of both, such technologies are awaited to be modeled

for Punjab’s Energy Policy.

4.5

Biomass as a Coal Substitute

Biomass Power technologies compete in niche applications as well as in direct

competition with Conventional Electricity sources in Centralized Electricity supply. In large

scale grid based applications, cost is the primary determinant of competitiveness. A power

plant with the capability of producing 8MW of electricity could cost up to 40 crore INR.

While annual maintenance (done for 1week twice a year) costs 50 Lakhs INR. Variable

expenses are related to price of Biomass cost (approx. 3500-5500 INR per metric ton) is

highly variable, depending upon the source, location etc while other expenses includemanpower wages [1].

Operating life of Biomass Power plant lies between 25 to 30 years. since the cost of

setting a biomass plant is high as compared to thermal plant but it has many advantages over

thermal power plant such as--a) Biofuels can be transported and store and allow for heat and

power generation on demand. b) The energy balance of biomass plants indicates that biomass

energy is 10 to 30 times greater than the energy input for fuel production and transport. c)

Accessibility in rural areas where commercial fuels and centralized electric grid are not

available. d) Greater employment for local populations. 5) Restoration of deforested and

degraded lands by energy plantations. e) Near-zero fuel costs (paid in local currency),

commercial use of a waste product, decentralized supply and increased fuel efficiency leading

to an increase in the economic. f) Cost of electricity per unit from biomass power plant is

lower than coal plan.

4.6 Environmental Criteria

Expanding the share of Renewable Energy in its Energy mix is one of the key pillars of

India’s low-carbon development strategy. The Biomass fuels are more suitable & promising

than coal due to its low carbon, sulphur and nitrogen content as depicted in Table 7. Since

CO2 and acidification of SO2 & NO2 are primarily responsible for global warming & coal

contain maximum value of these elements (Carbon, Nitrogen & Sulphur) as compared to

other Biomass.[1] So coal contributes more towards the Global warming. Moreover

depending upon the content of Carbon & Sulphur there is Environmental taxes (High Tax &

Low Tax). High tax scenario with $50 per ton of carbon tax and $400 per ton of Sulphur

dioxide tax. Low tax scenario with $25 per ton of carbon tax and $200 per ton of Sulphur




38

dioxide tax. The cost of delivered Electricity under the Low tax and High tax cases for Coal

Power increases by 1.4 and 2.8 cents/kWh, respectively.

Table -7: Estimated Analysis:(Where C-carbon, H-hydrogen,O-oxygen, N-nitrogen,S-sulphur)

C H O N S Ash content (%)

Coal 75 5 8 1.5 0.5 10

Rice husk 38.35 5.08 36.24 0.56 0.16 14.9

Wheat straw 48.54 5.73 40.71 0.81 0.17 8.5

Rice straw 43.36 5.44 39.03 0.87 0.10 19.2

Maize(stalk+cob) 49 4.9 - 0.6 -Bagasse(dry basis) 49 6.5 42.7 0.2 0.1 1.5

Cotton stalk 51 4.9 43.87 1.00 - 6.68

Further Biomass fuels have less reactive character as compared to Coal & Cogeneration

applications in agriculture processing industries typically achieve fuel efficiency of 40 to 45%

compared to 30% efficiency of the conventional technologies . Although the conversion of

Biomass to Electricity in itself does not emit more CO2 than is captured by the Biomass

through photosynthesis.

This analysis suggests that under, these advantages, together with more efficient and

versatile Biomass Electricity Generation with Modern Technologies, have led to the transition

of re-emergence of Biomass as a competitive and Sustainable Energy option in the Future

Energy Scenarios.

4.7 Conclusion

Significant conclusions of this paper are as follows:

a) Punjab has abundant capacity to produce reliable, price competitive and ecologically

sustainable Bio-energy to meet the energy demand of domestic and commercial sector. A

number of such Power Generation project have not only solved the rural electrification

problem for the remote villages, where infrastructural costs could have been quite high

for conventional electrification, but also the power generation cost has also been

relatively low.




39

b) In Biomass, Carbon, Nitrogen & Sulphur contents are low, which favours in lesser or

Zero Global warming. Moreover their quantity decides the Environmental tax, so for

biofuel we have to pay less tax as compared to Coal. Further, it is analyzed that

replacement of each KWh of Coal –based electricity by Biomass-based electricity is

likely to reduce CO2 by 1Kg.

c) Biomass based decentralized generation is likely to generate direct or indirect & skilled

or unskilled employment to about 84 people in rural areas.

d) Biomass based power plants helps in reducing the bio-waste disposal problem in effective

way.

e) Cogeneration applications in agriculture processing industries achieve fuel efficiency up

to 40 to 45% as compared to 30% efficiency of the conventional technologies.

Apart from having so many Economical & Environmental benefits, it also opens a new

door for future innovations in our Country.

Refereences

1. Buljit Buragohain, Pinkeswar Mahanta & Vijayanand S. Moholkal (2010) Biomass for

decentralized power generation :The India perspective . Science Direct ,14:73-92

2.

Sara Schuman & Alvin Lin(2012)China’s Renewable Energy Law & its impact on

renewable power in China. Energy policy ,51:89-101

3.

Jagtar Singh , B.S Panesar & S.K. Sharma (2008) Energy Potential through

agricultural biomass using geographical information system-A case study in Punjab.

Science Direct , 14:301-307

4. Load Generation Balance report 2013-2014 by Central Electricity Authority

5. Department of Agricultural Punjab (2013) National Conference on Agricultural for

Kharif Compaign.6. K.S.Sidhu .(2006) Director of Punjab state Electricity Board. Non Conventional

energy resources




40

CHAPTER 5

DEVELOPMENT OF QUALITY TESTING

METHODOLOGIES OF FUEL BRIQUETTES

Madhurjya Saikia and Bichitra Bikash

Abstract

Biomass material such as rice straw, banana leaves and teak leaves (Tectona grandis) could

be densified by means of wet briquetting process. Wet briquetting is a process of

decomposing biomass material up to a desired level under controlled environment in order to

pressurize to wet briquettes at a lower pressure. Upon drying these wet briquettes could be

used as solid fuels. This study is aimed at to develop methodologies to measure quality and

handling characteristics of these briquettes and burning characteristics as well.

Key words: Biomass, briquettes, durability, shear strength, impact resistance.

5.1 Introduction

India produces yearly a large amount of agro residue such as rice straw, rice husk,

coffee husk, jute stick, coir pith, bagasse, groundnut shells etc. Some part of this agro residue

is used as animal fodder. A large amount of agro residue is left on the paddy fields to be burnt

or decomposed [Ponnamperuma et al., 1983]. Both ways are means of pollution to

environment, as field burning of agro residue emits a lot of GHGs to environment and

decomposition of the agro residue too produces methane gas which is considered of the GHGs

[Campbell et al., 2002; Sokhansanj et al., 2006]. Instead of letting these agro residues to beburnt or decomposed, these could be converted to densified forms by briquetting method

[Stanely, 2003]. This will mitigate the problems of pollution while at the same time we would

be successful to trap this energy resource for industrial purposes. Briquettes are far better to

handle rather than loose biomass.

Biomass briquetting is the densification of loose biomass material to produce compact

solid composites of different sizes with the application of pressure. Three different types of

densification technologies are currently in use [Saikia and Baruah, 2013]. The first, called

pyrolizing technology relies on partial pyrolysis of biomass, which is added with binder and




41

then made into briquettes by casting and pressing. The second technology is direct extrusion

type, where the biomass is dried and directly compacted with high heat and pressure (Grover

and Mishra , 1994). The last type is called wet briquetting in which decomposition is used in

order to breakdown the fibers. On pressing and drying, briquettes are ready for direct burning

or gasification. These briquettes are also known as fuel briquettes [Stanley, 2003, Chou et al.,

2010, Saikia et al., 2013].

These fuel briquettes are the newest edition of briquettes manufactured at low pressure

unlike the others. As these briquettes are produced at relatively low pressure, a series of

quality test such as durability, compressive strength, shear strength, impact test and

combustion tests are needed to be done so that they could be more competitive with existing

types.

5.2 Parameters of Quality Assessment

Durability, shear strength and impact resistance of briquettes are the basic parameters to

assess the quality of briquettes in terms of handling and transportability of fuel briquettes

(Grover and Mishra, 1994). .

5.2.1 Durability

Durability of briquettes gives the mechanical handling of the solid fuel (Chou, 2009).

This is measured by standard procedure ASAE S269.4 [Kaylyan et al]. To measure durability,

100 g of sample is taken and sample is tumbled at 50 rpm for 10 minutes in a dust tight

enclosure of size 300mm×300mm. Metal cloth of aperture size 4mm is used to retain

crumbled briquettes after tumbling.

Briquettes durability index in % given by= !"#$ %&' &" $'*,

"& *"#$ %&',×100

Durability test set up: Working principle: The test set up is fabricated according to

specification of ASAE S269.4 (Temmerman et al., 2006). The set up consists of a box 300

mm× 300 mm × 125 mm mounted on a frame. The box can be rotated by wooden handle or a

motor at 50 rpm for 10 minutes which simulates the probable conditions of briquettes under

transport by truck or conveyer belt into furnace. Figure below shows a durability measuring

test set up made according to specification.

5.2.2 Shear Strength Test

To measure shear strength, a simple test is done. Briquette is sheared between two

planes and shear force developed is the shear strength of briquette (Saikia and Baruah, 2013).




42

Fig. 1 Durability test set up Fig. 2 Shear strength test set up

Shear strength test set up: To measure shear strength, shearing test set up has been

fabricated. The instrument consists of three wooden plates. The middle plate with a central

cylindrical hole of 45 mm diameter and 30 mm thickness slides over the bottom plate. In the

top plate, a cylindrical hole of same diameter as that of moving plate with 20 mm thickness is

being provided in such a way that it coincides with the one that is provided in the movable

plate when this plate is fully inserted between top and non moving bottom plate. The movableplate is connected to dead weights.

Shear strength, kPa =2F

π D2

Where F= Force causing shear in briquette, kN

D= diameter of briquette, m

5.2.3 Impact Resistance Test

This test simulates the forces encountered during emptying of densified products from

trucks onto ground, or from chutes into bins. Drop tests can be used to determine the safe

height of briquette production during mass production (Debdoubi A. et al., 2005). ASTM

D440-86 method is used to determine impact resistance index (Kaliyan N. and Morey R.V.,

2008).. In the drop test, briquettes are dropped twice from a height 1.83 m onto a concrete

floor. An impact resistance index (IRI) is calculated.

IRI =100×N

n

Where, N= Number of drops, n= Total number of pieces. The upper limit of IRI is 200 which

would result if the briquettes are not broken even after dropping twice.




43

5.3 Parameters of Combustion Characteristics of Briquettes

Proximate analysis and combustion rates of briquettes were done to assess effectiveness

of briquettes as cooking fuel.

5.3.1 Proximate Analysis

Moisture Content: The moisture content is determined according to the method described in

the Forestry Hand Book (Wenger, 1984). 10 g of sample is taken immediately upon sampling

and then air dried. This air- dried sample is taken immediately in an aluminum moisture box

and kept in an oven heated at 105º ± 3 ºC until constant weight is obtained. The difference of

the oven dry weight of the sample and the fresh weight of the sample is used to determine the

percentage of moisture content as follows:

Moisture content, % =-"% . /"0

-"% × 100

Ash content: For determination of ash content, ASTM Test No. D-271-48 is followed. At

first, an empty 25 ml. silica crucible as well as the sample is heated in a moisture oven.

Sample is weighed accurately to 2 g. The sample in the crucible is kept in muffle furnace with

the lid cover. Muffle furnace temperature is set at 550º ± 50ºC and kept for 6 hours. After 6

hours of burning crucible is removed from the furnace and placed in a desiccators and

weighed accurately. Percentage of ash content was calculated as follows:

Ash content, % =Weight of ash

Weight of sample × 100

Volatile matter: Volatile matter of samples is determined by ASTM Test No. D- 271- 48. A

clean platinum crucible of 10 ml. capacity is taken and heated in a furnace at 950ºC for 2

minutes and cooled in desiccators for 15 minutes. Crucible weighed to nearest 0.1 mg.

Sample filled crucible is weighed and heated in a furnace at 950ºC for 2 minutes. After

volatile matter escaped, the crucible is removed from furnace and cooled in air 2 to 5 minutes

and then in desiccators for 15 minutes. The percentage of weight loss of the samples is

reported as volatile matter as follows:

VM=Wt. loss of dry sample

Net wt.of dry sample × 100

VM=

Wt. loss of wet sample ×100

Gross wt. of wet sample

100- percent moisture × 100 (Wet samples)

Determination of fixed carbon content

Fixed carbon (FC) is determined by ASTM Test No. D- 271- 48

FC (on dry basis) = 100- (% of volatile matter +% of ash)




44

FC (on wet basis) = 100 – (% of volatile matter + % of ash + % of moisture)

5.3.2 Calorific Value

Calorific value is determined by using Automatic Microprocessor Calorimeter (5E-

AC/ML) (make- Changsha Kaiyuan, China) (Saikia M. and Baruah D., 2013). The briquette’s

material is nicely ground and pellets of 10 mm diameter are prepared from grounded material.

The pellets are placed inside a crucible so that tungsten wire touches the pellet. The calorific

value is analyzed by the Automatic Microprocessor Calorimeter or auto bomb calorimeter.

The system has facilities such as water cycling system, automatic water feeding, temperature

adjusting with a PT 500 temperature sensor, zero drift bridge temperature circuit to ensure

that temperature resolutions reach 0.0001 , auto-diagnose, remote data transfer and auto-weight entry.

Fig. 3 Auto bomb calorimeter for proximate analysis

5.3.3 Combustion Rate

Combustion rate or burning rate is the mass loss per unit time. The briquettes are dried

at 105ºC so that it does not affect on combustion or burning. Dried briquette is placed on a

steel wire mesh grid resting on three supports allowing free flow of air (Chaney J. O.et al. ,

2010) Now the whole system is placed on mass balance. Briquette is ignited from top and

mass loss data is taken at an interval of 30 seconds.




45

Fig. 4 Combustion rate determination set up for briquettes

5.4 Conclusion

With the help of this study we aim to standardize the practice of briquette making for

the commercial purpose. Generally, there are very few literatures on the standardization of

quality testing methodologies of briquettes and burning characteristics as well. Durability,

shear strength and impact resistance of briquettes could be determined easily as discussed in

the section II in order to assess the quality of briquettes in terms of handling and

transportability. Higher the durability, impact resistance and shear strength, higher will be the

handling characteristics. But we need to reach at an optimum value of these indexes in order

to produce quality briquettes at a cost effective way as quality always adds cost to production.

Similarly, we can also asses the burning rates of briquettes in room condition as discussed in

the section III. This generally helps to build a rough idea of performance of briquettes in

actual condition. Moreover, with the knowledge of burning rates, we can further manipulate

parameters of briquette manufacturing such a density and porosity of briquettes in order to

obtain a desired level of burning rate. Apart from that proximate analysis and calorific valuetests will so help us to give answer to some of the questions regarding the fuel such as

whether it produces too much harmful fly ash and unwanted gases which are general indoor

air pollutants many households and effectiveness of the fuel in terms of heat value. A fuel

without heat value would be useless as a lot of fuel will be needed during use for its lower

heat value.

References

1. Stanley R. (2003). Fuel Briquette making, Legacy Foundation.

Steel wire mesh

Three leg support

Electronic mass

balance

Briquette

Stop watch




46

2. Kaliyan N. and Morey R.V. (2008). Factors affecting strength and durability of

densified of biomass products, Biomass and Bioenergy, Vol 33, pg 337-359.

3. Wenger (1984). Forestry Hand Book.

4.

Chaney J. O., Clifford M. J., Wilson R. An experimental study of the combustion

characteristics of low-density biomass briquettes. Biomass magazine 2010, Vol 1.

5. Grover P.D. and Mishra S.K. (1994). Development of an appropriate biomass

briquetting technology suitable for production and use in developing countries Energy

for Sustainable Development, Vol 1.

6. Faborod M. 0 (1987).Optimizing the Compression/Briquetting of Fibrous Agricultural

materials, Journal of agricultural. Engineering Resources, Vol 38, pg 245-262.

7.

Chen L. (2010). The development of agro-residue densified fuel in China based on

energetics analysis, Waste Management, Vol 30, pg 808–813.

8. Suramaythangkoor T. and Gheewala S.H. (2010). Potential alternatives of heat and

power technology application using rice straw in Thailand, Applied Energy, Vol 87, pg

128–133.

9. Chou CS, Lin S.H., Peng C.C. and Lu W.C. (2010).The optimum conditions for preparing

solid fuel briquette of rice straw by a piston-mold process using the Taguchi method,

Fuel Processing Technology, Vol 90, pg 1041–1046.10.

Mania S., Tabil L.G. and Sokhansanj S. (2006). Effects of compressive force, particle

size and moisture content on mechanical properties of biomass pellets from grasses,

Biomass and Bioenergy, Vol 30, pg 648–654.

11. Chou C. S. (2009). Preparation and characterization of solid biomass fuel made from

rice straw and rice bran, Fuel Processing Technology, Vol 90, pg 980–987.

12.

Singh R.N. (2004). Equilibrium moisture content of biomass briquettes, Biomass and

Bioenergy, Vol 26, pg 251 – 253.13. Demirba A. (1997). Evaluation of biomass residue briquetting waste paper and wheat

straw mixtures, Fuel Processing Technology Vol 55, pg 175–183.

14. Parikh Jigisha, Channiwala S.A. and Ghosal G.K. (2005). A correlation for calculating

HHV from proximate analysis of solid fuels, Fuel, Vol 84 (2005), pg 487–494.

15. Bamgboye I. and Bolufawi S.J. (2009). Physical Characteristics of Briquettes from

Guinea Corn (sorghum bi-color) Residue, Agricultural Engineering International: the

CIGR Ejournal.

16. Husain Z., Zainac Z. and Abdullah Z. (2002). Briquetting of palm Fiber and shell from

the processing of palm nuts to palm oil, Biomass and Bioenergy, Vol 22, pg 505 – 509.




47

17. Kumar N., Patel K., Kumar R.N. and Bhoi1 R.K. (2009). An assessment of Indian fuel

wood with regards to properties and environmental impact, Asian Journal on Energy

and Environment, Vol 10(02), pg 99-107

18.

Saikia M. and Baruah D. (2013). Analysis of Physical Properties of Biomass Briquettes

Prepared by Wet Briquetting Method, International Journal of Engineering Research

and Development Volume 6, Issue 5 (March 2013), PP. 12-14.

19.

Bikash B. Bhowmik R. and Saikia M. (2013). Challenges of Wet Briquetting from

Locally Available Biomass with Special Reference to Assam, International Journal of

Computational Engineering Research , Vol, 03, Issue, 7.




48

Part II

Thermochemical Conversion




49

CHAPTER 6

THERMAL AND CATALYTIC CRACKING OF NON-EDIBLE

OIL SEEDS TO LIQUID FUEL

Krushna Prasad Shadangi and Kaustubha Mohanty

Abstract

Verities of edible and non-edible oil containing seeds are available in nature. Out of these, the

seeds containing edible oil habitually fulfill our food requirements. Hence edible seeds should

not be use as a feedstock for production of fuel as it will directly affect the food chain. The

seeds that are less edible or totally non-edible can be a considered as a feed stock for pyrolysis

for the production of bio-oil. The quality and yield of pyrolytic product depends on biomass

composition which include cellulose, hemicelluloses and lignin, oil and extractives. Higher

amount of cellulose and extractive content enhances the yield of oil whereas the presence of

lignin results in char during pyrolysis. The product yield and its quality is a function of

reactor type, physical and chemical properties of biomass, final pyrolysis temperature, gasresidence time in the reactor, pressure, ambient gas composition and catalyst types. The high

viscosity, acid and water content of the thermal pyrolytic oil as well as low stability retards its

use as an alternative fuel which can be overcome by cracking in presence of a suitable

catalyst. Literatures revealed that catalytic cracking of non-edible oil seeds such as mahua,

karanja, castor in presence of zeolite, Al2O3, CaO, Kaolin, Criterion-424 and BP 3189

enhanced the yield of pyrolytic oil and the fuel quality by altering the pH, reducing the water

content and viscosity. It has been observed that the catalytic activity varies with the types of

feedstock and its composition. Moreover non-edible seeds yield more oil compared to other

feedstocks and has closer fuel properties to that of diesel which indicates their suitability as an

alternative fuel for diesel engine.

Keywords: Non-edible seeds; Thermal pyrolysis; Catalytic pyrolysis; Fuel properties

6.1 Introduction

The depletion of the fossil fuel, increasing prices along with environmental

degradation is a major global problem. The utilization of energy also increased due to the

rapid industrialization and growth of population. Present sources of energy are not enough to




51

Therefore, it is always better to focus on non-edible oil seeds as feedstock to produce liquid

fuel as they are outside the food chain.

In this work, the use of non-edible seeds for the production of liquid fuel following

thermal and catalytic pyrolysis is reported. Various process parameters that affect the yield as

well as fuel properties of pyrolytic oil are discussed here. The role of catalyst during pyrolysis

on the yield and quality of pyrolytic oil along with some drawbacks of thermal pyrolytic oil

are also reported.

6.2 Pyrolysis and its Types

Biomass pyrolysis simply means the heating of selected biomass at the elevated

temperature in absence of oxygen. During pyrolysis of biomass, the biomass undergoes

various phases such as dehydration and depolymerization. Initially, within 100-200 oC most

of the moisture and water gets eliminated and at the elevated temperature depolymerization of

such chemical bonds associated with extractives hemicelluloses, cellulose and lignin takes

place respectively. At the lower temperature the weaker chemical bonds and at higher

temperature stronger chemical bonds breaks and the long chain chemical bond breaks to form

short chain compounds (Singh and Shadangi, 2011). Pyrolysis can be carried out thermally

where the operating parameter is only temperature and known as thermal pyrolysis. Pyrolysisin presence of catalyst is termed as catalytic pyrolysis. To overcome the drawbacks of thermal

process, pyrolysis is carried out in presence of several catalysts which is discussed latter.

6.3 Process parameters that affect the yield

6.3.1 Effect of temperature on products yields

One of the most important parameters that affect the yield of pyrolytic products is

temperature. The yield of pyrolytic liquid, gas and char varies with final pyrolysis

temperature. During non-edible seed pyrolysis the foremost aim is to produce high yield of

pyrolytic liquid/oil other than that of char and non-condensable gas. However, char and non-

condensable gases are the other by-products of biomass pyrolysis. The temperature at which

the yield of liquid is highest is considered as the optimum temperature. The thermal pyrolysis

of Pistacia khinjuk seed (Onay, 2007 a), rapeseed (Onay and Kockar, 2004), safflower seed

(Beis et al., 2002, Onay, 2007 b), pomegranate seed (Ucar and Karagoz, 2009), cherry seed

(Dumanet et al., 2010), rape seed (Sensoz et al., 2000), jatropha seed (Figueiredo et al., 2011),

tamarind Seed (Kader et al., 2011), mahogany seed (Kader et al., 2012), castor seed (Singh

and Shadangi, 2011), neem seed (Nayan et al., 2013), mahua (Shadangi and Mohanty, 2014 a)




52

and karanja seed (Shadangi and Mohanty, 2014 b) was carried out and the effect of

temperature parameter on yield is reported in Table 1. It was found that non-edible oil seeds

can be used as a suitable feed stock for the production of alternative fuel by pyrolysis. Table 1

also provides the optimum temperature for maximum yield of pyrolytic liquid of several non-

edible oil seeds. It was observed that the degradation temperature and the optimum

temperature for maximum liquid yield varied with the types of feedstock. The yield of

pyrolytic liquid increased with increasing temperature, whereas the yield decreased after a

certain temperature. The temperature beyond which the yield of liquid starts decreasing is

termed as optimum temperature for maximum liquid yield. Several researchers reported

similar findings for the decrease in the liquid products. At higher temperatures, the secondary

decomposition of char may also produce non-condensable gaseous products which would also

contribute to increase in gas yield with increase in pyrolysis temperature.

6.3.2 Effect of heating rate on yield

During pyrolysis the feed biomass is heated from room temperature to the

depolymerization temperature. Hence the required rate of heating plays an important role for

maximum liquid yield. Lower the heating rate, the yield of char is more and liquid is less

during pyrolysis. Higher heating rate breaks the heat and mass transfer limitation during

pyrolysis and resulted in maximum yield of oil. Onay (2007 a) reported that the yield of

pistacia khinjuk seed pyrolytic oil increased by 25 % when the rate of heating raised from 5

oC min-1 to 300 oC min-1. Similar results were also obtained by Onay (2007 b) during

pyrolysis of safflower seed, rape seed (Onay and Kockar, 2004) and safflower seed (Beis et

al., 2002).

6.3.3 Effect of sweeping gas flow rate on yield

Biomass pyrolysis may be carried out in an inert atmosphere or without flowing of anysweep gas. The yield of pyrolytic liquid does increase with supply of any sweep gas. In

general nitrogen is used as a sweep gas to maintain inert atmosphere during the process. The

important role of flow of sweep gas during pyrolysis is that it helps in reducing the formation

of char, which is controlled by mass transfer of tar molecules into the light gas species. Onay

and Kockar (2004) pyrolyzed rape seed in a fixed bed reactor with and without sweeping gas

atmosphere and observed that the oil yield increased from 41.4 % – 51.7%. Onay (2007 a) and

Onay and Kockar (2004) has reported that the effect of nitrogen flow rate on the liquid yieldwas negligible if nitrogen flow rate exceed more than 100 cm3 min-1. Similar conclusions




53

about the use of sweep gas flow rate are also reported by Kader et al. (2011) during pyrolysis

of mahogany seed and during pyrolysis of pistacia khinjuk seed (Onay, 2007 a).

Table 1. Thermal pyrolysis of non-edible seeds

Seeds

P i s t a c i a

k h i n j u k

R a p e s e e d

S a f f l o w e r

S a f f l o w e r

C h e r r y

R a p e s e e d

J a t r o p

h a

T a m a r i n

d

M a h o g a n y

C a s t o r

N e e m

M a h u a

K a r a n

j a

P o m e g r a n a

t e

Process Thermal pyrolysis

N2 flowrate, cm3

min-1

100 200 25 No 500 600 500 No 30

Reactor

type Fixed-bed

C o n

t i n u o u s

Fixed-bed Semi-batch reactor

Heatingrate,

oC min-1

300 5 40 10 At finaltemp.

25 5

Temp., oC 600 550 600 500 500 500 420 400 550 550 475

525 550 600

Total

liquidyield, % 57.6 68 54 44 44 46.1 23 45 49 64.4 38 57.75 55.17 54.2

Charyield, %

15.2 13.5 17.2

20.5

16.5

20.44

- 40 35 20.93

30 21.55

19.81 29.28

6.3.4 Effect of particle size on yield

The yield of pyrolytic liquid during pyrolysis is also affected by the particle size of the

feedstock. Larger feed size decreased the heat transfer rate and increased the pyrolysis time

which is liable for the formation of more char. In general, lower particle size of feed materials

enhances the pyrolytic yield of liquid. Kader et al. (2011) experimented pyrolysis on tamarind

seed and reported that liquid yield first increased up to a maximum value for feed size of 1.07

cm3 and subsequently decreased for larger feed size above 1.07 cm3. Pyrolysis experiments

performed by Onay and Kockar (2004) on rapeseed, Kader et al. (2012) on mahogany seed

and Onay et al. (2007b) on safflower seed suggested that particle size more than 1mm

decreased the yield of pyrolytic oil by producing more char.

6.4 Fuel properties of Seed Pyrolytic Oil

6.4.1 Calorific Value




54

The calorific values of several non-edible seeds are presented in Table 2. This

confirmed that non-edible seed pyrolytic oils have higher calorific value and closer to diesel.

The calorific value of pyrolytic oil is a function of feedstock, operating conditions and the

process used. Zhang et al. (2007) reported that non-edible seed pyrolytic oil having more

heating value in comparison with wood pyrolytic oil.

Table 2. Fuel properties of seed pyrolytic oil

Seeds Calorific value, MJ kg- Viscosity, cSt References

Pistacia khinjuk 39.84 -- Onay, 2007 a

Rapeseed 38.4 43 (50 oC) Onay and Kockar, 2004

Safflower 41 33 (50 oC) Onay, 2007 b

Cherry 38.4 -- Duman et al., 2011

Tamarind 19.1 6.51 (30o

C) Kader et al., 2011Mahogany 32.4 3.81 (26 oC) Kader et al., 2012

Castor 35.64 83.19 (25 oC) Singh and Shadangi, 2011

Neem 32.49 22.6 (40 oC) Nayan et al., 2013

Mahua 41 33.97(25 oC) Shadangi and Mohanty, 2014 a

Karanja 37.65 51.67(25 oC) Shadangi and Mohanty, 2014 b

Diesel 45-47 3.68 (40 oC)4.57 (25 oC)

--

6.4.2 Viscosity

High viscosity is one of the principal drawbacks of seed pyrolytic oils. Viscosity of

the pyrolytic oil is concerned with water content and water insoluble compounds. The higher

water content and less water insoluble components in the pyrolytic oil reduce the viscosity of

the pyrolytic oil. Table 2 shows the viscosities of various non-edible seed thermal pyrolytic

oils. It is concluded that viscosity of pyrolytic oil varies with biomass types and the process

type. The pyrolytic oil viscosity is much more higher compared to conventional diesel. Thus,

the direct use of thermal pyrolytic oil in combustion engine is not acceptable and it needs

upgradation to match the fuel properties.

6.4.3 pH of pyrolytic oil

Pyrolytic oil is acidic in nature having high pH value ranging from 3-5. The high acid

content is due to the presence of carboxylic acid, acetic acid and formic acid in the pyrolytic

oils. These acids are formed by the depolymerization of cellulose and hemicelluloses. High

cellulose content in the biomass produces more acid in the pyrolytic oil during pyrolysis. The

presence of various acids in the pyrolytic oil also reduces the thermal stability during storage.The direct use of seed pyrolytic oil as a transportation fuel may damage the engine. Table 3




55

confirmed that the non-edible oil seed pyrolytic oils are highly acidic in nature and also varies

with the feedstocks.

6.4.4 Water content

The presence of water content in pyrolytic oil reduces the heating value and the flame

temperature. Less water content decreases the viscosity which enhances the atomization

towards complete combustion and reduces the harmful emissions such as SOx and NOx. It is

confirmed from the literature that non-edible seed pyrolytic oil contains very less water in it

which can be observed from Table 3. It was reported that the water content of pyrolytic oil

varies from 0.33 % to 35 %. Since water is insoluble with organic pyrolytic oil most of the

produced water and the water soluble chemicals collected separately as aqueous pyrolytic

liquid (Shadangi and Mohanty, 2014 a, b). This confirmed that the non-edible seed pyrolytic

oil can be accepted as a future alternative fuel.

6.4.5 Pour point

Higher pour point is one of the disadvantages of pyrolytic oil. The pour point of seed

pyrolytic oil varies from 5-27 oC. Table 3 shows the pour point of several non-edible seed

pyrolytic oils. High pour point of pyrolytic oil reduces the flow ability in winter especially in

low temperature regions due to the formation of crystals which clogs the filter and reduces the

efficiency of the combustion engines.

Table 3. pH, water and pour point of some non-edible seed pyrolytic oil

Pyrolytic oil pH Water content, % Pour point, oC References

Neem 3.9 30-35 11 Nayan et al., 2013

Mahua 4.86 0.33 26.63 Shadangi and Mohanty, 2014 a

Karanja 4.05 1.33 12.05 Shadangi and Mohanty, 2014 b

Castor 3.7 -- 5 Singh and Shadangi, 2011

6.5 Catalytic Pyrolysis of Non-edible Seeds

6.5.1 Effect of Catalyst on Yield

Theoretically catalyst has an effect on the rate of reaction. The catalytic effect

enhances the rate of reaction which increases the conversion during pyrolysis. It might have

positive or negative impact. The influence of various catalysts on the pyrolytic yield of non-

edible oil seeds were studied by different researchers. Catalytic pyrolysis of Pistacia khinjuk

seed was carried out by Onay (2007 a) using BP3189 and Criterion-424 as catalyst and a

liquid yield of 66.5% and 69.2% was found for the two catalysts respectively while it was




56

only 57.6% without catalyst. Shadangi and Mohanty (2014 a,b) studied the catalytic pyrolysis

of Mahua and Karanja seed using CaO, Al2O3 and Kaolin and observed little increase in the

yield of pyrolytic oil (4-6%) over thermal pyrolysis.

6.5.2 Effect of catalyst on fuel properties of pyrolytic oil

Literature revealed that the use of catalyst altered the physical properties of non-edible

seed pyrolytic oils. The catalyst pyrolysis decreases the viscosity, increases the calorific value

and decrease the acidity of pyrolytic oil. Shadangi and Mohanty (2014 a) reported that the use

of CaO catalyst increased the calorific value from 41 to 43.15 MJ kg-1, reduced the viscosity

from 0.033 to 0.018 Pa s and alter the pH acidic to basic (4.86 to 8.58) for Mahua pyrolytic

oil. The effect of catalyst Al2O

3, CaO and Kaolin on pyrolysis of karanja seed was studied by

Shadangi and Mohanty (2014 b) and it was reported that CaO at 8:1 ratio produced less

viscous (0.019629 Pa-s) pyrolytic oil compared to other catalysts. The calorific value

increased for all the three catalytic pyrolysis with CaO (40.42 MJ kg-1), Al2O3 (41.21 MJ kg-1)

and Kaolin (39.04 MJ kg-1) compared to thermal pyrolysis (37.65 MJ kg-1).

6.6 Conclusion

The study of thermal and catalytic pyrolysis of non-edible seed confirmed that the

pyrolytic oil can be used as an alternative fuel. Non-edible seed produced more organic liquid

in comparison with other feed stocks due to its high extractive content. The thermal pyrolytic

oil is highly acidic in nature and very viscous and hence is not suitable for direct use in diesel

engine. Catalytic pyrolysis is one of the suitable processes which altered the pH, reduces the

viscosity and enhances the calorific value. Hence, catalytic pyrolytic oil from seeds will be

suitable as an alternative fuel. However, more emphasis should be given on the effect of

various catalysts to found out the most suitable catalyst which will enhance the fuel properties

and stability of pyrolytic oils.

References

1. Beis S.H., Onay O., Kockar O.M. (2002) Fixed-bed pyrolysis of safflower seed:

Influence of pyrolysis parameters on product yields compositions. Renewable Energy,

26: 21–32.

2. Duman G., Okutucu C., Ucar S., Stahl R., Yanik J. (2011) The slow and fast pyrolysis

of cherry seed. Bioresource Technology, 102: 1869-1878.




57

3. Figueiredo M.K.K., Romeiro G.A., Silva R.V.S., Pinto P.A., Damasceno R.N., D’Avila

L.A., Amanda P. (2011) Pyrolysis Oil from the Fruit and Cake of Jatropha curcas:

Produced Using a Low Temperature Conversion (LTC) Process: Analysis of a Pyrolysis

Oil-Diesel Blend. EPE., 3: 332-338.

4. Kader Md A., Islam M.R., Joardder M.U.H. (2011) Fast Pyrolysis for Better Utilization

of Tamarind Seed from Renewable Energy Point of View. Proceedings of the

International Conference on Mechanical Engineering and Renewable Energy.

Chittagong, Bangladesh., 22- 24.

5. Kader M.A., Joardder M.U.H., Islam M.R., Das B.K., Hasan M. (2012) Production of

Liquid Fuel and Activated Carbon from Mahogany Seed by Using Pyrolysis

Technology. In Green Chemistry for Sustainable Development. Bangladesh, Jessore:

July 14. Id code: 53696.

6. Nayan N.K., Kumar S., Singh R.K. (2013) Production of the liquid fuel by thermal

pyrolysis of neem seed. Fuel, 103: 437–443.

7. Onay O. and Kockar O.M. (2004) Fixed-bed pyrolysis of rapeseed (Brassica napus L.).

Biomass and Bioenergy, 26: 289–299.

8. Onay O. (2007a) Fast and catalytic pyrolysis of Pistacia khinjuk seed in a well-swept

fixed bed reactor. Fuel, 86: 1452–1460.

9. Onay O. (2007b) Influence of pyrolysis temperature and heating rate on the production

of bio-oil and char from safflower seed by pyrolysis, using a well-swept fixed-bed

reactor. Fuel Process Technology, 88: 523–531.

10. Shadangi K.P., and Mohanty K. (2014a) Thermal and catalytic pyrolysis of Karanja

seed to produce liquid fuel. Fuel, 115: 434–442.

11. Shadangi K.P., and Mohanty K.. (2014b) Comparison of yield and fuel properties of

thermal and catalytic Mahua seed pyrolytic oil. Fuel, 117 (30): 372–380. 12. Singh R.K., and Shadangi K.P. (2011) Liquid fuel from castor seeds by pyrolysis. Fuel,

90: 2538–2544.

13. Sensoz S., Angn D., Yorgun S. (2000) Influence of particle size on the pyrolysis of

rapeseed (Brassica napus L.): fuel properties of bio-oil. Biomass and Bioenergy, 19:

271-279.

14. Uçar S. and Karagöz S. (2009) The slow pyrolysis of pomegranate seeds: The effect of

temperature on the product yields and bio-oil properties, J. Analytical Applied

Pyrolysis, 84: 151-156.




58

CHAPTER 7

EVALUATION OF MICRO GASIFIER COOKSTOVE

PERFORMANCE WITH HANDMADE BIOMASS PELLETS

USING REGION-SPECIFIC FUELS AND ASSESSMENT OF

DEPLOYMENT POTENTIAL

Debkumar Mandal, Vikas Dohare, Vijay H. Honkalaskar, Anurag Garg, Upendra V.

Bhandarkar and Virendra Sethi

Abstract

In rural areas of developing countries, large populations depend on traditional biomass such

as wood, crop residues, and cattle-dung for cooking. Emissions from these activities have

been reported to cause regional environmental impacts and global climate change. While

engineered cook stove designs could be considered as a solution for reducing emissions, it is

not independent of the region and season specific availability of fuels. A design solution

based on gasification using engineered solid fuel from agricultural residues/coal powder is a

relatively new development, and accounts for both the design of the cookstove and the

physical property of the fuel. Work is needed to fine tune these gasifier cookstoves, quantify

emissions and fuel consumption with emphasis on the utilisation of region-specific fuels.

Micro-gasifier type cookstove have become more popular among small scale

commercial operations where replacement of LPG with biomass pellets is economically

profitable. However, in rural areas, where wood/agricultural residue is “freely” available as anatural resource, purchase of commercial pellet-based fuel is not affordable. The purpose of

the study was to determine how locally available biomass may be used to take advantage of

the micro-gasifier cookstove design. Further, it is also important to understand the

behavioural and cultural obstacles for deployment of these stoves, and the suitability for

ensuring reduction in indoor kitchen exposure. A field campaign was carried out in

Gawandwadi village in Maharashtra, to understand deployability based on cooking practices

and fuel availability. Experiments were conducted using local biomass residues (wheat husk,

rice husk and saw dust) with locally available binding materials (cow dung, wheat flour and

rice flour) in different proportions for making handmade “pellets”. Spherical balls were made




59

by pressing mixture (biomass residues with binding material) by hand. A simple “sewai”

making home machine was modified and also used for extrusion for making disk shaped

“pellets”. Standard Water Boiling Test was conducted to evaluate the performance of the

handmade “pellets” in micro-gasifier stove. Besides, locally available fuel woods in small

pieces were also used and the results were compared with the handmade “pellets”. The

evaluation was done on the basis of Input power (kW), fuel required per output power

(kg/kWh) and Efficiency (%). The efficiency and input power of the handmade “pellets”

ranged between 33% to 57% and 2.4 kW to 3.1 kW respectively.

Keywords: Micro-gasifier coookstove, Biomass, Handmade pellets

7.1. Introduction

Three billion people across the world use biomass for cooking and heating purposes.In

India, 82% of the population use solid fuels for cooking, of which rural and urban population

accounts for 88 % and 24.6% respectively (URL 1).The global use of biomass fuels from

traditional stoves (three stone, open fire) has been linked to adverse health effects, climate

change, and deforestation. In India, forest area grew by ~2% annually, but afforestation

quality is poor and deforestation persists due to urbanization across many parts in the country.

Encouraging use of non-solid fuel in improved cookstoves (such as, micro-gasifier cookstove)can play a crucial role in preventing land degradation and deforestation, particularly in areas

with negligible or negative afforestation rates.Improved cooking stoves have been shown to

reduce the amount of fuel used to cook food and the air pollution produced in kitchens.58% of

Indian population relies on fuel wood and 11% uses cow-dung for cooking purposes. On the

other hand only 0.4% uses other fuels which include agricultural residues for cooking (URL

1).

The traditional cookstoves used in the rural areas lead to the emission of unburnt gasessuch as CO,a toxic air pollutant in indoor air in rural as well as urban areas. The CO emission

factors ranged widely from 3.0×10-2 g/Kg for coal gas/traditional stoves to 2.8× 102 g/Kg for

the charcoal/ Angethi stoves (Zhang et al., 1999). Besides this, at a temperature of around

800-1000°C particulate matter (PM) is dominated by soot (carbon aggregate) in conventional

cookstoves (Bolling et al., 2009). The CO standards for residential and agricultural areas are 2

mg m-3 for an 8 hour average, or 16 h-mg m-3 exposure equivalent according to the WHO

guideline. These could be easily exceeded by CO exposure caused by traditional cookstovesusing biomass (Zhang et al., 1999).




60

To reduce these emission of CO and PM, improvements have been made in the

combustion devices by studying the combustion pattern of the raw biomass and its processed

form (in form of briquettes and pellets) so that less amount of gases and PM are released. A

gasifier stove is such an attempt in which the processed forms of biomass (i.e. briquettes or

pellets) are used. While studying the combustion pattern of fuel in many stoves it has been

found that if the ratio of fuel and air is maintained at certainlevel then clean combustion can

be achieved (Mukunda, 2011). Many of the fundamental phenomena observed in biomass

combustion, which are most relevant to cookstoves, have been extensively studied and mainly

focus on understanding how wood burns. It includes modelling of air-flow rates both on the

solid and gas phases and heat transfer (Burnham-Slipper, 2008 and Mukunda, 2011).

The present work deals with the fan based gasifier stove, Oorja, built by BP, India.

The main focus of the work is the issue of using different handmade pellets or briquettes

(using powdery biomass) and to check their adaptability for the Oorjagasifier stove.The Oorja

stove has two modes of air flow rates i.e. “low” and “high” modes. In the present study, the

biomass handmade pellets (using commonly available biomass) have been tested mainly

using the “high” mode. For most existing stoves, whether traditional stoves such as the three

stone fire or other stoves developed over the last two decades, the efficiency (determined

using water boiling tests) is between 15 to 35%. In contrast, the water boiling efficiency of the

Oorja stove (used with pellets designed for Oorja) has been found to be up to 60%

(Varunkumar, 2012).

7.2. Materials and Methods

Table 1 shows the equipment and materials used for this study. Several combinations

of biomass-binders were used to hand-make pellets and compacted “briquettes”. This section

describes the method followed for making of the region specific fuel to adapt in and Oorja

(gasification type) domestic scale stove.

7.2.1 Process for Making the Handmade Pellets

The making of the pellet without any kind of sophisticated pellet making machine

using the locally available biomass and the binder was done in three steps, which are

described in the following sub sections.

a)

Preparing the mixture of the biomass and the binder

Preparing the mixture for the pellet has been the most important step in the process to

ensure maximum energy density, by maximising the amount of biomass with the minimum




61

amount of binder. The local binder used in the preparation were cowdung, wheat flour and

rice flour while the biomass used were saw dust, rice husk and wheat husk. Sophisticated

pellet making machine (designed especially for the manufacturing of the pellets that are

commercially available in the market) generally the binder is not required as the lignin present

in the biomass, when compressed, at high temperature acts as the binder. For such cases high

degree of the compaction is needed which cannot be achieved by the hand pressing of the

mixture to make the pellets.

Table 1 Equipment and materials used in the present study

S.No Materials

Required

Quantity Remarks References

1Biomass fuel 4 types Oorja pellets, Rice Husk,

Wheat Husk and Saw DustAs per the requirement of theexperiment

2Binders 3 types Cow Dung, Rice Flour and

Wheat FlourAs per the requirement of theexperiment

3 Thermocouple 2 K- type Thermocouple tomeasure temperature up to1200˚ C

Varunkumar (2012)

4 Thermometer1

Thermometer of range up to100˚C

Water Boiling Test version4.1.2 (URL 2)

5 Cooking vessel 2 3 L Aluminium vessel Water Boiling Test version

4.1.2 (URL 2)6 Hood 1 Hood including exhaust Water Boiling Test version4.1.2 (URL 2)

7 WeighingBalance

1 Range 0-50 kg (accuracy ingrams)

As per the requirement for theexperiment

8 Stove 1 Oorja pellet gasifier Stove As per the requirement for theexperiment

9 Stirrer1

For stirring the water forconstant mixing

Water Boiling Test version4.1.2 and Varunkumar(2011)

10 Kerosene 2 l To ignite the stove As per the requirement for theexperiment

11 Bomb

Calorimeter

1 To determine the calorific

value of the pellets

As per the need for the

experiment

During the making of the pellet, it was first determined as to what amount of

minimum amount of binder could hold the maximum amount of biomass. Different binders

were mixed in equal proportions with the biomass, and its binding capacity was checked. The

biomass was then added continually until the binder could not hold more of the biomass. The

composition, in which the binder cannot hold more of the biomass was determined, was then

used as the mixture for the pellet making. Binding capacity of different binders for different

biomass varied, in which the cow dung was found to have the minimum binding capacity and

wheat flour with the maximum binding capacity. But, since the cow dung is available freely it




62

was preferred over the other binders, even though the binding property was low as compared

with the wheat and the rice flour.

Table 2 Different types of fuels and binders used for the pellet making

S.No. Biomass Binders

1 Saw dust Cow dung

2 Rice husk Rice flour

3 Wheat husk Wheat flour

b)

Compaction of the mixture for the making of the pellets

The compaction of the mixture was the second step in the making of the pellets

making by hand pressing into small “balls” as shown in Fig 1(a). Besides the hand pressingtechnique a commonly available snack making machine called as “sevai machine” was used.

This is a hollow cylinder fixed with a piston. The top of the piston is provided with a rotating

shaft. When this shaft is rotated the piston moves downwards and presses the mixture filled in

the cylinder. At a certain point shaft, cannot be rotated, at that particular point the piston is

removed and then compacted form of the mixture is recovered in the form of the discs as

shown in the Figure 1 (b). This compacted form of the mixture, which can also be called as

pellets are then put for drying.

c) Drying of the pellets

These wet pellets are dried until the moisture content of the pellet reaches ~12 % or

below (needed for the gasification of the pellets). The drying can either be done either in the

sun for over a week or they can also be dried in the hot air oven for 48 to 72 hours at the

temperature of 45°C. Figure 1 (a) and (b) shows the different pellets made by hand pressing

and using household sewai machine.

Table 3 Different types of the pellets and their composition (notation is indicated at the

bottom of the table)

S. No. Pellets Composition 1 CD+RH+SD (Balls) 400 g cow dung + 100 g rice husk + 100 g saw dust

2 CD+SD (Balls) 500 g cow dung + 60 g saw dust

3 SD+WF (Balls) 250 g Saw Dust + 270 g Wheat Flour + 375 g water

4 CD+SD+WH (Briquette) 1120 g Cow Dung + 245 g Saw Dust + 180 g Wheat Husk

5 CD+SD+WH+WF(Briquette)

650 g Cow Dung + 200 g Saw Dust + 150 g Wheat Husk +75 g Wheat Flour

6 WH+RF (Briquette) 340 g Wheat Husk + 110 g Rice Flour + 600 g Water

CD = Cow Dung, SD =Saw Dust, RH = Rice Husk, WF = Wheat Flour, WH = Wheat Husk andRF= Rice Flour




63

Figure 1(a) Disk shaped pellets made using the sewai machine

Figure 1(b) Ball shaped pellets made using hand pressing (a) Cow dung + Rice husk + Saw

dust (b) Cow dung + Saw dust, (c) Saw dust + Wheat flour and (d) Wheat husk + Wheat

flour

7.3 Results and Discussion

Stove Performance with Different Types of Handmade Biomass Pellets with the Oorja

Gasifier Stove

To evaluate the performance of the different compositions of the handmade biomass

pellets, the following parameters are discussed:

1. Determination of the calorific value

2. The input power for different types of pellets in Oorja stove

3. Specific fuel consumption for each fuel

4. Efficiency of the stove

The calorific values and densities of the fuels are shown in the following Table 4.




64

Table 4 Calorific values and the densities of the different biomass fuels

Biomass fuels Calorific Value (MJ/Kg) Density (Kg/m3)

CD+RH+SD (Balls) 15.66 244.3

CD+SD (Balls) 16.13 270.5

SD+WF (Balls) 15.4 423.2

CD+SD+WH (Briquette) 16.04 307.9

CD+SD+WH+WF (Briquette) 15.34 275.4

WH+RF (Briquette) 17.02 566.0

Oorja pellet 16.04 ___

Babool wood 17.3 ___

Ain wood 16.8 ___

To conduct the water boiling test, the standard 3 litrewater boiling test protocols were

taken into accounts with slight modification to test the biomass handmade pellets on “high”

mode in the domestic gasifier Oorja stove. Tests were conducted in a way only a batch

process was considered.The operational procedure does not require refuelling. Table 4 and 5

shows the result of water boiling tests and the performance of the stove respectively, for

biomass pellets.

Figure 2 plots the temperature of the boiling water as a function of time for each fuel.

From the plot, we can observe that Oorja pellets take only 15 minutes to boil the water and

complete the test in 49 minutes for one batch process, which is comparatively faster than the

other types of pellets. On the other hand, the small pieces of Babool wood takes only 10 min

to boil the water which is comparatively faster than the other types of biomass fuels but lasts

only 26 minutes for one batch process. For the other pellets, the water did not even reach the

boiling temperature, except for the ball shaped pellets made from Saw Dust and Wheat Flour

that took 50 minutes to reach the temperature of 99˚C.




65

Table 5 Result of Water Boiling Test (3 L Water) in Oorja Stove

Parameters CD+SD

(Balls)

CD+RH+

SD (Balls)

SD+WF

(Balls)

CD+SD+WH

(Briquette)

CD+SD+

WH+WF

(Briquette)

WH+RF

(Briquette)

Oorja

Pellet

(not

handma

Room Temp

(°C)

31 29 27 28 27 27 26

Initial water

Temp ( °C)

31 29 27 28 27 27 26

Final Water

Temp (°C)

58 69 96 63 89 78 99 (15

Cold Sta

99 (14

Hot Star

96 (20

Simmeri

Mass of

Pellet (g)

140 150 575 380 440 210 595

Duration of

Burning

(min.)

12 14 50 15 41 24 49

CD = Cow Dung, SD =Saw Dust, RH = Rice Husk, WF = Wheat Flour, WH = Wheat Husk and RF= Rice Fl




66

Table 6 Stove performance with different biomass fuels on Oorja stove

Sl. No PelletsInput Power

(kW)

Fuel required per

output power

(Kg/kWhr)

Efficiency (%)

1 CD+RH+SD (Balls) 3.1 0.44 54.1

2 CD+SD (Balls) 2.8 0.47 57.1

3 SD+WF (Balls) 2.9 0.73 42.9

4 CD+SD+WH (Briquette) 6.7 1.75 33.1

5 CD+SD+WH+WF(Briquette)

2.7 0.92 40.0

6 WH+RF (Briquette) 2.5 0.62 48.8

7 Oorja Pellet 2.2 1.17 32.3

8 Babool small wood pieces 3.7 0.59 58.9

9 Babool large wood pieces 6.7 0.87 37.5

10 Ain wood 8.1 1.35 24.9

Figure 2 Comparision of Temperature vs. Time profiles for different fuels in the Oorja stove

during the Water Boiling Test

0

20

4060

80

100

120

0 20 40 60

T e m p

( º C )

Time (min)

WBT of Biomass Fuels in OorjaCD+RH+SD(Balls)

SD+WF (Balls)

CD+SD+WH

(Briquette)CD+SD+WH+WF

(Briquette)

WH+RF(Briquette)

Oorja (Karjat)

sall !a!ool "ie#es




67

7.4 Conclusions

In this present work the analysis of gasification behaviour of the handmade pellets

made from different locally available biomass was carried out to address the issue of how

region specific fuel can be used for the clean combustion process, i.e., the gasification in the

already available Oorja pellet gasifier stove. It also focussed on what changes can be made in

the existing stove and what could be the alternate fuel that can be used by the rural people

without having to buy the commercially available pellets. During the study, it was found that

if the pellets are more densified with some better technique, the locally available biomass

could be pelletized and used in the forced draft gasifier stoves like Oorja.

References

1. Bolling, A.K., Pagels, J., Yttri, K. E., Barregard.,Sallesten, G., Schwarze, P.E., and

Boman, C. (2009) Health Effect of Residential Wood Smoke particles: The

Importance of Combustion Condition and Physiochemical Particle Properties, Particle

and Fibre Technology, 6:29.

2. Burnham-Slipper, H., Clifford, M.J. and Pickering, S.J. (2007) A simplifiedwood

combustion model for use in the simulation of cooking fires, in 5th InternationalConference on Heat Transfer, Fluid Mechanics and Thermodynamics.

3. Mukunda, H.S. (2011) Understanding Clean Energy and Fuels from Biomass, Wiley

India.

4. Varunkumar, S (2012) PhD Thesis- Packed bed gasification-combustion in biomass

domestic stove and combustion system, IISc, Bangalore.

5. Zhang, J., Smith, K.R., Uma, R., Ma, Y., Kishore, V.V.N., Lata, K., Khalil, M.A.K.,

Rasmussen, R.A. and Thorneloe, S.T. (1999) Carbon monoxidefromcookstoves in

developing countries: 1. emission factors, Chemosphere: Global Change Science, 1(1-

30: 353–366.

Web References

6. http://www.cleancookstoves.org/ (Last accessed on 17th November, 2013).

7. www.aprovecho.org/lab/component/rubberdoc/doc/231/raw (Last accessed on 15th

September, 2013).




68

CHAPTER 8

PRODUCTION OF HYDROCARBON LIQUID BY

PYROLYSIS OF CAMELLIA SINENSIS (TEA) SEED

DEOILED CAKE AND CHARACTERIZATION OF

PRODUCTS

Nabajit Dev Choudhury, Priyanko Protim Gohai, Bichitra Bikash, Sashi Dhar Baruah and

Rupam Kataki

Abstract

Increasing demand of the liquid fuel can be partially fulfilled by utilization of the bio-wastes

for biofuel production through pyrolysis. In this line, the present work aims to explore

Camellia Sinesis de-oiled cake (CSDC) for bio-fuel production. CSDC was pyrolysed for

obtaining liquid biofuel. The thermal pyrolysis of CSDC was carried out in a fixed-bed

reactor made up of stainless steel at temperature range from 400-600 oC and at heating rate

300Cmin-1. and at 150 mlmin-1 nitrogen gas flow rate to determine the yield and

characteristic of the liquid and solid product. The maximum bio-oil yield was 27.38% at

5000C. The chemical composition of the bio-oil was investigated using FTIR and GC/MS.

Results showed that the bio-oil obtained from deoiled cake of CSDC is a valuable source of

fuel and chemicals.

Keywords: Pyrolysis, Deoiled cake, Bio-oil, FTIR, GC-MS.

8.1 Introduction

Increasing energy consumption, depleting conventional fossil fuel and environmental

consideration intensified the research on renewable energy particularly on utilization of

biomass for the production relatively clean fuel due to its easy availability, easy process

ability and environment friendly nature in contrast to the fossil fuel. Biomass act as a sink for

greenhouse gases as it absorbs CO2 during its cultivation. Biomass therefore can be

considered as an alternative clean development mechanism (CDM) option for reducing

greenhouse gas emission [Mulligan et al., 2010]. The different thermo-chemical and bio-




69

chemical process such as pyrolysis, gasification, liquification and anaerobic digestion; are

used to convert biomass to fuel. Among these processes pyrolysis is one of the suitable

thermo chemical conversion processes to get maximum liquid product from biomass. Any

type of biomass can give liquid fuel after pyrolysis which can be used as energy fuel and also

for production of different chemicals [Bridgwater and Peacocke, 2004]. Pyrolysis is a

conversion technique in which biomass is heated to a desired temperature in an oxygen or air

free atmosphere to yield solid, liquid and gaseous products. There are many parameters that

influence the yield and quality of the products obtained through pyrolysis. These parameters

can be divided into processs parameters and non process parameters. The feedstocks

properties such as particle size, fixed carbon, cellulose, hemicelluloses, lignin, ash and

mineral content are non process parameters and heating rate, temperature, residence time,sweeping gas type and flow rate, raction time and type of catalysts used are process

paremeters. The advantage of the pyrolysis process is that the process parameters can be

controlled to maximize the production of either solid char, liquid or gaseous products.

Ususally, fast pyrolysis with high temperature and longer residence time favour conversion of

biomass into uncondensable gaseous product and moderate temperature with short residence

time favour the production of bio-oil. Slow pyrolysis is preferred when solid char is desirable

product.

Deoiled cake of non-edible oil seeds can serve as potential feedstocks for pyrolysis to

produce fuels including liquid and gas which can be used as substitute for petroleum or

natural gas for internal combustion engines, power station and heat supplies. These biofuel

are environment bening in contrast to fossil fuel as they contain low nitrogen and sulphur

content. Many researchers have done pyrolysis experiment on different de-oiled cakes such

as Jatropha [Raja et al., 2010], Soybean [Uzun et al., 2006], rapeseed cake [Ozcimen et al.,

2004] etc. In present study, a new source of de-oiled cake, Camellia Sinesis de-oiled cake(CSDC) was taken and pyrolysis experiment was done. The objective of th present work is to

determine the maximum bio-oil production condition in fixed bed reactor. The present work

also reports on the characterization of the bio-oil by FTIR and GC-MS for chemical

compositon.Thermogravimetric analysis (TGA) and was used to determine the thermal

behavior of CSDC. The physical properties of the bio-oil such as kinematic viscosity, flash

point, acid number, and pH were also determined.




70

8.2 Materials and Methods

8.2.1 Materials

In this study, the sample (Fig 1) was collected from Biomass Conversion &

Gasification Laboratory of Department of Energy, Tezpur University, India, after lipid

extraction with mechanical oil expeller [Malnad type oil expeller (Indus)]. CSDC were

ground using a Wiley mill to pass a 0.4 mm (40 mesh) screen (as per TAPPI T257 Om- 85

methods) to fine particles (420 micron) in order to eliminate heat transfer effects during

pyrolysis and then samples were oven dried and kept in a desiccator. The proximate and

ultimate analyses data for CSDC are given in Table 1.

Figure 1: Camellia Sinesis amd CSDC

8.2.2 Characterization of feed stocks and biochar

The proximate analysis of tea seed deolied cake and biochar obtained after pyrolysis

were done by ASTM D 3173-75 and ultimate analysis was done using CHN analyzer (Perkin

Elmer, 2400Series-II). The percentage of oxygen was determined by means of difference.

Higher heating value (HHV) was determined by 5E-1AC/ML, Auto bomb calorimeter

according to ASTM D2015. The thermal behavior of CSDC to 900oC at different heating

rates of 20oC/min were studied non-isothermally using Pyris Diamond TG/DTA analyzer

(PERKIN ELMER). A high purity N2 gas (99.99%) was used as a carrier gas at a flow rate of

100 ml min-1.

8.2.3 Pyrolysis set-up

The schematic diagram of the experimental setup is shown in Fig 2. The pyrolysis

setup consists of fixed bed reactor made of stainless steel with a length of 48 cm and an

internal diameter of 3 cm, equipped with an inert gas (nitrogen) connection. The reactor was

heated externally by an electric furnace, with the temperature being controlled by a Ni–Cr–Ni




71

thermocouple fitted inside the reactor. The thermocouple was connected to a PID controller for

controlling the temperature and heating rate. Before the experiments, the reactor was purged by

nitrogen gas for 10 min at a flow rate of 30 ml min-1 to remove the air inside. Then, nitrogen flow

rate was set to the desired value. A 10g of deoiled cake were loaded for each run of experiment.The liquid portion was recovered with diethylether washing. The aqueous phase was separated

from oil phase with a separating funnel. The bio-oil and solvent mixture was passed over dry

sodium sulphate to make it water free and then the solvent was evaporated from bio-oil by rotary

evaporator and the residual was weighed as bio-oil. The residual solid in the reactor was weighed

as char. The gas yield was calculated from the material balance. The reactions were carried out at

different temperatures ranging from 400 – 600oC.

Figure 2: Schematic diagram of experimental unit for pyrolysis

8.2.4 Characterization of bio-oil

Fourier Transformer Infrared spectroscopy (FTIR) of the pyrolytic oil obtained at

maximum yielding condition was taken with a Nicolet Impact I-410 model Fourier Transform

Infrared Spectrometer to chemical composition of the bio-oil. The components of the bio-oil

were analyzed using Perkin Elmer Clarus680 GC/600C MS. A capillary column coated with

a 0.25 µm film of DB-5 with length of 30 m and diameter 0.25 mm was used. The GC was

equipped with a split injector at 2000C with a split ratio of 1:10. Helium gas of 99.995%

purity was used as carrier gas at flow rate of 1.51 ml min-1. The oven initial temperature was

set to 70oC for 2 min and then increased to 290oC at a rate of 10oCmin-1 and maintained for 7

Gas

Thermocouple

N2 cylinder

F F

R

C

V 1 V 2

FC

Liquid product

F- Furnace

R- ReactorC- Condenser

V- ValveFC- Flow controller




72

min. All the compounds were identified by means of the NIST library. Mass spectrometer

was operated at an interface temperature of 200oC with ion source temperature of 180oC of

range 40–1000 m/z.

The physical properties such as density, kinematic viscosity , flash point and calorific

value were determined using standard test methods.

8.3 Result and Discussion

8.3.1 Characterization of feedstock and obtained biochar

The proximate and ultimate analysis of CSDC and biochar are shown in the Table 1.

The volatile matters and ash content of the sample in proximate analysis was found to be

80.7% and 5.34% respectively. Low ash content of deoiled cake indiacate that the sample is

good candidate for thermochemical conversion process. The volatile matter of the sample

was 80.7% which is reduced to 22.72% after pyrolysis. It indicates that a large portion of the

sample was converted to condensable and incondensable gases. As a result of significance

decrease in the volatilie matters the fixed carbon content of the solid material increased which

indicates relatively less liberation of the fixed carbon during pyrolysis. Moisture content play

an important role in selection of the conversion process. Sample with less moisture content

are suitable for thermal conversion while those with high moisture content are more suitablefor biochemical conversion process such as fermentation. In this regard, tea seed deoiled cake

with moisture content 4.61 % is a potential candidate for thermal conversion. Ultimate

analysis presented in the Table1 showed a significance increase in carbon content of biochar

whereas its oxygen content decreased in comparison to the oxygen content of the raw

material.

8.3.2 TGA and DTG analysis of CSDC

Thermogravimetric analysis (TGA) technique is applied in determination of thermal

stability of the sample in various ranges of temperatures. The TGA plot of CSDC at heating

rate of 200C min-1 under nitrogen atmosphere is shown in Fig 3(a). The TGA of the sample

shows that the temperature range from 133- 363oC associates with maximum weight loss. In

this region this zone of TGA can be referred to as active pyrolytic zone. The intial weight loss

in the temperature range 43- 133oC represents the evaporation of moisture contents physically

absorbed in the deoiled cake. There is also possibility of loss of some light volatile matters.

During the final stage of decomposition starts at 363

o

C, the rate of weight loss is very slow.In initial stage 7.24%, in the active pyrolytic zone or the second stage of decomposition,




73

52.76 % and in the final stage 40% weight loss was observed. During the Second stage, the

intermolecular associations and weaker chemical bonds are destroyed [Jinno et al., 2004;

Chan et al., 2009]. The side aliphatic chains may be broken and some small gaseous

molecules are produced at the lower temperature [Biswal et al., 2013]. During the Third stage

at higher temperature chemical bonds are broken and the parent molecular skeletons are

destroyed. As a result, the larger molecule decomposes to form smaller molecules.

DTG curve (Fig 3(b)) shows that there is only one major peak at 294oC which was

present in active pyrolytic zone at temperature range 133-363oC.

Table 1: Proximate and ultimate analysis of CSDC

Properties Tea seed deoiled

cake

Biochar obtained

at 5000C

Rapeseed char

(Ucar et al., 2008)Proximate analysis (wt%)

Moisture 4.34 ± 0.27 6.23 ± 0.34 -

Volatile matter 80.22 ± 0.48 22.45 ± 0.27 20.01

Fixed Carbon 10.66 ± 0.41 38.46 ± 0.02 16.41

Ash 4.78 ± 0.66 9.78 ± 0.41 18.54

Ultimate analysis (wt%)

Carbon 47.63 76.35 56.48

Hydrogen 7.34 3.76 3.22

Nitrogen 3.48 5.67 7.32

Oxygen 41.55 14.22 32.25Sulphur - - 0.23

H/C molar ratio (on ashfree basis)

1.849 0.591 0.68

Emperical formula CH1.849N0.062O0.654 CH0.591N0.063O0.139 CH0.08N0.114O0.43S0.001

Gross calorific value(MJ/kg)

19.65 27.92 23.88

8.3.3 Effect of temperature on the product distribution

The pyrolysis of the tea seed deoiled cake yielded three different products viz. liquid,

gas and solid residue (biochar). Results of mass balance of pyrolytic decomposition products

were presented in Table 2. The bio-oil yield increased from 22.25% to 27.38% with increased

in temperatures from 400-500oC but decreased from 27.38% to 23.74% in temperature range

500-600oC. The maximum char yield 37.43% was obtained at temperature 400oC. The yield

of char decreased with increasing temperature. The decrease in char yield with increase in

temperature may be due to the higher decomposition of the biomass sample in higher

pyrolytic temperature or may be due secondary decomposition of char residue [Horn and

William, 1996]. The yield of gas increased with increase in temperatures. This is may be due




74

to the secondary decomposition of the char and secondary cracking of the pyrolysis may

enrich the content of the gas product at higher temperature.

0 200 400 600 800 1000

0

20

40

60

80

100

W e i g h t r e m a i n i n g ( % )

Temperature (0C)

(a)

100 200 300 400 500 600 700 800 900

100.0

100.2

100.4

100.6

100.8

101.0

101.2

101.4

D e r i v a t i v e Y 1 ( m g / m i n )

Temperature (0C)

(b)

Figure 3: (a) TGA and (b) DTG plot of Camellia Sinesis deolied cake

Table 2: Effect temperatures on product distribution of pyrolysis

Temperature

(oC)

Biochar

(wt%)

Bio-oil (wt%) Aquous Phase

(wt%)

Gas (wt%)

400 37.43 22.25 18.87 20.31

450 32.23 25.46 17.81 23.67

500 29.13 27.38 17.36 25.43

550 27.67 24.51 16.54 30.67

600 25.47 23.74 15.23 35.28

8.3.4 FTIR of the bio-oil sample obtained at 500oC

Biomass pyrolytic oil is composed of wide range of complex organic compound.

FTIR analysis was performed to investigate the chemical structure of the bio-oil sample.

Figure 4 shows the FTIR spectra of tea seed deoiled cake. The O-H stretching vibrations at




75

frequency 3379 cm-1 indicates the presence of alcohol or phenols. The presence of alkanes is

detected at peaks around 2851 cm-1 and 2930 cm-1 with C-H stretching vibrations. C=O

stretching vibrations cause band at 1717 cm-1. The presence of alkenes was detected by C=C

stretching vibrations at 1610 cm-1

. The peaks in the range of 950–1300 cm-1

show the

presence of C–O stretching vibrations present in alcohol or ester. the C-H bending vibrations

frequency 812 cm-1 indicates the presence of phenyl ring substitution bands. The results were

found consistent when compared with the results of GC-MS.

4000 3500 3000 2500 2000 1500 1000 500

20

30

40

50

60

70

80

90

100

110

% T r a n s m i t t a n c e

Wavenum ber (cm-1

)

O-H

C-H

C=O,C=C

C-O,C-H

Figure 4: FTIR of CSDC bio-oil

8.3.5 GC-MS of the bio-oil sample

The GC-MS analysis (Fig.5) of the oil sample is summarized in Table 3. More than

40 peaks are displayed in the GC/MS chromatograph but because of the complex nature the

perfect separation of all the peaks are not possible and also the depending on strength of MS

library, 10 peaks are evaluated. Comparing the mass spectra fragmentation pattern with

Perkin Elmer NITS library and published data, the highest likelihood of compounds

identification were obtained. The carbon distribution of the identified compounds were in the

range of C5-C29.

Figure 5: GC-MS of CSDC bio-oil

ScanTI

1.78e

I-(b)_15-5-

0

%

10

10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0Tim

8.5

9.9

10.1

11.3

14.6

16.421.2

21.922.5

23.9

24.4




76

Table 3: Compound identified by GC-MS of CSDC bio-oil

S.No. TR (min) Tentative compounds Empirical

formulae

1 8.57 2-PYRIDINECARBOXYLIC ACID, METHYL ESTER C7H7NO2 2 9.93 DECANOIC ACID, 2-METHYL- C11H22O2

3 10.14 3-FURANMETHANOL C5H6O2

4 11.37 FURAN, 2,4-DIMETHYL- C6H8O

5 14.67 MEQUINOL C7H8O2

6 16.47 PHENOL, 2-METHOXY-4-METHYL- C8H10O2

7 21.28 N-TETRACOSANOL-1 C24H50O

8 21.94 1-HEPTACOSANOL C27H56O

9 22.53 METHYL OCTACOSANOATE C29H58O2

10 23.97 CYCLOTRISILOXANE, HEXAMETHYL- C6H18O3Si3

8.3.6 Physical properties of the bio-oil

Table 4 shows the results of the elemental analysis of the CSDC bio-oil. The result

shows that the bio-oil has higher heating value of 32.25 KJ/kg which is higher than some

bio-oils produced from different de-oiled cake such as Neem cake bio-oil (30 MJ/kg) [Volli

et al., 2012], Mustard de-oiled cake (25.2 MJ/kg) [Volli et al., 2012].

Table 4: Ultimate analysis of bio-oil

PARAMETERS CSDC BIO- OILC 71.35H 8.05N 4.65O 15.95

H/C 1.354O/C 0.167

EMPERICAL FORMULA

Higher Heating Value(MJ/kg)CH1.354 N0.055 O0.167

32.25

The comparison of the various necessary properties of the oil obtained from themustard de-oiled cake and diesel is shown in Table 5. The viscosity of the bio-oil is

comparatively higher than that of diesel which may lead to poor atomization and incomplete

combustion. Therefore the bio-oil is not suitable for direct use as a engine fuel but can be use

in moder diesel engine by blending with the diesel. The higher heating value of bio-oil is also

lower than that of diesel engine.




77

Table 5: Fuel properties of of CSDC pyrolyitic oil

Properties Standard test method Pyrolytic oil Diesel [Tuttle

et al. 2004]

Kinematic viscosity, 40

0

C ASTM D 445 28 2-5.5Flash point ASTM D 93 62 53-80

PH PH meter 3.24 -

Acid Number ASTM D664 33.56 -

Higher heating value (MJ/kg) - 32.25 42-45

Appearance - Dark brown

Chemical formula Identified C5-C29 C8-C25

8.4 Conclusion

Pyrolysis of Camellia Sinesis de-oiled cake was carried out in a fixed-bed reactor

made up of stainless steel at temperature range from 400oC to 600oC and at a rate of 30oC

min-1 to produce bio-fuel. The maximum yield of oil is 27.38% on wt. % basis for Camellia

Sinesis de-oiled cake, was obtained at a temperature of 500oC. The fuel and chemical analysis

of bio-oil reveals that these pyrolytic oils can be used as fuel and as a source of chemicals.

The carbon distribution in the chemical identified in bio-oil is in range C5-C29.

References

1. Biswal B., Kumar S. and Singh R.K.(2013).Production of hydrocarbon liquid by thermal

pyrolysis of paper cup waste. Journal of Waste Management, URL:

http://dx.doi.org/10.1155/2013/731858.

2. Bridgwater A.V. and Peacocke G.V.C., (2004).Fast pyrolysis: Fast pyrolysis processes

for biomass. Renewable and Sustainable Energy Reviews, 4:41-73.

3. Chan P.W., Atrey A., Howard R.B. (2009). Determination of pyrolysis temperature for

charring materials. Proceedings of the combustion Institute, 32: 2471-2479.

4. Horne P.A., Williams P.T. (1996). Influence of temperature on the products from the

flash pyrolysis of biomass. Fuel, 75: 1051-1059.

5. Jinno D., Gupta A.K.(2004). Determination of chemical kinetic parameters of surrogate

solid wastes. J.Eng. Gas. Turb. Power, 126: 126-685.

6. Mulligan C.J., Strezov L., Strezov V., (2010). Thermal decomposition of wheat straw

and mallee residue under pyrolysis condition. Energy and fuel, 24:46-52.




78

7. Ozcimen D, Karaosmanoglu F. (2004) Production and characterization of bio-oil and

biochar from rapeseed cake. Renew Energy; 29:779–87.

8. Raja S.A., Kennedy Z.R., Pillai B.C., Lee C.L.R.(2010). Flash pyrolysis of jatropha oil

cake in electrically heated fluidized bed reactor. Energy; 35:2819–23.

9. Tuttle J., Kuegelgen V., (2004). Biodiesel Handling and Use Guidelines, Third edition in

National Renewable Energy Laboratory.

10. Ucar S., Ozkan A.R. (2008). Characterization of products from the pyrolysis of rapeseed

oil cake. Bioresour Technol; 99:8771–6.

11. Uzun BB, Putun AE, Putun E. (2006) Fast pyrolysis of soybean cake: product yields and

compositions. Bioresour Technol; 97:569–76.

12. Volli V., Singh R.K. (2012). Production of bio-oil from de-oiled cakes by thermalpyrolysis. Fuel, 96: 579-585.




79

CHAPTER 9

COMPARATIVE STUDY OF DIFFERENT BIOMASS

COOKSTOVE MODEL: AN EXPERIMENTAL STUDY

K Pal, A K Pandey, P Gera and S K Tyagi

Abstract

This article presents the comparative experimental study of five different types of improved

biomass cookstoves models based on their thermal efficiency, power output rating and

emission reduction potential. All the cookstoves models are design and fabricated in thelaboratory based on the gasification principle except NIRE-02, which is the modified form of

the traditional cookstove. The performance of each cookstoves were evaluated following

water boiling test as per Bureau of Indian Standard (BIS) protocols, whereas the emission

reduction was calculated based upon the clean development mechanism (CDM) of United

Nation Framework Convention on Climate Change (UNFCCC). The overall performances of

these models were found to be much better than that of the traditional cookstoves being used

by the majority of population around the globe. The emission reductions potential from these

models were found to be between 2.0-3.0 tons per household annually, which not only shows

the good agreement with the experimental values available in the literature but also represent

a high potential for disseminating these cookstoves through CDM in the rural and remote area

of the country, especially, where woody biomass is the major consumption as the cooking

fuel.

Keywords: Energy efficiency, Improved biomass cookstove, Clean development mechanism.

9.1 Introduction

The past evidences of fire have been found as old as four lakh years i.e. during the

first ice age (Bronowski, 1973). However at that time fire was used for roast the meat.

Mastery of fire is considered to be an important step toward human development which took

off only about 12,000 years ago (FOA, 1993). A three stone fire arrangement was first time

was used by people of ancient time for cooking their food. The use of three stone fire

arrangement has many health and environment problems like exposure to heat as well as fire

hazard beside the problem of poor thermal efficiency. The requirement for a better cookstove




80

arose which gave rise to U-shaped mud cookstove/traditional cookstove. The traditional

cookstoves solved many of the technical problems such as, enhancement of thermal

efficiency by many folds. However, the health, socio-economic and cultural problems were

not solved completely.

The development of efficient cookstoves at International level was started in the

1940s (Anhalt and Holanda, 2013). However, in India the efforts for making improved

biomass cookstoves started during 1950s with the main objective to improve the design of

biomass-fired stoves and the widespread R&D started in 1970s. Later on, improved

cookstove programs (ICPs) were considered as a solution to the fuel wood crisis and to

reduce deforestation. A model of improved multiport stove was introduced by Raju in 1953

which was one of the high-mass and shielded-fire type and had a chimney to remove smoke

and adjustable metal dampers to regulate the fire. Singer (1961) in Indonesia conducted

cookstove efficiency tests on high mass mud stove with the main objective to increase

efficiency and save fuel, without considering the socio-economic and cultural aspects.

However, in 1990s the focus shifted more on the issues involving the Indoor air pollution and

its effect on human health (World Bank, 2011).

In addition to above factors, the comfort of cooking, smoke free kitchens,

convenience and safety of stove users were tested and considered to be the important aspects

as compared to the fuel savings. Smith et al., (2000) stated that the burning of biomass fuels

emits high levels of smoke, containing hazardous pollutants such as, respirable suspended

particulate matter (RSPM), carbon dioxide (CO2), carbon monoxide (CO), nitrogen oxides

(NOx), sulfur oxides (SOx) and some cancer causing organic compounds like Benzopyrene,

benzene and 1,3-Butadiene. Fullerton et al., (2008) found that different types of health risks

were associated with the indoor air pollution such as, respiratory infections like pneumonia,

tuberculosis, chronic obstructive pulmonary disease, birth defects, cataracts, cardiovascular

events which cause serious problems in the women and child. Kleeman et al., (2000) studied

the adverse health impacts of the particle size distribution and result of deposition of those

pollutants in different areas of lungs.

Keeping the above facts in mind, Ministry of New and Renewable Energy,

Government of India has initiated a National level program to develop next-generation

cleaner cookstoves and deploy them to the households where traditional cookstoves being

used for cooking and heating applications. The initiative has set itself the lofty aim of




81

providing energy service comparable to clean sources like LPG without changing the fuels.

Based on National surveys, published literature and assessments, and measurements of

cookstove performance around the globe, it was found that about 570,000 premature deaths

in poor women and children and over 4% of India's estimated greenhouse emissions could be

avoided if such an initiative were in place. Although, the current advanced biomass stoves

show substantial emissions reductions over the traditional stoves but there is a lot to be done

to reach the LPG-like emission levels.

WHO (2011) has estimated that every year indoor air pollution (IAP) is responsible

for the death of 1.8 million people around the globe which comes out to be one death every

20 seconds. It was further estimated that the exposure to smoke from simple act of cooking

constitutes the fifth worst risk factor for disease in the developing countries and causes

almost two million premature deaths per year, exceeding the deaths attributable to malaria or

tuberculosis (WHO, 2006). Ramanathan and Carmichael, (2008) recently found that the black

carbon is playing a major role in the global warming.

The Global Alliance for Clean Cookstoves (GACC), a new public–private partnership

led by the UN Foundation was established to create a thriving global market for clean and

efficient household cooking solutions (Global Alliance for Clean Cookstoves, 2011). The newly

cookstoves which are known as advanced biomass cookstoves are based on better design

principles; they have the better combustion efficiency and thus, reduce the fuel consumption

to a greater extent. These cookstoves can deal with both the emissions and health issues

resulting from cooking with open fires or traditional biomass cookstoves. These cookstoves

have the ability to get carbon credits (UNFCCC, 2013), not only because of their contribution

to climate-change mitigation but also they yield major co-benefits in terms of energy access

for the poor people. Although, the current advanced biomass stoves show substantial

emissions reductions over the traditional stoves, yet more improvements are needed to reach

the LPG-like emission levels.

9.2 Mathematical Modeling

An improved biomass cooking stoves have the ability to reduce indoor air pollution,

deforestation, climate change, and improve quality of life of rural peoples on a global scale.

The better design of these cookstoves can significantly impact their performance and

emissions. Although these improved biomass stoves have been studied for a long time

however, a theoretical understanding of their operating behavior and the development of




82

engineering tools for an improved cookstove based on natural convection is still lacking. This

section presents the mathematical modeling of improved biomass cookstove based on various

performance parameters as below:

9.2.1

Side feeding wood-burning cookstove

Agenbroad et al. (2011a) developed a simplified model for the understanding the

fundamental operating behaviour of these natural convection based biomass cookstoves. This

model was developed utilize the dimensional form of two equation system. This has been

further developed into a dimensionless form at a later stage (Agenbroad et al. 2011b). A

simplified model of the fundamental stove has been developed for predicting bulk flow rate,

temperature, and excess air ratio, based on stove geometry including chimney height,

chimney area, viscous and heat release losses and the operating firepower. The stove operator

decided the operating firepower of the stove by controlling the fuel feed rate, excess air ratio.

The resulting bulk flow rate, temperature, and excess air ratio etc. are the fundamental inputs

for stove performance. The processes are categorized into two basic and fundamental, (a)

heat addition from combustion, and (b) kinetic energy addition due to the chimney effect, and

the details are given as below:

9.2.2 Heat addition from combustion

The heat of combustion increased the temperature and decreased the density of bulk

flow passed over there. If we assume that the heat addition is efficient and instantaneous and

the system is isobaric with no mechanical work done on/by the system, having ideal gas

behavior, with constant potential and kinetic energies. The heat addition from the combustion

showed an enthalpy increase (hC to hH) distributed in the mass flow rate ( Am.

). The increased

bulk flow temperature is calculated for a given mass flow rate Am.

, heat release ratein

Q. , using

the constant pressure specific heat of air (Cp), as below (Kumar et al., 2013):

)(,

.)(

.)(

..

C T H T avg pC Am

H T

cT dT T pC Am H h

C h AminQ −=∫=−=

(1)

where T is the bulk flow temperature and the subscripts H and C denote the hot and cold

conditions, respectively.

9.2.3 Kinetic energy addition due to the chimney effect

The air flow through the stove depends on the chimney effect resulting from thebuoyant force of the decreased density of air after heat addition of combustion. The decreased




83

density of air in the chimney creates a lower pressure as compare to the ambient pressure. In

a fluid, the relationship between pressure variation with depth can be determined using the

hydrostatic equation and the net pressure difference (∆P) as given below (Kumar et al.,

2013):

∫=∆ dhhgP )( ρ (2)

where g is the acceleration due to gravity, and ρ is the density of medium which is a function

of chimney height (h). The pressure can be calculated as below (Kumar et al., 2013):

∫∫∫ −=+−+=∆−

2

3

2

33

1

3321 )())(()( dhhghgdhhgPdhgPP ambamb ρ ρ ρ ρ (3)

If chimney walls are assumed to be adiabatic, than the temperature and density, ρ of the flue

gases in the chimney (TH and ρH) remain constant, so Eq. (3) can be simplifies which

determined the chimney effect due to the pressure difference as (Kumar et al., 2013):

)(21 H amb

ghP ρ ρ −=∆−

(4)

The gain in kinetic energy from the stagnant ambient air can be calculated using the integral

form of Bernoulli's equation for the compressible flow, as (Kumar et al., 2013):

2

2

2

1)(

21vghP

H H amb ρ ρ ρ =−=∆

−

(5)

where H ρ is the density of hot gas and 2

v is the velocity use for calculating the kinetic

energy. Again assuming ideal gas behavior of the flue gases, the density is related to the

temperature by the ideal gas law. Using ideal gas equations and solving Eq. (5) for volume

and mass flow rate gives (Kumar et al., 2013):

2.

2

1)(

=−

A

V gh H H amb ρ ρ ρ

(6)

H

H ambghCAV ρ

ρ ρ −= 2

.

(7)

amb

amb H

T

T T ghCAV

−= 2

.

(8)

amb

amb H

H s

AT

T T gh

T R

PCAm

−×

= 2

1.

(9)




84

where C is the loss coefficient introduce to account for uncertainties and inefficiencies in the

chimney effect including viscous losses, chimney wall heat transfer, and the unrealistic ideal

point heat addition at state point 2 and its range is 0≤ C ≤1. The mass flow rate of fuel (F

m.

)

in the cookstove model can be calculated from the firepower (inQ

. ) and the heating value (HV)

of the fuel as (Kumar et al., 2013):

LHV Q

mF

..

= (10)

The air fuel ratio (AFR) and excess air ratio (EAR) can be determined as below (Kumar et

al., 2013):

.

.

F

A

m

m AFR = (11)

AFR

AFRstoich=Φ

(12)

Φ

Φ−=

%100).1(% EAR

(13)

where AFRstoich is the air furl ratio (AFR) for stoichiometric combustion. The lower heating

value (LHV) is used because the latent heat of the water vapor is significant, and seldomrecovered.

9.2.4

Dimensionless chimney effect equation

The advantages of working in the dimensionless form include the scale similarity and

reducing the number of independent parameters for experimentation independent of stove

geometry. The dimensionless temperature form can be determined by inspection and is given

as below (Kumar et al., 2013):

amb

amb H

T

T T T

−≡

* (14)

Using Eq. (14) and Eq. (9), the mass flow rate of air in the cookstove model can be

expressed as given below (Kumar et al., 2013):

)1(

2*

*.

+×

=

T

ghT

T R

PCAm

amb

A

(15)




85

Substituting P/RTamb as the ambient density ρamb in the above equation and rearranging it as

(Kumar et al., 2013):

1

2

*

*.

+

=T

T

ghCA

m

amb

A

ρ (16)

This dimensionless mass flow rate can also be defined as the ratio of the actual mass

flow rate to the characteristic natural convection and from Eq. (16), the dimensionless mass

flow rate of air for the given geometry given as below (Kumar et al., 2013):

ghambCA

Am Am ρ

.*.≡

(17)

Using the dimensionless mass flow rate*.

Am of Eq. (17), the final form of chimney

effect equation is given as below (Kumar et al., 2013):

1

2*

**

.

+=

T

T m A

(18)

9.2.5 Dimensionless heat addition equation

The mass burn rate of fuel is used instead of the firepower. The relation between the

firepower, mass burn rate and heating value is given as below (Kumar et al., 2013):

HV mQ F in

..

= (19)

Substituting Eq. (19) and Eq. (14) into Eq. (1) and rearranging it, a dimensionless heating

value group (HV*) is formed as (Kumar et al., 2013):

*..

T mT C

HV m A

amb p

F =

(20)

amb pT C

HV HV ≡*

(21)

This group defined as the ratio of the combustion heating energy to the initial thermal

energy of the flow. Substituting the dimensionless mass air flow rate as defined in Eq. (17),

into Eq. (20) which gives the relation between dimensionless heating value, dimensionless

mass flow rate of air and dimensionless temperature as (Kumar et al., 2013):




86

**.*.T ghambCA

Am HV

F m ρ =

(22)

A dimensionless mass burn rate similar to the dimensionless air flow rate of Eq. (17) is given

as below (Kumar et al., 2013):

ghCA

mm

amb

F F

ρ

.

*.

≡

(23)

Using the dimensionless mass burn rate*

.

F m , the final form of the dimensionless heat addition

equation becomes (Kumar et al., 2013):

**.

**.

T m HV m AF = (24)

9.2.6

Air/fuel ratio from dimensionless model

The air/fuel ratio (AFR) is defined as the ratio of mass flow rate of air to the mass

burn rate of fuel and can be given by rearranging Eq. (24) as shown below (Kumar et al.,

2013):

*

*

*.

*.

T

HV

F m

Am AFR ==

(25)

The dimensionless heating value (HV*) is considered as remain constant throughout the stove

operation. From the above equation a simple inverse linear relationship can be seen between

dimensionless temperature (T*) and the air fuel ratio (AFR).


A comparative study on modified traditional cookstove and three different improved

biomass cookstoves (NIRE-03, NIRE-05, NIRE-06) has been presented in this research

article. These improved cookstove work on the principle of down-draft gasifier where

pyrolysis, gasification and combustion of biomass are taking place simultaneously. These

cookstoves were made of mild steel whereas for insulation clay and wheat straw mixture was

used. The line diagram of NIRE-02, NIRE-03, NIRE-05 and NIRE-06 are shown in the figure

1-4.

9.3.1 Materials Use

The following instruments/equipments have been used during the experiments:

(a) Platform balance




87

(b) Glass cylinders for measuring water

(c) Aluminium vessels with lids of proper volumes as per BIS standard

(d) Kerosene oil to ignite the process

(e) Match stick

(f) Stopwatch

(g) Thermometer/Thermo-couple

(h) Bomb calorimeter

(i) Wood fuel in proper size

Space for flame

Air

200

260

190

200

40

35

35

260

Grate

Platform

Fig. 1: Line diagram of the modified traditional Cookstove, (NIRE-02)




88

Fig. 3: Line diagram of improved cookstove (NIRE-05)

410

25

253

Primary air inlet

5060

50

125

Wick

Handle

Grate

Pot support

Secondary air inlet

25

15

Insulation23

50

Top view

Bottom view

Handle for primary

Air adjustment

Handle for primary

Air adjustment

25

410Bottom view

Primary air inlet

Secondary air inlet

50

5060

50

150

Wick

Handle

Grate

Pot support

25

15

Insulation23

Top view

253





89

9.3.2 Methods

As per the Bureau of Indian Standards (BIS, 2013) water boiling test of all above

mentioned four cook stoves were performed for the measurement of thermal efficiency.

According to the BIS protocol stepwise procedure was follow for cookstove testing. At the

time of experimental run, the temperature of water, flame and cookstove body was measured

with the help of digital temperature sensor whereas the temperature of pot, plate and ambient

was measured with mercury-glass thermometer. The measured value of thermal efficiency of

each cookstove is compared with the open three stone fire cookstove which generally have

the thermal efficiency 8-10%. The stepwise procedure of fuel preparation, burning capacity

rate and water boiling test is as follow:

a) Fuel Preparation

The fuel wood cut from the same log into pieces of 3x3 cm square cross-section and

length of half the diameter/length of combustion chamber so as to be housed inside the

combustion chamber. The fuel pieces shall be oven dried by the following method (BIS,

2013):

a) Weigh total quantity of wood (say 'M' kg.).

b) Pick up one piece and mark 'X' by engraving and take its mass (say 'm' g.).

230

Secondary air inlet

Handle for primary

Air adjustment

Primary air inlet

25

5060

410

50

110

Wick

Handle

Grate

Pot support

25

15

Insulation45

50

Air gap

Bottom view

Top view





90

c) Raise the temperature of oven up to 105 ºC.

d) Stack the wood pieces in a honey comb fashion inside the oven.

e) Maintain the oven temperature at 105 ºC.

f) After 6 hours, remove the marked 'X' piece, weigh it and note reduction in mass from

'm' g, if any. If reduction is observed put the marked piece in the oven again and

repeat the weighing of 'X' marked piece after every subsequent 6 hours period till the

mass is constant and no further reduction in mass is observed.

g) At this stage, weigh the total quantity of wood and note loss of mass from 'M' kg.

h) Determine the calorific value of the prepared wood with the help of bomb calorimeter.

b) Burning Capacity Rate

Fuel burn per hour is known as the burning capacity rate of a cookstove, which can be

calculated as: Stack the combustion chamber with test fuel in honey comb fashion up to 3/4

of the height or in a pattern recommended by the manufacturer. Sprinkle 10 to 15 ml. of

kerosene on the fuel from the top of chulha/fire box mouth. Weigh the chulha with fuel; let

the mass be M1 kg. After half an hour of lighting weigh the chulha again and let the mass be

M2 kg. If the calorific value (CV) of the test fuel in kcal/kg then calculate the burning

capacity of the chulha as heat input per hour as follows (BIS, 2013):

Heat input: per hour = 2 (M1 - M2) x CV kcal/h (26)

c)

Water Boiling Test

a) Take the test fuel according to burning capacity rating for one hour. Let the mass be

'X' kg.

b) Stack the first lot of test fuel in the combustion chamber in honey comb fashion or as

indicated by the manufacturer.

c) Select and weigh the vessel with the lid in accordance with the table above. A

minimum of two such vessels in a set will be required. Put the recommended quantity

of water at 23 + 2 ºC (T1).

d) Sprinkle measured quantity 'X' ml. (say 10 - 15 ml.) of kerosene for easy lighting on

the test fuel and light. Simultaneously start the stop watch.

e) Feeding of fresh test fuel lot shall be done after every 15 minutes.

f) The water in the vessel shall be allowed to warm steadily till it reaches a temperature

of about 93 ºC, then stirring is commenced and continued until the temperature ofwater reaches 5 ºC below boiling point at a place. Note down time taken to heat the




91

water up to final temperature (less than 5 ºC below the boiling point) T2 ºC.

g) Remove the vessel of from the chulha and put the second vessel immediately on the

chulha. Prepare first vessel for subsequent heating.

h) Repeat the experiment by alternatively putting the two vessels taken till there is no

visible flame in the combustion chamber of the chulha. Note down the temperature of

the water in the last vessel.

9.4 Analysis of Cookstove

Improved biomass cookstoves can reduce indoor air pollution, deforestation, climate

change, and therefore, the quality of life can be improved on a universal scale. The better

design of these cookstoves can significantly impact their performance and emissions.

Although these improved biomass cookstoves have been studied for a long time however, a

theoretical understanding of their operating behaviour and the development of engineering

tools for an improved cookstove based on natural convection is still missing.

9.4.1 Thermal efficiency

Thermal efficiency of a cookstove may be defined as the ratio of heat utilized to the

heat produced by complete combustion of a given quantity of fuel based on the net calorific

value of the fuel and this can be written as below [Kumar et al., 2013, Kishore and Ramana,2002, Tyagi et al., 2013, BIS, 2013].

100ProducedHeat

UtilizedHeatηefficiencyThermal ×= (2)

Heat utilized= )T-8)(T4.186w+0.896)(WT-8)(T4.186w+0.8961)(W-(n 1312 ×××× kJ

Heat produced = /1000)c(V+)c(X84.186 21 ××× ρ kJ

100)1000 / (+)c(X84.186

)T-8)(T4.186w+0.896)(WT-8)(T4.186w+0.8961)(W(n-η

21

1312×

×

××××=

cV ρ

where w is the mass of water in vessel, W is the mass of vessel complete with lid and stirrer,

X is the mass of fuel consumed, c1 is the calorific value of wood, V is the volume of kerosene

consumed, c2 is the calorific value of kerosene, ρ is the density of kerosene, T1 is the initial

temperature of water, T2 is the final temperature of water, T3 is the final temperature of water

in last vessel at the completion of test, in °C; and n is the total number of vessels used.




92

9.4.2 Exergy efficiency

Exergy efficiency may be defined as the ratio of output exergy to the input exergy and

given by [Dincer, 2007, Tyagi et al., 2013]:

100

cdxη)T

T(1cm

)T

T)(1T(TCm)

T

T)(1T(TCm

100E

E100

inputExergy

outputExergyψ

2

fuel

a1wd

fp

aipfp1pApot

fw

aiwfwpw

in

o

×

××+×−

−−+−−

=

×=×= x

x

(3)

where mw is the mass water in the pot, Cp is the specific heat of water, Tfw is the final

temperature of water, Tiw is the initial temperature of water, mpot is the mass of pot, CpA1 is

the specific heat of Aluminium pot, Tfp is the final temperature of pot, Tip is the initial

temperature of pot, mwd is the mass of wood, c1 is the calorific value of wood, Ta is the

ambient temperature, Tfuel is the flame temperature, η theoretical efficiency, x is the volume

of kerosene, d is the density of kerosene and c2 is the calorific value of kerosene.

9.4.3 Power Output Rating

The power output rating of a cookstove is a measure of total useful energy produced

during one hour burning of fuel wood. It shall be calculated as follows (BIS, 2013):

kW,1003600

ηCVratingoutputPower

×

××=

m (4)

where m is the quantity of fuel wood burnt per hour, CV is the calorific value of fuel wood

and η is the thermal efficiency of the cookstove, as calculated above.

9.4.4 Emission reduction calculations

The emission reduction from each cookstove is based upon fraction of biomass thatcan be saved during the project year and the calorific value of the biomass used and this can

be calculated according to the formula given below (Global Alliance for Clean Cookstoves,

2011):

fossilfuel projected biomass y NRBsavings y y EF NCV f B ER _,, ×××= (5)

where y ER is emission reductions during the year y in tCO2 , savings y B ,

is quantity of woody

biomass that is saved in tonnes, y NRB f

, is the fraction of woody biomass saved by the project




93

activity in year y that can be established as non-renewable biomass, biomass NCV is the net

calorific value of the non-renewable woody biomass that is substituted (IPCC default for

wood fuel, 0.015 Tj/tonne), fossilfuel projected EF _ is emission factor for the substitution of non-

renewable woody biomass by similar consumers, use of value of 81.6 tCO2 /TJ andsavings y B ,

can be calculated using the following formula:

−=

new

old old savings y B B

η

η 1., (6)

Where Bold is Quantity of woody biomass used in the absence of the project activity in

tonnes, Ƞold is the efficiency of the baseline system being replaced, measured using

representative Sampling methods or based on referenced literature values (fraction), use

weighted Average values if more than one type of system is being replaced; a default value of

0.10 may be optionally used if the replaced system is the three stone fire or a Conventional

system with no improved combustion air supply or flue gas Ventilation system i.e., without a

grate or a chimney; for other types of systems a Default value of 0.2 may be optionally used

and Ƞnew is the efficiency of the system being deployed as part of the project activity

(fraction), as Determined using the water boiling test (WBT) protocol. Use weighted average

Values if more than one type of system is being introduced by the project activity.


Based on the biomass characteristics, the performance of different cookstoves has

been carried out using burning capacity rate, power output, thermal efficiency and carbon

emission reduction analysis at a typical location in India, while the discussion of results is

given as below:

Figure 5 shows the burning capacity rate and power output rating of the different

model of cookstoves. As shown from the figure the burning rate and power output of NIRE-

02 are 3.6 kg/hr and 4.80 kW respectively. The burning rate and power output of NIRE-03

are 2.42 kg/hr and 3.45 kW respectively. NIRE-05 has the burning rate 3.22 kg/hr with 5.23

kW power output rating and NIRE-06 has the value of these quantities 2.32 kg/hr and 3.22

kW respectively. The maximum power output is found with NIRE-05 with 3.22 kg/hr

burning rate which is the highest among all the other improved biomass cookstoves (NIRE-

03 and NIRE-06) and also from the modified traditional cookstove. These results are shows

that the combustion bf biomass in NIRE-05 is better than that of the other cookstove models.




94

Fig-5: Burning rate and power output of different cookstove model.

On the basis of results obtained from the water boiling test the thermal efficiency,

exergy efficiency and emission reduction of different cookstove models was calculated using

the equation (2)-(5) and the results obtained are presented in Fig 6. The thermal efficiency,

exergy efficiency and emission reduction potential of modified traditional cookstove (NIRE-

02) was found to be 25.31%, 1.86% and 2.08 tCO2 /year respectively. The performance of this

modified model (NIRE-02) is two to three folds higher that of the traditional three stone fire.

This cookstove model also has the good agreement with the carbon emission reduction as

shown in the figure. However the thermal efficiency of improved cookstove models NIRE-

03, NIRE-05 and NIRE-06 are 35.19%, 37.47%, and 32.26 percent respectively, exergy

efficiency 6.17%, 6.23% and 5.45% respectively and emission reduction potential from

different model was 2.46, 2.52, and 2.37 tCO2 /year respectively. Among all the improved

cookstove models, NIRE-05 has the maximum value of thermal efficiency, exergy efficiency

and as well as the CO2 emission reduction potential. This is because of the fact that the

burning of the fuel wood is very good in this cookstove with higher power output and

minimum heat loss into the environment. Based on the results obtained from different

cookstove models it is found that the NIRE-05 cookstove is a best designed model which is

steadied in this present study.

3.6

2.42

3.22

2.32

4.80

3.45

5.23

3.22

NIRE-02 NIRE-03 NIRE-05 NIRE-06

Cookstove Model

Burning Rate(kg/hr) Power Output(kW)




95

Fig-6: Thermal efficiency, Exergy efficiency and emission reduction of different cookstoveat burning capacity rate.

9. 6 Conclusions

Based upon the analysis of experimental data following conclusions are drawn:

• Based on the experimental observations the energy and exergy efficiency and as

well as the emission reduction potential for NIRE-02 model was found to be

best when both grate and top space was provided.

• The thermal efficiency 25.31% of modified traditional cookstove NIRE-02 was

found which is around two-three times higher than that of the open three stone

fire.

• The performance and the CO2 emission reduction potential of NIRE-05 model is

found to be higher than that of all other models for all set of operating

parameters.

• NIRE-05 model performs best and gives a large amount of emission reduction

value i.e. 2.52 tonnes of CO2 reduced per household per year which means if

approximately 10,000 NIRE-5 cookstove are provided to the consumers it will

save approximately 25,200 tonnes of CO2 in one year.

• Also energy efficiency was found to be always higher than that of exergy

efficiency which is due to the energy gained by the hot water at that particular

temperature.

25.31

35.19 37.47

32.26

1.86

6.17 6.23 5.452.08 2.46 2.52 2.37

NIRE-02 NIRE-03 NIRE-05 NIRE-06

Cookstove Model

Thermal Efficiency,η (%) Exergy Effficiency ,ψ(%) Emission Reduction (tCO2/year)




96

Acknowledgment

Authors gratefully acknowledge the financial assessment provided by Sardar Swaran

Singh National Institute of Renewable Energy, Kapurthala and Ministry of New and

Renewable Energy, New Delhi.

References

1. Anhalt J., and Holanda S. (2013) Policy for subsidizing efficient stoves. Project No.

10604030. Accessed from internet on 25.07.13, 10604030_2.pdf

2. Agenbroad J, DeFoort M, Kirkpatrick A, Kreutzer C, A simplified model for

understanding natural convection driven biomass cooking stoves—Part 1: Setup and

baseline validation, Energy for Sustainable Development 2011;15: 160–168.

3. Agenbroad J, DeFoort M, Kirkpatrick A, Kreutzer C, A simplified model for

understanding natural convection driven biomass cooking stoves-Part 2: With cook

piece operation and the dimensionless form, Energy for Sustainable Development

2011; 15: 169–175.

4. Bronowski J. (1973) The Ascent of Man, Little, Brow and Co., Boston.

5. Bureau of Indian Standards. www.bis.org.in. Accessed 2013.

6. Fullerton D.G., Brucec N. and Gordona S.B. (2008) Indoor air pollution from

biomass fuel smoke is a major health concern in the developing world, Transactions

of the Royal Society of Tropical Medicine and Hygiene; 102: 843-851.

7. FAO, (1993) Improved solid biomass burning cookstoves: A development manual,

Regional Wood Energy Development Program in Asia, file document 44; accessed

from internet from wgbis.ces.iisc.ernet.in/energy/HC270799/RWEDP/acrobat/fd44.

pdf on 4 September, 2013.

8. Global Alliance for Clean Cookstoves (2011) (http://www.cleancookstoves.org)

9. Household cookstove, Environment, health and climate change. The world bank,

2011 Accessed from internet on 13.09.13 climatechange.worldbank.org/…/

Household%20Cookstoves-web.pdf

10. http://cdm.unfccc.int/ accessed from internet on 30-08-13.

11. Ibrahim D. and Marc A. Rosen (2007) Exergy, energy, environment and sustainable

development (Elsevier).




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12. Kleeman M.J., Schauer J.J. and Cass G.R. (2000) Size and composition distribution

of fine particulate matter emitted from motor vehicles. Environmental Science &

Technology; 34 (7):1132-1142.

13. Kumar M., Kumar S. and Tyagi S.K. (2013) Design, development and technological

advancement in the biomass cookstoves: A review, Renewable and Sustainable

Energy Reviews, 26: 265-285.

14. Raju S.P. (1957) Smokeless Kitchens for the millions, Rev. edn., The Christian

Literature Society, Madras, India.

15. Ramanathan V, and Carmichael G (2008) Global and regional climate changes due

to black carbon, Nature Geo-science, 1:221–227.

16. Singer H. (1961) Improvement of fuel wood cooking stoves and economy in fuelwood consumption, Report to the Government of Indonesia, Report no. 1315,

Expanded Technical Assistance Program, FAO, Rome.

17. Tyagi S.K., Pandey A.K., Sahu S., Bajala V. and Rajput J.P.S. (2013) “Experimental

study and performance evaluation of various cookstove models based on energy and

exergy analysis,” Journal of Thermal Analysis and Calorimetry, 111 (3): 1791-1799.

18. Smith KR, Uma R, Kishnore VVN, Zhang J, Joshi V and Khalil MAK (2000)

Greenhouse implications of household stoves: an analysis for India. Annual Reviews

of Energy and the Environment, 25:741–763.

19. Kishore V.V.N. and Ramana P.V. (2002) Improved cookstoves in rural India: how

improved are they?: a critique of the perceived benefits from the National

Programme on Improved Chulhas (NPIC). Energy, 27: 47–63.

20. WHO (2006) Fuel for Life. Household Energy and Health, Geneva.




98

Part III

Biochemical Conversion




99

CHAPTER 10

BIOPROSPECTING HALOTOLERANT CELLULASE FROM

SALINE ENVIRONMENT OF BHITARKANIKA NATIONAL

PARK, ODISHA

Dash Indira, Sahoo Moumita, Dethose Ajay, C. S. Kar, R. Jayabalan

Abstract

Research interest on biofuels gained much attention with depleting fossil fuel reserves and

growing concern on environment. Bioethanol and biodiesel from biomass will fulfil the future

energy needs. Lignocellulosic and algal biomass are sustainable and cost-effective resources for

bioethanol production. However, algal biomass is preferred over lignocellulosic biomass due to

its high cost of pre-treatment and production of fermentation inhibitors. Systems, which use non-

potable water for biofuel production, are considered due to the future threats on availability of

potable water. Halotolerant enzymes are one of the promising candidates to be used in seawater

based systems. Halotolerant enzymes can also be used for saccharifying the biomass, which are

pre-treated with ionic liquids. Odisha being a coastal state with a coastline of 450 Km and with a

mangrove ecosystem has potential to evidence halotolerant microorganisms, which can produce

salt tolerant enzymes. In the present study five isolates were confirmed for cellulase production

using CMC agar plates and Congo red assay. Activity of FP-endoglucanase produced by the

isolates was assayed by determining the amount of reducing sugar formed from Cellulose by

methods recommended by IUPAC. Endoglucanase produced by halotolerant microorganisms,

named as BHK1, BHK2, BHK3, BHK4 and BHK5 which were isolated from soil and water

samples were having optimal pH 5.0, 4.0, 5.0, 5.0 and 6.0 and optimal temperature of action at

65°C, 45°C, 65°C, 55°C and 65°C respectively. Endoglucanase activity was found to be

enhanced several folds by use of 5 mM MnSO4 as cofactor. Identification of microorganisms and

further characterization of the endoglucanase are in progress.

Keywords: Cellulase, halotolerant, optimal temperature, optimal pH, cofactor.




100

10.1 Introduction

In 2008, 88% of global energy consumption was dependent on fossil fuels (Brennan andOwende, 2010). Fossil fuel however, now accepted as unsustainable resources due to its

depletion and increasing environmental concern due to accumulation of green house gases

(Schenk et al., 2008). To overcome the dependence on fossil fuels, maintenances of sustainable

economy and for global environmental concern, it is necessary to focus over and promote

renewable resources of energy (Brennan and Owende, 2010; Prasad et al., 2001 a,b; Singh et al.,

2010 a,b). Biomass is one of the most promising renewable resources for the production of

biofuels, such as bioethanol (John et al., 2011) and biodiesel (Ho et al., 2010). Most of thebioethanol production across the globe comes from sugar and starch crops and from

lignocellulosic biomass. However, with increasing demand of food, decreasing water availability

and cost involved in pre-treatment of lignocellulosic biomass, agriculture crops are losing

importance. By overcoming the drawbacks of lignocellulosic and sugar rich biomass, microalgae

has gained the title of third generation feedstock for biofuel production (Nigam and Singh, 2011).

Microalgal biomass rich in carbohydrate forms an excellent substrate for bioethanol production.

It grows faster and fixes carbon dioxide at higher rate than terrestrial plants (Ho et al., 2012),

also, rich in starch and cellulose and lacks lignin, which makes monosaccharide conversion

easier (Ho et al., 2012, John et al., 2011). The carbohydrates in the form of starch or cellulose or

complex polysaccharides are not fermentable and therefore are hydrolyzed to fermentable sugar

prior to fermentation (Hagerdal-Hahn et al., 2007). Acid or alkali hydrolysis are common

chemical methods used as they are faster, cheaper and easier however, they lead to release of

components that acts as fermentation inhibitors and as a result hampers the fermentation process

(Harun et al., 2010; Girio et al., 2010; Moxley and Zhang, 2007). Enzymatic hydrolysis process

is amiable to environment and gives higher glucose yield without production of fermentation

inhibitors. Cellulases are the key enzymes of saccaharification of both lignocellulosic and algal

biomass. However, it is interestingly observed that in saline system, which is independent of

fresh water, cellulase and other glucoamylases are completely inhibited thereby making them

unsuitable for industrial usage (Matsumoto et al., 2003). There is expanding knowledge on

halophilic enzymes and organisms capable of effectively treating marine biomass. A recent

review on industrial and environmental applications of halophilic microorganisms states that




101

demand of salt tolerant enzymes and microorganisms is still limited (Oren, 2010), nevertheless,

continuous efforts in this field and favourable results may change the scenario. Potential salt

tolerant enzymes have been isolated from hypersaline microorganisms, which basically includes

the halophilic α-amylase from Haloarcula hispanica (Vasisht et al., 2005), glucoamylases from

marine yeast Aureobasidium pullulans N13d (Oren et al., 2006), thermophilic and halophilic

amyloglucosidase from Halobacterium sodomense with optimal temperature between 66°C to

76°C and optimal activity between 8% to 22% of NaCl (Duan et al., 2006). An alkali-

halotolerant cellulase from Bacillus flexus NT, isolated from Ulva lactuca with residual activity

of about 70% at 15% NaCl concentration (Trivedi et al., 2010). Reports suggests that

commercially available accellerase-1500 (cocktail of different glycosidases) performs

depolymerisation of cellulose and avicel in reaction media containing 1X, 2X and 4X

concentration of seawater (Grande et al., 2012) . Regarding the fermentation of marine algal

biomass, minimal progress reported in the past 30 years after the reports of Clostridium

pasteurianum fermenting Dunaliella species to produce butanol, 1, 3-propanediol and ethanol in

presence of 10% NaCl. To overcome the salinity issues micro algae is either washed in deionised

water or grown in a less saline medium however, the salinity problem during fermentation can be

encountered by using halotolerant yeast. Citeromyces siamensis is novel halotolerant yeast

isolated from dry salted squid and fermented soybeans in Thailand (Nagatsuka et al., 2002)

which is tolerant to higher concentration of cations (3M NaCl and 0.8M LiCl) and osmotic

pressure (60% glucose). Pichia sorbitophila is also reported halotolerant yeast, which can grow

in 4M NaCl concentration when glucose and glycerol are sole carbon source (Lages and Lucas,

1995). The present work aimed to determine the effect of temperature, pH and metal ions on the

cellulase produced by salt tolerant cellulolytic bacteria isolated from Bhitarkanika National Park,

Odisha.

10.2 Materials and methods

10.2.1 Isolation of cellulase producing bacteria

Soil and water samples were collected from Bhitarkanika National Park, Odisha. Samples

were serially diluted and inoculated in Trypticase soy agar (TSA) media (15 g Tryptone, 5 g

Soytone, 5 g Sodium Chloride, 15 g agar/ 1 L Distilled water) and incubated at 37°C for 24

hours. Individual colonies were obtained by streaking the culture on same media. Confirmation




102

of cellulose-degrading ability of bacterial isolates was performed by streaking on the CMC agar

media (5 g CMC, 1 g NaNO3, 1 g K2HPO4, 1 g KCl, 0.5 g MgSO4, 0.5 g yeast extract, 1 g

glucose, 17 g agar/ 1L Distilled water). The use of Congo red (1 mg/ml) as an indicator for

cellulose degradation in an agar medium and 1 M NaCl as destaining solution provides the basis

for a rapid and sensitive screening test for cellulolytic bacteria (CB). Colonies showing

discoloration of Congo red was taken as positive cellulose-degrading bacterial colonies (Lu et al.,

2004) and only these were taken for further study.

10.2.2 Cellulase enzyme production

The selected cellulolytic bacterial isolates were cultured at 37°C at 150 rpm in 100 ml ofenzyme production media composed of 0.1 g NaNO3, 0.1 g KH2PO4, 0.0.1 g KCl, 0.5 MgSO4,

0.5 g yeast extract , 0.1 g glucose, 0.5 g filter paper at pH 6.8–7.2. Broth culture after three days

of incubation period was subjected to centrifugation at 5000 rpm for 15 min at 4°C. Supernatant

was collected and stored as crude enzyme preparation at 4°C for further enzyme assays (Tailliez

et al., 1989).

10.2.3 Cellulase assay

Filter paper cellulase activity was assayed by measuring the amount of reducing sugar

formed from soluble form of cellulose, CMC. Determination of enzyme activity is measured

using methods suggested by International Union of Pure and Applied Chemistry (IUPAC)

(Ghose, 1987). In these tests, 0.5 ml of crude enzyme extract is incubated with 0.5 ml of 2%

CMC in 10 mM citrate buffer at different temperatures and pH and the amount of reducing

sugars formed were estimated spectrophotometrically with 3,5-dinitrosalicylic acid using glucose

as standards (Miller, 1959). Then enzymatic activities of total endoglucanase were defined in

units. One unit of enzymatic activity is defined as the amount of enzyme that releases one

micromole reducing sugars (measured as glucose) per minute.

10.2.3.1 Effect of temperature on cellulase activity

The crude enzyme extract along with the substrate (2% CMC in 10 mM citrate buffer)

was incubated for 15 min at different temperatures such as 30, 40, 50, 60 and 70°C to study their

effect on cellulase activity and assayed spectrophotometrically for their effect of enzyme activity.

10.2.3.2 Effect of pH on cellulase activity




103

The pH of the test solution was adjusted to different pH range of 3.0, 4.0, 5.0, 6.0 and 7.0

with phosphate buffer (10 mM Na2HPO4.2H2O, 1.8 mM KH2PO4) at their respective optimal

temperature for 15 minutes. The test samples were then assayed to study their effect on enzyme

activity.

10.2.3.3 Effect of metals on cellulase activity

After getting optimum temperature and pH value different metal ions such as MgCl2,

ZnCl2, MnCl2, FeCl2, CoCl2, EDTA and PMSF were subjected to study their effect on enzyme

activity. They all were taken in a concentration of (5 mM) and assayed.

10.3 Result and discussion

10.3.1 Isolation and screening of cellulase producing bacteria

Five strains of cellulolytic bacteria were isolated from soil and water samples from

Bhitarkanika National Park, Odisha, India. Three bacterial isolates from soil (BHK1, BHK2,

BHK3) and two from water (BHK4 and BHK5) were confirmed for cellulase production on

CMC agar plates on aerobic incubation of 48 hours at 37°C by production of clear zone which

was identified using Congo red assay (Figure1).

10.3.2 Production of cellulase

All the five isolates (BHK1, BHK2, BHK3, BHK4 and BHK5) were cultured on enzyme

production medium containing filter paper as sole cellulosic substrate. All the isolates showed

cellulase activity on filter paper ranging between 0.93 EU/ml to 0.98 EU/ml. Highest cellulolytic

potential was exhibited by BHK1 with 0.98 EU/ml whereas lowest was recorded in BHK4 with

0.93 EU/ml (table 1).

10.3.3 Cellulase assay

Cell free supernatant collected from culture of BHK1, BHK2, BHK3, BHK4 and BHK5

was used as crude enzyme extract for determination of effect of temperature, pH and metal ions.

10.3.3.1 Effect of temperature on cellulase activity

Crude enzyme extract from all the five isolates (BHK1, BHK2, BHK3, BHK4 and

BHK5) were assayed at temperature ranging between 30°C to 70°C for 15 minutes. Maximum

enzyme activity was recorded by BHK1 at 65°C, BHK2 at 45°C, BHK3 at 65°C, BHK4 at 55°C




104

and BHK5 at 65°C (Figure3.a). The above result in correlation with the fact that most of the

cellulases have optimal temperature of action at 55°C.

Figure1: Clear zone formed on CMC agar plates by BHK1, BHK2, BHK3, BHK4 and BHK5

after 48 hours of incubation and stained with Congo red confirming the presence of extracellular

cellulase.

Table 1: Production of cellulase by BHK1, BHK2, BHK3, BHK4 and BHK5 in enzyme

production medium after 72 hours of incubation.

Cellulolytic bacterial strains Amount of cellulase produced after 72hours incubation(EU/ml)

BHK 1 0.98

BHK 2 0.95

BHK 3 0.96

BHK 4 0.93

BHK 5 0.93




105

10.3.3.2 Effect of pH on cellulase activity

Crude enzyme extract from all the isolates (BHK1, BHK2, BHK3, BHK4 and BHK5)

were assayed in pH ranging between 3.0 to 7.0. Optimal pH of action for all the five isolates

recorded as BHK1 at pH 5.0, BHK2 at pH 4.0, BHK3 at pH 5.0, BHK4 at pH 5.0 and BHK5 at

pH 6.0 (Figure3.b). Optimal pH of action ranging between 5.0 to 6.0 in most cases may be due to

the fact that all the isolates were from estuarine environment and not from purely saline sources.

10.3.3.3 Effect of metal ions on cellulase activity

With optimal temperature and pH value obtained from the above five cellulolytic isolates

(BHK1, BHK2, BHK3, BHK4 and BHK5) were assayed for effect of metal ions (5 mM

concentration each Of MgCl2, ZnCl2, MnCl2, FeCl2, CoCl2, EDTA and PMSF). In all cases,

enzyme activity is recorded maximum in presence of Mn2+. Other ions (Mg2+, Zn2+, Fe 2+, Co2+,

EDTA and PMSF) did not show any substantial effect on the enzyme activity except Co2+ in

BHK1, which enhanced the enzyme activity but not to such extent as Mg2+ (Figure 3.c and d). It

may be inferred that Mn2+ may act as cofactor or enhancer of enzyme activity in these filter paper

cellulases isolated from cellulolytic bacteria isolated from Bhitarkanika National Park, Odisha.

(a) (b)




106

(b) (d)

Figure 3. (a) Effect of Temperature on enzyme action, (b) Effect of pH on enzyme action, (c)

Effect of metal ions on enzyme action and (d) Comparative effect of Mn2+ on different strains of

cellulolytic bacteria.

10.4 Conclusion

The present study focused on screening of cellulase producing organisms from saline

environment of Bhitarkanika National Park, Odisha. Five isolates tested positive for production

of extracellular cellulase and utilized for production of cellulase using suitable enzymeproduction media with filter paper as raw source of cellulose. Crude enzyme so produced were

analysed for effect of temperature, pH and metal ions on their activity. All the potential cellulase-

producing organisms had enzyme ranging between 0.93EU/ml to 0.98 EU/ml. Optimal

temperature of enzyme action is between 45°C to 65°C suggests that the enzymes work at higher

temperature and at thermotolerant in nature. Optimal pH of action in the range 5.0 to 6.0 suggests

that strains from less saline environment. Endoglucanase activity was found to be enhanced

several folds in presence of 5 mM Mn2+

suggesting its role as cofactor or enhancer molecule.Identification of microorganisms and further characterization of the endoglucanase are in

progress. A further study also includes isolation of potential halotolerant cellulolytic bacteria

from more saline environment for their utilization in seawater-based medium.

Acknowledgement

Authors are very much thankful to MHRD and National institute of Technology,

Rourkela for the financial support for providing all research facilities and Office of the Principal




107

CCF (Wildlife) & Chief Wildlife Warden, Odisha for authorization of permission for sample

collection from Bhitarkanika National Park, Odisha.

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2. Schenk P. M., Thomas-Hall S. R., Stephens E., Marx U. C., Mussgnug J. H., Posten C.,

Kruse O., Hankamer B. (2008) Second generation biofuels: high-efficiency microalgae

for biodiesel production. Bioenergy Res., 1: 20–43.

3. Prasad S., Singh A., Jain N., Joshi H. C.( 2007) Ethanol production from sweet sorghum

syrup forutilization as automotive fuel in India. Energy Fuels., 21: 2415–2420.

4. Prasad S., Singh A., Joshi H. C. (2007) Ethanol as an alternative fuel from agricultural,

industrial and urban residues. Resour Conserv Recy., 50: 1–39.

5. Singh A., Pant D., Korres N. E., Nizami A. S., Prasad S., Murphy J. D. (2010) Key issues

in life cycle assessment of ethanol production from lignocellulosic biomass: challenges

and perspectives. Bioresour. Technol., 101: 5003–5012.

6. Singh A., Smyth B. M., Murphy J. D. (2010) A biofuel strategy for Ireland with an

emphasis on production of biomethane and minimization of land take. Renew. Sustain.

Energy Rev., 14: 277–288.

7. John R.P., Anisha G.S., Nampoothiri K.M., Pandey A. (2011) Micro and macroalgal

biomass: a renewable source for bioethanol. Bioresour. Technol., 102 (1): 186– 193.

8. Ho S.-H., Chen W.-M., Chang J.-S. (2010) Scenedesmus obliquus CNW-N as a potential

candidate for CO 2 mitigation and biodiesel production. Bioresour. Technol., 101 (22):

8725–8730.

9. Nigam P.S. and Singh A. (2011) Production of liquid biofuels from renewable resources.

Prog. Energ. Combust., 37 (1): 52–68.




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10. Ho S.-H., Chen C.-Y., Chang J.-S. (2012) Effect of light intensity and nitrogen starvation

on CO 2 fixation and lipid/carbohydrate production of an indigenous microalgaScenedesmus obliquus CNW-N. Bioresour. Technol., 113: 244–252.

11. Hahn-Hagerdal B., Karhumaa K., Fonseca C., Spencer-Martins I., Gorwa-Grauslund M.F.

(2007) Towards industrial pentose-fermenting yeast strains. Appl. Microbiol. Biotechnol.,

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12. Harun R., Danquah M.K., Forde G.M. (2010) Microalgal biomass as a fermentation

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13. Girio F.M., Fonseca C., Carvalheiro F., Duarte L.C., Marques S., Bogel-Lukasik R.(2010) Hemicelluloses for fuel ethanol: a review. Bioresour. Technol., 101 (13): 4775–

4800.

14. Moxley G. and Zhang Y.H.P. (2007) More accurate determination of acid-labile

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15. Matsumoto M., Yokouchi H., Suzuki N., Ohata H., Matsunaga T. (2003) Saccharification

of marine microalgae using marine bacteria for ethanol production. Appl BiochemBiotechnol., 105:247-254.

16. Oren A. (2010) Industrial and environmental applications of halophilic microorganisms.

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amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles, 9:487-495.

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110

CHAPTER 11

ISOLATION AND MOLECULAR CHARACTERIZATION OF

CELLULOLYTIC FUNGI USED FOR CONVERSION OF

SUGARCANE BIOMASS FOR BIOETHANOL PRODUCTION

Chetan, A. M., Harinikumar, K. M., Bhavani, P., Manoj Kumar, H. B., Madhu T., Ningaraj

Dalawai

Abstract

One of the major alternatives to fossil fuels that received major attention is bioethanol derived

from biomass. The cellulosic material are potential sources of ethanol. Cellulolytic fungi are

capable of degrading cellulose to smaller sugar components like glucose units. The aim of this

research was to isolate and screen fungi capable of producing cellulases and to convert pre-

treated lignocellulosic material to fermentable sugars for the production of ethanol using

Saccharomyces cerevisiae. The lignocellulosic material such as sugarcane bagasse and sugarcane

trash were used as substrates for ethanol production. Fungi were isolated from soil and compost

samples collected from various regions. The pure cultures were screened for the ability to

degrade cellulose. The cellulolytic activity was determined by measuring the clearing zone

created by the fungi. The cultures were further characterized using five random primers. The

fungi capable to produce cellulases were identified as Aspergillus niger, Aspergillus fumigatus,

Trichoderma viride, Trichoderma harzianum and Trichoderma reesei based on colony

characters, microscopic observation and identification at molecular level based on DNA coding

for 18S rRNA. The substrates were powdered and pretreated with fungal isolates using Mandels’

and Reese media. The substrates were used as a carbon source. Sugarcane bagasse and trash

treated with Trichoderma reesei have shown highest concentration of reducing sugars of

45.95mg/g and 40.56mg/g respectively and ethanol yield of 11.56g/l and 10.92g/l respectively.

From this study the fungal cultures having the potential to degrade cellulosic material were

identified and they can be used for bioethanol production from lignocellulosic wastes.




111

11.1 Introduction

The growth of population and the associated demand for fuel and food coupled with morerestrictive environmental regulations have intensified the research and development of renewable

energy feedstocks to substitute for and/or to complement fossil fuel sources (Pereira Jr. et al.,

2008). The transfer of crude oil-based refinery to biomass-based biorefinery has attracted strong

scientific interest which focuses on the development of cellulosic ethanol as an alternative

transportation fuel to petroleum fuel (Zheng, et al., 2009). In India, ethanol is primarily produced

from molasses which is a byproduct from sugar mills using Saccharomyces cerevisiae strains.

Sugarcane bagasse and trash, the left-over residue of leaves and tops, can be converted to ethanolby enzymatic or acid catalyzed hydrolysis and fermentation of the released monosaccharides

(Michael et al., 2006). Sugarcane, an important cash crop, is grown over an area of 4.2 million

hectares in India with an average productivity of 70 tons ha–1. Sugarcane converts approximately

2% of solar energy into chemical bonds of carbohydrates where in two third of these

carbohydrates are in the form of lignocelluloses. These lignocellulosic material are a potential

source of bioethanol production. There are three major steps to be employed in the conversion of

lignocellulosic to Bioethanol which are pretreatment for lignin breakdown, hydrolysis and

fermentation for Bioethanol production. The most challenging part is the hydrolysis process in

order to obtain the reducing sugars. Hydrolysis of lignocellulosic can be done in two ways, either

by using enzymatic or chemical methods. However, enzymatic hydrolysis is more environmental

friendly as compared to chemical hydrolysis. Although, the costs of using commercial enzymes

are expensive, however this problem can be overcome by using cellulose degrading organisms

(Zainan et al., 2011). This research work has been carried out in order to produce bio-ethanol

from lignocellulolytic wastes by using cellulolytic fungi.

11.2 Material and Methods

Present investigation was carried out to isolate and identify cellulolytic fungi for the

conversion of lignocellulolytic biomass into Bio-ethanol and to characterize them using ITS

primers (Internal transcribed spacer) and RAPD primers.

11.2.1 Isolation




112

The soil and compost samples were collected from various regions of Mandya. The

samples were inoculated on potato dextrose agar (PDA) medium. For the isolation of fungi,

dilution plate method was used. All the samples (i.e., three soil samples and two compost

samples) were serially diluted and the dilutions 10-2, 10-3, 10-4 were plated using spread plate

method.

11.2.2 Screening

From the various isolates, screening for cellulolytic fungi was made using selective media

(Mandels' and Reese agar medium) procedures to select the potential isolates that could

saccharify lignocellulosic wastes. Fungi determined to be cellulolytic were then cultured inMandels salt medium supplemented with cellulose. (Mandels & Reese, 1957). Cellulolytic fungi

create a clearing zone around the colony on the agar.

11.2.3 Molecular characterization

Molecular characterization was done using five random primers OPA-1; OPD-6; OPA-4;

A-5 and AA-11.

11.2.4 Extraction of DNA

DNA extraction protocol was followed according to Chakraborty et al., (2010). Isolation

of fungal genomic DNA was done by growing the fungi for 4 days. The quality and quantity of

DNA was analyzed both spectrophotometrically and in 0.8% agarose gel. The DNA from all

isolates produced clear sharp bands, indicating good quality of DNA.

11.2.5 PCR Amplification of ITS Region of fungal Isolates:

All fungal isolates were taken up for ITS-PCR amplification. Genomic DNA was

amplified by mixing the template DNA (50 ng), with the polymerase reaction buffer, dNTP mix,

primers and Taq polymerase. Polymerase Chain Reaction was performed in a total volume of 50

µl, containing Sample ~50ng of gDNA, 100ng of Forward Primer , 100ng of Reverse Primer, 2µl

of 10mM dNTPs mix, 5µl of 10X Taq Pol. Buffer, 3U Taq Polymerase enzyme, PCR grade water

to make the volume upto 50µl. PCR was programmed with an initial denaturing at 94°C for 5

min. followed by 30 cycles of denaturation at 94 °C for 30 sec, annealing at 52°C for 30 sec and

extension at 72°C for 1 min and the final extension at 72 °C for 3 min in a Thermocycler. After




113

PCR, all the amplified products were ran on 1.5% Agarose gel with 1X TAE buffer. Sequencing

was done at Amnion biosciences using ABI3730xl sequencer.

11.2.6 RAPD of Trichoderma Isolates:

For RAPD, six random primers i.e. OPA-1; OPD-6; OPA-4; A-5; AA-04 and AA- 11

were selected (Table-1). Polymerase Chain Reaction was performed in a total volume of 50 µl,

containing Sample ~25ng of gDNA, 400ng RAPD Primer, 2µl of 10mM dNTPs mix, 5µl of 10X

Taq Pol. Buffer, 3U Taq Polymerase enzyme, PCR grade water to make the volume upto 50µl.

PCR was programmed with an initial denaturing at 94°C for 5 min. followed by 45cycles of

denaturation at 94°C for 1 min, annealing at 38°C for 1 min and extension at 72°C for 2 min andthe final extension at 72°C for 5 min in a Thermocycler. After PCR, all the amplified products

were ran on 1.5% Agarose gel with 1X TAE buffer.

11.2.7 Scoring the data

The image of the gel the ribosomal RNA genes (rDNA) possess electrophoresis was

documented through gel documentation system and analysis software. All reproducible

polymorphic bands were scored and analysed following UPGMA cluster analysis protocol. The

RAPD patterns of each isolate was evaluated, assigning character state “1” to indicate the

presence of band in the gel and “0” for its absence in the gel. The scored band data (Presence or

absence) was subjected to cluster analysis-using STATISTICA.

Production of Bio-ethanol from Pretreated Sugarcane Biomass Using Saccharomyces

Cerevisiae

The two substrates, sugarcane bagasse and sugarcane trash were powered and pretreated

with the fungal cultures using Mandels' and Reese medium using the substrates as carbon source.

Culture conditions: 10g /l of each residue was taken in conical flask containing 200ml of

Mandle’s medium. The conical flasks were plugged with cotton and sterilized at 15lbs per

sq.inch for 20 minutes. Each flask was inoculated with 4-5 discs of different fungi. These flasks

were incubated at room temperature for 5days on an orbital shaker. After five days mycelium was

separated by filtration through Whatman filter paper No.1. The filtrate was used for further

studies (Kader et al., 1999).




114

Determination of total carbohydrate: The carbohydrate content of untreated and pretreated raw

material in the culture broth was measured by phenol sulphuric acid method with glucose as

standard (Dubois et al., 1956; Krishnaveni et al., 1984).

Determination of reducing sugars: Reducing sugars in untreated and pretreated raw material in

the culture broth were determined by dinitrosalicylic acid (DNS) method with glucose as

standard (Miller, 1972).

Fermentation: Culture filtrate was further inoculated with Saccharomyces cereviseae strain and

allowed for fermentation for seven days. After fermentation it was filtered and ethanol content

was determined.

Ethanol estimation: The ethanol was estimated by Colorimetric method as described by Caputi

et al., (1968).

11.3 Result and discussion

Nine fungal isolates were obtained using Potato Dextrose Agar medium from the soil and

compost samples collected. Among them five isolates were identified and pure cultures were

maintained on PDA plates. The pure fungal isolates were screened for cellulolytic ability.Highest cellulolytic activity was detected in three isolates of Trichoderma sp. Zone of clearance

was highest in Trichoderma reesei (2.10mm) followed by Trichoderma viride (2.00mm) and

Trichoderma harzianum (1.60mm). The enzymatic activity was considerably low in other fungi

such as Aspergillus sp the clearance zone was lesser in Aspergillus niger (1.50mm) and the least

was Aspergillus fumigatus (0.80mm) . The screened fungal strains were used for further studies.

The DNA isolated from the desired cellulose producing fungi tentatively identified as species of

Apergillus and Trichoderma species. When checked for purity exhibited that the DNA isolated

from the two sources was pure. The same DNA samples, when run on an agarose gel also,

confirmed to be pure as the bands of DNA are single and distinct. Traces of contaminants were

found when observed under the Gel doc and photographed. In the present study we focused on

the ITS region of ribosomal genes for identifying fungal species. ITS region of rDNA was

amplified using genes specific ITS-1 and ITS-4 Primers. Amplified products were of size in the

range 100bp to 180bp. The results are in accordance with Chakroborthy et al., (2010) who




115

studied the identification and genetic variability of fungal isolates. The ITS PCR has helped to

detect polymorphism at ITS region of rDNA among fungal isolates.

In this study, a set of 5 accessions were genotyped with 5 RAPD primers as these primers

are considered as superior for assessing genetic diversity. The genotypes were grouped into two

major clusters, cluster II was the largest with 3 genotypes i.e. Trichderma reesei, Trichoderma

viride and Trichoderma harzianum indicating that the three species are genetically closely related

followed by cluster I with 2 genotypes i.e. Aspergillus niger and Aspergillus fumigatus indicating

that the two species are closely related. The results obtained in the present study are also in

accordance with the cluster analysis results obtained by Chakraborthy et al., (2010). Total sugar,reducing sugar and nonreducing sugar content of fungal pretreated and untreated samples were

estimated. The results obtained are presented in Table 1. Autoclaving for sterilization has

affected and resulted in increase in sugar content. With fungal treatment still increase in the yield

of sugars was observed. The treated samples were subjected to distillation and the ethanol thus

obtained was estimated using potassium dichromate method and the concentration of ethanol

obtained is presented in Table 2. The results obtained were analyzed statistically using

completely randomized design. In Tables 1 and 2 mean values assigned with same superscript(s)

do not differ significantly (P=0.05). Among the two substrates sugarcane bagasse pretretaed with

Trichoderma reesei has given maximum reducing sugar content (45.95 mg/g) followed by

sugarcane trash pretreated with the same culture (40.56 mg/g). The substrates pretreated with

Trichoderma viride and Trichoderma harzianum have moderately increased the sugar content.

Pretreatment with Aspergillus niger and Aspergillus fumigatus have shown comparatively lesser

results. Among the two substrates sugarcane bagasse pretretaed with Trichoderma reesei has

given maximum ethanol yield (11.56g/l) followed by sugarcane trash pretreated with the same

culture 10.92g/l. The substrates pretreated with Trichoderma viride and Trichoderma harzianum

have moderately increased the sugar content. Pretreatment with Aspergillus niger and Aspergillus

fumigatus have shown comparatively lesser results.




116

Fig (a) Fig (b)




117

Fig (d) Fig (e)

Fig a: genomic DNA; Fig (b-f) Gel picture RAPD analysis of fungal isolates with OPA-1, OPD-6, OP

Trichoderma harzianum, Lane M: 100bp DNA ladder (100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 7

TV: Trichoderma viride, TR: Trichoderma reesei, AF: Aspergillus fumigatus, AN: Aspergillus niger




118

Table: 1: Concentration of reducing sugars, non reducing sugars and total sugars of the hydrolysates

Sl no. Cultures

Sugarcane bagasse Sugarcan

Reducing

sugar

(mg/g)

Non

reducing

sugar

(mg/g)

Total sugar

(mg/g)

Reducing

sugar

(mg/g)

Control Untreated 0.98f 1.27

e 2.25

f 0.88

f

1. Aspergillus niger 35.45d 23.55

c 59.00

d 31.12

d

2. Aspergillus fumigatus 33.32e 21.02

d 54.34

e 28.19

e

3. Trichoderma viride 39.22b 30.90

a 70.12

b 38.54

b

4. Trichoderma harzianum 37.98

c 28.00

b 65.98

c 32.22

c

5. Trichoderma reesei 45.95a 30.05

a 76.00

a 40.56

a

SEm± 0.212 0.220 0.107 0.171

CD at 1% 0.841 0.872 0.426 0.677




Tree Diagram for 5 Variables

Unweighted pair-group average

Squared Euclidean distances

Linkage Distance

TV

TH

TR

AN

AF

0 2 4 6 8 10 12 14 16 18

Table 2: Concentration of Ethanol obtained from hydrolysates of Sugarcane Biomass

Sl no. Cultures

Ethanol (g/l)

Sugarcane bagasse Sugarcane trash

1. Untreated 1.56f 1.20f

2. Aspergillus niger 7.32d 6.58d

3. Aspergillus fumigatus 6.10e 5.86e

4. Trichoderma viride 10.15b 9.78b

5.

Trichoderma harzianum 9.01c

8.77c

6. Trichoderma reesei 11.56a 10.92a

SEm± 0.112 0.066

CD at 1% 0.440 0.261




120

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CHAPTER 12

APPLICATION OF THERMOSTABLE CELLULASE IN

BIOETHANOL PRODUCTION FROM LIGNOCELLULOSIC

WASTE

Neha Srivatsava, Rekha Rawat and Harinder Singh Oberoi

Abstract

Lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical,

chemical, biological pretreatment operations and slow enzymatic hydrolysis makes the overall

process less economical than presently available fossil fuels. Cellulase is a group of enzymes,

which can be classified into several types based on the reactions they catalyze, including

endoglucanase (EG) or carboxymethyl cellulase (CMCase), exoglucanase or cellobiohydrolase

(CBH), cellobiase or β-glucosidase. Auxilliary enzymes like β-xylosidase, α-L-

arabinofuranosidase, and feruloyl esterase if present along with xylanases help in complete

conversion of hemicellulose to sugars like xylose and arabinose. The synergistic action of

these enzymes plays an important role in the hydrolysis of cellulosic and hemicellulosic

fractions. It is therefore desirable to have a consortium of all the cellulase and xylanase

components for effective hydrolysis of cellulosic biomass. The use of thermostable cellulases

for hydrolysis of cellulosic biomass have significant advantages, such as (i) improved

hydrolysis of cellulosic substrates because of the higher rate of reaction, (ii) higher mass-

transfer rates leading to improved substrate solubility, (iii) lowered risk of contamination, and

(iv) increased flexibility with respect to process design, thereby improving the overall

economics of the process. Therefore, research on developing thermostable cellulase

consortium for hydrolysis of cellulosic biomass is gaining momentum. Thus, the current

chapter covers sources and application of thermostable enzyme along with their characteristic

features. Limitations and possible approaches are also discussed.

Keywords: Bioethanol, Lignocellulosic biomass, Cellulases, Thermostability, Protein

engineering, Hydrolysis




122

12.1 Introduction

The production and utilization of bio-ethanol is gaining attention worldwide because

of many advantages like reducing global warming and improving global energy crises

(Chovau et al., 2013). Ethanol can be produced by fermentation of sugars from agro-industrial

waste materials. Lignocellulosic biomass is known as the most abundant polymer and

renewable source of energy which is finally converted into glucose and soluble sugars in

ethanol production process (Reese and Mandels, 1984). Lignocellulosic agricultural biomass

is used as substrate for the production of second generation biofuels. Lignocellulosic biomass

is a complex which is composed of cellulose, hemicellulose and lignin and the conversion

efficiency of lignocellulosic biomass into biofules depends upon the lignin content and degree

of polymerization (DP) in cellulose and hemicellulose (Oberoi et al., 2012). Cellulose and

hemicellulose are regarded as the potential sources of sugars for second generation biofuel

production and cover around two-third of the lignocellulosic biomass (Hamelinck et al.,

2005). Different pretreatment methods are applied to increase the accessibility of cellulosic

substrate which helps to open the lignin sheath (Alvira et al., 2010). Lignocellulose-degrading

enzymes, such as cellulases and hemicellulases are used to release fermentable sugars after

pretreatment of biomass.

Progressive research on cellulase enzymes started in the early 1950s due to their

potential in conversion of lignocellulosic biomass, the most abundant polymer and renewable

source of energy which is finally converted into glucose and soluble sugars in ethanol

production process (Reese and Mandels, 1984). Continued research on cellulases and

hemicellulases revealed their industrial potential in different sectors and industries, such as

bioenergy, food, brewery and wine, animal feed, textile and laundry, pulp and paper,

agriculture. Cellulases is a complex enzyme system which is divided into three major groups:endo-1,4-β-D-glucanases (EC 3.2.1.4) which cleaves β-linkages at random, commonly in the

amorphous parts of cellulose; exo-1,4-β-D-glucanases (EC 3.2.1.91) (or cellobiohydrolase)

which liberates cellobiose from the non-reducing or the reducing end, in general from the

crystalline parts of cellulose; and β-glucosidases (EC3.2.1.21) which releases glucose from

cellobiose and short-chain cello-oligosaccharides. Thermostable enzymes have number of

commercial applications as the paper processing industries are always interested in such type

of cellulases which can withstand higher temperatures. In addition, one of the most importantapplications of thermostable cellulase is in the bioconversion of cellulosic biomass into




123

fermentable sugars for bioethanol production at elevated temperature. Generally, enzymatic

hydrolysis reactions are carried out at 45oC-50oC which shows slow enzymatic hydrolysis

rates, low yield of sugars, and incomplete hydrolysis, more amounts of enzyme requirement

and is very sensitive to microbial contamination. These limitations could be resolved by using

thermostable enzymes (Yeoman et al., 2010; Viikari et al., 2007). Figure.1 is showing

possible advantages of thermostable cellulases in bioconversion of lignocellulosic waste.

Therefore, the current chapter focuses on thermostable cellulase enzymes and their

applications in biofuels production.

Figure-1 Application of thermostable cellulases in biofules production

12.2 Bioethanol production: Current production status and Challenges

The production of biofules has reached 105 billion liters in 2010 and increased up to

17% from the year 2009. Biofuels contributed about 2.7% of world’s fuel road transportation

Application of thermostable

cellulases in biofuels production

Complete

hydrolysis

Economically

viable

High reaction

rate

Less risk of

contamination

Fungi and bacteria are

the best source of

production of

thermostable cellulases

Less time

required

for

hydrolysis

reaction

High Sugars yield

Pretreatment

step can be

avoided




124

in which bioethanol and biodiesel are the most prominent. In 2010, global bioethanol

production reached about 86 billion liters. United States and Brazil are the top producers of

ethanol in the world accounting for 90% of the total global production (Kocar and Civas

2013). USA produces more fuel ethanol than any other country; Brazil is the second largest

producer of ethanol in the world. The US and Brazil put together produced a little over 86%

of the world’s fuel ethanol in 2010. Although production of bioethanol has improved using

new technologies, there are still some challenges that need further investigations. For

example, the cost of cellulase and ethanol distillation using lignocellulosic biomass as

substrate, account for 30 to 50% and 20% of the total cost, respectively. To achieve the

commercialization of cellulosic ethanol, a number of technological break throughs as well as

cost reductions in all the process steps are required (Chen and Qiu, 2010).

12.3 Microorganism for thermostable cellulase production

Thermophilic microorganisms are potential sources of highly active and thermostable

enzymes (Zambare et al., 2011; Liang et al., 2011; Yeoman et al., 2010). However, many

mesophilic microorganisms are also known for significant production of thermostable

cellulases (Gao et al., 2008; Lee et al., 2010). Numbers of bacteria and fungi have been

reported to produce thermostable cellulases. Fungi are the most studied organisms withrespect to degradation of cellulose and production of cellulolytic enzymes because bacteria

degrade cellulosic biomass slowly due to lack of penetrating ability like fungi (Swaroopa et

al., 2004). Thermophilic and mesophilic fungal genera belonging to Aspergillus, Rhizopus,

Trichoderma, Sclerotium and Sporotrichum thermophile etc. (Barnard et al., 2010) are known

for the production of cellulases. Synthesis of heat shock protein (HSPs) is a common

phenomenon for production of thermostable enzymes. In presence of cycloheximide, the

ability to produce thermostable enzyme and acquired thermo-tolerance is lost (Maheshwari etal., 2000). There are number of reports available showing HSPs synthesis in thermophillic

microbes along with rapid breakdown of pulse label proteins (Trent et al., 1994, Maheshwari

et al., 2000). Thermophilic microorganisms have specialised protein known as ‘chaperonins’

which help protein to refold and retain their native form and store their function even after

denaturation. The cell membranes of thermophiles are made up of saturated fatty acids which

create hydrophobic environment for cell and keeps it rigid so that it can survive at higher

temperatures (Haki and Rakshit, 2000). The DNA of thermophiles has reverse gyrase whichforms positive supercoils and hence enhances melting point. Besides this, thermophiles also




125

have electrostatic, disulphide bridge and hydrophobic interactions in their cell membranes like

thermotolerants which enhance the tolerant capacity of thermophiles at extremely higher

temperatures (Kumar and Nussinov, 2001). Many thermophilic fungal species have been

reported to produce cellobiose dehydrogenase and glycoside hydrolase 61 (GH61) family of

proteins (Dimarogona et al., 2012), addition of which enhance cellulase performance in

lignocellulose hydrolysis (Barakat et al., 2012; Harris et al., 2010). Thermophilic fungi

produce multiple forms of the cellulase components like mesophilic fungi. However, two

different strains of T. aurantiacus produced one form each of endoglucanase, exoglucanase,

and β-glucosidase, but the forms from the two strains had somewhat different properties

(Khandke et al., 1989). Several thermophilic bacteria, belonging to the genera Bacillus,

Geobacillus, Caldibacillus, Acidothermus, Caldocellum, and Clostridium produce

thermostable cellulases. Hyperthermostable lignocellulolytic enzymes (optima above 80oC)

have been isolated from Thermotoga anaerocellum (Evans et al., 2000). Most of the studies

reported that hyperthermophilic microbes did not degrade crystalline cellulose, when

temperatures increased beyond 75oC due to the lack of carbohydrate-binding modules (CBM)

(Bhalla et al., 2013, Graham et al., 2011; Maki et al., 2009) while presence of a multi-domain

hyperthermophilic cellulase in an archaeal enrichment, allowed maximum deconstruction of

lignocellulosic biomass beyond 90oC (Bhalla et al., 2013, Graham et al., 2011).

12.4 Thermostable enzymes

12.4.1 Thermostable cellulases

Cellulases are enzymes that catalyze the depolymerization of cellulose and work

synergistically to efficiently hydrolyze substrate. Endoglucanases and exoglucanases are

commonly referred to as cellulases (Blumer-Schuette et al., 2008). Endoglucanases having

carbohydrate-binding modules (CBM) are considered as primary cellulases and are

responsible for efficient utilization of crystalline cellulose. Trichoderma and Aspergillus sp.

are referred to as model for significant cellulase production. Several fungi and bacteria have

been reported earlier for the production of thermostable cellulases (Evans et al., 2000; Bok et

al., 1998) (Table-1). Thermostable enzymes are stable and active at high temperatures which

are higher than the optimum growth temperatures of the microorganisms. Endoglucanases (30

to 100 kDa) of thermophilic fungi are thermostable, with optimal activity between 55 and

80°C at pH 5.0 to 5.5 and with carbohydrate contents varying from 2 to 50%. Exoglucanases

(40 to 70 kDa) are optimally active at 50 to 75°C and are thermostable (Maheshwari et al.,




126

2000). Combination of thermostable enzyme with a wide pH range makes them suitable

candidates for bioprocessing. Hydrolysis of substrate with thermostable enzyme increases the

rate of reaction, decreases viscosity, increases diffusion coefficient, increases bioavailability

of organic compounds and the solubility of the substrate, resulting in complete hydrolysis and

less risk of contamination at elevated temperatures. Such types of enzymes can also be used

for models studies for the understanding of temperature stability and activity, which is helpful

for protein engineering (Haki and Rakhsit, 2003).

Table- Thermostable cellulases from different microorganisms

Organism Optimum

temperature

Stability References

Fusarium proliferatum 55oC 50% (Badal, 2002)Teheromyces lanuginosus (wildand mutant)

70oC 50% (Bakalova et al. 2002)

Bacillus subtilis 70oC 50% (Mawadza et al., 2000)Thermotoga neapoltana

(EndocellulaseA)95oC 50% (Bok et al., 1998)

Alicyclobacillus

acidocaldarius ATCC27009(Endoglucanases)

80oC 60% (Eckert andSchneider, 2003)

E. coli expressing endoglucanase

gene fromClostridium thermocellum

80oC 50% (Zverlov et al., 2005)

E. coli expressing endoglucanasegene fromGeobacillus sp. 70PC53

65oC 80% (Ng et al., 2009)

Geobacillus sp. WSUCF1(Endoglucanases)

70oC 50% (Rastogi et al., 2010)

E. coli expressing endoglucanasegene fromThermoanaerobacter

tengcongensis MB4

80oC 50% (Liang et al., 2011)

Aspergillus fumigates SK1 60oC 50% (Ang et al., 2013)

12.4.2 Thermostable xylanases

Xylan is the second most abundant polysaccharide in nature after cellulose. Complete

degradation of xylan needs the combined action of a group of hydrolytic enzymes: the

endoxylanases (EC 3.2.1.8), which cleaves β-1,4-linked xylose randomly (xylan backbone);

the β-xylosidases (EC 3.2.1.37), which converts xylobiose to monomeric xylose units ; and

the various side-branch splitting enzymes such as α-glucuronidase and α-arabinosidase, acetylxylan esterase, and acetyl esterase, which liberate other sugars (glucuronic acid arabinose) that




127

are attached as branches to the backbone (Bhalla et al., 2013, Biely., 1985). Thermostable

endo-β-1,4 xylanases (referred to as endoxylanases) produced by thermophilic and

hyperthermophilic bacteria such as Thermotaga, Acidothermus, Cellulomonas, Paenibacillus

,Thermoanaerobacterium, Actinomadura have been reported and are receiving considerable

attention because of their application in bio- bleaching of pulp in the paper industry, wherein

the enzymatic removal of xylan from lignin-carbohydrate complexes facilitates the leaching of

lignin from the fibre cell wall, avoiding the need for chlorine for pulp bleaching in the

brightening process. They also have applications in the pre-treatment of animal feed to

improve its digestibility. Production of xylnase has also been reported from marine algae,

protozoa, snails and insects but bacteria and fungi are the major producers of thermostable

xylanases (Collins et al., 2005). According to Brienzo et al., (2009), xylanses obtained from

bacteria are more diverse than fungi. Fungi including Laetiporus sulphureus (Lee et al., 2009),

Talaromyces thermophiles (Maalej et al., 2009), Thermomyces lanuginosus (Singh et al.,

2003), Rhizomucor miehei (Fawzi, 2011) produce thermostable xylanases. Thermostable and

alkalistable xylanases have also been reported from many fungi and bacteria, such as

thermoalkalophilic xylanase obtained from Enterobacter sp. MTCC 5112 retained its 90% of

enzyme activity after 0.66 h at 80oC and pH 9, and 85% and 64% of its activity after 18 h at

60 and 70oC, respectively, showing its potential for industrial applications (Bhalla et al., 2013,

Khandeparkar Bhosale, 2006).

12.4.3 Thermostable endoglucanses

Endoglucanase is responsible for formation of cellobiose (oligosaccharides) from

cellulose which is finally converted into glucose molecules by the action of β-glucosidase.

Several studies have reported production of thermostable endoglucanase production from

mesophillic and thermophillic microorganisms including fungi and bacteria both. Generally,

endoglucanases show high thermal stability, for example endoglucanases from T. aurantiacus

shows thermal stability at 70oC with half life of 98 h, (Gomes et al., 2000). In one of the

study, Parry et al. (2002) discussed about an endoglucanase with a molecular weight of 34

kDa (based on SDS-PAGE) along with a pI of 3.7. Endoglucanases, broadly with molecular

weight ranging from 30 to 100 kDa obtained from thermophilic fungi are thermostable, with

optimal activity between 55 and 80°C at pH 5.0 to 5.5 and with carbohydrate contents varying

from 2 to 50% (Maheshwari et al., 2000). Exoglucanases having molecular weight in the

range of 40 to 70 kDa, are optimally active at 50 to 75°C and are thermostable; these enzymes

fall into the category of glycoproteins (Brizeno et al., 2009).




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12.4.4 Thermostable β-glucosidase

Conversion of cellooligosacchrides into glucose is done by the help of enzyme β-

glucosidase (BGL). Several hyperthermophilic bacteria (Dion et al., 1999) such as

archaeabacteria (Kim et al., 2009) as well as fungi (Parry et al., 2001) are known for the

production of significant thermostable BGL which shows maximum optimal temperatures of

88, 90, and 80C, respectively (Handelsman J., 2005). In one of the study, Duex et al., (2012)

reported thermostability of BGL at 66oC. Now days, with the help of metagenomics, complete

microbial genomes may be screened and isolated directly from the natural environments (Feng

et al., 2009; 2010, Jiang et al., 2011). Number of BGL genes have been isolated by employing

metagenomic libraries of different samples, including rabbit cecum, marine samples (Jiang et

al., 2009), soil (Jiang et al., 2009, 2011), and termite gut (Scharf et al., 2010, Matteotti et al.,

2011).

12.5 Application of thermostable cellulases

The significant industrial importance of cellulases lies mainly in the bioenergy

development, textile, and detergent and cellulosic pulp paper industries. In the present

available industrial processes, cellulolytic enzymes are used in: (i) clarification of juices and

wines; (ii) detergents causing colour brightening and softening; (iii) pretreatment of biomassto improve nutritional quality of forage; and (iv) pretreatment of industrial wastes (Bhalla et

al., 2013, Brienzo et al., 2009, Haki and Rakshit, 2003, Bhat, 2000).

Currently, lignocellulosic biomass conversion into fermentable sugar is one of the

main thrust area for the production of biofuels. Some studies have shown that a long reaction

time is required for complete hydrolysis when cellulases do not have this property (Sassner et

al., 2008; Zhu et al., 2008; Borjesson et al., 2007). The use of thermostable enzymes like those

produced from T. aurantiacus help in improving the hydrolysis process. In one of the study,

Ang et al., (2013), reported optimum hydrolysis temperature of 60oC by using thermostable

enzyme obtained from fungus Aspergillus fumigates SK1. It is therefore important to have a

combination of all the forms of cellulases like exo-, endo-glucanases and β-glucosidases in a

consortium along with the xylanases and accessory enzymes capable of hydrolysis of biomass

at elevated temperatures, thereby giving a characteristic advantage to such a consortium.

Development of this kind of consortium is the need of an hour in improving the sugar

productivity and decreasing the enzyme cost in bioethanol production from lignocellulosic

biomass.




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12.6 Concluding remarks

The remarkable progress has been done in the production and development of

thermostable cellulases and has attracted worldwide attention for further research. Although

thermostable enzymes have many advantageous but for industrial scale processes, final

selection depends on the many factors such as energy consumption for overall process,

production cost, enzyme efficiency and environmental issues of the complete process of

lignocelluloses biomass conversion. Cultivation of thermophiles on commercial-scale for the

production of thermostable enzyme is still an economical challenge because of low cell yield

and might even increase the overall production cost. Based on this, there is need of more

research for development and optimization of thermophilic processes of lignocellulosic

biomass conversion to gain an economical industrial biofuel production comparable to

existing processes.

Acknowledgements

Authors thankfully acknowledge the financial assistance received from the NAIP sub-project

(4183) funded by World Bank through Indian Council of Agricultural Research, Government

of India for conducting a part of the study reported in this book chapter.

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CHAPTER 13

ENDOGLUCANASES: CHARACTERIZATION AND ITS

ROLE IN BIOCONVERSION OF CELLULOSIC BIOMASS

Rekha Rawat, Neha Srivastava, Harinder Singh Oberoi

Abstract

The growing global energy demand and negative environmental impacts created by growing

greenhouse gas emissions from fossil fuels have forced the global scientific community to

intensify research on the use of cellulases to perform enzymatic hydrolysis of the

lignocellulosic materials for the production of bioethanol. Endoglucanases are the major

component of cellulase enzyme involved in the initial stages of cellulose breakdown. These

are classified into11 glycoside hydrolase (GH) families, including GH5, 6, 7, 8, 9, 12, 44, 45,

48, 51, and 74 on the basis of different sequences, specificity and tertiary structure. The

application of this enzyme in biofuel industry requires identification of highly stable enzymes

that are able to perform at extreme values of pH and temperature. Several bacteria and fungi

are known to produce thermoacidophilic as well as thermoalkalophilic endoglucanases. The

present chapter focuses on the importance of extremophilic endoglucanases in order to

improve the existing biomass conversion processes. In addition to this, structural information,

protein dynamics and models for thermostable endogucanases are also discussed.

Key words: Endoglucanase, Structure, Classification, Catalytic Mechanism, Thermostability.

13.1 Introduction

Biofuels are drawing increasing attention worldwide as substitutes to petroleum-

derived transportation fuels to help address energy cost, energy security and global warming

concerns associated with liquid fossil fuels (Gray et al., 2006; Lee, 2011). It refers to energy

obtained from biomass which is the biodegradable fraction of products, or waste and residues

from agriculture, forestry and related industries, as well as the biodegradable fraction of

industrial and municipal waste. Lignocellulosic biomass is the most abundant renewable

organic material on earth which is composed of cellulose, hemicellulose and lignin. Cellulose,

which is an unbranched linear homopolymer of glucose molecules with β (1-4) linkages,




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generally accounts for 20- 45% of plant biomass. Cellulose is the main source of sugars for

biofuel production (Hamelinck et al., 2005).

Processing of lignocellulosic biomass into biofuel consists of four major unitoperations: pretreatment, hydrolysis, fermentation, and product recovery. A key step in the

production of biofuel is the hydrolysis of biomass into fermentable sugars that is facilitated by

cellulase enzyme. Cellulases comprise of three enzymes namely endo-1,4-β-glucanase (also

referred to as carboxymethylcellulase or CMCase; EC 3.2.1.4), exo-1,4-β-glucanase or

cellobiohydrolases (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) that synergistically convert

cellulose into soluble sugars and glucose (Lynd et al., 2002). Among these, endoglucanases

are cellulases that act synergistically in the initial attack on cellulose as they hydrolyze theβ-1,4 glycosidic bonds. The by-product is subsequently catalyzed by other enzymes, thereby

making endoglucanases crucial for the bioprocessing of plant biomass (Bhat, 2000).

Therefore, this chapter presents information about structure, mechanism of cellulose

hydrolysis and applications of endoglucanases. In addition, importance of thermostable

endoglucanases and factors affecting thermostability are also discussed.

13.2 Mechanism of cellulolysis

The hydrolysis of insoluble cellulose requires the synergistic action of three types of

enzyme: endoglucanase (1,4-β-D-glucan-4-glucanohydrolase; EC 3.2.1.4), exoglucanase (1,4-

β-D-glucan cellobiohydrolase; cellobiohydrolase; EC 3.2.1.91) and β-glucosidase (β-

glucosideglucohydrolase; cellobiase; EC 3.2.1.21) as mentioned previously. Endoglucanases

initiate cellulose hydrolysis by cleaving the cellulose polymer exposing both the reducing and

non-reducing ends, while cellobiohydrolases acts upon these reducing and non-reducing ends

to liberate cello-oligosaccharides and cellobiose units, and β-glucosidases cleave the

cellobiose to liberate glucose, thereby completing the hydrolysis process (Bhat and Bhat

1997). Endoglucanases are classified as endo-acting cellulases because they are thought to

cleave β-1,4-glycosidic bonds internally only and appear to have cleft-shaped open active

sites. They are typically active on the more soluble amorphous region of the cellulose crystal,

increase the concentration of chain ends and significantly decrease degree of polymerization

by attacking interior portions of cellulose molecules (Tomas et al., 2009). On the other hand,

cellobiohydrolases are classified as exo-acting cellulases as they cleaveβ-1,4-glycosidic bonds

from chain ends and have a tunnel-shaped closed active site that retains a single glucan chain

and prevents it from re-adhering to the cellulose crystal (Divne et al., 1998). They are usually




137

active on the crystalline regions of cellulose; shorten degree of polymerization incrementally

by binding to the chain ends releasing mainly cellobiose units. Thus, endoglucanase activity is

thought to be primarily responsible for chemical changes in solid-phase cellulose that occurover the course of hydrolysis, but plays a minor role in solubilization relative to exoglucanase,

while exoglucanase activity is thought to be primarily responsible for solubilization but plays

a minor role in changing the chemical properties of residual cellulose.

13.3 Classification of Endoglucanases

Based on sequence and 3-dimensional structure, endoglucanases (EGs) are grouped

into 11 glycoside hydrolase (GH) families, including GH5, 6, 7, 8, 9, 12, 44, 45, 48, 51, and

74 (Cantarel et al., 2009). Glycoside hydrolases (EC 3.2.1.) are a widespread group of

enzymes which hydrolyse the glycosidic bond between two or more carbohydrates or between

a carbohydrate and a non-carbohydrate moiety. The International Union of Biochemistry and

Molecular Biology (IUBMB) Enzyme nomenclature of glycoside hydrolases is based on their

substrate specificity and occasionally on their molecular mechanism; such a classification

does not reflect the structural features of these enzymes. A classification of glycoside

hydrolases in families based on amino acid sequence similarities has been proposed a few

years ago. Because there is a direct relationship between sequence and folding similarities,

such a classification:

(i) Reflects the structural features of these enzymes better than their sole substrate specificity,

(ii) Helps to reveal the evolutionary relationships between these enzymes, (iii) provides a

convenient tool to derive mechanistic information (Henrissat, 1991; Henrissat and Bairoch,

1993). The details of each family are shown in the table 1.

13.4 Structure of endoglucanases

Endoglucanase is made up of different domains. The main domain contains the large,

globular catalytic domain which expresses the active site. A loop of the protein chain forms a

tunnel that encloses the active site. It is attached at the O-glycosylated B block hinge region of

the catalytic domain to the smaller, globular cellulose binding domain (CBM) at its C-

terminal A block by a linker peptide (Nimlos, et al., 2007).The overall shape of the complex

looks like a tadpole, with the A and B blocks forming the extended tail and the catalytic

domain forming the head (Pilz, et al., 1990). These structural domains contain three types ofstructure folds depending upon the endoglucanases (Yennamalli et al., 2011).




138

Table 1: Classification of different endoglucanases and their properties

Family Mechanism StructureCatalytic

Nucleophile/Base

Catalytic proton

donor

GH5 Retaining (α / β)8 Glu Glu

GH6 Inverting - Asp Asp

GH7 Retaining β-jelly roll Glu Glu

GH8 Inverting (α / α)6 Asp Glu

GH9 Inverting (α / α)6 Asp Glu

GH12 Retaining β-jelly roll Glu GluGH44 Retaining (α / β)8 Glu Glu


GH48 Inverting (α / α)6 - Glu

GH51 Retaining (α / β)8 Glu Glu


13.4.1 (α / β)8 fold

This type of fold has an alternating pattern of eight α and β subunits in a single

domain, such that the eight parallel β strands on the inside are protected by eight α helices on

the outside. This very common fold has been reported to exhibit the highest diversity of

enzymatic functions (Wierenga, 2001).

13.4.2 β-jelly roll fold

This type of fold consists of 15 β-strands in two twisted antiparallelβ-sheets, named A

and B, that pack against each other. β-sheet A contains six antiparallel β-strands forming the

back, convex surface while β-sheet B contains nine anti-parallel β-strands arranged to form

the front, concave binding surface (Sandgren et al., 2005). Additionally two α-helices pack

against the back side of β-sheet B.

13.4.3 (α / α)6 fold




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The substrate binding cleft in this type of fold has a tunnel shape that is formed at the

N-termini of six central, parallel α-helices. These six helices are surrounded by six external α-

helices (Alzari et al., 1996).13.5 Mechanism of cellulose hydrolysis by endoglucanases

Endoglucanases uses two types of catalytic mechanisms for hydrolysis of glycosidic

bonds of cellulose:

13.5.1 Retaining mechanism

In this type of mechanism, the stereomeric configuration of the anomeric carbon is

retained in the β configuration after hydrolysis. A pair of Glu amino acids, act as the catalyticresidues: one as a nucleophile and the other as an acid-base donor. The first step in this double

displacement mechanism is glycosylation, where one residue plays the role of a nucleophile,

attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme

intermediate. At the same time the other residue functions as an acid catalyst and protonates

the glycosidic oxygen as the bond cleaves. The second step in this mechanism is

deglycosylation step, where water molecule acts as a nucleophile and the first residue’s

carboxylic group acts as a base. Once deprotonated, the water molecule is an activatednucleophile that then hydrolyzes the glycosyl-enzyme intermediate leading to a break in the

polymer (Yennamalli et al., 2011).

13.5.2 Inverting mechanism

In this type of mechanism, the configuration of the anomeric carbon is inverted; i.e.,

hydrolysis of β-glycosidic bond leads to α-configuration of carbon and vice versa. Contrary to

retaining method, this method involves single displacement mechanism. The reaction typically

occurs with general acid and general base assistance from two amino acid side chains,

normally glutamic or aspartic acids (Guimaraes et al., 2002; Guerin et al., 2002) and a water

molecule acts as a nucleophile. Utilizing the water molecule on the opposite side of the sugar

ring to stabilize the transition, these residues catalyze the glycosylation or deglycosylation in

one step. Unlike the retaining mechanism, this mechanism does not involve the glycosyl-

enzyme intermediate.

13.6 Microbial sources of endoglucanase enzyme




140

A variety of microorganisms including fungi, bacteria and actinomycetes produce

endoglucanases for utilizing cellulose as a source of carbon and energy. Among them, most

emphasis has been placed on fungi and bacteria because of their ability to produce plentifulamounts of endoglucanses. The list of microbes having potential for endoglucanase

production is given in Table 2.

13.7 Application of endoglucanases

Microbial endoglucanases can be used for the production of bioenergy and other value

added products and chemicals with great potential.

Table 2: Most extensively studied microbes employed for endoglucanase production

Group Organisms

Fungi Agaricus bisporus, Aspergillus niger, A. fumigatus, A. oryzae, A. terreus, A.

wentii, A.aculeatus, A. awamori, Chaetomium cellulyticum; C.

thermophilum, Daldinia eschscholzii, Fomitopsis sp., Fusarium solani, F.

oxysporum, Humicola insolens, H. grisea, Macrophomina phaseolina,

Melanocarpus albomyces, Mucor circinelloides, Paecilomyces inflatus,

Penicillium pinophilum, P. chrysogenum, P. occitanis, P. purpurogenum,

Phanerochaete chrysosporium, Phlebia gigantean, Pleurotus ostreatus,

Pyrenochaeta lycopersci, Rhizopus stolonifer, R. oryzae, Schizophyllum

commune, Trametes versicolor, Trichoderma reesei, T. atroviride, T.viridae,

T. harzianum, Thermoascus aurantiacus

Bacteria Acetivibrio cellulolyticus, Acinetobacter junii, A. amitratus, Anoxybacillus

sp., Bacillus subtilis, B. pumilus, B.amyloliquefaciens, B. licheniformis, B.

circulan, B. flexus, Bacillus agaradhaerens, Bacteriodes cellulosolvens,

Butyrivibrio fibrisolvens, Cellvibrio gilvus, Clostridium thermocellum; C.

cellulolyticum; C. acetobutylium; C. cellulofermentans, C. cellulovorans,

Eubacterium cellulolyticum, Geobacillus sp., Fibrobacter succinogenes,

Microbispora bispora, Paenibacillus campinasensis, P. polymyxa,

Pectobacterium chrysanthemi, Pseudomonas fluorescens, Rhodothermus

marinus, Ruminococcus albus, R. succinogenes, Thermotoga maritime

Actinomycetes Cellulomonas fimi, C. flavigena, C. cellulans, C. uda, Streptomyces

cellulyticus, S. aureofaciens, Thermomonospora fusca, T. curvata,




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Thermobifida fusca, T. cellulolytica

Kuhad et al., 2011

13.7.1 Biofuel industry

Bioconversion of lignocellulosic biomass into the fermentable sugars is the most

important application of endoglucanases. Bioconversion is carried out either enzymatically or

chemically using sulfuric or other acids (Wyman, 1999). However, when sulfuric acid is used,

it is necessary to remove the residual sulfuric acid from the hydrolyzing solution prior to yeast

fermentation. Furthermore, it produces toxic compounds that inhibit fermentation. The

advantages of enzymatic hydrolysis are the lower requirements for cooling water, gas,

electricity and disposal costs and no corrosion issues for equipment together with lower

environmental pollution (Sun and Cheng, 2002).

13.7.2 Textile and laundry

Endocellulases play a key role in textile and laundry because of their ability to modify

cellulosic fibres in order to improve the quality of fabrics. They especially help in biostoning

and biopolishing of cotton and fabrics. In bio-stoning of denim fabrics, endoglucanases

remove excess dye from denim fabrics and restore the softness of cotton fabrics withoutdamaging the fibre. Similarly, in biopolishing, they facilitate the removal of excess

microfibrils from the surface of cotton and non-denim fabrics. In addition to this, they

improve the detergent performance and allow the removal of small, fuzzy fibrils from fabric

surfaces and restore the appearance and color brightness (Galante et al., 1998a; Godfrey,

1996; Kumar et al., 1996).

13.7.3 Paper and Pulp industry

Paper and pulp industry also require endoglucanases for substantial energy savings

during mechanical pulping, improvements in hand-sheet strength properties and deinking of

fibre surface for improving brightness and freeness of the pulp by partial hydrolysis of

carbohydrate molecules (Akhtar, 1994; Leatham et al., 1990; Prasad et al., 1993).

13.7.4 Wine and Brewery Industry

Endoglucanases are also vital for fermentation processes to produce alcoholic

beverages including beers and wines. They improve color extraction, skin maceration, must

clarification, filtration, and finally the wine quality and stability. These enzymes can improve

both quality and yields of the fermented products (Singh et al., 2007; Galante et al., 1998b).




142

13.7.5 Other applications

Endoglucanases also find applications in clarification of fruit and vegetable juices, oil

extraction, and in improving the nutritive quality of bakery products and animal feed (Bhat,2000). The enzymes are used to improve cloud stability and texture and decrease viscosity of

the nectars and purees from tropical fruits, thus clarify the juices (Singh et al., 2007). The

enzyme causes degradation of β-glucan in feed which lowers the viscosity of the intestinal

contents and thus, improves the quality of the feed (Bedford, 1995).

13.8. Significance of thermostable endoglucanases

Currently, the bioconversion of lignocellulosic biomass into fermentable sugar is the

major concern for the production of biofuel. Thermostable endoglucanases are economically

important because they are able to contribute to the hydrolysis of cellulose at higher

temperatures compared to their mesostable homologs, reducing the number of processing

steps during biofuel conversion. Thus, lignocellulosic conversion using thermostable

endoglucanses have attracted much attention because of their several potential advantages

such as (i) higher reaction rates due to the increased solubility of substrates; (ii) higher

productivity as hydrolysis time is shortened; (iii) lessen the amount of enzyme needed; (iv)

decreased risk of contamination; (v) facilitated recovery of volatile products; (vi) decreased

cost of energy for cooling as the thermostable enzymes can be used directly after the heating

step without a pre-cooling step and (vii) loss of enzyme activity is low during processing, at

higher temperature used during pre-treatments (Zhang et al., 2011; Viikari et al., 2007; Bhalla

et al., 2013).

13.9 Factors responsible for thermal stability

Thermostability is ability of an enzyme to maintain its active structural conformationat higher temperature for a prolonged incubation period (Bhalla et al., 2013).There are

multiple factors that are responsible for increased thermostability of enzyme.

13.9.1 Amino acid composition

Positively charged residues (Lys, Arg and Glu) on the solvent accessible surface are

more significant in thermophiles than in mesophiles (Glyakina et al., 2007). Kumar et al.,

(2000) reported that Arg and Tyr are significantly higher whereas Cys and Ser are

significantly lower in thermophilic proteins. It was reported that a decrease in the number of

Gly residues in thermophilic proteins leads to greater stability at higher temperatures (Panasik




143

et al., 2000). Yennamalli et al., (2011) observed that amino acids Arg and Met are statistically

significant among thermophilic proteins, whereas Gln and Ser are statistically significant

among mesophilic proteins.13.9.2 Intramolecular interactions

For the thermophiles, only ionic interactions were significant, whereas for mesophiles,

no intramolecular interactions were significantly different from thermophiles (Yennamalli et

al., 2011). Comparison of intramolecular interactions showed that cation-π interactions are

highly significant in imparting thermophilicity (Chakravarty and Varadarajan, 2002).

13.9.3 Fold specificity:

Recently, Yennamalli et al., (2011) conducted the study on thermostable

endoglucanases and demonstrated fold specificity as a key factor for controlling

thermostability in endoglucanases. For the (α / β)8 fold, Arg and Pro (significant in

thermophiles) are replaced by polar amino acids whereas Leu is primarily replaced with

aromatic amino acids in the mesophilic counterpart. The absence of arginine amino acids

leads to a loss of ionic interactions in mesophiles, rendering them enzymatically inactive at

higher temperatures. In the β-jelly roll fold, the amino acids Glu, Arg, and His are substitutedwith polar, hydrophobic amino acids. Substitution to Pro is higher for Arg indicating the

potential for fewer salt-bridges in mesophiles whereas number of salt bridges is high in

thermophiles. For the Ser and Thr positions (significant among mesophiles) the thermophilic

protein has hydrophobic, acidic, and basic amino acids substituted. In the (α / α)6 fold Glu and

Val are replaced with polar amino acids and to a lesser extent with other amino acid groups.

Gln (significant in mesophiles) is substituted to a large extent by hydrophobic, acidic and to a

lesser extent with basic amino acid groups in thermophiles indicating that in the thermophilicprotein these substitutions contribute towards more intramolecular interactions and extend

stability to proteins at higher temperatures.

13.9.4 Other factors

These include a decrease in loop length and a concomitant increase in secondary

structure, an increase in aromatic stacking, increased hydrophobic interactions, increased

metal-binding capacity, and increased oligomerization (Yano and Poulas, 2003). Gromiha et

al., (1999) found Gibbs free energy change of hydration, long-range non-bonded energy, β-

strand tendency and average long-range contacts as factors responsible for imparting




144

thermostability. Several studies implicated partial, reversible folding of the protein as a factor

responsible for thermostability at high temperatures (Uversky, 2009).

13.10 Conclusion

Endoglucanases, also called primary cellulases, are synergistically involved in the first

stage of cellulose breakdown-a vital step in the bioprocessing of lignocellulosic biomass into

biofuel and other industrial bioprocesses. Despite possessing several advantages,

lignocellulosic biofuel has not yet been well implemented on a commercial scale because of

high costs of cellulolytic enzymes and lack of robust cellulases that can function efficiently at

high temperature. Thus, understanding the structure of enzyme, mechanism of reaction and

basis for thermostability helps in engineering the protein for enhanced activity. It can lead to

more cost effective processes for biofuels and other industrial applications by designing a

more efficient endoglucanase enzyme.

Acknowledgements

Authors thankfully acknowledge the financial assistance received from the NAIP sub-

project funded by the World Bank through the Indian Council of Agricultural Research

(ICAR) Government of India (PR/8488/PBD/26/68/2006) for conducting a part of the studyreported in this book chapter.

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CHAPTER 14

COMPARATIVE STUDY OF FERMENTATION EFFICIENCYFOR BIO-ETHANOL PRODUCTION BY ISOLATES

Richa Arora, Shuvashish Behera, Sachin Kumar

Abstract

Recent production of bio-ethanol through microbial fermentation as an alternative source has

renewed its research interest because of the increase in the fuel price and environmental

concern. Yeast strains are commonly associated with the ethanol production potential in sugar

rich environments. In the present study, isolation of various yeast strains were carried out from

different soil samples collected from dumping sites of sugar-mills. A total of four yeast strains

were isolated with the ethanol producing ability, which were used for the further study. An

attempt has been made to evaluate the pattern of sugar utilization and ethanol yield by the

yeast strains using the salt medium. The results obtained in this study showed a range of

ethanol production between 5.0± 0.2 and 22.0± 0.4 in all the strains. Two isolates NIRE K1

and NIRE K3 showed the highest ethanol yield of 0.49 and 0.41, respectively after 40 h of

incubation at 45oC. This study revealed the characteristics of the isolate NIRE K1 allow it to

ferment glucose efficiently to ethanol and have the potential to develop a bioprocess for

bioethanol production.

Key words: Bio-ethanol; Fermentation; Ethanol Yield; Thermotolerant.

14.1 Introduction

The global rise in energy consumption, predicted increase in energy demands in the

near future, the depletion of low extraction cost fossil fuel reserves, and climate change are

forcing the search for new and alternative energy sources (Agbor et al., 2011). A concern

about energy security, the environmental impact of energy production has led to the

implementation of policies designed to encourage the production and use of renewable

bioenergy (Glithero et al., 2013).




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An alternative fuel must be technically feasible, economically competitive,

environmentally acceptable, and readily available. Numerous potential alternative fuels have

been proposed, including bioethanol, biodiesel, methanol, hydrogen, natural gas, liquefiedpetroleum gas (LPG), Fischer–Tropsch fuel, electricity, and solar fuels (Limayem and Ricke,

2012). Bioethanol has been considered in all over the world as an alternative renewable fuel

with the largest potential to replace fossil derived fuels, responsible for a significant fraction

of greenhouse gas emissions (Dias et al., 2013).

Current bioethanol research focus on lignocellulosic feedstocks due to its abundance

and renewability; especially in relation to reduce the cost and increase the efficiency of the

key steps in the lignocelluloses-to-bioethanol process (e.g. lignocellulosic pre-treatment,enzymatic hydrolysis and fermentation) (Saratale and Oh, 2012; Mathew et al., 2013;

Matsushita et al., 2013). The main advantage of the production of second-generation biofuels

from lignocellulosic biomass is to reduce the limitation between food versus fuel competition

associated with first generation biofuels (Nigam and Singh, 2011; Singh et al., 2010).

Most of the potential ethanologens that are in industrial use belong to mesophillic

group (28-37oC). However, the bioethanol production from lignocellulosic biomass by

thermophillic/ thermotolerant species have some process advantages over mesophiles due to

high growth temperatures, require less energy for mixing and product recovery, higher

saccharification and fermentation rates, minimized contamination risk, lower costs of

pumping and stirring and no aeration and cooling problems (Georgieva&Ahring, 2007; Oberoi

et al., 2010; Frock & Kelly, 2012).

Considering the above, this study was carried out to compare the performance of the

thermotolerant yeast isolates for ethanol production. Further, the growth and fermentation

parameters of the isolates during fermentation were compared.


14.2.1 Microorganisms and culture conditions

Microorganisms were isolated from soil samples collected from dumping sites of

crushed sugarcane bagasse in Sugar Mills at 45oC. Four yeast isolates were compared for high

ethanol production rate and higher sugar consumption rate.

For growing the isolated strains, salt medium (SM) was used in g l-1, di-sodium hydrogen

ortho phosphate, 0.15; potassium di-hydrogen ortho phosphate, 0.15; ammonium sulphate,




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2.0; yeast extract, 1.0; glucose, 10.0 at pH 5.0. The cells were grown in 250-ml flasks in a

shaker at 45oC and 150 rpm for 24 h.

14.2.2 Fermentation conditions

The medium for fermentation was the same as that for the growth medium, except for

glucose 50 g l-1. Fermentation was carried out in 250-ml capped flasks at 100 rpm at 45oC.

14.2.3Analytical methods

At 4h intervals, fermented broths (in triplicate) were removed and the contents were

analyzed for biomass, sugar and ethanol. Glucose was analyzed by high-performance liquid

chromatography (HPLC) using Hi-Plex H column at 57oC with 1mM H2SO4 as the mobile

carrier at a flow rate 0.7 ml min-1 and detected by refractive index detector. Ethanol was

analyzed by gas chromatography using Nucon 5765 with a 2-m-long and 1/8-in. diameter

Porapak-Q column with mesh range 80/100. The sample was injected at an inlet temperature

150oC, and flame ionization detector 250oC using nitrogen gas as a carrier.

Dry cell weight (DCW) was determined in the broth by centrifuging 1 ml of broth in

pre-dry weighted Eppendorf tube using Eppendorf centrifuge 5430 R at 10,000 rpm for 5 min,

followed by washing twice with distilled water and drying in a vacuum oven at 80oC to a

constant weight.


The cultural characteristics of the isolates are shown in Table 1. In case of isolates

NIRE K1 and NIRE K3, the concentration of sugar dropped down in 24 h, with concomitant

production of ethanol; thereafter, the decline was gradual. At the end of 40 h of incubation,

the residual sugar concentration reached close to 5 g l-1with the ethanol concentration was 22g l-1.

In contrast to NIRE K1 and NIRE K3, there was a marginal difference in sugar

consumption albeit, there was no ethanol produced in 20 h NIRE K4 and NIRE K5. The

marginal decrease in sugar concentration might be due to their utilization for growth and

metabolism by NIRE K4 and NIRE K5. After 24 h, there was a gradual increase in ethanol

concentration over the incubation period with simultaneous decrease in sugar concentration.

At the end of 40 h of incubation, the ethanol production from NIRE K4 and NIRE K5 reached




152

8 and 5 g l-1, respectively. The pattern of sugar utilization and ethanol formation among the

strains is shown in Fig. 1.

The initial DCW of NIRE K1 and NIRE K3 was kept at 2.7 g l-1

and 2.8 g l-1

, whichwas 2.6 g l-1and 2.8 g l-1 after fermentation, and shows almost constant DCW throughout the

process, respectively. No significant change was observed during fermentation in the cell-

mass concentration, which means the ethanol formation is non-growth associated when using

NIRE K1 and NIRE K3.

Table 1 Cultural characteristic of four yeast isolates

Fig. 1Comparison of ethanol production between free cells of four yeast isolates at 45oC

Isolate Colour Margin Shape Opacity Elevation

NIRE-K1 White Entire Circular Transparent Flat

NIRE-K3 White Crenate Circular Opaque Raised

NIRE-K4 White Crenate Circular Opaque Flat

NIRE-K5 Creamish yellow Undulate Irregular Opaque Umbonate




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However, in case of isolates NIRE K4 and NIRE K5, the initial DCW was kept at 2.5

g l

-1

, which was 1.7 and 1.3 g l

-1

after fermentation, respectively. The DCW declined slightlywhen ethanol concentration increased in broth.

Table 2 shows the fermentation parameters evaluated among the isolates. The

maximum ethanol concentration was 22± 0.4 g l-1 on initial glucose concentration of 50 g l-1

with 89% of sugar conversion and ethanol yield of 96% of theoretical yield in 40 h. Banat et

al., 1992 reported the maximum ethanol concentration of 7.2% (w/v) with ethanol yield of

98% of theoretical yield and ethanol productivity 1.71 g l-1 h-1 on 140 g l-1 glucose by K.

marxianusIMB2 at 45oC. Cazetta et al., 2007 achieved an ethanol concentration of 55.57 g l-1

with an ethanol yield of 63.03% of theoretical yield and productivity of 1.16 g l-1 h-1 on

molasses containing 200g l-1 reducing sugar in 48 h at 30oC by using Zymomonasmobilis.

Table 2 Growth and Fermentation kinetics of free cells of four yeast isolates at 45oC in Salt

Medium

Parameters evaluated NIRE K1 NIRE K3 NIRE K4 NIRE K5

Final ethanol (P, g l-1) 22.0 ± 0.4* 12.0 ± 0.4 8.0 ± 0.1 5.0 ± 0.2

Ethanol Yield (Yp/s, g g-1) 0.49 0.41 0.21 0.17

Sugar utilisation (g l-1) 44.90 ± 0.04 29.27 ± 0.05 38.10 ± 0.07 29.41 ± 0.03

Conversion rate (%) into

ethanol89.80 58.54 76.19 58.82

Maximum specific growth

rate, h-1 0.51 0.43 0.16 0.14

*mean ± standard deviation

The ethanol yield (Yp/s = 0.49) obtained with the free cells of NIRE K1 was more than

that of free cells of NIRE K3 (Yp/s = 0.41) followed by NIRE K4 (Yp/s = 0.21) and NIRE K5

(0.17). Kumar et al., 2009 reported ethanol yield of 90% of theoretical yield on glucose by

Kluyveromyces sp. IIPE453 at 50oC and concluded thatthe strain has the ability to convert

hexose sugars to cell mass as well as ethanol during the growth phase. Similar effect was

found in our studies where NIRE K1 and NIRE K4 follow the Crabtree rather than the Pasteur

Effect. In this study, isolate NIRE K1 was found to be more efficient than the other 3 isolates

in ethanol production.




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14.4 Conclusion

The new isolated thermotolerant yeast strain NIRE K1 has shown the good

consumption of sugar for ethanol fermentation. The results showed that ethanol production byisolate NIRE K1 was the highest in comparison to other isolates. For all the isolates, the peak

ethanol concentration was obtained after 40 h of fermentation. This study revealed the

characteristics of the isolate NIRE K1 allow it to ferment glucose efficiently to ethanol. The

yield could be further increased after optimization of the fermentation parameters andhave the

potential to develop a bioprocess for bioethanol production.

Acknowledgments

The authors are thankful to Dr. Y. K. Yadav, Director, SSS-NIRE for all the possible

support and encouragement. The authors also acknowledge MNRE, Govt. of India for

providing financial assistanceship.

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156

CHAPTER 15

SWEET SORGHUM - AN IDEAL FEEDSTOCK FORBIOETHANOL PRODUCTION

Reetika Sharma1, Gurvinder Singh Kocher1 and Harinder Singh Oberoi

Abstract

Sweet sorghum can serve as a potential feedstock for ethanol production, largely because of

its ability to grow under hot and dry conditions; short duration, so that two crops could be

taken in a year in arid regions; low water requirement and high biomass yield. Since sweet

sorghum juice cannot be crystallized as cane sugar, it could be converted to ethanol through

fermentation. The sugar content in different varieties of sweet sorghum varies from 14-22 %.

After juice extraction, the bagasse could also be used for ethanol production. Although, the

ethanol production process from lignocellulosic biomass is cost and energy intensive, sweet

sorghum bagasse (SSB) contains relatively higher cellulose content (40 % or higher) as

compared to rice straw, sugarcane bagasse or wheat straw. This means that there is an

opportunity to obtain higher glucose concentration, which is essentially required for ethanol

production. In addition, relatively lower concentrations of lignin and ash in SSB in

comparison to other agricultural residues makes it easily amenable to pretreatment and

hydrolysis processes. Therefore, higher ethanol concentration obtained using both sweet

sorghum juice and bagasse could reduce the cost and energy required during downstream

processing, thereby making sweet sorghum as an ideal and cost-effective feedstock forbioethanol production for its use as biofuel.

Key words: Lignocellulosic biomass, Sweet sorghum, Pretreatment, Hydrolysis,

Fermentation, Ethanol.

15.1 Introduction

Fossil fuel limitations and constraints on carbon dioxide emissions have a high impact

in the market of bioethanol, which is the most commonly used biofuel for petrol substitution

in the world (Taherzadeh and Karimi, 2008). Ethanol can be produced from a variety of feed

stocks such as saccharine materials, starchy materials and many types of lignocellulosic




157

wastes (Sanchez and Cardona, 2008). Lignocellulosic biomass is considered a future

alternative as raw material for bioethanol production, because it is more abundantly available

and is much less expensive than food crops, especially when waste streams are used(Hamelink et al., 2005; Prasad et al., 2007b). Furthermore, the use of lignocellulosic biomass

is more attractive in terms of energy balances and green house gas emissions (Taherzadeh and

Karimi, 2007).

Sweet sorghum (Sorghum bicolor (L.) Moench) represents an analogous crop to

sugarcane with similar accumulation of sucrose but with a higher agronomic stability to

temperature fluctuations, less water requirement and better tolerance to salinity, alkalinity and

drought (Prasad et al., 2007a; Almodares and Hadi, 2009; Goshadrou et al., 2011). It contains43.6 - 58.2 % soluble sucrose, glucose and fructose in the stalk (Billa et al., 1997; Dolciotti et

al., 1998; Amaducci et al., 2004; Antonopoulou et al., 2008) and 22.6 - 47.8 % insoluble

cellulose and hemicellulose (Dolciotti et al., 1998; Rattunde et al., 2001; Antonopoulos et al.,

2008). In addition, it is an annual crop with a typical growing season of 3-5 months instead of

9-12 months required by sugarcane. Additionally, the sweet sorghum bagasse has a

comparatively higher nutritional value for ruminants because of its more favorable fiber

composition and is a better alternative for further hydrolysis and fermentation (Almodares andHadi, 2009). Because of its agronomic flexibility and productivity, sweet sorghum is viewed

as a viable feedstock option for ethanol production in some regions of the world. Sweet

sorghums have potential for specific tropical, subtropical and arid regions of the US, Mexico,

China, India, Southern Africa and other developing countries where the use of maize and

other crops for ethanol production is not feasible due to either economic, agronomic or social

considerations (Reddy et al., 2005; Chuck-Hernandez et al., 2009; Wu et al., 2010; Zhang et

al., 2010).

Sweet sorghum has a high a ratio of energy output to fossil energy input in comparison

to sugarcane, sugar beet, maize and wheat and its fermentation efficiency has been reported

higher than 90 % (Almodares and Hadi, 2009; Wu et al., 2010). Lastly, sorghum is known as

one of the most variable crops in terms of genetic resources and germplasm that allows the

breeding and development of new cultivars adapted to different regions around the globe

(Zhang et al., 2010). For all these reasons, bioethanol produced from sweet sorghum presents

a high environmental, economic and energetically sustainable biofuel which ascribes GHGs

saving upto 70-71 % (Liu et al., 2008).




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Despite these advantages, utilization of sweet sorghum as a potential energy crop

presents some major technical challenges which must be resolved before sweet sorghum is

widely planted to provide feedstock for ethanol biorefineries. These include, its highconcentration of soluble sugars, due to which it can be easily contaminated, limiting its

storage stability. Secondly, the lignocellulosic stalk cannot be easily hydrolyzed enzymatically

to fermentable sugars due to the presence of free sugars inhibiting the hydrolytic enzymes

action; thus, the sugars in the stalk should be removed before the enzymatic hydrolysis of the

cellulosic part of the plant (Taherzadeh and Karimi, 2007; Molaverdi et al., 2013). Short

harvest periods also increase the capital cost involved in building a central processing plant

that may be operated only seasonally. Finally, the common practice of utilizing sweetsorghum either involves a stage of sugars extraction and separate utilization of soluble sugars

and fiber fraction, which exhibits some technical difficulties, or involves a solid state

fermentation, which makes fermentation process and ethanol extraction more difficult,

leading to increased cost (Mei et al., 2009; Kundiyana et al., 2010, Wu et al., 2010, Matsakas

and Christakopoulos, 2013). Moreover, fermentation performance of sweet sorghum can be

affected by the microorganism used, bioreactor configuration, free amino nitrogen, sugar

content and composition of juices (Lui and Shen, 2008; Laopaiboon et al., 2009; Chohnan etal., 2011). For this reason, evaluation of different sweet sorghum cultivars for their bioethanol

production potential is critically important (Zhao et al., 2009; Davilla-Gomez et al., 2011).

15.2 Origin and biology of sweet sorghum

Cultivated sorghum (Sorghum bicolor spp. bicolor L. Moench) is in the sub-genus

Sorghum and originates from semi-arid regions of Africa. However, due to its adaptive

capacity, now- a -days it is cultivated on a wide spectrum of climates in every continent

(Saballos 2008). Sweet sorghum varieties belong to the grain, forage and broomcorn

sorghums. These names describe well the diversity of phenotypes, as well as the aim and

direction of selection. Sweet sorghum has been selected for accumulation of high amount of

sucrose in the stem (Murray et al., 2009). Sweet sorghum belongs to C4 crop with high

photosynthetic efficiency and high productivity. The plant grows to a height of about 14 feet.

Seeds are produced by self-pollination from the panicle at the top of the plant and contains the

male and female inflorescences. The plant lodging is more likely to occur in high population

fields because stalks become smaller in diameter due to competition. The plant can also be

blown down in strong winds due to its height (Nahar, 2011).




159

15.3 Cultivation and harvesting of sweet sorghum

Cultivation of sweet sorghum possesses the following advantages arising from the

physiology and biochemistry of C4 plants that usually generate high biomass yield with minimalinputs (Rooney et al., 2007; Saballos, 2008; Byrt et al., 2011):

1. High conversion efficiency of light into biomass (biomass yields competing with

switchgrass and miscanthus) resulting in high sugar, and thus ethanol yields;

2. High water use efficiency and thus low water requirement that is 25% of that needed for

sugar cane and 50-66% needed for maize production (no irrigation);

3. Drought tolerance - even though drought leads to reduction in plant growth, enhanced

accumulation of sucrose and starch was observed in drought stressed stems and thus

resulting in equal sugar yields (Massacci et al., 1996);

4. Reduced demand for fertilizer due to the high leaf nitrogen use efficiency and large

fibrous root system;

5. Modest demand for soil quality (that are not appropriate for corn or wheat)

6. High tolerance towards salinity and water-logging;

7. Pest and disease management is less complex,

8. Greater tolerance towards climate changes (e.g. temperature extremities, droughts).

9. Its cultivation is also possible on marginal lands and therefore, it can contribute to

sustainable ways to produce bioethanol, for instance, avoiding the food versus fuel

debate often related to bioethanol production (Prasad et al., 2009; Allen et al., 2011);

10. Its untapped genetic diversity as presented by its viability in almost every climate

condition, carries enormous potential for further breeding (Rooney et al., 2007; Saballos,

2008).

Cultivation Technology: The plant can be grown on soils ranging from heavy clay to light

sand. Loam and sandy loam soils generally allow the best syrup production. Good surface

drainage is preferred although sweet along sorghum can withstand long waterlogged condition;

clay loam is preferred with soil acidity not lower than pH 6.

Sowing: Sowing can be done on ridges or in furrows at a spacing of 60 cm between rows and

15 cm between plants. Three to four seeds are dibbled in each planting hole and the seedlings

are to be eventually thinned to one per hole. Sweet sorghum is not suitable for high density;recommended density is about 7000 plants/ha. The plant is ideally shown during June to




160

September, when soil can hold much water (deep).The crop does not prefer high rainfall as high

soils moisture or continuous heavy rain after flowering may decrease sugar content in plants.

Setting of Furrow: Two planting seasons are possible for sweet sorghum. During the wetseason, furrows are set 100 cm apart while in the dry season planting are set about 75 cm apart.

Fertilizer Application: The plant needs adequate nutrients to produce good yields. Quality of

syrup is also affected by the fertilizer applications. The recommended dose of fertilizer for sweet

sorghum is 80 kg of nitrogen, 60 kg of phosphorous and 40 kg of potassium per hectare. Half of

N and whole of P and K are applied as basal dose. Remaining N is top-dressed during 25-35

days after germination, following weeding and inter-cultivation. Nitrogen fertilizer should not

be applied in the field when sweet sorghum is grown immediately after a legume crop, as the

soil contains nitrogen.

Intercropping: Sweet sorghum is suitable to intercropping with early maturing crops for its

characteristics in growth and development. Sweet sorghum seedlings develop slowly at their

early stage. It can be intercropped with potato, maize, wheat etc. Adaptation and Yield: It is

relatively inexpensive to grow high yield sweet sorghum plants and can be used to produce a

range of high value added products like ethanol, energy and dried grains. It can produce

approximately 30 tons/ ha per year of biomass on low quality soils with low inputs of fertilizer,

half of that required by sugar beet and a third of the requirement for sugarcane or corn.

Harvesting: The plant varieties mature between 115-125 days after plantation. To obtain high-

quality syrup and high yields, the crop should be harvested when the seed is in the soft dough

stage. Stalks can be harvested either along with the grain, or 4-5 weeks after the grain harvest.

The ear-head and peduncle (between the base of the seed head and the top node) should be

removed before processing the stalks. Ear heads may be dried and threshed so the seeds can beused for the next year's crop.

15.4 Inherent advantages of sweet sorghum

Sorghum is an annual crop with a lot of varieties (at present estimated at about 4,600

approximately), and some of these have a very high sugar content in the stems. Its bioenergy

applications are numerous: sweet sorghum can be used to produce ethanol, but alternatively

also biogas through anaerobic digestion, fiber sorghum (pelletized or not) can supply

combined heat and power (CHP) plants, grain sorghum can be employed for food, feed and

energy needs of small isolated communities. Grain sorghum is not so suitable for the ethanol




161

production, because in these varieties, sugars are prevalently polymerized to starch, which

then require hydrolysis before the alcoholic fermentation. Sweet sorghum is a sugar cane-like

C4 plant, containing juice in the stem with large amount of sucrose (11-23 % brix, dependingon variety and conditions) that can be effectively extracted by squeezing and thereafter,

readily fermented to ethanol by yeast (Almodares and Hadi, 2009, Byrt et al., 2011). While

it is not well suited to the production of refined sugar, sweet sorghum has multiple inherent

advantages which have been represented in Table 1. Most important, sweet sorghum is a seed-

propagated annual crop, i.e., a crop established through sowing seed and which matures and is

harvested in a single season. These key characteristic impacts both its fit within current

production cycles as well as the pace of scale-up and on-going improvements to the varietiesthemselves. In addition, sweet sorghum can be utilized in rotation with other annual crops,

and potentially, with sugarcane itself, where sweet sorghum could be sown on fallow

sugarcane land, hectares destined for rotation and land where sugarcane yields are limited due

to marginal soils. This flexibility is due to the sorghum plant’s natural hardiness and rapid

growth.

Table 1: Inherent advantages of sweet sorghum over sugarcane

Sugarcane Sweet sorghum

Sugar quality Sucrose Mixed sugars

Establishment cost Vegetative propagation Seed propagation

Sugar yield (% fw) 13-15 8-13

Input requirements Limited by water, nitrogen 50% water, 60% nitrogen

Scale-up time Vegetative propagation Seed propagation

Biomass yield (tons/ha) 70-90 tons/ha 60-100 tons/ha

Marginal land Limited yield Significant yield

Season extension 12-18 months 70-120 days

Product development Perennial, 10-16 years Annual, 3-5 years

Source: Wu et al., 2008

Traditional uses of the juice include production of syrup, alcoholic beverages,

crystalline sugar and in some regions stalks are also consumed fresh (Saballos, 2008). The




162

leftover, built up of lignocellulose, is called bagasse. The main advantage of sweet sorghum

bagasse over other cellulosic biomass is relatively higher cellulose and low ash content (Table

2). Low ash content facilitates the use of milder and low energy intensive pretreatment, whilehigher cellulose content creates a possibility of obtaining higher quantity of glucose (Gao et

al., 2010).

Table 2: Comparative compositional analysis of sweet sorghum bagasse with other agro

residues

Agro Residue Cellulose

(%)

Hemicellulose

(%)

Lignin

(%)

Ash (%) Reference

Kinnow pulp 8.82±0.70 4.44±0.55 3.71±0.40 5.93±0.34 Oberoi et al.,

2010

Sunflower

Stalks

32.56±1.65 20.73±0.66 14.36±0.56 6.03±2.46 Díaz et al., 2011

Soybean hulls 33.49±3.18 17.15±0.04 9.88±0.01 4.71±0.07 Brijwani et al.,

2011

Wheat bran 7.57±0.17 31.19±0.30 4.06±0.09 6.53±0.01 Brijwani et al.,

2010

Sugarcane

bagasse

35.22±0.91 24.52±0.63 22.28±0.14 3.71±0.31 Rezende et al.,

2011

Sweet

sorghum

bagasse

44.6± 0.13 27.1±0.23 20.7±0.21 0.4±0.11 Kim and Day,

2011

Sorghum

bagasse

40.4±1.01 35.5±0.91 22.3±0.43 0.2±0.01 Dogaris et al.,

2009

Besides these uses, another possibility is gaining growing attention i.e. to cultivate it

as energy crop for bioethanol production (Almodares and Hadi 2009; Byrt et al., 2011;

Ratnavathi et al., 2011). Ethanol yields of 2100-8000 L/ha have been reported with oneharvest annually that significantly exceed the ethanol yields from starchy materials, such as




163

corn and wheat grains (Byrt et al., 2011; Balat and Balat, 2009). Furthermore, when

comparing the energy performance of wheat and sweet sorghum monocultures, a significantly

higher net energy gain for sweet sorghum was demonstrated in Northern Italy (Monti andVenturi, 2003). In Asia (China, India and the Philippines) and South America fermentation of

sweet sorghum juice is carried out on a small to medium scale (Saballos, 2008). Contrarily,

the conversion of sweet sorghum bagasse to ethanol is still in an experimental phase

(Ballesteros et al., 2004; Sipos et al., 2009; Li et al., 2010; Yu et al., 2010; Shen et al.,

2011; Ratnavathi et al., 2011). Economic evaluation of a sweet sorghum biorefinery for

ethanol production from bagasse has been studied under North Chinese circumstances

(Gnansounou et al., 2005), while the economy of juice processing has been investigatedmore deeply, for example, in Indian context (Prasad et al., 2007), upper Midwest of the USA

(Bennett and Anex, 2009), Zimbabwe (Woods, 2001) and Inner Mongolia (Wang and Liu,

2009).

15.5 Technical hurdles

Under moderate climate, the technological difficulty of sweet sorghum processing is

the short harvest period making the juice available only for 1-2 months annually. Due to this

reason, the juice cannot be stored because the microbes including its natural microbial flora

degrade the easily fermentable sugar content. Without any preservation, up to 12-30 % of the

fermentable sugar content can be lost in 3 days and 40-50 % in a week at room temperature

(Daeschel et al., 1981; Wu et al., 2010). Many methods have been proposed to elongate the

availability of the juice, for instance: refrigerating (Wu et al., 2010), evaporation (Hodúr et

al., 2008), ensiling the whole stalks (Bennett and Anex, 2009), proper harvest and

processing method (Lingle et al., 2012) and lowering the pH with the addition of different

acids (Hodúr et al., 2008). But these alternatives lead to elevated energy demands and/or

chemicals needs, thereby, influencing the overall process economy. The reports of Bennet

and Anex (2009) and Gnansounou et al. (2005) suggested that fermentation of sweet

sorghum juice under moderate climate could only be a complementary process, for example,

in a biorefinery concept.

15.6 Bioethanol production from sweet sorghum

Sweet sorghum is being considered as a SMART crop as it offers triple benefits of‘F’, i.e., food, fodder and fuel, without significant tradeoffs in any of these uses in the




164

production cycle. It’s relatively short vegetation cycle allows sweet sorghum to be grown in

double cropping systems based on water availability, which in turn can lead to greater agro-

biodiversity and a reduced demand for fertilizers and pesticides (Nahar, 2011). Sorghum hasthe potential to be an excellent diversified biofuel crop able to fill the needs of multiple

bioenergy conversion process across many environments with reduced energy requirements.

Besides environmental advantages, sorghum is one of the more acquiescent plant to genetic

modifications because it is highly variable in terms of genetic resources and germplasms. This

facilitates plant breeding and development of new cultivars adapted to different regions

around the globe (Ratnavathi et al., 2011). Overall scheme for production of bioethanol from

sweet sorghum involving various steps which include pretreatment, enzymatic hydrolysis andvarious fermentation methods is presented in Figure 1.

For the effective conversion of lignocellulosic material into ethanol, the following three major

steps are involved:

1. A thermo-chemical pretreatment is a pre-processing step that improves the access of

enzymes to cellulose and hemicellulose.

2. Enzymatic saccharification that breaks down cellulose and hemicellulose into simple

sugars.3. Fermentation of released sugars by specialized organisms.

It is clear from the figure that sweet sorghum bagasse (SSB) can be used as an ideal

substrate for production of cellulases which can further be used for saccharification of

pretreated biomass. Pretreatment is an important step for cellulose conversion processes, and

is one of the important factors affecting ethanol production from lignocellulosics. It is

essential for causing alterations in the structure of cellulosic biomass in order to facilitate the

enzymatic saccharification process. The goal here is to break the lignin seal and disrupt the

crystalline structure of cellulose (Agbor et al., 2011; Zhang and Shahbazi, 2011).

Once the cellulose and hemicellulose fractions are exposed after pretreatment and are

accessible to the action of cellulases and xylanases, a mixture of hexose as well as pentose

sugars are produced (Sun and Cheng, 2002). The sugars thus produced are fermented by the

fermenting micro-organisms, such as Saccharomyces cerevisiae, Pichia kudriavzevii or a

combination of hexose and pentose fermenting microbial strains to exploit the full potential ofthe sugars, thus produced. However, in absence of robust pentose fermenting micro-




165

organisms, generally the alcohol yields from the mixed sugar streams are low. Table 3

represents the different microorganisms used in the fermentation for ethanol production from

sweet sorghum along with their ethanol yields. However, the biological/biochemical route forethanol production from lignocellulosic biomass is a preferred method, because it is less

energy intensive as compared to the thermo-chemical conversion method and the

infrastructural and capital requirements are relatively lower.

Figure 1: Flow chart for the bioethanol production from sweet sorghum (Ratnavathi et al.,2011)

In one of our studies on production of ethanol from sweet sorghum bagasse, we foundthe ethanol concentration in the range of 35-40 g/l using crude cellulases and thermotolerant

Pichia kudriavzevii strain (unpublished data).

15.7 Energy ratio and environmental sustainability

The available data about the European Union (EU) model for the ethanol produced

from all components of sweet sorghum crop suggest an output/input ratio of 1.7 -7.3

(depending on the strategy chosen for the by-products exploitation), a high greenhouse gases

(GHGs) saving in accordance with the RES Directive and a low water footprint. The main




166

environmental advantage relative to the use of bioethanol in substitution of gasoline and/or

the use as bio-ETBE (ethyl tert-butyl ether) instead of fossil antiknocks is the abatement of

the transport sector contribution to the GHG emissions. For bioethanol produced using sweetsorghum, this assertion assumes an absolute validity because in this case, the saving

calculated with the methodology indicated by EU is 71% which is one of the most virtuous

values among the attainable ones. The use of sweet sorghum for bioethanol production

conciliates the production of sustainable bioethanol applying already mature technologies

with an involvement of the agricultural sector in the pathway.

Table 3: Micro-organisms used in the fermentation for ethanol production from sweetsorghum

Sr. No. Micro-organisms used in the

fermentation of:

Ethanol yield Reference

Juice

1 Saccharomyces cerevisiae CFTR 01 and SG 0.39-0.48 (g/g) Ratnavathi et al., 2010

2 Fermax yeast (Saccharomyces cerevisiae) 77.07-79.58(g/l) Kundiyana et al., 2010

3 Super start yeast (Saccharomyces cerevisiae) 73.18-76.95(g/l) Kundiyana et al., 2010

4 Saccharomyces cerevisiae TISTR 5048 0.42-0.48 (g/g) Laopaiboon etal.,2007

5 Saccharomyces Strains 29-87% (sugarconversioneffienciency)

Bulawayo et al., 1996

6 Saccharomyces cerevisiae (Nanayang) 91.61 % Liu et al., 2008

7 Saccharomyces cerevisiae 0.42-0.45 (g/g) Balint-Sipos et al.,2009

Bagasse

8 Saccharomyces cerevisiae Ethanol conc. -42.3 (g/l)

Li et al., 2010

9 Saccharomyces cerevisiae 0.147 (g/g) Ban et al., 2008

Source: Ratnavathi et al., 2011

15.8 Small-medium scale bioethanol production plant from sweet sorghum

Sweet sorghum could favour the diffusion of the bioethanol pathway in the local

agricultural sector, because the creation of small-medium plants involves the plant building




167

near the fields where the biomass is harvested (max 50 km approximately). This choice

allows an increase of sustainability of the bioethanol production, because the impacts of the

long distance transportation is reduced. The size of small-medium plants does not exceed15,000 t/y of ethanol capacity. Table 4 represents the comparative theoretical ethanol

production from sweet sorghum with other feedstocks. There are many criteria to select a

suitable bioethanol production technology, such as plant scale, investment and operation cost,

management operations, and conversion yield, which have a reflecting effect on the

production cost. Another important criterion is the energy balance that shows the efficiency of

processing energy used.

Pilot studies in India have indicated that ethanol production from sweet sorghum iscost-effective (Dayakar et al., 2004). The International Crops Research Institute for the Semi-

Arid Tropics (ICRISAT) located at Patancheru in Andhra Pradesh, India, has developed

several improved sweet sorghum lines with high stalk sugar content and a few of these lines

are being tested in pilot studies for sweet sorghum-based ethanol production in India, of

which, SSV 84, SSV 74 and NSSH 104 have been released (Reddy et al., 2008). Further,

sweet sorghum bagasse (SSB) has a higher biological value than the bagasse from sugarcane

when used as feed for cattle, as it is rich in micronutrients and minerals and is as good asstover in terms of digestibility (Panwar et al., 2000). In addition to the above characteristic

features, short duration of this crop and a yield in the range of 54-69 tons/ha renders SSB an

important and readily available substrate for ethanol production (Almodares and Hadi, 2009).

Thus, sweet sorghum represents a potential opportunity to improve the economic and

environmental sustainability of the bioethanol pathway.

Table 4: Theoretical ethanol production from multiple feedstocks (kg/ha, dry basis)

(Adapted from Kim and Day, 2011)

Component Sugarcane Energy cane Sweet sorghum

Feed stock 70,000 100,000 60,000

Juicea 0 53,600 43,140

Fiber 9,450 26,700 7,800

Sugar

Monomeric sugar from juice 0 5,253 5,091

Glucose from celluloseb 4,368 12,846 3,865

Xylose from hemicellulosec 2,695 7,221 2,402




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Ethanol

Ethanol from juiced 0 2,684 2,601

Ethanol from cellulosed 2,232 6,564 1,975

Ethanol from hemicellulosed 1,377 3,690 1,227

Total ethanol 3,609 12,938 5,804

a Juice is wet kilogramsb Glucose (kg)= Glucan (kg) × 1.11; 1.11 is a conversion factor considering water addition during hydrolysisc Xylose (kg) = Xylan (kg) × 1.14; 1.14 is a conversion factor considering water addition during hydrolysisd Ethanol (kg) = Glucose, fructose, sucrose or xylose (kg) × 0.511; 0.511 is a conversion factor for sugar to ethanolbased on stoichiometric biochemistry of yeast.

15.9 Conclusions

Sweet sorghum has distinct advantages over the other lignocellulosic biomass for

bioethanol production because of some of the inherent characteristics of this crop. Higher

biomass yield, ability of the crop to grow under drought and stress conditions, ability to get

two crops in a year makes sweet sorghum as an attractive contestant for ethanol production.

The juice extracted from sweet sorghum could be used directly for bioethanol production

through fermentation and the sweet sorghum bagasse could be converted to ethanol through a

series of steps, like, size reduction, pretreatment, hydrolysis and fermentation. Therefore,combining the ethanol yields from both the juice as well as bagasse confers a distinct

advantage to sweet sorghum over other lignocellulosic biomass, such as rice straw, cotton

stalks, bagasse, etc.

Acknowledgements

Authors thankfully acknowledge the financial assistance received from the project

funded by the Department of Biotechnology, Government of India

((PR/8488/PBD/26/68/2006) for conducting a part of the study reported in this book chapter.

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biofuel. Field Crops Res., 111: 55–64.




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CHAPTER 16

FERMENTATION OF GLUCOSE AND XYLOSE SUGARFOR THE PRODUCTION OF ETHANOL AND XYLITOL BY

THE NEWLY ISOLATED NIRE-GX1 YEAST

Shuvashish Behera , Richa Arora

, Nilesh Kumar Sharma and Sachin Kumar

Abstract Hemicellulose is the second most abundant polysaccharide after the cellulose available on the

earth in the form of lignocelluloses. The hemicellulose must utilized for the cost-efficient

production of ethanol. Xylan, the major component in the hemicellulose in plant biomass,

yields mainly xylose as pentose sugars on hydrolysis. The progress in fermentation of pentose

sugars has gone on slow pace as there are few microorganisms known, which are capable of

pentose metabolism. Therefore, this study was carried out to isolate and screen the yeasts from

soil samples collected from different dumping sites for the production of ethanol usingglucose and xylose sugar. About 16 yeast strains showed positive results in ethanol production

from glucose sugar. Four isolates designated NIRE-GX1, NIRE-GX2, NIRE-GX3, NIRE-

GX4 showed positive result in ethanol production from both glucose and xylose sugars.

Further study was carried out using the isolate NIRE-GX1 yeast, which showed more growth

and fermentation efficiency at a temperature of 40oC on both the sugars. Anaerobic batch

fermentations were carried out using the yeast strain from the individual glucose and xylose

sugar separately and further mixture of both at 40oC temperature. The strain also showedxylitol production from xylose. The strain showed maximum ethanol concentration of 7.1 ±

0.6 g l-1 with complete utilization of glucose (20 g l-1) in 24 h. However, in case of xylose

fermentation, the strain showed maximum ethanol concentration of 0.8 ± 0.08 g l-1 as well as

xylitol concentration of 0.64 ± 0.3 g l-1 in 72 h on initial xylose concentration of 20 g l-1. The

strain was capable of simultaneously using glucose and xylose in a mixture of glucose

concentration of 14 g l-1 and xylose concentration of 6 g l-1, achieving maximum ethanol and

xylitol concentration of 5.3 ± 0.5 g l-1 and 0.95 ± 0.32 g l-1, respectively in 72 h.

Key Words: Bio-ethanol; Fermentation; Glucose; Xylose.




176

16.1 Introduction

The largest potential feedstock for ethanol is lignocellulosic biomass, which includes

materials such as agricultural residues (corn stover, crop straws, sugarcane bagasse),herbaceous crops, short rotation woody crops, forestry residues, waste paper and other plant

wastes (Kim and Dale, 2005). It varies among plant species but generally consists of cellulose

(40–50%), hemicelluloses (25–30%), and lignin (10–20%) (Wiselogel et al., 1996). Complete

hydrolysis of cellulose and hemicellulose results in glucose and xylose respectively, which can

be ferment to ethanol by several microorganisms (Alvira et al., 2010; Sims et al., 2010).

Cellulose contains glucose and hemicellulose contains mainly pentoses, such as a xylose,

which are not fermentable by the natural industrial strains and can add up to 25% of theconstitution of lignocellulosic materials (Schell et al., 2004; Olofsson et al., 2008).

Yeasts that produce ethanol from D-xylose have been isolated from various locations,

including tree exudates (Ipsit et al., 2013), wood-boring insects (Suh et al., 2003), decaying

wood (Cadete et al., 2009), rotten fruit and tree bark (Rao et al., 2008). There are also several

yeast species such as Candida shehatae, Pachysolen tannophilus, Brettanomyces

naardenensis, C. tenuis, Pichia segobiensis, C. lyxosophila, C. intermedia, C. jeffriesii,

Spathaspora passalidarum, Spathaspora arborariae, C. prachuapensis, and Scheffersomyces

stipitis which has been reported as xylose fermenting yeasts (Barnett et al., 2000; Nguyen et

al., 2006; Cadete et al., 2009; Nitiyon et al., 2011). Candida and Pichia are the two naturally

occurring best ethanol producing organism from pentose. However, those yeast strains are

neither well adapted to ethanol production nor can tolerate to by-products of lignocellulosic

hydrolysates (Jeffries, 2006; Hahn-Hägerdal et al., 2007). Although Saccharomyces cerevisiae

combines several desired attributes such as high ethanol tolerance, general robustness and

operation experience which favored for industrial scale of ethanol production; the success isrestrained by the organism’s inability to naturally ferment pentose sugars (Helle et al., 2004;

Hahn-Hägerdal et al., 2007).

In a study, cocultivation of S. cerevisiae and S. stipitis strains to co-ferment glucose

and xylose was unsatisfactory, due to their difference in fermenting condition and ethanol

tolerance (Agbogbo et al., 2006). However, S. stipitis strain prefers to ferment glucose rather

than xylose having lower ethanol tolerance than S. cerevisiae strain (Watanabe et al., 2007).

Further, in order to assimilate xylose, some strains produce xylitol as the intermediate product

with the production of ethanol. Despite the existence of these microorganisms, it is still




177

challenging to reach high yields of ethanol from pentose sugars on a large scale (Hahn-

Hagerdal and Pamment, 2004) because no microorganisms that robustly convert pentose

sugars into ethanol at high yields while withstanding fermentation inhibitors have beenidentified (Chandel et al., 2011). Therefore, this study was carried out for a new search of

yeasts from soil samples collected from different dumping sites for the production of ethanol

using glucose and xylose sugar. Finally fermentation of both glucose and xylose sugars were

carried out using NIRE-GX1 yeast for the production of ethanol.


16.2.1 Sample collection

Samples for isolation of ethanol producing yeasts were obtained from the dumpyard at

Jalandhar, Punjab and from Wahid Sandhar Sugars Ltd., Punjab. Exploration for collecting

potential samples was carried out by walking-through within 60-90 min for each site. Soil

samples were put into plastic or polythene bags and finally sample details were recorded.

16.2.2 Isolation of yeast

To isolate yeast, 1g of each soil samples was suspended in 10 ml of sterile water by

vortexing for 2 minute on maximum speed, followed by a 10x serial dilution. About 0.1 ml ofeach dilution in the series was spread onto the surface of yeast extract-peptone-dextrose

(YPD) phytagel (yeast extract, 1%; peptone, 2%; dextrose, 2%; phytagel, 1.5%; ampicillin, 50

mg/ml; water, 1000ml; pH, 5.5) plates and incubated at 40o C for 24 hour. For isolation of

xylose utilizing yeasts, xylose sugar (2%) was added in replacement of dextrose to the above

medium. Various colonies were selected based on their morphology, size and color appear on

the phytagel plates. The selected colonies were then subcultured on to separate phytagel plates

to ensure their purity.

16.2.3 Microorganism and culture condition

The yeast culture was maintained on yeast extract-peptone (YP) medium (g/l): yeast

extract, 1%; peptone, 2%; phytagel, 1.5%; ampicillin, 50 mg/ml; water, 1000ml; pH, 5.5 and

sugar (glucose and xylose) was added according to the use by the isolates. The culture was

stored at 4±0.5oC for further use.

16.2.4

Preparation of inoculum




178

The inoculum was prepared in 100 ml YP growth medium (as mentioned above but

without phytagel) containing glucose and xylose as the sugar source, taken in sterilized (at

121

o

C for 20 min) 500 mL erlenmeyer flask and cotton plugged. The flask was inoculatedwith a loopful of the yeast culture and incubated for 24 h at 40o C at 120 rpm in an orbital

shaker incubator (Remi Pvt, Ltd, Bombay, India). The cells grown after 24 h were acts as the

inoculum for the fermentation medium.

16.2.5 Fermentation medium

Fermentation medium was prepared using salt medium (SM) in gl-1: di-sodium

hydrogen ortho phosphate, 0.15; potassium di-hydrogen ortho phosphate, 0.15; ammonium

sulphate, 2.0; yeast extract, 1.0; glucose, 10.0 at pH 5.5. The cells from the YP medium was

centrifuged at 7500 rpm for 10 min and added in the fermentation medium. The cells were

grown in 250-ml capped flasks in a shaker at 40oC and 120 rpm for 72 h.

16.2.6 Analytical methods

At 24 h interval, fermented broths (in triplicate flasks) were removed and the contents

were analyzed for total sugar, ethanol and xylitol production. Glucose was analyzed by high-

performance liquid chromatography (HPLC) using Hi-Plex H column at 57oC with 1mM

H2SO4 as the mobile carrier at a flow rate 0.7 ml min-1 and detected by refractive index

detector. The pH was measured by a pH meter (Systronics, Ahmadabad, India) using glass

electrode. The fermentation kinetics was studied as per the formulae described below (Bailey

and Ollis, 1986)

16.3 Results and discussion

About 16 yeast strains showed positive results in ethanol production from glucosesugar. Four isolates designated NIRE-GX1, NIRE-GX2, NIRE-GX3, NIRE-GX4 showed

positive result in ethanol production from both glucose and xylose sugars. The isolated strains

were carefully identified by morphological characteristics including color of the colony and

growth pattern studies with the help of microscope. Strain selection was based on anaerobic

growth on xylose as well as on high xylose uptake rate. In fermentation studies on defined

media the resulting NIRE-GX1 yeast was shown to possess the ability to grow on xylose

under anaerobic conditions and to ferment both xylose and glucose to ethanol in good yieldand productivity.




179

Therefore further study was carried out using the isolate NIRE-GX1 yeast, which

showed more growth and fermentation efficiency at a temperature of 40oC on both the sugars.

Anaerobic batch fermentations were carried out using the yeast strain from the individualglucose and xylose sugar separately and further mixture of both at 40oC temperature. The

strain also showed xylitol production from xylose. The strain showed maximum ethanol

concentration of 7.1 ± 0.6 g l-1 with complete utilization of glucose (20 g l-1) in 24 h.

However, in case of xylose fermentation, the strain showed maximum ethanol concentration

of 0.8 ± 0.08 g l-1 as well as xylitol concentration of 0.64 ± 0.3 g l-1 in 72 h on initial xylose

concentration of 20 g l-1. The strain was capable of simultaneously using glucose and xylose

in a mixture of glucose concentration of 14 g l

-1

and xylose concentration of 6 g l

-1

, achievingmaximum ethanol and xylitol concentration of 5.3 ± 0.5 g l-1 and 0.95 ± 0.32 g l-1, respectively

in 72 h (Table 1). Nigam et. al. (1985) found maximum ethanol concentration of 8.8, 10.9 and

9.8 g l-1 with initial D-xylose sugar concentration of 40, 60 and 80 g l-1.

Table 1. Ethanol production from glucose and xylose sugar using yeast isolate NIRE-GX1

Glucose Fermentation Xylose Fermentation Fermentation of mixture of sugar

Time (h) pH Sugar Consume

(g l-1)

Ethanol (g

l-1)

pH Sugar

Consumed

(g l-1

)

Ethanol

(g l-1)

Xylitol

(g l-1)

pH Sugar Consumed

(g l-1)

Ethanol (g

l-1)

Xylitol

(g l-1)

24 2.93 19.98±0.04 7.3±0.06 3.48 7.26±0.2 0.12±0.03 0.47±0.1 3.08 16.75±1.4 4.43±0.1 0.79±0.05

48 - - - 3.39 8.38±1.2 0.2±0.04 0.6±0.05 3.06 17.07±0.2 4.97±0.3 0.85±0.2

72 - - - 3.19 9.3±0.1 0.8±0.06 0.64±0.3 3.03 17.12±0.8 5.3±0.5 0.95±0.32

The growth and fermentation kinetics of free cells in presence of individual and mixed

of sugars were also studied (Table 2). The ethanol concentration (P) obtained with glucose

fermentation with NIRE-GX1 yeast (7.3±0.06 g l-1) was 89.04 % more than that of xylose

fermentation (0.8±0.06 l-1), where as the volumetric substrate uptake (Qs) was found to be

84.34 % more in case of glucose fermentation (0.83 g l-1 h-1) over xylose fermentation (0.13 g

l-1 h-1). The ethanol yield (Yp/s= 0.37 g g-1) and volumetric product productivity (Qp=0.3 g l-1

h-1) obtained with glucose fermentation was found to be 75.68 and 96.7 %, respectively higher

than that of Yp/s (0.09 g g-1) and Qp (0.01 g l-1 h-1) of xylose fermentation. However, the final

biomass (X) concentration in case of xylose fermentation (1.5 ± 0.2 g l-1) was considerably

lower than glucose fermentation (1.2 ± 0.2 g l-1), which is useful during product separation

and purification process (Diderich et al. 1999). Cadete1 et al. (2012) reported that the newly

isolated S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and




180

productivities (0.62 g l-1 h-1 to 0.75 g l-1 h-1). Also another strain Candida amazonensis

exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g g-1

to 0.59 g g

-1

), with concentrations up to 25.2 g l

-1

.Table 2. Growth & Fermentation Kinetics of Ethanol Production

Glucosefermentation

Xylosefermentation

Fermentation ofmixed sugar

Final ethanol (P, g l-1) 7.3±0.06 0.8±0.06 5.3±0.5

Final biomass concentration ( X , g

l-1)

1.9±0.2 1.5±1.2 1.8±0.7

Ethanol yield (Yp/s, g g-1) 0.37 0.09 0.3Volumetric substrate uptake (Qs, g

l-1 h-1)

0.83 0.13 0.24

Volumetric product productivity

(Qp, g l-1 h-1)

0.3 0.01 0.07

16.4 Conclusion

Efficient ethanol production from xylose is crucial for Bioethanol production from

lignocellulosic biomass. We attempted to enhance the ethanol productivity of the xylose-

fermenting yeast NIRE-GX1. The strain showed utilization both hexose and pentose sugar for

the production of bioethanol with a low amount of xylitol. Further studies like pentose sugar

uptake, presence of sugar transporter and inhibitor tolerance are required to increase the

uptake rate of pentose sugar.

Acknowledgment

We greatly acknowledge the Ministry of New and Renewable Energy, New Delhi,

Govt. of India for providing funds to carry out research work.

References

1. Agbogbo F.K., Coward-Kelly G., Torry-Smith M. and Wenger K.S. (2006)

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2. Alvira P., Tomas-Pejo E., Ballesteros M. and Negro M.J. (2010) Pretreatment

technologies for an efficient bioethanol production process based on enzymatic

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4. Barnett J.A., Payne R.W. and Yarrow D. (2000) Yeast Characteristics and

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5. Cadete R.M., Santos R.O., Melo M.A., Mouro A. and Gonc¸alves D.L. (2009)

Spathaspora arborariae sp. nov., a D-xylose-fermenting yeast species isolated from

rotting wood in Brazil. FEMS Yeast Res., 9:1338–1342.6. Cadete1 R.M., Melo1 M.A., Dussan K.J., Rodrigues R.C.L.B., Silva S.S., Zilli J.E.,

Vital M.J.S., Gomes F.C.O., Lachance M.A. and Rosa1 C.A. (2012) Diversity and

physiological characterization of D-xylose-fermenting yeasts isolated from the

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7. Diderich J.A., Schepper M., Van H.P., Luttick M.A., Van D.J.P. and Pronk J.T. (1999)

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Grauslund M.F. (2007) Towards industrial pentose-fermenting yeast strains. Appl.

Microbiol. Biotechnol., 74:937–953.

9. Helle S.S., Murray A., Lam J., Cameron D.R. and Duff S.J.B. (2004) Xylose

fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite

liquor. Bioresour. Technol., 92:163–171.

10. Ipsit H., Bidisha S., Anindita R. and Subhra K.M. (2012) Ethanol production fromxylose and enzymatic hydrolysate of hemicelluloses by a newly isolated yeast strain. J.

Microbiol. Biotechnol. Res., 08:54-58.

11. Jeffries T.W. (2006) Engineering yeast for xylose metabolism. Curr. Opin.

Biotechnol., 17: 320–326.

12. Kim S. and Dale B.E. (2005) Life cycle assessment of various cropping systems

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13. Nguyen N.H., Suh S.O., Marshall C.J. and Blackwell M. (2006) Morphological and

ecological similarities: wood boring beetles associated with novel xylose-fermenting

yeasts,Spathaspora passalidarum

gen. sp. nov. andCandida jeffriesii

sp. nov. Mycol.Res., 110:1232–1241.

14. Nigam J.N., Margaritis W.A. and Lachance M.A. (1985) Aerobic fermentation of D-

xylose to ethanol by Clavispora sp. Appl. Environ. Microbiol., 50:763-766.

15. Nitiyon S., Boonmak C., Am-In S., Jindamorakot S., Kawasaki H., Yongmanitchai W.

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22. Watanabe S., Saleh A.A., Pack S.P., Annaluru N., Kodaki T. and Makino K. (2007)

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183

CHAPTER 17

COMPARATIVE BIOETHANOL PRODUCTION BY S. cerevisiae AND Z. mobilis FROM SACCHARIFIED SWEET

POTATO ROOT FLOUR ( Ipomoea batata L) USING

IMMOBILIZED α- AMYLASES AND GLUCOAMYLASE

Preeti Krishna Dasha, Sonali Mohapatraa , Manas Ranjan Swainb, Hrudaya Nath Thatoi

Abstract

Sweet potato ( Ipomoea batatas L) represents an important biomass resource for fuel alcohol

production, because of its chemical composition which has a dry matter content of 30-40%

and high density of starch (70%), compared to other forms of biomass and thus promises as an

alternative bioresource for the production of ethanol through fermentation. The starch present

in sweet potato can be hydrolysed to simple sugars and can then be used by microorganisms in

fermentation process. Fermentation, by using immobilized enzyme is an advanced technology

which has several benefits as compared to free cell culture which include enhanced ethanol

yield, easy to separate, reduced percentage of contamination, better operational stability and

viability of enzyme actions for several cycles of fermentation. In this experiment batch

fermentation is carried out in flask condition to compare the bioethanol production by

Saccharomyces cerevisiae (S. cerevisiae) and Zymomonas mobilis ( Z. mobilis) from

saccharified sweet potato root flour (SPRF) using immobilized α-amylase and glucoamylase.

The ethanol yields were 485.5 g/ kg and 448.7 g/ kg of SPRF for S. cerevisiae and Z. mobilis

respectively after an incubation period of 96 hours maintained at pH 4.5 and 30oC

temperature. The study demonstrated that S. cerevisiae can produce 10.6% higher bioethanol

in comparison to Z. mobilis. The highest ethanol concentration was obtained after 96 hour of

fermentation. Thus, it was evident from the present study that Z.mobilis does not appear to be

an efficient microbial source for commercial bioethanol production, attributed to its lower

tolerance to temperature, ethanol and utilization of substrate range compared to S. cerevisiae.

Thus the use of S. cerevisiae has great scope for bioethanol production under enzyme

immobilization condition having potential for commercial application.




184

Keywords: Sweet potato, Saccharification, Immobilization, S. cerevisiae, Z. mobilis.

17.1 Introduction

The uses of ethanol as an alternative motor fuel has been steadily increasing around

the world for a number of reasons such as it decreases dependence on foreign oil, reduces

trade deficits, creates jobs in rural areas, reduces air pollution by replaceing aromatic and

sulfur-content in gasoline, it also reduce nitrogen oxide (NOx) emission to improve air quality

which can reduce urban smog and reduces global climate change carbon dioxide buildup

(Wheals et al., 1999). The high oxygen content in bioethanol could reduce the generation of

known hazardous volatile organic compounds (VOCs) and carbon monoxide in vehicle

exhaust (Wyman, 1996; Yoon et al., 2009).

Establishment of ethanol industry requires sufficient and cheap feedstock in order to

reduce the costs of production that has been recognized as a critical point (Cardona and

Sanchez, 2007). However, the transformation of some conventional raw materials (like corn,

wheat and rice etc) is not feasible, due to food security issues (Lin and Tanaka, 2006). With

high concentrations of starch, tuber crops are considered one of the most important sources for

bioethanol. The tuber crops viz. potato, sweet potato, cassava are most promising feed stock

due to their economic viability and availability. Sweet potato ( Ipomoea batata L.) is one of

the most important starch producing crops grown worldwide. The dry matter content in sweet

potato ranges from 21 to 30% of which about 80% starch (Zhang and Oates, 1999). Starch is a

complex carbohydrate which needs conversion into simpler sugars before being converted into

ethanol. Starchy materials required to be converted to glucose monomers by saccharification

process which can be achieved by enzyme treatments (by amylases and glucoamylases). After

conversion to simpler monomers (glucose) it is fermented to produce ethanol with help of

ethanol producing microorganisms (Lin and Tanaka, 2006).

Saccharomyces cerevisiae and Zymomonas mobilis are usually the first choice for

industrial ethanol production, because of their good fermentative capacity, high tolerance to

ethanol and the capacity to grow rapidly under the anaerobic conditions that are

characteristically established in large scale fermentation vessels (Agbogo and Kelly, 2008).

The concept of immobilization provides a promising strategy for the use of enzymes in a

bioreactor for easy scale up an industrial biomass conversion. Day by day number ofapplications of immobilized industrial important enzymes are increasing steadily.




185

Immobilized enzymes could be employed in the bioethanol production with the aim of

reducing the production cost by reusing the enzymes.

Considering the above facts, the present study is carried out to compare the bioethanolproduction by S. cerevisiae and Z. mobilis from saccharified sweet potato root flour (SPRF)

using immobilized α -amylase and glucoamylase.


17.2.1 Sweet potato

Freshly harvested and sweet potato (SP) roots (var. ST-13) (starch, 178 g/kg; total

sugar, 25 g/kg dry weight basis) were collected from the experimental farm of RegionalCentre of Central Tuber Crops Research Institute (CTCRI), Bhubaneswar during the month of

November, 2011 and used within 24 h after harvest. The fresh SP roots were chipped

manually, dried at 60 ºC to reduce the moisture level to ~ 12 % and finally grinded to flour by

dry milling and consist particles with diameter 0.2- 1.7 mm (95 % or more particles pass

through a 1.70 mm sieve).

17.2.2 Microorganisms and culture conditions

The Z. mobilis MTTC 92 strain was maintained on ZS ( Z. mobilis specific) medium

[(g/L) yeast extract ,10; glucose, 20; MgCl2, 10; NH4SO4 ,10 ; KH2PO4, 10; agar, 15 and pH

6-6.5] and S. cerevisiae (CET) was maintained on malt-extract-yeast extract-glucose-peptone

(MYGP) medium [(g/L): malt extract,3; yeast extract,5; peptone, 5; glucose, 20; agar, 15; pH

5.5 ]. Both the cultures were stored at 40C for further use. A loop of microbial culture ( Z.

mobilis and S. cerevisiae) was inoculated to a 250 ml Erlenmeyer flask which contained 100

ml sterile respective medium (as mentioned above) and were incubated at 30 0C in a rotary

shaker (120 rpm) for 24 h. The liquid culture had initial cell density (3×109 colony forming

unit (CFU)/ml).

17.2.3 Enzymatic saccharification of SPRF

SPRF (Sweet Potato Root Flour) (10%) slurry was prepared in 250 ml Erlenmeyer

flasks with a working volume of 100 ml by adding tap water in a ratio of 1:10 for experiment.

In first step the slurry was dextrinized by addition of 32 µl of immobilized Palkolase- ®HT at

pH 5.5 and 90°C for 1hour and then slurry was cooled down to room temperature. In second




186

step, immobilized Palkodex® (329.7 µl) was added to the dextrinized slurry at pH 4.5 and

incubated for 24 h at 60 °C for saccharification.

17.2.4 Immobilization of Enzymes

The immobilized beads of α amylase were prepared with 0.3% sodium alginate, 0.5%

glutaraldehyde, 0.3M CaCl2 solution and 0.2% starch concentration where as for glucoamylase

0.3% sodium alginate with 0.5% glutaraldehyde in 3M CaCl2 solution with starch

concentration of 0.2% were required.

17.2.5 Ethanol fermentation from saccharified SPRF

Ethanol fermentation was conducted by S. cerevisiae and Z. mobilis under anerobiccondition in an Erlenmeyer flask sealed with rubber stopper equipped with opening for CO2

venting. The immobilized enzyme hydrolysed SPRF (100ml) was inoculated with freshly

harvested Z. mobilis and S. cerevisiae starter cultures at [10 % v/v (3×109 CFU)/ml]

aseptically. The fermentation medium containing flasks (n=3) were incubated in an incubator-

cum shaker at 30±20C for 120 h with a constant shaking (100 rpm). The fermented broth was

distilled to recover ethanol using alcohol distillation apparatus (Borosil Glass Works Ltd.,

Mumbai, India).

17.2.6 Study of Fermentation parameter

(1) Incubation period: The enzyme enzyme hydrolyzed sweet potato slurry was inoculated

with 10 % (v/v) starter culture with pH 4.5 and incubated for 24-120h.

(2) Initial medium pH: The fermentation medium (saccharified 10% SPRF slurry) with pH

(3- 5.5) was inoculated with 10 % (v/v) starter culture and incubated for 96 h at 300C.

17.2.7 Analytical techniques

At appropriate time intervals, fermentation medium (in triplicates) were removed and

contents were analysed for sugar and ethanol. The ethanol concentration was determined

based on the density of the alcohol distillates obtained from the fermentation broth. Ethanol

concentration of the fermentation liquid was determined by measuring the specific gravity of

the distillate according to the procedure described by Amerine and Ough (1984). The pH was

measured using a pH meter (Systronics, Ahmadabad, India) fitted with a glass electrode. The

total sugars were assayed by Dinitrosalicylic acid and Anthrone’s method (1999).Fermentation kinetics was calculated using the formulae by Bailey and Ollis (1986).




187

17.2.8 Population count

Yeast and bacterial populations in the fermented mash were calculated by serially

diluting the substrate (fermented SPRF slurry) in sterile distilled water and plating in suitabledilution (104-105) on Petri plates containing ZSM and MYGP medium for Z. mobilis

(bacteria) and S. cerevisiae (yeast) respectively.

17.2.9 Calculations

The maximum theoretical ethanol yield from sugar was calculated according to the

stoichiometric relation represented by Eq. (1), i.e. 100 g of hexose produce 51.1 g of ethanol

and 48.9 g of CO2. Ethanol yields over total initial sugar (Y1) and average ethanol productivity

rate (Y2) were calculated according to Eqs. (2) and (3).

C2H12O6 →2CH3CH2OH + 2CO2 (1)

Y1 = ethanol produced in fermentation/ ethanol produced in theoretical x 100% (2)

Y2 = Final ethanol concentration/fermentation time (3)


17.3.1 Effect of incubation period

In this study, the immobilized enzyme liquefied SPRF hydrolysate was used for

subsequent fermentation (submerged) by yeast, S. cerevisiae and bacteria, Z. mobilis. A 10%

inoculums was added and cultivated at 30 ºC for both the organisms and the comparison of

sugar utilization and ethanol production profile of the two organisms are given in (Figure1 a

and b) respectively. After 96 h S. cerevisiae and Z. mobilis produced 487.1 and 445.3g/kg of

ethanol respectively. Both the organisms S. cerevisiae and Z. mobilis utilises 88.4 %, 84.7 %

total sugar and 94.3%, 86.7% of reducing sugar respectively. Z. mobilis

utilises less sugarcontent and produce less amount of ethanol with respect to Saccharomyces cerevisiae and

produces 10.4% less bioethanol with comparison to S. cerevisiae. The ethanol productivity

(1.95g/L/h, 1.77g/L/h), ethanol yield (0.614g/g, 0.573g/g) and final ethanol efficiency (98.7%,

94.4%) were obtained by S. cerevisiae and Z. mobilis respectively at optimum incubation

period (96 h).

In case of S. cerevisiae the concentration of total sugar decreased rapidly within 24 h

(73.08% over initial content) of fermentation with concomitant production of ethanol (231.3g/kg). Thereafter the decline of total sugar was gradual in between 24 and 96 h and then




188

decreases. After the end of 96 h incubation period, the residual total and reducing sugar

concentration was 100 g/kg and 47 g/kg respectively in culture broth. Similar ethanol

production and sugar utilization were observed for bacterium ( Z. mobilis

) but after 24 h but

the bioethanol production is 10.5% low in comparison to S. cerevisiae. The result shows that

Z. mobilis was significantly less efficient than S. cerevisiae (7.3%) [Fishers LSD test, P<0.05,

LSD between treatments is 0.65] in converting sugar into ethanol at least in the case of SPRF

hydrolysate.

A similar finding on bioethanol production from mahula flower usingS. cerevisiae and

Z. mobilis was reported by Behera et al., (2007). The S. cerevisiae strain showed 10.5% more

final ethanol production in comparison to Z. mobilis. Ethanol productivity, volumetric productproductivity and sugar to ethanol conversion rate (%) obtained by S. cerevisiae were found to

be 68.5%, 3.08%, 134% higher than Z. mobilis respectively after 96h of fermentation. In

another study for bioethnaol production from the molasses reported higher bioethanol

production by S. cerevisiae than Z. mobilis at sugar concentration above 15% (v/v) (Bansal

and Singh 2003).

17.3.2 Effect of pH

The effect of initial pH of SPRF hydrolysate on bioethanol fermentation is shown in

(Figure 2 a and b). The ethanol productivity increased up to 4.5 and decreased marginally

above 4.5. The maximum of ethanol concentration 477.5 and 449.3 g/kg were for S. cerevisiae

and Z. mobilis respectively. Ethanol productivity (2.001g), ethanol yield (0.632g/g) and final

ethanol efficiency (97.3%) were obtained in culture grown at pH 4.5. In the earlier study,

Roukas (1994) has studied the effect of pH on ethanol production from carob pod by S.

cerevisiae and found that the maximum ethanol concentration, ethanol yield and fermentation

efficiency were obtained at pH 4.5. Swain et al., (2006) previously reported optimum pH 5.5

for bioethanol production from mahula flowers using free cells of S. cerevisiae in submerged

fermentation. Yeast has a pH optimum between 4.0 and 6.0 although it can grow in a long pH

range 2.5 to 8.5 (Narendranath and Power, 2005).

In contrast to S. cerevisiae, a similar pattern of bioethanol and sugar utilization was

observed from Z. mobilis (Figure 2 b).The maximum ethanol concentration 429.3 g/kg was

obtained at medium. Ethanol productivity (1.788g), ethanol yield (0.591g/g) and final ethanolefficiency (93.7%) were obtained in culture grown at pH 4.5. Maiti et al., (23) reported




189

slightly higher pH 5.13 for optimum bioethanol production from sugar cane molasses. But in

another study, Z. mobilis produced optimum ethanol from mahula flower at pH 6.5 (Swain et

al., 2007). Both the organismS. cerevisiae

and Z. mobilis

utilised 87.1%, 83.1% total sugarand 93.5, 86.4 % reducing sugars respectively at optimum pH 4.5. However, the optimum

ethanol produced by S. cerevisiae is 10.8% higher [Fishers LSD test, P<0.05, LSD between

treatments is 0.30] than the Z. mobilis.

Figure (1): A comparative study for role of incubation period on ethanol concentration and

sugar consumption (total and reducing sugar) by S. cerevisiae [a] and Z. mobilis [b]

17.4 Importance of enzyme Immobilization

In this experiment the advantage of using enzyme immobilized beads was that the used

beads survived and were active on the support used for immobilization for four cycles offermentation, which could save considerable time and energy. In this study, the enzyme

A

B




190

immobilized beads were successfully recycled for four more times without much affecting the

final ethanol concentration. The cells not only survived but were also active physiologically

yielding ethanol 485±0.02, 483±0.08, 478±0.12 and 468±0.12 and 448.6±0.04, 444±0.02,436.2±0.06 and 428.2±0.06 g/kg of SPRF in 1st, 2nd, 3rd and 4th cycles of 96 h fermentation,

from S. cerevisiae and Z. mobilis, respectively. Both of this experiment bioethanol production

remains constant. The ethanol production is 10.6% higher in S. cerevisiae than that of Z.

mobilis.

Figure (2): A comparative study for effect of fermentation medium pH on ethanol

concentration and sugar consumption (total and reducing sugar) by S. cerevisiae [a] and Z.

mobilis [b].

B

A




191

17.5 Conclusion

The present experiment revealed that, enzyme immobilization is an advanced

technique for bioethanol production. In the present study, S. cerevisiae was found to be apromising microbial source for bioethanol production from immobilized enzyme saccharified

sweet potato flour as substrate in comparison to Z. mobilis. The study demonstrated that S.

cerevisiae can be able to produce 10.6% higher bioethanol than Z. mobilis, the peak ethanol

concentration was obtained after 96 hour of fermentation. Further, it is evident from the

present study Zymomonas does not appear to be an efficient microbial source for commercial

bioethanol production, attributed to its lower tolerance to temperature, ethanol and utilization

of substrate range compared to S. cerevisiae.

Acknowledgements

The Department of Science and Technology, Govt. of Odisha is gratefully

acknowledged for the financial support to carry out this work in the form of the research

project (Project no.3897/ST, dated 07/08/10). The authors are thankful to the M/s Maps

Enzymes Ltd, Ahamadabad, India for providing the enzymes. We thank the Principal, College

of Engineering and Technology for providing necessary facilities for this research work.

References

1. Agbogbo F.K. and Coward-Kelly G. (2008) Cellulosic ethanol production using the

naturally xylose-fermenting yeast, Pichia stipitis. Biotech Lett. 30: 1515–1524.

2. Amerine M.A. and Ough C.S. (1984) Wine and must analysis. New York, USA, Wiley.

3. Bailey J.E. and Ollis D.F. (1986) Biochemical Engineering Fundamentals. McGraw-Hill, New York.

4. Bansal R. and Singh R.S. (2003) A comparative study on ethanol production from

molasses using Saccharomyces cerevisiae and Zymomonas mobilis. Ind J Microbiol.,

43:261-264.

5. Behera, S., Kar, S., Mohanty, R.C. and Ray, R.C. (2009) Comparative study of

Bioethanol production from mahula ( Madhuca latifolia L.) flowers by Saccharomyces

cerevisiae cells immobilized in agar agar and Ca-alginate matrices. Appl. Energy.,87:96-100.




192

6. Busche, R.M., Scott, C.D. and Davison, B.H. (1992) Ethanol, the ultimate feed stock A

technoeconomic evaluation of ethanol manufacture in fluidized bed bioreactors

operating with immobilized cells. Appl Biochem Biotechnol., 34/35: 395-415.7. Cardona C.A. and Sanchez O. (2007).Fuel ethanol production: process design trends

and integration opportunities. Bioresour technol., 98:2415-2427.

8. Davis L., Rogers P., Pearce J. and Peiris P. (2006) Evaluation of Zymomonas-based

ethanol production from a hydrolyse waste starch stream. Biomass Bioenerg.. 30: 809–

814.

9. Hoover R. (2001) Composition, molecular structure, and physicochemical properties of

tuber and root starches: a review. Carbohydr Polym., 45:253–267.10. Lin Y. and Tanaka S. (2006).Ethanol fermentation from biomass resources: current state

and prospects. Appl Microbial Biotechnol., 69 :627-642 .

11. Mahadevan A. and Sridhar R. (1999) Methods in Physiological plant pathology. 5th ed,

Sivakami Publication, Madras: India.

12. Maiti, B., Rathore, A., Srivastava, S., Shekhawat, M. and Srivastava, P. (2011)

Optimization of process parameters for ethanol production from sugar cane molasses by

Zymomonas mobilis using response surface methodology and genetic algorithm. ApplMicrobiol Biotechnol., 90(1):385-395.

13. Narendranath N.V. and Power R. (2005) Relationship between pH and medium

dissolved solids in terms of growth and metabolism of lactobacilli and Saccharomyces

cerevisiae during ethanol production. Appl Environ Microbiol. 71:2239-2243.

14. O’Connor C. B. (1994) Rural Dairy Technology. ILRI training manual No.1.

International Livestock Center for Africa. Addis Ababa, Ethiopia 133 pp.

15. Roukas T. (1994) Solid- state fermentation of carob pods for ethanol production. ApplMicrobiol Biotechnol., 41: 296-301.

16. Shigechi H., Koh J., Fujita Y., Matsumoto T., Bito Y., Ueda M., Satoh E. and Kondo A.

(2004) Direct production of ethanol from raw corn starch via fermentation by use of a

novel surface engineered yeast strain co-displaying glucoamylase and alpha amylase.

Appl Environ Microbiol., 70:5037-5040.

17. Swain, M.R., Kar, S., Sahoo, A.K. and Ray, R.C. (2007) Ethanol fermentation of

mahula ( Madhuca latifolia L.) flowers using free and immobilized yeast Saccharomycescerevisiae.. Microbiol Res., 162:93-98 .




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18. Szambelan K., Nowak J. and Czarnecki Z. (2004).Use of Zymomonas mobilis and

Saccharomyces cerevisiae mixed with Kluyveromyces fragilis for improved ethanol

production from Jerusalem artichoke tubers. Biotechnol Lett., 26: 845-848.19. Watanabe S, Saleh A, Pack SP, Annaluru N, Kodaki T, Makino K. 2007. Ethanol

production from xylose by recombinant Saccharomyces cerevisiae expressing protein

engineered NADP+- dependent xylitol dehydrogenase. J Biotechnol. 130 :316–319

20. Wheals A.E., Basso L.C., Alves D.M.G. and Amorim H.V. (1999) Fuel ethanol after

25 years. Trends Biotechnol., 17: 482–487.

21. Wyman C.E., Dale B.E., Elander R.T., Holtzapple M., Ladisch M.R. and Lee Y.Y.

(2005) Co ordinated development of leading biomass pretreatment technologies.Bioresource Technol., 96: 1959–1966.

22. Yoon J. J., Kim Y. J., Kim, S. H., Ryu H. J., Choi J. Y., Kim G. S. and Shin M. K.

(2010). Production of polysaccharides and corresponding sugars from red seaweed. Adv

Mater Res., 93–94: 463–466.

23. Zhang T. and Oates C.G. (1999) Relationship between α-amylase degradation and

physicochemical properties of sweet potato starches. Food Chem., 65:157-163.




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CHAPTER 18

GENETIC MODIFICATION FOR SIMULTANEOUSUTILIZATION OF GLUCOSE & XYLOSE BY YEAST

Nilesh Kumar Sharma, Shuvashish Behera, Sachin Kumar

Abstract

In recent era, escalating utilization of ethanol as a blend in gasoline has increased the demand

in the world market due to its valuable contribution to the energy security and environment

protection. In recent decades, the emphasis has been given on the agro-based bioethanol

production due to shortage of conventional raw materials such as molasses. The major sugars

present in any lignocellulosic biomass are glucose and xylose and other sugars such as

mannose, galactose, arabinose are found at lower level. Xylose is the second major

fermentable sugar in the lignocellulosic hydrolysates after saccharification. For economical

production of agro-based ethanol, it is recommend to utilize both pentose and hexose sugars,

present in the lignocelluloses, simultaneously. The conventional ethanologens such as

Saccharomyces cerevisiae are not able to utilize both glucose and xylose for ethanol

production. Some pentose utilizing ethanologens such as Scheffersomyces stipitis, Candida

shehatae and Pachysolen tannophylus have also been used for ethanol fermentation. But, they

could not be commercialized due to inefficient in ethanol yield, glucose utilization, etc.

However, there are some reported yeasts such as Kluyveromyces sp., which are able to utilize

both pentose and hexose sugars simultaneously. But, they utilize pentose sugars for higher

yield of xylitol as compared to ethanol. We have isolated a yeast of same characteristics in our

laboratory. As the yeast has capability of utilizing both pentose and hexose sugars

simultaneously, the ethanol yield on xylose can be increased by using metabolic tools.

Keeping in mind, the proposed study is focused on the methods to identify the metabolic

inefficiency and probable solution including metabolic pathway identification, metabolic flux

balance, toxicity tolerance and solution to feedback inhibition.

Key words: Metabolic engineering, Pathway modelling, Xylose metabolism, metabolic fluxbalance.




195

18.1 Introduction

Rising concerns over the cost of petroleum and the prospect of global warming are

driving the development of technologies for the production of alternative fuels such as ethanol(Jeffries and Jin, 2004; Ragauskas et al., 2006; Behera et al., 2012). Currently, bioethanol is

produced mainly from sugar-containing or starchy biomass such as sugarcane and corn as the

raw material. However, lignocellulosic biomass, such as woods and agricultural residues, is

also an attractive feedstock for bioethanol production because of its large amount of

potentially fermentable sugars (Matsushika et al., 2009).

The main component of lignocellulosic biomasses is glucose, a hexose sugar derived

from cellulose and hemicellulose. Xylose is the second most abundant carbohydrate in nature

and its commercial fermentation to ethanol could provide an alternative fuel source for the

future (Cadete et al., 2012). Fermentation of the available sugars in cellulosic biomass

presents a unique challenge because of the presence of other sugars such as xylose and

arabinose (pentose sugars). The processes that efficiently utilize both the sugar component of

lignocellulose could significantly decrease the cost of bioethanol production (Bhatia et al.,

2012). The larger sizes, thicker cell walls, better growth at low pH, less stringent nutritional

requirements, and greater resistance to contamination give yeasts advantages over bacteria for

commercial fermentations. One of the most effective ethanol-producing yeast for hexose

sugars including glucose, mannose, and galactose is Saccharomyces cerevisiae, a yeast with

high ethanol productivity, high tolerance to ethanol, and tolerance to inhibitory compounds

present in the hydrolysate of lignocellulosic biomass (Kumar et al., 2009; Goshim et al.,

2013). However, it does not naturally use xylose as a substrate, however, and must be

engineered to both transport and ferment xylose. In contrast to the efficient glucose

fermentation in yeast, xylose fermentation has been challenging because only a few ethanol-

producing microorganisms can readily ferment xylose, though many microorganisms.

Developing an efficient organism to ferment the pentose sugars has been pursued for the past

few decades. Microbes such as yeasts and bacteria are essential for the fermentation of xylose

(Dien et al., 2003; Jeffries and Jin, 2004) (Table 1). Organisms to ferment the pentose sugars

in lignocellulosic biomass can be divided into two subgroups, namely naturally occurring and

genetically engineered microorganisms. The naturally-occurring microorganisms include

Pichia stipitis, Candida shehatae, and Pachysolen tannophilus. But the rate and yield of

ethanol production from xylose in these xylose-utilizing yeast strains are considerably low




196

compared to their glucose fermentation (Kahr et al., 2011; Villanueva et al., 2012).

Genetically-engineered organisms with pentose fermenting capabilities include

Saccharomyces cerevisiae

, E. coli

, Zymomonas mobilis

(Agbogbo and Kelly, 2008).Table 1. Comparative analysis of ethanol yield with different xylose utilizing strains.

Source: (Lisbeth et al., 1996)

A number of different approaches have been used to engineer yeasts for both transport

and ferment xylose, including modeling, flux analysis and expression analysis followed by the

targeted deletion or altered expression of key genes. Therefore this review is based on the

details about the genetic engineering of yeasts, problems, solutions and its future perspective.

18.2 Necessity of pentose (C5) sugar fermenting organisms

Many researchers are interested in economical production of fuel-grade ethanol from

cellulosic materials. They generally consist of 40% cellulose, 30% hemicellulose and 20%

lignin as main components and may be considered as a potential feedstock for the production

Strain Xylose ( g l-1) Ethanol ( g l-1) Yield (g g-1 )

Candida blankii ATCC 18735 50 5.1 0.10

C. famata 20 3.9 0.20

C. fructus JCM-1513 20 4.7 0.24

C. guilliermondii ATCC 22017 40 4.5 0.11

C. shehatae CSIR-Y492 90 26.2 0.29

C. tenius CBS 4435 (11) d 20 6.4 0.32

C. tropicalis KY 5014 (2) 20 2.8 0.14

Clavispora sp. UWO(PS) 83-877-1 (11) 20 5.9 0.30

Kluyveromyces cellobiovorus KV 5199 (3) 20 4.4 0.22

K. marxianus 20 5.6 0.28

P. tannophilus RL 171 20 6.2 0.31

Pachysolen tannophilus NRRL Y-2460 50 13.8 0.28

Pichia segobiensis CBS 6857 20 5.0 0.25

P. stipitis CBS 5773 (5) 20 5.9 0.30

P. stipitis CBS 5776 50 22.3 0.45

Schizosaccharomyces pombe ATCC

2478(8)

50 5.0 0.10




197

of ethanol by microbial fermentations (Enari et al., 1984). Hemicellulose constitutes up to

35% of hardwood species and other woody angiosperms by dry weight, with the aldopentose

D-xylose as major constituent of xylan, amounting up to 25% of dry biomass. The hydrolysisof cellulose and hemicelluloses, the major constituents of plant materials, yields a mixture of

sugars in which D-glucose and D-xylose are the major components. The economic feasibility

of ethanol production from these materials depends on the ability of microorganisms to fully

convert the available carbon sources. Industrial yeast strains normally ferment hexoses, but D-

xylose remains unutilized (Lin and Tanaka, 2006).

Efficient ethanol production from xylose is crucial for bioethanol production from

lignocellulosic biomass. An economic analysis of xylose fermentation concluded that a fixedsubstrate cost, the yield and final concentration of ethanol are the most important factors in the

cost of ethanol production (Hinman et al., 1989). For ethanol production from xylose to be

commercially viable, it was suggested that a microorganism should be capable of producing

50 to 60 g/L ethanol within 36 h with a yield of at least 0.4 g ethanol/g sugar. Developing an

efficient organism to ferment the pentose sugars has been pursued for the past few decades.

There are different organisms responsible for the production of different yield of ethanol from

pentose sugar which is depicted in (Table 1).

18.3 Problems with pentose (C5) sugar fermenting yeast

The general requirements of an organism for ethanol production from pentose sugar

hydrolysate should be high ethanol yield, high productivity, good tolerance against inhibitors

as well as high ethanol concentrations and ability to ferment at relatively low pH.S. cerevisiae

is one of the most commonly used yeasts for ethanol fermentation using glucose. However, it

does not have the ability of fermenting pentose sugars. In yeasts, the conversion is carried out

by two oxidoreductases; xylose reductase (XR) and xylitol dehydrogenase. XR is NADPH

cofactor specific whereas XDH is NAD+ cofactor specific. The difference in cofactor

preference of XR and XDH leads to the formation of xylitol under anaerobic conditions.

Xylitol is therefore a byproduct in ethanol fermentation and its production reduces the final

ethanol yield. However, one of the key problems in utilizing xylose for ethanol production is

the impairment of the redox balance that arises from the different coenzyme specificities in

the xylose metabolic pathway (Jeppsson et al., 2003). P. stipitis is one of the few types of

yeast that is able to ferment xylose to ethanol under anaerobic conditions because it possesses

both NADH and NADPH specific XR cofactor.




198

Generation of an NADH dependent XR by protein engineering could resolve this

problem. Decreasing the NADPH-preferring activity of Scheffersomyces stipitis XR (PsXR)

might therefore increase ethanol production inS. cerevisiae

(Watanabe et al., 2007, Zhang etal., 2013). The choice to produce ethanol or cell mass by P. stipitis depends on the O2 supply

to the cells (Agbogbo and Kelly, 2008, Zhang et al., 2013). The benefit of XR using NADH is

that there is a total cofactor balance when this cofactor is used, and therefore no xylitol is

produced. Kinetic studies indicate that NADPH is the preferred coenzyme because its affinity

is about double the value for NADH (Agbogbo and Kelly, 2008). Under anaerobic conditions,

xylose fermentation by P. stipitis must proceed by NADH-linked XR for a total cofactor

balance. The ability of P. stipitis and P. tannophilus to use NADH for XR provides theseyeasts with the ability to produce less xylitol in xylose conversion compared to other xylose

fermenting yeasts under anaerobic conditions (Agbogbo and Kelly, 2008).

18.4 Need of Genetic Engineering for xylose fermentation

In yeasts, filamentous fungi and other eukaryotes, conversion of D-xylose to D-

xylulose via a two-step reduction and oxidation, which are mediated by XR and XDH,

respectively. The cofactor requirements of these two reactions affect cellular demands for

oxygen, as explained in the text. S. cerevisiae can grow on xylulose, an isomer of xylose but

not in xylose. Therefore, the cloning of XR and XDH into S. cerevisiae would allow xylose to

be used as a substrate (Katahira et al., 2006; Hahn-Hagerdal et al., 2007). The yeast S.

cerevisiae has been extensively engineered for ethanolic fermentation of the pentose sugar

xylose either by introducing genes encoding XR and XDH, or by introducing the gene

encoding xylose isomerase (XI) (Hahn-Hägerdal et al., 2007; Van Vleet and Jeffries, 2009;

Matsushika et al., 2009) (Table 2).

Still xylose fermentation with recombinant S. cerevisiae is significantly less efficient

than hexose fermentation. Among others this has been attributed to the difference in cofactor

preference of XR and XDH, which results in xylose to xylitol conversion rather than ethanolic

fermentation. Protein engineering to alter the coenzyme specificities of XR and XDH is a

useful strategy for the development of yeast strains with improved xylose fermentation (Hou

et al., 2007; Klimacek et al., 2010; Krahulec et al., 2010). By changing one residue in the

putative phosphate pocket that binds NADPH, the resulting enzyme exhibited an

NADH/NADPH activity ratio of 23:1 (Watanabe et al., 2007; Van Vleet and Jeffries, 2009).




199

Genetic engineering can also improve the fermentative activities of some native

xylose-metabolizing yeast such as S. stipitis. A number of different approaches have been

used to engineer yeasts for this purpose, including modeling, flux analysis and expressionanalysis followed by the targeted deletion or altered expression of key genes. P. stipitis is

engineered by alteration in XR with altered coenzyme preference improves ethanolic xylose

fermentation (Liang et al., 2007, Bengtsson et al., 2009), some system biological approach has

also done including central carbon metabolic network of P. stipitis has been reconstructed on

the basis of Flux Balance Analysis (FBA) and Principal Component Analysis (PCA). Rather

than P. stipitis many approaches has been done on other xylose fermenting yeast like in

cloning and characterization of xyl1 gene, which encoding NADH preferring XR fromCandida perapsilosis, to Candida tropicalis. (Lee et al., 2003). Mutated Candida tropicalis

strains showing more XR activity in comparison with wild type (Rao et al., 2006) some

substantial factor also applied to increases activity of XR (Liu et al., 2012, Verduyn et al.,

1985). XDH obtained same interest as of XR. XDH were experimented by so many

approaches simultaneously as XR including change in substantial factor like temperature,

NADPH availability, UV treatment etc (Hughes et al., 2012). The genetic engineering

approach has been applied in different strains of microorganisms for xylose utilization whichis shown in Table 3.

18.5 Conclusion and future prospects

More than a decade of research has been devoted to the development of strains for

efficient pentose fermentation. The majority of the studies conducted used metabolic

engineering through a rational selection of genes to be manipulated for the development of

novel pentose-fermenting strains of yeast with varying levels of success. The increased

knowledge about pentose metabolism, substrate binding and cofactor specificity of pentose

assimilating enzymes and sugar transport has contributed to the improvement of yeast for

bioconversion of pentose sugars to bioethanol. However, the mechanisms by which pentose-

fermenting yeasts can accomplish an increased rate of ethanol production are still not fully

understood and the simultaneous co-fermentation of hexose and pentose sugars still

constitutes a major strain engineering challenge. More emphasis on host genome and

regulatory structure must occur in future projects to understand the full effect of biological

complexity on a pathway. In this regard, classic approaches combined with next-generation

technologies may be combined to allow simultaneous optimization of all steps in a pathway.




200

The idealized approach combines the power of many approaches including pathway

engineering, directed evolution, evolutionary engineering, and combinatorial genetics to

harness this cellular complexity.Table 2. Genetic engineering of Sacharomyces cerevisae in xylose utilization for ethanolproduction.

****** =100%, ****=80%, ***=60%, **=40%, *=20%

Gene from different

strain NADPH

Afinity

Redox

Imblance

Ethenol

Production

Xylitol

productionReference

XR(P. stipitis)

* *** * ***Watanabe et al., 2007Karhumaa et al.,, 2005Matsushika et al., 2009Bengtsson et al., 2009

XR

(P. stipitis)** ** * **

Jeppsson et al., 2003Bettiga et al., 2008Krahulec et al., 2010Runquist et al., 2010

XR(C. tenuis)

****

** **Bengtsson et al., 2009,Petschacher andNidetzky, 2008

XR(P. stipitis)

**** **

*Zeng et al., 2009Khattab et al., 2011aLee et al., 2012

XDH(P. Stipitis)

* *** * ***Karhumaa et al., 2007Bettiga et al., 2008Bengtsson et al., 2009

XDH(S. stipitis+

T. brockii)

** *** * *** Watanabe et al., 2007

XDH(P. stipitis)

** ****

*Khattab et al., 2011b

Gene from different

strain

Active at

higher temp

EthanolReference

XI(Piromyces sp.)

No * Kuyper et al., 2003

XI(T. thermophilus)

Yes * * Lonn and co-workers, 2002

XI(Piromyces sp.)

Yes * * Brat et al., 2009

XI(C. phytofermentans)

Yes * *Brat et al., 2009




201

Table 3. Comparative analysis of experimental study on xylose utilizing strains.

StrainXylose

Utilization

Ethanol

production

Redox flux

Imbalance

Xylitol

productionReference

Xylose Reductase (XR) Scheffersomyces

stipitis ***

** ** **

Liang et al.,2007,

Bengtsson et al.,2009,

Peng et al., 2012Candida

tropicalis ** ** ** **

Lee et al., 2003,Rao et al., 2006

Pachysolen

tannophilus

*** * ** ***

Sanchez et al.,2004

Verduyn et al.,1985

Liu et al., 2012Xylitol Dehydrogenase (XDH)

Scheffersomyces

stipitis *** * *** **

Bicho et al.,1988,

Slininger et al.,2011

Candida

tropicalis * ** ** **

Walther et al.,2001,

Lima et al., 2006

Kluyveromyces

sp. * ** ** **

Fonseca et al.,2008,

Lulu et al., 2013

Xylose Isomerase (XI)

Kluyveromyces

marxianus ** ** - -

Wang et al.,2013

Hansenula

polymorpha ** ** - -Dmytruk et al.,

2008

Piromyces ** *** - - Zhou et al., 2012

****** =100%, ****=80%, ***=60%, **=40%, *=20%

Acknowledgment

Authors are very much thankful to Ministry of New and Renewable Energy, New

Delhi, Govt. of India for the financial support and providing all research facilities to carry out

research work.




202

References

1. Agbogbo FK, Kelly GC. Cellulosic ethanol production using the naturally occurring

xylose-fermenting yeast, Scheffersomyces stipitis. Biotechnol. Lett. 2008., 30:1515–1524.

2. Behera S, Mohanty RC, Ray RC. Ethanol fermentation of sugarcane molasses by

Zymomonas mobilis MTCC 92 immobilized in Luffa cylindrica L. sponge discs and ca-

alginate matrices. Braz. J. Microbiol. 2012, 43:1499-1507.

3. Bengtsson O, Hahn-Hagerdal B, Gorwa-Grauslund MF. XR from Pichia stipitis with

altered coenzyme preference improves ethanolic xylose fermentation by recombinant

Saccharomyces cerevisiae. Biotechnol. Biofuels. 2009, 2: 9.4. Bettiga M, Hahn-Hägerdal B, Gorwa-Grauslund MF. Comparing the XR/XDHand

xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces

cerevisiae strains. Biotechnology for Biofuels 2008, 1:16

5. Bhatia L, Johri S, Ahmad R. An economic and ecological perspective of ethanol

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CHAPTER 19

TO OPTIMIZE THE PROCESS OF ALCOHOL PRODUCTIONFROM BANANA PEEL

Mohit Jain, Anand Kumar Gupta, Sayan Chatterjee

Abstract

With day by day increase in demand of fuel and price hike, has forced the world to use an

alternative source of fuel. There are two global biorenewable transportation fuels that might

replace oil derived gasoline and diesel fuel. These are bioethanol and biodiesel. Even in few

countries like Brazil, United States they are in use as a fuel alternative since 2006. Currently

the popular raw materials for preparation of biofuel are corn, sweet potato, potato, sugarcane

molasses etc. The aim of our study was to use banana waste as a raw material for the

production of alcohol which also targets another global problem of waste disposal. Using

banana peel for alcohol production provides a way to use waste material efficiently in addition

to that fuel produce through banana waste is ecofriendly, cheap and easily renewable. MTCC

178 strain of Saccharomyces cerevisiae yeast was used for fermentation to produce alcohol.

To optimize the production of alcohol from banana peel different conditions i.e. temperature,

pH and yeast concentration were varied with the help of Response Surface Methodology

(RSM). The results obtained from the design expert software shows that the production of

alcohol from the banana peel was optimized at 35.78 o C temperature, 3.81 pH, 9.57 yeast

concentration and the yield of alcohol obtained was 7.43% v/v.Keywords: Optimization, Fermentation, Banana peel, Response surface methodology (RSM),

bioethanol.

19.1 Introduction

Today our world has been surrounded with many problems and one of the most

important among them is fuel crisis. In recent years, with rising problems such as uncertain

fuel supply and efforts to reduce carbon dioxide emissions lead us to think about an

alternative source of fuel. There are two global biorenewable transportation fuels that might

replace oil derived gasoline and diesel fuel. These are bioethanol and biodiesel. From ages




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humans have been producing ethanol. According to the time flow, the area of ethanol has been

extending dramatically. The very first time, ethanol existed only in alcoholic drinks. After

establishment of some purification methods, the usage of ethanol highly extended and afterreviewing current scenario ethanol becomes highly attractive again.

Bioethanol is a high-octane fuel which was used primarily as a gasoline additive and

extender. It is seen as a good fuel alternative because the source crops such as sugarcane

molasses, corn etc can be grown renewably and in most climates around the world. In addition

bioethanol can be used as CO2 neutral i.e. during the growing phase of the source crop, CO2

absorbed by the plant and oxygen released in the equivalent volume to that CO2 produced in

the combustion of the fuel. This creates an obvious advantage over fossil fuels which onlyemit CO2 as well as other poisonous emissions (European Renewable Energy Council, 2006).

Only in the last few years due to rising environmental concerns and to the periodic

crises in some of the larger oil exporting countries, has made bioethanol a viable and realistic

alternative in the energy market. The United States produced 5.6 billion gallons per year as on

February 2007 (Renewable Fuels Association, 2007) from 3.4 billion gallons in 2004. In

2009, Brazil, produced 7.65 billion gallons of ethanol (Renewable energy world.com, 2009)

up from 4.0 billion gallons in 2004. Production of ethanol in Brazil utilizes sugarcane

molasses as a primary feedstock. Currently, corn is the primary feedstock being used in the

production process.

Fruits are among the most important foods of mankind as they are not only nutritive

but are also vital for the maintenance of health. Fruits both in fresh as well as in processed

form not only improve the quality of our diet but also provide essential ingredients like

vitamins, minerals, carbohydrates etc. India is the largest producer of fruits in the world.

Among the fruit crops, banana occupies the fourth world rank of the most significant

foodstuffs after rice, corn and milk. According to India Agricultural Research Data Book

2004, the estimated fruit and vegetable production in India was 150 million tones and the total

waste generated was 50 million tones. The extent of total losses in these commodities is

approximately estimated as 20-30% of the total production, amounting to a loss of Rs. 30,000

crore per annum.

Peels are the major by-products obtained during the processing of various fruits andthese were shown to be a good source of various bioactive compounds which posses various




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beneficial effects. But, significant quantities of fruit peels (20- 30% for banana) are discarded

as waste According to FAO (FAOSTAT, 2003), the total waste generated from fruits was

estimated as 3.36 million tones (MT) out of the total production of 16.8 MT and particularlyfor banana it was 6.4 MT. The solid wastes generated by fruit processing industries can serve

as potential raw materials for the production of secondary metabolites of industrial

significance by microorganisms such as bioethanol production and this could also be an

attractive alternate for disposal of the polluting residues (Wyman, 2001).

19.1.1 Response surface methodology

RSM method was introduced by G.E.P. Box and K.B. Wilson in 1951. Whereas Mead

and Pike stated origin of RSM was in 1930s with use of Response Curves (Myers et al., 1989).

Response surface methodology (RSM) is a collection of mathematical and statistical

techniques for empirical model building. It uses quantitative data from appropriate

experiments to simultaneously determine and solve multivariate equations thus providing an

optimum solution (Daramola et al., 2007). The objective of RSM is to optimize a response

(output variable) which is influenced by several independent variables (input variables) in

addition to that it aims at reducing the cost of expensive analysis methods. Response surface

methodology gives advantage over other methods as it reduces the number of experimental

trials need to evaluate multiple parameters, it is less laborious and less time-consuming.

RSM plays important role in designing, formulating, developing, and analyzing new

scientific studying and products. It is also efficient in improving existing studies and products.

The most common applications of RSM are in Industrial, Biological and Clinical Science,

Social Science, Food Science, and Physical and Engineering Sciences. It has been successfully

applied to optimize alcoholic fermentation and other fermentation media (Maddox &

Reichert, 1977; Zertuche & Zall, 1985; Coteron et al., 1993; Chen, 1996; Sunitha et al., 1998;

Ambati & Ayyanna, 2001; Ratnam, 2001; Ratnam et al., 2003).

19.2 Materials used

Musa acuminate variety of banana was procured from the local vendor in Delhi market

and banana peel was obtained. The culture used for fermentation was Saccharomyces

cerevisiae strain MTCC 178 was procured from Microbial Type Culture Collection (MTCC),

Institute of Microbial Technology (IMTECH), Chandigarh, India. Commercial cellulase and




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pectinase enzymes were obtained from Sigma company. Alcohol refractometer (0-80%) which

was ordered online through university portal.

19.3 Methodology

19.3.1 Enzyme Stock preparation

A stock of cellulase and pectinase enzyme was prepared in phosphate buffer. As

optimum working pH for pectinase enzyme is 3.4 so 100ml phosphate buffer (1M) was

prepared by mixing monobasic (KH2PO4) and dibasic (K2HPO4) potassium phosphate salts in

a particular ratio i.e. 13.795g and .008g respectively. Similarly the optimum working pH for

cellulase enzyme is 4.8 so monobasic and dibasic salts were mixed in a ratio of 13.68g and

.228g respectively.

19.3.2 Revival and Sub-culturing of yeast

Yeast strain MTCC 178 was initially in freeze dried form in an ampoule. After

procuring sample the content from the ampoule was poured into test tubes with 1.8 cm3

physiological solution. In order to activate the yeast cells, the test tube was put in a thermostat

at 300C for 30 minutes – time sufficient to restore their viability. For culturing and sub-

culturing of yeast strain a YPD media was used. For 100ml seed media yeast extract, dextrose,peptone and agar were mixed in an amount i.e. 1g, 2g, 2g and 2g respectively and volume was

makeup to 100ml using distilled water.

19.3.3 Experimental design

The statistical analyses were performed according to the response surface method

using Design Expert 8.0.7.0 (Stat-Ease Inc., 2009) trial version software. Central composite

experimental design (CCD) (Box & Wilson, 1951) was employed to study the combinedeffect of three variables namely temperature, pH and inoculum size. The response variable i.e.

alcohol content (%) was measured timely. A CCD has three groups of design points two-level

factorial or fractional factorial design points, axial points (sometimes called "star" points) and

center points.

19.3.4 Processing of Banana peel

Banana was thoroughly washed with running water and then with distilled water to

remove any dirt that could be on it. The peel was removed and cut into small pieces with

sterilized blade and it was left overnight in hot air oven at 65oC to dry it completely. The dried




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peel was then subjected to grinding in a mixer grinder machine. Then banana peel extract was

treated with pectinase and cellulase enzyme. After treating with enzymes a clarified juice was

obtained which works as an production media for anaerobic fermentation.19.4 Results & Discussion

Table 1 shows the run sheet prepared through Design Expert Software using Central

Composite Design. It provides 18 sets of 3 parameters i.e. temperature, pH and inoculum

concentration according to which experimental trials have been performed to find out the

response factor i.e. alcohol concentration for each set of trials. In relation to actual values

RSM also provided the coded values. Table 2 shows the minimum and maximum range

values for the parameters were coded as -1 & +1, mean value was coded as 0 and minimum

and maximum extreme values were coded as - α & + α, where α=1.68179. The second order

polynomial equation obtained from the analysis of multiple regression for estimation of

alcohol concentration in terms of coded value was: alcohol = +7.40 +0.10*A +0.029*B

+0.14*C +0.0000*A*B +0.0000*A*C -0.050*B*C -0.17*A2 -0.21B2 -0.071C2.

TABLE 1: Run sheet prepared by Design Expert Software

Run Temperature

(oC)

pH Inoculum conc.

(%)

Alcohol conc.

(%)1 37 3.33 5.0 6.82 23 3.33 10.0 7.03 30 3.83 7.5 7.44 30 3.83 3.3 7.05 37 3.33 10.0 7.26 30 3.83 11.7 7.47 30 4.67 7.5 6.88 37 4.33 5.0 7.09 30 3.83 7.5 7.4

10 30 3.83 7.5 7.4

11 30 3.83 7.5 7.412 37 4.33 10.0 7.213 23 4.33 5.0 6.814 30 3.83 7.5 7.415 23 3.33 5.0 6.616 30 2.99 7.5 6.817 30 3.83 7.5 7.418 23 4.33 10.0 7.0

Table 3 shows the design matrix evaluation for the quadratic model suggested by the

response surface methodology central composite design where A, B & C representstemperature, pH and inoculum concentration respectively.




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Variance inflation factor (VIF) is generally the reciprocal of tolerance in

computational language. In other terms it explains about the severity of multicollinearity

which have a direct influence on the analysis of regression. As VIF increases the severity ofmulticollinearity increases leading to poor estimation of model. The ideal value for VIF is 1.

Similarly Ri-squared value indicates about the correlation between the parameters and its

higher value possibly leads to poor model prediction.

TABLE 2: Showing the coded values for the parameters

Run Temperature

(oC)

pH Inoculum conc.

(%)

Alcohol conc.

(%)

1 1 -1 -1 6.8

2 -1 -1 1 7.03 0 0 0 7.4

4 0 0 - α 7.0

5 1 -1 1 7.2

6 0 0 α 7.4

7 0 α 0 6.8

8 1 1 -1 7.0

9 0 0 0 7.4

10 0 0 0 7.4

11 0 0 0 7.4

12 1 1 1 7.2

13 -1 1 -1 6.8

14 0 0 0 7.415 -1 -1 -1 6.6

16 0 - α 0 6.8

17 0 0 0 7.4

18 -1 1 1 7.0

TABLE 3: Evaluation of model generated through Response surface

Term Standard Error VIF Ri-squared

A 0.27 1.00 0.0000

B 0.27 1.00 0.0000

C 0.27 1.00 0.0000

AB 0.35 1.00 0.0000

AC 0.35 1.00 0.0000

BC 0.35 1.00 0.0000

A2 0.26 1.02 0.0179

B2 0.26 1.02 0.0179

C 0.26 1.02 0.0179




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Table 4 shows the overall effect of process parameters on the alcohol response. It

indicates that temperature (F =55.72, p<0.0001) and inoculum concentration (F =178.88,

p

<0.0001) had a significant effect on alcohol response whereas pH (F

=8.16, p

=0.0213) didnot had a significant effect on alcohol concentration. Through the software it has been

predicted that at 35.78oC temperature, 9.57% inoculum concentration and 3.81 pH alcohol

response was optimized and its value is 7.43391%. Figure 1 shows the 3D graph plotted

between two independent variables and a dependent variable or a response variable i.e.

alcohol is a response variable and two independent variables were pH and temperature and

inoculum concentration was constant at 9.57%.

TABLE 4: ANOVA result for response surface quadratic modelSource Sum of

squares

df* Mean

squares

F value p-value

(prob > F)

Model 1.29 9 0.14 99.55 <0.0001

A 0.080 1 0.080 55.72 <0.0001

B 0.012 1 0.012 8.16 0.0213

C 0.26 1 0.26 178.88 <0.0001

AB 0.000 1 0.000 0.000 1.0000AC 0.000 1 0.000 0.000 1.0000

BC 0.020 1 0.020 13.93 0.0058

A 0.110 1 0.110 73.64 <0.0001

B2 0.54 1 0.54 376.14 <0.0001

C2 0.060 1 0.060 41.79 0.0002

* degree of freedom




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Figure 1: 3 Dimensional representation of optimized results

19.5 Conclusion

The energy demands of the growing population can be somewhat fulfilled if steps are

taken to make use of the alternative renewable energy sources and their production is

optimized so that input cost incurred during their production can be minimized, and the raw

materials are fully utilized. Using agricultural waste for production of industrially important

metabolites also eliminates the risk associated with the disposal of such substances and

minimizes the chances of polluting the environment.

Through the help of Response Surface Methodology the effect of many independent

variables can be studied on the response variable with minimum experimental trials.

Acknowledgement

We would like to acknowledge Guru Gobind Singh Indraprastha University, Dwarka,

New Delhi; for providing facilities to carry out our experimental work and also like to

acknowledge the Dean of our department (University School of Biotechnology) Prof. P.C.

Sharma for his help and support throughout the project.




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15. Sunitha I., Subba Rao M.V. & Ayyanna C. (1998) Optimization of medium

constituents and fermentation conditions for the production of L-glutamic acid by theco-immobilized whole cells of Micrococus glutanicus and Pseudomonas reptilivora.

Bioprocess Engineering, 18, 353–359.

16. Wyman, Ch.E. (2001). Twenty years of trials, tribulations, and research progress in

bioethanol technology. Appl. Biochem. Biotech., 91: 5

17. Zertuche L. & Zall R.R. (1985) Optimizing alcohol production from whey using

computer technology. Biotechnology and Bioengineering, 27, 547–554.




218

CHAPTER 20

COMMERCIAL PRODUCTION OF BIO-CNG & ORGANIC

MANURE FROM PRESSMUD BIOMETHANATION

Preetam Holkar, A.V. Mohan Rao and K.K. Meher

Abstract

Increasing demand for fossil fuels and escalating prices of chemical fertilizers has been the

prime source of motivation for this project. The concept of anaerobic digestion is a promising

eco-friendly solution for the treatment of organic biomass, which leads to bioenergy production.

Press mud is a byproduct of sugar making process from sugar cane and accounts for 3.5 to 4

% of the total sugarcane crushed. About 5.2 million tons of press mud is produced in our

country every year. Generally, press mud is used as manure in agriculture after composting.

There are no reports on large commercial scale biomethanation of press mud. SREL has

established first commercial BIO-CBG and organic manure plant of 100 TPD capacity of

press mud feed at Warnanagar, in India. This plant is constructed with Indo-German technical

inputs and is in operation for the last 15 months. It is generation capacity is about 7000 to

7,500 kg of BIO-CNG and 40 to 45 tons of organic manure per day. Calorific value of BIO-

CNG and commercial CNG are comparable. While BIO-CNG is being supplied as industrial

fuel, organic manure as “spectrum digestate (liquid) and spectrum soil conditioner” are used

in agriculture. Application of these products improved soil physical properties, soil health,

moisture retaining capacity etc. Horticulture crops recorded 15 to 20% higher yields.

Key words: Biomethanation, Pressmud, BIO-CNG, Organic manure.

20.1 Introduction

In the current energy scenario, growing gap between demand and supply, increasing

prices of fossil fuels in international markets are putting tremendous pressure on the

economies of many countries. This gap can be reduced with the energy generation from

renewable resources. Biomass is one of the feasible renewable energy sources. In the recent

year’s interest in bio energy has increased and it represents approximately 14% of world final

energy consumption (1). Large quantities, about 150 million tones of biomass wastes such as

household and agro processing wastes are generated annually in India and are disposed in a




219

dispersed manner. Dumping results in serious environmental consequences such as air and

water pollution. In India biomass based power generation is developing into an industry. The

two promising pathways for biomass energy conversion are biological, Anaerobic Digestion(AD) route to generate biogas fuel and Thermo-Chemical (TC), biomass gasification route to

generate producer gas fuel. Biogas production technology has received attention worldwide

and remains attractive and promising in meeting future energy demands.

Globally, India is the second largest producer of sugarcane producing over 350 million

tons of sugar per year. In India Uttar Pradesh (37.2 %) is the biggest producer of sugarcane

followed by Maharashtra (2) with 23.5% (2011-12). Sugar industry (more than 500 mills) is

one of the major agro-industry in India. Several co-products of immense value are generatedduring sugar making. Press mud is one such product which accounts for about 3.5– 4.0 % of

the total sugarcane crushed. About 5.2 million tons of press mud is produced in our country

every year. It is a soft, spongy, amorphous and dark brown material containing 30 to 35 % dry

matter out of that 80% organic matter hence, it’s a very good feed stock for biogas generation.

There are very few reports on the anaerobic digestion of press mud, no large scale

biomethanation plants based on Press mud are reported. SREL has established first

commercial BIO-CNG and organic manure plant based on 100 TPD press mud feed capacity

at Warnanagar, Maharashtra in India. Press mud is selected as feed material due to its

abundant availability in sugar mills and tremendous scope for the expansion. During 2011-12

Warana sugar factory crushed about 13.8 lakh tons of sugar cane and generated about 42,000

tons of press mud. It contains rich organic matter, organic carbon, sugar, protein, enzymes,

micro (N, P, and K) and macro (Zn, Fe, Mg, Mn, Cu etc) nutrients and microbes (Yaduvanshi

and Yadav, 1990; Ranganathan and Parthasarathi, 1999; Parthasarathi and Ranganathan,

1998, 1999, 2000). The composition of the press mud indicates that it is a good source forextracting bioenergy (Table-1).

Press mud is procured from Warna sugar factory during the sugarcane crushing season

and used directly as it comes from the factory, during off season press mud stored in yard is

used. Total holding capacity of the storage yard is 18000 tons. Press mud is conveyed to

the feed tank by a conveyer belt. During off season other feed stocks such as cattle dung,

poultry waste, milk effluents etc are added as when needed.




220

20.2 Feed preparation

Press mud is mixed with the digesting slurry taken from digester to prepare feed slurry

by constantly mixing it with an agitator to have uniform feed slurry. Fresh water is added

to have 10 to 12% solids in the feed slurry. This slurry is pumped back into the respective

digesters using feed pump. Daily data on feed and effluent slurry pH, TS, VS, VFA,

Alkalinity, Temperature and gas composition etc are collected.

. Table-1: Press mud composition (karan et al.)

S.No Componenet %

1 Moisture 70

2 Volatile matter 76.6

3 C:N 14

4 Sugar 5.7

5 Cellulose 11.4

6 Hemi cellulose 9.3

7 Protein 15.5

8 Lignin 9.3

9 Wax 8.4

20.3 CSTR Digesters

Three CSTR digesters, each of 3400 cum capacity are built, these are equipped with

double layered gas capturing system to hold about 1000 cum of biogas in each. All the three

digesters are of the same design and are interconnected through pipes at the upper gas storage

area to have equal gas pressure. Hot water pipes are fitted in each digester. Hot water (60o

C)

from the generator is circulated through these pipes [Fig.1] for maintaining the optimum

digester temperature. Each digester is also provided with four agitators for mixing the digester

contents. All the digesters are fitted with safety valve to prevent over and under gas

pressureinside the gas capturing system

Biogas generated is continuously sucked by a blower and supplied to gas cleaning

system. In addition to the SREL biogas about 12000 cum of biogas from Warna sugar limited

is also processed by the gas cleaning system for making BIO-CNG. Biogas produced [Fig.2]

for 30 continuous days is considered in this article.




221

Figure 1. CSTR Digester

Table-2. Performance of the digesters

S.No Parameter Data

1 Daily Loading 100 tons

2 Total solids in feed 10 to 12%

3 Volatile solids in feed 70% of TS

4 pH of the feed slurry 5.5 ± 0.1

5 Biogas production(24 hrs) 8 000 to 9000 m3

6 Biogas production/ton of Press mud/day 80 to100 m

7 Methane in biogas produced 62 ± 2 %

8 pH of the effluent slurry 7.1 ± 0.1

9 Digesting slurry temp 36 ± 20C

10 TS degradation 42.8%

11 Biogas production /kg TS degraded/day 1.168 m

12 TVS degradation 76.1%

13 Biogas production/kg VS degraded/day 0.939 m




222

Figure 2. Biogas production from Spectrum and WSL biogas plants

20.4 Biogas cleaning process

Raw Biogas is taken to the gas header and its composition

Biogas pressure 2500 mm WC

Methane (CH4) 60 -62%

Carbon Dioxide(CO2) 35 % (Max)

Hydrogen Sulphide(H2S) 3 % (Max)

Biogas from Raw gas header is taken to H2S removing plant through a gas flow meter.

The “Bioskrubber”TM

treats H2S containing gases, where sulphide is biologically converted

to elemental sulfur. Biogas from bioskrubber is then passed through an adsorbent bed where

the H2S concentration is reduced to about 5 ppm. Online H2S analyzers are provided to

measure the H2S conc. H2S scrubbed biogas is stored to the clean gas holder. Biogas is

compressed to 7.5 kg/cm2 before sending it to the CO2 scrubber.

CO2 removal is by absorption process Soft water as solvent at high pressure is used in

a packed column. CO2 concentration is brought down from 35% to less than 5%. After

purification the biogas composition is comparable [Fig. 3] to that of commercial CNG and

hence it is named as BIO-CNG.

0

2000

4000

6000

8000

10000

1200014000

16000

18000

20000

1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930

Spectrum gasWSL gas

B i o g a s i n




223

20.5 BIO-CNG production & composition

After removing CO2 biogas is dried for moisture removal and stored in a buffer vessel,

where the gas pressure is 6 to 7 kg/cm2. From buffer vessel gas is taken for compression and

compressed to 200 bars and filled into the cylinders [Fig. 3].

Biogas composition after purification

Biogas pressure 6-7 kg/cm2

Methane (CH4) minimum 95 to 95.5 %

Carbon Dioxide(CO2) maximum 4.0 to 3.5 %

Hydrogen Sulphide(H2S) less than 5 ppm

Water vapor Nil

Oxygen Nil

Hydrogen 0.2 to 0.5%

Methanol/Glycerol Absent



Recent Adva

Online analyzers a

Apart from on line analy

Mumbai and analyzed.

20.6 Biogas utilization

The upgraded and

specially designed trucks

application in industrial fur

20.7 Biogas based pow

A 340 kW capacit

H2S removed Biogas. The

generator generates aroun

extracts exhaust heat from

the digester through heatin

20.8 Digestate (Organi

Importance of org

increasing costs and non

organic manure used by th

mixed with animal dung

manure developed by SR

anaerobic digestion Press

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

1 B i o g a s i n c u m & B i o C N i n

! g " # a $

ces in Bioenergy Research

224

Figure 3. BIO-CNG produced

nd flow meters are provided to measure

zers periodically BIO-CNG samples are

storage devices cascades

compressed BIOCNG storage cylinders (ca

nd shipped to the customer site, where it

naces in place of fossil fuels.

r

y generator is installed for power productio

generated power is used for captive cons

2.0 units per cum [Fig.4]. A heat recove

the generator. Heat recovered from these s

manifold and maintained optimum digester

Manure)

nic manure application in agriculture is g

vailability of chemical fertilizers. Farmyar

e man. It consists of litter; waste products f

nd urine and allowed to degrade biologica

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ud is subjected to intense microbial enzy

3 5 7 9 11 13 15 17 19 21 23 25

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ol. III 2014

he above parameters.

ent to ONGC lab at

scades) are placed on

will be used for their

n. It operates fully on

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urces is transferred to

s temperature.

aining ground due to

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27 29

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digestate is well stabilized,

as spectrum soil condition

action in soil. Humus helps

Table-3. SREL organic ma

Parameter

pH

Conductivity

Moisture

Dry matter

Organic carbo

C/N

Water holding

capacity

Nitrogen

Phosporous

Potash

Note: Spectrum Soil condi

with water in 1:3 ratios and

Figure 4. Biogas

0

1000

2000

3000

4000

5000

1 3 5

Biog

B i o g a s i n c u m & . e c t r i c i t $

i n + W / " # a


225

nutrient rich, ready to use soil conditioner (

r. When this manure is applied it helps in a

in improving soil fertility and water holding

ure analysis

Unit Spectrum- soil

conditioner

Spectru

7.92

Mmhos/cm 7.5

% 23.7

% 76.29

% 16.27

14.52

162.32

% 1.12

% 1.14

% 0.76

tioner is directly applied to the soil.Spectr

applied to the plants.

to Electricity generation per day in January2

9 11 13 15 17 19 21 23 25 2

as consume# ,cum- .&ectricit$generate# ,+

ol. III 2014

able-3), and marketed

tivating the microbial

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m digestate

.35

27.4

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10

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.15

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.47

o.45

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29 31

-




226

Being tropical country, Indian soils are often deficient in humus. Humus is an

extremely important material formed after decomposition of organic manure. The application

of Spectrum’s Pressmud based organic manure offers one of the best ways for thesimultaneous production of humus in soil. Humus imparts grey brown or slightly black color

to soil and thereby helps the soil to absorb greater amount of radiation. Humus also resists too

much fluctuation in soil temperature.

Spectrum’s Digestate contains micro and macro nutrient required for the plant growth

hence like other organic material it effect the physical, chemical and biological properties of

the soil (Tandon, 1995). Results show that with Spectrum’s organic manure increased yield

ranging from 15-20 per cent are observed. Still better results can be obtained when it is usedin combination with other cultural practices such as improved seeds, proper crop rotation,

good soil management and supplementary dose of chemical fertilizers.

20.9 Conclusions

In the current energy scenario of growing energy demand and dwindling fossil fuel

resources, shift to renewable energy source is an obvious option. Since most sugar mills are

situated in the rural areas producing press mud year after year in large quantities.

Development of Press mud based BIOCNG plants in association with sugar mills will help in

bringing decentralized renewable energy and organic manure producing units. If exploited

properly these units minimize the dependence on fossil fuels and helps in reducing the

environmental pollution. Continuous availability of organic manure helps farmers to plan and

adopt for seasonal organic farming. This will lead to less reliance on inorganic fertilizers and

boost the self confidence of the people in agriculture. Hence, setting up SREL type of biogas

plants helps in employment generation and in the economic development of small towns and

rural areas.

References

1. http://www.economywatch.com/energy-economy/scenario.html

2. Price policy for sugarcane the 2013 sugar season.PP-39.Commission for agricultural costs and

prices, Department of agriculture & cooperation, Ministry of agriculture, Govt of India, New

Delhi.




227

3. Yaduvanshi, N.P.S. and D.V.Yadav: Effects of sulphitation pressmud and nitrogen fertilizer on

biomass, nitrogen economy, plant composition in sugarcane and on soil chemical properties.

J.Agric. Sci., 1990,114, 259-263.

4. Ranganathan, L.S. and K.Parthasarathi: Precocious development of Lampito mauritii (Kinberg)

and Eudrilus eugeniae (Kinberg) reared in pressmud. Pedobiologia, 1999, 43, 904-908.

5. Parthasarathi, K. and L.S. Ranganathan : Pressmud vermicasts are the hot spots of fungi and

bacteria, Ecol. Environ. Conser., 1998, 4,81-86.

6. Parthasarathi, K. and L.S.Ranganathan: Longevity of microbial and enzyme activity and their

influence on NPK content in pressmud vermicasts, Eur. J. Soil. Biol., 1999, 35(3), 107-113.

7. Parthasarathi, K. and L.S.Ranganathan: Aging effect on enzyme activities in the pressmud

vermicasts of Lampito mauritii (Kinberg) and Eudrilus eugeniae (Kinberg). Biol. Fertil. Soils,2000, 30, 347-350.

8. Karan M. Agrawal, B. R. Barve and Shareena S. Khan: Biogas From Press mud, IOSR journal

of mechanical and civil engineering (IOSR-JMCE) ISSN:2278-1684, pp 37-41.

9. Tandon (1995). In: Waaste Recyling on Agriculture.Fertilizer Development Consultation

Organization, New Delhi.




228

CHAPTER 21

FEASIBILITY OF FILLING BIOGAS IN CYLINDERS

S.S. Sooch, Jasdeep Singh Saini

Abstract

A case study has been done for Ludhiana in Punjab State regarding feasibility of filling of

biogas in cylinders for cooking as well as industrial use. Biogas produced from Cattle dung in

the Corporation area can produce 30,000 m3

/day

of biogas which can cater the needs for

cooking of 85,000 people and in addition to solve the Waste Disposal Problem and also

provide High Quality Fertilizer.

Key words: Waste disposal, Biogas, Bottling of biogas

21.1 Introduction

There is acute shortage of conventional type of fuel like petrol, diesel, kerosene and

fire wood. It is, therefore, very essential that some alternate fuel may be located. One such

fuel which can be made available in rural as well as urban areas is methane gas produced from

anaerobic digestion of organic waste. As the population of the country is increasing perhaps

the largest single problem resulting from the recent increase in confinement rearing of

livestock and poultry involve waste handling and disposal. The odour and fly nuisance of the

waste, the decline of manure as a competitive fertilizer and encroachment of urban areas close

to production units complicate the problem of waste treating and handling. Anaerobic

digestion process of well known acid is often practiced by various municipal corporations in

the country for stabilizing organic material and for production of gas.

A case study has been done for Ludhiana in Punjab. It is observed that in addition to

seven lakhs inhabitants, there are more than fifty thousand milch cattle in the city. The result

is that Ludhiana city is facing lot of difficulty in disposal of solid wastes and one finds heaps

of rubbish in every vacant plot of land or on a wide corner of a road. Although municipal

corporation has made some arrangements for disposal of such waste even then the position is

no satisfactory from health point of view. The municipal corporation has made separate

arrangement of construction of dairy sheds out side the city. But no dairy owner has so far

shifted to the dairy area.




229

The question again arises, if cattle are shifted to the new area what will be the

arrangement for the disposal of waste produced from the animals. Simple answer for this

would be to dump at one open space or sell it directly as a raw fertilizer to the farmer. This isnot possible because such a customer will not be readily available. The authors have

suggested a suitable possible method in this paper by which the whole waste of dairy sheds

can be directly handled in a safe manner.


The methods recommended for safe handling is by anaerobic digestion of organic

waste by biological means. The end products would be biogas and very good organic manure

free from any odours and flies etc. The detailed plan of the scheme is as under:

The dung from the dairies be purchased by a contractor who would run the biogas

plant and should resell the end product to the farmer at a suitable rate. Such a method is beingpractised in some of the States of India.

The quantity of biogas available is calculated as under:

Number of cattle in the city = 50,000

Average dung produced = 10 to 20 kg per day (say 15 kg)

Total dung = 50,000 x 15 = 750000 kg

Biogas produced per kg of dung = 0.04 m

Total quantity of biogas produced/day = 750,000 x 0.04 = 30,000 m

DUNG FROM

CATTLE

ADDITION OF

EQUAL AMOUNT

OF WATER

ANAEROBIC

DIGESTION

DRYING

BEDS

GAS FOR

COOKING BIOGAS

FERTILIZER

FIELDSSEEPAGE

WATERPOWER

GENERATION

GAS

CYLINDERS




230

The gas produced may be used for cooking purposes at homes by laying distribution

pipe network in the nearby locality. The gas is sufficient to meet the demand of about 84,000

people. But the cost of network may be very high because of the cost of pipes. The otheralternative would be to generate power with the gas at the plant site to avoid losses. The

power may be supplied to the dairy sheds for light or for operation of tubewells. The power

expected from the gas is equal to 45,000 Kilo-watts per day.

The third use can be the filling of gas in cylinders which may be sold in the market at

usual rates like LP gas. This method seems to be more attractive as the increasing cost of

LPG. Although it looks very easy to fill gas in cylinders but there are many difficulties in

actual procedure such as:

1. Biogas is a mixture of methane, carbon dioxide and other gases in trace amounts like

hydrogen sulphide, carbon monoxide and ammonia. This is the reason that calorific value of

biogas is very low (4700 Kcal/ m3). Therefore, it is suggested that gas be purified from carbon

dioxide, H2S and moisture, at least, to increase its efficiency by some method to change it to

pure form of methane.

2. The other difficulty is that the gas cannot be liquefied at low pressure. It is reported that

methane gas can be liquefied at low pressure. It is reported that methane gas can be liquefied

at 162°C and 20,000 psi which is not practicable. Below is a chart showing the details

regarding volume of the cylinder to store 28 m3 at different temperatures and pressure.

From the table above it could be seen that 28 m3 gas can be stored in 5.593 ft

3 cylinder

and such cylinders are available in the market for filling gas like oxygen, carbon dioxide etc.

The same cylinder may be used for filling methane gas. The cylinder when filled with purified

biogas free from carbon dioxide and other impurities, with high calorific value, will besufficient for an average family of live persons for a period of 30 days.

Sr. No. Pressure (psi) Temperature (°C) Volume of cylinder (ft3.)

1. 1200 -70 3.195

2. 1500 -50 4.088

3. 2100 -25 4.1444. 2400 0.0 4.641

5. 2400 25 5.593




231

21.3 Discussion

The filling of gas in cylinders as mentioned above can be tried in our country to meet

the fuel shortage Although as mentioned above to fill 28 m3

gas a thick cylinder of about 3 cm

thickness is required. This can be managed easily as is done in the industries for the carriage

of oxygen cylinders etc. The cost of methane gas cylinders would be less and can be easily

available in the town as compared to LPG. Only draw back would be its weight because

atleast two men are required for lifting such cylinders. The cost of the cylinders is comparable

with LPG cylinder.

From the filling of gas a highly sophisticated arrangement is necessary to avoid

accidents. This would be quite costly and skilled labour is necessary to run the compressor.

From the Ludhiana town itself 1000 cylinders can be filled daily and supplied in the market.

To regulate the pressure of gas a regular similar in use for oxygen and carbon-dioxide is

necessary. Ordinary regulator used with LPG will not be suitable.

21.4 Conclusion

The method suggested in this paper would not only solve the waste disposal problem

of the Ludhiana city but also provide valuable fuel for 85,000 people. In addition high quality

fertilizer free form weeds, insects and odour would be available to the farmers for growing

crops.

References

1. Mittal, K.M. 1996 Biogas Systems: Principles and Applications, New Age International (P)

limited Publishers, New Delhi.

2. Biogas – A Rural Energy Source. (1985), Ministry of Non-Conventional Energy Sources

Publication, New Delhi.

3. Grewal N.S., Sooch S.S., Ahluwalia S and Brar G.S. 2000. Hand Book Biogas Tech, PAU,

Ludhiana.

4. Sooch S.S. 2010. Biogas Plants for Rural Masses. School of Energy Studies for Agriculture,

PAU, Ludhiana.

5. Akshyay Urja 2012. Energing the re way, Ministry of Renewable Energy Government of India,

5 (6). www.mnre.gov.in.




232

CHAPTER 22

EFFECT OF PRETREATMENT ON BIOCONVERSION OFWHEAT STRAW FOR THE PRODUCTION OF BIOGAS

Nishshesh Singh, Vivek Saini, Pranshu Gupta, Rajan Sharma, G Sanjay Kumar, Dr. Avanish

K. Tiwari

Abstract

Lignocelluloses are often a major or sometimes the sole components of different waste

streams from various industries, forestry, agriculture and municipalities. Hydrolysis of these

materials is the first step for either digestion to biogas (methane) or fermentation to ethanol.

However, enzymatic hydrolysis of lignocelluloses with no pretreatment is usually not so

effective because of high stability of the materials to enzymatic or bacterial attacks.

Pretreatment helps to improve the process of hydrolysis .In this work different methods of

pretreatment was studied.

The present work illustrates about the effect of acid, alkaline pretreatment on different

sizes of wheat straw and anaerobic digestion of treated biomass for the production of biogas

in batch stirred tank bioreactor at particular parameters. The quality of biogas formed was

analyzed by gas chromatography and quantity measured by water displacement method. The

untreated wheat straw gave a biogas yield of 104 ml/g and methane content of 64%. Acid

treated wheat straw gave biogas yield of 130, 140, 134 ml/g and methane content of 68%,

72%, 75% for respective 1%, 2%, 5% acid concentration. Similarly, for alkali treatment gave

biogas yield of 124, 128, 126ml/g and methane content of 66%, 69%, 71% for respective 1%,

2%, 5% NaoH concentration.

Key words: Pretreatment, Lignocelluloses, Biogas, Anaerobic Digestion, Batch Stirred Tank

Bioreactor.

22.1 Introduction

Biofuels are rapidly becoming a significant partner in our future energy needs. It can

provide means to mitigate deleterious impacts of greenhouse gas emissions. Production of

waste materials is an undeniable part of human society. For effective waste management




233

utilization of waste is necessary. Anaerobic digestion is the process by which the waste

treatment can be done easily and side by side it can be useful for the production of valuable

products such as Biogas and organic manure [Tsavkelova and Netrusov, 2012] .There aredifferent types of wastes such as industrial, forestry, agricultural and municipal waste which

can act as substrate for the Biogas production. Wheat straw is one of the most abundant

agricultural wastes produced in world [Wang, 2009]. However, due to its complex structure

and high lignin content, its degradability and gas yield are low. The degradabitity can be

improved by pre-treatment, making the material more accessible to microbial degradation

thus increasing the biogas yield.

The composition of wheat straw is - Cellulose-30%, Hemicellulose-50%, and Lignin-15%. Lignin is responsible for the integrity, structural rigidity and resistance to swelling of

lignocelluloses. Therefore a delignification processes can improve the rate and extent of

enzymatic hydrolysis from the matrix polymers. The reason for improved rate of hydrolysis

by removal of lignin might be related to a better surface accessibility for enzymes by

increasing the population of pores after removal of lignin.

Li Sun et al. [2013] investigated the microbial response to straw as a feed stock for

biogas production. The addition of straw, pre-treatment of straw and operating temperature all

affects the cellulose degrading community in biogas digesters, but there were no major

differences in the digester performance and gas yield.[Nekema et al. 2013] evaluated biogas

production in batch and Up-flow anaerobic sludge bed (UASB) reactors from pilot-scale acid

catalyzed steam pretreated and enzymatic hydrolyzed wheat straw. The results showed that

the pretreatment was efficient and, a sugar yield of 95% was obtained. The pretreatment

improved the methane yield compared to untreated straw. [R.Chandra et al.] studied

experimental methane fermentation on untreated, NaOH and hydrothermal pretreatedsubstrates of wheat straw. NaOH pretreated substrate produced 87.5% higher biogas

production and 111.6% higher methane production compared to the untreated wheat straw

substrate.

Pia-Maria Bondesson [2013] investigated wheat straw in two different process

alternatives with simultaneous saccharification and fermentation (SSF) to ethanol and

anaerobic digestion (AD) to biogas. In her study three types of pretreatment were done steam

pretreatment with water, acetic acid or phosphoric acid. The overall best yield was obtainedwhen using phosphoric acid as impregnation material. The highest methane yield, 754 ml




234

CH4 /g fed was achieved. [Prasad Kaparaju, 2009] investigated wheat straw hydro lysate for

biogas production in continuous stirred tank reactor (CSTR) and (UASB) reactors. In his

study methane yields increased with increase in hydro lysate concentration. However, heconcluded that, biogas process was affected by the reactor type and operating conditions.

None of above work has illustrated the effect of acid and alkali treatment on a particular

sieved size wheat straw particle.

The present work studies pretreatment of wheat straw with acid and alkali (1 vol%,

2vol %, and 5 vol% ) on mechanically grinded wheat straw (<75µm, 75µm, 150µm, 212µm

425 µm). It was shown that highest lignin content was extracted in 425 µm. The objective to

use 212 µm particle size wheat straw was due to its abundant production during grinding.Hence, process was economical.


Standards Chemicals used (conc. sulphuric acid, sodium hydroxide and distilled

water) were from Rankem. A Standard lignin used was from Merck. Standard biogas sample

were from Centurion scientific pvt ltd.

22.2.1 Preparation of standard lignin solution

Standard lignin samples of 1 and 10 ppm were prepared by dissolving weighed

quantity of pure lignin in appropriate volume of distilled water. The substrate wheat straw was

collected from Dehradun.

22.2.2 Experimental Method

22.2.2.1 Mechanical Treatment of wheat straw

Raw wheat straw procured from Bidholi, Dehradun was first ground. The powdered

sample was sieved. Five different sample sizes were collected: <75 µm, 75 µm, 150µm 212

µm, and 425µm.

22.2.2.2 Estimation of acid soluble lignin content

0.3 g of different particle size samples were taken mixed with 3ml of 72% conc.

H2SO4 [8] and kept in a water bath for 2hrs for estimation of acid soluble lignin. The solution

obtained was mixed with 84 ml water and autoclaved for 30 min at 121ºC and 15 psi.

Autoclaved solution was then filtered to separate residues and filtrate. Filtrate solutions were




235

diluted to 100 ppm and the same were studied under UV spectrophotometer at 205 nm for

lignin content. The process is shown in figure 1.

22.2.3 Pretreatment of Wheat Straw

Wheat Straw pretreatment was done by both acid and alkaline methods.

22.2.3.1 Acid pretreatment of wheat straw

50g of mechanically treated wheat straw of 212 µm was treated with 1% H2SO4. The

sample was mixed with 500 ml of 1% H2SO4 solution and kept in orbital shaker at 70ºC,

150rpm for 2 hrs. The solution was filtered and the residual biomass was washed twice with

water and then dried. Similar treatment was done with 2% and 5% H2SO

4 also. Figure 2

shows color variation of the sample after treatment with 1%, 5% H2SO4.

Figure 1. Schematic representation of estimation of soluble lignin from wheat straw

Figure 2. Acid(1%.5%) pretreated wheat straw

22.2.3.2 Alkali pretreatment of wheat straw

Wheat straw was treated in presence of 500 ml of 1%, 2%, 5% NaoH in an orbital

shaker at 150 rpm for 2 hours at 70ºC. Reaction mixture was filtered, washed twice with

water and then dried. The residual biomass was used for anaerobic digestion in batch reactorfor biogas production.




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22.2.4 Anaerobic digestion of pretreated wheat straw

Wheat straw after pretreatment was digested in a bio-reactor. The pH of the slurry was

maintained neutral. The cow dung was used as inoculums. The set-up is shown in Figure 3.

Gas was collected in glass collecting port via gas nozzle of digester. The quantity of biogas

produced was determined by the water displacement method. The reaction conditions

maintained in bioreactor are given in Table 1.

Table 1. Reaction specification

Reactor Batch

Working volume 500ml

Total volume 1 lt.

pH 6.8

Temperature 38º

HRT 4 days

22.2.5 Gas analysis

Biogas was analyzed by GC (Nucon 5700). GC conditions are given below:

• Injection volume – 100 µl

• Mobile phase - Argon

• Column Make – Stainless Steel

• Column ID - (HEYSEP. Q)

•

Run time - 10 minute

Figure 3. Batch type stirred tank reactor setup




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22.3.1 Delignification of Wheat Straw

The absorbance of lignin standard was determined at 1 ppm and 10 ppm as 0.791 and

3.261.

22.3.2 Estimation of acid soluble lignin content

The observations obtained from acid pre treatment of wheat straw are summarized in

Table 2 and Figure 4.

Table 2. Effect of particle size on lignin removal

Figure 4 Comparative graphical representation of estimated lignin

Particle size Absorbance of soluble

lignin (1 ppm)

Absorbance of soluble

lignin (10 ppm)

%lignin

(1 ppm)

%lignin

(10 ppm)

425nm 1.363 1.643 0.8 10.5

212nm 1.370 1.671 0.9 10.7

150nm 1.376 1.732 1.0 11.1

75 1.379 1.759 1.1 11.3

<75 1.387 1.836 1.2 11.8




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Absorbance is increased as the particle size of the wheat straw is decreased. It was

found that mechanical treatment had good impact on delignification. Particle (<75µm) yield

more lignin because particle has large surface contact with acid. 212µm yield 10.7% lignin.

22.3.3 Acid pretreatment of wheat straw

Above observation indicate that the acid soluble lignin removal was found more in 5%

H2SO4 reaction condition as shown in Table 3. But 2% H2SO4 was feasible to use for acid

hydrolysis and moreover lignin removal was found nearby to 5% H2SO4.

Table 3. Acid Treatment

% H2SO4 Absorbance of 100 ppm solution Lignin removal (g/lit)

1 3.205 2.836

2 3.312 2.930

5 3.506 3.102

22.3.4 Alkaline pretreatment of wheat straw

The observations obtained from alkaline treatment of wheat straw are summarized in Table-4.Results of alkaline pretreatment show that 2% NaOH stage is beneficial for the removal of

lignin, but more lignin was extracted out in the presence of 5% NaOH as shown in Figure 5.

Table 4. Alkali Treatment

%NaOH Absorbance 100 ppm solution Lignin removal(g/lit)

1 2.945 2.606

2 3.102 2.745

5 3.314 2.932

22.3.5 Biogas Production from Pretreated wheat straw:

A standard Biogas sample was run on GC. The composition was (CH4-62%), (CO2-36%).

This is used to calibrate the GC.

The chromatogram indicates percentage of methane content increases from 64% to

75% with increase in the conc. of sulphuric acid from 1% to 5%.As it already seen in fig 4

that 5% acid has good impact on lignin removal. Acid treatment also disrupts crystalline




239

structure of cellulose. Hence accessibility of microorganism to biomass surface increases the

2% acid pretreatment give better biogas methane yield comparable 5% sulphuric acid. Hence

preferred because it is found economical than 5% acid pretreated as require less alkalinesolution to maintain pH of pretreated slurry.

Figure 5. Comparative effect of alkali, acid treatments on lignin removal (g/lit)

Above figure indicates that acid treatment has more impact on lignin removal than alkali.

Figure 6. Comparative chromatogram of biogas after (1%, 2%, 5%) acid treatment.

Figure 7. Comparative chromatogram of biogas obtained after (1%, 2%, 5%) alkaline

treatment.




240

Above chromatograph indicates percentage methane content was found more after 5%

alkali treated wheat straw as compared to 2% NaoH. 5% alkali pretreatment extracts more

soluble lignin as compare to other.

Figure 8. Yield of biogas, methane/gm Figure 9. Yield of biogas, methane/g from

pretreated wheat straw. acid from alkali pretreated wheat straw.

The figures (8&9) indicates biogas yield/g was found more after 2%acid pretreated

and wheat straw while the rest of treatment also yield good volume of biogas per gm of

biomass. Methane yield was found more after 2%acid, 5% alkaline pretreated wheat straw.

22.4 Conclusions

Different pretreatment methods for delignification of wheat straw were tried. 5%

conc. H2SO4 yields more acid soluble lignin as compared to 2% conc. H2SO4, but 2% yield

more biogas. Alkaline pretreatment is also good method for delignification of wheat straw.

However, the methane content of biogas obtained from acid treated biomass was 10% more as

compared alkaline treated biomass.

References

1. Tsavkelova EA, Netrusov Al, (2012), Biogas production from cellulose-containing

substrates: a review. Appl Biochem Microbial 48:421-433.

2. Wang G, Gavala HN, Skiadas IV, Ahring BK, (2009), Wet explosion of wheat straw

and codigestion with swine manure: Effect on the methane productivity. Waste

Management 29:2830-2835

3. Li Sun, Bettina Müller and Anna Schnürer (2013), Biogas production from wheat

straw: community structure of cellulose-degrading bacteria, Sustainability and

Society, springer journal, 3:15.




241

4. Nkemka VN, Murto M (2013) Jan Biogas production from wheat straw in batch and

UASB reactors: the roles of pretreatment and seaweed hydro lysate as a co-substrate.

Bioresour Technol. 128:164-72.5. R. Chandra, H. Takeuchi, T. Hasegawa, R. Kumar ,(2012) , Improving

biodegradability and biogas production of wheat straw substrates using sodium

hydroxide and hydrothermal pretreatments , Energy 43 (2012) 273-282, 2012 Elsevier

Ltd.

6. Pia-Maria Bondesson, (2010), combined production of bioethanol and biogas from

wheat straw, Lund University.

7. Prasad Kaparaju, María Serrano, Irini Angelidaki, (2009), Effect of reactor

configuration on biogas production from wheat straw hydro lysate. Bio resource

technology ,100(24):6317-23.

8. Sluiter A., Hames B., Ruiz R., Scarlata C., Sluiter J., Templeton D., Crocker D.,

(2012), Determination of Structural Carbohydrates and Lignin in Biomass, National

Renewable Energy Laboratory.




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CHAPTER 23

ULTRASONIC PRETREATMENT TO ENHANCEBIOHYDROGEN PRODUCTION FROM FOOD WASTE

Abhijit Gadhe, Shriram Sonawane, Mahesh Varma

Abstract

The present size of human civilization in terms of population and footprint has never been

witnessed before. The sustenance of this civilization at the present standard of living requires

huge amount of resources in terms of agricultural biomass, minerals and fossil fuels

(petroleum and coal). Minerals and fossil fuels are non renewable and their mining,

processing and use cause considerable disruption to the delicate processes of this planet

system, responsible for the well being of the entire biosphere. Algal biomass is a huge

resource as biomaterial feedstock, waiting to be harvested not only for fuel production but

also as animal feed and even human food, protein and vitamins. First micro algae were

cultured at the laboratory by O. Warburg in 1919. We have isolated and identified four fresh

water microalgae from nature. Biomass collection from microalgae is a big challenge for all

of us. There are few techniques like centrifugation, filtration, electro coagulation, dispersed

air electro coagulation and salt precipitation. We have demonstrated new technique like

flocculation using starch grafted polymer, cationic guar gum plant source. These biomasses

have been further used for downstream processing like bio hydrogen production and lipid

estimations. We used TAP-S media for hydrogen production. Almost all the microalgae

produced hydrogen but Chlamydomonas sp CRP7 showed better result. The total lipid was

found increase with nitrogen deprivation. All the microalgae have been characterized using

SEM, ITS studies for their genus and species confirmation.

23.1 Introduction

Irreversible depletion of traditional fossil fuels because of the rapid development of

new growing economics coupled with accumulation of green house gases (GHG) derived

from there burning has not only created energy insecurity but also a major problem of global

warming to this planet [Amaro et al., 2012]. Comparing to other forms of renewable energy

(eg; wind, tidal and solar) the chemical energy of biodiesel make it more suitable to be used in




243

the existing diesel engines or after blending it to certain ratios with petrodiesel. First

generation biofuel i.e. biodiesel and bioethanol are derive from food and oil crops which

includes sugarcane, maize, palm, soybean, rape seed, canola etc. Although the productionmethods, conversion technologies, and markets are well established, producing the first

generation biodiesel at required scale have generated lots of issues like redirecting food crops,

arable land, freshwater, and fertilizer to fuel production. Second generation biofuel are

derived from non-food biofeed stocks like Jatropa, agriculture forestry and municipal solid

wastes, these fuel resolves food vs. fuel problem, more efficient and environmental friendly,

with less requirement of farm land but still it faces the scalable problem to meet our desired

requirement [de Boer et al., 2012]. These issues have resulted in the development of the third

generation biofuel derived from microalgae.

23.1.1 Advantages of microalgae for biodiesel production includes

Microalgae can be grown on non arable land and waste water in this way it resolves

food vs. fuel issues. Microalgae can be grown all the year round; therefore the oil production

from microalgae exceeds the best oil seed crops. Microalgae have high growth potential and

even the oil content of many species ranges from 20-50% dry weight of biomass.Algae do not

require any herbicide, pesticide and weedicide for their cultivation.Microalgal biomass

production fixes waste CO2 and therefore improves the air quality also.Even the metabolic

pathway of the the microalgae can be easily modulated by just varying the culture condition.

This phenomenon can be adopted for critical R&D to enhance the oil production from

microalgae [Brennan and Owende, 2009; Mata et al., 2010]. Not only the appropriate site and

the type of production system is only responsible for the better productivity but also selection

of specific algae also plays an critical role in determining the overall productivity both

qualitatively and quantitatively. Following are the criterias that are generally considered forthe selection of microalgae’s for the biodiesel production [Lam and Lee, 2012]:

• Lipid content in percentage dry biomass.

• Growth rate in per unit time in per unit volume.

• Resistance to environmental changes and contamination.

• Simplicity for biomass separation and processing.

• Possibility of getting other valuable compounds.

A major hurdle for large scale economical production for the biofuel and other valued

products is the harvesting of relatively dilute algae from the medium in which they are grown




244

as it is one of the major energy input step during the whole process. Numerous techniques

have been available to harvest algae from the media or solution. Centrifugation, filtration and

settling are commonly employed where centrifugation is highly effective, for the conventionalfiltration systems microalgae are too small as they clog filters [Schlesinger et al., 2012], both

centrifugation and filtration is costly and energy intensive and therefore economically not

feasible for large scale production, and settling is a very slow and time consuming process

which works on large diameter algae only [Scholz et al., 2011].

Flocculation either by natural means or by chemically induced is one of the promising

approaches for the harvesting of algae. In our research up we found that microalgaes have

shown excellent aggregation with cationic polymers derived from tamarind, cassia (data notpublished) and guar gum [Banerjee et al, 2013].

Most of the algal cells are small in size, even though they are larger than true colloids,

algae posses many surface properties similar to true colloids. Scattered algal cells form a

stable microbial suspension which possesses chemically reactive cellular surface charge

because of the ionizable functional group present on the cellular surface. Since the stability of

algal suspension depends on the forces interacting among algal cells and also between algal

cells and water, algae can be considered a hydrophilic biocolloids. A better flocculation of

such negatively charged colloidal practical by cationic polymer is possible. These polymer

molecules attach themselves to one or more site on the surface of algae and a part of the

polymer also attaches itself to other algae in this way it forms an algae polymer matrix and

gets flocculated [Tenney et al., 1969].

The variation in the flocculation efficiency differs not only with the different algae but

also with the dosage of polymer [Wyatt et al., 2012]. We have also studied the harvesting of

freshwater microalgae (Chlorella sp. CB4) biomass by using polyacrylamide grafted starch

(St-g-PAM) St-g-PAM [Banerjee et al., 2012a] has been synthesized by microwave-assisted

method (i.e. synthesis based on free radical mechanism using microwave radiation in

synergism with ceric ammonium nitrate, to generate free radicals on the starch backbone).

Various grades of the graft copolymer were synthesized and optimized by varying the ceric

ammonium nitrate (CAN) and acrylamide (monomer) concentration. The process of synthesis

involved microwave irradiation of the reaction mixture until it sets into a viscous gel like

mass. The flocculation efficacy of polyacrylamide grafted starch has been investigatedthrough standard ‘Jar test’ procedure, in algal culture solution. The flocculation efficacy has




245

been determined in terms of decrease in optical density (at 750 nm) of the supernatant

collected after completion of the jar test protocol and found 85% recovery of algal biomass

with just using 0.8 ppm dosage of St-g-PAM .The use of this polymer even does not interferewith the quality of the harvested algal biomass and can be used for extraction of desired

compounds on large scale. But for flocculating using St-g-PAM is time taking so another

flocculating technique using cationic guar gum was also studied.

Cationic guar gum was carried out by the insertion of cationic moiety N-(3-chloro-2-

hydroxyl propyl) trimethyl ammonium chloride (CHPTAC) onto the polysaccharide backbone

of base polymer, which has shown better flocculation activity then St-g-PAM.


23.2.1 Isolation and culturing

Chlorella sp CB4 and Chlamydomonas sp CRP7 were isolated through phototaxis

followed by plating in TAP agar medium (Banerjee et al., 2012b). Chlorella sp CB2 was

isolated by serial dilution technique and Desmodesmus sp CB1 was isolated by using

microscopic manipulation technique.

23.2.2 Scanning Electron Microscopy (SEM)

Scanning electron microscopy was performed using the isolated four microalgae. 500

µl of the algal solution was diluted with 500 µl of water. The cells were washed three times

using distilled water by centrifugation at 3000 rpm for 3 minutes. Fixative glutaraldehyde was

used and dehydrated using series of acetone [Kaur et al., 2012].

23.2.3 Isolation of DNA and ITS region amplification

Before DNA isolation the algal cells were pelleted at 3000 rpm and washed for 3 to 4

times with distilled water to remove the adhering salts and metabolites so that they don’t

inhibit in downstream processing of DNA. Algal DNA was isolated [Alvarez et al., 2006].

PCR amplification of ITS1, 5.8S, and ITS2 regions was performed by PCR using the

following primers: Forward: 5´-GAAGTCGTAACAAGGTTTCC-3´ and Reverse: 5´-

TCCTGGTTAGTTTCTTTTCC-3´ [Timmins et al., 2009] The amplification conditions were

95 ºC for 4 min followed by 35 cycles of 45 s denaturation at 94 ºC, 1 min at 60 ºC as

annealing temperature and 1 min extension at 72 ºC with a final extension of 72ºC for 10 min.

The amplified products were separated by size on 1.0 % agarose gels found to in the range of

700–800 bp and are purified according to pure link PCR purification kit (Invitrogen).




246

23.2.4 Hydrogen production

For hydrogen production all the isolated algae (Chlorella sp CB2, Desmodesmus sp

CB1, Chlamydomonas sp CRP7 and Chlorella sp CB4) were harvested at its mid log phase

during their growth. The culture was flushed with nitrogen for 15 minutes to remove all the

dissolve oxygen subsequently placed under light for next 7 days for the hydrogen gas

production. The culture was tightly sealed by parafilm to prevent the leakage of gas [Melis et

al., 2000].

23.2.5 Lipid analysis

The algal biomass was centrifuge (4000 rpm/5 min) after the hydrogen production was

washed twice with distilled water. This dried extract was weighed and used to calculate total

percentage of lipids by dry weight. The lipid was isolated using Bligh and dyer method [Bligh

and Dyer, 1959].

23.2.6 Flocculation Using St-g-PAM and Cationic guar gum

Synthesis of polyacrylamide grafted starch by microwave assisted technique method

was adapted from Mishra et al 2011. In short 1 gm of starch was dissolve in requisite amount

of distilled water. Subsequent Ceric ammonium nitrate and polyacrylamide was added andirradiated at 800 W.

Cationic guar gum was synthesized using protocol [Banerjee et al., 2013]. Algal

flocculation efficacy of the synthesized grades of St-g-PAM and Cationic guar gum was

studied with fresh water microalgae Chlorella sp. CB4 and other isolated algae respectively

through standard ‘Jar test procedure’ in flocculator respectively.


23.3.1 Isolation

Chlorella sp CB4 and Chlamydomonas sp CRP7 was isolated through phototaxis

followed by plating in TAP agar medium (Fig. 1).

23.3.2 Scanning Electron Microscopy

This analysis was performed to understand surface morphological characteristics.

SEM images have help to identify to its genus level (unpublished data).

23.3.3 Isolation of DNA, ITS region amplification and phylogenetic relationship




247

DNA isolated from the samples was of high molecular weight (~30 Kb) and were

largely intact with little or no shearing. Yield of DNA, as estimated from spectrometer as ratio

(A260/280) of 1.9 and 1.72 for Chlamydomonas sp

CRP7 , Chlorella sp

CB4 respectively. The

amplicon for ITS was found to lie between 700 to 800 bp. The ITS sequence were submitted

at NCBI (JQ408690, JQ710681, JQ710682, JQ710683). Blast was performed

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) using different four ITS sequence to find out the

closest similarity between the isolated species and deposited sequences in the database. The

Blast search result shows the closest similarity with Chlamydomonas and Chlorella and

Desmodesmus family. The phylogenetic tree (figure 2) was constructed by using Mega 5 by

NJ method with 1000 replicate.

Figure 1- Represents the different isolation step. (Arrow mark is for Chlorella sp CB4)

23.3.4 Hydrogen production

All the isolated and purified algae were tested for the hydrogen gas production by

transferring them into TAP-S medium from TAP medium. The headspace gas was analyzed

by GC and found to be pure hydrogen.

23.3.5 Lipid analysis

Lipid was found to increase three fold in N starved condition then compared to normal

condition Percentage lipid was calculated by gravimetrically and is as follows (Unpublished

result).

23.3.6 Flocculation Using St-g-PAM and Cationic guar gum




248

Various grades of St-g-PAM were synthesized (Table 1) and was used for flocculation

purpose (Figure 3).

Figure 2- Phylogenetic tree of isolated genus

Figure 3- Study of flocculation efficacy of various synthesized grades of St-g-PAM




249

Table 1- Synthesized grades of St-g-PAM

Among this St-g-PAM2 (907 %) shows best flocculation activity so, further

flocculation activity was checked in different pH (Figure 4) and found 85% recovery (Table

2) of algal biomass with just using 0.8 ppm at pH 10.5.

Table 2- Percentage recovery of algal biomass using St-g-PAM at an optimized dosage

Figure 4- The extent of flocculation of algal biomass With time, at different pH, using St-g-

PAM as a flocculant

23.3.7 Flocculation using cationic guar gum

The time to flocculate Chlorella sp CB4 was not suitable for harvesting large scale

biomass. In order to overcome this cationic guar was synthesized and used to flocculate all the

other four microalgae (Table 3).




250

Table 3- Flocculation characteristic with Cationic guar gum with four different algae.

23.4 Conclusions

In present study we have characterize different microalgal strains through Molecular,

microscopical, bioharvesting and lipid techniques. All the isolated strain is able to produce

hydrogen in TAP-S medium. Morphological features through light microscopy are usually not

reliable to identify the microalgae. Therefore, SEM studies and molecular characterization are

needed to identify into its genus level. A phylogenetic analysis was done on basis of ITS1-

5.8s-ITS2 region. Though, algal harvesting is also a challenging task for its further processing

towards many downstream applications. St-g-PAM and cationic guar gum (CGG) was

synthesized and used it for the flocculation purpose which bypass the energy consuming

centrifugation step. CGG neutralize negative charge of the algae cells resulting in

neutralization to form floc. The algal flocculation efficacy of CGG can be well explored for

commercial algal harvesting. Cationic guar gum was proved to be a better flocculant than St-

g-PAM towards harvesting.

References

1. Alvarez E.V., Lago-Leston, A., Pearson G.A., Serrao E.A., Procaccini G., Duarte C.M.

and Marba N (2006) Genomic DNA isolation from green and brown algae (Caulerpales

and Fucales) for microsatellite library construction. J Phycol., 42:741–745.

2. Amaro H.M., Macedo A.C. and Malcata F.X. (2012) Microalgae: An alternative as

sustainable source of biofuels? Energy, 44:158-166.

3.

Banerjee C., Bandopadhyay R. and Shukla P. (2012b) A simple novel agar diffusion

method for isolation of indigenous microalgae Chlamydomonas sp. CRP7 and Chlorella

Materia

l usedAlgae used Percentage

recovery

Biomass used

for flocculation

(gm/L)

Optimize

d dosage

(ppm)

pH Time

(min)

CGGChlorella sp.

CB494.5 0.78 40 7.52 30

CGGChlamydomonas

sp. CRP792.15 0.89 100 7.34 15




251

sp. CB4 from operational swampy top soil. Indian J Microbiol, ISSN: 0046-8991, DOI

10.1007/s12088-012-0295-6.

4.

Banerjee C., Ghosh S., Sen G., Mishra S., Shukla P. and Bandopadhyay R. (2013)Study of algal biomass harvesting using cationic guar gum from the natural plant source

as flocculant. Carbohydr. Polym., 92:675-681.

5. Banerjee C., Gupta P., Mishra S., Sen G., Shukla P. and Bandopadhyay R. (2012a)

Study of polyacrylamide grafted starch based algal flocculation towards applications in

algal biomass harvesting. Int J Biol Macromol, 51:456– 461.

6. Bhatnagar S.K., Saxena A. and Kraan, S. (2011) Algae based biofuels: A Review of

Challenges and Opportunities for Developing Countries. Food and Agriculture

Organization of the United Nations. Italy

7. Bligh E.G. and Dyer W.J. (1959) A rapid method of total lipid extraction and

purification. Can. J. Biochem. Physiol., 37:912–917.

8. Brennan L. and Owende P. (2009) Biofuels from microalgae—A review of technologies

for production, processing, and extractions of biofuels and co-products. Renewable

Sustainable Energy Rev., 14:557-577.

9. De Boer K., Moheimani N.R., Borowitzka M.A. and Bahri P.A. (2012) Extraction and

conversion pathways for microalgae.J Appl Phycol. DOI 10.1007/s10811-012-9835-z.

10. Kaur S., Sarkar M., Srivastava R.B., Gogoi H.K. and Kalita M.C. (2011) Fatty acid

profiling and molecular characterization of some freshwater microalgae from India with

potential for biodiesel production. New Biotechnol, 29:332-344.

11. Lam M.K. and Lee K.T. (2012) Microalgae biofuels: A critical review of issues,

problems and the way forward. Biotechnol Adv., 30:673–690.

12. Mata T.M., Martins A.M. and Caetano N.S. (2010) Microalgae for biodiesel production

and other applications: A review. Renewable Sustainable Energy Rev., 14:217–232.

13. Melis A., Zhang L., Forestier M., Ghirardi M.L. and Seibert M. (2000) Sustained

photobiological hydrogen gas production upon reversible inactivation of oxygen

evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol., 122:127–136.

14. Mishra S., Mukul A., Sen G. and Jha U. (2011) Microwave assisted synthesis of poly-

acrylamide grafted starch (St-g-PAM) and its applicability as flocculant for water

treatment. Int. J. Biol. Macromol., 48:106-111.

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Müller T., Philippi N., Dandekar T., Schultz J., Wolf M. (2007) Distinguishing species.

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16. Schlesinger A., Eisenstadt D., Bar-Gil A., Carmely H., Einbinder S. and Gressel J.

(2012) Inexpensive non-toxic flocculation of microalgae contradicts theories;

overcoming A major hurdle to bulk algal production Biotechnol Adv., 30:1023–1030.17. Scholz M., Hoshino T., Johnson D., Riley M.R. and Cuello J. (2011) Flocculation of

wall-deficient cells of Chlamydomonas reinhardtii mutant cw15 by calcium and

methanol. Biomass Bioenerg, 35: 4835–4840.

18. Tenney M.W., JR W.F.E.,.Schuessler R.G. and.Pavoni J.L.(1969) Algal Flocculation

with Synthetic Organic Polyelectrolytes. Appl Microbiol., 18:965–971.

19. Timmins M., Thomas-Hall S.R., Darling A., Zhang E., Hankamer B., Marx U.C. and

Schenk P.M. (2009) Phylogenetic and molecular analysis of hydrogen-producing green

algae. J. Exp. Bot., 60:1691-1702.

20. Wyatt N.B., Gloe L.M., Brady P.V., Hewson J.C., Grillet A.M., Hankins M.G. and Pohl P.I.

(2012) Critical Conditions for Ferric Chloride-Induced Flocculation of Freshwater Algae.

Biotechnol. Bioeng., 109:493–501.




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CHAPTER 24

Biological hydrogen production by facultative anaerobic bacteria Enterobacter aerogens (MTCC 8100)

Virendra Kumar, Richa Kothari and S.K.Tyagi

Abstract

Biological hydrogen is an efficient way for the degradation of organic matter to the useful

products with the help of microorganism. It basically deals with the organic matter present in

the solid or liquid substrates. Some bacteria like Clostridium, Enterobactor, and Escherichia

etc. are found to be potential to degrade the organic matter in the useful product such as

hydrogen and ethanol. Among the various process of biohydrogen production, dark

fermentation seems to be feasible in this aspect. A study of biological hydrogen production by

anaerobic facultative bacteria Enterobactor aerogens was investigated by using glucose as the

feedstock for finding the feasibility at experimental part. The growth period of the bacteria,

gas production, pH and glucose consumption was investigated during the study. In salt

medium with glucose the gas produced was comprises of 35 % hydrogen. The process shows

40% glucose consumption in 18 hours at 30°C with pH of 6.5. The study reveals the selected

species is suitable for biohydrogen production from carbon source.

Key words: Biological hydrogen production, Enterobacter aerogens

24.1 Introduction

Hydrogen is a promising alternative for carbon based fuel compared to others because

it is a clean renewable source of energy having calorific value 122 Kj/g and produces water as

only by product after burning [Chang et al., 2002]. Biological routes of energy production are

taking more attention nowadays because it generates energy as well as treat the waste without

harming the environment. Biological hydrogen production is a microbial mediated process.

The generation of hydrogen by biological means is not energy intensive compared with the

conventional thermo-chemical techniques, since the operating temperature and pressure are

not very high. The method of the dark fermentation has certain advantages compared with the

other biological processes. In contrast to bio-photolysis and photo fermentation, the process




254

needs no solar radiation, but the required energy is supplied by the organic substrates and

hence the process is not interrupted during the night. Moreover, the production rate of the H2

of the fermentative bacteria in comparison with the other biological processes is greater(Kumar et al., 2000; Nath et al., 2005). Fermentative hydrogen production also has good

properties for the biotechnological applications, due to the capability to exploit variety of

substrates (Nath and Das, 2003).

Dark fermentation of organic substrate is manifested by the majority of diverse group

of bacteria obligate as well as facultative anaerobes that perform several biochemical

reactions. Optimization of the performance of these bacteria increases the conversion

efficiency of organic matter to hydrogen. Facultative anaerobic bacteria are gram-negative,rods shaped, with relatively simple nutrient requirements (Schmauder, 1992). Among the

species that can produce H2, are Escherichia ( E. coli), Proteus (P. vulgaris), Enterobacter (E.

aerogenes). These bacteria ferment sugars to a variety of end products such as acetate,

formate, lactate, succinate, ethanol, CO2 and H2. The degradation of organic matter in

anaerobic environments by microbial consortia involves the cooperation of a population of

microorganisms that generate a stable, self-regulating fermentation [Das, 2001]. First,

hydrolytic bacteria hydrolyze polymeric proteins and sugars. Then, fermentative bacteria form

organic acids, H2 and CO2 from monomeric molecules (Fig. 1). At that point, H2 and acetate

can be utilized and/or produced by several microbial groups. Thus, acetate is generated during

acetogenesis from CO2 reduction and H2 by autotrophic acetogens via the Wood– Ljungdahl

pathway, a process named homoacetogenesis [Köpkea et al., 2012].

The process of hydrogen production by facultative anaerobes is given in figure 1[Jenni

et al. 2011].The organic substrate such as glucose, is degraded to pyruvate by the glycolysis.

Pyruate was further converted to acetyle coenzyme A (Co A) and formate by the enzymepyruate formate lyase (PFL). Formate is again converted to hydrogen and carbon dioxide by

the enzyme formate hydrogen lyase (FHL) complex. In this study the facultative anaerobic

bacteria Enterobacter aerogens, MTCC no 8100 is selected for biological hydrogen

production through anaerobic consortia.




255

Figure 1: Biohydrogen production process by facultative anaerobic bacteria


24.2.1 Microbial culture development

A bacterial culture of Enterobacter aerogens (MTCC no.8100) was brought from

Microbial Technology Culture Centre (MTCC), Chandigarh and grown on prescribed media.One litre of growth medium contains 1gm of beef extract, 2gm of yeast extract, 5gm of

peptone and 5gm of NaCl with distilled water. The culture was grown in media at 30oC

temperature in an incubator shaker (New Brunswick Scintific Innova 43) for 24 hours

incubation period.

(a) (b)

Figure 2. A microscopic view of Enterobactor aerogens (a) & (b)

24.2.2 Preparation of fermentation medium

Pyruvate

Acetyl-CoA Formate

H2

CO2

Ethanol Acetate

Glucose




256

Anaerobic liquid media used for experimental study contained, 10.0 g/l of Glucose ,

4.0 g/l of (NH4)2SO4, 4.0 g/l of KH2PO4, 4.0 g/l of Na2HPO4, 1.0 g/l of yeast extract, 0.20

g/l of MgSO4 and Trace elements solution 2.0 ml/l having composition of 1.0 ml/l of HCl,100 mg/l of MnCl2.4H2O, 70mg/l of ZnCl2, 60mg/l of H3BO3, 200mg/l of CoCl2.6H2O, 10

mg/l of CuCl2.2H2O, 20mg/l of NiCl2, 30 mg/l of Na2MoO4.2H2O.

24.2.3 Experimental set-up

Experiments were done in anaerobic jars with batch set-up plan (Walker et al., 2009).

Total volume of each jar 1000 ml was made for the study having working volume 600 ml.

The fermentation medium sample with 10% v/v microbial seed culture was kept for study

with adjusting the initial pH at 6.5.

Figure 3. Experimental Batch set-up for gas measurement.


Hydrogen content in the gas was determined with Gas Chromatograph (GC 5765

Nucon India Make ) equipped with Thermal Conductivity Detector (TCD) a stainless steel

column packed with porapak Q(80/100 mesh) . The operational temperature of the oven

injector port and detector were 70, 120 and 1200C respectively. Nitrogen was used as the

carrier gas at a flow rate of 20 ml/min. The sugar content was determined by DNS method

(Miller, 1959). Biomass was measured by turbidimetricaly at 600 nm using UV-Vis

Spectrophotometer (Perkin Elmer Lambda 35) also 1 ml of the sample was centrifuged and

dried to measure dry cell weight.




257


24.3.1 Growth of Bacteria

Most facultative anaerobes produce hydrogen through breakdown of glucose to

pyruate forwarded by volatile fatty acids formations during fermentation (Elsharnouby et al.,

2013). The growth pattern of the bacteria in growth medium was analyzed after each hour for

OD600 as well as for dry cell weight and it was found that in both cases the stationary phase

was achieved after 24 period of the growth.

24.3.2 Biochemical assay for the carbohydrate fermentation

The biochemical analysis of bacteria for the fermentation of Carbohydrate (Glucose

and Sucrose) was done in lab [Aneja, 2010]. The results obtained were given in Table 1. All

test tubes in duplicate for assay’s observation.

Figure 4. Growth curve of bacteria Growth medium (a) OD600 (b) Dry cell weight basis.

Table 1: Fermentation of sugar assay by selected microbial species

0

05

1

15

2

1 4 7 10 13 16 19 22 25 28

O D 6 0 0

Time (h)

0

50

100

150

200

1 3 5 7 9 11131517192123252729

B i o m

a s s ( m g / L )

Time (h)

Composition Results Observation

Media + Phenol red _ No colour change

Media + Bacteria + Phenol red _ No color change

Media + Bacteria + Phenol red

Glucose

+ Yellow color appeared +

Gas bubbles seen

Media + Bacteria + Phenol red

Sucrose

+ Yellow color appeared +

bubbles seen




258

24.3.3 Hydrogen Production

The maximum yield of facultative anaerobic bacteria by dark fermentation is 2 mol

H2 /mol glucose or 0.248L H2 /g glucose (2 mol H2×22.4 L/mol)/180g glucose) at standard

temperature and pressure [Hallenbeck, 2004]. In our study maximum 2g of glucose was

consumed and total water displacement was approximate 600ml. The concentration of H2 in

the gas was 35 % so H2 was 210 ml or 105 ml H2 /g glucose or 0 .105 L H2 /g glucose. This

low yield was due to the decrease in the pH of the medium during fermentation (figure 8) and

formation of volatile fatty acids that could not determined during course of experiment

however it was supported by literature available. [Byung et al., 1985; Debrock et al., 1992;

Khanal et al., 2004]. While the optimum yield was found on controlled pH 5.5 to 6.5 [Kumarand Das, 2000; Khanna et al., 2011]

(a) (b)

(c)

Figure 5. Biochemical assay of selected microorganism (a) initial phase (b) fermentation

phase (c) gas production phase.

24.3.4 Glucose Consumption

Initial concentration of Glucose 1% (10g/l) was taken as the feedstock for study.

During the fermentation, bacteria utilized only 40% of the glucose and rest was remained as

Volatile fatty acid. The facultative anaerobic bacteria generate acetyl coenzyme A and

formate by utilizing glucose. Formate is then converted to hydrogen and carbon dioxide. An




259

acidic condition exists due to the formation of other fermentation products such as lactate,

acetate formate, succinate, ethanol and butandiol [Hallenbeck and Benemann 2002].

Figure 6. Cumulative hydrogen production.

Figure 7. Glucose consumption and cumulative gas production of the bacteria during

hydrogen production.

24.3.5 Effect of pH

The pH effect on biohydrogen production was investigated that fall during the process

from 6.5 to 4.3. As resultant in the initial stage of the fermentation the production of gas was

high as at the pH 5 to 6.5 till the 7.5th

hours and it was decreased to the 4.3 in the 14th

hours so

as the gas production. However in case of batch with initial pH above 6.5 the yield decreased

0

50

100

150

200

250

0 15 3 45 6 75 9 105 12 135 15 165 18

H y d r o g e n P r o d u c t i o n ( m l )

Time (h)

0

1

2

3

4

5

6

0

100

200

300

400

500

600

700

0 1 5 3

4 5 6

7 5 9

1 0 5 1 2

1 3 5 1 5

1 6 5 1 8

u c o s e , g " L -

a s r o # u c t i o n , m

-

%ime ,/-

as

ro#uction,m&-

&ucose

,g"L-




260

drastically. Whereas some workers found the initial pH 6.5 was optimum for hydrogen

production (Wei et al., 2010 and Lin et al., 2010).

Figure 8. pH pattern and gas production in fermentation medium

24.4 Conclusions

From the above study it was concluded that the selected species Enterobactor

aerogens shows the feasibility to produce hydrogen from glucose. During the study it was

also observed that consumption of glucose was only 40% that causes low production of

hydrogen. The low production of hydrogen was due to the lowering of pH during the process.

These experiments showed that the measurement of metabolic gases i.e. the useful method of

monitoring bacterial fermentation, which could not only measure the productivity of H2 but

also reveal process unknown so far. Experiment also showed that the Enterobacter aerogens

utilizes glucose and convert it in to H2 and organic acids strongly dependent on the bacterial

isolate. Thus there is a possibility to use glucose as a substrate for H2 production, althoughprobably optimization of bacterial strains or their genetic modification will be desired.

Acknowledgement

The authors wish to thank Sardar Swarn Singh- National Institute of Renewable

Energy, Kapurthala Punjab. An autonomous institute of Ministry of New and Renewable

Energy, New Delhi., Dr. Sachin Kumar, Scientist ‘B’ and Dr. Subhashish Behra, Post Doc.

Fellow of the institute to provide us the laboratory and guidance during the course of study.

0

1

2

3

4

5

6

7

0

20

40

60

80

100

120

0 15 3 45 6 75 9 105 12 135 15 165 18

p

a s r o # u c t i o n , m -

%ime ,/-

as ro#uction

,m&-

p




261

References

1. Chang, J. S., Lee, K. S. and Lin, P. J. (2002) Biohydrogen production with fixed-bed

bioreactors. International Journal of Hydrogen Energy, 27:1167-1174.

2. Das, D., Veziroglu, T.N. (2001) Hydrogen production by biological processes: a survey

of literature. International Journal of Hydrogen Energy, 26:13–28.

3. Hallenbeck, P.C. and Benemann J. R. (2002) Biological hydrogen production

fundamental and limiting process. International Journal of Hydrogen Energy 27:1185-

1193

4. Hallenbeck, P. C. (2004) Fundamental of the fermentative production of hydrogen.

Water Science and Technology 52: 21-29.5. Jenni, J., Seppa, la., Jaakko A, Puhakka., Olli, Yli-Harja., Matti T, Karp., and Ville,

Santala,. (2011) Fermentative hydrogen production by Clostridium butyricum and

Escherichia coli in pure and co-cultures. International journal of Hydrogen Energy

36:10701 -08.

6. K. R. Aneja (2010) Experiments in Microbiology Plant Pathology and Biotechnology.

New age International (p) Limited Publishers, New Delhi.

7. Nath, K.., Muthukumarb, M., Kumarb, A. and Das D. (2008) Kinetics of two-stage

fermentation process for the production of hydrogen. International Journal of Hydrogen

Energy 33:1195–1203.

8. Lin, Y.H., Juan, M.L., and Hsien, H. J. (2010) Effects of temperature and initial pH on

biohydrogen production from food processing and wastewater using anaerobic mixed

cultures. Earth Environ. Sci., DOI: 10.1007/s10532-010-9427-2.

9. Walker, M.., Zhang,Y., Heaven, S. and Charles Banks.(2009) Potential errors in the

quantitative evaluation of biogas production in anaerobic digestion processes.

Bioresource Technology, 100: 6339–6346.

10. Köpke, M., Claudia, H., Hujer, S., Liesegang, H., Wiezer, A., Wollherr, A.,

Ehrenreich, A., Liebl, W., Gottschalk, G. and Dürre, P.(2012) Clostridium ljungdahlii

represents a microbial production platform based on syngas. Proceeding of National

Academy of Science of United State of America. 107: 13087–13092.

11. Khanna, N., Kotay, S.M., Gilbert, J.J. and Das D. (2011) Improvement of biohydrogen

production by Enterobacter cloacae IIT-BT 08 under regulated pH. Journal of

Biotechnology 152:9–15.




262

12. Kumar, N. and Das, D.(2000) Enhancement of hydrogen production by Enterobacter

cloacae IIT-BT 08,. Process Biochemistry 35:589–593.

13.

Nath, K., Das, D. (2003) Hydrogen from biomass. Current Science.85:265-271.14. Elsharnouby, O., Hafez

,H., Nakhla, G., Naggar, M. H. E. (2013) A critical literature

review on biohydrogen production by pure cultures. International Journal of Hydrogen

Energy Volume 38:4945–4966.

15. Schmauder, H.-P. (1992). Hans G. Schlegel, Allgemeine Mikrobiologie. Stuttgart–New

York 1992. Georg Thieme Verlag. 3-13-444607-3.

16. Wei, J., Liu, Z.T., Zhang, X., (2010) Biohydrogen production from starch waste water

and applications in fuel cell. International Journal of Hydrogen Energy 35: 2949–2952.




263

CHAPTER 25

ENHANCED BIOHYDROGEN PRODUCTION FROMGLYCEROL USING PRETREATED MIXED CULTURE

Anbalagan Krishnasamy, Mohanraj Sundaresan, Kodhaiyolii Shanmugam, Pugalenthi velan

Abstract

The anaerobic batch fermentation of glycerol was developed using pretreated dairy sludge as

mixed culture to enhance the biohydrogen production. The effect of pH and initial substrate

concentration on fermentative hydrogen production were also investigated using heat treated

mixed culture. The experimental results showed that the hydrogen yield from heat, acid, base

pretreated dairy sludge was 300, 250 and 120 mL of H2, respectively, and its corresponding

hydrogen content was 43 %, 38 % and 29 %. The results of the study demonstrate that the

heat treated dairy sludge enhanced the biohydrogen production when compared to other

pretreatment methods. The optimum values of pH and glycerol concentration for efficient

biohydrogen production were 6.0 and 30 g/L respectively. The highest biohydrogen

production and hydrogen content were 510 mL and 42 % respectively, under optimum

conditions.

Key words: Biohydrogen, Mixed culture, Glycerol, Pretreatment.

25.1 Introduction

Hydrogen is an ideal future energy carrier for alternative of fossil fuels because it releases a

huge amount of energy, and generates no air pollutants. Hydrogen is mainly used for many

applications such as fuel for automobile and electricity generation (Foglia et al., 2010; Wu et

al., 2011). 95% of the hydrogen produced by conventional methods is expensive and energy

intensive process (Chen et al., 2008; Foglia et al., 2010). Fermentative hydrogen process is

one of the alternative processes for conventional methods (Elsharnouby et al., 2013).

Biological methods for the biohydrogen production such as dark fermentation, photo

fermentation, and integrated system using both dark fermentation and photo fermentation

have been extensively studied (Prakasham et al., 2009; Pott et al., 2013; Chen et al 2008).




264

Among these processes, dark fermentative hydrogen production was faster than the other

processes and simple operation (Sinha and Pandey, 2013).

Biohydrogen is produced by dark fermentation from carbohydrates and other

renewable organic substrates (Li et al., 2008). Researchers have focused to develop the

hydrogen production from various organic wastes including agricultural, lingocellulosic,

industrial waste and wastewater (Elsharnouby et al., 2013). However, the biohydrogen

production from pure and waste glycerol received the great attention in recent years.

Worldwide, the development in biodiesel production sector glycerol has noticeably increased

quantity of waste (Kumar and Lin, 2013). Theoretically 3 mole of hydrogen can be produced

per mole of glycerol in suitable fermentative process. Few researchers carried outbiohydrogen production from pure glycerol and waste glycerol using pure cultures such as

Enterobactor aerogens (Markove et al., 2010), Klebsiella pneumoniae TR17 (Chookaewa et

al., 2012), Thermotoga neapolitana (Nago and Sim, 2011), and mixed cultures (Selembo et

al., 2009; Seifert et al., 2009).

Fermentative microorganisms exist enormously in natural habitat such as soil, sludge

and wastewater. These resources are used as possible source of inoculum for fermentative H2

production (Sinha and Pandey, 2011; Selembo et al., 2009). Microbial mixed cultures are

important alternatives to pure microbial cultures in fermentation processes because it can be

easily operated under unsterile conditions (Rafrafi et al., 2013). The main challenges in mixed

microbial for fermentative process are; microbial growth rate is increased when dark

fermentation using open environmental source (without pretreatment) resulted low hydrogen

yield and the pathway is shifted from acidogenesis to methanogenis. These environmental

mixed cultures consume H2 in two forms first one is consumption of NADH2 and another one

is consumption of molecular hydrogen (Saady, 2013). Therefore, pretreatment of sludge isone of the important features for selecting the efficient hydrogen producing microflora. The

pretreatment inoculum can control the methanogenesis and improve the activity of hydrogen

producers (Wang et al., 2011).

Pretreatment methods including heat, acid, alkali and ultrasonic treatment are widely

used for efficient fermentative hydrogen production (Cai et al., 2004; Ren et al., 2008; Wang

et al., 2011). Among these pretreatment methods, heat treatment process has been used to

create the constant inoculums for hydrogen production (Logan et al., 2002). The mostimportant issues for development of a fermentative hydrogen production process using mixed




265

culture require the large quantities of stable inoculums. Enrichment of mixed microbial

cultures gets considerable attention for stable inoculums in efficient hydrogen production

because they can get better reaction stability and efficiency of H2 production (Hawkes et al.,2002)

Hence, the efficient hydrogen production from glycerol by dairy sludge as mixed

cultures was investigated and different pretreatment methods including heat, acid and alkali

treatment methods were also studied to improve the fermentative hydrogen production. In

addition, effects of glycerol concentration and initial pH on hydrogen production were also

examined.


25.2.1 Pretreatment and enrichment of H2 producing mixed culture

The sludge inoculum was obtained from local dairy industry, Tiruchirappalli, Tamil

Nadu, India. Prior to use the sludge as a inoculums was pretreated by three methods including

heat treatment (105oC for 30 min), acid treatment (pH 3, 1 N HCl, 24 h) and alkali treatment

(pH 11, 1 N NaOH, 24 h) to inactivate the hydrogen consuming bacteria and facilitating the

growth of mixed culture. Further the pH was adjusted to 6.5 by the addition of NaOH and

HCl for acid and alkali treated cultures.

The enrichment of mixed culture was carried out by following medium compositions

per liter; 10 g of glycerol 3.4 g of K2HPO4, 1.3 g of KH2PO4, 4,2g of (NH4) 2SO4, 0.2 g of

MgSO4, 20 mg of CaCl2 and 5 mg FeSO4, according to Barbirato et al., (1995) The anaerobic

conditions were created by flushing the bottles with pure N2 for 10 min. 48 hours grown

mixed culture was transferred to fresh growth medium and enriched mixed cultures were

maintained and preserved at 4oC for further use.

25.2.2 Experimental setup

Biohydrogen production experiments were conducted in a 250 mL screw cap bottle in

a batch mode. 200 ml of fermentation medium containing 6 g (30 g/L) of glycerol was

inoculated with 2 mL of the pretreated mixed culture. The pH of the growth medium was

adjusted in the range from pH 6.0 to pH 6.5. Bottles were flushed with nitrogen for 10 min to

generate anaerobic condition and placed in a magnetic stirrer at 150 rpm. All the fermentative

experiments were carried out in duplicates, and control experiments were conducted in

parallel using the dairy sludge without pretreatment. The optimization experiments were




266

carried out for heat treated mixed culture with different initial substrate concentration from 10

to 50g/L and pH values from 4.0 to 8.0.The pH was adjusted on addition of 1N NaOH or HCl.


The quantity of biogas produced in dark fermentation was measured from time to time

by water displacement method. The hydrogen, methane and carbon dioxide content were

calculated by gas chromatograph (GC, SHIMADZU GC-2014). GC was equipped with a

thermal conductivity detector (TCD) and stainless column packed with Porapak Q (80/100

mesh). The injection port, column oven and detector were operated at 40oC, 40

oC, and 80

0C

respectively. Nitrogen was used as carrier gas at a flow rate of 20 mL/min. The pH values of

the fermentation media were analyzed by a pH meter (Elico Model –LI 615.)


25.3.1 Comparison of pretreatment methods

The batch experiments with dairy sludge pretreated by heat, acid and alkali were

investigated and the experiment without pretreated sludge was done as control for

comparison. As seen in the Figure 1, the cumulative hydrogen production of 300, 250 and 120

mL were heat, acid and alkali treated cultures, respectively and its corresponding hydrogencontent was 43, 38 and 29 %. On the other hand, untreated culture showed that the cumulative

hydrogen production and hydrogen content were 270 mL and 22% respectively. These

findings demonstrate that the relatively higher hydrogen production was obtained at heat

treated inoculums when compared to other pretreated cultures and control. Moreover, this

experiment indicated that the methanogenic activity in fermentative process was completely

inhibited for all pretreatment methods. This similar observation was also found by other

researchers (Wang et al., 2011). Recently, Rossi et al. (2011) reported that the heat

pretreatment was more efficient for biohydrogen production using mixed culture from

glycerol and also maximal substrate degradation were obtained than the other pretreatment

methods.

25.3.2 Eff ffff ff ect of glycerol concentration on hydrogen production using heat treated mixed

culture

Biohydrogen production from glycerol by heat treated mixed culture was evaluated in

this study to find out the enhancement efficiency. To evaluate the fermentative hydrogen

production, glycerol concentration was varied in the range from 10 to 50 g/L (Fig. 2). The




267

experimental results show that the hydrogen production was increased when glycerol

concentration was differed from 10 to 30g/L. Further the hydrogen production was decreased

at above 30 g/L. The possible reason for low hydrogen production may be due to theinhibition of higher substrate concentration or fermentative end product inhibition of this

process (O-Thong et al., 2008). The maximum cumulative H2 production of 510 ml and

hydrogen content of 42% were obtained at 30 g/L of glycerol concentration. However our

data showed that the optimum concentration of glycerol was important for efficient

fermentative hydrogen production. Similar result was observed by Sittijunda and Reungsang,

(2012). They reported that the maximum hydrogen production was found to be in the range of

25-50 g/L of glycerol concentration. Seifert et al. (2009) also reported that the hydrogen

production increased from 0.345 to 0.715 l H2 /L when the glycerol concentration was

increased from 5 to 30 g/L.

Fig. 1- Effect of Pretreatment methods on hydrogen production

Fig 2- Effect of glycerol concentration on hydrogen production




268

25.3.3 Effect of pH on hydrogen production using heat treated mixed culture

The eff ect of initial pH (4.0 to 8.0) on fermentative hydrogen production from glycerol

by heat pretreated mixed culture was studied. pH is an key factor for fermentative H 2

production, Kapdan and Kargi, (2006) reported that the pH in production medium influenced

the hydrogen production yield, composition of biogas content and specific hydrogen

production rate. Also, pH affected the hydrogenase activity as well as the fermentative

hydrogen producing pathway (Sinha and Pandey, 2011). From Figure 3, the experimental

result indicated that the low hydrogen production was obtained at initial pH 4.0 and hence the

lower pH was not suitable for this fermentative process. The possible reasons for lower H 2

production in fermentation were reported by (Mohan et al., 2007). They reported that the pHvalues below 5.0 inhibited the acidogenic metabolism and shifting the metabolic pathway to

solventogenesis which led to inhibition of H2 production. The cumulative hydrogen

production was increased at the initial pH from 4.0 to 6.0. Further, the hydrogen production

was decreased as the initial pH was increased from 6.0 to 8.0. The same effect was found in

hydrogen content at all the initial pH. The present study confirmed that the optimum initial

pH for efficient fermentative H2 production was 6.0. Similar result was found by other

researchers. Venkata Mohan et al. (2007) studied effect of pH and substrate concentration

using heat-treated mixed culture and they found that the optimal pH 6.0 was suitable for

fermentative hydrogen production. Yossan et al. (2012) also reported that the maximum

hydrogen production by heat treated palm oil mill lagoon sludge at pH 6.0.

Fig 3- Effect of pH on heat treated mixed culture




269

25.4 Conclusions

The efficient hydrogen production from glycerol using heat treated inoculums under

optimum condition was investigated. The experimental results indicated that the pretreatment

methods were effectively suppressed methanogenic activity and only the hydrogen and carbon

dioxide was present in evolved gas. Among the pretreatment methods, heat pretreatment was

the most effective method for hydrogen production from glycerol. The maximum hydrogen

production of 300 mL and hydrogen content of 42% were achieved by heat treated inoculums

experiment. In addition, the fermentative H2 production using heat treated dairy sludge

showed that the optimal condition was 30 g/L of glycerol at initial pH 6.0 for efficient

hydrogen production.

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21. Rossi D.M., Costa J.B., Souza E.A., Peralba M.C.R., Samios D. and Ayub M.A. (2011)

Comparison of different pretreatment methods for hydrogen production using

environmental microbial consortia on residual glycerol from biodiesel. Int. J. Hydrogen

Energy, 36:4814-4819.

22. Seifert K., Waligorska M., Wojtowski M. and Laniecki M. (2009) Hydrogen generation

from glycerol in batch fermentation process. Int. J. Hydrogen Energy, 34: 3671-3678.

23. Selembo P.A., Perez J. M., Lloyd W.A. and Logan B.E. (2009) Enhanced hydrogen and

1,3-propanediol production from glycerol by fermentation using mixed cultures.

Biotechnol. Bioeng. 104: 1098-106.

24. Sinha P. and Pandey. (2013) Biohydrogen production from various feeds tocks by

Bacillus firmus NMBL-03. Int. J. Hydrogen Energy, In press

25.

Sinha P. and Pandey A. (2011) An evaluative report and challenges for fermentative

biohydrogen production. Int. J. Hydrogen Energy, 36:7460-7478.

26. Sittijunda S. and Reungsang A. (2012) Media optimization for biohydrogen production

from waste glycerol by anaerobic thermophilic mixed cultures. Int. J. Hydrogen

Energy, 37:15473-15482.

27. Wang Y.Y., Ai P., Hu C.X. and Zhang Y.L. (2011) Effects of various pretreatment

methods of anaerobic mixed microflora on biohydrogen production and the

fermentation pathway of glucose. Int. J. Hydrogen Energy, 36:390-396.

28. Wu K., Lin Y., Lo Y., Chen C., Chen W. and Chang J. (2011) Converting glycerol into

hydrogen, ethanol, and diols with a Klebsiella sp. HE1 strain via anaerobic

fermentation. J. Taiwan. Inst. Chem. Eng., 42:20-25.

29. Yossan S., O-Thong S. and Prasertsan P. (2012) Effect of initial pH, nutrients and

temperature on hydrogen production from palm oil mill effluent using thermotolerant

consortia and corresponding microbial communities. Int. J. Hydrogen Energy,

37:13806-13814.




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Part IV

Chemical Conversion




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CHAPTER 26

ISOLATION AND CHARACTERIZATION OF FRESHWATER

MICROALGAE SCENEDESMUS FROM CONTAMINATED

FIELD SAMPLES FOR BIOENERGY GENERATION

Mayur M. Phukan and B. K. Konwar

Abstract

A green alga (chlorophyceae), representative of the microalgal genera Scenedesmus was

isolated from a eutrophic water body (26°40'10"N 92°47'52"E) near Tezpur University,

Assam-784028, India. A mixture of antibiotics (ampicillin, kanamycin and chloramphenicol)

and antifungals (bavistin and indofil) were used to obtain a unialgal culture from field

samples with substantial bacterial and fungal contamination. The purified microalga was

cultured in liquid media and the respective microalgal biomass was examined for its physical

and chemical characteristics using SEM, CHN, TGDTA and FTIR spectroscopy. The

microalgal biomass was also studied by FTIR spectroscopy, when grown under nitrogen

limited conditions. Scenedesmus biomass showed appreciable energy (15.18 MJ/Kg) and

carbohydrate (29%) content whereas low ash (7.3%) and a shorter thermal degradation

profile. The study high lightens the use of chemical agents of control (chemotherapeutic

agents) for obtaining unialgal cultures from contaminated field samples and also additionally

suggests the importance of microalgae Scenedesmus as a second generation bioenergy

feedstock.

Keywords: Scenedesmus, Algae, Biomass, Feedstock, FTIR.

26.1 Introduction

Escalating petroleum prices, phenomenal upsurge in global energy demands and

greater concerns for environmental protection has high lightened the importance of renewable

energy resources for the energy sector. In addition, to the sustainable favorability of

renewable energy sources, they are in general more evenly distributed over the surface of the

Earth than fossil fuels or uranium and may be exploited using less capital intensivetechnologies (Naik et al., 2010). Bioenergy today lies in the forefront of renewable energy




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research and can assist in anthropogenic endeavors for curbing down reliance on fossil fuels

which are at the brink of quick depletion.

Bio-energy (energy generated from biomass) is a feasible alternative in offering

potentially attractive solutions for addressing issues related to energy security, energy crisis

and sustainable development. The total availability of biomass in the world is 220 billion

oven-dry ton (odt) per year or 4500 EJ (1018 J) (Naik et al., 2010). Biomass has the potential

to become one of the most important global primary energy sources during the next century,

and moreover modern bioenergy systems are suggested to be important contributors to future

sustainable energy systems and sustainable development in industrialized and developing

nations (Berndesa et al., 2005). Bioenergy should play a crucial role in achieving targets to

replace petro-fuels with a viable alternative, and in reducing long-term carbon dioxide

emissions, provided environmental and economic sustainability are considered carefully

(Yuan et al., 2008). With a view to sincerely realize the importance of bioenergy and its future

implications in issues related to energy and environment, research in microalgae, cannot be

overruled.

Microalgae are sunlight driven biochemical factories which convert sunlight into food,

feed and high value bioactives (Akkerman et al., 2002; Chisti, 2007; Metzer and Largeau,

2005). They are a potential source of renewable energy, and can be converted into energy

such as biofuel oil and gas (Amin, 2009). Oleaginous microalgae are a source of renewable,

lipid-rich biomass that serves as feedstock for an emerging biofuel industry (Holguin and

Schaub, 2013). The concept of fuel production from microalgae is not new (Nagle and

Lemke, 1990; Sawayama et al., 1998), but it is now being taken seriously owing to

considerable hikes in price of petroleum and, more importantly, the emerging concern about

global warming associated with burning of fossil fuels (Gavrilescu and Chisti, 2005).

Additionally, microalgal research has also gained significant impetus as an elixir for the long

identified conundrum of Food Vs Fuel debate. They can serve as vessels for photosynthetic

biorefineries, which are believed to be the future solution for sustainable production of

various bioproducts (Xie et al., 2013). Microalgae usually have higher photosynthetic

efficiency, higher biomass production and faster growth rate, compared to lignocellulosic

biomass (Zou et al., 2009). In addition, microalgae can grow in both fresh water and saline

environments, can be cultivated on a large scale and do not require the use of agriculturally

productive or environmentally sensitive land (Ross et al., 2010). Microalgae in possession of




275

all these promising attributes have global prospects for the bioenergy sector and are likely to

foster sustainable economic development.

The present investigation aimed at isolation and purification of microalgae from

contaminated field samples via the agency of chemical agents, and subsequent investigation

of the respective microalgal biomass as a bioenergy feedstock.


26.2.1 Micro-organism and growth medium

Replicate water samples (50mL) were collected from a eutrophic water body

(26°40'10"N 92°47'52"E) near Tezpur University, Assam-784028, India. Compound

microscopic analysis of the water samples revealed the presence of representative genera ofthe microalgal species Scenedesmus and Spirogyra. But however, the later was not taken into

consideration for the present study due to loss of sample purity. The microalgae

(Scenedesmus) were subjected to purification by serial dilution followed by plating and

quadrant streaking. The individual colonies were isolated and inoculated into liquid medium.

The purity of the culture was established by repeated streaking and routine microscopic

examination. Modified BBM media with the following compositions (per liter): NaNO3 (400

mg), K2HPO4 (80 mg), KH2PO4 (30 mg), CaCl2.2H20 (20 mg), MgSO4.7H2O (80 mg), NaCl(25 mg), FeSO4 (1 mg) and EDTA (45 mg) was used as the growth media (both solid and

liquid culture) for the microalgae.

26.2.2 Antibiotics and Antifungal

Three antibiotics viz. kanamycin, ampicillin (50 mg/ml, stock) and chloramphenicol

(10mg/ml, stock) was added at a volume of 1µl/ml of the culture media to ward of bacterial

contamination. Prior to serial dilution the field sample of microalgae was subjected to gravity

precipitation. The upper water layer was decanted and the microalgal biomass was subjected

to three washings with distilled water via centrifugation at 3000 rpm for 15 min. The resultant

microalgal pellet was given three different treatments viz. incubation at ambient temperature

with 0.1% HgCl2 (w/v, 10 min), 1% AgNO3 (w/v, 10 min) and 1% bavistin (w/v, 30 min).

Additionally, Indofil M-45 (contact fungicide, Indofil Chemicals Company, Mumbai, India)

was added to the culture media of microalgae (0.5 µl/ml of the culture media) from a stock

concentration of 1 mg/µl. The antibiotics and antifungal (Indofil M-45) was added to the

autoclaved culture media at (~40°C) inside a laminar flow cabinet.

26.2.3 Culture conditions




276

The micro algal cultures were carried out in 500ml conical flasks (200ml media) with

shaking at 100 rpm; at 28±2°C, light intensity 1200 lux, and (16:8) light and dark cycle. A six

day old culture (exponential growth phase) was used as the inoculum at 10% (v/v) for all

subsequent experiments.

26.2.4 Growth evaluation

The growth of Scenedesmus sp. was monitored spectrophotometrically (CECIL 7400)

by reading the culture absorbance at 680nm.

26.2.5 Determination of Gross calorific value (GCV)

The Gross calorific value (GCV) of the biomass sample was calculated according to

Dulong formula (Huang et al., 2011):

Gross calorific value (MJ/kg) = 0.3383C + 1.442 (H- (O/8))

26.2.6 Determination of Net calorific value (NCV)

The NCV was calculated from the following equation as follows (Koppejan et al., 2008)

NCV = GCV ×1 − 100 − 2.444 ×

100 − 2.444 × 100 × 8.936

× 1 − 100 ; , . .

Where 2.444=Enthalpy difference between gaseous and liquid water at 25 0C.

8.936 = !"#!"

;$.%.&'% ()*%+*- (-// %*-&$) %&%% 5 -

Where,

NCV= Net calorific value

GCV= Gross calorific value

h= Concentration of hydrogen in weight%

w= Moisture content of the fuel in weight%

26.2.7 CHN and Proximate analysis

The content of major elements viz. carbon, hydrogen and nitrogen was analyzed in a

CHN analyzer (Perkin Elmer, 2400 Series-II) and finally the oxygen content was calculatedby difference. The moisture, volatile matter and ash content of the dry algal biomass were




277

determined according to ASTM D 3173, ASTM D 3175 and ASTM D 3174 protocols. The

fixed carbon content was calculated by difference

26.2.8 Determination of cell contents

The anthrone method was used for determination of total carbohydrates (Vyas and

Kohli, 2002). Protein content was determined by the Folin Lowry method (Lowry et al.,

1951) whereas the total lipid content was determined by the method of Bligh & Dyer (Bligh

and Dyer, 1959)

26.2.9 Scanning electron microscopy (SEM)

The microalgal cells were harvested by centrifugation at 10,000 rpm for 15 min,

washed with PBS (Phosphate buffered saline) and fixed with 0.5% glutaraldehyde in 0.1M

cacodylate buffer. Dehydration was carried out in an acetone series with 30 min changes (30,

50, 70, 90 and 100%). The scanning electron micrographs were acquired using a JEOL JSM-

6390LV (Oxford Instrumentation Ltd.) model Scanning electron microscope. For SEM

analysis, the dehydrated microalgal biomass was sprinkled on the carbon tape and then coated

with 30 nm platinum coat using JOEL auto fine coater (model no. JFC-1600). The SEM was

operated at 15KV and under 1 Pascal pressure with the spot size fixed at 36.

26.2.10 FTIR analysis

For FTIR spectroscopy, briefly known weight of the dried algal biomass (1mg) was

taken in a mortar and mixed thoroughly with 2.5 mg of dry potassium bromide (KBr) using a

pestle. The IR spectra was recorded at room temperature (28⁰C±2⁰C) in the mid infrared

range (4000-400 cm-1) using a PERKIN ELMER Spectrum 100 spectrometer.

26.2.11 Thermal analysis

For thermal analysis, microalgae were harvested by centrifugation at 10,000 rpm for

15 minutes. The pellet was washed twice with distilled water and then dried at 90 ⁰C for 24

hours. The dried microalgal biomass was pulverized in a mortar to fine particles, and then

finally stored in a desiccator. Thermogravimetric analysis (TGA) was done to study the

degradation profile of the biomass sample. The algal biomass was subjected to

thermogravimetric analysis in nitrogen atmosphere at heating rate of 10⁰C/min.

Approximately, 10 mg sample was heated at the preselected heating rate from ambient

temperature to 900⁰C in a Pyris diamond TG/DT analyzer (PERKIN ELMER). High puritynitrogen gas (99.99%) was fed at a constant flow rate of 50ml/min as an inert gas. The




278

continuous on-line records of weight loss and temperature were obtained to plot the TGA

curve and the derivative thermogravimetric analysis (DTG) curves.

26.3 Discussion

A microalga was isolated from a eutrophic water body (26°40'10"N 92°47'52"E) near

Tezpur University, Assam-784028, India. Compound microscopic analysis of the water

samples revealed the presence of representative genera two microalgal species viz.

Scenedesmus and Spirogyra, but however the second species was not taken into consideration

for the present investigation due to loss of sample purity. The microalga was purified using

standard microbiological techniques (serial dilution followed by plating and quadrant

streaking). Obtaining unialgal culture (a viable single species culture of algae, free from

undesirable contaminants) from contaminated field samples is the first major achievable

milestone in phycological research. Field samples might contain substantial bacterial and

fungal load. Bacteria generally produce small, limited colonies, and unialgal cultures can be

obtained if the plate has been properly streaked (Andersen and Kawachi, 2005). Fungus have

faster growth rates, they produce sporangia and spores that may hamper the microalgal

isolation processes, and moreover fungal contamination is generally worse than bacterial

contamination because fungi are harder to eliminate by physico-chemical methods, and the

fungus may also exhibit overgrowth over algae on prolonged incubation under suboptimal

conditions (Lorenz et al., 2005). Table 1 shows the treatment regimes for antibacterials and

antifungals to obtain unialgal culture of Scenedesmus sp.

Table 1- Treatment regimes for antibacterials and antifungals to obtain unialgal culture of

Scenedesmus sp

AB

1% AgNO3 0.1%

HgCl2

Bav Ind Bav +Ind

Amp # - BF HBMF +

Kan # - BF HBMF +

Cmp # - BF HBMF +

Amp + Kan # - MBHF MBMF +

Amp + Cmp # - MBHF MBMF +

Kan + Cmp # - MBHF MBMF +

Amp + Kan + Cmp # - HF MF U

AF




279

Where, AB= Antibacterial, AF= Antifungal, Amp= Ampicillin, Kan= Kanamycin, Cmp=

Chloramphenicol Bav= Bavistin, Ind= Indofil ,-= No growth, += Mild bacterial growth, U=

Unialgal growth, BF= Heavy bacterial and mild fungal growth, #= No algal viability with

fungal growth, MBHF= Mild bacterial and heavy fungal growth, HF=Heavy fungal growth,

MF = Mild fungal growth, HBMF =Heavy bacterial and mild fungal growth, MBMF= Mild

bacterial and mild fungal growth.

As evident from the Table 1 its quiet clear that AgNO3 and HgCl2 are highly toxic for

the microalgae. The microalga was not resistant to incubation with 1% AgNO3 and 0.1%

HgCl2 at minimal exposure duration of 10 min, leading to the death of the inoculum itself.

Both the antifungals viz. bavistin (1% w/v, incubation for 30 min) and indofil (added at a

volume of 0.5µl/ml of the culture media, from a stock concentration of 1mg/ µl) when used

individually were only supportive in reducing the fungal load but fungal free culture was

obtained only with a combination of the two. The experimental results also indicate that

individual application of either of the antibiotics were not efficient enough in warding off

bacterial contamination. But however, as expected there was a progressive decline in the

intensity of contamination (visually obvious from plating results) with a combination strategy

of two antibiotics. A combination of three antibiotics (ampicillin, kanamycin and

chloramphenicol) and two antifungals (bavistin and indofil) was necessary to obtain unialgal

culture of the species under investigation.

Scanning electron micrographs were acquired to study the surface morphology and

size of the Scenedesmus cells. Generally Scenedesmus colonies have 4 to 8 cells. Fig 1 shows

the scanning electron micrograph of Scenedesmus spp at 2000x. Harsh processing steps

during sample preparation resulted in elimination of certain cells from the Scenedesmus

colony. The micrographs reveal that Scenedesmus cells are arranged in a flat plate. The cells

are around 2-2.5 µm in diameter, with a thin cell wall. Smooth and "rough" cell walls were

observed in the Scenedesmus colony covered by an irregular network, but no spines were

visible. Boat shaped cavities were also observed on the upper side of the cells and apparently

they were of similar size.

The study aims to highlight fresh water Scenedesmus biomass as a potential bioenergy

feedstock. Significant physico-chemical properties of biomass must be taken into

consideration prior to selection of the biomass conversion technology. Biomass fuels have

significant differences with respect to chemical (volatile matter, ash content, fixed carbon)

and physical (moisture content) characteristics. These fuel characteristics are necessary for




280

choosing the proper biomass conversion technologies. Table 2 shows the biomass properties

of Scenedesmus sp. with regard to their average characteristic composition. The moisture

content of biomass may differ significantly, depending on the biomass type and its storage.

Moisture content is of great importance with regard to selection of biomass conversion

technology. Low moisture content in biomass is preferable since biomass fuels with elevated

moisture levels burns less readily and also generates less heat energy per unit mass of the

biomass being burnt. Generally biomass fuels with low moisture content are more suited for

thermal conversion technology while those with high moisture content are more suited for

biochemical processes such as fermentation conversion (Mckendry, 2002). On this basis,

Scenedesmus sp with a moisture content of 6.4% seems to be a potential candidate for

thermochemical conversion. Scenedesmus biomass had an appreciable quantum ofcarbohydrate (29%). A high percentage of carbohydrate in the biomass is suggestive of its

prospective candidature for biochemical conversion via fermentation for bio-alcohol

production (a part of planned future research activity). Scenedesmus biomass had a lipid

content of 15% which is appreciable from biodiesel point of view. The coupling of bio-

alcohol production with biodiesel production may be suggestive in reducing the economics of

feedstock utility.

Fig 1: Scanning electron micrograph of Scenedesmus spp at 2000x.

In this investigation, ash value was determined using ASTM D 3174 protocol. The ash

content was found to be 7.3% and volatile matter content was 72.14%. The high amount of

volatile matter in Scenedesmus sp biomass will strongly influence its combustion behavior

and thermal decomposition profile. The fixed carbon content was 14.16%. The content of

major elements viz. carbon, hydrogen, oxygen and nitrogen in the microalgal biomass was

41.75, 6.36, 44.98 and 6.91% respectively. The empirical formula of the algal biomass is




282

respectively. The band at 1740 cm-1 is associated with C=O of ester groups (mainly from fatty

acids and lipids). The most significant vibrations in the spectra of lipid are CH 2 stretching

vibrations which give rise to bands in the region of 3100-2800 cm -1 (Stuart, 2004). This

increase in the intensity of absorption in the range of 3100-2800 cm-1 is indicative of increase

in lipid quantum.

Fig 2: FTIR spectra of Scenedesmus biomass and its nitrogen deficient analogue

Thermogravimetric analysis was done in order to study the degradation profile of the

microalgal biomass. The thermal degradation products of the algal biomass consist of

moisture, volatiles and char.

As shown in Fig. 2 the TG-DTG profile of Scenedesmus sp biomass reveals an initial

weight loss between ambient temperature and about 130⁰C for 10⁰C/min .This could possibly

be explained by the elimination of physically absorbed water in the sample and/or

elimination of external or superficial water bounded by surface tension. This was followed by

continuous decrease in sample weight (region of main degradation) which ended by

approximately 460-470⁰C. The phase is characterized by devolatization (main pyrolysis

reaction) where most of the volatile matter evolved. This zone (130-470⁰C) has been referred

to as the zone of active pyrolysis. A very slow loss of weight occurred until 900⁰C which




283

indicates that there was further reaction involving char. The TG-DTG profile reveals that the

initial weight loss temperature, weight loss climax point, and final end temperature for

microalgal biomass pyrolysis for 10⁰C/min are 130, 312 and 470⁰C respectively.

36.4 Conclusion

With scientific momentum in bioenergy research gearing up for reasons connected

with energy security, energy crisis and environmental deterioration numerous biomass sources

are being investigated as potential source for biofuel production. Research endeavors in

microalgae is no exception and as evident from the present study, it can be concluded that thecharacterization of Scenedesmus biomass under the aegis of bioenergy research ensures that it

can be used as a renewable feedstock for both biochemical and thermo-chemical conversion

and may serve the exaggerating need for second generation biofuels. Scenedesmus offers

potential candidature for bioenergy generation with inexpensive nutrient regime for culture,

low ash content (5.93%), short thermal degradation profile, high energy (15.18 MJ/Kg) and

carbohydrate (29%). Scenedesmus can be efficiently harnessed as a fresh water bio-based

feedstock for biomass conversion thereby warranting future research endeavors. However, theinvestigation of Scenedesmus biomass for liquid fuel (biodiesel) production is in progress.




284

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CHAPTER 27

PROSPECTS OF BIODIESEL PRODUCTION FROM NON-

EDIBLE OIL SEEDS OF NORTH EAST INDIA: A REVIEW

Debashis Sut, Rupam Kataki

Abstract

Biodiesel, an alternative diesel fuel derived from vegetable oils, animal fats or used frying

oils, is gaining increased attention due to the twin crisis of fossil fuel depletion andenvironmental degradation. India, one of the fastest growing economies in the world is

becoming increasingly dependent on fossil fuel for its developmental needs. However, it is

necessary to supplement petro-based fuels with resources that are available locally for

production of liquid bio-fuel from the energy security point of view. NE region of India,

particularly Assam (24.30°N and 28.10°N latitude and 89.50°E and 96.10°E longitude) and

Arunachal Pradesh (26.28°N and 29.30°N latitude and 91.20°E and 97.30°E longitude) falls

in one of the richest biodiversity zones of the country. In the forests of Assam and ArunachalPradesh, a large variety of non-edible oil seed bearing trees and shrub species are available,

which can be utilised for production of biodiesel. In this paper, an attempt has been made to

review some of the non-edible oils found in Assam and Arunachal Pradesh viz. Nahar ( Mesua

ferrea), Yellow oleander (Thevetia peruviana), Jatropha ( Jatropha carcus), Koroch

(Pongamia glabra) etc. as feedstocks for biodiesel production. Further, some promising non-

edible oil bearing tree species available in the forests of NE India are discussed for their

potential as a source of feedstock for biodiesel production.Keywords: Non-edible oil, Biodiesel, Methyl ester, NE region of India.

27.1 Introduction

The declining fossil fuel reserves and the growing environmental concerns regarding

the green house gas emission and global warming have made renewable energy as an

alternative energy source for the future. During the past few decades due to the growing

human population and industrialization, worldwide petroleum consumption has increased,resulting in depletion of fossil fuel reserves and increase in petroleum price. On the other




287

hand, combustion of fossil fuels contributes to emissions of greenhouse gases, which

lead to pollution and global warming. About 98% of carbon emissions result from fossil

fuel combustion [Balat, 2011]. The climate change and energy security issues have gained

much higher priorities in recent times and the search for sustainable source of energy resulted

in biodiesel production as an alternative source of energy. Biodiesel, defined as the mono-

alkyl esters of long chain fatty acids derived from vegetable oils or animal fats and alcohol

with or without a catalyst, is one of the promising alternative fuel for diesel engine. It is

renewable, biodegradable, readily available, portable, non-toxic and ecofriendly fuel [Singh

and Singh, 2010]. The potential feed stocks for biodiesel production can be classified into

different categories. They are first generation feedstocks or edible vegetable oils , second

generation feedstocks or non-edible vegetable oils, third generation feedstock or microalgaeand others which include waste cooking oils[Atabani et al.,2013]. The price of biodiesel

depends mainly on the cost of feedstocks, which makes 70–95% of the total biodiesel

cost [Karmakar et al.,2010]. At present edible oils are the main resources for biodiesel

production (more than 95%) [Xue, 2013]. However, there are many difficulties in using

edible oils as feedstocks for biodiesel production. Recently the use of edible vegetable

oils has been of great concern as it results in food versus fuel crisis that might cause food

shortage in the developing countries and also environmental problems by utilizing much ofthe available land for cultivation. It can create serious ecological imbalances as the countries

are cutting down forests for plantation purposes resulting in deforestation and damage to the

wildlife. Therefore, focus is on non-edible resources, which are not suitable for human

nutrition and could grow in barren land. Microalgae have become the latest potential

feedstock for biodiesel production. They are very economical in comparison to edible oils. It

was reported that microalgae has the highest oil yield compared to other oil crops [Mata et al.,

2010]. Microalgae with high oil content have the potential to produce 25 times higher oilyield than the traditional biodiesel crops, such as palm oil [Ahmad et al., 2011].Other

feedstocks for biodiesel production include waste cooking oil, which can be considered as a

promising option with relatively lower price than fresh vegetable oil. [Demirbas, 2009] The

feedstocks for biodiesel production should be as diversified as possible, because relying on

certain sources could bring harmful influence in the long run depending on geographical

locations in the world. Non-edible vegetable oils have gain more attention as a promising

feedstock for the sustainable production of biodiesel. The use of non-edible oils can

improve the economy of biodiesel production and its commercialisation at the industrial

scale as they are cheaper than the edible oil. Non-edible oils are unsuitable for human




288

consumption due to the presence of toxic compounds [Ahmad et al., 2011]. There are

numerous oil bearing plants in nature all over the world. Besides, non-edible oil plants

can be easily cultivated in unproductive lands with much lower costs than the edible

oil crops cultivation [Gui et al., 2008]. Moreover, growing of these plants reduces CO2

concentration in the atmosphere [Karmakar et al., 2010]. However, the main drawback is

high free fatty acids (FFAs) content which increases the biodiesel production cost

[Bankovic-Ilic , 2012].

India is one of the fastest growing economies in the world. The Indian economy has

experienced unprecedented economic growth over the last decade. India with a population of

more than 1210 million is growing at an annual rate of 1.76% and has resulted in more energy

use. India is the third largest consumer of energy in the world after USA and China

[WEO,2012]. India like many other developing countries is a net importer of energy. The

consumption as well as import of crude oil has increased year by year. High dependence on

imported energy is expensive with the ever increasing energy prices; it also impinges

adversely on energy security. To reduce the country’s dependency on conventional fuel, the

government of India has taken initiative for the growth and development of renewable energy.

Government of India has announced its national biofuel policy in 2008 to meet 20% blending

of bio-diesel and bio-ethanol by 2017 [Anon, 2008]. Due to insufficient production of edible

vegetable oils in India, the country has emphasized on non-edible feedstocks for production of

biodiesel. According to a report of Greenpeace, renewable energy can successfully meet over

35% of power demand in India by 2030 [Kumar and Sharma, 2011].

In India where fossil fuel resources are limited, the production and use of biodiesel

from vegetable oils and fats may be a viable solution to supplement the growing demand of

fuel for diesel engine in the country. However while selecting the feedstocks for biodiesel

production, special consideration should be given to utilize the local non-edible oil bearing

tree species. Various oil bearing tree and shrub species are found in the forests of India

especially north-east India. This region is famous for its high ethnic and biological

biodiversity. After the Andaman and Nicobar Islands and the Western Ghats, it forms a range

of tropical forests mainly the species-rich tropical rain forests. North eastern region is also

considered as a significant part of the Indo-Myanmar biodiversity hotspot and presently

accepted as one of the 25 global biodiversity hotspots [Chakraborty et al.,2012]. Among the

north eastern states, a large variety of oil bearing trees and shrub species are found in the

forests of Assam (24.30°N and 28.10°N latitude and 89.50°E and 96.10°E longitude) and




289

Arunachal Pradesh (26.28°N and 29.30°N latitude and 91.20°E and 97.30°E longitude). The

seeds of some of these trees are generally not used for any useful purposes. In this present

paper, some of these non-edible oils found particularly in Assam and Arunachal Pradesh are

presented as potential feedstocks for biodiesel production that can replace the current

dependence on the edible oil resources. Various aspects such as overview of non-edible oil

resources, fatty acid composition and properties and characteristics of biodiesel associated

with these feedstocks have been reviewed from recent publications.

27.2 Non-edible vegetable oils resources

Non-edible vegetable oils are not appropriate for human consumption due to the

existence of some toxic elements in the oils. The selection of non-edible vegetable oils as

feedstocks for biodiesel production requires reviewing the existing works. From the reviews

of biodiesel production from various feedstocks, it is found that non-edible oils bear certain

advantages over edible oils. Biodiesel production from non-edible oils can overcome the

problems of food shortage and also economic as well as environmental and issues related to

edible oils [Gui et al., 2008]. In addition, non-edible oil crops can be grown in barren lands,

degraded forests, fallow lands and also along railways, roads and irrigation canals. Apart from

providing energy security, biodiesel production from non-edible oils could be converted into a

major poverty mitigation program for the rural poor and improvement of the rural non-farm

sector. Non-edible feedstocks for biodiesel production can be considered as sustainable and

alternative fuels [Sastry, 2009]. In the following section, a brief account of different types of

non-edible plant oils is provided.

27.2.1 Jatropha curcas L. [Family: Euphorbiaceae]

J. Curcas popularly known as Ratanjayot is considered as one of the most promising

oil source for biodiesel production in Central and South America, Africa, India and SouthEast Asia. Jatropha can grow under a wide variety of climatic conditions like arid and semi-

arid conditions. J. Curcas is a multipurpose, deciduous, drought resistant plant of 5–7 m

height, which belongs to the family Euphorbiaceae [Pant et al. 2006]. J. Curcas can be used

in a variety of purposes including fuel. Various parts of the plant have medicinal values. J.

curcas oil can be used as lubricant, in cosmetics industry, for making soap and lighting, apart

from biodiesel production. The oil content is about 40–60% in seeds and 46–58% in kernels

and varies depending on the types of species, climatic conditions and the altitude where it isgrown[Kumar and Sharma,2011]. The yield of Jatropha seed ranges from 0.1 to 15 tonnes ha-1




291

natural surfactant for washing the soils contaminant with organic compounds [Kumar and

Sharma, 2011]. Oil content of soapnut seeds is 23% of which 92% is triglycerides [Atabani et

al, 2013]. The oil from soapnut can be considered as promising feedstock for biodiesel

production.

27.2.4 Melia Azedarach [Family: Meliaceae]

M. azedarach is a deciduous tree with a height of 7- 12 m. It is native to India,

southern China, and Australia and belongs to the family Meliaceae. The oil content of dried

kernel is about 10 wt % [Sharma and Singh, 2009]. The fatty acid profile shows that the oil

contains a high percentage of unsaturated fatty acids such as oleic (21.8 wt.%) and linoleic

(64.1 wt.%) acids. The saturated fatty acids present are palmitic (10.1 wt.%) and stearic (3.5

wt.%) acids [Stavarache et al.,2008].

27.2.5 Thevetia peruviana [Family: Apocynaceae]

T. peruviana is an evergreen perennial shrub with a height of 4.5–6 m. It is native to

tropical America, particularly Mexico, Brazil and West Indies and naturalized in tropical

regions of the world.The leaves are deep green with linear sword shape and flowers are

funnel shaped (yellow, white or pinkish yellow in colour). The tree belongs to Apocynaceae

family and has various common names like yellow oleander, gum bush, bush milk, exile treein India, cabalonga in Puerto Rico, ahana in Guyana, olomi ojo by Yorubas in Nigeria

[Atabani et al, 2013]. The plant starts flowering after one and a half year and blooms thrice a

year [Balusamy and Manrappan, 2007]. Depending on the rainfall and plant age it produces

about 400–800 fruits per year [Ibiyemi et al., 1995]. Almost all parts of the plant are

poisonous and bear white latex. The kernel has very high oil content (67%) [Azam et al.,

2005] and the de-oil cake has protein content of 37% [Ibiyemi et al., 2002]. The plant is

generally used as an ornamental or fencing or wasteland plant. It has annual seed yield of52.5 t h-1 and around 1750 L of oil can be obtained from a hectare of waste land [Balusamy

and Manrappan, 2007]. The oil has a very good thermal stability and thus has a potential for

various uses like biodiesel production [Ibiyemi et al., 2002]

27.2.6 Michelia champaca [Family: Magnoliaceae]

M. champaca belongs to Magnoliaceae plant family. It is a tall handsome evergreen

tree with straight stem and smooth brown bark. It is found in Eastern Himalayas, Burma,

China, Assam, Western Ghats and throughout India. The fruits are dark brown. Its seeds have

a oil content of 45% [Atabani et al, 2013]. The perfumed oil obtained from M. champaca




292

flowers has useful application in pharmaceutical industries as well as in perfumery industries

[Hosamani et al., 2009].

27.2.7 Terminalia belerica [Family: Combretaceae]

Terminalia (Terminalia belerica Roxb.) is a large deciduous tree that grows up to a

height of 40 m and belongs to the family of Combretaceae. It is found in the northeastern

region of India. The plant can tolerate moderate draught and heavy rainfall. The tree bears

flowers that are greenish-white or greenish-yellow in colour. The plant starts bearing fruits

from about 10 years of age and survives for more than 80 years. Fruits become mature in

November and harvesting could be continued up to January. The fruit consists of 9% kernel,

30% mesocarp and 61% endocarp. The oil content of the kernel is about 43% [Chakraborty et

al., 2009]. The mesocarp portion of the fruit has medicinal value and used for commercial

purposes. It is regularly used as traditional medicine in India in the treatment of various

diseases like dyspepsia, chronic diarrhea, dysentery etc. Dried fruit pulp is an ingredient of

Triphala (Indian Ayurvedic medicine) which is used for the treatment of intestinal disorders,

liver disorders, pancreatic cancer and many other diseases. The oil could be a potential

feedstock for biodiesel production and terminalia planting would help to curb the

deforestation in the northeastern region of India [Basumatary, 2012].

27.2.8 Mesua ferrea [Family: Clusiaceae]

Nahar ( Mesua ferrea L.) is native to Sri Lanka but also grown in Assam,

southern Nepal, Indochina, and the Malay Peninsula [Sarma, 2006]. It is a moderate sized

tree known as Indian ironwood or Indian rose chestnut and belongs to Clusiaceae family.

Nahar trees can be planted on the wasteland, roadside and in the forests. The tree starts

flowering between April and July and fruits between October and November. The leaves are

linear with a length of 3-6.5 inches and white flowers from the uppermost leaf axils. Diameterof stem at the base is about one foot only and height about 40 feet. The timber from the Nahar

tree is one of the hardest and heaviest. The oil content of the seeds is very high (70-75%) and

is non-edible [Konwer et al. 1989]. Thus it is a promising feedstock for biodiesel production

and has immense scope in NE India [Basumatary, 2012].

27.3 Fatty acid profiles of the biodiesel

The fatty acid composition of the oil is a significant property to determine its

suitability as a raw material for biodiesel production. Generally the type and percentage

of fatty acid composition depends on the plant species and their growth conditions. Biodiesel




293

properties are determined by the fatty acid composition of the oil [Gui et al., 2008]. The most

common fatty acids present in the feedstocks are C16 and C18 acids. Table 1 shows the fatty

acid composition of some of the potential non-edible oils that can be used for production of

biodiesel. Table shows that palmitic (C16:0), stearic (C18:0), oleic (C18:1) and Linoleic acid

(C18:2) are the major fatty acids present in the feedstocks.

Table 1 Fatty acid profiles of the biodiesel

27.4 Properties of the biodiesel

The properties of biodiesel depend on the fatty acid composition of the feedstock which is

used for biodiesel production [Gui et al., 2008]. The quality of biodiesel is most important for

engine part of view and various standards have been specified to check the quality. The

ASTM D6751and EN 14214 are the two most commonly used standards for determining the

quality of biodiesel. In general, biodiesel standards help in identifying the parameters that a

pure biodiesel must have, before being used as a fuel for diesel engines. Some main properties

of biodiesel produced from non-edible oils are listed in Table 2. Most of the properties of

biodiesel meet the biodiesel standards ASTM D6751 and EN 14214.

Non-edible oil

FeedstockC16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 Reference

Mesua ferrea 15.9 - 9.5 52.3 22.3 - - -[Konwer et al.,

1989]

Jatropha curcas 15.6 - 9.7 40.8 32.1 - 0.4 -[Kumar and

Sharma, 2011]Pongamia

glabra11.30 - 9.80 45.25 24.75 2.90 1.75 3.20 [Basumatary,20

12]Thavetia

peruviana14.1 0.14 3.19 58.10 19.49 .088 1.58 0.1

[Oseni etal.,2012]

Terminalia

belerica32.8 0.5 6.4 31.3 28.8 - 0.3 -

[Chakraborty etal.,2009]

Sapindus

mukorossi4.67 0.37 1.45 52.63 4.73 1.94 7.02 1.45

[Jadon et al.,2012]

Melia

azedarach8.1 1.5 1.2 20.8 67.7 - - -

[ Kumar andSharma, 2011]Atabani et al,

2013

Michelia

champaca20.7 6.9 2.5 - - 42.5 - -

[Singh andSingh,2010]Atabani et al,

2013




294

Table 2 Properties of the biodiesel

27.5 Conclusion

North East India is considered as one of the richest biodiversity zones in India. In this

region particularly in Assam and Arunachal Pradesh, various oil seed bearing tree and shrub

species are available which can be used for biodiesel production. Considering availability,abundance, oil content and fuel properties, these feedstocks possess a huge potential for

biodiesel production. These species will be a sustainable alternative to conventional diesel

without affecting the world food crisis.

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Non-

edible oilFeedstock

Calorific

value(MJ kg

-1)

Density

at 15°C(kg m

-3)

Kinematic

Viscosity at40°C (mm

2 s

-1)

Cetane

number

Pour

point(°C)

Flash

point(°C)

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ferea42.23 898 6.2 54 3 112 0.01 0.25

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curcas41.17 880 4.4 57.1 - 163 - 0.3

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CHAPTER 28

A CRITICAL REVIEW OF ENZYMATIC

TRANSESTERIFICATION: A SUSTAINABLE TECHNOLOGY

FOR BIODIESEL PRODUCTION

Neetu Singh, M.K. Jha, A.K. Sarma

Abstract

Fossil fuels are exhaustible. Therefore transition to an economy that runs on sustainable

energy sources is both necessary and inevitable. An approach that focuses on high penetration

of renewables will provide a more effective path towards sustainable energy future. For that

liquid fuels mainly biodiesel is considered as the best sustainable energy source which is

renewable, ecofriendly. It can be produced by chemically or enzymatically via

transesterification reaction. Researchers are trying more efforts to make enzymatically

catalyzed biodiesel production process more feasible as it has certain advantages over

chemically catalyzed process. Like this process is less energy intensive, allows easy and pure

recovery of glycerol and major advantage include the transesterification of glycerides

containing high FFA contents. Although, it has some disadvantages also like high cost of

enzyme but it can be easily solvent by protein or genetic engineering and recombinant DNA

technology methods. These methods help in reducing the high cost of enzymes and also help

in making biodiesel production process more feasible for industrial purpose.

28.1 Introduction

World energy forum predicted that the fossil oil will be exhausted in less than ten

decades. And the main reason for the reduction of the energy resources is rapid population

increase and industrialization. According to the EIA’s report, the world's energy demands are

set to soar in the next 21 years, with developing countries leading the way. The report says

that energy demand will rise 44% by 2030 with 70% of demand increase coming from

developing countries; oil will go to $110 per barrel in 2015 and $130 per barrel in 2030.

World renewable energy use for electricity goes from 19% in 2015 to 21% in 2030; CO2

emissions will rise 39% unless new policies like cap and trade are implemented; world net

electricity generation increases by 77% from 18 trillion kWh (kilowatt hours) in 2006 to 23.2




299

trillion KWh in 2015 and 31.8 kWh in 2030; world coal consumption is projected to increase

from 127 quadrillion Btu in 2006 to 190 quadrillion Btu in 2030, an average annual rate of

1.7%. Electricity generation from nuclear power is projected to increase from about 2.7

trillion kWh in 2006 to 3.8 trillion kWh in 2030, as concerns about rising fossil fuel prices,

energy security, and greenhouse gas emissions support the development of new nuclear

generation capacity. Major urban areas in the non-OECD nations are expected to address

transportation congestion and strains on infrastructure with a variety of solutions, including

development of mass transit (bus and/or rail) and urban design that reduces vehicle-miles

traveled, among other improvements in transportation network [1].

So it is predicted that the renewable energy from combustible energy sources such as

biodiesel will enter the energy market in the near future to diversify the natural energy source.

28.2 Biodiesel

Biodiesel is the best alternative energy fuels which can be produced form the

vegetable oil and animal fats. Triglycerides are the main component of vegetable oil and

animal fat and are known as ester of fatty acids attached to glycerol moiety. They have

hydrophobic properties which mean that they are insoluble in water. Normally, the vegetable

oil and animal fats are obtained in crude form through solvent extracting or mechanicalpressing, containing a lot of impurities such as free fatty acids, sterols and water. Also they

posses high viscosity and low volatility which posed serious problems like deposition, ring

sticking etc. so they must be subjected to chemical reaction such as transesterification to

reduce the viscosity of oils. In this process, the triglyceride molecules of oils are converted

into fatty acid methyl ester or ethyl ester (based on alcohol source) in the presence of catalyst

with glycerol as byproduct [2].

Currently, biodiesel is mainly produced form soybean oil in U.S, rapeseed, sunfloweror soybean oil in EU, and palm oil in Southeast Asia. But the production of biodiesel from

human nutrition sources can cause a food crisis. So, the fuel versus food problem has led the

exploration of non-edible oil feedstocks to be used as source for biodiesel production.

The majority of researchers have focused on non-edible oils or waste cooking oil as

feed stock for the biodiesel production such as algal oil [3-5], microalgae [6-10], jatropha oil

[11] and grease oil [12] etc. Different feed stocks which can be used for biodiesel production

is shown in table 1.




300

Table 1: Different edible and non-edible feedstocks used for biodiesel production [13]

Conventional feedstock Non-conventional feedstock

Mahua Olive

Nile tilapia Jojoba oilPalm RapeseedPoultry CanolaSesame CopraSunflower SoybeanBarley BabassuCoconut Brassica napusTobacco seed Brassica carinataRubber plant GroundnutRice bran Cynara cardunculusCorn CottonseedUsed cooking oil PumpkinLinseed CamelinaMustard Peanut

Okra

Jatropha curcasPoultry fatFish oilBacteriaAlgaeFungiMicro-algaeLatexesSea mangoPalangaPonjamia pinnataTarpenesTallowLard

Biodiesel can be produced by many processes but the conventional production

technique is transesterification. It can be carried out by chemical and enzymatic approach.

Chemical technology is a well developed technology that has been commercialized

worldwide. Homogeneous alkali catalyzed process is extensively used for the large scale

synthesis of alkyl esters, due to the low cost of catalysts and their efficiency at low cost

concentrations [14]. Though great efforts have been placed in the improvement of this

process, it still suffers from high production costs and environmental concern like waste

water, chemical disposal and low quality of the glycerol co-product. This process is also

energy-intensive, requires several separation and purification steps and generates lots of waste

water which is to be treated. Additionally alkali catalyzed process accelerates the oil oxidation

which is a major drawback when dealing with the feedstock rich in high FFA content [15]. It

cannot handle conversion of free fatty acid (FFA) effectively and often leads to soapformation. On the other hand, acid catalyzed process is limited to only esterification reaction.

Thus an oil source which contains a large amount of FFA, only a combination of acid and

alkaline process can fully utilize the feedstock oil. Also, the capital investment is doubled as

well as the operating cost, leading biodiesel to cost higher than the petroleum diesel. Common

cost allocation for biodiesel production through the chemical approach shows that the 70-90%

is allocated for the feedstock and 20-25% for operating cost while 5% for other fees.




301

Thus to obtain a product (biodiesel) with same properties as diesel, an alternative method is to

apply simple reformulation or re-synthesis that results to clearly defined pure products such as

is the case with traditional or enzymatic biodiesel production [16].

Table 2: Comparison of enzyme v/s chemical technology for biodiesel production [17]

S.No.Parameter

Enzymatic

processChemical process

Alkaline process Acid process

1FFA content in the

raw materialFFA are converted

to biodieselSoap formation

FFA converted tobiodiesel

2 Water content inthe raw material

It is not deleteriousfor lipase

Soap formation,

Oil hydrolysisresulting more

soaps

Catalystsdeactivation

3 Biodiesel yieldHigh, usuallyaround 99%

High, usually>96%

High yields(>90%)only for high

alcohol to oil molarratio, high catalystconcentration andhigh temperature

4 Reaction rate Low HighSlower than foralkaline process

5 Glycerol recoveryEasy, high grade

glycerolComplex, lowgrade glycerol

Complex, low gradeglycerol

6 Catalyst recovery EasyDifficult,

neutralized by anacid

Difficult, catalystends up in the by-

products

7 ReusabilityReusability provedbut not sufficiently

studied

Partially lost inpost-processing

stepsNo reusable catalyst

8 Energy costs Low Medium High

9 Temperature 20-50°C 60-80°C >100°C

10 Catalyst cost High Low Low

11Environment

impact

Low: waste watertreatment not

needed

High: waste watertreatment needed

High: waste watertreatment needed

Enzymatic process is known to be clean and environment friendly technique forbiodiesel production as it can simultaneously convert both FFA and triglyceride into




302

biodiesel. Or it can be said that it is insensitive to water and FFA content. No by-product, easy

product removal, reusability without any separation step and mild reaction conditions like low

temperature and high purity products are the key advantages of this process [18]. Enzymatic

transesterification especially those using lipase has drawn researcher’s attention in last ten

years. There are different types of lipases that can be used as catalysts such as: Rhizaopus

oryzae, Candida rugosa, Psuedomonas fluorescens, Burkholderia, Cepacia, Aspergillusniger,

Thermomyces lanuginose and Rhizomucormiehei etc [2]. However, the use of lipase as

catalysts for biodiesel production is facing a lot of obstacles as their cost is very high which

makes the enzymatic process very expensive for industrial purpose and hence increases the

production cost of biodiesel. Moreover, their low stability is also an important drawback.

For these reasons, much effort has been directed for reducing the enzyme cost thereby

allowing the development of competitive enzymatic processes with potential for industrial

application. Production of lipases from new sources [19, 20], the development of techniques

for lipase immobilization [21] as well as to perform the enzyme-catalyzed reactions in

compressed or supercritical fluids such as propane, n-butane or carbon dioxide have appeared

as attractive alternatives for reaching these goals [22, 23]. Repeated use of the enzymes is

essential from the economic point of view, which can be achieved by using them in

immobilized form. In a continuous process using immobilized enzyme, the operational

stability, the exhaustion of enzyme activity, and inhibition by reactants and/or products play

vital roles. The use of membrane bioreactors for the enzymatic processing is increasingly

becoming more attractive, as such systems allow continuous separation of products and

prevent enzyme inhibition. Research attention is also focused on genetic engineering in

enzymes production. Recently, genes of various enzymes have successfully been cloned, and

more genes are promised to be cloned rapidly in the coming years. The use of recombinant

DNA technology to produce large quantities of recombinant enzymes will help lower theenzymes costs. In addition, protein engineering will help to create novel enzyme proteins that

are more resistant and highly thermo-stable. The introduction of a new generation of cheap

enzymes, with enhanced activities and resilience, should change the economic balance in

favor of enzyme use [24].

In light of the increasing interest in the development of alterative energy sources, the

aim of this article is to make an overview of enzymatic biodiesel production, focusing on a

sustainable process.




303

28.3 Lipases as biocatalysts in biodiesel synthesis

Lipases (triacylglycerol hydrolase, EC 3.1.1.3.) are the enzymes that catalyze the

hydrolysis of ester link in the triglyceride molecule. They have the catalytic activity not only

in the aqueous solution but also in nonaqueous solvents [25]. Their natural function is to

catalyze the hydrolysis of ester links but they can also catalyze the esterification (link between

alcohol hydroxyl groups and carboxyl groups of carboxylic acids. They have biotechnological

applications as they can catalyze hydrolysis, esterification, alcolysis and transesterification.

Lipases are highly specific as chemo-, region- and enantioselective catalysts. Among

lipases of plant, animal and microbial origins, most commonly used are microbial lipases.

Using microorganisms it is possible to achieve a higher yield of enzymes and to genetically

manipulate the producing strain in obtaining a low-cost lipase with desired properties for the

conversion of fats into biodiesel [26].

A large number of lipases from different sources have been utilized for biodiesel

synthesis shown in table 3.

28.4 Lipase immobilization

High cost of biocatalysts is the major obstacle for industrial application. Therefore,

immobilization of lipases allows their reusability and makes them more attractive for

industrial biodiesel processes. The aim of immobilization is to enhance lipases properties like

thermo stability and activity in non-aqueous media, and to improve handling, recovery and

recycling of biocatalyst. Recycling greatly reduces the cost of the production and makes the

enzymatic biodiesel production more competitive to chemical processes.

Immobilization is defined localization or confinement of an enzyme on to a solid

support or on a carrier matrix. Supports which can be used for immobilization should be

thermally stable, chemically durable and also then its properties depends upon the mechanical

strength, lipase type, type of the reaction system, ease of regeneration, loading capacity and

cost [40]. Methods for enzyme immobilization can be classified as physical adsorption,

entrapment, covalent bonding and encapsulation, each with its advantages and disadvantages

shown in table 4.




304

Table 3: Different lipase used for enzymatic biodiesel production

S.

No.Oil

Enzyme

catalystAlcohol

Reaction

conditionsSolvent

Yield

(%)

Refe

rence

1 Waste cookingpalm oil

Candida Antarctica B

methanol 4h, 200rpm

Tert-butanol 79.1% [27]

2 Soybean oilThermomyces

lanuginosaethanol

10h, 200rpm

n-hexane/solventfree

70-100%

[28]

3 Jatropha oil

Candida

rugosa,

pseudomonas

fluorescens

ethanol8h, 200

rpmSolvent free 98% [29]

4 Cotton seed oilCandida

Antarcticamethanol

24h, 200rpm

Tert-butanol 97% [30]

5 Soybean oil

Rhizomucor

miehei,

penicillium

cyclopium

methanol12h, 200

rpmSolvent-free

68-95%

[31]

6 Soybean oilCandida

Antarctica B

Methylacetate

14h,200rpm

Solvent-free 92% [32]

7

Sunflower,soybean &

waste cooking

oil

Thermomyces

lanuginosaMethanol

24h, 200rpm

Solvent-free90-97%

[33]

8 Rapeseed oil

Thermomyces

lanuginose,

Candida

Antarctica

Methanol12h, 200

rpmTert-butanol 95% [34]

9 Sunflower oilCandida

Antarctica

Methylacetate

12h, 200rpm

Solvent-free >95% [35]

10 Cotton seed oilCandida

Antarctica

Methanol,propanol,

butanol,amylalcohol

7h, 200rpm

Solvent-free 91.5% [36]

11 Rapeseed oilCandida

AntarcticaMethanol

24h, 200rpm

Solvent-free 91.1% [37]

12Jatropha,karanja,

sunflower oil

Candida

AntarcticaEthyl acetate

12h, 200rpm

Solvent-free >90% [38]

13 Sunflower oil Rhizomucor

miehei,Methanol

24h, 200rpm

n-hexane >80% [39]

14 Sunflower oil

Thermomyces

lanuginose,

Pseudomonas fluorescens

Methanol24h, 200

rpmSolvent-free >90% [39]




305

Table 4: Different immobilization methods with their advantages and disadvantages

28.5 Variables affecting the enzymatic transesterification

Factors which influence the enzymatic biodiesel synthesis are lipid source, reaction

temperature, choice of acyl acceptors, alcohol to oil molar ratio, amount of water in the

system or water activity, and presence of organic solvent in the mixture. Optimal parameters

for enzymatic transesterification vary depending on the origin and type of lipase, type of oil

source, and reactor type.

28.5.1 Reaction Temperature

S.NO.Immobilization

method

Examples of used

supportAdvantages disadvantages

Refere

nce

1 Adsorption

Textile membrane,alumina, ceramics,sepharose, sepadex,cellulose,hydrotalcite, zeolites,celite, silica gel,polyethylene,polypropylene etc.

Procedure iseasy, mildconditions,cheap method,involve notoxic chemical,support can beregenerated.No majoractivity loss

Enzyme deactivationas enzyme areattached to support byweak forces (Vanderwall, hydrophobicinteractions andhydrogen bond), notbest for industrialapplication

[41-43]

2

Entrapment(capture oflipase within amatrix ofpolymer)

Natural and organicsupport, alginate,agarose, gelatin,phyllosilicate sol-gelmatrix

more stable

thanadsorption,enablesubstrate andproductdiffusion

Poor diffusion andenzyme leakage, Lowconversion

[44,45]

3

Encapsulation(confinement ofenzyme within aporousmembraneforming abilayer)

Natural polymers likealginate andcarrageenan, synthetic(resins) and acrylicpolymers, hydrogels,microemulsion basedgels

Preventsleaching andprovide highlyreusablebiocatalyst,,stability ofenzyme is high

Diffusion occurs withincrease in substrateconcentration, mass

transfer problems,

[46.47]

4

Covalentbonding(irreversiblebonding of thelipase to supportmatrix)

Agarose beads

Preventsleaching ofenzyme, stable,high reactionactivity &better catalyticproperties

Complex process,requires an activatingagent

[48,49]




306

Reaction temperature may vary from 23 to 50°C. In general, increasing the

temperature leads to an increase of the reaction rate of biodiesel production. And when the

optimum temperature is reached, further increase in temperature leads to decrease in the

catalytic activity due to denaturation and inactivation. The researches have shown that

immobilization of enzymes shift temperature optimum to higher values in comparison to free

enzymes. It seems that immobilization provides a more rigid external backbone for lipase

molecule, leading to the increase of the temperature optima and higher reaction rates [50].

28.5.2 Water content

Another major factor for enzymatic ester synthesis is water content in the system.

Lipases need an optimal small amount of water to maintain the activity in the organic media.

Nevertheless, increased water concentration has an unfavorable effect on the equilibrium

conversion, since it promotes reverse reaction of hydrolysis. The amount of water in the

system should be a compromise between minimizing hydrolysis and maximizing lipase

activity for the transesterification reaction and it should be determined for a particular reaction

system [51, 52]. Many studies have shown that immobilized enzymes show highest activity in

low water system.

Also, the content of free fatty acids (FFA) in waste oils is more as compared to refinedoils. The esterification of FFA releases water, which can shift the reaction equilibrium

towards ester hydrolysis. In these cases, molecular sieves are used for the control of water

activity and increase ester yields by removing water produced by esterification. However,

when lipases are immobilized on hydrophilic support, the molecular sieves are not need since,

in that case, they have a negligible impact on methyl esters yield [44].

28.5.3 Oil source

Generally, the main feedstock for biodiesel production can be divided in: 1) Vegetable

oils such as sunflower oil [33, 38], soybean oil [28, 31], rapeseed oil [34, 37], jatropha oil

[29], cotton seed oil [30]; 2) Animal fats such as tallow, lard; 3) Waste cooking oils and

industrial waste oils [27].

However, edible oils are not in surplus supply and the cost of oil sources accounts for

a large part in biodiesel production. In order to make the biodiesel production viable, the

solution is to develop a production based on waste cooking oils where no competition with

food production takes place. But the amount of waste oils alone is not sufficient to meet

demands. The optimal solution is to use non-edible oils which can not be used for human




308

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CHAPTER 29

SINGLE STEP REACTION FOR BIODIESEL PRODUCTION

OF JATROPHA CURCUS SEEDS

Sanjaykumar N. Dalvi and Swati R. Sonawane

Abstract

In India, biodiesel prepared from non-edible oil seeds of Jatropha Curcus appeared as a

promising alternative energy source. The oil extraction from these seeds is a primary step forbiodiesel production. This oil is then transesterified to prepare the biodiesel. To escape the

step of oil extraction, a single step method could be used in which the Jatropha seeds crush is

directly converted into biodiesel which is fatty acid methyl & ethyl ester composition. The

objective of this study was to investigate the transesterification allowing direct production

biodiesel from Jatropha seed. This work show that the results obtained from characterization

of the product of single step reaction from non-edible oil seeds of Jatropha Curcus is

biodiesel.

Keywords Biodiesel; Jatropha Curcus; Gas chromatography-mass spectroscopy (GC-MS)

29.1 Introduction

Biodiesel is an alternative fuel made from renewable biological sources such as

vegetable oils both (edible and non-edible oil) and animal fats. Vegetable oils are usually

esters of glycol with different chain length and degree of saturation. It may be seen that

vegetable contains a substantial amount of oxygen in their molecules (Raja et al., 2011).

200 districts in 19 potential states have been identified on the basis of availability of

wasteland, rural poverty ratio, below poverty line (BPL) census and agro-climatic conditions

suitable for Jatropha cultivation. Each district will be treated as a block and under each block

15000 ha Jatropha plantation will be undertaken through farmers (BPL). Proposed to provide

green coverage to about 3 Million ha of wasteland through plantation of Jatropha in 200

identified districts over a period of 3 years (Raja et al., 2011).

Jatropha is a genus of over 170 plants from the Euphorbiaceae family, native to the

Central America but commonly found and utilized across most of the tropical and subtropical




314

regions of the world. It has a yield per hectare of more than four times that of soybean and ten

times that of corn (Nobrega. et al., 2007). Among the different species of Jatropha, Jatropha

curcas has a wide range of uses and promises various significant benefits to human and

industry. Extracts from this species have been shown to have anti-tumor activity (Juan et

al.,2003),the leaves can be used as a remedy for malaria and high fever, (Gubitz et al.,1999),(

Henning R. 1997 ), the seeds can be used in treatment of constipation and the sap was found

effective in accelerating wound healing procedure (Gubitz et al.,1999). Moreover, this plant

can be used as an ornamental plant, raw material for dye, potential feed stock, pesticide, soil

enrichment manure and more importantly as an alternative for biodiesel production(

Vasudevan et al., 2007), (Tiwari et al., 2007).

It appears very difficult to estimate unequivocally the yield of a Jatropha plant that is

able to grow in very different conditions. The yield is a function of water, nutrients, heat and

the age of the plant and other factors. Many different methods of establishment, farming and

harvesting are possible. The yield can be enhanced with right balance of cost, yield, labour

and finally cost per Mt Seed production ranges from about 2 tons-1 hectare-1 year to over

12.5t-1 ha-1 year, after five years of growth. Although not clearly specified, this range in

production may be attributable to low and high rainfall areas.

The seed reaches maturity 90 days after flowering when the capsules changes from

green to yellow and are harvested at this stage to ensure a high oil yield (Pimenta et al., 2009).

The seeds contain around 20% saturated fatty acids and 80% unsaturated fatty acids, and they

yield 25%–40% oil by weight. Estimates of Jatropha seed yield vary widely, due to a

perennial life cycle. Seed yields under cultivation can range from 1,500 to 2,000 kilograms-1

hectare, corresponding to extractable oil yields of 540 to 680 litres -1 hectare. In addition, the

seeds contain other chemical compounds, such assaccarose, raffinose, stachyose, glucose,

fructose, galactose, and protein. The oil is largely made up of oleic and linoleic acids

(Bangboye and Hansen, 2008).

One of the most promising processes to convert vegetable oil into methyl ester is the

transesterification, in which alcohol reacts with triglycerides of fatty acids (vegetable oil) in

the presence of catalyst. Jatropha vegetable oil is one of the prime non edible sources

available in India. The vegetable oil used for biodiesel production might contain free fatty

acids which will enhance saponification reaction as side reaction during the transesterification

process (Raja et al., 2011).



Recent Adva

Figure 1: J

Jatropha can also

and vegetables. Jatropha

biodiesel and also use in pr

The study of in-si

single step of biodiesel

techniques to detect the

should give structural infor

29.2 Material and Met

The In-situ transest

with 500 ml three necked

platinum resistance thermo

connected to a digital in

separating funnel with valv

The Jatropha seeds

using electric mixer with h

powder mixed with potassi

stirring by adjusting 500 r

(Dalvi et al., 2012). At ro

vacuum filtration. Solvent

and purify separate the w

used for further analysis. T


315

atropha Curcus seed bearing plant and Seed

e intercropped with other cash crops such

Curcus plant oil seeds are the highest y

oduction of fertilizer, soaps & cosmetics.

u transesterification reaction of Jatropha

preparation. Characterization the obtained

resence of various components of transest

mation of fatty acid alkyl ester of prepared b

ods

erification carried out in a reactor which c

round bottom flask with digital controlled

meter detector (RTD) temperature sensor wit

icator and a condenser. The product wa

e which placed at bottom.

were collected, dried, cleaned and separat

igh rpm seeds grind into fine powder form.

um hydroxide in methanol. The reaction mi

pm oscillations. The reaction carried out at

m temperature, the solid cake & mother liq

was separated by a rotary evaporator. The

ter soluble impurities. It was preserved in

he product is analysed by GC-MS techniques

ol. III 2014

owder

s coffee, sugar, fruits

ielding feedstock for

urcus seed crush for

product by GC-MS

erified product which

odiesel.

nsisted of an oil bath

mechanical stirrer, a

h an accuracy of ±1°C

collected through a

the seed coating. By

Twenty grams of seed

ture was continuously

60°C for 60 minutes

uor were separated by

product was separated

airtight container and

.




316


The Jatropha curcas is better feedstock for preparation of biodiesel. This seed contain

27-40% oil. The in-situ transesterification reaction is carried out at 60°C with

KOH(Potassium hydroxide). The reaction mixture continuously stirring at 500 rpm for 60

minutes.The product is anlysed by Infrared spectroscopy to determine the functional group of

product(FAME) figure 2. The final product fatty acid methyl ester gives the carbonyl

vibrational bond frequency at 1741.60 cm - 1 , carbon oxygen alkoxy bond of ester is at

1461.94 cm ¯ 1 while finger print region hows the long chain methylene carbon hydrogen

bond frequency at 729.04 cm¯ 1 & 694.33 cm- 1.

The product is characterized by Gas chromatography-mass spectroscopy technique.It

used to separate component of mixture of product (FAME) and to study molecular structure

of compound.There are 4 components were shown their presence in the GC-MS analysis. The

qualitative peaks are shown the figure 3. Following ester were found in the product which is

given with the percentage and Retention time (RT) in table 1.

The result shows that the in-situ transesterification of the jatropha curcus seeds can be

done successfully without extraction of oil from seeds. The methanol might be worked as

solvent in this reaction. There is no need to use the solvent like hexane for oil extraction ,which are not eco-friendly.

The method to prepare the biodiesel is triple stage reaction but in-situ

transesterification single step eco-friendly reaction.

Figure 2: IR Jatropha fatty acid alkyl ester




317

Figure 3: The qualitative peak of GC-MS of Jatropha Curcus in-situ transesterified product

Table1. Jatropha Curcas Biodiesel Components with the RT, Percentage and Name of theEster

Sr.No. R.T. %Ester Name of Ester

1 24.289 5.29 Tridecanoic acid methyl ester2 26.283 28.96 9,12-Octadicadienoic acid methyl ester(E,E)3 26.356 63.72 11-Octadecenoic acid methyl ester

4 26.496 2.03 Tridecanoic acid methyl ester

29.4 Conclusions

With the in-situ transesterification which is single step reaction one can producebiodiesel. The biodiesel content obtained from crushed seed powder of Jatropha seeds without

extraction of oil. The fatty acid methyl ester fuel characterization was done with GC-MS

techniques and results are tabulated. The non-edible oil seeds of Jatropha Curcus are better oil

yielding seeds for the preparation of biodiesel with in-situ transesterification.




318

References

1. Dalvi S., Sonawane S. and Pokharkar R. (2012) Preparation of Biodiesel of Undi seed

with In-situ Transesterification. Leonardo Electronic Journal of Practices and

Technologies, 20:75-182

2. Gubitz G., Mittelbach M. and Trabi M. (1999) Exploitation of the tropical oil seed plant

Jatropha curcas L. Bioresour. Technol., 67: 73-82.

3. Henning R. (1997) Fuel Production Improves Food Production: The Jatropha Project in

Mali. Proceedings from the symposium Jatropha, Managua, Nicaragua.

http://www.jatrophaworld.org

4. Juan L., Fang Y., Lin T. and Fang C. (2003) Antitumor effects of curcin from seeds of

Jatropha curcas. Acta Pharmacol. Sin. 24: 241-246.

5. Nahar K. and Ozores-Hampton M. (2011). Jatropha: An Alternative Substitute to Fossil

Fuel.(IFAS Publication Number HS1193). Gainesville: University of Florida, Institute

of Food and Agricultural Sciences.

6. Nobrega W. and Sinha A. (2007) Riding the Indian Tiger: Understanding India-the

World's Fastest Growing Market. John Wiley and Sons, pp: 272.

7. Raja S. A., Robinson D.S., and Lee C. L. (2011) Biodiesel production from jatropha oil

and its characterization. Research Journal of Chemical Sciences, 1: 81-87.

8. Sharma Y. C., Singh B. and Upadhyay S. N. (2008) Advancements in development

Characterization of biodiesel: A review. Fuel, 87: 2355-2373

9. Tiwari A.K., Kumara A. and Raheman H. (2007) Biodiesel production from jatropha oil

(Jatropha curcas) with high free fatty acids: An optimized process. Biomass Bioenergy,

31: 569-575.

10. Vasudevan P.T. and Briggs M. (2007) Biodiesel production-current state of the art and

challenges. J. Ind. Microbiol. Biotechnology, 35: 421-430.




319

CHAPTER 30

PRODUCTION OF BIODIESEL FROM EDIBLE AND NON-

EDIBLE OILS: A COMPARATIVE STUDY

Aman Deep Singh, Raman Rao, L. Bhanuprakash Reddy, Hemant Kr. Raghuvanshi,

Abdullahi Isaku Kankia, Hitesh Sharma, Soumya Srivastava, Debjani Mukherjee

Abstract

As per IEA, the use of fossil fuels will be increased 56% of world’s present population. Itis already a noted fact that the depletion of fossil fuels has reached a peak value. Based on

the examples of oil crisis, several researchers are eagerly waiting to search for the

alternative biodiesel sources in order to save the economy from the oil crisis in the near

future. Searching feedstock for biodiesel production has always been a challenging step for

the researchers, as the selected one should be non-conventional, with higher yield

content, eco-friendly, and economically feasible. Based on the potential usage of the

biodiesel, the current research provides a comparative study on production of biodieselfrom non-edible and edible oils like pongamia oil, mixed vegetable oil, coconut oil,

mustard oil, soybean oil. Also this work includes a better explanation on the trans-

esterification process and physico-chemical characterization with use of ASTM standards.

After performing the sequential experiments, we obtained the following results for

coconut, mustard, pongamia, waste vegetable and soy bean oil (percentage yield: 87.49%,

60.66%, 81.66%, 79.34%, 83.99%; pH: 6.5, 7.3, 7.2, 7.1, 7.0; carbon content:0.05gm,

0.1gm, 0.04gm, 0.08gm, 0.09gm; specific gravity:0.884, 0.872, 0.892, 0.876, 0.874; Acidvalue:0.34mg KOH/gm, 0.44mg KOH/gm, 0.39mg KOH/gm, 0.44mg KOH/gm, 0.34mg

KOH/gm; Viscosity at 40oC: 75.98, 80.38, 79.45, 76,58, 78.76; Moisture content:

5.06%, 5.75%, 7.06%, 6.09%, 4.94%; Density: 0.878gm/cm3 , 0.877 gm/cm3, 0.886

gm/cm3, 0.872 gm/cm3, 0.868 gm/cm3; Flash Point: 150oC 180oC, 160oC, 170oC,

160oC; Fire point: 170oC, 205oC, 180oC, 195oC, 200oC). Owing to impact of the results,

we are looking to optimize production scale of biodiesel from non-edible sources in order

to quit from food v/s fuel problems.

Key Words: Fossil fuels, Biodiesel, Non-edible oils, Edible oils and Trans-




320

esterification.

30.1 Introduction

According to the United State Standard Specification for Biodiesel (ASTM 6751),defines Biodiesel as a fuel comprising of mono alkyl esters of long chain fatty acids derived

from renewable biomass which can be used in diesel engines and heating systems ( Mittelbach

et al., 1983; Staat and Vallet, 1994). Biodiesel is considered as neat fuel in comparison with

diesel fuel; it is a renewable fuel, non toxic, safer to handle, biodegradable, requires no engine

modifications and reduces dependency on foreign oil imports (Gerpan JV, 2006 et al., 2004).

It also has favorable combustion and emission profiles. For instance, emissions of carbon

monoxide (CO) and particulate matter decease by 45%, Hydrocarbon (HC) 70% but NOx

emissions increases by 10% with 100% biodiesel (B100) as a fuel ( Anon et al., 2002). The

carbon cycle, time for fixation of co2 from biodiesel is quite small compared to mineral diesel

thus contributing more to the reduction of greenhouse gas emissions compared to fossil diesel

(Gerpan JV, 2006; Carraretto et al., 2004; Agarwal et al., 2003), found that biodiesel

provides good lubricating properties that can reduce component wear and enhance engine life.

Hence algal oil is a potential alternative for fossil to harmonize agriculture, economic

development and the environment. Biodiesel is a renewable, natural and domestic fuel made

from edible and non-edible oils. It holds no petroleum, is nontoxic and biodegradable. It is an

alternative fuel for diesel engines. In our modern life there is regular and very fast

consumption of petroleum oil but the resource of petroleum oil are limited so, there is a

necessity to invent an alternative for future which is most renewable, optimal, and easily

accessible in nature. As the crude fuel resource is limited and non-renewable so, the fuel price

at faster pace is continuously increases for regularly diminishing supply and fulfilment of

demand. By continuous consumption of fossil fuel or crude oil results, speedy decline in

reserve of fossil fuels. (Rattan et al., 2012). Biodiesel is produced by the trans esterification

(alcoholises) of vegetable oil or animal fat and alcohol to yield Fatty Acid Methyl Esters

(FAME) and glycerol (Freedman et al., 1996). Searching feedstock for biodiesel production

has always been a challenging step for the researchers, as the selected one should be

non-conventional, with higher yield content, eco-friendly, and economically feasible.

Based on the potential usage of the biodiesel, the current research provides a

comparative study on production of biodiesel from non-edible and edible oils like pongamia

oil, mixed vegetable oil, coconut oil, mustard oil, soybean oil. Test quantities of ethyl and




321

methyl esters of four renewable fuels which were processed, characterized and the

performance was tested.

30.2 Scope for the Study

Analysis of the current situation with respect to availability of bio fuel resources,

processing technology, end-use applications, government policies, markets for bio fuels. The

Potential of bio fuels, considering land availability, technology advancements, future demand-

supply scenario for transportation fuels, and infrastructure and investments requirements.

Discussion and analysis on the sustainability issues in bio fuel development, with respect to,

food security, social and economic sustainability and environmental sustainability. Biodiesel

value chain and discussion on bio fuel production and utilization models. Analysis on thenational and international implications of large-scale bio fuel development, on petroleum

imports, international trade, foreign exchange balance, global environment etc.

30.3 Research Methodology

30.3.1 Praperation of Catalyst

Weigh 0.85gm of NaOH. In a beaker, add 14.28 ml of methanol. Add NaOH in the

methanol and allow to mix the solution on magnetic stirrer to obtain methoxide, which act asa catalyst in the transesterification reaction. During mixing the beaker must be covered with

aluminium foil, to avoid the vaporisation of methanol, as it is volatile in nature.

30.3.2 Transesterification: Material Required

Measure the 85.72ml of oil in a beaker and put the beaker in hot water bath for 45-60

min at 60°C. This step is called pre-treatment of oil. After pre-treatment of oil, put the oil on

magnetic stirrer, add the methoxide to the oil and allow the mixing for 60-80 min at 55°C to

60°C. After the oil/methoxide mixture has reacted, pour the mixture into a separatory funnel

and allow the glycerol waste to settle down for a 24hrs. After settling of glycerol, drain out

the glycerol from the bottom of the funnel and pour the top layer (crude biodiesel) back into

the beaker. Observation of the temperature during the mixing of oil and methoxide must be

continues, to maintain the temperature constant.

30.3.3 Separation




322

After the oil and methoxide were mixed, then mixture was put into the separatory

funnel for separation of crude biodiesel from glycerol. Separation of biodiesel and glycerol

started during the mixing of oil and methoxide, but generally it took 24 hrs for separation.

30.3.4 Washing

After the removal of glycerol, warm distilled water was added to the separatory funnel

that contains crude biodiesel. Tight the cork of the seperatory funnels and gently shakes it to

mix water with crude biodiesel. Let the water settle down of the biodiesel for one day. Then

drain out the water. Repeat the washing process for 3 to 4 more times until the water was

completely become transparent. Transfer the biodiesel to a clean beaker. If the pH is too high,

washing process should be performed for 2 to 3 time more.

30.3.5 Characterization

The biodiesel obtained can be characterized by following methods: ph, percentage

yield, specific gravity, carbon content, acid value, sponification value, moisture content,

viscosity, and density, flash and fire point. The procedure for these methods can be followed

by as per ASTM (American Standard for Testing Materials).


30.4.1 Results

The results obtained by different characterization methods can be shown in following

tables:

Table 1: pH of biodiesel Obtained from different oils

Sr.no. Biodiesel samplepH

(before washing)

pH

(after washing)Biodiesel colour

1. Mustard 10.2 7.3 Dark yellow

2. Coconut 9.2 6.5 Pale white

3. Soybean 9.7 7 Pale white

4. Pongamia 9.6 7.2 Dark brown

5. Mix vegetable oil 9.8 7.1 Light yellow




323

Table 2: The Biodiesel obtained gives the different characterization Results

Table 3: Viscosity of the Biodiesel sample in redwood records

Sr. NoTemperature

0C

Mustard

Oil

Coconut

Oil

Soybean

Oil

Pongamia

Oil

Mix Veg

Oil

1 40 80.38 75.98 78.76 79.45 76.58

2 50 73.54 69.05 71.82 71.65 69.34

3 60 65.09 62.87 64.23 66.87 60.05

4 70 60.76 57.97 56.76 57.34 59.72

Table 4: Flash and Fire Point of Biodiesel

Sr. No. Biodiesel SampleFlash Point

Observed at

Fire Point Observed

at

1 Mustard Oil 180 205

2 Coconut Oil 150 170

3 Soybean Oil 160 200

4 Pongamia Oil 160 1805 Mix Vegetable Oil 170 195

Sr.

No.

Biodiesel

Sample

Percentage

yield (%)

Carbon

content

(g)

Specific

Gravity

Acid value

(mgKOH/g

oil)

Moisture

Content

(%)

Density

(M/V)

1Mustard

Oil60.66 0.1 0.872 0.44 5.75 0.877

2Coconut

Oil87.49 0.05 0.884 0.34 5.06 0.878

3Soybean

Oil83.99 0.09 0.874 0.34 4.94 0.868

4 PongamiaOil 81.66 0.04 0.892 0.39 7.06 0.886

5Mix Veg

Oil79.34 0.08 0.876 0.44 6.09 0.872




324

30.4.2 Graphs

Graph 1: Yield of biodiesel Graph 2: pH of biodiesel

Graph 3: Specific Gravity of biodiesel Graph 4: Acid Value of biodiesel

Graph 5: Moisture content of the biodiesel Graph 6: Density of biodiesel

0

20

40

60

80

100Yield

Oil

sample(ml)

Yield(ml)

Percentage

yield(%)

0

2

4

6

8

10

12

pH

pH befre

!as"ing

pH after

!as"ing

0#86

0#8$

0#88

0#8

0#

Specific gravity

&pecific

gra'ity 0

0#1

0#2

0#

0#4

0#

Acid value

*cid 'al+e

0124

6$8

,nitial !eig"t

f

bidesel(gm

)

-inal !eig"t

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.ensity




325

30.4.3 Discussion

In this project, various tests were performed for the production and characterization of

the biodiesel from coconut oil, mustard oil, pongamia oil and mix vegetable oil. Thepercentage yield of the biodiesel was 60.66% from mustard oil, 87.49% from coconut oil,

83.99% from soybean oil, 81.66% from pongamia and 79.34% from mix vegetable oil. The

percentage yield of biodiesel from coconut oil comes out to be maximum amongst. The pH of

the biodiesel from mustard oil is 7.3, pH of the coconut oil is 6.5, pH of the soybean oil is 7,

pH of the pongamia oil is 7.2 and the pH of the mix vegetable oil is 7.1 after washing. The

carbon content is 0.1g for mustard oil, 0.05g for coconut oil, 0.09g for soybean, 0.04g for

pongamia oil and 0.08g for mix vegetable oil. Specific gravity for the biodiesel from mustard

oil is 0.872, form coconut oil is 0.884, from soybean oil is 0.874, from pongamia oil is 0.892,

and from mix vegetable oil is 0.876. Acid value of standard biodiesel must be 0.5-0.7mg

KOH/gm oil. The acid value for the biodiesel from mustard oil is 044mg KOH/gm, from

coconut oil is 0.34 mg KOH/gm, from soybean oil is 0.34 mg KOH/gm, from pongamia is

0.39 mg KOH/gm and from mix vegetable oil is 0.44 mg KOH/gm. Viscosity of the samples

calculated at 400C and the unit is redwood seconds, viscosity of biodiesel from mustard oil is

80.38, from coconut is 75.98, from soybean is 78.76, pongamia is 79.45 and mix vegetable oil

76.58. Moisture content of the samples were 5.75% for mustard B100, 5.06% for coconut

B100, 4.94% for soybean B100, 7.06% for pongamia B100 and 6.09% for mix vegetable

B100. Standard biodiesel fuel have density ranging 0.86-0.90gm/cm3, and the density of the

samples were 0.877, 0.878, 0.868, 0.886 and 0.872 for mustard B100, coconut B100, soybean

B100, pongamia B100 and mix vegetable B100 respectively. Flash point of standard biodiesel

must be between 130-210°C. The flash point for the samples were calculated to be 180 0C,

1500C, 1600C, 1600C, 1700C for mustard B100, coconut B100, soybean B100, pongamia

B100 and mix vegetable B100 respectively. Fire point for the samples were calculated to be

2050C, 1700C, 2000C, 1800C and 1950C for mustard B100, coconut B100, soybean B100,

pongamia B100 and mix vegetable B100 respectively.

References

1. Bannikov M.G., Combustion and emission characteristics of mustard biodiesel.

2. 6th International Advanced Technologies Symposium. (IATS 11), 132-136.

3. Bari1 M.A.A., Ali H., Rahman M., and Hossain R. Prospect of Bio-diesel Production

from soybean oil: An Alternative and Renewable Fuel for Diesel Engines. International

Journal of Mechanical Engineering, ISSN: 2277-7059, Volume 2.




326

4. Bobade S.N. and Khyade V.B (2012). Detail study on the Properties of Pongamia

Pinnata (Karanja) for the Production of Biofuel. Research Journal of Chemical

Sciences, Vol. 2(7), 16-20.

5. Bunyakiat K., Makmee S., Sawangkeaw R., Ngamprasertsith S., Cao W., Han H., and

Zhang J (2005). Continuous Production of Biodiesel via Transesterification from

Vegetable Oils in Supercritical Methanol. Fuel, 84: 347-351.Chandrasekhar L.A.,

Mahesh N.S., Gowda B., and Hall W (2012). Life cycle assessment of biodiesel

production from Pongamia oil in rural Karnataka. CIGR Journal, Vol. 14, No.3.

6. C.L. Peterson., D.L. Reece., B.L. Hammond., J. Thompson., and S.M. Beck (1997).

Processing, Characterization, and Performance of Eight Fuels from Lipids. Applied

Engineering in Agriculture, 13(1): 71-79.7. Cloin J., Courty P., Deamer T., and Vaitilingom G (2004). Coconut oil as a biofuel in

Pacific Island, South Pacific Applied Geosciences Commission, SOPAC, 1-5.

8. Hasib Z.M., Hossain J., Biswas S., Islam A (2011). Bio-Diesel from Mustard Oil: A

Renewable Alternative Fuel for Small Diesel Engines. Modern Mechanical

Engineering, (1): 77-83.

9. Husain A. B. M. S., Nasrulhaq B.A., Salleh A., and Chandra S (2010). Biodiesel

production from waste soybean oil biomass as renewable energy and environmentalrecycled process. African Journal of Biotechnology, Vol. 9(27): 4233-4240.

10. Issariyakul T., Dalai A.K., and Desai P (2011). Evaluating ester derived from mustard

oil (sinapis Alba) as potential diesel additives. Journal of America oil chemist, Society

88: 391-402.

11. Rattan R. Kumar M (2012). Biodiesel (a renewable alternative fuel) production from

mustard oil and its performance on domestic small diesel engines. International

Referred Research Journal, ISSN- 0975-3486, VoL. III: 45-51.12. Scott P.T., Pregelj L., Chen N., Hadler J.S., Djordjevic M.A., Gresshoff P.M (2008).

Pongamia pinnata: An Untapped Resource for the Biofuels Industry of the

Future.Bioenergy. Res, Vol. 1, 2–11.

13. Sharma S.K., Kalra K.L., and Grewal H.S (2002). Fermentation of enzymatically

saccharified sunflower stalks for ethanol production and its scale up. Bioresource

Technology, 85: 31–33.




327

CHAPTER 31

PRODUCTION OF BIODIESEL FROM NEEM OIL USING

SYNTHESIZED IRON NANOCATALYST

Mookan Rengasamy, Sundaresan Mohanraj, Krishnasamy Anbalagan, Shanmugam

Kodhaiyolii, Velan Pugalenthi

Abstract

The predicted shortage of fossil fuels encourages the search for substitutes of petroleumderivatives. The production of biodiesel has been greatly increasing due to its environmental

benefits. In this present study, the production of biodiesel by transesterification of neem oil

was carried out using synthesized iron nanoparticle as a catalyst. Iron nanoparticles were

synthesized from ferric chloride solution to enhance the transestrification process for the

production of biodiesel. Ferric ions were rapidly reduced by the aqueous sodium borohydride,

leading to the formation of iron nanoparticle. Synthesized iron nanoparticles were analysed by

UV–Visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) andtransmission electron microscopy (TEM). XRD results showed that the peaks were indexed

to the face-centered cubic (fcc) phase (1 1 0) of iron nanoparticles. TEM image confirms that

the formations of iron nanoparticles were predominantly cubical in shape and the size of an

iron nanoparticle was found to be within the range of 40 to 70 nm. The reaction conditions

were 60oC of reaction temperature, 3:1 molar ratio of methanol to oil, and 1wt % of

synthesized iron nanoparticles for the production of biodiesel. The determined values of

specific gravity, viscosity, flash point, cloud point, water content, carbon residue and coppercorrosion for biodiesel were 0.875, 4.9 cp at 40 oC, 145 oC, 3 oC, 0.01 vol %, 0.03 wt %

and 1b respectively. These findings confirm that the obtained values of physical and chemical

properties are in accordance with the specifications of biodiesel as per standard ASTM

D6751. The emission characteristics were also carried out for the blend B20 of neem oil

methyl ester and the results were compared with the hydrocarbon diesel. The results of the

study conclude that the emission characteristics of produced biodiesel were found to be less

when compared to hydrocarbon diesel and hence the produced biodiesel was found to be

superior.




328

Key Word: Iron nanoparticle, Biodiesel, Neem oil methyl ester, Emission characteristics of

biodiesel.

31.1 Introduction

The rapid depletion of fossil fuel resources and increasing environmental concerns,

biodiesel has great attention in recent years as the renewable and eco-friendly fuel. Biodiesel

is a clean and reliable alternative fuel and it has been considered as the fuel substitute for

hydrocarbon diesel (Leung et al., 2010). Biodiesel is generally produced from vegetable oils

and animal fats. Commonly edible vegetable oils are used for the production of biodiesel.

However, increasing the cost of vegetable oil is the main challenges to produce the cost

effective biodiesel. In order to reduce the production cost, low cost feed stocks such as non-edible oil, waste cooking oil and fats have been used as the raw materials (Bhatti et al., 2008).

Conventionally, biodiesel manufacturing processes employ strong acids or bases as catalysts.

But, separation of the catalyst and the by-product glycerol from the product ester is too

expensive to justify the product use as an automobile fuel (Deng et al., 2011). Hence,

heterogeneous catalysts are used since they offer exciting possibilities for improving the

economics of biodiesel synthesis. However, the preparation of heterogeneous catalysts are

more complicated and quiet expensive, which limits there industrial application (Qiu et al.,2011). Therefore, it is necessity to find cheap and efficient catalysts to carry out the

commercial biodiesel production from non-edible oil.

Recently, nanomaterial has been considerable attention as a catalyst for biodiesel

production, owing to its large specific surface area and high catalytic activity. In addition, it

exhibits high resistance to saponification and good rigidity (Hu et al., 2011). Especially,

metallic nanoparticles have various characteristics including catalytic, magnetic, and optical

properties (Neh et al., 2006; Woo et al., 2004; Zhou et al., 2006). The metal nanoparticles areused for various applications such as sensors, environmental remediation, catalysts and energy

storage (Lopeza et al., 2004; Akagia et al., 2012; Noubactep et al 2012; Zhao et al., 2012).

Recently, Qiu et al., (2011) reported that the transesterification from soyabean oil and

methanol was catalyzed by zirconia nanoparticles loaded with potassium bitartrate for the

production of biodiesel. Hence nanoparticles are gaining significant interest to use as a

catalyst for transesterification process to produce efficient biodiesel.

Several methods including physical, chemical and biological were available for thesynthesis of metallic nanoparticles. The main drawbacks of physical methods are inferior




329

quality of the products and also this method requires costly vacuum systems to prepare the

nanoparticles (Umer et al., 2012). Recently, biological methods such as plant and

microorganisms have been extensively studied for the synthesis of iron nanoparticle (Prasad

et al., 2007; Ahamed et al., 2011). However, many of the biological routes to produce metal

nanoparticles are time consuming process and agglomerations of the nanoparticles are the

main challenges. In order to reduce the time, chemical methods are preferred to synthesize

nanoparticles. Chemical synthesis is relatively faster than the physical and biological

methods. The main advantages of this method are to control over the size and shape of the

nanoparticles. Hence, the chemical synthesis was used in the present investigation to synthesis

iron nanoparticles.

In this present study, iron nanoparticles were synthesized using a single step, instant

chemical approach by reducing ferric chloride solution with aqueous sodium borohydride as

reducing agent. The synthesized iron nanoparticles were characterized by UV-Visible spectra,

FTIR, XRD, SEM-EDX and TEM. In addition, synthesized iron nanoparticles were used as

catalyst to investigate the transesterification of neem oil with methanol to produce efficient

biodiesel.


31.2.1 Materials

Analytical grade Ferric chloride (FeCl3) salt, sodium borohydride and methanol were

purchased from Merck Specialties Private Ltd, India. Neem oil was purchased from local

supplier in Tiruchirappalli, Tamil Nadu, India.

31.2.2 Synthesis of iron nanoparticles

Iron nanoparticles were synthesized using sodium borohydride as a reducing agent

with some modifications as reported by Uzum et al., (2008). A 100 mL of 2.5 mM ferric

chloride solution was prepared using sterile double distilled water and stirred at 400 rpm in a

500 mL beaker. To reduce ferric ion into iron nanoparticle, 100 mL of 2.5 mM aqueous

sodium borohydride solution was added at a rate of 0.625 mL/s. The black color was formed

after completing the reaction. The reaction mixture was centrifuged at 11000 rpm for 15 min

and then washed with distilled water for several times to remove impurities. To avoid the

oxidation of iron nanoparticles, it was stored in methanol until further use.

31.2.3 Production of biodiesel




330

The batch experiments were conducted in 250 mL three necked flask connected with

agitator, reflex condenser and thermometer for biodiesel production. 50 g of neem oil was

added to the flask and it was allowed to heat in the water bath at 60 ºC under stirred

conditions. Then, 1 wt % of iron nanoparticle (relative to neem oil weight) was added to the

flask as a catalyst. 3:1 molar ratio of methanol: oil was taken for this study. The reaction was

carried out for a period of 2 hr at 300 rpm. After the completion of the reaction, the mixture

was cooled to the room temperature and transferred into a separating funnel. The glycerol was

allowed to separate from the mixture. The crude fatty acid methyl esters (FAME) were

washed several times with 1:1 volume of warm distilled water about 50°C in a separating

funnel until the neutral pH was obtained.

31.2.4 Characterization of iron nanoparticles

The reduction process of the iron nanoparticles was monitor using Shimadzu UV-2450

UV–Visible spectroscopy. The dried iron powders were subjected to X-ray diffraction

analysis using PAN analytical- XPERTPRO diffractometer system, Netherlands.

Morphological studies of iron nanoparticles were performed using scanning electron

microscopy (SEM) equipped with an EDX detector (Model No: JSM – 6390LV) at a

magnification of 5000X. The size of iron nanoparticle was analyzed by transmission electron

microcopy (TEM) made by Philips, Netherland (Model: Tecnai 10).

31.2.5 Characterization of biodiesel

The quality of biodiesel was expressed in terms of the physicochemical properties

including specific gravity, viscosity, flash point, cloud point, water content, carbon residue

and copper corrosion. These properties of the biodiesel were determined as per the methods of

American Standards for Testing Material (ASTM) and compared with the specification as per

ASTM D6751. A single cylinder, air-cooled, four-stroke, direct injection Kirloskar, dieselengine was used to measure gaseous emissions including CO, CO2 and NOx for biodiesel

blend B20 (20 % biodiesel and 80 % regular diesel by volume) and regular diesel.


31.3.1 UV- Visible spectrum of iron nanoparticle

The synthesis of iron nanoparticles were monitored with the color changes and UV-

Visible spectroscopy. It was observed that, the reaction mixture was turned into brownish

black within 15 mins from its original pale yellow colour, when the slow addition of 2.5 mM

of aqueous sodium borohydride into 2.5 mM aqueous FeCl3 solution was done. As seen in the




331

Fig.1, the appearance of brownish black color indicated the reduction of Fe3+ ions into the

formation of iron nanoparticles. Similar results on visual observations have been reported for

the formation of iron nanoparticle using tea extract by Hoag et al., (2009). In addition, the

UV-Visible spectra were recorded for the reduction of Fe3+ ion and the results are presented in

Fig. 2. The UV-visible spectra of FeCl3 without reducing agent did not show a distinct peak,

whereas the appearance of peak at 260 nm after the addition of reducing agent into aqueous

FeCl3 revealed that the complete reduction of the Fe3+ ion was confirmed and led to the

formation of iron nanoparticles.

Fig 1. Visual observation of Ferric ChlorideReduction in stages

200 400 600 800

0.00

0.25

0.50

0.75

1.00

1.25

A b s o r b a n c e

Wave length (nm)

F e1

F e2

F e3

F e6

F e7

F e8

Fe10

Fe12

Fig 2. UV–Visible absorption spectra of

FeCl3 before and after reduction

31.3.2 X-Ray Diffraction

The crystalline nature of synthesised iron nanoparticles were confirmed

using X-ray diffraction analysis (Fig.3). The result indicates that the iron nanoparticles were

cubic and observed the characteristic diffraction peak of plane at (110). The obtained peak

value of iron nanoparticles were confirmed with JCPDS 85 -1410. The similar results were

reported by Lee et al., (2004) and Sixin et al. (2006). The average particle size of the iron

nanoparticles were determined using Scherer equation and the size of the nanoparticle was in

the range of 40 to 70 nm.

31.3.3 Scanning electron microscopy and EDX

The iron nanoparticles synthesized using chemical method was analyzed in Scanning

Electron Microscope and shown in Fig.4. Typical SEM image of the synthesized iron




332

nanoparticles clearly shows that the morphology of particles was observed to be a non-

uniform shape under the tested conditions.

Fig.3. XRD Pattern of iron nanoparticle

Energy dispersive X-ray (EDX) analysis was used for identifying the elemental

composition of the iron nanoparticle. The quantitative elements present in the synthesized ironnanoparticle are shown in Fig 5. The obtained quantitative percentage of iron nanoparticles

was found to be 43.75.

Fig. 4. SEM images of iron nanoparticles (A)

Magnification: 5,000×, inset bar: 1 µm.Fig. 5. EDX image of iron nanoparticle

Position [°2Theta]10 20 30 40 50 60 70 80

Counts

0

10

20

Fe -chem




333

31.3.4 Transmission electron microscopy

Transmission electron microscopy (TEM) was used to determine the core size and

shape of the iron nanoparticles. TEM image of the synthesised iron nanoparticles is shown in

Fig 6. It was observed that the iron nanoparticles were polydisperse with different shapes

including spherical, cubical triangular and hexagonal. Average particle size was found to be

the range from 40 to 70 nm. The size obtained using Scherer equation is consistent with the

size observed using TEM.

Fig.6.a. TEM images of iron nanoparticles

at 200 nm scale Fig.6.b. TEM images of iron nanoparticles

at 100 nm scale

31.3.5 Physical and Chemical properties of biodiesel

Transestrification from neem oil and methanol was catalyzed by iron nanoparticles for

the production of biodiesel. The fuel properties of produced biodiesel were analyzed using

ASTM test method and the obtained results are given in Table 1. The experimental results

were compared with ASTM D6751 standard and the values were found to be within the

specification range.

The important fuel properties of biodiesel including specific gravity, viscosity, flash

point, cloud point, water content, carbon residue content and copper corrosion test were analyzed

using ASTM method. The results showed that the properties of produced biodiesel were

relatively closer to that of regular diesel. Similar findings were reported by Hanna (1999);

Antolin et al., (2002). Since the viscosity of the biodiesel is closer to that of diesel, no hardware

modifications are required in the existing engine for handling this biodiesel. The cloud point is

the temperature at which the material ceases to flow when the fuel is cooled under prescribed

conditions. The cloud point of the produced biodiesel was determined to be 3 oC. The result

suggests that the higher value of cloud point could work in cold conditions when compared to

that of regular diesel.




334

Water content in fuels imposes engine corrosion problems or reacts with glycerides to

produce soaps and glycerin. In our experimental results, water content was about 0.01 vol%.

This finding indicates the better performance of the corrosion resistance of diesel engine.

Carbon residue of the biodiesel is an indication of carbon deposition tendencies on diesel

engine. Presence of impurities like free fatty acids, glycerides, soaps, polymers and inorganic

contents are indicators of higher carbon residue (Meher et al., 2006). The result showed that

the biodiesel obtained in this study was less impurities. Copper corrosion test results indicated

that the biodiesel was superior quality when compare to normal diesel. The value of copper

corrosion for the produced biodiesel was found to be 1b. This result indicates that the

biodiesel has good resistance to copper corrosion.

Table 1. Fuel properties produced biodiesel from neem oil using iron nanoparticle

Physiochemical

Properties

ASTM Test

Method

Biodiesel

Specification

as per

ASTM D6751

Values of produced

Biodiesel

Specific gravity D4052 0.81-0.90 0.875

Kinematic Viscosity,40 °C (mm2 /s)

D445 1.9-6.0 4.9

Flash point (°C) D93 ≥ 130 145Cloud point (°C) D2500 -3 to 12 3

Water content(% vol)

D2709 ≤ 0.05 0.01

Carbon residue(% mass)

D4530 ≤ 0.05 0.03

Copper stripcorrosion

D130 No.3 max. 1b

31.3.6. Emission Characteristics

The CO emission characeristics of B20 biodiesel and normal diesel was studied by

varying the load (0, 2.5, 5.0, 7.5 and 10 Kgs) of diesel engine and the results are shown in

Fig. 7. It was observed that 30 to 40 % of CO emission for B20 biodiesel was lower than the

normal diesel. The possible reason may be due to the presence of high oxygen content in B20

biodiesel, which enhanced the combustion process. This result confirms that CO emission was

inhibited by reducing the incomplete combustion process of diesel engine. Similar results

were obtained by Park et al., (2009).




335

Figure 7: CO Emission of B20 biodiesel blend and regular diesel

The emission levels of CO2 for B20 biodiesel blend and normal diesel at various loadsof diesel engine are shown in Figure 8. The experimental result revealed that the CO2

emission for B20 biodiesel were less as compared to normal diesel at all loads due to presence

of lower carbon content. The CO2 emission was increased with increasing the load, because of

the higher fuel entered into the diesel engine. Similar observations were made by Ekrem

Buyukkaya. (2010).

Figure 8: CO2 Emission of B20 biodiesel blend and regular diesel

The emission of NOx for B20 biodiesel and normal diesel is shown in Fig.9. NOx are

generally formed at high temperature since the exhaust gas temperature is higher. In the

present study, the NOx emission of B20 biodiesel was slightly higher than the regular diesel

for all the loads. Similar results were made by Lin et al.,(2007).

0

0#02

0#04

0#060#08

0#1

0#12

0#14

0 2 4 6 8 10 12

C O E m i s s i o n

i n V o l % e

Load in Kgs

diesel

20%bidiesel

0

0#1

0#2

0#

0#4

0#

0#6

0#$

0#8

0 2 4 6 8 10 12

C O 2 E m i s s i o n i n % V o l

Load in Kgs

diesel

20%bidiesel




336

Figure 9: NOx Emission of B20 biodiesel blend and regular diesel

31.4 Conclusions

Iron Nanoparticles was synthesized by liquid phase reduction method from ferric

chloride solution using sodium borohydride as reducing agent. The size of the synthesized

iron nanoparticle was in the range of 40 – 70 nm. Transesterification reaction was carried out

from neem oil: methanol mixture using iron nanoparticle as a catalyst for the production of

biodiesel. The properties of produced biodiesel from neem oil were closer to normal diesel.

The results of the study conclude that B20 biodiesel had lesser emissions of CO and CO 2,

when compared to the regular diesel. The emission of oxides of nitrogen from the engine was

found to be slightly higher as compared to regular diesel. Hence the produced biodiesel can be

considered as an alternative to the regular diesel.

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3. Chicgoua Noubactep, Sabine Caré and Richard Crane (2012) Nanoscale Metallic Iron

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Shaped Gold Nanoparticles. Nano Letters Vol. 6, No. 4, 683-688.

5. Dennis Y.C. Leung, Xuan Wu and M.K.H. Leung (2010) A review on biodiesel

production using catalyzed transesterification, Applied Energy 87, 1083–1095.

6. Ekrem Buyukkaya (2010) Effects of biodiesel on a DI diesel engine performance,

emission and combustion characteristics, Fuel, 89, 3099–3105.

7. Fangrui, M and. M.A, Hanna (1999) Biodiesel production: A Review, Bioresource

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biodiesel production, Bioresource Technology, 102, 4150–4156.9. G Antolin, FV Tinaut, Y Briceno, V Castano, C Perez and AI Rameriz. (2002)

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10. George E. Hoag John B. Collins Jennifer L. Holcomb Jessica R. Hoag Mallikarjuna N.

Nadagouda and Rajender S. Varma (2009) Degradation of bromothymol blue by

‘greener’ nano-scale zero-valent iron synthesized using tea polyphenols, Journal of

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339

CHAPTER 32

INFLUENCE OF FREE FATTY ACIDS CONTENT IN

CATALYTIC ACTIVITY OF [BSMIM] Cl IONIC LIQUID FOR

BIODIESEL PRODUCTION FROM NON EDIBLE ACIDIC

OILS

Subrata Das, Ashim Jyoti Thakur, Dhanapati Deka

Abstract

A Brønsted acidic ionic liquid 1-(1-butylsulfonic)-3-methylimidazolium chloride

([BSMIM]Cl) was synthesised and applied as a catalyst in the pretreatment of high free fatty

acid (FFA) containing various non edible acidic oils such as: Jatropha curcas, Pongamia

pinnata, Mesua ferrea L. and Thevetia peruviana. The ionic liquid showed high esterification

activity with a FFA conversion 91-94% depending on the oil under the optimum reaction

conditions of 10wt% catalyst,1:12 oil methanol molar ratio at 70ºC in 6 h. Our results showed

that both the esterification and transesterification activity of the ionic liquid catalyst was

affected by the FFA content of the oil, an increased activity was observed with the increase in

FFA content. For Mesua ferrea L. oil having the highest FFA content of 20.68wt%, the

maximum methyl ester yield 52.63% was obtained out of which 19.06% and 33.56% was due

to esterification and transesterification respectively. In this particular paper a method to

determine the individual yield due to esterification and transesterification separately was

reported.

Keywords: Ionic liquid, FFA, esterification, transesterification.

32.1 Introduction

Biodiesel is obtained from the triacylglycerols of vegetable oils or animal fats and free

fatty acids (FFAs) by transesterification and esterification respectively (Borugadda and

Goudn, 2012). The edible oil feedstocks accounts up to 60-75% of the total biodiesel

production cost and hence makes the biodiesel more expensive (Ma and Hanna, 1999). The

utilization of non edible oils such as crude acidic oils(AOs) as feedstocks can be a solution to

this problem due to its low cost and availability (Atabania et al., 2013). However, the standard




340

biodiesel production by applying the conventional alkaline catalysts cannot be allowed to

these feedstocks due to the presence of FFAs which produces soaps with alkalis (Ma and

Hanna, 1999).Therefore the FFA level in the crude oils should be reduced to 2 wt% prior to

transesterification by an esterification or pretreatment step (Lam et al.,2010).Liquid inorganic

acids and solid acids are widely used in the esterification step. However, the homogenous

mineral acids such as H2SO4 have serious drawbacks such as no recovery, corrosivity and

generation of waste water in washing and neutralizing the acids (Dokic et al., 2012).On the

other hand, solid acids have reduced activity, deactivated easily and adsorbed the products

into their fine powders therefore makes the separation process difficult (Zhang et al.,

2009).Therefore an alternative and environment friendly catalyst is always necessary. In this

respect the application of ionic liquids (ILs) as catalysts in this process has become a worthyarea of investigation (Zhao and Baker, 2013).

ILs is molten salts with tunable physical and chemical properties and composed of

only cations and anions (Welton, 2004). Room temperature ILs (RTILs) are applied as

catalysts because some of its unique features such as wide liquid range, large range of

solubility, variable Lewis and Brønsted acidity, high polarity, non flammability, high thermal

stability, large electrochemical window, negligible vapor pressure and potential for

recyclability and reusability (Zhao et al., 2002; Bourbigou et al., 2010). RTILs have high

catalytic activity and recyclability and thus have the potential to replace the homogenous

catalysts such as sulphuric acid, p-toluene sulphonic acids and heterogeneous solid acids in

esterification reactions (Joseph et al., 2005).

In present investigation, an acidic IL[BSMIM]Cl was used for the pretreatment or

esterification of crude Jatropha curcas oil(JCO), Pongamia pinnata oil(PPO),Mesua ferrea

L.oil(MFO) and Thevetia peruviana oil(TPO) and the effect of initial FFA content in theesterification and transesterification activity of the catalyst was studied. This IL has high

catalytic performances in esterification of FFAs of the said crude AOs. Additionally the

synthetic procedure of the ionic liquid is simple, easy, requires no additional solvent and atom

efficient since the generation of byproducts does not arise.


32.2.1 Materials

Local dried seeds of Jatropha, Pongamia pinnata, Mesua ferrea L. and Thevetia

peruviana were purchased from the Kaliabor Nursery, Kaliabor, Assam, and India. 1-




341

Methylimidazole (99%), 1, 4-Butane Sultone (≥99%), 2-propanol (anhydrous, 99.5%) were

bought from Sigma Aldrich, India. Methanol (≥99%, GC grade), hexane (fraction from

petroleum), HCl (35% for analysis), KOH (Analytical grade) were procured from Merck India

Limited. All the chemicals were used as received without any further purification.

32.2.2 Instruments

1H and 13 C NMR spectra were recorded in a 400 MHz NMR spectrophotometer

(JEOL, JNM ECS) using tetramethylsilane (TMS) as the internal standard and coupling

constants are expressed in Hertz.

32.2.3 Extraction and purification of JCO

The extraction of JCO, PPO, MFO and TPO were carried out by Soxhlet extraction

method by taking hexane (boiling point 65-70ºC) as solvent according to the AOAC method

2003.06. The solvent was separated from oil by rotary vacuum evaporator. The collected oils

were filtered and then heated at 100ºC for 10 min to remove the moisture (Jain and Sharma,

2010).

32.2.4 Preparation of IL

IL was prepared according to the method reported earlier (Kore and Srivastava, 2012).

32.2.5. Reaction procedure and calculations

The laboratory scale esterification reactions were carried out by adding 10 gm of AOs,

12:1 methanol oil molar ratio and 10wt% of [BSMIM]Cl IL catalyst in a round bottom flask

which was equipped with chilled water cooled condenser and a hot plate with magnetic

stirrer. After the reactions were performed for 6h at 60ºC with vigorous stirring the reaction

mixtures were cooled and centrifuged. After centrifugation of the respective cooled mixtures

for five minutes two layers were obtained. The esterified oil was in the upper layer andseparated by simple decantation while the lower layer was the mixture of ionic liquid, excess

methanol and water generated during the reaction. Pretreated oils were heated, dried over

anhydrous Na2SO4 and subjected to the determination of acid value by titration against

standard KOH solution. Conversions of FFAs due to esterification were calculated based on

acid value determination by using the following equation (Corro et al., 2013):

CEst (%) =

Where, Ai is the initial acid value and At is the acid value at time t.

Ai—At

Ai × 100………………………… (1)




342

The acid values were determined by using the following equation (Corro et al., 2013):

A=

Where, A=acid value (mg KOHgm¯1), N=strength of KOH solution (in normality),

V=volume of the KOH solution consumed, W= weight of the sample.

Acid values were measured by titration of small amounts (0.2-0.5g) of either AOs or

pretreated oils in 2-propanol with standard KOH solution. Accordingly the FFA (wt%)

content of the both acidic and pretreated oils were also determined from the equation

below(Jang et al.,2012):

Acid value= …………………….. (3)

Following this the analysis of the pretreated oils were analysed by 1H NMR and the

total methyl ester yields were evaluated from the NMR analysis by using the following

equation (Tariq et al., 2011):

Where, AMe = integral of methoxy methyl ester peak at 3.6 ppm and

Aα-CH2 = integral of α-methylene peak at 2.3 ppm.

The yields due to transesterification were determined by subtracting the esterification

yield (in terms of methyl ester).


32.3.1 Determination of acid values of AOs

Acid values and FFA content of the acidic oils were determined according to the

equation (2) and (3) and the results are described in Table 1.

C Tot (%) =2AMe

3Aα-CH2 × 100

(56.1×N×V)

W

………………………… (4)

………………………… (2)

FFA

2




343

Table 1. Acid values of the AOs

AOs Acid value(mg KOHgm¯1) FFA(wt%)

JCO 16.32 8.20

PPO 29.38 14.76

MFO 41.16 20.68

TPO 18.98 9.53

32.3.2. Influence of FFA content on catalytic activity

Different AOs with variable FFA contents (~8.20-20.68wt%) were subjected to

pretreatment or esterification reactions under the reaction conditions mentioned above and the

results are given in the table 2 and figure 1.

Table 2. Results of pretreatment reactions of AOs

aReaction conditions: catalyst=10wt%, methanol oil molar ratio=12:1, reaction

temperature=60ºC,reaction time=6h.

The FFA content of the AOs reduced below the permissible limit of 2wt% for the

alkali catalysed transesterification and hence the IL catalyst can be effectively applied in the

pretreatment of AOs (table 2).From the figure and the table 2 it was evidenced that both the

esterification and transesterification activity of the IL catalyst increases with the increase in

acid value and FFA content of the acidic oils. The most acceptable explanation of the

AOs Acid value FFAC

Est(%)a Methyl ester yield(%) due toC

Tot(%)a

InitialFina

lInitial

Final

EsterificationTransesterificatio

n

JCO 16.32 1.01 8.20 0.50 93.8 7.69 1.84 9.53

TPO 18.98 1.10 9.53 0.55 94.2 8.97 2.29 11.26

PPO 29.38 2.57 14.76 1.29 91.2 13.46 32.51 45.97

MFO 41.16 3.22 20.68 1.61 92.1 19.04 33.59 52.63




344

observation lying on the fact that the solubility of methanol was more in high FFA content

AOs than in low (Lakhya et al.,2013). Also the increments in transesterification yields were

more as compared to esterification yields for higher FFA content AOs and vice versa. The

proper explanation of this behavior may be due to the fact that in higher FFA content AOs

methanol was more soluble and can interact with the triglycerides molecules more effectively

than at lower FFA content AOs. At lower FFA content AOs methanol due to its low solubility

remains in the FFAs and gets less interaction with the triglycerides molecules and involved in

esterification of FFAs only.

Figure 1.Variation of esterification and transesterification activities of [BSMIM] Cl catalyst

with respect to FFA content of acidic oils

32.4 Conclusion

The synthetic method of the [BSMIM] Cl IL is very easy and green since no

additional solvent is necessary and also no byproducts are generated during the synthesis. IL

catalyst is very much active in the pretreatment of AOs. Under the mild reaction conditions

(10wt% of IL catalyst, 12:1 methanol oil molar ratio for 6h at 60ºC) 91-94% of the FFAs of

AOs esterified.IL catalyst lowered the FFA level of AOs below 2wt% and made the oils

suitable for alkali based transesterification. Thus this catalyst makes the way to produce

biodiesel from low cost AOs and hence makes the overall biodiesel production less expensive.

The high esterification and transesterification activity of the IL catalyst for high FFA content




345

AOs paves the way for the generation new types of IL from this parent IL for the production

of biodiesel from AOs and our group is actively involved in this field.

Acknowledgements

This work was financially supported by DST Green Chemistry Project

(No.INT/FINLAND/P-02).

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9. Konwar L.J., Das R., Thakur A.J., Salminen E., Mäki-Arvela P., Kumar N., Mikkola

J.P. and Deka D.(2013) Biodiesel production from acid oils using sulfonated carbon

catalyst derived from oil-cake waste. J. Mol. Catal. A-Chem. (Accepted).




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enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil)

to biodiesel: A review. Biotechnol. Adv., 28:500-518.

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Nasir Khalid N. and Mir Ajab Khand M.A.(2011) Identification, FT-IR, NMR (1H

and13C) and GC/MS studies of fatty acid methyl esters in biodiesel from rocket seed oil.Fuel Process. Technol., 92:336–341

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synthesis: a review. J. Chem.Technol. Biot. 88: 3–12.




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CHAPTER 33

ANALYSIS OF PHYSICAL PROPERTIES AND BIODIESEL

PRODUCTION FROM DIFFERENT ACCESSIONS OF

JATROPHA CURCAS

Dheeraj Singh, Chiranjib Banerjee, Animesh Sinha, Diwaker Prasad Nirala, Santosh Prasad,

Rajib Bandopadhyay

Abstract

India is expected to at least double its fuel consumption in the transportation sector by 2030.

To contribute to the fuel supply, renewable energies such as Jatropha appear to be an

attractive resource for biodiesel production in India as it can be grown on waste land and does

not need intensive water supply. Hence biodiesel will act as future perspective to cope up with

the energy crisis for domestic as well as industrial purpose. Biodiesel, which is environment

friendly, non-toxic and biodegradable used in diesel engine. The jatropha oil can be used for

soap production and cosmetics production in rural areas. The oil is a strong purgative, widelyused as an antiseptic for cough, skin diseases and as a pain reliever from rheumatism. The

production of Jatropha biodiesel will be done by mainly three process- acid catalyzed, base

catalyzed, and enzyme catalyzed. Acid catalysis is generally preferred due to presence of

moisture and free fatty-acid. Although the process of biodiesel production is slow but it will

resist the production of soap during trans-esterification process and hence good quality of

biodiesel will be produced. During the trans-esterification process, generation of fatty acid

methyl ester (biodiesel) will require the methanol to react with fatty acid in the proportion of5:1. The TLC has been performed to separate the different fatty acid including TAG

(Triacylglycerol). Different accessions of Jatropha seed were collected and their biochemical

parameters for biodiesel production were evaluated. The transesterified fatty acids were

checked through gas chromatography, leading to analysing the different fatty acid

composition. Further the major physico-chemical properties of Jatropha biodiesel will be

compared with diesel fuel. The physico-chemical properties of the produced biodiesel will be

characterized according to the ASTM D6751 standards. The physico-chemical propertiessuch as Cetane number, Iodine value and Saponification value, will be further analyzed. In

future 10% blending will be performed to test the engine efficiency.




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33.1 Introduction

The increasing economy of India has relatively increased its dependency on fossil

fuels where petro-based fuels account for about 95% of India’s

transportation energy demand (National Policy on Biofuels, 2009). Present analysis shows

that current growth in economy fulfils only 24% of the total oil demand so the dependency on

foreign crude oil is increasing rapidly (US EIA, 2013). According to the present scenario,

within two decades the country will nearly cross the limit of 500 million vehicles with current

of 150 million (Sharma, 2011). This transient enhancement in the fuel dependency on

imported energy causes the negative effect attributed to petro-fuel utilization such as

greenhouse gas emission.

Since biofuels can be easily mixed with fossil fuel and used in usual engines. As

biofuel seems to be challenging source of renewable energy hence, among few possible

surrogate substitutes some parts of fossil fuels are used in transportation sector. As a

legislative attempt to supplement the share of biofuels in India, the Ministry of New and

Renewable Energy set up a National Policy which defined an indicative target of 20%

blending of biofuels -whether biodiesel or bioethanol by 2017 (Ajayebi et al., 2013).

Biofuel is a renewable diesel, which is normally transformed by the transesterificationreaction of non-edible oil, vegetable oil, waste cooking oil and fats with smaller chain of

alcohols (generally methanol or ethanol). The reaction generally occurs in the presence of

acid, base or enzyme (Leduce, et al., 2009). In the absence of enzyme, high temperature and

pressure will be required while in the presence of enzyme low temperature and pressure is

required. Generally methanol is used in place of ethanol for transesterification reaction due to

its cheap cost. Usually base catalyzed reaction i.e. NaOH, KOH or corresponding alkoxides is

used for the reaction because it is faster than acid catalyzed reaction (Ataya et al., 2007).

Presence of high free fatty acid content in the seed oil results in lesser production of biodiesel.

It also causes the saponification reaction, hence sodium or potassium ions reacts with it and

form the soap which requires several washing and increase the cost of the production. So, for

commercial process people generally used cheaper feed stock (Baroi et al., 2009).

Biodiesel is a mixture of long fatty-acid chain of mono-alkyl ester which is derived

from the lipid content feed-stocks such as non-edible oil, animal fat, vegetable oil,

lignocellulosic material as an alternative fuel for internal combustion engines. Generallybiodiesel consists of long chain of fattyacids; the chain length is between C14- C22. And the

ester should be of either methanol or ethanol. The first diesel engine developed by Rudolf




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Diesel, in 1900, was run with ground nut oil hence biodiesel is the best material for internal

combustion engines. Diesel engines got the vogue due to its higher thermal efficiency and

also have more power to weight ratio. Therefore it is largely used in the industries and

automobiles for energy generation. Hence transesterified oil is widely accepted biofuel.

33.2 Source: Jatropha Curcus

Jatropha curcus L. (commonly known as physic nut, purging nut, Barbados purging

nut, ratanjot etc.) oil is known to be the most useful biofuel production. Jatropha belongs to

the family Euphorbiaceae. The fruit of the Jatropha is generally used for the treatment of

some sexually transmitted disease, dysentery, small pox, infertility and some skin infections.

In many tropical countries, soap and cosmetics were prepared by the seed oil. Several

components derived from the leaves and latex of the Jatropha have properties of wound

healing, anti-inflammatory and also act as relief to the patients suffering from rheumatism.

Due to increase in demand of biofuel, many industries have started using vegetable oil

for the production of biofuel which caused hike in the price of vegetable oil and enhanced the

rate of malnutrition. Hence, Jatropha curcus can act as an important feed stock for biofuel

production. Jatropha curcus has wide range of adaptability in many ecological environments

and require less nutrient for development, it can also grow on non-arable land which showsthat it does not compete with food production. It can also be useful in reclamation of marsh

land. Due to its simple agricultural process, non-toxic, ecofriendly, renewable, and bio-

degradable is gaining interest of Government of India for Jatropha (Ratanjot) mission

(Chouhan & Sarma, et al., 2013).

The oil content of the Jatropha biodiesel from seed varies from 29 – 50%, depending

upon different geographical condition. Out of which 21% is of saturated fatty acid and 79% is

of unsaturated fatty acid. Jatropha curcus oil is rich in Nitrogen, Phosphorus and Potassiumhence used as organic manure (see Table 1 & 2).

Table 1: Content of Jatropha curcus seed

Ingredient %Composition

Moisture 06.20Protein 18.00

Fat 38.00Carbohydrate 17.00

Fibre 15.50Ash 05.30




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Table 2: Detailed Fatty Acid composition of Jatropha curcus seed

Fatty acid %Composition

Palmitic acid 21.0Stearic acid 12.2Oleic acid 30.0

Linoleic acid 36.7Others 01.4* Source: Prepared from William Elliott, 2013.

33.2.1 Advantages of Jatropha

1. Irrigation of Jatropha plant on marginal land with less nutrient demand and moisture

content.

2. Jatropha plant can be cultivated on low as well as high rainfall region.

3. It can be propagated through branch pruning and seed.

4. It cannot be compete with useful agricultural land.

5. It is pest as well as disease resistant.

6. The by-product generated during chemical process can be used for other commercial

purpose.

7. After oil extraction, the seed cake generated is used as fertilizer in the field.

8. Due to presence of some toxic material it is not fit for human to use, hence Jatropha

oil eliminates tie competition with feed oil.

9. Jatropha plantation can create occupation in rural area and hence help in holding the

degraded land (Atabani et al., 2013).

33.2.2 Disadvantages of Jatropha plantation

1. It is planted in scattered location and generally mixed with the other shrubs present inthe forest region hence its collection is very much tedious job.

2. Different geographical conditions at different places causes different oil content. This

has lead to non-availability of proper quality of seed.

3. Due to improper marketing channel it has limited period of availability.

4. Improper technology for post- cultivation process and their processing.

5. There is huge fissure between actual production and competent production.

6. There is lack of Government funding and proper incentives to promote the biodiesel

production.




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33.3 Different criteria which effect the biodiesel production

33.3.1 Effect of free fatty acid and moisture

For transesterification reaction, the moisture content and free fatty acid should havelow values because it will cause the formation of soap and hence become difficult to separate

glycerol from it. It also increases the viscosity of the FAME.

33.3.2 Effect of molar ratio

The most important unsteady state which create problem in the ester yield is the molar

ratio of the triglycerides and alcohol. The stoichiometry shows that three moles of alcohol is

required for the proper conversion of triglyceride to three moles of fatty acid methyl ester and

one mole of glycerol. The molar ratio is generally depend upon the type of catalyst used, in

case of acid catalyzed reaction, the molar ratio should be of 9:1, because higher the reactant

more forward the reaction will proceed, according to Le- Chatlier principle (Manzetti, 2011).

33.3 Effect of catalyst

Generally there are three types of catalyst used, namely acid catalyst, base catalyst,

and enzyme catalyst. Alkali catalyzed reaction is more faster than acid catalyzed reaction but,

if the glyceride has higher content of free fatty acid and moisture content then it should be

preferred to perform the acid catalysis process because it prevents the formation of soap,

hence viscosity should be decreased.


33.4.1 Collection of Jatropha seed

Jatropha was planted during the end of the year 2010 at Nagri, Ranchi (23°21.388' N;

85°14.661' E and 685m above sea level) in a total area of 14 hectare. At present, plants are

three years old. According to evaluation report conducted by the funding agency (Department

of Biotechnology, Government of India, New Delhi), the performance of the trial plots is one

of the best plots in the Jatropha network project funded by DBT. For the present work, the

Jatropha seeds were procured from Institute of Forest Productivity (IFP), Lalgutwa, Ranchi,

(Jharkhand).

33.4.2 Climate and Soil condition of the study location




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The temperature in summer is normally vary from 20 °C to 42 °C and in winter it is

vary from 0 °C to 25 °C. The average relative humidity of the experimental site is 73%. The

annual precipitation on the experimental site is 1430mm (Singh et al., 2013).

Soil at the experimental location consists of mixture of laterite and red soil having

high content of iron. Laterites are soil types rich in iron and aluminium, formed in hot and wet

tropical areas. Nearly all laterites are rusty-red because of iron oxides. They are developed by

intensive and long-lasting weathering of the underlying parent rock. Tropical weathering

(laterization) is a prolonged process of chemical weathering which produces a wide variety in

the thickness, grade, chemistry and ore mineralogy of the resulting soils. The majority of the

land area containing laterites is between the tropics of Cancer and Capricon. The texture of

Red soils varies from sand to clay, the majority being loams. Their other characteristics

include porous and friable structure, absence of lime, kankar and free carbonates, and small

quantity of soluble salts. Their chemical composition include non-soluble material 90.47%,

iron 3.61%, aluminium 2.92%, organic matter 1.01%, magnesium 0.70%, lime 0.56%,

carbon-dioxide 0.30%, potash 0.24%, soda 0.12%, phosphorus 0.09% and nitrogen 0.08%. So

the pH of the soil is generally acidic due to presence of iron in large quantity and hence it

varies between the ranges of 4.5 to 5.1.

33.4.3 Collection of Jatropha accession

IFP, Ranchi collected seeds from different parts of India through DBT, GOI. Among

them, two different accessions were selected for the preliminary study. One sample is from

Lucknow Biotech Park, UP whose accession number is IC550449, having the oil content of

40%. The other sample was collected from Ruchi, Indore, MP whose accession number is

IC569131, having the oil content of 29.64%.

33.4.4 Cultivation method and treatment

Six month old cuttings were planted at the spacing of 3×3 m. The normal pruning

practise is done three times in a year to make sure that more branches can grow to increase the

fruiting phenomena. Plant seedling was collected from Lucknow, having accession number

IC550449 were treated with Nitrogen, Phosphorus and Potassium fertilizer constant but the

treatment time is once in a year and twice in a year. Plant collected from Indore having

accession number IC569131 were treated with fertilizer, thrice in a year with sulphur content

and other plant were treated with vermi-compost and biofertilizers twice in a year.

33.4.5 CHNS analysis




353

Different elements were analysed (Carbon, Nitrogen, Hydrogen, Sulphur) in the seed

of Jatropha by ELEMENTAL ANALYZER (Germany, VarioEL III). Which is having the

combustion temperature between 950-12000 C and the carrier gas used for this purpose is

Helium.

33.5 Production of Biodiesel

33.5.1 Oil extraction

For oil extraction from the Jatropha seeds, 1 g of seed was taken and decorticated and

then (Kalbande et al., 2008) crushed using Mortar pestle to make a fine powdered form to

extract oil. Solvent extraction process was used to extract oil from it. Chloroform and

Methanol was added in the proportion of 1:2 and volume makeup was done upto 20 ml forproper extraction of oil (Bligh and Dyer, 1959). Chloroform is a non-polar solvent and

methanol is polar solvent hence both the solvent use their property in which like dissolves

like, non-polar lipid dissolves in chloroform and polar lipid dissolve in methanol. The

solution was kept for 48 hours at room temperature for maximum oil extraction. The sample

is then centrifuged at 3000 rpm for 6 minute to settle down the non-dissolved substance. The

solution was extracted and 0.9% of NaCl solution was added to separate the upper methanol

phase and lower chloroform phase. The lower chloroform phase containing non-polar lipidwas taken out. The chloroform phase was concentrated either by flushing by rotatory

evaporator (Buchi, Germany). After that the percentage of dry lipid per dry biomass (g/g) was

calculated. Finally dried lipid was again dissolving in 1 ml of chloroform for further

processing (Figure 3).

33.5.2 Transesterification

Transesterification of total lipid into Fatty Acid Methyl Ester (FAME) has been done

by acid catalyzed reaction (fig.1). To 1 ml of concentrated lipid extract, 5 ml of solvent

Methanol-Benzene-Sulphuric acid in the proportion of 8.6:1.0:0.5 respectively was added by

volume. Sample was incubated at 60 °C for at least 4 hours for proper FAME conversion.

Then 7 ml of warm water was mixed well to remove the impurities and glycerol formed. After

that 5 ml of hexane was added and mixed carefully so that the transesterified lipid comes into

the hexane phase by solvent extraction method. The upper layer were properly collected and

placed in new vial. Little amount of sodium sulphate was added to absorb the water. Nitrogen

gas was flushed to concentrate the FAME mixture. All the chemicals were used of analyticalgrade (AR).




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33.5.3 Quantitative analysis

Qualitative analysis was done by Thin Layer Chromatography (TLC), it was prepared

by mixing 12 gram of Silica gel G-50 (MERCK, U.S.A) in 30 ml of distilled water and was

poured on clear TLC glass plate. After drying at room temperature the plate was finally

activated by keeping the plate in hot air oven (RIVOTEK, India) for 3 hours before use.

The transesterified mixture was run with FAME standard by using solvent system

Petroleum ether: Diethyl ether: Acetic acid in the proportion of 80:20:2 to remove unwanted

pigment from the FAME. The FAME part was scratched out from the TLC. Silica fraction

was eluted with 2 ml of hexane and eluted fraction was collected in a GC vial to which a little

sodium sulphate was added.

Fig.1. Transesterification of triglyceride with alcohol (adopted from Hanna et al., 1999).


Two Physical characters of the seed are shown below in table 3 and figure 2.

CHNS element analyser showed the different content of the nitrogen, carbon, sulphur and

hydrogen present in the different accessions of the Jatropha seed. The inference concluded

with this result shows that sample S4 which was grown adding of Sulphur as an extra nutrient

during manuring which may cause increase in C/N ratio and it may increase in the lipidcontent of the seed.

Scanning Electron Microscopy

The below figure 2 (a) and (b) shows the SEM analysis of the seed coat which tells

about the presence of lignocellulosic fibrils inside and smooth outside coating.

After rotatory vaccum evaporator of the extracted lipid, the weight of the lipid

percentage was calculated (Table 4 & Figure 3).




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Table 3: CHNS element analysis

S.No. Accession No. C/N ratio Content (%)

1 IC550449 18.39 N: 2.850

C: 52.40S: 0.284

H: 9.616

2 IC569131 13.04 N: 4.124

C: 53.77

S: 0.282

H: 10.02

3 IC569131 21.35 N: 2.427

C: 51.82

S: 0.154H: 8.935

4 IC550449 34.27 N: 1.583

C: 54.25

S: 0.126

H: 9.588

5 1C550449 25.44 N: 2.426

C: 61.72

S: 0.136

H: 11.43

Figure: 2 (a) & (b): SEM analysis of Jatropha seed.

The figure 3 illustrates the transesterified lipid present in the seed of the Jatropha

curcus. The figure 4 shows the TLC analysis of Fatty Acid Methyl Ester on Silica gel G-50.




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Table 4: Lipid content of different accessions

S. No. Accession No. Lipid %age

1

2

3

4

5

IC550449

IC569131

IC569131

IC550449

IC550449

28.85

41.27

33.20

38.08

37.58

Fig.3: Transesterified oil of Jtaopha seeds Fig.4: TLC analysis of transesterified oil of

Jatropha seed

33.7 Conclusion

Transesterification of natural oils and fats is preferred over several methods available

for biodiesel production. This process is chosen to lower the viscosity of oil. While blending

of oil and other solvents diminishes the viscosity, engine performance, but problems, such as

carbon deposit and lubricating oil contamination, still exists. The seed coat consists oflignocellulosic material hence biomass recovered after oil extraction will help in production

of biogas and also used as manure after pretreatment. Biodiesel can be used most efficiently

as a supplement to other energy forms, and not only as a primary source. Biodiesel is

particularly useful in mining and marine situations where maintenance of low pollution levels

is essential.

Acknowledgement

DS would like to acknowledge M. Tech. fellowship support from Centre of Excellence

(COE), TEQIP, PhaseII (Ref .No NPIU/TEQIP II/FIN/31/158; dated 16th April, 2013). AS is

Fatty Acid MethylEster




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highly thankful to DBT (Ref No. BT/PR 11962/PBD/26/2009 for financial support to collect

clonal materials and maintenance at IFP, Ranchi. All the authors acknowledge to HOD,

Biotechnology and Central Instrument Facilities (CIF) for providing the instrument facilities

and support during conducting this work. CB gratefully acknowledges the financial support as

Senior Research Fellow (SRF) from Council of Scientific & Industrial Research (CSIR)

[09/554/(0036)/2013 EMR-I],Government of India

References

1. Ajayebi, A., Gnansounou, E., Raman, K.J. (2013) Comprehensive life cycle assessment

of biodiesel from algae and Jatropha: a case study, Bioresource Technology (In-press).2. Atabani, A.E., Silitonga, A.S., Ong, H.C., Mahlia, T.M.I., Masjuki, H.H., Badruddin,

I.A., Fayaj, H. (2013) Non-edible vegetable oils: A critical evaluation of oil extraction,

Fatty acid composition, Biodiesel production, characteristics, engine performance and

emissions production, Renewable and sustainable energy reviews, 18, 211-245.

3. Ataya, F., Dube, M.A., Ternam, M. (2007) Acid catalyzed transesterification of canola

oil to biodiesel under single and two phase reaction, Energy and Fuels, 21, 2450-2459.

4. Baroi, C., Yanful, E.K., Bergougnou, M.A. (2009) Biodiesel production from Jatrophacurcus oil using potassium carbonate as an unsupported catalyst, IJCRE,7, ISSN 1542-

6580.

5. Bligh, E.G. and Dyer, W.J. (1959). A rapid method for total lipid extraction and

purification.

6. Can.J. Biochem. Physiol., 37, 911-917.

7. Chouhan, A.P.S., Sarma, A.K. (2013) Biodiesel production from Jatropha curcus L. oil

using Lemna perpusilla Torrey ash as heterogenous catalyst, Biomass & Bioenergy, 55,

386-389.

8. Elliott, W. (2013) Biodiesel production from Jatropha oil and its characterization,

European Journal of Earth and Environment ISSN (paper) 2668-327X ISSN (online)

9. Kalbande, S.R., More, G.R., Nadre, R.G. (2008) Biodiesel production from non-edible

oils of Jatropha and Karanj for utilization in electrical generator, Bioenergy Resource, 1,

170-178.

10. Leduce, S., Natarajan, K., Dotzauer, E., McCallum, I., Obersteiner, M. (2009)

Optimization of Biodiesel production in India, Applied Energy, 86, 5125-5131.




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11. Ma, F., Hanna, M.A. (1999) Biodiesel production: a review, Bioresource Technology,

70, 1-15.

12. Manzetti, S. (2011) Renewable Energy Driven by Le Chatelier’s Principle,Enzyme

Function, and Non-Additive Contributions to Ion Fluctuations: A Hypothesis in

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359

CHAPTER 34

ANALYSIS OF EXHAUST EMISSION FROM A DIESEL

ENGINE FUELED WITH TRANSESERTIFIED VEGETABLE

OILS

Hemanandh.J. and Narayanan.K.V

Abstract

In this study, the emissions from Kirloskar Direct Injection 4-stroke Diesel engine, single

cylinder air-cooled, 4.4 kW, constant speed at 1500 rpm, compression ratio 17.5:1, with

different blends of diesel in comparison with Refined Corn methyl ester (CF) and Refined

sunflower methyl ester (SF) has been analyzed. Refined Corn oil and Refined Sunflower oil

was transesterified using sodium Meth oxide before blending with diesel. The main objective

of this study is to analyze and compare the CO, HC, CO2, NOx, and Smoke emissions by

varying the injection pressure and the load with pure diesel. The experiments were conducted

with various blends of diesel (10% CF+90% PD, 30%CF+70% PD, and 40% CF+ 60% PD,

10%SF+90% PD, 30%SF+70% PD, and 40%SF+ 60% PD) at different Injection pressures

(210 bar & 240 bar) and at different loads (0%, 25%, 50%, 75%, 100%). A 3- hole nozzle was

used to inject the fuel. The Emission results were studied using AVL gas analyzer. CF

decrease in CO and HC were noticed as 50% and 18.75% and the same for SF was measured

as 23.07% and 33.33%. On comparing diesel for CO2 emission is insignificant for SF and 6%

decrease was observed for CF, at 240 bar with 30% blend, with the marginal increase of NOx.

Keywords: Kirloskar Di – Diesel Engine, Injection pressure, CF – Refined Corn flower

methyl ester, SF – Refined Sunflower methyl ester, 3 – hole Nozzle, Combustion Emission

characteristics.

34.1 Introduction

In the Present Indian scenario, an alternate fuel becomes most important due to the

continuous increase in the diesel fuel price and increasing pollution in the environment due to

diesel Engine exhaust emissions. Many types of Biodiesel can be used in Diesel Engines.




360

Biodiesel or Vegetable oil reduce the greenhouse emissions and is environmental friendly.

Biodiesel is a very good alternative for fossil fuels and is available in plenty.

The vegetable oils cannot be used directly along with diesel, since it is highly viscous.

Transesterification process is done in the presence of methanol, and added with Sodium meth

oxide as catalyst for Refined Sunflower oil and Refined Corn Oil. This improves the

performance of the engine and reduces the emissions.

34.2 Background

Similar experiments on biodiesel were conducted by many researchers. Mahin pey.N

et al., [1] explains, for transportation and safe handling low sulphur content with neutral CO 2

is essential. Jewel A. Capunitan et al. [2] conducted the experiments, one of the valuableenergy of fuels is the chemical stock produced from pyrolysis processed of corn stover. The

various studies made by, Ilknur Demiral et al.,[3] on chromatographic and spectroscopic on

bio-oil reveals that corncob stock can be classified as a renewable fuel. Significant reduction

of about 52.1% in green house gas emissions is evident [Nathan Kauffman] [4]. The literature

on production of raw material for biodiesel revealed by Xiao Huang et al., [5] that a corn

stove hydrolysate as fermentation feedstock for preparing microbial liquid reduces Nitrogen

content. N.N.A.N. Yusuf et al., [6] showed that compared with petroleum diesel reduction inemissions of biodiesel, on CO2, SO2, particulate, CO and the HC and increase of about 10%

NOx are noticed. However blending biodiesel with petroleum diesel reduces NOx emission

with slight increase in other values but of acceptable criteria. The experiments on the DI

diesel perkinson engine were conducted by Dorado MP et, al, [7] by using reused olive oil

methyl ester to study the effect on combustion efficiency. As a result, oxygen concentration

was increased and accelerated the combustion. It was also found that the rate of combustion

efficiency in the use of reused olive oil, methyl esters, and the rate of combustion efficiency

remains almost constant as in the use of diesel oil. A lower energy rate was showed in the

palm oil combustion, done by Tashtoush G et, al, [8]. It was more efficient and higher rate of

combustion (66%) seen in burning biodiesel, when compared with the diesel combustion that

is (56%). This is because of the properties like high viscosity, less volatility and density.

Sudhir C.V. et, al, [9] conducted test on Diesel Engine, The rate of combustion temperature

and pressure was low in the operation of biodiesel, and the NOx emissions was also equal to

that of diesel. The sulphate emission was very low due to the lesser level of sulphur. The pilot

combustion caused the pre-combustion. The observation was that the blending ratio of 15%

resulted in reduced smoke opacity. The test conducted in DI stationary engine by yusuf .T. F.




361

et, al, [10] showed that as the blend increases, the brake power and CO increases in variable

speed which was less than 1800 rpm. A review was done by shareena et, al, [11] using

catalyst along with methanol in the transesterification process, which results in varying fatty

acid content of the biodiesel. This could be a good alternative fuel for diesel. Natraj.M, et, al,

[12] conducted an experiment by varying the design parameters of the DI, 4S, diesel engine

like Nozzle spray hole, Piston head clearance, Nozzle protrusion, Starting of injection timing,

Injection control pressure, Swirl level. By varying these parameters, the emissions were

reduced and analyzed by taguchi method. The method of varying the engine displacement by

Valentin Mickunaitis, et, al, [13] showed the result of mass increase by 6.5% in petrol and

7.5% in diesel. Hence, there is an increase in fuel consumption and CO2 emission.

34.3 Methodology

The Density, Kinematic viscosity of the CF & SF is within the limits of the Biodiesel

Standards. The calorific value of the CF and SF is slightly less when compared to diesel. The

Engine requires a modification to improve better reduction in emissions. The flash point of

the CF and SF is high compared with pure diesel and is safe to store and transport.

The aim of the work is to analyze and compare the emissions, and to study the

performance of the diesel engine by using biodiesel. This has been done by varying theinjection pressure, fuelled with transesterified refined Corn oil and refined sunflower oil

combined with pure diesel at different blends.

34.3.1 Nomenclature

CF Refined Corn oil Methyl ester PD Pure Diesel

SF Refined Sunflower Methyl ester K No. of cylinders

ρ Density, kg/m3 L Length, mm

BIS Bureau of Indian standards A Area of the piston, mm2

BP Brake power, kW N Engine running speed, rpm

T Torque, N- m CV Calorific Value of the fuel, kJ

R Radius of the drum, mm ASTMAmerican standards of Testing and

Materials




362

34.3.2 Engine Specifications

Table 1: Specification of Test Engine

Type Kirloskar Vertical, 4S, Single acting, High speed, C.I. DieselEngine

Combustion Direct Injection

Rated Power 4.3 kW

Rated Speed 1500 rpm

Compression Ratio 17.5: 1

Injector type Single 3 hole jet injector

Fuel injection pressure 210 bar

Dynamometer Eddy currentDynamometer arm length 200 mm

Bore 87.5 mm

Stroke 110 mm

Connecting Rod 200 mm

Cubic Capacity 661.5 cm

Maximum Torque 0.030 kN – m (full load @1500 rpm)

Fuel tank Capacity 6.5 litresInjection pump type Single cylinder flange mounted without camshaft

Governor type Mechanical centrifugal type

34.3.3 Details of Measuring Systems

Table 2 - Details of Measuring Systems

AVL Pressure Transducer GH 12 D

Software Version V 2.0 AVL 617 Indi meter

Data Analyzer from Engine AVL PIEZO CHARGE AMPLIFIER

To measure pressure AVL 364 Angle Encoder

Smoke meter AVL 437 C Smoke

5 Gas Analyzer ( NOx, HC, CO, CO2, O2) AVL DIGAS 444 Analyzer



Recent Adva

34.3.4 Experimental Setu

A stationary kirl

specification of the engine

Electrical loading (Dynam

the engine for various loa

from the engine was meas

and the smoke opacity w

Encoder was used to meas

Fig.

1 - Kirloskar Vertical C.I.Electrica

.,0&01

2*34

5060

O,1 2

7P8 9

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) 6*&

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363

p

skar 4S, DI Diesel Engine was used

is given in Table – 1. The load on the en

meter). The Eddy current dynamometer fo

ing ( 0%, 25%, 50%, 75%, 100% ). The

ured using AVL DIGAS 444 Analyser (N

as measured using AVL 437C smoke m

re the pressure and crank angle.

1 Schematic Diagram of Experimental set-u

Diesel Engine, 2 - Fuel Tank, 3 – AVL 43l loading device, 5 – Engine temperature mo

*/10

34

4,;1O&4*; .,0&01

036,30

01072;

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in experiments. The

ine was applied using

loading is coupled to

xhaust gas emissions

x, HC, CO, CO2, O2 )

ter. AVL 364 Angle

C Smoke meter, 4 –itor

,7*1

6 O;

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364

34.3.5 Test Procedure

The experiments were conducted at different load conditions, with different pressure

at different blends viz ( 10% CF + 90% PD ), ( 30% CF + 70 % PD), (40% CF + 60% PD)

(10% SF + 90% PD ), ( 30% SF + 70 % PD) & (40% SF + 60% PD) as fuel. The test was

conducted at a constant speed of 1500 rpm. The engine was allowed to run at No load for 10

minutes, using each proportion of the blend before applying the load. The loads were

increased gradually for each blend in steps of 25 % at constant speed of 1500 rpm at different

pressures. The test was conducted to compare and analyze the emissions based on the above

conditions.

Table 3 - Comparison of properties of Diesel, Biodiesel standards, Refined palmolein oil &

Refined corn oil

S.

No.

Properties Diesel BIS

Standard

Bio Diesel

ASTM D – 6751

(IS 15607:2005)

SF CF

1. Cetane Index (min) 46 51 - 38 35

2. Density at 15 ° C kg / m 820 – 845 860 – 900 (860-900 kg/m ) 923 923

3. Kinematic Viscosity at 40

° C cst

2 – 4.5 2.5 – 6 1.9 – 6 mm /s 4.4 5.02

4. Flash point ° C min 35 ° C 262 ° C 130 ° C min 254 282

5. Calroific Value kJ/kg 44,000 - - 39,284

36,824

34.4 Results & Discussions

The emissions of CF oil and SF oil (CO, HC, NOx, CO2, and smoke) and its blends are

compared with diesel for a modified diesel engine are analyzed below.

34.4.1 Carbon mono oxide (CO)

Figure 2 shows that exhaust emissions of carbon mono oxide at different injection

pressures under constant speed of 1500 rpm with various blend ratios of the SF and CF

compared with diesel. The CO emissions with CF decreases by 24% at 210 bar and 30%

blend at 75% load condition whereas emission of CO with SF is same as that of diesel at 75%

of load. At 240 bar and 30% blend there is decrement in CF and SF by 50% and 18.75%

respectively at full load condition. This might be due to the availability of excess oxygen

content that leads to better combustion of the fuel droplets that travel from jet tip to cylinder




365

wall. The literature by krahl J et al., [14] shows that the emissions of CO is decreased by 50%

with rapeseed oil containing ultra low sulphur diesel.

Fig – 2 Variation of CO with respect to 30% blend of SF, CF, PD at 240 bar

34.4.2 Hydrocarbon (HC)

The fig. 3 shows the emissions of Hydrocarbon. The HC is increased at full load

condition with various blends at 210 bar for CF and SF, whereas HC decreases at 240 bar and

30% blend compared to pure diesel. It is observed that the increase in HC for CF between

20% load to 60% load, and thereafter decreases. CF and SF decrease by 23.07% and 33.33%at full load condition. This could be due to the rich air-fuel mixture leads to the better

combustion.

Fig – 3 Variation of HC with respect to 30% blends of SF, CF, PD at 240 bar

34.4.3 Carbon – di – oxide (CO2)

The CO2 emissions of the CF and SF as shown in fig.4. There is a marginal decrease

observed at 210 bar and 30% blend. Where as at 240 bar with 30% blend emission of CO2 is

0

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366

same as diesel up to 60% of load and decreases by 6% with CF and very marginal decrease

in SF at full load conditon. AL- widyan MI et al., [15] reviels that, the ethyl ester of waste

palm oil reduces the CO2 and 50:50 blend ratio. The reduction in CF is due to more oxygen

content leads to better combustion.

Fig – 4 Variation of CO2 with respect to 30% blends of SF, CF, PD at 240 bar

34.4.4 Nitrogen Oxide (NOx)

Fig. 5 & 6, shows the NOx emissions. The SF and CF is same as diesel upto 50% load

at 210 bar. The 30% blend at 210 bar and 240 bar shows the marginal increase in NOx for SF

and CF at full load condition,. This could be due to the reduction in flame temperature at

lower injection pressure.

Fig – 5 Variation of NOx with respect to 30%blends of SF, CF, PD at 210 bar

Fig – 6 Variation of NOx with respect to 30%blends of SF, CF, PD at 240 bar

01

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400

600

800

1000

1200

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367

34.4.5 Smoke

From fig. 7 & 8. it is noticed that, the Smoke increases at initial load condition and as

load increases the smoke decreases by 8.89 % for CF at 30% blend at both the injection

pressures when compared to pure diesel, Whereas the marginal increase in SF was observed

with the above conditons. This might be due to presence of oxygen atoms leading to better

combustion with CF and similar reason given by Kalam MA et al., [16].

Fig – 7 Variation of Smoke with respect to30% blends of SF, CF, PD at 240 bar

Fig – 8 Variation of Smoke with respect to30%blends of SF, CF, PD at 210 bar

34.5 Conclusion

The experiments conducted by varying the injection pressures on 4S diesel engine,

with the Refined Sunflower oil and Refined Corn oil and the emissions were studied are

listed below.

1. The CO decreases at 240 bar and 30% blend in CF and SF by 50% and 18.75% at

full load condition.2. The HC decreases for CF and SF with a decrease value of 23.07% and 33.33% at full

load condition at higher injection pressure as compared with pure diesel.

3. With the NOx increase, the CO2 decrease at 240 bar with 30% blend by 6% for CF and

marginal decrease for SF.

Future Scope of work

0

10

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368

The above analysis can be performed by changingthe the nozzle orifice with various

profiles and sizes like Elliptical, semi elliptical, etc., in addition to varying the number of

nozzles against different blend ratio which could yield higher efficiency and lower emissions.

References

1. Mahinpey N, Murugan P, Mani.T, Raina.R (2009) Analysis of bio oil gas and bio char

from pressurized pyrolysis of wheat straw using a tubular reactor energy, Fuel, 2736 –

42.

2. Jewel A. Capunitan,sergio C. Capareda (2010) Assesing the potential fr biofuel

production of corn stover pyrolysis sing a pres.surized batch reactor, Fuel, 563 -572.

3. Ilknur Demiral, Alper Eryazlci, sevgi sensoz (2012) Bio oil production from pyrolysis

of cron cob ( Zea Mays.L), Sciverse Science Direct, 43 -49.

4. Nathen Kauffman, Dermot Hayes, Robert Brown (2011), A life cycle assessemnt of

advanced biofuel production froma hectare of corn, Fuel, 3306 – 3314.

5. Xioa Huang, Yumei wang, Wei Liu, Jie Bao (2011) Biological removal of inhibitors

leads to the improved lipid production in the lipid fermentation of corn stoverhydrolysate by trichasporan cutaneum, Bioresources Technology, 9705 – 9709.

6. N.N.A.N.Yusuf, S.K. Kamarudin, Z.yaakub (2011) Overview on the current trends in

biodiesel productions, Energy Conversion and management, 2741 – 2751.

7. Daroda.M.P, Ballesteras E, Arnal JM, GOMEZJ, Lopez RJ (2003), Exhaust Emissions

from a Diesel Engine Fueled with Transesteified olive oil. Fuel, 1311 – 1315.

8. Tashtoush G, Al-widyan MI, AI – shyoukh (2003) AOCombustion performance and

Emissions of methyl esters of a waste vegetable oil in water cooled furnace. Applied

Thermal Engineering, 285-93

9. Sudhir CV, Sharma NY, Mohanan .P (2007) Potential of Waste Cooking oils as

biodiesel feed stock, Emirates Journal for Engineering Research,69 -75.

10. Yusaf T.F, yosif .B.F, Elawad.M.M (2011), Crude palm oil fuel for diesel engines:

Experimental and ANN simlation approaches, Energy, 4871 - 4878.

11. Shereena,K.M, Thangaraj.T, Biodiesel (2009) An alternate fuel produced fromvegetable oils by Transesterification, European Journal of Biochemistry, 67 – 74.




369

12. M.Natraj, V.P. Arunachalam, & N. Dhandapani,(2005) Optimizing diesel engine

parametes for low emissions using Taguchi method variation risk analysis approach –

Part – I, (ijems), Indian Journal of Engineering of Material Research, 169 – 181.

13. Valentinas Mickunaitis, Alvydas pikunas, Ignor Mackoit, (2007) Reducing fuel

consumption and CO2 emission in motor cars, Transport , 160-163.

14. Krahl J, Munack A, Schröder O, Stein H, Bünger J. (2003) Influence of biodiesel and

different designed diesel fuels on the exhaust gas emissions and health effects.SAE

paper.

15. Al-WidyanMI,TashtoushG,Abu-QudaisM. (2002) Utilization of ethyl ester of waste

vegetable oil as fuel in diesel engines. Fuel Proc Technol, 91–103.

16. Kalam MA, Masjuki HH. (2004) Emission and deposit characteristics of a small diesel

engine when operated on preheated crude palm oil. Biomass Bioenergy, 289–97.




370

CHAPTER 35

GENETIC ENHANCEMENT OF PONGAMIA PINNATA FOR

BIO-ENERGY

M.V.R. Prasad

Abstract

Pongamia pinnata or Millettia pinnata or Derris indica (n=2x=22) is a non-edible oil-seed

yielding ever green leguminous tree adapted to be grown under conditions of marginal soils,

under rain-fed conditions. The tree is native of India and Southeast Asia and is found in

Oceania too. The Carbon sequestration potential of Pongamia pinnata during the 10 to 15

years of its growth was found to be many folds higher than that of several other tree species.

Pongamia was found to sequester around 45 to 50 kg of C per tree per annum as against 28 to

35 kg of Neem (Azadirachta indica), 23 to 26kg of Mahua (Madhuca latifolia) and 11 to 15 kg

in respect of Tendu (Diospyros melanoxylon).Pongamia produces its own nitrogen through

Symbiotic Nitrogen Fixation, thereby displacing approx $200 / ha/year of nitrates applied as

compound fertiliser. Soils under Pongamia stands of +15 years’ age accumulate almost 1100

to 1300 kg of Nitrogen per hectare per annum, apart from enhancing the soil organic matter

and consequent positive soil biological activity.

Considering its oil yielding potential based on the earlier work carried out at the

Directorate of Oilseeds Research under the aegis of ICAR in the decades of late nineteen

eighties and early nineteen nineties, a program of genetic enhancement of Pongamia for seed

and consequent oil yield was launched by VAYUGRID.

Based on the learning and experience gained in the genetic improvement of cashew in

Africa, the genes controlling some crucial canopy characters exhibiting consistency and

stability of expression were employed in the development of basic criteria for selection of the

elite trees of Pongamia. Around 800 elite trees of Pongamia possessing significantly higher

levels of kernel yield of the range of over 45 kg / tree were selected based on the certain stable

canopy attributes strongly associated with seed yield. The yield data collected over two

seasons (2010-2011 and 2011-2012) demonstrated consistently higher yield levels of the

selected trees. The seed oil content of the selected trees ranged from 33 to 39% as against oil




371

content of 28 to 32% in normal and unselected trees. A certain number of elite Pongamia trees

exhibited higher contents of oil and karanjin, a flurano flavanol of industrial value.

The propagation and multiplication of the selected elite trees had been carried out

adopting novel vegetative propagation and nursery technologies that ensured 90 to 100%

success rate in the recovery of the clones (VayuSaps™) of the elite trees. At present

VAYUGRID possesses 8, 27,065 VayuSaps™ containing the elite characteristics contributing

to high yields.

VayuSap™ starts yielding by the end of the third year following planting with a

progressive increase in the yield every year. Normal unselected trees of Pongamia on the

other hand, start yielding poorly after the 6 th of 7th year only. It is estimated that VayuSaps™

planted over an area of one acre would yield around one ton of oil by the 5th year following

planting. VayuSap™ plantation technology is a low input; but highly rewarding one well

suited to wide range of agro-ecological situations including those characterized by marginal

and problem soils with low rainfall.

VayuSap™ plantations have been established with success at Mahindra & Mahindra

World City near Chennai, private farms near Hyderabad & Bangalore and Jindal Lignite

Mining Company (SWML) at Barmer (Rajasthan) in India and also in Ethiopia and Djiboutiin Africa.

Key words: Pongamia pinnata, genetic improvement, oil yield, karanjin

35.1 Intorduction

Pongamia pinnata or Millettia pinnata or Derris indica (n=2x=22) is a non-edible

oil-seed yielding ever green leguminous tree adapted to be grown under conditions of

marginal soils under rain-fed conditions. The tree is native of India and Southeast Asia and is

found in Oceania too. The annual seed production of Pongamia in India is estimated to be

130,000 tonne (Rattansi et al., 1997) most of which is obtained as an aggregation from avenue

and forest trees and those grown on field bunds and marginal soils without any care and

maintenance worth the name. There are no organised plantations of Pongamia in India despite

its manifold uses including as an ancient source of energy and in native medicine.

Considering its oil yield potential and suitability to be grown on marginal lands under

rain-fed conditions based on the earlier work carried out at the Directorate of Oilseeds

Research under the aegis of ICAR (Prasad, 1994) in the decades of late nineteen eighties and




372

early nineteen nineties, a program of genetic enhancement of Pongamia for seed and

consequent oil yield was launched by VAYUGRID.

35.2 Carbon Sequestration by Pongamia

The Carbon sequestration potential of Pongamia pinnata during the 10 to 15 years of

its growth was found to be many folds higher than that of several other tree species.

Pongamia was found to sequester around 45 to 50 kg of C per tree per annum as against 28 to

35 kg of Neem ( Azadirachta indica), 23 to 26kg of Mahua ( Madhuca latifolia) and 11 to 15

kg in respect of Tendu ( Diospyros melanoxylon). (Table1) (Chaturvedi et al., 2011; Jesse

More, 2009; Gupta, 2008; Kamarkar et al., 2012; Gera and Chauhan, 2010 and Reddy et al.,

2009)

Table 1: Carbon Sequestration by Some Popular Tree Species.

Tree typeC sequestration / tree of

15yrs (kg)/ annumC Sequestration (t)/ ha

Pongamia (Pongamiapinnata)

45-50 23.5****

Tendu(Diospyrosmelanoxylon)

11-15 6.0***

Mahua (Madhuca latifolia) 23-26 8.5**Neem (Azadirachta indica) 28-35 13.0*

• C - Sequestration by Neem is reported as 1.5 t/ha/yr from natural tree stands. He above

figure of 13.0 was arrived at based on the population density (450/ha) that would be

recommended for a regular plantation.

• All the above values are for regular plantation model with appropriate tree densities per

unit area.

** Tree density of 354 / ha

*** Tree population of 460/ha

**** Tree population of 500 / ha

The current trends of global warming are of great concern. It is estimated that an

increase of 2.5% of global temperature would reduce agricultural productivity in USA by 6%;

but by 38% in India. (Sengupta 2013) It is therefore necessary to come up with appropriate

technologies involving tree species that are efficient in cutting down global warming andbring about perceptible levels of C sequestration in addition to oil yield. Pongamia is one




373

such species that need attention in this regard. It is absolutely necessary that the tree types

grown for bio-fuels, must not occupy fertile irrigated lands to avoid competition with regular

food and commercial crops. Pongamia pinnata meets the above condition and as such forms

an excellent feed stock for bio-fuel production.

35.3 Sybiotic Nitrogen Fixation and Soil Amelioration by Pongamia

Pongamia produces its own nitrogen through Symbiotic Nitrogen Fixation, thereby

displacing approx $200 / ha/year of nitrates applied as compound fertiliser. Soils under

Pongamia stands of +15 years’ age accumulate almost 1100 to 1300 kg of Nitrogen per

hectare per annum, apart from enhancing the soil organic matter and consequent positive soil

biological activity (Elevitch and Wilkinson, 1999).

Table 2 - Soil Analysis Data

Parameter Under Pongamia

(6yr old trees)

Barren soil

pH 6.5 6.7

Bulk density 4.75 4.87

Oxidisable C 1.6 0.45

Organic matter 3.27 1.16N 0.091 0.027

P 0.0093 0.0030

K 0.086 0.032

The soil under six year old trees of Pongamia exhibited marked improvements not

only soil organic matter and Nitrogen, but also in other plant nutrients viz., Phosphorous and

Potash (Table 2).

35.4 Selection of Elite Trees

Based on the learning and experience gained in the genetic improvement of cashew in

Africa (Prasad et al., 2000), genes controlling some crucial canopy characters exhibiting

consistency and stability of expression were employed in the development of basic criteria for

selection of the elite trees of Pongamia. The procedure adopted by VAYUGRID for the

identification of genetically elite trees of Pongamia pinnata for consistent higher productivity

levels is based on the selection of characters that are stable in their expression with their close




374

association with kernel yield. The selection is not based on total yield alone, which could vary

from year to year.

The characters chosen are as follows:

1. Appropriate canopy architecture with extended canopy area

2. Intensive branching pattern with higher proportion of pod bearing branches.

3. Higher number of Pod bearing clusters

4. Cluster bearing habit

5. Higher shelling percentage

6. Resistance to pod-malformation and

7. Higher kernel yield per tree

Around 800 elite trees of Pongamia possessing significantly higher levels of kernel

yield of the range of over 45 kg / tree were selected based on the certain stable canopy

attributes strongly associated with seed yield. The yield data collected over two seasons

(2010-2011 and 2011-2012) demonstrated the consistently higher yield levels of the selected

trees. (Tables 3 & 4).The seed oil content of the selected trees ranged from 33 to 39% as

against oil content of 28 to 32% in normal and unselected trees (CFTRI 2012) (Table 5). A

certain number of elite Pongamia trees exhibited higher contents of oil and karanjin, a flurano

flavanol of industrial value (CFTRI 2012). Genetically elite tree of Pongamaia

pinnaGGGEBEGta FITREE (Figure1).

35.5 Vegetative Propagation

The propagation and multiplication of the selected elite trees had been carried out

adopting novel vegetative propagation and nursery technologies that ensured 90 to 100%success rate (Swamy 1992) in the recovery of the clones (VayuSaps™) of the elite trees. At

present VAYUGRID possesses 8, 27,065 VayuSaps™ containing the elite characteristics

contributing to high yields.

35.6 Vayusap Plantations

VayuSaps™ are to be planted at the rate of 200 saplings per acre in pre-dug pits each

of 45 to 50 cubic cm at spacing of 5m X 4m, with a mixture of dug-out top-soil, 4 to 5 kg

farm yard manure and 200 g of single super phosphate per pit with the onset of rains. The

trees start yielding by the end of the third year following planting with a progressive increase




375

in the yield every year. Normal unselected trees of Pongamia on the other hand, start yielding

poorly after the 6th of 7th year only. It is estimated that VayuSaps™ planted over an area of

one acre would yield around one ton of oil by the 5 th year following planting. VayuSap™

plantation technology is a low input; but highly rewarding one well suited to wide range of

agro-ecological situations including those characterized by marginal and problem soils with

low rainfall.

Table 3 - Yield of Selected Elite Trees in Hiriyur of Distt.Chitradurga, Karnataka

Location Tree

NumberTotal yield of the tree 2010-2012

District Village seed kernal yield

in kg (2010-2011)

seed/kernal yield

in kg(2011/2012)

Chitradurga Mayasandra 100 31 45

Chitradurga Kyathadevaranahatti 135 35 42

Chitradurga Bhaburu 139 44 41


Chitradurga Beemanabande 142 31 69




Chitradurga Hosahalli 171 30 42

Chitradurga Hosayalandu 174 31 32

Chitradurga Hosayalandu 177 30 37

Chitradurga VV.pura 181 44 29

Chitradurga AV.kottige 189 33 52


Chitradurga Bhaburu 236 60 63Chitradurga Shivapura 237 53 45

Chitradurga Shivapura 238 40 44

Chitradurga Kurubarahalli 265 30 34

Chitradurga Kurubarahalli 267 32 32

Chitradurga Doddaghatta 279 46 41

unselected control 9 8




376

Table 4. Yield Of Selected Elite Trees From Tumkur District Of Karnataka

LocationTree Number Total yield of the tree 2010-2012

District Village

seed kernal yield

in kg (2010-2011)

seed/kernal yield in

kg(2011/2012)

Thumakuru Hosahalli 312 34 44

Thumakuru Hosahalli 314 47 47

Thumakuru Chikkasarangi 316 51 46

Thumakuru Chikkasarangi 317 48 42

Thumakuru Chikkasarangi 322 47 42Thumakuru Kitthaganahalli 344 49 40

Thumakuru Vaddarahalli 358 51 59

Thumakuru Honnenahalli 360 54 42

Thumakuru Yallapura 411 38 35

Thumakuru Arekere 412 49 42

Thumakuru Obalapura 413 59 63

Thumakuru Obalapura 414 48 48

UnselectedControl

9 11

Figure 2. Productive Canopy of ElitePongamiaTree of VAYUGRID.

Figure 3. VayuSap™ at VAYUGRIDPongamia Nursery




377

Table 5. Fat and Karanjin Contents Of Some Elite Pongamia Trees From Chtradurga District

For The Year 2010-2011

Elite Tree No. Fat (%) In Seed Karanjin (%) In Oil

02 38.6 5.97

05 36.2 5.79

06 37.5 5.62

08 38.0 6.10

09 37.2 5.98

11 38.7 5.84

12 36.2 5.33

17 37.4 5.45

22 36.9 4.91

35 37.1 5.54

40 33.5 6.02

55 37.6 6.0058 38.0 5.72

61 38.6 5.17

83 33.5 6.17

125 38.4 5.98

181 36.1 5.78

202 37.0 5.38

203 36.0 4.99

211 33.4 6.04

218 32.8 6.07

236 38.2 5.21

239 37.7 5.38

Control 28.7 3.9




378

VayuSap™ plantations have been established with success by VAYUGRID at

Mahindra & Mahindra World City near Chennai, private farms near Hyderabad and

Bangalore and Jindal Lignite Mining Company (SWML) at Barmer (Rajasthan) in India and

also in Ethiopia and Djibouti in Africa.

Acknowledgement

The author thanks all his colleagues in VAYUGRID for their help, cooperation and

encouragement in carrying out this work.

References

1. CFTRI (2012) Consolidated Project Report on Studies on Karanja oil from Karanja

seeds for VAYUGRID, Bangalore, India. Central Food Technological Research

Institute, Mysore, India. 20.

2. Chaturvedi, R.K., Raghubanshi A.S.and Singh, J.S.(2011) Carbon density and

accumulation in woody species of tropical dry forest in India, Forest Ecology and

Management, 262(8): 1576–1588

3. DAFF (2008) Carbon Storage in Australian Forests. ABARES Fact Sheet.

4. Elevitch, C.R. and Wilkinson, K.M. (1999) Nitrogen fixing Tree Startup Guide.

SustainableAgricultural Research. USDA. Permanent Agricultural Resources, Holualoa,

HI96726, USA.

5. Gupta, H.S. (2009) Forest as Carbon Sink: Temporal Analysis for Ranchi district .

Indian Forester, 32 (1): 7-11

6. Jesse More (2009) Carbon Sequestration Potential of the MillionTrees.NYC Initiative ,

Biofuels and Bio-Based Carbon Mitigation

7.

Kamarkar, A., Kamarkar,S and Mukherjee,S. (2012)Biodiesel production from neem

towards feed stock diversification, Renewable and Sustainable Energy Reviews ,

16:1050–1060

8. Mohit Gera and Suresh Chauhan (2010) Opportunities for C sequestration benefits from

growing trees of medicinal importance on Farm lands of Haryana. Indian Forester

(2010): 287-300

9. Prasad, M.V.R. (1994) Minor Oil bearing Species of Forest Origin for Diversification of

Vegetable Oil Production. In: Prasad, M.V.R. et al(Ed). Sustainability in Oilseeds.




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Indian Society of Oilseeds Research, Directorate of Oilseeds Research, Hyderabad.

Pp91-98.

10. Prasad, M.V.R., Langa, A. and Consolo, J.P. (2000). Selection of superior genotypes of

cashew in Nampula zone of Mozambique. The Cashew, Jan-March,2000 :8-23

11. Rattansi, R., Dikshit, M, Rattansi, R and Dikshit, M. (1997) Protease inhibitors and in

vitro digestibility of Karanja (Pongamia glabra) oil seed residue. A comparative study

of various treatments. Journal of the American Oil Chemists' Society 1997. 74 (9):

1161-1164

12. Reddy, N.S., Ramesh, G. and Suryanarayana, B. (2009) Evaluation of Tree species

under different land use systems for higher Carbon sequestration. Indian J. Dryland

Agri. Res. & Dev.24 (2):74-78.13. Sengupta, R.P. (2013) Ecological Limits and Economic Development. Oxford

University Press, NewDelhi.

14. Swamy, K.R.M. (1992) Clonal Propagation of Elite planting materials. Soft-wood

Grafting, a novel technique. Proc. National Workshop on Cashew, Kanpur. National

Research centre for Cashew. ICAR. 76-79.




380

Part V

Electrochemical Processes




381

CHAPTER 36

EVALUATION OF ELECTRICAL PROPERTIES UNDERDIFFERENT OPERATING CONDITIONS OF BIO-

ELECTROCHEMICAL SYSTEM TREATING THIN

STILLAGE

Ghosh Ray, S., Ghangrekar, M.M.

Abstract

Edible ethanol production industries are generating wastewater with very high concentration

of organic matter and nitrogen; hence, if not properly treated it can adversely affect the

receiving environment. Low pH, high total COD of 61 g/l and TSS of 26.4 g/l due to the

presence of dietary fiber of the thin stillage made the mesophilic conversion more challenging,

and thus leading to lesser reduction of COD. Theoretically this industrial effluent has 238

kJ/m3 of energy present in the form of organic matter, which can be harvested in the form of

electricity by employing microbial fuel cell (MFC). In this study performance of MFC treating

thin stillage was investigated at organic loading rate of 1.5 g COD/l.d. Performance of this

Bio-electrochemical system (BES) is characterized by polarization behavior, exhibiting a

maximum power density of 22.16 mW/m2 and current density of 699.18 mA/m2 at 30 Ω

internal resistance. Maximum COD removal of 48% and 88% protein hydrolysis in anode

chamber was observed giving 2.91% Coulombic Efficiency (CE). Activation overpotential

was calculated to assess the kinetic properties during bio-electrochemical conversion. Charge

transfer coefficients (α) of 0.2 and 0.68, as well as the exchange current densities (i0) of 0.13

mA/cm2 and 0.1 mA/cm2 in the equilibrium state for cathode and anode, respectively were

observed. Further studies are required for breakdown of complex structure carbohydrate by

different physical and microbiological pretreatments to enhance the COD reduction, by

increasing the availability of acetate and to improved power generation.

Keywords: Bio-electrochemical system, Charge transfer coefficient, Exchange current

density, Mesophilic conversion, Microbial fuel cell, Thin stillage.




382

36.1 Introduction

The growing concern in continuous depletion of natural resources has germinated the

thought in researchers to invent alternative sources of energy. India is striving hard to achieve

required per capita energy consumption and expecting increased Gross Domestic Product

(GDP) from 4% to 7.5% with annual 8% growth in energy demand. Increasing population and

a high rate of economic growth proportionately increase the environmental pollution with the

emission of Green House Gas (GHG). Gradual diminution of natural resources and limited

commencement of renewable energy recovery arise questions on sustainability of country’s

energy security. An interest has been evolved in production of bio-fuels from nonconventional

substrate, thus, promises a new horizon of better future. Till date, 13% of country’s

renewable-based electricity capacity comes from biomass.

India has only 0.5% of world’s oil reserves. So, the authority has set a target to blend

ethanol with gasoline, initially at 5% in 2006, aiming to increase it up to 20% by 2017, to

step-down the discharge of harmful gasses to protect the environment. An aspiring venture has

been taken by many industries using non-food wastes like spent wash of sugar industries,

broken and uneatable grains for the commercial ethanol production. Distillery industry waste

is a major source of pollution among the 17 industrial wastes and thus having immense

potency in becoming alternative source of energy (MNRE, 2011).

Grain based alcohol has not been much popular in India; however, this can be a better

alternative as cultivation of malt producing grain (rice, barley, wheat, maize, jowar) is much

easier in Indian climate. Even though, starchy, cellulosic sources are being proven as a better

alternative, emerging technologies are insufficient to dissolve all the ailments. Moreover,

much concern has been generated to lower per liter ethanol production presently from 20 to 23INR. According to the CPCB report (2012), 100 grain-based distillery units are presently

running having the net capacity of 1.8 billion liters per annum. Mostly broken rice, kinki,

pearl millet and sorghum or mixed grains are being used mostly as raw material in Indian

distillery units. Increase in production of all type of rice to 12.7% from the last decade

consequently shows 9.8% increase in export quantity than the last five years. Only 5% of the

total produced rice is exported presently (AIREA, 2012). According to the statistics, around

12-24% of the annual total rice production in India is the broken rice, which is the by-productof rice mill, also known as brewer’s rice. Broken rice has been used for many years for




383

commercial alcohol production. Being of almost similar biochemical composition as whole

rice, 90% of the total dry weight is composed of α-glucans.

The high yield of paddy cultivation, especially in the eastern part of India generatesmore interest in distillers to utilize the cellulosic feedstock as the origin of ethanol production.

Some challenges possibly can be encountered as these small grains contain high concentration

of Non-Starchy Polysaccharides (NSPs), which have high water binding capacity, leading to

increased mash viscosity. NSP covers wide variety of polysaccharides excluding α-glucan

(starch). Hemicelluloses, such as arabinoxylans, xylans and xyloglucans are mostly found in

rice; the amorphous structure can be hydrolyzed easily to yield a complex coordination which

can hold water. The main cause of high viscosity mass is the presence of β-glucans andpentosans (Choct, 1997; Houston and Kohler, 1970).

During anaerobic respiration, it is difficult for micro-organisms to break down the

composite structure of NSPs, and hence Chemical Oxygen Demand (COD) reduction cannot

be achieved more than 70%. After rectification of spirit, the generated wastewater is having

COD ranging 55,000-60,000 mg/l, TSS around 13,000-15,000 mg/l and acidic pH (~3.6-3.8),

and it is 9-10 times more in volume than the total capacity of the plant. The wastewater,

typically having high concentration of yeast biomass is centrifuged and thin stillage fraction is

taken for wastewater treatment and several byproduct recoveries (Khannal, 2009). The highly

proteinaceous distillery grain or the solid fraction is used to produce cattle feed. Yeast

biomass increases the protein content of the wastewater. Therefore, pretreatment is necessary

for this wastewater to reduce suspended solids and organic matter. Fungal treatment of raw

wastewater can be done to produce Single Cell Protein (SCP), enzymes and biopolymers,

which are having high commercial value (Wilkie et al., 2000).

Minimization of organic load is been achieved by Indian industries in conventional

ways. Concentration of thin stillage followed by incineration reduces organics but this process

needs higher capital cost. Anaerobic digestion is another method where biogas generation

recoups more energy than that has been spent. As, none of the methods can suitably achieve

the distillery waste disposal standards, most recently bio-electrochemical systems (BES) are

developed that can utilize the post methanated effluent (PME) for the production of electricity

and hydrogen gas (Rabaey, 2010; Katuri and Scott, 2011; Kunusch et al., 2010). In microbial

fuel cell, microbial metabolism is utilized to convert carbohydrate to electrical energy. It is

proposed that, the utilization of remaining organics with high COD in such a manner can




384

successfully serve the purpose by producing alternative energy as well as reduction of

organics. The process is cost effective, although, it is not been established commercially and

many research is yet to be done (Rabaey and Verstraete, 2005). Energy conversion from PME,consisting complex carbohydrates, yeast cell biomass, several metabolites of fermentation and

very less residual mono-sugar and its derivatives, into electricity is the utmost challenge that

has been faced by the researchers to make this system commercially viable. Not much work

has been pursued for complex real wastewater to sustainable energy conversion (Lu et al.,

2009; Behera et al., 2010; Huang et al., 2011). The objective of the present study is to

elucidate the prospects of producing electricity from residual sugar and soluble proteins in raw

thin stillage, as well as simultaneous reduction of carbonaceous and nitrogenous organics bybiologically active heterotrophs. Furthermore, the study contributes to the knowledge about

feasibility of secondary treatment of high organic real wastewater.


36.2.1 Wastewater characterization

Thin stillage was acquired from the distillery unit of IFB Agro Ind. Ltd., Kolkata and

kept at 4° C. Ethanol has been produced in this industry from broken rice. Enzyme treated

cellulosic mash is subjected for fermentation with specific yeast strain and ethanol is

produced. Highly viscous wastewater is generated after rectification of ethanol. Centrifuged

wastewater known as thin stillage is collected and characteristic studies are done. Estimation

of total solids (TS), total volatile solids (TVS), total suspended solids (TSS), volatile

suspended solids (VSS), COD, biological oxygen demand (BOD), NH3 –N, total protein,

lignin and phenol were done following the standard methods (APHA, 1998). Acetate, nitrate

and phosphate content are estimated by Ion Chromatograph (Albright, 2008; APHA, 1998).

36.2.2

BES operation principle and reactor fabrication

A triple chambered BES is fabricated where two chambers are assigned as anode and

cathode and the third chamber is designed as nitrification chamber. Anode contains high

organic loading of cellulose, starch and proteinaceous wastewater which is inoculated with

heat treated sludge of calculated amount for removal of organic carbon under anaerobic

condition (Jadhav and Ghangrekar, 2008; Mohanakrishna, 2010). Sludge contains mostly

mesophilic range of bacteria, having the ability to hydrolyze and ferment organic compoundsto simple sugar, amino acids and fatty acids. In the next step acidogenic microorganisms




385

convert the yields to fatty acid, acetate, CO2 and H2 through different fermentative pathway.

The low molecular weight carboxylic acids are utilized by H2 producing syntrophs and acetate

is generated. Homoacetogens also produce acetate from fermented yields. The treated effluentcontaining degraded organic matter and organic proteins are mostly converted to ammonium

ions by de-nitrifiers (Virdis et al., 2010). Nitrification chamber was seeded with sludge from

anode. The effluent from anode is fed to nitrification chamber where oxygen is purged to

oxidize ammonium species by facultative autotrophic bacteria converting it to nitrite and then

to nitrate by nitrifiers in that same chamber where further degradation of complex

carbohydrate is also possible. Electrons generated by anode mechanism migrates through

external metallic circuit towards cathode while, H

+

ion passes through a clay separator tocathode and in presence of diffused oxygen it gets reduced to complete the electrochemical

cycle (Logan, 2008; Watanabe, 2008; Pant et al., 2010; Wen et al., 2010). Carbon felt is used

as the electrode material providing large effective surface area to grow the bio-film. Distance

between cathode and anode is reduced by attaching the electrodes close to separator to achieve

maximum efficiency of the cell (Rismani-Yazdi et al., 2008). The schematic diagram of C and

N mass transfer within the reactor is shown in figure 1.

R

AnodeCathode

PEM

NH4+

NO3-

Nitrification

Chamber

O2

Final

Effluent

O2

PEM Proton E!chan"e Membrane

#reated Effluent

Com$le!

$rotein

Pol%-&acc.

AcetateNH4

+

Feed

1.15 l

1.15 l

1.5 l

Figure 1: Schematic diagram of BES for simultaneous carbonaceous and nitrogenous thin

stillage treatment. Three chambers anode, cathode and nitrification chamber (NC) are labeled.

The volume of two half cells were kept similar as 1.15 l and NC as 1.5 l. The system is

maintained with 1.5 kg COD/m

3

.d

of organic loading rate in each cycle of 48 h of conversion.Phosphate buffer (50 mM) is applied to increase the feed pH from acidic range to near neutral




386

range. Reactor is fed by gravity in an up-flow mode with 5.75*10 -4 m3 /day flow rate. The

system is kept at 37±4° C to proliferate particularly mesophilic range of bacteria.

36.2.3

Treatment efficiency

The Coulombic Efficiency (CE) is directly related to degradation of complex waste

and electron migration:

CE (%) = (1)

where, Ms is 59 gmol-1 for acetate to transfer anodic electrons, b=8 (number of electrons

exchanged per mole of acetate), VAnode is the volume of anodic liquid, t is change in time over

which the measurement is taken and ∆COD is change in COD over that time period.Simplification of carbonaceous waste and peptide breakdown of organic nitrogen happens at

anode due to secretion of glycolytic and proteolytic enzymes by wide range of mesophilic

microorganisms. Electrons released during their metabolism are transferred to anode which

increases the flow of electron to the external circuit.

36.2.4

Calculation of thermodynamic entities

Half cell reactions:

Forward: CH3COO- + 4H2O 2HCO3- + 9H+ + 8e- (E0A = 0.300 V) (2)

Backward: O2 + 4H+ + 4e- 2H2O (E0C = 0.805 V) (3)

The thermodynamic units are calculated considering these half cell equation to prove

the spontaneity of the overall reaction. Free energy calculation of the fuel cell is calculated as:

- ∆G = nFE emf (4)

Where ‘n’ is the no. of electrons involved in the above reaction 1 and 2, ‘F’ is the Faraday’s

constant (96,485 C/e-mol), Eemf is the electromotive force considering the overall reaction and

E0A, E0C are equilibrium reaction potential of anode and cathode respectively. The maximum

energy efficiency and performance related to open circuit are characterized by polarization

curves where cell voltage is plotted as a function of current density to obtain kinetic data from

the steady-state current-voltage measurement.

E 0

Cell = E emf + E C - E A (5)

From equation 3 and 4 overall free energy of the system is calculated where E emf

represents the difference between cathode and anode working potential (EC and EA) under no

current flow condition. From the above equation EA and EC can be derived as:




387

E A = E 0A -

(6)

And, E C = E 0C -

(7)

Where, represent the concentration of proton involved in forward and backward

reaction which can be calculated from the pH value of the reaction (Manohar al., 2008).

36.2.5 Consideration of overpotentials:

A non-linear model is represented in order to characterize the electrical behavior of the

BES. From the polarization behavior, overall potential losses can be considered in a more

comprehensive way by accounting kinetic overpotential, ohmic loss, mass transfer limitation

and loss due to internal current. Kinetic overpotential occurs due to loss of activation energy

to overcome the slowest step of the electrochemical reaction by showing changes in electrode

potential (Bard and Faulkner, 1980). Tafel analysis evaluates Exchange current (I0), passing

through the electrode surface in the equilibrium state to emphasize activation potential by

deducing from Butler-Volmer Equation:

For cathodic reaction-

(8)

For anodic reaction-

(9)

Where I stands for total current density, when reactions take place at 37°C, α is called as

charge transfer coefficient (A- for anode and C –for cathode) which depends upon the reaction

involved and electrode material must lie between 0 to1. IA0 and IC0 are respective exchange

current passing through anode and cathode. and represent the overpotential observed for

anode and cathode, respectively.

36.3 Experimental results and discussion

36.3.1 Wastewater characteristics and removal of nutrients

The acidic wastewater comprises considerable level of non-fermentable organic

nutrients. Total COD and TSS range was high in between 55,000 and 60,000 mg/l and 26,000

and 28,000 mg/l, respectively (Table 1). Secondary treatment in BES is applied to reduce

COD and TSS. High level of suspended particulate matter obstructs the reaction dynamics of




388

the system by physical clogging or by increasing the viscosity of the effluent. Diluted

wastewater of pH 6.97±0.03 is fed to anodic chamber. Organic N is hydrolyzed resulting in

formation of organic acids and ammonium ion, which is eventually responsible for increasedalkalinity due to formation of ammonium bicarbonate. Increase in in-situ alkalinity by 30.8%

resulted due to conversion of organic matter, showing gradual increase in pH to 7.13±0.03

range. Catholyte pH of 7.59±0.05 is solely due to migration of ammonium ion through

separator. Increase in conductivity with increased TDS level of anolyte was observed, which

might have occurred due to breakdown of complex organic matter by extracellular microbial

enzymes.

Table 1 Characteristics of thin stillageParameter Value

pH 3.54

Conductivity (mS/cm) 2.679

TS (mg/l) 43,500

TSS (mg/l) 26,160

VSS (mg/l) 19,816

TDS (mg/l) 2704

TVS (mg/l) 40,776

Total COD (mg/l) 61,295

BOD5 (mg/l) 28,120

NH3 –N (mg/l) 610

Acetate (mg/l) 1599

Total Protein (mg/l) 4256Nitrate (mg/l) 0.8147

Orthophosphate (mg/l) 1670

Lignin (%) 5.586

Phenol (%) 0.532

The maximum COD removal is found to be 48±3 % over 12 weeks of operation. The

presence of less free acetate and because of complex nature of the waste or possibly due toNSP, the average COD reduction in anode chamber is not more than 50%, although the higher




389

effective surface area of carbon felt encourages bio-film growth to create considerable

potential. System performance due to charge transfer is calculated by equation 1, where

coulombic efficiency is more than 3%. Nitrogenous structure of cell wall gets hydrolyzed dueto bacterial metabolism rising ammonium ion concentration in anode due to simplification by

proteolytic enzymes. Moreover, the carbonaceous waste escaping from anode goes to

nitrification chamber, where it gets digested aerobically with simultaneous nitrification of

increased ammonium ion in presence of oxygen available through air purging. Conversion of

ammonium ion to its oxidized species such as nitrate and nitrite is recorded as 85±6 % by

ammonia oxidizing bacteria present in nitrification chamber.

36.3.2

Analysis of thermodynamic parameters

Theoretically the collected effluent has 238 kJ/m3 of energy present in the form of

organic matter. Thermodynamic entities are evaluated from equation 4 and 5 where theoretical

cell voltage, E0, at no current flow condition hence no loss condition is 1.009 V, however the

obtained open circuit potential were EA 0.532 V and EC 0.050 V, during no current flow

condition. From equation 6 and 7, the ratio of concentration of anodic element of real

wastewater, can be calculated which eventually assists to prove the spontaneity of

the reaction.

36.3.3 Analysis of electrical parameters

Polarization behavior i.e., voltage as a function of current and change in current

density over power density under different operating conditions (10,000-10 Ω external

resistance) is demonstrated to understand electrical properties in Figure 2. Maximum

volumetric power density is exhibited as 22.16 mW/m2, when the current density is plotted as

699.18 mA/m2 at 120 Ω external resistance. Internal resistance under maximum power point

was 30 Ω. Spontaneity of the overall reaction depends on reduction efficiency in cathode.

Theoretically 0.427 V more reduction potential can be produced for maximum electricity

generation by minimizing overpotential at cathode and enhancing oxygen reduction reaction.

From the polarization study we can interpret the loss in overall cell potential. Due to

voltage reduction in low current region of the graph, steep slope has been seen at the initial

stage which is solely because of activation polarization due to charge transfer. As the current

increases the slope becomes less steep as in this region loss occurs on account of the

resistivity of proton exchange membrane and cell electrolytes. Polarization of electrode




390

happens due to shift in cell potential from equilibrium potential and accordingly, this

overpotential can be measured to justify actual cell performance. Kinetic overpotential is

measured by Tafel plot shown in Figure 3.

Figure 2. Polarization behavior of BES

Figure 3. Tafel plot and charge transfer kinetics of working BES for (a) anode and (b) cathode

Exchange current density (I0) shows the current flow in absence of net electrolysis at

equilibrium, which is quantified as 0.13 mAcm-2 and 0.1 mAcm-2 considering α values as 0.2

and 0.68 during reduction and oxidation for cathode and anode, respectively, using equation 8

and 9. Charge transfer efficiency is the function of organic decomposition by micro-organisms

in negative electrode. The performance analysis is based on the availability of simple organics

and presence of exoelectrogens.

36.4 Conclusion

Post fermented rice distillery wastewater shows considerable potential as a source to

produce alternative energy. Although, this process emphasized a lot of challenges due to the

complex nature of the wastewater and high level of suspended solids, hindering the normalflow of the bio-electrochemical cell. Hence, emergence of different pretreatments to reduce




392

9. Jadhav G. S., and Ghangrekar M. M. (2009). Performance of microbial fuel cell

subjected to variation in pH, temperature, external load and substrate concentration.

Bioresource Technology, 100(2):717-723.10. Katuri K. P., and Scott K. (2011). On the dynamic response of the anode in microbial

fuel cells. Enzyme and microbial technology, 48(4):351-358.

11. Khannal S. (2009). Anaerobic biotechnology for bioenergy production: principles and

applications. John Wiley & Sons.

12. Kunusch C., Puleston P. F., Mayosky M. A., and Moré J. J. (2010). Characterization

and experimental results in PEM fuel cell electrical behaviour. Int. J Hydrogen

Energy., 35(11):5876-5881.13. Logan B. E. (2008). Microbial fuel cells. John Wiley & Sons. New Jersey.

14 . Lu N., Zhou S., Zhuang L., Zhang J., and Ni J. (2009). Electricity generation from

starch processing wastewater using microbial fuel cell technology. Biochem. Engg. J.,

43(3):246.

15. Manohar A. K., Bretschger O., Nealson K. H., and Mansfeld F. (2008). The

polarization behavior of the anode in a microbial fuel cell. Electrochimica acta,

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N. (2013). Bioelectricity generation using two chamber microbial fuel cell treating

wastewater from food processing. Enzyme and microbial technol., 52(6-7):352-357.

17. MP and MNRE (Year) Strategic Plan for New and Renewable Energy

Sector for the Period. Ministry of Power and Ministry of New and

Renewable Energy, Government of India. 2011-7.

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desalination along with power generation. J. hazardous materials., 177(1):487-494.

19. Pant D., Van Bogaert G., Diels L., and Vanbroekhoven K. (2010). A review of the

substrates used in microbial fuel cells (MFCs) for sustainable energy production.

Bioresour. Technol., 101(6):1533-1543.

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energy generation. Trends in Biotechnol., 23(6):291-298.




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21. Rismani-Yazdi H., Carver S. M., Christy A. D., and Tuovinen O. H. (2008). Cathodic

limitations in microbial fuel cells: an overview. J. Power Sources., 180(2):683-694.

22. Strategic Plan for New and Renewable Energy Sector for the Period 2011-17. (2011).Ministry of New and Renewable Energy. Government of India.

23. Srikanth S., and Venkata Mohan S. (2012). Change in electrogenic activity of the

microbial fuel cell (MFC) with the function of biocathode microenvironment as

terminal electron accepting condition: Influence on overpotentials and bio-electro

kinetics. Bioresour. Technol., 119:241-251.

24. Virdis B., Rabaey K., Rozendal R. A., Yuan Z., and Keller J. (2010). Simultaneous

nitrification, denitrification and carbon removal in microbial fuel cells. Water research,44(9):2970-2980.

25. Wen Q., Wu Y., Zhao L., and Sun Q. (2010). Production of electricity from the

treatment of continuous brewery wastewater using a microbial fuel cell. Fuel,

89(7):1381-1385.

26. Watanabe K. (2008). Recent developments in microbial fuel cell technologies for

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394

CHAPTER 37

EFFECT OF SALINITY, ACETATE ADDITION ANDALTERATION OF SEDIMENT ON PERFORMANCE OF

BENTHIC MICROBIAL FUEL CELLS

Jadhav D.A., Ghangrekar M.M.

Abstract

Sediment/benthic microbial fuel cell (SMFC) is an attractive approach for bioelectricity

production alongwith sediment bioremediation in water bodies. This study mainly focus on

operational factors like salinity of catholyte, acetate addition and alteration of sediment

properties, which mainly affect performance of SMFC in terms of power production. The

SMFC consisting of graphite electrodes with equal surface area of 28 cm2 for each anode and

cathode and separated by 8 cm distance between them was used. Freshwater sediment was

collected from Lower Saxony, Germany. With increase in salinity level by NaCl addition inthe water, the cell voltage increased double upto salinity of about 7 mS/cm and further

increase in salinity decreased the performance of SMFC. Conductivity of freshwater is a main

limiting parameter in SMFCs performance. The acetate can be used as the carbon source to

induce power production by electroactive bacteria. An increment was reported in cell voltage

due to addition of acetate upto 2 mM and further addition decreased the power from MFC.

While evaluating performance of SMFC with different sediments, the result shows that the

operating voltage of SMFC varies not only with locations from where sediment was collectedbut also upstream to downstream of same catchment area due to variation in total organic

carbon present in the sediment and microbial activity. In future, research should be focused on

the designing of SMFCs with increasing size and scale for increased power output and

reducing operational problems.

Keywords: Sediment microbial fuel cells, Conductivity, Sediment alteration, Acetate.




395

37.1 Introduction

Extensive research toward developing reliable microbial fuel cells (MFCs) is mostly

focused on selecting suitable operating conditions, organic and inorganic substances whichcan be used as sources of energy. Sediment microbial fuel cells (SMFCs) is important to

generate power for operating under sea devices like sensors, hydrophones (Bond et al., 2002).

Organic-rich sediment in freshwater and marine environments can be considered an abundant

potential source of renewable energy in SMFCs. The naturally occurred sediment water

interface serve as separator for anodic and cathodic chamber in SMFC (Rabaey et al., 2009).

The anode is placed in anaerobic sediment and connected it through an external load; the

cathode is placed in the overlying water (Fig. 1). Freshwater sediments and river water can beused as a renewable source to generate power using sediment microbial fuel cells (Sajana et

al., 2013; Lovely, 2006). The performance of SMFC is mainly governed by the characteristics

of sediment, operating conditions like salinity, electrode spacing, electrode material and its

properties, etc. Sediment-based MFCs were first explored in 2001 by Reimer et al., and it was

demonstrated that power could be generated from the microbes and the nutrients found within

the sediment alone (De Schamphelaire et al., 2008; Nielson, 2008).

Fig.1 Mechanism of electron transfer in SMFC

Previously, Logan et al. (2006) reported that dominant ohmic losses through the

electrolyte can be reduced by increasing the electrical conductivity (EC), which results in

higher power production from SMFCs in marine environments than in freshwater

environments.

Current and power densities increase with the increasing salinity of the aquatic

medium and therefore with increasing ionic strength and conductivity (De Schamphelaire et




396

al. 2010; Tender et al., 2002). Hidalgo et al. (2010) noted that the increase in NaCl

concentration resulted in an increase in both power density and Coulombic efficiency (CE) as

long as NaCl concentration remained below 2 mM. At latter value a maximum power and CEwere obtained (82.1 W/m2 and 15.91% respectively).

Particularly in marine environments, Geobacteraceae species are involved in electron

transferring process. Acetate served as the fuel in an MFC using marine sediment as the

inoculum (Lee et al., 2003). Song et al., 2010 found that maximum power density from the

SMFC with addition of 0.2% cellulose reached to 11.2 mW/m2 on 160 days of operation in

freshwater environment. Addition of cellulose causes increase in performance of SMFC after

few days of operation. The alteration of physico-chemical properties of sediment organicmatter (SOM) was studied by Hong et al., 2010 and reported that the physico-chemical

properties of SOM were considerably altered when electrical current was produced from

freshwater sediments in the MFC systems.

In the present study, SMFC was used to enrich electrochemically active microbes on

acetate as the sole electron donor. To date little effort has been directed towards quantifying

and predicting the effects of properties of sediment on the anodic reaction during electricity

generation from SMFC. The specific objective in this study was to evaluate the performance

of MFC under change in properties of sediments collected from different locations. The effect

of salinity or conductivity of water on performance of SMFC was also studied to find

optimum salinity limit.


37.2.1 Sediment as source of inoculum

The sediment [S0] was collected from downstream at Sandbach catchment area near

Hordorf and Schandelah, Lower Saxony, Germany. This site was located next to the

municipal wastewater treatment plant and not affected by any effluent contamination. The

sampling depth was restricted to 5 cm from the upper layer of the sediment in order to sample

mainly sediment under aerobic conditions (OECD, 2002). The sediment was then placed in

plastic container and covered with native water. Water/sediment samples were characterized

(Table 1) according to the OECD guideline (OECD, 2002, Mohamad, 2010).




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Table 1 Characteristics of sediment collected for study (Mohamad, 2010)

Sediment Properties Values

Sediment type SandySand 53 %

Silt 20%

Clay 27%

TOC (Total organic carbon) 0.4% (dry substrate)

WHC (Water holding capacity) 30 %

pH 7.3

Eh (Redox potential) 220 mVSIR (Substrate induced respiration) 1.90 mg O2 /100 g h

37.2.2 MFC design and Construction

This study was carried out with four laboratory scale SMFCs having an anodic volume

of 5 l. The sediment was collected from downstream at the Sandbach catchment area near

Hordorf and Schandelah, Lower Saxony, Germany. The SMFC was constructed with the

cathode and anode of graphite material having a total cross sectional area of 28 cm2 placed

parallel to each other (Fig. 2). The bottom half portion of the SMFC was filled with

homogeneously mixed sediment to work as anolyte and upper half portion with freshwater to

work as a catholyte. The spacing between electrodes was maintained at 8 cm. Both electrodes

were held in SMFC by a polyvinyl chloride (PVC) cylindrical pipe holder. The connection

between wire and electrode was made using conductive and watertight mixture of resin,

hardener and carbon powder to make it conductive. Air was introduced in the form of fine air

bubbles by using air diffuser and a air pump (ELITE 799, Hagen, Mansfield, MA) in the

freshwater on cathodic side. All connections were made with watertight connectors using

silicon and flexible parafilm. Over the period of the experimental run, the water loss occurred

due to evaporation was compensated by adding additional either freshwater from river or

distilled water daily and a constant water level in MFC was maintained. SMFC was operated

by adding wet sediment to the anodic side without giving any pre-treatment. Within a day, the

sediment re-settled at the bottom of the bucket. After stabilization, 100 ohm external

resistance (unless stated otherwise) is connected in circuit.




398

Fig. 2 Experimental set-up of lab scale SMFC

37.2.3 Measurement and analysis

The potentials generated by the cells were measured with a digital multimeter with

data acquisition unit (model 2700 and 2701, Integra Up and Running, Keithley Instruments,

Data Acquisition, Ohio, U.S.A). The voltage difference between each electrode pair was

recorded at 5 min interval on a spreadsheet using ExceLINXTM (Keithley). Power was

calculated according to the formula: P = IV , where ‘P’ = power, ‘ I ’ = current and ‘V ’ =

voltage. Power density (mW/m2) was calculated as power production per m2 surface area of

anode and the power output is usually normalized to the anode surface area because the anodeis where the biological reaction occurs (eq. 1, 2).

P A = E * j … (1)

j = …

(2)

where, P A = power density, E = voltage generated, R = external resistance, Aa = area of anode,

j = current density, P = actual power generated.

The highest voltage produced in a SMFC is the open circuit voltage (OCV), which was

monitored after allowing the circuit to remain in open condition (no current flow) till the

potential value is stabilized. The anode potential, cathode potential, OCV and operating

voltage (OV) was also monitored daily during experiment under different operating

conditions. Cathode potential was measured by using Ag/AgCl reference electrode (Sat. KCl,

sensor technique, Meinsberg GmbH, Germany). The cell EMF can be calculated as eq. 3

E emf = E cathode – E anode … (3)




399

Conductivity of water in SMFC was measured daily by using a conductivity probe

(GLF 100, Greisinger electronics, Germany). The pH of water was measured by using pH

meter (GPHR 1400A, Greisinger electronic, Germany). Temperature during experiment wasmaintained nearly constant about 18 ± 0.5 °C in winter and 20 ± 0.5 °C in summer season.

37.2.4 Effect of Salinity

The electrical conductivity (EC) of freshwater in SMFC [with sediment S0, Table 2] is

about 1030-1270 µS/cm. Freshwater typically contains less than 1% sodium chloride. To

study effect of EC, salinity of water changed from 1000 to 10500 µS/cm using sodium

chloride (NaCl) (added stepwise) while considering bacterial sensitivity to sodium salt.

37.2.5 Effect of Acetate addition

Acetate is the most abundant fatty acid in anaerobic ecosystems and is used as an

electron donor by anaerobic respiratory bacteria. To study the microbial response, acetate was

added and performance of SMFC with 8 cm electrode spacing (with sediment S 3, Table 2)

performance was evaluated. Acetate was added as 0 to 15 mM at regular interval of time to

enhance activity of microorganism. The sodium acetate was added considering volume of

anodic side to maintain this concentration. However, there is possibility of diffusion of acetatethat occurs through sediment water interface. The pH of acetate solution was neutralised using

glacial acetic acid (CH3-COOH). In SMFC, acetate injected at different points in sediment

using syringes so that it distributed evenly in the sediment. Over the course of operational

period, bioelectrical response in association with external acetate addition was observed.

37.2.6 Alteration of sediment properties

The sediment properties changes from location to location due to different natural and

human activities so that performance of SMFC also varies. However, the relationships

between organic carbon decomposition, microbial diversity and density, and power production

remain largely unknown. In this study, aim is to find how sediment from different location

affects on cell performance. For this study, sediments were collected from Sandbach

catchment area (52°16'7.16"N, 10°40'51.81"E) [S0] from downstream, [S3] from upstream of

same catchment, Wiedigsteich Lake (52°16'18.28"N, 10°34'47.26"E) [S1] and Kreuzteich

Lake (52°16'19.33"N, 10°34'39.82"E) [S2] in Braunschweig, Lower Saxony, Germany. River

sediment was collected from shallow river bank. The prototype of SMFCs using these




400

sediments were constructed similar to SMFC as mentioned previously with maintaining 8 cm

spcing between the electrodes. The characteristics of sediments are given below in Table 2.

Table 2. sediment characteristics of SMFC collected at different locations in study areaSediment Properties Values of sediment characteristics from different locations

S0 S1 S2 S3

Location in

Braunschweig

Sandbach

catchment (d/s)

Wiedigsteich

Lake

Kreuzteich

Lake

Sandbach

catchment (u/s)

pH 7.3 7.62 7.51 8.03

Conductivity (µS/cm) 1060-1276 1170-1265 918-1090 910- 1575

Other Sandy type More watercontent

*** Loamy type

*** Values not determined


37.3.1 Effect of Salinity

To study effect of salinity, EC was increased from 1000 to 10500 µS/cm by adding

sodium chloride (NaCl) stepwise. Increase in EC of freshwater caused decrease in ohmic

losses and increase in power output. It was noted that high salinity of the water provides good

ion conductivity between the electrodes. No change in the microbes present in the sediment

was done, while changing the salinity by NaCl addition. It was reported that cell voltage

increases with increasing salinity of water (Logan et al., 2006). However, cell voltage

decreases after certain limit of salinity because of possible adverse effect on bacterial

metabolism at higher salt concentration. Bacteria can produced current only within certain

salinity limits, and thus solution conductivity cannot be increased beyond certain value.

SMFCs shows critical salinity limit at 7000 µS/cm. This critical limit is helpful to determine

level of salinity of river water for feeding it to an SMFC system which also limit use of SMFC

for highly saline seafloor.

There was small apparent change in cell performance in SMFC with NaCl addition of

upto 90 mM, then cell voltage increases rapidly (Fig.3). Increasing the catholyte conductivity

decreases the ohmic resistance, and it has been shown that power density can be substantially

increased by adding NaCl upto 444 mM of to the solution. Further increase in salt

concentration reduced power production by inhibiting bacterial growth because microbes are




401

sensitive to certain salinity limits (Jeffrey, 2008). Also, high salt concentrations are known to

adversely affect the physiology of anaerobic microbial consortia. However, at high salt

concentration, cations present in the solution compete with protons in their migration towardscathode which results into reduction in voltage produced by the cell (Wang et al., 2011).

During this experiment, cathode potential was found to be negative in some cases because of

electrical energy provided to the cell is being used to decompose organic compounds present

in sediment. The cell voltage increased from 3.81 to 7.38 mV upto maximum salinity limit

(about 7 mS/cm) when operated at 100 Ω resistance (Fig. 3). The cell voltage decreased with

further increase in salinity. This suggests that EC (salinity) of water is main limiting factor in

SMFC performance which increased cell voltage by 93% due to reduction in ohmic losses,when the salinity was at optimum.

1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000

4,0

4,5

5,0

5,5

6,0

6,5

7,0

7,5

C e l l V

o l t a g e ( m V )

Salinity (µS/cm)

Cell Voltage

Fig 3 Effect of salinity on SMFC performance

37.3.2 Effect of acetate addition

To understand the effect of presence of organic matter in sediment on performance of

MFC, acetate was used as the external carbon source. Microbes like geobacter oxidizes

acetate to carbon dioxide and water while reducing compounds such as sulphur and some

metals including iron oxides, with quantitative transfer of electrons to an anode. Acetate

added get oxidised faster due to faster oxidation after some time. Small change in voltage

occurred during acetate addition initially at low concentration (Fig. 4; Fig. 5). Acetate was

used as carbon source to induce power production by electroactive bacteria (Kim et al., 2000)

because of its inertness towards alternative microbial conversions at room temperature (Zhao

et al., 2012).




402

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

2

4

6

8

10

12

14

16

C e l l v o l t a g e ( m V )

Acetate addition (mM)

8 cm A

8cm B

8cm C

Fig 4. Effect of acetate addition on cell voltage

87 90 93 96 99 102 105 108 111

0,000

0,002

0,004

0,006

0,008

0,010

0,012

0,014

0,016

0,018

0,020

Cell voltage

Power density

Time (days)

C e l l v o l t a g e ( V )

0.3mM

0.33 mM

1mM

2mM

5mM

10mM

15mM

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

P o w e r d e n

s i t y ( m W / m 2 )

Fig. 5. Voltage produced and power density vs. time at different acetate concentration

Once the current reached a stable level, acetate was consumed completely, and currentgeneration was dependent on the acetate supply. During consumption of acetate, cell voltage

increased and then started decreasing along with time period (Fig. 5). Addition of acetate

stimulates growth of bacteria in the MFC. Power density also increased upto 2 mM acetate

addition and then decreased. Addition of 5 mM also drastically changed electrode potential.

The results suggest that there is about ten fold increment in cell voltage due to addition

of acetate (2 mM) in sediment. There was slight increase in cathode potential after 5 mM

addition (Fig. 4); however, the potential started to decrease slowly. After 10 mM acetate

addition, cell voltage decreased further and subsequent additions of acetate typically did not




403

increase voltage output. The reduction in power production at higher acetate addition could be

attributed to higher concentration of acetate near cathode, which had adversely affected

cathodic reaction (Zhang et al., 2006) and reducing overall performance of the cell. There is apossibility of diffusion of sodium acetate (CH3-COONa) through sediment water interface

during injection in sediment zone.

37.3.3 Alteration of sediment in SMFC

The sediment properties are useful for determining the microbial biomass and

microbial activities in the sediment. This gave hint for high porosity of river sediments as it

consists of mainly coarse to fine sand grains. Power generation by SMFCs was dependent on

the particulate sediments, sediment structure and texture. In general, rivers in Braunschweig

contain less organic matter. Due to this, large fish and other aquacultural animals found less in

quantities in these rivers.

In addition, assuming that temporal organic pollution has occurred in a water body

(rivers), this study examined the possibility of harvesting current by utilizing the organic

matter coexisting in water and sediment phases. Results showed that the most important

parameters that control SMFC’s power output were total organic carbon in the sediment and

activity of geobacter . It was found that sediment properties had strong effect on SMFC’s

performance, and Fe(III) contents in sediments were significantly related to voltage values

produced (Song et al., 2012). The environmental conditions in the sediments were suitable for

enrichment and growth of microbes. However, the activity and competitive ability of

exoelectrogenic microorganisms in sediments in different locations might show variance

(Table 1; Table 2), as the composition of sedimentary microbial communities were

significantly affected by local environmental conditions, large intra-lake variability in the

composition and the relative abundance of microbial communities in sediment was always

observed (Cordova- Kreylos et al., 2006).

During initial period of operation, SMFC with loamy sediment (S3) showed current

density of 4.18 mA/m2 which is quite higher than other sediments (Fig. 6); because the

sediment S3 belonging to this SMFC contained more mud and humic substances as compared

to the other SMFCs (with sediments S0, S2 and S1) consisting of more fine to coarse sand as

major sediment type and some other pollutants (Table 2). The performance of SMFC utilizingS2 sediment was slightly lesser than SMFC with S3 sediment. Over the course of experimental




404

period, stable OCV’s were observed from SMFCs. Due to high percent of sand, SMFC with

sediment S0 showed lower voltage (about 0.69 mV). In this MFC, due to higher porosity of

sand, there is possibility of oxygen intrusion to the anodic side. It shows that the physicalproperties of sediments and the dynamics of natural environments are a special set of factors

that affect the performance of SMFCs. These results suggest that site selection for sediment

collection is important constraints before starting the experiments on SMFC. Certainly, the

values representing physical and chemical properties of sediment of the Sandbach catchment

area (upstream) has been highly humified in comparison with humus materials found in other

sediment type used in the study. Therefore, the presence of more humic substances in

sediments may boost current production in freshwater SMFC.The sediment S1 contains more water than others sediments used in the study. Hence,

SMFC with sediment S1 showed lots of noise in measurement during operation over period of

time (not shown in Fig. 6). The sediment properties changes from one location to other

locations on same water bodies. So, it’s not correct way to consider same SMFC power

performance to all along the bank of river as noted in case of Sandbach catchment (Table 3).

The electric signal produced in SMFCs reflectes the activity and competitive ability of

exoelectrogenic microbes in sediments present at that location. Thus, these observationssupport that the current production induced by microorganisms and sediment organic matter

(SOM) was dependent on the sediment characteristics: type of organic matter and diversity of

microbial populations (Hong et al., 2009).

Fig. 6 Power performance of SMFC with different sediment




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37.4 Future perspectives

It seems that SMFCs are effective technique to remove organic matter from sediment

(sediment bioremediation) and also useful for energy harvesting. With few improvements, inorder to scale it up to an appropriate size, SMFC could really make a difference in the world

energy sector for future generations. The potential for energy generation from the seafloor is

large, although the accessibility will often pose a problem. Furthermore, there are many

microorganisms yet to be discovered that might be beneficial for electricity production. The

cell design in future tests should more closely reflect environmental conditions. Sediment type

fuel cells show potential for low power generation from marine contaminated and

uncontaminated sediment. Future research should be focused on maximizing of power outputand minimizing construction and operation cost of SMFCs.

Table 3 Summary of performance of SMFC with different sediment characteristics

SMFC

with

sediments

Operating

characteristics

Locations in

Braunschweig

Power

density

µW/m2

Current

Density

mA/m2

Cell Voltage

at 100 ohm

(mV)

S0 8 cm ES Sandbach

catchment (d/s)

1.7 2.46 0.69

S1 8 cm ES Wiedigsteich 1.12 2.01 0.56

S2 8 cm ES Kreuzteich 4.56 4.04 1.13

S3 8 cm ES Sandbach

catchment (u/s)

4.89 4.18 1.17

37.5 Conclusions

Power performance in cell increases by 93 % with increase in the salinity of water

from 1.03 to 7 mS/cm in SMFC. Our investigation also revealed that power density varies

with sediment properties as well. Increase in concentration of acetate increases the power

density till 2 mM and further increment reduce the power production from the SMFC. The

power output for SMFC using sediment from Sandbach catchment area (upstream) is higher

than other SMFC from remaining sediments used for study. Thus, these results support that

the current production induced by microorganisms and sediment organic matter was

dependent on the sediment characteristics: type of organic matter and diversity of microbial

populations. These results can be helpful for site selection to install scale up SMFC for power




406

harvesting from freshwater sediment. It was noticed that cell voltage varies with locations

from where sediment is collected for the same catchment area.

Acknowledgement

The authors wish thanks to the German Academic Exchange Service ‘ DAAD’ for

providing scholarships during research stay in Braunschweig, Germany. The authors thank

Prof. Dr. Uwe Schröder, Harnisch F., Carmona-Martinez A.A. and the team of Institute

of Environmental and Sustainable Chemistry, Technical University of Braunschweig,

Germany for inspiring guidance and valuable advice throughout the period of research work.

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1. Bond, D.R., Holmes, D.E., Tender, L.M. and Lovley, D.R. (2002). Electrode-reducing

microorganisms that harvest energy from marine sediments. Science., 295: 483–485.

2. Cordova-Kreylos, A.L., Cao, Y., Green, P.G., Hwang, H.M., Kuivila, K.M.,

LaMontagne, M.G., van de Werfhorst, L.C., Holden, P.A. and Scow, K.M., (2006).

Diversity, composition, and geographical distribution of microbial communities in

California salt marsh sediments, Appl. Environ. Microbiol. 72: 3357-3366.3. De Schamphelaire L., Rabaey K., Boeckx P., Boon N. and Verstraete W. (2008).

Outlook for benefits of sediment microbial fuel cells with two bio-electrodes, Microb.

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4. De Schamphelaire, L., Boeckx, P. and Verstraete, W. (2010). Evaluation of biocathodes

in freshwater and brackish sediment microbial fuel cells. Appl. Microbiol. Biotechnol.

87: 1675–1687.

5. Hidalgo, P. and Jeison, D. (2010). Production of electricity from a mixed microbialconsortium using microbial fuel cells, paper presented in 2nd International Workshop

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Pucón, Chile, 27-29, 19.

6. Hong S. W., Chang I. S., Choi Y. S. and Chung T. H. (2009). Experimental evaluation

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Bioresour Technol., 100: 3029–3035.

7. Hong, S.W., Kim, H.S. and Chung, T.H. (2010). Alteration of sediment organic matterin sediment microbial fuel cells. Environ. Pollut. 158(1):185-191.




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8. Jeffrey Roshan De Lile (2008). Energy harvest by benthic microbial fuel cell: Influence

of salinity. M.Sc. thesis, University of Applied Science, Cologne, Germany.

9. Kim, N., Choi, Y., Jung, S., Kim, S. (2000). Effect of initial carbon sources on theperformance of microbial fuel cells containing Proteus vulgaris. Biotechnology and

Bioengineering, 70(1): 109-114.

10. Lee J., Phung N.T., Chang I.S., Kim B.H. and Sung H.C. (2003). Use of acetate for

enrichment of electrochemically active microorganisms and their 16S rDNA analyses,

FEMS Microbiol. Lett. 223: 185-191.

11. Logan, B.E. Hamelers, B. Rozendal, R.A. Schroder, U. Keller, J. Freguia, S. Aelterman,

P. Verstraete, W. and Rabaey, K., (2006). Microbial Fuel Cells: Methodology andTechnology, Environ. Sci. Technol, 5181–5192.

12. Lovley D.R. (2006). Bug juice: harvesting electricity with microorganisms. Nature

Reviews Microbiology 4: 497-508.

13. Mohamad A. (2010). Environmental chemical analysis related to drug industries: fate

monitoring of Diclofenac in water/sediment systems. Ph.D. Thesis, Technical University

of Braunschweig, Germany.

14. Nielson M.E. (2008). Utilization of natural and supplemental biofuels for harvestingenergy from marine sediments. Ph.D. thesis, Oregon State University, USA.

15. OECD (2002). Guideline for testing of chemicals No. 308. Aerobic and anaerobic

transformation in aquatic sediment system. Organisation for Economic Co-operation

and Development, Paris, France.

16. Rabaey, K., Angenent, L., Schroder, U. and Keller, J. (Eds.) (2009). Bioelectrochemical

Systems: from Extracellular Electron Transfer to Biotechnological Application, IWA

publishing, London, New York.17. Reimers, C.E., Tender, L.M., Fertig, S. and Wang, W. (2001). Harvesting Energy from

the Marine Sediment-Water Interface, Environ. Sci. Technol (35): 192-195.

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freshwater sediment microbial fuel cell for electricity generation. BioprocessBiosystems Eng., 34: 621–627.




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20. Song, T.S., Cai, H.Y., Yan, Z.S., Zhao, Z.W. and Jiang, H.L. (2012). Various voltage

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Pilobello K., Fertig S.J. and Lovely D.R. (2002). Harnessing microbially generated

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23. Zhang E., Xu W., Diao G. and Shuang, C. (2006). Electricity generation from acetateand glucose by sedimentary bacterium attached to electrode in microbial-anode fuel

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CHAPTER 38

BIOHYDROGEN PRODUCTION USING SINGLE-CHAMBERMEMBRANE-FREE MICROBIAL ELECTROLYSIS CELL

WITH A STAINLESS STEEL CATHODE

Sundaresan Mohanraj, Krishnasamy Anbalagan, Kodhaiyolii Shanmugam, Velan Pugalenthi

Abstract

Single-chamber membrane-free microbial electrolysis cell was designed and used to

investigate the hydrogen production from glucose by mixed culture at 37°C. In the present

study, the low cost anode and cathode materials were carbon graphite (1.9 cm diameter x 3.2

cm length) and stainless steel mesh (3.5 cm diameter X 4.8 cm length), respectively. In batch

experiments, the hydrogen production rate was increased from 1.42 to 1.64 ml/h when the

applied voltage was varied from 0.2 V to 0.6 V with an interval of 0.1V. Further the hydrogen

production rate was decreased with increase of applied voltage at above 0.6 V. A membrane-less system with close electrode spacing (1.74 cm) showed that a maximum hydrogen

production rate of 1.64 ml/h was obtained at an applied voltage of E (ap) = 0.6 V. In

comparison, the control experiment (without applied voltage) indicated that the hydrogen

production rate was 1.07 ml/h. The maximum cumulative hydrogen production and hydrogen

content at 0.6 V were 149 mL and 41 %, respectively. These results suggest that the low cost

cathode was able to increase the hydrogen recoveries and hydrogen production rates. In

conclusion, the current density and volumetric hydrogen production rate of this system can beenhanced by further modification of electrode with increasing the ratio of electrode surface

area.

Key words: Microbial Electrolysis Cell; SS cathode, mixed culture; applied voltage

38.1 Introduction

Hydrogen is an important role in a future energy fuel, owing to its high energy content

and the ability to produce from various organic substrates. Among the hydrogen productiontechnologies, hydrogen production from organic matter by fermentative bacteria have been




410

extensively studied (Liu et al., 2010). However, the stoichiometric potential of 12 mol H2 /mol

glucose from fermentative process has not been achieved. Mostly, fermentative processes are

limited to a maximum yield of 4 mol H2 /mol glucose and remaining of the energy is convertedto organic acids and alcohols. Therefore, further processes are required to convert

fermentative effluent (organic acids) to hydrogen (Lu et al., 2009). Recently, microbial

electrolysis cell (MEC) has been developed a new method for hydrogen production from

fermentative end products and other organic substrate (Liu et al., 2005; Rozendal et al., 2006;

Call and Logan, 2008; Logan et al., 2008). In microbial electrolysis cell, a substrate is

oxidized by exoelectrogens and released electrons to the anode. Further, the electrons are

transferred from anode to cathode through an external circuit and combined with protons toproduce hydrogen at cathode (Call and Logan, 2008). However, a small voltage is required to

the circuit for allowing hydrogen production at the cathode when compared to water

electrolysis. A voltage of >0.2V in practice has been required for hydrogen production from

acetate as a substrate (Cheng and Logan, 2007; Hu et al., 2008). This voltage has considerably

lower than the water electrolysis process for hydrogen production (Lu et al., 2009).

Recent years, the microbial electrolysis cell performance has been considerably

improved. However, in practice, the high cost of the reactor and low production rates are mainissues for its application (Cheng and Logan, 2007, Rozendal et al., 2007). Therefore, its recent

advances in reactor design (Call and Logan, 2008, Tartakovsky et al., 2008 and Hu et al.,

2008) and operation (Lalaurette et al., 2009 and Selembo et al., 2009) still need to be

developed for realizing the applications of this technology. Also, the high cost of cathode is

one of the most critical ones. Pt based cathodes have been widely used in MEC studies (Liu et

al., 2005) owing to its effecient catalytic properties and popularity in microbial fuel cell

(MFC) studies (Logan et al., 2008). The alternatives to Pt based cathode in MECs have beeninvestigated recently. Selembo et al. (2009) studied the nickel oxide catalysts through cathodic

electrodeposition of NiSO4 and (NH4)2SO4 on a metal sheet in MECs. Harnisch et al. (2009)

reported that the electrocatalytic behavior of synthesized tungsten carbide powder in MECs by

pasting the powder onto graphite disc with Nafion. However the main challenges associated in

an MEC for hydrogen production including low volumetric efficiency and the use of an

expensive catalyst (platinum). Hence, the researchers have led to examine non-precious

metals and alternatives for Pt based cathodes.




411

A single-chamber membrane less MEC with cost effective cathode has been attention

recently. Lack of membrane in MECs reduce the lower the internal resistance, eliminate the

pH gradient across the membrane, and enhance the hydrogen production rate (Hu et al., 2008,Lu et al., 2009). However, in membrane less MEC, hydrogen reoxidization by exoelectrogens

and methanogens are main challenges. The present study attempts to the develop the efficient,

stable and cost-effective stainless steel cathode for hydrogen production in MECs. This

electrode performance was also examined for hydrogen production in membrane less single-

chamber MECs.


38.2.1 Reactor construction

Single-cell MEC was constructed with graphite brush (1.9cm diameter x 3.2cm length)

as anode. Stainless steel mesh (3.5 cm diameter X 4.8cm length) was used as cathode. The

distance from the middle of brush anode to the cathode was 1.75 cm. Both anode and cathode

chambers were connected to electrode and outside circuit. A power source was supplied at a

steady voltage (0.2 –1.0 V) between anode and cathode electrode. A power source (DC-3002,

Beetech, India) was connected to the circuit for providing voltage, and an external resistor

(1000 Ω) was to calculate the current (Fig.1).

Fig.1. Photograph of MECs with stainless steel cathode

38.2.2 Start-up and operation

MEC were first operated in microbial fuel cell (MFC), with the cathode exposed to air

as mentioned previously (Lu et al., 2009). MFC was inoculated with sewage sludge, collected

from the local wastewater treatment plant, in a nutrient buffer solution (NBS) (Na 2HPO4, 4.58

g/l; NaH2PO4·H2O, 2.45 g/l; NH4Cl, 0.31 g/l; KCl, 0.13 g/l; trace mineral; 50:50

(v:v)wastewater and buffer) (Call and Logan, 2008) containing 1 g/l glucose. During each fed-




413

Hydrogen yields was calculated as YH2 = g H2 /g glucose or mol H2 /mol glucose and

coulombic efficiency, CE (%), was calculated as CE = CT /CC, where CT is the total coulombs

calculated by integrating the current over time, and CC is the total charge consumed. Cathodichydrogen recoveries (rcat, e− to H2 in the cathode), overall hydrogen recovery (RH2 = CErcat),

and maximum volumetric hydrogen production rates (mL/h) were calculated as previously

described assuming standard biological conditions (T=298.15K, P = 1 bar, pH 7) (Logan et

al., 2008).


38.3.1 Biohydrogen production from glucose

Hydrogen production rates were substantially increased from 1.42 (at 0.2 V) to 1.64

ml/h (0.6 V) using buffered fermentation effluent by increasing the applied voltage (Fig. 2).

Further, the hydrogen production rate was decreased from 1.64 ml/h to 0.43 ml/h, when the

Eap was varied from 0.6 to 1.0 V. The control experiment (without applied voltage) showed

that the hydrogen production rate was 1.47 ml/H. On the other hand, hydrogen content was

found to be same in Eap upto 0.5V and control experiment. The maximum hydrogen content

of 41 % was obtained at 0.6 V. Furthermore, the hydrogen content was decreased from 41 to

31 % as the Eap was varied from 0.6 to 1.0V. The maximum cumulative hydrogen volume of

149 ml was observed at 0.6V. The hydrogen production remained decreased with the further

increasing of applied voltage upto 1.0 V. These findings indicate that the hydrogen production

was considerably enhanced at an optimum applied voltage (0.6V) in single-chamber

membrane-less MEC using mixed culture when compared to the control experiment (110 mL)

(Fig.3).




414

Fig.2. Hydrogen production rate, hydrogen content and cumulative hydrogen production with

different applied voltage

Fig.3. Cumulative hydrogen production with different time interval of control and 0.6V

experiment

38.3.2 Coulombic efficiency and current density

As seen in the Figure 4, the Coulombic efficiencies (CE) were increased from 5.4 to

33.7 % by varying the applied voltage from 0.2 to 0.7V. Further it was decreased at above 0.7

V. At low applied voltages of 0.2 and 0.4 V, CE values were found to be low due to

methanogenesis. Similar results were obtained by Tartakovsky et al., (2008). As the Eap was

increased from 0.7 to 1.0 V, the anode potential was increased and the CE was decreased (Fig.

4). The decrease of CE at the highest applied voltage resulted in a reduction of hydrogen

recovery (RH2). However, the current density was gradually increased from 0.04 to 2.32

mA/m2 when the applied voltage was varied from 0.2 to 1.0 V. According to literature, a

higher resistor increased the output voltage of the power supply and shared a higher voltage

on it (Hu et al., 2012). For the MEC, the hydrogen production rate was partially dependent on

the resistor. In spite of this, a constant resistor (1000 Ω) was used in this study and the

maximum output current density of the external power supply was reached to 2.32 mA/m2.

Consequently, the high hydrogen production rate was achieved (Fig.2). Similar observation

was obtained by Chae et al. (2008).

38.3.3 Hydrogen Recoveries




415

Factors affecting the hydrogen yield were observed to be current density (normalized

to electrodes area), coulombic efficiency, and cathodic hydrogen recovery. From Figure 4, the

cathodic hydrogen recoveries were increased from 37.5 to 79.3 % as the applied voltage wasvaried from 0.2 to 1.0 V. In addition, the overall hydrogen recovery was similar to the

cathodic hydrogen recoveries. The overall hydrogen recoveries were remarkably increased

remarkably from 34.7 to 75.9 % as the applied voltage was differed from 0.2 to 1.0V (Fig. 3).

These results demonstrate that cathodic hydrogen recovery at below 0.5 V was very low. The

high hydrogen recovery was achieved at above 0.5V of applied voltage. These results show

that the low current density and coulombic efficiency reduced the hydrogen production rate.

Further study requires on optimizing the architecture to lower the internal resistance forimproving the hydrogen production.

Fig.4. Current density and Coulombic efficiency at different applied voltage

Fig.5. Hydrogen recoveries and glucose utilization at different applied voltage




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38.4 Conclusions

The hydrogen recovery was improved in a low cost single-chamber membrane less

MEC with stainless steel cathode. In addition, small electrode spacing and lack of membraneused in this reactor contruction reduced the pH gradients and proton diffusion resistance

which led to high hydrogen production rate. The maximum hydrogen production rate of 1.64

ml/h and overall hydrogen recovery of 69.7 % were achieved when the applied voltage was

0.6 V. In comparison, the control experiment (without applied voltage) indicated that the

hydrogen production rate was 1.47 mL/h and hydrogen recovery was 31.5%. Further,

optimizing the internal resistance and increasing the current density would improve the

hydrogen production rate in this MEC.

Acknowledgements

This research was funded by the Department of Biotechnology (Ref. No.

BT/PR12051/PBD/26/213/2009, dated 19th November 2010), New Delhi, India. Author

Mohanraj gratefully thank Ministry of New and Renewable Energy, New Delhi, India for

Senior Research Fellowship (NREF–SRF).

References

1. Call D. and Logan B.E. (2008) Hydrogen production in a single chamber microbial

electrolysis cell lacking a membrane. Environ. Sci. Technol., 42:3401–6.

2. Chae K.J., Choi M., Ajayi F.F., Park W., Chang I.S., Kim I.S. (2008) Biohydrogen

production via biocatalyzed electrolysis in acetate-fed bioelectrochemical cells and

microbial community analysis. Int. J. Hydrogen Energy, 33:5184–92.

3. Cheng S. and Logan B.E. (2007) Sustainable and efficient biohydrogen production viaelectrohydrogenesis. Proc. Natl. Acad. Sci., 104:18871–3.

4. Harnisch F., Sievers G., Schroder U. (2009) Tungsten carbide as electrocatalyst for the

hydrogen evolution reaction in pH neutral electrolyte solutions. Appl Catal B: Environ.,

89; 455–458.

5. Hu H., Fan Y. and Liu H. (2008) Hydrogen production using single-chamber membrane

free microbial electrolysis cells. Water Res., 42:4172–4178.

6. Liu H., Grot S. and Logan B.E. (2005) Electrochemically assisted microbial productionof hydrogen from acetate. Environ. Sci. Technol, 39:4317–20.




417

7. Lalaurette E., Thammannagowda S., Mohagheghi A., Maness P.C. and Logan B.E.

(2009) Hydrogen production from cellulose in a two-stage process combining

fermentation and electrohydrogenesis. Int. J. Hydrogen Energy, 34:6201–10.8. Liu H., Hu H., Chignell J. and Fan Y. (2010) Microbial electrolysis: novel technology

for hydrogen production from biomass. Biofuels., 1:129–142.

9. Logan B.E., Call D., Cheng S., Hamelers H.V.M., Sleutels T.H.J.A., Jeremiasse A.W.

and Rozendal R.A. (2008) Microbial electrolysis cells for high yield hydrogen gas

production from organic matter. Environ. Sci. Technol., 42:8630–8640.

10. Lu L., Rena N., Xing D. and Logan B.E. (2009) Hydrogen production with effluent from

an ethanol–H2-coproducing fermentation reactor using a single-chamber microbialelectrolysis cell. Biosen. Bioelect., 24:3055–3060.

11. Rozendal R.A., Hamelers H.V.M. and Buisman C.J.N. (2006) Effects of membrane

cation transport on pH and microbial fuel cell performance. Environ. Sci. Technol.,

40:5206–11.

12. Rozendal R.A., Hamelers H.V.M., Euverink G.J.W., Metz S.J. and Buisman C.J.N.

(2006) Principle and perspectives of hydrogen production through biocatalyzed

electrolysis. Int. J. Hydrogen Energy., 31:1632–40.13. Rozendal R.A., Hamelers H.V.M., Molenkamp R.J. and Buisman C.J.N. (2007)

Performance of single chamber biocatalyzed electrolysis with different types of ion

exchange membranes. Water Res., 41:1984–94.

14. Selembo P.A., Merrill M.D. and Logan B.E. (2009) The use of stainless steel and nickel

alloys as low-cost cathodes in microbial electrolysis cells. J. Power Sources., 190, 271–

278.

15. Tartakovsky B., Manuel M.F., Neburchilov V., Wang H. and Guiot S.R. (2008)Biocatalyzed hydrogen production in a continuous flow microbial fuel cell with a gas

phase cathode. J. Power Sources., 182:291–7.




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CHAPTER 39

FEASIBILITY OF INTERLINKING TWO TECHNOLOGIESFOR SIMULTANEOUSLY TWO BIOENERGIES

GENERATION

Prashant Pandey, Vikas Shinde, S.P. Kale, R.L. Deopurkar

Abstract

Microbial fuel cell for bioelectricity generation rapidly extending interdisciplinary area, has

attracted great research efforts to fulfill the demand of renewable energy. Here, we studied

bioelectricity generation using food waste leachate obtained from aerobic predigestion tank of

two-stage aerobic-anaerobic sequential biogas reactor (Nisargruna Biogas Technology).

Volatile fatty acids content of food waste leachate was 8000 mgl-1; it inhibits the methanogens

for methane production in anaerobic reactor. Hence, after electrochemical evaluation of MFC

for food waste leachate, it was observed that maximum open circuit voltage (OCV) 0.830 V

was obtained for the COD load of 1000 mgl -1. At optimum condition, MFC current density

was reached up to 5.897 Am-2 and maximum power density 2.379 Wm-2 along with substrate

removal more than 80% in terms of COD. This study infers that simultaneously bioelectricity

and biogas production can be done using food waste by linking bioelectrochemical technology

(Microbial fuel cell) to Nisargruna Biogas technology.

Key words: Microbial fuel cell (MFC), Electricity, Biogas, Wastewater treatment

39.1 Introduction

Three E’s (Energy, Environment and Economy) are the prime and inescapable concern

of modern society. Society is always ultimate beneficiary of scientific research, knowledge

derived and its understanding. Biotechnology is employed for the betterment three E’s in the

well being of human beings.

The first E i.e. Energy resources scenario if considered, current global energies in use

are coal, gas, oil, nuclear and renewable energies. There are limits of non-renewable

resources. Oil reserves will not appreciably run out for at least 100 years or more, demand for




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food waste is utilized as resource in two-stage aerobic-anaerobic sequential biogas reactor

(Nisargruna Biogas Technology) but it produces acidogenic food waste leachate in anaerobic

digestion or methane production process. High content of volatile fatty acids in acidogenicfood waste leachate has adverse effect on growth of methanogens resulting in reduced amount

of biogas production. Microbial fuel cell is promising sustainable technology for bioelectricity

generation, wastewater treatment and resource recovery.

Our aim is to evaluate the feasibility of interlinking of Nisargruna Biogas technology

(anaerobic digester) to microbial fuel cell technology (anodic compartment) so that

simultaneously both energies i.e. Methane and Bioelectricity generation can be done.

Therefore, efficacy of acidogenic food waste leachate obtained through the Autothermalanaerobic digestion of Nisargruna Biogas plant predigestion tank’s food waste is examined as

microbial fuel cell resource for bioelectricity generation.


39.2.1 Acidogenic food waste leachate : Production

Food waste was collected from the predigester tank of Nisargruna plant based on

kitchen waste from Symbiosis International University, S.B. road, Pune (India) developed byBhabha Atomic Research Centre (BARC), Navi Mumbai, (India). Batch mode digestion was

carried out in a simulated Autothermal thermophillic aerobic digester (ATAD). The total

volume of the conical flask was 1000 ml with effective volume of 600 ml. The temperature of

45°C in the digester was maintained using water bath for 144 h. High amount of VFAs causes

reduction in Biogas production (Wang et al., 2009) in Nisargruna Biogas plant. Therefore,

Volatile fatty acids were used as indicators of process imbalance in anaerobic digesters as

suggested by Ahring et al., 1995. After analysis of VFAs at regular intervals for 144 hrs, itwas observed that with progression of time, the volume of leachate was increased with

increased in COD. pH of leachate was depleted day by day due to accumulation of VFAs. hrs

of digestion at 45°C gave maximum concentrations for all VFAs under observation. (Figure1).

Then the Acidogenic food waste leachate digested at hrs at 45°C and was stored at 4 0C for

use as substrate in anodic compartment.

39.2.2

Microbial innocula:

Two types of inocula: 1) municipal wastewater 2) anaerobic sludge from a sequential

two stage aerobic-anaerobic digester, were mixed in ratio of 3:7 (100 ml). First inocula were




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collected from Sangam Bridge, Pune, Maharashtra, India. Second inocula was collected from

sequential two stage aerobic-anaerobic digester (Nisargruna Biogas technology), Matheran,

Maharashtra, India.

Figure 1: Variation in concentration of VFAs with respect to ATAD digestion time.

39.2.3 Preparation of Substrate:

3.36 g of acidogenic food waste leachate diluted to 300 ml (COD, 1000 mgl -1), then

the 100 ml of mixed inocula were added, making the working volume of the reactor 400 ml.

The prepared fuel is ultrasonically treated using Ultrasonic Processor (Piezo –U- Sonic brand)

having constant supplied power of 100 W, frequency of 53 kHz for 5 minutes. This substrate

was used in MFC’s anodic compartment. In Cathode, 400 ml of 100 mM potassium

ferricyanide in phosphate buffer solution (pH 7.1) was inoculated.

39.2.4 Microbial fuel cell reactor configuration and operation

Laboratory scale dual chamber MFC made of transparent Plexiglas material was used

in this study. The basic concept of design was taken from Logan et al., 2006. The two

chambers were designed with about 0.500 l capacities. The working volume of each chamber

was 0.400 l. The anode compartment was maintained anaerobic condition. The anode and




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cathode chamber had two openings connected through a pipe for inlet and outlet and for

circulation of fuel. Two Pure carbon brush electrodes were used with 16*12.5*2 mm in

dimensions in anodic compartment having 1.0 cm apart from each other. In cathode, a purecarbon brush electrode having dimensions of 16*12.5*2 mm was used in opposite side of the

CEM in between anodic compartment electrodes. Electrodes were placed equal distance from

membrane. The internal distance of each electrode was fixed i.e. 2.0 cm from membrane. The

terminal of each electrodes were connected with ultrathin copper conceal wires. The effective

surface area of both carbon electrodes in anode compartment was 0.00102 m2. Both the

compartments were connected with CEM having 7 cm in diameter (Nafion T117; Dupont,

Wilmington, Delaware ) by using rubber coupling arrangement. The two chambers withmembrane coupling assembly were fixed with nut- bolts. The anode chamber was washed

with nitrogen gas to remove traces of oxygen to maintain the anaerobic condition. The

systematic diagram of dual chambered MFC is given in Figure 2 (A).

Figure 2: Systematic diagram (A) and Actual figure (B) of dual chambered MFC.

MFC was operated in batch fed mode. For inoculation and start up of MFC, the

synthetic wastewater was prepared with NH4Cl (0.31gl-1), KCl (0.13 gl-1), NaH2PO4.H2O

(2.69 gl-1), Na2HPO4 (4.33 gl-1), Acetate (10mM), Trace element solution (12.5 ml), Vitamin

solution (12.5 ml). At the starting condition, MFC was operated in open circuit voltage (OCV)

mode. The pH of the solution influent was 4.70 in anode and 7.01 in cathode. The MFC was

operated once with synthetic wastewater. After stable voltage generation, MFC was switched

for different food waste leachate concentration to decide the optimum organic influent rate.

The influent concentration was tested for 500 mg l-1, 1000 mg l-1 and 2000 mg l-1 of organic




423

matter as COD. The MFC was kept in incubator at 30°C. To prevent the sedimentation of fuel

a magnetic bead was kept in anode chamber and placed at magnetic stirrer (REMI brand) at

170 rpm throughout the operation. The MFC operating condition is given in the figure 2 (B).39.2.5 Data collection, Analysis and Calculation

Voltage was verified directly by using digital multimeter (MASTECH brand Digital

Multimeter 10 A DC) for every hour. Current (I) and Power (P = IV) were recorded. The

power density was normalized to anode surface area and anode void volume. The polarization

curve was prepared for measuring stable voltage for various external resistances (39-470

ohms). The curve then used to calculate the maximum power density. The current density (Id =

V/RA) was calculated, where V (V) is voltage, R (Ω) is resistance and A (m2), the geometric

anode surface area. The power density (Pd = V2 /RA) was calculated. The columbic efficiency

(CE) was calculated for batch fed mode system by using the formula (Logan et al., 2006) as:

where ∆ COD is the substrate concentration for batch fed mode system by the time ( tb ) , F is

the faradays’ constant, ʋan liquid volume in anode chamber, I, is the current in ampere. pH and

conductivity was measured by probes (HACH, USA). COD (including both soluble and

particulate) was determined using a standard dichromate oxidation (open reflux) method and

VS, SCOD was analyzed by the standard method (APHA, 2005). To quantify VFA 1ml

filtrate sample of the supernatant was inserted into glass syringe and inserted into the

Shimadzu GC -2010 with a flame ionization detector. Before the assay was started, the

machine was calibrated and 1ml of standard VFA solution was added. The sample injection

volume was 1ml. Nitrogen was the carrier gas. The injection port and detector were

maintained at 180°C respectively. The determination result of VFA constituents were

respectively expressed in the form of height of the peaks in the graph and their occupied area

was used to calculate their respective concentration in mgml-1. Carbohydrate estimation was

done by Anthrone’s method (Scott and Melvin, 1953). Protein content was measured by

Biuret method (Layne, 1955).

39.3

Results

39.3.1 Acidogenic food waste leachate characterization




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Before anaerobic digestion raw material has pH of 6.1 that after anaerobic digestion

gradually decreases with increase in VFA concentration. After 72 hrs of digestion the

characterization of Acidogenic food waste leachate is given in the table 1.Table 1: Characterization of Acidogenic food waste leachate after 72 hrs digestion.

Parameter Unit Food waste leachate ( after 72 hrs digestion )

pH - 4.40

Soluble COD mgO2 l-1 12400

Conductivity mScm-1 19.39

VS gl-1 78.3

VFA mgl-1 8010

Temperature °C 45

Carbohydrate mgml-1 11.80

Protein mgml-1 7.61

39.3.2 Pretreatment of Anodic Substrate

For the selective enrichment of specific group of bacteria, pre-treatment of parent

inoculum was carried out to use in anode chamber of a MFC. Symbiotic relationship within

population and composition of the substrates (wastewater) strongly affects the composition of

the bacterial associations in the anode chamber. Inocula in present study are a mixture of

different consortia of bacteria and therefore the susceptibility of this to ultrasonication

depends on the resistance of the predominant bacterial groups. Resistant microorganisms to

ultrasonication stress have more possibility to survive and become a dominant species in the

mixed culture. Ultrasonication can suppress activity of Grampositive methanogens by

retaining Gram-negative bacteria. Major electrogens are gram-negative bacteria.

Methanogenic bacteria lacks protective spores forming capability in restrictive environment

such as high temperature, extreme acidity and alkalinity, and also have a slower growth rate

unlike other hydrogen producing and electrogenic bacteria (Zhu and Beland, 2006).

Therefore, the application of moderate duration ultrasonic pre-treatment to inoculums

might have enhanced the activity of electrogenic bacteria and somewhat change in leachatecomposition, which resulted in enhanced current production. The pretreatment of ultrasonic




425

waves at 53 kHz at 100 W for 5 minutes ( Figure 4 ) causes reversible damage although 2, 10

and 15 minutes treatment produces poor results. Ultrasonication of fuel (acidogenic food

waste leachate and mixed inocula) produced initial open circuit voltage of 0.130 V withrespect to untreated 0.92 V only. Thus, with pretreatment initial OCV is increased by 41.30%.

Figure 4: Microscopic image of ultrasonically treated inocula before and after ultrasonic

treatment (53 Htz, 100W for 5 minutes) at 1000X magnification.

39.3.3 MFC performance:

39.3.3.1 Determination of Organic Loading Rate (OLR)

During start up, it was decided to operate MFC almost for 12 days to reach the stable

OCV voltage of approx 0.536 V for 0.400 l of anode solution. It means that anode and

cathode were considered fully enriched (Zhang et al., 2011). MFC was freshly re-inoculated

with food waste leachate solution of varying concentration of 500, 1000 and 2000 mgl -1 of

COD. It was observed that, at different COD input, MFC gave different OCV. For leachate

concentration of 500, 1000, 2000 mgl-1 as COD, OCV was 0.631 V, 0.830 V, 0.638 V

respectively.

The MFC gave maximum voltage of 0.830 V with carbon brush electrode for 1000

mgl-1 COD. After that, voltage was reduced for further substance load. This was because of

high COD concentration may affect the microbial activity of anode chamber. It also leads to

increase in internal resistance of anode chamber and may increase the charge transfer




427

According to Ohm’s law, at maximum power density, the internal and external resistances are

equal. Thus, at Maximum Power density (2.379 Wm-2) MFC external resistance equal to its

internal resistance i.e. 220Ω

at 3.258 Am

-2

current density. MFC current density was reachedup to 5.897 Am-2. The trend of Voltage and Power density as function of current density is

given in the figure 7.

Figure 6: Closed circuit Voltage at a range of external resistances (39 to 470 Ω).

Figure 7: The polarization behavior of MFC and Power density.

39.3.3.3 Columbic efficiency and Substrate removal efficacy




428

Columbic efficiency (CE) is the ratio of the coulombs obtained in a MFC to the

theoretical coulombs if all the substrate oxidized produces current. CE percentage was

obtained 24 %. MFC demonstrated the high COD removal efficacy of 83%.

39.4 Discussion

The application of low-frequency ultrasonication was reported to be very effective in

decreasing the amount of bacterial population in sewage sludge. As the frequency and time

period increases, the bacterial population decreases (Kesari and Behari, 2008). However,

with the prolonged exposure time, the flaw may expand and the structure of cell wall will be

destroyed, which leads to the decrease of bacterial activity (Xie et al., 2009). Effect of

pretreated anaerobic sludge was reported by More and Ghangrekar, 2009. They reported

maximum 0.745 V OCV (40 kHz, 120 W for 5 minutes) with simple synthetic wastewater as

substrate rather in present study high molecular wastewater is used and maximum OCV of

0.830 V (53 kHz,100 W for 5 minutes) is produced.

The effects of three different inocula (domestic wastewater, activated sludge, and

anaerobic sludge) on the treatment of acidic food waste leachate in MFCs were evaluated by

Li et al., 2013. Using 1000 mgl-1COD food waste leachate (pH 4.76) as the substrate highest

power (0.432 Wm-3) was obtained using anaerobic sludge inoculum. COD removal and CE%

was obtained >87% and 20% respectively. In another experiment, using 5000 mgl -1 COD

food waste leachate produced maximum power density 15.14 Wm -3 with COD removal

efficacy of 90% and columbic efficacy of 66.40% (Rikame et al., 2012).

Rather in present study, although the OCV is less than Rikame et al., 2012 but it is

greater than Li et al., 2013. Volumetric power density is less than Rikame et al., 2012 and Li

et al., 2013 because large volume to anode surface area ratio. If both power density isconverted to Wm-2 (with respect to anode surface area) then there is no any literature

available as best of my knowledge until date reporting such high power density with

Acidogenic food waste leachate. Columbic efficacy and COD removal efficacy although is

lesser than all of above because of lesser surface area of anode and respectively large anodic

chamber working volume.

39.5

Conclusion

This study infers the feasibility of interlinking of Nisargruna Biogas technology

(anaerobic digester) to microbial fuel cell technology (anodic compartment) is possible with




429

further research and improvement of Power density, Current density and Voltage, so that

simultaneously both energies i.e. Biogas and Bioelectricity generation can be done.

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Part VI

Hybrid Systems




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CHAPTER 40

DEVELOPMENT OF NANO BASED THERMIC FLUID:

RHEOLOGICAL ASPECTS OF NEW ENERGY SYSTEM

Vijay Juwar, Shriram Sonawane

Abstract

Nanofluids are emerging as highly efficient thermic fluids due to its enhanced thermal

conductivity, to used nanofluids as heating or cooling media on industrial level it becomes

mandatory to study the flow related aspects of nanofluids. The viscosity of nanofluid plays

decisive role in economic viability nanofluids as thermal fluid. Variation of viscosity of

nanofluid with temperature and concentration of nanoparicle directly affects pressure drop

which in turn affects pumping cost in heat exchange equipments. Present study deals with

measurement of viscosity nanofluid, prepared by dispersing Fe3O4 nanoparticles in ethylene

glycol. Viscosity is measured both as function of volume concentration and temperature. Pure

base fluid displays Newtonian behaviour in the experimental temperature range. After

addition of nanoparticles nanofluid retains Newtonian behaviour. Our study shows that

viscosity of nanofluid increases with increase in volume concentration and decreases with

increase in temperature

Key Words: Nanofluid, Thermic fluid Nanofluid viscosity

40.1 Introduction

Nanotechnology is emerging field, to which entire world is looking forward as a

solution to many issues. Amongst those, energy and environmental issues are occupying the

topmost attention. Fossil fuels are depleting and creating environmental problems like air

pollution, green house effect etc. so need of the hour is to design the system which can

efficiently utilize the conventional fuel with least environmental issues like air pollution or

search of alternative which replace the existing fuel which can be economic and

environmental friendly.

Nanofluid is one such potential alternative which can be used to reduce thedependence on the conventional fuel and also reduce load of pollution. Nanofluid is colloidal




435

viscosity was done with the help of AR G2 Rheometer. Plate cone geometry with diameter 40

mm and A 1 was used. A gap of 32 was maintained when geometry acts as parallel

plate.It has facility to raise temperature with help of peltier plate that offers temperature range

of -40C to 200C with typical heating rates up to 50C/min and accuracy of 0.1C. They

incorporate four Peltier heating elements to cover an 80mm diameter plate surface. These

Peltier elements are placed directly in contact with thin copper disc with an externally rugged,

hardened chrome surface. A platinum resistance thermocouple, PRT is placed at exact centre

ensuring accurate temperature measurement and control. The unique design provides for

rapid, precise and uniform temperature control over entire 80mm diameter surface allowing

standard geometries up to 60mm in diameter.The viscosity of nanofluid was measured with

increasing shear rate in the range of 1-500 . Each sample of different volume concentration

was taken for measurement of viscosity in temperature range 100-50

0C.

40.2.1 Modeling of Viscosity

After experimentation it becomes necessary to correlate results with the effective

viscosity of nanofluid. In theoretical point of view to understand the properties of nanofuid is

another challenge. Since nanofluid is two phase fluid, must have common features of solid

liquid mixture. Application of relation of solid liquid mixture to nanofluid is still doubtful to

predict the properties of nanofluid.

Some of the widely used models are mentioned below:

Einstein’s model is used for relatively low volume fraction ( ≤0.02) which is given as

= (1+2.5)

Where, is base fluid viscosity.

Brinkman et al (1952) extended Eienstein’s model for the use of moderate volume fraction as

=( 1 − ).

Batchelor (1977) considered Brownian motion of particles on the bulk stress of an isotropic

suspension of spherical particles and the expression for viscosity is

=1+2.5

+6.5

Maiga et al (2004) proposed another model




436

= 306-0.19 + 1

44.2.2 Temperature dependence of viscosity

Generally it is observed that fluids are having low viscosity near to their boiling point

and high viscosity near freezing point. To study the effect of temperature on viscosity of

nanofluid various models are proposed. Some widely used are given in a table1

Table 1 Models for temperature dependence of viscosity.

Authors Equations

White(2004) ln

= a + b(

) + c(

)

Reid et al (1987) =Aexp(BT)

Yaws (1977) Log() =A + B + CT +D

Kulkarni et al (2006) ln= A- B

Namburu et al (2007) Log = A!"


The viscosity of nanofluid as function of shear rate is shown in figure 1. It may be

seen that that viscosity of nanofluid remains independent of shear rate as the shear rate is

increased; at lower shear rate viscosity sharply declines and for further increase in shear rate

remains constant under experimental temperature range of 100C to 50

0C.

This indicates the

Newtonian behavior of nanofluid in experimental temperature and shear rate range of 01/s to

500 l/s.

Figure 2 shows relationship between shear rate and shear stress of nanofluid, a linear

relationship is observed with zero intercept in experimental range, indicates Newtonian

behavior of nanofluid.

In figure 3 the effect of concentration of nanoparticles and temperature on the

viscosity of nanofluid is shown. The viscosity of nanofluid increases with increase in

concentration of nanoparticle and decreases with increase in temperature of nanofluid.




437

A

B

C

D

Figure 1 Nanofluid viscosity as a function of shear rate: (A)1%; (B) 2%; (C)3%(D)4%

0

0.01

0.02

0.03

0.04

0.05

0 100 200 300 400 500 600

N a n o f l u i d V i s c o s i t y ( P a . s

)

Shear Rate (1/S)

283 K 293 K 303 K 313 K 323

0

0.01

0.02

0.03

0.04

0.05

0.06

0.1 100.1 200.1 300.1 400.1 500.1 N a n o f l u i d V i s c o s

i t y ,

( P a . s

)

Shear Rate, (1/S)

283K 293K 303 K 313 K 323 K

0

0.05

0.1

0.15

0.2

0.25

0 100 200 300 400 500 600 N a n o f l u i d V i s c o s

i t y ( P a . s

)

Shear Rate (1/S)

283 K 293 K 303 K 313 K 323 K

0.00E+00

5.00E-02

1.00E-01

1.50E-01

2.00E-01

2.50E-01

3.00E-01

3.50E-01

4.00E-01

4.50E-01

0 100 200 300 400 500 600

N a n o f l u i d V i s c o s i t y ( P a . s

)

Shear Rate (1/s)283 K 293 K 303 K 313 K 323 K




438

A

B

C

Figure 2 Relation between shear stress and shear strain : (A) 1% (B) 2% (C) 4%

0

2

4

6

8

10

12

14

0 200 400 600

S H E A R S T R E S S ( P a . )

SHEAR RATE (1/S)

283 K 293 K 303 K 313 K 323 K

0

2

4

6

8

10

12

14

16

0 100 200 300 400 500 600

S h e a r S t r e s s ( P a . s

)

Shear Rate (1/S)293 K 293 K 303 K 313 K 323 K

0.00E+00

2.00E+00

4.00E+00

6.00E+00

8.00E+00

1.00E+01

1.20E+01

0 100 200 300 400 500 600

S h e a r S t r e s s ( P a )

Shear Rate (1/S)

283 K 293 K 303 K 313 K 323 K




439

Figure 3 Effect of concentration and temperature on nanofluid viscosity

Figure 4 discuss model studies of Brinkman, (1952) ,Batchelor (1977)and

Maiga,(2004). All of these models consider the effect of nanoparticle concentration on

viscosity of nanofluids. At lower temperature all models over predict the viscosity ofnanofluid and higher temperature all models under predicts the viscosity of nanofluid.

In figure 5 temperature dependence of nanofluid viscosity is shown. Models shown in

table 1 are proposed for prediction of nanofluid viscosity at different temperatures. Out of all

mentioned models only Namburu’s model fits well to the experimental data.

Log = A!" where A and B are constants, T is temperature in Kelvin and

is nanofluid viscosity in centipoises. Values of curve fit constants A and B are shown in table

2.

0

0.005

0.01

0.015

0.02

0.025

0.03

280 290 300 310 320 330

N a n o f l u i d i s c o s i t y ( ! a . s

)

Te"!erature (#)

4%

3%

2%

1%




440

A

B

C

D

E

Figure 4 Study of Models of viscosity at constant temperature: (A) 283 K (B) 293K (C)

303K (D)313K (E) 323K

0

0.5

1

1.5

0 0.002 0.004 0.006 R e l a t i e V i s c o s i t y

Volu"e $raction

Experimental

BrinkmanBatchelor

ai!a

0.00E+00

2.00E-01

4.00E-01

6.00E-01

8.00E-01

1.00E+00

1.20E+00

0 0.002 0.004 0.006

R e l a t i e i s c o s i t y

olu"e fraction

experimental

Brinkman

Batchelor

ai!a

0.00E+00

5.00E-01

1.00E+00

1.50E+00

0 0.002 0.004 0.006


olu"e fraction

Experimental

Brinkman

Batchelor

ai!a

0.00E+00

2.00E-01

4.00E-01

6.00E-01

8.00E-01

1.00E+00

1.20E+00

0 0.002 0.004 0.006


olu"e fraction

Experimental

Brinkman

Batchelor

ai!a

0.00E+00

5.00E-01

1.00E+00

1.50E+00

2.00E+00

0 0.002 0.004 0.006


olu"e fraction

Experimental

Brinkman

Batchelor

ai!a




441

Table 2 Curve fit values

NP volume conc. (v/v)

2% 3% 4%A 54.49996 54.49996 56.77083

B 0.013077 0.013372 0.012931

A

B

C

Figure 5 Effect of temperature on nanofluid viscosity by Naburu’s model: (A) 2%(B) 3% (C)

4%

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

280 300 320 340

# $ % (

# $ % (

# $ % (

# $ % ( & && & ' '' '

) )) )

Te"!erature (#)

"am#$r$&

Experimental

'inear

("am#$r$&)

0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

280 300 320 340

# $ % (

# $ % (

# $ % (

# $ % ( & && & ' '' '

) )) )

Te"!erature (#)

Experimental

"am#$r$&

'inear ("am#$r$&)

0

0.2

0.4

0.6

0.8

11.2

1.4

1.6

280 300 320 340

# $ % ( &

# $ % ( &

# $ % ( &

# $ % ( & ' '' '

) )) )

Te"!erature (#)

Exprimental

"am#$r$&

'inear ("am#$r$&)




442

40.4 Conclusion

F3O4 Nanoparicles in ethylene glycol shows Newtonian behavior in temperature range

of 100C to 50

0C and in volume concentration of 1% to 4%. The shear rate was increased from

1s-1

to 500s-1

. The viscosity of nanofluid is increased when volume concentration of

nanoparticle is increased. At temperature of 500C approximately 63.1% increase in base fluid

viscosity is observed at 4% volume concentration of nanoparticle. As the temperature

increases the viscosity of nanofluid decreases exponentially.

References

1. Aladag B., Halelfadl S., Doner N., Maré T., Duret S. and Estellé P. (2012) Experimental

investigations of the viscosity of nanofluids at low temperatures. Applied Energy.,

97:876–880

2. Batchelor G.K. (1977) The effect of Brownian motion on the bulk stress in asuspension

of spherical particles, J. Fluid Mech., 83:97–117.

3. Brinkman H.C. (1952) The viscosity of concentrated suspensions and solution, J. Chem.

Phys. 20:571–581

4. Duangthongsuk W. and Wongwises S. (2009) Measurement of temperature-dependent

thermal conductivity and viscosity of TiO2-water nanofluids Experimental Thermal and

Fluid Science., 33:706–714

5. Kole M. and Dey T.K. Viscosity of alumina nanoparticles dispersed in car engine

coolant, Experimental Thermal and Fluid Science 34 (2010) 677–683

6. Kulkarni D.P., Das D.K. and Chukwu G.A. (2006) Temperature dependent rheological

property of copper oxide nanoparticles suspension, J. Nanosci. Nanotechnol., 61150–

1154.

7. Lee S.W., Park S.D., Kang S, Bang I , KimG.H. (2011) Investigation of viscosity and

thermal conductivity of SiC nanofluids for heat transfer applications International

Journal of Heat and Mass Transfer 54:433–438

8. Maiga E.B.,.Nguyen C.T, Galanis N. and G. Roy. (2004) Heat transfer behaviors of

nanofluids in a uniformly heated tube, Super lattices and Microstructures,35:543 -557

9. Namburu P.K., Kulkarni D.P., Misra D. and D.K. Das. (2007) Viscosity of copper oxide

nanoparticles dispersed in ethylene glycol and water mixture, Exp. Ther. Fluid Sci.

32:397–402




443

10. Prasher R., Song D., Wang J. and Phelan P. (2006)Measurements of nanofluid viscosity

and its implications for thermal applications, Applied Physics Letters., 89:133108–

133111

11.

Reid R.C., Prausnitz J.M. and Sherwood T.K. (1987)The Properties of Gases and

Liquids, forth ed., McGraw Hill, New York

12. Sundar L. S, Singh M.K, and Antonio C.M. Sousa , (2013) Investigation of thermal

conductivity and viscosity of Fe3O4nanofluid for heat transfer applications

International Communications in Heat and Mass Transfer, 44 7–14

13. White F., (2005) Viscous Fluid Flow, third ed., McGraw Hill, New York,

14. Yaws C.L. (1977) Physical Properties – A Guide to the Physical, Thermodynamic and

Transport Property Data of Industrially Important Chemical Compounds, McGraw Hill,

New York.




444

CHAPTER 41

CONVERSION OF PLASTIC WASTES INTO LIQUID FUELS

– A REVIEW

Arun Joshi, Rambir and Rakesh Punia

Abstract

Various technologies are being developed to overcome the drawback of plastics, namely, theirnon-biodegradability. Though work has been done to make futuristic biodegradable plastics,

there have not been many conclusive steps towards cleaning up the existing problem.

Recycling waste plastics into reusable plastic products is a conventional strategy followed to

address this issue for years. However this technique has not given impressive results as

cleaning and segregation of waste plastics was found difficult. Over a 100 million tones of

plastics are produced annually worldwide, and the used products have become a common

feature at overflowing bins. Plastics is placed in a landfill, it becomes a carbon sink ,

Incineration, blast furnace, gasification are not much appreciated solution to the problem, as

toxic gases are produced and their cost of production is quite high. Pyrolysis of waste plastics

into fuel is one of the best means of conserving valuable petroleum resources in addition to

protect the environment. This process involves catalytic degradation of waste plastic into fuel

range hydrocarbon i.e. petrol, diesel and kerosene etc. A catalytic cracking process in which

waste plastic were cracked at very high temperature, the resulting gases were condensed to

recover liquid fuels. Type of plastics also effect the rate of conversion of into fuel and the

results of this process are found to be better than other alternate methods which are used for

the disposal of waste plastic.

Key words: waste plastics, thermal degradation, pyrolysis, catalyst degradation.

41.1 Introduction

Plastics play an important role in day- today life. It is unique material because of their

toughness, light weight, resistance to water and chemicals, resistant to heat and cold, low

electrical and thermal conductivity, ease of fabrication, remarkable color range, more design

flexibility, durability and energy efficiency. Due to above properties it is used in packaging




445

materials, agriculture, construction, insulation, automobile sector, electronic devices, textiles

and sports equipment and toys.

Plastics constitutes in two main categories. It is thermoplastics and thermoset plastics.

Thermoplastics make up 80% of the plastics and thermoset plastics make up of remaining 20

% of plastics produced today (Birley et al, 1988), etc. Thermo plastics can re-melt or re-

mould and therefore it recyclable easily but thermoset plastics cannot re-melt or reshape and

therefore it is difficult to recycling. Use of different type of some thermo plastics is given in

table1 below. Plastics are relatively cheap, easy available, easy to manufacture and their

versatility replace to conventional materials.

Plastic waste management is biggest problem now due to their non- biodegradability

nature. Now plastics manage by plastics recycling technologies.

Table 1:Uses of different types of plastics.

Type of Plastics Uses

Polyester Textile fiber

PET Carbonated drink bottles, plastics film

PE Supermarket bags, plastics bottle

HDPE Milk jugs, detergent bottles, thicker

Plastics film, pipes

LDPE Floor tiles, shower curtains, cling film

PVC Agriculture (fountain) pipe, guttering

Pipe, window frame, sheets for

building material

PS foam use for insulation of roofs and

walls, disposal cups, plates, foodContainer, CD and cassette box.

PP Bottle caps, drinking straws,

Bumper, house ware, fiber carpeting and rope.

41.1.1 Plastics in environment

The quantum of solid waste is ever increasing due to increase in population,

developmental activities, changes in life style, and socio-economic conditions, Plastics wasteis a significant portion of the total municipal solid waste (MSW). In India generation of




446

plastics are increased from about 2.6 MT in 2003 to about 3.6 MT in 2007(MOEF, 2007).

Also it is estimated that approximately 10 thousand tons per day (TPD) of plastics waste is

generated i.e. 9% of 1.20 lacks TPD of MSW in the India(CPCB, 2003). 32 million of plastics

were generated in 2011 in America, representing 12.7 percent of total MSW (EPA, 2011). It is

estimated that 100 million tones of plastics are produced each year with PE, PS, PVC and PP

amounting to more than 65% of total produced. The average European throws away 36kg of

plastics each year. Discarded plastic products and packaging materials make up a growing

portion of municipal solid waste. Plastics packaging totals 42% of total consumption and very

little of this is recycled (Vogler et al, 1984), etc. Only 8 percent of the total plastic waste

generated in 2011 was recovered for recycling (EPA, 2011).

Plastics waste may grow in India in future because more and other countries like as

U.S, China and U.K will comes in Indian market. There is a much wider scope for recycling

in developing countries mainly in India due to low labor cost, plastics consumption increase

and therefore raw materials increase.

41.1.2 Environmental hazards due to mismanagement of plastics waste

Plastics are no biodegradable material. It takes time to biodegrade is 300-500 years

and therefore environmental hazards due to improper manage include following aspect:

1. Littered plastics spoils beauty of the city and choke drains and make important public

places dirty.

2. Garbage containing plastics, when burnt may cause air pollution by emitting polluting

gases.

3. Garbage mix with plastics gives problem in landfill operation.

4. Lack of recycling plant to posing unhygienic problem to environment

41.1.3 Side Effect of plastics in nature

1. Durability and chemical structure greatly influences the biodegradability of some

organic compounds therefore an increased number of functional groups (groups of

atoms) attached to the benzene ring in an organic molecule usually hinders microbial

attack.

2. Instead of biodegradation, plastics waste goes through photo-degradation and turns

into plastic dusts which can enter in the food chain and can cause complex health

issues to earth habitants.




447

3. Plastics are produced from petroleum derivatives and are composed primarily of

hydrocarbons but also contain additives such as antioxidants, colorants, and other

stabilizers.

4.

However, when plastic products are used and discarded, these additives are

undesirable from an environmental point of view.

5. Burning of plastics give NOX, COX, SOX, particulate, dioxins, furans and fumes to

increase air pollution with result acid rain and increase global warming.

6. Plastics in land fill area leaching of toxins into ground water.

41.2. Target of waste plastics into liquid fuel

41.2.1 Recycling Technologies

1. Mechanical Recycling of waste plastics into reusable product is difficult and

unfeasible due to contamination of plastics, difficulty to identifying and separating

different type of plastics.

2. Uncontrolled incineration of plastics at higher temp above 850 deg Celsius to

produces polychlorinated dibenzo-p-dioxins, a carcinogen (cancer causing chemical).

Open-air burning of plastic occurs at lower temperatures, and normally releases such

toxic fumes and many oxide gases. So flue gases treatment use for protectenvironment and health problems in incineration plant.

3. Chemical recycling could lead to useful raw materials via by degradation and

monomerization of plastics waste, but no method of this primary recycling currently

available. The degradation of some plastics into chemicals has been reported in

research level.

Gasification and blast furnace of plastics waste to produce gases that are carbon

dioxide, nitrogen, carbon mono oxide, hydrogen and methane at higher temp above 800 deg.

Celsius.

41.2.2 Biodegradability

Plastics are non biodegradable material that resists microbial attack. Though work has

been done to make futuristic biodegradable plastics, there have not been many conclusive

steps towards cleaning up the existing problem because prices of biodegradable plastics is

more than petrochemicals based plastics. It may be due to high cost of production and low

availability or high cost of raw materials. Some degradable plastics have been developed, but




449

41.4.1 Raw materials

Type of Plastics as raw materials and its contents in table 3 is below.

Table 3: Type of plastics and its content.Type of plastics contents

PE (HDPE, LDPE), PP, PS hydro carbons

PET, PVA, PF hydro carbons with oxygen

PVC, PVCD hydrocarbons with chlorine

Nylon (polyamide), PU hydrocarbons with nitrogen

Polyphenylene sulfide hydrocarbons with sulfur

41.4.2 Effect of raw material as plastics in production

If PE, PS, PP with other plastics gives flue gas pollution and contaminated to reactor

by making other unexpected compound. In contamination to reactor resulting liquid may

contain alcohol, waxy hydrocarbons and inorganic substance. Type of plastics and their

product in table 4 is below.

Table 4: Effect of plastics in production.

Type of plastics Product

PET terephthalic acid and benzoic acid

PVA water and alcohol

PVC, PVDC HCL gas and carbonous compound

PU, PF, NYLON carbonous product

PE, PS, PP liquid fuels

(UNEP, 2009)

41.4.3 Pyrolysis

It is thermal degradation process in the absence of oxygen. It prevent of formation of

C0X, NOX, SOX due to absence of oxygen. It breaks large hydrocarbon chain into smaller

ones, but this type of pyrolysis requires higher temperature and high reaction time. Also

resulting fluid have low octane value, higher pour point of diesel and high residue content.

41.4.4 Catalytic Pryolysis

Pyrolysis of waste plastics in presence of catalyst lower the pyrolysis temp and

reaction time, increase conversion rate of waste plastics into fuel, increase the yield of fuel

and satisfying diesel, petrol quality of fuel by increase octane value of petrol and decrease




450

pour point of diesel. Catalyst use for this purpose is solid acids such as silica, alumina,

zeoliteβ, zeoliteY, mordenite, HZSM-5, MCM-41. Acidic catalysts (HZSM-5, Zeolitey,

mordenite and so on) have greater efficiency than less acidic ones, for example amorphous

alumina silicate. The pore size and structure of catalyst determine their performance on

cracking reaction as well as production, for example mordenite size( about 7x8Ȧ) larger give

large product molecules while HZSM-5 have smaller pore size(5x5Ȧ) give small product

molecules.(P.A. Parikh and Y.C. Rotliwala, 2008)

41.4.5 Process of formation

Collect waste plastics and separate that clean and recyclable. Store the waste plastics

that can’t separate. Shredding of waste plastics to reduce volume of its. Shredded plastics is

treated in a cylindrical reactor at temperature of 300ºC – 350ºC(Pawar harshal and Lawankar

Shailendra, 2013).Plastics waste further cracked with catalyst and resulting hydrocarbons are

condensed from water cool condenser and collected in receiver. Then liquid fuel fractionates

to get diesel, kerosene, petrol etc.

Gases produced are toxic, corrosive with non toxic gases. For example hydrogen

chloride, hydrogen sulfide etc is toxic and non toxic is butanes, methane, ethane and

propylene. So all the gases are treated from this process before it discharge into atmosphere.Therefore flue gas treated through scrubbers and water/ chemical treatment for neutralization

i.e. Solution of methanol amine is use in hydrogen sulfide absorption. Treated flue gas can

incinerate use in dual Fuel diesel-generator set for generation of electricity. After process

remove the formed carbonous substance or residue in reactor to work as insulator for

maintaining the efficiency of process. The block diagram of process is given in figure1.

41.4.6 Yield

The average percentage yield of various fuel fractions by fraction distillation

depending on composition of waste plastics are Gasoline (60% ) and Diesel (30%). The

percentage of liquid distillate is mentioned in terms of weight by volume (Antony Raja and

Advaith Murali 2011).

41.5. Advantages of process of fuel production

41.5.1 Eco-friendly

The fuel satisfies quality of liquid fuel with low sulfur content and low carbon residue.

The properties of waste plastic pyrolysis oil and diesel in table 5.




451

collection and segregation of plastic waste

storing of plastic waste

shredding of plastic waste

Figure 1- Conversion waste plastics into liquid fuel (Pawar Harshal and lawankar, 2013)Table 5: Properties of Waste Plastic Pyrolysis Oil and Diesel.

Sr. No. Properties WPPo Diesel

1. Density(kg/m2) 793 850

2. Ash content (%) <1.01%wt 0.045

3. Calorific value(kJ/kg) 41,800 42,000

4. Kinematic viscosity @ 2.149 3.05

40C(cst)

5. Cetane number 51 55

6. Flash point oC 40 50

7. Fire point oC 45 56

8. Carbon residue (%) 0.01%wt 0.20%

9. Sulphur content (%) <0.002 <0.035

10. Pour point oC -4 3-15

(Pawar Harshal and Lawankar, 2013)

feeding into hopper

Flow of waste into heating vessel in absence of oxygen and presence of catalyst

movement of liquid-vapor into condenser vessel tarry waste

Tapping of liquid fuel

Fractionation of liquid fuel to obtain diesel, petrol, kerosene etc.




452

41.5.2 Feasibility

Process of conversion of waste plastics into liquid fuels is feasible. Also the rate of

fuel does not vary widely along the period. The cost for per kg of input and related output in

table 1.6 is below.

Table 6: cost for 1 kg of input and the yield, cost of output.

Input Qty Kg Rate per Kg Amount (Rs) Output Qty (l) Rate per liter

Amount (Rs)

Plastic 1.00 12.00 12.00 Petrol 0.600 37.50 22.50

Labour 5.00 Diesel 0.300 25.50 7.65

Service

Charge 2.50 Lube oil 0.100 15.00 1.50

Total 1.00 19.50 1.00 31.65

(Antony Raja and Advaith Murali, 2011)

40.5.3 Good performance

Liquid fuels from petroleum is diesel, petrol, kerosene require to mix various additives

for improving burner and engine performance but fuel from waste plastics does not require to

add these additives for work on burner and engines. Tarry waste or residue in reactor can useas solid fuel.

40.6 Conclusion and recommendation

Based on review papers, waste plastics liquid fuel is good alternative method for

obtaining new energy resource and eliminate greater problem of plastics waste management.

In India 3.6 million ton of plastics waste generated in 2007. Improper management of plastics

gives hazardous problem to human and environment. Mechanical recycling is not effective to

reduce to problem of plastics waste. Incineration, gasification , blast furnace is other method

does not effectively eliminate to this problem due to air pollution, economical unfeasibility

compare to waste plastics fuel method. Biodegradable plastics are not meet at same rate as

petroleum based plastics.

Growth of energy demand due to urbanization, population, industrialization and also

increased price of fuel need to reduce to this demand and increased rate of fuel. Waste plastics

fuel is eco friendly due to low content of pollutants, good performance characteristics on

engine, burner with no added any additives like as lubricants and good feasibility with earning

profit.




453

Abbreviation

PET- polyethylene terephthalate

HDPE- high density polyethylene

LDPE- low density polyethylene

PS- polystyrene

PVC- polyvinyl chloride

PP- polypropylene

PF- phenol formaldehyde

PU- poly urethane

PVA- poly vinyl alcohol

PVDC- polyvinylidene chloride

References

1. Antony Raja and Advaith Murali, 2011 Conversion of Plastic Wastes into Fuels

Journal of Materials Science and Engineering B 1 (2011) 86-89

2. Birley, A. W., Heath, R. J., and Scott, M. J. (1988) Plastics Materials. Blackie, 2nd ed.

Introductory scientific textbook.

3.

Central Pollution Control Board. Study on solid waste management CPCB Delhi.

(2003).

4. Environment Protection Agency, U.S.A. Study on solid waste management (2011).

5. Ministry Of Environment and Forest. News letter on solid waste management, New

Delhi, (2007)

6. Pawar Harshal R. and Lawankar Shailendra M.(2013) Waste plastic Pyrolysis oil

Alternative Fuel for CI Engine – A Review Research Journal of Engineering Sciences

ISSN 2278 – 9472 Vol. 2(2), 26-30, February (2013)

7. P.K Parikh PhD, Y.C Rotliwala (2008) DOI: 10.1680/warm.2008.161.2.85

ISSN : 1747-6526

8. S Rao, Dr. B.B Parulekar (2012) Energy Technology (NONCONVENTIONAL,

RENEWABLE & CONVENTIONAL), Khanna Publishers, ISBN NO. 81-7409-040-1

9. Tiwari D.C., Ejaz Ahmad, Kumar Singh K.K. Catalytic degradation of waste plastic

into fuel range hydrocarbons International Journal of Chemical Research, ISSN: 0975-

3699, Volume 1, Issue 2, 2009, pp-31-3610. UNEP, Converting Waste Plastics into Resource, (2009).




454

11. Vogler, Jon, Small-scale recycling of plastics. Intermediate Technology Publications

1984. A book aimed at small-scale plastics recycling in developing countries




455

CHAPTER 42

KINETICS OF NOX REDUCTION IN BIODENOX PROCESS

WATER: EFFECT OF TEMPERATURE AND IRON

CHELATE

B. Chandrashekhar, Heena Tabassum, Nidhi Sahu, Padmaraj Pai, R. A. Pandey

Abstract

The aqueous solution of various iron chelates viz. FeIIEDTA, Fe

IINTA has been utilized for

NOx absorption from gaseous emissions using wet-scrubbers. In order to make the spent

solution recyclable to the scrubber, the BioDeNOx process involves treatment of the spent

scrubber solution wherein the reduction of NOx adduct of FeIIEDTA takes place by

employing denitrifying bacteria. The influence of temperature on batch NOx reduction

process using different concentration of FeIIEDTA was investigated and modelled. The

specific NOx reduction rates in 0 to 30 mM FeIIEDTA solution were estimated in the

temperature range of 298-313 K. The values of Arrhenius factor (Ar) and activation energy

(Ea) were determined using the Arrhenius equation. The values of Ar and Ea were highest in

the absence of FeIIEDTA (319.54 µmoles gVSS

-1 L

-1 h

-1and 88.298 J/mol respectively).

Addition of 5 mM FeIIEDTA to the solution decreased the values of Ar and Ea, which

however increased with further increase of FeIIEDTA concentration. The sensibility of NOx

reduction rates to temperature was modeled using the equation R = R293 x 10K(T-293)

and the

values of temperature constant (KT) was predicted, which was found to be highest in 30 mM

FeIIEDTA solution (0.0034 K-1). The temperature coefficient (QT) was also calculated at each

FeIIEDTA concentration to determine the sensitivity of reduction rate to temperature.

Keywords: Nitrogen oxides, Ferrous EDTA, Denitrification, NOx reduction, Arrhenius

Equation

42.1 Introduction

The adducts of chelating agents and metals such as Ferrous (FeII) EDTA (ethylene

diamine tetra acetic acid) and Ferrous NTA (nitrilotriacetic acid) have been used to enhancethe absorption rate of NOx from into aqueous phase in wet scrubbers (Gambardella et al.,




456

2006; Demmink et al., 1997). FeIIEDTA is the most commonly used chelates for absorption

of NOx. In order to make absorption process economical, the spent metal–ligand solution has

to be regenerated and recycled which is done by the biological denitrification process in

which the NO adduct of FeIIEDTA is reduced to molecular nitrogen. It is reported that

reduction of NO is enzymatically catalyzed by potential denitrifying bacterial strains viz.

Bacillus azotoformans (Kumaraswamy et al., 2005), Pseudomonas (Zhang et al., 2007),

Paracoccus denitrificans (Li et al., 2012) and anaerobic sludge (Van der Maas et al., 2008;

Dilmore, 2004) which use an organic electron donor as a reducing agent. The regenerated

adduct solution therefore is recycled back to the scrubber where it continuously absorbs NOx

from the emission gases. This process is also known as BioDeNOx (van der Maas, 2008;

2005) and a general schematic of the process is shown in Fig-1.

Fig. 1 General schematic of the BioDeNOx concept of NOx removal from gaseous emissions

In order to develop a full scale engineered process for biological NOx removal using

FeII EDTA solution, it is necessary to evaluate the kinetic parameters associated with NOx

reduction under the operating conditions. The emission gases containing NOx have high

temperatures; therefore, scrubbing of such gases with the scrubber solution would also lead to

a rise in temperature of the solution, which consequently would affect the biological reduction

process. In the present investigation, the biomass from a wastewater denitrification process

was evaluated for NOx reduction in different concentrations of FeIIEDTA solutions using

ethanol as carbon source as well as electron donor. The bioreduction rates (R) were

determined and the impact of temperature on the reduction rate was investigated in the range




457

of 298-313 K. Arrhenius equation was applied for the calculation of activation energy (EA)

and Arrhenius factor (Ar). Also, a kinetic model was applied to determine the value of

temperature constant at each FeIIEDTA constant.


42.2.1 Source of biomass for conducting NOx reduction experiments

The bacteria used for this study were obtained from the sludge obtained from a

denitrification tank of an effluent treatment plant. The biomass was cultivated for a period of

30–35 days in a 1 L batch reactor with a medium containing 0.020 M FeIIEDTA with

nutrients and trace elements. NaNO2 (as a source of NOx) was added to the medium along

with ethanol and trace element solution from a concentrated feed stock whenever required.

The unit was operated in anaerobic conditions to allow bacterial growth. The temperature of

unit was controlled at 37 *C the help of a water bath. The inoculum was prepared by

harvesting the cultivated bacteria from the medium by centrifugation (8000 rpm, 10 min). The

pellet obtained was again washed with 0.1 N phosphate buffer saline and used as inoculum for

the batch experiments.

42.2.2 Media composition and batch experiments

All the batch experiments for NOx reduction were conducted in 250 mL Erlenmeyer

flasks containing 200 mL media. The flasks were sealed with silicon stoppers and kept in a

shaking incubator at 100 rpm. The experiments were conducted in order to find out the rate at

which FeIIEDTA-NO was reduced by the biomass at different Fe

IIEDTA concentration.

Hence, the assays were conducted by varying the FeIIEDTA (0 - 30 mM) in a medium

containing: NaNO2 – 1 mM, K2HPO4 – 3 mM, KH2PO4 – 4 mM, MgCl2 – 0.002 mM,

MgSO4.7H2O – 0.4 mM, Na2SO3 – 0.5 mM, FeSO4 – 0.06 mM, CuSO4.5H2O – 0.03 mM,

Na2- MoO4 – 0.02 mM and Ethanol- 4.5 mM. FeIIEDTA-NO medium was prepared by

mixing equimolar concentrations (0 - 30 mM) of FeCl2 and Na2H2EDTA to the above

medium under anoxic conditions according to a method described by van der Maas et al

(2005). The media as well as head space was flushed with pure nitrogen gas in order to

maintain anaerobic conditions. The pH of the media was adjusted to 6.5 by adding NaOH or

HCl for all the experiments. The biomass was inoculated to the medium at initial volatile

suspended solid (VSS) concentration of about 2.0 g VSS L-1

. A medium without the biomass

inoculation was kept as control for each experiment. The NOx concentration (N) after




458

different time intervals was monitored in the media and the specific NOx reduction rate (R)

was calculated as follows-

R = - (lnN2 - lnN1)/(t2 - t1) * x ……… (1)

where, x is the initial VSS concentration in the medium.

To study the impact of temperature NOx reduction at each FeIIEDTA concentration,

the experiments were set up as previously described and conducted at 25, 30 and 35 and 40 +

0.5 °C under anoxic conditions as most of the denitrifying bacteria are mesophiles that are

known to grow in the range of 20-45 °C. The kinetics of the reduction reaction included the

estimation of activation energy (EA) and the temperature coefficient (K) determination that

determine the process sensibility and the change of the NOx reduction rates along with the

change in temperature. EA measures the change in the potential energy that is required to

begin the reaction to convert the reactants into products. The reactions with a low E A are less

sensitive to the change of temperature. The change of the NOx reduction rates along with the

temperature was given by the Arrhenius equation

R = Ar×e-(Ea/Rg.T)

……… (2)

where R is the specific NOx reduction rate, Ar is the Arrhenius factor (mM.g-1

.h-1

), EA is the

activation energy (J mol-1

), Rg is the gas constant (8.314 J mol-1

K-1

) and T is temperature

(K). A linear form of the equation obtained after taking logarithm is shown below-

lnR = lnAr − EA /Rg·T ...…… (3)

The graphic plot of lnR versus 1/T is a straight line with a slope of - EA /Rg and

intercept of lnAr which enables the estimation of EA and Ar. The sensitivity of the reaction to

increasing temperature was expressed by the following equation [18]:

R = R293 × 10KT (T-293)

……… (4)

Where, KT is the temperature constant. The temperature coefficient (QT), which determines

the change of the reduction rate along with the change of temperature, can be calculated as the

ratio of the specific reduction rates at different temperatures as shown below-

QT = R(t++t) /Rt ……… (5)

Where R(T++T) and RT are the specific NOx reduction rates at times T++T and T respectively.




459

42.3 Analytical methods

The concentration of NOx in the media was measured in the form of NO2- in all the

experiments. In case of the medium without FeIIEDTA, NO2

- was measured directly by standard

protocol using sulphanilamide and NED reagent (APHA, 2005). In case of media with

FeIIEDTA, some amount of NOx was present in the form of NO adduct of Fe

IIEDTA,

therefore the samples were oxidized by 0.1 N NaOH (to convert NO to NO 2-) before

subjecting to nitrite estimation by the above method. All the analyses results reported in this

paper are average values of duplicate samples.


42.4.1 Specific NOx reduction rates under various conditions

The biomass used for the batch NOx reduction experiments were initially cultivated

for 30 days in a medium containing 20 mM FeIIEDTA along with NOx, using ethanol as

electron donor. The acclimatized biomass thus possessed the ability to grow in the presence of

FeIIEDTA using NOx as the available electron acceptor. It has been earlier reported that

FeIIEDTA has the potential to act as an electron donor for the chemical reduction of NOx (van

der Maas et al, 2008). The objectives of the batch experiments were to investigate the effect

of FeIIEDTA concentration on NOx reduction under different temperatures of incubation. The

experiments were designed to simultaneously determine the effect of FeIIEDTA as well as

temperature on NOx reduction rates. The batch NOx reduction experiments were carried out

in a medium containing 1 mM NOx using ethanol as the organic electron donor. The biomass

concentration in each experiment was kept constant (2 mg VSS/ml). Four sets of reduction

media with varying FeIIEDTA concentration were prepared; each set was incubated at

different temperature viz. 25, 30, 35 and 40 *C and monitored for NOx concentration at

regular intervals.

42.4.1.1 Effect of FeII

EDTA concentration

The NOx reduction curves obtained at 298 K for different concentration of FeIIEDTA

in the media are shown in Fig (2).

Similar curves were obtained for different incubation temperature, and the specific

NOx reduction rate for each experimental condition was calculated using Eq. 1. The specific

reduction rates using various concentration of FeIIEDTA at different temperatures (298 – 313

K) are depicted in Fig (2). It was observed that highest NOx reduction rates (R) were obtained

in the absence of FeIIEDTA. However, addition of 5 mM Fe

IIEDTA drastically reduced the




460

specific reduction rate, which indicated that FeIIEDTA inhibited the biological reduction of

NOx. However, it was also observed that with further increase in FeIIEDTA concentration, the

specific NOx reduction rates also increased. Therefore it can be suggested that though the

biological reduction process was inhibited at higher FeIIEDTA concentration, the chemical

reduction of NOx by FeIIEDTA occurred. The chemical reduction of NOx by Fe

IIEDTA is

shown in the Eq. 6-7 (van der Maas et al., 2008). The reactions clearly show that 2 moles of

FeIIEDTA

2- is required for complete reduction of each mole of NO2

- to N2. As more unbound

FeIIEDTA would be available at higher concentration of Fe

IIEDTA in the medium for the

chemical reaction to occur, this provides the reason for the enhanced reduction rates.

2FeIIEDTA

2--NO + 2H

+ N2O + H2O + 2Fe

IIIEDTA

- ………….. (6)

N2O + FeIIEDTA

2- + 2H

+ N2 + H2O + 2Fe

IIIEDTA

- …………. (7)

Fig. 2 NOx (as NO2-) reduction curves obtained at 298 K with various concentration of

FeIIEDTA

42.4.1.2 Effect of temperature on NOx reduction rates

The temperature range selected for the investigation was based on the fact that most

denitrifying bacteria grow under mesophilic conditions and also that the stability of the

FeIIEDTA-NOx complex would be lower at temperatures higher than 323 K (Gambardella et

al, 2006). Therefore, the batch experiments were carried out at 298 – 313 K. The percentage

0.000

0.200

0.400

0.600

0.800

1.000

0 5 10 15 20 25 30 35 40 45

N % & ' ( "

' 1 )

Ti"e (h)

0.000 ,eE/ 0.005 ,eE/ 0.010 ,eE/

0.020 ,eE/ 0.030 ,eE/




461

NOx reduction at different concentration of FeIIEDTA incubated for 40 hours at various

temperatures is summarized in Table-1.

Table 1 NOx reduction (%) at various temperatures and FeIIEDTA concentrations after 40

hours of incubation

% NOx reduction

FeII

EDTA

concentration (mM)

Temperature (K)

298 303 313 323

0 85.92 8.65 89.38 88.25

5 6.68 6.96 68.24 66.63

10 3.81 4.9 5.6 5.38

20 80.59 81.30 82.00 81.22

30 80.82 82.58 84.33 84.41

Also, it is evident from Fig (3) that at all FeIIEDTA concentrations used in the present

investigation, the specific reduction rates increased with temperature.

Fig. 3 Specific NOx reduction rates at different FeIIEDTA concentrations and temperatures

The impact of temperature on reduction rate can be studied by applying Arrhenius equation,

which enables to calculate the Arrhenius factor (A) and activation energy (Ea). The change ofreduction rates along with temperature is given by Eq. 3. A linear plot drawn between 1/T

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0 5 10 15 20 25 30 35

S ! e c i f i c N % * r e d u c t i o n r a t e ( R )

" o l e s + ' 1 h ' 1

$eE-TA ("oles '1)

25 e!ree& 35 e!ree& 40 e!ree&




462

and lnR showed good degree of linearity and was used to determine the values of A and Ea for

each concentration of FeIIEDTA as shown in Fig. 4. The values of Ea and A obtained for each

concentration of FeIIEDTA are summarized in Table 2.

Fig. 4 Plots between log values of specific NOx reduction rate (ln R) and inverse of

temperature (1/T) to determine the value of activation energy and Arrhenius factor at each

FeIIEDTA concentration

Table 2 Activation energy and Arrhenius factor for NOx reduction at different FeIIEDTA

concentration

$eE-TA

(m)

Arrhenius factor, Ar

(m!h)

Actiation Ener+y, Ea

(mol)

0 319.54 38.43

5 28.21 3.39

10 34.83 4.19

20 5.14 9.0430 25.21 30.94

7 -89.08x + 2.9418

: 0.9938

2.64

2.64

2.65

2.65

2.65

2.66

2.66

0.0031 0.0032 0.0033 0.0034

l n R

1/T (#'1)

. " $eE-TA

7 -200.54x + 3.5504

: 0.9625

2.8

2.88

2.89

2.90

2.91

2.92

0.0031 0.0032 0.0033 0.0034

l n R

1/T (#'1

)

1 " $eE-TA

7 -3.12x + 4.3193

: 0.549

3.04

3.06

3.08

3.10

3.12

0.0031 0.0032 0.0033 0.0034

l n R

1/T (#'1)

& " $eE-TA

7 -21.03x + 5.5499

: 0.93

3.12

3.16

3.20

3.24

3.28

0.0031 0.0032 0.0033 0.0034

l n R

1/T (#'1)

0 " $eE-TA




463

It was observed that similar to the specific NOx reduction rates, the values of both Ea

and A were highest in the absence of FeIIEDTA, and lowest in the presence of 5 mM which

gradually increased along with FeIIEDTA concentration. Generally, reactions with low

activation energies are less sensitive to temperature. The sensitivity of the reaction to

increasing temperature can be also expressed by the Eq. 4. The values of R obtained at

various temperatures (298-313 K) for 0 - 30 mM FeIIEDTA concentration was modeled using

this equation by Solver function as shown in Fig. (5), and the predicted values of temperature

constant (KT) are summarized in Table 3.

Table 3 Temperature constants of NOx reduction at different FeIIEDTA concentration

$eE-TA (") R&10 (m!h)

#T

0 26.21 0.0033

5 13.98 0.00041

10 1.59 0.00094

20 20.38 0.002

30 22.00 0.00339

Since, higher values of KT signifies higher sensitivity of the reaction to temperature, it

can be suggested from the results that NOx reduction in the medium without FeIIEDTA

(completely biological reaction) was highly sensitive to temperature.

On the other hand, lesser values of KT obtained in the presence of 5- 10 mM

FeIIEDTA indicate that the process was less sensitive to temperature. Further increase in

FeIIEDTA concentration increased the value of KT, which means increasing the concentration

of FeIIEDTA increases the chemical reduction of NOx and also renders the reduction reaction

to be more temperature sensitive. The temperature coefficient determines the change in

reaction rate along with change in temperature. The temperature coefficient (+t = 10 K) for

each FeIIEDTA concentration was calculated from the predicted values of R, using Eq. 5 and

as shown in Fig. 5, value of Q also slightly increased along with FeIIEDTA concentration.

However the value of Q was found to be very less as it is well accepted that that a 10 K rise in

temperature results in doubling of the reaction rate.

The effect of temperature on reduction of NOx in the presence of FeIIEDTA has not

been reported elsewhere. The effect of temperature on biological reduction of NOx in the

absence of FeIIEDTA has been studied previously (Pfenning and McMohan, 1996; Rusmana,

2007; Carrera et al, 2003; Kadlec, 2001). Casey (1997) and Foglar et al (2010) have studied




464

the effect of temperature on bio-denitrification rates and reported similar values of KT, as

reported in the present investigation. However, higher values of temperature coefficient (2.03)

within the same temperature range has been reported by Foglar et al (2010; 2005) and

Delanghe et al (1994).

Fig. 5 Modeled NOx reduction rates as a function of temperature at various concentration of

FeIIEDTA

26.0

2.0

28.0

29.0

30.0

31.0

290 300 310 320

R ( " . +

' 1 h ' 1 )

Te"!erature (#)

" $eE-TA

14.00

14.05

14.10

14.15

14.20

14.25

14.30

295 300 305 310 315

R ( " . +

' 1 h ' 1 )

Te"!erature (#)

. " $eE-TA

1.

1.9

18.1

18.3

18.5

290 300 310 320

R ( " . + ' 1 h ' 1 )

Te"!erature (#)

1 " $eE-TA

20.5

21.0

21.5

22.0

22.5

23.0

23.5

295 300 305 310 315

R

( " . + ' 1 h ' 1 )

Te"!erature (#)

& " $eE-TA

22.5

23.0

23.5

24.024.5

25.0

25.5

26.0

26.5

295 300 305 310 315

R ( " . +

' 1 h ' 1 )

Te"!erature (#)

0 " $eE-TA




465

42.5 Conclusion

The biological reduction of NOx in the presence of FeIIEDTA is a slow process, since

the growth of bacteria in FeIIEDTA medium is inhibited. Biological reduction of NOx is

therefore the limiting step of the two stage BioDeNOx process for NOx removal. The results

obtained from the present investigation indicate that chemical reduction of NOx by 30 mM

FeIIEDTA show comparable results with biological NOx reduction (in the absence of

FeIIEDTA) under the same conditions. Using higher concentrations of Fe

IIEDTA can make

the NOx reduction process more sensitive to temperature. It can be suggested that chemical

reduction of NOx by FeIIEDTA itself can be enhanced by optimizing the concentration of

FeIIEDTA (i.e. ratio of Fe

IIEDTA/NOx) and temperature in order to decrease the process cost

and improve NOx removal efficiency of the process.

Acknowledgements

The authors are thankful to Director, CSIR-NEERI, to give kind permission to present

this research work. The financial support extended by Department of Biotechnology and

Council of Scientific and Industrial Research, Ministry of Science & Technology,

Government of India, for execution of this project work is duly acknowledged.

References

1. American Public Health Association (APHA) (2005). Standard Methods for the

Examination of Water and Wastewater, nineteenth ed. American Public Health

Association, Washington, DC, USA

2. Demmink, J.F., Van Gils, I.C.F., Beenackers, A.A. (1997). Absorption of nitric oxide

into aqueous solutions of ferrous chelates accompanied by instantaneous reaction. Ind.

Eng. Chem. Res. 36:4914–4927.

3. Dilmore, R. (2004). Evaluation of the kinetics of biologically catalyzed treatment and

regeneration of NOx scrubbing process waters. Doctoral Thesis. University of

Pittsburgh, 224pp

4. Gambardella, F., Winkelman, J.G.M., Heeres, H.J. (2006) Experimental and modeling

studies on the simultaneous absorption of NO and O2 in aqueous iron chelate solutions.

Chem. Eng. Sci. 61: 6880–6891




466

5. Rusmana I. (2007) Effects of Temperature on Denitrifying Growth and Nitrate

Reduction End Products of Comamonas testosteroni Isolated from Estuarine Sediment,

Microbiology Indonesia, 43-47.

6.

Carrera J, Vicent T and Lafuente FJ (2003) Influence of temperature on denitrification

of an industrial high-strength nitrogen wastewater in a two-sludge system Water SA, 29

(1).

7. Pfenning K.S., McMahon P.B. (1996) Effect of nitrate, organic carbon, and temperature

on potential denitrification rates in nitrate-rich riverbed sediments Journal of Hydrology

187: 283-295.

8. Kadlec, R.H. and Reddy, K.R. (2001). Temperature effects in treatment wetlands. Water

Environment Research, 73 (5): 543-557.

9. Kumaraswamy, R., van Dongen, U., Kuenen, J.G., Abma, W., van Loosdrecht, M.C.M.,

Muyzer, G. (2005). Characterization of microbial communities removing nitrogen

oxides from flue gas: the BioDeNOx process. Appl. Environ. Microbiol. 71: 6345–6352

10. Li, N., Zhang, Y., Chen, M., Dong, X., Zhou, J. (2012). Reduction of Fe(II)EDTA–NO

using Paracoccus denitrificans and changes of Fe(II)EDTA in the system. J. Chem.

Technol. Biotechnol..http://dx.doi.org/10.1002/jctb.3833

11.

van der Maas, P. (2005). Chemically enhanced biological NOx removal from flue gases

– nitric oxide and ferric EDTA reduction in BioDeNOx reactors. Doctoral Thesis.

Wageningen, The Netherlands, 224pp

12. Van der Maas, P., Manconi, I., Klapwijzk, B., Lens, P. (2008). Nitric oxide reduction in

BioDeNox reactors: kinetics and mechanism. Biotechnol. Bioeng. 100 (6): 1099–1107

13. Zhang, S.H., Li, W., Wu, C.Z., Chen, H., Shi, Y., 2007. Reduction of Fe (II) EDTA–NO

by a newly isolated Pseudomonas sp. strain DN-2 in NO scrubber solution. Appl.

Microbiol. Biotechnol. 76: 1181–1187




467

CHAPTER 43

STATUS OF WASTE TREATMENT, UTILIZATION AND

MANAGEMENT IN AGRO PROCESSING

Yogender Singh and Y. K. Yadav

Abstract

The present situation of increase in world population brought severe problems to the

sustainable agriculture with simultaneous food scarcity and climatic change. In the near

future the management of food and agricultural systems will play an important role in the

conservation of the natural resources. Agro processing comprise of techno-economic

activities associated for conservation and handling of agricultural produce and to

make it usable for feed, food, byproduct utilization and industrial raw material.

Agricultural and food processing industrial waste contains many reusable substances of high

value. The agricultural residue occurs at the time of harvesting, handling transportation,

storage, marketing and processing resulting in waste. Depending on availability of an

adequate technology this residual matter can be converted into commercial products either as

raw material for further secondary processes, as operating supplies or as ingredients of new

products. Efficient management of these wastes can help in preserving vital nutrients of our

foods, feeds and byproducts utilization, bringing down the cost of production of processed

foods, besides minimizing pollution hazards. It causes serious pollution problem if not

utilized or disposed off appropriately. A clear, concise and consistent policy is necessary to

establish and set up waste management systems and backed by legislations for all kinds ofviolations. Implementation and monitoring of the different waste management rules should

also be identified at a regular interval, to study the effects of inappropriate disposal of waste

on the environment. This is essential for guiding the management of waste in a manner that is

environmentally responsible and which minimizes danger to public health. Thus, it is

recommended that phrase of “Reduce, Reuse, Recycle” should be used for waste

management.

43.1 Introduction

The rapid increasing of world population brought serious food, health and




468

environmental problems such as global food availability and climatic changes. In the near

future the management of food and agricultural wastes will play an important role in the

conservation of the natural resources in many countries, including India. Agro processing is

a set of techno- economic activities carried out for conservation and handling of

agricultural produce and to make it usable for feed and food or industrial raw material.

Agro processing industries occupy an important position economically and generate large

volumes of mostly biodegradable wastes. Large amount of wastes is generated every year

from the industrial processing of agricultural raw materials. Agro-industrial wastes can be

used as solid support, carbon and/or nutrient source for the production of a variety of value-

added products. The presence of carbon sources, nutrients and moisture in these wastes

provides conditions suitable for the development of microorganisms and their possibilities of

reuse. The generation of biodegradable waste increased linearly with the growth and

development of agro and food processing industries. A huge amount of waste in solid and

liquid form is produced in agro processing industries is valuable but biodegradable natural

resources with large economic prospects. It causes serious pollution problem if not utilized or

disposed off appropriately. However, hazardous wastes are also occasionally causes

depending on situations, as contamination by pesticides/herbicides and pathogens.

“Wastes” are materials which are discarded after use at the end of their intended life

period. Waste also defined as materials that are not prime products for which the

generator has no further use for their own purpose of production and consumption,

which discarded or intends to discard. Wastes may be generated during the extraction

and processing of raw materials to intermediate and final products during consumption

and any other human activity. Food wastes and effluents are rich in biodegradable

components with high biological oxygen demand (BOD) and chemical oxygen

demand. If they remained untreated their uncontrolled decomposition is hazardous to

the environment due to the production of methane and toxic materials (Waldron, 2004).

Wastes derived from agro processing industries are categorized into three groups:

(a) manufacturing losses,

(b) food products thrown away as municipal solid waste (MSW), and

(c) discarded wrappers and containers.

The lack of pilot scale testing of the developed technologies, negative attitude of the

industrialists and perhaps, less helping hand from the government sector are the major

constraints in utilization of the waste.




469

Due to poor postharvest management, the post harvest losses of farm produce in

India have been assessed of a very high order. According to Indian Agricultural Research

Data Book estimated post production losses in food commodities to the tune of INR

0.75 to 1.0 lakh crore per annum. The estimated loss includes losses during handling, storage

and processing. The extent of losses could be drop down to less than 50% of the estimated

existing level on adopting proper agro processing technology. For reducing the maximum

losses new initiatives need to be searched. It would be the long term interest of the

economy to developing suitable infrastructure processing systems to avoid the losses.

43.1.1 Agro processing industrial wastes

Agro processing industrial wastes are generated during the industrial processing of

agricultural and animal products. The agricultural activities includes the waste materials such

as straw, stalk, stem, leaves, husk, peel, shell, seed, pulp/stubble from fruits, legumes and

cereals, bagasses produced from sugarcane or sorghum milling, brewers spent grains and

many others. These wastes are generated in large amounts throughout the year with the

most abundant renewable resources on the earth. They are mainly composed of fibres, sugars,

proteins and minerals which are major compounds of industrial interest.

Due to the large availability and composition rich in compounds that could be used inother refining processes, there is a great potential on the reuse of these wastes on economical

and environmental aspects. The economical aspect is based on the fact that such wastes may be

used as low-cost raw materials for the production of other value addition of compounds, with

the expertise of reducing the production and input costs. The agro-industrial wastes may

contain phenolic compounds and other compounds of toxic potential, which may cause

deterioration of the environment when the waste is discharged to the nature.

43.1.2 Solid Wastes

Solid wastes are generated in food industries can be categories in two groups. One

group is organic residual wastes such as sludge from wastewater treatment and food wastes or

garbage accompanied with consumption. For organic food wastes, the options of feed use,

biogas then composting and heat recovery is adopted for reuse and recycling. Another type of

waste relating to food industries is the waste originating from packaging materials. These

wastes comprise of a large portion of municipal solid waste. Among these wastes, plastic

wastes in particular should be focused on from an environmental standpoint.

43.1.3 Agricultural Wastes




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Cereals are a major source of agricultural waste in many countries. Part of plant

residues (utilized/non-utilized) are considered as agricultural wastes. The amount of wastes

from livestock and especially of liquid manure is also included in wastes. Liquid manure

contains different microorganisms that are dangerous for people as well as for animals. On

the other hand, the manure has high energy potential and it is a significant source of

renewable energy.

43.1.4 Food Wastes

Food-processing wastes are those end products of various food-processing industries

that have not been recycled or used for other purposes. Food industry produces large volumes

of solids and liquid wastes from the production, processing and consumption of product.

These wastes results increasing disposal and potentially severe pollution problems and

represent a loss of valuable biomass and nutrients. In general, wastes from the food-

processing industry have the following characteristics (Litchfield, 1987):

1. Large amounts of organic materials

2. Varying amounts of suspended solids

3. High biochemical oxygen demand or chemical oxygen demand

43.1.5 Hazardous Wastes

Food that has been accidentally contaminated by, herbicides, pesticides and fumigants

may be treated as hazardous waste. For hygienic cleaning chlorine is frequently used in food

processing and daily operations. Hence, chlorinated organic compounds should be observed

in the wastewater treatment of industries. The wastewater may also contain certain levels of

trihalomethane and other related compounds. Food products contaminated with pathogenic

microbes or food poisoning sometimes result in hazardous wastes.

43.2 Agro processing industrial wastes treatment/utilization

The utilization and treatment of product specific waste is not easy due to its

inadequate biological stability, potentially pathogenic nature, high water content, potential for

rapid autoxidation as well as its high level of enzymatic activity. The two general methods of

traditional waste utilization have been to use the waste as either animal feed (e.g., spent

grains, distiller’s wash) or fertilizer (filtration sludge, carbonation sludge). Disposal of this

waste can be difficult for the following reasons (Russ and Meyer-Pittroff, 2004):

- Biological stability




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- High water content

- Rapid autoxidation

- Changes due to enzymatic activity

There are three general methods of waste disposal which not associated with

agricultural practices are incineration, anaerobic fermentation and composting, but very little

of value (e.g., energy, fertilizer) can be recovered using these processes, it is often

necessary to pay for these kinds of disposal.

43.2.1 Solid State Fermentation

Solid state fermentation consists of the microbial growth and product formation on

solid particles in almost absence of water. However, the substrate may contain sufficient

moisture to allow the microorganism growth and metabolism. This bioprocess has been subject

of several studies and it has been proved that solid state fermentation has the important

advantage of leading to higher yields and productivities or better product characteristics than

submerged fermentation, which is characterized by the cultivation of the microorganisms in a

liquid medium. Another great advantage is the lower capital and operating costs due to the

utilization of low cost agricultural and agro-industrial wastes as substrates (Nigam, 2009).

43.2.2 Ethanol production

Bioethanol production by agro-industrial wastes have been considered as an excellent

alternative for reusing these wastes with additional technological and economic advantages,

since this process is of easy operation and save energy. The waste can be subjected to solid

state fermentation for the production of ethanol for several uses. It can be used as a liquid fuel

supplement and as a solvent in many industries. Ethanol production by SSF using grape and

sugar beet pomaces (Rodriguez et al., 2010), and apple pomace (Joshi and Devrajan, 2008)

as solid substrates, has been recently evaluated.

43.2.3 Biogas Production

The constituents of the Biogas are methane (CH4), carbon dioxide (CO2) making up

approximately 90%. Other impurities such as nitrogen, hydrogen sulphide and oxygen

complete the unrefined fuel source (Zinoviev et al., 2007). Biomass consisting of agricultural

crop residues, solid as well as liquid wastes from industries and sludge can also be utilized

for production of biogas through microbial technology.

Biogas is produced by anaerobic digestion of agricultural and horticultural produce

wastes. Methanotropic bacteria can utilize CO2 from waste materials results production of




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methane. The process follows the complex polymers are first hydrolyzed into simple

substances by acid forming bacteria and finally these are digested anaerobically by

methanotropic bacteria and methane gas is liberated. Thus, the waste from agro processing

wastes in real sense is not a waste as everything can be potentially recycled and utililzed in

other form as food, feed or fodder. Thus, proper waste utilization will add to the wealth of the

nation and will benefit all involved in the process.

43.2.4 Byproducts Utilization

The new methods of byproduct utilization focus on certain contents of the food and

agricultural waste. The content of fibrous material (soluble and insoluble) in food is gaining

importance in human nutrition. To produce building materials, fibrous materials from spent

grains can be used as well, as filler and structural material in fiber board. The fibers of the

used up grains of brewery industry waste increase the strength of bricks before they are

kilned, which increases the bricks’ ability to thermal insulator as well. Pectin, a soluble

fiber, can be extracted from apples, citrus fruit, and beet waste through another extraction

process. Pomace can be used as fertilizer after it has been subjected to a special fermentation

process. Single cell proteins can be produced from dried and pectin extracted apple pomace

by using Aspergillus niger and Trichoderma viride. The grape apple pulp wastes have also

been employed as a substrate for Aspergillus niger to generate crude protein and cellulose.

The waste obtained from processing of fruits and vegetables is rich in fibre with poor quality

of protein. Fermented potato waste has been successfully used as animal feed purpose. Apple

pomace after fermentation followed by drying makes the produce enriched with proteins,

vitamins amd minerals which can be used for animal feed.

Waste from wineries, breweries and distilleries can be used for feeding livestock.

Natural colors of blue grapes skin, kokum (Garcinia indica), Phalsa (Grewia subin acqualis),

and Jamun (Syzygium cumini) have been thoroughly investigated for their nature,

concentration, extractability, stability and suitability as food colors. Fat is partially removed

from slaughterhouse waste, and is then used as a basis for many products in the chemical and

cosmetic industries.

43.3 Waste water in agriculture and food processing

Wastewater is the most serious environmental problem in the manufacturing and

processing of foods. Whenever and wherever food, in any form, is handled, processed, packed

and stored, there will always be an unavoidable generation of wastewater. Most of the volume




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of wastewater comes from cleaning operations at almost every stage of food processing and

transportation operations. Wastewater from food processing operations is defined by the food

itself. There are common pollutants present in the majority of food and agricultural

wastewater and effluents from each stage of the typical waste water treatment processes, they

are free and emulsified oil/grease, suspended solids, organic/inorganic colloids, acidity or

alkalinity, and sludges. The effluents from fruit and vegetable processing operations consist of

mainly carbohydrates, sugars, pectins, vitamins, and other components of the cell walls

components that have been severed during processing.

43.3.1 Waste water Treatment

Different sources contribute to the generation of wastewater in agro processing

industries, including fruits and vegetable processing, cereal processing, dairy products, meat

processing, seafood and fish processing, sugar processing and alcoholic/non-alcoholic

beverages. Wastewaters released from these industries are turbid, with high concentrations

of bio-chemical oxygen demand, Fats, oils/grease and suspended solids. The main parameters

for physicochemical and biological treatment of wastewater are pH, solids content (suspended

solids and dissolved solids), temperature, odor, biochemical oxygen demand and total organic

carbon.

Usually it is desirable to group wastewater as high, medium and low concentration.

High concentration wastewater sometimes may be concentrated further, treated and

recycled/disposed as solid wastes. Medium concentration wastewater may be treated on site.

Low concentration wastewater such may be discharged without any treatment. Activated

sludge processes are generally employed in food processing industries. Sometimes various

advanced treatment systems are used such as coagulation and filtration.

43.3.2 Waste management

Waste management is a collective activity involving segregation, collection,

transportation, re-processing, recycling and disposal of various types of wastes. Sustainable

waste management involves managing waste in an environmental, socially satisfactory and a

technological economic viable manner. Waste management differs for different types of

wastes and for wastes in different geographical locations such as urban, rural and hilly areas.

While the management of non-hazardous domestic waste is the joint responsibility of the

government, industrial and hazardous waste is the responsibility of the waste generators i.e.

commercial and industries establishments.




43.4 Importance of waste management

Waste management allows to save money on commodities, labor, energy and disposal

costs. Waste leads to considerable carbon emission in the environment. In the case of food

industry waste, farm inputs, storage and transportation each require additional input costs and

landfill disposal leads to production of methane gas, by decreasing this, can reduce various

environmental hazard.

43.5 Challenges in Waste management

1. Segregation at source

2. Quality of waste (High moisture content, low calorific value)

3.

Poor quality of landfill

4. Lack of information on substrate specific biocatalyst

5. High lag period of biomethanation process,

6. Process parameters control

Sustainable waste management can be achieved through strategic planning,