biological pretreatment and dry digestion processes for ...1141448/fulltext01.pdf · research...

Thesis for the Degree of Doctor of Philosophy

Biological pretreatment and dry digestion processes for biogas production

Regina Jijoho Patinvoh

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Biological pretreatment and dry digestion processes for biogas production

Copyright 2017 © Regina Jijoho Patinvoh

Swedish Centre for Resource Recovery Faculty of Textiles, Engineering and Business University of Borås SE-501 90 Borås, Sweden

Digital version: http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-12575 ISBN 978-91-88269-60-7 (printed) ISBN 978-91-88269-61-4 (PDF) ISSN 0280-381X Skrifter från Högskolan i Borås, nr. 83 Cover photo: Europe’s largest dry-AD, in Italy (Kompogas® plant) Picture source: Hitachi Zosen Inova

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ABSTRACT

Biogas technology has been used quite extensively to generate renewable energy from organic

wastes while also recycling nutrients in the wastes and reducing harmful emissions. However,

the challenges of low biogas yield from recalcitrant and inhibitory solid wastes together with

high construction and operation costs of bioreactors have impeded optimal performance

through this process. Additionally, solid organic wastes with total solids (TS) contents greater

than 20 % are produced daily in enormous amounts; treating these wastes in conventional wet

anaerobic digestion processes has required the addition of water, leading to large reactor

volumes, high energy costs for heating, and costly dewatering processes for the digestate

residue. In this study, the challenges mentioned above were addressed by using biological

pretreatment and dry anaerobic digestion processes. Pretreatment, non-pretreatment strategies,

biogas bioreactors design were also studied since this will aid optimizing its economy.

The suitability of a novel textile bioreactor for biogas production was accessed in dry

digestion process (dry-AD) treating manure bedded with straw (22 – 30 %TS of feedstock).

The 90-L textile bioreactor was robust and simple to operate; it can be accessed easily by

developing countries where required expertise may not be available. Methane yield from the

manure with straw was 290 NmlCH4/gVS using acclimatised bacteria; the digestate residue

was affirmed suitable as bio-fertiliser. The efficiency of continuous plug flow reactor for dry

anaerobic digestion of manure bedded with straw was also investigated at 22 %TS. Organic

loading rates up to 4.2 gVS/L/d with retention time of 40 days gave better process stability.

Recalcitrant structure of chicken feather wastes was altered using Bacillus sp. C4 (Bacillus

pumilus) and this pretreatment improved methane yield by 124 % compared to the untreated.

Considering the fact that easily degraded feedstocks are not highly available and some

problems associated with pretreatments: dry anaerobic co-digestion of citrus wastes with

chicken feathers, wheat straw and manure bedded with straw, was investigated at 20 %TS in

batch process. The best mixing ratio enhanced methane yield by 14 % compared to the

expected yield from individual fractions. Process performance at different organic loading

rates (OLR) was then investigated in continuous plug flow reactors at 21 %TS and 32 %TS of

feedstock. Stability of the process decline as OLR was increased to 3.8 gVS/l/d resulting in

high total volatile fatty acids (VFA), VFA/alkalinity ratio and reduction in methane yield.

Keywords: Solid wastes; Dry anaerobic digestion; Textile bioreactor; Plug flow bioreactor; Digestate; Process stability; Pretreatment; Co-digestion; Mesophilic

iii

ABSTRACT

Biogas technology has been used quite extensively to generate renewable energy from organic

wastes while also recycling nutrients in the wastes and reducing harmful emissions. However,

the challenges of low biogas yield from recalcitrant and inhibitory solid wastes together with

high construction and operation costs of bioreactors have impeded optimal performance

through this process. Additionally, solid organic wastes with total solids (TS) contents greater

than 20 % are produced daily in enormous amounts; treating these wastes in conventional wet

anaerobic digestion processes has required the addition of water, leading to large reactor

volumes, high energy costs for heating, and costly dewatering processes for the digestate

residue. In this study, the challenges mentioned above were addressed by using biological

pretreatment and dry anaerobic digestion processes. Pretreatment, non-pretreatment strategies,

biogas bioreactors design were also studied since this will aid optimizing its economy.

The suitability of a novel textile bioreactor for biogas production was accessed in dry

digestion process (dry-AD) treating manure bedded with straw (22 – 30 %TS of feedstock).

The 90-L textile bioreactor was robust and simple to operate; it can be accessed easily by

developing countries where required expertise may not be available. Methane yield from the

manure with straw was 290 NmlCH4/gVS using acclimatised bacteria; the digestate residue

was affirmed suitable as bio-fertiliser. The efficiency of continuous plug flow reactor for dry

anaerobic digestion of manure bedded with straw was also investigated at 22 %TS. Organic

loading rates up to 4.2 gVS/L/d with retention time of 40 days gave better process stability.

Recalcitrant structure of chicken feather wastes was altered using Bacillus sp. C4 (Bacillus

pumilus) and this pretreatment improved methane yield by 124 % compared to the untreated.

Considering the fact that easily degraded feedstocks are not highly available and some

problems associated with pretreatments: dry anaerobic co-digestion of citrus wastes with

chicken feathers, wheat straw and manure bedded with straw, was investigated at 20 %TS in

batch process. The best mixing ratio enhanced methane yield by 14 % compared to the

expected yield from individual fractions. Process performance at different organic loading

rates (OLR) was then investigated in continuous plug flow reactors at 21 %TS and 32 %TS of

feedstock. Stability of the process decline as OLR was increased to 3.8 gVS/l/d resulting in

high total volatile fatty acids (VFA), VFA/alkalinity ratio and reduction in methane yield.

Keywords: Solid wastes; Dry anaerobic digestion; Textile bioreactor; Plug flow bioreactor; Digestate; Process stability; Pretreatment; Co-digestion; Mesophilic

iv

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I dedicate this thesis to the Almighty God and the blessed memories of my beloved late father

and mother: Mr Akotonayon Godonu and Mrs Bernice Godonu whom I painfully lost during

the course of this study

v

I dedicate this thesis to the Almighty God and the blessed memories of my beloved late father

and mother: Mr Akotonayon Godonu and Mrs Bernice Godonu whom I painfully lost during

the course of this study

vi

vii

LIST OF PUBLICATIONS

This thesis is based on the results presented in the following articles:

I. Patinvoh, R. J., Feuk-Lagerstedt, E., Lundin, M., Sárvári Horváth, I., Taherzadeh, M.

J. (2016). Biological pretreatment of chicken feather and biogas production from total

broth. Applied Biochemistry and Biotechnology 180(7): 1401–1415.

II. Patinvoh, R. J., Kalantar Mehrjerdi, A., Sárvári Horváth, I., Taherzadeh, M. J.

(2017). Dry fermentation of manure with straw in continuous plug flow reactor:

Reactor development and process stability at different loading rates. Bioresource

Technology 224: 197–205.

III. Patinvoh, R. J., Osadolor, O. A., Chandolias, K., Sárvári Horváth, I., Taherzadeh, M.

J. (2017). Innovative pretreatment strategies for biogas production. Bioresource


IV. Patinvoh, R. J., Osadolor, O. A., Sárvári Horváth, I., Taherzadeh, M. J. (2017). Cost

effective dry anaerobic digestion in textile bioreactors: Experimental and economic

evaluation. Bioresource Technology 245(Part A): 549-559

V. Patinvoh, R. J., Arulappan, J. V., Johansson, F., Taherzadeh, M. J. (2017). Biogas

digesters: from plastics and bricks to textile bioreactor—A review. Submitted.

VI. Patinvoh, R. J., Lundin, M., Taherzadeh, M. J., Sárvári Horváth, I. (2017). Dry

anaerobic co-digestion of citrus wastes with keratin-rich and lignocellulosic solid

organic wastes: Batch vs. Continuous Processes. Submitted.

vii

LIST OF PUBLICATIONS

This thesis is based on the results presented in the following articles:

I. Patinvoh, R. J., Feuk-Lagerstedt, E., Lundin, M., Sárvári Horváth, I., Taherzadeh, M.

J. (2016). Biological pretreatment of chicken feather and biogas production from total

broth. Applied Biochemistry and Biotechnology 180(7): 1401–1415.

II. Patinvoh, R. J., Kalantar Mehrjerdi, A., Sárvári Horváth, I., Taherzadeh, M. J.

(2017). Dry fermentation of manure with straw in continuous plug flow reactor:

Reactor development and process stability at different loading rates. Bioresource


III. Patinvoh, R. J., Osadolor, O. A., Chandolias, K., Sárvári Horváth, I., Taherzadeh, M.

J. (2017). Innovative pretreatment strategies for biogas production. Bioresource


IV. Patinvoh, R. J., Osadolor, O. A., Sárvári Horváth, I., Taherzadeh, M. J. (2017). Cost

effective dry anaerobic digestion in textile bioreactors: Experimental and economic

evaluation. Bioresource Technology 245(Part A): 549-559

V. Patinvoh, R. J., Arulappan, J. V., Johansson, F., Taherzadeh, M. J. (2017). Biogas

digesters: from plastics and bricks to textile bioreactor—A review. Submitted.

VI. Patinvoh, R. J., Lundin, M., Taherzadeh, M. J., Sárvári Horváth, I. (2017). Dry

anaerobic co-digestion of citrus wastes with keratin-rich and lignocellulosic solid

organic wastes: Batch vs. Continuous Processes. Submitted.

viii

STATEMENT OF CONTRIBUTIONS

My contributions to each of the publications follow:

Paper I: Responsible for part of the idea, performed all experimental work, analysed the data,

wrote the manuscript, and executed its revision

Paper II: Responsible for part of the idea, performed all experimental work, analysed the

data, wrote the manuscript, and executed its revision

Paper III: Responsible for part of the idea, the literature survey, and the data collection,

wrote the major part and revision of the manuscript

Paper IV: Responsible for part of the idea, performed all the experimental work and the data

analysis, wrote and revised the manuscript, except for the economic evaluation aspect of the

work

Paper V: Responsible for part of the idea, the literature survey, and the data collection, wrote

the major part of the manuscript

Paper VI: Responsible for part of the idea, performed all the experimental work, analysed the

data, and wrote the manuscript

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RESEARCH JOURNEY

I started my academic career as a graduate assistant at Lagos State University (LASU),

Nigeria. I obtained a Master’s of Science degree in chemical engineering at the University of

Lagos and Lagos State University, and my focus since that time has been to provide solutions

to environmental, health, and energy challenges in Nigeria. I decided to pursue my doctorate

degree to enhance my career goals, and my sincere desire to pursue this degree abroad

(Sweden) came from an aspiration to become more skilful in carrying out research in my field

and providing solutions to some of the challenges facing my beloved country, Nigeria.

At the University of Borås, Sweden, I decided to focus on biogas production from solid

wastes because this technology can generate energy while also reducing harmful emissions

that affect human health and the ecosystem. As a doctoral student, my first task was to carry

out a literature review on dry anaerobic digestion (dry-AD) of solid wastes for biogas

production, after which I gave a short presentation providing a general overview of dry-AD

processes, previous research findings, and research gaps.

I started my first experimental work, with a former Ph.D. student (Dr. Maryam M. Kabir), on

biogas production from manure with straw in a textile bioreactor and dry anaerobic digestion

of lignocellosic and protein wastes in a batch process.

In the latter part of my first year, I began experimental work using a synthetic medium for

biogas production. Thereafter, we started a study on the use of chicken feather for biogas

production (Paper I). Chicken feathers are readily available solid waste, and more than 90 %

of chicken feather is keratin (insoluble protein), which has great potential for biogas

production if converted to soluble protein. This potential motivated our study into the use of

chicken feathers for biogas production, but because chicken feathers are recalcitrant, we

needed to perform a pretreatment prior to biogas production. We focused on a biological

pretreatment method that would be sustainable and cost effective. Additionally, we carried out

a general study on pretreatment and non-pretreatment strategies to overcome the challenges

associated with the use of recalcitrant and inhibitory feedstock for biogas production (Paper

III).

During my second and third years, I focused on dry digestion of solid wastes using novel

bioreactors. Plug flow reactors had been designed and developed for continuous dry digestion

ix

RESEARCH JOURNEY

I started my academic career as a graduate assistant at Lagos State University (LASU),

Nigeria. I obtained a Master’s of Science degree in chemical engineering at the University of

Lagos and Lagos State University, and my focus since that time has been to provide solutions

to environmental, health, and energy challenges in Nigeria. I decided to pursue my doctorate

degree to enhance my career goals, and my sincere desire to pursue this degree abroad

(Sweden) came from an aspiration to become more skilful in carrying out research in my field

and providing solutions to some of the challenges facing my beloved country, Nigeria.

At the University of Borås, Sweden, I decided to focus on biogas production from solid

wastes because this technology can generate energy while also reducing harmful emissions

that affect human health and the ecosystem. As a doctoral student, my first task was to carry

out a literature review on dry anaerobic digestion (dry-AD) of solid wastes for biogas

production, after which I gave a short presentation providing a general overview of dry-AD

processes, previous research findings, and research gaps.

I started my first experimental work, with a former Ph.D. student (Dr. Maryam M. Kabir), on

biogas production from manure with straw in a textile bioreactor and dry anaerobic digestion

of lignocellosic and protein wastes in a batch process.

In the latter part of my first year, I began experimental work using a synthetic medium for

biogas production. Thereafter, we started a study on the use of chicken feather for biogas

production (Paper I). Chicken feathers are readily available solid waste, and more than 90 %

of chicken feather is keratin (insoluble protein), which has great potential for biogas

production if converted to soluble protein. This potential motivated our study into the use of

chicken feathers for biogas production, but because chicken feathers are recalcitrant, we

needed to perform a pretreatment prior to biogas production. We focused on a biological

pretreatment method that would be sustainable and cost effective. Additionally, we carried out

a general study on pretreatment and non-pretreatment strategies to overcome the challenges

associated with the use of recalcitrant and inhibitory feedstock for biogas production (Paper

III).

During my second and third years, I focused on dry digestion of solid wastes using novel

bioreactors. Plug flow reactors had been designed and developed for continuous dry digestion

x

processes. I began a preliminary experiment with the newly developed plug flow reactor and

thereafter started a study on continuous dry fermentation of manure bedded with straw at 22

%TS with different organic loading rates using this plug flow reactor (Paper II). The reactor

was monitored continuously to avoid failure of the process and to identify the critical organic

loading rate, above which instability can occur in the reactor. Thereafter, we carried out a

study on dry fermentation of solid wastes in a textile bioreactor (Paper IV). The plug flow

bioreactor studied earlier is suitable for dry-AD processes, but it requires expert skills and

constant monitoring, so there is a need for simple technologies that can be accessed easily by

developing countries where the required expertise may not be available. Therefore, we

worked on the dry digestion of solid wastes in the textile bioreactor and discovered that it is

very simple to operate; the reactor is a robust and cost effective solution for rural and

developing countries. We also proposed an industrial-scale concept for dry digestion of solid

wastes using the textile bioreactor for farmers. Additionally, we carried out a general study on

biogas bioreactors and the challenges associated with them while introducing a novel textile

bioreactor (Paper V).

In view of the fact that easily degraded feedstocks are not highly available and some problems

associated with pretreatments, we focused on dry anaerobic co-digestion of solid wastes in

batch and continuous processes using plug flow reactors during my fourth year (Paper VI).

Working on biogas production from solid wastes under various conditions using textile and

plug flow bioreactors has been very interesting. I am looking forward to setting up a biogas

laboratory in a prominent university in Nigeria and subsequently a biogas plant.

xi

NOMENCLATURE

AD Anaerobic digestion

ATP Adenosine triphosphate

BMP Biomethane potential

C/N Carbon to nitrogen ratio

COD Chemical oxygen demand

CSTR Continuous stirred tank reactor

GHG Greenhouse gas

HMF 5-hydroxymethylfurfural

HRT Hydraulic retention time

LCFA Long chain fatty acids

MSW Municipal solid waste

MP-AES Microwave plasma-atomic emission spectrometer

OLR Organic loading rate

PF Plug flow

STR Solid retention time

TS Total solids

UASB Upflow anaerobic sludge blanket

VFA Volatile fatty acids

VS Volatile solids

xi

NOMENCLATURE

AD Anaerobic digestion

ATP Adenosine triphosphate

BMP Biomethane potential

C/N Carbon to nitrogen ratio

COD Chemical oxygen demand

CSTR Continuous stirred tank reactor

GHG Greenhouse gas

HMF 5-hydroxymethylfurfural

HRT Hydraulic retention time

LCFA Long chain fatty acids

MSW Municipal solid waste

MP-AES Microwave plasma-atomic emission spectrometer

OLR Organic loading rate

PF Plug flow

STR Solid retention time

TS Total solids

UASB Upflow anaerobic sludge blanket

VFA Volatile fatty acids

VS Volatile solids

xii

xiii

TABLE OF CONTENTS

ABSTRACT .............................................................................................................................................................. iii

LIST OF PUBLICATIONS ..................................................................................................................................... vii

RESEARCH JOURNEY ......................................................................................................................................... ix

NOMENCLATURE .................................................................................................................................................. xi

1. Introduction .................................................................................................................................................. 1

1.1 Aims of studies .......................................................................................................................................... 2

1.2 Thesis structure ......................................................................................................................................... 2

1.3 Research ethics and social aspects ....................................................................................................... 3

2. Solid wastes as potential biomass resources ......................................................................... 5

2.1 Emissions from solid wastes ................................................................................................................. 5

2.2 Potential of solid wastes ......................................................................................................................... 6

2.2.1 Agricultural residues ........................................................................................................................ 6

2.2.1.1 Chicken feathers...................................................................................................................... 7

2.2.1.2 Animal manure ........................................................................................................................ 8

2.2.1.3 Forest and crop residues ....................................................................................................... 8

2.2.2 Municipal solid wastes .................................................................................................................... 9

2.2.3 Industrial residues ............................................................................................................................. 10

3. An overview of Biogas production ................................................................................................. 13

3.1 Basic reactions involved in the anaerobic digestion process ....................................................... 14

3.2 Challenges of biogas production and possible solutions............................................................... 15

3.2.1 Temperature fluctuations ................................................................................................................ 15

3.2.2 Feedstock composition .................................................................................................................... 16

3.2.2.1 Organic content and nutrients ................................................................................................. 16

3.2.2.2 Impurities in feedstock ............................................................................................................. 17

3.2.2.3 Long chain fatty acid content ................................................................................................. 17

3.2.2.4 Total solid content...................................................................................................................... 18

3.2.3 Foam formation .................................................................................................................................. 19

3.2.4 Process instability .............................................................................................................................. 20

3.2.5 Reactor design .................................................................................................................................... 21

3.3 Biogas in Nigeria ...................................................................................................................................... 25

3.3.1 Biogas challenges and pioneered projects in Nigeria ............................................................. 26

xiii

TABLE OF CONTENTS

ABSTRACT .............................................................................................................................................................. iii

LIST OF PUBLICATIONS ..................................................................................................................................... vii

RESEARCH JOURNEY ......................................................................................................................................... ix

NOMENCLATURE .................................................................................................................................................. xi

1. Introduction .................................................................................................................................................. 1

1.1 Aims of studies .......................................................................................................................................... 2

1.2 Thesis structure ......................................................................................................................................... 2

1.3 Research ethics and social aspects ....................................................................................................... 3

2. Solid wastes as potential biomass resources ......................................................................... 5

2.1 Emissions from solid wastes ................................................................................................................. 5

2.2 Potential of solid wastes ......................................................................................................................... 6

2.2.1 Agricultural residues ........................................................................................................................ 6

2.2.1.1 Chicken feathers...................................................................................................................... 7

2.2.1.2 Animal manure ........................................................................................................................ 8

2.2.1.3 Forest and crop residues ....................................................................................................... 8

2.2.2 Municipal solid wastes .................................................................................................................... 9

2.2.3 Industrial residues ............................................................................................................................. 10

3. An overview of Biogas production ................................................................................................. 13

3.1 Basic reactions involved in the anaerobic digestion process ....................................................... 14

3.2 Challenges of biogas production and possible solutions............................................................... 15

3.2.1 Temperature fluctuations ................................................................................................................ 15

3.2.2 Feedstock composition .................................................................................................................... 16

3.2.2.1 Organic content and nutrients ................................................................................................. 16

3.2.2.2 Impurities in feedstock ............................................................................................................. 17

3.2.2.3 Long chain fatty acid content ................................................................................................. 17

3.2.2.4 Total solid content...................................................................................................................... 18

3.2.3 Foam formation .................................................................................................................................. 19

3.2.4 Process instability .............................................................................................................................. 20

3.2.5 Reactor design .................................................................................................................................... 21

3.3 Biogas in Nigeria ...................................................................................................................................... 25

3.3.1 Biogas challenges and pioneered projects in Nigeria ............................................................. 26

xiv

3.3.2 Strategies for improved biogas technology ............................................................................... 28

4. Dry anaerobic digestion (dry-AD) ................................................................................................... 31

4.1 Comparison of wet and dry anaerobic digestion ............................................................................. 31

4.2 Start-up phase in dry anaerobic digestion ......................................................................................... 32

4.3 Dry anaerobic digestion – Batch process........................................................................................... 33

4.3.1Dry digestion in textile bioreactor ................................................................................................. 34

4.3.2 BEKON dry digestion technology ............................................................................................... 36

4.4 Dry anaerobic digestion – Continuous process ................................................................................. 36

4.4.1 Plug flow reactor for continuous dry digestion process .......................................................... 37

4.4.2 Continuous dry digestion of manure bedded with straw ......................................................... 37

4.4.3 Continuous dry co-digestion of solid wastes .............................................................................. 39

4.4.4 Continuous dry-AD technologies .................................................................................................. 40

5. Improving biogas production from solid wastes ................................................................... 43

5.1 Pretreatment to overcome biomass recalcitrance ............................................................................ 44

5.1.1 Mechanical pretreatment ................................................................................................................. 43

5.1.2 Thermal pretreatment ....................................................................................................................... 44

5.1.3 Chemical pretreatment ..................................................................................................................... 44

5.1.4 Biological pretreatment ................................................................................................................... 44

5.1.4.1 Hydrolysis of chicken feathers for biogas production ..................................................... 45

5.2 Co-digestion to achieve balance nutrient ............................................................................................ 46

5.3 Acclimatisation of microorganisms to substrates ............................................................................. 47

6. Digestate as biofertiliser ....................................................................................................................... 49

6.1 Composition and quality of the digestate ......................................................................................... 49

6.2 Benefits of digestate ............................................................................................................................... 50

6.3 Dewatering process ................................................................................................................................. 51

6.4 Digestate from dry anaerobic digestion in textile reactor ............................................................ 52

7. Concluding remarks and future directions 7.1 Conclusions ............................................................................................................................................... 55

7.2 Future directions ...................................................................................................................................... 56

ACKNOWLEDGEMENT ........................................................................................................................................ 57

REFERENCES ........................................................................................................................................................ 61

1

Chapter 1

Introduction

World population is growing rapidly, and this explosion has led to rapid consumption of oil

resources and a tremendous increase in the volume of wastes generated. Globally, about 17

billion tonnes of total solid wastes are generated per year [1], and the amount is estimated to

reach 27 billion tonnes in 2050 [2]. Continuous emissions of carbon dioxide, methane, and

other greenhouse gases from these waste streams and the burning of fossil fuels has led to a

global environmental crisis. The intensive agriculture practised to produce food also damages

the environment through the use of chemical fertilisers. Additionally, about 16 % of the

global population does not have access to electricity and about 38 % of the population [3, 4]

uses solid wastes (forest residue, animal manure, crop and other wastes residues) for

residential heating and cooking in poorly ventilated areas, which results in environmental and

health hazards. Concerns about these environmental pressures and energy insecurity have

increased the need for research on energy generation from renewable sources. The

undervalued and abundant solid wastes that are generated have great potential as sources of

biomass for energy production if properly harnessed and could lead to reduced environmental

pollution and increased renewable energy production.

Biogas production through anaerobic digestion is a resource effective way of managing the

large volume of organic waste generated. It is a well-established and sustainable process for

energy generation from organic wastes. In addition to the biogas produced for energy

generation, the digestate residue can be utilised as bio-fertilizer. Anaerobic digestion of

organic wastes for biogas production is generally done through both wet and dry digestion

processes. In industry, biogas production from wastes with high water content is most

common process; however the utilization of solid wastes from agricultural, municipal, and

industrial activities, including forest and crop residues, is becoming more and more important

as well. Solid wastes usually have a solid content of between 15 % and 50 %; hence

conventional wet anaerobic digestion treatment of these kinds of wastes requires a lot of

water. Therefore, processing these waste streams for biogas production using dry-AD

technology is a better option making it possible to manage waste streams with low moisture

content.

1

Chapter 1

Introduction

World population is growing rapidly, and this explosion has led to rapid consumption of oil

resources and a tremendous increase in the volume of wastes generated. Globally, about 17

billion tonnes of total solid wastes are generated per year [1], and the amount is estimated to

reach 27 billion tonnes in 2050 [2]. Continuous emissions of carbon dioxide, methane, and

other greenhouse gases from these waste streams and the burning of fossil fuels has led to a

global environmental crisis. The intensive agriculture practised to produce food also damages

the environment through the use of chemical fertilisers. Additionally, about 16 % of the

global population does not have access to electricity and about 38 % of the population [3, 4]

uses solid wastes (forest residue, animal manure, crop and other wastes residues) for

residential heating and cooking in poorly ventilated areas, which results in environmental and

health hazards. Concerns about these environmental pressures and energy insecurity have

increased the need for research on energy generation from renewable sources. The

undervalued and abundant solid wastes that are generated have great potential as sources of

biomass for energy production if properly harnessed and could lead to reduced environmental

pollution and increased renewable energy production.

Biogas production through anaerobic digestion is a resource effective way of managing the

large volume of organic waste generated. It is a well-established and sustainable process for

energy generation from organic wastes. In addition to the biogas produced for energy

generation, the digestate residue can be utilised as bio-fertilizer. Anaerobic digestion of

organic wastes for biogas production is generally done through both wet and dry digestion

processes. In industry, biogas production from wastes with high water content is most

common process; however the utilization of solid wastes from agricultural, municipal, and

industrial activities, including forest and crop residues, is becoming more and more important

as well. Solid wastes usually have a solid content of between 15 % and 50 %; hence

conventional wet anaerobic digestion treatment of these kinds of wastes requires a lot of

water. Therefore, processing these waste streams for biogas production using dry-AD

technology is a better option making it possible to manage waste streams with low moisture

content.

2

A significant drawback regarding some of these solid wastes fractions is their recalcitrant

nature, which limits their biological degradation and results in slow processes and low biogas

production. Therefore, it is necessary to overcome the recalcitrant structure and make them

accessible to microorganisms for improved biogas production [5], so there is a need for a

suitable pretreatment prior to biogas production. Additionally, dry anaerobic digestion of solid

wastes for biogas production has proven to be very challenging for industries and developing

nations due to problems with feeding, mixing, and obtaining the appropriate technology for

operation. Hence, there is great need for reactors that are simple to operate, robust in nature,

and cost effective. Agricultural sectors, communities, and industries can install on-site

anaerobic digesters to treat solid wastes and produce energy. Anaerobic digestion of these

wastes is sustainable and can play an important role in reducing greenhouse gas emissions and

the continuing dependence on fossil fuels.

1.1 Aims of studies

The main goals of this study were to improve biogas yield from recalcitrant and inhibitory

solid wastes and to assess dry anaerobic digestion of solid wastes through the development

and use of novel bioreactors. To attain this goal, this study was divided into four parts:

Biological pretreatment of solid wastes (chicken feathers) prior to biogas production

(Paper I)

Study of pretreatment and non-pretreatment strategies for improved biogas production

from recalcitrant and inhibitory solid wastes (Paper III)

Investigation of the potential of dry anaerobic digestion (dry-AD) using novel biogas

bioreactors: plug flow and textile bioreactors (Papers II, IV, and VI)

Study of conventional biogas bioreactors and the challenges associated with them

while introducing a novel textile bioreactor (Paper V)

1.2 Thesis structure

This thesis is divided into seven main chapters as follows:

Chapter 1 introduces the thesis, motivation, and the main objectives of this research.

Chapter 2 provides information on emissions from solid wastes, the potentials of

solid wastes, and the challenges associated with the processing of these wastes

fractions for biogas production.

3

Chapter 3 presents a general overview of biogas production, the reactions involved,

biogas production challenges and possible solutions. It also presents an overview of

the potential of biogas in Nigeria and strategies for advancement of biogas technology

in Nigeria.

Chapter 4 describes dry anaerobic digestion (dry-AD) for biogas production and its

advantages over wet fermentation. It also presents the start-up phase for dry digestion

processes, the batch dry digestion process in a textile bioreactor, dry digestion in

continuous plug flow reactors, and available continuous dry digestion technologies in

the industries (Papers II, IV, V and VI).

Chapter 5 describes pretreatment and non-pretreatment strategies to improve biogas

production from recalcitrant and inhibitory solid wastes (Papers I and III).

Chapter 6 presents a short review on the digestate residue after biogas production and

discusses the composition and quality. Suitability of digestate residue obtained after

dry digestion of manure with straw in a textile bioreactor was also presented (Paper

IV).

Chapter 7 provides the main conclusions of research and discusses potential future

work.

1.3 Research ethics and social aspects

Access to a safe ecosystem and undamaged natural resources is a public right because human

health depends on the quality of the surrounding environment. Many people die prematurely,

especially in developing countries, as a result of improper management of solid wastes, which

leads to air pollution, flooding, health and environmental challenges. It is estimated that 38 %

of the global population [3, 4] uses solid biomass (wood, forest debris, manure, crop, and

other waste residues) for home heating and cooking in poorly ventilated areas. My research on

biogas production from wastes will help improve quality of life by reducing environmental

pollution and increasing renewable energy production.

Biogas production sometimes requires a lot of water, and this use of water should be reduced

if possible because access to water is an essential human right. Biogas production for the

generation of energy should not be at the expense of this essential right. Therefore, this

research was focused on dry anaerobic digestion, in which a reduced amount of water is used

(Papers II, IV, and VI).

3

Chapter 3 presents a general overview of biogas production, the reactions involved,

biogas production challenges and possible solutions. It also presents an overview of

the potential of biogas in Nigeria and strategies for advancement of biogas technology

in Nigeria.

Chapter 4 describes dry anaerobic digestion (dry-AD) for biogas production and its

advantages over wet fermentation. It also presents the start-up phase for dry digestion

processes, the batch dry digestion process in a textile bioreactor, dry digestion in

continuous plug flow reactors, and available continuous dry digestion technologies in

the industries (Papers II, IV, V and VI).

Chapter 5 describes pretreatment and non-pretreatment strategies to improve biogas

production from recalcitrant and inhibitory solid wastes (Papers I and III).

Chapter 6 presents a short review on the digestate residue after biogas production and

discusses the composition and quality. Suitability of digestate residue obtained after

dry digestion of manure with straw in a textile bioreactor was also presented (Paper

IV).

Chapter 7 provides the main conclusions of research and discusses potential future

work.

1.3 Research ethics and social aspects

Access to a safe ecosystem and undamaged natural resources is a public right because human

health depends on the quality of the surrounding environment. Many people die prematurely,

especially in developing countries, as a result of improper management of solid wastes, which

leads to air pollution, flooding, health and environmental challenges. It is estimated that 38 %

of the global population [3, 4] uses solid biomass (wood, forest debris, manure, crop, and

other waste residues) for home heating and cooking in poorly ventilated areas. My research on

biogas production from wastes will help improve quality of life by reducing environmental

pollution and increasing renewable energy production.

Biogas production sometimes requires a lot of water, and this use of water should be reduced

if possible because access to water is an essential human right. Biogas production for the

generation of energy should not be at the expense of this essential right. Therefore, this

research was focused on dry anaerobic digestion, in which a reduced amount of water is used

(Papers II, IV, and VI).

4

Biogas production also becomes a human rights issues when it destroys ecosystems and

natural resources that are critical to the health and subsistence of people [6], hence, the

residue remaining after biogas production must be free of pathogens, viruses, and toxic

compounds that could contaminate soil, air, or water.

Methane, which is the major product of biogas production, is a greenhouse gas. It is the

second most important greenhouse gas after CO2, causing roughly 25 % of contribution to

greenhouse warming [7]. Emissions of methane during biogas production contribute to global

warming, so precautions should be taken to reduce methane emissions to the barest minimum.

Methane can also reduce the amount of life-sustaining oxygen in the air, especially in

confined spaces.

Additionally, genetically modified bacteria can have an effect on natural ecosystems [8] when

the digestate residue from biogas production is released into the environment. In this research,

a wild strain of bacteria (Bacillus pumilus), which is sustainable and ecologically safe, was

used for the biological pretreatment prior to biogas production (Paper I).

Biogas production should be environmentally sustainable to preserve life, protect our

ecosystem, improve air quality, and ameliorate current climate problems.

5

Chapter 2

Solid wastes as potential biomass resources

A tremendous amount of solid wastes is generated on a daily basis, and this amount will

increase due to population growth and increasing rates of consumption. A large amount of

these wastes is still being disposed with limited recycling, combustion without energy

recovery, and landfilling without gas control [9], resulting in global environmental issues.

These wastes are potential sources for new material and renewable energy generation if

harnessed properly.

2.1 Emissions from solid wastes

As solid wastes from municipal communities and agricultural and industrial activities are

disposed, the organic fraction of these wastes is degraded over a period of time releasing CH4,

CO2, and other gases to the atmosphere and thereby contributing to global warming. The

estimated global emissions from solid waste sites are in the range of 20 to 40 million tonnes

of CH4 per year [10] and livestock sector also contributes significantly to anthropogenic

greenhouse gas emissions.

Solid wastes are often disposed in landfills, which results in emissions of methane and nitrous

oxides and thus contribute to the greenhouse effect. Another common methods for the

treatment of these waste streams are composting and incineration; composting results in

emissions of volatile compounds (ketones, aldehydes, ammonia, and methane) [11] and

incineration often leads to significant releases of dioxins to the environment if the exhaust gas

is not treated properly. The environmental impacts of these conventional management

methods are listed in Table 2.1.

Anaerobic digestion is a promising technology for reducing emissions from solid wastes: it is

a cost effective, resourceful, and viable method for solving waste problems. Studies have

shown that anaerobic digestion of these wastes is sustainable and has a great advantage over

aerobic treatment because of its improved energy balance [11].

5

Chapter 2

Solid wastes as potential biomass resources

A tremendous amount of solid wastes is generated on a daily basis, and this amount will

increase due to population growth and increasing rates of consumption. A large amount of

these wastes is still being disposed with limited recycling, combustion without energy

recovery, and landfilling without gas control [9], resulting in global environmental issues.

These wastes are potential sources for new material and renewable energy generation if

harnessed properly.

2.1 Emissions from solid wastes

As solid wastes from municipal communities and agricultural and industrial activities are

disposed, the organic fraction of these wastes is degraded over a period of time releasing CH4,

CO2, and other gases to the atmosphere and thereby contributing to global warming. The

estimated global emissions from solid waste sites are in the range of 20 to 40 million tonnes

of CH4 per year [10] and livestock sector also contributes significantly to anthropogenic

greenhouse gas emissions.

Solid wastes are often disposed in landfills, which results in emissions of methane and nitrous

oxides and thus contribute to the greenhouse effect. Another common methods for the

treatment of these waste streams are composting and incineration; composting results in

emissions of volatile compounds (ketones, aldehydes, ammonia, and methane) [11] and

incineration often leads to significant releases of dioxins to the environment if the exhaust gas

is not treated properly. The environmental impacts of these conventional management

methods are listed in Table 2.1.

Anaerobic digestion is a promising technology for reducing emissions from solid wastes: it is

a cost effective, resourceful, and viable method for solving waste problems. Studies have

shown that anaerobic digestion of these wastes is sustainable and has a great advantage over

aerobic treatment because of its improved energy balance [11].

6

Table 2.1. Environmental impacts of conventional solid waste management methods [12]

Landfill Composting Incineration Air Emissions of methane (CH4)

and carbon monoxide (CO) Emissions of methane (CH4) and carbon monoxide (CO)

Emissions of SO2, NOx, HCl, HF, CO, CO2, N2O, dioxins, furans, heavy metals (Zn, Pb, Cu, As)

Water Leaching of salts, heavy metals, and biodegradable and persistent organics to groundwater

N/A Deposition of hazardous substances on surface water

Soil Accumulation of hazardous substances in soil

N/A Landfilling of ashes and scrap

Landscape Soil occupancy; restriction on other land uses

Soil occupancy; restriction on other land uses

Visual intrusion; restriction on other land uses

Ecosystem Contamination and accumulation of toxic substances in the food chain

Contamination and accumulation of toxic substances in the food chain

Contamination and accumulation of toxic substances in the food chain

Urban areas Exposure to hazardous substances

N/A Exposure to hazardous substances

2.2 Potential of solid wastes

Solid wastes are potential biomass sources because they are readily available and their

conversion to energy through biological processes is feasible with low capital investment.

Biomass comes from a range of sources, as shown in Fig. 2.1, which can be classified

according to the activities generating these wastes or the locations where these wastes are

generated [13]. Solid wastes generated by agricultural sectors, communities, and industries

along with their potential as renewable energy sources are discussed in this section. Table 2.2

shows the major characteristics of solid wastes used during this study.

2.2.1 Agricultural residues

Agricultural wastes account for the largest potential feedstock, and wide varieties of these

wastes can be used as sources of biomass energy. The most common sources (chicken

feathers, animal manure, forest and crop residues) are discussed in this section.

7

AGRICULTURAL RESIDUES

MUNICIPAL SOLID WASTES

INDUSTRIAL RESIDUES

FOREST RESIDUES

ANIMAL RESIDUES

CROP RESIDUES

SOLID WASTES BIOMASS SOURCES

Fig. 2.1. Solid wastes; potential sources of biomass

2.2.1.1 Chicken feathers (CF)

Currently, the world’s average stock of chicken is about of 22 billion live chickens [14], and

about 5–7 % of the total weight of a normal chicken is feathers [15]. Therefore, chicken

feathers are available in tremendous amount around the world, causing environmental

challenges. The major component of feather is β-keratin [16], which is an insoluble structural

protein. The polypeptides chain, as shown in Fig. 2.1, is tightly packed and highly cross-

linked with disulphide bonds, hydrogen bonds, and hydrophobic interactions [17]. This

structure makes the protein insoluble and resistant to enzymatic attack, which is a major

limitation to the biological processing of these wastes [5]. Chicken feather is a potential

source of biomass for energy generation if an appropriate pretreatment method is applied prior

to anaerobic digestion. The methane potential of protein wastes is as high as 0.496 Nm3/kgVS

[18] if all insoluble proteins are converted to protein. In this research, chicken feathers were

used as feedstock for methane production at various concentrations, and the recalcitrant nature

of feathers were altered prior to anaerobic digestion using biological pretreatment (Paper I).

7

AGRICULTURAL RESIDUES

MUNICIPAL SOLID WASTES

INDUSTRIAL RESIDUES

FOREST RESIDUES

ANIMAL RESIDUES

CROP RESIDUES

SOLID WASTES BIOMASS SOURCES

Fig. 2.1. Solid wastes; potential sources of biomass

2.2.1.1 Chicken feathers (CF)

Currently, the world’s average stock of chicken is about of 22 billion live chickens [14], and

about 5–7 % of the total weight of a normal chicken is feathers [15]. Therefore, chicken

feathers are available in tremendous amount around the world, causing environmental

challenges. The major component of feather is β-keratin [16], which is an insoluble structural

protein. The polypeptides chain, as shown in Fig. 2.1, is tightly packed and highly cross-

linked with disulphide bonds, hydrogen bonds, and hydrophobic interactions [17]. This

structure makes the protein insoluble and resistant to enzymatic attack, which is a major

limitation to the biological processing of these wastes [5]. Chicken feather is a potential

source of biomass for energy generation if an appropriate pretreatment method is applied prior

to anaerobic digestion. The methane potential of protein wastes is as high as 0.496 Nm3/kgVS

[18] if all insoluble proteins are converted to protein. In this research, chicken feathers were

used as feedstock for methane production at various concentrations, and the recalcitrant nature

of feathers were altered prior to anaerobic digestion using biological pretreatment (Paper I).

8

Fig. 2.2. Secondary structure of β-keratin; tightly packed structure (recalcitrance) of chicken

feathers (Modified from [19])

2.2.1.2 Animal manure

Farm animals produce billions of tons of manure annually, and the amount of manure bedded

with straw increases as the number of housed dairy animals increases. Storing these immense

quantities of manure can lead to significant anthropogenic emissions of methane and N2O

[20]. Additionally, untreated manure can also contain pathogens and viruses that can lower

the productivity of livestock farms [21] and pose a threat to human health when applied to

agricultural land. However, the large volume of animal manure generated by agricultural

sectors is a great source for energy production; methane from the manure can be captured and

used as a source of energy, thereby preventing the environmental harm caused by these waste

streams. Freshness of manure is required for effective conversion to energy through anaerobic

digestion process: the older the manure, the higher the degree of previous decomposition and

the lower the amount of undecomposed matter available for conversion to methane [22].

Cattle manure bedded with straw at 22 %TS, 27 %TS, and 30 %TS was used as feedstock for

biogas production through the dry anaerobic digestion process in this study (Papers II, IV,

and VI).

2.2.1.3 Forest and crop residues

Forest and crop residues are lignocellulosic biomass with a yearly production of

approximately 200 billion tons [23]. They are the most abundant renewable source for energy

production. Forest residues include dead trees, litter fall, and wastes generated in the process

of culling and logging. Crop residues include straw, husk, leaf, stem, stalk, shell, cob, and

bagasse, which come from cereals, cotton, groundnut, fruits, legumes, palm oil, and sugar

9

cane. These waste streams are composed of polysaccharide-rich walls, as shown in Fig. 2.3.

They are difficult to break down as a result of the matrix polymers (hemicellulose, pectins,

and lignin) surrounding the cellulosic microfibrils in the plant cell wall [24]. The degree of

resistance of these wastes to microbial attack varies depending on the type of lignocellulose,

various compartments of the cell wall, cell age, and processing phenomena such as drying and

heating [25].

Effective biological processing of this biomass for energy production requires an appropriate

pretreatment to open the structure and increase the accessibility for the cellulose degrading

microorganisms. Wheat straw was used in co-digestion of cattle manure to increase the C/N

of the substrate and thereby enhance the performance of the anaerobic digestion process

(Paper IV). It was also used in dry co-digestion of different solid waste fractions i.e. chicken

feathers, citrus wastes, wheat straw, and manure with straw to reduce the inhibitory effect of

citrus waste (Paper VI).

Fig. 2.3. Different layers in cell wall of a wood fiber or tracheid including middle lamella,

primary wall, and secondary wall (S1, S2, S3). Cellulosic microfibrils in Plant Cell wall

surrounded by matrix polymers (reprinted with permission) [26].

2.2.2 Municipal solid wastes (MSW)

Municipal solid wastes usually include all the wastes generated in a community, such as

residential, commercial, and institutional wastes. They are the most concentrated of all waste

fractions, containing food wastes, paper, cardboard, textiles, plastics, glass, metals, yard

9

cane. These waste streams are composed of polysaccharide-rich walls, as shown in Fig. 2.3.

They are difficult to break down as a result of the matrix polymers (hemicellulose, pectins,

and lignin) surrounding the cellulosic microfibrils in the plant cell wall [24]. The degree of

resistance of these wastes to microbial attack varies depending on the type of lignocellulose,

various compartments of the cell wall, cell age, and processing phenomena such as drying and

heating [25].

Effective biological processing of this biomass for energy production requires an appropriate

pretreatment to open the structure and increase the accessibility for the cellulose degrading

microorganisms. Wheat straw was used in co-digestion of cattle manure to increase the C/N

of the substrate and thereby enhance the performance of the anaerobic digestion process

(Paper IV). It was also used in dry co-digestion of different solid waste fractions i.e. chicken

feathers, citrus wastes, wheat straw, and manure with straw to reduce the inhibitory effect of

citrus waste (Paper VI).

Fig. 2.3. Different layers in cell wall of a wood fiber or tracheid including middle lamella,

primary wall, and secondary wall (S1, S2, S3). Cellulosic microfibrils in Plant Cell wall

surrounded by matrix polymers (reprinted with permission) [26].

2.2.2 Municipal solid wastes (MSW)

Municipal solid wastes usually include all the wastes generated in a community, such as

residential, commercial, and institutional wastes. They are the most concentrated of all waste

fractions, containing food wastes, paper, cardboard, textiles, plastics, glass, metals, yard

10

trimmings, and others. Globally, the volume of MSW generated is about 1.3 billion tonnes per

year, and this volume is expected to increase to about 2.2 billion tonnes per year in 2025 [27].

MSW usually contains an easily biodegradable organic fraction of up to 40 %, and about 60

% of the dry weight of the average municipal solid waste is composed of lignocelluloses [28].

A major challenge in the biological processing of these wastes is the composition; there are

varieties of different fractions involved, depending on the source of the wastes, which can

have adverse effects on the digestion process. Therefore, MSW has to be mechanically,

magnetically, or hand-sorted to remove large stones, plastics, glass, metals, and other debris

and then shredded to reduce its particle size to be able to access and process the organic

fraction.

2.2.3 Industrial residues

Major organic industrial residues are those generated from food and fruit processing

industries. These industries produce large volumes of solid wastes, such as peels, pulp,

molasses, seeds, skins, pomace, spoiled food, and fruits. The composition and quantity of the

solid wastes generated varies from plant to plant and also depends on the technology

employed by the industry. Solid wastes generated from these industries contain large amounts

of organic matter such as carbohydrates, proteins, and lipids, which makes them potential

sources of biomass for energy production through biological processes. The major limitation

is that some of these wastes contain chemicals or substances that inhibit the activity of

microorganisms and thereby have an adverse effect on energy generation through anaerobic

digestion processes [29, 30]. Therefore, the wastes often need to be pre-treated or co-digested

with other substrates to reduce the inhibitory effect on biogas yield. Installation of anaerobic

digesters can be an integral part of industry services to treat solid wastes while also meeting

the industry’s energy demands. In this research, citrus waste was co-digested with chicken

feather, wheat straw, and cattle manure to reduce the inhibitory effect of D-limonene on

methane yield and enhance the buffering capacity of the digestion process (Paper VI).

11

Table 2.2: Major characteristics of solid wastes used during this study (mean values and

standard deviations based on triplicate measurements) (modified from Paper VI)

Solid wastes

pH Bulk density (g/L)

TS (%) VS ٭(%)

Ash ٭(%)

Total carbon ٭(%)

TKN ٭(%)

C/N COD gCOD /gVSsubstrate

Chicken feathers

ND ND 93.47 ± 0.06

99.26 ± 0.01

0.74 ± 0.01

55.14 ± 0.01

15.50 ± 0.30

3.6 ND

Citrus wastes

3.24 753.60 ± 24.32

23.42 ± 2.23

96.07 ± 0.74

3.93 ± 0.74

53.37 ± 0.41

0.99 ± 0.08

55.5 1.03

Wheata straw

ND 190.47 ± 6.93

89.07 ± 0.08

94.96 ± 0.14

5.04 ± 0.14

52.76 ± 0.08

0.83 ± 0.08

69 0.82

Manure with straw

8.01 542.00 ± 26.87

25.84 ± 0.92

77.21 ± 2.74

22.79 ± 2.74

42.89 ± 1.52

2.26 ± 0.04

19 0.89

aExtractives 8.27 %٭, Total lignin 16.52 %٭, Cellulose 42.74 %٭, Hemicellulose 27.99 %٭

dry basis. ND – not determined. C/N – carbon to nitrogen ratio. COD – chemical oxygen demand٭

11

Table 2.2: Major characteristics of solid wastes used during this study (mean values and

standard deviations based on triplicate measurements) (modified from Paper VI)

Solid wastes

pH Bulk density (g/L)

TS (%) VS ٭(%)

Ash ٭(%)

Total carbon ٭(%)

TKN ٭(%)


Chicken feathers

ND ND 93.47 ± 0.06

99.26 ± 0.01

0.74 ± 0.01

55.14 ± 0.01

15.50 ± 0.30

3.6 ND

Citrus wastes

3.24 753.60 ± 24.32

23.42 ± 2.23

96.07 ± 0.74

3.93 ± 0.74

53.37 ± 0.41

0.99 ± 0.08

55.5 1.03

Wheata straw

ND 190.47 ± 6.93

89.07 ± 0.08

94.96 ± 0.14

5.04 ± 0.14

52.76 ± 0.08

0.83 ± 0.08

69 0.82

Manure with straw

8.01 542.00 ± 26.87

25.84 ± 0.92

77.21 ± 2.74

22.79 ± 2.74

42.89 ± 1.52

2.26 ± 0.04

19 0.89


dry basis. ND – not determined. C/N – carbon to nitrogen ratio. COD – chemical oxygen demand٭

12

13

Chapter 3

An overview of biogas production

Biogas is produced from organic wastes through anaerobic digestion processes. This reduces

the effect of feedstock costs on biogas production and makes biogas production and utilisation

a good solution for addressing both waste and energy challenges [31]. Waste and residues are

also considered the most abundant renewable resources, and their use as feedstock for biogas

production reduces the competition between fuel and food, which is important for the long-

term success of biofuel [32]. The process of biogas production includes preparation of the

feedstock for digestion and the anaerobic digestion process, which consists of four basic steps,

hydrolysis, fermentation, acetogenesis, and methanogenesis, as explained in Fig. 3.1. In this

process, the first stage is very essential because microorganisms cannot use large molecules

directly unless they are disintegrated into smaller molecules; the rate of disintegration during

this stage depends on the nature of the substrate. During fermentation, the products from the

previous stage are used as substrates except fatty acids released during the decomposition of

fats and aromatic structures [33]. The third stage is very complex because it requires

interaction between the acetogens and the methanogens; the process is closely linked to the

concentration of hydrogen gas [33] and will stop if the hydrogen produced is not continuously

consumed. The most important substrates for methane formation are H2, CO2 and acetate but

some methanogens can also use methanol, formate and methylamines [7]. Additionally,

optimal conditions such as neutral pH, constant temperature, balanced nutrient composition,

and consistent feed rate are required for effective conversion of organic wastes to biogas.

Biogas produced through this process is a mixture of gases, consisting mainly of methane

(usually between 50 and 80 % [34]), carbon dioxide (between 20 and 45 %), and some

quantities of water vapour, hydrogen sulphide, ammonia, and traces of other gases. The

composition of the biogas depends on types of substrates, process condition, activities of the

microorganism during the digestion process [35], and various technical designs of the plant

[36]. Biogas can be used for heating, generation of electricity, and as a fuel for transportation,

and the nutrient rich residue remaining after biogas production can be used as a bio-fertiliser.

The sub-sections of this chapter outline the basic reactions, challenges of biogas production,

and assess biogas in Nigeria.

13

Chapter 3

An overview of biogas production

Biogas is produced from organic wastes through anaerobic digestion processes. This reduces

the effect of feedstock costs on biogas production and makes biogas production and utilisation

a good solution for addressing both waste and energy challenges [31]. Waste and residues are

also considered the most abundant renewable resources, and their use as feedstock for biogas

production reduces the competition between fuel and food, which is important for the long-

term success of biofuel [32]. The process of biogas production includes preparation of the

feedstock for digestion and the anaerobic digestion process, which consists of four basic steps,

hydrolysis, fermentation, acetogenesis, and methanogenesis, as explained in Fig. 3.1. In this

process, the first stage is very essential because microorganisms cannot use large molecules

directly unless they are disintegrated into smaller molecules; the rate of disintegration during

this stage depends on the nature of the substrate. During fermentation, the products from the

previous stage are used as substrates except fatty acids released during the decomposition of

fats and aromatic structures [33]. The third stage is very complex because it requires

interaction between the acetogens and the methanogens; the process is closely linked to the

concentration of hydrogen gas [33] and will stop if the hydrogen produced is not continuously

consumed. The most important substrates for methane formation are H2, CO2 and acetate but

some methanogens can also use methanol, formate and methylamines [7]. Additionally,

optimal conditions such as neutral pH, constant temperature, balanced nutrient composition,

and consistent feed rate are required for effective conversion of organic wastes to biogas.

Biogas produced through this process is a mixture of gases, consisting mainly of methane

(usually between 50 and 80 % [34]), carbon dioxide (between 20 and 45 %), and some

quantities of water vapour, hydrogen sulphide, ammonia, and traces of other gases. The

composition of the biogas depends on types of substrates, process condition, activities of the

microorganism during the digestion process [35], and various technical designs of the plant

[36]. Biogas can be used for heating, generation of electricity, and as a fuel for transportation,

and the nutrient rich residue remaining after biogas production can be used as a bio-fertiliser.

The sub-sections of this chapter outline the basic reactions, challenges of biogas production,

and assess biogas in Nigeria.

14

STAGE I STAGE II STAGE IVSTAGE III

Hydrolysis (conversion of sugars,

fats and proteins to simple sugars, fatty

acids and amino acids)

Fermentation(conversion of sugars,

amino acids etc. to organic acids, alcohols,

ammonia, CO2 and H2

Acetogenesis(conversion of fatty

acids and alcohols to hydrogen gas, acetate

and CO2)

Methane formation (conversion of

hydrogen gas, acetate and CO2 to methane

and CO2)

FERMENTATIVE BACTERIA

ACETOGENIC BACTERIA METHANOGENSHYDROLYTIC

BACTERIA

Fig. 3.1. Stages of the anaerobic digestion process for active biogas production and required

different microbial communities

3.1 Basic reactions involved in the anaerobic digestion process

Themelis and Kim [37] showed that the mixture of organic wastes can be approximated by the

chemical formula C6H10O4, excluding nitrogen and sulphur because they are relatively minor

and occur principally in mixed food wastes. The hydrolysis reaction can then be written as

shown in equation 1.

(1)

The hydrolysed organic compounds (sugars and amino acids) are converted to alcohols and

organic acids by fermentative bacteria, as written in equations 2 and 3 [38]

(2)

(3)

All of the organic acids, except the acetic acids [39], are consumed by the acetogenic bacteria

and thereafter converted to acetic acid and hydrogen, as written in equation 4 [38].

(4)

The last step is the formation of methane, in which methanogens consume acetic acid,

hydrogen, and carbon dioxide to produce methane. Some methanogens can feed on methanol

and several other substrates, including methylated compounds [7]. The basic reactions

involved in methane formation are written in equations 5, 6, 7, and 8 [38].

15

(5)

(6)

(7)

(8)

3.2 Challenges of biogas production and possible solutions

Despite the benefits of biogas production, a negative effect on any of the processes explained

in Fig. 3.1 has a direct effect on the other processes and the biogas process can become

unsteady or fail completely [40]. The major challenges discussed in this subsection are those

related to temperature fluctuations, feedstock composition, foam formation, process

instability, and reactor design.

3.2.1 Temperature fluctuations

Microorganisms require optimum temperature for growth and active performance during

biogas process and can be divided into psychrophilic (optimum temperature around 10 °C),

mesophilic (optimum temperature around 37 °C), thermophilic (optimum temperature around

55 °C) and hyper thermophilic (optimum temperature around 85 °C) [33]. Temperature

fluctuations affect the performance of a biogas process adversely because the activity of the

microorganism is reduced if temperature is above or below their optimum range. A decrease

in temperature may result in a reduced volatile fatty acid production rate, substrate

decomposition rate, and metabolic rate of the microorganism [41, 42]. Navickas, Venslauskas

[43] investigated the influence of temperature variations on the performance of anaerobic

digestion of industrial wastes and observed that a change in temperature from 52 °C to 57 °C

(at constant total solids concentration, organic load, and pH) resulted in a 24 % reduction in

biogas yield. The degradation rate is higher thermophilic processes, however they are more

sensitive to changes in temperature [44] and moreover, they require increased energy input to

maintain a stable temperature. In this study, all experiments were operated at mesophilic

temperature, at 37 °C (Papers I, II, and VI) and at 25 °C for dry digestion of cattle manure

with straw in a textile bioreactor (Paper IV), to check the suitability of the process in tropical

regions, but the reactor can be heated from below to attain the desired temperature in cold

15

(5)

(6)

(7)

(8)

3.2 Challenges of biogas production and possible solutions

Despite the benefits of biogas production, a negative effect on any of the processes explained

in Fig. 3.1 has a direct effect on the other processes and the biogas process can become

unsteady or fail completely [40]. The major challenges discussed in this subsection are those

related to temperature fluctuations, feedstock composition, foam formation, process

instability, and reactor design.

3.2.1 Temperature fluctuations

Microorganisms require optimum temperature for growth and active performance during

biogas process and can be divided into psychrophilic (optimum temperature around 10 °C),

mesophilic (optimum temperature around 37 °C), thermophilic (optimum temperature around

55 °C) and hyper thermophilic (optimum temperature around 85 °C) [33]. Temperature

fluctuations affect the performance of a biogas process adversely because the activity of the

microorganism is reduced if temperature is above or below their optimum range. A decrease

in temperature may result in a reduced volatile fatty acid production rate, substrate

decomposition rate, and metabolic rate of the microorganism [41, 42]. Navickas, Venslauskas

[43] investigated the influence of temperature variations on the performance of anaerobic

digestion of industrial wastes and observed that a change in temperature from 52 °C to 57 °C

(at constant total solids concentration, organic load, and pH) resulted in a 24 % reduction in

biogas yield. The degradation rate is higher thermophilic processes, however they are more

sensitive to changes in temperature [44] and moreover, they require increased energy input to

maintain a stable temperature. In this study, all experiments were operated at mesophilic

temperature, at 37 °C (Papers I, II, and VI) and at 25 °C for dry digestion of cattle manure

with straw in a textile bioreactor (Paper IV), to check the suitability of the process in tropical

regions, but the reactor can be heated from below to attain the desired temperature in cold

16

regions. This is a good strategy because mesophilic processes are less sensitive to temperature

changes, although they may require longer degradation time.

One of the ways of overcoming the major challenge of temperature fluctuations is using a

two-phase anaerobic digestion process in which the hydrolysis/acidogenesis stage is operated

under thermophilic conditions and the methanogenic stage is operated under mesophilic

conditions [44, 45]. Another way is avoiding heat loss by insulating the reactor with low cost

materials such as Styrofoam [46]; this material was used to insulate the plug flow reactors

used in this study (Paper II and VI). Previous research has also shown that between 12 %

and 50 % of the biogas produced, depending on system design characteristics, is used in

heating the reactor and maintaining constant temperature within the system [47, 48].

Therefore, the use of solar thermal energy to heat the reactor is a better option with low

operating cost and better optimisation of biogas yield [49]. Additionally, dry-AD is a good

solution as it requires less energy for heating due to low water content. The textile biogas

bioreactor is also an appropriate technology for tropical countries because it does not require

heating and the reactor is UV treated and weather resistant.

3.2.2 Feedstock composition

The biogas yield from anaerobic digestion of organic solid wastes depends on factors such as,

the organic and nutrient contents, impurities in the feedstock, the presence of possible

inhibitors (e.g. antibiotics, disinfectants, solvents, herbicides, salts, and heavy metals) [50],

the total solids content as well as the long chain fatty acids content.

3.2.2.1. Organic content and nutrients

The organic content of a feedstock determines the theoretical yield of biogas that can be

produced from it. It is necessary to determine the methane potential of the feedstock in order

to estimate the extent to which the specific feedstock can be degraded [51, 52]. The organic

content and the nutrients present in the feedstock affect the rate of growth and the activity of

microorganisms. Microorganisms are dependent on macro and micro nutrients, trace

elements, and vitamins for their growth, and these are vital for effective conversion of organic

matter to methane [7, 53]. The most important nutrients are carbon and nitrogen, and the C/N

ratio of feedstock is a vital factor for the choice of feedstock. Excess availability of nitrogen

during the degradation leads to the formation of NH3, which inhibits microbial growth at

higher concentrations [36, 54], and its deficiency causes the biogas process to fail.

17

Feedstocks used in this study were prepared according to Zupančič and Roš [55] by blending

1g of the sample with water (dilution factor of 50) to allow homogenization [56]. The

theoretical methane potential of the feedstocks were then calculated from the amount of

feedstock used and the chemical oxygen demand (COD) concentration using equation (9)

[57], assuming that the equation is valid for any substance or product [58].

, (9)

where:

BMPthCOD (theoretical yield under laboratory condition); R (gas constant, 0.082 atm L/mol

K); T (working temperature, 310 K); P (atmospheric pressure, 1 atm); VSadded (volatile solids

of the substrate added, g); nCH4 (methane produced, mol) determined according to equation

(10)

(10)

3.2.2.2 Impurities in the feedstock

Clean feedstock is important among others for the quality of the digestate residue and for the

overall efficiency of the anaerobic digestion process. Inadequate preparation of feedstock

before feeding can lead to blockage of gas pipes and the formation of foam in the reactors,

thereby causing a significant reduction in biogas yield and a great effect on the overall

digestion process [59]. Additionally, inhibitors (e.g. antibiotics, disinfectants, solvents,

herbicides, salts, and heavy metals) that enter the reactor through the feedstock can slow the

process, or the accumulation of inhibitors in the reactor can lead to the death of the

microorganisms at high concentrations.

3.2.2.3. Long chain fatty acid content

Lipid-rich wastes such as wastes from slaughterhouses, oil processing industry, dairy product

industry, and wool scouring contain high methane content compared to carbohydrates and

proteins-rich wastes [60]. These waste streams are easily degraded to glycerol and LCFAs;

excessive amount of LCFAs could inhibit the activity of the microorganisms thereby resulting

into low biogas yield or failure of the process. Angelidaki et al. [61] investigated the effect of

long chain fatty acids in cattle manure under a thermophilic biogas process and observed that

low concentrations of oleate and stearate acid inhibited all steps of the anaerobic digestion.

17

Feedstocks used in this study were prepared according to Zupančič and Roš [55] by blending

1g of the sample with water (dilution factor of 50) to allow homogenization [56]. The

theoretical methane potential of the feedstocks were then calculated from the amount of

feedstock used and the chemical oxygen demand (COD) concentration using equation (9)

[57], assuming that the equation is valid for any substance or product [58].

, (9)

where:

BMPthCOD (theoretical yield under laboratory condition); R (gas constant, 0.082 atm L/mol

K); T (working temperature, 310 K); P (atmospheric pressure, 1 atm); VSadded (volatile solids

of the substrate added, g); nCH4 (methane produced, mol) determined according to equation

(10)

(10)

3.2.2.2 Impurities in the feedstock

Clean feedstock is important among others for the quality of the digestate residue and for the

overall efficiency of the anaerobic digestion process. Inadequate preparation of feedstock

before feeding can lead to blockage of gas pipes and the formation of foam in the reactors,

thereby causing a significant reduction in biogas yield and a great effect on the overall

digestion process [59]. Additionally, inhibitors (e.g. antibiotics, disinfectants, solvents,

herbicides, salts, and heavy metals) that enter the reactor through the feedstock can slow the

process, or the accumulation of inhibitors in the reactor can lead to the death of the

microorganisms at high concentrations.

3.2.2.3. Long chain fatty acid content

Lipid-rich wastes such as wastes from slaughterhouses, oil processing industry, dairy product

industry, and wool scouring contain high methane content compared to carbohydrates and

proteins-rich wastes [60]. These waste streams are easily degraded to glycerol and LCFAs;

excessive amount of LCFAs could inhibit the activity of the microorganisms thereby resulting

into low biogas yield or failure of the process. Angelidaki et al. [61] investigated the effect of

long chain fatty acids in cattle manure under a thermophilic biogas process and observed that

low concentrations of oleate and stearate acid inhibited all steps of the anaerobic digestion.

18

Dasa, Westman [62] also reported that palmitic and oleic acids with concentrations of 3.0 and

4.0 g/l, respectively, resulted in > 50 % inhibition of biogas production and that stearate acids

had an even greater inhibitory effect.

3.2.2.4. Total solids content

The total solids content of the feedstock can affect gas yield because there is limited mass

transfer if the total solids content is high and, as a result, the microorganisms are only able to

decompose the substrate in their immediate environment. At very high contents ≥ 40 %,

digestion can come to a complete halt as there is insufficient water available for the growth of

the microorganisms [36, 63]. Additionally, a high content of total solids can cause problems if

inhibitors are present in the feedstocks as these are present in concentrated forms because of

the low water content. In this study, feedstocks with total solid contents between 20 % and 32

% were investigated in batch and continuous dry digestion processes (Paper II, IV and VI).

To overcome the challenges with feedstock composition, the feedstock can be sorted

mechanically, magnetically, or by hand to remove debris and then shredded to reduce its

particle size. Pretreatment of the feedstock is also vital, especially for lignocellulosic and

keratin-rich wastes, because it helps to increase the availability of the feedstock to the

microorganisms and thereby increase the biogas yield [64-66]. Ammonia inhibition from

protein-rich wastes can be resolved by low loading or by dilution [67]. Membrane

applications have also been reported to be effective in protecting the microorganisms from the

toxic effects of substrate and from product inhibition [62, 68]. Dry digestion processes allow

flexibility in the choice of feedstocks, less pre-treatment, and are better options for treating

fibre-rich feedstocks compared to wet digestion processes. Additionally, nutrient medium

should be used during anaerobic digestion unless these nutrients are present in the feedstock

or the inoculum used [53]. The nutrient medium is necessary to supply the macronutrients,

trace elements, and vitamins needed for growth and microbial activity. In this study, the

nutrient medium described by Angelidaki, Alves [53] was used (Papers I, II, and IV), except

in the dry co-digestion of solid wastes (Paper VI), in which four different substrates (citrus

wastes, chicken feather, cattle manure, and wheat straw) were used and it was presumed that

all nutrients needed would be supplied by the individual substrates.

19

3.2.3. Foam formation

Foam is a dispersion of gas in liquid consisting of a large proportion of gas, and its formation

in anaerobic digestion processes is a major challenge for plant operators [69]. The presence of

surface active substances such as volatile fatty acids, oil, grease, detergents, proteins,

particulate matter (grit, metals, sand, etc.) [69-71] is the major cause of foam formation.

Reactors operated with a high organic loading rate are more prone to foaming because at

higher loading rate the organic compounds are not fully degraded [72]. This incomplete

degradation results in accumulation of these surface-active agents and by-products that

promote foaming. The organic loading rate for mesophilic anaerobic digestion of municipal

sludge varies from 0.7 kgVS/m3/d to 7.2 kgVS/m3/d [70], but it has been reported that

operating reactors at organic loading rates higher than 4.5 kgVS/m3/d can result in foaming

even though these rates are still within the recommended range [71]. Excessive mechanical

mixing also increases the amount of bubbles in the bulk phase, enhancing the attachment of

surface active and hydrophobic compounds and thereby causing foaming [73]. On the other

hand, insufficient mixing during digestion results in stratification, which causes the surface

active agents and other non-degraded hydrophobic materials to rise to the surface of the bulk

phase and thus cause foaming [70].

Foaming can be overcome by identifying a critical organic loading threshold above which

foaming can occur in each reactor [74]. Overloading of reactors should be avoided by daily

monitoring of organic loading and process performance. Ensuring sufficient but not excessive

mixing by monitoring reactors regularly and preventing grit accumulation is a good

preventive measure against foaming. Foam formation is a common challenge in wet digestion

processes, so dry-AD is a better technology to avert this problem. In this study, continuous

plug flow reactors were used (Papers II and VI). The impeller in this reactor allows minimal

mixing by moving the reactor contents manually to the inlet and back to the outlet for better

performance of the process. Increasing the C/N ratio of feedstock is also an option for

reducing foaming in reactors. Tanimu, Mohd Ghazi [75] investigated the effect of feedstock

C/N ratio on foam formation in anaerobic digestion and found that increasing the C/N ratio of

food waste from 17 to 26 and 30 by adding other wastes reduced foaming in the reactors by

up to 60 %.

19

3.2.3. Foam formation

Foam is a dispersion of gas in liquid consisting of a large proportion of gas, and its formation

in anaerobic digestion processes is a major challenge for plant operators [69]. The presence of

surface active substances such as volatile fatty acids, oil, grease, detergents, proteins,

particulate matter (grit, metals, sand, etc.) [69-71] is the major cause of foam formation.

Reactors operated with a high organic loading rate are more prone to foaming because at

higher loading rate the organic compounds are not fully degraded [72]. This incomplete

degradation results in accumulation of these surface-active agents and by-products that

promote foaming. The organic loading rate for mesophilic anaerobic digestion of municipal

sludge varies from 0.7 kgVS/m3/d to 7.2 kgVS/m3/d [70], but it has been reported that

operating reactors at organic loading rates higher than 4.5 kgVS/m3/d can result in foaming

even though these rates are still within the recommended range [71]. Excessive mechanical

mixing also increases the amount of bubbles in the bulk phase, enhancing the attachment of

surface active and hydrophobic compounds and thereby causing foaming [73]. On the other

hand, insufficient mixing during digestion results in stratification, which causes the surface

active agents and other non-degraded hydrophobic materials to rise to the surface of the bulk

phase and thus cause foaming [70].

Foaming can be overcome by identifying a critical organic loading threshold above which

foaming can occur in each reactor [74]. Overloading of reactors should be avoided by daily

monitoring of organic loading and process performance. Ensuring sufficient but not excessive

mixing by monitoring reactors regularly and preventing grit accumulation is a good

preventive measure against foaming. Foam formation is a common challenge in wet digestion

processes, so dry-AD is a better technology to avert this problem. In this study, continuous

plug flow reactors were used (Papers II and VI). The impeller in this reactor allows minimal

mixing by moving the reactor contents manually to the inlet and back to the outlet for better

performance of the process. Increasing the C/N ratio of feedstock is also an option for

reducing foaming in reactors. Tanimu, Mohd Ghazi [75] investigated the effect of feedstock

C/N ratio on foam formation in anaerobic digestion and found that increasing the C/N ratio of

food waste from 17 to 26 and 30 by adding other wastes reduced foaming in the reactors by

up to 60 %.

20

3.2.4 Process instability

Process instability in biogas reactors occurs due to temperature fluctuations, nutrient

unavailability, feeding problems, high organic loading rate, and the presence of inhibitors in

feedstocks, as explained above. Possible good operation practises to overcome instability in

biogas reactors are explained in this section.

The initial start-up phase is essential for continuous processes. This phase enables the

microbial community to adapt to the feedstock and the conditions in the reactor. A moderate

amount of substrate should be fed at the initial stage and then the loading can be increased

gradually to reach the optimum value for the digestion process. If the organic loading rate

(OLR) is too high, there can be volatile fatty acid (VFA) accumulation, which causes

instability by a decrease in pH which can lead to inhibition of methanogens resulting into

process failure. Early indicators of process instability are VFA concentration, alkalinity ratio

(VFA/alkalinity ratio), hydrogen concentration (should be typically less than 100 ppm) and

redox potential (should be lower than -300 mV) [40]. In this study (Papers II and VI), VFA

and VFA/alkalinity ratio were used for monitoring the biogas process. The VFA/alkalinity

ratio is a two way titration measurement; titration is carried out until a pH of 5.0 is reached

(bicarbonate alkalinity) and then until pH of 4.4 (alkalinity caused by VFA) and was

calculated based on the Nordmann method [76]. The VFA/alkalinity ratio lower than 0.3 is

generally considered to indicate stable processes [40]. A continuous feed rate is vital for the

stability of the process [77] because variations in feed rate often result in variations in biogas

production rates, which can affect the biogas yield. Alterations in the energy content of two

different batches of the feedstock can also result in biogas production instability, even though

the feed rate remains the same [40].

The process performance of a continuous plug flow reactor treating manure with straw at 22

%TS was monitored by regular measurement of the VFA/alkalinity ratio, as shown in Fig. 3.2

(Paper II). The figure shows the variation of pH and VFA/alkalinity ratio at different OLRs.

At 2.8 gVS/L/d (OLR 1), the VFA/alkalinity ratio was lower than the reported limit of 0.3,

indicating stable process conditions. When the OLR was increased to 4.2 gVS/L/d (OLR 2),

the VFA/alkalinity ratio was still below 0.3. However, increasing the organic loading rate to 6

gVS/L/d (OLR 3) led to a gradual increase in the VFA/alkalinity ratio. The process started to

show signs of instability during the first retention time of 28 days with fluctuations in the

daily biogas production (Paper II). Continuing the loading at the same conditions (6

21

gVS/L/d) into the second retention time caused considerable disturbances in the system, with

a sharp increase in VFA/alkalinity ratio, which is a typical sign of overloading. However, the

process could be restored back to stability by reducing the organic loading rate back to 4.2

gVS/L/d (OLR 2).

Fig. 3.2. pH and VFA/Alkalinity ratio variation at different organic loading rate (OLR) during

experiments. The symbols represent pH (◊), VFA/Alkalinity ratio (□). Presented values are

mean values of duplicate measurements with error bars as standard deviation between the two

values. (Paper II)

3.2.5 Reactor design

Anaerobic reactors for biogas production also pose some challenges to the effectiveness of the

biogas process depending on the process configurations and operating conditions of the

reactors. A reactor may be suitable and economical for a particular type of feedstock or co-

substrate but may not be suitable for another. Therefore, for overall effectiveness of the

biogas production process, reactors must be selected with consideration of the feedstock

composition, amount of feedstock to be treated, desired product, and process economy [78].

For an anaerobic reactor to accommodate high loading rates, the following basic conditions

must be met [79]:

High retention of viable microorganisms in the reactor under operational conditions

Viable microorganisms that are sufficiently adapted and/or acclimatised

Sufficient contact between viable microorganisms and the feedstock

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

6,2

6,4

6,6

6,8

7

7,2

7,4

7,6

7,8

8

8,2

8,4

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230

VFA/

Alk

alin

ity

pH

Time (days)

OLR 1 2.8 gVS/L/d

OLR 2 4.2 gVS/L/d

OLR 3 6 gVS/L/d

OLR 2 4.2 gVS/L/d

Start-up period

21

gVS/L/d) into the second retention time caused considerable disturbances in the system, with

a sharp increase in VFA/alkalinity ratio, which is a typical sign of overloading. However, the

process could be restored back to stability by reducing the organic loading rate back to 4.2

gVS/L/d (OLR 2).

Fig. 3.2. pH and VFA/Alkalinity ratio variation at different organic loading rate (OLR) during

experiments. The symbols represent pH (◊), VFA/Alkalinity ratio (□). Presented values are

mean values of duplicate measurements with error bars as standard deviation between the two

values. (Paper II)

3.2.5 Reactor design

Anaerobic reactors for biogas production also pose some challenges to the effectiveness of the

biogas process depending on the process configurations and operating conditions of the

reactors. A reactor may be suitable and economical for a particular type of feedstock or co-

substrate but may not be suitable for another. Therefore, for overall effectiveness of the

biogas production process, reactors must be selected with consideration of the feedstock

composition, amount of feedstock to be treated, desired product, and process economy [78].

For an anaerobic reactor to accommodate high loading rates, the following basic conditions

must be met [79]:

High retention of viable microorganisms in the reactor under operational conditions

Viable microorganisms that are sufficiently adapted and/or acclimatised

Sufficient contact between viable microorganisms and the feedstock

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

6,2

6,4

6,6

6,8

7

7,2

7,4

7,6

7,8

8

8,2

8,4

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230

VFA/

Alk

alin

ity

pH

Time (days)

OLR 1 2.8 gVS/L/d

OLR 2 4.2 gVS/L/d

OLR 3 6 gVS/L/d

OLR 2 4.2 gVS/L/d

Start-up period

22

High reaction rates and absence of mass transfer limitations

Favourable environmental conditions for the microbial communities in the reactor

under the operating conditions

Plug flow reactors were used for continuous dry digestion processes in this study (Papers II

and VI), and these were effective for dry digestion of manure bedded with straw and dry co-

digestion of different solid waste fractions. The textile bioreactor newly developed by FOV

Fabrics AB (Borås, Sweden) was also used in this study (Paper IV) for dry batch digestion of

manure bedded with straw, and the reactor was suitable and efficient for this process. The

basic reactors used in biogas production processes and the major challenges associated with

these reactors are presented in Table 3.1.

23

Table 3.1 Overview of biogas reactors and possible challenges

Basis of Classification Reactor Challenges

Feeding mode Batch Larger volume required Insufficient mixing can induce dead zones, which make the digestion process inefficient Limited opportunity to control the process Lower biogas yield Possibility of explosions during methane-to-air transition phase when unloading the reactor

Continuous Longer start-up times required Increased VFA accumulation Require high skill in operation

Solids content Wet Larger amounts of water and energy required Dilute digestate residue requiring costly dewatering processes Frequent clogging of nozzles Sedimentation at the bottom creating extensive operational problems Foam formation

Dry Feedstock handling and mixing are difficult Longer retention time required Might be prone to substrate/product inhibition due to low water content

Operating temperature Mesophilic Lower reaction rates, low enzyme activity Digestate may contain higher pathogen concentration

Thermophilic Higher heat energy demand Prone to temperature fluctuations, so the process is less stable Higher risk of ammonia nitrogen toxicity Higher odour formation due to higher VFA concentration

Process stage Single-stage Higher retention time Formation of foam

Multi-stage Higher energy consumption; high investment cost. More complex process, required high skill in operation

Reactor design/material of construction

Plug flow Reactor has to be emptied if impeller requires servicing Feedstock can lead to blockage of gas pipes

Start-up may take a longer time CSTR Prone to foam formation. Intermixing and

process control is more complex as size increases. High capital and operational cost

23

Table 3.1 Overview of biogas reactors and possible challenges

Basis of Classification Reactor Challenges

Feeding mode Batch Larger volume required Insufficient mixing can induce dead zones, which make the digestion process inefficient Limited opportunity to control the process Lower biogas yield Possibility of explosions during methane-to-air transition phase when unloading the reactor

Continuous Longer start-up times required Increased VFA accumulation Require high skill in operation

Solids content Wet Larger amounts of water and energy required Dilute digestate residue requiring costly dewatering processes Frequent clogging of nozzles Sedimentation at the bottom creating extensive operational problems Foam formation

Dry Feedstock handling and mixing are difficult Longer retention time required Might be prone to substrate/product inhibition due to low water content

Operating temperature Mesophilic Lower reaction rates, low enzyme activity Digestate may contain higher pathogen concentration

Thermophilic Higher heat energy demand Prone to temperature fluctuations, so the process is less stable Higher risk of ammonia nitrogen toxicity Higher odour formation due to higher VFA concentration

Process stage Single-stage Higher retention time Formation of foam

Multi-stage Higher energy consumption; high investment cost. More complex process, required high skill in operation


Plug flow Reactor has to be emptied if impeller requires servicing Feedstock can lead to blockage of gas pipes

Start-up may take a longer time CSTR Prone to foam formation. Intermixing and

process control is more complex as size increases. High capital and operational cost

24

Table 3.1 Overview of biogas reactors and possible challenges (Cont’d.) Basis of Classification Reactor Challenges


UASB Requires constant monitoring to avoid washout of cells Sensitive to sudden temperature changes resulting in granule disintegration Start-up may take a longer time, especially when reactor is inoculated with sludge instead of dense granules Prone to shock loading and risk of odour problems. Effluent requires further treatment

Fixed dome Requires high technical skill for construction Prone to leakage Utilisation of gas produced is less effective

because the gas pressure fluctuates substantially

Anaerobic filter Requires expert design and construction Requires constant monitoring to ensure good functioning and a water tight system Effluent require further treatment Risk of clogging, depending on pre- and primary treatment Cleaning the clogged filter is cumbersome Long start-up and risk of foul odour

Floating drum Steel drum is expensive and requires rigorous maintenance to avoid leakage Not suitable for fibrous substrates Gas holder can get stuck in the resulting floating scum

Covered lagoon Longer retention time and lower degradation rate Poor interaction between substrate and microorganisms. Seasonal variation in biogas production

Polyethylene tubular

reactors Susceptible to mechanical damage Short lifespan

Sensitive to sunlight and deteriorate rapidly due to seasonal changes

Plastic tanks Expensive Not suitable for large-scale biogas plants

Reinforced plastic reactors

Expensive

Floating where underground water level persists

References: [36, 39, 44, 80-91]

25

3.3 Biogas in Nigeria

Nigeria, with a growing population of about 186 million [92], has the largest economy among

the nations in Africa with a gross domestic product (GDP) of about $405 billion, according to

World bank 2016 [93], yet Nigeria continues to face energy and environmental challenges.

About 96 % of the population of Nigeria is connected to the national grid, but only 18 % of

the functioning connections have a reliable supply of electricity [94], which is a major

challenge that depletes the energy and enterprises necessary for the country’s development.

Also, continuous burning of fossil fuels and solid wastes has resulted in 94 % of the

population of Nigeria being exposed to air pollution levels (measured in PM2.5) that exceed

the WHO guidelines and air pollution damage costs of about 1 % post of gross national

income [95]. Water and soil pollution is also a challenge due to improper management of

human sewage and the tremendous amounts of solid wastes generated. As water, soil, and air

pollution as well as energy poverty pose great challenges to human health and the

environmental and economic development of the country, the need for renewable energy

cannot be overemphasised.

Renewables accounted for about 62 % of the net additions to global power generating

capacity in 2016, and the vast majority of renewable energy for heating was supplied by

biomass, with smaller contributions from solar thermal and geothermal energy [4]. Biogas

production is an appropriate technology needed in Nigeria to ease the nation’s energy and

environmental challenges. According to Ngumah, Ogbulie [96] Nigeria generates about 542.5

million tonnes of total wastes per annum (livestock wastes, human excreta, crop residues, and

municipal solid wastes). This tremendous amount of wastes has the potential to produce an

estimated 25.53 billion m3 of biogas and 88.19 million tonnes of bio-fertilisers annually, as

shown in Table 3.2. The biogas produced can be used for heating, cooking, transportation, and

generation of electricity if properly harnessed, and the residue remaining after biogas

production is suitable for improving agricultural development in the country. Biogas can

augment the conventional energy sources in the country, thereby improving the quantity and

quality of the energy supply while also reducing environmental pollution.

25

3.3 Biogas in Nigeria

Nigeria, with a growing population of about 186 million [92], has the largest economy among

the nations in Africa with a gross domestic product (GDP) of about $405 billion, according to

World bank 2016 [93], yet Nigeria continues to face energy and environmental challenges.

About 96 % of the population of Nigeria is connected to the national grid, but only 18 % of

the functioning connections have a reliable supply of electricity [94], which is a major

challenge that depletes the energy and enterprises necessary for the country’s development.

Also, continuous burning of fossil fuels and solid wastes has resulted in 94 % of the

population of Nigeria being exposed to air pollution levels (measured in PM2.5) that exceed

the WHO guidelines and air pollution damage costs of about 1 % post of gross national

income [95]. Water and soil pollution is also a challenge due to improper management of

human sewage and the tremendous amounts of solid wastes generated. As water, soil, and air

pollution as well as energy poverty pose great challenges to human health and the

environmental and economic development of the country, the need for renewable energy

cannot be overemphasised.

Renewables accounted for about 62 % of the net additions to global power generating

capacity in 2016, and the vast majority of renewable energy for heating was supplied by

biomass, with smaller contributions from solar thermal and geothermal energy [4]. Biogas

production is an appropriate technology needed in Nigeria to ease the nation’s energy and

environmental challenges. According to Ngumah, Ogbulie [96] Nigeria generates about 542.5

million tonnes of total wastes per annum (livestock wastes, human excreta, crop residues, and

municipal solid wastes). This tremendous amount of wastes has the potential to produce an

estimated 25.53 billion m3 of biogas and 88.19 million tonnes of bio-fertilisers annually, as

shown in Table 3.2. The biogas produced can be used for heating, cooking, transportation, and

generation of electricity if properly harnessed, and the residue remaining after biogas

production is suitable for improving agricultural development in the country. Biogas can

augment the conventional energy sources in the country, thereby improving the quantity and

quality of the energy supply while also reducing environmental pollution.

26

Table 3.2 Estimated potential of waste biomass in Nigeria (modified from [96])

Biomass resource

Total biomass generated (million tons/year)

Estimated biogas potential (billion m3/year)

Biomethane potential (BMP) (billion m3/year)

Energy potential (Terajoules/year)

Estimated bio-fertiliser (dry) potential (million tons/year)

Cattle excreta

197.60 6.52 3.65 142350 25.69

Sheep & goat excreta

39.60 2.30 1.61 62790 3.71

Pig excreta 15.30 0.92 0.55 21450 1.68

Poultry excreta

32.60 2.50 1.65 64350 1.89

Abattoir waste

83.30 4.42 2.65 103350 6.12

Human excreta

52.00 2.60 1.69 65910 6.45

Crop residue

83.00 4.98 3.00 117000 36.20

Municipal solid wastes

39.10 1.29 0.85 33150 6.45

Total 542.50 25.53 15.65 610350 88.19

3.3.1 Biogas challenges and pioneered projects in Nigeria

The fixed-dome reactor is one of the commonly used biogas reactors in Nigeria because of its

long lifespan, but the technology is expensive, labour intensive, and requires skilled

supervision [86]. The lack of government commitment and poor continuity of previous biogas

programme initiatives through successive governments is a major factor limiting the

advancement of this technology [97]. An additional challenge is corruption, which increases

the investment costs for biogas implementation and thereby reduces the rate of return for the

investment [98]. Many biogas plants have failed due to poor planning, non-suitable biogas site

selections, poor reactor construction technology, poor maintenance, non-skilful operators of

the digestion process, and unavailability of spare parts.

In spite of these challenges, there are some existing biogas plants in Nigeria: less than 20 pilot

projects, including the United Nations Development Programme (UNDP) project in Kano

state, have been established [99]. The Cows-to-Kilowatts Project, located in Ibadan, the

27

capital of Oyo State, was begun in May 2008 [100] with collaboration among the Global

Network for Environment and Economic Development Research, Nigeria (NGO), the Biogas

Technology Research Centre, KMUTT, Thonburi, Thailand (research institute), the Centre for

Youth, Family and the Law, Nigeria (community-based organisation), and the Sustainable

Ibadan Project, Nigeria (UN-HABITAT Programme) [101]. The biogas plant, shown in Fig.

3.3, employs an anaerobic fixed-film reactor with a volume of 3000 m3 for treatment of

abattoir waste to produce biogas and organic fertiliser. The Bodija market in Ibadan

slaughters about 1000 cows per day, and the plant is capable of producing about 1500 m3 of

biogas (900 m3 of methane) [102], which is sufficient to fill 5400 cylinders of cooking gas per

month. The plant is also capable of producing about 1500 litres of bio-fertiliser per day for

farmers.

NASRUN Nig. Ltd. was established in 2002 as a crop and livestock enterprise. It has a biogas

plant (Kutunku Farms Biogas Plant) of about 140 m3 located in Minna, Niger State, Nigeria

[103], where a fixed-dome reactor was designed to use farm waste feedstock for biogas

production.

Fig. 3.3. Biogas plant in Nigeria for the treatment of abattoir wastes from Bodija market,

Ibadan (reprinted with permission) [101]

Another fixed-dome bio-digester of 20 m3 which was built by the Energy Commission of

Nigeria ECN-SERC/UDU in 1998 is fed with cow dung. The reactor is located at the

Mayflower Secondary School, Ikenne Ogun state [100], and produces gas for cooking and

bio-fertiliser for farmers. Additionally, the National Centre for Energy Research and

Development, University of Nigeria Nsukka (NCERD/UNN) built a biogas plant of 10 m3 for

27

capital of Oyo State, was begun in May 2008 [100] with collaboration among the Global

Network for Environment and Economic Development Research, Nigeria (NGO), the Biogas

Technology Research Centre, KMUTT, Thonburi, Thailand (research institute), the Centre for

Youth, Family and the Law, Nigeria (community-based organisation), and the Sustainable

Ibadan Project, Nigeria (UN-HABITAT Programme) [101]. The biogas plant, shown in Fig.

3.3, employs an anaerobic fixed-film reactor with a volume of 3000 m3 for treatment of

abattoir waste to produce biogas and organic fertiliser. The Bodija market in Ibadan

slaughters about 1000 cows per day, and the plant is capable of producing about 1500 m3 of

biogas (900 m3 of methane) [102], which is sufficient to fill 5400 cylinders of cooking gas per

month. The plant is also capable of producing about 1500 litres of bio-fertiliser per day for

farmers.

NASRUN Nig. Ltd. was established in 2002 as a crop and livestock enterprise. It has a biogas

plant (Kutunku Farms Biogas Plant) of about 140 m3 located in Minna, Niger State, Nigeria

[103], where a fixed-dome reactor was designed to use farm waste feedstock for biogas

production.

Fig. 3.3. Biogas plant in Nigeria for the treatment of abattoir wastes from Bodija market,

Ibadan (reprinted with permission) [101]

Another fixed-dome bio-digester of 20 m3 which was built by the Energy Commission of

Nigeria ECN-SERC/UDU in 1998 is fed with cow dung. The reactor is located at the

Mayflower Secondary School, Ikenne Ogun state [100], and produces gas for cooking and

bio-fertiliser for farmers. Additionally, the National Centre for Energy Research and

Development, University of Nigeria Nsukka (NCERD/UNN) built a biogas plant of 10 m3 for

28

Women at Achara, Nsukka, Enugu state. The biogas plant is fed with domestic animal wastes,

cassava peels and wastes from the milling of cowpea, and bambara nuts from a food

processing plant [104]. The Sokoto Energy Research Centre (SERC) also constructed a 30 m3

biogas reactor at the National Animal Production Research Institute (NAPRI) in Zaria. The

reactor is fed with human excreta, and the biogas produced is used for cooking at the Zaria

prison.

3.3.3 Strategies for improved biogas technology in Nigeria

Successful implementation of biogas technology in Nigeria requires innovative strategies to

motivate investment in biogas production, which will lead to economic growth, environmental

safety, and national security. Strategies that have been used by developed countries for

successful implementation are discussed in this section. Nigeria needs to adopt some of these

strategies to stimulate the growth of biogas in the country.

Technology: The new textile bioreactor used in this study for biogas production (Paper IV) is

a promising technology for developing countries such as Nigeria, where the biogas process

should be cost-effective. The laboratory- and pilot-scale results demonstrated the suitability of

this technology for advancement of global energy production (Paper V). Farmers and

individual households can easily install such reactors and produce biogas for heating and

cooking. A number of reactors can be run in parallel for continuous gas production,

depending on the amount of wastes generated. In this way, farmers can become self-sufficient

in generating renewable energy while also producing bio-fertiliser for enhancement of

agricultural development in the country.

Funding: One major problem for successful large-scale implementation of biogas technology

in Nigeria is funding. Financial support from the government and from non-governmental

organisations is needed for significant improvements in the technology. In Sweden, there have

been significant advances in biogas production for energy generation and after upgrading it’s

used as fuel for vehicles, and this development has been made possible by many years of

central government funding through local investment programs (LIP) and climate investment

programs (KLIMP) [105]. In Denmark, the first plant built in Vester Hjermitslev, with the

goal of making the town energy self-supporting [106], was implemented with almost

complete governmental funding: a 4 million DKK grant from the government and an 8.4

million DKK loan from the North Jutland County Council [107]. There have also been

29

investment grants for centralised biogas plants (up to 40 % of costs) and loan schemes with

long-term low interest rates.

Policies: There is need for policies that will support the development of biogas production for

energy generation in the country. The government should develop state-of-the-art policies at

all levels (federal, state, and local government) that will attract investment and encourage

flexibility in energy generation. Policies such as feed-in tariffs, net metering, virtual net

metering, and tax incentives have provided great support for renewable energy in developed

countries [4]. Policies in Sweden supporting renewable transport fuel include tax exemptions

or reductions of taxes applied on fossil fuels and laws mandating refilling stations to provide

at least one renewable fuel [108].

Awareness campaigns and education: People should be educated on the importance of proper

management of the wastes generated daily and the benefits of biogas produced from these

waste fractions. There is need for continuous counselling and training in communities,

schools, and market places about how to manage their wastes effectively, reduce the amount

of wastes generated as much as possible, reuse for a longer time, recycle to new products, and

recover energy from wastes.

Research development: The government should support research development by ensuring

strong collaboration between universities and industries in Nigeria. Pilot projects can be

conducted in universities to evaluate the feasibility of the technology and obtain optimum

conditions for successful implementation and thereafter the technology can be adopted by

industries.

29

investment grants for centralised biogas plants (up to 40 % of costs) and loan schemes with

long-term low interest rates.

Policies: There is need for policies that will support the development of biogas production for

energy generation in the country. The government should develop state-of-the-art policies at

all levels (federal, state, and local government) that will attract investment and encourage

flexibility in energy generation. Policies such as feed-in tariffs, net metering, virtual net

metering, and tax incentives have provided great support for renewable energy in developed

countries [4]. Policies in Sweden supporting renewable transport fuel include tax exemptions

or reductions of taxes applied on fossil fuels and laws mandating refilling stations to provide

at least one renewable fuel [108].

Awareness campaigns and education: People should be educated on the importance of proper

management of the wastes generated daily and the benefits of biogas produced from these

waste fractions. There is need for continuous counselling and training in communities,

schools, and market places about how to manage their wastes effectively, reduce the amount

of wastes generated as much as possible, reuse for a longer time, recycle to new products, and

recover energy from wastes.

Research development: The government should support research development by ensuring

strong collaboration between universities and industries in Nigeria. Pilot projects can be

conducted in universities to evaluate the feasibility of the technology and obtain optimum

conditions for successful implementation and thereafter the technology can be adopted by

industries.

30

31

Chapter 4

Dry anaerobic digestion (dry-AD)

The anaerobic digestion process can be defined according to the total solid content of the

feedstock: wet digestion for total solid contents between 0.5 % and 15 % [109] and dry

digestion for total solid contents greater than 20 % [110, 111]. In industries, wet anaerobic

digestion processes are most common, but recently there has been great concern about the

large amount of water used while treating organic solid wastes for biogas production and the

huge water content of the digestate residue. In addition, the use of solid wastes (total solid

content between 15 % and 50 %) from agricultural, municipal, and industrial activities,

including forest and crop residue, is becoming more attractive. Therefore, the impetus for

developing dry anaerobic digestion processes is increasing in both research and industry.

4.1 Comparison of wet and dry anaerobic digestion

Wet anaerobic digestion of solid wastes requires a lot of water, which is a challenge for

countries with water shortage. Presently, there is global water scarcity; about 1.4 billion

people live in river basins in which water use rates exceed recharge rates [112]. It is obvious

that there will be competition for water as population increases and industrial development

progresses. Also, the digestate residue remaining after biogas production through the wet

process contains a lot of water, and dewatering of the digestate requires high energy

consumption and results in loss of nutrients. Therefore, dry anaerobic digestion is a promising

technology to avert these problems. Compared with the wet anaerobic digestion processes,

dry-AD provides better economic feasibility because reactor volume is minimised due to the

reduced volume of water [113, 114]. It is also beneficial because it is more robust, flexible in

its acceptance of feedstocks which requires less pre-treatment [114].

The total solid content of feedstocks affects the performance of anaerobic digestion processes,

and the microbial morphology in the reactor changes as the total solid content changes [63,

111]. The microbial communities that are dominant in dry digestion processes may differ

from those in wet digestion processes as the TS content increases [115, 116]. Yi, Dong [111]

investigated microbial communities using pyrosequencing technology while treating food

waste with TS contents from 5 % to 20 % under the mesophilic condition. The result showed

31

Chapter 4

Dry anaerobic digestion (dry-AD)

The anaerobic digestion process can be defined according to the total solid content of the

feedstock: wet digestion for total solid contents between 0.5 % and 15 % [109] and dry

digestion for total solid contents greater than 20 % [110, 111]. In industries, wet anaerobic

digestion processes are most common, but recently there has been great concern about the

large amount of water used while treating organic solid wastes for biogas production and the

huge water content of the digestate residue. In addition, the use of solid wastes (total solid

content between 15 % and 50 %) from agricultural, municipal, and industrial activities,

including forest and crop residue, is becoming more attractive. Therefore, the impetus for

developing dry anaerobic digestion processes is increasing in both research and industry.

4.1 Comparison of wet and dry anaerobic digestion

Wet anaerobic digestion of solid wastes requires a lot of water, which is a challenge for

countries with water shortage. Presently, there is global water scarcity; about 1.4 billion

people live in river basins in which water use rates exceed recharge rates [112]. It is obvious

that there will be competition for water as population increases and industrial development

progresses. Also, the digestate residue remaining after biogas production through the wet

process contains a lot of water, and dewatering of the digestate requires high energy

consumption and results in loss of nutrients. Therefore, dry anaerobic digestion is a promising

technology to avert these problems. Compared with the wet anaerobic digestion processes,

dry-AD provides better economic feasibility because reactor volume is minimised due to the

reduced volume of water [113, 114]. It is also beneficial because it is more robust, flexible in

its acceptance of feedstocks which requires less pre-treatment [114].

The total solid content of feedstocks affects the performance of anaerobic digestion processes,

and the microbial morphology in the reactor changes as the total solid content changes [63,

111]. The microbial communities that are dominant in dry digestion processes may differ

from those in wet digestion processes as the TS content increases [115, 116]. Yi, Dong [111]

investigated microbial communities using pyrosequencing technology while treating food

waste with TS contents from 5 % to 20 % under the mesophilic condition. The result showed

32

better VS reduction and methane yield in reactors with higher TS content and that

Methanosarcina was dominant in three reactors showing an increasing trend with increasing

TS content. Li, Chu [117] reported that Anaerococcus species are abundant in solid-state

anaerobic digestion (dry-AD) and that these species are responsible for improved degradation

efficiency and methane yield.

However, dry-AD requires pumps specifically designed for handling high-solid slurries,

which may be more expensive than the centrifugal pumps used in wet digestion systems [114,

118]. It also requires a longer start-up period and is sometimes prone to inhibition because

inhibitors that enter the reactor through feedstocks are in higher concentration due to the

reduced water content.

4.2 Start-up phase in dry anaerobic digestion

In dry-AD, there is minimal or no mixing during the digestion process due to the high solid

content in the reactor. This necessitates the need for proper conditioning of the feedstock. The

start-up phase is important for the generation of microbial cultures adapted to the feedstock

and can be very long due to the low growth rates of the anaerobic microorganisms. If

inoculum adapted to feedstock similar to the one of interest is available then start-up can be

shorter, just a few days otherwise it can take longer period up to 1 or 2 months or longer if the

right microorganisms are not acclimatised. In this study, the start-up period took 40 days

(Paper II) for dry digestion of manure with straw and 28 days (Paper VI) for initial start-up

in dry co-digestion of chicken feathers, citrus wastes, wheat straw, and manure with straw in

plug flow reactors, as shown in Fig. 4.1.

The inoculum (anaerobic sludge) should be collected from an active anaerobic digester,

preferably from a dry digestion plant because inoculum with high solid content is needed.

Obtaining inoculum with high solid content is vital because centrifuging the inoculum at high

speed to obtain as many bacteria as possible (solid inoculum) may result in significant

reductions in bacteria viability [119], which can have a potential effect on the outcome of the

experiment. Furthermore, appropriate inoculum to substrate ratio is needed; the amount of

solid inoculum needed depends on the substrate and the feedstock of the previous digester

from which the inoculum is taken. In this study, an inoculum-to-substrate ratio (VSinoculum to

VSsubstrate) of 1 was used in a textile batch reactor treating manure with straw, but this ratio

was reduced to 0.5 after an acclimatisation period of 232 days (Paper IV). This study also

33

used, an inoculum-to-substrate ratio of 2 in the co-digestion of chicken feathers, wheat straw,

and citrus wastes in continuous plug flow reactors, but after a start-up period of about 1 month

(adaptation period), this ratio was reduced to 0.4 to increase the solid content in the reactor

(Paper VI). The inoculum must be mixed with the substrate prior to placing them into the

reactor, but the mixing must not be too rigorous. Rigorous mixing can results rapidly

overwhelmed methanogenic areas by acid inhibition [120].

Fig. 4.1. Daily biogas production and accumulated methane yield obtained during the initial

startup phase carried out in batch mode for the digestion of the best mixing ratio at 20 %TS

(1:1:6 referring to chicken feather: citrus wastes: wheat straw) in plug flow reactors; reactor 1

(R1) and reactor 2 (R2). Colours represent: blue for reactor 1 (R1) and red for reactor 2 (R2).

Symbols represent: ◊ and □ for daily biogas production and ∆ and x for cumulative methane

yield. (Paper VI).

4.3 Dry anaerobic digestion – Batch process

In the batch digestion processes, an appropriate amount of inoculum is mixed with the

substrate, the mixture is loaded into the reactor, the reactor is closed, and the substrate is

digested under anaerobic conditions. The reactor remains closed until degradation is

completed. Usually little or no mixing is possible inside the reactor. Biogas production starts

slowly, increases gradually to a peak, and then declines until there is no more gas production.

0

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100

150

200

250

300

0

1000

2000

3000

4000

5000

6000

7000

8000

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Cu

mu

lati

ve m

eth

ane

yiel

d (

Nm

l/gV

S)

Dai

ly b

iog

as p

rod

uce

d (

Nm

l/d)

Time (days)

33

used, an inoculum-to-substrate ratio of 2 in the co-digestion of chicken feathers, wheat straw,

and citrus wastes in continuous plug flow reactors, but after a start-up period of about 1 month

(adaptation period), this ratio was reduced to 0.4 to increase the solid content in the reactor

(Paper VI). The inoculum must be mixed with the substrate prior to placing them into the

reactor, but the mixing must not be too rigorous. Rigorous mixing can results rapidly

overwhelmed methanogenic areas by acid inhibition [120].

Fig. 4.1. Daily biogas production and accumulated methane yield obtained during the initial

startup phase carried out in batch mode for the digestion of the best mixing ratio at 20 %TS

(1:1:6 referring to chicken feather: citrus wastes: wheat straw) in plug flow reactors; reactor 1

(R1) and reactor 2 (R2). Colours represent: blue for reactor 1 (R1) and red for reactor 2 (R2).

Symbols represent: ◊ and □ for daily biogas production and ∆ and x for cumulative methane

yield. (Paper VI).

4.3 Dry anaerobic digestion – Batch process

In the batch digestion processes, an appropriate amount of inoculum is mixed with the

substrate, the mixture is loaded into the reactor, the reactor is closed, and the substrate is

digested under anaerobic conditions. The reactor remains closed until degradation is

completed. Usually little or no mixing is possible inside the reactor. Biogas production starts

slowly, increases gradually to a peak, and then declines until there is no more gas production.

0

50

100

150

200

250

300

0

1000

2000

3000

4000

5000

6000

7000

8000

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

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mu

lati

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eth

ane

yiel

d (

Nm

l/gV

S)

Dai

ly b

iog

as p

rod

uce

d (

Nm

l/d)

Time (days)

34

The reactor is opened and the digestate is replaced with a new batch. The tank is then closed

again and is ready for operation [121]. Batch processes are best operated in parallel, so at least

one reactor is always producing biogas. The principle of the batch digestion process is

technically simple, but supplying the substrates to the microbial communities at once can

repress the digestion process. Additionally, insufficient mixing can induce settlement layers

making the digestion process inefficient. Thus, the conditions in the process change

continually due to cell metabolism and lack of control; hence steady biogas production is not

attainable. The following subsections describe the experimental batch digestion processes

carried out in this study (textile bioreactor) as well as the BEKON dry digestion technology.

4.3.1 Dry anaerobic digestion in textile bioreactor

Textile biogas reactor technology is designed to make biogas technology accessible and

attractive to developing countries. This new bioreactor is made of textiles, which makes it less

combustible, resistant to tearing, light-weight, and environmentally benign. This reactor is UV

treated to prevent easy degradation due to exposure to sunlight and weather resistant, which

increases its lifespan. The reactor is also composed of coated polymers as a protection against

corrosive gases such as H2S. Textile biogas reactors range in size from 1 m3 to 1000 m3 and

are suitable for both small- and large-scale biogas processes. Conventional biogas reactors,

such as the fixed-dome, floating-drum, and plastic biogas reactor, along with the challenges

associated with these reactors were studied while introducing the textile biogas bioreactor

(Paper V). Textile bioreactors are a promising technology for dissemination, and this will

enhance global advancement of biogas technology, especially in developing countries.

Dry digestion of manure bedded with straw was investigated using the newly developed

textile bioreactor (Paper IV). Figure 4.2 shows a schematic sketch of the experimental set-up.

Repeated batches were carried out. During the first digestion process, the total feedstock with

22 %TS was inoculated with the initial inoculum (unacclimatised inoculum) maintaining a

volatile solids (VS) ratio of inoculum to substrate at 1:1 and thereby having a total TS of 10 %

in the reactor. In order to increase the TS in the textile reactor and investigate the response of

microorganisms after acclimatisation, a second batch digestion (acclimatised low TS) was

performed; the TS of the substrate was increased to 27 %, thereby increasing the TS in the

reactor to 12 %. To increase the TS in the reactor further, a third batch digestion process

(acclimatised high TS) was also performed. The VS ratio was reduced to 0.5 and the TS of the

substrate was increased to 30 %, thereby increasing the TS in the reactor to 17 %.

35

Fig. 4.2. Schematic sketch of the experimental set-up (Paper IV).

It was found that the methane yield increased from 183 to 290 NmlCH4/gVS with decreasing

degradation time from 136 to 92 days after a long-term acclimatisation period of 232 days, as

shown in Fig. 4.3. The bioreactor worked successfully for more than 324 days without any

gas leakage or major maintenance.

Fig. 4.3. Cumulative methane yield and production rate obtained during the digestion process

with gradual increase of total solid (TS) content of the feedstock and reactor mixture. 22 %,

27 %, and 30 % (TS of feedstock); 10 %, 12 %, and 17 % (TS in reactor) (Paper IV).

35

Fig. 4.2. Schematic sketch of the experimental set-up (Paper IV).

It was found that the methane yield increased from 183 to 290 NmlCH4/gVS with decreasing

degradation time from 136 to 92 days after a long-term acclimatisation period of 232 days, as

shown in Fig. 4.3. The bioreactor worked successfully for more than 324 days without any

gas leakage or major maintenance.

Fig. 4.3. Cumulative methane yield and production rate obtained during the digestion process

with gradual increase of total solid (TS) content of the feedstock and reactor mixture. 22 %,

27 %, and 30 % (TS of feedstock); 10 %, 12 %, and 17 % (TS in reactor) (Paper IV).

36

4.3.2 BEKON dry digestion technology

The BEKON process allows digestion of solid wastes with high solid content. It is suitable for

processing wastes with total solid contents of up to 50 % [122]. The solid waste is inoculated

with fermented substrate, loaded into the reactor, closed in the reactor, and digested under the

anaerobic condition. The process is a single-step batch process; all digestion steps, as

explained in Fig. 3.1, take place in the same reactor. There is no addition of feedstock or

mixing inside the reactor applied during the digestion. The fermentation process runs at the

mesophilic condition (34 – 37 °C), and the temperature is regulated through heated floor and

walls. To allow continuous inoculation and to provide moisture for the microorganisms during

the digestion, percolation liquid is collected and recirculated back continuously into the

reactor. This is a good practise because recirculation of the liquid can facilitate the growth of

the microorganisms [123] and thereby improve methane yield while also reducing degradation

time. However, liquid recirculation sometimes can result in a high risk of acidification,

thereby leading to instability in the process. BEKON technology allows several reactors to

run simultaneously, and the biogas produced is used for electricity generation. A detailed

illustration of the process is shown in Fig. 4.4.

Fig. 4.4. BEKON dry batch digestion technology (reprinted with permission [122])

4.4 Dry anaerobic digestion – Continuous process

In the continuous digestion process, the substrate is supplied to the microorganisms

continuously in small amount, which minimises toxic effects of the substrate or product on the

cells. The substrate is fed continuously into the reactor, and an equal amount is removed; this

is done to maintain a stable working volume and steady state conditions. A moderate amount

37

of the substrate is fed at the initial stage and then the loading rate is increased gradually to

reach the target. This continuous supply of substrate without alteration results in steady biogas

production, and the process can be controlled easily. Plug flow bioreactors are commonly

used in the industry for continuous digestion processes.

4.4.1 Plug flow reactors for continuous dry digestion processes

Horizontal plug flow reactors, as shown in Fig. 4.5, for continuous dry anaerobic digestion

processes were used in this study. The reactor was operated under mesophilic conditions (37

°C), and the temperature was sustained by circulating water from a heater, a thermostatic

water bath through a water jacket surrounding the reactor, and 10-mm-thick styrofoam was

used to insulate the reactor against heat loss. The plug flow reactor has three main sections,

namely the inlet zone, main zone, and outlet zone as shown in Fig. 4.5.

Fig. 4.5. Plug flow reactors used in this study for continuous dry anaerobic digestion

processes

4.4.1.1 Continuous dry digestion of manure bedded with straw

Cattle manure bedded with straw was used as feedstock and digested at 22 %TS using the

continuous plug flow reactor (Fig. 4.5). Figure 4.6 shows the details of the experimental set-

up and other accessories.

Inlet zone

Main zone

Outlet zone

37

of the substrate is fed at the initial stage and then the loading rate is increased gradually to

reach the target. This continuous supply of substrate without alteration results in steady biogas

production, and the process can be controlled easily. Plug flow bioreactors are commonly

used in the industry for continuous digestion processes.

4.4.1 Plug flow reactors for continuous dry digestion processes

Horizontal plug flow reactors, as shown in Fig. 4.5, for continuous dry anaerobic digestion

processes were used in this study. The reactor was operated under mesophilic conditions (37

°C), and the temperature was sustained by circulating water from a heater, a thermostatic

water bath through a water jacket surrounding the reactor, and 10-mm-thick styrofoam was

used to insulate the reactor against heat loss. The plug flow reactor has three main sections,

namely the inlet zone, main zone, and outlet zone as shown in Fig. 4.5.

Fig. 4.5. Plug flow reactors used in this study for continuous dry anaerobic digestion

processes

4.4.1.1 Continuous dry digestion of manure bedded with straw

Cattle manure bedded with straw was used as feedstock and digested at 22 %TS using the

continuous plug flow reactor (Fig. 4.5). Figure 4.6 shows the details of the experimental set-

up and other accessories.

Inlet zone

Main zone

Outlet zone

38

Fig. 4.6. Schematic sketch of experimental set-up and other accessories (Paper II)

The feedstock was inoculated and the experiment started in batch mode until no further gas

production was monitored (40-day digestion start-up period). The continuous feeding

operation began after the start-up period and was investigated at organic loading rates of 2.8

gVS/L/d (OLR 1), 4.2 gVS/L/d (OLR 2), and 6 gVS/L/d (OLR 3) with corresponding

retention times of 60, 40, and 28 days. Figure 4.7 shows the biogas production at the various

stages. Overloading was observed when the OLR of 6 gVS/L/d was applied, so the procedure

of this OLR was repeated to check the stability of the process under this condition. However,

the daily gas production remained unstable leading to increased VFA/alkalinity ratio which is

an indication of instability (explained in section 3.3.4). However the process could be restored

back to stability by reducing the OLR to 4.2 gVS/L/d with the corresponding retention time of

40 days.

39

Fig. 4.7. Biogas production at different organic loading rates (OLRs) during continuous

experiments. The symbols represent daily biogas production (◊) and cumulative biogas

production (□). OLR 1 (2.8 gVS/L/d), OLR 2 (4.2 gVS/L/d) and OLR 3 (6 gVS/L/d). (Paper

II).

4.4.1.2 Continuous dry co-digestion of citrus wastes with chicken feathers and wheat

straw

Dry co-digestion of citrus wastes with chicken feathers, wheat straw and manure bedded with

straw was investigated in batch digestion assay (Paper VI). The long term effects in process

performance were investigated for the best mixing ratio indicating synergy, determined

through the batch assays, in continuous plug flow reactors at different organic loading rates.

Reactor 1 (RTS21) was fed with a feedstock having 21 %TS and reactor 2 (RTS32) was fed with

a feedstock having 32 %TS. The OLR was kept first at 2.0 gVS/L/d (OLR 1) and then it was

increased to 3.8 gVS/L/d (OLR 2) with corresponding retention times of 50 and 30 days,

respectively. During the feeding appropriate amounts of the digestate residue withdrawn

every day from the reactors was mixed with the fresh feedstock in a ratio of 1:2

(feedstock:digestate; wet basis). Process parameters such as volume biogas produced,

methane content of the biogas, pH, TS, VS, VFA/Alkalinity ratio, total VFA and total

ammonia concentration were monitored regularly during the digestion process. At OLR of 2

gVS/l/d, the methane yield of 362 NmlCH4/gVSadded was obtained from RTS21 which is 13.5 %

higher than the yield obtained from RTS32 (319 NmlCH4/gVSadded) (Paper VI). However, the

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ioga

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OLR 1 OLR 2 OLR 3 OLR 2

39

Fig. 4.7. Biogas production at different organic loading rates (OLRs) during continuous

experiments. The symbols represent daily biogas production (◊) and cumulative biogas

production (□). OLR 1 (2.8 gVS/L/d), OLR 2 (4.2 gVS/L/d) and OLR 3 (6 gVS/L/d). (Paper

II).

4.4.1.2 Continuous dry co-digestion of citrus wastes with chicken feathers and wheat

straw

Dry co-digestion of citrus wastes with chicken feathers, wheat straw and manure bedded with

straw was investigated in batch digestion assay (Paper VI). The long term effects in process

performance were investigated for the best mixing ratio indicating synergy, determined

through the batch assays, in continuous plug flow reactors at different organic loading rates.

Reactor 1 (RTS21) was fed with a feedstock having 21 %TS and reactor 2 (RTS32) was fed with

a feedstock having 32 %TS. The OLR was kept first at 2.0 gVS/L/d (OLR 1) and then it was

increased to 3.8 gVS/L/d (OLR 2) with corresponding retention times of 50 and 30 days,

respectively. During the feeding appropriate amounts of the digestate residue withdrawn

every day from the reactors was mixed with the fresh feedstock in a ratio of 1:2

(feedstock:digestate; wet basis). Process parameters such as volume biogas produced,

methane content of the biogas, pH, TS, VS, VFA/Alkalinity ratio, total VFA and total

ammonia concentration were monitored regularly during the digestion process. At OLR of 2

gVS/l/d, the methane yield of 362 NmlCH4/gVSadded was obtained from RTS21 which is 13.5 %

higher than the yield obtained from RTS32 (319 NmlCH4/gVSadded) (Paper VI). However, the

0

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600

700

800

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0 10 20 30 40 50 60 70 80 90 100110120130140150160170180190200210220230

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Dai

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ioga

s Pr

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(L/d

)

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start-up period

OLR 1 OLR 2 OLR 3 OLR 2

40

stability of the process declined for both RTS21 and RTS32 as OLR was increased to 3.8 gVS/l/d

resulting in high total VFA and VFA/alkalinity ratio as shown in Fig. 4.8.

Fig. 4.8. Variation in pH, VFA/alkalinity ratio, total VFA and total ammonia concentration in

reactor 1 (RTS21) and reactor 2 (RTS32) during continuous operation; startup (35 days), OLR1

(2 gVS/L/d for 50 days) and OLR2 (3.8 gVS/L/d for 30 days). Colours represent: blue for

reactor 1 (R1) and red for reactor 2 (R2). Symbols represent: (a) ◊ and □ pH, ∆ and x

VFA/alkalinity ratio (b) ◊ and □ total VFAs concentration, ∆ and x total ammonia

concentration. (Paper VI).

4.4.2 Continuous dry-AD technologies

Väst Blekinge Miljö AB (VMAB) in Sweden uses a technology designed for dry digestion of

household wastes to produce biogas fuels for transportation. The process uses plug flow

reactors for continuous dry digestion of solid wastes with a total solid content of about 35 %.

The reactor is capable of treating 15,000 tons of wastes per year, and the wastes typically

41

consist of 75 % sorted household wastes and 25 % garden wastes [124]. The household

wastes are pre-treated by crushing and separation of non-digestive wastes. The feedstock is

stored for 1 – 3 days in a buffer tank and thereafter pumped continuously into the plug flow

reactors for anaerobic digestion. This process runs at thermophilic conditions (55 °C), and

horizontal impellers inside the reactor, shown in Fig. 4.9, allow minimal mixing in the reactor.

Presently, the continuous dry fermentation process works properly without liquid

recirculation, which is similar to the process used in this research study (Paper II).

Fig. 4.9. Plug flow reactor with impeller inside the reactor for flow of material and mixing

[124]

DRANCO technology which was developed in Belgium, is a single-stage system for

treatment of solid wastes, mostly municipal solid wastes, with total solid content of 15 % to

40 % in the reactor [125]. It is a vertical plug flow reactor, as shown in Fig. 4.10a. Feedstock

is pumped to the top of the reactor through feeding tubes and extracted through a conical

outlet at the bottom. There is no mixer inside the reactor, but mixing is enhanced by recycling

the digestate (one part of fresh wastes mixed with six parts of the digestate) [118]. Manual

recycling of the digestate at feedstock to digestate ratio of 1 to 2 was also investigated in this

research for dry co-digestion of solid wastes (Paper VI).

STRABAG technology is designed for the treatment of solid waste with TS content between

15 % and 45 % using horizontal plug flow reactors, as shown in Fig. 4.10b. The reactors are

equipped with agitators arranged transversely to the flow direction to prevent formation of

floating scum and settlement of materials [126].

41

consist of 75 % sorted household wastes and 25 % garden wastes [124]. The household

wastes are pre-treated by crushing and separation of non-digestive wastes. The feedstock is

stored for 1 – 3 days in a buffer tank and thereafter pumped continuously into the plug flow

reactors for anaerobic digestion. This process runs at thermophilic conditions (55 °C), and

horizontal impellers inside the reactor, shown in Fig. 4.9, allow minimal mixing in the reactor.

Presently, the continuous dry fermentation process works properly without liquid

recirculation, which is similar to the process used in this research study (Paper II).

Fig. 4.9. Plug flow reactor with impeller inside the reactor for flow of material and mixing

[124]

DRANCO technology which was developed in Belgium, is a single-stage system for

treatment of solid wastes, mostly municipal solid wastes, with total solid content of 15 % to

40 % in the reactor [125]. It is a vertical plug flow reactor, as shown in Fig. 4.10a. Feedstock

is pumped to the top of the reactor through feeding tubes and extracted through a conical

outlet at the bottom. There is no mixer inside the reactor, but mixing is enhanced by recycling

the digestate (one part of fresh wastes mixed with six parts of the digestate) [118]. Manual

recycling of the digestate at feedstock to digestate ratio of 1 to 2 was also investigated in this

research for dry co-digestion of solid wastes (Paper VI).

STRABAG technology is designed for the treatment of solid waste with TS content between

15 % and 45 % using horizontal plug flow reactors, as shown in Fig. 4.10b. The reactors are

equipped with agitators arranged transversely to the flow direction to prevent formation of

floating scum and settlement of materials [126].

42

KOMPOGAS technology is similar to STRABAG technology. It is aided by slow rotation of

impellers inside the reactors for homogenisation and degassing [118]. The process requires

careful adjustment of the solid content in the reactor to around 23 %TS; higher TS values may

cause excessive resistance to flow [118].

Fig 4.10. Reactor designs for adequate mixing of solid wastes (a) DRANCO technology [125]

(b) STRABAG technology

(a) (a)

43

Chapter 5

Improving biogas production from solid wastes

Anaerobic digestion of agricultural, municipal, and industrial solid wastes often results in low

biogas yields and slow degradation rates because some of these wastes have recalcitrant

molecular structures that make them difficult to degrade by microorganisms and some contain

chemicals/substances that can inhibit microbial growth. Strategies to overcome the challenges

associated with these wastes in order to ascribe value to them are discussed in this section.

5.1 Pretreatment to overcome biomass recalcitrance

Pretreatment of the substrate is needed either for making it easier to handle at the biogas plant

or for altering its structure for easy degradation, hence enhancing its methane potential. There

are different pretreatment methods that can be used depending on the types of substrates and

the goals of the pretreatment. The most suitable pretreatment methods for agricultural,

municipal, and industrial solid wastes are discussed in this section.

5.1.1 Mechanical pretreatment

Mechanical pretreatment is required for biomass with high total solids content (e.g. forest

residue, crop residue, chicken feather wastes) to reduce the particle size and crystallinity of

the feedstock. It can also reduce viscosity in biogas reactors making mixing easier [127].

Milling is commonly used, and optimising the size reduction process is important for

improving biogas yield. Lindner, Zielonka [128] pretreated non-degraded digestate by ball

milling in order to feed it back into the fermentation process, and they observed that ball

milling pretreatment enhanced the methane yield of two-stage maize silage digestate by 9 %

and 17 % using two-stage hay/straw digestate, respectively. However, the electrical demand

of this pretreatment method is very high, so it is not viable as a stand-alone pretreatment for

industrial applications but can be combined with other pretreatments that are cost effective

[5]. In Paper I, the chicken feather used was chopped to particle sizes between 1 and 10 mm

and then biologically pretreated prior to biogas production. The untreated wheat straw used in

Papers IV and VI was milled to particle sizes between 0.5 mm and 2 mm prior to use as a

co-substrate for biogas production.

43

Chapter 5

Improving biogas production from solid wastes

Anaerobic digestion of agricultural, municipal, and industrial solid wastes often results in low

biogas yields and slow degradation rates because some of these wastes have recalcitrant

molecular structures that make them difficult to degrade by microorganisms and some contain

chemicals/substances that can inhibit microbial growth. Strategies to overcome the challenges

associated with these wastes in order to ascribe value to them are discussed in this section.

5.1 Pretreatment to overcome biomass recalcitrance

Pretreatment of the substrate is needed either for making it easier to handle at the biogas plant

or for altering its structure for easy degradation, hence enhancing its methane potential. There

are different pretreatment methods that can be used depending on the types of substrates and

the goals of the pretreatment. The most suitable pretreatment methods for agricultural,

municipal, and industrial solid wastes are discussed in this section.

5.1.1 Mechanical pretreatment

Mechanical pretreatment is required for biomass with high total solids content (e.g. forest

residue, crop residue, chicken feather wastes) to reduce the particle size and crystallinity of

the feedstock. It can also reduce viscosity in biogas reactors making mixing easier [127].

Milling is commonly used, and optimising the size reduction process is important for

improving biogas yield. Lindner, Zielonka [128] pretreated non-degraded digestate by ball

milling in order to feed it back into the fermentation process, and they observed that ball

milling pretreatment enhanced the methane yield of two-stage maize silage digestate by 9 %

and 17 % using two-stage hay/straw digestate, respectively. However, the electrical demand

of this pretreatment method is very high, so it is not viable as a stand-alone pretreatment for

industrial applications but can be combined with other pretreatments that are cost effective

[5]. In Paper I, the chicken feather used was chopped to particle sizes between 1 and 10 mm

and then biologically pretreated prior to biogas production. The untreated wheat straw used in

Papers IV and VI was milled to particle sizes between 0.5 mm and 2 mm prior to use as a

co-substrate for biogas production.

44

5.1.2 Thermal pretreatment

The feedstock is heated to a temperature between 125 and 190 °C under pressure and

maintained at that temperature for one hour [127, 129]. Autoclaves and microwaves are

commonly used in the laboratory for this method. The method’s effectiveness depends on the

feedstock, pretreatment temperature, and amount of time. Thermal pretreatment of solid

wastes requires additional water. The presence of heat and water causes swelling of the

biomass by disrupting the hydrogen bonds between cellulose and matrix polymers

(hemicellulose and lignin) [129]. Higher pretreatment temperature can enhance the solubility

of organic matter [130], although this might not result in increased biogas production [131].

Liquid hot-water pretreatment at lower temperature is effective for hydrolysis of

hemicellulose [65], and moderate pretreatment of biomass is required for improved anaerobic

digestion. This process requires a lower amount of chemicals for neutralisation of the

hydrolysate and produces a lower amount of neutralisation residue compared to dilute-acid

pretreatment [64].

5.1.3 Chemical pretreatment

The chemical pretreatment method involves the use of acids and bases at different

concentrations and conditions for hydrolysis of recalcitrant biomass. It is most suitable for

forest and crop residues (lignocellulosic biomass) but can be used for hydrolysis of chicken

feathers as well [132]; it enhances the degradation rate of this biomass and improves biogas

production. Dilute acid pretreatment under controlled conditions is generally used for

solubilising hemicellulose from this lignocellulose biomass and thereby increasing the

accessibility of cellulose to the microorganisms [5]. High temperature and high acid

concentration, can however cause the release of inhibitory products [133]. Acid pretreatment

of wheat straw and sugarcane bagasse was studied by Bolado-Rodríguez, Toquero [134], and

the result showed acid solubilisation of high concentrations of sugars generating furfural and

HMF with strong inhibition effects. However, Xiao and Clarkson [135] reported 80 % lignin

solubilisation when newsprint was pretreated with 35 % acetic acid and 2 % nitric acid,

resulting in a three times higher methane yield than the yield obtained from the untreated

material, although there was no effective recovery of the acids used.

45

5.1.4 Biological pretreatment

Biological pretreatment makes use of microorganisms (mostly bacteria and fungi) to degrade

recalcitrant biomass for improved biogas production. Studies have shown that white-rot fungi

produce enzymes that are capable of extensive degradation of lignin and that brown-rot fungi

break down cellulose and hemicellulose resulting in increased biomass digestibility [136],

although there is a loss of cellulose and hemicellulose during this process [137]. Based on the

enzyme production patterns of white-rot fungi, Hatakka [138] suggested three categories of

fungi for lignin degradation, namely a lignin-manganese peroxidase group (e.g. P.

chrysosporium and Phlebia radiata), manganese peroxidase-laccase group (e.g. Dichomitus

squalens and Rigidoporus lignosus), and lignin peroxidase-laccase group (e.g. Phlebia

ochraceofulva and Junghuhnia separabilima). Additionally, the soft rot ascomycetal fungus

(Trichoderma viride) has been applied to MSW (kitchen wastes, garden wastes, grass

clippings, foliage, and wood pellets) in an aerobic upstream process prior to anaerobic

digestion [139]. The results showed a threefold increase in methane yield compared to the

case of untreated MSW.

Bacteria and fungi have also been reported capable of producing enzymes that degrade

keratin-rich wastes. Some species of Bacillus [66, 140-144] and some species of saprophytic

and parasitic fungi [145, 146] have been reported effective for hydrolysing keratin-rich

wastes. Commercial enzymes (cellulase, hemicellulase, proteases, amylases, and lipases) are

also suitable for effective hydrolysis of complex biomass. Multiple commercial enzymes

(consortium) are more effective than single commercial enzymes because they act

synergistically to enable efficient conversion of the substrate by the concerted action of

several species [65]. Commercial enzymes are very expensive, but the enzymes can be

produced on-site from cheap feedstocks, which makes the process cost effective [147]. The

biological pretreatment method is usually carried out at low temperatures and pressures

without expensive equipment or chemical reagents. This method is also sustainable,

ecological, and cost effective; however, the enzymatic reactions are slow and as such require

longer pretreatment times compared to other pretreatments method [148].

5.1.4.1 Hydrolysis of chicken feathers for biogas production

The keratin-degrading bacterium used in this study was a naturally occurring strain of

Bacillus isolated from compost and identified as Bacillus sp. C4 (2008) [141]. Chicken

45

5.1.4 Biological pretreatment

Biological pretreatment makes use of microorganisms (mostly bacteria and fungi) to degrade

recalcitrant biomass for improved biogas production. Studies have shown that white-rot fungi

produce enzymes that are capable of extensive degradation of lignin and that brown-rot fungi

break down cellulose and hemicellulose resulting in increased biomass digestibility [136],

although there is a loss of cellulose and hemicellulose during this process [137]. Based on the

enzyme production patterns of white-rot fungi, Hatakka [138] suggested three categories of

fungi for lignin degradation, namely a lignin-manganese peroxidase group (e.g. P.

chrysosporium and Phlebia radiata), manganese peroxidase-laccase group (e.g. Dichomitus

squalens and Rigidoporus lignosus), and lignin peroxidase-laccase group (e.g. Phlebia

ochraceofulva and Junghuhnia separabilima). Additionally, the soft rot ascomycetal fungus

(Trichoderma viride) has been applied to MSW (kitchen wastes, garden wastes, grass

clippings, foliage, and wood pellets) in an aerobic upstream process prior to anaerobic

digestion [139]. The results showed a threefold increase in methane yield compared to the

case of untreated MSW.

Bacteria and fungi have also been reported capable of producing enzymes that degrade

keratin-rich wastes. Some species of Bacillus [66, 140-144] and some species of saprophytic

and parasitic fungi [145, 146] have been reported effective for hydrolysing keratin-rich

wastes. Commercial enzymes (cellulase, hemicellulase, proteases, amylases, and lipases) are

also suitable for effective hydrolysis of complex biomass. Multiple commercial enzymes

(consortium) are more effective than single commercial enzymes because they act

synergistically to enable efficient conversion of the substrate by the concerted action of

several species [65]. Commercial enzymes are very expensive, but the enzymes can be

produced on-site from cheap feedstocks, which makes the process cost effective [147]. The

biological pretreatment method is usually carried out at low temperatures and pressures

without expensive equipment or chemical reagents. This method is also sustainable,

ecological, and cost effective; however, the enzymatic reactions are slow and as such require

longer pretreatment times compared to other pretreatments method [148].

5.1.4.1 Hydrolysis of chicken feathers for biogas production

The keratin-degrading bacterium used in this study was a naturally occurring strain of

Bacillus isolated from compost and identified as Bacillus sp. C4 (2008) [141]. Chicken

46

feathers at total solid concentrations of 5, 10, and 20 % (dry weight of feathers) were

biologically pretreated. The increase in feather concentration from 5 to 20 %TS caused a

suppression of keratinase activity, which resulted in a decrease in the percentage of feathers

degraded, as shown in Fig. 5.1a.

Fig. 5.1 (a) Substrate degradation at different feather concentrations (b) Soluble crude protein from TKN value (modified from Paper 1)

After 8 days of degradation, the amount of soluble crude protein increased from an average of

4.13 g/l to 35.97 g/l, 36.97 g/l, and 45.94 g/l at initial feather concentrations of 5, 10, and 20

%TS, respectively, as shown in Fig. 5.1b.

Both the hydrolysate and the whole broth of the pretreated feathers were investigated as

substrates for biogas production using anaerobic sludge and bacteria granules as inocula

(Paper 1). Pretreatment improved methane yield by 292 % and 105 % when anaerobic sludge

and granules were used on the feather hydrolysate, respectively. Bacteria granules worked

effectively on the total broth, yielding 445 NmlCH4/gVS methane, which is 124 % more than

that obtained with the same type of inoculum from untreated feather.

5.2 Co-digestion to achieve balance nutrient

Co-digestion of different feedstocks with an appropriate mixing ratio is a possible solution to

overcome the challenges associated with mono-digestion of potential feedstocks and obtain

improved biogas yield. Mono-digestion of protein-rich wastes (animal manure and chicken

feathers) results in low biogas yield due to high nitrogen content, which may inhibit

methanogens. Lignocellulosic wastes are seasonal biomass with high carbon content but very

0

10

20

30

40

50

60

70

80

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Subs

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otei

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Time of filtrate collection (days)

5 %TS10 %TS20 %TS

(b)

47

low nitrogen content, but digestion of these feedstocks alone also results in a slow process

leading to low biogas yield. Additionally, some industrial wastes such as fruit and food wastes

may contain chemicals and substances that inhibit methanogens and thereby result in low

biogas yield. Choosing an appropriate mixing ratio is vital for effective performance of the

co-digestion process, and it is important to consider the C/N ratio, biodegradability, possible

inhibitors, and the TS of the individual feedstocks [5]. Considering the fact that easily

degraded feedstocks are not highly available and the problems associated with pretreatments,

co-digestion is a good strategy for improving biogas production because it favours

synergisms, dilutes harmful compounds, optimises biogas production, and increases digestate

quality [149]. Co-digestion of different solid wastes has been reported by various researchers

to have substantial impacts on anaerobic digestion processes, as shown in Table 51.

In this study, a methane yield of 163 NmlCH4/gVS was obtained from manure bedded with

straw at 22 %TS and C/N ratio of 16.8 using the plug flow reactor (Paper II). However,

digestion of manure bedded with straw at 22 %TS and C/N ratio of 25 due to the addition of

untreated wheat straw in the textile reactor resulted in a 12 % increase in methane yield due to

the increased C/N produced by the wheat straw (Paper IV). Additionally, co-digestion of

citrus wastes with chicken feathers, wheat straw and manure bedded with straw was

investigated at 20 %TS content to reduce the inhibitory effect of the citrus waste (Paper VI).

The best mixing ratio (1:1:6; referring to chicken feathers: citrus wastes: wheat straw)

enhanced the methane yield by 14 % compared to the expected yield calculated from the

methane potentials obtained for the individual fractions.

5.3 Acclimatisation of microorganisms to substrates

One strategy for overcoming the problem of low methane yield due to substrate or product

inhibition and preventing a lag phase is long-term acclimatisation of microbial communities

to the feedstock and conditions in the reactor. Studies have shown changes in microbial

communities compared with the initial inoculum due to adaptation [150-152]. Carucci,

Carrasco [153] investigated the effect of acclimatised inoculum on co-digestion of fresh

vegetables and sludge mixture and observed that inhibition of methanogenesis was overcome

by a long acclimatisation period of 120 days. Adaptation of the microbial community to

supplied feedstock during a long-term (337 days) anaerobic digestion of maize and sugar beet

silage has also been reported [152]. This shows that acclimatisation of microorganisms to

feedstock and conditions in the reactor is vital for improved biogas production. In this study, a

47

low nitrogen content, but digestion of these feedstocks alone also results in a slow process

leading to low biogas yield. Additionally, some industrial wastes such as fruit and food wastes

may contain chemicals and substances that inhibit methanogens and thereby result in low

biogas yield. Choosing an appropriate mixing ratio is vital for effective performance of the

co-digestion process, and it is important to consider the C/N ratio, biodegradability, possible

inhibitors, and the TS of the individual feedstocks [5]. Considering the fact that easily

degraded feedstocks are not highly available and the problems associated with pretreatments,

co-digestion is a good strategy for improving biogas production because it favours

synergisms, dilutes harmful compounds, optimises biogas production, and increases digestate

quality [149]. Co-digestion of different solid wastes has been reported by various researchers

to have substantial impacts on anaerobic digestion processes, as shown in Table 51.

In this study, a methane yield of 163 NmlCH4/gVS was obtained from manure bedded with

straw at 22 %TS and C/N ratio of 16.8 using the plug flow reactor (Paper II). However,

digestion of manure bedded with straw at 22 %TS and C/N ratio of 25 due to the addition of

untreated wheat straw in the textile reactor resulted in a 12 % increase in methane yield due to

the increased C/N produced by the wheat straw (Paper IV). Additionally, co-digestion of

citrus wastes with chicken feathers, wheat straw and manure bedded with straw was

investigated at 20 %TS content to reduce the inhibitory effect of the citrus waste (Paper VI).

The best mixing ratio (1:1:6; referring to chicken feathers: citrus wastes: wheat straw)

enhanced the methane yield by 14 % compared to the expected yield calculated from the

methane potentials obtained for the individual fractions.

5.3 Acclimatisation of microorganisms to substrates

One strategy for overcoming the problem of low methane yield due to substrate or product

inhibition and preventing a lag phase is long-term acclimatisation of microbial communities

to the feedstock and conditions in the reactor. Studies have shown changes in microbial

communities compared with the initial inoculum due to adaptation [150-152]. Carucci,

Carrasco [153] investigated the effect of acclimatised inoculum on co-digestion of fresh

vegetables and sludge mixture and observed that inhibition of methanogenesis was overcome

by a long acclimatisation period of 120 days. Adaptation of the microbial community to

supplied feedstock during a long-term (337 days) anaerobic digestion of maize and sugar beet

silage has also been reported [152]. This shows that acclimatisation of microorganisms to

feedstock and conditions in the reactor is vital for improved biogas production. In this study, a

48

long-term acclimatisation period of 232 days resulted in a 58 % increase in methane yield

when manure bedded with straw was digested in the textile bioreactor (Paper IV).

Table 5.1 Co-digestion of solid wastes for improved biogas production (modified from [147])

Feedstocks Effect of co-digestion Influencing factor Reference

Food, vegetable, fruit, leaf and paper wastes

Reduced ammonia-nitrogen inhibition

C/N ratio [154]

Solid energy crops and pig manure

Enhanced process performance

High buffering capacity

[155]

Increased plant capacity

Crops (grass silage, oat straw, and sugar beet tops) and cow manure

Increased methane yield Increased VS removal

Mixing ratio [156]

Cattle manure with wheat straw

Increased methane yield C/N ratio [157] Paper IV

Fresh vegetable and precooked food wastes

Increased methane production yield and rate

Dilution to non-inhibiting initial concentration Synergetic effect

[153]

Yard and food wastes Increased methane yield and volumetric productivity

Mixing ratio (C/N ratio)

[158]

Increased VS reduction

MSW, manure, crop residues, and slaughterhouse wastes

Increased methane yield High buffering capacity (synergetic effect)

[159]

Paper tube residues and nitrogen-rich substrate mixture

Stabilized the process Reduced HRT Reduced VFA accumulation

High buffering capacity

[160]

Crop silage and cow manure Increased methane yield Mixing ratio [161] Allowed higher organic loading rate

Citrus wastes, chicken feathers and wheat straw

Improved methane yield

Buffering capacity and C/N ratio

Paper VI

Cassava silage and excess sludge

Reduced propionate concentration. Increased methane yield

C/N ratio [162]

C/N = carbon to nitrogen ratio, VS = volatile solids, HRT = hydraulic retention time, VFA = volatile fatty acids

49

Chapter 6

Digestate as biofertiliser

Digestate is the residue remaining after biogas production by anaerobic digestion of organic

wastes. The organic wastes used as feedstocks for biogas production contain some

macronutrients (nitrogen, phosphorus, potassium, calcium, sulphur, and magnesium) and

micronutrients (boron, chlorine, manganese, iron, zinc, copper, molybdenum, and nickel), and

additional nutrients/trace elements are sometimes added to enhance the digestion process. As

these feedstocks degrade during the digestion process, the nutrients are released and

concentrated in the residue [33]; therefore, the digestate residue is a valuable fertiliser which

contains most of the macronutrients and minor nutrients in plant-available form. This residue

after biogas production can result in similar or even better crop yields than obtained by using

commercial fertilisers [163].

6.1 Composition and quality of the digestate

The composition of the digestate determines its quality, and this varies from one biogas

reactor to another. The nutrients content of the digestate i.e. its composition depends on the

composition of the feedstock, TS content of the feedstock, process conditions, and pre-

treatment methods.

Feedstock composition: Most of the nutrients in the feedstock end up in the digestate [163].

The nutrients content of the digestate can be controlled by regulating the composition of the

feedstock and as such the feedstock should be free of heavy metals (Cadmium, Lead and

Mercury) which in higher concentration are toxic to plant growth [164, 165]. The most

suitable feedstocks for biogas production with digestate used as fertiliser are animal manure,

crops, the organic fraction of MSW, vegetable by-products and residues, and wastes from

agriculture, horticulture, and forestry. Furthermore, during co-digestion of different

feedstocks, there may be variations in the digestate nutrients compared to those of mono-

digestion [166].

TS content of the feedstock: The higher the TS of the feedstock, the higher the TS of the

digestate residue. Digestate with higher TS contains larger amounts of carbon and nitrogen

that can be broken down further in the soil, which in the long-run results in the release of

more nutrients [33, 166].

49

Chapter 6

Digestate as biofertiliser

Digestate is the residue remaining after biogas production by anaerobic digestion of organic

wastes. The organic wastes used as feedstocks for biogas production contain some

macronutrients (nitrogen, phosphorus, potassium, calcium, sulphur, and magnesium) and

micronutrients (boron, chlorine, manganese, iron, zinc, copper, molybdenum, and nickel), and

additional nutrients/trace elements are sometimes added to enhance the digestion process. As

these feedstocks degrade during the digestion process, the nutrients are released and

concentrated in the residue [33]; therefore, the digestate residue is a valuable fertiliser which

contains most of the macronutrients and minor nutrients in plant-available form. This residue

after biogas production can result in similar or even better crop yields than obtained by using

commercial fertilisers [163].

6.1 Composition and quality of the digestate

The composition of the digestate determines its quality, and this varies from one biogas

reactor to another. The nutrients content of the digestate i.e. its composition depends on the

composition of the feedstock, TS content of the feedstock, process conditions, and pre-

treatment methods.

Feedstock composition: Most of the nutrients in the feedstock end up in the digestate [163].

The nutrients content of the digestate can be controlled by regulating the composition of the

feedstock and as such the feedstock should be free of heavy metals (Cadmium, Lead and

Mercury) which in higher concentration are toxic to plant growth [164, 165]. The most

suitable feedstocks for biogas production with digestate used as fertiliser are animal manure,

crops, the organic fraction of MSW, vegetable by-products and residues, and wastes from

agriculture, horticulture, and forestry. Furthermore, during co-digestion of different

feedstocks, there may be variations in the digestate nutrients compared to those of mono-

digestion [166].

TS content of the feedstock: The higher the TS of the feedstock, the higher the TS of the

digestate residue. Digestate with higher TS contains larger amounts of carbon and nitrogen

that can be broken down further in the soil, which in the long-run results in the release of

more nutrients [33, 166].

50

Process conditions: The retention time for the feedstock inside the reactor, at constant process

temperature, influences the digestate quality [167]. If the organic loading rate of the anaerobic

digestion process is high and the retention time is short, the digestate might contain a large

amount of undigested organic matter, which is not economical [168]. In continuous processes,

it is possible that fractions of feedstock (and the impurities contained in them) may escape

through the digestate. Appropriate process temperatures and retention times must be selected

during the digestion process to ensure a quality digestate. A thermophilic process increases

the rate of pathogen reduction in the digestate, but a mesophilic digestion process alone may

not be adequate [168]. Pasteurisation (70 °C, 60 min) is required before or after anaerobic

digestion, especially when animal by-products are used as substrates [169]. The waste may

sometimes be heated at a lower temperature but for a longer time prior to mesophilic or

thermophilic digestion aiming to achieve similar effects [170].

Pre-treatment methods: Recalcitrant feedstocks are often pretreated to enhance digestibility

and thereby improve AD performance. However, the pretreatment method should be mild and

must not generate inhibitors or retain chemicals that can have a negative effect on the quality

of the digestate.

6.2 Benefits of digestate

Improved quality of soil: Digestate has some active microorganisms which increase the

biological activity of the soil and enhance the formation of microscopic biofilms within the

soil. The microorganisms in soil are mostly heterotrophic (use organic carbon as a source of

carbon and energy), and the use of digestate stimulates the growth of these microorganisms in

soil, thereby facilitating nutrient mineralisation for plant uptake and protection of plants

against disease [33, 171]. Digestate increases the buffering capacity of soil and enhances

retention of water and air in the soil profile [33].

During anaerobic digestion of organic wastes, organic nitrogen is converted to ammonium

nitrogen, so the digestate residue contains a high proportion of mineralised nitrogen,

especially in the form of ammonium, which is available for plants. It also contains other

macro- and micro-elements essential for plant growth [172, 173]. In addition, the digestate

contains a certain amount of carbon and nitrogen, which is broken down further in soil

resulting in the release of more nutrients in the long run. In this way, digestate improves crop

yield.

51

During anaerobic digestion of organic wastes, the volatile solid content and viscosity of the

waste is reduced, which makes the digestate easier to spread on agricultural land than

untreated manure. The use of digestate as bio-fertiliser decreases the cost of crop production,

and the income of farmers can be increased through the selling of their bio-fertiliser when

they have a surplus of nutrients on their farm.

6.3 Dewatering process

The digestate is usually separated into solid and liquid fractions to reduce the volume, making

the solid fraction easier to handle and reducing the cost of transportation. The liquid fraction

usually contains higher amounts of ammonium and potassium [174], but phosphorus is

retained in the solid fraction. The liquid fraction of the digestate can be used directly as

organic fertiliser. The solid fraction can also be used on soil to improve soil structure, retain

nutrients, and prevent leaching, or the solid fraction can be post-treated using an aerobic

composting process thereby closing the production cycle [175-177]. The digestate is usually

separated into different fractions using the following techniques.

Screw press: The screw press separates the digestate into liquid and solid fractions. The press

can be set to the desired amount of total solid content needed in the solid fraction [178]. This

method can achieve between 30 % to 38 % total solid contents in the solid fraction. There is

also a belt press that functions as a screw press but has a higher separation efficiency [179].

Centrifuge: This method uses centrifugal force to separate the digestate. In this research, the

digestate was separated into solid and liquid fractions by centrifuging at 5,000g for 10 min

prior to analysis (Paper IV).

Evaporation: This method utilises thermal energy and is applied to concentrate the digestate

and increase the total solid content. Concentrations ranging up to 20 % total solids can be

achieved, but at high temperature ammonia can be released. This can be avoided by

decreasing the pH of the digestate prior to evaporation [180].

Chemical separation: This method involves chemical precipitation, adsorption, and removal

of major nutrients in the liquid fraction. Concentrated nitrogen, potassium, and phosphorus

can be obtained in solid form from the liquid fraction [179]. Another separation technique is

membrane filtration and reverse osmosis. This technique is still new, but it can allow total

separation of the solid fraction to obtain a clean liquid.

51

During anaerobic digestion of organic wastes, the volatile solid content and viscosity of the

waste is reduced, which makes the digestate easier to spread on agricultural land than

untreated manure. The use of digestate as bio-fertiliser decreases the cost of crop production,

and the income of farmers can be increased through the selling of their bio-fertiliser when

they have a surplus of nutrients on their farm.

6.3 Dewatering process

The digestate is usually separated into solid and liquid fractions to reduce the volume, making

the solid fraction easier to handle and reducing the cost of transportation. The liquid fraction

usually contains higher amounts of ammonium and potassium [174], but phosphorus is

retained in the solid fraction. The liquid fraction of the digestate can be used directly as

organic fertiliser. The solid fraction can also be used on soil to improve soil structure, retain

nutrients, and prevent leaching, or the solid fraction can be post-treated using an aerobic

composting process thereby closing the production cycle [175-177]. The digestate is usually

separated into different fractions using the following techniques.

Screw press: The screw press separates the digestate into liquid and solid fractions. The press

can be set to the desired amount of total solid content needed in the solid fraction [178]. This

method can achieve between 30 % to 38 % total solid contents in the solid fraction. There is

also a belt press that functions as a screw press but has a higher separation efficiency [179].

Centrifuge: This method uses centrifugal force to separate the digestate. In this research, the

digestate was separated into solid and liquid fractions by centrifuging at 5,000g for 10 min

prior to analysis (Paper IV).

Evaporation: This method utilises thermal energy and is applied to concentrate the digestate

and increase the total solid content. Concentrations ranging up to 20 % total solids can be

achieved, but at high temperature ammonia can be released. This can be avoided by

decreasing the pH of the digestate prior to evaporation [180].

Chemical separation: This method involves chemical precipitation, adsorption, and removal

of major nutrients in the liquid fraction. Concentrated nitrogen, potassium, and phosphorus

can be obtained in solid form from the liquid fraction [179]. Another separation technique is

membrane filtration and reverse osmosis. This technique is still new, but it can allow total

separation of the solid fraction to obtain a clean liquid.

52

6.4 Digestate from dry digestion of manure with straw in textile bioreactor

In this study, the digestate residue remaining after dry anaerobic digestion of manure bedded

with straw was analysed for its suitability as a bio-fertiliser (Paper IV). The digestate residue

was separated into liquid and solid fractions, and the available macro and minor nutrients,

heavy metals were quantified using MP-AES (Agilent technologies), as shown in Table 6.1.

Table 6.1 Composition of digestate from dry anaerobic digestion of manure bedded with

straw (Paper IV)

Parameter Digestate residue

pH 8.01 – 8.06

Total carbon (%)36.23 – 34.36 ٭

TS (%)39 .13 – 7.97 ٭

VS (%)65.15 – 61.84 ٭ Unit Liquid fractiona Solid fractiona

Total nitrogen g/L 2.45 – 2.8 6.77 – 9.22 (mg/g)

Ammonium nitrogen

g/L 1.25 – 1.55 ND

Potassium g/L 1.19 – 4.29 0.025 – 0.051

Phosphorus mg/L 10.03 – 30.54 14.71 – 36.52

Calcium mg/L 15 – 50.50 48.33 – 64.33

Magnesium mg/L 3.35 – 60 13.4 – 16.73

Copper mg/L 0.13 – 0.19 0.14 – 0.35

Iron mg/L 3 – 5.5 ND

Zinc mg/L 0.11 – 0.19 0.33 – 0.69

Nickel mg/L 0.28 – 0.37 0.01

Chromium mg/L < 0.001 0.02 – 0.05

Cadmium mg/L < 0.001 < 0.001

a Fresh matter; ND = not determined; ٭ dry basis

The pH of the digestate was slightly alkaline, which should increase the buffering capacity of

soil because most agricultural lands are somewhat acidic. The liquid fraction had higher

nitrogen and potassium contents compared to the solid fraction, but the phosphorus content

was low in both fractions. Studies have shown a loss of some amount of phosphorus during

anaerobic digestion processes [173, 181]. Phosphorus can be added as a supplement to avoid

53

phosphorus deficiency in the soil; however, nutrient requirements vary from one soil to

another, so the digestate can be supplied to soil with major deficiencies of nitrogen and

potassium. Some amounts of Ca, Mg, Cu, Fe, Zn, and Ni were found in the digestate, but

heavy metals that are toxic to plants, such Cd and Cr had insignificant concentrations, as

shown in Table 6.1.

53

phosphorus deficiency in the soil; however, nutrient requirements vary from one soil to

another, so the digestate can be supplied to soil with major deficiencies of nitrogen and

potassium. Some amounts of Ca, Mg, Cu, Fe, Zn, and Ni were found in the digestate, but

heavy metals that are toxic to plants, such Cd and Cr had insignificant concentrations, as

shown in Table 6.1.

54

55

Chapter 7

Concluding remarks and future directions

Dry anaerobic digestion (dry-AD) is an appropriate technology for processing solid organic

waste; potentials of textile and plug flow bioreactors for biogas production through dry-AD

were assessed in this study. The major solid wastes treated for biogas production in this study

were chicken feather, manure bedded with straw, wheat straw, and citrus wastes.

7.1 Conclusions

The major conclusions of this research are presented below.

Anaerobic digestion of chicken feather wastes with bacteria granules is possible even

without any pretreatment. Bacteria granules used as inoculum resulted in almost two

time more methane production from the untreated feather than when anaerobic sludge

was used.

Pretreatment improved methane yield by 124 % and 237 % compared to the untreated

when bacteria granules and anaerobic sludge were used on the total broth of pretreated

feathers, respectively. Anaerobic sludge as inoculum performed better on the feather

hydrolysate, whereas bacteria granules worked better on the total broth of the

pretreated feathers.

The plug flow reactor was efficient for dry-AD of manure bedded with straw at 22

%TS with increasing organic loading rates of 2.8, 4.2, and 6 gVS/L/d and

corresponding retention times of 60, 40, and 28 days. Organic loading rates of up to

4.2 gVS/L/d with a corresponding retention time of 40 days produced better process

stability with methane yields up to 0.163 LCH4/gVSadded/d, which is 56 % of the

theoretical yield.

The textile bioreactor is robust and simple to operate. The reactor worked successfully

for the dry digestion of manure with straw at 22 – 30 % TS (solid content of the

feedstock). A long-term acclimatisation period of 232 days resulted in a 58 % increase

in methane yield.

The digestate residue after biogas production from manure with straw in the textile

bioreactor was determined to be suitable as a bio-fertiliser.

55

Chapter 7

Concluding remarks and future directions

Dry anaerobic digestion (dry-AD) is an appropriate technology for processing solid organic

waste; potentials of textile and plug flow bioreactors for biogas production through dry-AD

were assessed in this study. The major solid wastes treated for biogas production in this study

were chicken feather, manure bedded with straw, wheat straw, and citrus wastes.

7.1 Conclusions

The major conclusions of this research are presented below.

Anaerobic digestion of chicken feather wastes with bacteria granules is possible even

without any pretreatment. Bacteria granules used as inoculum resulted in almost two

time more methane production from the untreated feather than when anaerobic sludge

was used.

Pretreatment improved methane yield by 124 % and 237 % compared to the untreated

when bacteria granules and anaerobic sludge were used on the total broth of pretreated

feathers, respectively. Anaerobic sludge as inoculum performed better on the feather

hydrolysate, whereas bacteria granules worked better on the total broth of the

pretreated feathers.

The plug flow reactor was efficient for dry-AD of manure bedded with straw at 22

%TS with increasing organic loading rates of 2.8, 4.2, and 6 gVS/L/d and

corresponding retention times of 60, 40, and 28 days. Organic loading rates of up to

4.2 gVS/L/d with a corresponding retention time of 40 days produced better process

stability with methane yields up to 0.163 LCH4/gVSadded/d, which is 56 % of the

theoretical yield.

The textile bioreactor is robust and simple to operate. The reactor worked successfully

for the dry digestion of manure with straw at 22 – 30 % TS (solid content of the

feedstock). A long-term acclimatisation period of 232 days resulted in a 58 % increase

in methane yield.

The digestate residue after biogas production from manure with straw in the textile

bioreactor was determined to be suitable as a bio-fertiliser.

56

Co-digestion of citrus wastes with chicken feather and wheat straw with mixing ratio

of 1:1:6 gave the best performance in batch assays

Best mixture investigated in continuous dry digestion experiments using plug flow

reactors. At OLR of 2 gVS/l/d and retention time of 50 days, the methane yield of 362

NmlCH4/gVSadded was obtained from RTS21, which is 13.5 % higher than the yield

obtained from RTS32 (319 NmlCH4/gVSadded). Higher OLR of 3.8 gVS/l/d and retention

time of 30 days shows process instability in both reactors

7.2 Future directions

Dry-AD is gaining interest due to reduced amount of water needed, less pretreatment, more

flexibility in acceptance of waste and easy handling of digestate residue. However, there are

still some challenges associated with this process for biogas production, such as longer start

up and retention time, inhibition problems due to reduced amounts of water, problems of

mixing, process instability and suitability of the digestate as bio-fertiliser. The following

future research will be necessary to overcome these challenges.

Dry-AD in this study was carried out under mesophilic condition. It will be necessary

to investigate dry-AD under the thermophilic condition to shorten the start-up period

as well as the retention time. This will probably also improve the methane yield.

The methane yield obtained from dry-AD using acclimatised inoculum under this

condition was higher than that obtained from the original inoculum (unacclimatised

inoculum). It will be interesting to investigate the dominant microbial communities in

dry-AD and compared with wet-AD.

Considering the problem of mixing in the dry-AD process, it is necessary to

investigate the significance of liquid, digestate, and biogas recirculation for

optimising the performance of dry-AD.

Acceptability of the digestate residue after biogas production is important for the

agricultural sector. Therefore, investigating the influence of single substrates, co-

substrates, process design, and pre-treatment methods on composition and quality of

the digestate is essential.

Further investigations on the synergistic and antagonistic effects of co-digestion of

different solid wastes with an appropriate mixing ratio, considering the C/N ratio,

inhibitors, feedstock biodegradability, and total solid content will be of great value.

57

ACKNOWLEDGEMENTS

Thank God for wisdom, provision, and grace to finish well; great is HIS faithfulness and

mercy towards me. Thank you Lord Jesus.

Although it was quite challenging, pursuing my doctoral studies in Sweden was a great

opportunity for me, and I would like to express my deepest gratitude to those who contributed

to the successful completion of my studies.

I am grateful to Lagos State University (LASU), Nigeria, for its collaboration with the

University of Borås and most grateful to Emeritus Prof. Sikiru A. Sanni. I extend special

thanks to Emeritus Prof. Alfred A. Susu and Dr Emmanuel S. Asapo for their encouragement

and advice during my studies. The financial assistance from 4P-BUILDBLOC Consulting,

Ltd., Nigeria, was highly appreciated. I am also grateful to the Research and Educational

Board of the University of Borås, Sweden, for its support.

I am most grateful to my admirable supervisor, Professor Mohammad J. Taherzadeh. Working

with you was quite demanding, but you brought out the best in me and made the journey

faster than I thought it could be. I appreciate your prompt responses to email and your quick

revisions of my manuscripts, despite your very tight schedule. Learning under you has been

great. I learned to be self-dependent, more creative, and to meet targets. Thank you so much

for your guidance.

I also thank Dr. Ilona Sárvári Horváth (my dear co-supervisor). I really appreciate your

support, guidance, and constant encouragement throughout my doctoral studies. Thank you so

much for being there at my most confusing moments. You taught me to be calm at difficult

times; ‘it will be good’, as you always told me.

To my examiner, Professor Tobias Richard, thank you for keeping a watchful eye on my

progress and for the many valuable comments during my studies.

I am grateful to all of the lecturers at the University of Borås for the knowledge that they

shared: Dr. Anitta Pattersson, Dr. Magnus Lundin, Prof. Jan Nolin, Prof. Gustaf Nelhans,

Helena Francke, Dr. Elisabeth Feuk-Lagerstedt, Prof. Christine Räisänen, and Prof. Lars

Sandman. Thank you all.

57

ACKNOWLEDGEMENTS

Thank God for wisdom, provision, and grace to finish well; great is HIS faithfulness and

mercy towards me. Thank you Lord Jesus.

Although it was quite challenging, pursuing my doctoral studies in Sweden was a great

opportunity for me, and I would like to express my deepest gratitude to those who contributed

to the successful completion of my studies.

I am grateful to Lagos State University (LASU), Nigeria, for its collaboration with the

University of Borås and most grateful to Emeritus Prof. Sikiru A. Sanni. I extend special

thanks to Emeritus Prof. Alfred A. Susu and Dr Emmanuel S. Asapo for their encouragement

and advice during my studies. The financial assistance from 4P-BUILDBLOC Consulting,

Ltd., Nigeria, was highly appreciated. I am also grateful to the Research and Educational

Board of the University of Borås, Sweden, for its support.

I am most grateful to my admirable supervisor, Professor Mohammad J. Taherzadeh. Working

with you was quite demanding, but you brought out the best in me and made the journey

faster than I thought it could be. I appreciate your prompt responses to email and your quick

revisions of my manuscripts, despite your very tight schedule. Learning under you has been

great. I learned to be self-dependent, more creative, and to meet targets. Thank you so much

for your guidance.

I also thank Dr. Ilona Sárvári Horváth (my dear co-supervisor). I really appreciate your

support, guidance, and constant encouragement throughout my doctoral studies. Thank you so

much for being there at my most confusing moments. You taught me to be calm at difficult

times; ‘it will be good’, as you always told me.

To my examiner, Professor Tobias Richard, thank you for keeping a watchful eye on my

progress and for the many valuable comments during my studies.

I am grateful to all of the lecturers at the University of Borås for the knowledge that they

shared: Dr. Anitta Pattersson, Dr. Magnus Lundin, Prof. Jan Nolin, Prof. Gustaf Nelhans,

Helena Francke, Dr. Elisabeth Feuk-Lagerstedt, Prof. Christine Räisänen, and Prof. Lars

Sandman. Thank you all.

58

I appreciate Sari Sarhamo, Susanne Borg, Jonas Edberg, and Irene Lammassaari for their

wonderful administrative work. I also extend sincere gratitude to Peter Therning (Section

Head), Dr. Tomas Washnström (Director of Ph.D. Studies), Louise Holmgren (economist),

Thomas Södergren, and Dan Åkesson for their support.

I express special thanks to Kristina and Marlen for supplying all of the things needed in the

laboratory and for their understanding. Thank you so much Dr Jorge for the time you shared

with me to answer my questions while I was working in the laboratory and for the great

knowledge that you shared about biotechnology. I thank Adib Kalantar Mehrjerdi for his

technical assistance when solving problems with the plug flow reactors. To Dr Mofoluwake

Ishola, thank you for the wonderful time that we spent together and for your encouragement

and prayers. Thank you Alex Osagie Osadolor for your encouragement and our successful

collaboration. To my wonderful office mates, Mostafa, Adib, Konstantinos, Babak Nemat,

and Dr Kehinde Oluoti, special thanks for the wonderful times we spent together. Grateful to

the following Doctors Paivi, Patrik, Akram, Supansa, Julius Akinbomi, Fatima Bakare,

Solmaz, Maryam, Swarnima, Karthik, Jhosane, Wilkan, Ramkumar, and other Ph.D. students

Amir, Forough, Kamran, Lukitawesa, Stephen, Pedro, Rebecca, Andreas, and Veronica for

creating a pleasant working environment and assisting in the laboratory when necessary.

I express my deepest gratitude to Pastor Hans Dahlgren and all of the members of the Borås

Baptist Church (Bodakrykan); I really appreciate your prayers, love, and care throughout my

studies in Sweden. I am most grateful to Christina Sahlman, Ann-Marie Lakhall, Elisabeth

Freij, Brita Erlandsson, and several others. I am also very grateful to Rev. S. A. Oyebanji and

all my beloved reverends, pastors, and friends in the Lord in Nigeria for their encouragement

and prayers.

I express my heartfelt thanks to Marianne Hennichs (dear Mama). Thank you so much for

letting me be a part of your family during my stay in Sweden. You lessened my burden

through your care. I also send special thanks to your beloved daughters (Monica and

Catherina) for the wonderful time we spent together.

My special thanks go to Thorbjörn and Ragnhild Svalander (beloved Papa and Mama). I am

very grateful for your support, love, and care. You made the journey easier by your support.

Thank you so much. Thank you sincerely Anna Svalander and the entire Svalander family for

the moments we shared.

59

I appreciate all my siblings and relatives (the entire Akotonayon Godonu family) for their

love, encouragement, and prayers. I am grateful to my dear sister Josephine Odita and her

beloved husband Pastor Ikechukwu Odita for taking good care of my family during my

studies in Sweden. I express special thanks to Rev. & Pastor (Mrs.) J. N. Patinvoh and all my

in-laws (the entire Patinvoh family) for their understanding, encouragement, and prayers.

Finally, I am most grateful to my dear family: beloved husband Daniel T. Patinvoh and our

children Emmanuel Sewanu Patinvoh and Samuel Seyon Patinvoh. Thank you so much my

SPECIAL for this great opportunity. This study would not have been completed without your

continuous motivation and very active support. I love you very much; you are truly special to

me. Thank you so much our beloved children for your understanding; we love you

immensely. As arrows are in the hands of a mighty warrior, so you are unto us in Jesus’ name.


December 2017

59

I appreciate all my siblings and relatives (the entire Akotonayon Godonu family) for their

love, encouragement, and prayers. I am grateful to my dear sister Josephine Odita and her

beloved husband Pastor Ikechukwu Odita for taking good care of my family during my

studies in Sweden. I express special thanks to Rev. & Pastor (Mrs.) J. N. Patinvoh and all my

in-laws (the entire Patinvoh family) for their understanding, encouragement, and prayers.

Finally, I am most grateful to my dear family: beloved husband Daniel T. Patinvoh and our

children Emmanuel Sewanu Patinvoh and Samuel Seyon Patinvoh. Thank you so much my

SPECIAL for this great opportunity. This study would not have been completed without your

continuous motivation and very active support. I love you very much; you are truly special to

me. Thank you so much our beloved children for your understanding; we love you

immensely. As arrows are in the hands of a mighty warrior, so you are unto us in Jesus’ name.


December 2017

60

61

REFERENCES

1. Chattopadhyay, S., A. Dutta, and S. Ray, Municipal solid waste management in Kolkata,

India–A review. Waste Management, 2009. 29(4): p. 1449-1458. 2. Karak, T., R.M. Bhagat, and P. Bhattacharyya, Municipal Solid Waste Generation,

Composition, and Management: The World Scenario. Critical Reviews in Environmental Science and Technology, 2012. 42(15): p. 1509-1630.

3. IEA, World Energy Outlook. International Energy Agency (IEA). http://www.worldenergyoutlook.org/resources/energydevelopment/energyaccessdatabase/ Accessed 2016/11/21. 2016.

4. REN21, Renewable Energy Policy Network for the 21st Centuery (REN21). Renewables 2017 Global Status Report (Paris: REN21 Secretariat) ISBN 978-3-9818107-6-9. 2017.

5. Patinvoh, R.J., et al., Innovative pretreatment strategies for biogas production. Bioresour. Technol., 2017. 224: p. 13-24.

6. NuffieldCouncilonBioethics, Biofuels: ethical issues http://nuffieldbioethics.org/wp-content/uploads/Biofuels_one_page_summary.pdf Accessed 2017/05/16. 2011.

7. Ferry, J.G., Methanogenesis: ecology, physiology, biochemistry & genetics. 2012: Springer Science & Business Media.

8. Smith, E., J.D. Van Elsas, and J.A. Van Veen, Risks associated with the application of genetically modified microorganisms in terrestrial ecosystems. FEMS Microbiology Reviews, 1992. 8(3-4): p. 263-278.

9. Weitz, K.A., et al., The Impact of Municipal Solid Waste Management on Greenhouse Gas Emissions in the United States. Journal of the Air & Waste Management Association, 2002. 52(9): p. 1000-1011.

10. Jensen, J.E.F. and R. Pipatti. CH4 emissions from solid waste disposal. Background paper on Good Practice Guidance and Uncertainty Management in National Greenhouse Gas Inventories http://www.ipcc-nggip.iges.or.jp/public/gp/bgp/5_1_CH4_Solid_Waste.pdf Accessed 2017/09/07 2002 [cited 2017/09/07]; Available from: http://www.ipcc-nggip.iges.or.jp/public/gp/bgp/5_1_CH4_Solid_Waste.pdf.

11. Mata-Alvarez, J., S. Macé, and P. Llabrés, Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresour. Technol., 2000. 74.

12. Strange, K., Overview of waste management options: their efficacy and acceptability. Issues in Environmental Science and Technology, 2002. 18: p. 1-52.

13. Kreith, F. and F. Kreith, Handbook of solid waste management. 1999. 14. FAOSTAT, Live animals. . 2013: Food and Agriculture Organization of the United Nations. FAO

Statistics Division 2016 . http://faostat.fao.org/site/573/DesktopDefault.aspx?PageID=573#ancor Accessed 2016/02/18.

15. Matikevičienė, V., D. Masiliūnienė, and S. Grigiškis. Degradation of keratin containing wastes by bacteria with keratinolytic activity. in Environment. Technology. Resources. Proceedings of the International Scientific and Practical Conference. 2015.

16. Suh, H. and H. Lee, Characterization of a Keratinolytic Serine Protease from Bacillus subtilis KS-1. Journal of Protein Chemistry, 2001. 20(2): p. 165-169.

17. Daroit, D.J., A.P.F. Corrêa, and A. Brandelli, Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus. Int. Biodeter. Biodegr., 2009. 63(3): p. 358-363.

18. Angelidaki, I. and W.T.M. Sanders, Assessment of the anaerobic biodegradability of macropollutants. Rev. Environ. Sci. Biotechnol., 2004. 3(2): p. 117-129.

61

REFERENCES

1. Chattopadhyay, S., A. Dutta, and S. Ray, Municipal solid waste management in Kolkata,

India–A review. Waste Management, 2009. 29(4): p. 1449-1458. 2. Karak, T., R.M. Bhagat, and P. Bhattacharyya, Municipal Solid Waste Generation,

Composition, and Management: The World Scenario. Critical Reviews in Environmental Science and Technology, 2012. 42(15): p. 1509-1630.

3. IEA, World Energy Outlook. International Energy Agency (IEA). http://www.worldenergyoutlook.org/resources/energydevelopment/energyaccessdatabase/ Accessed 2016/11/21. 2016.

4. REN21, Renewable Energy Policy Network for the 21st Centuery (REN21). Renewables 2017 Global Status Report (Paris: REN21 Secretariat) ISBN 978-3-9818107-6-9. 2017.

5. Patinvoh, R.J., et al., Innovative pretreatment strategies for biogas production. Bioresour. Technol., 2017. 224: p. 13-24.

6. NuffieldCouncilonBioethics, Biofuels: ethical issues http://nuffieldbioethics.org/wp-content/uploads/Biofuels_one_page_summary.pdf Accessed 2017/05/16. 2011.

7. Ferry, J.G., Methanogenesis: ecology, physiology, biochemistry & genetics. 2012: Springer Science & Business Media.

8. Smith, E., J.D. Van Elsas, and J.A. Van Veen, Risks associated with the application of genetically modified microorganisms in terrestrial ecosystems. FEMS Microbiology Reviews, 1992. 8(3-4): p. 263-278.

9. Weitz, K.A., et al., The Impact of Municipal Solid Waste Management on Greenhouse Gas Emissions in the United States. Journal of the Air & Waste Management Association, 2002. 52(9): p. 1000-1011.

10. Jensen, J.E.F. and R. Pipatti. CH4 emissions from solid waste disposal. Background paper on Good Practice Guidance and Uncertainty Management in National Greenhouse Gas Inventories http://www.ipcc-nggip.iges.or.jp/public/gp/bgp/5_1_CH4_Solid_Waste.pdf Accessed 2017/09/07 2002 [cited 2017/09/07]; Available from: http://www.ipcc-nggip.iges.or.jp/public/gp/bgp/5_1_CH4_Solid_Waste.pdf.

11. Mata-Alvarez, J., S. Macé, and P. Llabrés, Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresour. Technol., 2000. 74.

12. Strange, K., Overview of waste management options: their efficacy and acceptability. Issues in Environmental Science and Technology, 2002. 18: p. 1-52.

13. Kreith, F. and F. Kreith, Handbook of solid waste management. 1999. 14. FAOSTAT, Live animals. . 2013: Food and Agriculture Organization of the United Nations. FAO

Statistics Division 2016 . http://faostat.fao.org/site/573/DesktopDefault.aspx?PageID=573#ancor Accessed 2016/02/18.

15. Matikevičienė, V., D. Masiliūnienė, and S. Grigiškis. Degradation of keratin containing wastes by bacteria with keratinolytic activity. in Environment. Technology. Resources. Proceedings of the International Scientific and Practical Conference. 2015.

16. Suh, H. and H. Lee, Characterization of a Keratinolytic Serine Protease from Bacillus subtilis KS-1. Journal of Protein Chemistry, 2001. 20(2): p. 165-169.

17. Daroit, D.J., A.P.F. Corrêa, and A. Brandelli, Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus. Int. Biodeter. Biodegr., 2009. 63(3): p. 358-363.

18. Angelidaki, I. and W.T.M. Sanders, Assessment of the anaerobic biodegradability of macropollutants. Rev. Environ. Sci. Biotechnol., 2004. 3(2): p. 117-129.

62

19. Properties of Macromolecules I-Proteins. Accessed 2017/07/15. [cited; Available from: https://wikispaces.psu.edu/display/Biol230WCE/Properties+of+Macromolecules+I-Proteins.

20. USEPA, US Environmental Protention Agency. Inventory of U.S. Greenhouse Gas Emissions and Sinks: 1990 - 2005 2007.

21. Lukehurst, C.T., P. Frost, and T. Al Seadi, Utilisation of digestate from biogas plants as biofertiliser. IEA bioenergy, 2010: p. 1-36.

22. Risser, P.G., Agricultural and Forestry Residues, in Biomass Conversion Processes for Energy and Fuels, S.S. Sofer and O.R. Zaborsky, Editors. 1981, Springer US: Boston, MA. p. 25-47.

23. Zhang, Y.H.P., Reviving the carbohydrate economy via multi-product lignocellulose biorefineries. J. Ind. Microbiol. Biotech., 2008. 35(5): p. 367-375.

24. Himmel, M.E. and S.K. Picataggio, Our Challenge is to Acquire Deeper Understanding of Biomass Recalcitrance and Conversion, in Biomass Recalcitrance. 2009, Blackwell Publishing Ltd. p. 1-6.

25. Taherzadeh, M.J. and A. Jeihanipour, Recalcitrance of Lignocellulosic Biomass to Anaerobic Digestion, in Biogas Production: Pretreatments for enhanced Anaerobic Technology A. Mudhoo, Editor. 2012. p. 27 - 54.

26. Plomion, C., G. Leprovost, and A. Stokes, Wood Formation in Trees. Plant Physiology, 2001. 127(4): p. 1513-1523.

27. Hoornweg, D. and P. Bhada-Tata, What a Waste: A Global Review of Solid Waste Management. Urban Development Series. Urban Development & Local Government Unit. World Bank in Urban Development Series 2012.

28. Holtzapple, M.T., et al., Pretreatment of lignocellulosic municipal solid waste by ammonia fiber explosion (AFEX). Appl. Biochem. Biotechnol., 1992. 34(1): p. 5-21.

29. Chen, Y., J.J. Cheng, and K.S. Creamer, Inhibition of anaerobic digestion process: A review. Bioresour. Technol., 2008. 99(10): p. 4044-4064.

30. Wikandari, R., et al., Inhibitory effects of fruit flavors on methane production during anaerobic digestion. Bioresour. Technol., 2013. 145: p. 188-192.

31. Curry, N. and P. Pillay, Biogas prediction and design of a food waste to energy system for the urban environment. Renewable Energy, 2012. 41: p. 200-209.

32. Baker, E. and J.M. Keisler, Cellulosic biofuels: Expert views on prospects for advancement. Energy, 2011. 36(1): p. 595-605.

33. Schnürer, A. and A. Jarvis, Microbiological Handbook for Biogas Plants. Swedish Waste Management U2009:03 2010. p. 1 - 138.

34. Vintila, T., S. Neo, and C. Vintilă, Biogas Production Potential from Waste in Timis County. Animal Science and Biotechnologies, 2012. 45(1): p. 366 - 373.

35. Sárvári Horváth, I., et al., Recent updates on biogas production - a review. Biofuel Research Journal 2016. 10: p. 394-402.

36. Friehe, J., P. Weiland, and A. Schattauer, Guide to Biogas From production to use c.r.e. 5th, Gülzow, 2010 Editor. 2010.

37. Themelis, N.J. and Y.H. Kim, Material and energy balances in a large-scale aerobic bioconversion cell. Waste management & research, 2002. 20(3): p. 234-242.

38. Madigan, M., et al., Brock biology of microorganisms. 14th ed. Vol. 11. 2015: Pearson Education. 4.

39. Kothari, R., et al., Different aspects of dry anaerobic digestion for bio-energy: An overview. Renew. Sust. Energ. Rev., 2014. 39: p. 174-195.

40. Drosg, B., Process Monitoring in Biogas Plants. IEA Bioenergy 978-1-910154-03-8. 2013: IEA Bioenergy 978-1-910154-03-8. p. 1-38.

41. Bowen, E.J., et al., Low-temperature limitation of bioreactor sludge in anaerobic treatment of domestic wastewater. Water Science and Technology, 2014. 69(5): p. 1004-1013.

42. Wu, M.-C., K.-W. Sun, and Y. Zhang, Influence of temperature fluctuation on thermophilic anaerobic digestion of municipal organic solid waste. Journal of Zhejiang University. Science. B, 2006. 7(3): p. 180-185.

63

43. Navickas, K., et al., Influence of temperature variation on biogas yield from industrial wastes and energy plants. Engineering for Rural Development, 2013: p. 405-410.

44. Song, Y.-C., S.-J. Kwon, and J.-H. Woo, Mesophilic and thermophilic temperature co-phase anaerobic digestion compared with single-stage mesophilic- and thermophilic digestion of sewage sludge. Water Research, 2004. 38(7): p. 1653-1662.

45. Mao, C., et al., Review on research achievements of biogas from anaerobic digestion. Renewable and Sustainable Energy Reviews, 2015. 45: p. 540-555.

46. Pham, C.H., et al., Factors Affecting Process Temperature and Biogas Production in Small-scale Rural Biogas Digesters in Winter in Northern Vietnam. Asian-Australasian Journal of Animal Sciences, 2014. 27(7): p. 1050-1056.

47. Verma, S., Anaerobic digestion of biodegradable organics in municipal solid wastes. 2002, Columbia University.

48. Krich, K., et al., Production of Biogas by Anaerobic Digestion in Biomethane from Dairy Waste: A Sourcebook for the Production and Use of Renewable Natural Gas in California 2005. p. 29 - 46.

49. Pollard, A.M. and R.E. Blanchard. A hybrid biogas system for Kolkata. in Non Conventional Energy (ICONCE), 2014 1st International Conference on. 2014.

50. Friehe, J., P. Weiland, and A. Schattauer, Fundamentals of anaerobic digestion. in: Guide to Biogas From Production to Use. Fachagentur Nachwachsende Rohstoffe e. V. (FNR). 5th revised edition. . 2010.

51. Angelidaki, I., et al., Defining the biomethane potential (BMP) of solid organic wastes and energy crops: A proposed protocol for batch assays. Water Science & Technology, 2009. 59(5): p. 927 - 934.

52. Nielfa, A., R. Cano, and M. Fdz-Polanco, Theoretical methane production generated by the co-digestion of organic fraction municipal solid waste and biological sludge. Biotechnology Reports, 2015. 5: p. 14-21.

53. Angelidaki, I., et al., Defining the biomethane potential (BMP) of solid organic wastes and energy crops: A proposed protocol for batch assays. Water Sci. Technol., 2009. 59(5): p. 927 - 934.

54. Bachmann, N., Sustainable biogas production in municipal wastewater treatment plants. 2015.

55. Zupančič, G.D. and M. Roš, Determination of Chemical Oxygen Demand in Substrates from Anaerobic Treatment of Solid Organic Waste. Waste Biomass Valor., 2012. 3: p. 89–98.

56. Raposo, F., et al., Anaerobic digestion of solid organic substrates in batch mode: An overview relating to methane yields and experimental procedures. Renew. Sust. Energ. Rev., 2012. 16(1): p. 861-877.

57. Nielfa, A., R. Cano, and M. Fdz-Polanco, Theoretical methane production generated by the co-digestion of organic fraction municipal solid waste and biological sludge. Biotechnol. Rep., 2015. 5: p. 14-21.

58. Tarvin, D. and A.M. Buswell, The Methane Fermentation of Organic Acids and Carbohydrates1,2. J. Am. Chem. Soc., 1934. 56(8): p. 1751-1755.

59. Barjenbruch, M., et al., Minimizing of foaming in digesters by pre-treatment of the surplus-sludge. Wat Sci Tech, 2000. 42.

60. Hanaki, K., T. Matsuo, and M. Nagase, Mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process. Biotechnology and Bioengineering, 1981. 23(7): p. 1591-1610.

61. Angelidaki, I. and B.K. Ahring, Effects of free long-chain fatty acids on thermophilic anaerobic digestion. Applied Microbiology and Biotechnology, 1992. 37(6): p. 808-812.

62. Dasa, K.T., et al., Inhibitory Effect of Long-Chain Fatty Acids on Biogas Production and the Protective Effect of Membrane Bioreactor. BioMed Research International, 2016. 2016: p. 9.

63. Motte, J.-C., et al., Total solids content: a key parameter of metabolic pathways in dry anaerobic digestion. Biotechnology for Biofuels, 2013. 6: p. 164-164.

63

43. Navickas, K., et al., Influence of temperature variation on biogas yield from industrial wastes and energy plants. Engineering for Rural Development, 2013: p. 405-410.

44. Song, Y.-C., S.-J. Kwon, and J.-H. Woo, Mesophilic and thermophilic temperature co-phase anaerobic digestion compared with single-stage mesophilic- and thermophilic digestion of sewage sludge. Water Research, 2004. 38(7): p. 1653-1662.

45. Mao, C., et al., Review on research achievements of biogas from anaerobic digestion. Renewable and Sustainable Energy Reviews, 2015. 45: p. 540-555.

46. Pham, C.H., et al., Factors Affecting Process Temperature and Biogas Production in Small-scale Rural Biogas Digesters in Winter in Northern Vietnam. Asian-Australasian Journal of Animal Sciences, 2014. 27(7): p. 1050-1056.

47. Verma, S., Anaerobic digestion of biodegradable organics in municipal solid wastes. 2002, Columbia University.

48. Krich, K., et al., Production of Biogas by Anaerobic Digestion in Biomethane from Dairy Waste: A Sourcebook for the Production and Use of Renewable Natural Gas in California 2005. p. 29 - 46.

49. Pollard, A.M. and R.E. Blanchard. A hybrid biogas system for Kolkata. in Non Conventional Energy (ICONCE), 2014 1st International Conference on. 2014.

50. Friehe, J., P. Weiland, and A. Schattauer, Fundamentals of anaerobic digestion. in: Guide to Biogas From Production to Use. Fachagentur Nachwachsende Rohstoffe e. V. (FNR). 5th revised edition. . 2010.

51. Angelidaki, I., et al., Defining the biomethane potential (BMP) of solid organic wastes and energy crops: A proposed protocol for batch assays. Water Science & Technology, 2009. 59(5): p. 927 - 934.

52. Nielfa, A., R. Cano, and M. Fdz-Polanco, Theoretical methane production generated by the co-digestion of organic fraction municipal solid waste and biological sludge. Biotechnology Reports, 2015. 5: p. 14-21.

53. Angelidaki, I., et al., Defining the biomethane potential (BMP) of solid organic wastes and energy crops: A proposed protocol for batch assays. Water Sci. Technol., 2009. 59(5): p. 927 - 934.

54. Bachmann, N., Sustainable biogas production in municipal wastewater treatment plants. 2015.

55. Zupančič, G.D. and M. Roš, Determination of Chemical Oxygen Demand in Substrates from Anaerobic Treatment of Solid Organic Waste. Waste Biomass Valor., 2012. 3: p. 89–98.

56. Raposo, F., et al., Anaerobic digestion of solid organic substrates in batch mode: An overview relating to methane yields and experimental procedures. Renew. Sust. Energ. Rev., 2012. 16(1): p. 861-877.

57. Nielfa, A., R. Cano, and M. Fdz-Polanco, Theoretical methane production generated by the co-digestion of organic fraction municipal solid waste and biological sludge. Biotechnol. Rep., 2015. 5: p. 14-21.

58. Tarvin, D. and A.M. Buswell, The Methane Fermentation of Organic Acids and Carbohydrates1,2. J. Am. Chem. Soc., 1934. 56(8): p. 1751-1755.

59. Barjenbruch, M., et al., Minimizing of foaming in digesters by pre-treatment of the surplus-sludge. Wat Sci Tech, 2000. 42.

60. Hanaki, K., T. Matsuo, and M. Nagase, Mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process. Biotechnology and Bioengineering, 1981. 23(7): p. 1591-1610.

61. Angelidaki, I. and B.K. Ahring, Effects of free long-chain fatty acids on thermophilic anaerobic digestion. Applied Microbiology and Biotechnology, 1992. 37(6): p. 808-812.

62. Dasa, K.T., et al., Inhibitory Effect of Long-Chain Fatty Acids on Biogas Production and the Protective Effect of Membrane Bioreactor. BioMed Research International, 2016. 2016: p. 9.

63. Motte, J.-C., et al., Total solids content: a key parameter of metabolic pathways in dry anaerobic digestion. Biotechnology for Biofuels, 2013. 6: p. 164-164.

64

64. Taherzadeh, M.J. and K. Karimi, Pretreatment of Lignocellulosic Wastes to Improve Ethanol and Biogas Production: A Review. Int. J. Mol. Sci., 2008. 9(9): p. 1621-1651.

65. Johnson, D.K. and R.T. Elander, Pretreatments for Enhanced Digestibility of Feedstocks, in Biomass Recalcitrance. 2009, Blackwell Publishing Ltd. p. 436-453.

66. Patinvoh, R.J., et al., Biological Pretreatment of Chicken Feather and Biogas Production from Total Broth. Applied Biochemistry and Biotechnology, 2016. 180(7): p. 1401-1415.

67. DaSilva, E.J., Biogas generation: developments, problems, and tasks - an overview, in Division of Scientific Research and Higher Education,: Unesco, Paris, France.

68. Youngsukkasem, S., et al., Biogas production by encased bacteria in synthetic membranes: protective effects in toxic media and high loading rates. Environ. Technol., 2013. 34(13-14): p. 2077-2084.

69. Vardar-Sukan, F., Foaming: Consequences, prevention and destruction. Biotechnology Advances, 1998. 16(5): p. 913-948.

70. Ganidi, N., S. Tyrrel, and E. Cartmell, Anaerobic digestion foaming causes – a review. Bioresource Technol, 2009. 100.

71. Brown, S. and R. Sale, Operating a High-Rate Digester: The Southern Water Experience. Water and Environment Journal, 2002. 16(2): p. 116-120.

72. Moen, G., Anaerobic digester foaming: Causes and solutions, in Water Environment and Technology. 2003, Water Environment Federation: Alexandria. p. 70-73.

73. Scardina, P. and M. Edwards, Fundamentals of Bubble Formation during Coagulation and Sedimentation Processes. Journal of Environmental Engineering, 2006. 132(6): p. 575-585.

74. Ganidi, N., S. Tyrrel, and E. Cartmell, The effect of organic loading rate on foam initiation during mesophilic anaerobic digestion of municipal wastewater sludge. Bioresource Technol, 2011. 102.

75. Tanimu, M.I., et al., Effects of feedstock carbon to nitrogen ratio and organic loading on foaming potential in mesophilic food waste anaerobic digestion. Applied Microbiology and Biotechnology, 2015. 99(10): p. 4509-4520.

76. Lossie, U. and P. Pütz, Targeted control of biogas plants with the help of FOS/TAC. Practice Report, Laboratory Analysis Titration FOS/TAC. HACH LANGE in Laboratory Analysis Titration FOS/TAC. 2008. p. pp 1-4.

77. Gallert, C. and J. Winter, Propionic acid accumulation and degradation during restart of a full-scale anaerobic biowaste digester. Bioresource technology, 2008. 99(1): p. 170-178.

78. Patinvoh, R.J., et al., Dry fermentation of manure with straw in continuous plug flow reactor: Reactor development and process stability at different loading rates. Bioresour. Technol., 2017. 224: p. 197-205.

79. Lettinga, G., Anaerobic digestion and wastewater treatment systems. Antonie van leeuwenhoek, 1995. 67(1): p. 3-28.

80. Karthikeyan, O.P. and C. Visvanathan, Bio-energy recovery from high-solid organic substrates by dry anaerobic bio-conversion processes: a review. Reviews in Environmental Science and Bio/Technology, 2013. 12(3): p. 257-284.

81. Kardos, L., et al., Comparing of mesophilic and thermophilic anaerobic fermented sewage sludge based on chemical and biochemical tests. Applied Ecology and Environmental Research, 2011. 9(3): p. 293-302.

82. Rodríguez-Gómez, R., et al., A model to describe the performance of the UASB reactor. Biodegradation, 2014. 25(2): p. 239-251.

83. Abbasi, T. and S.A. Abbasi, Formation and impact of granules in fostering clean energy production and wastewater treatment in upflow anaerobic sludge blanket (UASB) reactors. Renewable and Sustainable Energy Reviews, 2012. 16(3): p. 1696-1708.

84. Boe, K. and I. Angelidaki, Serial CSTR digester configuration for improving biogas production from manure. Water Research, 2009. 43(1): p. 166-172.

65

85. Morel, A. and S. Diener, Greywater Management in Low and Middle-Income Countries, Review of Different Treatment Systems for Households or Neighbourhoods. . 2006: Dübendorf, Switzerland.

86. Kossmann, W., et al., Biogas Digest, Volume II–Biogas–Application and Product Development. Information and Advisory Service on Appropriate Technology, Eschborn, 1999.

87. Rajendran, K., et al., Experimental and economical evaluation of a novel biogas digester. Energy Convers. Manage., 2013. 74: p. 183-191.

88. Nazir, M., Biogas plants construction technology for rural areas. Bioresource Technology, 1991. 35(3): p. 283-289.

89. Nzila, C., et al., Multi criteria sustainability assessment of biogas production in Kenya. Applied Energy, 2012. 93: p. 496-506.

90. Rakotojaona, L., Domestic biogas development in developing countries http://www.enea-consulting.com/wp-content/uploads/2015/05/Open-Ideas-Domestic-biogas-projects-in-developing-countries.pdf Accessed 2017/08/27. 2013.

91. Cheng, S., et al., A review of prefabricated biogas digesters in China. Renewable and Sustainable Energy Reviews, 2013. 28: p. 738-748.

92. bank, T.W. Population [cited 2017/08/17]; Available from: http://data.worldbank.org/indicator/SP.POP.TOTL?locations=NG

93. bank, T.W. GDP (Gross domestic product) [cited 2017/08/17]; Available from: http://data.worldbank.org/country/nigeria?view=chart.

94. Atlas. The disparity in connections and reliable power supply in Africa. Africa, electricity [cited 2017/08/21]; Available from: https://www.theatlas.com/charts/HJ3w68Gl.

95. WorldBank, The Little Green Data Book. 2015: Washington, DC: World Bank. doi:10.1596/978-1-4648-0560-8. License: Creative Commons Attribution CC BY 3.0 IGO.

96. Ngumah, C.C., et al., Biogas potential of organic waste in Nigeria. Journal of Urban and Environmental Engineering, 2013. 7(1).

97. Akinbomi, J., et al., Development and dissemination strategies for accelerating biogas production in Nigeria. BioResources, 2014. 9(3): p. 5707-5737.

98. Taherzadeh, M.J. and K. Rajendran, Factors affecting development of waste management. Waste Management and Sustainable Consumption: Reflections on Consumer Waste, 2014: p. 67.

99. ECN and UNDP, Renewable Energy Master Plan. Final Draft Report. Energy Commission of Nigeria (ECN), United Nations Development Programme (UNDP) 2005.

100. Dahlquist, E., Technologies for Converting Biomass to Useful Energy: Combustion, Gasification, Pyrolysis, Torrefaction and Fermentation. 2013: Taylor & Francis.

101. Cows to Kilowatts. 2005 [cited 2017/08/21]; Available from: https://www.seed.uno/images/documents/461/W05_Nigeria_Cows%20to%20kilowatts.pdf.

102. Cows to Kilowatts, Nigeria: Turning Waste into Energy and Fertiliser Innovative for Sustainable Development: Local Case Studies from Africa [cited 2017/08/18]; Available from: http://www.un.org/esa/sustdev/publications/africa_casestudies/kilowatts.pdf.

103. Biogas - NASRUN Nig. Ltd. [cited 2017/08/15]; Available from: http://www.nasrun.safsms.com/?p=218.

104. Dioha, E. and B. Nfor, Operating and Financing a Family Biogas Plant. Journal of Energy Technologies and Policy, 2012. 2(5): p. 25-37.

105. Held, J., Anders Mathiasson, and Anders Nylander, Biogas for energy and the environment. 2008: Swedish Gas Centre, Swedish Gas Association and Swedish Biogas Association. Stockholm, Sweden. .

106. Al Seadi, T., Danish centralised biogas plants–plant descriptions. Bioenergy Department, University of Southern Denmark, 2000: p. 28.

107. Geels, F.W. and R. Raven, Socio-cognitive evolution and co-evolution in competing technical trajectories: biogas development in Denmark (1970–2002). The International Journal of Sustainable Development & World Ecology, 2007. 14(1): p. 63-77.

65

85. Morel, A. and S. Diener, Greywater Management in Low and Middle-Income Countries, Review of Different Treatment Systems for Households or Neighbourhoods. . 2006: Dübendorf, Switzerland.

86. Kossmann, W., et al., Biogas Digest, Volume II–Biogas–Application and Product Development. Information and Advisory Service on Appropriate Technology, Eschborn, 1999.

87. Rajendran, K., et al., Experimental and economical evaluation of a novel biogas digester. Energy Convers. Manage., 2013. 74: p. 183-191.

88. Nazir, M., Biogas plants construction technology for rural areas. Bioresource Technology, 1991. 35(3): p. 283-289.

89. Nzila, C., et al., Multi criteria sustainability assessment of biogas production in Kenya. Applied Energy, 2012. 93: p. 496-506.

90. Rakotojaona, L., Domestic biogas development in developing countries http://www.enea-consulting.com/wp-content/uploads/2015/05/Open-Ideas-Domestic-biogas-projects-in-developing-countries.pdf Accessed 2017/08/27. 2013.

91. Cheng, S., et al., A review of prefabricated biogas digesters in China. Renewable and Sustainable Energy Reviews, 2013. 28: p. 738-748.

92. bank, T.W. Population [cited 2017/08/17]; Available from: http://data.worldbank.org/indicator/SP.POP.TOTL?locations=NG

93. bank, T.W. GDP (Gross domestic product) [cited 2017/08/17]; Available from: http://data.worldbank.org/country/nigeria?view=chart.

94. Atlas. The disparity in connections and reliable power supply in Africa. Africa, electricity [cited 2017/08/21]; Available from: https://www.theatlas.com/charts/HJ3w68Gl.

95. WorldBank, The Little Green Data Book. 2015: Washington, DC: World Bank. doi:10.1596/978-1-4648-0560-8. License: Creative Commons Attribution CC BY 3.0 IGO.

96. Ngumah, C.C., et al., Biogas potential of organic waste in Nigeria. Journal of Urban and Environmental Engineering, 2013. 7(1).

97. Akinbomi, J., et al., Development and dissemination strategies for accelerating biogas production in Nigeria. BioResources, 2014. 9(3): p. 5707-5737.

98. Taherzadeh, M.J. and K. Rajendran, Factors affecting development of waste management. Waste Management and Sustainable Consumption: Reflections on Consumer Waste, 2014: p. 67.

99. ECN and UNDP, Renewable Energy Master Plan. Final Draft Report. Energy Commission of Nigeria (ECN), United Nations Development Programme (UNDP) 2005.

100. Dahlquist, E., Technologies for Converting Biomass to Useful Energy: Combustion, Gasification, Pyrolysis, Torrefaction and Fermentation. 2013: Taylor & Francis.

101. Cows to Kilowatts. 2005 [cited 2017/08/21]; Available from: https://www.seed.uno/images/documents/461/W05_Nigeria_Cows%20to%20kilowatts.pdf.

102. Cows to Kilowatts, Nigeria: Turning Waste into Energy and Fertiliser Innovative for Sustainable Development: Local Case Studies from Africa [cited 2017/08/18]; Available from: http://www.un.org/esa/sustdev/publications/africa_casestudies/kilowatts.pdf.

103. Biogas - NASRUN Nig. Ltd. [cited 2017/08/15]; Available from: http://www.nasrun.safsms.com/?p=218.

104. Dioha, E. and B. Nfor, Operating and Financing a Family Biogas Plant. Journal of Energy Technologies and Policy, 2012. 2(5): p. 25-37.

105. Held, J., Anders Mathiasson, and Anders Nylander, Biogas for energy and the environment. 2008: Swedish Gas Centre, Swedish Gas Association and Swedish Biogas Association. Stockholm, Sweden. .

106. Al Seadi, T., Danish centralised biogas plants–plant descriptions. Bioenergy Department, University of Southern Denmark, 2000: p. 28.

107. Geels, F.W. and R. Raven, Socio-cognitive evolution and co-evolution in competing technical trajectories: biogas development in Denmark (1970–2002). The International Journal of Sustainable Development & World Ecology, 2007. 14(1): p. 63-77.

66

108. Lönnqvist, T., Biogas in Swedish transport–a policy-driven systemic transition. 2017, KTH Royal Institute of Technology.

109. Li, Y., S.Y. Park, and J. Zhu, Solid-state anaerobic digestion for methane production from organic waste. Renew. Sustainable Energy Rev., 2011. 15(1): p. 821-826.

110. Bolzonella, D., et al., Semi-dry thermophilic anaerobic digestion of the organic fraction of municipal solid waste: focusing on the start-up phase. Bioresource technology, 2003. 86(2): p. 123-129.

111. Yi, J., et al., Effect of Increasing Total Solids Contents on Anaerobic Digestion of Food Waste under Mesophilic Conditions: Performance and Microbial Characteristics Analysis. PLoS ONE, 2014. 9(7): p. e102548.

112. Watkins, K., Human Development Report 2006-Beyond scarcity: Power, poverty and the global water crisis http://www.undp.org/content/dam/undp/library/corporate/HDR/2006%20Global%20HDR/HDR-2006-Beyond%20scarcity-Power-poverty-and-the-global-water-crisis.pdf Accessed 2017/09/07. 2006.

113. Karthikeyan, O.P. and C. Visvanathan, Bio-energy recovery from high-solid organic substrates by dry anaerobic bio-conversion processes: a review. Rev. Environ. Sci. Bio/Technol., 2013. 12(3): p. 257-284.

114. Rapport, J., et al., Current anaerobic digestion technologies used for treatment of municipal organic solid waste. 2008, Sacramento, CA: California Environmental Protection Agency.

115. Li, A., et al., A pyrosequencing-based metagenomic study of methane-producing microbial community in solid-state biogas reactor. Biotechnology for biofuels, 2013. 6(1): p. 3.

116. Di Maria, F., et al., Solid anaerobic digestion batch with liquid digestate recirculation and wet anaerobic digestion of organic waste: Comparison of system performances and identification of microbial guilds. Waste Management, 2017. 59: p. 172-180.

117. Li, A., et al., A pyrosequencing-based metagenomic study of methane-producing microbial community in solid-state biogas reactor. Biotechnol Biofuels, 2013. 6.

118. Vandevivere, P., L. De Baere, and W. Verstraete, Types of anaerobic digester for solid wastes, in Biomethanization of the organic fraction of municipal solid wastes. 2003, Iwa Publishing. p. 111-140.

119. Pembrey, R.S., K.C. Marshall, and R.P. Schneider, Cell surface analysis techniques: what do cell preparation protocols do to cell surface properties? Applied and Environmental Microbiology, 1999. 65(7): p. 2877-2894.

120. Kusch, S. Understanding and Managing the Start-up Phase in Dry Anaerobic Digestion. in Proceedings in Research Conference in Technical Disciplines. 2013.

121. Samer, M., Biogas plant constructions. Cairo University, Faculty of Agriculture, Department of Agricultural Engineering,Egypt, in Biogas. 2012, InTech.

122. Innovative Solutions for Cities and Agriculture. BEKON Energy Technologies GmbH & Co. KG., in Bioenergy via Dry Fermentation http://www.cityofpaloalto.org/civicax/filebank/documents/19875 Accessed 2014/03/03.

123. Kusch, S., H. Oechsner, and T. Jungbluth, Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates. International Journal of Environment and Waste Management, 2012. 9(1-2): p. 69-88.

124. Tyrberg, L. VMAB Biogas at Mörrum waste treatment plant in Mörrum, Karlshamn municipality. http://www.res-chains.eu/wp-content/uploads/2014/09/M%C3%B6rrum-biogas-final.pdf Accessed 2017/07/29 2013 [cited; Available from: http://www.res-chains.eu/wp-content/uploads/2014/09/M%C3%B6rrum-biogas-final.pdf

125. Baere, L.D. The DRANCO technology: a unique digestion technology for solid organic wastes. http://www.ows.be/wp-content/uploads/2013/02/The-DRANCO-technology-2012.pdf Accessed 2017/09/16 2012 [cited.

126. GmbH, S.U. Dry digestion. STRABAG Umweltanlagen GmbH [cited 2017/09/16]; Available from: http://www.strabag-

67

umwelttechnik.com/databases/internet/_public/files.nsf/SearchView/A9C0D56F88B4274EC125774600504FB5/$File/3_4%20Trockenvergaerung_e%20d.pdf.

127. Montgomery, L.F.R. and G. Bochmann, Pretreatment of feedstock for enhanced biogas production. IEA Bioenergy, 2014: p. 1-20.

128. Lindner, J., et al., Effects of mechanical treatment of digestate after anaerobic digestion on the degree of degradation. Bioresour. Technol., 2015. 178: p. 194-200.

129. Bochmann, G., et al., 4 - Storage and pre-treatment of substrates for biogas production A2 - Wellinger, Arthur, in The Biogas Handbook. 2013, Woodhead Publishing. p. 85-103.

130. Papadimitriou, E.K., Hydrolysis of organic matter during autoclaving of commingled household waste. Waste Management, 2010. 30(4): p. 572-582.

131. Tampio, E., et al., Anaerobic digestion of autoclaved and untreated food waste. Waste Management, 2014. 34(2): p. 370-377.

132. Forgács, G., et al., Methane Production from Feather Waste Pretreated with Ca(OH)2: Process Development and Economical Analysis. Waste and Biomass Valorization, 2014. 5(1): p. 65-73.

133. Taherzadeh, M.J., et al., Characterization and Fermentation of Dilute-Acid Hydrolyzates from Wood. Ind. Eng. Chem. Res., 1997. 36(11): p. 4659-4665.

134. Bolado-Rodríguez, S., et al., Effect of thermal, acid, alkaline and alkaline-peroxide pretreatments on the biochemical methane potential and kinetics of the anaerobic digestion of wheat straw and sugarcane bagasse. Bioresour. Technol., 2016. 201: p. 182-190.

135. Xiao, W. and W.W. Clarkson, Acid solubilization of lignin and bioconversion of treated newsprint to methane. Biodegradation, 1997. 8(1): p. 61-66.

136. Adney, W.S., et al., Understanding the Biomass Decay Community, in Biomass Recalcitrance. 2009, Blackwell Publishing Ltd. p. 454-479.

137. Hatakka, A.I., Pretreatment of wheat straw by white-rot fungi for enzymic saccharification of cellulose. Eur. J. Appl. Microbiol. Biotechnol., 1983. 18(6): p. 350-357.

138. Hatakka, A., Lignin-modifying enzymes from selected white-rot fungi: Production and role from in lignin degradation. FEMS Microbiol Rev, 1994. 13.

139. Mutschlechner, M., P. Illmer, and A.O. Wagner, Biological pre-treatment: Enhancing biogas production using the highly cellulolytic fungus Trichoderma viride. Waste Management, 2015. 43: p. 98-107.

140. Cai, C.-g., et al., Purification and characterization of keratinase from a new Bacillus subtilis strain. Journal of Zhejiang University. Science. B, 2008. 9(9): p. 713-720.

141. Fellahi, S., et al., A Bacillus Strain Able to Hydrolyze Alpha- and Beta-Keratin. J. Bioprocess. Biotech., 2014. 4(7): p. 7.

142. Forgács, G., et al., Biological treatment of chicken feather waste for improved biogas production. J. Environ. Sci., 2011. 23(10): p. 1747-1753.

143. Zaghloul, T.I., A.M. Embaby, and A.R. Elmahdy, Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: Scaling up in a laboratory scale fermentor. Bioresour. Technol., 2011. 102(3): p. 2387-2393.

144. El-Shora, H.M., S.A. Ashour, and A.A. Ghanem, Purification and characterization of keratinase from B. circulans, B. licheniformis, and B. megaterium. Journal of Environmental Sciences, 1993. 6: p. 89-103.

145. Mazotto, A.M., et al., Degradation of feather waste by Aspergillus niger keratinases: Comparison of submerged and solid-state fermentation. Inter. Biodeter. Biodegr., 2013. 85: p. 189-195.

146. Hirschman, D.J., J.M. Zametkin, and R.E. Rogers, The utilization of wool by four saprophytic microorganisms in the presence of added nutrients. Am. Dyestuff Reptr, 1944. 33: p. 353-359.

147. Luque, R., et al., Handbook of Biofuels Production. 2016: Elsevier Science. 148. Amarasekara, A.S., Handbook of Cellulosic Ethanol. 2013: Wiley. 149. Mata-Alvarez, J., et al., A critical review on anaerobic co-digestion achievements between

2010 and 2013. Renew. Sust. Energ. Rev., 2014. 36: p. 412-427.

67

umwelttechnik.com/databases/internet/_public/files.nsf/SearchView/A9C0D56F88B4274EC125774600504FB5/$File/3_4%20Trockenvergaerung_e%20d.pdf.

127. Montgomery, L.F.R. and G. Bochmann, Pretreatment of feedstock for enhanced biogas production. IEA Bioenergy, 2014: p. 1-20.

128. Lindner, J., et al., Effects of mechanical treatment of digestate after anaerobic digestion on the degree of degradation. Bioresour. Technol., 2015. 178: p. 194-200.

129. Bochmann, G., et al., 4 - Storage and pre-treatment of substrates for biogas production A2 - Wellinger, Arthur, in The Biogas Handbook. 2013, Woodhead Publishing. p. 85-103.

130. Papadimitriou, E.K., Hydrolysis of organic matter during autoclaving of commingled household waste. Waste Management, 2010. 30(4): p. 572-582.

131. Tampio, E., et al., Anaerobic digestion of autoclaved and untreated food waste. Waste Management, 2014. 34(2): p. 370-377.

132. Forgács, G., et al., Methane Production from Feather Waste Pretreated with Ca(OH)2: Process Development and Economical Analysis. Waste and Biomass Valorization, 2014. 5(1): p. 65-73.

133. Taherzadeh, M.J., et al., Characterization and Fermentation of Dilute-Acid Hydrolyzates from Wood. Ind. Eng. Chem. Res., 1997. 36(11): p. 4659-4665.

134. Bolado-Rodríguez, S., et al., Effect of thermal, acid, alkaline and alkaline-peroxide pretreatments on the biochemical methane potential and kinetics of the anaerobic digestion of wheat straw and sugarcane bagasse. Bioresour. Technol., 2016. 201: p. 182-190.

135. Xiao, W. and W.W. Clarkson, Acid solubilization of lignin and bioconversion of treated newsprint to methane. Biodegradation, 1997. 8(1): p. 61-66.

136. Adney, W.S., et al., Understanding the Biomass Decay Community, in Biomass Recalcitrance. 2009, Blackwell Publishing Ltd. p. 454-479.

137. Hatakka, A.I., Pretreatment of wheat straw by white-rot fungi for enzymic saccharification of cellulose. Eur. J. Appl. Microbiol. Biotechnol., 1983. 18(6): p. 350-357.

138. Hatakka, A., Lignin-modifying enzymes from selected white-rot fungi: Production and role from in lignin degradation. FEMS Microbiol Rev, 1994. 13.

139. Mutschlechner, M., P. Illmer, and A.O. Wagner, Biological pre-treatment: Enhancing biogas production using the highly cellulolytic fungus Trichoderma viride. Waste Management, 2015. 43: p. 98-107.

140. Cai, C.-g., et al., Purification and characterization of keratinase from a new Bacillus subtilis strain. Journal of Zhejiang University. Science. B, 2008. 9(9): p. 713-720.

141. Fellahi, S., et al., A Bacillus Strain Able to Hydrolyze Alpha- and Beta-Keratin. J. Bioprocess. Biotech., 2014. 4(7): p. 7.

142. Forgács, G., et al., Biological treatment of chicken feather waste for improved biogas production. J. Environ. Sci., 2011. 23(10): p. 1747-1753.

143. Zaghloul, T.I., A.M. Embaby, and A.R. Elmahdy, Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: Scaling up in a laboratory scale fermentor. Bioresour. Technol., 2011. 102(3): p. 2387-2393.

144. El-Shora, H.M., S.A. Ashour, and A.A. Ghanem, Purification and characterization of keratinase from B. circulans, B. licheniformis, and B. megaterium. Journal of Environmental Sciences, 1993. 6: p. 89-103.

145. Mazotto, A.M., et al., Degradation of feather waste by Aspergillus niger keratinases: Comparison of submerged and solid-state fermentation. Inter. Biodeter. Biodegr., 2013. 85: p. 189-195.

146. Hirschman, D.J., J.M. Zametkin, and R.E. Rogers, The utilization of wool by four saprophytic microorganisms in the presence of added nutrients. Am. Dyestuff Reptr, 1944. 33: p. 353-359.

147. Luque, R., et al., Handbook of Biofuels Production. 2016: Elsevier Science. 148. Amarasekara, A.S., Handbook of Cellulosic Ethanol. 2013: Wiley. 149. Mata-Alvarez, J., et al., A critical review on anaerobic co-digestion achievements between

2010 and 2013. Renew. Sust. Energ. Rev., 2014. 36: p. 412-427.

68

150. Gomez-Romero, J., et al., Selective adaptation of an anaerobic microbial community: Biohydrogen production by co-digestion of cheese whey and vegetables fruit waste. International Journal of Hydrogen Energy, 2014. 39(24): p. 12541-12550.

151. Liu, C., et al., Evolution of microbial community along with increasing solid concentration during high-solids anaerobic digestion of sewage sludge. Bioresour. technol., 2016. 216: p. 87-94.

152. Klang, J., et al., Dynamic variation of the microbial community structure during the long-time mono-fermentation of maize and sugar beet silage. Microbial Biotechnology, 2015. 8(5): p. 764-775.

153. Carucci, G., et al., Anaerobic digestion of food industry wastes: effect of codigestion on methane yield. Journal of Environmental Engineering, 2005. 131(7): p. 1037-1045.

154. Zeshan, O.P. Karthikeyan, and C. Visvanathan, Effect of C/N ratio and ammonia-N accumulation in a pilot-scale thermophilic dry anaerobic digester. Bioresource Technology, 2012. 113: p. 294-302.

155. Lindorfer, H., et al., Doubling the organic loading rate in the co-digestion of energy crops and manure – A full scale case study. Bioresour. Technol., 2008. 99(5): p. 1148-1156.

156. Lehtomäki, A., S. Huttunen, and J.A. Rintala, Laboratory investigations on co-digestion of energy crops and crop residues with cow manure for methane production: Effect of crop to manure ratio. Resources, Conservation and Recycling, 2007. 51(3): p. 591-609.

157. Patinvoh, R.J., et al., Cost effective dry anaerobic digestion in textile bioreactors: Experimental and economic evaluation. Bioresour. Technol. In Press, 2017.

158. Brown, D. and Y. Li, Solid state anaerobic co-digestion of yard waste and food waste for biogas production. Bioresource Technology, 2013. 127: p. 275-280.

159. Pagés Díaz, J., et al., Co-digestion of different waste mixtures from agro-industrial activities: Kinetic evaluation and synergetic effects. Bioresour. Technol., 2011. 102(23): p. 10834-10840.

160. Teghammar, A., et al., Improved Anaerobic Digestion by the Addition of Paper Tube Residuals: Pretreatment, Stabilizing, and Synergetic Effects. Energy Fuels, 2013. 27(1): p. 277-284.

161. Comino, E., M. Rosso, and V. Riggio, Investigation of increasing organic loading rate in the co-digestion of energy crops and cow manure mix. Bioresour. Technol., 2010. 101(9): p. 3013-3019.

162. Wang, W., et al., Biohydrogen and methane production by co-digestion of cassava stillage and excess sludge under thermophilic condition. Bioresour. Technol., 2011. 102(4): p. 3833-3839.

163. Debosz, K., et al., Evaluating effects of sewage sludge and household compost on soil physical, chemical and microbiological properties. Applied Soil Ecology, 2002. 19(3): p. 237-248.

164. Kupper, T., et al., Heavy metals in source-separated compost and digestates. Waste Management, 2014. 34(5): p. 867-874.

165. Govasmark, E., et al., Chemical and microbiological hazards associated with recycling of anaerobic digested residue intended for agricultural use. Waste management, 2011. 31(12): p. 2577-2583.

166. Risberg, K., Quality and function of anaerobic digestion residues. Doctoral thesis. Vol. 2015. 2015.

167. Al Seadi, T. and C. Lukehurst, Quality management of digestate from biogas plants used as fertiliser. IEA Bioenergy, 2012: p. 4-36.

168. Makádi, M., A. Tomócsik, and V. Orosz, Digestate: a new nutrient source–review. Energy, 2012. 4(7.5): p. 8.7.

169. Sahlström, L., A review of survival of pathogenic bacteria in organic waste used in biogas plants. Bioresource Technology, 2003. 87(2): p. 161-166.

170. Bendixen, H.J. Hygienic Safety–Results of scientific investigations in Denmark (Sanitation requirements in Danish biogas plants). in IEA BioEnergy Workshop: Hygienic and environmental aspects of AD: Legislation and experiences in Europe. 1999.

69

171. Odlare, M., M. Pell, and K. Svensson, Changes in soil chemical and microbiological properties during 4 years of application of various organic residues. Waste management, 2008. 28(7): p. 1246-1253.

172. Makádi, M., A. Tomócsik, and V. Orosz, Digestate: A New Nutrient Source - Review, in Biogas, D.S. Kumar, Editor. 2012, InTech.

173. Möller, K. and T. Müller, Effects of anaerobic digestion on digestate nutrient availability and crop growth: A review. Engineering in Life Sciences, 2012. 12(3): p. 242-257.

174. Möller, K., R. Schulz, and T. Müller, Substrate inputs, nutrient flows and nitrogen loss of two centralized biogas plants in southern Germany. Nutr. Cycl. Agroecosys., 2010. 87(2): p. 307-325.

175. Fouda, S.E.-S., Nitrogen availability of biogas residues. 2011, PhD Dissertation, Technical University of München. .

176. Poggi-Varaldo, H.M., et al., Quality of anaerobic compost from paper mill and municipal solid wastes for soil amendment. Water Sci. Technol., 1999. 40(11-12): p. 179-186.

177. Meissl, K. and E. Smidt, High quality composts by means of cocomposting of residues from anaerobic digestion. Compost Sci. Util., 2007. 15(2): p. 78-83.

178. Kompogas® dry anaerobic digestion Energy from organic waste. [cited 2017/09/16]; Available from: http://www.hz-inova.com/cms/wp-content/uploads/2015/06/HZI_Kompogas_10_engl_RZ_3_WEB.pdf.

179. Digestate in Practical guide to anaerobic digestion. [cited 2016/09/19]; Available from: http://adbioresources.org/wp-content/uploads/2013/06/81-92_chapter6_v3.pdf.

180. Frischmann, P., Enhancement and treatment of digestates from anaerobic digestion 2012. 181. Massé, D.I., F. Croteau, and L. Masse, The fate of crop nutrients during digestion of swine

manure in psychrophilic anaerobic sequencing batch reactors. Bioresource Technology, 2007. 98(15): p. 2819-2823.

69

171. Odlare, M., M. Pell, and K. Svensson, Changes in soil chemical and microbiological properties during 4 years of application of various organic residues. Waste management, 2008. 28(7): p. 1246-1253.

172. Makádi, M., A. Tomócsik, and V. Orosz, Digestate: A New Nutrient Source - Review, in Biogas, D.S. Kumar, Editor. 2012, InTech.

173. Möller, K. and T. Müller, Effects of anaerobic digestion on digestate nutrient availability and crop growth: A review. Engineering in Life Sciences, 2012. 12(3): p. 242-257.

174. Möller, K., R. Schulz, and T. Müller, Substrate inputs, nutrient flows and nitrogen loss of two centralized biogas plants in southern Germany. Nutr. Cycl. Agroecosys., 2010. 87(2): p. 307-325.

175. Fouda, S.E.-S., Nitrogen availability of biogas residues. 2011, PhD Dissertation, Technical University of München. .

176. Poggi-Varaldo, H.M., et al., Quality of anaerobic compost from paper mill and municipal solid wastes for soil amendment. Water Sci. Technol., 1999. 40(11-12): p. 179-186.

177. Meissl, K. and E. Smidt, High quality composts by means of cocomposting of residues from anaerobic digestion. Compost Sci. Util., 2007. 15(2): p. 78-83.

178. Kompogas® dry anaerobic digestion Energy from organic waste. [cited 2017/09/16]; Available from: http://www.hz-inova.com/cms/wp-content/uploads/2015/06/HZI_Kompogas_10_engl_RZ_3_WEB.pdf.

179. Digestate in Practical guide to anaerobic digestion. [cited 2016/09/19]; Available from: http://adbioresources.org/wp-content/uploads/2013/06/81-92_chapter6_v3.pdf.

180. Frischmann, P., Enhancement and treatment of digestates from anaerobic digestion 2012. 181. Massé, D.I., F. Croteau, and L. Masse, The fate of crop nutrients during digestion of swine

manure in psychrophilic anaerobic sequencing batch reactors. Bioresource Technology, 2007. 98(15): p. 2819-2823.

Biological pretreatment of chicken feather and biogas production from total broth. Applied Biochemistry and Biotechnology 180(7): 1401–1415.

Pap

er I

Biological Pretreatment of Chicken Feather and BiogasProduction from Total Broth

Regina J. Patinvoh1 & Elisabeth Feuk-Lagerstedt1 &

Magnus Lundin1 & Ilona Sárvári Horváth1 &

Mohammad J. Taherzadeh1

Received: 12 April 2016 /Accepted: 20 June 2016 /Published online: 27 June 2016# Springer Science+Business Media New York 2016

Abstract Chicken feathers are available in large quantities around theworld causing environmentalchallenges. The feathers are composed of keratin that is a recalcitrant protein and is hard to degrade.In this work, chicken feathers were aerobically pretreated for 2–8 days at total solid concentrations of5, 10, and 20 % by Bacillus sp. C4, a bacterium that produces both α- and β-keratinases. Then, theliquid fraction (feather hydrolysate) as well as the total broth (liquid and solid fraction of pretreatedfeathers) was used as substrates for biogas production using anaerobic sludge or bacteria granules asinoculum. The biological pretreatment of feather waste was productive; about 75 % of feather wasconverted to soluble crude protein after 8 days of degradation at initial feather concentration of 5%.Bacteria granules performed better during anaerobic digestion of untreated feathers, resulting inapproximately two times more methane yield (i.e., 199 mlCH4/gVS compared to 105 mlCH4/gVSwhen sludge was used). Pretreatment improved methane yield by 292 and 105 % when sludge andgranules were used on the hydrolysate. Bacteria granules worked effectively on the total broth,yielded 445 mlCH4/gVS methane, which is 124 % more than that obtained with the same type ofinoculum from untreated feather.

Keywords Chicken feather . Pretreatment . Bacillus substilis strain . Keratinase . Biogasproduction .Mesophilic . Hydrolysate . Total broth . Bacteria granules

Introduction

The world’s average stock of chicken is about 22 billion live chickens [1], and about 5–7% of thetotal weight of a normal chicken are feathers [2]. The poultry industries produce about six billiontons of chicken feathers annually. Landfilling is a major practice of disposing feather wastes in the

Appl Biochem Biotechnol (2016) 180:1401–1415DOI 10.1007/s12010-016-2175-8

* Regina J. [email protected]

1 Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden

world today, resulting in landfill gas including methane and nitrous oxides. The second largesttreatment of feather waste is incineration [3]; however, a minor portion of the feathers areconverted to feather meals used in animal feed preparation and in organic fertilizer throughhydrothermal or chemical pretreatment methods. Nevertheless, this product has a low nutritionalvalue due to the denaturing of the amino acid content [4, 5] and, moreover, the process is capitalintensive.

The organic fraction of solid organic wastes and materials (agricultural wastes, sludge, animalby products, energy crops, and other substrates) is used for biogas production, but the use ofkeratin wastes such as chicken feathers, wools, hair, or nails is not yet fully explored. Thesewastes, if properly pretreated, can serve as a valuable feedstock for biogas production. It isimportant to alter the recalcitrant structure of the keratin to enhance bio-digestibility of the wastesfor biogas production resulting in improved biogas yields [6]. The high degree of crosslinking ofthe polypeptide chain caused by extensive formation of disulfide bonds is a major challenge inprocessing these wastes. This strong covalent bondmakes them insoluble in polar solvents [7] andresistant to proteases [8, 9].

Current processing of these wastes is based on strong acid or alkaline hydrolysis and otherphysicochemical methods such as hydrothermal and superheated water treatments. These treat-ment methods result in severe degradation and destruction of feather keratins [10]; however, theenergy and/or chemicals demand related to these methods leads to economical or sustainabilitychallenges. Enzymatic hydrolysis using keratinases is also an option, but the high cost of enzymesmakes this method limited for industrial applications. Hence, the recent research focuses onbiological pretreatment methods, which are sustainable, ecological, and cost effective forextracting soluble keratins. Several microorganisms (bacteria and fungi), such as Bacillus [4,11, 12] and Aspergillus sp. [13–15] were reported to degrade keratins (chicken feathers). Hence,introducing these kinds of pretreatment can be promising and therefore there is a need to explorethese effectively for their use prior to biogas production. In a previous study [16], a recombinantBacillus megaterium strain was used for pretreatment of chicken feathers. An initial featherconcentration of 4 % was pretreated prior to biogas production and then only the hydrolysate ofthe pretreated feather was further investigated as a biogas substrate.

In this study, the goal was to use a wild strain of bacteria for the pretreatment, and then thewhole broth of the pretreated feathers including the bacteria was investigated as a substrate toproduce biogas, a concept that is closer to be applied in future industrial applications. Chickenfeathers at total solid concentrations of 5, 10, and 20 % (dry weight of feathers) were biologicallypretreated with a naturally occurring Bacillus strain and thereafter the total broth of pretreatedfeathers, as well as only the feather hydrolysate for a comparison, was used for biogas production.This protein-degrading bacteria was isolated from compost and identified as Bacillus sp. C4

(2008) [4]. Moreover, this paper assesses investigations at higher initial total solid concentrationsin order to minimize reactor volume and utilize larger amount of wastes. Additionally, the effectsof incubation time, as well as the type of the inoculum on the biogas yield were also evaluated.

Experimental

Biological Treatment of Chicken Feather

White chicken feathers were collected from a slaughterhouse (Håkantorp Slakteri AB,Genomfatrsvägen, Sweden). The feathers were washed in dilute soap solution immediately after

1402 Appl Biochem Biotechnol (2016) 180:1401–1415

collection and rinsed with tap water and then distilled water. The washed feathers were air-driedfor 3 days and then chopped into particle sizes between 1 and 10 mm, thereafter stored at roomtemperature before use.

The keratin-degrading bacteria used in this study was a Bacillus sp., isolated from compostand identified as Bacillus sp. C4 (2008) [4], stored at −80 °C in 50 % glycerol. The bacteriawere incubated at 37 °C for 18 h on agar plates containing 1.5 % bacteriological agar in Luria-Bertani broth. It was then inoculated in 100 ml of Luria-Bertani broth (tryptone 1 % (w/v),yeast extract 0.5 % (w/v), and NaCl 0.5 % (w/v)) and incubated in a water bath at 37 °C andshaking with 160 rpm overnight (18 h).

The feathers with different total solid concentrations of 5, 10, or 20 % (dry weight offeather) were mixed with 90, 84, or 73 ml, respectively, of a modified basal medium (NH4Cl0.5 g/l, NaCl 0.5 g/l, K2HPO4 0.5 g/l, KH2PO4 0.4 g/l, MgCl2.6H2O 0.1 g/l, yeast extract1.5 g/l, peptone 1 g/l), and the pH was adjusted to 7.0 using K2HPO4 or KH2PO4 (1 M) in 500-ml Erlenmeyer flasks, sterilized and inoculated with 5 ml of the overnight culture, i.e., 5 % (v/v) of Bacillus sp. C4 (2008), making a total volume of 100 ml. The flasks were then placed in awater bath, and all of the cultivations were running under aerobic conditions at 37 °C, and160 rpm for 2, 4, 6, or 8 days. All experimental setups were carried out in duplicates.

The remaining feathers in each culture was removed by vacuum filtration using 10-μmpaper filters, and the percentage of feather degradation was determined according to themethod presented by Park and Son [17]. After removing the un-degraded feathers, 15 ml ofthe liquid fraction (called hydrolysate) was used to measure the total crude protein andkeratinase activity. The hydrolysate was then stored at −20 °C prior to use for biogasproduction.

Another setup of the experiments was carried out with identical conditions as theabovementioned one, except that the un-degraded feathers were not separated after the biologicalpretreatment, so that the total broth of pretreated feathers was stored at −20 °C and then used forbiogas production.

Soluble Keratin Preparation and Keratinase Activity Assay

Soluble keratin was prepared from chicken feathers according to Wawrzkiewicz et al. [18] withslight modifications and used as substrate for the keratinase activity measurements. Thechicken feathers (2 g) were treated at 100 °C for 2 h with 100 ml of dimethyl sulfoxide(DMSO) using a reflux condenser. Then, 300 ml of acetone cooled at −20 °C for 2 h wasadded to 100 ml of cooled keratin solution in DMSO and left in a refrigerator for 1 h for betterprecipitation. The solution was then centrifuged at 4000g for 10 min. The precipitate (cake)was washed with distilled water, and then the acetone was allowed to evaporate from theuncapped tube at room temperature for 30 min and finally the cake was suspended in 50 mldistilled water.

This prepared soluble keratin was used as substrate for the enzyme activity determination,and the assay was performed according to the method of Wawrzkiewicz et al. [18], with slightmodifications. The prepared soluble keratin suspension (0.5 ml) was mixed with 3.5 ml of0.1 M phosphate buffer pH 7.5 and 1 ml of sample solution containing the enzyme. Thereafter,the reaction mixture was incubated for 5 h at 45 °C, followed by stopping the enzymaticreaction using 3 ml of 10 % (w/v) trichloroacetic acid (TCA) and left for 30 min at 4 °C. In thecontrol test, the reaction was stopped first with 3 ml of 10 % w/v TCA before adding 0.5 ml ofprepared soluble keratin suspension. The test and control suspensions were centrifuged at

Appl Biochem Biotechnol (2016) 180:1401–1415 1403

4000g for 10 min at 4 °C, and the absorbance of supernatant was measured at 280 nm using aspectrophotometer. One unit (U) keratinase activity was defined as the amount of enzymecausing 0.01 absorbance increase between the sample and the control at 280 nm under theconditions above [17].

Anaerobic Batch Digestion Assays

Anaerobic batch digestion assays were carried out using either feather hydrolysate or totalbroth of biologically pretreated chicken feathers as substrates, performed in accordance to themethod described by Angelidaki et al. [19]. The assays were carried out under mesophilicconditions (37 ± 1 °C) using 118-ml serum glass bottles as reactors with active volume of56 ml and headspace of 62 ml. The inoculum was either granulated bacteria obtained from aUASB reactor treating municipal wastewater (Hammarby Sjöstad, Stockholm, Sweden) ordigested sludge from a digester treating activated sludge and operating at mesophilic condi-tions at a municipal wastewater treatment plant (Getteröverket, Varberg, Sweden). Theinoculants were filtered through a 2-mm porosity sieve to remove large and undigestedparticles, and then acclimated for 5 days in an incubator at 37 °C prior to use. Substrates(feather hydrolysate or total broth) with loading of 0.25 gVS (amount of solubilized volatilesolids for feather hydrolysate and volatile solids of both solubilized and undegraded feathersfor total broth) was used, and then inoculum corresponding to 0.5 gVS was added, keeping aVS ratio (VSsubstrate to VSinoculum) at 1:2 in all setups. The pH in each reactor was adjusted to7.0 using hydrochloric acid solution (2 M), and the nutrient composition was kept according toAngelidaki et al. [19]. Inoculum and water instead of substrate were used as blank to discloseany methane production by the inoculum itself. The reactors were then sealed with rubbersepta and aluminum caps, and the headspace was flushed with a gas mixture of 80 % N2 and20% CO2 for 2 min to create anaerobic environment in each setup [19]. The reactors were thenplaced in an incubator at 37 ± 1 °C, and they were shaken manually once a day during theincubation period of 55 days. All experimental setups were performed in duplicates. Gassamples were taken twice a week at the beginning and once a week towards the end of thedigestion period from the headspace of each reactor using a pressure-tight syringe (VICI,precious sampling Inc., USA) and then they were analyzed using a gas chromatograph (GC).Gas measurement and analysis were carried out as described previously [20]. All methanevolumes are presented at standard conditions (0 °C and 1 atm).

Analytical Methods

Moisture content, pH, total nitrogen, total solids, and volatile solids were determined accordingto biomass analytical procedures [21]. Total nitrogen contents were measured using theKjeldahl method [22], and then the total crude protein content was calculated by multiplyingthe Kjeldahl nitrogen content by 6.25. The total organic carbon was obtained by correcting thetotal dry weight carbon value for the ash content [23]. The fat content was determined usingthe Soxhlet extraction procedure [24].

The methane produced was determined by a GC (Perkin-Elmer, USA) equipped witha packed column (6′ × 1.8″ OD, 80/100, Mesh, Perkin Elmer, USA) and a thermalconductivity detector (Perkin-Elmer, USA), with an inject temperature of 150 °C. Thecarrier gas was nitrogen operated with a flow rate of 20 ml/min at 60 °C. A 250-μlpressure-lock gas syringe (VICI, precious sampling Inc., USA) was used for taking


samples for the gas analysis, and the accumulated methane production was calculatedaccordingly [20].

Statistical Analysis

The experiments were randomized during biogas production analysis using statistical software,MINITAB® (version 17.1.0). Randomization was used in order to ensure that equal treatmentwas given to all reactors during gas sampling and analysis.

Factor effects, pretreatment impacts, confidence intervals, and standard deviations wereanalyzed for the anaerobic digestion experiments. The set of experimental runs was analyzedusing general linear model analysis of variance (ANOVA) with accumulated methane yield asresponse variable, with three different factors (pretreatment time, initial percentage of feathers, andtype of inoculum used with either hydrolysate or total broth of pretreated feathers) and respec-tively four, three, and four factor levels. The interaction effects between pretreatment time andtype of inoculum used, as well as initial percentage of feathers were also evaluated. ANOVAwasused to analyze data, which generate confidence intervals and the significance difference betweenthe different factors considered in the anaerobic digestion experiments. The factors were consid-ered significant when the probability (p value) was less than or equal to 0.05.

Results and Discussion

Untreated Chicken Feathers and Inoculants Characterization

The feathers used in this study contained 92.05 % dry matter of which 97.59 % was organicmatter (Table 1). Crude protein constituted 92.65 % of the feathers, and due to high nitrogencontent in the feathers, the C/N ratio was low (3.66). The fat content was also very low (0.87 %TS). The characteristics of the two different inoculants, i.e., anaerobic sludge and granularsludge, used for the anaerobic batch digestion assays are as shown in Table 1. The anaerobicsludge used in this work contains 62.18 % of total solid as organic matter, which is slightly

Table 1 Characteristics of untreated feather and anaerobic digestion inoculants

Parameters Chicken feather Anaerobic sludge Bacteria granules

Total solids (%) 92.05 ± 0.17 3.53 ± 0.08 10.20 ± 0.40

Volatile solids (%TS) 97.59 ± 0.95 62.18 ± 0.55 68.00 ± 0.19

Moisture (%) 7.95 ± 0.17 96.47 ± 0.08 89.80 ± 0.40

Total organic carbon (%TS) 54.21 ± 0.53 34.54 ± 0.31 37.78 ± 0.11

Total nitrogen (%TS) 14.82 ± 0.13 4.16 ± 0.15 4.83 ± 0.11

Crude protein (% TS) 92.65 ± 0.13 26.00 ± 0.15 30.18 ± 0.11

C/N 3.66 ± 0.04 8.30 ± 0.07 7.82 ± 0.02

Fat content (% TS) 0.87 ± 0.19 ND ND

Bulk density (kg/m3) ND 965.3 ± 17.38 1012.7 ± 14.18

pH ND 8.21 ± 0.01 6.96 ± 0.03

ND not determined

±Standard deviation based on at least duplicate measurements


lower than that measured in the bacteria granules (68 %). C/N ratio was almost the same inboth inoculants used, and the total solids in granules were substantially higher than that in thesludge.

Bacteria Growth and Keratinase Activity

The feather broth was sterilized prior to aerobic pretreatment with Bacillus sp. in orderto deactivate all forms of biological agents. Bacillus sp. prefers yeast extract andpeptone as nutrients for keratinase enzyme production [4] and therefore the mediumsupplemented with yeast extract and peptone. These materials will be degraded by thebacteria during the biological pretreatment. However, the excess amount of the yeastextract and peptone can be further assimilated in the subsequent digestion leading tomore biogas production.

The Bacillus sp. grew well in the medium and produced keratinase resulting indegradation of feathers to soluble crude protein. The pH of the medium increased from7.0 to 8.9, 8.6 and 8.7 for 5, 10, and 20 % concentrations, respectively, during the 8 days ofdegradation. This increase in pH was likely due to the production of ammonia and otheralkaline compounds during the degradation of feathers as reported by Cai et al. [11].Bacteria growth reached a maximum of 9.0 × 109, 14.3 × 109, and 1.6 × 109 CFU/ml onday 4 for 5, 10, and 20 % feather concentrations, respectively. This shows an increase inbacteria population as the feather concentration increased from 5 to 10 %. However, thepopulation of the bacteria was reduced at higher feather concentration of 20 %, probablybecause at this concentration, the contact between the bacteria and its substrate waslimited, since at this high initial feather concentration, larger aggregated feather particleswere observed reducing the accessibility of feathers. An increase in pH from 7.5 to 8.5 wasreported by Suntornsuk and Suntornsuk [25]. They found maximum bacteria growth of8.3 × 108 CFU/ml after 3 days of incubation, and these values also increased whenconcentration of feathers increased using feather concentrations between 1 and 5 %.

The keratinase activity of the enzyme produced by the Bacillus sp. is presented inFig. 1. There was a mass loss of 77, 38, and 24 %, respectively for 5, 10, and 20 % ofinitial feather concentrations, determined after 8 days of degradation, which is an indica-tion of the ability of the produced keratinase to degrade feathers [26]. The activity of theenzyme produced increased from day 2 to day 6 when 5 % initial concentration of featherswas applied and then decreased slightly after 6 days of incubation. However, at higherinitial feather concentrations of 10 and 20 %, a sharp decrease in the enzyme activity was

0

5

10

15

20

25

30

35

86420

Ker

atin

ase

activ

ity (U

)

time of degradation (days)

Fig. 1 Enzyme activity atsubstrate concentrations of 5 %TS(blue diamond suit), 10 %TS(orange square), and 20 % TS(grey up-pointing triangle)


observed already after 4 days of degradation (Fig. 1). Ghasemi et al. [27] also reported adecrease in keratinase activity after 3 days of incubation, when 1 % of feather was used,and this was linked to feedback inhibition of the enzyme by its product.

Increase in feather concentrations from 5 to 20 % caused a suppression of keratinaseactivity, which resulted in a decrease in the percentage of feathers degraded (Fig. 2). At5 % initial concentration, a maximum activity of 31.9 U was observed at day 6, corre-sponding to 72.3 % of substrate degradation. Furthermore, a maximum activity of 28.4 Uwas observed at day 4 when 10 % initial concentration of feathers was applied of which25.0 % was degraded. Finally, a maximum enzyme activity of 26.2 U was determined for20 % initial feather concentration at day 4 resulting in 24.9 % degradation of the substrate.Similarly, Cai et al. [11] reported reduction in keratinase activity as feather concentrationincreased from 1 to 20 g/l.

Degradation of Feather to Soluble Crude Protein

Feather degradation during the bacterial pretreatment was observed by visualizing the disap-pearance of feathers into the culture medium and measuring the amount of feathers thatdisappeared. At different initial concentrations of 5, 10, and 20 %, 77, 38, and 24 %,respectively, (Fig. 2) of the total initial amount of feathers were dissolved after 8 days ofdegradation. The percentage of feather degraded increased as the duration of the degradationwere prolonged; however, there was no significant difference in the amount of degradedfeathers after 6- and 8-day-long degradation times, and this was observed at all the differentinitial feather concentrations. Park and Son [17] reported 100 % feather degradation when theinitial feather concentration was 0.1 % after 10 days of treatment with B. megaterium F7–1. Onthe other hand, Deivasigamani and Alagappan [28] reported 85 % degradation for initialconcentration of 1 % feathers and after 5 days of treatment with Bacillus sp. FK 46, whileBálint et al. [29] obtained 75 % degradation when 4 % TS feather concentration was appliedafter 138 h (approximately 6 days) of treatment with Bacillus licheniformis KKl. Moreover,similar results were reported by Suntornsuk and Suntornsuk [25], where 1 to 5 % of featherconcentrations were investigated. These previously reported results are all in accordance withour observations, where the percentage of feathers degraded decreased as the initial featherconcentration increased, which is possibly an indication of product inhibition for the enzyme.Moreover, as it was mentioned earlier at the highest initial feather concentration, i.e., at 20 %,the bacterial growth itself was also limited leading to less overall biomass concentrationcompared to those achieved at lower concentration feathers.

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7 8 9

subs

trat

e de

grad

ed (%

)

�me (days)

Fig. 2 Substrate degradation atfeather concentrations of 5 %TS(blue diamond suit), 10 %TS(orange square), and 20 % TS(grey up-pointing triangle)


On day zero, the amount of soluble crude protein was very low (an average of 4.13 g/l), butthis amount increased as days of degradation increased, which is an indication that the feathersmost probably converted to soluble crude protein by the keratinase enzyme produced (Fig. 3).Beside soluble proteins, other intermediate degradation products, such as amino acids, ammo-nia, and sulfate can also be formed during feather degradation [26].

After 8 days of degradation, the amount of soluble crude protein increased from an averageof 4.13 to 35.97 g/l, 36.97 and 45.94 g/l at initial feather concentration of 5, 10, and 20 %,respectively (Fig. 3). If we compare these values with the feather degradation shown on Fig. 2,it is clear that the main products formed during feather degradation are soluble proteins. Thesoluble crude protein increased slightly up to day 8, when total solid concentrations of 5 and10 % were applied. However, this increased only up to day 4 in the case of 20 % initialconcentration of feathers and thereafter it was approximately the same, as shown in Fig. 3. Ourresults are in line with the results obtained by Deivasigamani and Alagappan [28], where 75 %of the feather keratin was converted to soluble crude protein after 5 days of incubation when1 % of feather concentration was used.

Anaerobic Digestion of Hydrolysate of Pretreated Feathers

Accumulated methane profiles from digesters using hydrolysates as substrate are shown inFig. 4. Bacteria granules performed better during anaerobic digestion of untreated feathers,resulting in approximately two times more methane yield (i.e., 199 mlCH4/gVS compared to105 mlCH4/gVS when anaerobic sludge was used). However, the highest methane yield ofaround 430 mlCH4/gVS was achieved from the hydrolysate of the pretreated feathers inde-pendently of which kind of inoculum was applied during the anaerobic batch digestion assays(Fig. 4). Hence, the methane yield was improved by 292 % after 55 days of anaerobicdigestion when sludge was used as inoculum and, respectively, the yield was improved by105 % when granules were used as inoculum, showing that pretreatment with Bacillus sp. C4

significantly improved the digestibility in both cases.At 5 % initial concentration of feathers, increasing the pretreatment time did not give any

significant effect on the methane production from the hydrolysate irrespective of the inoculumused. An average of 433 ± 12 mlCH4/gVS yield was observed, reaching the maximum yieldafter 19 days of digestion in the case of using sludge as inoculum (Fig 4a). However, when thegranules were used, the maximum average methane production of 426 ± 22 mlCH4/gVS wasachieved only after 45 days (Fig. 4d).

0

10

20

30

40

50

60

0 2 4 6 8

solu

ble

cru

de

pro

tein

(g

/l)

time of filtrate collection (days)

Fig. 3 Soluble crude protein fromtotal Kjeldahl nitrogen (TKN) val-ue of 5 %TS (blue bar), 10 %TS(orange bar), and 20 % TS (greybar)


At higher initial feather concentrations of 10 and 20 %, the accumulated methane yieldfrom hydrolyzates obtained after 2 days of pretreatment was lower and it reached its maximumof 331 ± 3 mlCH4/gVS after 19 days when sludge was used (Fig. 4b, c) and a maximum yieldof 324 ± 20 mlCH4/gVS after 45 days when granules were used (Figs. 4e, f) as inoculum.

Time (days)

0

100

200

300

400

500Y C

H4

(Nm

lCH

4/gVS

)5% (a) 5% (d)

0

100

200

300

400

500

Y CH

4(N

mlC

H 4/g

VS)

10% (b) 10% (e)

0

100

200

300

400

500

Y CH

4(N

mlC

H 4/g

VS)

20% (c)

0 10 20 30 40 50 600 10 20 30 40 50 60

20% (f)

Fig. 4 Effect of inoculum (sludge (a, b, c) and granules (d, e, f)) and initial feather concentrations (5, 10, or20 %) on accumulated methane yield of hydrolysate of pretreated feathers at different days of degradationcompared with the untreated. Day 2 (blue diamond suit), day 4 (red square), day 6 (green up-pointing triangle),day 8 (Х), and untreated (ж)


However, as pretreatment time increased from day 4 to day 8, the accumulated methane yieldwas not significantly different in cases of when either sludge or granules were used asinoculum. The reason behind this is probably the fact that at higher concentrations of 10and 20 %, the percentage of soluble crude protein (Fig 3) in the total feather degraded (Fig. 2)at day 2 was slightly lower compared to longer pretreatment times, but at lower concentrationof 5 %, the percentage of soluble crude protein in total feather degraded was almost the sameindependently on the length of the pretreatment time. These results are in accordance withwhat was found by Forgács et al. [16] who used 4 % of feather concentration in thepretreatment prior to biogas production.

Regarding the degradation rate achieved during the first 10 days of the digestion period, itwas higher when anaerobic sludge was used as inoculum compared to that when granules wereused (Fig. 4). The maximum methane yield at all conditions could be reached within 19 dayswhen sludge was used while it was reached only after 45 days in the case of granules (Fig. 4).Though the free cells had a faster rate than the bacteria granules, the same overall yield wasobtained in the end of the digestion time (Fig. 4).

Anaerobic Digestion of Total Broth of Pretreated Feathers

Accumulated methane profiles from digesters when the total broth of pretreated feathers wasused as substrate are shown in Fig. 5. Overall, lower methane yields and slower degradationrates were obtained using anaerobic sludge as inoculum, compared to those determined incases of granules as inoculum. For example, the accumulated methane yield of total broth offeathers pretreated for 2 days was 49 % higher when bacteria granules were used than thatwhen sludge was used as inoculum. These results show that the granular sludge was probablyable to adapt to possible inhibitors such as ammonia and sulfate [26, 30] in the total broth ofpretreated feathers better than the sludge. When granules were used, the remaining un-degraded feathers were broken down better; and thereafter the intermediate products couldbe converted to methane on a higher rate than those obtained in the case of the free cellspresent in the anaerobic sludge (Fig. 5). The same trend is shown during anaerobic digestion ofthe untreated feathers. The methane yield from the untreated feather was 89 % higher whengranules were used as inoculum compared to that when sludge was applied (Figs. 4 and 5).This suggests that granules perform better both on the untreated feather and on total brothcontaining a larger amount of un-degraded feathers than the sludge.

Furthermore, in the cases of anaerobic sludge as inoculum, the methane yield was slightlylower after 2-day-long pretreatment times at all initial concentration of feathers, compared tothose obtained after the longer treatments (Fig. 5a, b, c). However, there was no significantdifference in the methane yields obtained (an average of 445 ± 12 mlCH4/gVS) as pretreatmenttime increases in cases when granules were used as inoculum (Fig. 5d, e, f).

The result from the statistical analysis (Table 2) supports our results, showing that pretreat-ment duration of 2 days has significant effect on the accumulated methane yield but thissignificant effect could be observed only at higher initial concentrations of 10 and 20 % offeathers (Fig. 6). This also shows that in the case of hydrolysate as substrate, the shortestpretreatment time of 2 days was not enough to obtain higher methane yields at higher initialconcentrations irrespective of which kind of inoculum was used. The statistical analysis alsoshows that using total broth of pretreated feather instead of hydrolysate as substrate, gave asignificant effect (p = 0.000) on the accumulated methane yields (Table 2). It is also shown onFig. 6 that the pretreatment time had no significant effect on the accumulated methane yield


Time (days)

0

100

200

300

400

500Y C

H4

(Nm

lCH

4/gV

S5% (a) 5% (d)

0

100

200

300

400

500

Y CH

4(N

mlC

H4/g

VS

10% (b) 10% (e)

0

100

200

300

400

500

Y CH

4(N

mlC

H4/g

VS

20% (c)

0 10 20 30 40 50 600 10 20 30 40 50 60

20% (f)

Fig. 5 Effect of inoculum (sludge (a, b, c) and granules (d, e, f)) and initial feather concentrations (5, 10, or20 %) on accumulated methane yield of total broth of pretreated feathers at different days of degradationcompared with the untreated. Day 2 (blue diamond suit), day 4 (red square), day 6 (green up-pointing triangle),day 8 (Х), and untreated (ж)


Table 2 General linear model ANOVA on anaerobic digestion of hydrolysate and total broth of pretreatedfeathers with accumulated methane yield as response variable

Factors Coef SE Coef T value p value

Constant 405.30 3.75 107.99 0.000

Pretreatment time

2 −33.18 6.50 −5.10 0.000

4 12.15 6.50 1.87 0.065

6 8.73 6.50 1.34 0.183

8 12.30 6.50 1.89 0.062

Initial % of feather

5 5.19 5.31 0.98 0.331

10 1.08 5.31 0.20 0.839

20 −6.27 5.31 −1.18 0.241

Type

HG 2.07 6.50 0.32 0.751

HS 7.82 6.50 1.20 0.232

TBG 39.56 6.50 6.09 0.000

TBS −49.44 6.50 −7.61 0.000

HG hydrolysate with granules, HS hydrolysate with sludge, TBG total broth with granules, TBS total broth withsludge

p ≤ 0.05 (95%CI) denoted significance. S = 36.7744, R-sq. = 53.50 %; R-sq.(adj) = 49.23 %

TBSTBGHSHG

460

440

420

400

380

360

340

320

300

Type

Mea

n

2

46

8

TimePret.

Interaction Plot for Methane yield

20105

430

420

410

400

390

380

370

360

350

340

Initial %

Mea

n

24

6

8

Time

Pret.

Interaction Plot for Methane yield

Fig. 6 General linear model ANOVA interaction plot of pretreatment time with type and initial %, accumulatedmethane yield as response variable. HG (hydrolysate with granules), HS (hydrolysate with sludge), TBG (totalbroth with granules), and TBS (total broth with sludge)


when total broth was used as substrate together with bacteria granules as inoculum, but theaccumulated methane yield was highly affected by the length of the pretreatment time whentotal broth was digested with anaerobic sludge.

The results of the statistical analysis also support our experimental results determined fromtotal broth of pretreated feather using bacteria granules as inoculum, which show that theaccumulated methane yield was basically constant after different pretreatment times andirrespective of the initial feather concentrations used.

Conclusions

1. Increase in feather concentration from 5 to 20 % did not result in significant increase in theamount of feathers degraded. Pretreatment of feather wastes with Bacillus sp. C4 (2008) wassuccessful, as an average of 75.5 % of the feather keratin was converted to soluble crudeprotein by the enzyme produced after 8 days of degradation.

2. Chicken feather wastes can effectively be used for biogas production. Anaerobic digestion ofthese wastes is possible even without any pretreatment; the granules used as inoculum resultedin almost two times more methane production from the untreated feather (an average yield of199 mlCH4/gVS) than when anaerobic sludge was used as inoculum (an average yield of105mlCH4/gVS).However, the finalmethane yield observed from samples treated biologicallyby using naturally occurring bacteria that can produce both α- and β-keratinases reachedapproximately the same level. Accordingly, the methane yield could be increased by 292 %when sludge was used as inoculum on the hydrolysate of the pretreated feather (initialconcentration of 5 %) after 55 days of anaerobic digestion and respectively by 105 % whengranules were used as inoculum on the hydrolysate compared to those obtained from untreatedfeathers. The same trend was observed in the case of anaerobic digestion of total brothof treated feathers, an increase by 237 % was obtained when sludge was used asinoculum and, respectively, an increase by 124 % was achieved when bacteriagranules were applied as inoculum.

3. Sludge used as inoculum performed better on the feather hydrolysate, while granules workedbetter on total broth of the pretreated feathers. A pretreatment duration of 2 days isrecommended when 5 % initial feather concentration is applied while 4 days are recom-mended for higher, i.e., 10% and 20%, initial concentrations, when hydrolysate of pretreatedfeathers is used for methane production. Total broth of pretreated feathers can be usedeffectively with granules for methane production at shorter pretreatment duration of 2 daysirrespective of the initial feather concentrations in between the investigated range of 5–20 %.

Acknowledgments The authors greatly acknowledge Håkantorp Slakteri AB, Genomfatrsvägen, Sweden forsupplying the chicken feathers and Alex Osagie Osadolor for helpful discussion.

References

1. FAOSTAT. (2013) Live animals. Food and Agriculture Organization of the United Nations. FAO StatisticsDivision 2016. http://faostat.fao.org/site/573/DesktopDefault.aspx?PageID=573#ancor. Accessed 18 Feb2016.


2. Matikevičienė, V., Masiliūnienė, D. and Grigiškis, S. (2015) Degradation of keratin containing wastes bybacteria with keratinolytic activity. Environment. Technology. Resources. Proceedings of the InternationalScientific and Practical Conference, pp. 284–289.

3. Wang, L., Xin, J., Li, X., & Wang, Y. (2015). The variability of biomass burning and its influenceon regional aerosol properties during the wheat harvest season in North China. AtmosphericResearch, 157, 153–163.

4. Fellahi, S., Zaghloul, T. I., Feuk-Lagerstedt, E., & Taherzadeh, M. J. (2014). A bacillus strain ableto hydrolyze alpha- and beta-keratin. Journal of Bioprocessing and Biotechniques, 4, 7.

5. Johnson, D. K. and Elander, R. T. (2009), In Biomass Recalcitrance, Blackwell Publishing Ltd.,pp. 436–453.

6. Taherzadeh, M. J., & Karimi, K. (2008). Pretreatment of lignocellulosic wastes to improve ethanol andbiogas production: a review. International Journal of Molecular Sciences, 9, 1621–1651.

7. Zhao, W., Yang, R., Zhang, Y., & Wu, L. (2012). Sustainable and practical utilization of featherkeratin by an innovative physicochemical pretreatment: high density steam flash-explosion. GreenChemistry, 14, 3352–3360.

8. Korniłłowicz-Kowalska, T., & Bohacz, J. (2011). Biodegradation of keratin waste: theory and practicalaspects. Waste Management, 31, 1689–1701.

9. Onifade, A. A., Al-Sane, N. A., Al-Musallam, A. A., & Al-Zarban, S. (1998). A review:potentials for biotechnological applications of keratin-degrading microorganisms and their enzymesfor nutritional improvement of feathers and other keratins as livestock feed resources. BioresourceTechnology, 66, 1–11.

10. Barone, J. R., Schmidt, W. F., & Gregoire, N. T. (2006). Extrusion of feather keratin. Journal of AppliedPolymer Science, 100, 1432–1442.

11. Cai, C.-G., Lou, B.-G., & Zheng, X.-D. (2008). Keratinase production and keratin degradation bya mutant strain of Bacillus subtilis. Journal of Zhejiang University. Science. B, 9, 60–67.

12. Zaghloul, T. I., Embaby, A. M., & Elmahdy, A. R. (2011). Biodegradation of chicken feathers wastedirected by Bacillus subtilis recombinant cells: scaling up in a laboratory scale fermentor. BioresourceTechnology, 102, 2387–2393.

13. Kanchana, R., & Mesta, D. (2013). Native feather degradation by a keratinophilic fungus. InternationalJournal of Chemtech Research, 5, 2947–2954.

14. Kim, J.-D. (2003). Preliminary characterization of keratinolytic enzyme of aspergillus flavus K-03and its potential in biodegradation of keratin wastes. Mycobiology, 31, 209–213.

15. Mazotto, A. M., Couri, S., Damaso, M. C. T., & Vermelho, A. B. (2013). Degradation of featherwaste by Aspergillus niger keratinases: comparison of submerged and solid-state fermentation.International Biodeterioration & Biodegradation, 85, 189–195.

16. Forgács, G., Alinezhad, S., Mirabdollah, A., Feuk-Lagerstedt, E., & Horváth, I. S. (2011). Biologicaltreatment of chicken feather waste for improved biogas production. Journal of Environmental Sciences,23, 1747–1753.

17. Park, G.-T., & Son, H.-J. (2009). Keratinolytic activity of Bacillus megaterium F7-1, a feather-degradingmesophilic bacterium. Microbiological Research, 164, 478–485.

18. Wawrzkiewicz, K., Łobarzewski, J., & Wolski, T. (1987). Intracellular keratinase of Trichophyton gallinae.Journal of Medical and Veterinary Mycology, 25, 261–268.

19. Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J. L., Guwy, A. J., Kalyuzhnyi, S.,Jenicek, P., & Van Lier, J. B. (2009). Defining the biomethane potential (BMP) of solid organic wastes andenergy crops: a proposed protocol for batch assays. Water Science and Technology, 59, 927–934.

20. Teghammar, A., Yngvesson, J., Lundin, M., Taherzadeh, M. J., & Horváth, I. S. (2010).Pretreatment of paper tube residuals for improved biogas production. Bioresource Technology,101, 1206–1212.

21. APHA-AWWA-WEF. (2005) Standard methods for the examination of water and wastewater 21st Edition.21st Edition ed., American Public Health Association, 800 I Street, NW, Washington, DC 20001–3710.

22. LABCONCO. (2015) A guide to Kjeldahl nitrogen determination. Methods and Apparatus. LABCONCO, AnIndus t r y Se rv i c e Pub l i c a t i on . Acce s s ed 8 th o f Apr i l . h t t p : / /www.expo t echusa .com/catalogs/labconco/pdf/KJELDAHLguide.PDF. ed.

23. Haug, R. T. (1993), In The Practical Handbook of Compost Engineering., Taylor & Francis, pp. 247–25724. Carpenter, D. C. (2010), In Food Analysis Laboratory Manual: Food Science Texts Series, (Nielsen, S. S.,

ed.), Springer US, pp. pp 29–37.

25. Suntornsuk, W., & Suntornsuk, L. (2003). Feather degradation by Bacillus sp. FK 46 in submergedcultivation. Bioresource Technology, 86, 239–243.

26. Korniłłowicz-Kowalska, T. (1997). Studies on the decomposition of keratin wastes by saprotrophicmicrofungi. I. Criteria for evaluating keratinolytic activity. Acta Mycologica, 32, 51–79.


27. Ghasemi, Y., Shahbazi, M., Rasoul-Amini, S., Kargar, M., Safari, A., Kazemi, A., & Montazeri-Najafabady, N. (2012). Identification and characterization of feather-degrading bacteria fromkeratin-rich wastes. Annals of Microbiology, 62, 737–744.

28. Deivasigamani, B., & Alagappan, K. M. (2008). Industrial application of keratinase and soluble proteinsfrom feather keratins. Journal of Environmental Biology, 29, 933–936.

29. Bálint, B., Bagi, Z., Tóth, A., Rákhely, G., Perei, K., & Kovács, K. (2005). Utilization of keratin-containing biowaste to produce biohydrogen. Applied Microbiology and Biotechnology, 69, 404–410.

30. Deublein, D. and Steinhauser, A. (2011) Biogas from waste and renewable resources. ed. Wiley-VCH.


Pap

er II

Dry fermentation of manure with straw in continuous plug flow reactor: Reactor development and process stability at different loading rates. Bioresource Technology 224: 197–205.

Dry fermentation of manure with straw in continuous plug flow reactor:Reactor development and process stability at different loading rates

Regina J. Patinvoh ⇑, Adib Kalantar Mehrjerdi, Ilona Sárvári Horváth, Mohammad J. TaherzadehSwedish Centre for Resource Recovery, University of Borås, Sweden

h i g h l i g h t s

� New plug flow reactor developed forcontinuous dry digestion processes.

� Reactor worked successfully for230 days using untreated manurewith straw at 22% TS.

� Methane yield of 56% of thetheoretical value was obtained at OLRof 4.2 gVS/L/d.

� OLR of 6 gVS/L/d caused processinstability with 41% VS removalefficiency.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 October 2016Received in revised form 1 November 2016Accepted 3 November 2016Available online 5 November 2016

Keywords:Dry fermentationPlug flow reactorContinuous processProcess stabilityReactor development

a b s t r a c t

In this work, a plug flow reactor was developed for continuous dry digestion processes and its efficiencywas investigated using untreated manure bedded with straw at 22% total solids content. This newlydeveloped reactor worked successfully for 230 days at increasing organic loading rates of 2.8, 4.2 and6 gVS/L/d and retention times of 60, 40 and 28 days, respectively. Organic loading rates up to 4.2 gVS/L/d gave a better process stability, with methane yields up to 0.163 LCH4/gVSadded/d which is 56% ofthe theoretical yield. Further increase of organic loading rate to 6 gVS/L/d caused process instability withlower volatile solid removal efficiency and cellulose degradation.

� 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic digestion of organic wastes for biogas production hasbeen successfully applied but there is a need for improved pro-cesses and design of less expensive reactors. In this vein the drivefor dry anaerobic digestion processes is increasing both in researchand the industry because of technical simplicity in design, togetherwith low construction and operational costs (Karthikeyan &

Visvanathan, 2013; Kothari et al., 2014). Dry anaerobic digestiontechnology is designed to process more organic wastes per reactorvolume with total solids (TS) content greater than 20% (Demirer &Chen, 2008; Fernández et al., 2008) treating food wastes, manurebedded with straw, garden wastes and other high solid waste frac-tions. In comparison with the wet anaerobic digestion process, thisprocess allows higher organic loading rates (OLR), less pretreat-ment and gives better economic feasibility (Karthikeyan &Visvanathan, 2013) since the reactor volume is minimized and itis easier to handle the digestate residue.

Animal wastes and crop residues are one of the most abundantwaste fractions generated worldwide, the amount of manure

http://dx.doi.org/10.1016/j.biortech.2016.11.0110960-8524/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: [email protected] (R.J. Patinvoh).

Bioresource Technology 224 (2017) 197–205

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Dry fermentation of manure with straw in continuous plug flow reactor:Reactor development and process stability at different loading rates

Regina J. Patinvoh ⇑, Adib Kalantar Mehrjerdi, Ilona Sárvári Horváth, Mohammad J. TaherzadehSwedish Centre for Resource Recovery, University of Borås, Sweden

h i g h l i g h t s

� New plug flow reactor developed forcontinuous dry digestion processes.

� Reactor worked successfully for230 days using untreated manurewith straw at 22% TS.

� Methane yield of 56% of thetheoretical value was obtained at OLRof 4.2 gVS/L/d.

� OLR of 6 gVS/L/d caused processinstability with 41% VS removalefficiency.

g r a p h i c a l a b s t r a c t



Keywords:Dry fermentationPlug flow reactorContinuous processProcess stabilityReactor development

a b s t r a c t

In this work, a plug flow reactor was developed for continuous dry digestion processes and its efficiencywas investigated using untreated manure bedded with straw at 22% total solids content. This newlydeveloped reactor worked successfully for 230 days at increasing organic loading rates of 2.8, 4.2 and6 gVS/L/d and retention times of 60, 40 and 28 days, respectively. Organic loading rates up to 4.2 gVS/L/d gave a better process stability, with methane yields up to 0.163 LCH4/gVSadded/d which is 56% ofthe theoretical yield. Further increase of organic loading rate to 6 gVS/L/d caused process instability withlower volatile solid removal efficiency and cellulose degradation.


1. Introduction

Anaerobic digestion of organic wastes for biogas production hasbeen successfully applied but there is a need for improved pro-cesses and design of less expensive reactors. In this vein the drivefor dry anaerobic digestion processes is increasing both in researchand the industry because of technical simplicity in design, togetherwith low construction and operational costs (Karthikeyan &

Visvanathan, 2013; Kothari et al., 2014). Dry anaerobic digestiontechnology is designed to process more organic wastes per reactorvolume with total solids (TS) content greater than 20% (Demirer &Chen, 2008; Fernández et al., 2008) treating food wastes, manurebedded with straw, garden wastes and other high solid waste frac-tions. In comparison with the wet anaerobic digestion process, thisprocess allows higher organic loading rates (OLR), less pretreat-ment and gives better economic feasibility (Karthikeyan &Visvanathan, 2013) since the reactor volume is minimized and itis easier to handle the digestate residue.

Animal wastes and crop residues are one of the most abundantwaste fractions generated worldwide, the amount of manure


⇑ Corresponding author.E-mail address: [email protected] (R.J. Patinvoh).





bedded with straw produced increases daily as the number ofhoused dairy herd increases. Cattle manure bedded with strawusually have a TS greater than 20%, farmers can use low cost anaer-obic digesters to convert these enormous waste streams to biogas,thereby improve water quality, reduce methane and nitrous oxideemissions and improve soil fertility. Dry anaerobic digestion istherefore a better option for processing these wastes. Since biogasproduction involves a complex biological process, monitoring ofthe process is essential to avoid process instability and failure ofthe digester (Drosg, 2013). For enhanced performance of dry anaer-obic digestion processes, a suitable reactor is required; consideringthe substrate composition, amount of substrate to be treated, andprocess economy of the reactor.

Plug flow reactors have been reported to be efficient for dryanaerobic digestion processes. These reactors are inexpensiveand easy to build which make them a suitable technology toimprove the livelihoods of farmers (Lansing et al., 2010, 2008). Plugflow reactors have also been reported to have the highest successrate in the United States, where 42% out of the 242 anaerobicdigesters operating at livestock farms in 2015 were plug flowdesigns (USEPA, 2016). Nevertheless, some shortcomings, such aslower mass transfer due to lack of mixing, thermal stratificationand solid sedimentation problems have been reported (Lansinget al., 2010). These problems can be minimized by the use of impel-lers in plug flow reactors. The impellers allow minimal mixing forbetter performance in the reactors. In high solid digestion pro-cesses, however, continuous mixing have been reported to indicateunstable performance at high OLR and it was observed that thecontinuously mixed unstable reactor became stable when the mix-ing level was reduced (Stroot et al., 2001). Researchers have alsoinvestigated the effectiveness of plug flow reactors on manureand other substrates with solid content in the range of 11–14%TS (Adl et al., 2012; Cantrell et al., 2008). There have been studieson dry digestion of different substrates in batch reactors but littleinformation is available on dry digestion in continuous plug flowreactors. The innovation of this paper in meeting this gap wasthe development of a novel type solid-state plug-flow laboratoryreactor to treat substrates at higher TS levels, i.e. greater than 20%.

The efficiency of this reactor was then investigated in a contin-uous dry anaerobic digestion process treating manure bedded withstraw at 22% TS content. The main objective was to identify thecritical OLR, above which instability can occur in the reactor. Theprocess was monitored by measuring VFA/Alkalinity ratio, pH,volatile fatty acid (VFA) and total ammonia nitrogen concentra-tions regularly. This is vital because the cost of starting the entireprocess over again far outweighs monitoring of the process.

2. Materials and methods

2.1. Reactor design

A horizontal plug flow reactor (Fig. 1) for continuous dry anaer-obic digestion process was designed and made-up at the Universityof Borås, Sweden. This reactor has 9.2 L total volume, 1565 mmlength and maximum inside pressure of 10 kPa. It was mountedon a base surface with which a clamp to the two edges of the reac-tor was associated for suspending the reactor. Mesophilic (37 �C)conditions were maintained by circulating water from a heater, athermostatic water bath (GD 100, Grant instruments Ltd., Cam-bridgeshire, UK), through a water jacket (150 mm outer diameterwith 4 mm wall thickness and capacity of 5 L) surrounding thereactor. The reactor was then shielded with a 10 mm thick-Styrofoam to avoid heat loss.

The reactor was sub-divided into 3 zones as follows. Inlet zone –this part was made of polyethylene material with sealed buffer

system to avoid the release of gas and air entering the system. Thispart can be considered as an inactive zone in the inlet system. Ashutoff valve was connected at the inlet with a piston rod (madegas tight by O-rings). The inlet storage volume is about 0.256 Lwith 0.0053 L of feedstock per rotation.

Main Zone – the main zone of the reactor was made of a Poly-methylmethacrylate (PMMA) material with 110 mm outer diame-ter and 5 mm thickness for easy handling and to make the reactortransparent. An impeller, installed on a hexagonal shaft that runsthrough the reactor connected the inlet to the outlet. The hexago-nal shaft had two oil seals that prevented leakage of materials andseveral O-rings making the different parts gas tight. The impellerallowed mixing of the feedstock at the bottom part of the inlet,and transported the materials very slowly towards the outlet tak-ing several rotations: 100 cm3 per rotation but it can also dependon the viscosity of feedstock and working volume of reactor.

Outlet zone – this part was also made of polyethylene materialwith an outlet pipe for the collection of the digestate residue. Thematerial was transferred from inlet towards outlet at the end of thereactor by rotating of the impeller shaft; as a result the digestedresidue was discharged through the outlet pipe while new materi-als were added. The impeller handle was connected to the outletfor manual rotation. In this zone, the gas outlet was also connectedwhere the daily volume of biogas produced was measured by thetipping device in the automated methane potential test system(AMPTs); meanwhile with an inserted thermocouple the tempera-ture of the reactor was controlled.

Safety measures – a construction was made with a polyethylenematerial at the inlet and outlet with specific rubber connectors forsealing. If pressure inside the reactor would increase beyond 10 kPadue to blockage in the gas outlet or for any reason, this part of thereactor could open up avoiding an explosion. All interior parts werecarefully selected in order to avoid any chemical reaction with theexperimental materials as well as any corrosion during the test.

Fig. 1 shows a schematic diagram of the reactor and the exper-imental set up together with other accessories, such as heater andwater bath for maintaining the temperature, sampling point forbiogas composition analysis and the AMPTs system for measuringthe biogas produced.

2.2. Substrates and inoculum

The substrate, cattle manure bedded with straw, was collectedfrom a cattle farm outside Borås (Sweden) and used as feedstockfor the continuous anaerobic digestion process. During the experi-mental period two different batches, with similar content of totalsolids (TS) and volatile solids (VS) were obtained from the samefarm. The manure was shredded manually to reduce the particlesize of straw; then it was characterized, weighted and stored inplastic containers at �20 �C to prevent biodegradation until furtheruse. During experiment, weighted frozen substrate was defrostedat room temperature and thoroughly mixed to gain a homogenizedfeed before use. Sludge used as inoculum was obtained from adigester treating waste water sludge and operating at mesophilicconditions (Vatten and Miljö i Väst AB, Varberg, Sweden). Theinoculum was filtered through a 2 mm porosity sieve to removesand, plastic and other unwanted particles after which it was accli-mated for five days in an incubator at 37 �C prior to use. The inocu-lumwas centrifuged at 10,000g for 10 min to obtain a TS content of7.8 ± 0.24%. Table 1 shows the most important characteristics ofthe substrate and the inoculum used during the investigations.

2.3. Experimental procedure

The substrate was inoculated with inoculum to start-up thereactor, keeping a volatile solids (VS) ratio (VSsubstrate to VSinoculum)

198 R.J. Patinvoh et al. / Bioresource Technology 224 (2017) 197–205

at 1:2 in the reactor. The pH of the mixture was adjusted to 7.47 by2 M HCl solution. Furthermore, a nutrient solution with composi-tion according to Angelidaki et al. (2009) was also added into thereactor. The experiment was started at batch mode until no furthergas production was monitored (40 days of digestion period). Afterthat the continuous feeding operation was started with an OLR of2.8 gVS/L/d. The experiments continued with gradually increasingOLRs, thereby decreasing retention times with the aim of main-taining a fixed working volume. The OLR values investigated were2.8 gVS/L/d (OLR 1), 4.2 gVS/L/d (OLR 2) and 6 gVS/L/d (OLR 3) with

corresponding retention times of 60, 40 and 28 days. For the exper-imental period (start-up and OLR1) the first batch of manure wasutilized, while another batch of the feedstock was used duringthe experimental period of OLR 2 and OLR 3. Each OLR conditionwas kept until a period of at least one corresponding retentiontime. The reactor was fed regularly in every second day and thedigestate residue was withdrawn before feeding, and kept for anal-ysis, while biogas composition was monitored daily. Processparameters, such as pH, VFA/Alkalinity ratio, VFA and total ammo-nia nitrogen concentration, TS, VS, as well as lignin, cellulose andhemicellulose contents in the digestate residue were also mea-sured to monitor the digestion process. The reactor was mixedmanually (once daily) with the impeller by moving its content tothe inlet and back to the outlet to minimize stratification in thereactor. Overall, the experiment lasted for 230 days.

2.4. Theoretical BMP of experimental feedstock

The feedstock being solid was prepared according to Zupancicand Roš (2012) by blending 1 g of the sample with water (dilutionfactor of 50) to reduce the particle size and allow homogenization(Raposo et al., 2012). Thereafter, theoretical methane potential ofthe substrate used during digestion process was calculated fromthe amount and chemical oxygen demand (COD) concentration ofthe actual feeding using Eq. (1) (Nielfa et al., 2015) assuming thatthe equation is valid for any substances or products (Tarvin &Buswell, 1934).

BMPthCOD ¼ nCH4RTpVSadded

ð1Þ

Fig. 1. Schematic diagram of developed reactor and experimental set-up.

Table 1Characteristics of Substrate and Inoculum used during experiment (standard devi-ation based on at least duplicate measurements).

Parameters Manure with Straw Anaerobic sludge

Total solids (%) 22.29 ± 2.78 7.80 ± 0.24Volatile solids (%)a 70.44 ± 2.68 40.46 ± 1.12Moisture (%) 77.72 ± 2.78 92.20 ± 0.30Ash (%)a 29.56 ± 2.68 59.54 ± 1.12Fat content (%)a 1.37 ± 0.24 NDTotal carbon (%)a 39.14 ± 1.49 22.48 ± 0.62Kjeldahl Nitrogen (%)a 2.33 ± 0.30 4.16 ± 0.15C/N 16.80 ± 0.64 5.4 ± 0.15pH 8.81 ± 0.45 8.19 ± 0.30Bulk density (kg/m3) 967.20 ± 14.18 1001.33 ± 17.38Protein Content (%TS) 14.56 ± 0.30 26.00 ± 0.15Total COD (gCOD/gVSadded) 0.73 ± 0.02 NDBMPtheoretical (LCH4/gVSadded) 0.290 ± 0.01 ND

ND = Not determined; COD = chemical oxygen demand; C/N = carbon nitrogenratio.

a Dry basis.

R.J. Patinvoh et al. / Bioresource Technology 224 (2017) 197–205 199

at 1:2 in the reactor. The pH of the mixture was adjusted to 7.47 by2 M HCl solution. Furthermore, a nutrient solution with composi-tion according to Angelidaki et al. (2009) was also added into thereactor. The experiment was started at batch mode until no furthergas production was monitored (40 days of digestion period). Afterthat the continuous feeding operation was started with an OLR of2.8 gVS/L/d. The experiments continued with gradually increasingOLRs, thereby decreasing retention times with the aim of main-taining a fixed working volume. The OLR values investigated were2.8 gVS/L/d (OLR 1), 4.2 gVS/L/d (OLR 2) and 6 gVS/L/d (OLR 3) with

corresponding retention times of 60, 40 and 28 days. For the exper-imental period (start-up and OLR1) the first batch of manure wasutilized, while another batch of the feedstock was used duringthe experimental period of OLR 2 and OLR 3. Each OLR conditionwas kept until a period of at least one corresponding retentiontime. The reactor was fed regularly in every second day and thedigestate residue was withdrawn before feeding, and kept for anal-ysis, while biogas composition was monitored daily. Processparameters, such as pH, VFA/Alkalinity ratio, VFA and total ammo-nia nitrogen concentration, TS, VS, as well as lignin, cellulose andhemicellulose contents in the digestate residue were also mea-sured to monitor the digestion process. The reactor was mixedmanually (once daily) with the impeller by moving its content tothe inlet and back to the outlet to minimize stratification in thereactor. Overall, the experiment lasted for 230 days.

2.4. Theoretical BMP of experimental feedstock

The feedstock being solid was prepared according to Zupancicand Roš (2012) by blending 1 g of the sample with water (dilutionfactor of 50) to reduce the particle size and allow homogenization(Raposo et al., 2012). Thereafter, theoretical methane potential ofthe substrate used during digestion process was calculated fromthe amount and chemical oxygen demand (COD) concentration ofthe actual feeding using Eq. (1) (Nielfa et al., 2015) assuming thatthe equation is valid for any substances or products (Tarvin &Buswell, 1934).

BMPthCOD ¼ nCH4RTpVSadded

ð1Þ

Fig. 1. Schematic diagram of developed reactor and experimental set-up.

Table 1Characteristics of Substrate and Inoculum used during experiment (standard devi-ation based on at least duplicate measurements).

Parameters Manure with Straw Anaerobic sludge

Total solids (%) 22.29 ± 2.78 7.80 ± 0.24Volatile solids (%)a 70.44 ± 2.68 40.46 ± 1.12Moisture (%) 77.72 ± 2.78 92.20 ± 0.30Ash (%)a 29.56 ± 2.68 59.54 ± 1.12Fat content (%)a 1.37 ± 0.24 NDTotal carbon (%)a 39.14 ± 1.49 22.48 ± 0.62Kjeldahl Nitrogen (%)a 2.33 ± 0.30 4.16 ± 0.15C/N 16.80 ± 0.64 5.4 ± 0.15pH 8.81 ± 0.45 8.19 ± 0.30Bulk density (kg/m3) 967.20 ± 14.18 1001.33 ± 17.38Protein Content (%TS) 14.56 ± 0.30 26.00 ± 0.15Total COD (gCOD/gVSadded) 0.73 ± 0.02 NDBMPtheoretical (LCH4/gVSadded) 0.290 ± 0.01 ND

ND = Not determined; COD = chemical oxygen demand; C/N = carbon nitrogenratio.

a Dry basis.


where:BMPthCOD = theoretical yield under laboratory conditionR = gas constant (R = 0.082 atm L/mol K)T = working temperature (310 K)P = atmospheric pressure (1 atm)VSadded = volatile solids of the substrate added (g)nCH4 = methane produced (mol) determined according to Eq. (2)

nCH4 ¼ COD64 g

mol

� � ð2Þ

2.5. Analytical procedure

Moisture content, pH, total nitrogen, TS and VS were deter-mined according to biomass analytical procedures (APHA-AWWA-WEF, 2005). Total nitrogen contents were measured usingthe Kjeldahl method, and the protein content was estimated bymultiplying the Kjeldahl nitrogen content with a factor of 6.25according to Gunaseelan (2009). The total carbon was obtainedby correcting the total dry weight carbon value for the ash content(Haug, 1993; Zhou et al., 2015). The fat content was determinedusing the Soxhlet extraction procedure (Carpenter, 2010). Alkalin-ity measured as the total inorganic carbonate and was determinedby the Nordmann titration method according to Lossie and Pütz(2008). Samples were centrifuged at 4000g for 15 min and then5 ml of the supernatant was titrated with 0.1 N sulfuric acid topH 5, the titration was then continued until pH 4.4 was reachedin order to determine the VFA concentration measured as aceticacid equivalent. Digestate samples for total ammonia nitrogen con-centration were centrifuged (15 min at 4000g); supernatantdiluted 50 times in deionized water to a final volume of 5 ml. Sam-ples were then analyzed using Ammonium 100 test kit (Nanocolor,MACHEREY-NAGEL GmbH & Co. KG. Germany) and concentrationmeasured using Nanocolor 500D Photometer (MACHEREY-NAGELGmbH & Co. KG. Germany). The free ammonia concentration wascalculated from the total ammonia nitrogen concentration andpH values of samples according to Kayhanian (1999).

Total cellulose, hemicellulose and lignin content of the solidfraction of the digestates were determined according to NREL pro-tocols (Sluiter et al., 2011). The digestate was centrifuged at 4000gfor 15 min and the solid fraction was washed with 100 ml distilledwater and then air dried until moisture content was less than 10%.The samples were then hydrolyzed using 72% H2SO4 in a waterbath at 30 �C for 60 min, samples were stirred every 5 min toensure uniform hydrolysis, and then a second hydrolysis was per-formed using 4% H2SO4 in an autoclave at 121 �C for 60 min. Mono-meric sugars contained in the hydrolysis liquid were determinedby HPLC (Waters 2695, Waters corporation, Milford, MA, USA).Mannose, glucose, galactose, xylose and arabinose were analyzedusing Aminex HPX-87P column (Bio-Rad) at 85 �C and 0.6 mL/min ultrapure water as eluent. Acid soluble lignin (ASL) was deter-mined using a UV spectrophotometer (Libra S60, Biochrom, Eng-land) at 320 nm. Acid insoluble lignin (AIL) was gravimetricallydetermined as residual solid after hydrolysis corrected with ashcontent. The ash content was determined as the remaining residueafter keeping the samples in the muffle furnace at 575 �C for 24 h.The percentage of cellulose degraded was calculated according toZhou et al. (2015).

The daily volume of biogas produced was measured by the tip-ping device in the Automatic Methane Potential Testing System(AMPTS, Bioprocess control AB, Lund, Sweden), which is based onthe principle of water displacement buoyancy. The composition(methane and carbon dioxide) of the produced gas was determinedusing a GC (Perkin-Elmer, USA) equipped with a packed column(60 � 1.800 OD, 80/100, Mesh, Perkin Elmer, USA), and a thermal

conductivity detector (Perkin-Elmer, USA), with an inject tempera-ture of 150 �C. The carrier gas was nitrogen operated with a flowrate of 20 ml/min at 60 �C. A 250-ll pressure-lock gas syringe(VICI, precious sampling Inc., USA) was used for taking samplesfor the gas composition analysis.

VFAs in the digestate filtrates were measured using a high-performance liquid chromatograph (HPLC, Waters 2695, WatersCorporation, Milford, MA, USA) equipped with an RI detector(Waters 2414, Waters Corporation Milford, MA, USA) and abiohydrogen-ion exchange column (Aminex HPX-87H, Bio-Rad,Hercules, CA, USA) operating at 60 �C. A UV absorbance detector(Walters 2487), operating at 210 nmwavelength was used in serieswith a refractive index (RI) detector (Walters 2414) operating at60 �C.

3. Results and discussion

3.1. Reactor development

During preliminary studies a syphon mechanism was appliedinitially at the inlet of the reactor to make it gas tight and avoidentering of air during feeding. This mechanism worked well atlower TS rates up to 11%. However, it was very troublesome whenworking with higher TS concentrations; i.e. when feedstock with13% TS was applied, this completely blocked the syphon systemfor the feed. After that, the inlet part of the reactor was modifiedto a sealed buffer system described in Section 2.1 and shown inFig. 1. The shutoff valve connected at the inlet was always closedat the start of feeding to avoid gas leakage. When materials werefed in, the piston rod was inserted and tightened, to avoid therelease of any gas. After this, the valve was opened, and the mate-rial was pressed down into the reactor via the piston rod and thenthe valve was closed again. The impeller in this reactor allows min-imal mixing by moving the reactor content manually to the inletand back to the outlet for better performance of the process. Thisnew modification worked well even with an inlet feed of 22% TSwithout any blockage at the inlet. The developed plug flow reactorworked successfully treating cattle manure bedded with straw at22% TS for 230 days using organic loading rates of 2.8, 4.2, and6 gVS/L/d at retention times of 60, 40 and 28 days respectively.

3.2. Substrate characterization

The most important characteristics of the substrate as collectedfrom the cattle farm are shown in Table 1. The substrate contains22.29% TS of which 70.44% are organic matter. The carbon nitrogen(C/N) ratio was 16.8:1 which is within the required range for stableanaerobic digestion process. Previously, optimal C/N ratiosbetween 20:1 and 35:1 have been stated (Habiba et al., 2009;Kayhanian, 1999), also, other researchers, i.e. Friehe et al. (2010),Pagés Díaz et al. (2011) reported a wider range of between 10:1and 30:1 as optimal C/N ratios. However, the availability of carbonand nitrogen for the microorganisms is more significant than theactual C:N ratio calculated on the basis of elementary compositionof the feedstock. Hence the C:N ratio of straw might be overesti-mated due to the limited availability of carbon fraction in the lig-nocellulosic structure of straw (Herrmann et al., 2016). Thetheoretical methane potential of the feedstock used in this exper-iment was calculated to be 0.290 LCH4/gVSadded.

3.3. Biogas production

The startup batch digestion period ran for 40 days, the daily gasproduction was high at the beginning; hence the digestion processstarted up properly and the feedstock were degraded until the end


of this batch digestion period. The methane content in the pro-duced gas reached 63% ± 3.86 at the end of the startup (batch) per-iod as shown in Fig. 2b. This start-up period was allowed until gasproduction nearly stopped (40 days) after which the continuousdigestion mode with feeding and withdrawing in every secondday was started.

Fig. 2a illustrates the daily biogas production at different OLR.Experiments were carried out using progressive organic loadingrates; the loading rate of 2.8 gVS/L/d (OLR 1) produced only about1 L/d in the initial days. Then it gradually increased to around 3 L/dafter 36 days and remained at the same level until the end of theOLR1 (2.8 gVS/L/d) period. Similar pattern was observed for theperiod when OLR 2, (4.2 gVS/L/d) was applied, it started from3.5 L/d daily methane production initially which increased

gradually to around 5 L/d after 18 days, and thereafter it remainedstable. The process performed differently when OLR 3, i.e. 6 gVS/L/d was introduced. There were larger fluctuations observed in thedaily gas production and only a slightly stable period could beachieved after 20 days maintaining a daily biogas production ofaround 7 L/d. The reactor was then fed keeping the OLR 3 (6 gVS/L/d) for an additional corresponding retention time of 28 days tocheck the stability of the process under this condition. However,the daily gas production could not be kept stable with decreasingvalues towards the end of this period. In parallel, the VFA concen-tration continued to increase (Fig. 4a) leading to increased VFA/alkalinity ratios with a maximum of around 0.9 at the end of theperiod (Fig. 3a) showing instability in the process. To examine ifVFA could be reduced and restore stability in the process, the

Fig. 2. Biogas production (a) and biogas composition (b) at different organic loading rate (OLR) during experiments. The symbols represent daily biogas production (}),cumulative biogas production (h), methane composition (r) and carbon dioxide composition (j).


OLR was then reduced to 4.2 gVS/L/d (OLR 2) again. As a conse-quence of the reduced load the biogas production reduced gradu-ally and remained stable after 16 days maintaining a daily biogasproduction around 4.2 L/d which is slightly lower than thatobtained when the same loading rate (OLR 2) was used previously.

The average methane content in the biogas was 64.9%, 65.1%and 63.3% for OLR of 2.8, 4.2 and 6 gVS/L/d respectively. Overall,OLR up to 4.2 gVS/L/d gave a better process stability with methaneyield of 0.163 LCH4/gVSadded accounting around 56% of the theoret-ical methane yield of 0.290 LCH4/gVSadded as shown in Table 2. Themethane yield at OLR 2.8 gVS/L/d was slightly lower than the yieldat OLR 4.2 gVS/L/d probably due to slight differences in the feed-stock composition, since these two experimental periods were per-formed with two different batches of the substrate. This lowermethane yield could be associated with the presence of untreated

straw in the cattle manure. Hydrolysis of the cellulose in untreatedstraw has been reported to be the rate limiting step (Noike et al.,1985) especially when it is mixed with cattle manure (Myint &Nirmalakhandan, 2006). However, our result is similar to themethane yield of 0.170 LCH4/gVSadded obtained by Kusch et al.(2008) when the digestion of horse dung with straw was investi-gated in batch-operated solid phase digestion.

3.4. Process performance

Fig. 3a shows the variation of pH and VFA/Alkalinity ratio at dif-ferent OLR while Fig. 4a illustrates the variation of total and indi-vidual VFA for all the experiments carried out. At 2.8 gVS/L/d(OLR 1), the pH was increased slightly to around 8 initially butdropped to around 7.5 after 4 days of digestion and it remains

Fig. 3. pH and VFA/Alkalinity ratio variation (a) and composition of cellulose, hemicellulose and lignin in digestate (b) at different organic loading rate (OLR) duringexperiments. The symbols represent pH (}), VFA/Alkalinity ratio (h), total lignin (r), cellulose (j) and hemicellulose (N). Presented values are mean values of duplicatemeasurements with error bars as standard deviation between the two values.


steady afterwards which is within the favourable pH range of 7–8for anaerobic digestion (Drosg, 2013). The VFA/Alkalinity ratio waslower (Fig. 3a) than the reported failure limit value of 0.3 whichshows that the process was stable during these conditions.

Anaerobic digestion process has been reported to be workingfavourably without acidification possibility when this ratio is lessthan 0.3 (Drosg, 2013). When the OLR was increased to 4.2 gVS/L/d, the pH remained still within the favourable range around 7.6throughout this period and the VFA/Alkalinity ratio was below0.3. So, an appropriate buffering capacity and high stability of theprocess was observed for OLR of 2.8 and 4.2 gVS/L/d and at corre-sponding retention times of 60 and 40 days.

No VFA accumulation was observed during the process applying2.8 gVS/L/d (OLR 1) and 4.2 gVS/L/d (OLR 2) as shown in Fig. 4a.This signifies the complete degradation of the intermediary pro-duced volatile fatty acids. Fast consumption of VFAs was alsoreported by Liu et al. (2007) as a proof of stable and properly func-tioning process without danger of failure. However, when the OLRwas increased to 6 gVS/L/d with corresponding decreased reten-tion time of 28 days the stability of the system deteriorated. At this6 gVS/L/d loading (OLR 3), the process started to show signs forinstability already during the first retention time of 28 days,

Fig. 4. Total and individual volatile fatty acids (VFA) variation (a) and Total ammonia nitrogen with free ammonia concentration (b) at different organic loading rate (OLR)during experiments. The symbols represent Total VFA (}), Acetic (h), Butyric (4), isobutyric (�), isovaleric (⁄), propionic (s), valeric (|), Total ammonia nitrogen (r) and freeammonia (j).

Table 2Experimental Results at different loading rate (values are averages with standarddeviation).

Parameter OLR 1 OLR 2 OLR 3

Loading rate (gVS/d) 13.80 20.70 29.90Organic Loading rate (OLR)

(gVS/L/d)2.80 4.20 6.00

Retention time 60 40 28Biogas produced (L/gVSadded) 0.23 ± 0.01 0.25 ± 0.01 0.23 ± 0.01Average methane content (%) 64.90 ± 1.75 65.10 ± 2.81 63.30 ± 2.06Methane yield (LCH4/gVSadded) 0.15 ± 0.01 0.163 ± 0.01 0.146 ± 0.01Initial VS concentration (%) 17.17 17.17 17.17Final VS concentration (%) 4.31 ± 0.64 7.31 ± 1.19 10.10 ± 0.82VS removal efficiency (%) 74.87 ± 3.75 57.42 ± 6.96 41.17 ± 4.80


steady afterwards which is within the favourable pH range of 7–8for anaerobic digestion (Drosg, 2013). The VFA/Alkalinity ratio waslower (Fig. 3a) than the reported failure limit value of 0.3 whichshows that the process was stable during these conditions.

Anaerobic digestion process has been reported to be workingfavourably without acidification possibility when this ratio is lessthan 0.3 (Drosg, 2013). When the OLR was increased to 4.2 gVS/L/d, the pH remained still within the favourable range around 7.6throughout this period and the VFA/Alkalinity ratio was below0.3. So, an appropriate buffering capacity and high stability of theprocess was observed for OLR of 2.8 and 4.2 gVS/L/d and at corre-sponding retention times of 60 and 40 days.

No VFA accumulation was observed during the process applying2.8 gVS/L/d (OLR 1) and 4.2 gVS/L/d (OLR 2) as shown in Fig. 4a.This signifies the complete degradation of the intermediary pro-duced volatile fatty acids. Fast consumption of VFAs was alsoreported by Liu et al. (2007) as a proof of stable and properly func-tioning process without danger of failure. However, when the OLRwas increased to 6 gVS/L/d with corresponding decreased reten-tion time of 28 days the stability of the system deteriorated. At this6 gVS/L/d loading (OLR 3), the process started to show signs forinstability already during the first retention time of 28 days,

Fig. 4. Total and individual volatile fatty acids (VFA) variation (a) and Total ammonia nitrogen with free ammonia concentration (b) at different organic loading rate (OLR)during experiments. The symbols represent Total VFA (}), Acetic (h), Butyric (4), isobutyric (�), isovaleric (⁄), propionic (s), valeric (|), Total ammonia nitrogen (r) and freeammonia (j).

Table 2Experimental Results at different loading rate (values are averages with standarddeviation).

Parameter OLR 1 OLR 2 OLR 3

Loading rate (gVS/d) 13.80 20.70 29.90Organic Loading rate (OLR)

(gVS/L/d)2.80 4.20 6.00

Retention time 60 40 28Biogas produced (L/gVSadded) 0.23 ± 0.01 0.25 ± 0.01 0.23 ± 0.01Average methane content (%) 64.90 ± 1.75 65.10 ± 2.81 63.30 ± 2.06Methane yield (LCH4/gVSadded) 0.15 ± 0.01 0.163 ± 0.01 0.146 ± 0.01Initial VS concentration (%) 17.17 17.17 17.17Final VS concentration (%) 4.31 ± 0.64 7.31 ± 1.19 10.10 ± 0.82VS removal efficiency (%) 74.87 ± 3.75 57.42 ± 6.96 41.17 ± 4.80


though the pH was still within the favourable range, i.e. an averageof 7.7. However, the VFA/Alkalinity ratio increased to 0.4 and theacetic acid concentration increased to 2100 mg/l which signifiesa slightly unstable process (Drosg, 2013). There was fluctuationsin the daily biogas production (Fig. 2a) as well at this loading rate(OLR3). Continuing the loading at the same conditions, i.e. 6 gVS/L/d, into the second retention time period caused considerably dis-turbances in the system. As shown in Fig. 3a, the VFA/Alkalinityratio increased up to 0.9 and the total VFA content also increasedgradually up to around 7000 mg/L (Fig. 4a) which is a typical signfor overloading. The acetic acids increased up to around 3800 mg/Land the propionic acid concentration increased slightly to around1000 mg/L during the process. Propionic acid accumulation withinthe range of 250–1000 mg/L has been reported as an indication ofinstability in the process (Drosg, 2013) and concentrations greaterthan 1000 mg/L has been reported as a first sign for overloading inthe system (Björnsson et al., 1997). Running the process at a higherOLR (6 gVS/L/d) led to a shorter retention time of 28 days, since thereactor was kept at a fixed working volume during the wholeexperimental period. This shorter retention time could cause theremoval of the slow-growing methanogenic community, whichled to process instability as well as overloading of the reactor.

Despite the increase in VFA the pH still remained within therange of 7.6 and 8 during the second retention period of 6 gVS/L/d (OLR3). Weiland (2010) has also reported that manure can havea surplus of alkalinity which stabilizes the pH value even at higherVFA accumulation and as such will not always result in pH drop. Toexamine if the VFA could be reduced and restore the process backto stability, the loading rate was reduced again to 4.2 gVS/L/d (OLR2). It was observed that the VFA/Alkalinity ratio reduced graduallyto 0.2 (Fig. 3a) and the VFA accumulation reduced drastically(Fig. 4a) and so the process was restored back to stability.

During digestion, at OLR of 2.8 and 4.2 gVS/L/d the total ammo-nia nitrogen concentration was between 1350 and 2000 mg/l(Fig. 4b) and the VFA/Alkalinity ratio was below 0.3 which showsgood buffering capacity. However, at OLR of 6 gVS/L/d, the totalammonia nitrogen concentration was slightly reduced to around1000 mg/l probably due to reduced time for feedstock degradationand as such the buffering capacity was slightly lower. This togetherwith the VFA accumulation resulted to higher VFA/Alkalinity ratioshowing instability in the process. The free ammonia concentrationthroughout the process at different OLR was between 47 and145 mg/l which shows that the reported tolerable free ammoniaconcentration of 150 mg/l (Kayhanian, 1999) was not exceeded,hence the process was not inhibited by free ammonia.

3.5. Volatile solids removal efficiency

An important factor for assessing the efficiency of an anaerobicdigestion process is following up the reduction in VS. As shown inTable 2, 2.8 gVS/L/d (OLR 1) has the highest VS removal efficiency,an average of 74.87 ± 3.75%, while 6 gVS/L/d (OLR 3) has the lowestwith an average of 41.17 ± 4.80%. It was observed that as VFAs get-ting accumulated the removal efficiency reduces. The removal effi-ciency decreases as the OLR increases, since with increasing OLRthe retention time in the digester decreases, so that the organicmatter loaded into the digester has not enough time for the degra-dation, especially not if difficult to digest fractions, as untreatedstraw, is present in the feedstock.

3.6. Degradation of lignocelluloses

As shown in Fig. 3b, the amount of cellulose and hemicellulosedecreases gradually compared to those of the initial composition inthe feed during the digestion process, while the lignin contentincreases. This shows that the carbohydrates were consumed

during the process at all loading rates examined. However, thehighest cellulose degradation was obtained at organic loading rateof 2.8 gVS/L/d (OLR 1); an average of 60.9% ± 6.37, while at 4.2 gVS/L/d (OLR 2) an average of 48.8% ± 6.73 cellulose degradation wasobserved, and finally 6 gVS/L/d (OLR 3) having the lowest cellulosedegradation of an average of 30.1% ± 7.05. For the same reason asexplained above the amount of cellulose degraded decreases asthe loading rate increases and with reducing retention time.

4. Conclusions

The new plug flow reactor developed can operate successfullyfor continuous dry digestion of manure bedded with straw at22% TS when operated at OLR of 2.8 and 4.2 gVS/L/d with retentiontime of 60 and 40 days respectively. OLR of 6 gVS/L/d and retentionof 28 days favoured process instability decreasing the VS removalefficiency and cellulose degradation. Digestion of manure beddedwith straw without pretreatment at 22% TS was successful,methane yield 0.163 LCH4/gVSadded counting around 56% of thetheoretical yield with 57% VS removal efficiency was obtained atorganic loading rate of 4.2 gVS/L/d.

Acknowledgements

This work was financially supported by Västra Götalandsregio-nen (Sweden).

References

Adl, M., Sheng, K.C., Xia, Y.H., Gharibi, A., Chen, X., 2012. Examining a hybrid plug-flow pilot reactor for anaerobic digestion of farm-based biodegradable solids.Int. J. Environ. Res. 6 (1), 335–344.

Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J.L., Guwy, A.J.,Kalyuzhnyi, S., Jenicek, P., Van Lier, J.B., 2009. Defining the biomethane potential(BMP) of solid organic wastes and energy crops: a proposed protocol for batchassays. Water Sci. Technol. 59 (5), 927–934.

APHA-AWWA-WEF, 2005. Standard Methods for the Examination of Water andWastewater. American Public Health Association (APHA), American WaterWorks Association (AWWA), Water Environment Federation (WEF),Washington DC.

Björnsson, L., Mattiasson, B., Henrysson, T., 1997. Effects of support material on thepattern of volatile fatty acid accumulation at overload in anaerobic digestion ofsemi-solid waste. Appl. Microbiol. Biotechnol. 47 (6), 640–644.

Cantrell, K.B., Ducey, T., Ro, K.S., Hunt, P.G., 2008. Livestock waste-to-bioenergygeneration opportunities. Bioresour. Technol. 99 (17), 7941–7953.

Carpenter, C., 2010. Determination of fat content. In: Nielsen, S.S. (Ed.), FoodAnalysis Laboratory Manual. Springer, US, pp. 29–37.

Demirer, G.N., Chen, S., 2008. Anaerobic biogasification of undiluted dairy manurein leaching bed reactors. Waste Manage. 28 (1), 112–119.

Drosg, B., 2013. Process Monitoring in Biogas Plants. IEA Bioenergy 978-1-910154-03-8.

Fernández, J., Pérez, M., Romero, L.I., 2008. Effect of substrate concentration on drymesophilic anaerobic digestion of organic fraction of municipal solid waste(OFMSW). Bioresour. Technol. 99 (14), 6075–6080.

Friehe, J., Weiland, P., Schattauer, A., 2010. Fundamentals of anaerobic digestion. In:Guide to Biogas From Production to Use, 5th revised ed., FachagenturNachwachsende Rohstoffe e. V. (FNR).

Gunaseelan, N.V., 2009. Biomass estimates, characteristics, biochemical methanepotential, kinetics and energy flow from Jatropha curcus on dry lands. BiomassBioenergy 33 (4), 589–596.

Habiba, L., Hassib, B., Moktar, H., 2009. Improvement of activated sludgestabilisation and filterability during anaerobic digestion by fruit andvegetable waste addition. Bioresour. Technol. 100 (4), 1555–1560.

Haug, R.T., 1993. Feed conditioning – physical and chemical. In: The PracticalHandbook of Compost Engineering. Taylor & Francis, pp. 247–257.

Herrmann, C., Kalita, N., Wall, D., Xia, A., Murphy, J.D., 2016. Optimised biogasproduction from microalgae through co-digestion with carbon-rich co-substrates. Bioresour. Technol. 214, 328–337.

Karthikeyan, O.P., Visvanathan, C., 2013. Bio-energy recovery from high-solidorganic substrates by dry anaerobic bio-conversion processes: a review. Rev.Environ. Sci. Bio/Technol. 12 (3), 257–284.

Kayhanian, M., 1999. Ammonia inhibition in high-solids biogasification: anoverview and practical solutions. Environ. Technol. 20 (4), 355–365.

Kothari, R., Pandey, A.K., Kumar, S., Tyagi, V.V., Tyagi, S.K., 2014. Different aspects ofdry anaerobic digestion for bio-energy: an overview. Renewable SustainableEnergy Rev. 39, 174–195.


Kusch, S., Oechsner, H., Jungbluth, T., 2008. Biogas production with horse dung insolid-phase digestion systems. Bioresour. Technol. 99 (5), 1280–1292.

Lansing, S., Martin, J.F., Botero, R.B., da Silva, T.N., da Silva, E.D., 2010. Methaneproduction in low-cost, unheated, plug-flow digesters treating swine manureand used cooking grease. Bioresour. Technol. 101 (12), 4362–4370.

Lansing, S., Víquez, J., Martínez, H., Botero, R., Martin, J., 2008. Quantifyingelectricity generation and waste transformations in a low-cost, plug-flowanaerobic digestion system. Ecol. Eng. 34 (4), 332–348.

Liu, G., Zhang, R., Sun, Z., Li, X., Dong, R., 2007. Research progress in anaerobicdigestion of high moisture. Agric. Eng. Int. CIGR J. 9 (13), 1–10.

Lossie, U., Pütz, P., 2008. Targeted control of biogas plants with the help of FOS/TAC.Practice Report, Laboratory Analysis Titration FOS/TAC. HACH LANGE.

Myint, M., Nirmalakhandan, N., 2006. Evaluation of first-order, second-order, andsurface-limiting reactions in anaerobic hydrolysis of cattle manure. Environ.Eng. Sci. 23 (6), 970–980.

Nielfa, A., Cano, R., Fdz-Polanco, M., 2015. Theoretical methane productiongenerated by the co-digestion of organic fraction municipal solid waste andbiological sludge. Biotechnol. Rep. 5, 14–21.

Noike, T., Endo, G., Chang, J.E., 1985. Characteristics of carbohydrate degradationand the rate-limiting step in anaerobic digestion. Biotechnol. Bioeng. 27 (10),1482–1489.

Pagés Díaz, J., Pereda Reyes, I., Lundin, M., Sárvári Horváth, I., 2011. Co-digestion ofdifferent waste mixtures from agro-industrial activities: kinetic evaluation andsynergetic effects. Bioresour. Technol. 102 (23), 10834–10840.

Raposo, F., De la Rubia, M.A., Fernández-Cegrí, V., Borja, R., 2012. Anaerobicdigestion of solid organic substrates in batch mode: an overview relating tomethane yields and experimental procedures. Renewable Sustainable EnergyRev. 16 (1), 861–877.

Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2011.Determination of Structural Carbohydrates and Lignin in Biomass. LaboratoryAnalytical Procedure. NREL/TP-510-42618.

Stroot, P.G., McMahon, K.D., Mackie, R.I., Raskin, L., 2001. Anaerobic codigestion ofmunicipal solid waste and biosolids under various mixing conditions—I.Digester performance. Water Res. 35 (7), 1804–1816.

Tarvin, D., Buswell, A.M., 1934. The methane fermentation of organic acids andcarbohydrates 1, 2. J. Am. Chem. Soc. 56 (8), 1751–1755.

USEPA, 2016. US Environmental Protention Agency. AgSTAR Data and Trends –Anaerobic digester facts and trends <https://www.epa.gov/agstar/agstar-data-and-trends#adfacts> Accessed 16/10/18.

Weiland, P., 2010. Biogas production: current state and perspectives. Appl.Microbiol. Biotechnol. 85 (4), 849–860.

Zhou, C., Liu, Z., Huang, Z.-L., Dong, M., Yu, X.-L., Ning, P., 2015. A new strategy forco-composting dairy manure with rice straw: addition of different inocula atthree stages of composting. Waste Manage. 40, 38–43.

Zupancic, G.D., Roš, M., 2012. Determination of chemical oxygen demand insubstrates from anaerobic treatment of solid organic waste. Waste BiomassValor. 3, 89–98.


Innovative pretreatment strategies for biogas production.Bioresource Technology 224: 13–24.

Pap

er II

I

Review

Innovative pretreatment strategies for biogas production

Regina J. Patinvoh, Osagie A. Osadolor, Konstantinos Chandolias, Ilona Sárvári Horváth,Mohammad J. Taherzadeh ⇑

Swedish Centre for Resource Recovery, University of Borås, SE 50190 Borås, Sweden

h i g h l i g h t s

� Substrates for biogas might be indigestible, hard to digest, toxic materials.� Indigestible materials can be gasified to syngas and then fermented to biogas.� Recalcitrant biomass can go through a variety of pretreatments.� Toxic substrate can be digested with or without a pretreatment.� Right choice of digesting reactor and criteria can resolve some of these challenges.



Keywords:BiogasPretreatment strategiesLignocellulosic residueSyngasFruit and food wasteKeratin waste

a b s t r a c t

Biogas or biomethane is traditionally produced via anaerobic digestion, or recently by thermochemical ora combination of thermochemical and biological processes via syngas (CO and H2) fermentation.However, many of the feedstocks have recalcitrant structure and are difficult to digest (e.g., lignocellu-loses or keratins), or they have toxic compounds (such as fruit flavors or high ammonia content), ornot digestible at all (e.g., plastics). To overcome these challenges, innovative strategies for enhancedand economically favorable biogas production were proposed in this review. The strategies consideredare commonly known physical pretreatment, rapid decompression, autohydrolysis, acid- or alkali pre-treatments, solvents (e.g. for lignin or cellulose) pretreatments or leaching, supercritical, oxidative or bio-logical pretreatments, as well as combined gasification and fermentation, integrated biogas productionand pretreatment, innovative biogas digester design, co-digestion, and bio-augmentation.


Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141.1. Biochemistry of biogas production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.1.1. Biological route. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141.1.2. Combined thermochemical and biological route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151.1.3. Theoretical yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2. Digestion of potential feedstocks for biogas production and their challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.1. Indigestible feedstocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.2. Hard-to-digest feedstocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.2.1. Lignocellulosic wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.2.2. Keratin-rich wastes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.3. Feedstock with inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.3.1. Inhibitors as part of the feedstock-fruit wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.3.2. Inhibitors released during digestion – wastes from food processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.3.3. Inhibitors released during digestion – ammonia inhibition from protein wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.3.4. Inhibitors released during gasification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3. Pretreatment methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18


⇑ Corresponding author.E-mail address: [email protected] (M.J. Taherzadeh).





3.1. Gasification of indigestible feedstock. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183.2. Hard-to-digest feedstocks and feedstocks with inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.2.1. Accessible surface area and decrystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.2.2. Hydrolysis of hemicellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.2.3. Lignin removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.2.4. Hydrolysis of keratin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.2.5. Removal of inhibitors present in the feedstocks-fruit flavors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4. Innovative non-pretreatment strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204.1. Integrated biogas production strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214.2. Innovative digester design strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214.3. Co-digestion strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224.4. Bio-augmentation strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

1. Introduction

Biogas production through anaerobic digestion technology hasadvanced tremendously over the years. Presently, due to highenergy demand and environmental concerns as the world’s popu-lation increases, the drive for anaerobic digestion processes is gain-ing momentum within research and the industry for sustainableenergy generation. In this vein, there is an increasing focus on bet-ter feedstock utilization for improved biogas production. Neverthe-less, the challenges of low biogas yield, high retention time, andhigh investment cost impede the maximum performance of biogasproduction in anaerobic digestion systems. These bottlenecks arehighly dependent upon the availability, composition, and degrad-ability of the feedstock used for biogas production. Great potentiallies in biogas production from various feedstocks such as crop resi-dues, livestock residues, municipal waste, landfill waste, foodwaste, aquatic biomass, keratin waste, and lignocellulosic feed-stocks because of their availability and abundance. However, mostof these feedstocks have slow degradation rates and as suchrequire longer retention times. In addition, some of these feed-stocks form toxic intermediates or contain toxic compounds, whichinhibit the biogas production process. Nevertheless, the abundanceand thus low cost of these feedstocks confirm that there is a needfor new strategies for a better utilization of such kinds of wastestreams.

Theoretically, biogas can be produced from the organic fractionof any material, such as wood, crop residue, textile wool, chickenfeathers, lignocellulosic waste, industrial food waste, fruit waste,etc. However, today, biogas is typically produced only from feed-stocks that are easily utilizable by the microbial communityresponsible for transforming these feedstocks into biogas. How-ever, these easily digestible feedstocks, i.e., crop and livestock resi-dues, source sorted municipal waste, food waste, waste water withhigh organic content, etc., are not as abundant or readily availablefor biogas production, thereby, limiting the amount of biogas thatcan be produced. Nevertheless, the development of innovativetechnologies aiming for the utilization of feedstocks that are read-ily available but not easily degradable would result in an increasein biogas production.

The major reasons why some feedstocks are not ideal for biogasproduction are: (a) they cannot be digested by microorganisms, (b)digestion by microorganisms is very difficult to achieve, (c) diges-tion could be achieved but in a very slow way, and (d) the presenceof inhibitors in the feedstock or the production of inhibitory com-pounds during microbial degradation. The goal of the ‘‘pretreat-ment” is to facilitate the digestion process by removing thesebarriers and to make the organic content of the substrate easilyaccessible and utilizable by the microbial community. There havebeen several approaches toward pretreatment, which can be

classified as physical, chemical, physicochemical, and biological(Taherzadeh and Karimi, 2008). This review considers some inno-vative strategies, helping the utilization of indigestible, slow, hardto digest, and inhibitory feedstocks for biogas production. The idealpretreatment to be employed for processing these feedstocksshould be cost-effective, increase feedstock accessibility tomicroorganisms, should not use or produce substances that inhibitbiogas production, should not demand high energy, and should notgenerate by-products that are harmful to the environment. A shortreview on the biochemistry of biogas production, theoretical yield,available potential feedstocks, and the challenges associated withthese feedstocks will help in understanding how innovative strate-gies toward pretreatment can be implemented.

1.1. Biochemistry of biogas production

1.1.1. Biological routeFeedstock for biogas production is prepared prior to digestion

by removing contaminants such as grits, metals, and other debrisdepending on the source of feedstock. Moreover, the size of thefeedstock may be reduced (for feedstock whose available surfacearea is not accessible by hydrolyzing bacteria), and inhibitors fromfeedstocks such as fruit flavors and the oil from palm oil mill efflu-ent (POME) can be removed.

Organic feedstocks undergo different degradation steps duringthe anaerobic digestion (AD) process (Fig. 1), and they are brieflydiscussed as follows. Step one is hydrolysis, here the feedstock isdisintegrated by the action of a diverse community of hydrolyticbacteria producing exoenzymes. The products of this first stepare simple sugars, amino acids, and fatty acids. This step has beenreported as being the rate limiting step for hard-to-digest biomass(Fernandes et al., 2009) such as lignocellulose and keratin-richwastes. Moreover, some toxic by-products can be formed duringthis step (Neves et al., 2006). Step two is acidogenesis, in this stepmonomers from the hydrolysis are converted into short chainorganic acids, alcohols, a few organic-nitrogen and organic-sulfurcompounds, together with hydrogen and carbon dioxide. This stephas been reported to be the fastest step in the AD process (Vavilinet al., 1996). If the feedstock has a low buffering capacity and theorganic loading rate is high, the accumulation of volatile fatty acidscan result in a pH drop, which would inhibit the methanogens thatproduce methane in the final step. Step three is the acetogenesis,here the homoacetogenic microorganisms reduce hydrogen andcarbon dioxide to acetic acid (Deublein and Steinhauser, 2011).In this step, the acetogenic bacteria can only survive at a verylow hydrogen concentration, so excessive production of hydrogenfrom the acidogenesis step can inhibit these bacteria (Deubleinand Steinhauser, 2011). Step four is methanogenesis, wheremethane production takes place under strict anaerobic conditions.


3.1. Gasification of indigestible feedstock. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183.2. Hard-to-digest feedstocks and feedstocks with inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.2.1. Accessible surface area and decrystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.2.2. Hydrolysis of hemicellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.2.3. Lignin removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.2.4. Hydrolysis of keratin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.2.5. Removal of inhibitors present in the feedstocks-fruit flavors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4. Innovative non-pretreatment strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204.1. Integrated biogas production strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214.2. Innovative digester design strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214.3. Co-digestion strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224.4. Bio-augmentation strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

1. Introduction

Biogas production through anaerobic digestion technology hasadvanced tremendously over the years. Presently, due to highenergy demand and environmental concerns as the world’s popu-lation increases, the drive for anaerobic digestion processes is gain-ing momentum within research and the industry for sustainableenergy generation. In this vein, there is an increasing focus on bet-ter feedstock utilization for improved biogas production. Neverthe-less, the challenges of low biogas yield, high retention time, andhigh investment cost impede the maximum performance of biogasproduction in anaerobic digestion systems. These bottlenecks arehighly dependent upon the availability, composition, and degrad-ability of the feedstock used for biogas production. Great potentiallies in biogas production from various feedstocks such as crop resi-dues, livestock residues, municipal waste, landfill waste, foodwaste, aquatic biomass, keratin waste, and lignocellulosic feed-stocks because of their availability and abundance. However, mostof these feedstocks have slow degradation rates and as suchrequire longer retention times. In addition, some of these feed-stocks form toxic intermediates or contain toxic compounds, whichinhibit the biogas production process. Nevertheless, the abundanceand thus low cost of these feedstocks confirm that there is a needfor new strategies for a better utilization of such kinds of wastestreams.

Theoretically, biogas can be produced from the organic fractionof any material, such as wood, crop residue, textile wool, chickenfeathers, lignocellulosic waste, industrial food waste, fruit waste,etc. However, today, biogas is typically produced only from feed-stocks that are easily utilizable by the microbial communityresponsible for transforming these feedstocks into biogas. How-ever, these easily digestible feedstocks, i.e., crop and livestock resi-dues, source sorted municipal waste, food waste, waste water withhigh organic content, etc., are not as abundant or readily availablefor biogas production, thereby, limiting the amount of biogas thatcan be produced. Nevertheless, the development of innovativetechnologies aiming for the utilization of feedstocks that are read-ily available but not easily degradable would result in an increasein biogas production.

The major reasons why some feedstocks are not ideal for biogasproduction are: (a) they cannot be digested by microorganisms, (b)digestion by microorganisms is very difficult to achieve, (c) diges-tion could be achieved but in a very slow way, and (d) the presenceof inhibitors in the feedstock or the production of inhibitory com-pounds during microbial degradation. The goal of the ‘‘pretreat-ment” is to facilitate the digestion process by removing thesebarriers and to make the organic content of the substrate easilyaccessible and utilizable by the microbial community. There havebeen several approaches toward pretreatment, which can be

classified as physical, chemical, physicochemical, and biological(Taherzadeh and Karimi, 2008). This review considers some inno-vative strategies, helping the utilization of indigestible, slow, hardto digest, and inhibitory feedstocks for biogas production. The idealpretreatment to be employed for processing these feedstocksshould be cost-effective, increase feedstock accessibility tomicroorganisms, should not use or produce substances that inhibitbiogas production, should not demand high energy, and should notgenerate by-products that are harmful to the environment. A shortreview on the biochemistry of biogas production, theoretical yield,available potential feedstocks, and the challenges associated withthese feedstocks will help in understanding how innovative strate-gies toward pretreatment can be implemented.

1.1. Biochemistry of biogas production

1.1.1. Biological routeFeedstock for biogas production is prepared prior to digestion

by removing contaminants such as grits, metals, and other debrisdepending on the source of feedstock. Moreover, the size of thefeedstock may be reduced (for feedstock whose available surfacearea is not accessible by hydrolyzing bacteria), and inhibitors fromfeedstocks such as fruit flavors and the oil from palm oil mill efflu-ent (POME) can be removed.

Organic feedstocks undergo different degradation steps duringthe anaerobic digestion (AD) process (Fig. 1), and they are brieflydiscussed as follows. Step one is hydrolysis, here the feedstock isdisintegrated by the action of a diverse community of hydrolyticbacteria producing exoenzymes. The products of this first stepare simple sugars, amino acids, and fatty acids. This step has beenreported as being the rate limiting step for hard-to-digest biomass(Fernandes et al., 2009) such as lignocellulose and keratin-richwastes. Moreover, some toxic by-products can be formed duringthis step (Neves et al., 2006). Step two is acidogenesis, in this stepmonomers from the hydrolysis are converted into short chainorganic acids, alcohols, a few organic-nitrogen and organic-sulfurcompounds, together with hydrogen and carbon dioxide. This stephas been reported to be the fastest step in the AD process (Vavilinet al., 1996). If the feedstock has a low buffering capacity and theorganic loading rate is high, the accumulation of volatile fatty acidscan result in a pH drop, which would inhibit the methanogens thatproduce methane in the final step. Step three is the acetogenesis,here the homoacetogenic microorganisms reduce hydrogen andcarbon dioxide to acetic acid (Deublein and Steinhauser, 2011).In this step, the acetogenic bacteria can only survive at a verylow hydrogen concentration, so excessive production of hydrogenfrom the acidogenesis step can inhibit these bacteria (Deubleinand Steinhauser, 2011). Step four is methanogenesis, wheremethane production takes place under strict anaerobic conditions.


This step has been reported as the rate limiting step for easilydegraded and those with low buffering capacity feedstocks (Rozziand Remigi, 2004). There are mainly two groups of methanogenicbacteria that can be distinguished, i.e., hydrogenotrophic methano-gens and acetotrophic methanogens converting hydrogen and car-bon dioxide, and acetic acid, respectively, to methane. The balancebetween the hydrogen-forming and hydrogen-consuming microor-ganisms is very important, since anaerobic oxidation, i.e., acetateformation can only take place at a low partial pressure of hydrogenbecause of thermodynamic reasons.

1.1.2. Combined thermochemical and biological routeThe increasing generation of indigestible organic waste

increases the interest in the combination of thermochemical andbiochemical processes such as gasification and fermentation. Bothgasification and fermentation consist of several process steps. Ini-tially, the feedstock is fed to the gasifier in which temperatureincreases to approx. 1200 �C. As the temperature rises, the feed-stock’s water evaporates (at 100 �C), tar and char are produced,and pyrolysis gases are generated. The main reaction steps at thispoint are the water gas and the Boudouard reaction (Eqs. (1) and(2)). Moreover, gases react with each other (gas-phase reactions)and with the carbon (gas-solid reactions). Methane can also be pro-duced by the reaction of carbon and hydrogen.

Water gas reaction : H2Oþ C $ H2 þ CO; DHSTP ¼ 131:3 kJ=mol

ð1Þ

Boudouard reaction : CO2 þ C $ 2CO; DHSTP ¼ 172:5 kJ=mol

ð2Þ

The gas produced from the gasification contains fermentablehydrogen, carbon monoxide, and carbon dioxide. This gas mixtureis fed into a fermenter where it is converted into methane. Morespecifically, carbon dioxide and hydrogen are converted intomethane and water by hydrogenotrophic methanogens, while ace-totrophic methanogens convert carbon monoxide and water intomethane and carbon dioxide.

1.1.3. Theoretical yieldThe organic content of a feedstock determines the theoretical

yield of biogas production. When the elemental composition isknown, the theoretical methane production can be calculatedusing Eq. (3) (Symons and Buswell, 1933):

CcHhOoNnSs þ yH2O ! xCH4 þ nNH3 þ sH2Sþ ðc� xÞCO2 ð3aÞ

Moles of methane produced ðxÞ¼ 1=8ð4cþ h� 2o� 3n� 2sÞ ð3bÞIf carbohydrates (C6H10O5), protein (C5H7O2N), or fat

(C57H104O6) are used as substrates, the theoretical yield will be0.42, 0.50, and 1.01 N m3-CH4/kgVS respectively (Schnürer andJarvis, 2010). When both the elemental composition and the pro-portion of carbohydrates, proteins, and fats are not known, the the-oretical methane yield can also be calculated from the chemicaloxygen demand of the feedstock using Eq. (4):

CH4 þ 2O2 ! CO2 þ 2H2O ð4ÞFrom this equation, 2-kmols of O2 (or 64 kg COD) are needed for

the complete oxidation of 1-kmol of methane, so 1 kg COD isequivalent to 1/64-kmol of methane or 0.35 m3 CH4 at standard

Complex PolymersCarbohydrates, Fats, Proteins etc.

Monomers(Fatty acids, Sugars, Amino acids)

Hydrolytic bacteria

Fermentative Bacteria

Hydrogen + Carbondioxide Acetate

Acetate-oxidizing bacteria

Homoacetogenic bacteria

Acetotrophic methanogens

Methane + Carbondioxide

Hydrogenotrophic methanogens

Intermediates products(Volatile fatty acids and Alcohols)

Fig. 1. Biochemistry of anaerobic digestion processes (modified from Mkoma and Mabiki (2011)).


temperature and pressure. Using the calculations from Eqs. (3) and(4), the theoretical methane yields of some substrates are summa-rized in Table 1.

2. Digestion of potential feedstocks for biogas production andtheir challenges

As discussed earlier, biomass that is easily processed is mainlyused as feedstock for anaerobic digestion process. Common, easilyprocessed, feedstocks include livestock manure, food-processingwastes, and sewage sludge. On the other hand, biomass that is dif-ficult to process is in high abundance and accumulates tremen-dously. This biomass, when properly pretreated, can be avaluable feedstock for biogas production, thereby reducing envi-ronmental pollution and enhancing the recovery of renewableenergy. For instance, lignocellulosic wastes have about 40–60% cel-lulose and 20–40% hemicellulose (Kang et al., 2014), which is agood potential carbon source for biogas production if made acces-sible to the microorganisms. In addition, keratin-rich wastes (e.g.,feathers) are composed of about 91–93% crude protein (Patinvohet al., 2016; Salminen et al., 2003), which is an insoluble proteinand when converted into soluble oligomers will be of great poten-tial for biogas production. Moreover, approximately 91% of thevolatile solids of fruit wastes are degradable, depending on thefruit species (Schnürer and Jarvis, 2010), and food processingwastes also have good potentials for biogas production. The biogasproduction potential of these feedstocks varies depending on theircomposition, pretreatment process, and concentration ofbiodegradable material. Therefore, some of these feedstocks whichare indigestible, hard to digest, slow to digest, or contain inhibitorsare discussed as potential feedstocks for anaerobic digestion. Chal-lenges associated with these feedstocks are discussed as well.

2.1. Indigestible feedstocks

A large fraction of the globally produced waste is composed oforganic indigestible materials, which are not biologically degrad-able or their degradation is extremely slow and cost-inefficient.

Landfills contain many indigestible compounds such as processedpaper and impregnated wood. Because of the indigestible andheterogeneous nature of these wastes, their volumes increaseexponentially posing an environmental threat. According to theworld bank, these materials make up the highest proportion ofmunicipal solid waste in high income countries, while theyaccounted for 27 % of the global solid waste generated in 2009(TheWorldBank, 2010).

Indigestible wastes contain components that can be used bymicrobial catalysts during the biogas production. Recently, somestudies were conducted on the biological degradation of tradition-ally indigestible feedstock for biogas production. For example, anovel bacterium, Ideonella sakaiensis 201-F6, was proven todegrade polyethylene tetraphthalate (PET) (Yoshida et al., 2016).Furthermore, the digestion of cotton/polyester textiles was investi-gated in another study (Jeihanipour et al., 2013). However, theseprocesses are premature, and more research is required for scalingup. One way to convert indigestible waste into fermentable feed-stock is to apply gasification step before AD. During gasification,the feedstock is converted into a gas mix called syngas, which isthereafter converted into methane by anaerobic microbes. Thecombination of two already industrial processes gathers manyadvantages such as efficient know-how and existing infrastruc-tures. However, the main bottleneck of syngas AD or fermentationis the low gas-liquid mass transfer, especially of hydrogen and car-bon monoxide. However, the use of a gas dispenser such as aninnovative hollow fiber membrane can drastically increase thegas diffusion rate (Shen et al., 2014). In addition, the diffusion ofsyngas in a more economical way would be a very positive stepfor scaling up the process.

2.2. Hard-to-digest feedstocks

The recalcitrant nature of some feedstocks limits their biologi-cal degradation for biogas production. This recalcitrant nature isa result of several properties such as the crystalline structure ofthe feedstock, different layers of the plant cell wall, and their inter-actions with other polymers (Taherzadeh and Jeihanipour, 2012).

Table 1Wastes from food processing industries (Russ and Meyer-Pittroff, 2004) and theoretical methane yield from the different substrates (Schnürer and Jarvis, 2010).

Industry Application Waste

Diary Diary processing Diluted whole milk, whey, separated milk, cleaning waterYoghurt processing Diluted milk, cleaning water, wheyCheese processing Dry milk particle, whey, cleaning water

Oil Palm oil processing Palm kernel shell, empty fruit bunches, palm oil mill effluentOlive oil processing Vegetable water, olive husk and skinVegetable oil processing Oil seed cakeCoconut oil processing Outer coat fiber (coir), desiccated coconut meat (copra)

Meat Slaughterhouse Slaughterhouse waste water, slaughterhouse bloodBrewery Beer making Malt dust, spent grain, kieselguhr sludge, yeastGrain Grist or flour mill Bran, middlings, shells, husk, broken grain, ergot

Oat husking Bran, huskRice mill Rice bran, brown rice wasteMalt processing Malt dust, grain separator waste

Sugar Sugar cane processing Bagasse, molassesSugar beet processing Molasses, exhausted beet pulp, carbonation sludge

Substrate Methane yield (m3/ton-vs)Food waste 400–600Fruit waste 200–500Grass 200–400Straw 100–320Municipal sludge 160–350Protein wastes 496Manure (pigs, cattle, chickens) 100–300Slaughterhouse waste 700Cereals 300–400


These complex polymers are tightly packed and highly crosslinkedwith hydrogen bonds, disulfide bonds (for keratin wastes), andhydrophobic interactions (Daroit et al., 2009). Hydrolysis is therate-limiting step for these feedstocks; therefore, it is necessaryto overcome the recalcitrant nature and make them accessible tothe microorganisms for effective biogas production. Some of thesefeedstocks and their challenges as well as how to overcome thesechallenges are discussed below in order to ascribe value to them.

2.2.1. Lignocellulosic wastesLignocellulosic wastes are one of the most abundant renewable

organic resources on earth with a yearly production of approxi-mately 200 billion tons (Zhang, 2008). The increasing expansionof the agricultural industry and its activities has led to an increasein the production and the consequent accumulation of a large vol-ume of these wastes. Forestry by-products, woody crops, andmunicipal solid waste also contribute to an enormous amount ofthese waste streams. About 60% of the dry weight of the averagemunicipal solid waste is composed of lignocelluloses (Holtzappleet al., 1992). The abundance of lignocellulosic wastes makes thema potential feedstock for biogas production, and it has beenreported that these kinds of wastes can add up to a significantenergy value of approximately 1500 MJ/year (Kang et al., 2014)depending on the yield from the biomass, total cultivable area,and the technology used. Although these wastes are rich in fibers,readily available, and have a significant energy value, they have alow biogas yield due to their resistance to microbial attack.

Lignocellulosic wastes are difficult to digest as a result of matrixpolymers (hemicellulose, pectins, and lignin) surrounding the cel-lulosic microfibrils in the plant cell wall (Himmel and Picataggio,2009). The degree of resistance of these wastes to microbial attackvaries depending on the type of lignocellulose, various compart-ments of the cell wall, and cell age, while it is also affected by pro-cessing phenomena such as drying and heating (Taherzadeh andJeihanipour, 2012). However, effective use of lignocellulosic bio-mass for biogas production requires a hydrolysis step in order toopen up the structure and increase the accessibility for cellulosedegrading microorganisms. It is only after this step that the mate-rial will be ready for conversion. The rate and extent to which cel-lulose can be hydrolyzed depends on the connection between thelignin, hemicellulose, and cellulose and also the degree of crys-tallinity in the cellulose itself. A potential solution is an innovativepretreatment method to increase the digestibility and ascribevalue to the lignocellulosic wastes.

2.2.2. Keratin-rich wastesKeratin-rich wastes such as chicken feather, wool, hair, nails,

horns, hooves, and claws are produced worldwide by the poultry,wool, meat, and fish industries. For instance, the world’s averagestock of chicken is about 22 billion (FAOSTAT, 2013); thus, thepoultry industries produce a tremendous amount of feathersyearly. Moreover, the global production of wool amounts to 2.1million tons per year (IWTO, 2015). Assuming that all the insolubleprotein (keratin) are converted into soluble protein, the methanepotential of keratin wastes is as high as 0.496 Nm3/kgVS(Angelidaki and Sanders, 2004). However, the methane yieldobtained is usually low due to the recalcitrant structure of keratin.Keratin is an insoluble structural protein where the polypeptideschain is tightly packed and highly cross-linked with disulfidebonds, hydrogen bonds, and hydrophobic interactions (Daroitet al., 2009). This structure makes the protein insoluble and resis-tant to enzymatic attack, which is a major hindrance in the biolog-ical processing of these wastes. The keratin in hair, wool, and hornis tightly packed in an a-helix, and it is different from the keratin inthe chicken feathers (b-helix) because it contains a higher amountof cysteine (Moreira et al., 2007), which makes a-keratins even

more difficult to hydrolyze than b-keratins. An appropriate pre-treatment is required, therefore, prior to biogas production forkeratin-rich wastes to be utilized for biogas production.

2.3. Feedstock with inhibitors

Inhibitors are compounds or substances that slow down or stopthe ability of enzymes, catalysts, or microorganisms to transformreactants to desired products. From the biogas pretreatment per-spective, inhibitors could be classified based on their biodegrad-ability or the point at which they enter the biogas productionprocess. Based on the biodegradability, some inhibitors such as fur-fural, long chain fatty acids, and organic pollutants are biodegrad-able, while others such as heavy metals are non-biodegradable(Chen et al., 2008). Under optimal conditions, microbes in thedigesters can digest feedstocks containing low concentrations ofbiodegradable inhibitors, while the accumulation of non-biodegradable inhibitors can lead to the death of microbes in thedigester (Chen et al., 2008). The other categories of inhibitorseither come into the digester as part of the feed (such as fruit fla-vors, aromatic compounds) or are released during digestion or pre-treatment (such as NH3, long chain fatty acids). Feedstocks withinhibition based on point of entry or release are discussed in thefollowing subsections.

2.3.1. Inhibitors as part of the feedstock-fruit wastesThe Food and Agricultural Organization (FAO) of the United

Nations 2013 statistics showed that the global fruit productionhas increased by 3% annually in the past decade, with 804.4 milliontons produced in 2012 (FAO, 2013). The FAO also reported that 36–56 % of the produced fruits end up as waste (FAO, 2011). Fruitwaste could be generated as a result of poor management or stor-age, diseases or infestation as well as mechanical damage duringharvesting or fruit processing. Fruit processing generates two typesof waste: solid waste that consists of peels, skin, seed, stones, etc.,and liquid waste from juice and wash-waters (Gustavsson et al.,2011). The average VS of fruit waste consists of 78.3% carbohy-drates, 8.5 % protein, and 6% fat (Schnürer and Jarvis, 2010), sothe theoretical yield of methane from fruit waste is 0.43 Nm3-CH4/kg-VS or 0.04 Nm3-CH4/kg fruit waste. Using the estimatedglobal fruit production for the past decade, the estimated fruit pro-duction in 2016 would be 905.4 million tons, and if 46% ends up aswaste, 16.7 � 109 Nm3 of methane could be produced from thefruit waste. However, the actual methane production from fruitwaste is much less than this theoretical yield because of the pres-ence of inhibitory flavor compounds affecting the AD process neg-atively (Wikandari et al., 2013).

These flavor compounds can be classified into esters, alcohols,aldehydes, ketones, lactones, and terpenoids. Compounds likegeraniol, thymol, carvacrol, etc. belonging to the terpenes familyhave been reported as cytotoxic (Rosato et al., 2007). Wikandariet al. (2013) found that 0.5% of terpenoids, aldehydes, and alcoholsin an AD feedstock can reduce the methane production by 99 %.Limonene, a terpenoid and a major component of peel oil fromcitrus fruits, has been reported to cause failure in a continuousmesophilic AD process at a concentration of 400 lL/L (Mizukiet al., 1990), while at a concentration of 450 lL/L it can lead tothe failure of a thermophilic AD process (Forgács, 2012). Due tothese flavor compounds, it is essential for fruit waste to be pre-treated before it is used for biogas production.

2.3.2. Inhibitors released during digestion – wastes from foodprocessing

Wastes from the food processing industries are a good choicefor biogas production because the waste produced consists mostlyof the organic fraction of the raw materials used by the industry.


These complex polymers are tightly packed and highly crosslinkedwith hydrogen bonds, disulfide bonds (for keratin wastes), andhydrophobic interactions (Daroit et al., 2009). Hydrolysis is therate-limiting step for these feedstocks; therefore, it is necessaryto overcome the recalcitrant nature and make them accessible tothe microorganisms for effective biogas production. Some of thesefeedstocks and their challenges as well as how to overcome thesechallenges are discussed below in order to ascribe value to them.

2.2.1. Lignocellulosic wastesLignocellulosic wastes are one of the most abundant renewable

organic resources on earth with a yearly production of approxi-mately 200 billion tons (Zhang, 2008). The increasing expansionof the agricultural industry and its activities has led to an increasein the production and the consequent accumulation of a large vol-ume of these wastes. Forestry by-products, woody crops, andmunicipal solid waste also contribute to an enormous amount ofthese waste streams. About 60% of the dry weight of the averagemunicipal solid waste is composed of lignocelluloses (Holtzappleet al., 1992). The abundance of lignocellulosic wastes makes thema potential feedstock for biogas production, and it has beenreported that these kinds of wastes can add up to a significantenergy value of approximately 1500 MJ/year (Kang et al., 2014)depending on the yield from the biomass, total cultivable area,and the technology used. Although these wastes are rich in fibers,readily available, and have a significant energy value, they have alow biogas yield due to their resistance to microbial attack.

Lignocellulosic wastes are difficult to digest as a result of matrixpolymers (hemicellulose, pectins, and lignin) surrounding the cel-lulosic microfibrils in the plant cell wall (Himmel and Picataggio,2009). The degree of resistance of these wastes to microbial attackvaries depending on the type of lignocellulose, various compart-ments of the cell wall, and cell age, while it is also affected by pro-cessing phenomena such as drying and heating (Taherzadeh andJeihanipour, 2012). However, effective use of lignocellulosic bio-mass for biogas production requires a hydrolysis step in order toopen up the structure and increase the accessibility for cellulosedegrading microorganisms. It is only after this step that the mate-rial will be ready for conversion. The rate and extent to which cel-lulose can be hydrolyzed depends on the connection between thelignin, hemicellulose, and cellulose and also the degree of crys-tallinity in the cellulose itself. A potential solution is an innovativepretreatment method to increase the digestibility and ascribevalue to the lignocellulosic wastes.

2.2.2. Keratin-rich wastesKeratin-rich wastes such as chicken feather, wool, hair, nails,

horns, hooves, and claws are produced worldwide by the poultry,wool, meat, and fish industries. For instance, the world’s averagestock of chicken is about 22 billion (FAOSTAT, 2013); thus, thepoultry industries produce a tremendous amount of feathersyearly. Moreover, the global production of wool amounts to 2.1million tons per year (IWTO, 2015). Assuming that all the insolubleprotein (keratin) are converted into soluble protein, the methanepotential of keratin wastes is as high as 0.496 Nm3/kgVS(Angelidaki and Sanders, 2004). However, the methane yieldobtained is usually low due to the recalcitrant structure of keratin.Keratin is an insoluble structural protein where the polypeptideschain is tightly packed and highly cross-linked with disulfidebonds, hydrogen bonds, and hydrophobic interactions (Daroitet al., 2009). This structure makes the protein insoluble and resis-tant to enzymatic attack, which is a major hindrance in the biolog-ical processing of these wastes. The keratin in hair, wool, and hornis tightly packed in an a-helix, and it is different from the keratin inthe chicken feathers (b-helix) because it contains a higher amountof cysteine (Moreira et al., 2007), which makes a-keratins even

more difficult to hydrolyze than b-keratins. An appropriate pre-treatment is required, therefore, prior to biogas production forkeratin-rich wastes to be utilized for biogas production.

2.3. Feedstock with inhibitors

Inhibitors are compounds or substances that slow down or stopthe ability of enzymes, catalysts, or microorganisms to transformreactants to desired products. From the biogas pretreatment per-spective, inhibitors could be classified based on their biodegrad-ability or the point at which they enter the biogas productionprocess. Based on the biodegradability, some inhibitors such as fur-fural, long chain fatty acids, and organic pollutants are biodegrad-able, while others such as heavy metals are non-biodegradable(Chen et al., 2008). Under optimal conditions, microbes in thedigesters can digest feedstocks containing low concentrations ofbiodegradable inhibitors, while the accumulation of non-biodegradable inhibitors can lead to the death of microbes in thedigester (Chen et al., 2008). The other categories of inhibitorseither come into the digester as part of the feed (such as fruit fla-vors, aromatic compounds) or are released during digestion or pre-treatment (such as NH3, long chain fatty acids). Feedstocks withinhibition based on point of entry or release are discussed in thefollowing subsections.

2.3.1. Inhibitors as part of the feedstock-fruit wastesThe Food and Agricultural Organization (FAO) of the United

Nations 2013 statistics showed that the global fruit productionhas increased by 3% annually in the past decade, with 804.4 milliontons produced in 2012 (FAO, 2013). The FAO also reported that 36–56 % of the produced fruits end up as waste (FAO, 2011). Fruitwaste could be generated as a result of poor management or stor-age, diseases or infestation as well as mechanical damage duringharvesting or fruit processing. Fruit processing generates two typesof waste: solid waste that consists of peels, skin, seed, stones, etc.,and liquid waste from juice and wash-waters (Gustavsson et al.,2011). The average VS of fruit waste consists of 78.3% carbohy-drates, 8.5 % protein, and 6% fat (Schnürer and Jarvis, 2010), sothe theoretical yield of methane from fruit waste is 0.43 Nm3-CH4/kg-VS or 0.04 Nm3-CH4/kg fruit waste. Using the estimatedglobal fruit production for the past decade, the estimated fruit pro-duction in 2016 would be 905.4 million tons, and if 46% ends up aswaste, 16.7 � 109 Nm3 of methane could be produced from thefruit waste. However, the actual methane production from fruitwaste is much less than this theoretical yield because of the pres-ence of inhibitory flavor compounds affecting the AD process neg-atively (Wikandari et al., 2013).

These flavor compounds can be classified into esters, alcohols,aldehydes, ketones, lactones, and terpenoids. Compounds likegeraniol, thymol, carvacrol, etc. belonging to the terpenes familyhave been reported as cytotoxic (Rosato et al., 2007). Wikandariet al. (2013) found that 0.5% of terpenoids, aldehydes, and alcoholsin an AD feedstock can reduce the methane production by 99 %.Limonene, a terpenoid and a major component of peel oil fromcitrus fruits, has been reported to cause failure in a continuousmesophilic AD process at a concentration of 400 lL/L (Mizukiet al., 1990), while at a concentration of 450 lL/L it can lead tothe failure of a thermophilic AD process (Forgács, 2012). Due tothese flavor compounds, it is essential for fruit waste to be pre-treated before it is used for biogas production.

2.3.2. Inhibitors released during digestion – wastes from foodprocessing

Wastes from the food processing industries are a good choicefor biogas production because the waste produced consists mostlyof the organic fraction of the raw materials used by the industry.


These wastes usually have a high biological oxygen demand (BOD)and a high chemical oxygen demand (COD), with varying contentsof suspended solids depending on their source (Russ and Meyer-Pittroff, 2004). The wastes from food processing are either solidor liquid, and most of the solid part can be classified as a lignocel-lulosic residue (Table 1). As lignocellulosic wastes have been dis-cussed in Section 2.2.1, in this section only the liquid fraction ofthe waste from food processing which is associated with a highvolume of production and negative environmental effects, will bediscussed. More specifically, the liquid waste from the oil industryhas attracted a lot of attention because of its potential for biogasproduction and its negative effect on the environment.

Global vegetable oil production increased from 90.5 � 106 MTin 2000 to 160.59 � 106 MT in 2012 (Statista, 2016) with oil-palm, soybean, rapeseed, and sunflower accounting for 75% ofthe global oil production (FAO, 2013). Over this period, the palmoil production increased from 21.75 � 106 MT to 51.74 � 106 MTmaking palm oil to be the most produced vegetable oil in theworld. The production of vegetable oil is usually associated withthe production of wastewater. Taking palm oil as an example, forevery ton of crude palm oil produced, 2.5–3.75 tons of palm oilwastewater called palm oil mill effluent (POME) is produced(Chin et al., 2013). POME is a thick brownish liquid generated fromthe clarification and sterilization of palm oil during the oil extrac-tion process in the palm oil mills. It is considered to be one of themost polluting effluents from the agricultural industry because ofits properties such as high COD (15–100 g/L), high BOD (10.25–43.75 g/L), and acidic pH (3.4–5.2) (Ahmad et al., 2011). Biogas pro-duction from POME could solve the problem of treating POME forthe environment and also generating an additional valuable pro-duct (biogas) for the oil-palm industries. Using 50 g/L as an averageCOD of POME, every 1 m3 of POME can produce 18.78 Nm3 of CH4

or 37.56 Nm3 of biogas. Taking into account the production of3.125 tons of POME per ton of the palm oil, theoretically2.9 � 1015 Nm3 of CH4 or 5.8 � 1015 Nm3 of biogas could have beenproduced from the palm oil produced globally in 2012. With thistremendous potential, it is important both from energy and envi-ronmental perspectives that POME is properly utilized.

Despite the tremendous potential of producing biogas fromPOME or other waste water from oil production, in reality mostAD digesters fail when fed continuously with daily COD load ofbetween 35 and 65 g/L (Zinatizadeh et al., 2007), which is challeng-ing as the COD of these feedstocks could be as high as 100 g/L(Ahmad et al., 2011). One of the reasons for this failure is thatPOME or other waste water from the oil production contains poly-meric fat, which are broken down during hydrolysis to long chainfatty acids such as stearic acid, oleic acid, and arachidic acid, andlong chain fatty acid accumulation has been shown to inhibit theAD process (Angelidaki and Ahring, 1992). Another reason for thefailure when using POME is the accumulation of volatile fatty acids(VFAs) in the digester. Thus, to fully harness the potential of wastewater from the oil production, such as POME, for biogas produc-tion, it is important to come up with innovative strategies.

2.3.3. Inhibitors released during digestion – ammonia inhibition fromprotein wastes

Presently, about 1 million tons of protein-rich wastes are pro-duced annually (Kovács et al., 2013), and about 30% of the weightof animals processed for food ends up as slaughterhouse waste(Kovács et al., 2015). These wastes mainly contain manure andblood; they are highly rich in protein, readily available, and canbe easily digested, which makes them a potential feedstock for bio-gas production. The theoretical methane potential of proteinwastes is 0.496 Nm3/kgVS (Angelidaki and Sanders, 2004), andslaughterhouse wastes have a theoretical potential of about0.70 Nm3 CH4/kgVS (Schnürer and Jarvis, 2010); however, since

they have a low C/N ratio, digestion of these feedstocks as a singlesubstrate is usually unsuccessful due to the accumulation ofammonia during digestion. During the breakdown of protein-richwastes, depending on the pH and temperature, free ammonia(NH3) or ammonium ion (NH4

+) are produced, which at higher con-centrations inhibit the anaerobic digestion process (Yenigün andDemirel, 2013). Studies have shown that inhibition is due to thepresence of free ammonia rather than ammonium ion(Kayhanian, 1999), with a free ammonia nitrogen (FAN) concentra-tion of 150 mg/L causing complete inhibition (Yenigün andDemirel, 2013). Therefore, proper treatment of these feedstocksbefore digestion and control of ammonia concentration duringthe digestion process is necessary to maximize the potential ofthese wastes for biogas production.

2.3.4. Inhibitors released during gasificationDuring feedstock gasification, several inhibitors can be gener-

ated mainly because of the feedstock composition and the operat-ing conditions. Some of these inhibitors are char, H2S, NH3, NOx,ethane, ethylene, and acetylene as well as heavy metals and parti-cles. A high concentration of these components is known to causecell dormancy, enzyme inhibition, low cell growth, and limitedhydrogen uptake. The amounts of these components can be limitedby cleaning the syngas with cyclones, reforming, and by controllingthe operating conditions and the feedstock composition.

3. Pretreatment methods

3.1. Gasification of indigestible feedstock

During gasification, the feedstock is gasified by exposure to hightemperatures (1000–1200 �C) and an oxidizing agent. Steam, oxy-gen, and air are mainly used as oxidizing streams. The producedgas (syngas) is mainly composed of H2, CO, and CO2, which canthereafter be fermented for biogas production. In addition, at theend of the gasification, a residual ash, in the form of slang, is leftin the gasifier. The composition of the syngas and the ash isaffected greatly by the feedstock composition and the process tem-perature. For example, feedstocks with high amount of carbon gen-erate syngas with high CO content. Furthermore, at loweroperating temperatures below 1000 �C, the gas produced containshigher amounts of impurities. The remaining ashes, from thermo-chemical processes, have been mainly used as building material inroad construction. However, ashes consist of toxic componentssuch as heavy metals, which pose an environmental threat. There-fore, alternative uses have been considered. For example, it hasbeen reported that the addition of ash enhanced the biogas produc-tion, benefitted the alkalinity, and pH of anaerobic digesters (Banksand Lo, 2003).

One of the main advantages of gasification as a pretreatmentprocess is that it can treat a wide variety of heterogeneous feed-stocks. Furthermore, gasification has a high conversion rate, andboth syngas and the remaining ash can be used for biogas produc-tion (Banks and Lo, 2003). In addition, syngas production can beeconomically efficient if parameters such as pressure, gasificationagent, syngas and feedstock composition are taken into considera-tion. More specifically, an economic assessment showed that it ispossible to produce syngas with an approximate cost of $ 0.25/Nm3 based only on biomass feedstock. Moreover, the productioncosts can almost be reduced by half when considering co-gasification of biomass and coal (Trippe et al., 2011). On the otherhand, another study argues that challenges such as high opera-tional costs should be addressed in order to upgrade the combinedgasification and fermentation process to a commercial scale(Richter et al., 2013).


These wastes usually have a high biological oxygen demand (BOD)and a high chemical oxygen demand (COD), with varying contentsof suspended solids depending on their source (Russ and Meyer-Pittroff, 2004). The wastes from food processing are either solidor liquid, and most of the solid part can be classified as a lignocel-lulosic residue (Table 1). As lignocellulosic wastes have been dis-cussed in Section 2.2.1, in this section only the liquid fraction ofthe waste from food processing which is associated with a highvolume of production and negative environmental effects, will bediscussed. More specifically, the liquid waste from the oil industryhas attracted a lot of attention because of its potential for biogasproduction and its negative effect on the environment.

Global vegetable oil production increased from 90.5 � 106 MTin 2000 to 160.59 � 106 MT in 2012 (Statista, 2016) with oil-palm, soybean, rapeseed, and sunflower accounting for 75% ofthe global oil production (FAO, 2013). Over this period, the palmoil production increased from 21.75 � 106 MT to 51.74 � 106 MTmaking palm oil to be the most produced vegetable oil in theworld. The production of vegetable oil is usually associated withthe production of wastewater. Taking palm oil as an example, forevery ton of crude palm oil produced, 2.5–3.75 tons of palm oilwastewater called palm oil mill effluent (POME) is produced(Chin et al., 2013). POME is a thick brownish liquid generated fromthe clarification and sterilization of palm oil during the oil extrac-tion process in the palm oil mills. It is considered to be one of themost polluting effluents from the agricultural industry because ofits properties such as high COD (15–100 g/L), high BOD (10.25–43.75 g/L), and acidic pH (3.4–5.2) (Ahmad et al., 2011). Biogas pro-duction from POME could solve the problem of treating POME forthe environment and also generating an additional valuable pro-duct (biogas) for the oil-palm industries. Using 50 g/L as an averageCOD of POME, every 1 m3 of POME can produce 18.78 Nm3 of CH4

or 37.56 Nm3 of biogas. Taking into account the production of3.125 tons of POME per ton of the palm oil, theoretically2.9 � 1015 Nm3 of CH4 or 5.8 � 1015 Nm3 of biogas could have beenproduced from the palm oil produced globally in 2012. With thistremendous potential, it is important both from energy and envi-ronmental perspectives that POME is properly utilized.

Despite the tremendous potential of producing biogas fromPOME or other waste water from oil production, in reality mostAD digesters fail when fed continuously with daily COD load ofbetween 35 and 65 g/L (Zinatizadeh et al., 2007), which is challeng-ing as the COD of these feedstocks could be as high as 100 g/L(Ahmad et al., 2011). One of the reasons for this failure is thatPOME or other waste water from the oil production contains poly-meric fat, which are broken down during hydrolysis to long chainfatty acids such as stearic acid, oleic acid, and arachidic acid, andlong chain fatty acid accumulation has been shown to inhibit theAD process (Angelidaki and Ahring, 1992). Another reason for thefailure when using POME is the accumulation of volatile fatty acids(VFAs) in the digester. Thus, to fully harness the potential of wastewater from the oil production, such as POME, for biogas produc-tion, it is important to come up with innovative strategies.

2.3.3. Inhibitors released during digestion – ammonia inhibition fromprotein wastes

Presently, about 1 million tons of protein-rich wastes are pro-duced annually (Kovács et al., 2013), and about 30% of the weightof animals processed for food ends up as slaughterhouse waste(Kovács et al., 2015). These wastes mainly contain manure andblood; they are highly rich in protein, readily available, and canbe easily digested, which makes them a potential feedstock for bio-gas production. The theoretical methane potential of proteinwastes is 0.496 Nm3/kgVS (Angelidaki and Sanders, 2004), andslaughterhouse wastes have a theoretical potential of about0.70 Nm3 CH4/kgVS (Schnürer and Jarvis, 2010); however, since

they have a low C/N ratio, digestion of these feedstocks as a singlesubstrate is usually unsuccessful due to the accumulation ofammonia during digestion. During the breakdown of protein-richwastes, depending on the pH and temperature, free ammonia(NH3) or ammonium ion (NH4

+) are produced, which at higher con-centrations inhibit the anaerobic digestion process (Yenigün andDemirel, 2013). Studies have shown that inhibition is due to thepresence of free ammonia rather than ammonium ion(Kayhanian, 1999), with a free ammonia nitrogen (FAN) concentra-tion of 150 mg/L causing complete inhibition (Yenigün andDemirel, 2013). Therefore, proper treatment of these feedstocksbefore digestion and control of ammonia concentration duringthe digestion process is necessary to maximize the potential ofthese wastes for biogas production.

2.3.4. Inhibitors released during gasificationDuring feedstock gasification, several inhibitors can be gener-

ated mainly because of the feedstock composition and the operat-ing conditions. Some of these inhibitors are char, H2S, NH3, NOx,ethane, ethylene, and acetylene as well as heavy metals and parti-cles. A high concentration of these components is known to causecell dormancy, enzyme inhibition, low cell growth, and limitedhydrogen uptake. The amounts of these components can be limitedby cleaning the syngas with cyclones, reforming, and by controllingthe operating conditions and the feedstock composition.

3. Pretreatment methods

3.1. Gasification of indigestible feedstock

During gasification, the feedstock is gasified by exposure to hightemperatures (1000–1200 �C) and an oxidizing agent. Steam, oxy-gen, and air are mainly used as oxidizing streams. The producedgas (syngas) is mainly composed of H2, CO, and CO2, which canthereafter be fermented for biogas production. In addition, at theend of the gasification, a residual ash, in the form of slang, is leftin the gasifier. The composition of the syngas and the ash isaffected greatly by the feedstock composition and the process tem-perature. For example, feedstocks with high amount of carbon gen-erate syngas with high CO content. Furthermore, at loweroperating temperatures below 1000 �C, the gas produced containshigher amounts of impurities. The remaining ashes, from thermo-chemical processes, have been mainly used as building material inroad construction. However, ashes consist of toxic componentssuch as heavy metals, which pose an environmental threat. There-fore, alternative uses have been considered. For example, it hasbeen reported that the addition of ash enhanced the biogas produc-tion, benefitted the alkalinity, and pH of anaerobic digesters (Banksand Lo, 2003).

One of the main advantages of gasification as a pretreatmentprocess is that it can treat a wide variety of heterogeneous feed-stocks. Furthermore, gasification has a high conversion rate, andboth syngas and the remaining ash can be used for biogas produc-tion (Banks and Lo, 2003). In addition, syngas production can beeconomically efficient if parameters such as pressure, gasificationagent, syngas and feedstock composition are taken into considera-tion. More specifically, an economic assessment showed that it ispossible to produce syngas with an approximate cost of $ 0.25/Nm3 based only on biomass feedstock. Moreover, the productioncosts can almost be reduced by half when considering co-gasification of biomass and coal (Trippe et al., 2011). On the otherhand, another study argues that challenges such as high opera-tional costs should be addressed in order to upgrade the combinedgasification and fermentation process to a commercial scale(Richter et al., 2013).


3.2. Hard-to-digest feedstocks and feedstocks with inhibitors

Due to the recalcitrant nature and strong elasticity, recalcitrantfeedstock can only result in a low biogas yield. Furthermore, feed-stocks such as fruit wastes are easily degraded but also result inlow yield due to the presence of inhibitors. Therefore, these wastesare pretreated prior to the biogas production. The main purpose ofpretreatment for lignocellulosic wastes is to break the lignin layerthat protects the cellulose and hemicellulose, in order to make thefeedstock more accessible for digestion. Pretreatment also helps todecrease the crystallinity of cellulose and to increase the porosity.For other feedstocks like keratin-rich wastes, if the crosslinking

between the polypeptides chain breaks, the keratin is more acces-sible and easier to digest. Another purpose of pretreatment is toremove the inhibitors, which are present in potential feedstocks.Several pretreatment methods have been reported in detail aimingto make these feedstocks viable to digestion by microorganisms,thereby increasing the biogas yield (Table 2). Some pretreatmentslead to changes in the feedstock composition by dissolving thehemicellulose or lignin or both, while some other pretreatmentsresult in changes in the cellulose crystallinity, molecular weight,biomass porosity, and particle size (Johnson and Elander, 2009).Specific pretreatment methods suitable for (i) increasing accessiblesurface area, (ii) decrystallization of cellulose, (iii) hydrolysis of

Table 2Conventional pretreatment processes for biogas production.

Pretreatment Processes Function Economic challenges

Physicalpretreatmenta,b,c

Milling, irradiation, pyrolysis, ultrasonic Increase of surface area and pore sizeDisruption of biomass structure and decrease ofcrystallinityReduction of degree of polymerization

Consumes high electricenergyp

Equipment repairs can bevery expensive

Rapid decompressionpretreatmentd,e

High pressure steaming, steam explosionammonia fibre explosion (AFEX)CO2 explosion, SO2 explosion

Partial hydrolyzation of hemicelluloseDisruption and defibration of biomassPartial decrystallizationPartial lignin solubilizationIncrease of hydrolysis rate

Effective recovery ofammonia can be expensivec

Autohydrolysispretreatmente

Liquid hot water batch, liquid hot water percolation Hydrolyzation of cellulose at very high andlower temperatureHigh removal of hemicellulose and lignin

Energy cost is relativelymoderatec

Acid pretreatmente,f Sulfuric, hydrochloric, phosphoric, nitric and carbonicacid

Enhancement of hemicellulose hydrolysis Recovery of acid isexpensiveq

Corrosion-resistantequipment is costlyr

Alkaline pretreatmenta,e Sodium hydroxide, ammonia, lime, potassium hydroxide Increase of internal surface areaReduction in degree of polymerization,DecrystallizationDisruption of lignin structureBreaking of structural linkages between ligninand Carbohydrates

High cost of bases

Solvent pretreatmentg,h Organic solvents e.g. methanol, ethanol, acetone,ethylene glycol and hydrofurfuryl alcoholCellulose-dissolving solvents e.g. cadozen, concentratedmineral acids, DMSO*, NMMO** and zinc chloride

Solubilization of lignin High cost of solvents

Effective recovery of solventis costly

Leachingi Organic solvents e.g hexane, diethyl ether,dichloromethane or ethyl acetate

Removal of inhibitors in citrus waste

Supercritical fluidpretreatmentj

Utilizing water, CO2 or ammonia Removal of ligninIncrease of biomass digestibility

Highly expensivec

Oxidative pretreatmentd Oxidizing compounds e.g hydrogen peroxide orperacetic acid

Removal of hemicellulose and lignin. Increaseof cellulose accessibility

High cost of chemicalt

Biologicalpretreatmentf,g,k,l,m,n,o

White rot fungi, brown rot fungi, Enzyme products(cellulase, hemicellulase and b-glucosidase)Bacillus sp., Aspergillus sp. and alkaline endopeptidase

Degradation of lignin, degradation of celluloseand hemicelluloseHydrolyzation of keratin

High cost of enzymesr

* Dimethyl sulfoxide.** N-Methylmorpholine N-oxide.a Bochmann et al. (2013).b Onyeche et al. (2002).c Taherzadeh and Karimi (2008).d Hendriks and Zeeman (2009).e Johnson and Elander (2009).f Singh et al. (2014).g Hsu (1996).h Sun and Cheng (2002).i Wikandari et al. (2015).j Li and Kiran (1988).k Fellahi et al. (2014).l Forgács et al. (2011a).

m Mazotto et al. (2013).n Patinvoh et al. (2016).o Zaghloul et al. (2011).p Wang et al. (2016).q Xiao and Clarkson (1997).r Kumar and Murthy (2011).s Kabir et al. (2015).t Li et al. (2013).


hemicellulose, (iv) removal of lignin, (v) hydrolysis of keratin, and(vi) removal of inhibitory compounds are discussed in the follow-ing subsections.

3.2.1. Accessible surface area and decrystallizationComminution, such as ball milling, is a physical pretreatment

method that increases the feedstock surface area and decrystallizescellulose. Silva et al. (Silva et al., 2012) investigated the effect ofgrinding on wheat straw. The results showed that ball milling sam-ples yield 46% total carbohydrates and 72% glucose as a result ofthe reduction in the cellulose crystallinity from 22% to 13%. Ballmilling was also applied to non-degraded digestate in order to feedit back into the digestion process (Lindner et al., 2015). Enhancedmethane production by 9% was reported in the case of two-stagemaize sillage digestate, and an increase of 17% was detected whenusing two-stage hay/straw digestate. Irradiation is also an effectivepretreatment method that disrupts the structure of the biomasscell wall and decreases the crystallinity of the cellulose (Johnsonand Elander, 2009). The effect of microwave pretreatment wasevaluated on the solubilization of microalgae as a substrate for bio-gas production, and it was observed that at a specific energy of21.8, 43.6, and 65.4 MJ/kgTS, the pretreatment increased the biogasproduction rate by 27–75% and the biogas yield by 12–78%, accord-ing to the results applied from the BMP assays. These pretreat-ments are suitable methods for increasing the surface area anddecrystallizing recalcitrant feedstocks; however, they are not eco-nomically feasible due to the high energy consumption. Therefore,they are not viable as stand-alone pretreatments for industrialapplications but can be combined with other pretreatments thatare cost effective.

3.2.2. Hydrolysis of hemicelluloseDilute acid pretreatment at controlled conditions can solubilize

almost all the hemicellulose from a biomass and thereby increasethe accessibility of microorganisms to cellulose. This methodbreaks the intramolecular bonds between the lignin, hemicellu-lose, and cellulose in the cell wall and hydrolyzes the hemicellu-lose (Taherzadeh and Jeihanipour, 2012). However, at a hightemperature and acid concentration, undesirable dehydrationoccurs resulting in the hemicellulose and cellulose being degradedinto different types of inhibitory compounds, such as furfural(Taherzadeh et al., 1997). Hence, it is necessary to carry out thepretreatment at mild conditions to prevent excessive sugar degra-dation. Un-catalyzed steam explosion is also an alternative pre-treatment method for the hydrolysis of hemicellulose. However,the xylose yield from the hemicellulose is usually not higher than65% of the theoretical yield due to the excessive sugar degradation(Brownell and Saddler, 1987). Liquid hot water pretreatment at alower temperature (200–230 �C) has also been reported as aneffective method for the hydrolysis of hemicellulose (Johnsonand Elander, 2009). A higher yield of soluble sugars is possible withthis method compared to the un-catalyzed steam explosionmethod. However, the liquid hot water pretreatment method liber-ates the sugars in the oligometric form (Johnson and Elander,2009).

3.2.3. Lignin removalAlkaline pretreatment is a suitable method for solubilizing the

lignin; it can be carried out at different concentrations of lime,sodium hydroxide, or ammonia. This method causes swelling ofthe fibers, which results in an increased accessible surface area,reduced degree of polymerization, and decrystallization of the cel-lulose. Alkaline treatment breaks the intramolecular bondsbetween the lignin, hemicellulose, and cellulose and disrupts thelignin structure (Hsu, 1996). Lime (Ca(OH)2) pretreatment in thepresence of water (9 ml/g dry biomass) was investigated for 2 h

on switchgrass at 100–120 �C with 0.1 g Ca(OH)2/g dry biomassloading (Chang et al., 1997). The results showed that 29% ligninwas solubilized. Moreover, Sambusiti et al. (2013) investigatedthe effect of alkaline (NaOH) pretreatment on ensiled sorghum for-age in semi-continuous digesters. It was observed that pretreat-ment with 10 g NaOH/100gTS increased the methane yield by25% compared to untreated sorghum and did not cause any inhibi-tion of the process. Organic or organic-aqueous solvent mixturesutilizing ethanol, benzene, ethylene glycol, or butanol have alsobeen reported as being effective for lignin removal (Taherzadehand Karimi, 2008). Studies have also shown that white-rot fungiproduce enzymes that are capable of extensive degradation of lig-nin. As a result, the biomass digestibility is increased (Adney et al.,2009), although there is a loss of cellulose and hemicellulose dur-ing this process (Hatakka, 1983).

3.2.4. Hydrolysis of keratinPretreatment of keratin-rich waste using a strong acid, alkaline

hydrolysis, and other physicochemical methods results in severedegradation and destruction of the keratin (Barone et al., 2006).Hence, recent research focuses on the biological pretreatmentmethods, which have been reported as successful for keratindegradation. Microorganisms (bacteria and fungi) produceenzymes that can break down the keratin and make it soluble.The keratin-rich feedstock is inoculated with appropriate bacteriaor fungi cultures and incubated for days or weeks depending onthe activity of the microorganism used and the properties of thesubstrate. Several microorganisms such as Bacillus sp. (Cai et al.,2008; Fellahi et al., 2014; Forgács et al., 2011a; Patinvoh et al.,2016; Zaghloul et al., 2011) and Aspergillus sp. (Mazotto et al.,2013) have been investigated and reported as being effective forhydrolyzing keratin.

3.2.5. Removal of inhibitors present in the feedstocks-fruit flavorsInhibitory compounds that are part of the feedstocks used for

the biogas production such as flavor compounds present in fruitshave been shown to be adversely affecting the anaerobic digestionprocess even at low concentrations. Steam explosion has beenreported to successfully remove these types of inhibitors fromcitrus waste, while also breaking up the lignocellulose structurein the feedstock (Forgács et al., 2011b). Steam explosion is a ther-mal pretreatment method that requires a very high pressure of upto 60 bar and a temperature of up to 150 �C. Combined chemicaland steam explosion pretreatment methods have been found toreduce the required temperature and residence time of the pre-treatment, while also increasing the degradation rate in contrastto when chemical or steam explosion methods are used separately(Forgács et al., 2011b).

4. Innovative non-pretreatment strategies

Although several pretreatment methods have emerged over thepast few years, certain challenges such as high cost, harmful envi-ronmental by-products, low biogas yield, and high energy require-ment still persist. To address these challenges, some innovativenon-pretreatment strategies aiming to improve the use of the read-ily available and low cost feedstocks for biogas production are dis-cussed in this section. The strategies discussed include integratedbiogas production, innovative digester design, co-digestion, andbio-augmentation. An integrated biogas production strategyaddresses the cost challenge, the innovative digester design strat-egy addresses the inhibition challenge, and the co-digestion strat-egy addresses both the challenge of low yield and inhibitors, whilethe bio-augmentation strategy addresses the challenge ofinhibitors.


hemicellulose, (iv) removal of lignin, (v) hydrolysis of keratin, and(vi) removal of inhibitory compounds are discussed in the follow-ing subsections.

3.2.1. Accessible surface area and decrystallizationComminution, such as ball milling, is a physical pretreatment

method that increases the feedstock surface area and decrystallizescellulose. Silva et al. (Silva et al., 2012) investigated the effect ofgrinding on wheat straw. The results showed that ball milling sam-ples yield 46% total carbohydrates and 72% glucose as a result ofthe reduction in the cellulose crystallinity from 22% to 13%. Ballmilling was also applied to non-degraded digestate in order to feedit back into the digestion process (Lindner et al., 2015). Enhancedmethane production by 9% was reported in the case of two-stagemaize sillage digestate, and an increase of 17% was detected whenusing two-stage hay/straw digestate. Irradiation is also an effectivepretreatment method that disrupts the structure of the biomasscell wall and decreases the crystallinity of the cellulose (Johnsonand Elander, 2009). The effect of microwave pretreatment wasevaluated on the solubilization of microalgae as a substrate for bio-gas production, and it was observed that at a specific energy of21.8, 43.6, and 65.4 MJ/kgTS, the pretreatment increased the biogasproduction rate by 27–75% and the biogas yield by 12–78%, accord-ing to the results applied from the BMP assays. These pretreat-ments are suitable methods for increasing the surface area anddecrystallizing recalcitrant feedstocks; however, they are not eco-nomically feasible due to the high energy consumption. Therefore,they are not viable as stand-alone pretreatments for industrialapplications but can be combined with other pretreatments thatare cost effective.

3.2.2. Hydrolysis of hemicelluloseDilute acid pretreatment at controlled conditions can solubilize

almost all the hemicellulose from a biomass and thereby increasethe accessibility of microorganisms to cellulose. This methodbreaks the intramolecular bonds between the lignin, hemicellu-lose, and cellulose in the cell wall and hydrolyzes the hemicellu-lose (Taherzadeh and Jeihanipour, 2012). However, at a hightemperature and acid concentration, undesirable dehydrationoccurs resulting in the hemicellulose and cellulose being degradedinto different types of inhibitory compounds, such as furfural(Taherzadeh et al., 1997). Hence, it is necessary to carry out thepretreatment at mild conditions to prevent excessive sugar degra-dation. Un-catalyzed steam explosion is also an alternative pre-treatment method for the hydrolysis of hemicellulose. However,the xylose yield from the hemicellulose is usually not higher than65% of the theoretical yield due to the excessive sugar degradation(Brownell and Saddler, 1987). Liquid hot water pretreatment at alower temperature (200–230 �C) has also been reported as aneffective method for the hydrolysis of hemicellulose (Johnsonand Elander, 2009). A higher yield of soluble sugars is possible withthis method compared to the un-catalyzed steam explosionmethod. However, the liquid hot water pretreatment method liber-ates the sugars in the oligometric form (Johnson and Elander,2009).

3.2.3. Lignin removalAlkaline pretreatment is a suitable method for solubilizing the

lignin; it can be carried out at different concentrations of lime,sodium hydroxide, or ammonia. This method causes swelling ofthe fibers, which results in an increased accessible surface area,reduced degree of polymerization, and decrystallization of the cel-lulose. Alkaline treatment breaks the intramolecular bondsbetween the lignin, hemicellulose, and cellulose and disrupts thelignin structure (Hsu, 1996). Lime (Ca(OH)2) pretreatment in thepresence of water (9 ml/g dry biomass) was investigated for 2 h

on switchgrass at 100–120 �C with 0.1 g Ca(OH)2/g dry biomassloading (Chang et al., 1997). The results showed that 29% ligninwas solubilized. Moreover, Sambusiti et al. (2013) investigatedthe effect of alkaline (NaOH) pretreatment on ensiled sorghum for-age in semi-continuous digesters. It was observed that pretreat-ment with 10 g NaOH/100gTS increased the methane yield by25% compared to untreated sorghum and did not cause any inhibi-tion of the process. Organic or organic-aqueous solvent mixturesutilizing ethanol, benzene, ethylene glycol, or butanol have alsobeen reported as being effective for lignin removal (Taherzadehand Karimi, 2008). Studies have also shown that white-rot fungiproduce enzymes that are capable of extensive degradation of lig-nin. As a result, the biomass digestibility is increased (Adney et al.,2009), although there is a loss of cellulose and hemicellulose dur-ing this process (Hatakka, 1983).

3.2.4. Hydrolysis of keratinPretreatment of keratin-rich waste using a strong acid, alkaline

hydrolysis, and other physicochemical methods results in severedegradation and destruction of the keratin (Barone et al., 2006).Hence, recent research focuses on the biological pretreatmentmethods, which have been reported as successful for keratindegradation. Microorganisms (bacteria and fungi) produceenzymes that can break down the keratin and make it soluble.The keratin-rich feedstock is inoculated with appropriate bacteriaor fungi cultures and incubated for days or weeks depending onthe activity of the microorganism used and the properties of thesubstrate. Several microorganisms such as Bacillus sp. (Cai et al.,2008; Fellahi et al., 2014; Forgács et al., 2011a; Patinvoh et al.,2016; Zaghloul et al., 2011) and Aspergillus sp. (Mazotto et al.,2013) have been investigated and reported as being effective forhydrolyzing keratin.

3.2.5. Removal of inhibitors present in the feedstocks-fruit flavorsInhibitory compounds that are part of the feedstocks used for

the biogas production such as flavor compounds present in fruitshave been shown to be adversely affecting the anaerobic digestionprocess even at low concentrations. Steam explosion has beenreported to successfully remove these types of inhibitors fromcitrus waste, while also breaking up the lignocellulose structurein the feedstock (Forgács et al., 2011b). Steam explosion is a ther-mal pretreatment method that requires a very high pressure of upto 60 bar and a temperature of up to 150 �C. Combined chemicaland steam explosion pretreatment methods have been found toreduce the required temperature and residence time of the pre-treatment, while also increasing the degradation rate in contrastto when chemical or steam explosion methods are used separately(Forgács et al., 2011b).

4. Innovative non-pretreatment strategies

Although several pretreatment methods have emerged over thepast few years, certain challenges such as high cost, harmful envi-ronmental by-products, low biogas yield, and high energy require-ment still persist. To address these challenges, some innovativenon-pretreatment strategies aiming to improve the use of the read-ily available and low cost feedstocks for biogas production are dis-cussed in this section. The strategies discussed include integratedbiogas production, innovative digester design, co-digestion, andbio-augmentation. An integrated biogas production strategyaddresses the cost challenge, the innovative digester design strat-egy addresses the inhibition challenge, and the co-digestion strat-egy addresses both the challenge of low yield and inhibitors, whilethe bio-augmentation strategy addresses the challenge ofinhibitors.


4.1. Integrated biogas production strategy

Process integration refers to combining production systemseither at the level of individual processes or whole factories withthe goal of efficient utilization of resources, while reducing the costand environmental emissions (Hallale, 2001). Although researchersagree that having biofuel production facilities close to each othercan reduce the costs associated with the waste treatment, energyutilization, and shared logistics, the economics of integrating dif-ferent biofuel production facilities have not been fully explored.Despite this, there have been successful instances where integrat-ing an additional process route into an already established biofuelproduction facility resulted in better economics for the biofuel pro-duction company. As an example, Dererie et al. (2011) used ther-mochemically pretreated oat straw for ethanol production andafter the removal of ethanol, the residue was used for biogas pro-duction. The results showed that sequential ethanol and biogasproduction resulted in 28–34% higher energy yield than direct bio-gas digestion. Biogas production was also faster with fermentationresidues compared to direct digestion, showing that enzymaticsaccharification and ethanol fermentation make the feedstockmore digestible.

Detailed techno-economic analysis of this kind of integrationhas not been investigated, however a brief understanding of howintegration affects the economics can be investigated by compar-ing the production cost with the revenue. The cost of methane pro-duction from wheat straw using steam pretreatment wasestimated as $ 0.85/m3, with the cost from pretreatment as $0.11/m3 CH4 (13% of the total cost) (Shafiei et al., 2013). The costof ethanol production from lignocellulose residue in Californiawas estimated as $ 0.43/L ethanol (Williams et al., 2007), excludingpretreatment (as the pretreatment cost has been factored into thebiogas production cost), the cost could drops to $ 0.33/L ethanol.Using the current selling price of ethanol ($ 0.4/L) (USEIA, 2016)and the experimental results from Dererie et al. (2011), integratingethanol and biogas production facility could increase the prof-itability of the biogas facility by $ 0.014/kg-dry matter of wheatstraw than when pretreated wheat straw is used to produce onlybiogas. This shows that the ethanol production process can be usedfor pretreating biogas feedstocks, while also bringing the benefitsof process integration such as increased productivity.

Considering citrus waste as an example of fruit waste, the highcosts associated with the pretreatment could be a deterrent fromusing it for biogas production. However, integrating pretreatedcitrus waste into already established ethanol and biogas produc-tion facilities could make the process more economically attrac-tive, because in addition to ethanol and biogas, limonene andpectin would be valuable products that could generate additionalrevenue to offset the cost of pretreatment. Pourbafrani et al.(2010) found that from a ton of citrus waste at 20% dry weight,39.64 L of ethanol, 45 m3 of methane, 8.9 L of limonene, and38.8 kg of pectin can be produced. Furthermore, oranges, a typeof citrus waste, accounted for 8.5% of the total global fruit wastein 2012 (FAO, 2013). Putting this amount of orange waste intothe results found by Pourbafrani et al. (2010) would result in theproduction of 2.7 � 106 m3 of ethanol, 3.1 � 109 Nm3 of biogas,6.1 � 105 m3 of limonene, and 2.7 � 106 tons of pectin.

Apart from the fruit waste, the integration concept can beapplied to the other readily available feedstocks. From the biorefin-ery principles, diverse valuable products or energy can be gener-ated from these feedstocks. For example, lignocellulosic wasteconversion into ethanol and biogas has gained a lot of attentionfrom researchers, but in addition to this, the US Department ofEnergy (DOE) identified fifteen high value building block chemicalsthat can be attained from lignocellulosic sugar conversion (Werpyet al., 2004). Integrating any of the identified high value chemicals

production with biogas production could enhance the economicsof biogas production from lignocellulosic waste. A similar analogyapplies to other easily available feedstocks. Thus, process integra-tion could be vital in making the economics of biogas productionmore favorable.

4.2. Innovative digester design strategy

Conventionally, a biogas digester design could be simple ordetailed depending on the scale of application, the nature of thefeedstock as well as the microorganisms involved. The design usu-ally aims for the digester to provide a good environment thatenables efficient contact between the microorganisms and the sub-strate. However, in case of some pretreatment methods or the useof certain chemicals, the AD process can be inhibited, even thoughthese methods can successfully release dissolved intermediates forthe AD process (Bolado-Rodríguez et al., 2016). To make thesemethods beneficial for methane production, it is important todevelop a new digester concept that shields the microorganismsfrom the inhibitors, and in this way allow for a higher biogas yieldto be achieved.

One way for this is to allow for a good contact between themicroorganisms and the substrate, while selectively removingthe inhibitors. In a recent study (Ezeji et al., 2013), a bioreactordesign was applied, called continuous bioreactors with simultane-ous product recovery and bleeding, to overcome the inhibitionchallenges in the bio-butanol production. This design allowed fora good contact between the microorganisms and the substrate,while bleeding away the inhibitory materials and recovering thebio-butanol to prevent an inhibitory concentration. With thisdesign, 25 times higher production of butanol was achieved thanin the traditional bioreactor used as a control (Ezeji et al., 2013).Apart from the inhibitory by-products or chemicals, feedstock suchas POME could be inhibitory to the AD process. POME is producedin large volumes, so to minimize the costs associated with a largedigester volume, a digester design that can allow for a shorthydraulic retention time (HRT) is necessary. Najafpour et al.(2006) used a modified form of an up-flow anaerobic sludge blan-ket (UASB) reactor called an up-flow anaerobic sludge fixed film(UASFF) digester and in this way they were able to achieve 90%reduction in the COD of POME at an organic loading rate (OLR) of23.15 g-COD/d and HRT of 1.5 days. This modification of the UASBconcept was essential since POME contains suspended and col-loidal components. Furthermore, these components consist of fat,protein, or cellulose, which can lead to bed failure and washoutof microorganisms from the traditional UASB system(Zinatizadeh et al., 2007). Hence, an innovative digester designcan be helpful, both in overcoming inhibition challenge and forreducing the retention time needed for the biogas production.

Another innovative design is the use of two- or multi-stagedigesters. Separating the methanogenesis step from the acidogen-esis step during the AD process has been found to give optimalconditions to the different microorganisms involved in the AD pro-cesses. Moreover, it has achieved a better process control and abil-ity to handle higher OLRs than that in the single digesters (Demireland Yenigün, 2002). This is due to the difference in the nutritionalneeds, environmental sensitivity, and the growth rate of the acido-genesis microorganism from those of the methanogens. Theoreti-cally, using the two-stage AD system means that optimalconditions can be provided to the rate limiting steps within theAD process, leading to a better methane yield or shorter retentiontime, especially for the difficult-to-digest substrates. Using 20 g/Lcellulose loading, Jeihanipour et al. (2013) found that a two-stagedigester system resulted in 80% increase in the maximummethaneproduction and a decrease in the lag phase from 15 to 4 days. How-ever, the cost and the added complexities of the two-stage AD sys-


tems have been hindering the wide scale establishment of com-mercial two-stage AD facilities (Blumensaat and Keller, 2005).

The use of membranes for biological processes, referred to asmembrane bioreactor, is another interesting application of theinnovative digester strategy for biogas production. Membranebioreactor applications have been reported to be effective in pro-tecting the microorganisms from the toxic effect of substrate andproduct inhibition (Youngsukkasem et al., 2013). Youngsukkasemet al. (2013) reported that using a membrane bioreactor enabledbiogas production from a substrate with 3% D-limonene at a load-ing rate of 15 g-COD/L, while the control digester with free cellsfailed at 7.5 g-COD/L loading. The use of a membrane is also a suit-able option for handling inhibition caused by ammonia accumula-tion. The use of a hollow fiber membrane was reported tosuccessfully reduce the free ammonia nitrogen concentration byabout 70% (Lauterböck et al., 2012). The ability of the membranebioreactors to shield the microorganisms from inhibitors meansthat the pretreatment of inhibitory feedstock and the costs associ-ated with it could be avoided.

4.3. Co-digestion strategy

Mono-digestion of the recalcitrant feedstocks, highly protein-rich substrates, or feedstocks with harmful compounds oftenresults in a slow process and a low biogas yield. These restrictionscan be overcome by the co-digestion of different feedstocks withan appropriate mixing ratio, considering the C/N ratio, inhibitors,feedstock biodegradability, and total solid content. This is an effec-tive strategy for reducing the ammonia inhibition during the diges-tion process since it aims at favoring synergisms, dilutes harmfulcompounds, optimizes the biogas production, and increases thedigestate quality (Mata-Alvarez et al., 2014). Comino et al. (2010)investigated the co-digestion of chopped crop silage with cowmanure at different feeding rates. It was observed that feedingwith 70% VS of crop in the feedstock increased the methane yieldby 109% compared to manure mono-culture. Co-digestion of vari-ous substrates (manure, slaughterhouse wastes, municipal solidwastes, and crop residues) at different mixing ratios was investi-gated by Pagés Díaz et al. (2011). Synergetic effects were reported,giving rise to up to 43% more methane yield, compared to that cal-culated from the methane yields of individual feedstocks. Papertube residuals added to a nitrogen-rich substrate mixture used inan industrial digestion plant was also reported as having stabiliz-ing effects on the digestion process (Teghammar et al., 2013). Paperaddition prevented the accumulation of VFAs, increased themethane yield by 15–34%, and decreased the hydraulic retentiontime by five days. Considering the fact that easily degraded feed-stocks are not highly available and the problems associated withpretreatments, co-digestion is a good strategy to increase theamount of produced biogas, while avoiding the costs and chal-lenges associated with the pretreatments. Nevertheless, moreresearch is necessary in this area for a better understanding ofthe appropriate mixing ratios, interaction effects, and the impactof co-digestion.

Another benefit of co-digestion is the reduction of ammoniaaccumulation when feedstocks with high protein content are beingdigested. Zeshan et al. (2012) reported that adjustment of the C/Nratio to 32 resulted in 30% less ammonia formation when foodwastes, fruit and vegetable wastes, garden and paper wastes wereco-digested in a dry digestion process.

4.4. Bio-augmentation strategy

Biological pretreatment of lignocellulose biomass and keratin-rich wastes has been reported to be successful thereby, improvingthe biogas production (Forgács et al., 2011a; Patinvoh et al., 2016;

Rouches et al., in press). Nevertheless, the need for two-stage pro-cesses or for pretreatment makes these options unfeasible forindustrial applications. A promising option is bio-augmentationwith anaerobic cellulolytic bacteria and with keratinase enzymesto enhance hydrolysis and improve the biogas yield, which has pre-viously been demonstrated by several researchers. Peng et al.(2014) applied the bio-augmentation strategy to improve thehydrolysis of wheat straw using Clostridium cellulolyticum. Accord-ing to the results, the biochemical methane potential of wheatstraw was improved. Bio-augmentation with 10% inoculation ofAcetobacteroides hydrogenigenes on the anaerobic digestion of cornstraw has also been reported (Zhang et al., 2015) leading to anincrease of 19–23% of the methane yield. Forgács et al. (2013)investigated the co-digestion of the organic fraction of municipalsolid waste (OFMSW) with chicken feathers bio-augmented withsavinase, an alkaline endopeptidase (0.53 ml/gVSfeathers). The addi-tion of this anaerobic enzyme resulted in a methane yield of 0.485Nm3/kgVS/d with stable reactor performance, while the processwithout bio-augmentation resulted in a low yield and accumula-tion of un-degraded feathers.

Another bioaugmentation strategy that could be helpful in han-dling the inhibitory feedstocks and some inhibitory byproductsformed during the pretreatment is the use of flocculating bacteria(flocs). Flocculation has been shown as an effective way for yeastcells to overcome the inhibitory effect of some by-products (e.g.,furfural), which are formed during the pretreatment of lignocellu-losic biomass (Westman et al., 2014). The formation of the localhigh cell density by flocs allows the outer cells to shield the cellsinside the flocs from the inhibitory surrounding, thus, allowingthe flocs to utilize the biodegradable content of the feedstock. Inanother similar work, flocculating cells were used by Najafpouret al. (2006) to treat POME with a pH of between 3.8–4.4, whichcould otherwise be lethal to methanogens. Apart from pH, ammo-nia toxicity can also be handled by using flocs. Koster and Lettinga(1988) studied the anaerobic digestion of potato juice at extremeammonia concentrations using flocs, showing that methanonogen-sis can occur even at 11.8 g ammonia-nitrogen/L concentration.The flocs were adapted to a high concentration of ammonia nitro-gen, and after the adaptation processes the maximum tolerableammonia concentration was 6.2 times higher than the initial toxi-city threshold level. Bioaugmentation, either in the form of addi-tion of new microbial community or the formation of high localcell density (flocs), is a promising strategy for possible industrialapplications utilizing recalcitrant and inhibitory feedstocks for bio-gas production.

5. Conclusion

The feedstock for biogas production may contain indigestible,hard-to-digest, and inhibitory compounds. Therefore, there is aneed for pretreatment or other innovative methods in order tofacilitate the biological digestion. The indigestible materials canbe treated by a combined gasification-fermentation process. More-over, hard-to-digest compounds such as recalcitrant lignocellu-loses or keratin can be pretreated by thermal, chemical,physicochemical, or biological methods. In addition, feedstock withinhibitors can be pretreated by several methods such as steamexplosion, or non-pretreatment methods developed to facilitatetheir digestion, such as innovative digesters and/or bio-augmentation.

References

Adney, W.S., van der Lelie, D., Berry, A.M., Himmel, M.E., 2009. Understanding thebiomass decay community. Biomass Recalcitrance, 454–479. BlackwellPublishing Ltd..


Ahmad, A., Ghufran, R., Wahid, Z.A., 2011. Bioenergy from anaerobic degradation oflipids in palm oil mill effluent. Rev. Environ. Sci. Bio/Technol. 10 (4), 353–376.

Angelidaki, I., Ahring, B.K., 1992. Effects of free long-chain fatty acids onthermophilic anaerobic digestion. Appl. Microbiol. Biotechnol. 37 (6), 808–812.

Angelidaki, I., Sanders, W.T.M., 2004. Assessment of the anaerobic biodegradabilityof macropollutants. Rev. Environ. Sci. Biotechnol. 3 (2), 117–129.

Banks, C.J., Lo, H.-M., 2003. Assessing the effects of municipal solid wasteincinerator bottom ash on the decomposition of biodegradable waste using acompletely mixed anaerobic reactor. Waste Manage. Res. 21 (3), 225–234.

Barone, J.R., Schmidt, W.F., Gregoire, N.T., 2006. Extrusion of feather keratin. J. Appl.Polym. Sci. 100 (2), 1432–1442.

Blumensaat, F., Keller, J., 2005. Modelling of two-stage anaerobic digestionusing the IWA Anaerobic Digestion Model No. 1 (ADM1). Water Res. 39 (1),171–183.

Bochmann, G., Montgomery, L.F.R., Murphy, J., Baxter, D. 2013. 4 – Storage and pre-treatment of substrates for biogas production A2 - Wellinger, Arthur. In: TheBiogas Handbook, Woodhead Publishing, pp. 85–103.

Bolado-Rodríguez, S., Toquero, C., Martín-Juárez, J., Travaini, R., García-Encina, P.A.,2016. Effect of thermal, acid, alkaline and alkaline-peroxide pretreatments onthe biochemical methane potential and kinetics of the anaerobic digestion ofwheat straw and sugarcane bagasse. Bioresour. Technol. 201, 182–190.

Brownell, H.H., Saddler, J.N., 1987. Steam pretreatment of lignocellulosic materialfor enhanced enzymatic hydrolysis. Biotechnol. Bioeng. 29 (2), 228–235.

Cai, C.-G., Lou, B.-G., Zheng, X.-D., 2008. Keratinase production and keratindegradation by a mutant strain of Bacillus subtilis. J. Zhejiang Univ. Sci. B 9(1), 60–67.

Chang, V.S., Burr, B., Holtzapple, M.T., 1997. Lime pretreatment of switchgrass. Appl.Biochem. Biotechnol. 63–5, 3–19.

Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: areview. Bioresour. Technol. 99 (10), 4044–4064.

Chin, M.J., Poh, P.E., Tey, B.T., Chan, E.S., Chin, K.L., 2013. Biogas from palm oil milleffluent (POME): Opportunities and challenges from Malaysia’s perspective.Renewable Sustainable Energy Rev. 26, 717–726.

Comino, E., Rosso, M., Riggio, V., 2010. Investigation of increasing organic loadingrate in the co-digestion of energy crops and cow manure mix. Bioresour.Technol. 101 (9), 3013–3019.

Daroit, D.J., Corrêa, A.P.F., Brandelli, A., 2009. Keratinolytic potential of a novelBacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus.Int. Biodeter. Biodegr. 63 (3), 358–363.

Demirel, B., Yenigün, O., 2002. Two-phase anaerobic digestion processes: a review. J.Chem. Technol. Biotechnol. 77 (7), 743–755.

Dererie, D.Y., Trobro, S., Momeni, M.H., Hansson, H., Blomqvist, J., Passoth, V.,Schnürer, A., Sandgren, M., Ståhlberg, J., 2011. Improved bio-energy yields viasequential ethanol fermentation and biogas digestion of steam exploded oatstraw. Bioresour. Technol. 102 (6), 4449–4455.

Deublein, D., Steinhauser, A., 2011. Biogas from Waste and Renewable Resources.Wiley-VCH.

Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2013. Microbial production of a biofuel(acetone–butanol–ethanol) in a continuous bioreactor: impact of bleed andsimultaneous product removal. Bioprocess Biosyst. Eng. 36 (1), 109–116.

FAO.,2011. Global food losses and food waste – Extent, causes and preventionhttp://www.fao.org/docrep/014/mb060e/mb060e.pdf Accessed 2016/09/20.

FAO, 2013. FAO Statistical Yearbook 2013,World Food and Agriculture http://www.fao.org/docrep/018/i3107e/i3107e.PDF Accessed 2016/09/15.

FAOSTAT, 2013. Live animals. Food and Agriculture Organization of the UnitedNations. FAO Statistics Division 2016. http://faostat.fao.org/site/573/DesktopDefault.aspx?PageID=573#ancor Accessed 2016/02/18.

Fellahi, S., Zaghloul, T.I., Feuk-Lagerstedt, E., Taherzadeh, M.J., 2014. A bacillus strainable to hydrolyze alpha- and beta-keratin. J. Bioprocess. Biotech. 4 (7), 7.

Fernandes, T.V., Klaasse Bos, G.J., Zeeman, G., Sanders, J.P.M., van Lier, J.B., 2009.Effects of thermo-chemical pre-treatment on anaerobic biodegradabilityand hydrolysis of lignocellulosic biomass. Bioresour. Technol. 100 (9), 2575–2579.

Forgács, G., 2012. Biogas production from citrus wastes and chicken feather:pretreatment and co-digestion. Chalmers University of Technology.

Forgács, G., Alinezhad, S., Mirabdollah, A., Feuk-Lagerstedt, E., Sárvári Horváth, I.,2011a. Biological treatment of chicken feather waste for improved biogasproduction. J. Environ. Sci. 23 (10), 1747–1753.

Forgács, G., Pourbafrani, M., Niklasson, C., Taherzadeh, M., Sárvari Horváth, I., 2011b.Methane production from citrus wastes: process development and costestimation. J. Chem. Technol. Biotechnol. 87 (2), 250.

Forgács, G., Lundin, M., Taherzadeh, M., Sárvári Horváth, I., 2013. Pretreatment ofchicken feather waste for improved biogas production. Appl. Biochem.Biotechnol. 169 (7), 2016–2028.

Gustavsson, J., Cederberg, C., Sonesson, U., Van Otterdijk, R., Meybeck, A., 2011.Global Food Losses and Food Waste. Food and Agriculture Organization of theUnited Nations, Rom.

Hallale, N., 2001. Burning bright: trends in process integration. Chem. Eng. Prog. 97(7), 30–41.

Hatakka, A.I., 1983. Pretreatment of wheat straw by white-rot fungi for enzymicsaccharification of cellulose. Eur. J. Appl. Microbiol. Biotechnol. 18 (6), 350–357.

Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility oflignocellulosic biomass. Bioresour. Technol. 100 (1), 10–18.

Himmel, M.E., Picataggio, S.K., 2009. Our challenge is to acquire deeperunderstanding of biomass recalcitrance and conversion. BiomassRecalcitrance, 1–6. Blackwell Publishing Ltd..

Holtzapple, M.T., Lundeen, J.E., Sturgis, R., Lewis, J.E., Dale, B.E., 1992. Pretreatmentof lignocellulosic municipal solid waste by ammonia fiber explosion (AFEX).Appl. Biochem. Biotechnol. 34 (1), 5–21.

Hsu, T.A. 1996. Pretreatment of biomass. In: Wyman, C.E. (Ed.), Handbook onBioethanol: Production and Utilization, Taylor & Francis, London, pp. 179–212.

IWTO, 2015. Wool the Natural Fibre, International Wool Textile Organisation(IWTO) http://www.iwto.org/wool/the-natural-fibre/ Accessed 2016/09/17.

Jeihanipour, A., Aslanzadeh, S., Rajendran, K., Balasubramanian, G., Taherzadeh, M.J.,2013. High-rate biogas production from waste textiles using a two-stageprocess. Renewable Energy 52, 128–135.

Johnson, D.K., Elander, R.T., 2009. Pretreatments for enhanced digestibility offeedstocks. Biomass Recalcitrance, 436–453. Blackwell Publishing Ltd..

Kabir, M.M., Rajendran, K., Taherzadeh, M.J., Sárvári Horváth, I., 2015. Experimentaland economical evaluation of bioconversion of forest residues to biogas usingorganosolv pretreatment. Bioresour. Technol. 178, 201–208.

Kang, Q., Appels, L., Tan, T., Dewil, R., 2014. Bioethanol from lignocellulosic biomass:current findings determine research priorities. Sci. World J. 2014, 13.

Kayhanian, M., 1999. Ammonia inhibition in high-solids biogasification: anoverview and practical solutions. Environ. Technol. 20 (4), 355–365.

Koster, I.W., Lettinga, G., 1988. Anaerobic digestion at extreme ammoniaconcentrations. Biol. Wastes 25 (1), 51–59.

Kovács, E., Wirth, R., Maróti, G., Bagi, Z., Rákhely, G., Kovács, K.L., 2013. Biogasproduction from protein-rich biomass: fed-batch anaerobic fermentation ofcasein and of pig blood and associated changes in microbial communitycomposition. PLoS ONE 8 (10).

Kovács, E., Wirth, R., Maróti, G., Bagi, Z., Nagy, K., Minárovits, J., Rákhely, G., Kovács,K.L., 2015. Augmented biogas production from protein-rich substrates andassociated metagenomic changes. Bioresour. Technol. 178, 254–261.

Kumar, D., Murthy, G.S., 2011. Impact of pretreatment and downstream processingtechnologies on economics and energy in cellulosic ethanol production.Biotechnol. Biofuels 4 (1), 27.

Lauterböck, B., Ortner, M., Haider, R., Fuchs, W., 2012. Counteracting ammoniainhibition in anaerobic digestion by removal with a hollow fiber membranecontactor. Water Res. 46 (15), 4861–4869.

Li, L., Kiran, E., 1988. Interaction of supercritical fluids with lignocellulosicmaterials. Ind. Eng. Chem. Res. 27 (7), 1301–1312.

Li, Z., Chen, C.H., Hegg, E.L., Hodge, D.B., 2013. Rapid and effective oxidativepretreatment of woody biomass at mild reaction conditions and low oxidantloadings. Biotechnol. Biofuels 6 (1), 119.

Lindner, J., Zielonka, S., Oechsner, H., Lemmer, A., 2015. Effects of mechanicaltreatment of digestate after anaerobic digestion on the degree of degradation.Bioresour. Technol. 178, 194–200.

Mata-Alvarez, J., Dosta, J., Romero-Güiza, M.S., Fonoll, X., Peces, M., Astals, S., 2014.A critical review on anaerobic co-digestion achievements between 2010 and2013. Renewable Sustainable Energy Rev. 36, 412–427.

Mazotto, A.M., Couri, S., Damaso, M.C.T., Vermelho, A.B., 2013. Degradation offeather waste by Aspergillus niger keratinases: comparison of submerged andsolid-state fermentation. Int. Biodeter. Biodegr. 85, 189–195.

Mizuki, E., Akao, T., Saruwatari, T., 1990. Inhibitory effect of Citrus unshu peel onanaerobic digestion. Biol. Wastes 33 (3), 161–168.

Mkoma, S.L., Mabiki, F.P., 2011. Theoretical and practical evaluation of jatropha asenergy source biofuel in Tanzania. In: Bernardes, M.A.D.S. (Ed.), Economic Effectof Biofuel Production. InTech, pp. 181–200.

Moreira, F.G., de Souza, C.G.M., Costa, M.A.F., Reis, S., Peralta, R.M., 2007.Degradation of keratinous materials by the plant pathogenic fungusMyrothecium verrucaria. Mycopathologia 163 (3), 153–160.

Najafpour, G.D., Zinatizadeh, A.A.L., Mohamed, A.R., Isa, M.H., Nasrollahzadeh, H.,2006. High-rate anaerobic digestion of palm oil mill effluent in an upflowanaerobic sludge-fixed film bioreactor. Process Biochem. 41 (2), 370–379.

Neves, L., Ribeiro, R., Oliveira, R., Alves, M.M., 2006. Enhancement of methaneproduction from barley waste. Biomass Bioenergy 30 (6), 599–603.

Onyeche, T.I., Schläfer, O., Bormann, H., Schröder, C., Sievers, M., 2002. Ultrasoniccell disruption of stabilised sludge with subsequent anaerobic digestion.Ultrasonics 40 (1–8), 31–35.

Pagés Díaz, J., Pereda Reyes, I., Lundin, M., Sárvári Horváth, I., 2011. Co-digestion ofdifferent waste mixtures from agro-industrial activities: kinetic evaluation andsynergetic effects. Bioresour. Technol. 102 (23), 10834–10840.

Patinvoh, R.J., Feuk-Lagerstedt, E., Lundin, M., Sárvári Horváth, I., Taherzadeh, M.J.,2016. Biological pretreatment of chicken feather and biogas production fromtotal broth. Appl. Biochem. Biotechnol. 180 (7), 1401–1415.

Peng, X., Börner, R.A., Nges, I.A., Liu, J., 2014. Impact of bioaugmentation onbiochemical methane potential for wheat straw with addition of Clostridiumcellulolyticum. Bioresour. Technol. 152, 567–571.

Pourbafrani, M., Forgács, G., Horváth, I.S., Niklasson, C., Taherzadeh, M.J., 2010.Production of biofuels, limonene and pectin from citrus wastes. Bioresour.Technol. 101 (11), 4246–4250.

Richter, H., Loftus, S.E., Angenent, L.T., 2013. Integrating syngas fermentation withthe carboxylate platform and yeast fermentation to reduce medium cost andimprove biofuel productivity. Environ. Technol. 34 (13–14), 1983–1994.

Rosato, A., Vitali, C., De Laurentis, N., Armenise, D., Milillo, M.A., 2007. Antibacterialeffect of some essential oils administered alone or in combination withNorfloxacin. Phytomedicine 14 (11), 727–732.

Rouches, E., Zhou, S., Steyer, J.P., Carrere, H., in press. White-Rot Fungi pretreatmentof lignocellulosic biomass for anaerobic digestion: impact of glucosesupplementation. Process Biochem.


Rozzi, A., Remigi, E., 2004. Methods of assessing microbial activity and inhibitionunder anaerobic conditions: a literature review. Rev. Environ. Sci. Bio/Technol. 3(2), 93–115.

Russ, W., Meyer-Pittroff, R., 2004. Utilizing waste products from the foodproduction and processing industries. Crit. Rev. Food Sci. Nut. 44 (1), 57–62.

Salminen, E., Einola, J., Rintala, J., 2003. The methane production of poultryslaughtering residues and effects of pretreatments on the methane productionof poultry feather. Environ. Technol 24 (9), 1079–1086.

Sambusiti, C., Ficara, E., Malpei, F., Steyer, J.P., Carrère, H., 2013. Benefit of sodiumhydroxide pretreatment of ensiled sorghum forage on the anaerobic reactorstability and methane production. Bioresour. Technol. 144, 149–155.

Schnürer, A., Jarvis, A. 2010. Microbiological Handbook for Biogas Plants. SwedishWaste Management U2009:03.

Shafiei, M., Kabir, M.M., Zilouei, H., Horváth, I.S., Karimi, K., 2013. Techno-economical study of biogas production improved by steam explosionpretreatment. Bioresour. Technol. 148, 53–60.

Shen, Y., Brown, R., Wen, Z., 2014. Syngas fermentation of Clostridiumcarboxidivoran P7 in a hollow fiber membrane biofilm reactor: evaluating themass transfer coefficient and ethanol production performance. Biochem. Eng. J.85, 21–29.

Silva, G.G.D., Couturier, M., Berrin, J.-G., Buléon, A., Rouau, X., 2012. Effects ofgrinding processes on enzymatic degradation of wheat straw. Bioresour.Technol. 103 (1), 192–200.

Singh, R., Shukla, A., Tiwari, S., Srivastava, M., 2014. A review on delignification oflignocellulosic biomass for enhancement of ethanol production potential.Renewable Sustainable Energy Rev. 32, 713–728.

Statista, 2016. Vegetable oil production worldwide 2000-2016 https://www.statista.com/statistics/263978/global-vegetable-oil-production-since-2000-2001/Statista Accessed 2016/09/12.

Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanolproduction: a review. Bioresour. Technol. 83 (1), 1–11.

Symons, G.E., Buswell, A.M., 1933. The methane fermentation of carbohydrates 1, 2.J. Am. Chem. Soc. 55 (5), 2028–2036.

Taherzadeh, M.J., Jeihanipour, A. 2012. Recalcitrance of lignocellulosic biomass toanaerobic digestion. In: Mudhoo, A. (Ed.), Biogas Production: Pretreatments forenhanced Anaerobic Technology, pp. 27–54.

Taherzadeh, M.J., Karimi, K., 2008. Pretreatment of lignocellulosic wastes toimprove ethanol and biogas production: a review. Int. J. Mol. Sci. 9 (9), 1621–1651.

Taherzadeh, M.J., Eklund, R., Gustafsson, L., Niklasson, C., Lidén, G., 1997.Characterization and fermentation of dilute-acid hydrolyzates from wood.Ind. Eng. Chem. Res. 36 (11), 4659–4665.

Teghammar, A., Castillo, M.D.P., Ascue, J., Niklasson, C., Sárvári Horváth, I., 2013.Improved anaerobic digestion by the addition of paper tube residuals:pretreatment, stabilizing, and synergetic effects. Energy Fuels 27 (1), 277–284.

TheWorldBank, 2010. Waste Composition. Urban Development Series – KnowledgePapers http://siteresources.worldbank.org/INTURBANDEVELOPMENT/Resources/336387-1334852610766/Chap5.pdf Accessed 2016/09/10.

Trippe, F., Fröhling, M., Schultmann, F., Stahl, R., Henrich, E., 2011. Techno-economicassessment of gasification as a process step within biomass-to-liquid (BtL) fueland chemicals production. Fuel Process. Technol. 92 (11), 2169–2184.

USEIA, 2016. Today In Energy, Prices. USEIA (U.S. Energy Information Admistration).https://www.eia.gov/todayinenergy/prices.php Accessed 2016/11/14.

Vavilin, V.A., Rytov, S.V., Lokshina, L.Y., 1996. A description of hydrolysis kinetics inanaerobic degradation of particulate organic matter. Bioresour. Technol. 56 (2),229–237.

Wang, F., Zhang, D., Wu, H., Yi, W., Fu, P., Li, Y., Li, Z., 2016. Enhancing biogasproduction of corn stover by fast pyrolysis pretreatment. Bioresour. Technol.218, 731–736.

Werpy, T., Petersen, G., Aden, A., Bozell, J., Holladay, J., White, J., Manheim, A., Eliot,D., Lasure, L., Jones, S. 2004. Top value added chemicals from biomass. Volume1-Results of screening for potential candidates from sugars and synthesis gas.DTIC Document.

Westman, J.O., Mapelli, V., Taherzadeh, M.J., Franzén, C.J., 2014. Flocculation causesinhibitor tolerance in Saccharomyces cerevisiae for second-generationbioethanol production. Appl. Environ. Microbiol. 80 (22), 6908–6918.

Wikandari, R., Gudipudi, S., Pandiyan, I., Millati, R., Taherzadeh, M.J., 2013.Inhibitory effects of fruit flavors on methane production during anaerobicdigestion. Bioresour. Technol. 145, 188–192.

Wikandari, R., Nguyen, H., Millati, R., Niklasson, C., Taherzadeh, M.J., 2015.Improvement of biogas production from orange peel waste by leaching oflimonene. BioMed Res. Int. 2015, 1–6.

Williams, R., Jenkins, B., Gildart, M., 2007. Ethanol production potential and costsfrom lignocellulosic resources in California. In: 15th European BiomassConference & Exhibition, pp. 7–11.

Xiao, W., Clarkson, W.W., 1997. Acid solubilization of lignin and bioconversion oftreated newsprint to methane. Biodegradation 8 (1), 61–66.

Yenigün, O., Demirel, B., 2013. Ammonia inhibition in anaerobic digestion: a review.Process Biochem. 48 (5–6), 901–911.

Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., Toyohara, K.,Miyamoto, K., Kimura, Y., Oda, K., 2016. A bacterium that degrades andassimilates poly(ethylene terephthalate). Science 351 (6278), 1196–1199.

Youngsukkasem, S., Akinbomi, J., Rakshit, S.K., Taherzadeh, M.J., 2013. Biogasproduction by encased bacteria in synthetic membranes: protective effects intoxic media and high loading rates. Environ. Technol. 34 (13–14), 2077–2084.

Zaghloul, T.I., Embaby, A.M., Elmahdy, A.R., 2011. Biodegradation of chickenfeathers waste directed by Bacillus subtilis recombinant cells: scaling up in alaboratory scale fermentor. Bioresour. Technol. 102 (3), 2387–2393.

Zeshan Karthikeyan, O.P., Visvanathan, C., 2012. Effect of C/N ratio and ammonia-Naccumulation in a pilot-scale thermophilic dry anaerobic digester. Bioresour.Technol. 113, 294–302.

Zhang, Y.H.P., 2008. Reviving the carbohydrate economy via multi-productlignocellulose biorefineries. J. Ind. Microbiol. Biotech. 35 (5), 367–375.

Zhang, J., Guo, R.-B., Qiu, Y.-L., Qiao, J.-T., Yuan, X.-Z., Shi, X.-S., Wang, C.-S., 2015.Bioaugmentation with an acetate-type fermentation bacteriumAcetobacteroides hydrogenigenes improves methane production from cornstraw. Bioresour. Technol. 179, 306–313.

Zinatizadeh, A.A.L., Mohamed, A.R., Mashitah, M.D., Abdullah, A.Z., Isa, M.H., 2007.Optimization of pre-treated palm oil mill effluent digestion in an up-flowanaerobic sludge fixed film bioreactor: a comparative study. Biochem. Eng. J. 35(2), 226–237.


Cost effective dry anaerobic digestion in textile bioreactors: Experimental and economic evaluation. Bioresource Technology 245(Part A): 549-559

Pap

er IV



journal homepage: www.elsevier.com/locate/biortech

Cost effective dry anaerobic digestion in textile bioreactors: Experimentaland economic evaluation

Regina J. Patinvoha,b,⁎, Osagie A. Osadolora, Ilona Sárvári Horvátha, Mohammad J. Taherzadeha

a Swedish Centre for Resource Recovery, University of Borås, SE 501 90 Borås, Swedenb Department of Chemical and Polymer Engineering, Faculty of Engineering, Lagos State University, Lagos, Nigeria

A R T I C L E I N F O

Keywords:Dry anaerobic digestionTextile bioreactorSolid waste managementDigestateEconomic evaluation

A B S T R A C T

The aim of this work was to study dry anaerobic digestion (dry-AD) of manure bedded with straw using textile-based bioreactor in repeated batches. The 90-L reactor filled with the feedstocks (22–30% total solid) and in-oculum without any further treatment, while the biogas produced were collected and analyzed. The digestateresidue was also analyzed to check its suitability as bio-fertilizer. Methane yield after acclimatization increasedfrom 183 to 290 NmlCH4/gVS, degradation time decreased from 136 to 92 days and the digestate compositionpoint to suitable bio-fertilizer. The results then used to carry out economical evaluation, which shows dry-AD intextile bioreactors is a profitable method of handling the waste with maximum payback period of 5 years, netpresent value from $7,000 to $9,800,000 (small to large bioreactors) with internal rate of return from 56.6 to19.3%.

1. Introduction

Solid wastes from agricultural, municipal and industrial activitiesare major sources of environmental pollution. Large volumes of thesewastes are being generated and are increasing immensely due to po-pulation growth and high consumption rate. Currently, people generateglobally about 17 billion tonnes of total solid wastes annually(Chattopadhyay et al., 2009) and it is estimated to reach 27 billiontonnes in 2050 (Karak et al., 2012). These wastes streams usually havea solid content between 15% and 50%. Landfilling is a major practice ofdisposing solid wastes resulting in emissions of methane and nitrousoxides which contribute to greenhouse effect. Composting and in-cineration are also common methods of treating these wastes; com-posting results in emissions of volatile compounds (ketones, aldehydes,ammonia and methane) (Mata-Alvarez et al., 2000) while incinerationcan lead to significant release of dioxin to the environment (Strange,2002) if the exhaust gas is not treated properly. There are regulationsand standards for management of these wastes, but in addition to this itis essential that other waste management options which are both en-vironmentally friendly and economical are explored. Biogas productionthrough anaerobic digestion is an interesting waste management optionfor handling the organic fraction of solid waste, as biogas productionusually leads to reduced pollution and increased energy production.Studies have shown that anaerobic digestion of these wastes is sus-tainable and has a great advantage over aerobic treatment because of its

improved energy balance (Mata-Alvarez et al., 2000). In addition tobiogas production, digestate residue from anaerobic digestion usuallycontains high content of phosphate and nitrogen, especially in the formof ammonium which is available for plants; it also contains othermacro-nutrients and trace elements essential for plant growth (Makádiet al., 2012).

Anaerobic digestion of organic wastes is carried out generallythrough wet (feedstock with solid concentration between 0.5% and15%) (Li et al., 2011) and dry (feedstock with solid concentrationgreater than 20%) (Bolzonella et al., 2003) anaerobic digestion pro-cesses. When treating solid wastes in wet anaerobic digestion processes,fresh and recycled water must be added while large reactor volume,high energy for heating and costly dewatering process of the digestate isrequired. Therefore, processing of solid wastes for biogas productionthrough dry-AD is a better option since they usually have low moisturecontent. However, for enhanced performance of dry-AD process, asuitable reactor is required; considering the substrate composition,amount of substrate to be treated, and process economy of the reactor(Patinvoh et al., 2017). It also requires an appropriate technology foroperation. In this vein, several continuous and batch reactors for dry-AD processes have been employed such as plug flow reactors (Denget al., 2016; Patinvoh et al., 2017) for continuous dry-AD processes, andcompletely mixed reactor (Guendouz et al., 2010) for batch dry-ADprocesses. However, some of these reactors required expert design andconstructions, constant monitoring, high capital and operational cost.

http://dx.doi.org/10.1016/j.biortech.2017.08.081Received 30 June 2017; Received in revised form 11 August 2017; Accepted 14 August 2017

⁎ Corresponding author at: Swedish Centre for Resource Recovery, University of Borås, SE 501 90 Borås, Sweden.E-mail address: [email protected] (R.J. Patinvoh).


Available online 19 August 20170960-8524/ © 2017 Elsevier Ltd. All rights reserved.



journal homepage: www.elsevier.com/locate/biortech

Cost effective dry anaerobic digestion in textile bioreactors: Experimentaland economic evaluation

Regina J. Patinvoha,b,⁎, Osagie A. Osadolora, Ilona Sárvári Horvátha, Mohammad J. Taherzadeha

a Swedish Centre for Resource Recovery, University of Borås, SE 501 90 Borås, Swedenb Department of Chemical and Polymer Engineering, Faculty of Engineering, Lagos State University, Lagos, Nigeria

A R T I C L E I N F O

Keywords:Dry anaerobic digestionTextile bioreactorSolid waste managementDigestateEconomic evaluation

A B S T R A C T

The aim of this work was to study dry anaerobic digestion (dry-AD) of manure bedded with straw using textile-based bioreactor in repeated batches. The 90-L reactor filled with the feedstocks (22–30% total solid) and in-oculum without any further treatment, while the biogas produced were collected and analyzed. The digestateresidue was also analyzed to check its suitability as bio-fertilizer. Methane yield after acclimatization increasedfrom 183 to 290 NmlCH4/gVS, degradation time decreased from 136 to 92 days and the digestate compositionpoint to suitable bio-fertilizer. The results then used to carry out economical evaluation, which shows dry-AD intextile bioreactors is a profitable method of handling the waste with maximum payback period of 5 years, netpresent value from $7,000 to $9,800,000 (small to large bioreactors) with internal rate of return from 56.6 to19.3%.

1. Introduction

Solid wastes from agricultural, municipal and industrial activitiesare major sources of environmental pollution. Large volumes of thesewastes are being generated and are increasing immensely due to po-pulation growth and high consumption rate. Currently, people generateglobally about 17 billion tonnes of total solid wastes annually(Chattopadhyay et al., 2009) and it is estimated to reach 27 billiontonnes in 2050 (Karak et al., 2012). These wastes streams usually havea solid content between 15% and 50%. Landfilling is a major practice ofdisposing solid wastes resulting in emissions of methane and nitrousoxides which contribute to greenhouse effect. Composting and in-cineration are also common methods of treating these wastes; com-posting results in emissions of volatile compounds (ketones, aldehydes,ammonia and methane) (Mata-Alvarez et al., 2000) while incinerationcan lead to significant release of dioxin to the environment (Strange,2002) if the exhaust gas is not treated properly. There are regulationsand standards for management of these wastes, but in addition to this itis essential that other waste management options which are both en-vironmentally friendly and economical are explored. Biogas productionthrough anaerobic digestion is an interesting waste management optionfor handling the organic fraction of solid waste, as biogas productionusually leads to reduced pollution and increased energy production.Studies have shown that anaerobic digestion of these wastes is sus-tainable and has a great advantage over aerobic treatment because of its

improved energy balance (Mata-Alvarez et al., 2000). In addition tobiogas production, digestate residue from anaerobic digestion usuallycontains high content of phosphate and nitrogen, especially in the formof ammonium which is available for plants; it also contains othermacro-nutrients and trace elements essential for plant growth (Makádiet al., 2012).

Anaerobic digestion of organic wastes is carried out generallythrough wet (feedstock with solid concentration between 0.5% and15%) (Li et al., 2011) and dry (feedstock with solid concentrationgreater than 20%) (Bolzonella et al., 2003) anaerobic digestion pro-cesses. When treating solid wastes in wet anaerobic digestion processes,fresh and recycled water must be added while large reactor volume,high energy for heating and costly dewatering process of the digestate isrequired. Therefore, processing of solid wastes for biogas productionthrough dry-AD is a better option since they usually have low moisturecontent. However, for enhanced performance of dry-AD process, asuitable reactor is required; considering the substrate composition,amount of substrate to be treated, and process economy of the reactor(Patinvoh et al., 2017). It also requires an appropriate technology foroperation. In this vein, several continuous and batch reactors for dry-AD processes have been employed such as plug flow reactors (Denget al., 2016; Patinvoh et al., 2017) for continuous dry-AD processes, andcompletely mixed reactor (Guendouz et al., 2010) for batch dry-ADprocesses. However, some of these reactors required expert design andconstructions, constant monitoring, high capital and operational cost.

http://dx.doi.org/10.1016/j.biortech.2017.08.081Received 30 June 2017; Received in revised form 11 August 2017; Accepted 14 August 2017

⁎ Corresponding author at: Swedish Centre for Resource Recovery, University of Borås, SE 501 90 Borås, Sweden.E-mail address: [email protected] (R.J. Patinvoh).


Available online 19 August 20170960-8524/ © 2017 Elsevier Ltd. All rights reserved.

The most commonly used reactors in developing countries are fixed-dome reactors, floating-drum reactors and polyethylene tubular re-actors (usually modelled as plug flow reactors) (Rowse, 2011). The costof a fixed-dome reactor is relatively low and the reactor is usuallyconstructed underground which makes it less sensitive to seasonaltemperature changes but this reactor requires high technical skill forconstruction (Kossmann et al., 1999). It is also prone to leakages(Kossmann et al., 1999; Rajendran et al., 2013) (porosity and cracks)and utilization of gas produced is less effective as the gas pressurefluctuates substantially. Floating-drum reactor is easy to operate, itprovides gas at constant pressure but the steel drum is relatively ex-pensive and requires rigorous maintenance (Kossmann et al., 1999). Itrequires constant removal of rust and regular painting to avoid gasleakage, this reactor is not also suitable for fibrous substrates becausethe gas-holder can get stuck in the resulting floating scum (Kossmannet al., 1999). Hence, there is a need for reactors that are robust innature, easy to maintain, suitable for dry digestion processes and costeffective. An innovative textile-based bioreactor which is durable, costeffective and easy-to-operate was developed for biogas production, andit was found to be effective and economical for wet anaerobic digestionof organic fraction of municipal solid wastes (Rajendran et al., 2013).

In this work, the potential of dry anaerobic digestion in the textile-based bioreactor was studied in batch process, and its technical andeconomic aspects were evaluated. Manure bedded with straw was usedas an example of the substrate abundantly available at farms. Repeatedbatch digestion processes were investigated, the TS in the reactor wasgradually increased in order to acclimatize the inoculum to the sub-strates under solid state condition and thereafter increase the amount ofwastes treated. The residue after biogas production was analysed tocheck its suitability as bio-fertilizer. Additionally, the economic ana-lysis was carried out to evaluate this technology.

2. Materials and methods

2.1. Bioreactor

A new design of innovative textile reactor at laboratory scale wasdeveloped and supplied by FOV Fabrics AB (Borås, Sweden). It wasmade of advanced textiles and sophisticated coated polymers whichmakes the bioreactor durable, resistant to digesting bacteria and che-micals, easily transportable and highly resistant to UV light and hightemperature (up to 80 °C) (Rajendran et al., 2013). The reactor is madeof high quality polyester PVC coated fabrics; it is a horizontal bioreactorof 184 cm length, 54 cm breadth and had a total volume of about 90 L.The bioreactor was maintained at 25 °C by circulating water through

water jacket. This bioreactor had an air tight zip which was opened forfeeding and closed after feeding; it also had an opener for gas outlet.The reactor was examined for leakages by filling with water and airbefore starting the experiment. Fig. 1 shows the schematic sketch of theexperimental set up together with other accessories, such as gas col-lection bag, sampling point for biogas compositional analysis and thegas flow meter (Drum-type gas meter, TG 05 Model 5, Ritter, Germany)for measuring the produced biogas volume.

2.2. Substrate and inoculum preparation

Cattle manure bedded with straw and untreated wheat straw wasobtained from a farm outside Borås (Rådde Gård, Sweden). During theexperimental period, two different batches of the substrates were ob-tained; the amount of straw in manure was higher in the second batchcollected compared to the first batch. The first batch was used for firstexperiment while second batch was used for the second and third ex-periments. The manure was shredded manually to reduce the particlesize of straw in the manure. The untreated wheat straw was milled toparticle size of 0.5–2 mm, after which the feedstocks were character-ized.

Inoculum was obtained from a digester treating wastewater sludgeand operating at mesophilic conditions (Vatten and Miljö i Väst AB,Varberg, Sweden). The inoculum was filtered through a 2-mm porositysieve to remove sand, plastic and other unwanted particles after whichit was acclimatized for five days prior to use. The inoculum was cen-trifuged at 7,600×g using continuous centrifuge to obtain a TS contentof 7.0 ± 0.24%. The inoculum from wet fermentation with TS contentof 3.53 ± 0.01% was centrifuged in order to reduce its water contentsince the textile reactor is studied under dry anaerobic digestion. Theinoculum after centrifuge having TS and VS content of 7% and 4%respectively was then used for the first batch experiment.

2.3. Experimental procedure

Manure bedded with straw was mixed with required amount ofuntreated wheat straw to increase the C/N ratio; the C/N ratio was keptbetween 20:1 and 25:1 throughout the experiment. The amount ofuntreated straw needed to increase the C/N was calculated according toEq. (1) (AAFRD, 2005) and the required amount added is shown inTable 3. During the first digestion process (unacclimatized inoculum),the total feedstock with 22% TS was inoculated with the initial in-oculum (7%TS and 4%VS) keeping a volatile solids (VS) ratio(VSinoculum to VSsubstrate) at 1:1 thereby having total TS of 10% in thereactor. A nutrient solution with composition according to Angelidaki

Fig. 1. Schematic sketch of the experimental set up.

R.J. Patinvoh et al. Bioresource Technology 245 (2017) 549–559

550

et al. (2009) was added only at this start-up period and the pH of themixture was adjusted to 7.45 using 2 M HCl solution. The reactor wasmaintained at 25 °C by circulating water through water jacket arrangedunder the reactor as shown in Fig. 1 and there was no mixing inside thereactor during the digestion process. The biogas produced was collectedin a gas bag and volume was measured once the gas collection bag wasfilled. Biogas samples were taken through the gas sampling point forcompositional analysis. The first digestion process lasted for 136 days.In order to increase the TS in the textile reactor and investigate theresponse of bacteria after acclimatization, the second batch digestion(acclimatized low TS) was performed. Second digestion process wasperformed the same way as in the first one, except that the digestateresidue from the first digestion was used as inoculum (7.97%TS and5.19%VS) and no nutrient was added. The volatile solids (VS) ratio(VSinoculum to VSsubstrate) was kept at 1:1 and TS of the substrate wasincreased to 27% thereby increasing the TS in the reactor to 12%. Tofurther increase the TS in the reactor thereby treating more wastes, athird batch digestion process (acclimatized high TS) was also per-formed. The digestate residue from the second digestion was used asinoculum (9.6%TS and 6.2%VS). The volatile solids (VS) ratio(VSinoculum to VSsubstrate) was reduced to 0.5 and TS of the substrate wasincreased to 30% thereby increasing the TS in the reactor to 17%. Thecompositions in different batch digestion setup are summarized inTable 2.

= × −−

× −−

a NN

R RR R

MM

%%

11

b

a

b

a

b

a (1)

where:

a = total weight of strawb = total weight of manure with strawMa = moisture content of strawMb = moisture content of manure with straw% Na = % nitrogen of straw on dry basis% Nb = % nitrogen of manure with straw on dry basisRa = C/N ratio of strawRb = C/N ratio of manure with strawR = desired C/N of mixture

2.4. Analytical methods

Moisture content, pH, total nitrogen, TS and VS were determinedaccording to biomass analytical procedures (APHA-AWWA-WEF,2005). Total nitrogen contents were measured using the Kjeldahlmethod, and the protein content was estimated by multiplying theKjeldahl nitrogen content with a factor of 6.25 according to Gunaseelan(2009). The total carbon was obtained by correcting the total dryweight carbon value for the ash content (Haug, 1993; Zhou et al.,2015). The bulk density was determined according to Zhang et al.(2012).

Extractives in untreated straw samples were determined accordingto NREL protocols (Sluiter et al., 2008) using Soxhlet method withwater and ethanol extraction for 24 h. Total carbohydrate and lignincontent of extractive free straw samples were determined according toNREL protocols (Sluiter et al., 2011). The extractive free straw sampleswere air-dried until moisture content was less than 10%. Samples werethen hydrolyzed using 72% H2SO4 in a water bath at 30 °C for 60 minwhile samples were stirred every 5 min to ensure uniform hydrolysis,and then a second hydrolysis was performed using 4% H2SO4 in anautoclave at 121 °C for 60 min. Monomeric sugars obtained during thehydrolysis were determined by HPLC. Mannose, glucose, galactose,xylose and arabinose were analyzed using Aminex HPX-87 P column(Bio-Rad) at 85 °C and 0.6 mL/min ultrapure water as eluent. Acid so-luble lignin (ASL) was determined using a UV spectrophotometer (LibraS60, Biochrom, England) at 320 nm. Acid insoluble lignin (AIL) wasgravimetrically determined as residual solid after hydrolysis corrected

with ash content. The ash content was determined as the remainingresidue after keeping the samples in the muffle furnace at 575 °C for24 h.

The biogas volume was measured with Drum-type gas meter (TG 05Model 5, Ritter, Germany), and then it was corrected for reporting atstandard temperature and pressure (0 °C, and 1 bar) using ideal gas law.The methane composition of the produced biogas was determined usinga GC (Perkin-Elmer, USA) equipped with a packed column (6′ × 1.8′'OD, 80/100, Mesh, Perkin Elmer, USA), and a thermal conductivitydetector (Perkin-Elmer, USA), with an inject temperature of 150 °C. Thecarrier gas was nitrogen operated with a flow rate of 20 ml/min at60 °C. A 250-µl pressure-lock gas syringe (VICI, precious sampling Inc.,USA) was used for taking samples for the gas composition analysis. Thedegree of digestion was calculated using Eq. (2) according to Schnürerand Jarvis (2010);

= ∗ − ∗ ∗ ∗Degree of Digestion TS VS TS VS TS VS((( ) ( ))/( )) 100in in out out in in

(2)

2.5. Digestate analysis

The digestate residue was centrifuged at 5,000×g for 10 min andthe liquid fraction was passed through 0.2 µm filter prior to analysis.0.2g of the solid fraction was dissolved in 2 ml concentrated HNO3

(65%), 6 ml concentrated HCl (37%), 1 ml concentrated HF (48%) and1 ml H2O2 (30%). Thereafter, it was digested at 200 °C (800 W) for20 min in the microwave oven. Dissolved samples were then dilutedwith milli-Q water to 25 ml. The available heavy metals, trace elementsand macro-nutrients in both the liquid and solid fraction of the diges-tate were quantified using microwave plasma-atomic emission spec-trometer (Agilent Technologies 4200 MP-AES). The liquid fraction ofthe digestate was analyzed for ammonium nitrogen concentration usingAmmonium 100 test kit (Nanocolor, MACHEREY-NAGEL GmbH& Co.KG. 10 Germany) and concentration measured using Nanocolor 500DPhotometer (MACHEREY-NAGEL GmbH&Co. KG. Germany).

2.6. Economic analysis

The annual cost of producing biogas or any of the fermentativebiofuels in US$/Volume can be estimated using Eq. (3) (Bergeron,1996). A 15-year biogas production lifetime was assumed for the textilebioreactor (Rajendran et al., 2013). The interest rate (i) used for theanalysis was 7.8% which is the average deposit interest rate in Nigeria(a tropical country) from 2010 to 2016 (World Bank, 2017a).

= + + −Annual biogas production cost ACBP FC Y ACC Ye EC( ) / ( OC) .(3)

where FC = feedstock cost ($/tonne), Y = yield (Nm3/tonne), AC-C = annual capital cost ($/Nm3), CC = Capital cost, i = interest rate,OC = operating cost ($/Nm3), Ye = electricity yield (kWh/L), EC = -electricity credit ($/kWh), n = 15 (years).

Economic analysis was performed on the cost of replacing agri-cultural solid waste generation by collection through waste manage-ment providers and by composting with dry-AD at three different dry-AD scenarios which are; no acclimatization of the inoculum using solidwaste with a density of 907 kg/m3 (scenario A), short acclimatization(136 days) of the inoculum using solid waste with a density of 542 kg/m3 (scenario B) and longer acclimatization (232 days) of the inoculumusing solid waste with a density of 542 kg/m3 (scenario C). The cal-culations were performed at three different waste generation levels; 3tons/year for a typical small scale farm with animal husbandry andvegetable cultivation or 2 mature cows (Ho et al., 2013), 5200 tons/year for a medium mechanized farm or over 100 mature cows and51,000 tons/year for a large scale mechanised farm or over 1000 ma-ture cows (Chen, 2016; Hoornweg et al., 1999). The unit cost of com-positing from a study in Taiwan (which is similar to many tropical or


551

et al. (2009) was added only at this start-up period and the pH of themixture was adjusted to 7.45 using 2 M HCl solution. The reactor wasmaintained at 25 °C by circulating water through water jacket arrangedunder the reactor as shown in Fig. 1 and there was no mixing inside thereactor during the digestion process. The biogas produced was collectedin a gas bag and volume was measured once the gas collection bag wasfilled. Biogas samples were taken through the gas sampling point forcompositional analysis. The first digestion process lasted for 136 days.In order to increase the TS in the textile reactor and investigate theresponse of bacteria after acclimatization, the second batch digestion(acclimatized low TS) was performed. Second digestion process wasperformed the same way as in the first one, except that the digestateresidue from the first digestion was used as inoculum (7.97%TS and5.19%VS) and no nutrient was added. The volatile solids (VS) ratio(VSinoculum to VSsubstrate) was kept at 1:1 and TS of the substrate wasincreased to 27% thereby increasing the TS in the reactor to 12%. Tofurther increase the TS in the reactor thereby treating more wastes, athird batch digestion process (acclimatized high TS) was also per-formed. The digestate residue from the second digestion was used asinoculum (9.6%TS and 6.2%VS). The volatile solids (VS) ratio(VSinoculum to VSsubstrate) was reduced to 0.5 and TS of the substrate wasincreased to 30% thereby increasing the TS in the reactor to 17%. Thecompositions in different batch digestion setup are summarized inTable 2.

= × −−

× −−

a NN

R RR R

MM

%%

11

b

a

b

a

b

a (1)

where:

a = total weight of strawb = total weight of manure with strawMa = moisture content of strawMb = moisture content of manure with straw% Na = % nitrogen of straw on dry basis% Nb = % nitrogen of manure with straw on dry basisRa = C/N ratio of strawRb = C/N ratio of manure with strawR = desired C/N of mixture

2.4. Analytical methods

Moisture content, pH, total nitrogen, TS and VS were determinedaccording to biomass analytical procedures (APHA-AWWA-WEF,2005). Total nitrogen contents were measured using the Kjeldahlmethod, and the protein content was estimated by multiplying theKjeldahl nitrogen content with a factor of 6.25 according to Gunaseelan(2009). The total carbon was obtained by correcting the total dryweight carbon value for the ash content (Haug, 1993; Zhou et al.,2015). The bulk density was determined according to Zhang et al.(2012).

Extractives in untreated straw samples were determined accordingto NREL protocols (Sluiter et al., 2008) using Soxhlet method withwater and ethanol extraction for 24 h. Total carbohydrate and lignincontent of extractive free straw samples were determined according toNREL protocols (Sluiter et al., 2011). The extractive free straw sampleswere air-dried until moisture content was less than 10%. Samples werethen hydrolyzed using 72% H2SO4 in a water bath at 30 °C for 60 minwhile samples were stirred every 5 min to ensure uniform hydrolysis,and then a second hydrolysis was performed using 4% H2SO4 in anautoclave at 121 °C for 60 min. Monomeric sugars obtained during thehydrolysis were determined by HPLC. Mannose, glucose, galactose,xylose and arabinose were analyzed using Aminex HPX-87 P column(Bio-Rad) at 85 °C and 0.6 mL/min ultrapure water as eluent. Acid so-luble lignin (ASL) was determined using a UV spectrophotometer (LibraS60, Biochrom, England) at 320 nm. Acid insoluble lignin (AIL) wasgravimetrically determined as residual solid after hydrolysis corrected

with ash content. The ash content was determined as the remainingresidue after keeping the samples in the muffle furnace at 575 °C for24 h.

The biogas volume was measured with Drum-type gas meter (TG 05Model 5, Ritter, Germany), and then it was corrected for reporting atstandard temperature and pressure (0 °C, and 1 bar) using ideal gas law.The methane composition of the produced biogas was determined usinga GC (Perkin-Elmer, USA) equipped with a packed column (6′ × 1.8′'OD, 80/100, Mesh, Perkin Elmer, USA), and a thermal conductivitydetector (Perkin-Elmer, USA), with an inject temperature of 150 °C. Thecarrier gas was nitrogen operated with a flow rate of 20 ml/min at60 °C. A 250-µl pressure-lock gas syringe (VICI, precious sampling Inc.,USA) was used for taking samples for the gas composition analysis. Thedegree of digestion was calculated using Eq. (2) according to Schnürerand Jarvis (2010);

= ∗ − ∗ ∗ ∗Degree of Digestion TS VS TS VS TS VS((( ) ( ))/( )) 100in in out out in in

(2)

2.5. Digestate analysis

The digestate residue was centrifuged at 5,000×g for 10 min andthe liquid fraction was passed through 0.2 µm filter prior to analysis.0.2g of the solid fraction was dissolved in 2 ml concentrated HNO3

(65%), 6 ml concentrated HCl (37%), 1 ml concentrated HF (48%) and1 ml H2O2 (30%). Thereafter, it was digested at 200 °C (800 W) for20 min in the microwave oven. Dissolved samples were then dilutedwith milli-Q water to 25 ml. The available heavy metals, trace elementsand macro-nutrients in both the liquid and solid fraction of the diges-tate were quantified using microwave plasma-atomic emission spec-trometer (Agilent Technologies 4200 MP-AES). The liquid fraction ofthe digestate was analyzed for ammonium nitrogen concentration usingAmmonium 100 test kit (Nanocolor, MACHEREY-NAGEL GmbH& Co.KG. 10 Germany) and concentration measured using Nanocolor 500DPhotometer (MACHEREY-NAGEL GmbH&Co. KG. Germany).

2.6. Economic analysis

The annual cost of producing biogas or any of the fermentativebiofuels in US$/Volume can be estimated using Eq. (3) (Bergeron,1996). A 15-year biogas production lifetime was assumed for the textilebioreactor (Rajendran et al., 2013). The interest rate (i) used for theanalysis was 7.8% which is the average deposit interest rate in Nigeria(a tropical country) from 2010 to 2016 (World Bank, 2017a).

= + + −Annual biogas production cost ACBP FC Y ACC Ye EC( ) / ( OC) .(3)

where FC = feedstock cost ($/tonne), Y = yield (Nm3/tonne), AC-C = annual capital cost ($/Nm3), CC = Capital cost, i = interest rate,OC = operating cost ($/Nm3), Ye = electricity yield (kWh/L), EC = -electricity credit ($/kWh), n = 15 (years).

Economic analysis was performed on the cost of replacing agri-cultural solid waste generation by collection through waste manage-ment providers and by composting with dry-AD at three different dry-AD scenarios which are; no acclimatization of the inoculum using solidwaste with a density of 907 kg/m3 (scenario A), short acclimatization(136 days) of the inoculum using solid waste with a density of 542 kg/m3 (scenario B) and longer acclimatization (232 days) of the inoculumusing solid waste with a density of 542 kg/m3 (scenario C). The cal-culations were performed at three different waste generation levels; 3tons/year for a typical small scale farm with animal husbandry andvegetable cultivation or 2 mature cows (Ho et al., 2013), 5200 tons/year for a medium mechanized farm or over 100 mature cows and51,000 tons/year for a large scale mechanised farm or over 1000 ma-ture cows (Chen, 2016; Hoornweg et al., 1999). The unit cost of com-positing from a study in Taiwan (which is similar to many tropical or


551

subtropical country) was $67/ton for highly mechanised privatizedlarge scale compost production facility, $97/ton for mid scale and$404/ton for small scale manual government affiliated composting fa-cility or farms (using a conversion factor of 1 NT$ = $0.033 and 53%conversion of waste to compost) (Chen, 2016; Hoornweg et al., 1999).For the waste collection by waste managers option, the estimated costof collecting waste for lower mid income countries in 2010 was be-tween $30–$75 per ton of solid waste (World Bank, 2016). Additionallyfarms that employ waste disposal by waste managers also have topurchase compost or organic fertilizer (Ruggieri et al., 2009) and thiscost an additional $211–$356 per ton (Chen, 2016), making the totalcost handling solid waste by waste managers to be from $241 to $431per ton.

The annual profit (AP) was calculated by substracting the annualcost of biogas production (Eq. (3)) from the annual cost of handlingwaste through other means plus the revenue generated from biogassales as shown in Eq. (4);

= − +AP Annual expenditure on waste management ACBP Revenue (4)

The revenue (R) from biogas was gotten by multiplying the currentcommercial selling price of natural gas of $0.222/1000-Nm3 (Sellingprice in Febrauary 2017) by the annual methane production from thedry anearobic digestion divided by the methane fraction in natural gas(95%) (EIA, 2017). The revenue calculations (Eq. (5)) were done usingthe methane production values for the experiments performed.

= ×R Vol of methane produced"$"0.000234 (Nm ) (Nm )3 3 (5)

The payback period (PBP) was calculated using Eq. (6), where theinitial project delay time was the time needed for completing the firstanaerobic digestion batch. The net present value (NPV) was calculatedusing Eq. (7), with the profit from the first year different from those ofthe other years, and the internal rate of return which makes the NPVzero was also calculated.

= +PBP CC AP initial project delay time/ (6)

∑= − ++=

NPV C APi n(1 )n

n

01 (7)

where C0 = the initial capital investment.The capital cost for the biogas production was based on the volume

of the textile bioreactor needed for biogas production per year and thecost of other auxiliary equipment such as shredders, shovels and trac-tors (for mid and large scale). The volumes of the textile bioreactorsneeded for the different scenerious investigated were calculated usingthe data from the experimental section by dividing the mass of thewaste and innoculum by their respective densities while accounting forduration of the dry-AD, feedstock storage, gas storage and digestatestorage. Details on how to perform this kind of calculations and optimalreactor sizing can be found elsewhere (Mähnert and Linke, 2009). Using3 tons/year waste generation without acclimatization as an example,the digestion period is 136 days so storage for 1.12 tons (i.e. 3tons × 136/365) of waste is needed as the waste is produced con-tinuously. The reactor volume needed for storing the daily producedwaste is 1.23 m3 (i.e. mass/density). This is also going to be the volumeneeded for storing the digestate as the amount digestate removed fromthe reactor would be equal to the amount of waste to be feed to thereactor. The volume needed for the digestion will be equal to the sum of1.23 m3 (the volume of the waste needed for the dry-AD), 5.24 m3 forthe inoculum (i.e. inoculum mass/density) and 2 m3 to provide some airspace which gives 8.47 m3. Using the assumption that the producedbiogas will be sold monthly, the textile bioreactor volume needed forone month storage is 8 m3 making the total required volume for allactivities to be 19 m3. Calculations similar to this where performed forthe other scenarios and scales, and the required reactor volume areshown in Table 2.

The annual operating cost associated with the textile reactor was

assumed to be 5% of its capital cost (Rajendran et al., 2013), while thatfor the auxiliary equipment were; $1.95/ton for shredders, $0.12/tonfor tractors and $0.14/ton for shovels (using 1€ = 1.09 $) (Ruggieriet al., 2009). The investment cost for the first year for the shredders,shovels and tractors were $20/ton, $1.5/ton and $1.2/ton respectivelyaccounting for the work-time used only feedstock preparation related tobiogas production (Ruggieri et al., 2009) and they were assumed to lastthrough the 15 years of the project.

2.7. Sensitivity analysis

To determine the flexibility of the results of the economic evalua-tion, sensitivity analysis were carried out using cost of the textile re-actor, cost of auxiliary equipment and annual tons of waste produced.For the cost of the textile reactor and auxiliary equipment, a± 20%change in the investment and operation cost were used to determinehow the calculated NPV and IRR would vary, while a± 20% change inthe annual tons of produced waste was used to determine the effect ofvarying amount of waste generation on NPV and IRR. For the textilereactor cost, in the base case, the investment cost of the textile reactorwas $150/volume for small scale (in volume of 2 m3) (Rajendran et al.,2013), $100/volume for mid-scale (volume in 100s of m3) and $70/volume for large-scale (volume in 1000s of m3) (Osadolor et al., 2014),while the operation cost was 5% of the investment cost. For the cost ofthe auxiliary equipment, in the base condition, the investment cost forthe shredders, shovels and tractors were $20/ton, $1.5/ton and $1.2/ton respectively, while the operation cost was $1.95/ton for shredders,$0.12/ton for tractors (used only mid and large scale evaluations) and$0.14/ton for shovels. For the amount of produced waste, in the basecase, 3 tons/y was used for small scale, 5200 tons/y for mid-scale and51,000 tons/y for large scale.


The batch dry anaerobic digestion (dry-AD) of manure bedded withstraw was carried out in a textile-based bioreactor. The reactor is builtfrom textile (Rajendran et al., 2013) and has undergone drag, tears andpressure testing prior to the experimental study and was found verydurable. It was used successfully for over 324 days without any gasleakage or major maintenance. The first batch anaerobic digestion ofthe feedstock took longer degradation time and the methane yield wasslightly lower. Subsequent batch digestion processes resulted to im-proved methane yield and reduced degradation time. The digestateafter biogas production was analyzed as supplement for soil deficient ofmajor nutrients. Then economic analysis of replacing waste collectionby waste managers or composting with dry-AD in a textile bioreactorwas carried out using payback period, net present value and internalrate of return as profitability criteria. A sensitivity analysis of whateffect changes in key cost and waste generation factors could have onthe profitability of dry-AD in the textile bioreactor was also in-vestigated.

3.1. An industrial concept for dry-AD in rural areas

The results obtained in this work would be beneficial for rural areas,farmers, some industrial applications and developing countries where acost-effective and simple solution is needed. The solid waste withhigher TS is treated while generating energy and bio-fertilizer. Dry-ADof solid wastes in the textile-based bioreactors function on a simpleprinciple (Fig. 2a) as a few reactors should run in parallel. Fresh solidwastes are mixed with inoculum (needed only at the initial stage), fedto the bioreactor (with shovel for small scale or tractor/pumps for largescale) and then the bioreactor is closed. As the materials inside thebioreactor have higher TS, and it is not mixed, it has low heat transferso it can perform better than wet digestion in colder climates. However,it can be heated underneath to attain desired temperature in cold


552

regions, but this is not needed for tropical regions. The bioreactor isclosed until degradation time is completed while gas produced is col-lected continuously in a gas holder. When there is no more gas pro-duction, the bioreactor is opened and a portion of the digestate will bepumped or moved for storage in a separate bioreactor, while a portionof the digestates remains inside the bioreactor, mixed with fresh wastesand the bioreactors is closed and sealed for the next run of gas pro-duction. The number of the bioreactors needed in parallel depends onthe feeding time and the gas production duration in each batch. Forexample, if 3 months would be the gas production period in each batchand 15 days would be enough for emptying, and refilling the bior-eactors, 6 bioreactors can work in parallel to fulfil continuous receivingof the fresh wastes. The volume of the textile bioreactor needed tohandle different amount of waste is shown in Table 2, and they can bearranged as shown in Fig. 2a.

3.2. Feedstock characterization

The characteristics of the feedstocks used in this work are shown in

Table 1. The result showed that the substrates have different moisturecontent at different batches, probably because they were collectedunder different storage conditions. The C/N of the manure bedded withstraw was the same but the C/N ratio of wheat straw was considerablydifferent from one batch to another. The variation in the C/N ratio ofwheat straw could be due to the age of the straw; the younger the tis-sues of the straw, the lower the C/N ratio (Hicks, 1928). Cellulose andhemicellulose content of wheat straw were similar for the two batches.The cellulose was between the range of 39–43% and hemicellulose wasbetween the ranges of 28–32% but not all these carbon will be acces-sible during the anaerobic digestion process. The manure bedded withstraw collected has a total solid content between 18% and 26% whichmakes it suitable for dry digestion compared to other means of hand-ling. This waste also has a C/N of 19:1 which means it can be useddirectly by farmers for the digestion process; the C/N can also be ad-justed slightly using untreated wheat straw to enhance the buffer ca-pacity of the process and also reduce the possibility of substrate orproduct inhibition (Wang et al., 2011).

Fig. 2. (a) Typical dry-AD technology in textile bioreactors – industrial concept for rural areas. (b) Cumulative methane production and methane production rate obtained duringdigestion process with gradual increase of total solid (TS) content of the feedstock and reactor mixture. 22%, 27% and 30% (TS of feedstock); 10%, 12% and 17% (TS in reactor).


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3.3. Methane yield and production rate

Fig. 2b shows the cumulative methane production for the three (3)batch digestion processes during the whole experiment and the me-thane production rates were estimated from the cumulative methaneproduction as shown. For the first digestion process (22% TS of feed-stock; 10% TS in reactor), there was a lag phase of 10 days and after-wards the methane production increased steadily indicating the be-ginning of the exponential growth. The digestion process lasted for136 days and methane yield of 183 NmlCH4/gVS was obtained.

Patinvoh et al. (2017) reported yield of 163 NmlCH4/gVS using plugflow reactor when manure bedded with straw at 22%TS and C/N of16.8 was used. However, in the current study using textile reactor therewas slight increase of 12% in the methane yield obtained, probably dueto increase in C/N by addition of untreated wheat straw. Wang et al.(2013) also indicated that addition of even small amounts of co-sub-strates altered the bacteria communities within the reactors, andthereafter increased the yield. This shows that increasing the C/N ratioslightly with addition of fresh wheat straw is more favorable to thedigestion process. During the second digestion process (27% TS and12% TS in feedstock and reactor respectively), the digestion startedalmost immediately (no lag phase) showing the acclimatization of mi-crobial communities to the feedstock resulting in an increase in me-thane yield by about 13% compared to that during the first digestion,while the degradation time was reduced from 136 days to 96 days.After a long-term acclimatization of 232 days (first and second diges-tion), the VS ratio for inoculum to feedstock was reduced from 1 to 0.5thereby increasing the TS in the reactor to 17%. The biogas productionstarted almost immediately even here, although there was a slight re-duction in the methane yield at the beginning compared to that duringthe second digestion. Then, there was steady increase in the methaneyield resulting in about 58% increase compared to that obtained in thefirst digestion. Accordingly, the degradation time was reduced from136 days to 92 days. Furthermore, the methane production rates in-creased with gradual increase in the TS which shows gradual acclima-tization of microbial communities to the conditions in the reactor andprobably new predominant microbial community for high solid-statedigestion were formed (Li et al., 2013). However, the degree of diges-tion was low; only 29% of the organic matter was broken down andconverted to biogas during the digestion period. This could be due tolack of internal mixing and slow degradation rate of the feedstocks(manure with straw and untreated wheat straw). An approximate de-gree of digestion of 35% has been reported for cattle manure(Schnürer & Jarvis, 2010) which is low compared to readily degradablesubstrates. The residue can undergo post storage stage where biogasproduction can continue over a long period of time.

The results showed that farmers can only treat small volume ofwastes at the beginning which will also result to low biogas yield andlonger degradation time. Then, the volume of wastes can be increasedand subsequent digestion processes will results to higher methane yieldand shorter degradation time.

Table 1Characteristics of substrates used during the experiments.

Parameters Manure bedded with straw Wheat straw

First BatchTotal solids (%) 17.81 ± 0.16 91.77 ± 0.02Volatile solids (%)* 80.23 ± 0.55 92.89 ± 0.11Moisture (%) 82.19 ± 0.16 8.23 ± 0.02Ash (%)* 19.77 ± 0.55 7.11 ± 0.11Total Carbon (%)* 44.57 ± 0.31 51.60 ± 0.06Total Nitrogen (%)* 2.33 ± 0.03 0.37 ± 0.15Protein content (%) 14.56 ± 0.03 2.31 ± 0.15C/N 19 139Bulk density (kg/m3) 907.1 ± 7.83 89.5 ± 2.89Extractives (%)* ND 12.17Total Lignin (%)* ND 15.27 ± 0.71Cellulose (%)* ND 39.25 ± 0.03Hemicellulose (%)* ND 31.99 ± 0.08

Second BatchTotal solids (%) 25.84 ± 0.92 89.07 ± 0.08Volatile solids (%)* 77.21 ± 2.74 94.96 ± 0.14Moisture (%) 74.16 ± 0.92 10.93 ± 0.08Ash (%)* 22.79 ± 2.74 5.04 ± 0.14Total Carbon (%)* 42.89 ± 1.52 52.76 ± 0.08Total Nitrogen (%)* 2.26 ± 0.04 0.77 ± 0.01Protein content (%) 14.13 ± 0.04 4.81 ± 0.01C/N 19 69Bulk density (kg/m3) 542 ± 26.87 190.47 ± 6.93pH 8.72 ± 0.83 NDExtractives (%)* ND 8.27Total Lignin (%)* ND 16.52 ± 0.38Cellulose (%)* ND 42.74 ± 0.06Hemicellulose (%)* ND 27.99 ± 0.01

Standard deviation based on at least duplicate measurements.* Dry basis; ND = Not determined; C/N = carbon nitrogen ratio.

Table 2Composition and experimental results at different batch digestion setup Volume and cost of textile reactor needed for the different scenarios and scales.

Setup Feedstock (g) Inoculum (g) Total Feedstock Reactor Mixture TS (%) Reactor Mixture VS (%)* CumulativeMethane yield

Manure withstraw

Wheat straw TS (%) C/N In Out In Out (Nml/gVS)

Unacclimatizedinoculum

3478 235 17425 22 25 10 7.97 ± 0.02 66 65.10 ± 0.15 182.94

Acclimatized (LowTS)

5000 86 20617 27 20 12 9.60 ± 0.12 69.2 64.60 ± 0.06 207.03

Acclimatized (HighTS)

7021 473 14516 30 25 17 13.39 ± 1.15 72.4 65.5 ± 0.16 289.86

Volume of textile reactor (m3) Cost of textile reactor ($)

Scenario 3 tons/year 5200 tons/year 51000 tons/year 3 tons/year 5200 tons/year 51000 tons/yearUnacclimatized

inoculum19 33 687 330 392 2 915 3 368 698 23 127 405

Acclimatized (LowTS)

14 24 202 237 367 2 094 2 420 208 16 615 659

Acclimatized (HighTS)

9 14 871 145 853 1 287 1 487 124 10 209 675

* Dry basis.


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3.4. Digestate quality

The pH of the digestate was slightly alkaline as shown in Table 3;this will increase the buffering capacity of the soil as many agriculturallands are mostly acidic. The liquid fraction of the digestate has highernitrogen and potassium content compared to the solid fraction as shownin Table 3. Möller et al. (2010), also reported high nitrogen and po-tassium content in liquid fraction after separation of the biogas effluent.The digestate (liquid fraction) contains an average of 2.63 g/L of totalnitrogen and 1.4 g/L of ammonium nitrogen which is a major benefit ofusing digestate as bio-fertilizer because the ammonium nitrogen can bedirectly taking up by the plants for their growth (Börjesson & Berglund,2007; Svensson et al., 2004). The digestate (liquid fraction) contain ahigher amount of potassium (an average of 2.74 g/L) which is also amajor macronutrient needed for plant growth. However, the phos-phorus content of the digestate is low probably part of the phosphorushas been lost during the digestion process (Möller &Müller, 2012) andas such phosphorus or phosphate can be added as supplement to avoidphosphorus deficiency in the soil or the digestate supplied to soillacking majorly nitrogen and potassium. Additionally, concentration ofheavy metals (Cadmium, Chromium, Nickel, Zinc and Copper) in thedigestate is low; the concentration did not exceed the quality standards(Al Seadi & Lukehurst, 2012) and as such the digestate could be used asbio-fertilizer. The solid fraction can as well be used on the soil to im-prove the soil structure; retain nutrients and avoid leaching. Therefore,the whole digestate can be used as bio-fertilizer or the solid fractionpost treated using aerobic composting process thereby closing theproduction cycle (Fouda, 2011; Meissl & Smidt, 2007; Poggi-Varaldoet al., 1999).

3.5. Economic evaluation

For biogas to be used as a means for handling manure and strawwaste, it has to be more profitable than other possible methods ofhandling the waste. Several techno-economic analysis have been donein comparing the profitability of biogas production against othermethods such as; replacing kerosene or LPG utilization with biogas athousehold level (Rajendran et al., 2013), biogas for electricity genera-tion (Gebrezgabher et al., 2010), biogas for combined heat and powergeneration (Trendewicz & Braun, 2013). However, economic analysis ofreplacing composting or waste collection by waste managers with dry-AD has not yet been reported in literature. In this work, economicprofitability was measured by the payback period, net present value

and the internal rate of return. The economics associated with replacingcomposting and waste collection by waste managers with dry anaerobicdigestion was investigated for three different dry digestion scenarios;no acclimatization of the inoculum and solid waste with a density of907 kg/m3 (scenario A), short acclimatization of the inoculum and solidwaste with a density of 542 kg/m3 (scenario B) and longer acclimati-zation of the inoculum and solid waste with a density of 542 kg/m3

(scenario C). The analyses were performed at three different scalescorresponding to solid waste generation from typical small, mid andlarge scale farms.

The volume of the textile bioreactors needed for the different scalesand scenarios were calculated as discussed in Section 2.6 and are shownin Table 2. How the reactor volumes can be used for gathering waste,dry-AD, digestate storage and biogas storage is shown in Fig. 2a andwas discussed in Section 3.1. Building on that, this is how the reactorvolumes will be allocated for different purposes: For the first anaerobicdigestion batch; 6 % (scenario A), 16% (scenario B), 32% (scenario C)of the total reactor volume is needed for storing the daily producedfarm waste prior to dry anaerobic digestion at all scales, while 6%(scenario A), 10% (scenario B), 16% (scenario C) of the total reactorvolume is needed for storing the digestate after the completion of thedry-AD. For the subsequent batches 6% (scenario A), 10% (scenario B),16% (scenario C) of the total reactor volume would be needed forstoring the daily produced waste, while 6% (scenario A), 10% (scenarioB), 16% (scenario C) of the total reactor volume would be needed forstoring the digestate after the dry-AD. Details on how to perform thiskind of calculations and optimal reactor sizing can be found in litera-ture (Mähnert & Linke, 2009). The investment cost of the textile bior-eactor (Table 2) were calculated using; $150/volume for small scale (involumes of 2 m3) (Rajendran et al., 2013), $100/volume for mid-scale(volume in 100s of m3) and $70/volume for large-scale (volume in1000s of m3) (Osadolor et al., 2014). The total capital investmentneeded for dry-AD for the different scenarios and scales with the annualcost associated with managing the waste either through composting orthrough waste managers is shown in Table 4.

Table 3Analysis of digestate from dry anaerobic digestion.


pH 8.01–8.06Total carbon (%)* 34.36–36.23TS (%)* 7.97–13. 39VS (%)* 61.84–65.15

Unit Liquid fractiona Solid fractiona

Total Nitrogen g/L 2.45–2.8 6.77–9.22 (mg/g)Ammonium nitrogen g/L 1.25–1.55 NDPotassium g/L 1.19–4.29 0.025–0.051Phosphorus mg/L 10.03–30.54 14.71–36.52Calcium mg/L 15–50.50 48.33–64.33Magnesium mg/L 3.35–60 13.4–16.73Copper mg/L 0.13–0.19 0.14–0.35Iron mg/L 3–5.5 NDZinc mg/L 0.11–0.19 0.33–0.69Nickel mg/L 0.28–0.37 0.01Chromium mg/L <0.001 0.02–0.05Cadmium mg/L <0.001 <0.001

a Fresh matter; ND = not determined.* Dry basis.

Table 4Cost associated different waste management option for handling solid waste and revenuefrom biogas sales.

Cost associated with different solid waste management approach

Total Investment cost needed for biogas production ($)

Scenario Small scale Mid-scale Large-scale

A 2980 3 518 040 24 257 700B 3032 3 518 040 24 957 700C 1967 2 318 040 16 557 700Total annual operation cost needed for biogas production ($)


A 152 170 007 1 155 007B 155 170 007 1 190 007C 102 110 007 770 007Revenue from biogas sales ($)


A 0 42 409B 0 53 519C 0 83 817Annual composting cost 1200 504000 3417000Annual cost when waste managers are

used1300 1250000 12300000

A dense substrate without innoculum acclimatization; B less dense substrate with shortinnoculum acclimatization; C less dense substrate with long innoculum acclimatization.


555

3.4. Digestate quality

The pH of the digestate was slightly alkaline as shown in Table 3;this will increase the buffering capacity of the soil as many agriculturallands are mostly acidic. The liquid fraction of the digestate has highernitrogen and potassium content compared to the solid fraction as shownin Table 3. Möller et al. (2010), also reported high nitrogen and po-tassium content in liquid fraction after separation of the biogas effluent.The digestate (liquid fraction) contains an average of 2.63 g/L of totalnitrogen and 1.4 g/L of ammonium nitrogen which is a major benefit ofusing digestate as bio-fertilizer because the ammonium nitrogen can bedirectly taking up by the plants for their growth (Börjesson & Berglund,2007; Svensson et al., 2004). The digestate (liquid fraction) contain ahigher amount of potassium (an average of 2.74 g/L) which is also amajor macronutrient needed for plant growth. However, the phos-phorus content of the digestate is low probably part of the phosphorushas been lost during the digestion process (Möller &Müller, 2012) andas such phosphorus or phosphate can be added as supplement to avoidphosphorus deficiency in the soil or the digestate supplied to soillacking majorly nitrogen and potassium. Additionally, concentration ofheavy metals (Cadmium, Chromium, Nickel, Zinc and Copper) in thedigestate is low; the concentration did not exceed the quality standards(Al Seadi & Lukehurst, 2012) and as such the digestate could be used asbio-fertilizer. The solid fraction can as well be used on the soil to im-prove the soil structure; retain nutrients and avoid leaching. Therefore,the whole digestate can be used as bio-fertilizer or the solid fractionpost treated using aerobic composting process thereby closing theproduction cycle (Fouda, 2011; Meissl & Smidt, 2007; Poggi-Varaldoet al., 1999).

3.5. Economic evaluation

For biogas to be used as a means for handling manure and strawwaste, it has to be more profitable than other possible methods ofhandling the waste. Several techno-economic analysis have been donein comparing the profitability of biogas production against othermethods such as; replacing kerosene or LPG utilization with biogas athousehold level (Rajendran et al., 2013), biogas for electricity genera-tion (Gebrezgabher et al., 2010), biogas for combined heat and powergeneration (Trendewicz & Braun, 2013). However, economic analysis ofreplacing composting or waste collection by waste managers with dry-AD has not yet been reported in literature. In this work, economicprofitability was measured by the payback period, net present value

and the internal rate of return. The economics associated with replacingcomposting and waste collection by waste managers with dry anaerobicdigestion was investigated for three different dry digestion scenarios;no acclimatization of the inoculum and solid waste with a density of907 kg/m3 (scenario A), short acclimatization of the inoculum and solidwaste with a density of 542 kg/m3 (scenario B) and longer acclimati-zation of the inoculum and solid waste with a density of 542 kg/m3

(scenario C). The analyses were performed at three different scalescorresponding to solid waste generation from typical small, mid andlarge scale farms.

The volume of the textile bioreactors needed for the different scalesand scenarios were calculated as discussed in Section 2.6 and are shownin Table 2. How the reactor volumes can be used for gathering waste,dry-AD, digestate storage and biogas storage is shown in Fig. 2a andwas discussed in Section 3.1. Building on that, this is how the reactorvolumes will be allocated for different purposes: For the first anaerobicdigestion batch; 6 % (scenario A), 16% (scenario B), 32% (scenario C)of the total reactor volume is needed for storing the daily producedfarm waste prior to dry anaerobic digestion at all scales, while 6%(scenario A), 10% (scenario B), 16% (scenario C) of the total reactorvolume is needed for storing the digestate after the completion of thedry-AD. For the subsequent batches 6% (scenario A), 10% (scenario B),16% (scenario C) of the total reactor volume would be needed forstoring the daily produced waste, while 6% (scenario A), 10% (scenarioB), 16% (scenario C) of the total reactor volume would be needed forstoring the digestate after the dry-AD. Details on how to perform thiskind of calculations and optimal reactor sizing can be found in litera-ture (Mähnert & Linke, 2009). The investment cost of the textile bior-eactor (Table 2) were calculated using; $150/volume for small scale (involumes of 2 m3) (Rajendran et al., 2013), $100/volume for mid-scale(volume in 100s of m3) and $70/volume for large-scale (volume in1000s of m3) (Osadolor et al., 2014). The total capital investmentneeded for dry-AD for the different scenarios and scales with the annualcost associated with managing the waste either through composting orthrough waste managers is shown in Table 4.

Table 3Analysis of digestate from dry anaerobic digestion.


pH 8.01–8.06Total carbon (%)* 34.36–36.23TS (%)* 7.97–13. 39VS (%)* 61.84–65.15

Unit Liquid fractiona Solid fractiona

Total Nitrogen g/L 2.45–2.8 6.77–9.22 (mg/g)Ammonium nitrogen g/L 1.25–1.55 NDPotassium g/L 1.19–4.29 0.025–0.051Phosphorus mg/L 10.03–30.54 14.71–36.52Calcium mg/L 15–50.50 48.33–64.33Magnesium mg/L 3.35–60 13.4–16.73Copper mg/L 0.13–0.19 0.14–0.35Iron mg/L 3–5.5 NDZinc mg/L 0.11–0.19 0.33–0.69Nickel mg/L 0.28–0.37 0.01Chromium mg/L <0.001 0.02–0.05Cadmium mg/L <0.001 <0.001

a Fresh matter; ND = not determined.* Dry basis.

Table 4Cost associated different waste management option for handling solid waste and revenuefrom biogas sales.

Cost associated with different solid waste management approach

Total Investment cost needed for biogas production ($)


A 2980 3 518 040 24 257 700B 3032 3 518 040 24 957 700C 1967 2 318 040 16 557 700Total annual operation cost needed for biogas production ($)


A 152 170 007 1 155 007B 155 170 007 1 190 007C 102 110 007 770 007Revenue from biogas sales ($)


A 0 42 409B 0 53 519C 0 83 817Annual composting cost 1200 504000 3417000Annual cost when waste managers are

used1300 1250000 12300000

A dense substrate without innoculum acclimatization; B less dense substrate with shortinnoculum acclimatization; C less dense substrate with long innoculum acclimatization.


555

3.5.1. Payback periodThe payback period for replacing composting and using waste

managers are shown in Fig. 3 for the different scenarios considered. Theinitial project delay time for the first anaerobic digestion batch com-pletion for scenario A was 0.37 years (calculated by dividing thenumber of needed for batch completion by 365), 0.64 for scenario B and0.89 for scenario C. Replacing composting or the use of waste managers

by dry-AD in the textile reactor always resulted in a payback period lessthan the proposed project life of 15 years. In addition, it can be ob-served that the longer the acclimatizing time of the inoculum, theshorter the payback period. Another possible explanation for the re-duction in the payback period is that more amount of waste per year isprocessed in scenario B compared to A, and scenario C processes themost amount of waste per year. Considering the result for small scale,

-

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B Fig. 3. Payback period if dry anaerobic digestion is used toreplace compost (A) or waste collection by waste managers(B) when there is; no inoculum acclimatization and densefeedstock (■), short inoculum acclimatization with lessdense feedstock ( ) and long inoculum acclimatization withless dense feedstock ( ).

0

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-1 0 1 2 3 4 5 6 7 8

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A

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Fig. 4. The net present value and the corresponding internalrate of return analysis for replacing waste collection by wastemanagers and composting with dry anaerobic digestion for;small scale (A), mid-scale (B) and large-scale(C) when is; noinoculum acclimatization and dense feedstock (■), shortinoculum acclimatization with less dense feedstock ( ) andlong inoculum acclimatization with less dense feedstock ( ).


556

the payback period for all dry anaerobic digestion scenarios consideredwere all less than 3.5 years, indicating that using the textile bioreactorfor dry digestion is more profitable than composting or waste collectionby waste managers for small scale farmers. As the farm scale increased,dry anaerobic digestion in the textile bioreactor would still be the mostprofitable method of handling solid waste, particularly dry anaerobicdigestion after long inoculum acclimatization which had a payback

period of 5 years or less for all cases considered.

3.5.2. Net present value and internal rate of returnThe result for the net present value (NPV) and internal rate of return

(IRR) calculated for the different scenarios using an interest rate of7.8% are shown in Fig. 4. For the first year, the annual profit wasmultiplied by 0.627 for scenario A (1 – delay fraction used for PBP

Table 5Sensitivity analysis on replacing composting with dry anaerobic digestion.

Replacing composting with dry anaerobic digestion

Base case

Net present value (NPV) ($) Internal rate of return (IRR) (%)

Small scale Mid-scale Large scale Small scale Mid-scale Large scale

Scenario A 5320 −733,125 −5,948,401 31.44 4.00 3.32Scenario B 6152 428,723 1,874,533 40.83 10.47 9.48Scenario C 6959 1,553,297 9,783,711 56.62 20.53 19.31

20% less textile reactor cost

NPV ($) IRR (%)


Scenario A 6086 151,842 127,233 39.86 8.7 7.91Scenario B 6698 1,059,041 6,373,146 50.09 15.38 14.37Scenario C 7291 1,937,377 12,420,569 67.1 26.02 24.48

20% more textile reactor cost

NPV ($) IRR (%)


Scenario A 4555 −1,618,091 −12,024,036 25.47 0.26 −0.37Scenario B 5607 −201,595 −2,281,603 34.2 6.68 5.95Scenario C 6627 1,169,216 7,146,853 48.95 16.37 15.35

20% less auxiliary equipment cost

NPV ($) IRR (%)



20% more auxiliary equipment cost

NPV ($) IRR (%)



20% less waste generation

NPV ($) IRR (%)



20% more waste generation

NPV ($) IRR (%)



A dense substrate without innoculum acclimatization; B less dense substrate with short innoculum acclimatization; C less dense substrate with long innoculum acclimatization.


557

the payback period for all dry anaerobic digestion scenarios consideredwere all less than 3.5 years, indicating that using the textile bioreactorfor dry digestion is more profitable than composting or waste collectionby waste managers for small scale farmers. As the farm scale increased,dry anaerobic digestion in the textile bioreactor would still be the mostprofitable method of handling solid waste, particularly dry anaerobicdigestion after long inoculum acclimatization which had a payback

period of 5 years or less for all cases considered.

3.5.2. Net present value and internal rate of returnThe result for the net present value (NPV) and internal rate of return

(IRR) calculated for the different scenarios using an interest rate of7.8% are shown in Fig. 4. For the first year, the annual profit wasmultiplied by 0.627 for scenario A (1 – delay fraction used for PBP

Table 5Sensitivity analysis on replacing composting with dry anaerobic digestion.

Replacing composting with dry anaerobic digestion

Base case

Net present value (NPV) ($) Internal rate of return (IRR) (%)



20% less textile reactor cost

NPV ($) IRR (%)


Scenario A 6086 151,842 127,233 39.86 8.7 7.91Scenario B 6698 1,059,041 6,373,146 50.09 15.38 14.37Scenario C 7291 1,937,377 12,420,569 67.1 26.02 24.48

20% more textile reactor cost

NPV ($) IRR (%)


Scenario A 4555 −1,618,091 −12,024,036 25.47 0.26 −0.37Scenario B 5607 −201,595 −2,281,603 34.2 6.68 5.95Scenario C 6627 1,169,216 7,146,853 48.95 16.37 15.35

20% less auxiliary equipment cost

NPV ($) IRR (%)



20% more auxiliary equipment cost

NPV ($) IRR (%)



20% less waste generation

NPV ($) IRR (%)



20% more waste generation

NPV ($) IRR (%)



A dense substrate without innoculum acclimatization; B less dense substrate with short innoculum acclimatization; C less dense substrate with long innoculum acclimatization.


557

calculation), 0.36 for scenario B and 0.11 for scenario C. Consideringwaste disposal through waste managers, irrespective of inoculum ac-climatization, dry anaerobic digestion is more profitable as evident bythe positive NPV’s. In addition, the IRR for all scales and scenariosconsidered were from 30.1 to 69.6%, implying that dry anaerobic di-gestion using the textile reactor would remain the most profitable op-tion for all farm scale with or without inoculum acclimatization untilthe interest rate in that country exceeds the IRR. Currently there areonly three countries in the world where the interest rate exceeds 30%,and there is no country with an interest rate greater than 50% (WorldBank, 2017b) so the odds of solid waste management by using wastemanagers becoming more profitable than dry anaerobic digestion in thetextile reactor is rear.

Comparing dry-AD to composting (Fig. 4), both the scale of wastegeneration and the duration inoculum acclimatization influences whatoption is more profitable. For small scale, irrespective of inoculumacclimatization or the nature of the feed, dry-AD is more profitable thancomposting as evident by the positive NPV’s and IRR greater than 33%.As the scale of waste generation increases, the profitability of dry-AD inthe textile reactor over composting increases with the duration of theacclimatization of the inoculum used, as evident by the increasing po-sitive NPV’s and the increasing IRR from short to long inoculum ac-climatization time at all scales. The minimum IRR for replacing com-posting with dry anaerobic digestion when long acclimatization is usedwas 19.3%, this indicates that the NPV would remain positive over ahigh interest rate range, with it only becoming negative when the in-terest rate in that country exceeds the IRR. In the cases where com-posting was found to be a better option than anaerobic digestion be-cause of negative NPV, the IRR were 4% or less indicating that the NPVat those cases is sensitive to the interest rate assumed for the calcula-tions, if an interest rate of 3% was used for the calculations all theNPV’s will be positive. A 3% interest rate is feasible because there aremany countries whose current interest rate are 3% or less (World Bank,2017b), using the interest rate of any of those countries would makeNPV of replacing composting with dry anaerobic digestion positive forall scale with or without inoculum acclimatization.

3.6. Sensitivity analysis

The result from the sensitivity analysis calculations for replacingcomposting with dry-AD is shown in Table 5. At small scale, the NPVswere insensitive to a± 20% change in reactor cost, equipment cost andthe amount of waste generated. For cases that had a positive or negativeNPV in the base scenario, a± 20 % change in the cost of equipment andamount of waste generated was not sufficient in changing the signnotation of the NPV of any of the base cases. So, the profitability criteriawere not sensitive to a± 20% change in the cost of equipment andamount of waste generated.

The profitability criteria for replacing composting with dry anae-robic digestion at mid and large scale was sensitive to changes in thecost of the textile reactor when short or no inoculum acclimatizationtime was used. For the base case, the NPV of using scenario A for re-placing composting with dry anaerobic digestion were negative at mid(IRR = 4 %) and large scale (IRR = 3.32%), however, a 20% reductionin the cost of the textile reactor made the NPV positive for both mid(IRR = 8.7%) and large scale (IRR = 7.91%) scenarios. A 20% increasein the cost of the textile reactor resulted in the positive NPV at the basecase of scenario B at mid (IRR = 10.47) and large scale (IRR = 9.48)becoming negative for both mid (IRR = 6.68%) and large scale(IRR = 5.95%). The sensitivity of the textile reactor cost for mid- andlarge-scale scenario A and B cases would mean that using other reactorsmore expensive than the textile reactor for dry-AD would not be eco-nomical when compared with composting. However, replacing com-posting with dry anaerobic digestion after long acclimatization (sce-nario C) was not sensitive to a 20% change in the cost of the textilereactor at all scales, as seen from the positive NPV in all cases and high

IRR values ranging from 15.35% to 67.1%. This indicates that dryanaerobic digestion of solid farm waste in the textile reactor after a longperiod of inoculum acclimatization is a more economical way ofhandling the waste than composting.

4. Conclusion

Textile-based bioreactor is a cost-effective solution and the tech-nology is simple to operate. It can be accessed easily by developingcountries where required expertise may not be available. For small scalefarm, irrespective of inoculum acclimatization or the nature of the feed,dry-AD is more profitable than composting as evident by the positiveNPV’s and IRR greater than 33%. A long acclimatization period makesdry-AD in the textile reactor more profitable than composting for small,mid and large scale farms when handling solid waste as evident by thepositive NPV’s and IRR greater than 19%.

Acknowledgements

This work was financially supported by Västra Götaland Region(Sweden), Smart textile (Sweden) and FOV Fabrics, AB (Sweden). Theauthors acknowledge Mostafa Jabbari for assisting in making the heatexchanger for the reactor.

References

AAFRD, 2005. Manure Composting Manual. Alberta Agriculture, Food and RuralDevelopment. Edmonton, Alberta, Canada T6H 5T6. http://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/agdex8875/$file/400_27-1.pdf?OpenElement,Accessed 2015/09/21.

Al Seadi, T., Lukehurst, C., 2012. Quality management of digestate from biogas plantsused as fertiliser. IEA Bioenergy 4–36.

Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J.L., Guwy, A.J.,Kalyuzhnyi, S., Jenicek, P., Van Lier, J.B., 2009. Defining the biomethane potential(BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays.Water Sci. Technol. 59 (5), 927–934.

APHA-AWWA-WEF, 2005. Standard Methods for the Examination of Water andWastewater 21st ed, American Public Health Association (APHA), American WaterWorks Association (AWWA), Water Environment Federation (WEF), Washington DC.

Bergeron, P., 1996. Bioethanol market forces. In: Wyman, C. (Ed.), Handbook on bioe-thanol: production and utilization. CRC Press, pp. 61–88.

Bolzonella, D., Innocenti, L., Pavan, P., Traverso, P., Cecchi, F., 2003. Semi-dry ther-mophilic anaerobic digestion of the organic fraction of municipal solid waste: fo-cusing on the start-up phase. Bioresour. Technol. 86 (2), 123–129.

Börjesson, P., Berglund, M., 2007. Environmental systems analysis of biogassystems—Part II: the environmental impact of replacing various reference systems.Biomass Bioenergy 31 (5), 326–344.

Chattopadhyay, S., Dutta, A., Ray, S., 2009. Municipal solid waste management inKolkata, India–a review. Waste Manage. 29 (4), 1449–1458.

Chen, Y.-T., 2016. A cost analysis of food waste composting in Taiwan. Sustainability 8(11), 1210.

Deng, L., Chen, C., Zheng, D., Yang, H., Liu, Y., Chen, Z., 2016. Effect of temperature oncontinuous dry fermentation of swine manure. J. Environ. Manage. 177, 247–252.

Fouda, S.E.-S., 2011. Nitrogen availability of biogas residues (PhD Dissertation),Technical University of München.

Gebrezgabher, S.A., Meuwissen, M.P.M., Prins, B.A.M., Lansink, A.G.J.M.O., 2010.Economic analysis of anaerobic digestion—a case of Green power biogas plant in TheNetherlands. NJAS-Wagen. J. Life Sci. 57 (2), 109–115.

Guendouz, J., Buffière, P., Cacho, J., Carrère, M., Delgenes, J.P., 2010. Dry anaerobicdigestion in batch mode: Design and operation of a laboratory-scale, completelymixed reactor. Waste Manage. 30 (10), 1768–1771.

Gunaseelan, N.V., 2009. Biomass estimates, characteristics, biochemical methane po-tential, kinetics and energy flow from Jatropha curcus on dry lands. BiomassBioenergy 33 (4), 589–596.

Haug, R.T., 1993. Feed conditioning – physical and chemical. In: The Practical Handbookof Compost Engineering. Taylor & Francis, pp. 247–257.

Hicks, P.A., 1928. Distribution of carbon/nitrogen ratio in the various organs of the wheatplant at different periods of its life history. New Phytol. 27 (2), 108–116.

Ho, T.L.T., Cao, T.S., Luong, D.A., Vu, D.T., Kurosawa, K., Egashira, K., 2013. Evaluationof water pollution caused by different pig–farming systems in Hungyen province ofVietnam. Kyushu Univ. Int. Reposit 58, 159–165.

Hoornweg, D., Thomas, L., Otten, L. 1999. Composting and its applicability in developingcountries. World Bank working paper series, 8.

Karak, T., Bhagat, R.M., Bhattacharyya, P., 2012. Municipal solid waste generation,composition, and management: the world scenario. Crit. Rev. Environ. Sci. Technol.42 (15), 1509–1630.

Kossmann, W., Pönitz, U., Habermehl, S., Hoerz, T., Krämer, P., Klingler, B., Kellner, C.,Wittur, W., Klopotek, F., Krieg, A. 1999. Biogas Digest, Volume II–Biogas–Application


558

and Product Development. Information and Advisory Service on AppropriateTechnology, Eschborn.

Li, A., Chu, Y., Wang, X., Ren, L., Yu, J., Liu, X., Yan, J., Zhang, L., Wu, S., Li, S., 2013. Apyrosequencing-based metagenomic study of methane-producing microbial commu-nity in solid-state biogas reactor. Biotechnol Biofuels 6.

Li, Y., Park, S.Y., Zhu, J., 2011. Solid-state anaerobic digestion for methane productionfrom organic waste. Renew. Sustainable Energy Rev. 15 (1), 821–826.

Makádi, M., Tomócsik, A., Orosz, V., 2012. Digestate: a new nutrient source – review. In:Kumar, D.S. (Ed.), Biogas. InTech.

Mata-Alvarez, J., Macé, S., Llabrés, P., 2000. Anaerobic digestion of organic solid wastes.An overview of research achievements and perspectives. Bioresour. Technol. 74.

Meissl, K., Smidt, E., 2007. High quality composts by means of cocomposting of residuesfrom anaerobic digestion. Compost Sci. Util. 15 (2), 78–83.

Mähnert, P., Linke, B., 2009. Kinetic study of biogas production from energy crops andanimal waste slurry: effect of organic loading rate and reactor size. Environ. Technol.30 (1), 93–99.

Möller, K., Müller, T., 2012. Effects of anaerobic digestion on digestate nutrient avail-ability and crop growth: a review. Eng. Life Sci. 12 (3), 242–257.

Möller, K., Schulz, R., Müller, T., 2010. Substrate inputs, nutrient flows and nitrogen lossof two centralized biogas plants in southern Germany. Nutr. Cycl. Agroecosys. 87 (2),307–325.

Osadolor, O.A., Lennartsson, P.R., Taherzadeh, M.J., 2014. Introducing textiles as ma-terial of construction of ethanol bioreactors. Energies 7 (11), 7555–7567.

Patinvoh, R.J., Kalantar Mehrjerdi, A., Sárvári Horváth, I., Taherzadeh, M.J., 2017. Dryfermentation of manure with straw in continuous plug flow reactor: reactor devel-opment and process stability at different loading rates. Bioresour. Technol. 224,197–205.

Poggi-Varaldo, H.M., Trejo-Espino, J., Fernandez-Villagomez, G., Esparza-Garcia, F.,Caffarel-Mendez, S., Rinderknecht-Seijast, N., 1999. Quality of anaerobic compostfrom paper mill and municipal solid wastes for soil amendment. Water Sci. Technol.40 (11–12), 179–186.

Rajendran, K., Aslanzadeh, S., Johansson, F., Taherzadeh, M.J., 2013. Experimental andeconomical evaluation of a novel biogas digester. Energy Convers. Manage. 74,183–191.

Rowse, L.E., 2011. Design of small scale anaerobic digesters for application in rural de-veloping countries. University of South Florida.

Ruggieri, L., Cadena, E., Martínez-Blanco, J., Gasol, C.M., Rieradevall, J., Gabarrell, X.,Gea, T., Sort, X., Sánchez, A., 2009. Recovery of organic wastes in the Spanish wineindustry. Technical, economic and environmental analyses of the composting process.

J. Clean. Prod. 17 (9), 830–838.Schnürer, A., Jarvis, A. 2010. Microbiological Handbook for Biogas Plants. Swedish Waste

Management U2009:03.Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2011.

Determination of structural carbohydrates and lignin in biomass. laboratory analy-tical procedure. NREL/TP-510-42618.

Sluiter, A., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008. Determination ofExtractives in Biomass. Laboratory Analytical Procedure (LAP). NREL/TP-510-42619.

Strange, K., 2002. Overview of waste management options: their efficacy and accept-ability. Issues Environ. Sci. Technol. 18, 1–52.

Svensson, K., Odlare, M., Pell, M., 2004. The fertilizing effect of compost and biogasresidues from source separated household waste. J. Agric. Sci. 142 (4), 461–467.

Trendewicz, A.A., Braun, R.J., 2013. Techno-economic analysis of solid oxide fuel cell-based combined heat and power systems for biogas utilization at wastewater treat-ment facilities. J. Power Sources 233, 380–393.

U.S Energy Information Administration. 2017. U.S. Price of Natural Gas Sold toCommercial Consumers. https://www.eia.gov/dnav/ng/hist/n3020us3m.htm,Accessed 2017/05/02.

Wang, W., Xie, L., Chen, J., Luo, G., Zhou, Q., 2011. Biohydrogen and methane pro-duction by co-digestion of cassava stillage and excess sludge under thermophiliccondition. Bioresour. Technol. 102 (4), 3833–3839.

Wang, W., Xie, L., Luo, G., Zhou, Q., 2013. Enhanced fermentative hydrogen productionfrom cassava stillage by co-digestion: The effects of different co-substrates. Int. J.Hydrogen Energy 38 (17), 6980–6988.

World Bank Deposit interest rate: Nigeria, 2017. http://data.worldbank.org/indicator/FR.INR.RINR?end=2015 & locations=NG& page=3& start=2008 & view=chart,Accessed 2017/05/01.

World Bank, 2016. A Global Review of Solid Waste Management; Annex E: EstimatedSolid Waste Management Costs. http://siteresources.worldbank.org/INTURBANDEVELOPMENT/Resources/336387-1334852610766/AnnexE.pdf,Aceesed 2017/05/02.

World Bank. 2017b. Rear interest rate. http://data.worldbank.org/indicator/FR.INR.RINR?end=2015 & start=1997 & view=map, Accesed 2017/05/05.

Zhang, Y., Ghaly, A.E., Li, B., 2012. Physical properties of wheat straw varieties cultivatedunder different climatic and soil conditions in three continents. Am. J. Eng. Appl. Sci.5 (2), 98–106.

Zhou, C., Liu, Z., Huang, Z.-L., Dong, M., Yu, X.-L., Ning, P., 2015. A new strategy for co-composting dairy manure with rice straw: Addition of different inocula at three stagesof composting. Waste Manage. 40, 38–43.


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Biogas digesters: from plastics and bricks to textile bioreactor — A review. Submitted.

Pap

er V

1

Biogas digesters: from plastics and bricks to textile bioreactor – A review

Regina J. Patinvoha,b, Joseph V. Arulappanc, Fredrik Johanssond and Mohammad J. Taherzadeha*

aSwedish Centre for Resource Recovery, University of Borås, 50190 Borås, Sweden.

bDepartment of Chemical and Polymer Engineering, Faculty of Engineering, Lagos State

University, Lagos, Nigeria

cFOV Biogas, India, Velachery, Chennai-42 , India

dFOV Fabrics AB, 50113 Borås, Sweden

Corresponding author

Tel: +46 33 435 5908

Fax: +46-33-435-4008

E-mail: [email protected]

2

Abstract

Biogas technology and anaerobic digestion is a well-established process for energy generation

but there are still technical and economic barriers to advancement of this technology especially

in developing countries. Considering the relatively long retention time of the materials inside

the bioreactors, the material of construction of the bioreactor is important for commercial

applications and should be selected considering cost, lifespan, effectiveness, design and

operation of the bioreactor. The design and operation of particularly household and smaller

bioreactors with their challenges are reviewed in this study. A particular attention is made to

the new textile bioreactor that are relatively new in the market. The laboratory and pilot scale

results shows that the innovative textile bioreactor is a promising technology for global energy

generation advancement.

Keywords:

Biogas digesters; materials of construction; plastic bioreactors, textile bioreactors;

2

Abstract

Biogas technology and anaerobic digestion is a well-established process for energy generation

but there are still technical and economic barriers to advancement of this technology especially

in developing countries. Considering the relatively long retention time of the materials inside

the bioreactors, the material of construction of the bioreactor is important for commercial

applications and should be selected considering cost, lifespan, effectiveness, design and

operation of the bioreactor. The design and operation of particularly household and smaller

bioreactors with their challenges are reviewed in this study. A particular attention is made to

the new textile bioreactor that are relatively new in the market. The laboratory and pilot scale

results shows that the innovative textile bioreactor is a promising technology for global energy

generation advancement.

Keywords:

Biogas digesters; materials of construction; plastic bioreactors, textile bioreactors;

3

1. Introduction

Renewable energy is gaining widespread acceptance; renewables accounted for about 62 % of

net additions to global power generating capacity in 2016 and majority of renewable heat were

supplied by biomass with smaller contributions from solar thermal and geothermal energy

(REN21, 2017). Biogas technology has been used quite extensively in developed countries and

some developing countries for renewable energy generation thereby reducing environmental

impacts of wastes disposal. The technology has improved significantly through support from

government and non-governmental organizations (Dahlquist, 2013; Geels & Raven, 2007;

Nilsson et al., 2012). However, many countries are still under environmental pressures and

energy insecurity which has led to increased interest in biogas technology around the world.

This technology is currently the most sustainable way to utilize the energy content of organic

wastes while also recycling the nutrients and reducing harmful emissions (Luostarinen et al.,

2011). Acceptance of the digestate residue as bio-fertilizer, ban of land-filling and the limitation

of incineration of organic waste are possible factors favoring the development of this

technology around the globe (Torrijos, 2016).

During the last decades, a number of reactors have been developed and examined for small-

and large-scale biogas production, but there are still technical barriers for successful operation

of biogas reactors which impede the development of this technology. In addition, the cost of

many biogas reactors (capital, operational and maintenance costs) is high; most farmers and

many developing countries are not financially buoyant to install such reactors despite increased

interest in biogas technology. Therefore, improving energy efficiency worldwide through

biogas technology requires innovative strategies and initiatives to motivate investment in biogas

production; this will lead to increased economic growth, environmental safety and national

security.

4

Biogas reactor is at the heart of any process plant; its demand and performance are usually the

most important factors in the design of a whole plant; and the performance of the reactor is

important for economic assessment of the technology as a whole (Peacock & Richardson,

2012). A suitable bioreactor for biogas production should be easy to operate, effective in

producing biogas, easy to install and cost effective in order to promote development of biogas

technology. Additionally, for an anaerobic reactor to accommodate high loading rates, basic

conditions must be met such as high retention of viable microorganisms in the reactor under

operational conditions, sufficient contact between viable microorganisms and the feedstock,

high reaction rates, and favourable environmental conditions for the microbial communities in

the reactor under the operating conditions (Lettinga, 1995). Therefore, for overall effectiveness

of the biogas production process, reactors must be selected putting into consideration the design

of the reactor, its mode of operation (Peacock & Richardson, 2012) as well as feedstock

composition, amount of feedstock to be treated, desired product and process economy of the

reactor (Patinvoh et al., 2017a). A biogas reactor must be well designed since the technical

conception of a typical biogas plant is towards achieving an optimum biogas yield considering

the overall economy of the process (Werner et al., 1989).

This work aims at reviewing conventional biogas reactors and challenges associated with them

while introducing an advanced textile-based reactor for biogas production, which is relatively

new in the market. Detailed knowledge of the textile bioreactor technology for small- and large-

scale biogas plants will contribute to development of biogas technology and aid optimizing its

economy.

2. Conventional Biogas reactors

Anaerobic reactors are devices designed for microbial degradation of organic wastes in oxygen-

free condition for biogas production; such reactors must be water tight and gas tight.

Additionally, biogas reactors must be protected against UV light, chemicals, corrosive gases,

5

and insulated against extreme weather conditions. There are different types of anaerobic

reactors; what is common to them all is that they produce biogas for energy generation and bio-

fertilizer for soil amendment in agriculture. They are, however differentiated either by mode of

operation, solid content, operating temperature, process stages, reactor design or material of

construction. Commonly used materials for construction of biogas reactors include steel,

stainless steel, bricks, concrete and plastics (Cheng et al., 2013; Cheng et al., 2014a; ISSF,

2012). Major factors for the choice of material are technical suitability, availability,

maintenance and cost-effectiveness (Kuria & Maringa, 2008); reactors are designed for small

and large scale biogas production.

The size of the biogas reactor depends on the amount of organic wastes to be treated, site

location, demand of natural gas and consumption pattern, and technical skill of staff regarding

operation of the biogas plant (Samer, 2012). Small scale reactors convert organic wastes such

as kitchen wastes, animal manure, human excreta and garden wastes to biogas for household

and community use; commonly used reactors are fixed-dome, floating-drum and rubber-balloon

(Rowse, 2011). Large scale reactors are typically obtainable in developed countries but also

gaining interest in developing countries (DeBruyn et al., 2006) due to environmental pollution

and energy poverty. These reactors are used for conversion of large volume of wastes such as

municipal solid wastes, agricultural wastes, industrial wastes and sludge from wastewater

treatment plantto biogas (Werner et al., 1989). The biogas produced is usually fed into public

grid (De Mes et al., 2003) for heating and electricity generation; it can also be upgraded and

used as transportation fuel. The most commonly used large scale reactors are plug flow,

continuous stirred tank, upflow anaerobic sludge blanket (UASB), and complete mix reactors;

these types of reactors are complex in design and operation (usually require skillful personnel).

Search for new technologies to enhance global advancement of biogas technology has led to

invention of plastics and textile-based biogas reactors. A short review on conventional biogas

5

and insulated against extreme weather conditions. There are different types of anaerobic

reactors; what is common to them all is that they produce biogas for energy generation and bio-

fertilizer for soil amendment in agriculture. They are, however differentiated either by mode of

operation, solid content, operating temperature, process stages, reactor design or material of

construction. Commonly used materials for construction of biogas reactors include steel,

stainless steel, bricks, concrete and plastics (Cheng et al., 2013; Cheng et al., 2014a; ISSF,

2012). Major factors for the choice of material are technical suitability, availability,

maintenance and cost-effectiveness (Kuria & Maringa, 2008); reactors are designed for small

and large scale biogas production.

The size of the biogas reactor depends on the amount of organic wastes to be treated, site

location, demand of natural gas and consumption pattern, and technical skill of staff regarding

operation of the biogas plant (Samer, 2012). Small scale reactors convert organic wastes such

as kitchen wastes, animal manure, human excreta and garden wastes to biogas for household

and community use; commonly used reactors are fixed-dome, floating-drum and rubber-balloon

(Rowse, 2011). Large scale reactors are typically obtainable in developed countries but also

gaining interest in developing countries (DeBruyn et al., 2006) due to environmental pollution

and energy poverty. These reactors are used for conversion of large volume of wastes such as

municipal solid wastes, agricultural wastes, industrial wastes and sludge from wastewater

treatment plantto biogas (Werner et al., 1989). The biogas produced is usually fed into public

grid (De Mes et al., 2003) for heating and electricity generation; it can also be upgraded and

used as transportation fuel. The most commonly used large scale reactors are plug flow,

continuous stirred tank, upflow anaerobic sludge blanket (UASB), and complete mix reactors;

these types of reactors are complex in design and operation (usually require skillful personnel).

Search for new technologies to enhance global advancement of biogas technology has led to

invention of plastics and textile-based biogas reactors. A short review on conventional biogas

6

reactors and challenges associated with them will advance the implementation of textile-based

bioreactors for small and large scale biogas production.

2.1 Fixed-dome and floating-drum reactors

A typical fixed-dome reactor consists of a cylindrical or hemispherical chamber constructed

underground; it is made of bricks and cement (An et al., 1997; Samer, 2012; Sasse, 1988). It

has both mixing and overflow tanks connected to the inlet and outlet valves of the reactor

respectively. The biogas produced is collected in upper chamber (fixed gas holder) and as

pressure increases due to continuous gas production the digestate is discharged through the

outlet to the overflow tank. The use of fixed-dome reactors for biogas production is an

established technology in many parts of the world especially in China, India, Nepal, Vietnam,

Bangladesh, Cambodia, Pakistan and Tanzania (Cheng et al., 2013; Ghimire, 2013).

Fixed dome reactors are commonly used for rural households because of their long lifespan

(Ghimire, 2013). However, the technology is expensive (Pérez et al., 2014), the construction is

labor intensive and required skilled supervision (Kossmann et al., 1999). It is also prone to

porosity and cracks as a result of atmospheric temperature fluctuations or earthquake. Cheng et

al. (2014b) reported only 53 % of fixed dome reactors in good operation during a survey of 94

household plants in Nepal; most plants are not functioning well due to plumbing problems,

damages at the slurry chamber and cracks in the reactor (Tomar, 1994). Utilization of the gas

produced is less effective as the gas pressure fluctuates and possibility of explosion of the fixed

gas holder due to excessive biogas pressure (Kuria & Maringa, 2008). Additionally, large fixed-

dome plants always require a separate gas holder and an agitator; floating gas holders are

sometimes used in cold region which is more expensive (Sasse, 1988).

Floating-drum reactors are similar to the fixed-dome reactors in operation; the major difference

with floating drum reactor is the floating drum on top of the reactor that separates gas

6

reactors and challenges associated with them will advance the implementation of textile-based

bioreactors for small and large scale biogas production.

2.1 Fixed-dome and floating-drum reactors

A typical fixed-dome reactor consists of a cylindrical or hemispherical chamber constructed

underground; it is made of bricks and cement (An et al., 1997; Samer, 2012; Sasse, 1988). It

has both mixing and overflow tanks connected to the inlet and outlet valves of the reactor

respectively. The biogas produced is collected in upper chamber (fixed gas holder) and as

pressure increases due to continuous gas production the digestate is discharged through the

outlet to the overflow tank. The use of fixed-dome reactors for biogas production is an

established technology in many parts of the world especially in China, India, Nepal, Vietnam,

Bangladesh, Cambodia, Pakistan and Tanzania (Cheng et al., 2013; Ghimire, 2013).

Fixed dome reactors are commonly used for rural households because of their long lifespan

(Ghimire, 2013). However, the technology is expensive (Pérez et al., 2014), the construction is

labor intensive and required skilled supervision (Kossmann et al., 1999). It is also prone to

porosity and cracks as a result of atmospheric temperature fluctuations or earthquake. Cheng et

al. (2014b) reported only 53 % of fixed dome reactors in good operation during a survey of 94

household plants in Nepal; most plants are not functioning well due to plumbing problems,

damages at the slurry chamber and cracks in the reactor (Tomar, 1994). Utilization of the gas

produced is less effective as the gas pressure fluctuates and possibility of explosion of the fixed

gas holder due to excessive biogas pressure (Kuria & Maringa, 2008). Additionally, large fixed-

dome plants always require a separate gas holder and an agitator; floating gas holders are

sometimes used in cold region which is more expensive (Sasse, 1988).

Floating-drum reactors are similar to the fixed-dome reactors in operation; the major difference

with floating drum reactor is the floating drum on top of the reactor that separates gas

7

production and collection (Shi, 2014). They are made of mild steel and the reactor walls and

bottom are made of bricks; the reactor has a moveable gas holder, cylindrical digestion chamber

with the inlet and outlet pipes connected (Kumar et al., 2015). These types of reactors are easy

to operate and the drum can provide gas at constant pressure. However,

the steel drum is relatively expensive and requires constant removal of rust and regular painting

to avoid gas leakage (Kossmann et al., 1999). Recently the steel drum is replaced with fiber

reinforced plastics gas holders to solve the problem of corrosion but they are more expensive;

sometimes high density polyethylene (HDPE) is used (Mostajir et al., 2013).

2.2 Plastic biogas reactors

Construction of biogas reactors from plastics is an alternative option to solve challenges

associated with fixed-dome and floating drum reactors; the technology evolved from the need

for new materials that are non-corrosive, good insulator, cheaper and easier to construct (Kumar

& Bai, 2005).

Flexible and rigid plastics materials have been used for construction of biogas reactors, gas

holders, digestate storage and membrane covers; polymeric materials used include polyethylene

(PE), polyvinylchloride (PVC), polypropylene (PP), polystyrene (PS), polymethyl methacrylate

(PMMA), acrylonitrile butadiene styrene (ABS) and fiber reinforced plastics (Cheng et al.,

2013). Several plastic biogas reactors have been developed and tested in countries like China,

India Vietnam, Kenya, Bolivia, Bangladesh, Rwanda, Taiwan, Tanzania, Ethiopia, Columbia

and Honduras (An et al., 1997; Cheng et al., 2013; Cheng et al., 2014a; GTZ/EnDev, 2010;

Nzila et al., 2012; Rakotojaona, 2013). These reactors and challenges associated with them are

discussed in subsequent subsections.

2.2.1 Polyethylene tubular reactors

8

The first plastic reactor was a tubular reactor; an inexpensive red mud PVC reactor of 15 m3

total volume introduced in Taiwan and evaluated by Pound et al. (1981) using plug flow

principles; the content of the reactor was unmixed and unheated. This model was later

simplified in Ethiopia and then Colombia (An et al., 1997). Polyethylene tubular reactors also

known as balloon tubular reactors were introduced within the project supported by FAO and

SIDA (college of agriculture and forestry in Ho Chi Minh City) (An et al., 1994). Since 1990s

(Rakotojaona, 2013), several of these reactors have been implemented and commercialized for

small scale biogas plants due to low cost and simplicity of installation. Biogas produced is either

stored in the upper part of the digester (Nzila et al., 2012) or in a similar rubber tubes made

from polyethylene material (Nazir, 1991) and used through the gas vent when required. The

first-generation plastic reactors are susceptible to mechanical damage, have short lifespan (2 to

5 years), sensitive to sunlight and deteriorate rapidly due to seasonal changes (Kossmann et al.,

1999; Nazir, 1991; Nzila et al., 2012).

2.2.2 Plastic tanks

Rigid plastic tanks are developed for biogas production and they are made of hard polymeric

materials such as hard polyvinylchloride (PVC), high density polyethylene (HDPE) and

modified plastics except reinforced plastics (Cheng et al., 2013). They are composed of two

pre-built rigid plastic tanks (Rakotojaona, 2013); the first tank for anaerobic digestion and the

second tank for gas storage. They are easy to install, longer lifespan compared to polyethylene

tubular reactors and quick biogas production start-up (3 to 4 days) (Rakotojaona, 2013) but they

are expensive and not suitable for large scale biogas plants. Currently there are very few plastic

tanks installed.

2.2.3 Latest generation plastic reactors

The problem of low reliability (40 %) (Nzila et al., 2012) with the low-cost polyethylene tubular

reactors led to modified plastic biogas reactors. Several PE tubular reactors installed in Kenya

8

The first plastic reactor was a tubular reactor; an inexpensive red mud PVC reactor of 15 m3

total volume introduced in Taiwan and evaluated by Pound et al. (1981) using plug flow

principles; the content of the reactor was unmixed and unheated. This model was later

simplified in Ethiopia and then Colombia (An et al., 1997). Polyethylene tubular reactors also

known as balloon tubular reactors were introduced within the project supported by FAO and

SIDA (college of agriculture and forestry in Ho Chi Minh City) (An et al., 1994). Since 1990s

(Rakotojaona, 2013), several of these reactors have been implemented and commercialized for

small scale biogas plants due to low cost and simplicity of installation. Biogas produced is either

stored in the upper part of the digester (Nzila et al., 2012) or in a similar rubber tubes made

from polyethylene material (Nazir, 1991) and used through the gas vent when required. The

first-generation plastic reactors are susceptible to mechanical damage, have short lifespan (2 to

5 years), sensitive to sunlight and deteriorate rapidly due to seasonal changes (Kossmann et al.,

1999; Nazir, 1991; Nzila et al., 2012).

2.2.2 Plastic tanks

Rigid plastic tanks are developed for biogas production and they are made of hard polymeric

materials such as hard polyvinylchloride (PVC), high density polyethylene (HDPE) and

modified plastics except reinforced plastics (Cheng et al., 2013). They are composed of two

pre-built rigid plastic tanks (Rakotojaona, 2013); the first tank for anaerobic digestion and the

second tank for gas storage. They are easy to install, longer lifespan compared to polyethylene

tubular reactors and quick biogas production start-up (3 to 4 days) (Rakotojaona, 2013) but they

are expensive and not suitable for large scale biogas plants. Currently there are very few plastic

tanks installed.

2.2.3 Latest generation plastic reactors

The problem of low reliability (40 %) (Nzila et al., 2012) with the low-cost polyethylene tubular

reactors led to modified plastic biogas reactors. Several PE tubular reactors installed in Kenya

9

were damaged (burst) and as such reactors were modified to PE/PVC; inner layer PE and outer

layer UV treated PVC and further strengthen by synthetic mesh to avoid failure due to stress

and high pressure (GTZ/EnDev, 2010). The new model of reinforced plastics in China is made

of unsaturated polyester resin, gel-coated resin, chopped strand mat and high-quality glass fiber

cloth (Cheng et al., 2013). About 200,000 of these modified reactors have been installed in

China with volume ranges from 2.5 m3 to 10 m3. Reinforced plastic reactors are developed for

biogas production in order to make them weather or UV resistant (Kumar et al., 2015) thereby

increasing their lifespan. Research has shown that methane content of biogas produced using

reinforced plastics is 15.3% higher than that obtained when concrete reactors are used (Cheng

et al., 2013). However, these modified reactors are expensive and floating where underground

water lever persist (Cheng et al., 2013).

Reactor covers are used to store gas produced, insulate the reactor and prevent emission of odor

and gases such H2S, CH4, NH3, N2O, CO2, and volatile organic compound; reactors covers must

be able to withstand exposure to moisture, corrosive gases and digestion feedstock (Stenglein

et al., 2011). There are new reactor covers made from plastics which are different from

conventional fixed and floating covers. Plastic covers are made of high density polyethylene

(HDPE), linear low density polyethylene (LLDPE), polyvinylchloride (PVC). Recently plastic

covers are made of composites polymeric materials to reduce permeability, reinforced and

strengthen the membrane and also to provide protection against UV light and chemicals

(Stenglein et al., 2011).

3. Textile biogas reactor technology

3.1 History and development

Plastic biogas reactor technology was introduced to solve some of the problems associated with

conventional biogas reactors as discussed in section 2. However, the lifespan of the material is

short due to easy degradation and plastic biogas reactors are not robust for large scale

10

application (Chen et al., 2012; Cheng et al., 2013). GTZ/EnDev (2010) also reported several of

biogas plants installed failing due to low quality of plastic reactor bags. These shortcomings led

to the development of a textile biogas reactor for reliable biogas production considering

efficiency, economy and large-scale application of the reactor. This invention aims at making

the biogas technology accessible and attractive to several countries especially developing

countries that may not have substantial subsidy support for biogas implementation.

This new biogas reactor is made of textiles which makes it less combustible, resistant to tearing,

light weight and environmentally friendly. The textile reactor is UV treated to prevent easy

disintegration due to exposure to sunlight; they are also insulated against extreme weather

conditions thereby increasing lifespan. Additionally, the reactor is composed of sophisticated

coated polymers as a protection against corrosive gases such as H2S. Textile biogas reactors

range in size from 1 m3 to 1000 m3 and they can be used for small and large scale biogas plants

treating sludge from wastewater treatment plants, industrial wastes, agricultural and organic

fraction of municipal solid wastes.

3.2 Laboratory studies

The first textile biogas reactor developed was of a pyramid shape as shown in Fig.1; it was a

continuous reactor with a total volume of about 112 L. The inlet and outlet pipes were fixed on

opposite sides of the reactor, the gas outlet was fitted on the upper part and opener fixed to the

reactor for discharging the digestate residue when desired. The efficiency of this reactor was

investigated by Rajendran et al. (2013) for biogas production using synthetic medium and

treating organic fraction of municipal solid waste; the reactor was operated at room temperature.

The goal of this study was to check the suitability and efficiency of this new reactor since textile

material has never been used for constructing biogas reactor. The results showed a stable biogas

operation with OLR of 1.0 gVS/L/d yielding 570 L/kgVS/d when the synthetic medium was

used. The same yield was obtained when organic fraction of municipal solid waste was treated

11

with an increasing OLR from 0.1gVS/L/d to 1.0 gVS/L/d; this shows that the reactor was

appropriate for effective degradation of the treated waste. Rajendran et al. (2013) also carried

out an economic evaluation of the process; the result shows the sum of investment and 15 years

operational cost of the reactor was 656 USD which is a positive investment when compared to

1455 USD for subsidized LPG and 975 USD for kerosene.

A horizontal textile bioreactor was thereafter produced for batch anaerobic digestion process as

shown in Fig. 2. The reactor has a volume of about 90 L with a gas outlet and a zip for opening

and closing the reactor; it was operated at a temperature of 25°C. The potential of this reactor

for dry anaerobic digestion was evaluated by Patinvoh et al. (2017b) treating manure bedded

with straw at 22 % to 30 %TS of feedstock. Additional goal of this study was to evaluate the

technical and economic aspect of this new reactor technology. The reactor was very easy to

operate and worked successfully for over 324 days without leakage or major maintenance.

Patinvoh et al. (2017b) reported methane yield of 290 NmlCH4/gVS at 30 %TS after long

acclimatization of the inoculum and the digestate residue analyzed showed suitability as bio-

fertilizer; this shows there was no reaction between the material of construction and the

digestate. The economic evaluation on the experimental results also shows dry-AD with this

reactor a profitable method of handling the waste with maximum payback period of 5 years,

net present value from $7,000 to $9,800,000 (small to large bioreactors) with internal rate of

return from 56.6 to 19. 3 %.

3.3 Stress analysis and test for large scale application

A biogas reactor is liable to failure if the gas pressure exceeds a limit that the reactor can

withstand; it is therefore important to understand and quantify stress in the reactor (Kaminski,

2005) as a safety measure. GTZ/EnDev (2010) reported some of the plastic biogas reactors

installed in Kenya were damaged (burst) due to stress or high pressure in the reactor.

12

The developed textile biogas reactor is made of unique composite materials to increase the

tensile strength of the reactor for large scale application. However, for safety measures the stress

associated with this reactor was determined using curvature and numerical analysis by Osadolor

et al. (2016); the analysis was performed for large scale application of bioreactors with volume

from 100 m3 to 1000 m3. The calculated tensile stress in a 1000 m3 reactor was reported to be

14.2 MPa. The result showed that using textile material can increase the tensile strength more

than 14.2 MPa thereby preventing failure while also reducing cost. The strength of the reactor

was also tested for large scale application; this reactor was filled up to half of its capacity

(Rajendran, 2015) as shown in Fig.2 and there was no damage to the reactor after the test.

4 Biogas plant in India

India has a long history in development of small scale biogas plants; there were several biogas

plants installed in India during the past 30 years. Most commonly used biogas reactors are fixed

dome reactors and floating drum reactors of which there has not been much innovation in the

design during the past several years. Additionally, these reactors require high technical skill for

construction and also prone to leakages. The steel drum is relatively expensive and requires

rigorous maintenance; it requires constant removal of rust and regular painting to avoid gas

leakage. After studying operations of existing reactors and challenges involved, FOV fabrics

developed plug flow textile-based reactors as shown in Fig. 4 which are easy to install and

operate, cost effective, portable, modular in nature and easy to maintain. FOV fabrics have

successfully installed more than 20 textile reactors for biogas production in India treating food

wastes, cow dung, human excreta and mixed feedstocks.

4.1 Process Description

Total volume of the textile reactor is about 100 m3 and the biogas plant operates on continuous

process; the complete system of the plant is shown in Fig. 5. Initially the reactor is loaded with

inoculum for a start-up and thereafter fed with the substrates. One MT of cow dung (sometimes

13

food wastes) was mixed with 1 m3 of water to make fluent slurry; 2 m3 of slurry was fed into

the reactor everyday through the inlet and the same amount withdrawn. Part of the slurry is

recycled back along with fresh cow dung as replacement for water; this is done in order to

minimize the volume of water used. The retention time of the process is about 30 days and

under optimized condition generates about 40 m3 of biogas per day. Biogas produced

accumulates at the upper part of the reactor and flows through the gas vent to a separate gas

storage tank and used afterwards for cooking or for power generation using biogas generator.

Digestate residue after biogas production is projected to be used as organic fertilizer to improve

the quality of agricultural soil thereby generating additional revenue. The projected economic

remunerations from the biogas plant are shown in Table 1.

5. Conclusions Bioreactor design for biogas production are getting better in order to reduce cost and

construction time of biogas installations; advanced textile bioreactors are promising technology

for dissemination. This will enhance global advancement of biogas technology especially in

developing countries.

References

An,B.X.,Preston,T.R.,Dolberg,F.1997.Theintroductionoflow-costpolyethylenetubebiodigestersonsmallscalefarmsinVietnam.LivestockResearchforRuralDevelopment,9(2),27-35.

An,B.X.,VanMan,N.,Khang,N.D.,Anh,N.D.,Preston,T.1994.Installationandperformanceoflow-costpolyethylene tubebiodigesterson small scale farms inVietnam.Proceedings NationalSeminar-workshopinSustainableLivestockProductiononLocalFeedResources.AgriculturalPublishingHouse.HoChiMinhcity.pp.95-103.

Chen,L.,Zhao,L.,Ren,C.,Wang,F.2012.TheprogressandprospectsofruralbiogasproductioninChina.EnergyPolicy,51,58-63.

Cheng,S.,Li,Z.,Mang,H.-P.,Huba,E.-M.2013.AreviewofprefabricatedbiogasdigestersinChina.RenewableandSustainableEnergyReviews,28,738-748.

Cheng,S.,Li,Z.,Mang,H.-P.,Huba,E.-M.,Gao,R.,Wang,X.2014a.Developmentandapplicationofprefabricated biogas digesters in developing countries.Renewable and Sustainable EnergyReviews,34,387-400.

Cheng,S.,Li,Z.,Mang,H.-P.,Neupane,K.,Wauthelet,M.,Huba,E.-M.2014b.Applicationoffaulttreeapproachfortechnicalassessmentofsmall-sizedbiogassystemsinNepal.AppliedEnergy,113,1372-1381.

14

Dahlquist,E.2013.TechnologiesforConvertingBiomasstoUsefulEnergy:Combustion,Gasification,Pyrolysis,TorrefactionandFermentation.Taylor&Francis.

DeMes,T.Z.D.,Stams,A.J.M.,Reith,J.H.,Zeeman,G.2003.Methaneproductionbyanaerobicdigestionofwastewaterandsolidwastes.Bio-methane&Bio-hydrogen,58-102.

DeBruyn,J.,House,H.,Rodenburg,J.2006.OntariolargeherdoperatorsEuropeananaerobicdigestiontour report. Germany, Denmark and the Netherland. August 21-29. Ontario Ministry ofAgriculture,foodandRuralAffairs.

Geels, F.W., Raven, R. 2007. Socio-cognitive evolution and co-evolution in competing technicaltrajectories: biogas development in Denmark (1970–2002). The International Journal ofSustainableDevelopment&WorldEcology,14(1),63-77.

Ghimire,P.C.2013.SNVsupporteddomesticbiogasprogrammesinAsiaandAfrica.RenewableEnergy,49,90-94.

GTZ/EnDev. 2010. Low Cost Polyethylene Tube Digester in Rwanda (Final mission report)https://energypedia.info/wiki/File:Low_Cost_Polyethylene_Tube_Digester_Rwanda.pdf.Accessed2017/08/28.

ISSF.2012.StainlessSteelinBiogasProduction.ASustainableSolutionforGreenEnergy.InternationalStainless Steel Forum (ISSF). Rue Colonel Bourg 120. 1140 Brussels, Belgiumhttp://www.worldstainless.org/Files/ISSF/non-image-files/PDF/ISSF_Stainless_Steel_in_Biogas_Production.pdfAccessed2017/08/27.

Kaminski,C.2005.StressAnalysis&PressureVessels.UniversityofCambridge.Kossmann,W., Pönitz,U.,Habermehl, S.,Hoerz, T., Krämer, P., Klingler,B., Kellner,C.,Wittur,W.,

Klopotek, F., Krieg, A. 1999. Biogas Digest, Volume II–Biogas–Application and ProductDevelopment.InformationandAdvisoryServiceonAppropriateTechnology,Eschborn.

Kumar,A.,Mandal,B.,Sharma,A.2015.AdvancementinBiogasDigester.Kumar, K.V., Bai, R.K. 2005. Plastic biodigesters–a systematic study. Energy for Sustainable

Development,9(4),40-49.Kuria,J.,Maringa,M.2008.Developingsimpleproceduresforselecting,sizing,schedulingofmaterials

andcostingofsmallbio-gasunits. International Journal forServiceLearning inEngineering,HumanitarianEngineeringandSocialEntrepreneurship,3(1).

Lettinga,G.1995.Anaerobicdigestionandwastewatertreatmentsystems.Antonievanleeuwenhoek,67(1),3-28.

Luostarinen,S.,Normak,A.,Edström,M.2011.Overviewofbiogas technology.BalticmanureWP6Energypotentials.,47.

Mostajir,B.,Floc'h,L.,Mas,S.,Pete,R.,Parin,D.,Nouguier,J.,Fouilland,E.,Vidussi,F.2013.Anewtransportable floating mesocosm platform with autonomous sensors for real-time dataacquisition and transmission for studying thepelagic foodweb functioning. Limnology andOceanography:Methods,11(7),394-409.

Nazir,M.1991.Biogasplantsconstructiontechnologyforruralareas.BioresourceTechnology,35(3),283-289.

Nilsson,A.E.,GergerSwartling,Å.,Eckerberg,K.2012.KnowledgeforlocalclimatechangeadaptationinSweden:challengesofmultilevelgovernance.LocalEnvironment,17(6-7),751-767.

Nzila,C.,Dewulf, J.,Spanjers,H.,Tuigong,D.,Kiriamiti,H.,VanLangenhove,H.2012.Multi criteriasustainabilityassessmentofbiogasproductioninKenya.AppliedEnergy,93,496-506.

Osadolor,O.A., Lundin,M., Lennartsson,P.R., Taherzadeh,M.J. 2016.Membrane stressanalysisofcollapsibletanksandbioreactors.BiochemicalEngineeringJournal,114,62-69.

Patinvoh,R.J.,KalantarMehrjerdi,A.,SárváriHorváth,I.,Taherzadeh,M.J.2017a.Dryfermentationofmanurewithstrawincontinuousplugflowreactor:Reactordevelopmentandprocessstabilityatdifferentloadingrates.Bioresour.Technol.,224,197-205.

Patinvoh, R.J., Osadolor, O.A., Horváth, I.S., Taherzadeh,M.J. 2017b. Cost effective dry anaerobicdigestionintextilebioreactors:Experimentalandeconomicevaluation.Bioresour.Technol.InPress.

15

Peacock, D.G., Richardson, J.F. 2012. Chemical Engineering, Volume 3: Chemical and BiochemicalReactorsandProcessControl.Elsevier.

Pérez,I.,Garfí,M.,Cadena,E.,Ferrer,I.2014.Technical,economicandenvironmentalassessmentofhouseholdbiogasdigestersforruralcommunities.RenewableEnergy,62,313-318.

Pound,B.,Bordas,F.,Preston,T.R.1981.THECHARACTERISATIONOFPRODUCTIONANDFUNCTIONOFA15m3RED-MUDPVCBIOGASDIGESTER.TropicalAnimalProduction,6(2),146-153.

Rajendran,K.2015.IndustrialBioprocessDevelopmentsforBiogasandEthanolProduction,HögskolaniBorås,Ph.D.thesis.

Rajendran, K., Aslanzadeh, S., Johansson, F., Taherzadeh,M.J. 2013. Experimental and economicalevaluationofanovelbiogasdigester.EnergyConvers.Manage.,74,183-191.

Rakotojaona, L. 2013. Domestic biogas development in developing countries http://www.enea-consulting.com/wp-content/uploads/2015/05/Open-Ideas-Domestic-biogas-projects-in-developing-countries.pdfAccessed2017/08/27.

REN21. 2017. Renewable Energy PolicyNetwork for the 21st Centuery (REN21). Renewables 2017GlobalStatusReport(Paris:REN21Secretariat)ISBN978-3-9818107-6-9.

Rowse,L.E.2011.Designofsmallscaleanaerobicdigestersforapplicationinruraldevelopingcountries.UniversityofSouthFlorida.

Samer,M.2012.Biogasplantconstructions.CairoUniversity,FacultyofAgriculture,DepartmentofAgriculturalEngineering,Egypt.in:Biogas,InTech.

Sasse,L.1988.Biogasplants.Vieweg&Sohn.Wiesbaden.Shi,F.2014.ReactorandProcessDesigninSustainableEnergyTechnology.ElsevierScience.Stenglein,R.M.,Clanton,C.J.,Schmidt,D.R.,Jacobson,L.D.,Janni,K.A.2011.Impermeablecoversfor

odorandairpollutionmitigationinanimalagriculture:atechnicalguide.AirQualityEducationin Animal Agriculture. Accessed 2017/08/30https://articles.extension.org/sites/default/files/Impermeable%20covers%20FINAL.pdf

Tomar,S.S.1994.StatusofbiogasplantinIndia.RenewableEnergy,5(5),829-831.Torrijos, M. 2016. State of development of biogas production in Europe. Procedia Environmental

Sciences,35,881-889.Werner, U., Stöhr, U., Hees, N. 1989. Biogas plants in animal husbandry. Deutsches Zentrum für

Entwicklungstechnologien-GATE.

16

Figure Captions

Fig. 1. Schematic sketch of the first generation textile bioreactor (Rajendran et al., 2013)

Fig. 2. Schematic sketch of textile bioreactor for dry-AD (Patinvoh et al., 2017b)

Fig. 3 Strength testing for a pilot textile reactor (Rajendran, 2015)

Fig. 4. Plug flow textile-based reactors installed in India (a) 100 m3 (b) 10 m3

Fig. 5. Scheme of complete textile biogas reactor plant in India

17

Table 1: Economic estimate of products from the biogas plant in India

LPG Savings by setting up a 1 Mt/day of cow dung based textile biogas reactora

Annual savings on LPG Cylinder

Number of kg cow dung generated per day 1000

Gas generated (m3/day) 40

Number of kg of LPG saved per day 20

Savings on LPG per day in Rs Rs 1, 600

Savings in no. of kg of LPG per year 7, 300

Annual Savings on LPG in Rs Rs 5, 84, 000

Electricity Savings by setting up a textile biogas reactorb

Annual savings on Electricity

Number of kgs’ cow dung waste generated per day 1000

Power generated (Kw/day) 40

Savings on electricity per day in Rs Rs 400

Power generated in Kw annually 14, 600

Annual savings on Electricity in Rs Rs 1, 46, 000

Savings from organic fertilizer obtained from the reactor

Annual savings on Organic Manure

Number of liters of diluted waste fed in per day 2000

Organic manure obtained from bio-slurry per day in kg 160

Savings on organic manure per day in Rs Rs 800

Organic manure obtained from bio-slurry per year in kg 58, 400

Annual savings on Organic Manure in Rs Rs 2, 92, 000 a average cost of commercial LPG is considered at Rs. 80 per kg. b average cost of electricity is considered at Rs. 10 per KW.

18

Fig. 1.

Inlet

Outlet

Opener

Gas outlet

19

Fig. 2.

20

Fig. 3.

21

Fig. 4.

(a) (b)

21

Fig. 4.

(a) (b)

22

Storageandmixing

FOVBiogas

Waste

Waterfordilution

Biogas

Cleaning,ifrequired

DisposalFertilizer

Storageclosetokitchen

Stoveandkitchen

Fig. 5.

Dry anaerobic co-digestion of citrus wastes with keratin-rich and lignocellulosic solid organic wastes: Batch vs. Continuous Processes. Submitted.

Pap

er V

I

1

Dry anaerobic co-digestion of citrus wastes with keratin-rich and

lignocellulosic solid organic wastes: Batch vs. Continuous Processes

Regina J. Patinvoha,b*, Magnus Lundina, Mohammad J. Taherzadeha and Ilona Sárvári

Horvátha

aSwedish Centre for Resource Recovery, University of Borås, 50190 Borås, Sweden.

bDepartment of Chemical and Polymer Engineering, Faculty of Engineering, Lagos State

University, Lagos, Nigeria

*Corresponding author:

Tel: +46 33 435 4625

E-mail: [email protected]

2

Abstract

Dry anaerobic co-digestion of citrus wastes (CW) with chicken feather (CF), wheat straw

(WS) and manure bedded with straw (MS) was investigated in batch and continuous

processes. Experiments were designed with different mixing ratios considering the inhibitory

effect of citrus wastes, C/N ratio, and total solid content of individual feedstocks. With the

best mixing ratio (CF:CW:WS:MS) of 1:1:6:0, enhanced methane yield by 14 % was obtained

compared to the expected yield calculated according to the methane yields obtained from the

individual fractions. The process performance of this mixture was then investigated in

continuous plug flow reactors at different organic loading rates (OLR) using feedstock total

solid contents of 21 %TS (RTS21) and 32 % TS (RTS32). At OLR of 2 gVS/L/d, methane yield

of 362 NmlCH4/gVSadded was obtained from RTS21, which is 13.5 % higher than the yield

obtained from RTS32 (319 NmlCH4/gVSadded). However, it was not possible to achieve a stable

process when the OLR was further increased to 3.8 gVS/L/d, which resulted in high total

VFAs concentrations, VFA/alkalinity ratios and a decline in the biogas production.

Keywords:

Solid wastes; Dry co-digestion; Batch process; Continuous process; Process stability

3

1. Introduction

Organic fraction of solid wastes from agricultural and industrial activities degrades over a

period of time. This process releases CH4, CO2 and other gases into the atmosphere; leading

to global warming. Global emissions from solid wastes are estimated between 20 to 40

million tonnes of CH4 per year (Jensen and Pipatti 2002). Waste streams like chicken feathers,

solid manure and wheat straw account for the largest potential feedstock as sources of

biomass energy and are readily available. Converting them into energy through anaerobic

digestion is a feasible option with low capital investment. Chicken feather is a good potential

source of nitrogen with over 90 % of crude protein content (Patinvoh et al. 2016; Salminen et

al. 2003) and wheat straw is a potential source of carbon with a composition of about 35 - 43

% of cellulose and 21 – 32 % of hemicellulose (Kabir et al. 2015; Karthikeyan and

Visvanathan 2013; Patinvoh et al. 2017c). Additionally, fruit wastes contain large amount of

organic matter that are easily degraded; about 78.3 % carbohydrates, 8.5 % protein, and 6 %

fat (Schnürer and Jarvis 2010), which make them potential biomass source for energy

production. Over 40 million tons of industrial citrus wastes are generated globally (Sharma et

al. 2017).

Notably, because of the recalcitrant structure of most agricultural solid wastes such as chicken

feathers and wheat straw, they degrade slowly: resulting in low biogas. Several research

works have been conducted to improve methane yield from keratin-rich and lignocellulosic

solid wastes by chemical pretreatment (Aslanzadeh et al. 2011; Bolado-Rodríguez et al. 2016;

Chandra et al. 2012; Forgács et al. 2014; Lohrasbi et al. 2010), steam explosion pretreatment

(Theuretzbacher et al. 2015), and biological pretreatment (Forgács et al. 2011; Patinvoh et al.

2016). Additionally, the presence of peel oil with D-limonene as main ingredient in citrus

wastes inhibit microbial growth (Mizuki et al. 1990), which leads to low biogas yield. This

inhibition challenges in citrus wastes for biogas production have been addressed by leaching

(Wikandari et al. 2015), steam explosion pretreatment (Forgács et al. 2012) and membrane

technology (Wikandari et al. 2014). Although these methods have been reported effective in

solving problems with low biogas yield, there are still problems of high energy consumption,

expensive technology, harmful environmental by-products, and release of inhibitors during

some of the pretreatment methods (Patinvoh et al. 2017b). Other problem associated with the

digestion of these waste streams is the high nitrogen (i.e. chicken feather) or carbon (i.e. straw

and citrus waste) content leading to nutrient imbalance when they are digested as sole

substrate (mono-digestion).

3

1. Introduction

Organic fraction of solid wastes from agricultural and industrial activities degrades over a

period of time. This process releases CH4, CO2 and other gases into the atmosphere; leading

to global warming. Global emissions from solid wastes are estimated between 20 to 40

million tonnes of CH4 per year (Jensen and Pipatti 2002). Waste streams like chicken feathers,

solid manure and wheat straw account for the largest potential feedstock as sources of

biomass energy and are readily available. Converting them into energy through anaerobic

digestion is a feasible option with low capital investment. Chicken feather is a good potential

source of nitrogen with over 90 % of crude protein content (Patinvoh et al. 2016; Salminen et

al. 2003) and wheat straw is a potential source of carbon with a composition of about 35 - 43

% of cellulose and 21 – 32 % of hemicellulose (Kabir et al. 2015; Karthikeyan and

Visvanathan 2013; Patinvoh et al. 2017c). Additionally, fruit wastes contain large amount of

organic matter that are easily degraded; about 78.3 % carbohydrates, 8.5 % protein, and 6 %

fat (Schnürer and Jarvis 2010), which make them potential biomass source for energy

production. Over 40 million tons of industrial citrus wastes are generated globally (Sharma et

al. 2017).

Notably, because of the recalcitrant structure of most agricultural solid wastes such as chicken

feathers and wheat straw, they degrade slowly: resulting in low biogas. Several research

works have been conducted to improve methane yield from keratin-rich and lignocellulosic

solid wastes by chemical pretreatment (Aslanzadeh et al. 2011; Bolado-Rodríguez et al. 2016;

Chandra et al. 2012; Forgács et al. 2014; Lohrasbi et al. 2010), steam explosion pretreatment

(Theuretzbacher et al. 2015), and biological pretreatment (Forgács et al. 2011; Patinvoh et al.

2016). Additionally, the presence of peel oil with D-limonene as main ingredient in citrus

wastes inhibit microbial growth (Mizuki et al. 1990), which leads to low biogas yield. This

inhibition challenges in citrus wastes for biogas production have been addressed by leaching

(Wikandari et al. 2015), steam explosion pretreatment (Forgács et al. 2012) and membrane

technology (Wikandari et al. 2014). Although these methods have been reported effective in

solving problems with low biogas yield, there are still problems of high energy consumption,

expensive technology, harmful environmental by-products, and release of inhibitors during

some of the pretreatment methods (Patinvoh et al. 2017b). Other problem associated with the

digestion of these waste streams is the high nitrogen (i.e. chicken feather) or carbon (i.e. straw

and citrus waste) content leading to nutrient imbalance when they are digested as sole

substrate (mono-digestion).

4

In view of the fact that easily degraded feedstocks are not readily available and the problems

associated with some of the pre-treatments methods, co-digestion can be an alternative

strategy for improving the biogas yield from these types of solid wastes. This concept favours

synergisms, dilutes harmful compounds, optimizes the biogas production, and increases the

digestate quality (Mata-Alvarez et al. 2014). Co-digestion of these solid wastes with an

appropriate mixing ratio will lead to a balanced nutrient supply and will enhance the buffering

capacity of the digestion system. In choosing appropriate mixing ratio, it is important to

consider the C/N ratio, biodegradability, possible inhibitors and total solid content of

individual feedstocks (Patinvoh et al. 2017b). Although nitrogen is an essential nutrient for

the microorganisms, its excess leads to the formation of ammonia during the degradation;

which inhibits microbial growth at higher concentrations (Bachmann 2015; Friehe et al.

2010b). Additionally, when the amounts of easily degradable carbon is too high, the process

tends to be susceptible to acids accumulation (Liu et al. 2008). Co-digestion of crop residues

(i.e. carbon-rich) with livestock residues (i.e. nitrogen-rich) could balance the C/N ratio,

thereby improve the biogas yield (Comino et al. 2010a). A considerable wider C/N ratio, e.g.

between 10:1 and 30:1, has been reported in the literature for a stable digestion process

(Friehe et al. 2010a; Pagés Díaz et al. 2011). Nevertheless, the composition of feedstocks

(Gunaseelan 2007), the availability of carbon and nitrogen to the microbial community

(Herrmann et al. 2016) and different operation parameters during the digestion process are

also significant for an effective anaerobic digestion.

Furthermore, with a total solid (TS) greater than 20 %, there has been great concern about the

large amount of water usually used while treating these solid wastes and which consequently

leads to huge amounts of digestate residue with high water content to handle. To combat this

problem, recent studies have been focused on dry anaerobic digestion of these waste streams

both in pilot and large-scale applications (Baere 2012; Patinvoh et al. 2017a; Patinvoh et al.

2017c; Tyrberg 2013; Zeshan et al. 2012).

The main purpose of this work was to investigate co-digestion of citrus wastes with chicken

feathers, wheat straw and manure bedded with straw in dry anaerobic digestion process.

Experiments were designed with different mixing ratios; the synergetic and antagonistic

interactions, which might arise in different mixtures, were evaluated through anaerobic batch

digestion assays. In addition, the long term effects in process performance were investigated

for the best mixture indicating synergy in continuous plug flow reactors at different organic

loading rates with feedstock total solid contents of 21 %TS and 32 %TS.

5

2. Materials and Methods

2.1 Substrates and inoculum

Different substrates used during this study were chicken feathers (CF), citrus wastes (CW),

wheat straw (WS) and cattle manure bedded with straw (MS). The chicken feather waste was

collected from a slaughterhouse (Håkantorp Slakteri AB, Håkantorp, Sweden) and prepared

according to Patinvoh et al. (2016) prior to use. Citrus wastes were obtained from Brämhults

juice AB (Borås, Sweden) and chopped manually. Wheat straw and cattle manure bedded

with straw were obtained from a farm outside Borås (Rådde Gård, Sweden). The manure was

shredded manually to reduce the particle size of straw in the manure and the wheat straw was

milled to particle size of 0.5 to 2 mm. Chicken feather wastes and wheat straw were stored at

room temperature, while citrus wastes and manure bedded with straw were stored in plastic

containers at -20 °C to prevent biodegradation until further use. During the experiment,

weighted frozen substrates were thawed at room temperature and thoroughly mixed to gain a

homogenized feed before use.

Prior to analyses, the feedstocks being solids were prepared according to Zupančič and Roš

(2012) by blending 1g of the sample with water (dilution factor of 50) to reduce the particle

size and allowing homogenization (Raposo et al. 2012). Thereafter, the theoretical methane

potential (BMPthCOD) of the feedstocks used were calculated related to their chemical oxygen

demand (COD) content as described previously (Patinvoh et al. 2017a), after determining the

COD content for the individual feedstocks. However, the COD for chicken feathers was not

detectable; therefore, the theoretical methane yield of 496 Nml methane produced per g

volatile solids (VS) for proteins (Angelidaki and Sanders 2004) was used for the calculations,

which is applicable if all insoluble protein (keratin) are converted to soluble protein.

6

Anaerobic sludge used as inoculum was obtained from a digester treating wastewater sludge

and operating at mesophilic conditions (Vatten and Miljö i Väst AB, Varberg, Sweden). The

inoculum was incubated at 37 °C for 2 weeks prior to use. The inoculum with 3.8 %TS and

2.7 %VS was then centrifuged at 10,000g for 10 min to increase its TS content to 9.43 % and

VS content to 6.44 %. Table 1 shows the characteristics of the substrates and the anaerobic

sludge (inoculum) used during the experiments.

2.2 Statistical design

The experiment was designed with statistical software MINITAB® (version 17.1.0), using 3-

factor simplex lattice design, consisting of pure substrates and mixtures of two, three and four

substrates at wet weight ratios. The chicken feather (CF) fraction was kept constant to

maintain the C/N ratio of the mixtures between 12 and 21. The experiments were replicated

according to the same setups two times and methane yield was used as response variable. The

linear mixing model without synergetic or antagonistic interaction was used to obtain the

expected methane yield from all mixtures which correspond to VS fraction from individual

substrates while the quadratic model was used to obtain the predicted methane yield from all

mixtures with synergetic and antagonistic interaction between VS fractions of individual

substrates. Table 2 shows the mixture compositions following the simplex lattice design.

2.3 Batch anaerobic dry digestion of individual substrates and mixtures

Anaerobic batch digestion tests on individual substrates and the co-digestion mixtures were

performed according to the method described by Angelidaki et al. (2009). The assays were

carried out under mesophilic conditions (37 ± 1°C) using 118 ml serum glass bottles as

reactors; each reactor contains 39 ml of inoculum and 1.25 gVS of individual substrates and

mixtures keeping a VS ratio (VSsubstrate to VSinoculum) at 1:2 in all setups. The total solid content

of individual substrates and mixtures were adjusted to 20 %TS thereby having a final TS of

11 % in all reactors; the mixture compositions used in the various set ups are shown in Table

7

2. Inoculum and water was used as blank to disclose any methane production by the inoculum

itself. The pH in all reactors were adjusted to 7.0 ± 1 using 2M NaOH and HCl solution, then

the reactors were sealed with rubber septa and aluminum caps, and the headspace was flushed

with a gas mixture of 80 %N2 and 20 %CO2 for 2 minutes to create anaerobic environment in

each setup. The reactors were placed in an incubator at 37 ± 1°C and were shaken manually

once a day during the incubation period of 82 days. All experimental setups were performed

in duplicates, and gas samples taken twice in a week during the process and once a week

towards the end of the digestion period.

2.4 Co-digestion of the best mixture in plug flow reactors

The best mixing ratio, i.e. the mixture with highest methane yield, as determined during the

batch assays, was applied in the continuous co-digestion process using plug flow reactors. The

reactors worked as described previously (Patinvoh et al. 2017a) with a working volume of 5

L. The first part of the experiment was to carry out batch digestion in plug flow reactors with

the same condition used in serum bottles (118 ml); this was done to adapt the inoculum to the

feedstock (best mixing ratio) and examine the performance in plug flow reactors. The plug

flow reactors were loaded with the feedstock at 20 %TS and inoculated with anaerobic sludge.

VS ratio (VSsubstrate to VSinoculum) was kept at 1:2 thereby having a TS of 11 % in the reactors.

The process was operated in batch mode for 28 days while the TS in the bioreactors were

reduced to around 3 to 4 %TS. Since, the co-digestion process was studied under dry

digestion; the TS in the reactors were increased to 15 % by adding fresh feedstock to the

acclimatized inoculum achieving a VS ratio (VSsubstrate to VSinoculum) of 2.5:1. Thereafter, the

continuous feeding operation began after a startup phase of 32 days. Reactors were fed every

day; reactor 1 (RTS21) was fed with a feedstock having 21 %TS with bulk density of 511.7g/L

and reactor 2 (RTS32) was fed with a feedstock having 32 %TS with bulk density of 322.9 g/L.

The OLR was kept first at 2.0 gVS/L/d (OLR 1) and then it was increased to 3.8 gVS/L/d

7

2. Inoculum and water was used as blank to disclose any methane production by the inoculum

itself. The pH in all reactors were adjusted to 7.0 ± 1 using 2M NaOH and HCl solution, then

the reactors were sealed with rubber septa and aluminum caps, and the headspace was flushed

with a gas mixture of 80 %N2 and 20 %CO2 for 2 minutes to create anaerobic environment in

each setup. The reactors were placed in an incubator at 37 ± 1°C and were shaken manually

once a day during the incubation period of 82 days. All experimental setups were performed

in duplicates, and gas samples taken twice in a week during the process and once a week

towards the end of the digestion period.

2.4 Co-digestion of the best mixture in plug flow reactors

The best mixing ratio, i.e. the mixture with highest methane yield, as determined during the

batch assays, was applied in the continuous co-digestion process using plug flow reactors. The

reactors worked as described previously (Patinvoh et al. 2017a) with a working volume of 5

L. The first part of the experiment was to carry out batch digestion in plug flow reactors with

the same condition used in serum bottles (118 ml); this was done to adapt the inoculum to the

feedstock (best mixing ratio) and examine the performance in plug flow reactors. The plug

flow reactors were loaded with the feedstock at 20 %TS and inoculated with anaerobic sludge.

VS ratio (VSsubstrate to VSinoculum) was kept at 1:2 thereby having a TS of 11 % in the reactors.

The process was operated in batch mode for 28 days while the TS in the bioreactors were

reduced to around 3 to 4 %TS. Since, the co-digestion process was studied under dry

digestion; the TS in the reactors were increased to 15 % by adding fresh feedstock to the

acclimatized inoculum achieving a VS ratio (VSsubstrate to VSinoculum) of 2.5:1. Thereafter, the

continuous feeding operation began after a startup phase of 32 days. Reactors were fed every

day; reactor 1 (RTS21) was fed with a feedstock having 21 %TS with bulk density of 511.7g/L

and reactor 2 (RTS32) was fed with a feedstock having 32 %TS with bulk density of 322.9 g/L.

The OLR was kept first at 2.0 gVS/L/d (OLR 1) and then it was increased to 3.8 gVS/L/d

8

(OLR 2) with corresponding retention times of 50 and 30 days, respectively. During the

feeding equivalent amounts of the digestate residue was withdrawn every day from the

reactors; digestate was mixed with fresh feedstock in a ratio of 1:2 (feedstock: digestate; wet

basis) prior to feeding. The reactors were then mixed manually (once daily) with the impeller

by moving its content to the inlet and back to the outlet to minimize stratification in the

reactor. Process parameters such as volume of biogas produced, methane content of produced

biogas, pH, TS, VS, VFA/Alkalinity ratio, total VFA and total ammonia concentration were

monitored regularly during the digestion process.

2.5 Analytical methods

Total solids (TS), volatile solids (VS), pH, and ash content were determined according to

biomass analytical procedures (APHA-AWWA-WEF 2005). The total nitrogen (TKN) was

determined using the Kjeldahl method. The total carbon was obtained by correcting the total

dry weight carbon value for the ash content (Haug 1993; Zhou et al. 2015) and the bulk

density was determined according to Zhang et al. (2012). COD concentrations were analyzed

using a COD test kit (Nanocolor, MACHEREY-NAGEL GmbH & Co. KG. Germany) and

the concentrations were then measured using Nanocolor 500D Photometer (MACHEREY-

NAGEL GmbH & Co. KG. Germany). Extractives in wheat straw samples were determined

according to the NREL protocol (Sluiter et al. 2008) using Soxhlet method with water and

ethanol extraction for 24 h. Total carbohydrate and total lignin content of extractive free straw

samples were then determined according to NREL protocols (Sluiter et al. 2011). Monomeric

sugars obtained during the hydrolysis were determined by HPLC. Mannose, glucose,

galactose, xylose and arabinose were analyzed using Aminex HPX-87P column (Bio-Rad) at

85 °C and 0.6 mL/min ultrapure water as eluent. Acid soluble lignin (ASL) was determined

using an UV spectrophotometer (Biochrom Ltd, Cambridge, England) at 320 nm. Acid

insoluble lignin (AIL) was gravimetrically determined as residual solid after hydrolysis

9

corrected with the ash content. The ash content was determined as the remaining residue after

keeping the samples in the muffle furnace at 575 °C for 24 h.

Samples of digestates obtained from reactors in the continuous experiment were centrifuged

(5,000 g for 10 min), and then the supernatant diluted 50 times were analyzed for total

ammonia concentration using Ammonia rapid test kits (Megazyme, Megazyme International

Ireland, Ireland) and the concentrations were measured at 340 nm wavelength using a

spectrophotometer (Biochrom Ltd, Cambridge, England). Alkalinity was measured as the total

inorganic carbonate and was determined by the Nordmann titration method according to

Lossie and Pütz (2008). Digestate samples were centrifuged at 5,000 g for 10 min and then 5

ml of the supernatant was titrated with 0.1 N sulfuric acid to pH 5, the titration was then

continued until pH 4.4 was reached in order to determine the VFA concentration measured as

acetic acid equivalent. Total VFAs in the digestate filtrates were measured using a high-

performance liquid chromatograph (HPLC, water 2695, Waters Corporation, Milford, MA,

USA) equipped with an RI detector (Waters 2414, Waters Corporation Milford, MA, USA)

and a biohydrogen-ion exchange column (Aminex HPX-87H, Bio-Rad, Hercules, CA, USA)

operating at 60 oC. A UV-absorbance detector (Walters 2487), operating at 210 nm

wavelength was used in series with a refractive index (RI) detector (Walters 2414) operating

at 60 oC.

Gas samples, taken from the headspace of each batch reactor using a 250-µl pressure-lock gas

syringe (VICI, precious sampling Inc., USA), were analyzed with gas chromatograph (Perkin-

Elmer, USA) equipped with a packed column (6'×1.8'' OD, 80/100, Mesh, Perkin Elmer,

USA), and a thermal conductivity detector (Perkin-Elmer, USA) with an inject temperature of

150 °C. The carrier gas was nitrogen operated with a flow rate of 20 ml/min at 60 °C. Gas

measurement and analysis were carried out as described previously (Teghammar et al. 2010).

The biogas volume from the continuous plug flow reactors was measured with Drum-type gas

9

corrected with the ash content. The ash content was determined as the remaining residue after

keeping the samples in the muffle furnace at 575 °C for 24 h.

Samples of digestates obtained from reactors in the continuous experiment were centrifuged

(5,000 g for 10 min), and then the supernatant diluted 50 times were analyzed for total

ammonia concentration using Ammonia rapid test kits (Megazyme, Megazyme International

Ireland, Ireland) and the concentrations were measured at 340 nm wavelength using a

spectrophotometer (Biochrom Ltd, Cambridge, England). Alkalinity was measured as the total

inorganic carbonate and was determined by the Nordmann titration method according to

Lossie and Pütz (2008). Digestate samples were centrifuged at 5,000 g for 10 min and then 5

ml of the supernatant was titrated with 0.1 N sulfuric acid to pH 5, the titration was then

continued until pH 4.4 was reached in order to determine the VFA concentration measured as

acetic acid equivalent. Total VFAs in the digestate filtrates were measured using a high-

performance liquid chromatograph (HPLC, water 2695, Waters Corporation, Milford, MA,

USA) equipped with an RI detector (Waters 2414, Waters Corporation Milford, MA, USA)

and a biohydrogen-ion exchange column (Aminex HPX-87H, Bio-Rad, Hercules, CA, USA)

operating at 60 oC. A UV-absorbance detector (Walters 2487), operating at 210 nm

wavelength was used in series with a refractive index (RI) detector (Walters 2414) operating

at 60 oC.

Gas samples, taken from the headspace of each batch reactor using a 250-µl pressure-lock gas

syringe (VICI, precious sampling Inc., USA), were analyzed with gas chromatograph (Perkin-

Elmer, USA) equipped with a packed column (6'×1.8'' OD, 80/100, Mesh, Perkin Elmer,

USA), and a thermal conductivity detector (Perkin-Elmer, USA) with an inject temperature of

150 °C. The carrier gas was nitrogen operated with a flow rate of 20 ml/min at 60 °C. Gas

measurement and analysis were carried out as described previously (Teghammar et al. 2010).

The biogas volume from the continuous plug flow reactors was measured with Drum-type gas

10

meter (TG 05 Model 5, Ritter, Germany), and was corrected for reporting at standard

temperature and pressure (0 °C, and 1 atm) using the ideal gas law. The methane content of

biogas produced was determined with gas chromatograph as described above.


3.1 Substrates and Inoculum composition

Characteristics of the feedstocks examined in this study are presented in Table 1; all the

substrates had a solid content of between 23 % and 93 %, which make them suitable for dry

anaerobic digestion. Chicken feather had the lowest C/N ratio (3.6) due its high nitrogen

content while wheat straw had the highest C/N ratio (69) as a result of its high carbon content.

Citrus wastes also contained high carbon, but very low nitrogen, while manure bedded with

straw had higher nitrogen content compared to that in citrus waste and wheat straw.

Additionally, citrus wastes was the only acidic waste of all the substrates investigated with its

pH of 3.24 and moreover, it had previously been reported to contain 3.78 % of limonene

(Pourbafrani et al. 2010) inhibiting methanogens (Mizuki et al. 1990). Several studies

reported that mono digestion of these solid wastes had resulted in low biogas yields or total

failure due to acid accumulation (Liu et al. 2008) or ammonia inhibition (Resch et al. 2011) or

inhibition by D-limonene (Forgács et al. 2012; Wikandari et al. 2014).

All the different mixing ratios examined in this study had a C/N ratio between 12 and 21 as

shown in Table 2; this is within the optimal C/N range required for a stable anaerobic

digestion process (Friehe et al. 2010a; Pagés Díaz et al. 2011).

3.2 Methane yield from individual substrates

The theoretical maximum methane potential of the individual feedstocks calculated based on

the COD content, together with the experimental results of methane yields obtained during the

batch anaerobic digestion assays are presented in Fig. 1. Mono-digestion of citrus wastes

11

resulted in methane yield of 137 NmlCH4/gVS, which is 33 % of the theoretical maximum

yield. This is in accordance with methane yields of 102 NmlCH4/gVS (Forgács et al. 2012)

and 131 NmlCH4/gVS (homogenized) (Wikandari et al. 2015) for similar substrate obtained

earlier under thermophilic wet digestion conditions. Citrus wastes degrade easily but the

methane yield is low due to the presence of D-limonene which is inhibitory to the microbial

community as reported previously by several researchers (Mizuki et al. 1990; Wikandari et al.

2014). Furthermore, the methane yield from chicken feathers was 132 NmlCH4/gVS

corresponding to 27 % of the theoretical yield. Previously, Patinvoh et al. (2016) and Forgács

et al. (2011) found methane yield of 105 (mesophilic condition) and 180 (thermophilic

condition), respectively. These yields are low due to the slow degradation of chicken feathers

as a result of its recalcitrant structure. The major component of chicken feather is β-keratin

(Suh and Lee 2001), which is an insoluble structural protein. Experimental methane yield of

191 NmlCH4/gVS was obtained from the cattle manure bedded with straw, which is 54 % of

the theoretical maximum yield, while 65 % of the theoretical methane yield was obtained

from wheat straw, as shown in Fig. 1. Other studies reported methane yields between 100 to

300 NmlCH4/gVS for wheat straw and manure bedded with straw obtained under different

conditions (Aslanzadeh et al. 2011; Kusch et al. 2008; Patinvoh et al. 2017a; Patinvoh et al.

2017c; Schnürer and Jarvis 2010).

3.3 Methane yield from mixtures – synergetic and antagonistic interactions

Fig. 2 shows the cumulative methane production curves obtained for anaerobic dry co-

digestion of chicken feather (CF), citrus waste (CW), wheat straw (WS) and manure bedded

with straw (MS) during 82 days of digestion period. The compositions of the different

mixtures are presented in Table 2. The co-digestion of the various substrate mixtures resulted

in methane yields ranging from 165 to 238 NmlCH4/gVS, with the mixing ratio

(CF:CW:WS:MS) of 1:1:6:0 (M4) giving the highest methane yield. Table 3 shows the

12

comparison between the experimental values and the response variables (methane yield)

obtained from the linear and quadratic mixing models.

In this work, the expected and predicted methane yield from all mixtures were modeled using

the linear mixing model (without synergetic or antagonistic interaction between individual

substrates) and the quadratic mixing model (including synergetic and antagonistic interactions

between individual substrates), respectively. Corresponding methane yields are presented in

Table 3 and Fig. 3. Of the mixing models, mixing ratios (CF:CW:WS:MS) of 1:1:5:1 (M2)

and 1:1:6:0 (M4) corresponding to C/N ratios of 19 and 21, respectively, gave the best

performance, i.e. the methane yield was increase by 12 % and 14 %, respectively, compared

to values calculated with the linear model. On the other hand, mixing ratios of 1:0:5.8:1.2

(M16) and 1:0:7:0 (M18) having the same C/N ratios of 19 and 21 showed antagonistic

effects, since the measured methane yields, and calculated methane yields using the quadratic

model were 9 % and 11 % lower compared to values calculated according to the linear model,

as shown in Table 3 and Fig.3.

Co-digestion of different substrates often results in synergetic effects due to the balance of

necessary nutrients achieved in the mixture (Mata-Alvarez et al. 2000). The experimental and

statistical results from the mixing ratios show that the higher the amount of citrus wastes and

wheat straw in the mixture, the better the methane yield. The results obtained did show

synergistic and antagonistic effects but these were not statistically significant (p-value ˃

0.05). Chicken feather and manure bedded with straw are the once contributing least to the

methane yield, however, individual fractions in the mixing ratios may have provided macro

nutrients and trace elements needed by the microorganisms for improved methane yield

(Mata-Alvarez et al. 2014; Pagés-Díaz et al. 2014). Furthermore, addition of chicken feather

may have contributed to buffering the digestion system by releasing ammonium cations (Lal

2005). Co-digestion of citrus wastes with chicken feather and wheat straw with mixing ratio

13

of 1:1:6:0 (M4) gave the highest methane yield, it was therefore chosen for further

investigations in continuous processes.

3.4 Best mixing ratio in continuous process

3.4.1 Initial phase (adaptation period)

This phase was carried out in batch mode using the best mixing ratio; this allows microbial

adaptation and conditioning feedstock prior to the continuous process. This phase lasted for

28 days and the daily as well as the accumulated biogas production is presented in Fig. 4. The

anaerobic digestion of the best mixing ratio 1:1:6:0 (M4) in the plug flow reactors; reactor 1

(R1) and reactor 2 (R2) was stable. The cumulative methane yield was 270 and 282

NmlCH4/gVS in R1 and R2, respectively. During the 28 days long digesting period, 57 l and

60 l of biogas was produced in R1 and R2 resulting in a TS reduction of 64 % and 73 %,

respectively. The methane content was 63 % ± 2.5 in R1 and 62 % ± 2.9 in R2. The methane

yield obtained during this adaptation phase in the plug flow reactors was between 13 % and

18 % higher than that obtained from this mixing ratio (M4) during the batch digestion test.

Minimal mixing in the plug flow reactor by moving its content with impeller to the inlet and

back to the outlet provides good contact between the microorganisms and the feedstock,

thereby improving degradation rate and methane yield. After this adaptation period and since

the co-digestion process was aimed to be studied at dry digestion conditions, the TS in the

reactors was increased to 15 % by adding fresh feedstock to the acclimatized inoculum

keeping a VS ratio (VSsubstrate to VSinoculum) at 2.5:1.

3.4.2 Process performance at different loading rates

The continuous digestion process started after 32 days of a startup at 15 %TS. By the end of

this startup phase, the TS content decreased to 7.6 % in RTS21 and to 8 % in RTS32. Then,

continuous operations were carried out at organic loading rates of 2.0 gVS/L/d (OLR 1) and

thereafter at 3.8 gVS/L/d (OLR 2) in both reactors. During this operation the TS in the

14

digestate increased with the increase in OLR both in RTS21 and RTS32. At OLR1 (2.0 gVS/L/d)

the TS content of digestate was 9.8 % ± 0.34 in RTS21 and 11.12 ± 0.69 in RTS32. As the OLR

increased to 3.8 gVS/L/d, the TS content of the digestate increased to 10.02 ± 0.55 % in RTS21

and to 12.1± 0.45 % in RTS32. Additionally, reactor 2 (RTS32) which was fed with a feedstock

with 32 %TS was blocked towards the end of this period (OLR 2); there were problems both

with the feeding and with the discharge of the digestate residue.

The daily biogas production and methane content in the produced biogas during continuous

operation at different organic loading rates is presented in Fig. 5. During OLR 1, a biogas

production of about 4 L/d was obtained from both reactors at the beginning, which increased

slightly and became stable towards the end of the digestion period at OLR1 showing an

average of 6.1 L/d in RTS21 and 5.7 L/d in RTS32. The methane proportion in the biogas was 60

% in RTS21 and 57 % in RTS32. The methane yield in RTS21 was 362 NmlCH4/gVSadded, which is

13.5 % higher than the yield obtained from RTS32 (319 NmlCH4/gVSadded). Chen et al. (2015)

also reported a decrease in the biogas yield during continuous dry digestion of swine manure

as the TS in the feedstock was increased from 20 % to 35 %. However, when the organic

loading rate was increased to 3.8 gVS/L/d (OLR 2), the daily biogas production increased

slightly at the beginning but this could not be sustained and the biogas production dropped as

feeding continued. The methane content also dropped slightly to an average of 58 % in RTS21

and 56 % in RTS32.

The pH was stable, between 7.4 and 7.8 for both reactors during the digestion period at OLR1

(2.0 gVS/L/d), as shown in Fig. 6, in both reactors. Nevertheless, the pH decreased slightly as

OLR was increased to 3.8 gVS/L/d but did not drop below 7.0, hence it was within the

favorable pH range of 6.8 to 8 for the anaerobic digestion processes (Drosg 2013; Lahav and

Morgan 2004), although according to Jabeen et al. (2015) this can vary with substrates and

digestion techniques. At OLR of 2.0 gVS/L/d (OLR 1), the VFA/alkalinity ratio was below

15

0.3 for both reactors which is also within the optimum range required for a stable operation

(Drosg 2013; Liu et al. 2012). Furthermore, the total VFA was also below 1 g/l during this

organic loading rate signifying a stable process. However, the stability of the process declined

as the OLR increased to 3.8 gVS/L/d with corresponding retention time of 30 days. The total

VFA concentration increased to 8 g/l in R1 and to 4.4 g/l in R2 by the end of the digestion

period together with the VFA/alkalinity ratio, which increased sharply, indicating problems in

both reactors (Fig. 6). This resulted in a decline in biogas production as shown in Fig. 5. In

this study, process instability was observed at lower organic loading rate of 3.8 gVS/L/d

compared to OLR of 6 gVS/L/d reported previously for instability while treating manure

bedded with straw at 22 % TS using the continuous plug flow reactor (Patinvoh et al. 2017a).

This can be attributed to the presence of citrus waste in the co-digestion mixture in this study.

For rapidly biodegradable substrates, such as citrus wastes, the acidogenic reactions can occur

quickly resulting in VFA accumulation (Comino et al. 2010b). Co-digestion of fruit, vegetable

and food wastes have been reported less stable at OLR ˃ 2.0 gVS/L/d (Shen et al. 2013)

because of the accumulation of propionate. In addition, total ammonia concentration was

slightly higher in RTS32 than that in RTS21 as shown in Fig. 6; this could be the result of the

moisture content reduction in RTS32. Chen et al. (2015) also reported high ammonia nitrogen

concentration when TS concentration of feedstock was increased from 20 % to 35 % in

continuous dry fermentation of swine manure.

4. Conclusion

Co-digestion of citrus wastes with chicken feather and wheat straw with mixing ratio of 1:1:6

gave the best performance in batch assays, with a methane yield of 238 Nml/gVS. This

mixture was therefore chosen for further investigations in plug flow reactors at different TS

contents in the feed. The methane yields obtained during the adaptation phase in the plug flow

reactors (batch mode using the same condition as in serum glass bottles) were between 13 %

6

Anaerobic sludge used as inoculum was obtained from a digester treating wastewater sludge

and operating at mesophilic conditions (Vatten and Miljö i Väst AB, Varberg, Sweden). The

inoculum was incubated at 37 °C for 2 weeks prior to use. The inoculum with 3.8 %TS and

2.7 %VS was then centrifuged at 10,000g for 10 min to increase its TS content to 9.43 % and

VS content to 6.44 %. Table 1 shows the characteristics of the substrates and the anaerobic

sludge (inoculum) used during the experiments.

2.2 Statistical design

The experiment was designed with statistical software MINITAB® (version 17.1.0), using 3-

factor simplex lattice design, consisting of pure substrates and mixtures of two, three and four

substrates at wet weight ratios. The chicken feather (CF) fraction was kept constant to

maintain the C/N ratio of the mixtures between 12 and 21. The experiments were replicated

according to the same setups two times and methane yield was used as response variable. The

linear mixing model without synergetic or antagonistic interaction was used to obtain the

expected methane yield from all mixtures which correspond to VS fraction from individual

substrates while the quadratic model was used to obtain the predicted methane yield from all

mixtures with synergetic and antagonistic interaction between VS fractions of individual

substrates. Table 2 shows the mixture compositions following the simplex lattice design.

2.3 Batch anaerobic dry digestion of individual substrates and mixtures

Anaerobic batch digestion tests on individual substrates and the co-digestion mixtures were

performed according to the method described by Angelidaki et al. (2009). The assays were

carried out under mesophilic conditions (37 ± 1°C) using 118 ml serum glass bottles as

reactors; each reactor contains 39 ml of inoculum and 1.25 gVS of individual substrates and

mixtures keeping a VS ratio (VSsubstrate to VSinoculum) at 1:2 in all setups. The total solid content

of individual substrates and mixtures were adjusted to 20 %TS thereby having a final TS of

11 % in all reactors; the mixture compositions used in the various set ups are shown in Table

17

Comino, E., Rosso, M., and Riggio, V. (2010b). "Investigation of increasing organic loading rate in the co-digestion of energy crops and cow manure mix." Bioresour. Technol., 101(9), 3013-3019.

Drosg, B. (2013). "Process Monitoring in Biogas Plants. IEA Bioenergy 978-1-910154-03-8." IEA Bioenergy 978-1-910154-03-8.

Forgács, G., Alinezhad, S., Mirabdollah, A., Feuk-Lagerstedt, E., and Sárvári Horváth, I. (2011). "Biological treatment of chicken feather waste for improved biogas production." J. Environ. Sci., 23(10), 1747-1753.

Forgács, G., Niklasson, C., Sárvári Horváth, I., and Taherzadeh, M. J. (2014). "Methane Production from Feather Waste Pretreated with Ca(OH)2: Process Development and Economical Analysis." Waste and Biomass Valorization, 5(1), 65-73.

Forgács, G., Pourbafrani, M., Niklasson, C., Taherzadeh, M. J., and Hováth, I. S. (2012). "Methane production from citrus wastes: process development and cost estimation." J. Chem. Technol. Biotechnol., 87(2), 250-255.

Friehe, J., Weiland, P., and Schattauer, A. (2010a). "Fundamentals of anaerobic digestion. in: Guide to Biogas From Production to Use. Fachagentur Nachwachsende Rohstoffe e. V. (FNR). 5th revised edition. ."

Friehe, J., Weiland, P., and Schattauer, A. (2010b). "Guide to Biogas From production to use ". Gunaseelan, V. N. (2007). "Regression models of ultimate methane yields of fruits and vegetable solid

wastes, sorghum and napiergrass on chemical composition." Bioresource Technology, 98(6), 1270-1277.

Haug, R. T. (1993). "Feed Conditioning - Physical and Chemical." The Practical Handbook of Compost Engineering., Taylor & Francis, 247-257

Herrmann, C., Kalita, N., Wall, D., Xia, A., and Murphy, J. D. (2016). "Optimised biogas production from microalgae through co-digestion with carbon-rich co-substrates." Bioresour. Technol., 214, 328-337.

Jabeen, M., Zeshan, Yousaf, S., Haider, M. R., and Malik, R. N. (2015). "High-solids anaerobic co-digestion of food waste and rice husk at different organic loading rates." International Biodeterioration & Biodegradation, 102, 149-153.

Jensen, J. E. F., and Pipatti, R. (2002). "CH4 emissions from solid waste disposal." Background paper on Good Practice Guidance and Uncertainty Management in National Greenhouse Gas Inventories http://www.ipcc-nggip.iges.or.jp/public/gp/bgp/5_1_CH4_Solid_Waste.pdf Accessed 2017/09/07.

Kabir, M. M., Taherzadeh, M. J., and Sárvári Horváth, I. (2015). "Dry anaerobic digestion of lignocellulosic and protein residues." Biofuel Research Journal, 2(4), 309-316.

Karthikeyan, O. P., and Visvanathan, C. (2013). "Bio-energy recovery from high-solid organic substrates by dry anaerobic bio-conversion processes: a review." Rev. Environ. Sci. Bio/Technol., 12(3), 257-284.

Kusch, S., Oechsner, H., and Jungbluth, T. (2008). "Biogas production with horse dung in solid-phase digestion systems." Bioresour. Technol., 99(5), 1280-1292.

Lahav, O., and Morgan, B. E. (2004). "Titration methodologies for monitoring of anaerobic digestion in developing countries—a review." Journal of Chemical Technology & Biotechnology, 79(12), 1331-1341.

Lal, B. (2005). Building the future we want: trends and technologies (Second edition), Teri Press. Liu, X., Gao, X., Wang, W., Zheng, L., Zhou, Y., and Sun, Y. (2012). "Pilot-scale anaerobic co-digestion

of municipal biomass waste: Focusing on biogas production and GHG reduction." Renewable Energy, 44(Supplement C), 463-468.

Liu, X., Liu, H., Chen, Y., Du, G., and Chen, J. (2008). "Effects of organic matter and initial carbon–nitrogen ratio on the bioconversion of volatile fatty acids from sewage sludge." Journal of Chemical Technology & Biotechnology, 83(7), 1049-1055.

Lohrasbi, M., Pourbafrani, M., Niklasson, C., and Taherzadeh, M. J. (2010). "Process design and economic analysis of a citrus waste biorefinery with biofuels and limonene as products." Bioresource Technology, 101(19), 7382-7388.

18

Lossie, U., and Pütz, P. (2008). "Targeted control of biogas plants with the help of FOS/TAC. Practice Report, Laboratory Analysis Titration FOS/TAC. HACH LANGE ".

Mata-Alvarez, J., Dosta, J., Romero-Güiza, M. S., Fonoll, X., Peces, M., and Astals, S. (2014). "A critical review on anaerobic co-digestion achievements between 2010 and 2013." Renew. Sust. Energ. Rev., 36, 412-427.

Mata-Alvarez, J., Macé, S., and Llabrés, P. (2000). "Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives." Bioresour. Technol., 74.

Mizuki, E., Akao, T., and Saruwatari, T. (1990). "Inhibitory effect of Citrus unshu peel on anaerobic digestion." Biol. Wastes, 33(3), 161-168.

Pagés-Díaz, J., Pereda-Reyes, I., Taherzadeh, M. J., Sárvári-Horváth, I., and Lundin, M. (2014). "Anaerobic co-digestion of solid slaughterhouse wastes with agro-residues: Synergistic and antagonistic interactions determined in batch digestion assays." Chemical Engineering Journal, 245(Supplement C), 89-98.

Pagés Díaz, J., Pereda Reyes, I., Lundin, M., and Sárvári Horváth, I. (2011). "Co-digestion of different waste mixtures from agro-industrial activities: Kinetic evaluation and synergetic effects." Bioresour. Technol., 102(23), 10834-10840.

Patinvoh, R. J., Feuk-Lagerstedt, E., Lundin, M., Sárvári Horváth, I., and Taherzadeh, M. J. (2016). "Biological Pretreatment of Chicken Feather and Biogas Production from Total Broth." Applied Biochemistry and Biotechnology, 180(7), 1401-1415.

Patinvoh, R. J., Kalantar Mehrjerdi, A., Sárvári Horváth, I., and Taherzadeh, M. J. (2017a). "Dry fermentation of manure with straw in continuous plug flow reactor: Reactor development and process stability at different loading rates." Bioresour. Technol., 224, 197-205.

Patinvoh, R. J., Osadolor, O. A., Chandolias, K., Sárvári Horváth, I., and Taherzadeh, M. J. (2017b). "Innovative pretreatment strategies for biogas production." Bioresour. Technol., 224, 13-24.

Patinvoh, R. J., Osadolor, O. A., Horváth, I. S., and Taherzadeh, M. J. (2017c). "Cost effective dry anaerobic digestion in textile bioreactors: Experimental and economic evaluation." Bioresour. Technol. In Press.

Pourbafrani, M., Forgács, G., Horváth, I. S., Niklasson, C., and Taherzadeh, M. J. (2010). "Production of biofuels, limonene and pectin from citrus wastes." Bioresour. Technol., 101(11), 4246-4250.

Raposo, F., De la Rubia, M. A., Fernández-Cegrí, V., and Borja, R. (2012). "Anaerobic digestion of solid organic substrates in batch mode: An overview relating to methane yields and experimental procedures." Renew. Sust. Energ. Rev., 16(1), 861-877.

Resch, C., Wörl, A., Waltenberger, R., Braun, R., and Kirchmayr, R. (2011). "Enhancement options for the utilisation of nitrogen rich animal by-products in anaerobic digestion." Bioresource Technology, 102(3), 2503-2510.

Salminen, E., Einola, J., and Rintala, J. (2003). "The methane production of poultry slaughtering residues and effects of pretreatments on the methane production of poultry feather." Environ. Technol, 24(9), 1079-1086.

Schnürer, A., and Jarvis, A. (2010). "Microbiological Handbook for Biogas Plants. Swedish Waste Management U2009:03 ".

Sharma, K., Mahato, N., Cho, M. H., and Lee, Y. R. (2017). "Converting citrus wastes into value-added products: Economic and environmently friendly approaches." Nutrition, 34(Supplement C), 29-46.

Shen, F., Yuan, H., Pang, Y., Chen, S., Zhu, B., Zou, D., Liu, Y., Ma, J., Yu, L., and Li, X. (2013). "Performances of anaerobic co-digestion of fruit & vegetable waste (FVW) and food waste (FW): Single-phase vs. two-phase." Bioresource Technology, 144(Supplement C), 80-85.

Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., and Crocker, D. (2011). "Determination of Structural Carbohydrates and Lignin in Biomass. Laboratory Analytical Procedure. NREL/TP-510-42618."

19

Sluiter, A., Ruiz, R., Scarlata, C., Sluiter, J., and Templeton, D. (2008). "Determination of Extractives in Biomass. Laboratory Analytical Procedure (LAP). NREL/TP-510-42619 ", Laboratory Analytical Procedure (LAP). NREL/TP-510-42619

Suh, H., and Lee, H. (2001). "Characterization of a Keratinolytic Serine Protease from Bacillus subtilis KS-1." Journal of Protein Chemistry, 20(2), 165-169.

Teghammar, A., Yngvesson, J., Lundin, M., Taherzadeh, M. J., and Horváth, I. S. (2010). "Pretreatment of paper tube residuals for improved biogas production." Bioresour. Technol., 101(4), 1206-1212.

Theuretzbacher, F., Lizasoain, J., Lefever, C., Saylor, M. K., Enguidanos, R., Weran, N., Gronauer, A., and Bauer, A. (2015). "Steam explosion pretreatment of wheat straw to improve methane yields: Investigation of the degradation kinetics of structural compounds during anaerobic digestion." Bioresour. Technol., 179, 299-305.

Tyrberg, L. (2013). "VMAB Biogas at Mörrum waste treatment plant in Mörrum, Karlshamn municipality." http://www.res-chains.eu/wp-content/uploads/2014/09/M%C3%B6rrum-biogas-final.pdf Accessed 2017/07/29.

Wikandari, R., Millati, R., Cahyanto, M. N., and Taherzadeh, M. J. (2014). "Biogas Production from Citrus Waste by Membrane Bioreactor." Membranes, 4(3), 596-607.

Wikandari, R., Nguyen, H., Millati, R., Niklasson, C., and Taherzadeh, M. J. (2015). "Improvement of Biogas Production from Orange Peel Waste by Leaching of Limonene." BioMed Res. Int., 2015, 1-6.

Zeshan, Karthikeyan, O. P., and Visvanathan, C. (2012). "Effect of C/N ratio and ammonia-N accumulation in a pilot-scale thermophilic dry anaerobic digester." Bioresource Technology, 113, 294-302.

Zhou, C., Liu, Z., Huang, Z.-L., Dong, M., Yu, X.-L., and Ning, P. (2015). "A new strategy for co-composting dairy manure with rice straw: Addition of different inocula at three stages of composting." Waste Manage., 40, 38-43.

Zupančič, G. D., and Roš, M. (2012). "Determination of Chemical Oxygen Demand in Substrates from Anaerobic Treatment of Solid Organic Waste." Waste Biomass Valor., 3, 89–98.

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Figure Captions

Figure 1. Cumulative methane yield during batch anaerobic digestion and theoretical

maximum methane potential obtained from COD compositions of individual substrates.

Symbols represent: grey (■) experimental yield and black (■) theoretical maximum potential

Figure 2. Cumulative methane yield obtained during dry co-digestion of the various mixing

ratios in batch experiments. The composition of the different mixtures (i.e., M1…M18 can be

found in Table 2.

Figure 3. Synergetic and antagonistic effect of the various mixtures investigated. Symbols

represent: grey (■) obtained methane yield from quadratic mixing model (with synergetic and

antagonistic effect) and black (■) obtained methane yield from linear mixing model (without

synergetic and antagonistic interactions). The composition of the different mixtures (i.e.,

M1…M18 can be found in Table 2.

Figure 4. Daily biogas production and accumulated methane yield obtained during the initial

stage (adaptation period) carried out in batch mode for digestion of the best mixing ratio (M4)

in plug flow reactors. The digestion period lasted for 28 days and the initial load was 20 %TS

of feedstock in both reactors; reactor 1 (R1) and reactor 2 (R2). Colours represent: blue for

reactor 1 (R1) and red for reactor 2 (R2). Symbols represent: ◊ and □ for daily biogas

production and ∆ and x for cumulative methane yield.

Figure 5. Daily biogas production and the methane content of the produced biogas in reactor

1 (RTS21) and reactor 2 (RTS32) during continuous operation; startup (35 days) OLR1 (2

gVS/L/d for 50 days) and OLR2 (3.8 gVS/L/d for 30 days). Colours represent: blue for

reactor 1 (R1) and red for reactor 2 (R2). Symbols represent: ◊ and □ for daily biogas

production and ∆ and x for methane content of the biogas.

21

.

Figure 6. Variation in pH, VFA/alkalinity ratio, total VFA and total ammonia concentration

in reactor 1 (RTS21) and reactor 2 (RTS32) during continuous operation; startup (35 days), OLR1

(2 gVS/L/d for 50 days) and OLR2 (3.8 gVS/L/d for 30 days). Colours represent: blue for

reactor 1 (R1) and red for reactor 2 (R2). Symbols represent: (a) ◊ and □ pH, ∆ and x

VFA/alkalinity ratio (b) ◊ and □ total VFAs concentration, ∆ and x total ammonia

concentration.

22

Table 1 Characteristics of substrates and inoculum (mean values and standard deviations based on triplicate measurements)

Substrates pH Bulk density (g/L)

TS (%) VS ٭(%)

Ash ٭(%)

Total carbon ٭(%)

TKN ٭(%)


Chicken feather

ND ND 93.47 ± 0.06

99.26 ± 0.01

0.74 ± 0.01

55.14 ± 0.01

15.50 ± 0.30

3.6 ND

Citrus wastes

3.24 753.60 ± 24.32

23.42 ± 2.23

96.07 ± 0.74

3.93 ± 0.74

53.37 ± 0.41

0.99 ± 0.08

55.5 1.03

Wheata straw

ND 190.47 ± 6.93

89.07 ± 0.08

94.96 ± 0.14

5.04 ± 0.14

52.76 ± 0.08

0.83 ± 0.08

69 0.82

Manure with straw

8.01 542.00 ± 26.87

25.84 ± 0.92

77.21 ± 2.74

22.79 ± 2.74

42.89 ± 1.52

2.26 ± 0.04

19 0.89

Anaerobic sludge

8.19 1006.7 ± 5.52

9.43 ± 0.09

68.29 ± 0.01

29.43 ± 0.35

39.20 ± 0.19

4.73 ± 0.18

8.3 ND


dry basis. ND – not determined. C/N – carbon to nitrogen ratio. COD – chemical oxygen٭demand

23

Table 2 Mixture compositions used in batch anaerobic co-digestion assays.

Mixing ratiosa Mixtures C/N Mixing compositions Chicken feather (CF)

Citrus wastes (CW)

Wheat straw (WS)

Manure with straw (MS)

% b gadded

% VSadded

% b gadded

% VSadded

% b gadded

% VSadded

% b gadded

% VSadded

1:1:1:5 M1 14 3.4 12.5 14.0 12.5 3.7 12.5 78.9 62.5 1:1:5:1 M2 19 6.6 12.5 27.0 12.5 35.9 62.5 30.5 12.5

1:1:0:6 M3 13 3.0 12.5 12.5 12.5 0 0 84.5 75

1:1:6:0 M4 21 8.5 12.5 35.2 12.5 56.2 75 0 0

1:0.5:2.2:4.3 M5 15 3.9 12.5 8.1 6.25 9.3 27.1 78.8 54.2

1:0.5:4.3:2.2 M6 17 5.6 12.5 11.5 6.25 26.6 54.2 56.3 27.1

1:0.5:1.1:5.4 M7 14 3.4 12.5 7.0 6.25 4.0 13.5 85.6 67.7

1:0.5:5.4:1.1 M8 19 7.1 12.5 14.7 6.25 42.3 67.7 35.9 13.5

1:0.5:0:6.5 M9 13 3.0 12.5 6.2 6.25 0 0 90.8 81.3

1:0.5:6.5:0 M10 21 9.8 12.5 20.2 6.25 70 81.3 0 0

1:0.25:2.25:4.5 M11 14 3.9 12.5 4.1 3.125 9.7 28.1 82.3 56.3

1:0.25:4.5:2.25 M12 17 5.7 12.5 5.9 3.125 28.3 56.3 60 28.1

1:0.25:0:6.75 M13 12 3.0 12.5 3.1 3.125 0 0 93.9 84.4

1:0.25:6.75:0 M14 21 10.6 12.5 10.9 3.125 78.5 84.4 0 0

1:0:1.2:5.8 M15 13 3.4 12.5 0 0 4.3 14.6 92.3 72.9

1:0:5.8:1.2 M16 19 7.8 12.5 0 0 49.9 72.9 42.3 14.6

1:0:0:7 M17 12 3.0 12.5 0 0 0 0 97 87.5

1:0:7:0 M18 21 11.5 12.5 0 0 88.5 87.5 0 0 a ratios of CF:CW:WS:MS respectively; the mixing ratios are based on VSadded. b wet basis.

24

Table 3 Experimental values and response variable (YCH4) obtained from all mixtures investigated (mean values and standard deviations based on duplicate samples)

Mixtures Experimental YCH4 (Nml/gVSadded)

Quadratic modelb YCH4 (Nml/gVSadded)

Linear modelb YCH4 (Nml/gVSadded)

M1 170.26 (± 13.71) 186.19 (± 6.88) 181.82 (± 22.69) M2 232.52 (± 5.75) 226.60 (± 6.88) 201.61 (± 22.69) M3 188.18 (± 5.71) 174.36 (± 9.12) 176.91 (± 26.43) M4 238.05 (± 11.36) 234.98 (± 9.12) 206.55 (± 26.43) M5 164.65 (± 37.01) 192.07 (± 4.92) 190.98 (± 21.32) M6 226.51 (± 9.78) 206.41 (± 4.92) 201.69 (± 21.32) M7 175.98 (± 12.82) 183.69 (± 4.98) 185.63 (± 24.16) M8 221.64 (± 4.21) 212.36 (± 4.98) 207.04 (± 24.16) M9 191.22 (± 3.87) 174.49 (± 6.82) 180.27 (± 28.28) M10 200.24 (± 38.51) 217.50 (± 6.82) 212.39 (± 28.28) M11 198.34 (± 5.89) 188.40 (±4.31) 193.08 (± 22.02) M12 192.02 (± 30.79) 199.37 (± 4.31) 204.20 (± 22.02) M13 168.53 (± 5.57) 173.93 (± 6.67) 181.96 (± 29.27) M14 217.70 (± 13.21) 206.83 (± 6.67) 215.32 (± 29.27) M15 180.04 (± 13.76) 178.97 (± 6.40) 189.41 (± 25.86) M16 177.84 (± 18.69) 193.58 (± 6.40) 212.47 (± 25.86) M17 166.51 (± 8.85) 172.97 (± 8.90) 183.64 (± 30.31) M18 189.76 (± 38.02) 194.88 (± 8.90) 218.24 (± 30.31) b response variable (YCH4) from linear and quadratic mixing models. YCH4 = methane yield

25

Fig. 1

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Chicken feather Citrus wastes Wheat straw Manure with straw

Met

han

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(N

mlC

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Individual substrates

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Fig. 2

27

Fig. 3

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Fig. 4

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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

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Fig. 5

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%)

Daily

bio

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/d)

Time (days)

Startup period OLR 1 OLR 2

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Fig. 6

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