enhancing biogas production in two phase ......enhancing biogas production in two phase anaerobic...
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ENHANCING BIOGAS PRODUCTION IN TWO PHASE ANAEROBIC DIGESTION
(TPAD) USING BIOCHAR AND PREPARATION OF BIOCHAR LOADED
ORGANIC FERTILISERS
Nimas Mayang Sabrina Sunyoto
This thesis is presented for the degree of Doctor of Philosophy of
The University of Western Australia
School of Engineering
Centre for Energy
2019
i
THESIS DECLARATION
I, Nimas Mayang Sabrina Sunyoto, certify that:
This thesis has been substantially accomplished during enrolment in this degree.
This thesis does not contain material which has been submitted for the award of any other
degree or diploma in my name, in any university or other tertiary institution.
In the future, no part of this thesis will be used in a submission in my name, for any other
degree or diploma in any university or other tertiary institution without the prior approval
of The University of Western Australia and where applicable, any partner institution
responsible for the joint-award of this degree.
This thesis does not contain any material previously published or written by another
person, except where due reference has been made in the text and, where relevant, in the
Authorship Declaration that follows.
This thesis does not violate or infringe any copyright, trademark, patent, or other rights
whatsoever of any person.
The work described in this thesis was funded by Australian Research Council under the
ARC Linkage Projects Scheme (Project Number: LP100200137). Financial and other
supports have also been provided by the Australian Commonwealth Government
through the Australia Awards Scholarship.
This thesis contains published work and/or work prepared for publication, some of
which has been co-authored.
Signature:
Date: 06-08-2019
ii
ABSTRACT
The conventional anaerobic digestion (AD) is a mature technology to both manage
putrescible organic waste and generate biogas for energy services. However, the biogas it
produces is often of a low quality and the process is slow and operates over very long
durations. Two phase anaerobic digestion (TPAD) has been conceptualised to improve the
conventional AD. The advantage of the TPAD is the production of hydrogen (H2) from
the first phase to be mixed with the methane (CH4) generated from the second phase to
increase the overall quality of the biogas without sophisticated and expensive gas
processing. The TPAD also produced an increased nutrient availability for organic
fertiliser preparation for agricultural applications. However, improvements to further
enhance biogas production, increasing H2 and CH4 yields, operating TPAD in pilot scale
and adding value to the beneficial utilisation of the effluent are still required. Biochar with
its beneficial characteristics for a wide range of applications has become an innovative
aspect of this study. The present PhD thesis research was aimed to investigate the
utilisation of biochar in (1) the first and second phases of batch TPAD, (2) start-up
performance of a TPAD process demonstration unit (PDU) and (3) preparation of biochar-
loaded organic fertiliser from TPAD effluent.
The specific objectives of this thesis work included a systematic study on the effect of
biochar on the performance of the bench-scale TPAD and an investigation into the
working mechanisms of biochar through experimentation and process optimisation studies
in bench scale TPAD. In addition, a demonstration of the operation of TPAD PDU added
with biochar and evaluation of biochar-loaded organic fertiliser prepared from the effluent
of TPAD PDU and biochar. To accomplish these objectives, an investigation on the
individual effects of biochar addition ratio on the gas yield, gas production rate and
metabolic products in each phase of TPAD was conducted in a batch operation. The
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combined effects of biochar, pH and temperature on the H2 and CH4 production were
systematically studied and optimised using the response surface methodology (RSM). The
start-up performance of the TPAD PDU with the optimum biochar addition suggested
from the batch scale operation of TPAD was then conducted. The biogas production,
biogas composition, pH and metabolic products during the start-up operation were
investigated. Finally, the TPAD effluent was loaded on to biochar with different addition
ratios to prepare organic fertilisers. The biochar-loaded fertilisers were characterised, then
evaluated using seed germination assay.
Bench-scale studies of the first phase of TPAD showed that biochar addition shortened
the lag phase by 21.4 to 35.7 %, increased the maximum H2 production rate by 32.4% and
H2 production potential by 14.2 to 31 %. Biochar addition was shown to substantially
increase H2 yield (YH), especially at lower pH and higher temperatures. The RSM analysis
showed that the maximum YH of 1,331 mL.L-1 and H2 production rate (RH) of 763 mL.L-
1.day-1 were achieved under the optimum conditions of biochar addition ratio 10.1 g.L-1,
initial pH 6.4 and temperature 32C. The biochar initiated the biofilm formation, as
observed with scanning electron microscopy (SEM) images, and provided macro and
nutrients in the culture, enriching the microbial population. Biochar also acted as a pH
buffer of the cultures, preventing the cultures from sharp pH drop caused by acid
accumulation during hydrolysis, enhancing the H2 production.
Bench-scale studies of the second phase of TPAD showed that the biochar addition also
shortened the lag phase, by 41 to 45 %, increased the maximum CH4 production rate by
23.0-41.6% and CH4 production potential by 1.9 to 9.6%. A moderate level of biochar
addition, mesophilic temperature and neutral to alkaline pH were shown to benefit CH4
production in the second phase of TPAD. The effect of biochar addition was more
profound at higher pH. The optimum biochar addition, temperature and initial pH were
found to be 12.5 g.L-1, 36.2oC and 7.8, respectively. Under the optimum condition, the
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CH4 yield (YM) and CH4 production rate (RM) were 1755 mL.L-1 and 500.9 mL.L-1.day-1.
SEM images suggested an establishment of methanogenic biofilm in the pore and surface
of biochar. It was hypothesised that methanogenic biofilm enriched methanogens and
enhanced CH4 production. The alkalinity of biochar, however, was found to be
insignificant in promoting CH4 production in the second phase.
The investigation into the transient performance during the start-up of a TPAD PDU
treating food waste with biochar addition was conducted. A fed-batch followed by
semicontinuous operation strategy was found to be effective in starting up the TPAD PDU.
The fed-batch operation allowed sufficient time for microbial enrichment and adaptation.
Under semi-continuous operation, the peak H2 composition and yields in the first phase
were 49% and 46 L.kg volatile solids (kg VS)-1, respectively. CH4 production with the
composition of up to 59% and yield of 301 L.(kg VS)-1 were attained in the second phase.
The addition of biochar showed a potential to buffer the pH of culture and initiate biofilm
formation, which supported the successful start-up in both the reactors, supported by the
findings observed in bench scale studies of TPAD with biochar addition.
Finally, the biochar loaded organic fertilisers were successfully prepared and subject to
the seed germination assay. It was found that the addition of biochar significantly
increased water holding capacity (WHC) with no significant pH changes. It also increased
essential elements for germination and plant growth, such as potassium (K), calcium (Ca),
magnesium (Mg), sodium (Na), iron (Fe), manganese (Mn), nickel (Ni) and chromium
(Cr), compare to the TPAD effluent alone. The prepared fertilisers were tested using
germination bioassay and compared to the control without fertiliser addition. Fertilisers
with 0-90% biochar addition gave the positive effects on the seed germination, while the
pure biochar significantly reduced germination index (GI). However, despite the low GI,
the pure biochar, and the rest of the fertilisers tested, resulted in the increased sums of root
and shoot length compared to the control. The maximum sums of root and shoot (153.4±8
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and 62.3±5 cm) was achieved with the fertiliser prepared of 10% of TPAD effluent and
90% of biochar. The improved macro and micronutrients in the fertilisers were observed,
contributing to the good seed germination results with the biochar loaded fertilisers.
The outcomes of the current research have contributed new knowledge and useful
experimental data on the utilisation of biochar to enhance H2 and CH4 production in TPAD
operation. The working mechanisms biochar involved in each phase of TPAD have been
proposed. The applications of biochar in pilot scale TPAD and as organic fertiliser for
agriculture applications have also been demonstrated.
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TABLE OF CONTENTS
THESIS DECLARATION ................................................................................................ i
ABSTRACT ..................................................................................................................... ii
TABLE OF CONTENTS ................................................................................................ vi
ACKNOWLEDGMENTS ............................................................................................. xix
AUTHORSHIP DECLARATION: CO-AUTHORED PUBLICATIONS .................. xxii
Chapter 1 Introduction ................................................................................................... 1
1.1 Background and motivation.................................................................. 1
1.2 Scope and aims .................................................................................... 3
1.3 Thesis structure .................................................................................... 3
Chapter 2 Literature Review ......................................................................................... 6
2.1 Introduction .......................................................................................... 6
2.2 Anaerobic digestion (AD) ................................................................... 6
2.2.1 Application of AD for waste treatment and energy utilisation ... 6
2.2.2 Fundamental principles of AD .................................................... 7
2.2.3 Drawbacks of conventional AD ................................................ 10
2.3 Two-phase anaerobic digestion (TPAD) ............................................ 11
2.3.1 Mechanisms and system of TPAD ............................................ 11
2.3.2 Factors influencing TPAD operation ........................................ 14
2.4 Biochar ............................................................................................... 21
2.4.1 Physical characteristics ............................................................. 22
2.4.2 Chemical characteristics ............................................................ 23
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2.4.3 Biological properties ................................................................. 24
2.5 The effect of biochar in the AD system .............................................. 25
2.5.1 Biochar application in biogas production .................................. 25
2.5.2 Biochar application in pilot scale operation of TPAD .............. 32
2.5.3 Organic fertiliser production from AD effluent and biochar .... 36
2.5.4 Biochar application in biogas cleaning ..................................... 38
2.6 Summary of literature review and specific research objecti .............. 43
Chapter 3 Methodology, Approach and Techniques ...................................................... 47
3.1 Overall research strategies .................................................................. 47
3.2 Bench scale experimentation of TPAD .............................................. 48
3.2.1 Materials .................................................................................... 48
3.2.2 Experimental set up ................................................................... 49
3.2.3 Experimental procedure ............................................................ 50
3.2.4 Analysis ..................................................................................... 58
3.3 TPAD Process Demonstration Unit (PDU) ........................................ 62
3.3.1 Principles of TPAD PDU .......................................................... 62
3.3.2 Experimental set up ................................................................... 63
3.3.3 System monitoring and control ................................................. 68
3.4 Preparation, characterisation and evaluation of biochar-
added organic fertiliser ....................................................................... 69
3.4.1 Experimental set up ................................................................... 69
3.4.2 Analysis ..................................................................................... 71
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3.5 Data analysis and modelling ............................................................ 72
3.5.1 Analysis of variant (ANOVA) .................................................. 72
3.5.2 The modified Gompertz Model ................................................. 72
3.5.3 Response surface methodology (RSM) ..................................... 72
Chapter 4 Effect of Biochar Addition on Hydrogen Production .................................... 74
4.1 Introduction ...................................................................................... 74
4.2 Hydrogen production without biochar ............................................. 74
4.3 Hydrogen production with biochar .................................................. 76
4.3.1 Response surface analysis ......................................................... 80
4.3.2 Hydrogen yield .......................................................................... 85
4.3.3 Hydrogen production rate .......................................................... 87
4.4 Effect of biochar on H2 production via anaerobic digestion
as compared to other solid additives: role of acidity ....................... 89
4.5 Mechanisms ..................................................................................... 95
4.6 Summary .......................................................................................... 98
Chapter 5 Effect of Biochar Addition on Methane Production .................................... 100
5.1 Introduction .................................................................................... 100
5.2 Methane production without biochar addition ............................... 100
5.3 Methane production with biochar addition .................................... 101
5.3.1 Response surface analysis ....................................................... 106
5.3.2 Methane yield .......................................................................... 108
5.3.3 Methane production rate .......................................................... 110
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5.4 Methane production in single phase anaerobic digestion ................ 111
5.5 Mechanisms ...................................................................................... 112
5.6 Summary .......................................................................................... 114
Chapter 6 Transient Performance during Start-up of Two-Phase Anaerobic Digestion
Process Demonstration Unit ........................................................................ 116
6.1 Introduction ...................................................................................... 116
6.2 The start-up performance of the first phase ..................................... 116
6.3 The start-up performance of the second phase ................................. 121
6.4 Summary .......................................................................................... 130
Chapter 7 Preparation, Characterisation and Evaluation of Biochar-loaded Organic
Fertiliser ....................................................................................................... 131
7.1 Introduction ...................................................................................... 131
7.2 Characteristics of biochar-loaded organic fertilisers ........................ 131
7.3 Soil less petri dish bioassay .............................................................. 137
7.4 Summary .......................................................................................... 142
Chapter 8 Evaluation and Practical Implications.......................................................... 143
8.1 Introduction ...................................................................................... 143
8.2 Integration of Experimental Findings .............................................. 143
8.3 Evaluation against the Specific Research Objectives ...................... 144
8.4 Evaluation against the Literature ..................................................... 145
8.4.1 Effect of Biochar Addition on Hydrogen Production .......... 145
8.4.2 Effect of Biochar Addition on Methane Production ............ 147
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8.4.3 Mechanisms of biochar in enhancing H2 and CH4 production in
TPAD.................................................................................... 148
8.4.4 Transient Performance during Start-up of TPAD PDU ....... 155
8.4.5 Preparation, Characterisation and Evaluation of Biochar-loaded
Organic Fertiliser .................................................................. 156
8.5 Practical Implications .................................................................... 159
Chapter 9 Conclusions and Recommendations ............................................................ 161
9.1 Introduction .................................................................................... 161
9.2 Conclusions .................................................................................... 161
9.2.1 Effect of Biochar Addition on Hydrogen Production in the
Bench Scale Experiment ...................................................... 161
9.2.2 Effect of Biochar Addition on Methane Production in the Bench
Scale Experiment .................................................................. 162
9.2.3 Transient Performance during Start-up of TPAD PDU ...... 163
9.2.4 Preparation, Characterisation and Evaluation of Biochar-loaded
Organic Fertiliser .................................................................. 163
9.3 Recommendations .......................................................................... 164
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LIST OF FIGURES
1.1 Thesis map ........................................................................................... 5
2.1 Steps of anaerobic digestion ................................................................. 8
2.2 Schematic diagrams of SPAD ............................................................ 10
2.3 Schematic diagrams of TPAD ............................................................ 12
2.4 The TPAD utilisation for combined heat and power (CHP)
system ................................................................................................. 14
2.5 The characteristics, possible functions and applications of
biochar ................................................................................................ 23
3.1 Research strategies ............................................................................ 47
3.2 Schematic of the experimental set-up (a) and bench scale
TPAD in the incubator (b) .................................................................. 50
3.3 Schematics of the experimental procedure for TPAD
employed in this study ....................................................................... 51
3.4 Schematics of the experimental procedure for second phase
TPAD ................................................................................................. 56
3.5 Schematic set up of water displacement method for gas
volume measurement ........................................................................ 59
3.6 A typical gas chromatogram of gas collected from the first
phase of TPAD .................................................................................. 60
3.7 Typical standard curves for (a) acetic and (b) butyric acids
analysis .............................................................................................. 61
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3.8 Schematic diagram of TPAD PDU of the Centre for Energy
of University of Western Australia .................................................. 63
3.9 Preparation of organic fertiliser from TPAD effluent and
biochar ............................................................................................ 69
3.10 Experimental set up of soil-less petri dish bioassay ........................ 70
4.1 Cumulative yields and production rates of H2 without
biochar addition ............................................................................... 74
4.2 Cumulative H2 yields at different biochar addition ratios ............... 76
4.3 VFA profiles during H2 production in culture with (a) 0; (b)
8.3; (c) 16.6; (d) 25.1 and (e) 33.3 g.L-1 biochar addition
ratios................................................................................................. 79
4.4 Response surface and contour plots of cumulative H2 yield
(YH) over 8 days of operation as a function of: (a) initial pH
and biochar addition ratio at 32C and (b) temperature and
biochar addition ratio at initial pH 6 ................................................ 86
4.5 Response surface and contour plots of maximum H2
production rate (RH) as function of: (a) initial pH and
biochar addition ratio at 32C; and (b) temperature and
biochar addition ratio at initial pH 6.4 ............................................. 88
4.6 The pH of liquid culture with the addition of the additives
before the pH adjustment ................................................................. 90
4.7 Production rates of H2 from cultures with different types of
additives ...............................................................................................
91
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4.8 Cumulative yields of H2 from cultures with addition of
different additives ............................................................................... 92
4.9 The pH evolution of the cultures with different types of
additives ............................................................................................. 93
4.10 The mechanisms of biochar in promoting H2 and CH4
productions in TPAD from food waste .............................................. 95
4.11 SEM images of (a) biochar and (b) final effluent of H2
production .......................................................................................... 96
5.1 Cumulative yields and production rates of CH4 without
biochar addition ................................................................................ 100
5.2 Cumulative CH4 yields at different biochar addition ratios ............. 102
5.3 VFA profiles during CH4 production in culture with (a) 0;
(b) 8.3; (c) 16.6; (d) 25.1 and (e) 33.3 g.L-1 biochar addition
ratios ................................................................................................. 104
5.4 Response surface and contour plots of CH4 yield (YM) as
function of: (a) biochar addition and temperature at initial
pH 7.8 and (b) biochar addition and initial pH at temperature
36.2ºC ............................................................................................... 109
5.5 Response surface and contour plots of maximum CH4
production rate (RM) as function of: (a) biochar addition and
temperature at initial pH 7.8 and (b) biochar addition and
initial pH at temperature 36.2ºC ....................................................... 110
5.6 SEM images of (a) the fresh biochar and (b) biochar after 40
days of incubation at 35ºC ............................................................... 113
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6.1 (a) Biogas production, (b) biogas composition, and (c) H2
and CH4 production during the start-up of the first phase ............. 117
6.2 VFA of the first phase .................................................................... 118
6.3 pH and temperature during the start-up of the first phase ............. 119
6.4 TS and VS of the first phase .......................................................... 120
6.5 (a) Biogas production, (b) biogas composition, and (c) H2
and CH4 production during the start-up of the second phase ........ 122
6.6 VFA of the second phase ............................................................... 123
6.7 pH and temperature during the start-up of the second phase ......... 125
6.8 TS and VS of the second phase ..................................................... 125
6.9 SEM images of a liquid sample taken from (a) R1 on day 18
and (b) R2 on day 77 of the start-up operation .............................. 127
7.1 Photographs of germination of rocket seed with various
organic fertilisers conducted in the soil-less petri dish
bioassay .......................................................................................... 138
7.2 The effect of various organic fertilisers on root length of
germinated rocket seed conducted in the soil-less petri dish
bioassay .......................................................................................... 140
7.3 The effect of various organic fertilisers on shoot length of
germinated rocket seed conducted in the soil-less petri dish
bioassay .......................................................................................... 141
7.4 The effect of various organic fertilisers on shoot to root ratio
of germinated rocket seed conducted in the soil-less petri
dish bioassay .................................................................................. 141
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8.1 A schematic representation of the mechanisms of the
working of biochar in promoting H2 and CH4 productions in
TPAD from food waste .................................................................... 149
8.2 H2 and CH4 production in pilot scale TPAD using different
feedstock .......................................................................................... 156
8.3 Germination index of petri dish bioassay using different
plants and fertilisers ........................................................................ 160
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LIST OF TABLES
2.1 The studies of the AD application for wastewater treatment
and energy generation ........................................................................ 7
2.2 Major reactions in acidogenesis step ................................................. 8
2.3 Major reactions in acetogenesis ......................................................... 9
2.4 Fuel Properties ................................................................................. 11
2.5 General reactions of H2 and CH4 generation in ............................... 12
2.6 Hydrogenases and methanogens in typical inoculums .................... 18
2.7 Studies on pH effect on anaerobic digestion ................................... 19
2.8 Optimum temperature range for microbial growth .......................... 20
2.9 Existing studies on the biochar utilisation in H2 and CH4
production via AD P ........................................................................ 28
2.10 Studies on the pilot scale operation of TPAD.................................. 35
2.11 Biogas impurities ............................................................................. 39
2.12 Study of biochar and carbon-based materials to remove gas
impurities ......................................................................................... 41
3.1 Characteristics of feedstock, inoculum and biochar ........................ 49
3.2 Central composite design for H2 production.................................... 53
3.3 The characteristics of the additives .................................................. 54
3.4 Parameters and levels of Box Behnken Design ............................... 57
3.5 Box Behnken design for CH4 production ........................................ 57
3.6 Start-up strategy of the first phase of TPAD PDU .......................... 66
xvii
3.7 Start-up strategy of the second phase of TPAD PDU ........................ 67
3.8 Fertiliser composition ......................................................................... 70
4.1 Profiles of the first phase of TPAD without biochar .......................... 75
4.2 One-way ANOVA and post hoc analysis on cumulative H2
production in different biochar addition ratios ................................... 77
4.3 The results of calculation using the modified Gompertz
fitting equation on H2 production with different biochar
addition ratios ..................................................................................... 78
4.4 Central composite design and experimental results for H2
production .......................................................................................... 81
4.5 ANOVA analysis and the fitting model for YH .................................. 82
4.6 ANOVA analysis and the fitting model for RH .................................. 83
4.7 Model validation results ..................................................................... 84
4.8 The results of calculation using the modified Gompertz
fitting equation on hydrogen production with different
additives ............................................................................................. 93
5.1 Profiles of the second phase of TPAD without biochar ................... 101
5.2 One-way ANOVA and post hoc analysis on cumulative CH4
production in different biochar addition ratios ................................. 102
5.3 The results of calculation using the modified Gompertz
fitting equation on CH4 production with different biochar
addition ratios ................................................................................... 103
xviii
5.4 Box Behnken design and experimental results for CH4
production ............................................................................................
106
5.5 ANOVA analysis and the fitting model for YM and RM ................ 108
6.1 Studies on the start-up of pilot scale TPAD PDU ......................... 129
7.1 Main characteristics and composition of the organic
fertiliser with different percentages of biochar addition ................ 133
7.2 The concentrations (mg.kg-1) of elements in the organic
fertiliser with different percentage of biochar addition ................. 137
7.3 Germination index (GI) as percentage of germinated seeds
in the assay to the control .............................................................. 139
8.1 A comparison of the current experimental results with the
literature data on the batch H2 production with the addition
of different types of biochar........................................................... 147
8.2 A comparison of the current experimental results with the
literature data on the batch CH4 production with the addition
of different types of biochar........................................................... 148
8.3 Profiles of biochar and proposed mechanisms in H2
production of different studies ...................................................... 153
8.4 Profiles of biochar and proposed mechanisms in CH4
production of different studies ....................................................... 154
8.5 Characteristics of AD effluent and biochar prepared as
organic fertiliser ............................................................................. 158
xix
ACKNOWLEDGMENTS
This study was undertaken at the Centre for Energy, University of Western Australia,
Perth, Western Australia. I wish to acknowledge everyone who was very supportive in my
journey to complete the PhD study.
I would first like to express my sincere gratitude to my principal supervisor, Dongke
Zhang. I sincerely thank him for taking me under his wing and providing as much as
possible facilities and supports for my research. I thank him for being so patient and
considerate during my study. His supervision is not only limited to the academic aspect
but also extended to life philosophy and art of educating. I have and continue to learn from
him as an excellent role model with an intelligent brain, strong character and commitment.
To my co-supervisor, Mingming Zhu, I cannot thank him enough for his patient and
valuable guidance throughout my course of study. He always trains me to think practically,
logically and critically. I will treasure these values and lessons in my heart and apply it in
my future career as a researcher and educator.
I would also acknowledge the financial support for this research provided by ARC
Linkage Projects Scheme (Project Number: LP100200137). I acknowledge Australian
Commonwealth Government through the Australia Awards Scholarship for providing my
PhD scholarship and University of Brawijaya, my home institution for the permission and
support.
xx
I am grateful for wonderful support by Debra Basanovic and Celia Seah, Krystina Haq for
assisting the thesis writing, Peta Clode and Lyn Kirilak (CMCA UWA) for helping with
SEM analysis and interpretations, and Ross Duffield (Woodman Point Wastewater
Treatment Plant) for providing the sludge inoculum.
I also extend my gratitude to Zhezi Zhang for the advice on the technical aspects of my
research, Yii Leng Chan and Carina Wang for always taking care of us. To Zhijian Wan,
Hendrix Setyawan, and Jesse Sheng, I thank them for helping with the technical issues
during the experiments and sharing general PhD experiences, struggle and jokes to help
me survived the journey. Isabelle Jones, for always being so generous with her time and
experience, helping me with the fertiliser characterisation, writing and preventing me from
burning the laboratory.
Special thanks to my fellow PhD candidate, Yusron Sugiarto; TPAD FYP students 2014-
2018, and Tian Zhang, for being very warm and supportive teammates and also for the
time we spent together inside and outside the TPAD PDU. To my labs-mates: Pengfei Liu,
Jorge Preciado, Herry Lesmana, Juan Zhang, Qian Zhang and Chimeika Okoye and all
CFE families, thank you for sharing everything from labs tools to your cultures. I also
thank to Emily, Tine, Juwita and Dina for multiple PhD sharing sessions, it meant a lot.
Also, my office mates in Room 1.96 for being good companies during our long days and
nights. Terima kasih, xiexie, muchas gracias. I also thank Ezmieralda Melissa, Sigit Pria
Perdana, Reliana Lumban Toruan, and other Indonesian students’ cohort 2014, Maroonah,
Warneds, Liqo Rania, Liqo Matilda Bay and AIPSSA for the friendship and always
making me feel at home.
Finally, I would also like to thank my husband, Gilang Agus Setiyono, for being the best
supporter and enjoying the roller coaster of the PhD journey together. It would have been
impossible for me to finish this work without his continuous support and encouragement.
xxi
I thank my mother, Ibu Tri Sayekti, my parents-in-law, my sisters and brothers for their
endless prayer, love and support so I can complete the work. And I dedicate this thesis to
my late Father, Totok Sunyoto.
xxii
AUTHORSHIP DECLARATION: CO-AUTHORED
PUBLICATIONS
This thesis contains work that has been [published and/or prepared for publication].
Details of the work:
Sunyoto, N. M., Zhu, M., Zhang, Z., & Zhang, D. (2016). Effect of biochar addition
on hydrogen and methane production in two-phase anaerobic digestion of aqueous
carbohydrates food waste. Bioresource Technology, 219, 29-36.
Location in thesis: Chapter 4
Student contribution to work:
• The work was supported by and part of ARC Linkage project grant held by Dongke
Zhang
• The candidate designed, conducted and analysed the results of the experiment with
assistance from Zhezi Zhang and Mingming Zhu, and under supervision of Dongke
Zhang
• The candidate drafted the manuscript and worked closely with Dongke Zhang and
Mingming Zhu to critically review it
Details of the work:
Sunyoto, N. M., Zhu, M., Zhang, Z., & Zhang, D. (2017). Effect of Biochar Addition
and Initial pH on Hydrogen Production from the First Phase of Two-Phase Anaerobic
Digestion of Carbohydrates Food Waste. Proceedings of the 8th International
Conference on Applied Energy, Beijing.
Location in thesis: Chapter 4
Student contribution to work:
• The work was supported by and part of ARC Linkage project grant held by Dongke
Zhang
• The candidate designed, conducted and analysed the results of the experiment with
assistance from Zhezi Zhang and Mingming Zhu, and under supervision of Dongke
Zhang
xxiii
• The candidate drafted the manuscript and worked closely with Dongke Zhang and
Mingming Zhu to critically review it
• The candidate presented the paper at the conference
Details of the work:
Sunyoto, N. M., Zhu, M., Zhang, Z., & Zhang, D. (2017). Effect of Biochar Addition
and Initial pH on Hydrogen Production from the First Phase of Two-Phase Anaerobic
Digestion of Carbohydrates Food Waste. Energy Procedia, 105, 379-384.
Location in thesis: Chapter 4
Student contribution to work:
• The work was supported by and part of ARC Linkage project grant held by Dongke
Zhang
• The candidate designed, conducted and analysed the results of the experiment with
assistance from Zhezi Zhang and Mingming Zhu, and under supervision of Dongke
Zhang
• The candidate drafted the manuscript and worked closely with Dongke Zhang and
Mingming Zhu to critically review it
Details of the work:
Sunyoto, N. M. S., Zhu, M., Zhang, Z., & Zhang, D. (2018). Effect of Biochar Addition
and Temperature on Hydrogen Production From the First Phase of Two-Phase
Anaerobic Digestion of Carbohydrates Food Waste. Proceedings of the 42nd
Clearwater Clean Energy Conference, Florida
Location in thesis: Chapter 4
Student contribution to work:
• The work was supported by and part of ARC Linkage project grant held by Dongke
Zhang
xxiv
• The candidate designed, conducted and analysed the results of the experiment with
assistance from Zhezi Zhang and Mingming Zhu, and under supervision of Dongke
Zhang
• The candidate drafted the manuscript and worked closely with Dongke Zhang and
Mingming Zhu to critically review it
Details of the work:
Sunyoto, N. M. S., Zhu, M., Zhang, Z., & Zhang, D. (2018). Effect of Biochar Addition
and Temperature on Hydrogen Production From the First Phase of Two-Phase
Anaerobic Digestion of Carbohydrates Food Waste. Journal of Energy Resources
Technology, 140(6), 062204.
Location in thesis: Chapter 4
Student contribution to work:
• The work was supported by and part of ARC Linkage project grant held by Dongke
Zhang
• The candidate designed, conducted and analysed the results of the experiment with
assistance from Zhezi Zhang and Mingming Zhu, and under supervision of Dongke
Zhang
• The candidate drafted the manuscript and worked closely with Dongke Zhang and
Mingming Zhu to critically review it
Details of the work:
Sunyoto, N., Zhu, M., Sugiarto, Y., & Zhang, D. (2018). Effect of biochar on hydrogen
production via anaerobic digestion as compared to other solid additives: Role of acidity.
Chemeca 2018, 165.
Location in thesis: Chapter 4
Student contribution to work:
• The work was supported by and part of ARC Linkage project grant held by Dongke
Zhang
xxv
• The candidate designed, conducted and analysed the results of the experiment with
assistance from Zhezi Zhang and Mingming Zhu, and under supervision of Dongke
Zhang
• The candidate drafted the manuscript and worked closely with Dongke Zhang and
Mingming Zhu to critically review it
• The candidate presented the paper at the conference
Details of the work:
Sunyoto, N. M. S., Zhu, M., Sugiarto, Y., & Zhang, D. (2018). Effect of Biochar
Addition, Initial pH and Temperature on Methane Production in Two Phase Anaerobic
Digestion of Carbohydrates Food Waste. Proceedings of the 43rd Clearwater Clean
Energy Conference, Florida.
Location in thesis: Chapter 5
Student contribution to work:
• The work was supported by and part of ARC Linkage project grant held by Dongke
Zhang
• The candidate designed, conducted and analysed the results of the experiment with
assistance from Zhezi Zhang and Mingming Zhu, and under supervision of Dongke
Zhang
• The candidate drafted the manuscript and worked closely with Dongke Zhang and
Mingming Zhu to critically review it
Details of the work:
Sunyoto, N., Zhu, M., Sugiarto, Y., & Zhang, D. Transient Performance during Start-
up of a Two-Phase Anaerobic Digestion Process Demonstration Unit Treating
Carbohydrate-rich Waste with Biochar Addition. International Journal of Hydrogen
Energy (Submitted – 2018)
Location in thesis: Chapter 6
Student contribution to work:
xxvi
• The work was supported by and part of ARC Linkage project grant held by Dongke
Zhang
• The candidate planned and designed the experiment under supervision of Dongke
Zhang and Mingming Zhu
• The candidate carried out the experiments, and analysed results with assistance
from Zhezi Zhang and Mingming Zhu
• The manuscript was drafted by the candidate and critically reviewed by Dongke
Zhang and Mingming Zhu
1
Chapter 1 Introduction
1.1 Background and motivation
Anaerobic digestion (AD) is a good means of putrescible organic waste treatment and utilisation
due to its ability both as a system to minimise the environmental effects of waste disposal and
to provide energy service. The AD has been widely applied to convert all types of putrescible
organic waste and wastewater to produce biogas. The AD is preferred over aerobic digestion
process because of its higher effectiveness to reduce high solids and organic materials in
wastewater and convert these materials into the valuable form of energy, the biogas (mainly
CH4), without emissions of harmful volatile organic compounds (VOC). It is also efficient due
to lower energy and smaller space requirement leads to lower total operating costs. It also brings
more economic benefits for its ability to generate biogas [1]. Although AD has been well
applied for some decades, several drawbacks need to be overcome; such as slow conversion
process, long retention time, sub-optimum process condition and low biogas quality [2, 3]. AD
produces typical biogas which has low energy density and less ideal as fuel for gas engines [4].
The UWA Centre for Energy has advocated a new concept of two-phase anaerobic digestion
(TPAD), which is an improvement over the conventional AD. TPAD is a system consisting of
two anaerobic digesters designed to separate hydrogen (H2) production in the first phase from
methane (CH4) production in the second phase. This separation allows the enrichment of
specific microbes and optimisation of operation condition in each reactor [5]. These
improvements increase the effectiveness of anaerobic process by enhancing biogas production
and improving the stability of the system [6, 7]. At the end of the process, a mixture of H2 and
CH4 are produced from TPAD. The presence of H2 in the otherwise low-quality biogas helps
to improve ignition quality thus better for gas engine applications. This system has been trialed
2
in several waste streams including dairy wastewater [8-10], food waste [11-13] and sewage
sludge [14, 15]. To improve the performance, TPAD requires further innovations.
Biochar, a carbon-rich material derived from the thermal decomposition of biomass, has
attracted significant attention due to its potential as an additional feedstock and an ideal packing
material for biofilm formation that enhance the production of biogas. A study conducted by
Mumme et al. (2014) report that biochar addition in single phase AD improves CH4 yield by
32% and prevent the system from mild ammonia inhibition [3]. There are also some studies
investigating the effects of operation conditions on biogas production and composition,
specifically in single-phase anaerobic digestion [16, 17] but not in TPAD. Therefore, it is
essential to study biogas production and composition in TPAD under different biochar addition
and operation conditions.
Typically, impurities such as CO2, H2S and NH3 are also produced during the AD. The
impurities lower the caloric content of biogas and cause utility corrosion. Therefore, attempts
to remove impurities from raw biogas are also needed. One promising method in the removal
of biogas impurities is adsorption using biochar [18]. Different types of biochar such as sewage
sludge [18], shell-derived, camphor-derived, bamboo, rice hull [19, 20] and leaf based [21] have
been used in the study of the removal of single stream biogas impurities such as H2S. However,
up to now, there are limited studies on the removal of biogas impurities especially in NH3 using
biochar. Biogas cleaning using biogas, therefore, is recommended for future studies.
AD generates high nutrient content-effluent as a by-product at the end of the process [22, 23],
thus can be used as an organic fertiliser. It has been studied in a wide range of plants, such as
tomato [24], wheat [25] and grassland [26]. Likewise, studies of biochar as fertiliser have been
conducted in several plants cultivation, for instance, soybean, tea tree, rice, cowpea wheat grass
and oats [27, 28]. However, the results of those studies vary and inconclusive, and only a few
of them examined the “synergistic effect” of the effluent of anaerobic digestion and biochar as
3
fertilisers for agriculture applications [29]. In this study, a method to prepare organic fertiliser
by treating the TPAD effluent with biochar is studied.
1.2 Scope and aims
The present PhD thesis research was aimed to investigate the utilisation of biochar in (1) the
first and second phase of batch TPAD, (2) start-up of TPAD process demonstration unit (PDU),
(3) preparation of biochar loaded organic fertiliser from TPAD effluent. The individual effects
of biochar addition ratio on the gas yield, gas production rate and metabolic products in each
phase of TPAD were investigated in a batch operation. The combined effects of biochar, pH
and temperature on the H2 and CH4 production were further studied and optimised.
The start-up TPAD PDU with the optimum biochar addition suggested from the batch scale
operation of TPAD was conducted. The biogas production, biogas composition, pH and
metabolic product during the start-up operation were investigated. The effluent of TPAD with
different addition percentage of biochar was used to prepare organic fertiliser. The prepared
fertilisers were characterised, and the effects on the seed germination were studied.
1.3 Thesis structure
A schematic map of the thesis is presented in Figure 1.1. There are eleven chapters in this thesis
as outlined below:
Chapter 1 TPAD as an innovation to improve biogas quality is introduced. Biochar
potentials to improve biogas production, purify biogas and prepare organic
fertiliser are identified. The scope of research, overall aims and thesis
structure are defined.
Chapter 2 Fundamental knowledge on AD and TPAD and existing studies of biochar
utilisation in the AD, biogas cleaning, and organic fertiliser preparation are
4
reviewed. Knowledge gaps are identified, and specific objectives of the
thesis are defined.
Chapter 3 To achieve the aims in Chapter 2, methodology, approach and technique
are determined. Experimental set up of bench scale and demonstration
operations of TPAD, biogas cleaning and organic fertiliser preparation are
described.
Chapter 4 The effects of biochar addition, initial pH and temperature on H2
production, composition, and metabolic products in the bench scale
experimentation are presented. The optimisation of each factor on the batch
operation of the first phase is suggested.
Chapter 5 The effects of biochar addition, initial pH and temperature on CH4
production, composition, and metabolic products in the bench scale
experimentation are presented. The optimisation of each factor on the batch
operation of the second phase is suggested.
Chapter 6 The operation of TPAD in a demonstration-scale unit is reported. Practical
considerations of the unit operation are outlined.
Chapter 7 The preparation of organic fertiliser from TPAD effluent and biochar is
described. The effect of each type of fertiliser on the seed germination is
explained.
Chapter 8 The results from the present work are integrated. The findings are evaluated
against the objectives and the literature data. The practical implications are
also identified.
Chapter 9 The new and significant findings are identified. The new knowledge gaps
are recommended.
5
Figure 1.1 Thesis map
6
Chapter 2 Literature Review
2.1 Introduction
Chapter 2 reviews the fundamentals and state of knowledge relating to AD and TPAD.
Specifically, this chapter emphasises on the biochar utilisation as the centre of the study. The
biochar characteristics, utilisation of biochar in the AD and TPAD, including pilot-scale
operation of TPAD, and organic fertiliser preparation are also reviewed. At the end of the
review, the gaps of studies are suggested and specific objectives of this study are appointed.
2.2 Anaerobic digestion (AD)
2.2.1 Application of AD for waste treatment and energy utilisation
Anaerobic digestion (AD) is a series of complex biochemical conversions of organic content in
a material carried out by different groups of microbes in the absence of oxygen [30]. AD serves
as a means to treat waste/wastewater because the process results in reduced solids and organic
content of materials as to achieve an approved quality to be discharged to the environment. AD
also plays a role to generate valuable forms of energy in the form of biogas [31]. Single phase
AD typically produces CH4 (65-77%), CO2 (19-50%), N2 (0-5%) of and trace gases (H2S: 3-
20,000 ppm; NH3:50-100 ppm) [32]. After removal of the CO2 and other trace gases, the biogas
is upgraded to a pipeline quality bio-CH4 and ready to be used as an engine and vehicle fuel
[32]. At the end of the process, the AD also recovers various chemicals and nutrients to produce
useful by-products, such as nitrogen, phosphorus, potassium, alcohol and volatile fatty acids
(VFA) [1, 30, 31, 33]. AD has been applied to a wide variety of putrescible organic feedstocks
for some decades, including lignocellulosic biomass, animal manure, sewage sludge, industrial
and food waste [34, 35]. Table 2.1 shows the report of previous studies using various waste and
wastewater as feedstock in AD and its effectiveness in treating the waste and producing biogas.
7
The listed studies suggest that the AD removes up to 81% of the volatile solids in the waste and
converts each gram volatile solids of the waste into 0.26 – 0.46 litre of CH4.
Table 2.1 The studies of the AD application for wastewater treatment and energy
generation
Feedstock VS removala (%) CH4 yield
(l CH4.gr VS-1)
Ref.
Lignocellulosic biomass (Zea
mays L.)
72 0.30 [36]
Chicken manure 81 0.43 [37]
Sewage sludge 42 0.27 [38]
Dairy industry waste N.A 0.26 [39]
Food waste N.A. 0.46 [40]
a VS removal: volatile solids removal
b COD removal: chemical oxygen demand removal
2.2.2 Fundamental principles of AD
According to the microbial activities and metabolic products generated, the AD consists of 4
major steps namely: hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Figure 2.1)
[41].
Hydrolysis
In the hydrolysis stage, complex materials such as carbohydrate, protein and fats are broken
down into simpler compounds/monomers (short-chain sugars, amino acids and peptides,
glycerine and fatty acids). Wide ranges of hydrolytic bacteria are involved in the hydrolysis
process, including Pseudomonas sp, Clostridia, Micrococci [42]. These hydrolytic bacteria
secrete extracellular hydrolysis enzymes, such as cellulose, amylases, proteases and lipase that
are required during the hydrolysis process. The general reaction on hydrolysis is as follow [42]:
8
C6H10O4 + 2H2O → C6H12O6 + 2H2 (R2.1)
Figure 2.1 Steps of anaerobic digestion [42-45]
Acidogenesis
Table 2.2 Major reactions in acidogenesis step [42]
Product Reaction
Ethanol C6H12O6 2CH3CH2OH + 2CO2 (R2.2)
Propionate C6H12O6 2CH3CH2COOH + 2H2O (R2.3)
Acetic acid C6H12O6 → 3CH3COOH (R2.4)
In acidogenesis stage, different anaerobic bacteria degrade the monomers into short-chain
organic acids, alcohols, H2S, H2 and CO2. The major reactions of acidogenesis are shown in
Table 2.2. The fermentative microorganism, such as Lactobacillus sp; Propionibacterium sp,
contributes a significant role in the reactions [43]. These microbes grow ten times faster than
methanogenic archaea, therefore acidogenesis is the fastest process in an AD. The optimum
value of pH for acidogenesis bacteria is the range of 5.2 and 6.5 [43].
Process Reaction Microbes
Carbohydrate Protein Lipid
Pseudomonas sp,
Clostridia, Micrococci
Hydrolysis
Acidogenesis
Sugars Amino acids Free long fatty
acids and glycerol Lactobacillus sp;
Propionibacterium sp
Volatile fatty
acids, alcohol Acetogenesis
Ammonia
Acetobacter sp;
Syntrobacter wolinii
Methanogenesis
Acetic acid Hydrogen, carbon
dioxide
Methane, carbon
dioxide
a Methanomicrobiales,
a Methanobacteriales
(b) (a)
9
Acetogenesis
In the acetogenesis step, the compounds formed are converted to acetate, formate, CO2 and H2
[30]. Acetogens, bacteria involving in acetogenesis, are slow growing, strictly anaerobic and
work optimally in pH around 6 [42]. Included in this group are Acetobacter sp and Syntrobacter
wolinii [42]. Common reactions in acetogenesis are listed in Table 2.3.
Table 2.3 Major reactions in acetogenesis [42]
Substrate Reaction
Acetate H3CH2COO- + 3H2O CH3COO- + H++ HCO3- + 3H2 (R2.5)
Glucose C6H12O6 + 2H2O 2CH3COOH + 2CO2 + 4H2 (R2.6)
Ethanol CH3CH2OH + 2H2O CH3COO- + 2H2 + H+ (R2.7)
Methanogenesis
In the methanogenesis phase, methanogens transform acetic acid and H2/CO2 to CH4 and CO2.
There are two major pathways for CH4, production namely hydrogenotrophic methanogenesis
and acetoclastic methanogenesis. The first pathway converts CO2 and H2 to CH4 in this
following equation:
CO2 + 4H2 → CH4 + 2H2O (R2.8)
Acetoclastic methanogenesis produces CH4 from acetate (R2.9).
CH3COOH → CH4 + CO2 (R2.9)
In the conventional AD operation, the four steps are conducted in a single reactor (Figure 2.2).
Therefore conventional AD is often referred to as a single phase AD (SPAD)[41]. The SPAD
converts prepared organic materials into methane-rich biogas. The biogas can be used as biofuel
10
after the purification [20, 46]. The remaining volatile fatty acids (VFA) and solids in the final
effluent are also generated [41].
Figure 2.2 Schematic diagrams of SPAD [41]
2.2.3 Drawbacks of conventional AD
Despite its effectivity in treating waste and producing biogas, previous studies report several
drawbacks of SPAD.
a. Sensitive to inhibitions of metabolic products
The overall operation of the AD is “occurred” in a single reactor [41]. Various biochemical
steps requiring different growing conditions are exposed to the same conditions. In the early
stages of SPAD, the accumulation of acid often decreases the pH, while the inhibitory level of
ammonia, long chain fatty acid and sulphide are increased significantly. These factors disrupt
the stability of the SPAD and may lead to reactor failure [47].
b. Slow rate of biological reactions
AD reactions run slowly, especially at a higher organic loading rate [41]. AD requires a longer
period to degrade high organic loading of feedstock. When the treating time is too short, AD
faces an imbalanced process [48]. A conventional AD treating industrial wastewater is reported
to be unfeasible at a loading rate of 2 gr VS.l-1.day-1 or higher and in the hydraulic retention
time 10 days or shorter.
11
c. Low-quality biogas
Table 2.4 Fuel Properties [4]
Fuels Energy density Heating value
(1 atm and 15C) (MJ/kg)
Flame speed (cm/s)
LPG 2.26 45.7 38.25
Natural gas 0.79 50 34
H2 0.08 120 275
Biogas 1.2 17 25
Biogas produced from SPAD has low energy density and the heating value and flame speed are
much lower than liquid petroleum gas (LPG), natural gas, and H2 (Table 2.4) [4]. Therefore, to
optimise the AD and improve biogas quality, innovative alternatives are required.
2.3 Two-phase anaerobic digestion (TPAD)
As an alternative to overcome the drawbacks of the SPAD, TPAD has been developed. TPAD
is a system proposed by Pohland and Ghosh in 1971 to treat high solid organic loading rate and
to produce higher CH4 [49]. TPAD employs separate stages for acidogenesis and
methanogenesis under different operating conditions. This section reviews the mechanism and
operating consideration.
2.3.1 Mechanisms and system of TPAD
Figure 2.3 shows TPAD system. Unlike SPAD, the two main reactions in the AD are separated
into two different vessels. The first phase of TPAD is also called as acidogenesis phase,
accommodates the hydrolysis, acidogenesis and acetogenesis which produce H2, CO2 and VFA.
Meanwhile, the second phase is also known as methanogenesis phase because the primary
reaction is CH4 generation which produces CH4 and CO2. General reactions of H2 and CH4
generation in TPAD are listed in Table 2.5. The Tables 2.2, 2.3 and 2.5 are meant to explain
12
the individual reaction that may occur during AD, although in the reality, the reactions are
complex and occur simultaneously.
Figure 2.3 Schematic diagrams of TPAD [41]
Table 2.5. General reactions of H2 and CH4 generation in TPAD [50]
Phase Reaction
First C6H12O6 + 2 H2O → 4 H2 + 2 CH3COOH +2 CO2 (R2.10)
Second 2 CH3COOH → 2 CH4 + 2 CO2 (R2.11)
TPAD allows the separation of acidogenesis and methanogenesis into two separate reactors,
based on their optimum operating conditions [51]. The independent control of both reactors
optimises the growth and activity of each group for bacteria involved in each step [7, 52].
Typically, the first phase is operated in a relatively shorter period (0.5-1.5 days) than the second
phase (≥ 3 days)[51]. It washes out the CH4-forming bacteria and removes the inhibitory effect
of CH4, thus higher production of H2 is achieved. The first reactor buffers the system by
avoiding a sudden pH drop caused by the accumulation of volatile fatty acid (VFA) which
typically hinders the methanogenesis phase and sometimes leads to reactor failure [49]. The
effluent from the first phase is then used as the feedstock for the second phase with more
suitable characteristics to enhance CH4 production. Therefore, the separation of the phases
improves the stability and resilience of the overall AD system [53].
13
The separation of both phases also improves the efficiency of solubilisation and saccharification
of the feedstock in the first phase of TPAD. The operation time is reduced significantly [52].
Ghosh et al. (1985) [48] proved that TPAD provides “superior performance” over the current
SPAD by comparing the two system in treating wastewater from the soft drink industry. In this
experiment, TPAD outweighs SPAD regarding CH4 production rate (seven times higher),
organic loading rates (OLR) (7.6 times higher) and hydraulic retention time (HRT) (almost a
half-time shorter).
The innovative aspect of the TPAD is the harvest of H2 from the first phase to be mixed with
the CH4 generated from the second phase to increase the overall quality of the biogas [41]. Liu
et al. (2013) state that the H2 present in biogas improves its fuel and heat efficiency by
enhancing flame speed, reducing combustion time and quenching distance, and extending the
flammability of CH4[52]. The H2 also increases the H/C ratio thus decreases greenhouse gas
(GHG) emission [52, 54]. Zhang et al. (2017) report that high efficiency and low emission can
be achieved when the biogas generated from TPAD was used in spark ignition (SI) engine [55].
Lee and Chung (2010) also evaluated that TPAD is more promising in recovering energy than
SPAD [56]. The energy recovery of TPAD outnumbers SPAD by around 18-20% [53, 57]. This
is mainly due to the flexibility in controlling the operation conditions of each reactor [57] and
the increased availability of readily available of VFA that enhances the CH4 production in the
second phase of TPAD [53]. TPAD also reduces ammonia inhibition thus reduces the gas
cleaning requirement [41, 58].
The TPAD principles can be applied in a remote area as an integrated system of TPAD, biogas
cleaning and conditioning (Figure 2.4). The food and agriculture waste are a potential feedstock
for TPAD to produce biogas for household and TPAD operation. The TPAD also produces an
increased nutrient availability in the effluent for organic fertiliser preparation for agricultural
application [41]. This scope of this thesis covers the study on TPAD operation, gas cleaning
unit and fertiliser preparation from TPAD effluent.
14
Figure 2.4 The TPAD utilisation for combined heat and power (CHP) system [41]
2.3.2 Factors influencing TPAD operation
Each phase of TPAD should be operated in appropriate conditions to produce an optimum
amount of biogas. This section elaborates factors affecting the operation of TPAD; including
nutrients, microbial sources, pH and temperature.
Nutrients
According to Weiland (2010) and Wellinger et al. (2013), biogas can be produced from any
biomass which is rich in carbohydrates, proteins, fats, cellulose and hemicellulose [58, 59].
These types of biomass are applicable as feedstock both for SPAD and TPAD. Wide ranges of
feedstock have been used in TPAD, such as food waste [56, 60], industrial wastewater [61-63],
animal manure [64] and agricultural waste [65-67].
The adequacy of nutrients in the feedstock should be evaluated to achieve a good productivity
of the AD. This evaluation of the gas potential of feedstock is suggested to be based on ratio
volatile solids (VS), Chemical Oxygen Demand (COD) or C/N ratio. The organic loading rate
(OLR) of the anaerobic system should be designed between 3.2 and 40 g VS.l-1.day-1[68, 69].
Some researchers suggested that ideal C/N ratio of feedstock is in the range of 15-32 [58, 59,
68]. In TPAD operation, the range of C/N for hydrolysis step is suggested to be 10-45 and for
CH4 production is 20-30 [59, 70].
15
In addition to macronutrients such as C, H, O and N, the micronutrients and trace elements such
as sodium (Na), magnesium (Mg), zinc (Zn), nickel (Ni) and iron (Fe) are also required by
microbes inside TPAD due to their significant role in enzyme synthesis and activity [71-74].
For example, Fe and Ni is reported to be important in hydrogenase synthesis for the hydrolysis
step [71, 74]. The addition of trace elements contained in additives such as granular activated
carbon, biochar and industrial Fe was reported to improve fatty acid consumption and biogas
production in AD [40, 72]. Other studies have claimed that in TPAD, the trace elements give a
more profound effect during methanogenesis than during acidogenesis [71, 75]. However, a
higher concentration of trace elements can cause AD inhibition. For example, it is reported that
the addition of more than 350 mg.L-1 of Na inhibited mesophilic methanogenesis. While the
optimum K addition is less than 400 mg.l-1 at both mesophilic and thermophilic AD [76].
Therefore, a study of dosage optimisation of different additives to enhance AD is crucial.
Microbes
The presence of microorganisms in TPAD is required to degrade feedstock and converts
nutrients into a valuable form of biogas and effluent with reduced solids and organic content.
There are two major groups of microbes involved in TPAD, namely hydrogenanses in the first
phase, and methanogens in the second phase [41]. The separation of the two groups of bacteria
is a central consideration in TPAD [41, 77]. Therefore the understanding of the characteristic
of each group of microorganisms is crucial [41].
The hydrogenases are also known as H2 producing bacteria (HPB). Clostridium and
Enterobacter are a common genera identified in H2 production [50, 78]. Clostridium is
characterised as rod-shaped, strictly anaerobic, and gram-positive. Enterobacter is also rod-
shaped, but facultative anaerobic and gram-negative [78].
Methanogens belong to the Archaea domain, which differs from bacteria by their genomic and
biological signatures [41, 77]. Methanogens are typically rod-shaped or coccoid-rod and strictly
16
anaerobic. Some methane producing microbes are H2 consuming and convert H2 to CH4
(hydrogenotrophic) [44, 77], while others are acetoclastic, producing CH4 from acetate [44, 45].
Ruggeri et al. (2015) suggest the principal difference between the HPB and H2 consuming
archaea [77]:
• HPB can grow in a broader pH range (4.5 - 7), while H2 consuming archaea have a more
limited pH range (7-8).
• The growth kinetics of HPB are faster than H2 consuming archaea.
• HPB can survive extreme conditions such as high temperature and extreme acidity or
alkalinity by forming endospores and germinating back in a favourable environment, while
H2 consuming archaea cannot.
Generally, two types of microbial culture are used in TPAD, namely pure culture and mixed
culture [41]. TPAD with pure culture only employs a single specific strain of microbes [50].
Mixed culture, on the other hand, consists of more than one group of bacteria which can be
found easily in the environment [41]. The mixed culture is typically obtained from AD sludge,
sewage sludge, wastewater treatment plant, compost and soil [41, 78]. The application of mixed
culture is often regarded to as more practical than pure culture as it is easier to operate and less
sensitive to changes on operating conditions. However, since mixed culture consists of H2
producing bacteria (HPB) and H2 consuming archaea, the first phase of TPAD requires selective
enrichment of HPB and the elimination of the H2 consuming archaea [78]. The methods to
selectively enrich the HPB include thermal treatment (heat shock at 80-110ºC for 20-60 mins),
use of chemicals (chloroform), aerobic stress, and kinetic selection (controlling pH, organic
loading rate /OLR and hydraulic retention time/HRT during the operation) [77, 78].
Several studies on TPAD used mixed culture obtained from different sources. The further
microbial studies identified the possible hydrogenases and methanogens involved in the first
17
and second phase of TPAD, respectively. Table 2.6 enlists the corresponding results of the
microbial identification studies.
Table 2.6 suggests the dominance of microbial groups mainly depends on the feedstock and
operating condition of AD. For example, the microbes from genus Lactobacillus are identified
in the first phase of TPAD treating food waste [79, 80]. Thermoanaerobacterium
thermosaccharolyticum, a typical thermophilic bacterium is enriched at the thermophilic first
phase [81]. The knowledge on the microbial profile of each stage of TPAD is useful for a better
process control to achieve optimum biogas production [82].
Several studies highlight low biogas production and process stability may be caused by the
sensitivity of microbes employed in AD [75, 83]. Reports suggest several methods to enrich
microbial growth and activity in AD, including through the molecular engineering of the
HPB[84] and the addition of microbial carrier to facilitate microbial immobilisation [83, 85].
Microbial immobilisation is reported to enhance H2 production rate and retain more microbes
in the reactor[84]. Studies on the suitability of additives for microbial immobilisation,
especially those of lower cost are required [83].
pH
An optimum pH is a crucial factor for AD operation [86] since it is directly correlated to
activities and metabolic pathways of the AD microorganism [41, 86]. During feedstock
degradation, acidic and alkaline compounds are produced. The equilibrium of these compounds
is a function of pH of the digester, therefore, the changes of these compounds will alter the pH
of the system [45].
18
Table 2.6 Hydrogenases and methanogens in typical inoculums
Feedstock Source of inoculum Operating conditions Identified microorganism Ref.
1st phase 2nd phase
Municipal
wastewater
AD sludge treating local
wastewater
Both reactors: Continuous
1st phase: pH 5.5/35ºC
2nd phase: pH 7.0/35ºC
Flavobacteriales, Clostriales Methanobacteriales,
Methanosarcinaceae
[82]
WAS and
OFMSW*)
Wastewater treatment
plant sludge
Both reactors: Semicontinuous
1st phase: pH: NA/55ºC
2nd phase: pH: NA/55ºC
Clostridium sp. Methanosaeta [87]
Food recycling
wastewater
AD sludge Both reactors: Continuous
1st phase: pH: no control/ 35ºC
2nd phase: pH: no control / 35ºC
Lactobacillus acetotolerans-
and Lactobacillus kefiri-like
organisms
Methanosarcina-like
organisms
[79]
Brown water
and food waste
Mesophilic wastewater
treatment plant sludge
Both reactors: Continuous
1st phase: pH: no control/ 35ºC
2nd phase: pH: no control / 35ºC
Firmicutes, Lactobacillus.
amylovorus and Acetobacter
peroxydans
Methanosaeta [80]
Palm oil mill
effluent (90ºC)
AD treating palm oil
waste sludge
Both reactors: batch
1st phase: pH: 5.5/ 55ºC
2nd phase: pH: 7.5 / 28-34ºC
Thermoanaerobacterium
thermosaccharolyticum
Methanoculleus
sp.
[88]
*) WAS and OFMSW: Waste activated sludge and organic fraction of municipal solid waste
19
Generally, pH stability of CH4 phase (6.7-7.4) is narrower than H2 phase (6.5-9.0) [89].
However, Cooney et al. [7] stated that the H2 phase is typically operated at lower pH (5-6). And
although it is common that methanogens are active between pH 6.7 to 7.4, some studies have
reported methanogenic activity at pH 5.8 [82, 90]. The inconsistency of the suggested optimum
pH has led to several studies to understand the effect of pH and determine the optimum pH in
the AD/TPAD using different feedstock (Table 2.7).
Table 2.7 Studies on pH Effect on Anaerobic Digestion
Feedstock AD System pH range Optimum pH Ref.
Glucose SPAD (H2) 4.00-7.00 5.50 [91]
Brewery wastewater SPAD (H2) 5.00-7.00 5.50 [92]
Food waste SPAD (H2) 4.70-7.00 5.30 [93]
Sugarcane bagasse hydrolysate SPAD (H2) 4.50-9.00 6.50 [94]
Swine manure and maize stalk SPAD (CH4) 6.00-8.00 6.81 [86]
Synthetic feed media TPAD 1st: 4.50-6.50
2nd: 6.00-7.70
1st: 5.50
2nd: 6.80
[95]
Kitchen waste TPAD 1st: 5.00-11.00 1st: 7.00 [96]
In conclusion, it seems that the optimum pH for H2 and CH4 production of the previous studies
were feedstock dependant. Moreover, it is also suggested by Ruggeri et al. (2015) that there is
no definite optimum pH value in fermentation due to other factors and environmental conditions
during the process, including the characteristics of feedstock [77]. In order to maintain the pH
of each reactor, several studies have applied methods such as water recirculation and the
addition of the alkaline additives (NaOH, lime, biochar) are often applied [41, 97, 98]. More
studies are required to investigate the efficiency of the methods to adjust the pH of the system
to enhance biogas production with minimum operation cost.
20
Temperature
Table 2.8 Optimum temperature range for microbial growth [45, 99-101]
Phase of AD Temperature
range
Genus Optimal temperature
(°C)
Acidogenesis Psycrophilic Rahnella 20
Mesophilic Bacillus 35
Thermotogales 35
Thermophilic Thermoanaerobacterium 60
Desulfotomaculum 55
Methanogenesis Psychrophilic Methanogenium 20
Mesophilic Methanococcus 35-40
Methanococcoides 30-35
Thermophilic Methanohalobium 50-55
Methanosarcina 50-55
Similar to pH, temperature is also found to be an essential factor in AD operation due to its
effect on the essential enzyme activity of bacteria. Generally, there are three temperature
regimes in fermentation operation, namely psychrophilic (5-25C), mesophilic (30-40C),
thermophilic (50-60C) and hyperthermophiles operation (> 65C). However, the mesophilic
and thermophilic operation is more common in AD operation [45, 77]. Normally, the first phase
is operated at thermophilic condition for enhanced substrate degradation while the second phase
is operated under mesophilic condition for higher CH4 production [41]. Higher temperature
may be beneficial for the reaction kinetics, but it is typically followed by a rapid pH decrease
which inhibits especially H2 production [102]. Appropriate temperature allows optimum
germination, acclimatisation of bacteria to substrate used in the system, carbon consumption
rate and partial pressure of the produced gas [41, 77]. Optimum temperatures for microbial
growth in H2 and CH4 production are listed in Table 2.8.
In TPAD operation, various results on temperature effect on system performance were found.
Using corn straw and pig manure in mesophilic and thermophilic condition, Yang et al. (2014)
21
proposed that mesophilic system is better than thermophilic TPAD because of the less energy
requirement with similar biogas production [103]. On the contrary, Kim et al. (2002) [104]
found that TPAD operation in both mesophilic and thermophilic temperature has no impact in
improving the performance of both systems. An interesting result was reported by Parawira et
al. (2007) [105]. When TPAD operated in mesophilic temperature, higher CH4 yield was
reached, while at the thermophilic conditions, shorter HRT and OLR were achieved. Therefore,
this study suggested operating TPAD according to the final aim of the system. If the higher CH4
yield is the purpose, the mesophilic condition is preferable, while the thermophilic condition is
appropriate for shorter digestion period and higher organic loading rate. The variation of the
optimum temperature may be caused by the origin of the inoculum and the type of feedstock
used [102].
To sum up, it is well known that there is different optimum temperature for H2 producing
bacteria and CH4 producing archaea. However, there are conflicting results on the optimum
temperature of each phase of TPAD treating different feedstock. Therefore, it is suggested to
investigate the effect of temperature on the TPAD operation especially when the study is
conducted using different feedstock.
2.4 Biochar
The existing literature suggests the need to improve the performance and biogas yields of
TPAD. Supplementation of micronutrients and trace elements is required to enhance enzyme
activation and improve microbial activity [106, 107]. The initiation of microbial colonisation
using supportive materials is also thought to enrich and increase the robustness of the microbial
groups under different operating conditions [83]. The addition of neutraliser materials to buffer
the pH of TPAD at the favourable range is needed [98]. One possible solution to address the
above-mentioned requirements is by utilising biochar in TPAD operation.
22
Biochar, also known as charcoal, is a carbon-rich solid residue produced from biomass
pyrolysis. A typical pyrolysis heats organic materials as feedstock at between 300 and 800°C
under very low or preferably no oxygen conditions [108, 109]. The process yields biochar, bio
oil and syngas [110]. In comparison with activated carbon (AC), biochar is more cost effective
[3, 111]. Unlike AC, biochar is neither pyrolysed at a very high temperature nor activated using
steam or carbon dioxide (CO2). Thus energy consumption and operation cost are lower than
that of AC [109]. Also, biochar is generally produced from low-cost and abundant materials
such as agricultural waste, sewage sludge, solid waste and animal droppings and wood chips
[108, 111].
It is well known that the origin and the operating condition of pyrolysis affect the physical,
chemical and biological characteristics of the biochar [27, 108, 112-114] (Figure 2.5).
2.4.1 Physical characteristics
The important physical characteristics of biochar are specific surface area (SSA) and porosity.
These characteristics are affected by different feedstock and operating condition of pyrolysis
[113, 115]. During the pyrolysis, the C mass is removed while the pores in material is created,
influencing the surface area of the biochar [27, 116]. The surface area of the wood increased to
ten to several hundreds of m2.g-1 after the pyrolysis [27]. In general, the structure of feedstock
also plays a role. For example, under the same highest treatment temperature (HTT), the biochar
produced from sewage sludge has lower SSA (38 m2.g-1) than that from sawdust (511 m2.g-1)
[113]. It implies that the lignin-rich biomass is an ideal feedstock to derive the biochar with
high SSA [111, 113].
Larger SSA reflects the higher porosity in the biochar [115]. The pores contribute to the high
adsorptive capacity of biochar for the small molecules such as common solvent and gases [27,
111, 117]. In addition, biochar offers suitable dimension, increased surface area and porosity
as an ideal habitat for bacteria, archaea and fungi [27, 111]. It is also suggested that the porosity
23
and structure of biochar may interact with soil structure therefore directly impacts the soil
structure [27].
Biochar
Physical
- high specific
surface area
- high porosity
- bulk density
CharacteristicsPossible
applications
Anaerobic
digestion
Biogas cleaning
Fertiliser
Possible
functions
Liming agent
Adsorbent
Microbial carrier
Micronutrient
additives
Chemical
- surface
functional groups
- high alkalinity
Biological
- provision of
nutrients
- high water
holding capacity
Soil conditioner
Figure 2.5 The characteristics, possible functions and applications of biochar
A related physical property of biochar is bulk density. Biochar produced from woody biomass
has 0.30 to 0.43 g.m-3 bulk density [27, 118]. It is in an ideal range of bulk density for gas
adsorption, which is suggested in between 0.40 and 0.50 g.cm-3 [27, 119].
2.4.2 Chemical characteristics
Pyrolysis alters the elemental compositions of the raw materials. Generally, after being charred,
the carbon (C) composition increases, while hydrogen (H), nitrogen (N) and oxygen (O)
decrease [111]. Changes in the surface chemical compositions also occur [111]. An abundance
of oxygen-containing functional groups, such as OH, COO and C=O in rice hull biochar
prepared at 400ºC has been reported [19]. At temperature higher than 600ºC, neutral or basic
aromatic groups were formed [109]. The formation of more functional groups (such as
24
carbonyl, carboxylate and hydroxyl) may contribute to the higher cation exchange capacity
(CEC) of biochar [111]. The CEC reflects the total capacity of biochar in adsorbing and
exchanging species with positive charge [114]. Various functional groups and surface charge
of biochar suggest a potential utilisation of biochar as an adsorbent [111]. However, further
study using various sorbates, compounds to be adsorbed, under different operating conditions
are required to understand the sorption behaviour of different types of biochar [27, 111].
The pH of biochar is strongly influenced by the organic functional group and ash content [113,
114]. Biochar produced from slow pyrolysis is alkaline, while most hydrochar produced at a
lower temperature of pyrolysis is slightly acidic [111, 115]. It is believed that the higher HTT
leads to more oxygenated functional groups being consumed and/or deprotonated to the
conjugate bases [114]. The addition of biochar into deionised water increases the pH of the
solution due to the leaching of alkali salts released from feedstock during the pyrolysis [111].
In an AD system, the alkalinity of biochar is believed to buffer the pH of the culture, improve
microbial activity and biogas production [98]. Biochar also adjusts the pH of soil when it is
used for soil amendment [27, 115]. In addition, the alkalinity of biochar also plays a role in gas
adsorption, especially for the removal of acid gases, such as H2S [120]. For example, in a study
conducted by Shang et al (2013), rice hull biochar with the highest pH value (10.6) performed
the highest H2S removal [19]. It is suggested that the removal of H2S is governed by the local
pH in the pore system. Alkaline pH is reported to enhance the dissociation of H2S and its
oxidation to sulphur [19].
2.4.3 Biological properties
Biochar provides an ideal habitat for bacteria, archaea and fungi because of its high surface area
[27]. The high porosity of biochar also allows the biochar to retain moisture. A study by
Pietikainen et al. (2000) suggests that biochar produced from humus and wood has a higher
water holding capacity (WHC) (2.9 ml.gr-1) than AC (1.5 ml.gr-1) and pumice (1 ml.gr-1) [27,
121]. The increased amount of water retained by the biochar and soil may improve the
25
habitability for soil microorganisms. An increase in WHC of biochar improves the overall soil
WHC when it is applied as a soil amendment agent. These characteristics are important for
utilisation of biochar in biological applications, such as fertiliser and as a microbial carrier.
In addition, remaining bio-oils and volatile matters, including water-soluble compounds such
as sugars, alcohols, acids, aldehydes, and ketones, in the biochar surface after pyrolysis may be
utilised by microbes. Producing biochar at the lower temperature is reported to be better for
retaining nutrients for microbes [3, 113]. Some nutrients such as K, P, Na, Mg and Ca also
remained [114]. The provision of the macro and micronutrients in the biochar are expected to
be useful for enhancing the biogas production and supporting plant growth when biochar is
used for organic fertiliser [29, 114, 122].
To sum up, biochar has beneficial physicochemical and biological characteristics that may be
useful for applications in AD, biogas cleaning and soil amendment [20, 28, 108, 123] (Figure
2.5). However, some compounds associated with biochar like polycyclic aromatic hydrocarbon
(PAH), formaldehyde and other toxic compounds may be inhibitive for microorganisms.
Therefore, detailed studies on the utilisation of biochar on the above mentioned applications
are required [111].
2.5 The effect of biochar in the AD system
Biochar with its aforementioned beneficial characteristics for a wide range of applications
therefore becomes an innovative aspect of this study. This thesis investigates the utilisation of
biochar in the (1) TPAD, both bench and demonstration scale, (2) the gas cleaning and (3)
preparation of fertiliser from the TPAD effluent (see Figure 2.3). This section reviews the
existing literature on the utilisation of biochar on these three applications.
2.5.1 Biochar application in biogas production
This section reviews the existing studies on biochar utilisation in AD and the possible
mechanisms by which it enhances biogas production. SSA, nutrient content and pH of biochar
26
has been proposed as useful for H2 production via an AD [3, 113]. It is also reported that biochar
addition in single phase AD prevents the system from mild ammonia inhibition and improves
CH4 yield by 32% [3]. Several studies investigated the effect of biochar on H2 or CH4
production via AD (Table 2.8).
As can be seen from Table 2.9, more studies of the biochar addition were conducted for CH4
production, while only a few studies have investigated the H2 production and TPAD operation.
The existing literature suggests possible mechanisms for the role of biochar in improving AD,
although some studies state that the mechanisms are still unclear. The possible mechanisms by
which biochar in enhances H2 and CH4 are elaborated as follows.
1. Bacterial immobilisation
Bacterial immobilisation is an important strategy in the AD. Bacterial immobilisation is the
attachment of bacteria to the surface of the solid material to establish microbial colonisation. In
the context of bacterial immobilisation, additives are often referred to as supporting material or
microbial carrier. Bacterial immobilisation benefits AD in two ways. Firstly, a microbial carrier
provides an increased available surface for microbial growth. The increased surface allows
more intense cross feeding, co-metabolism and H2 and proton transfer, which then enriches
microbial growth and initiates microbial colonisation [124]. The enriched microbial population
has a greater potential for a higher biogas production. Secondly, microbial immobilisation
increases the robustness of the AD system. With microbes attached to a supporting material,
the AD system has a lower probability of experiencing microbial washout, where the system
cannot retain the microbes when the AD effluent is discharged. The use of a microbial carrier
will better maintain microbes inside the reactor thus improving the robustness of AD. This then
allows the system to operate with shorter hydraulic retention time (HRT) and higher organic
loading rate (OLR) [125].
27
One natural process of bacterial immobilisation is biofilm formation which allows the
colonisation of bacteria, fungi and protozoa on polymerised solid surfaces [123, 126]. The
biofilm formation rate depends on the characteristics of the microbial carrier, including pore
size, SSA, porosity and surface roughness [125, 127].
Luo et al. studied the effect of particle size (75-150 µm, 0.5-1 mm and 2-5 mm) of fruitwoods
biochar on the CH4 production from synthetic wastewater. The result shows that biochar
addition enriched Archea, achieving a higher amount than that of control without biochar.
Biofilm formation is known to depend on the particle size. Biochar selectively enriches the
Archea by tightly binding Methanosarcina in the larger biochar particles, loosely binding
Methanosaeta in all particle sizes and tightly binding it in smaller particle sizes [83].
28
Table 2.9 Existing studies on the biochar utilisation in H2 and CH4 production via AD
System Feedstock Type of biochar Optimum biochar
addition (g.L-1)
Operating
conditions
H2 yield
(mL.g VS-1)
CH4 yield
(mL.g VS-1)
Ref.
SPAD (H2) Glucose Corn bran
residue
0.6 Batch 37ºC
Initial pH: 7
204 mg H2.g
glucose-1)
- [73]
SPAD (H2) OFMSW Woody mass 12.5 Batch 37ºC
Initial pH: 5.5
80 - [128]
SPAD (H2) DAS* and food
waste
Saw dust 10 Batch 35ºC
Initial pH: 5.5
81 - [113]
SPAD (CH4) AD sludge Paper sludge
and wheat husk
20 Batch 42ºC
Initial pH: N.A*
- 158 [3]
SPAD (CH4) Glucose Fruit woods 10 Batch 35ºC
Initial pH: 7
- 13.7 mmol CH4.g
glucose-1
[83]
SPAD (CH4) Glucose + 7 g-
N.L-1
Fruit woods 10 Batch 35ºC
Initial pH: 7
- 16.7 mmol CH4.g
glucose-1
[129]
SPAD (CH4) Citrus peel Coconut shell N.A Batch 35ºC
Initial pH: 7
- 186 [130]
SPAD (CH4) Food waste Fruit woods 5 Batch 35ºC
Initial pH: 7
- 500 [131]
SPAD (CH4) OFMSW Rice straw 5 Batch 35ºC
Initial pH: N.A*
- 167 [132]
SPAD (CH4) Kitchen waste Vermi-compost 50 Batch 35ºC - 175±5 [98]
29
System Feedstock Type of biochar Optimum biochar
addition (g.L-1)
Operating
conditions
H2 yield
(mL.g VS-1)
CH4 yield
(mL.g VS-1)
Ref.
Initial pH: 6.5
SPAD (CH4) Dairy manure Dairy manure 10 Batch 35ºC
Initial pH: 7.7
- 467 [133]
TPAD (CH4) Primary sludge
and WAS
Corn stover 0.25 – 1 g.day-1 Semicontinuous
Batch 55ºC
1st phase: 6.5
2nd phase:
uncontrolled
- 340 [134]
*DAS = dewatered activated sludge
NA = not available
30
Sharma et al (2017) also reported that woody mass-biochar initiated biofilm formation in H2
production from OFMSW using co-culture of Enterobacter aerogenes and Escheria coli [128].
It contributed to the shorter lag phase, waiting period for the system to produce H2, by 25%
compared to control and improved H2 production rate [128].
2. Source of macro- and micro-nutrients
Biochar contains a stable and labile fraction of carbon. Previous studies report that the presence
of a labile fraction of biochar can be used by AD microbes to kick-start the production of biogas
[3]. Especially in the biochar produced at the low temperature (~200°C), often referred to as
hydrochar, there is the readily digestible carbon such as sugar and volatile matters remaining
after the pyrolysis. This portion of carbon is reported to be useful for CH4 production and
improved the production by 32% compared with the control [3]. Jang et al. (2018) reported that
the nutrients in dairy manure-derived biochar (Ca ~ 9.1%; Mg 3.6%; N ~ 1.3% and P ~ 0.14%)
supported the CH4 production of dairy manure at three different temperature regimes tested,
namely psychrophilic, mesophilic and thermophilic condition by 28, 32 and 36% compared to
the control [133].
3. Buffer the pH of the AD system
H2 production is a pH-dependent process. The pH influences the microbial activity and
activation of H2 producing-enzymes. The addition of biochar into the AD system is believed to
buffer the pH of the culture, enhancing the production of biogas. Zhang et al. (2017) reported
the buffering effect of biochar in AD to produce H2. The study used biochar prepared from corn
barn residue (pH 8.92) to produce H2 using glucose as the substrate [73]. In the study, the culture
containing biochar at 0.6 g.L-1 produced higher H2 than the control by 29% with a maintained
higher final pH than the control.
31
Wang et al. (2018) also studied the effects of different feedstock and preparation temperature
of biochar on H2 production. The results suggested that among other factors, the pH buffering
capacity served as the primary factor in improving H2 production to mitigate the pH drop as a
result of VFA accumulation during the process. Biochar contains alkaline compounds (-COOH
and -OH) that may neutralise and act as a buffer when the culture’s pH drops as a result of VFA
accumulation during the AD process [123]. The buffered pH provides a better environment for
microbial growth and activity thus allowing the culture to produce higher H2 [73, 113].
Using a type of biochar derived from vermicompost (VCBC), Wang et al. (2017) investigated
the capacity of biochar in buffering the pH and alleviating the adverse effect of acid
accumulation in the AD system producing CH4 [98]. The results show that the addition of
VCBC improved the buffering capacity to acetic, butyric, propionic and valeric acid. The metal
alkaline content of VCBC (Na and K) and alkaline-earth metals (Ca and Mg) are proposed to
be responsible for the improved buffering capacity, following equation (R2.12).
Ca(Mg)CO3 + CxHyCOOH [CxHyCOO]2Ca(Mg) + H20 + CO2 (R2.12)
The addition of VCBC supported the culture in SPAD with a high loading of chicken manure
(CM) (50 g TS.kg-1) by initiating the biogas production 10 days faster and preventing the system
from rapid pH drop during the operation [98].
On the other hand, Luo et al. (2015) claimed that the role of buffering capacity of biochar is
insignificant in a CH4 production system [83]. Instead, the porosity of biochar which served as
a microbial carrier is thought to be the central role of biochar in CH4 production [83]. Therefore,
it is believed that biochar enhances the H2 and CH4 production through a different mechanism.
Thus, additional experiments using different feedstock, biochar type and operating conditions
are required to investigate the possible mechanisms of biochar effects in AD.
32
4. Adsorption of inhibitors
There is an indication that carbonaceous materials, such as biochar, have a potential to be used
as an adsorbent to remove contaminants [123]. The pores in biochar exhibit an adsorptive
capacity to remove compounds, such as ammonium [128]. Biochar is suggested to adsorb both
ionic and organic compounds by electrostatic forces and Van der Waals forces, respectively.
These mechanisms are similar to those of AC and zeolite, however, biochar is relatively cheaper
than these additives [123, 128].
Sharma et al. (2017) studied the effect of biochar addition on the different addition of
ammonium to investigate the ability of biochar to adsorb ammonium in AD producing H2 from
the organic fraction of municipal solid waste (OFMSW)[128]. At a low concentration, ammonia
is required by microbes for microbial growth; however, it may be harmful at a higher
concentration. The results show that the addition of biochar (2.5 – 35 g.L-1) in the system
mitigated the ammonium inhibition which allowed the culture to produce a higher amount of
H2 than the control by up to four-fold. It is believed that the ability of biochar to adsorb
ammonium ions from matrices which accounts for the improved performance of the culture
with biochar addition [128]. Biochar also has a significant effect in mitigating ammonium
inhibition in a CH4 producing reactor. Mumme et al. (2014) report that the addition of biochar
mitigated the mild ammonia inhibition (up to 500 mg N.kg-1) [3].
2.5.2 Biochar application in pilot scale operation of TPAD
A study on the pilot scale operation of TPAD is necessary to confirm the results of the
bench/laboratory studies [135] and to investigate the technical challenges and the related trouble
shooting on a real full-scale application [56]. An initial study has been done in a demonstration
scale unit developed by the Centre for Energy of the University of Western Australia. The unit
consists of two reactors; the first reactor is an H2 producing reactor with the capacity of 150 L,
while the second reactor accommodates CH4 generation with 500 L of total volume. Prior to
the operation, commissioning and trial runs were conducted [41]. Several studies also focused
33
on the pilot scale operation of co-production of H2 and CH4 via TPAD using different types of
feedstock (Table 2.10).
Most of the studies were conducted in a range of 0.2 – 5 m3 capacity for H2 production and
larger reactor capacity for CH4 production phase (0.76 – 50 m3). Some studies focused on the
sole CH4 production [135-137], while others also took H2 production into account depending
on the scope of the study.
Despite the success in some pilot scale trials, several technical aspects are required to be
considered before and during the operation of the pilot scale TPAD. Cavinato et al. (2012)
reported that the short HRT and high OLR were effective in eliminating the methanogens from
the first phase reactor [138]. However, too high OLR may lead to the reactor shock load and
microbial wash out [85]. In this condition, it is advisable to allow some period for the adaptation
to the increased OLR, achieving a normal H2 production [56]. In some cases, the pH of the
culture of each reactor has to be automatically adjusted at the optimum pH, being at 5.5 and 7.0
-7.5 in the first and second phase, respectively [56]. Effluent recirculation from the second
reactor into the first reactor was also suggested as an alternative to increasing the pH of the first
reactor [138]. However, the methanogenic contamination and ammonium accumulation will be
the consequences [97]. Therefore, other solutions are required. The addition of biochar into the
system is one alternative to overcome the problems.
34
Table 2.10 Studies on the pilot scale operation of TPAD
Feedstock Operating conditions Gas production
Ref. 1st reactor 2nd reactor H2 CH4
Kitchen waste and
fruit/vegetable
waste
• V: 2.0 m3
• pH: N.A.
• T: 35ºC
• OLR: 2 – 10 g (VS)/L/day
• HRT: 10 days
• V: 4.0 m3
• pH: N.A.
• T: 35ºC
• OLR: 1 – 5 g (VS)/L/day
• HRT: 20 days
• Composition (%):
N.A
• Yield:
N.A
• Composition (%):
65 - 67
• Yield: 0.46 – 0.64 L/g
VS
[135]
Solid municipal
waste
• V: 0.5 m3
• pH: 5.3±0.2
• T: 33±4ºC
• OLR: 12.3 – 71.3 g-
COD/L/h
• HRT: 21 – 66 hours
• V: 2.3 m3
• pH: 7.4±0.3
• T: 36±4ºC
• OLR: 2.7 – 6.4 g
(VS)/L/day
• HRT: 3.9 – 6.4 days
• Composition (%):
60
• Yield:
1.8 – 2.2 L/L/day
• Composition (%):
60
• Yield:
4.6 – 5.4 L/L/day
[56]
Biowaste • V: 0.2 m3
• pH: 3.5 – 5.4
• T: 55ºC
• OLR: 16 – 21 g (VS)/L/day
• HRT: 3.3 – 6.6 days
• V: 0.76 m3
• pH: 7.6 – 8.2
• T: 55ºC
• OLR: 4 – 10 g (VS)/L/day
• HRT: 12.6 days
• Composition (%):
19 - 37
• Yield:
3 - 51 L/kg VS
• Composition (%):
60 - 65
• Yield:
377 - 410 L/kg VS
[139]
35
Feedstock Operating conditions Gas production
Ref. 1st reactor 2nd reactor H2 CH4
Food waste • V: 0.2 m3
• pH: 5.7±0.3
• T: 55ºC
• OLR: 16.8 g (TVS)/L/day
• HRT: 3.3 days
• V: 0.76 m3
• pH: 7.6 – 8.2
• T: 55ºC
• OLR: 1.3 – 4.8 g
(TVS)/L/day
• HRT: 12.6 days
• Composition (%):
38.5±9.7
• Yield:
0.067 m3/kg VS
• Composition (%):
67±3.7
• Yield:
0.48 m3/kg VS
[138]
Food waste • V: 0.2 m3
• pH: 4.6±0.3
• T: 55ºC
• OLR: 3.5 g (TVS)/L/day
• HRT: 20 days
• V: 0.76 m3
• pH: 8.0±0.1
• T: 55ºC
• OLR: 3.5 g (TVS)/L/day
• HRT: 20 days
• Composition (%):
N.A
• Yield:
N.A
• Composition (%):
55.2
• Yield:
0.55 m3/kg VS
[137]
Food waste
leachate
• V: 5.5 m3
• pH:
• T: 38.7 – 42.8 ºC
• OLR: 2.18 – 2.45
• HRT: 3 days
• V: 50 m3
• pH: 7.3
• T: 35.6 – 37.7 ºC
• OLR: 2.36 kg VS/m3/day
• HRT: 27 days
• Composition (%):
N.A
• Yield:
N.A
• Composition (%):
57 - 65
• Yield:
0.39 – 0.85 Nm3/kg
VSremoved
[136]
36
As explained, alkalinity of biochar contributes in buffering the pH of the system and alleviating
the effect of VFA accumulation as a result of high OLR and short HRT [98, 113]. Also it acts
as a microbial carrier which immobilises the microbes and prevents microbial wash out [83, 85,
140].
Despite growing interest on biochar utilisation in AD, the study on biochar addition in
demonstration or full-scale operation is rare. Cooney et al. (2015) investigated the start-up
period of CH4 production using 1.5 m3 demonstration scale of SPAD with the addition of
biochar. A successful start-up was achieved after 59 days, showing a COD removal of 68% and
CH4 composition of more than 60%. The AD almost achieved the maximum value of theoretical
methane production per kilogram of COD reduced [140]. There has been limited investigation
on the application of biochar in a biogas demonstration scale unit, so more studies are required.
In particular, a study of biochar application in a demonstration scale TPAD has not been
explored to date.
2.5.3 Organic fertiliser production from AD effluent and biochar
At the end of the AD process, a final effluent is generated. AD effluent (also called as digestate,
biogas residues, or biogas slurry) is an attractive material for soil improvement and restoration
due to its organic materials content [141]. This effluent contains nutrients useful for plant
growth [22, 23]. Generally, AD effluent contains 5-7% N, 30-50% P and 70-100% K required
in the first year of the growth of pasture plant [142]. Therefore, AD effluent can be used as an
organic fertiliser to improve soil fertility [143]. Several studies have investigated the utilisation
of AD effluent as fertiliser. Alburquerque et al. (2012) found that two types of AD effluent
produced from (1) combined pig manure and slaughterhouse waste effluent and (2) combined
cattle manure and maize-oat silage effluent positively affected lettuce seed germination. It can
37
be categorised to have a growth stimulant attributes as they exceeded a threshold value of 125%
of germination index (GI) (of the control) [144].
Similarly, biochar also has many beneficial characteristics, for example, high SSA, ability to
improve pH, high WHC, and good affinity for micro and macronutrients for plant growth [29,
122]. These characteristics enable biochar to impact soil fertility by increasing the content of
carbon (C), nitrogen (N) and aggregate stability as well as providing a beneficial environment
for microbes and bioremediation of soil. Also, biochar has a relatively lower cost than other
materials, estimated being 10 times cheaper than AC (2 USD/kg) [141]. Solaiman et al. (2013)
reported a positive effect of adding appropriate biochar on germination and early growth of
several plant seeds. The study used a soil-less petri-dish assay to investigate the potential
toxicity of lignin based-biochar (Oil Mallee, Rice husk, New Jarrah, Old Jarrah, Wheat Chaff)
on wheat, mung bean and subterranean clover seed germination. All biochar addition generally
increased the root length especially in the additional dose of 10, 20 and 50 t/ha. At the 100 t/ha,
biochar addition showed adverse or no significant effect on the root length. It is probably due
to the inhibitive effect of trace elements in the biochar when it is applied above the acceptable
agronomic rate [28].
Another positive effect of biochar on plant growth is also reported by Zhang et al. (2017). In
the study, sulphur-enriched AD-sludge biochar (SulfaChar) generated from biogas cleaning unit
was used. Compared to the control (synthetic S fertiliser), sulphur- enriched biochar achieved
a marked increase of plant biomass ranging from 31 – 49% in Zea mays L after 90-day of
greenhouse study. The study proposed that the SulfaChar may supply the macro (N,
phosphorus/P, potassium/K, calcium/Ca and magnesium/Mg) and micronutrients (zinc/Zn,
manganese/Mn and boron/B) or promoted the uptake of those nutrients [145].
Combining AD effluent and biochar for organic fertiliser may improve their fertiliser properties
and reduce the nutrient leaching from AD effluent [141, 146]. A willow biochar is proven to
38
reduce the P and K leaching from the soil, reduce the toxicity of sewage sludge and simulate
the growth of the tested organisms. The fertilising characteristic is shown by an increased root
growth of grass cress (L. sativum) when the rate of biochar addition increased [147].
On the other hand, Sun et al. (2014) reported that biochar (hickory wood, bagasse and bamboo
prepared at 200, 300, 450 and 600ºC) had an insignificant effect on the germination of brown
top millet seed. The hickory wood hydrochar (prepared at 200ºC) even had a lower germination
rate (45%) compared to the control (78%). It is estimated that the acidic pH of the hydrochar
(pH 5.3) inhibited the seed generation. Note that hydrochar is produced at a lower temperature
of pyrolysis [113]. The biochar dose and plant species were also believed to influence the result
[115]. Cardelli et al. (2018) also reported the fertiliser prepared from AD effluent and biochar
limited the amount and activity of the microbial in the soil, probably because the biochar
reduced the soluble organic compounds (dissolved organic C - DOC and phenols)[148].
In conclusion, the results of studies on the utilisation of AD effluent and biochar for fertiliser
are varied. Therefore, further studies are required.
2.5.4 Biochar application in biogas cleaning
During the AD, trace gases are produced during the H2 and CH4 production. These biogas
impurities, which generally consist of CO2, water vapour, hydrogen sulphide (H2S) and
ammonia (NH3) [149] limit the utilisation of CH4-H2 mixture as biofuel. For example, the water,
H2S and NH3 lead to the corrosion of utilities and CO2 caused a decrease in biogas caloric
content [19, 149, 150]. The types, quantity in biogas and possible effects of each impurity are
listed in Table 2.11.
The removal of H2S from biogas must be undertaken to achieve the H2S standard limit for safety
and fuel application concerns. Safe work Australia suggested 10 and 15 ppm as the 8 hours
average and short-term exposure limits for H2S, respectively. Biogas-fuelled generator engines
39
can tolerate to 200 ppm of H2S, while the H2S concentration limit for biogas boiler is 1000 ppm
[151]. The presence of NH3, which is typically generated in biogas from the nitrogen-containing
feedstock, can vary from a few hundred to 30,000 ppm. The NH3 may provoke NOx emissions
when the biogas is used as fuel in turbines, gas engines and burners [46]. Therefore, before the
utilisation of biogas as a fuel for engine operation requires the gas impurities to be removed.
Table 2.11 Biogas impurities [149, 152, 153]
Component Composition in
Biogas
Possible Effects
CO2 15-60% - Decreases caloric value
- If the gas is wet, it causes corrosion
H2S 0-2% - Provokes corrosion
- Generates SO2 emission in case of imperfect
combustion
- Spoils catalysts
NH3 <1% - Causes corrosion when dissolved in water
- NOx emission
- Increases anti-knock properties of engines
Water vapour 1-10% - Causes corrosion of facilities
- Causes water condensation
- Rises risk of freezing of piping and nozzles
Dust >5 µm - Interfere nozzles
N2 0-5% by volume - Decrease calorific value
- Improve engine’s anti-knock properties
Siloxanes 0-50 mg Nm-3 - Causes engine failure
There are several well-known methods to remove H2S from biogas. Among them are
adsorption, wet scrubbing, absorption with liquids, membrane separation, selective catalytic
oxidation and biological filtration. While for removing NH3, washing using diluted nitric and
sulfuric acid is a common method. Each of the methods has its own merits and limitations.
Chemical processes may generate hazardous compounds while biological processes can be slow
and more sensitive to operating conditions. Therefore, physical treatment such as adsorption is
40
an alternative method. The utilisation of a gas adsorption unit filled with carbon-based materials
is also suggested to remove both H2S and NH3 [21]. The replacement rather than regeneration
of these materials is suggested because of its their low cost and possible utilisation to prepare
sulphur-rich fertiliser [145, 149].
One promising material that can be used in biogas impurities removal is biochar [18]. Biochar
has been extensively used in the study of the removal of single stream biogas impurities such
as H2S both during [3] and after anaerobic digestion [18-20]. Biochar has an excellent adsorbing
ability to remove organic contaminants from soil and is also reported to be a potential gas
adsorbent [19, 20, 145].
Biochar is known to have strong alkalinity buffering ability that is favourable in removing
biogas impurities, particularly H2S [18]. The H2S is an acidic gas, therefore it is easier for H2S
to be adsorbed when it is in contact with an alkaline surface [20]. Xu et al., (2014) investigated
swine manure and sewage sludge-based biochar utilisation to remove H2S. This study proposes
a mechanism of H2S removal by biochar as follow:
H2S(gas) → H2S(ads) → H2S(ads-liq) (R2.13)
H2S(ads-liq) → OH- → HS-(ads) + H2O (R2.14)
HS-(ads) + O2 → S0 (R2.15)
HS-(ads) + O2 + H2O → SO4
2- (R2.16)
It is suggested that the caustic presence in the gas cleaning system catalyses the oxidation of
H2S to the elemental sulphur until the base is exhausted. The results suggest that there was only
a small decrease in pH in the treatment with biochar because of its basic characteristics with
higher quantities of oxygen-containing functional groups. The FTIR analysis confirmed the
presence of some surface structure such as OH, COO, and C=O[18]. On the other hand, in H2S
41
removal using AC having acidic pH, the base was exhausted because of the formation of
sulfuric acid [19].
Another study suggests that a high pH is believed to be the reason for the higher adsorption
capacity performed by biochar derived from rice hull (SR) in removing 10 – 50 ppm of H2S
[19]. The SR was superior to the rest of the materials, namely AC, biochar derived from bamboo
(SB), and camphor (SC), because of its highest pH value [19].
Table 2.12 Study of biochar and carbon-based materials to remove gas impurities
Material Type of
gas
Concentration
(ppm)
Adsorption
capacity
(mg.g-1)
References
Woodchips biochar H2S 1020 273 [153]
Green waste biochar H2S 2000 6.5 [151]
Camphor-derived biochar H2S 50 1.2-121.4 [20]
Activated carbon H2S 10 – 50 35.6 [19]
Rice hull derived biochar H2S 10 – 50 382.7 [19]
Switch grass-derived
biochar
H2S 150 8 [46]
Woody biochar
(Cryptomeria japonica)
NH3 100 RE* = 90% [154]
Switch grass-derived
biochar
NH3 300 8 [46]
*RE = Removal efficiency
Results of previous studies using biochar and carbon-based material to remove gas impurities
are summarised in Table 2.12. Kanjanarong et al. (2016) investigated the biochar made of the
mixture of 80% of woodchip (spruce, pine and fir) and AD residue mixture-based (20%) in
removing low strength of H2S (105, 510 and 1020 ppm) from biogas. The study reports the high
efficiency of H2S removal (up to 98% removal) caused by the high alkalinity of pH (7.98) and
moisture content during operation (80-85). Also, the presence of carboxylic and hydroxide
42
radical groups also contributed to the process [153]. Using a higher H2S concentration of 2000
ppm, Skerman et al. (2017) examined five different materials for H2S removal, namely,
commercial iron-oxide H2S scavenger (cg5), green waste-biochar, granulated steel furnace slag,
red soil, manure-based compost and granulated activated carbon (GAC). Although the biochar
breakthrough time was significantly shorter than the cg5 and red soil, biochar achieved the
second-highest breakthrough capacity (6.5 g S.kg medium-1) [151]. The studies show the
potential of biochar as an ideal H2S adsorbent.
Sahota et al. (2018) used leaf biochar prepared at three different temperatures (200, 300 and
400ºC) to remove 500 – 1300 ppm of H2S. The results suggest that the biochar prepared at
400ºC achieved the higher H2S removal efficiency of 84% owing to its higher pH which allowed
the increased H2S dissociation and elemental sulphur conversion rates. It is also believed that
the higher SSA, carbonisation temperature and surface mineral element of biochar play a role
[21].
While the utilisation of biochar to remove acid gases such as H2S and CO2 are popular, the
studies of biochar use in NH3 cleaning remain scarce. Iyobe et al. (2004) compared woody
biochar and activated carbon (AC) to remove 100 ppm of NH3. At 20ºC, the woody biochar
removed higher NH3 than AC, achieved ca. 90% removal efficiency compared to 15% at 24
hours of operation. It is well known that NH3 is an alkaline and polar gas [154]. It is reported
that biochar was more suitable for this type of gas, while AC performs better in removing
volatile organic carbon (VOC). Also, the presence of acidic functional groups such as carboxyl
and phenolic hydroxyl groups in this woody biochar prepared at 400ºC led to a maximum
absorbability for a base gas like NH3. The study reports that the surface acidity of the biochar
than its specific surface area, was more significantly affected the NH3 removal [154].
A contradictive result was reported by Bhandari et al. (2013) who conducted the study using
biochar and other materials namely activated carbon, acidic surface activated carbon, and mixed
43
metal oxide [46] to remove H2S, NH3 and toluene simultaneously. In the study, biochar was
found to be a moderate material in removing NH3, adsorbing 8 mg NH3.g-1 with 100 min of
breakthrough time. It is thought that the NH3 removal occurred via surface and micro-pore
filling. The study suggested that the mechanism of biochar in removing NH3 via adsorption
follows the following reactions [46]:
2NH3 → N2 + 3H2 (R2.17)
4NH3 + 3O2 → 2N2 + 6H2O (R2.18)
However, the result was significantly lower when compared with the treatment using AC which
adsorbed 0.03 g-NH3.g-1. It is believed that the acidic surface of the AC that sustained the NH3
removal to achieve higher removal efficiency [46].
The existing literature suggests the promising potential of biochar in removing H2S. However,
the results on the utilisation of biochar in NH3 removal are inconsistent, depending on the
feedstock and preparation condition of biochar. Therefore, further investigations are
recommended for future studies.
2.6 Summary of literature review and specific research objectives
The AD is a mature technology to both manage waste and generate energy. However, it
produces a low quality of biogas and operates in a sub-optimum condition. The development
of TPAD is idealised to improve the AD. The innovative aspect of the TPAD is the harvest of
H2 from the first phase to be mixed with the CH4 generated from the second phase to increase
the overall quality of the biogas. The TPAD principles can be applied in a remote area as an
integrated system of TPAD, biogas cleaning and conditioning. The food and agriculture waste
are a potential feedstock for TPAD to produce biogas for household and TPAD operation. The
TPAD also produced an increased nutrient availability for organic fertiliser preparation for the
44
agricultural application. However, optimisation in term of further enhancing biogas production,
improving H2 and CH4 yield, removing impurities from biogas generated from TPAD, and
adding value to the beneficial utilisation of the effluent is still required.
Biochar with its aforementioned beneficial characteristics for a wide range of application,
therefore, become the innovative aspect of this study. The reviews of current literatures suggest
the gaps on the knowledge of the application of biochar on the utilisation of biochar in the (1)
TPAD, (2) the gas cleaning and (3) preparation of fertiliser from the TPAD effluent as follows.
First, to the best of our knowledge, in a TPAD, where phases of the AD are separated into two,
the studies on the effects of biochar addition and operation condition on H2 and CH4 production
are limited. Therefore, it is essential to study the H2 and CH4 generation on TPAD under
different biochar addition. In addition, the majority of the studies of the biochar addition in AD
were conducted under single operating condition. As a result, the information on the interactive
effect of biochar addition and operating conditions in AD are scarce. Therefore, this thesis
investigated the effect of biochar addition under the different operating condition, such as
different initial pH and temperature. This thesis is also aimed to reveal the mechanism of
biochar in enhancing H2 and CH4 production in the separate phases of TPAD.
Second, despite the growing interest on the biochar utilisation in AD, the study on the biochar
addition in demonstration or full-scale operation is rare. To date, the study on the biochar
application in demonstration scale of TPAD has never been explored. The study on the
demonstration scale operation of TPAD is necessary to apply the results of the bench/laboratory
studies and to investigate the practical considerations on the biochar addition in the
demonstration scale application. A commissioning and start up period are important steps
before the continuous operation of demonstration scale TPAD.
45
Furthermore, for further study, it is suggested to investigate the impurities removal using
biochar derived from other sources of waste/feedstock [19]. Moreover, the inconsistent results
from the existing studies of NH3 removal using biochar imply that further studies are important.
Finally, the existing literature suggests that the AD effluent, biochar and the combination of
both materials have shown a possible utilisation both for soil amendment and fertiliser, although
some reports also show their negative effects on plant growth, depending on their
characteristics, application rate and the species of the tested plant [28, 147, 155]. Therefore, the
further characterisation, determination of the appropriate proportion of AD effluent and
biochar, and its fertilising character are in a high necessity to be conducted before the
application of AD effluent as fertiliser or stabiliser [144, 147].
It is expected that this study will fill the gaps in the literature about the biochar application on
the integrated TPAD system. The thesis investigates into the utilisation of biochar in the (1)
TPAD, (2) the gas cleaning and (3) preparation of fertiliser from the TPAD effluent. Overall
aims of this study are to study and reveal the mechanism of the biochar effects on TPAD process
for H2 and CH4 production using laboratory scale bench-top bioreactors, to demonstrate and
optimise performance of TPAD through the PDU with the addition of biochar, as well as to
devise a method to prepare organic fertilisers by treating the TPAD effluent with biochar.
To achieve these overall aims, the following specific objectives have been identified for this
thesis work:
1. To investigate the effect of biochar addition on the gas and metabolic products generation
as a function of time in the first and second phases of the bench-scale TPAD and establish
the working mechanisms of biochar in TPAD;
46
2. To study the effect of operating condition (initial pH and temperature) in the biochar-
added TPAD process on biogas production and composition in the first and second phases
of the bench-scale TPAD;
3. To determine the optimum value of the biochar addition, initial pH and temperature in
the first and second phases of TPAD;
4. To demonstrate the performance of the TPAD Process Demonstration Unit (PDU) using
selected feedstock over a sufficiently long period of operation;
5. To study the effect of biochar addition in the performance in the TPAD PDU operation;
6. To prepare and characterise biochar-loaded fertiliser prepared from TPAD effluent;
7. To assess the fertilising characteristic of the biochar-loaded fertiliser by using
germination assay.
47
Chapter 3 Methodology, Approach and Techniques
The chapter presents the detailed methodology, approach and techniques to achieve the study
objectives. Experimental set up of bench scale and demonstration operations of TPAD, biogas
cleaning and organic fertiliser preparation are described.
3.1 Overall research strategies
The experimental works was focused on the effects of biochar addition and operation in bench
scale TPAD, both first and second phase TPAD to produce H2 and CH4, respectively.
Experiment on the operation of TPAD PDU, fertiliser preparation and biogas cleaning were
also accommodated. Figure 3.1 shows the schematic diagram of the research strategies for the
experimental study.
Experiments
Modelling
Bench scale TPAD
Demonstration scale
TPAD
Preparation of
organic fertiliser
Biogas cleaning
First phase
Second phase
Gompertz model
Research strategies
Figure 3.1 Research strategies
48
3.2 Bench scale experimentation of TPAD
It is known that there is a standardised method to investigate an anaerobic biodegradability of
a given substrate and its ultimate methane potential, namely bio-methane potentials (BMP)
[156-158]. It is also essential for assessing the activity, inhibition and bio stability of an
anaerobic assay [156, 157]. The information then can be used for a feasibility study of design,
economic and full scale of AD operation [157, 158]. A similar method for bio-hydrogen
potential (BHP) is also develop for investigating potential of a certain substrate to produce H2
[159]. A combined assay of BHP and BMP is proposed to simulate a batch TPAD operation
[158, 159]. In this study, the combined BHP and BMP was used to assess the performance of
bench scale TPAD with the addition of biochar under different operating conditions.
3.2.1 Materials
To ensure the consistency in the feedstock quality and characteristics, white bread obtained
from a local supermarket was used to simulate carbohydrate-rich food waste in an aqueous
environment for TPAD. The bread was shredded to ca.1 mm in size before being used in the
TPAD experimentation.
The source of inoculum was a sludge obtained from Woodman Point Wastewater Treatment
Plant, Western Australia. For hydrogen production, the sludge was heated and stirred at the 95
C for 30 minutes to eliminate methanogens. A biochar obtained from pyrolysis of pine sawdust
using an indirectly fired kiln reactor at 650C with a retention time of ca. 20 min. The biochar
was ground and sieved to a size fraction of 3.5-25.9 µm. The biochar sample was dried in an
oven at 105C prior to use.
Elemental (C, H, N and S), total solids (TS) and volatile solids (TS) analysis the food waste,
inoculum and biochar used in the study were conducted according to the ASTM standards
(D3176) and US Environmental Protection Agency (EPA Methods 1694) [160]. Particle size
49
distribution was measured using Cilas 1180 particle size analyser. BET surface area and pore
volume were determined using TriStar II 3020 surface area and porosimeter analyser.
Characteristics of the feedstock, inoculum and biochar are listed in Table 3.1.
Table 3.1 Characteristics of feedstock, inoculum and biochar [161]
Parameter Food waste Inoculum Biochar
Carbon (%) 42.7 ND 77.9
Nitrogen (%) 2.3 ND 0.3
Hydrogen (%) 9.1 ND 3.7
Sulphur (%) 0.3 ND 0.1
Oxygen (%) 45.6 ND 18
C/N ratio 18.7 ND 255.0
pH 4.9 7.3 9.6
Total solids (%) 61.2 2.5 95.2
Volatile solids (%) 59.5 1.8 81.1
Particle size distribution (µm) ND* ND 3.5-25.9
BET surface area (m2.g-1) ND ND 130.0
Pore volume (cm3.g-1) ND ND 0.0138
ND: not determined
3.2.2 Experimental set up
Figure 3.2 shows the experimental set up of bench scale TPAD study. A serum botte with 100
mL total volume was used as the reactor. The working volume was 60 mL, containing sludge,
bread and water. The bottle was airtight sealed with rubber lid and aluminium crimp to ensure
the anaerobic condition and prevent the reactor from gas leaking. The typical bottles were then
placed in incubator at the designed temperature and duration.
50
Gas sample taken for
GC analysis
Liquid sample taken for
VFA analysis
Inoculum
Bread
Water
(a)
(b)
Figure 3.2 Schematic of the experimental set-up (a) and bench scale TPAD in the incubator
(b)
3.2.3 Experimental procedure
3.2.3.1 The first phase of TPAD
The effect of biochar under a fixed initial pH and temperature
The TPAD experimentation was conducted in batch mode. Schematic diagrams showing the
experimental set-up and the typical experimental procedure are shown in Figure 3.3. For H2
production, 8 g VS.L-1 of bread, 10 ml of heated sludge and water were added in a 100 ml serum
bottle with a working volume of 60 ml. The initial pH was adjusted to 5 by adding an
appropriate amount of hydrochloric acid (HCl) or sodium hydroxide (NaOH). Prior to an
experimental run, the reactors were flushed with high purity nitrogen (> 99.99 %) at 10 l.min-1
for one minute and then carefully sealed with rubber plugs and secured with aluminium caps
[156].
51
Figure 3.3 Schematics of the experimental procedure for TPAD employed in this study
The bottle was then placed in an incubator maintained at 35 C until the gas production stopped,
within approximately 8 days. The bottle was shaken once a day prior to sampling the gas for
composition measurement [156]. A sample of accumulated biogas was taken daily from each
reactor using a gas tight syringe and a 1 ml liquid sample was taken on day 2 and 8, respectively,
for further analysis. In order to study the effect of the amount of biochar addition on the
performance of the H2 production process, reactors with different biochar addition ratios of 8.3,
16.6, 25.1 and 33.3 g.l-1 were set up. These experiments were run following the same procedure
Heated sludge Distillate water Bread Biochar
Mix in 100 ml
serum bottle
Bring to initial pH
5±0.1
Purge with nitrogen
(1 minute)
Take initial sample (1 ml)HCl 1 M or NaOH 1 M
Close the serum
bottle with rubber
and alumunium lid
Place in incubator
(35°C) for 8 days
Add unheated
sludge into serum
bottle
Bring to initial pH
7±0.1
Take initial sample (1 ml)HCl 1 M or NaOH 1 M
Close the serum
bottle with rubber
and alumunium lid
Place in incubator
(35°C) for 39 days
Purge with nitrogen
(1 minute)
• Stir the bottles once a day
• Measure gas production
every day
• Take gas sample every day
• Take liquid sample (1 ml)
periodically for VFA analysis
• Stir the bottles once a day
• Measure gas production
every day
• Take gas sample every day
• Take liquid sample (1 ml) on
day 2 and 8 for VFA analysis
Hydrogen
production
Methane
production
52
as detailed above. Each set of experiments was repeated under identical conditions three times,
involving a total of fifteen serum bottle reactors.
The gas volume and composition were analysed every day according to the methods explained
in section 3.2.4.1 and 3.2.4.2, respectively. Liquid samples were taken initially, on day 2 and 8
for VFA analysis (section 3.2.4.3). Initial and final pH values were also recorded.
The effect of biochar under different initial pH and temperature
The experiment was according to the first phase experimental procedure as in section 3.3.1.1.
The batch experimentation of H2 production was designed based on a CCD [162]. A typical
CCD comprises of three different sets of experimental runs, namely a centre, factorial and axial
point runs. The centre point runs combine the medium levels of each factor examined in this
study, namely initial pH, temperature and biochar addition ratio.
The factorial point runs accommodate the upper and lower levels of each factor, and the axial
point runs typically cover slightly upper and lower levels of each factor [163]. Thus a CCD
explores the representative points of the experiments while keeps the number of total test runs
to a minimum.
The three independent factors examined in this study were the initial pH (X1), temperature (X2)
and biochar addition ratio (X3) and the responses were the cumulative H2 yield (YH) and
maximum H2 production rate (RM), respectively. The levels of initial pH were 4, 6 and 8, the
levels of temperature were 25, 35 and 45C and levels of the biochar addition ratio were 5, 10
and 15 g.l-1. The level of biochar addition ratio was determined based on our previous study
[161], in which a biochar addition ratio of 16.6 g.l-1and higher showed no beneficial effect on
H2 production. The total runs of the experiments were 20, consisting of 6 replications of centre
point runs, 8 sets of factorial runs and 6 sets of axial runs (Table 3.2).
53
Table 3.2 Central composite design for H2 production
Run Factor 1
A: Biochar addition (g.L-1)
Factor 2
B: Temperature (oC)
Factor 3
C: initial pH
1 4 25 15
2 8 25 15
3 6 35 10
4 8 25 5
5 6 35 10
6 9.4 35 10
7 4 45 5
8 4 25 5
9 6 35 10
10 6 35 10
11 6 35 18.4
12 8 45 15
13 6 35 10
14 6 51.8 10
15 2.6 35 10
16 8 45 5
17 6 18.2 10
18 6 35 10
19 4 45 15
20 6 35 1.6
Each experimental run was triplicated. The cumulative H2 yield (YH) and maximum daily
volumetric H2 production rate (RM) achieved during the experimentation were examined as the
targeted responses to factors, i.e. the treatment parameters: initial pH, temperature and biochar
54
addition ratio [164]. Additional experiments at biochar addition ratios ranging from 0 – 18.4
g.l-1 at the initial pH 35 and temperature 35C were performed to investigate a typical H2
production under different biochar addition.
The daily volumetric gas and composition were used to calculate the cumulative H2 yield (YH)
and maximum H2 production rate (RH). The YH and RH are the responses of the CCD. The
responses then analysed using response surface methodology explained in section 3.6.3.
Effect of Biochar on H2 Production via Anaerobic Digestion as Compared to Other Solid
Additives: Role of Acidity
The additives with different acidity and surface structure used in the study were pinewood
biochar [161], alumina and zeolite. Glass with a pH of 7 and very small surface area was also
used for a comparison purpose. The additives were crushed to have a particle size less than 50
μm. The pH and BET surface area of the additives are listed in Table 3.3. It is seen that the
biochar is alkaline and alumina is neutral while the zeolite is very acidic. Zeolite has the highest
BET surface area of 538 m2.g-1 followed by biochar (361 m2.g-1) and alumina (153 m2.g-1).
Table 3.3 The characteristics of the additives
Parameter
Additives
Biochar Glass Alumina Zeolite
pH 8.6 7.1 6.9 4.2
BET surface area (m2.g-1) 361 0.26 153 538
The feedstock (1 gram) and 10 mL of heated sludge and 10 g.L-1 of an additive are mixed in a
100 mL serum bottle and distillate water was added to occupy the working volume of 60 mL.
The initial pH was adjusted to 6.4, according to an optimum pH suggested by the optimisation
study, using hydrochloric acid (HCl) and sodium hydroxide (NaOH). The bottles were then
purged with high purity nitrogen and sealed with a rubber lid and aluminium crimp. The bottles
55
were then incubated at 32 °C, as suggested by the optimisation study, for 7 days. A set of control
without any biochar addition was also prepared. Each treatment was conducted in triplicate.
The daily volume and composition of biogas produced were monitored daily. Liquid sampling
was taken periodically for further analysis. A set of identical bottles of each treatment was also
prepared to be opened daily to monitor the pH and VFA changes during the experiment.
3.2.3.2 The second phase of TPAD
The effect of biochar under a fixed initial pH and temperature
This study is the continuation of the preliminary study explained in the section 3.2.3.1. After 8
days, the remaining culture in a reactor was used as the feed for CH4 production in the second
phase (Figure 3.3). The bottle tip was opened and 10 ml of unheated sludge was added to the
bottle to bring the pH to 7. The bottle was then sealed and incubated at 35 C for 39 days. Gas
sampling was conducted daily and liquid samples were taken periodically for further analysis.
The effect of biochar under different initial pH and temperature
The feedstock used in the experiment was the effluent from the first phase of TPAD of food
waste. It was prepared according to optimum biochar addition, initial pH and temperature of
our previous study, which is briefly described as follows. The feedstock was prepared in a one
litre Duran bottle. The heated sludge (100 mL), 10 g volatile solid.L-1 (g VS.L-1) of white bread,
10.1 g.L-1 of biochar and 500 mL of distillate were mixed in one litre bottle and set at optimum
initial pH 6.4. The culture was then incubated at an optimum temperature of 32C until it
stopped producing biogas (7 days). This effluent was used as the feedstock in the second phase
of TPAD to produce methane.
56
Sludge (10 ml) First phase effluent (50 ml) Biochar (as designed)
Mix in 100 ml
serum bottle
Bring to initial pH
(as designed)
Purge with nitrogen
(1 minute)
1M HCl or 1M NaOH
Seal the serum
bottles with rubber
and aluminum lids
Place the bottles in
incubator at
designed
temperature for 35
days
• Stir the bottles once daily
• Measure gas production daily
• Gas sampled and analysed
daily
Figure 3.4 Schematics of the experimental procedure for second phase
The experimental set up is illustrated in Figure 3.4. In a typical experimental run, 50 mL of the
feedstock and 10 mL of the seed sludge were mixed in a 100 mL serum bottle. The amount of
fresh sludge added was determined based on the result of the preliminary study. HCl and NaOH
were added to bring the pH of the liquid to the designed pH. The reactor was then purged with
nitrogen to create an anaerobic environment and closed with a rubber and aluminium lid and
incubated for 30 days. The effect of biochar addition (5, 10, and 15 g.L-1(w/v)), initial pH (5, 7
and 9) and temperature (25, 35 and 45 C) on methane yield (YM) and methane production rate
(RM) was examined in turn using a Box Behnken experimental design method. A control
experiment without biochar addition incubated at an initial pH of 7 and temperature of 35 C
was also conducted.
57
Table 3.4 Parameters and levels of Box Behnken Design
Variable Parameters Level
A Biochar addition (g.l-1) 5 10 15
B Temperature (ºC) 25 35 45
C Initial pH 5 7 9
The batch experimentation for methane production was designed based on a Box Behnken
design (BBD) [165]. The three independent factors examined in this study were the biochar
addition ratio (A), temperature (B) and initial pH (C) and the responses were the cumulative
methane yield (YM) and maximum methane production rate (RM), respectively. The levels of
initial pH, temperature and biochar addition ratio are listed in Table 3.4.
Table 3.5 Box Behnken design for CH4 production
Run Factor 1
A: Biochar addition (g.l-1)
Factor 2
B: Temperature (oC)
Factor 3
C: Initial pH
1 10 35 7
2 10 45 9
3 10 45 5
4 10 25 5
5 15 35 5
6 5 45 7
7 15 35 9
8 10 35 7
9 15 45 7
10 10 35 7
11 5 25 7
58
Run Factor 1
A: Biochar addition (g.l-1)
Factor 2
B: Temperature (oC)
Factor 3
C: Initial pH
12 5 35 9
13 15 25 7
14 10 35 7
15 10 35 7
16 5 35 5
17 10 25 9
The level of biochar addition ratio was determined based on our previous study [161], in which
a biochar addition ratio of 25.5 g.l-1and higher decreased CH4 production. The total runs of the
experiments were 17 with 5 replications of the centre point runs (Table 3.5). A control treatment
without biochar addition at the initial pH 7 and temperature 35C were also performed. Each
experiment was run in triplicate.
The daily volumetric gas and composition were used to calculate the cumulative CH4 yield (YM)
and maximum CH4 production rate (RM). The YM and RM are the responses of the BBD. The
responses then analysed using response surface methodology explained in section 3.6.3.
3.2.4 Analysis
3.2.4.1 Biogas volume
Water displacement is a typical method for volumetric gas measurement (Figure 3.5) [157,
158]. Thus in this current study, daily volumetric biogas production was measured by a water
displacement method [23] and then converted to volumetric biogas production under the STP
condition (273K and 1 atm pressure) according to the ideal gas law [161].
59
Figure 3.5 Schematic set up of water displacement method for gas volume measurement
3.2.4.2 Biogas composition
Studies use gas chromatography (GC) methods with flame ionization (FID) and thermal
conductivity detector (TCD) to analyse the composition of H2, CH4 and CO2 in biogas produced
by the assay [102, 156, 158]. In this study, gas composition was analysed using a Gas
Chromatograph (GC; Agilent 7980) facilitated with a flame ionisation detector (FID) (heater
200 C, hydrogen flow: 30 ml.min-1, air flow: 350 ml.min-1, make up flow: 35.5 ml.min-1), a
back detector thermal conductivity detector (TCD) (heater 250 C, reference flow: 10 ml.min-
1, make up flow: 5 ml.min-1) and a TCD (heater 200 C, reference flow: 7 ml.min-1, make up
flow: 3 ml.min-1) with the oven temperature of 90 ºC. Figure 3.6 shows a typical gas
chromatogram of gas collected from the first phase of TPAD.
60
Figure 3.6 A typical gas chromatogram of gas collected from the first phase of TPAD
The daily volumetric hydrogen or methane production was calculated by multiplying the
volumetric biogas production by the H2 or CH4 percentage as determined from the GC analysis
[156]. The total cumulative H2 or CH4 yield was then obtained by adding daily volumetric H2
or CH4 production ºC. The cumulative H2 yield (YH) and CH4 yield (YM) were the total
volumetric H2 production over the whole duration of an experimental run, calculated according
to the ideal gas law (in STP ml) and normalised to the volume of reactor [161]. The specific
gas production was the cumulative H2 yield relative to the gram VS of the added [53, 156, 161].
3.2.4.3 Volatile fatty acids
Volatile fatty acid (VFA) analysis is important to understand the metabolic products generated
from the assay, for example butyric, acetic and propionic acids [158, 159]. The metabolic
products may give the insight on the possible dominance metabolic pathway of an assay[113].
For the sample preparation, 100 µl of liquid sample was diluted with 300 µl deionised water in
a microcentrifuge tube. As much as 100 µl of 2-ethylbutyric acid (10 mmol.L-1) as internal
standard (IS) was also added. The pH of the sample was reduced by adding 50 µl of 6 N HCl.
To extract the volatile acid, 500 µl of diethyl ether was added. The tubes were then centrifuged
using microcentrifuge (Eppendorf centrifuge 5415D) at 3000 rpm for 10 minutes. After
61
centrifugation, the upper layer containing acid extract in the ether was transferred into GC vials
for VFA analysis. A GC (Agilent 7980) using DB-WAX column (30 m x 250 mm x 0.25 mm),
flame ionisation detector (FID) (heater 250 C, H2 flow: 35 ml.min-1, air flow: 350 ml.min-1,
make up flow 4 ml.min-1, total run time 13 minutes) was used to determine the volatile fatty
acid (VFA) of liquid samples taken from both phases. The calculation of each acid was
determined using the response factor of each acid generated from the standard curve developed
from the VFA analysis using a 10 mmol.L-1 mixed acid standard (Sigma Aldrich). The area of
the Figure 3.7 shows typical standard curves for some selected acids. The acid calculation was
based on the following equation.
𝐶𝑜𝑛𝑐. 𝐻𝑥 =𝐴𝑟𝑒𝑎 𝐻𝑥 𝑥 𝐶𝑜𝑛𝑐. 𝐼𝑆
𝐴𝑟𝑒𝑎 𝐼𝑆 𝑥 𝑅𝐹 (R3.1)
(a) (b)
Figure 3.7 Typical standard curves for (a) acetic and (b) butyric acids analysis
3.2.4.4 pH
A TPS AQUA-pH pH mater (Rowe Scientific) was used to measure the pH of liquid samples.
Prior to the test, two points calibration with pH 4.0 and 7.0 standard buffer solution (Rowe
Scientific) was conducted.
62
3.2.4.5 Scanning Electron Microscopy
In an effort to reveal the working mechanisms of biochar, the morphology of biochar before
and after experimentation was examined using Scanning Electron Microscopy (SEM). Liquid
sample (1 mL) containing biochar particles were taken at the end of the experiment and added
with fixative solution (glutaraldehyde 2.5%). In order to prevent the damage of the samples, the
critical point drying method (CPD) was conducted prior to the SEM analysis. The liquid sample
was washed twice using phosphate buffer solution (PBS) and set at BioWave for 30 second
each. The samples were then dehydrated using gradually increased concentration of ethanol
(50, 70, 90, and 100% in water) and microwaved for 30 second each. [85] The dried samples
were then coated with Au and analysed using SEM Zeiss 55 with SE or InLens detector.
3.3 TPAD Process Demonstration Unit (PDU)
3.3.1 Principles of TPAD PDU
A TPAD process demonstration unit (PDU) operated by the Centre for Energy of the University
of Western Australia consists of two reactors; the first reactor is an H2 producing reactor with
the capacity of 150 L, while the second reactor accommodates methane generation with 500 L
of total volume [41]. Before the operation, the feedstock is milled (where necessary) and diluted
with water to achieve the designed total solids (TS) in storage tank. The feedstock is then
pumped into the first phase of TPAD (R1). R1 1 is operated at low pH (4-6) and mesophilic
temperature to produce H2. Typical hydraulic retention time (HRT) of the R1 is 3 days. The
effluent from the R1 is pumped to the buffer tank to adjust the pH of effluent to around 7. The
effluent is transferred to the second reactor (R2) to produce methane-rich biogas. The HRT of
the R2 is around 7-14 days. The heating pump circulated heated water to double jackets of each
reactor to maintain the temperature. The effluent from the R2 is flown to discharge tank for
further treatment.
63
3.3.2 Experimental set up
TPAD PDU system
Figure 3.8 shows the schematic diagram of TPAD PDU of the CFE UWA. The components of
the unit are the process controller, feedstock preparation, digesters, heating system, gas
collection and effluent conditioning.
Figure 3.8 Schematic diagram of TPAD PDU of the Centre for Energy of University of
Western Australia
Process control
TPAD PDU is controlled by a programmable logic controller (PLC), with sensors measuring
pH, temperature and gas flow rate. The instructions for agitation, heating and pumping activities
across the facilities can be automatically executed via the PLC. The data obtained from the
sensors were recorded for further analysis.
64
Feedstock preparation
The unit consists of a mill and a storage tank. The mill is used to mechanically reduce the size
of the feedstock for easier access of microbes into the nutrient contents in the feedstock [41].
The storage tank (volume 200L) is needed for feedstock mixing and dilution, achieving
maximum total solids (TS) of 30% (w/w) in the AD system [166]. Size reduction and dilution
of feedstock also prevents jamming in the valves, pumps and pipes [41]. Both mill and storage
tank were safety guarded that are controlled by micro-switches. The motor of equipment stops
operating when the lid is open.
Digesters
The digester contains two reactors, first for H2 production (150 L) and second for CH4
production (500 L), and one buffer tank (100 L) installed between the two reactors. Both
reactors are continuous stirred tank reactor (CSTR), which comprises of a tank body for the
main reaction, heating jacket for adjusting temperature, an agitator to stir the culture, a stage
inlet and an outlet for effluent discharge. Both reactors are equipped with temperature, pH and
oxidation-reduction potential (ORP) sensors to monitor the operating conditions. The buffer
tank with a pH probe is used for pH adjustment of the effluent from the first phase before being
fed into the second reactor.
Heating system
A 250 L tank is used for water storage. The water is pumped to a 250 L heating tank and the
heated water is circulated by a heating pump and hot water pipe line to the heating jacket of
each reactor. The designed temperature is set and controlled by the PLC. Both the first and
second reactors can be heated up to 50ºC.
65
Gas and effluent collection unit
A wet flowmeter was used to measure daily biogas production from each reactor. A cautious
inspection should be conducted to monitor water level in the flowmeter as an incorrect level of
liquid may cause a flowmeter malfunction. When a low water level is found, additional liquid
should be added into the wet flowmeter and subsequent flow meter calibration needs to be
executed. The biogases produced from the first and second tank are mixed in a 1 m3 gas bag.
Gas can be transferred into a D size gas tank using a gas pressurisation system. The effluent
from the second tank is discharged to the disposal tank for further treatment or utilisation.
Feedstock, inoculum and biochar
White bread from a local supermarket was used as a simulated carbohydrate waste for TPAD
feedstock. Microbial inoculum was effluent from anaerobic digestion at the Woodman Point
Wastewater Treatment Plant, Western Australia. Biochar was prepared by heating pine saw
dust at 650 ºC for ca. 20 minutes in a kiln reactor [167]. Biochar was ground and sieved to a
particle size 3.5-25.9 µm. Elemental (C, H, N and S), total solids (TS) and volatile solids (TS)
analysis of the food waste, inoculum and biochar used in the study were conducted according
to ASTM standards (D3176) and US Environmental Protection Agency (EPA Methods 1694)
[160]. Particle size distribution was measured using Cilas 1180 particle size analyser. BET
surface area and pore volume were determined using TriStar II 3020 surface area and
porosimeter analyser. Characteristics of the feedstock, inoculum and biochar are listed in Table
3.1.
Start-up strategy of the TPAD PDU
First phase: An effective sterilisation and sludge heating were required prior to the start-up to
ensure the elimination of methanogens in the R1. The first tank was thoroughly washed and
sterilised with 70% of ethanol to prevent methanogens contamination. In our bench scale study,
66
the sludge was heated at 95ºC for 30 minutes [161]. However, when the same method was used
in TPAD PDU, the methanogens contamination occurred in R1. Therefore, a longer heating at
95ºC for one hour was conducted, which has been found to be effective in sterilise R1.20 L of
the heated sludge was added once in the beginning of the operation. Prior to the feeding, the
bread was blended with water according to the designed substrate concentration (Table 3.6). A
fed-batch strategy was applied in the start-up period. The feeding with the mixture of the
feedstock, biochar and water was conducted gradually until the working volume of 100 L was
occupied. There were six stages during the start-up of R1, with the HRT of 3 days for each
stage. The fed-batch was conducted during stage 1 – 4 and followed by semi-continuous
operation at stages 5-6 (Table 3.6).
Table 3.6 Start-up strategy of the first phase of TPAD PDU
Stage Sludge
Volume (L)
Water
volume
(L)
Bread Biochar
(g)
HRT
(days) (g) (g VS)
1 20 20 170 100 400 3
2 - 20 340 200 200 3
3 - 20 1000 600 200 3
4 - 20 1350 800 200 3
5 - 20* 1350 800 - 3
6 - 20* 1350 800 - 3
Note: *: used recirculated liquid from tank 1
The biochar was stepwise added with the total biochar addition ratio of 10 g.L-1 of working
volume of the reactor, as suggested by the result of bench scale study on the first phase TPAD
[160]. At stage 4, the designed working volume of the reactor (100 L) was achieved. In stages
5 and 6, 20 litres of the culture was discharged from the reactor and then blended with 1350
67
gram of white bread and recirculated back into the reactor. The first tank was operated under
ambient temperature. 1% (v/v) of 1 M NaOH was added into the reactor at the beginning of
stages 4, 5 and 6 to adjust the pH above 4.
Second phase: R2 was operated under fed-batch conditions by feeding the sludge, white bread,
biochar and diluting water gradually for the first 5 stages until a total working volume of 300 L
was achieved (Table 3.7).
Table 3.7 Start-up strategy of the second phase of TPAD PDU
Stage
Sludge
Volume
(L)
Water
volume
(L)
Bread
Biochar
(g)
HRT
(days) (g) (g VS)
1 15 60 500 300 940 7
2 15 60 1000 600 940 7
3 15 60 1500 900 940 7
4 15 30 2000 1200 375 7
5 - 30 2500 1500 375 7
6 60 - - - 750 21
7 - 20* 2000 1200 - 7
8 - 20* 2000 1200 - 7
9 - 20* 2000 1200 - 7
Note: *: used recirculated liquid from tank 2
In order to enhance CH4 production, 60 L of untreated sludge was added into the reactor at stage
6 [140, 168, 169]. Biochar at with the total addition ratio of 12.5 g.L-1 was applied as suggested
by our previous study [13]. A tiny amount of NaOH (1% v/v) was added into the system in
stages 1 to 5 to adjust pH. NaOH was not required after stage 5 since the pH of the culture
remained above 6. The whole start-up operation was conducted at ambient temperature. In the
68
beginning of start-up, the manhole and sampling ports in both reactors were tightly sealed and
flushed with N2 to check any gas leaking and ensure the anaerobic condition of the tanks.
3.3.3 System monitoring and control
The daily volumetric biogas production from the first and second phase of TPAD was
monitored using a wet gas meter (LMF-1, CCAF Company, Changchun, China), respectively.
The gas samples of each reactor were taken once daily using foil gas bags. The gas composition
of the gas samples were analysed using a gas chromatography (GC) Agilent 7980 (Shanghai,
China) which is equipped with flame ionisation (FID) (H2 flow: 30 ml.min-1, make up flow:
35.5 ml.min-1, air flow: 350 ml.min-1, heater temperature 200ºC), thermal conductivity detector
(TCD) (reference flow: 7 ml.min-1, make up flow: 3 ml.min-1, heater temperature 200ºC) and
back TCD (reference flow: 10 ml.min-1, make up flow: 5 ml.min-1, air flow: 350 ml.min-1, heater
temperature 250ºC) [167]. The daily H2 and CH4 production were calculated by multiplying the
daily biogas production and H2 and CH4 composition, respectively. A liquid sample was also
taken daily from each reactor for measurement of pH and VFA concentrations [161]. A TPS
AQUA-pH pH meter (Rowe Scientific) was used to measure the pH of liquid samples. For VFA
analysis, 2-ethylbutyric acid (10 mmol.L-1) was used as an internal standard (IS) and diethyl
ether was used to extract the volatile acid. A GC (Agilent 7980) using DB-WAX column (30
m x 250 mm x 0.25 mm), flame ionisation detector (FID) (heater 250 C, H2 flow: 35 ml.min-
1, air flow: 350 ml.min-1, make up flow 4 ml.min-1, total run time 13 minutes) was used to
determine the concentrations of VFA of liquid samples taken from both phases [161].
69
3.4 Preparation, characterisation and evaluation of biochar-added organic
fertiliser
3.4.1 Experimental set up
TPAD effluent Biochar
Add to container
according to the
designed percentages
Mixed
thourougly
Close the container with
airtight lid
Set at least
24 hours
Figure 3.9 Preparation of organic fertiliser from TPAD effluent and biochar
Figure 3.9 shows the experimental set up of the preparation of organic fertiliser from TPAD
effluent and pinewood biochar. The TPAD effluent was generated form the R2 of TPAD PDU
on the 57th days of the operation. The pinewood biochar prepared at 650ºC was milled to the
particle size c.a. 25 µm.
The organic fertiliser was prepared by mixing TPAD effluent and biochar according to the
designed percentage of each treatment (Table 3.8). Each combination TPAD effluent and
biochar were thoroughly mixed, kept in an airtight container and labelled according to the
sample names in Table 3.8. The organic fertiliser was set for at least 24 hours to reach the WHC
equilibrium of biochar before use [170].
70
Table 3.8 Fertiliser composition
Sample name TPAD effluent (%) Biochar (%)
BC00 100 0
BC10 90 10
BC20 80 20
BC30 70 30
BC40 60 40
BC50 50 50
BC60 40 60
BC70 30 70
BC80 20 80
BC90 10 90
BC100 0 100
Figure 3.10 Experimental set up of soil-less petri dish bioassay
BC90
71
In a petri dish (diameter 8.5 cm), one gram of the organic fertiliser, 50 rocket seeds and 20 ml
of water were added. It is important to make sure that the biochar and the seeds were evenly
distributed across the petri dish. Each type of the fertiliser was tested triplicated. The petri
dishes were then incubated at 25ºC for 72 hours. At the end of the 72 hours, a photograph of
each treatment was taken. The seeds were counted as germinated when the radicle had emerged
[171]. The germinated seed was counted, the root and shoot length were measured, and
root/shoot ratio and germination index (GI) were calculated.
3.4.2 Analysis
Characterisation of the organic fertiliser
The pH analysis of the fertiliser was according to Luo et al. [83]. The analysis of dry matter and
WHC were based on Foster et al. [170]. The macronutrients required for plant growth including
phosphorus (P), potassium (K), sulphur (S), calcium (Ca) and magnesium (Mg), while the
micronutrients are iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), boron (B), chlorine (Cl),
and molybdenum (Mo), and nickel (Ni). P, K and micronutrients analysis was conducted using
ICP-EOS (iCAP 7000 Series). The preparation of the fertiliser samples for the micronutrients
used the modified methods according to USEPA methods 3050B for acid washing the sludge
sample and Enders and Lehman (2012) for biochar [172, 173].
Germination bioassays
The germination, root and shoot measurement were conducted according to Solaiman et al.
(2012) [28]. The germination index (GI) was calculated using the following formula [144].
𝐺𝐼 = 𝐺𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑𝑠 𝑖𝑛 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑡𝑒𝑠𝑡
𝐺𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑𝑠 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑥 100% (E3.1)
Adopting the compost toxicity test, there are three criteria of toxicity based on GI, (1) GI
≥125%: the compost considered to possess plant nutrient and growth stimulants; (2) GI ≥ 80%:
72
the compost free of any toxic and (3) GI ≤ 50%: compost contains strong toxicity [144, 174,
175].
3.5 Data analysis and modelling
3.5.1 Analysis of variant (ANOVA)
To compare the mean of hydrogen and methane yields from each phase of all treatments, the
data were also statistically analysed using Analysis of Varians (ANOVA). Post hoc tests were
carried out using the least squares difference (LSD).
3.5.2 The modified Gompertz Model
In order to obtain the lag phase, maximum production potential and rate of both hydrogen and
methane production in each treatment, the following modified Gompertz [25] model was
employed:
(E3.2)
where G(t) is the cumulative hydrogen or methane production [ml.l-1], t the time [days], P the
maximum hydrogen or methane production potential [ml.l-1] , Rmax the maximum hydrogen or
methane production rate [ml.l-1.per day] and λ is the lag phase [days] defined as a delayed period
of a culture in responding to a new environment and starting to produce hydrogen or methane
[26]. The cumulative hydrogen and methane production results were fitted using the model
3.5.3 Response surface methodology (RSM)
The response surface methodology (RSM) is a popular method to investigate the effects of key
operational factors such as additive concentration, initial pH and temperature on a measured
response, for example biogas yield and biogas production rate [176]. The optimisation of such
( )
+−
−= 1expexp)(
maxt
P
eRPtG
73
factor provides crucial information for the full scale operation [177]. Using RSM, the number
of experiment can be significantly reduced since it only selects representative levels of each
factor involved in the process [178].
To determine the interactions between the factors and the optimum operating conditions, a
quadratic model (Equation 1) was used to fit the experimental data [179].
Y = 𝛼0 + 𝛼1𝑋1 + 𝛼2𝑋2 + 𝛼3𝑋3 + 𝛼12𝑋1 𝑋2 + 𝛼13𝑋1 𝑋3 + 𝛼23𝑋2 𝑋3 + 𝛼11𝑋12 + 𝛼22𝑋2
2+
𝛼33𝑋32 (E3.3)
where X1, X2 and X3 are the actual values of the three factors, Y is the response (YH or RM,
respectively), 0 is a fitting intercept, 1, 2, 3 are linier coefficients, 12, 13 and 23 are
coefficient of interactivity between factors, and 11, 22 and 33 are quadratic coefficients.
The significance of the fitting model, linear, interactive and quadratic terms in the fitting models
was then determined by conducting ANOVA analysis. The optimisation of the model was
conducted using the “Design Expert” software (Design-Expert 10, State-Ease, Inc.,
Minneapolis MN, USA).
74
Chapter 4 Effect of Biochar Addition on Hydrogen Production
4.1 Introduction
Chapter 4 presents the results of bench scale experiments on the H2 production in the first phase
of TPAD. Typical H2 production without biochar addition is reported in Section 4.2. Section
4.3 discussed the effect of biochar addition on H2 production under varying operating conditions
(initial pH and temperature). Investigation into the role of acidity of biochar on H2 production
is presented in Section 4.4, followed by the discussion of possible mechanisms of biochar in
enhancing H2 production (Section 4.5).
4.2 Hydrogen production without biochar
Figure 4.1 Cumulative yields and production rates of H2 without biochar addition
The potential H2 production from AD of food wastes simulated by bread was first examined
under the tested conditions. Figure 4.1 shows the cumulative yields and production rates of H2.
The H2 production started on day 2 and completely stopped on day 8. The highest H2 production
rate was reached on day three with 304±33 mL.L-1.per day. The maximum cumulative H2
75
production was 751±3 mL.L-1. The H2 composition ranged from 49-65 vol% (Table 4.1). The
remaining gas mainly consisted of carbon dioxide (CO2). No CH4 was detected in the first
phase, confirming that the heating of sludge prior to the inoculation to the system was effective
in eliminating methanogens from the culture. The yields and contents of H2 resulted in the
current study were comparative to the data reported in the literature using potato starch, bagasse
fermentation residue and food waste as feedstocks [88, 180].
Table 4.1 Profiles of the first phase of TPAD without biochar
Parameters Unit Value
Cumulative H2 production mL.L-1 751.1±3
Gas composition
H2 % 49-65
CH4 % 0
CO2 % 35-51
Volatile fatty acids (VFA) mg.L-1
Acetic acid mg.L-1 1063± 351
Butyric acid mg.L-1 1172±60
Propionic acid mg.L-1 7±7
Final pH 3.0
During the operation, H2 production was accompanied by the generation of VFAs. The
dominant fatty acids were butyric and acetic acids, as shown in Table 4.1. The accumulation of
2,242 mg.L-1 of VFA occurred at the end of the phase. The pH of the remaining culture dropped
from 5.0, as set at the beginning of the experiment, to 3.0. The accumulation of acid in the end
of H2 production was identified to be the cause of the pH drop.
76
4.3 Hydrogen production with biochar
Figure 4.2 Cumulative H2 yields at different biochar addition ratios
Figure 4.2 shows the cumulative H2 production over 8 days of operation in the first phase with
and without biochar addition. All cultures started to produce H2 after day one. It is clear that all
cultures with biochar addition produced higher cumulative H2 than that without biochar.
However, when the biochar addition was higher than 8.3 g.L-1, the H2 yield started to decrease
slightly. The highest H2 production of 945 ml.L-1 was achieved at 8.3 g.L-1 of biochar addition.
To determine whether the effect of the biochar addition on the H2 yield was statistically
significant, a statistical analysis was conducted by data-add on of Microsoft Excel 2010. The
procedure consisted of a one-way analysis of variance (ANOVA) and post hoc test analysis
[88]. A statistical level of significance was defined by a (p) value less than or equal to 0.05
which indicates that the mean value of at least one of the H2 production was not equal to the
others. Statistical non-significance was defined by (p) value greater than 0.05. The statistical
analysis results are presented in Table 4.2 including the differences in mean values of the final
H2 production of any two compared cultures, standard errors and the associated p value. It can
77
be seen that the differences between mean values of H2 yield of the control system and systems
with biochar additions less than 25.5 g.L-1 were greater than 100 mL with standard deviations
of less than 68.0 and p values of less than 0.05. This suggests that the H2 yields in the culture
with 8.3; 16.6 and 25.5 g.L-1 biochar additions were statistically significantly higher than that
from the control system. However, it is also evident that there was no significant difference
between the H2 yields of the system with the 33.3 g.L-1 biochar addition and the control system.
Table 4.2 One-way ANOVA and post hoc analysis on cumulative H2 production in
different biochar addition ratios
Biochar addition
(g.L-1)
Average cumulative H2
yields (mL.L-1)
Standard deviation P value
0 750.4a 55.5 0.006
8.3 944.5c 38.6 0.006
16.6 859.5b 68.0 0.006
25.1 858.0b 24.3 0.006
33.3 831.0a 32.0 0.006
Note: Different notation indicates a significant difference from post hoc analysis
The experimental data of H2 production in each culture was fitted using the Gompertz model
and the fitted curves were also presented in Figure 4.2. The coefficients of determination (R2)
of all of the fittings ranged from 0.96 to 0.98, suggesting that the model fits the experimental
data very well. The parameters, namely, the lag phase (λ), maximum H2 potential (P), and
maximum H2 production rate (Rmax) against operation time, were derived from the model and
the results are presented in Table 4.3. First of all, the λ changed from 1.4 days for the system
without biochar addition to 0.9 – 1.1 days with biochar additions. The Rmax of all cultures with
biochar (400 mL.L-1.per day) were higher than that of the control (302 mL.L-1.per day).
78
Similarly, the P of all cultures with any biochar addition was also higher than that of the control.
The highest P value was reached by the culture with 8.3 g.L-1 of biochar (981±24 mL.L-1).
Table 4.3 The results of calculation using the modified Gompertz fitting equation on H2
production with different biochar addition ratios
Biochar addition
(g.L-1)
λ (day) Rmax
(mL.L-1.per day)
P
(mL.L-1)
R2
0.0 1.4±0.1 302±32 749±22 0.96
8.3 0.9±0.1 400±37 981±24 0.97
16.6 1.1±0.1 400±41 884±23 0.97
25.1 1.0±0.1 400±31 886±17 0.98
33.3 0.9±0.1 400±36 855±18 0.97
Figure 4.3 shows the yields of VFA during H2 production phase on Days 2 and 8, respectively.
Generally, the cultures with biochar additions generated increasingly more VFA during H2
production than the control, starting from Day 2. However, towards the end of the
experimentation (day 8), the total VFAs showed little differences between the cultures with and
without biochar addition. It can be seen that the dominant VFA in the control and the culture
with 8.3 g.L-1 addition were different from those with higher biochar addition ratios (16.6; 25.1
and 33.3 g.L-1). The culture without biochar and with 8.3 g.L-1 biochar addition produced
butyric and acetic acids as dominant VFAs, while the cultures with higher biochar addition also
produced propionic acid in addition to butyric and acetic acids. This indicates that the dominant
pathway in the control and the culture with 8.3 g.L-1 biochar addition in H2 production was
butyric type of fermentation, while the other cultures, especially the culture added with 33.3
g.L-1 biochar were likely to follow the propionic pathway [181]. It is believed that the shift of
the reaction pathway was due to the organic overloading in the cultures with biochar addition.
At higher biochar additions (16.6; 25.1 and 33.3 g.L-1), the system will achieve a higher rate of
79
acidogenesis that leads to higher nicotinamide adenine dinucleotide (NADH/NAD+/). To
maintain a balanced NADH/NAD+ ratio, propionic acid fermentation was spontaneously
activated since more NAD+ was produced in propiogenesis than in butyrogenesis [180].
(a)
(b)
(c)
(d)
(e)
Figure 4.3 VFA profiles during H2 production in culture with (a) 0; (b) 8.3; (c) 16.6; (d)
25.1 and (e) 33.3 g.L-1 biochar addition ratios
Sharma et al. observed similar finding that the predominant acids were butyrate and acetate at
biochar addition ratios less than 12.5 g.L-1. The higher addition ratios of biochar (12.5 - 35 g.L-
80
1) was reported to increase propionic acid while decrease H2 production [128]. It is suggested
that the propionic acid follows H2 consuming pathway thus decrease H2 [128, 182].
The previous sections suggest that the addition of biochar enhanced H2 and VFA generation,
depending on the addition ratio. Other crucial factors affecting H2 production in the first phase
of TPAD are temperature and pH, because of their major influences on the microbial
metabolism [183, 184]. Understanding the interactions between biochar and operation
conditions including temperature and pH, therefore, is essential to optimise the AD process
[177, 178].
Extending the investigation reported in the previous section, this section reports systematic
examination of the effect of biochar addition on H2 production the first phase of the TPAD
under various initial pH and temperature conditions. The experiments were designed using the
central composite design (CCD) and response surface methodology (RSM) in order to obtain
the optimised condition [185, 186]. Scanning Electron Microscopy (SEM) analysis is used to
observe the morphological changes in the biochar before and after the experiment to reveal the
possible mechanism of biochar addition in enhancing H2 production and reported in Section
4.5.
4.3.1 Response surface analysis
The experimental data as listed in Table 4.4 were fitted using the quadric model as shown in
Equation 3.3 (Chapter 3) leading to the following correlations, respectively:
𝑌𝐻 = −4665.4 + 1138.9𝐴 + 119.3𝐵 + 92.2𝐶– 3.7𝐴𝐵– 1.3𝐴𝐶– 0.00625𝐵𝐶– 78.9𝐴2– 1.5𝐵2– 4.1𝐶2
(4.1)
81
𝑅𝐻 = −3649.3 + 563.9𝐴 + 121.3𝐵 + 94.2𝐶– 2.8𝐴𝐵 – 1.5𝐴𝐶 + 0.007𝐵𝐶– 31.8𝐴2– 1.6𝐵2– 4.2𝐶2
(4.2)
Table 4.4 Central composite design and experimental results for H2 production
Run Initial
pH
Temperature
(°C)
Biochar addition ratio
(g.L-1)
YH (mL.L-1) RH (mL.L-1.day-1)
1 4 25 15 800 334
2 8 25 15 950 582
3 6 35 10 1363 700
4 8 25 5 1060 662
5 6 35 10 1344 755
6 9.4 35 10 709 679
7 4 45 5 681 212
8 4 25 5 794 294
9 6 35 10 1310 690
10 6 35 10 1300 751
11 6 35 18.4 1094 459
12 8 45 15 539 278
13 6 35 10 1118 653
14 6 51.8 10 881 263
15 2.6 35 10 81 27
16 8 45 5 588 297
17 6 18.2 10 846 265
18 6 35 10 1424 858
19 4 45 15 624 195
82
Run Initial
pH
Temperature
(°C)
Biochar addition ratio
(g.L-1)
YH (mL.L-1) RH (mL.L-1.day-1)
20 6 35 1.6 901 371
Table 4.5 ANOVA analysis and the fitting model for YH
Source
Degree of
Freedom
Regression
coefficient
F
Value
p-value
Model 9 8.84 0.0011
Lack of fit 5 3.58 0.0938
R2 =0.89
A-pH 1 137.79 4.99 0.0494
B-temperature 1 -65.37 3.70 0.0834
C-biochar 1 5.30 0.039 0.8469
AB 1 -56.13 1.80 0.2093
AC 1 -15.24 0.059 0.8131
BC 1 0.36 3.174E-005 0.9956
A2 1 -127.17 58.43 < 0.0001
B2 1 -158.65 13.20 0.0046
C2 1 -105.25 6.18 0.0322
The ANOVA analysis was performed to evaluate the adequacy of the quadratic models and the
results are presented in Table 4.5 for YH and Table 4.6 for RH. As can be seen, the p-values of
both YH (p= 0.0010) and RH (p= 0.0006) are less than 0.05, indicating Equation 4.1 and
Equation 4.2 represent the experimental data very well [177]. The “lack of fit” describes the
fitness between the model predictions and the experimental data. The p-values of “lack of fit”
lower than 0.05 show a significant “lack of fit” of the model. Thus, a value > 0.05 is desired. In
this study, the p-values for both YH (p=0.09) and RH (p=0.11) are higher than 0.05 [187],
83
showing the “lack of fit” of the models were not significant. The R2 (squared regression
statistics) values of both fittings of YH and RH are 0.89 and 0.90, respectively. These values are
relatively high [179], showing that the quadratic models are sufficiently good to predict YH and
RH.
Table 4.6 ANOVA analysis and the fitting model for RH
Source
Degree of
Freedom
Regression
coefficient
F
Value
p-value
Model 9 10.16 0.0006
Lack of fit 5 3.16 0.1164
R2 = 0.90
A-pH 1 94.87 24.16 0.0006
B-temperature 1 -81.62 5.44 0.0419
C-biochar 1 8.41 0.036 0.8538
AB 1 -74.43 2.35 0.1563
AC 1 -13.47 0.17 0.6861
BC 1 -0.31 9.528E-005 0.9924
A2 1 -315.91 21.72 0.0009
B2 1 -150.17 33.80 0.0002
C2 1 -102.76 14.88 0.0032
Equations 4.1 and 4.2 derived from the quadratic model were further validated against the
experimental data. The experiments were performed under random combinations of the three
operation factors. The experimental conditions and the results of YH and RH are presented in
Table 4.7. YH and RM were also predicted using Equations 4.1 and 4.2 and the predicted results
are listed in Table 4.7. As seen from Table 4.7, the deviations between the experimental data
84
and the calculated results are less than 8% for YH while less than 10% for RM. This proves that
the quadratic model is adequately capable of predicting the H2 production.
The optimum biochar addition, initial pH and temperature were obtained using the numerical
optimisation function of Design Expert Software ver. 11 [188]. According to the optimisation
analyses, the optimum conditions with 0.93 desirability was found to be at the biochar addition
ratio 10.1 g.L-1, initial pH 6.4 and temperature 32C, reaching the maximum values of 1,330.7
mL.L-1 for YH and of 762.5 mL.L-1.day-1 for RH. The optimum addition of biochar at the range
tested was 10 g.L-1, which is at range of optimum biochar suggested by other studies. The
optimum biochar addition at the range of 10 -12.5 g.L-1 was reported to enhance H2 production
in several studies [113, 128]. Lin and Lay reported that in their study, H2 production was
occurred between pH 4 and 7, however, the maximum H2 was found to be at pH value between
6 and 7 [189], as suggested in this current study (pH 6.4).
Table 4.7 Model validation results
pH
Tempe-
rature
(°C)
Biochar
(g.L-1)
Cumulative YH (mL.L-1) RH (mL.L-1.day-1)
Predicted Experiment
Deviation
(%)
Predicted Experiment
Deviation
(%)
4 35 10 898 910 1 468 499 7
4 45 15 659 678 3 216 195 10
6 35 18.4 1033 1094 6 445 459 3
6 35 10 1309 1300 1 733 751 2
6.4 32 10.1 1331 1225 8 763 706 8
Normally, the first phase is operated at thermophilic condition for enhanced substrate
degradation [41]. Higher temperature may be beneficial for the reaction kinetics, but it is
typically followed by a rapid pH decrease which inhibits especially H2 production [102].
Appropriate temperature allows optimum germination, acclimatisation of bacteria to substrate
85
used in the system, carbon consumption rate and partial pressure of the produced gas [41, 77].
Other study suggested that at operating temperature range of 25-45C, the maximum H2
production was achieved at 35C. The reactor produced the minimum production at 25C and
inhibited at 45C [77]. In this current study, the similar optimum temperature was suggested
(32C).
4.3.2 Hydrogen yield
The response surface plots from the RSM analysis showing the influence of interactions
between variable on the YH are presented in Figure 4.4. Figure 4.4(a) illustrates the effect of
biochar addition ratio on YH at various pH values at 32C. The two-dimensional contour curves
beneath the 3D surface show a clear elongated running diagonally shape. This indicates a
significant interaction between biochar and initial pH [179]. It is evident that, at each pH, YH
increased first and then decreased with increasing biochar addition ratio from 1.6 to 18.4 g.L-1,
reaching a maximum value at biochar addition ratio 10g.L-1. The maximum YH increased with
increasing initial pH, peaking at pH 6 and then decreased as the initial pH was further increased
to 9.4.
As can be seen from Figure 4.4(a), the effect of biochar in enhancing YH was more profound at
a lower pH. For instance, at pH 4 and temperature 35C, the 10 g.L-1 biochar addition increased
YH by 57% compared to biochar addition 1.6%, while at pH 6, the increase was 30%. Starting
at low initial pH, the culture experienced delay in H2 production caused by longer adaptation
period [160, 190, 191]. Also, it is reported that at the pH below 4, the hydrogenase activity
declined [183]. The biochar used in the study, contains 10wt% of volatile matter and
micronutrients (Na, K, Mg, Fe) which was suspected to enriched the microbial growth and
initiated H2 production. In addition, our previous study showed that biochar addition prevented
a sharp pH decrease caused by accumulation of the acids produced in the first phase of TPAD
[98, 131, 161].
86
Figure 4.4 Response surface and contour plots of cumulative H2 yield (YH) over 8 days of
operation as a function of: (a) initial pH and biochar addition ratio at 32C and
(b) temperature and biochar addition ratio at initial pH 6.4
Figure 4.4 (b) shows the effect of biochar addition on YH at different temperatures while at a
constant initial pH 6.4. It is clear that, at each temperature, YH increased first with increasing
biochar addition ratio, reached the maximum at the biochar addition ratio 10 g.L-1, and then
decreased as biochar addition ratio rose to 18.4 g.L-1. As the temperature changed from 18 to
52 C, the maximum YH increased first and then decreased, reaching a peak value at temperature
(b)
(a)
87
35 C. It is noted that the influence of biochar addition ratios on YH at various temperatures was
similar, suggesting that the biochar effect on YH was not sensitive to changes in temperature. It
is estimated that the addition of biochar from 1.6 to 10 g.L-1 increased the YH around 30-39%
at temperature range 25 – 35%. Temperature was found to more significantly affected RH as
discussed in Section 4.3.3.
4.3.3 Hydrogen production rate
Figure 4.5 shows the 3D surface responses and the contour plots of combined effects of (a)
biochar addition and initial pH (b) biochar addition and temperature on RH. As seen from Figure
4.5 (a), it is clear that the effect of biochar on RM was significantly affected by initial pH of the
cultures. At each initial pH, RH reached a maximum value at the 10g.L-1 biochar addition ratio.
However, the maximum RH changed significantly at different initial pH. For instance, with the
addition of 10 g.L-1, RH increased drastically from 27 mL.L-1 at initial pH 2.6 to 733 mL.L-1 at
initial pH 6, remained almost constant at pH 8 (744mL.L-1), and decreased gradually at pH 9.4
(600 mL.L-1).
This implies that the effect of biochar in enhancing RH was more profound at lower pH. For
example, at initial pH 4, as the biochar addition ratio increased from 1.6 to 10 g.L-1, RH
increased by almost three folds, whereas at initial pH 6, RH only increased by 83%. At higher
pH, it has been reported that RH of the cultures plummeted three folds as the initial pH increased
from 6 to 7 [91] due to the poor ability for the cultures to degrade glucose used in their study.
However, at the optimum biochar addition ratio 10 g.L-1, as observed in this study, RH remained
almost stable while pH was increased from 6 to 8. This indicates that biochar could help stabilise
the activity of the cultures in degrading feedstock to acids and converting it to H2, at higher
initial pH.
88
Figure 4.5 Response surface and contour plots of maximum H2 production rate (RH) as
function of: (a) initial pH and biochar addition ratio at 32C; and (b) temperature
and biochar addition ratio at initial pH 6.4
Figure 4.5(b) shows the effect of biochar addition ratios on RH at different temperatures at initial
constant pH 6.4. At each temperature tested, the RH peaked at 10 g.L-1 addition of biochar.
However, the value of maximum RH varied significantly, increasing from 395 mL.L-1.day-1 at
temperature 18C to 733 mL.L-1.day-1 at the temperature 35C, and decreased to 176 mL.L-
1.day-1 at 52C. The microbes in cultures were more sensitive to higher temperatures. This is
consistent with literature reports that too high temperatures could deactivate and denature
(a)
(b)
89
enzyme and related proteins [192]. It is clear that with the biochar addition, RM substantially
increased regardless of temperature. However, such enhanced behaviour due to biochar addition
was more remarkable at higher temperatures. For example, the RH increased by 150% when the
biochar addition ratio increased from 1.6 to 10 g.L-1 at 45C, compared to only 72% at 35C
with the same addition ratio of biochar. The possible mechanisms of biochar promoting H2
production will be elaborated in Section 4.4 and 4.5.
4.4 Effect of biochar on H2 production via anaerobic digestion as
compared to other solid additives: role of acidity
In the previous sections, it has found that biochar addition significantly increased the H2 yield
by 31% in the first phase of a two-phase anaerobic digestion [161]. It was hypothesised that the
acidity and surface structure of the biochar play an important role. The high accumulation of
volatile fatty acid (VFA) in AD would reduce the pH of the culture supressing H2 production.
Alkaline additives have been proposed to add into AD to maintain or increase the alkalinity of
the system [193]. Several alkaline additives such as lime mud from paper making and calcined-
red mud showed positive effects on the H2 production [73, 194, 195]. Biochar can be alkaline
depending on the source of biomass and the pyrolysis temperature [193]. This section reports a
systematic investigation into the possible working mechanisms of biochar in enhancing H2
production in the first phase of TPAD. In order to achieve this objective, the effect of biochar
and several other solid additives with different acidity as references, namely alumina, glass and
zeolite, were tested and compared. The effect of these additives on the cumulative H2
production (YH), H2 production rate (RH) and pH during the operation were monitored.
90
Figure 4.6 The pH of liquid culture with the addition of the additives before the pH
adjustment
When the additives were mixed with the feedstock and sludge, they affected the pH of the
culture significantly. Figure 4.6 shows the pH of the culture with the addition of the additives
before it was adjusted to initial pH 6.4. The pH of the control and the culture with glass addition
were similar at 6.7. It is clear that the addition of glass particles into the cultures did not alter
the initial pH because the pH of the glass is neutral. The addition of alumina and biochar brought
the pH of the culture higher than that of the control. The pH of the culture dropped significantly
to 6.3 with the zeolite addition.
Figure 4.7 shows the daily H2 production rate (RH) of the cultures with addition of different
additives. The culture started to produce H2 after 12 hours of incubation. The culture with
alumina addition produced the highest rate of H2 on the first day, followed by the cultures with
additions of biochar. The glass bead addition did not change the H2 production rate, as expected.
The zeolite addition slowed down the H2 production. Most of the cultures reached the peak
production rate on the first day except the culture with zeolite addition, which experienced a
significant delay of reaching the peak value on day 2. The H2 production of all cultures with
91
and without the addition of the additives dropped on the day 2.5 and stopped producing H2 on
day 3.
Figure 4.7 Production rates of H2 from cultures with different types of additives
Figure 4.8 shows the comparison of cumulative H2 yields (YH) of the cultures with addition of
different additives during 7 days of incubation. The cultures with biochar and alumina addition
produced similar YH, which was much higher than the other cultures. The glass particle addition
did not affect YH while the zeolite addition showed slightly higher YH compared to the control.
It is clear that the cultures with addition of biochar and alumina had shorter lag phase than the
rest of the treatment, while the culture with zeolite addition experienced a delay in H2
production.
92
Figure 4.8 Cumulative yields of H2 from cultures with addition of different additives
The experimental data was then fitted using Gompertz model to obtain the maximum H2
production potential (P), maximum H2 production rate (Rmax) and lag phase () [196]. Table
4.8 shows a good agreement of the experimental data with the model fitting with the R2 of 0.97-
0.99. The fitting curves are also shown in Figure 4.8. The P values of the cultures with biochar
and alumina additions were predicted to be 1,234±14 and 1,216±14 mL H2.L-1, being higher
than the control by ca. 10%. However, the addition of glass particle and zeolite did not change
the P. The addition of different additives also significantly affected the Rmax of the culture. The
Rmax of the cultures with biochar and alumina additions were projected to be 1,148±98 and
1,076±120 ml H2.l-1.day-1, which was 32 and 24% higher than that of the control, respectively.
The calculated suggests that the zeolite addition would delay the hydrogen production, which
is consistent with the experimental observations.
93
Table 4.8 The results of calculation using the modified Gompertz fitting equation on
hydrogen production with different additives
Parameter Control Biochar Alumina Glass Zeolite
R2 0.99 0.99 0.97 0.99 0.99
P (ml H2.l-1) 1118±12 1234±14 1216±20 1127±8 1144±12
Rmax (ml H2.l-1.day-1) 870±60 1148±98 1076±120 969±54 957±67
λ (days) 0.59±0.05 0.59±0.05 0.51±0.07 0.62±0.03 0.65±0.05
Figure 4.9 The pH evolution of the cultures with different types of additives
The changes of pH of each treatment during the whole period of incubation are shown in Figure
4.9. It is clearly seen that the addition of biochar and alumina brought the initial pH of the
culture from 6.7 to 7.0 and 7.5, respectively, while the zeolite addition reduced the pH of the
culture. Prior to the incubation, the pH of all cultures was adjusted to 6.4. During the incubation,
the pH of all the cultures dropped significantly on day 1 and further decreased on day 2 but
slightly increased on the final day. However, the pH of the cultures with biochar and alumina
additions had higher pH than that of the control except on day 1. The culture with the zeolite
94
addition also showed a slightly higher pH than the control although zeolite itself had a very low
pH value.
The results suggest that the alumina and biochar addition in the culture generally increased both
RM and YH compared to the control while the zeolite addition suppressed H2 production. The
possible working mechanisms of biochar are discussed as follows. The biochar used in current
study is a pine wood biochar with a pH of 8.6. It is known that high pH promoted H2 production
in the AD [190, 197]. The biochar and alumina addition significantly increased the pH value of
the culture. Therefore, in the beginning of the incubation, the cultures with biochar and alumina
addition produced significantly higher amount of H2 compared to the control. The rapid H2
production usually results in quick accumulation of acids, decreasing the pH of the culture [14].
This explains why all the cultures showed a pH drop and the cultures with biochar and alumina
addition experienced even a greater pH drop compared to the control on day 1 (Figure 4.9).
However, as the incubation time increased, the cultures with biochar and alumina additions
produced more H2 but also exhibited higher pH in comparison with the control. This suggests
that the biochar and alumina acted as a pH buffer of the cultures. The biochar contains some
alkaline groups that may act as neutralizer for certain acids [73]. Due to the high acidity, the
addition of zeolite reduced the pH of the culture significantly (Figure 4.9 - initial). This explains
why the culture with the zeolite addition produced a very low amount of H2 on the first day and
it took a longer time for the culture to adapt the environment (Figure 3). Due to the slow H2
production rate, the amount of acids generated during the incubation process was low, which
was inadequate to cause dramatic pH drop as experienced by the rest of the treatment as
observed in Figure 4.9.
95
4.5 Mechanisms
The preceding discussion has shown that biochar was beneficial in enhancing H2 in TPAD. A
mechanism of the working of biochar in enhancing H2 production in TPAD has been proposed,
as shown in Figure 4.10, and is discussed as follows.
Figure 4.10 The mechanisms of biochar in promoting H2 and CH4 productions in TPAD from
food waste
The results show that the biochar addition in the culture promoted H2 production at all initial
pH and temperature tested. In the H2 production process, it is believed that biochar enhanced
biofilm formation [3], provided temporary nutrients for microbial growth and buffering pH of
the culture. A possible mechanism of biochar in enhancing H2 is that the biochar provides
surface area to initiate biofilm formation to immobilise microbes. Biochar has high surface area
(130 m2.g-1), facilitating microbial colonisation in the anaerobic digestion process [27]. Figure
4.10 shows the SEM images of the biochar samples before and after anaerobic digestion of
culture with 10 g.L-1 addition, at initial pH 6 and 35ºC. It is observed that there was the clear
development of biofilm on the surface of the biochar and the rod shape of bacteria on the surface
of biofilm was also evident.
The length of bacteria ranges from 2-5 µm, a typical characteristic of H2 producing bacteria
(HPB) as reported by Yin et al [198]. The development of biofilm in the biochar is explained
as follow. The biochar used in this study has a high specific surface area (130 m2g-1) which
provides the surface area for the initial attachment of the microbes. It is known that the microbes
First phase
▪ Promoting biofilm formation
▪ Providing temporary nutrients
▪ Buffering pH
Biochar
Results:
▪ Shorter lag phase
▪ Faster VFA generation
▪ Higher H2 production rate
▪ Higher cumulative H2 production
96
could produce extracellular polymeric substances (EPS), which consist of polysaccharides,
proteins, nucleic acids and lipids [85]. EPS is responsible for the nutrient supply, stability of
the bacteria and act as polymer bridges between microbes [85, 126, 199]. Once the microbes
were attached on the surface of the biochar, the microbial division, growth and colonisation
happened. The enriched microbial population in the microbial carrier has reported to enhance
biogas production [124, 199], which was confirmed by the results of the current study.
Figure 4.11 SEM images of (a) biochar and (b) final effluent of H2 production
HPB
Biofilm
Biochar
(a)
(b)
97
In addition, as Lehmann [27] stated that biochar produced from low temperature pyrolysis
contains temporary substrate to support microbial metabolism and growth [27]. Our previous
study showed that the biochar used in this study contained 10 wt% of volatile matter that would
serve as temporary nutrients in the beginning of H2 production [200]. It also contains some
beneficial temporary micronutrients that may be useful for microbial growth and enzyme
activation. Our previous study has found that the biochar leached sodium (Na) (183 mg.kg
biochar-1) and potassium(K) (1639 mg.kg biochar-1) at a leaching temperature of 30ºC for 24
hours [110]. Light metals such as Na+ and K+ are necessary for microbial growth [123, 201].
The Na+, especially, is useful for cofactor of bacterial enzyme, transport processes, the
formation of adenosine phosphate (ADH) and dehydrogenase [123, 201]. Lin et al reported that
Na, together with magnesium (Mg), zinc (Zn) and iron (Fe) were important trace elements in
H2 production from synthetic substrate using anaerobic sewage sludge as the inoculum and
incubated at 35ºC [201, 202]. It is also suggested that at moderate concentration (< 400 mg.l-1),
K stimulates AD process, promoting H2 production [41, 125, 203]. The provision of temporary
nutrients and trace element supported microbial growth and activity in the H2 production. It is
evident that biochar addition shortened the lag phase and produced higher VFA in cultures.
Finally, it is also worth noticing that at the end of the AD experiment (Section 4.1), the pH of
the control was 3.0 while the pH of the cultures with 16.6; 25.1 and 33.3 g.L-1 biochar additions
was 3.5; 3.7 and 3.7, respectively. This pH stability prevented the culture from significant
decrease in the pH caused by VFA accumulation, which is known to inhibit anaerobic digestion
[204]. This allowed the systems with biochar addition to achieve a higher H2 production rate
and accumulation.
Also, the biochar addition increased the YH in all pH tested in bench scale experiment under
different operating conditions, particularly at the lower pH. At the lower pH, the culture usually
produced lower amount of H2 since it prevents a further use of feedstock for H2 production due
to rapid acid inhibition and pH drop [190, 205]. The biochar used in this study is an alkaline
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additive (pH 8.6) which help alleviate the negative effect of pH drop due to the accumulation
of volatile fatty acid (VFA) [205]. Biochar also contains some alkaline groups that may act as
neutraliser for certain acids [73], thus supports the culture to produce higher H2 production.
Further experiment suggests that biochar behaves similarly with the alumina as a reference. The
biochar and alumina addition significantly increased the pH value of the culture. Therefore, the
cultures with biochar and alumina addition produced significantly higher amount of H2 and a
greater pH drop compared to the control, in the beginning of the incubation. However, the
cultures produced more H2 but also exhibited higher pH in comparison with the control during
the rest of the operation. This suggests that the biochar and alumina acted as a pH buffer of the
cultures, enhancing the H2 production.
4.6 Summary
The effect of biochar addition on the H2 and CH4 production in laboratory scale TPAD of
simulated carbohydrate food waste was studied systematically. Biochar addition was shown to
shorten the lag phase by 21.4 to 35.7 %, increased the maximum production rate by 32.4% and
H2 production potential by 14.2 to 31 % of H2.
Further investigation on the effect of biochar addition under different initial pH and temperature
on the cumulative H2 yield (YH) and maximum daily production rate (RH) in the first phase of
TPAD was conducted and optimised using the response surface methodology (RSM). The RSM
analysis showed that the maximum YH of 1,331 mL.L-1 and RH of 763 mL.L-1.day-1 could be
achieved under the optimum conditions of biochar addition ratio 10.1 g.L-1, initial pH 6.4 and
temperature 32C. Biochar addition was shown to substantially increase YH, especially at lower
pH and higher temperatures.
It is observed that the biochar initiates the biofilm formation and provides temporary nutrients
in the culture, enriching the microbial population. Also, the addition of biochar was observed
99
to bring the condition of the culture from acidic to alkaline at the beginning of operation and
prevented significant pH drop during incubation. As a result, the cultures with biochar additions
generated more H2 at faster rates.
100
Chapter 5 Effect of Biochar Addition on Methane Production
5.1 Introduction
Chapter 5 reports the experimental study on the bench scale of the CH4 production. Section 5.2
discusses the CH4 production without biochar addition. Section 5.3 presents the results on the
effect of biochar addition under different initial pH and temperature according to Box Behnken
Design (BBD) and response surface methodology (RSM). The result on additional experiment
on the single phase anaerobic is reported in Section 5.4. Possible mechanisms of the biochar in
enhancing CH4 in the second phase of TPAD are proposed in Section 5.5.
5.2 Methane production without biochar addition
Figure 5.1 Cumulative yields and production rates of CH4 without biochar addition
As shown in Figure 5.1, in the second phase, the culture started to produce a substantial amount
of CH4 on day 9. The CH4 production continued and achieved the peak production rate on day
15 with 139 mL.L-1 of CH4 followed by a significant drop on day 19. The CH4 production
completely stopped on day 39. The maximum cumulative CH4 production in the end of
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operation was 1070±3 mL.L-1. CH4 composition in the biogas produced from the system at the
conclusion of an experimental run was found to be in the range of 55-78% (Table 5.1). The H2
production was not observed in the second phase. The fresh sludge containing methanogenesis
added into the second phase converted the acetic acids produced in the first phase to CH4.
Therefore, there was no H2 was detected in the second phase. Low amount of VFA remained in
the final effluent of the second phase. The acetic acid decreased from 1063 to 78 mL.g-1 at the
end of the first phase, while butyric decreased from 1172 to 44 mL.g-1.
Table 5.1 Profiles of the second phase of TPAD without biochar
Parameters Unit Value
Cumulative CH4 production mL.L-1 1070±3
Gas composition
H2 % 0
CH4 % 55-78
CO2 % 22-45
VFA mg.L-1
Acetic acid mg.L-1 78±34
Butyric acid mg.L-1 44±15
Propionic acid mg.L-1 119±0
Final pH 6.7±0.1
5.3 Methane production with biochar addition
The cumulative CH4 production of the cultures with and without biochar is shown in Figure
5.2. In the beginning, all cultures with biochar addition produced higher CH4 yields than the
control. However, from day 29 until the end of the experiment, the cultures with 25.1 and 33.3
g.L-1 biochar additions started to show less CH4 yields and production rate than the control. At
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the end of the experiment, the highest cumulative CH4 production was achieved by the culture
with 8.3 g.L-1 biochar addition, while the culture with 33.3 g.L-1 biochar addition exhibited the
lowest cumulative CH4 production, even lower than that of the control.
Figure 5.2 Cumulative CH4 yields at different biochar addition ratios
Table 5.2 One-way ANOVA and post hoc analysis on cumulative CH4 production in
different biochar addition ratios
Biochar addition
(g.L-1)
Average cumulative CH4
yields (mL.L-1)
Standard deviation P value
0 1070.0b 5.1 0.002
8.3 1136.6b 7.0 0.002
16.6 1057.3b 8.7 0.002
25.1 956.2a 4.9 0.002
33.3 931.7a 1.8 0.002
ANOVA analysis on the cumulative CH4 production data shows a significant difference in
methane production among the various treatments as listed in Table 5.2. However, the LSD
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post hoc test suggested that the cumulative CH4 yields of cultures with 0, 8.3 and 16.6 g.L-1
biochar addition were not significantly different between each other but they were significantly
higher than that of cultures with 25.1 and 33.33 g.L-1 biochar addition according to a post hoc
analysis.
Table 5.3 The results of calculation using the modified Gompertz fitting equation on CH4
production with different biochar addition ratios
Biochar
addition (g.L-1)
λ (day)
Rmax
P (mL.L-1) R2
(mL.L-1.per day)
0 10.0±0.2 113±5 1027±9 0.98
8.3 5.9±0.2 156±7 1126±7 0.98
16.6 5.7±0.1 160±3 1046±3 0.99
25.1 5.5±0.1 145±3 949±2 0.99
33.3 5.7±0.2 138±8 918±6 0.98
The experimental data of cumulative methane production was also fitted using the Gompertz
model and the fitted curves were presented in Figure 5.2. The R2 of all of the fittings ranged
from 0.98 to 0.99, suggesting a good fitting between experimental data and the modelling
results, thus the feasibility of using the Gompertz model for the calculation of λ, P and Rmax.
The λ, P, and Rmax against operation time derived from the model are presented in Table 5.3.
As shown in Table 5.3, the λ of the cultures with various biochar additions were in the range of
5.5-5.9 days, while the lag phase of the control was 10.0 days. It is also clear that the Rmax of
the cultures with biochar additions were higher than that of the control. However, the Rmax
increased as the biochar addition increased from 0 to 16.6 g.L-1 biochar and then decreased as
the biochar addition was further increased to 33.3 g.L-1.The P of the cultures with 8.3 and 16.6
g.L-1 additions were higher than that of the control, whereas, the cultures with higher biochar
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additions showed lower P. The highest P was achieved for the culture with 8.3 g.L-1 biochar
addition. A proposed detailed mechanism of biochar effects on CH4 production are explained
in section 5.2.3.
(a)
(b)
(c)
(d)
(e)
Figure 5.3 VFA profiles during CH4 production in culture with (a) 0; (b) 8.3; (c) 16.6; (d)
25.1 and (e) 33.3 g.L-1 biochar addition ratios
The VFA profiles of the cultures on Day 7, 14, 21, and at the end of the experiment were
illustrated in Figure 5.3. The results confirm that the dominant VFA were acetic, acetate and
105
propionic acids [180]in all cultures. All cultures with biochar addition degraded VFA faster
than without biochar during the first 14 days of the experiment. VFA of all cultures significantly
degraded by Day 21 and propionic acid accumulated in all cultures. The propionic acid content
became higher with increasing biochar addition. In the end of the experiment, the cultures with
25.1 and 33.3 g.L-1 biochar addition had more propionic acid accumulation. This might be the
cause of lower CH4 production of these two cultures.
In addition, as shown in Figure 5.3, final cultures with 25.1 and 33.3 g.L-1 biochar remained in
the reactors, which were used as influent for methane production, had lower quantities of VFA
and higher amounts of propionic acid. According to Wang et al (2009) and Amani et al (2011),
the energy to anaerobically oxidize propionic acid to acetate (+76.1 kJmol-1) is almost doubled
as compared to that for butyrate (+48.1 kJ.mol-1). Therefore, the accumulation of propionic acid
in the culture resulted in a slower acetogenic rate of propionic acid [180, 206], as a consequence,
a lower CH4 production rate. The accumulation of propionic acid also led to low pH. When the
pH is reduced to below 7.0 methanogenic activity is often inhibited [58].
The pH and temperature are the two important factors affecting CH4 production in the second
phase of TPAD due to their influences on microbial growth and activity and metabolic products
generated [86]. It is well known that the optimal conditions for CH4 production are in the pH
range from 6.7 to 7.4 [207] and more stable under mesophilic conditions [2, 105]. However,
the behaviour of the cultures with biochar addition under different initial pH and temperature
regimes is still not clear. Therefore, the present chapter reports the examination of the effect of
biochar addition on CH4 production in the second phase of TPAD, utilising the pre-digested
feedstock from the first phase of TPAD (as prepared in Section 3.2.3.2), under different initial
pH and temperature conditions.
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5.3.1 Response surface analysis
The experimental data as listed in Table 5.4 were fitted using the quadric model as shown in
Equation 3.3 resulting in the following equations:
𝑌𝑀 = −7047.15 − 54.13𝐴 + 290.33𝐵 + 863.49𝐶 − 1.58𝐴𝐵 + 7.60𝐴𝐶 + 11.51𝐵𝐶 +
3.54𝐴2 − 4.97𝐵2 − 83.70𝐶2
(5.1)
𝑅𝑀 = −1837.63 − 12.75𝐴 + 45.08𝐵 + 323.08𝐶 − 0.33𝐴𝐵 + 0.59𝐴𝐶 + 5.31𝐵𝐶 +
0.83𝐴2 − 0.97𝐵2 − 31.61𝐶2
(5.2)
Table 5.4 Box Behnken design and experimental results for CH4 production
Run Factor 1
A: Biochar
addition (g.L-1)
Factor 2
B: Temperature
(oC)
Factor 3
C: initial
pH
Response
1 YM
(mL.L-1)
Response 2 RM
(mL.L-1. day-1)
1 10 35 7 1532.8 388.4
2 10 45 9 1392.2 525.1
3 10 45 5 0.3 0.2
4 10 25 5 582.2 137.134
5 15 35 5 1183.8 212.5
6 5 45 7 1457.9 616.3
7 15 35 9 1770.6 492.1
8 10 35 7 1771.2 508
9 15 45 7 1325.4 494.6
10 10 35 7 1655.2 474.3
11 5 25 7 877.4 216.6
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Run Factor 1
A: Biochar
addition (g.L-1)
Factor 2
B: Temperature
(oC)
Factor 3
C: initial
pH
Response
1 YM
(mL.L-1)
Response 2 RM
(mL.L-1. day-1)
12 5 35 9 1348.3 460.7
13 15 25 7 1060.3 160.3
14 10 35 7 1490.1 442
15 10 35 7 1492.1 429
16 5 35 5 1065.6 205
17 10 25 9 1052.7 237
Table 5.5 lists the results of ANOVA analysis of the fitting models of both YM and RM. The
probability (p value) of the quadratic models for both YM and RM were less than 0.05, indicating
the significance of the quadratic model in fitting and representing the experimental data. The
R2 (squared regression statistics) values of both fittings of YM and RM are 0.89 and 0.90,
respectively. These values are relatively high [179], showing that the quadratic models are
sufficiently good to predict YM and RM.
Using Design Expert ver. 11, the optimum biochar addition, temperature and initial pH were
then optimised. The calculation suggested the optimum biochar addition; temperature and
initial pH were 12.5 g.L-1, 36.2oC and 7.8, respectively. In the optimum condition, the YM and
RM were estimated to be 1755.3 mL.L-1 and 500.9 mL.L-1.day-1. The optimum addition of
biochar at the range tested was 12.5 g.L-1, which is at range of optimum biochar suggested by
other studies.
Literature suggest that the optimum biochar was at 5- 70 g.L-1 depending on the AD feedstock
and the type of biochar [3, 131]. It is well known that the optimum pH range for CH4 production
is 6.7 – 7.4, which is slightly lower than the optimum pH suggested by the current study (7.8).
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Normally, the second phase is operated under mesophilic condition for higher CH4 production
[41]. Optimum temperatures for microbial growth in typical mesophilic CH4 production are
listed at range 30 – 40C, depending on the source of inoculum and microbial genus, which also
reported in this study (36.2C) [45, 99-101].
Table 5.5 ANOVA analysis and the fitting model for YM and RM
Source
YM RM
Degree of
freedom
Regression
coefficient
p-
value
Degree of
freedom
Regression
coefficient
p-
value
Model 9 0.0126 9 0.0153
R2 0.8885 0.8814
A-biochar
addition
1 -54.13 0.3883 1 -12.75 0.6045
B-temperature 1 +290.33 0.3790 1 +45.08 0.0107
C-pH 1 +863.39 0.0038 1 +323.08 0.0027
AB 1 -1.58 0.5099 1 -0.33 0.7291
AC 1 +7.60 0.5247 1 +0.60 0.8989
BC 1 +11.52 0.0821 1 +5.31 0.0516
A2 1 +3.54 0.4497 1 +0.83 0.6544
B2 1 -4.97 0.0028 1 -0.97 0.0641
C2 1 -83.70 0.0193 1 -31.61 0.0243
5.3.2 Methane yield
The response surface plots from the RSM analysis showing the influence of interactions
between variable on the YM are presented in Figure 5.4. Figure 5.4 (a) shows the effect of
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biochar on the YM under different temperatures at optimum pH 7.8. Regardless of the
temperature, the YM increased slightly with the increase of biochar from 5 to 15 g.L-1. It is
evident that the temperature affected the YM significantly. The value of YM increased as the
temperature increased, reaching its peak at 35 ºC then slightly decreased as the temperature was
further increased to 45ºC. The YM peaked at temperature 35 ºC regardless of biochar addition.
(a)
(b)
Figure 5.4 Response surface and contour plots of CH4 yield (YM) as function of: (a) biochar
addition and temperature at initial pH 7.8 and (b) biochar addition and initial pH
at temperature 36.2ºC
110
Figure 5.4 (b) illustrates the effect of biochar on the YM at varying initial pH at temperature
36.2 ºC. It is clear that at low initial pH, biochar at various addition ratios gave similar YM. At
high initial pH, a higher biochar addition ratio resulted in higher YM. It is also noted that initial
pH greatly affected the YM. The YM was similar at initial pH 7 and pH 9, while the YM at pH 5
was very low.
5.3.3 Methane production rate
Figure 5.5 Response surface and contour plots of maximum CH4 production rate (RM) as
function of: (a) biochar addition and temperature at initial pH 7.8 and (b) biochar
addition and initial pH at temperature 36.2ºC
111
Figure 5.5 illustrates the response surface plots of RM as a function of various factors. Figure
5.5 (a) shows that the increase in temperature significantly increased the RM. Regardless of the
biochar addition ratio, the highest value of RM was always achieved under the thermophilic
conditions.
It is noted from Figure 5.5 (b) that higher initial pH led to greater RM regardless of biochar
addition ratio. This suggests that alkaline conditions support CH4 production in the culture. It
has been reported [208] that alkaline conditions promote activity of methanogens and a
shortened lag phase.
Comparing the YM and RM among cultures, the higher temperature led to the higher RM of the
cultures. However, while the higher initial pH and temperature led to the highest RM, it is noted
that at the end, they produced a similar amount of YM to the cultures set at neutral pH and
mesophilic temperature. Parawira et al [105] reported in their study of TPAD of potato waste,
thermophilic condition is to be preferred to shorten the operation duration, while mesophilic
condition supports the system to achieve the higher CH4 yield.
5.4 Methane production in single phase anaerobic digestion
A single-phase AD simulation was conducted using the same feedstock concentration and
operating conditions as the experiment in Section 5.3.2. The single phase of AD was added with
10 (g VS). L-1 of fresh bread, 10 g.L-1 of biochar set at initial pH 7 and incubated at temperature
35C.
The result shows that with the addition of 10 (g VS). L-1 of white bread, the culture produced
H2 instead of CH4. The reactor started producing H2 on the first day, achieving the peak of 730
mL.L-1. The H2 production then gradually decreased and stopped on day 4. The total cumulative
H2 production was 1079 mL.L-1. The experiment was extended until 14 days, however the CH4
production was not observed until the end of experiment. The result suggests that the SPAD
112
simulated in the study may suffer from feedstock overloading. In the early stages of SPAD, the
accumulation of acid often decreases the pH (4.2), while the inhibitory level of VFA is increased
significantly. These factors disrupt the stability of the SPAD thus led reactor to a failure [47].
A conventional AD treating industrial wastewater is reported to be unfeasible at a loading rate
of 2 gr VS.l-1.day-1 or higher [48]. The high feedstock concentration in this study disrupted the
culture, thus it failed producing CH4.
The culture treating the effluent from the first phase TPAD showed CH4 production was 1533
mL.L-1 (Table 5.4). The separation of both phases allowed the hydrolysis and acidogenesis to
occur in the first phase, therefore the second reactor was prevented from the acid accumulation.
The VFA produced in the first reactor was then converted into CH4 in the second phase. The
result of this experiment suggests that the separation of the acidogenesis and methanogenesis
allowed the TPAD to produce CH4 particularly in the high concentration of feeding.
5.5 Mechanisms
In the CH4 production process, it is believed that biochar mostly acted as a good microbial
carrier in enhancing methane production. According to recent studies of the effect of biochar
addition on CH4 production in conventional single phase AD [3, 83], the porous structure of
biochar acted as a microbial carrier and favoured biofilm development, accommodating a wide
range of microbial population including acido, aceto and methanogens [140].
Figure 5.6 shows the SEM images of the biochar samples before and after 40 days of anaerobic
digestion at initial pH 7 and temperature 35 ºC. It is clear that there was biofilm on the surface
of the biochar. The rod (bacillus) shape of bacteria on the surface of biofilm was also observed
as shown on Figure 5.6 (b). Biofilm allows the colonisation of bacteria, fungi and protozoa
[123, 126], which benefits CH4 production in two ways. Firstly, it provides more surface area
available for microbial growth. The biofilm allows more intense cross feeding, co-metabolism
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and H2 and proton transfer, which then enrich microbial growth and initiate the microbial
colonisation [124]. Secondly, it immobilises microbes, increasing the robustness of the AD
system. With a microbe attached to the biofilm, it has a lower probability of loss of microbes.
The promoted formation of biofilm is assumed to enhance microbial activity, leading to a
shorter lag phase, faster VFA degradation and CH4 production in the cultures with biochar.
Figure 5.6 SEM images of (a) the fresh biochar and (b) biochar after 40 days of incubation
at 35ºC
(b)
(a)
114
Note that pH values of the control (6.72) and cultures with biochar additions (6.96) were similar
(experiment Section 5.1). Therefore, it is assumed that unlike in the first phase, the pH buffering
capacity of biochar did not directly affect the pH condition of the cultures in the second phase
of TPAD. In other words, the pH buffering capacity of biochar is not a critical factor in
promoting CH4 production in the second phase.
5.6 Summary
A preliminary study on the effect of biochar addition on the CH4 production in laboratory scale
TPAD of simulated carbohydrate food waste was conducted. The biochar addition shortened
the lag phase by 41 to 45 %, increased the maximum production rate by 23.0-41.6% and CH4
production potential by 1.9 to 9.6% of CH4. Furthermore, the effect of biochar addition on CH4
production in the second phase of TPAD under varying initial pH and temperature conditions
has been studied. A moderate level of biochar addition, mesophilic temperature and neutral to
alkaline initial pH were shown to benefit CH4 production in the second phase of TPAD. In a
typical CH4 production process at initial pH 7 and 35 C, 10 g.L-1 of biochar addition increased
YM by 14% relative to cultures without biochar addition.
The effect of biochar addition was more profound at higher pH. The optimum biochar addition;
temperature and initial pH were found to be 12.5 g.L-1, 36.2oC and 7.8, respectively. Under the
optimum condition, the YM and RM were 1755 mL.L-1 and 500.9 mL.L-1.day-1. The SEM
analysis revealed the development of biofilm and the accumulation of the bacillus rods of
bacteria on the surface of biochar. The promoted methanogenic biofilm formation in the second
phase of TPAD may contribute to the enriched methanogens and enhanced CH4 production
An additional experiment of single-phase AD using fresh food waste was conducted. The result
suggests that at a high concentration of feedstock, the single-phase AD suffered from acid
accumulation thus failed to produce CH4, while the second phase TPAD successfully produced
115
substantial amount of CH4. It is due to the separation of acidogenesis and methanogenesis,
which favour CH4 production in the second phase AD.
116
Chapter 6 Transient Performance during Start-up of Two-Phase
Anaerobic Digestion Process Demonstration Unit
6.1 Introduction
Although there is a growing interest in biochar utilisation in AD, there are very limited reports
on the effect of biochar addition in a demonstration or pilot scale operation [140]. The studies
on the pilot scale operation of TPAD are necessary to confirm the results of bench/laboratory
studies and to investigate the technical challenges and the related troubleshooting required for
a real full-scale application [56]. Chapter 6 reports strategy and transient performance of the
TPAD PDU during the start-up period with biochar addition since this is a key step to achieve
a successful operation. During the start-up, rate, amount and composition of both H2 and CH4
and the production of metabolic products were studied [209]. The outcomes of this work are
expected to provide technical advices on the strategy of the start-up of TPAD PDU and
evidences on the beneficial effect of biochar in enhancing H2 and CH4 production in
demonstration scale [209, 210].
6.2 The start-up performance of the first phase
Figure 6.1 shows the gas production, composition and H2 yield of the first reactor during the
start-up period. The reactor experienced a delay for three days in producing biogas during stage
1. At the stages 2 and 3, the reactor started to produce biogas but the percentage of H2 was
negligible (Figure 6.1(b)). H2 was produced at the end of stage 3. Both the biogas production
and H2 composition continued to increase during stages 4 and 5, reaching up to gas production
of 63 L.day-1 of and H2 composition of 49% at the end of stage 5. At the stage 6, the gas
production decreased but the H2 composition in the biogas continued to increase. It is noted that
there was no trace of CH4 in the reactor during the whole start-up period, showing the
effectiveness of the sterilisation and sludge heating.
117
Figure 6.1 (a) Biogas production, (b) biogas composition, and (c) H2 and CH4 production
during the start-up of the first phase
(a)
(b)
(c)
118
Figure 6.1(c) shows the H2 yield during each stage. The H2 yield was negligible at the stages 1
to 2, reaching ca. 2 L H2 per gram VS of bread added at the end of stage 3. The H2 yield increased
significantly during stage 4 and fluctuated at a range between 22 - 46 L.kg VS-1 afterwards. The
decline in H2 yield in stage 6 of the first phase was believed to have been caused by VFA
accumulation, as discussed below [113, 161, 190].
The H2 production is typically accompanied with the generation of metabolic products, such as
VFA [190, 211]. The yield of H2 is significantly affected by the metabolic products and
microbial metabolism pathway [212, 213]. The investigation into VFA during the start-up gives
an insight on the possible metabolism pathway carried out by the culture in producing H2 [212].
A theoretical 4 moles of H2 is produced when the final product is acetic acid (HAc) and 2 moles
of H2 when butyric acid (HBu) is produced [214]. Thus molar ratio of acetate to butyrate
(HAc/HBu) has been used as an indicator of the efficiency of H2 production [213].
Figure 6.2 VFA of the first phase
Figure 6.2 presents the VFA profile during the start-up period of the first reactor. The culture
did not produce VFA at stage 1 and started to produce a low amount of acetic and butyric acid
at stage 2. The VFA production increased during stages 3 and 4 and reached the highest at stage
5 followed by a slight decrease at stage 6. This follows a similar trend to the H2 production as
119
shown in Figure 6.1(c). For instance, an increased H2 production was observed at stage 4 when
the culture produced higher VFA than the previous stages. At stage 5, the culture achieved the
highest H2 production and yields when the VFA production was also the highest. The HAc/HBu
during stages 5 – 6 was 1.3 – 1.6, which indicates a relatively high H2 production efficiency
compared to the literature reports [27]. In this study, the predominant VFAs were acetic and
butyric acids with very low propionic acid (HPr), indicating that the culture underwent a
favourable pathway for H2 production [213]. Propionic acid (HPr) is unfavourable for H2
production, thus a low amount of HPr in this study suggested an efficient H2 production [213].
Figure 6.3 pH and temperature during the start-up of the first phase
The temperature profile and evolution of pH during the start-up is shown in Figure 6.3. It is
evident that the temperature remained almost constant during the whole start-up period. It
should be mentioned that the temperature was slightly low in the first two stages due to the cold
weather, which might have delayed the biogas production at the beginning of the trial. The pH
changed significantly during the start-up. At stage 1, the pH remained at around 8 because the
culture was in an adaptation period with no VFA production. At stages 2 and 3, the culture
started producing VFA thus the pH decreased gradually. At the end of stage 3, the pH was 5.0.
At the beginning of stages 4, 5 and 6, the pH was fixed at around 6.0. This is due to the addition
120
of a base solution (4M NaOH, 1% (v/v)) in order to provide a favourable pH for the H2
producing bacteria. This suggests that in a typical pilot scale operation of H2 producing reactor,
an automatic addition of base solution, such as NaHCO3 for pH adjustment is needed [211]. In
the present study, it is observed that the pH was still in a favourable range for H2 production
during stages 1-3 and only a very small amount of base solution was required in the beginning
of stages 4-6. One possible reason may be due to the beneficial effect of biochar. The addition
of biochar has shown a potential to buffer the pH of the culture in the reactor [113, 161].
Figure 6.4 TS and VS of the first phase
The TS of the culture in R1 was 2.5% in the beginning of the trials and increased to 4.6% at the
end of operation. In addition, the VS (as a percentage of the TS) also slightly increased from
81.5 to 83.6% (Figure 6.4). The increase of TS and VS may be due to the accumulation of TS
and VS of the feedstock when the recirculation water was used for feeding in stage 5 and 6. The
increased TS and VS at stages 4 – 6 may also contribute to the increased H2 and VFA production
at those stages.
121
6.3 The start-up performance of the second phase
Figure 6.5 shows the biogas production during the start-up of the second reactor. The reactor
started to produce biogas at the end of stage 1. During stages 2 to 5, the biogas production
achieved the highest at the beginning and then dropped significantly towards the end at each
stage. This is believed to be caused by feedstock overloading (0.65 (g VS).L-1.day-1) and high
accumulation of VFA (up to 8200 mg.L-1) as discussed below.
The biogas production almost stopped at the end of stage 5 but resumed at stage 6 after 60 L of
sludge and 12.5 g.L-1 of biochar were added into the reactor. The biogas production fluctuated
at the range of 55 – 207 L.day-1 at stages 7 to 9. The CH4 production was negligible during
stages 1 to 5 and small amount of H2 was produced. The CH4 production increased while H2
composition decreased during the recovery period (stage 6). CH4 composition fluctuated during
stages 7-9 at the range between 35 – 59% with trace amount of H2 in the biogas. After the
recovery period of stage 6, the CH4 yield increased from 222 at stage 7 to 301 L.kgVS-1 at stage
9. There was no H2 data after stage 6 because H2 production potential had exhausted after stage
6 during this start-up run. In stage 6, the feeding was temporarily stopped, leading to the absence
of hydrolysis and thus H2 production ceased. After stage 6 (stages 7-9), when the feeding was
resumed, H2 produced during the hydrolysis was converted to CH4 quickly. This indicates the
effect of the addition of untreated sludge which is rich in methanogens in supporting CH4
production by converting the VFA accumulated in the beginning of stage 6 and the fresh
feedstock added at the beginning of stages 7-9 [215].
122
Figure 6.5 (a) Biogas production, (b) biogas composition, and (c) H2 and CH4 production
during the start-up of the second phase
(c)
(b)
(a)
123
Figure 6.6 VFA of the second phase
Figure 6.6 presents the VFA production during the start-up. At stage 1, the dominant VFA were
HBu and HAc. HPr was produced at stage 2. During stages 3 to 6, production of all acids
significantly increased. The accumulation of the VFA reached the highest at the end of stage 5,
with the concentration of each VFA following this order: HAc>HBu>HPr. The VFA
concentration was then decreased during the recovery period (stage 6). At the end of each stage
after recovery (stages 7 to 9), the VFA concentration was slightly increased, but showed much
lower than the peak observed at the end of stage 5. It is also observed that the concentration of
HPr was higher than HBu and HAc at stage 9.
During stages 1-5, the very low production of biogas and CH4 was mainly due to feedstock
overloading and the high accumulation of VFA. At the stage 5, the concentration of HAc and
HPr were 3310 and 1553 mg.L-1 which were much higher than their inhibitive concentration
which reported to be 2500 and 800, respectively [216, 217]. At the recovery stage (stage 6), the
VFA decreased for two reasons. First, feeding was stopped to prevent further accumulation of
the VFA. Second, untreated sludge, which is believed to contain abundant HAc consuming
microbes, was added into the tank [140, 168, 169]. At the end of the stage 6, HAc concentration
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reduced by around 50% (Figure 6.6) and consequently the CH4 production increased from 0.38
to 33 L.day-1 (Figure 6(c)). HAc and HBu are the main precursors for CH4 production. Around
65-95% of CH4 was converted from HAc [218]. It is evident that HAc and HBu were
significantly decreased at stages 7- 9 along with a high production of CH4. The high percentage
of HPr at the stages 7-9 may be due to its inhibited degradation. The degradation of HPr was
inhibited when its concentration more than 800 mg.L-1 with pH 7 and required a longer time
compared to the degradation of HAc and HBu at the same concentrations [216]. Clearly, the
stepwise addition of unheated sludge during the operation was required. It is necessary to
convert the accumulated VFA to CH4. Some studies suggested the stepwise addition of acetate
metabolising microbes during the start-up period [140, 168, 169]. This strategy is also found to
be effective in the current study.
Figure 6.7 describes the changes of pH and temperature during start-up of R2. Similar to the
R1, an adaptation period occurred during the stage 1 of the trial. During stages 1 to 3, the pH
was at the range of 5 – 7.5. At stages 4 and 5, extreme pH drops occurred caused by a high VFA
accumulation (Figure 6.6), leading to significant drop in the production of biogas and CH4.
Although, 4M of NaOH (1% (v/v)) was added to increase the pH at the beginning of stage 5,
the biogas production remained very low. The possible reason was the inhibition caused by
VFA (acetic, butyric, propionic acids) accumulation [216]. After the addition of 750 gr of
biochar and 60 L of unheated sludge in the beginning of stage 6, the pH of the reactor remained
stable at 6.8 – 7.1. The system seemed to experience a recovery period with the stable pH
condition and a gradual increase of biogas production at stage 6. The pH decreased at the
beginning of stage 7, 8, and 9, but it then increased towards the end of each stage. The
experiment was conducted under ambient temperature without a control of temperature. The
temperature during the trial fluctuated at a range between 13 – 29ºC. The slight decrease in
temperature at the end of the start-up was due to the natural variations in the ambient
125
temperature. However, such a decrease in the temperature did not influenced CH4 production
significantly as evident on Figure 6.5.
Figure 6.7 pH and temperature during the start-up of the second phase
Figure 6.8 TS and VS of the second phase
Figure 6.8 shows the changes in TS and VS during the operation. The increase of TS and VS
occurred during stage 1 to 5, and then decreased after the recovery stage until the end of the
start-up. Both TS and VS increased by 25% compared to the initial values at stages 1 to 5. This
is similar to the observation of R1. When the reactor was under fed-batch operation, the TS and
VS increased. At the end of stage 9, the TS and VS decreased by 25 and 35%, respectively,
126
compared to the TS and VS on the stage 5. At the recovery stage 6 when the unheated sludge
and biochar were added, the TS and VS decreased. It seems that the sludge addition not only
increased the CH4 production but also decreased the TS and VS of the culture.
The strategies applied in the current study led to a successful start-up of TPAD PDU. The H2
composition in this study is higher than the results of several pilot scale TPAD studies treating
bio and food wastes which were in the range of 19-39% [138, 139, 211]. The H2 yield (1.6 – 46
L H2.kg VS-1) was comparable to similar studies (3 - 67 L H2.kg VS-1) [138, 139]. The
composition of CH4 was at the range of the typical studies on pilot scale TPAD, which varies
from 55 – 67% [135, 137-139]. The CH4 yield, however, was slightly lower than similar studies.
A longer operation and of the second tank may lead to the higher CH4 yields which achieved
by other studies (377 – 550 L.kgVS-1) (Table 6.1) [138, 139].
127
Figure 6.9 SEM images of a liquid sample taken from (a) R1 on day 18 and (b) R2 on day
77 of the start-up operation
(a)
(b)
128
Table 6.1 Studies on the start-up of pilot scale TPAD PDU
Feedstock Operating conditions Gas production Ref.
1st reactor 2nd reactor H2 CH4
Biowaste • V: 0.2 m3
• pH: 3.5 – 5.4
• T: 55ºC
• OLR: 16 – 21 g
(VS)/L/day
• HRT: 3.3 – 6.6 days
• V: 0.76 m3
• pH: 7.6 – 8.2
• T: 55ºC
• OLR: 4 – 10 g (VS)/L/day
• HRT: 12.6 days
• Composition: 19 – 37
%
• Yield: 3 - 51 L/kg VS
• Composition : 60 – 65%
• Yield: 377 - 410 L/kg
VS
[139]
Food waste • V: 0.2 m3
• pH: 5.7±0.3
• T: 55ºC
• OLR: 16.8 g
(TVS)/L/day
• HRT: 3.3 days
• V: 0.76 m3
• pH: 7.6 – 8.2
• T: 55ºC
• OLR: 1.3 – 4.8 g
(TVS)/L/day
• HRT: 12.6 days
• Composition: 39 %
• Yield: 67 L/kg VS
• Composition: 67%
• Yield: 480 L/kg VS
[138]
Food waste • V: 0.2 m3 • V: 0.76 m3 • Composition : N.A • Composition: 55.2% [137]
129
• pH: 4.6±0.3
• T: 55ºC
• OLR: 3.5 g (TVS)/L/day
• HRT: 20 days
• pH: 8.0±0.1
• T: 55ºC
• OLR: 3.5 g (TVS)/L/day
• HRT: 20 days
• Yield: N.A
• Yield: 550 L/kg VS
Food waste • V: 0.15 m3
• pH: > 4
• T: ambient
• OLR:
• HRT: 3 days
• V: 0.5 m3
• pH: > 6
• T: ambient
• OLR:
• HRT: 7 – 21 days
• Composition :
10 – 49%
• Yield: 24 – 46
L.kg VS-1
• Composition :
35 – 59%
• Yield: 222 – 301
L.kg VS-1
This
study
130
6.4 Summary
Transient performance during the start-up of a TPAD PDU treating food waste with biochar
addition was investigated. A fed-batch followed by semicontinuous operation strategy was
found to be effective in starting up the TPAD PDU. The fed-batch operation allowed sufficient
time for microbial enrichment and adaptation. Under semi-continuous operation, the peak H2
composition and yields in the first phase were 49% and 46 L.(kg VS)-1, respectively. CH4
production with composition of up to 59% and yield of 301 L.(kg VS)-1 were attained in the
second phase. The addition of biochar has shown a potential to buffer the pH of culture and
initiate biofilm formation, which supported the successful start-up in both the reactors. This
provides evidences on the beneficial effect of biochar in enhancing H2 and CH4 production in
demonstration scale.
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Chapter 7 Preparation, Characterisation and Evaluation of
Biochar-loaded Organic Fertiliser
7.1 Introduction
At the end of the operation of TPAD PDU, the final effluent containing biochar was generated,
which can be utilised as a fertiliser for agriculture applications. The aim of this Chapter is to
prepare organic fertilisers from TPAD effluent from the TPAD PDU (reported in Chapter 6)
with and without addition of biochar and evaluate their potential for rocket seeds germination.
The TPAD PDU effluent was collected first and mixed with biochar with the biochar ratio
ranging from 10 to 100 wt% to prepare the biochar loaded-organic fertilisers. The fertilisers
prepared were then characterised with the results reported and discussed in Section 7.2. The
effect of the organic fertilisers on the rocket seed germination using the soil-less petri dish
bioassay experimentation was evaluated and the results were presented in Section 7.3.
7.2 Characteristics of biochar-loaded organic fertilisers
Table 7.1 shows the characteristics and nutrient composition of TPAD effluent and biochar
loaded fertilisers. It is evident that TPAD effluent (BC00) is alkaline, which is probably due to
the consumption of VFA and the addition of biochar, which is alkaline, into the reactor. It is
also reported that ammonia produced during the second phase of TPAD also brought the
effluent into alkaline condition [144]. Unfortunately, the ammonia production was not
monitored during this study. With 20% of moisture (dry matter = 80%), the water holding
capacity (WHC) of the TPAD effluent was low (0.2±0.02 mL.g-1).
The major macronutrients were P and Ca, followed by S, K and Mg. In terms of micronutrients,
Na was the most abundant, followed by Fe and others. The values of the macronutrients were
considered lower compared to AD effluent used by other studies [142, 144]. The AD effluent
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cannot be considered as a balanced fertiliser, therefore in the application, it is suggested to be
complemented with other materials [144].
Table 7.1 Main characteristics and composition of the organic fertiliser with different
percentages of biochar addition
Treatment
Biochar
content
(%)
pH
(H2O)
Dry
matter
(%)
WHC1
(mL.g-1)
P2 K3 S4 Ca5 Mg6
(mg.kg-1)
BC00 0 8.59±0.1 80±1 0.2±0.02 447.3 152.4 207.2 420.7 118.9
BC10 10 8.58±0.0 84±0 0.2±0.00 344.8 332.7 180.2 638.1 182.2
BC20 20 8.58±0.1 85±0 0.2±0.00 309.8 650.2 228.5 1191.3 345.7
BC30 30 8.60±0.0 87±1 0.8±0.07 302.0 1033.1 285.0 2254.8 670.5
BC40 40 8.49±0.0 89±1 0.8±0.00 303.0 1423.6 289.6 2425.3 706.1
BC50 50 8.57±0.0 90±0 0.9±0.02 322.0 1517.3 254.8 2366.6 682.8
BC60 60 8.55±0.1 95±4 0.9±0.08 228.6 1687.7 267.0 2639.5 778.4
BC70 70 8.60±0.1 94±0 1.1±0.08 214.3 1417.6 203.7 2558.7 737.9
BC80 80 8.52±0.0 97±0 1.2±0.02 150.5 1806.2 189.9 2436.3 690.2
BC90 90 8.48±0.0 97±0 1.4±0.08 211.1 2592 256.5 4450.9 1160.8
BC100 100 8.54±0.0 98±0 1.3±0.01 211.3 2702 207.9 4645.5 1211.8
Note: 1 WHC: water holding capacity; 2 P: Phosphorus; 3 K: Potassium; 4 S: Sulphur; 5Ca:
Calcium; 6 Mg: Magnesium; * trace: under detection limit
Both TPAD effluent (BC00) and biochar (BC100) are alkaline. Therefore, the mixtures between
the two materials are also characterised as alkaline. There are no significant pH differences
among the fertiliser, ranging at pH 8.48 – 8.60. The TPAD effluent is in a slurry form, with the
dry matter (DM) 80%, while the pure biochar is solid with DM of 98%. The DM of the fertilisers
prepared from the TPAD effluent and biochar mixtures range from 80 – 98%. The DM
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increased with the higher percentage of biochar addition. The fertilisers with 0-30% of biochar
formed watery fertilisers, fertilisers with 40-60% were sludge-like while the fertilisers with 70-
100% of biochar were solid with little trace of water.
According to Table 7.1, generally, the higher percentage of biochar addition increased the
WHC. The WHC of BC00 - BC20 were significantly lower than others. At the biochar addition
of 30 – 60% (BC30-BC60), the WHC increased to 0.8 – 0.9 mL. g-1. WHCs of BC70-BC100
were more than 1 mL.g-1. The addition of biochar into the mixture seems to provide an increased
porosity to retain higher moisture. The WHC of BC90 was 1.4 mL.g-1, comparable to the WHC
of biochar used by other studies (Wheat Chaff (1.4 mL.g-1), New Jarrah (1.6 mL.g-1)), activated
carbon (AC) (1.5 mL.g-1) and pumice (1 mL.g-1) [27, 28, 121]. The increased amount of water
retained by the biochar and soil may improve the habitability for soil microorganisms. An
increase in WHC of biochar improves the overall soil WHC when it is applied for soil
amendment [27].
The macronutrients in the prepared fertilisers, such as P, K, Ca and Mg are shown in Table 7.1
(standard deviation = 0.04 – 4%). The P concentration of the TPAD effluent was significantly
higher (447.3 mg.kg-1) than the biochar (211.3 mg.kg-1). Therefore, the P content of the fertiliser
decreased as higher percentage of biochar was added. However, the P value was still
comparable with other studies using AD effluent and pine/woody biochar [144, 219]. It is
reported that the P content reduced during pyrolysis [219].
On the contrary, the K content of the pure biochar was significantly higher than the TPAD
effluent. The addition of 10% biochar in BC10 doubled the K content compared to that of BC00.
The K kept increasing with the higher percentage of biochar addition, reaching the highest at
100% biochar addition (BC100). The highest K value of the fertiliser was higher than the K
content of pine chips-biochar prepared at 500±70ºC (1,450 – 2,354 mg.kg-1) [219, 220]. K
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activates enzymes required by plant metabolisms and growth. Also, it contributes to the cell
division, especially the turgor pressure driven [221, 222].
Low sulphur contents range at 180 - 290 mg.kg-1were detected in the TPAD effluent and the
fertiliser. Although lower than other macronutrients, the S contents was higher than that Gaskin
et al which found very low S content on the biochar produced from pine-chips (60 mg.kg-1)
[219]. The low content of S may be caused by the S loss during the pyrolysis. As much as 35 –
50% of S is released during the pyrolysis at 400-500ºC [27]. The biochar in this study was
prepared at 650ºC, therefore the higher loss of S might be occurred.
Ca was found to be one of the highest constituents of both TPAD effluent and pure biochar.
The addition of biochar significantly improved the Ca content of the fertiliser. Ca content in the
TPAD effluent was 421 mg.kg-1, while that in the pure biochar was 4646 mg.kg-1. At 90%
biochar addition (BC90), the Ca content was ten times higher (4451 mg.kg-1) than that of BC00.
Ca content of biochar derived from the same materials were varied, being closer to the Ca
content of biochar derived from willow and demolition wood [27, 219]. Ca plays a vital role in
cell expansion and enhances seed germination and growth, thus Ca deficiency may inhibits root
growth [221, 223].
Clearly, the content of Mg also extremely increased with the addition of biochar (Table 7.1).
The Mg contents improved from 182 to 1161 mg.kg-1 with the increased addition of biochar
from 10 – 90% (BC10 to BC90). Typical woody biochars contain Mg at 360 – 2,107 mg.kg-1
[27, 219, 224]. Mg acts as enzyme activators and osmoregulator and supports the root and
shoots growth [222, 223].
Table 7.2 enlists the micronutrients contents, including Na, Fe, Mn, and Cu, and heavy metals
(Cr, Ni) in the fertilisers. Generally, all micronutrients increased with the higher percentage of
biochar addition, except that of Na. Although the Na of the pure biochar is lower than that of
TPAD effluent, it is still in the range of typical study [27, 219].
135
Fe was a most abundant micronutrient in the pure biochar. The high content of Fe in biochar
significantly increased the Fe content of fertilisers with biochar addition. In woody biochar, the
Fe content range from 350 – 10,000 mg.kg-1[27]. Mn and Cu concentrations were much lower
than other micronutrients.
Heavy metals such as Ni and Cr were also observed in low concentrations. Generally, Ni (1.7
– 31 mg.kg-1) and Cr (3.3 – 60 mg.kg-1) concentrations also increased with the higher percentage
of biochar addition. It is interesting that the concentration of both Ni and Cr peaked at the
addition of 90% of biochar then decreased at 100% biochar. The value of Ni and Cu of the
fertiliser investigated in the current study are lower than the limits allowed for the utilisation of
AD effluent [144].
136
Table 7.2 The concentrations (mg.kg-1) of elements in the organic fertiliser with different percentage of biochar addition
Parameter
Biochar (%)
0 10 20 30 40 50 60 70 80 90 100
Na (mg.kg-1) 1850.12 1491.44 1638.75 1562.29 1822.58 1457.44 1290.91 773.05 804.74 994.56 818.52
Fe (mg.kg-1) 570.38 1133.71 2224.71 4551.88 5077.93 5211.22 6453.77 6191.43 6342.63 10718.19 10229.94
Mn (mg.kg-1) 5.99 14.54 30.99 68.86 75.18 79.15 95.22 87.92 88.19 156.12 151.49
Cu (mg.kg-1) 8.50 52.71 88.56 216.97 190.88 135.33 102.20 104.98 86.73 158.69 162.93
Ni (mg.kg-1) 1.73 3.23 5.77 12.91 21.89 10.82 17.55 12.54 13.22 30.65 12.68
Cr (mg.kg-1) 3.26 4.82 9.70 17.77 30.78 20.42 33.32 19.94 18.43 59.60 27.98
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7.3 Soil less petri dish bioassay
Figure 7.1 Photographs of germination of rocket seed with various organic fertilisers conducted in the soil-less petri dish bioassay
40%
70% 80% 90%
BC40 BC10 BC00 Control BC20 BC30
BC50 BC60 BC70 BC80 BC90 BC100
138
Figure 7.1 shows the photographs of the germination of rocket seeds with the applications of
various organic fertilisers. Visual observation suggests that the rocket seeds were succesfuly
germinated after 72 hours of incubation under 25 ºC and dark condition. The length of root and
shoot, however, varied among the treatments.
Table 7.3 Germination index (GI) as percentage of germinated seeds in the assay to the
control
Treatment GI (%) S.D.
Control 100 0
BC00 102 11
BC10 99 12
BC20 97 4
BC30 99 3
BC40 87 2
BC50 91 6
BC60 103 5
BC70 99 2
BC80 87 16
BC90 100 10
BC100 68 4
Typical indicators of the assay are percent of germination (and/or germiantion index), lengths
of radicle (root) and shoot [28, 225]. Table 7.4 shows the germination index (GI) calculated to
compare the seed germination of each treatment to the control (as percentage). The GIs of
treatment BC00 to BC 90 ranged from 87±16 to 103±5% of the control. The treatment of BC100
appeared to decrease the seed germination. It is noted that the GIs of almost all tested treatment
were > 80% of the control, except the BC100 which GI was 68%.
139
The nutrient micro and micronutrients content of the TPAD PDU is reported to have a strong
contribution to the seed germination. The P, as the major macronutrient in the effluent, has
reported to be an important element required during germination and seedling growth [226].
The P holds an important role in cellular metabolism and enhances root growth [223, 226]. The
lowest GI of the BC100 indicates that it was toxic that reduced the GI. The high amount of K,
Cu in BC100 may contribute to the toxicity. Other elements such as Cl and Zn which may also
contribute to the reduced GI, shoot and root length [144].
Figure 7.2 The effect of various organic fertilisers on root length of germinated rocket seed
conducted in the soil-less petri dish bioassay
The entire structure of plants is represented by the root and shoots. The sum of the root length
of each treatment was shown in Figure 7.3. It is clear that the sum of root length increased with
the percentage of biochar in the fertilisers increased, but then decreased when the pure biochar
(BC100) was used. However, it is worth mentioning that the sum of root length of BC100 was
still higher than the control. The highest sum of root length was achieved with the treatment
with BC90 (153±8 cm), being three times higher than the control (52±15 cm).
140
Figure 7.3 The effect of various organic fertilisers on shoot length of germinated rocket
seed conducted in the soil-less petri dish bioassay
Figure 7.4 The effect of various organic fertilisers on shoot to root ratio of germinated
rocket seed conducted in the soil-less petri dish bioassay
Similar trend was observed in the sum of shoot length regarding the effect of the biochar-loaded
organic fertiliser, as shown in Figure 7.3. The sum of shoot length increased with the higher
biochar percentage in the fertilisers achieving the highest with the BC90 treatment, attaining
141
63±5 cm. The shoot length became smaller with the BC100 treatment compared with the BC90
treatment. The ratios of shoot to root ratio calculated and presented in Figure 7.4. It is clear that
the shoot to root ratio tended to increase with the higher biochar percentage in the fertilisers.
The higher sum of root and shoot in the treatment with higher biochar percentage in the
fertilisers may attribute to higher nutrient contents. As suggested in Section 7.2, the addition of
biochar in the TPAD PDU significantly increased the macro and micronutrients required for
seed germination and plant growth, such as K, Ca, Mg and Fe. It is known that K, Mg and Fe
are cofactor of enzymes required by plant metabolisms and growth. Also, K contributes to the
cell division, especially the turgor pressure driven [221, 222]. Ca plays a vital role in cell
expansion and enhances seed germination [221, 223]. Mg also acts as enzyme activators and
osmoregulator, and supports the root and shoot growth [222, 223].
Root to shoot ratio indicates the allocation of the nutrient to the part of the plants [227]. Figure
7.4 suggests that the nutrients contained in the fertiliser enhanced root growth than shoot. The
abundance of Fe and Ca in biochar, may contribute to the enhance root growth [221-223].
It is worth noticing that the BC100 performed better than the control in term of shoot and root
length, despite the decrease in GI [171]. Similar result is reported in a study of germination
assay of watercress using biochar derived from compost-like output (CLO) mixed with sewage
sludge [171]. It was observed that although the GI reduced, the nutrient availability in the
biochar promoted the root and shoot once the seed germinated [171, 225]. The similar results
were found by Rogovska et al., (2012) and Stephenson et al., (1997). This suggests that using
GI alone as an indicator to evaluate the potential of biochar-loaded fertilisers appeared to be
insufficient. It is proposed to use lengths of root and shoot, together with GI, as indicators for
the assay [225, 228].
142
7.4 Summary
The TPAD PDU effluent was loaded onto biochar with different biochar loading ratios to
prepare the organic fertilisers. The biochar-loaded fertilisers were characterised and evaluated
using seed germination assay. The results suggest that the addition of biochar significantly
increased essential elements for germination and plant growth, such as K, Ca, Mg, Na, Fe, Mn,
Ni and Cr, compare to the treatment with the TPAD effluent alone. Biochar addition also
increased WHC of the fertiliser with no significant pH change. Fertilisers with 0-90 wt%
biochar loading gave the positive effects on the seed germination, while the pure biochar
reduced germination index (GI). However, despite the low GI, the pure biochar, and the rest of
the fertilisers tested, resulted in the increased sums of root and shoot length compared to the
control without the fertiliser. The maximum sums of root and shoot (153.4±8 and 62.3±5 cm)
was achieved with the fertiliser prepared of 10% of TPAD effluent and 90% of biochar. The
improved macro and micronutrients in the biochar-loaded fertilisers contributed to the good
seed germination results.
143
Chapter 8 Evaluation and Practical Implications
8.1 Introduction
In Chapter 8, the findings from the entire thesis work as presented in the previous four chapters
are integrated and evaluated against the specific objectives and literature data, respectively. In
addition, the practical implications are identified.
8.2 Integration of Experimental Findings
The study has provided strong, convincing and systematic evidence that the biochar addition
improves H2 and CH4 production both in the first and second phases of TPAD. It shortens the
lag phase, increases the maximum H2/CH4 production rate, and H2/CH4 production potential. It
was further observed and verified that the volatile fatty acids (VFA) generation and conversion
are also enhanced in the first and second phase, respectively. The beneficial characteristics of
biochar observed to contribute to the enhanced H2 include the high surface area for the initiation
of biofilm formation and provision of additional nutrients, enriching the microbial growth and
population. The pH buffering ability of biochar was also found to be essential in enhancing H2
production. In the second phase, biochar plays a significant role in biofilm formation which
increased microbial population and activity. The buffering ability of biochar, however, was an
insignificant factor.
A fed-batch followed by semicontinuous operation strategy was found to be effective in starting
up both phases of the TPAD process demonstration unit (PDU) at a pilot scale. Confirming the
findings in the bench scale study, biochar addition shows potential buffering capacity and
microbial immobilising ability, which contributed to the successful start-up of the TPAD PDU.
Finally, the biochar-loaded organic fertilisers were successfully prepared, characterised and
evaluated. The biochar addition significantly increased water holding capacity (WHC) and
144
micro and macronutrients with no significant pH changes compared to the TPAD effluent alone,
thus enhanced the seed germination.
8.3 Evaluation against the Specific Research Objectives
The objectives stated in Chapter 2 were achieved through a systematical and thorough study
according to the methodology explained in Chapter 3 then reported in Chapters 4, 5, 6 and 7.
The first three objectives were achieved by investigating the effect of biochar in bench scale
TPAD operating the two separate phases individually. Biochar was found to exert profound
beneficial effects on both the first and second phase of TPAD (Chapters 4 and 5). The biochar
used in the study, however, was only limited to one type of biochar, which was the pine sawdust
biochar, prepared at 650C. Considering the different characteristics which may be possessed
by different types of biochar prepared under different operating conditions, their effects and
mechanisms in TPAD may be different. There could well be some biochars that may not show
the same or similar beneficial effects as the pine biochar studied in the present work does and
it would be scientifically interesting and practically useful to clearly define the key
characteristics of biochar and relate them to the biochar effects in enhancing H2 and CH4
production in TPAD operations. Therefore, further studies using different types of biochar in
TPAD are recommended. In addition, in the current study, the presence of typical AD microbes
and the establishment of biofilm on the pore and surface of biochar were observed as evidenced
in the SEM images. However, a further study on the bacterial and biofilm identification using
molecular biological tools is necessary to give a better understanding of the interaction between
biochar, TPAD microbes and operating conditions.
The investigations into the effect of biochar on H2 and CH4 production were focused on the
batch operation in a bench scale AD. Very limited studies examined the biochar addition in
long term continuous operations on large scales. An attempt to achieve the fourth and fifth
objectives of the current study, demonstrating the operation of H2 and CH4 production with the
145
biochar addition in a larger scale was conducted, as reported in Chapter 6. The operation of
TPAD PDU with the addition of biochar was successfully conducted. However, it was limited
to the start-up period under fed-batch and semicontinuous operation. Further study on the
biochar addition in long term continuous operation of pilot scale TPAD is suggested.
The final specific objectives namely the preparation, characterisation and evaluation of biochar
and TPAD effluent as fertiliser were successfully achieved as detailed in Chapter 7. The biochar
addition improved the characteristics of TPAD effluent alone. The results also suggest the
potential utilisation of combined biochar and effluent as fertiliser as shown by seed germination
assay results. Further studies on the utilisation of the fertilisers in pot trials and using different
application ratio were necessary to provide confidence before the field application.
8.4 Evaluation against the Literature
8.4.1 Effect of Biochar Addition on Hydrogen Production
The current study was systematically conducted to examine the beneficial effect of biochar on
H2 production. The results show that the biochar addition significantly increased H2 production.
The results from the present study are compared with the literature data. Similar studies report
the beneficial effects of biochar on H2 production (Table 8.1); although some studies report its
negative effects. While some types of biochar are reported to have adverse effects on H2
production [113], in the present study, the pine sawdust biochar at all ratios tested gives positive
effects in H2 production. It was found that the biochar also enhances VFA generation [73, 128].
In addition, it has been found that the effect of biochar addition in H2 production is dependent
on the type and addition ratio of biochar, the feedstock of AD and operating conditions. The
present work was able to explain the wide variations in the optimal biochar addition ratio
ranging from 0.6 to 12.5 g. L-1 (Table 8.1). It implies the difficulty to achieve agreement on the
146
quantitative data among the literature data since different types and addition of biochar,
feedstock and operating conditions were used in each study [128].
Table 8.1 A comparison of the current experimental results with the literature data on the
batch H2 production with the addition of different types of biochar
Feedstock
of AD
Feedstock
of biochar
Biochar addition
(g.L-1) Operating
conditions
H2 yields
increase
compared to
control
Ref. Studied
range
Optimal
OFMSW1 Woody
mass
2.5 – 35 12.5 Batch 37ºC
Initial pH: 5.5
1.4 - 3.3
times
[128]
Glucose Corn bran
residue
0.2 – 0.8 0.6 Batch 37ºC
Initial pH: 7
11- 29% [73]
DAS2 and
food
waste
Sawdust 10 10 Batch 35ºC
Initial pH: 5.5
0-13% [113]
DAS2 and
food
waste
Wheat bran 10 10 Batch 35ºC
Initial pH: 5.5
6 – 13% [113]
DAS2 and
food
waste
Sewage
sludge
10 10 Batch 35ºC
Initial pH: 5.5
(-10) - 6% [113]
DAS2 and
food
waste
Peanut
shell
10 10 Batch 35ºC
Initial pH: 5.5
(-44) - (-
10)%
[113]
Current
study
Pine
sawdust
8.3 – 33.3 10 Batch 35ºC
Initial pH: 5.0
14– 31% [161]
Note: 1: OFMSW: organic fraction of municipal solid waste
2: DAS: dewatered activated sludge
Finally, while most of the literature focuses on the study of type and addition ratio of biochar
under a fixed set of operating conditions, the current study also examined the effect of biochar
addition under various operating conditions of initial pH values and temperatures. The
147
beneficial effect of biochar on H2 production was found to be more profound at lower pH and
higher temperatures.
8.4.2 Effect of Biochar Addition on Methane Production
Table 8.2 A comparison of the current experimental results with the literature data on the
batch CH4 production with the addition of different types of biochar
Feedstock
of AD
Feedstock
of biochar
Biochar addition
(g.L-1) Operating
conditions
CH4 yields
improvement
compared to
control (%)
Ref. Studied
range
Optimal
AD sludge Paper
sludge and
wheat husk
20 20 Batch 42ºC
Initial pH: N.A
31 [3]
Glucose Fruit
woods
10 10 Batch 35ºC
Initial pH: 7
21 [83]
Glucose Fruit
woods
10 10 Batch 35ºC
Initial pH: 7
11.5 [129]
Citrus
peel
Coconut
shell
9.7 9.7 Batch 35ºC
Initial pH: 7
13 [130]
Food
waste
Fruit
woods
2 - 10 2 Batch 35ºC
Initial pH: 7
39 - 44 [131]
Dairy
manure
Dairy
manure
1 - 10 10 Batch 35ºC
Initial pH: 7.7
5 – 24.5 [133]
Food
waste and
DAS
Sawdust 2 - 15 6 Batch 35C
Initial pH: NA
(-2) - 4 [229]
Current
study
Pine
sawdust
8.3 –
33.3
8.3 Batch 35C
Initial pH: 7
(-10) – 10 [161]
*DAS = dewatered activated sludge
NA = not available
The present work into the second phase of TPAD suggested strong evidence of the enhancement
of CH4 production with the addition of biochar addition, in good agreement with other studies
148
in the literature using various wastes as the feedstock (Table 8.2). The results of the current
study are within the range of the findings of other studies. At the appropriate ratios of biochar
additions, the CH4 yields increased by 4 – 44% compared to the control, depending on the types
and addition ratio of biochar, the feedstock of AD and operating conditions [3, 83, 129-131,
133, 229]. Similar to the results in H2 productions, the optimal biochar additions are varied in
the existing studies, which range from 2 to 20 g.L-1 (Table 8.2).
In addition, the current study observed that at the addition ratio of 25.5 – 33.3 g.L-1, the CH4
yields were lower than control due to the biochar overdose. However, the CH4 production rate
increased (by 23 – 42%). Similar results were reported by Wang et al. that the CH4 yield
decreased with the higher addition of biochar addition, while the CH4 production rate increased
at all biochar addition ratios tested [229]. Finally, further study on the biochar addition under
various operating condition (initial pH and temperature) suggested that the effect of biochar
additions was greater at the higher pH.
8.4.3 Mechanisms of biochar in enhancing H2 and CH4 production in TPAD
Figure 8.1 A schematic representation of the mechanisms of the working of biochar in
promoting H2 and CH4 productions in TPAD from food waste
First phase
▪ Promoting biofilm formation
▪ Buffering pH
▪ Providing temporary nutrients
Biochar
Results:
▪ Shorter lag phase
▪ Faster VFA generation
▪ Higher H2 production rate
▪ Higher cumulative H2
production
Second phase
▪ Promoting biofilm formation
Results:
▪ Shorter lag phase
▪ Faster VFA degradation
▪ Higher CH4 production rate
149
The possible mechanisms of the biochar effects in H2 and CH4 production have been proposed
in the current study (Figure 8.1). It is observed that in the first phase, biochar promoted biofilm
formation, buffered the pH and provided temporary nutrients. Meanwhile, in the second study,
the biofilm formation initiated by biochar was observed to be the main mechanism of biochar
in enhancing CH4. The proposed mechanisms were supported by other studies (Table 8.3 and
8.4) and discussed as follow.
Previous studies suggested that different types of biochar affected H2 production with different
mechanisms. First, Sharma et al. and Shang et al. found that biochar enhanced H2 production
due to the high surface area of biochar which facilitated microbial immobilisation by biofilm
formation, although there was no direct evidence of the establishment of biochar presented in
their studies [73, 128]. In the current study, a clear establishment of biofilm in biochar was
observed in the SEM images (Figure 4.11). The microbial immobilisation is reported to play a
critical role in increasing the microbial density in the culture compared to the suspended culture
without microbial immobilisation [85, 230]. The increased density of microbes favours H2
production [85, 230].
In addition, biochar was also shown to be an efficient pH buffer in this study as supported by
the findings in the study comparing biochar with other additives with different acidity as
references (Section 4.4). This finding is supported by other studies [73, 113]. Zhang et al. and
Wang et al. [113] reported an increased H2 with the increased in final pH which corresponds to
the higher addition of biochar, which was also observed in this study (Section 4.4 and 4.5).
Yet another mechanism was proposed that the provision of additional nutrients due to biochar
addition may be useful for microbial activity in enhancing H2. The biochar used in this study
contains 10 wt% of volatile matter that was available as a temporary microbial nutrient [27].
This mechanism is also reported in CH4 production using biochar produced at the lower
production temperature, where the remaining nutrient was readily available [3].
150
In addition, biochar used in this study contained micronutrients, such as Fe (10,230 mg.kg-1)
(Table 7.2). Zhang et al. found that the addition of 50 – 300 mg.L-1 of Fe2+ increased the H2 by
17 - 38 % compared to the control. H2 production increased from 158 to 212 mg.g glucose-1
with the addition of 0 – 100 mg.L-1 then gradually decreased to 184 mg.g glucose-1 with further
Fe addition to 300 mg.L-1[73]. Similar results were found in the current study, using the biochar
ranged at 8.3 – 33.3 g.L-1, which corresponded to 85 – 341 mg. L-1 of Fe. The H2 production
increased from 749 to 981 mL.L-1 with the addition of 8.3 g.L-1, and then decreased to 855
mL.L-1 when the biochar was increased to 33.3 g.L-1. This agreement shows that the nutrient
contained in biochar is essential for H2 production. Particularly Fe, it is required by the H2
producing bacteria (HPB) for hydrogenase protein redox, which directly correlates to microbial
growth and biogas release, however toxic when it is overdosed [73, 231, 232].
In the second phase of TPAD, the possible mechanisms of biochar effects in CH4 production
proposed in the current study were supported by other studies and discussed as follows (Table
8.4). A significantly shorter lag phase than control (41 – 45% shorter) and higher maximum
production rate (23 – 42%) was observed in the current study (Table 5.3). These observations
imply that the addition of biochar enhanced the microbial growth and activity. Thus the culture
started the CH4 production faster. It was observed that the microbial immobilisation was
facilitated with biochar addition (Figure 5.6). The promoted formation of biofilm has been
reported to enhance microbial activity, leading to a shorter lag phase, faster VFA degradation
and CH4 production in the cultures with biochar.
Some literature reported the beneficial effect of biochar in buffering the pH of the culture [133,
229]. The role of biochar as pH buffer, however, was found to be insignificant in the current
study (Section 5.5). The separation of acidogenesis from the methanogenesis was observed to
be effective in preventing the culture from extreme pH drop and high accumulation of VFA, as
151
reported in Section 5.4. Therefore, the pH drop was no longer an issue in the second phase.
Thus the pH buffer capability of biochar insignificantly affected the CH4 production.
152
Table 8.3 Profiles of biochar and proposed mechanisms in H2 production of different studies
References
Biochar
Proposed mechanisms
Feedstock pH SSA (m2.g-1)
Sharma et al. [128] Woody biomass N.A 125 - Biochar facilitated biofilm formation
- Biochar mitigated ammonia inhibition
Zhang et al. [73] Corn-bran residue 8.92 58 - Biochar acted as a microbial support carrier
- Biochar acted as pH buffers
Wang et al. [113] Sawdust 7.27 – 10.07 15.3 – 511.3 Biochar acted as pH buffers
Wang et al. [113] Wheat bran 7.37 – 10.33 4.2 – 45.9 Biochar acted as pH buffers
153
Table 8.4 Profiles of biochar and proposed mechanisms in CH4 production of different studies
References
Biochar
Proposed mechanisms
Feedstock pH SSA (m2.g-1)
Mumme et al. [3] Paper sludge and
wheat husks
9.3 N.A - Hydrochar provides anaerobically degradable carbon
- Biochar prevents ammonia inhibition
Luo et al. [83] Fruitwoods 8.6 N.A - Biochar selectively enriched methanogens
- Biochar improved the resistance of the microbes to highly
acidic conditions
Lu et al. [129] Fruitwoods 8.6 N.A - Fine biochar enhanced acid generation which can be useful
for H2 production
- Biochar mitigated ammonia and acid stress
- Biochar initiates microbial colonisation
Fagbohungbe et al.
[130]
Coconut shell 8.3 NA - Biochar adsorbed an inhibitive compound in the AD
feedstock
- Biochar immobilises methanogens
154
References
Biochar
Proposed mechanisms
Feedstock pH SSA (m2.g-1)
Cai et al. [131] Fruit woods 8.6 N.A - Biochar was a favourable material during easily degradable
feedstock
Jang et al. [133] Dairy manure N.A 6.3 - Biochar served as a pH buffer
Wang et al. [229] Sawdust 9.2 248.6 - Biochar provides excellent pH buffering capacity
- Biochar enriched specific methanogens for direct
interspecies electron transfer
155
8.4.4 Transient Performance during Start-up of TPAD PDU
The results of the TPAD PDU study are compared with the literature data of pilot scale TPAD
operation. Figure 8.2 shows studies on pilot scale TPAD using different feedstock, reactors and
operating conditions. All studies presented in Figure 8.2 were conducted without biochar
addition, except for the current study. The volume H2 and CH4 producing reactors were 150 –
200 L and 42 – 1,300 L, respectively. Some literature report both H2 and CH4 production, while
the others only considered the CH4 production. The results of the current study were comparable
to the results from other studies, where the H2 yield range at 51 to 68 L.(kg VS)-1 and the range
of CH4 yield are at 179 – 550 L.(kg VS)-1. A longer and continuous operation of the TPAD
PDU in the future study is expected to improve the H2 and CH4 production.
Figure 8.2 H2 and CH4 production in pilot scale TPAD using different feedstock
There is limited research work on the biochar addition on the pilot scale AD. To the best of
author’s knowledge, to date, there is only one study reporting biochar addition of biochar in
pilot scale single AD producing CH4 (reactor volume 900 – 1500 L) [140]. The study reports
the correlation between the maturity of biofilm with the pH changes during the operation. It
was observed that towards the end of the 59 days operation, the culture approached pH 7 more
156
rapidly, which implied the enhanced biofilm maturation [140]. Similar trends were also
observed in the current study (Figure 6.7), suggesting a mature establishment of methanogenic
biofilm as also suggested SEM images (Figure 6.9).
8.4.5 Preparation, Characterisation and Evaluation of Biochar-loaded Organic
Fertiliser
The characterisation of TPAD effluent and biochar in this study was conducted and evaluated
against literature data (Table 8.5). The pH of the TPAD effluent and biochar are consistent with
those of effluent from other studies. The macro and micronutrient depend on the feedstock of
the AD and biochar. Compared to other AD effluents, the nutrients of TPAD were relatively
lower, except for the Fe and Na. Therefore, the supplementation of biochar is aimed to increase
the essential macro and micronutrients for agricultural applications.
Figure 8.3 shows the comparison of the minimum and maximum germination index (GI) of the
current study and other studies. The GI presents the germination of the tested treatment relative
to the control without the addition of the tested fertilisers [144]. The GI of rocket seed used in
the current study ranged at 68 – 103 %, which is at the range of other studies. It is comparable
with the GI of those experiments using (1) pig slurry added with crop waste and (2) New Jarrah
biochar. Also, it is significantly higher than that of AD effluent (cattle slurry) added with
glycerol. The GI of the assay with AD effluent (pig slurry) added with animal waste and AD
effluent (cattle slurry) added with agriculture waste are higher than that of others. It is likely
that the significant content of P and K, that are higher than that of our study, play an essential
role in increasing the GI of cress used in the study by Albuquerque et al. [144].
157
Table 8.5 Characteristics of AD effluent and biochar prepared as organic fertiliser
Parameter
Feedstock of AD/TPAD effluent Feedstock of biochar
Food
waste
Pig slurry and animal by-
product
Cattle slurry and
agriculture waste
Pinewood sawdust Pine chips
New Jarrah
pH 8.59 7.95 7.50 8.54 8.30 9.52
P (mg.kg-1) 447.3 500 800 211.3 140 N.A
K (mg.kg-1) 152.4 2200 3100 2702 1450 N.A
S (mg.kg-1) 207.2 219 457 207.9 60 N.A
Ca (mg.kg-1) 420.7 799 4026 4645.5 1850 N.A
Mg (mg.kg-1) 118.9 324 698 1211.8 590 N.A
Fe (mg.kg-1) 570.4 51 301 10229.94 50 N.A
Na (mg.kg-1) 1850 696 746 818.52 13 N.A
Mn (mg.kg-1) 5.99 11.4 27.5 151.49 258 N.A
Cu (mg.kg-1) 8.50 14.3 10.8 162.93 9 N.A
Ni (mg.kg-1) 1.73 N.A. N.A. 12.68 3 N.A
Cr (mg.kg-1) 3.26 N.A. N.A. 27.98 18 N.A
158
Parameter
Feedstock of AD/TPAD effluent Feedstock of biochar
Food
waste
Pig slurry and animal by-
product
Cattle slurry and
agriculture waste
Pinewood sawdust Pine chips
New Jarrah
Reference This
study
[144] [144] This study [219] N.A
Note: N.A; Not available
159
Figure 8.3 Germination index of petri dish bioassay using different plants and fertilisers
The results from this current study and literature suggest strong evidence that the TPAD effluent
and biochar are potential materials for soil amendment for agricultural application. Studies
using germination assay are required for the TPAD effluent and biochar utilisation derived from
the different feedstock.
8.5 Practical Implications
The results and new findings from the present research significantly contribute to the practical
applications of TPAD in processing wastes for biogas production with the use of biochar in
enhancing H2 and CH4. Although TPAD is more promising in recovering energy than single
phase AD [56], it is still challenging to achieve stable operation and high production rates and
yields of both H2 and CH4.
The findings of the study provided strong evidence that biochar addition enhanced H2 and CH4
production. First, the enhanced H2 and CH4 production rates lead to the shorter time of
operation. Thus more waste/wastewater can be treated. Furthermore, with higher production of
H2 and CH4, the unit required less addition of buffering solutions due to the buffering ability of
160
biochar. Also, the strong establishment of biofilm on biochar can prevent the microbial wash
out in the real application of continuous operation of pilot scale AD [85]. Finally, the
experimental results from TPAD PUD provide valuable data and reference for the design,
operation and economic evaluation of TPAD in commercial scale with the use of biochar.
The findings suggest a very promising solution to the management of AD effluent, which can
sometimes be challenging. The TPAD effluent containing biochar with and without further
biochar addition provides nutrients for plants growth. The TPAD effluent loaded with biochar
has shown positive effects in the germination of the tested seed. The biochar-loaded organic
fertilisers can be used for agriculture, industry and commercial applications.
As a whole, the thesis has proposed an approach to the practical implication of integrated
pyrolysis and TPAD operation to achieve a sustainable bioenergy generation and bio-wastes
management, particularly in remote areas. Woody biomass or agricultural wastes are potential
feedstock for biochar production via pyrolysis. The biochar produced is an ideal material for
enhancing H2 and CH4 production via TPAD treating animal manure, food and agricultural
wastes. The effluent from the TPAD can be utilised for the agricultural applications.
161
Chapter 9 Conclusions and Recommendations
9.1 Introduction
The outcomes of the present thesis research have contributed new knowledge and useful
experimental data on the utilisation of biochar to enhance H2 and CH4 production in bench and
demonstration scale of TPAD operation, as well as the utilisation of the AD effluent and biochar
as fertilisers. The conclusions and evaluation of the current research as elaborated in Chapter 8
can naturally lead to recommendations for future studies. This Chapter summarises the key
conclusions and recommendations as follows.
9.2 Conclusions
9.2.1 Effect of Biochar Addition on Hydrogen Production in the Bench Scale Experiment
• The effect of biochar addition on the H2 production in laboratory scale TPAD of simulated
carbohydrate food waste was studied systematically. Biochar addition was shown to
shorten the lag phase by 21.4 to 35.7 %, increased the maximum production rate by 32.4%
and H2 production potential by 14.2 to 31 % of H2.
• Further investigation of the effect of biochar addition under different initial pH and
temperature on the cumulative H2 yield (YH) and maximum daily production rate (RH) in
the first phase of TPAD was conducted and optimised using the response surface
methodology (RSM). Biochar addition was shown to substantially increase YH, especially
at lower pH and higher temperatures.
• The RSM analysis showed that the maximum YH of 1,331 mL.L-1 and RH of 763 mL.L-
1.day-1 could be achieved under the optimum conditions of biochar addition ratio 10.1 g.L-
1, initial pH 6.4 and temperature 32C.
162
• The working mechanisms of biochar in enhancing H2 production were proposed. It is
observed that the biochar initiates the biofilm formation and provides temporary nutrients
in the culture, enriching the microbial population. Also, the addition of biochar was
observed to bring the condition of the culture from acidic to alkaline at the beginning of
the operation and prevented significant pH drop during incubation. As a result, the cultures
with biochar additions generated more H2 at faster rates.
9.2.2 Effect of Biochar Addition on Methane Production in the Bench Scale Experiment
• A preliminary study on the effect of biochar addition on the CH4 production in laboratory
scale TPAD of simulated carbohydrate food waste was conducted. The biochar addition
shortened the lag phase by 41 to 45 %, increased the maximum production rate by 23.0-
41.6% and CH4 production potential by 1.9 to 9.6% of CH4.
• Furthermore, the effect of biochar addition on CH4 production in the second phase of
TPAD under varying initial pH and temperature conditions has been studied. A moderate
level of biochar addition, mesophilic temperature and neutral to alkaline initial pH were
shown to benefit CH4 production in the second phase of TPAD. The effect of biochar
addition was more profound at higher pH.
• The optimum biochar addition; temperature and initial pH were found to be 12.5 g.L-1,
36.2oC and 7.8, respectively. Under the optimum condition, the YM and RM were 1755
mL.L-1 and 500.9 mL.L-1.day-1.
• The possible working mechanism of biochar in enhancing CH4 is related to the high surface
area of biochar, which initiates biofilm formation. The SEM analysis revealed the
development of biofilm and the accumulation of the bacillus rods of bacteria on the surface
of biochar. The promoted methanogenic biofilm formation in the second phase of TPAD
may contribute to the enriched methanogens and enhanced CH4 production. The pH
buffering capacity of biochar, however, was found to be insignificant.
163
• An additional experiment of single-phase AD using fresh food waste was conducted. The
result suggests that at a high concentration of feedstock, the single phase AD suffered from
acid accumulation thus failed to produce CH4, while the second phase TPAD successfully
produced a substantial amount of CH4. It is due to the separation of acidogenesis and
methanogenesis, which favour CH4 production in the second phase AD.
9.2.3 Transient Performance during Start-up of TPAD PDU
• Transient performance during the start-up of a TPAD PDU treating food waste with biochar
addition was investigated. A fed-batch followed by semicontinuous operation strategy was
found to be effective in starting up the TPAD PDU. The fed-batch operation allowed
sufficient time for microbial enrichment and adaptation.
• Under semi-continuous operation, the peak H2 composition and yields in the first phase
were 49% and 46 L.(kg VS)-1, respectively. CH4 production with the composition of up to
59% and yield of 301 L.(kg VS)-1 were attained in the second phase.
• The addition of biochar has shown a potential to buffer the pH of culture and initiate biofilm
formation, which supported the successful start-up in both the reactors. This provides
evidence on the beneficial effect of biochar in enhancing H2 and CH4 production in
demonstration scale
9.2.4 Preparation, Characterisation and Evaluation of Biochar-loaded Organic
Fertiliser
• The TPAD PDU effluent was loaded onto biochar with different biochar loading ratios to
prepare the organic fertilisers. The biochar-loaded fertilisers were characterised and
evaluated using seed germination assay. The results suggest that the addition of biochar
significantly increased essential elements for germination and plant growth, such as K, Ca,
Mg, Na, Fe, Mn, Ni and Cr, compare to the treatment with the TPAD effluent alone.
Biochar addition also increased WHC of the fertiliser with no significant pH change.
164
• Fertilisers with 0-90 wt% biochar loading gave the positive effects on the seed germination,
while the pure biochar reduced germination index (GI). However, despite the low GI, the
pure biochar, and the rest of the fertilisers tested, resulted in the increased sums of root and
shoot length compared to the control without the fertiliser.
• The maximum sums of root and shoot (153.4±8 and 62.3±5 cm) were achieved with the
fertiliser prepared of 10% of TPAD effluent and 90% of biochar. The improved macro and
micronutrients in the biochar-loaded fertilisers contributed to the good seed germination
results.
9.3 Recommendations
The overall objectives of the present study have been achieved. However, various new gaps
have also been identified during the evaluation of the findings of the current research. The new
gaps of knowledge are suggested in this Recommendations section.
• The biochar used in the study, however, was only limited to one type of biochar, which
was the pine sawdust biochar (prepared at 650C) and one type of feedstock (carbohydrate
food waste). Considering the different characteristics, which may be possessed by different
types of biochar and feedstock, their different effects and mechanisms may be found in the
AD. Therefore, further studies using different types of biochar and other feedstock in
TPAD are recommended.
• In the current study, the presence of typical AD microbes and the establishment of biofilm
on the pore and surface of biochar were observed in the SEM images. However, a further
study on the bacterial and biofilm identification using molecular biological tools is
necessary to give a better understanding of the interaction between biochar TPAD, H2 and
CH4 microbes and operating conditions.
• The operation of TPAD PDU with the addition of biochar was successfully conducted.
However, it was limited to the start-up period under fed-batch and semicontinuous
165
operation under ambient temperature and uncontrolled pH. Therefore, further study on the
biochar addition in long term continuous operation of pilot scale TPAD with controlled
operating conditions is suggested.
• The results also suggest the potential utilisation of combined biochar and effluent as
fertiliser as shown by seed germination assay results. Further studies on the utilisation of
the fertilisers in pot trials and using different application ratio were necessary to provide
confidence before the field application.
166
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