ENHANCING BIOGAS PRODUCTION IN TWO PHASE ......ENHANCING BIOGAS PRODUCTION IN TWO PHASE ANAEROBIC...

ENHANCING BIOGAS PRODUCTION IN TWO PHASE ANAEROBIC DIGESTION

(TPAD) USING BIOCHAR AND PREPARATION OF BIOCHAR LOADED

ORGANIC FERTILISERS

Nimas Mayang Sabrina Sunyoto

This thesis is presented for the degree of Doctor of Philosophy of

The University of Western Australia

School of Engineering

Centre for Energy

2019

i

THESIS DECLARATION

I, Nimas Mayang Sabrina Sunyoto, certify that:

This thesis has been substantially accomplished during enrolment in this degree.

This thesis does not contain material which has been submitted for the award of any other

degree or diploma in my name, in any university or other tertiary institution.

In the future, no part of this thesis will be used in a submission in my name, for any other

degree or diploma in any university or other tertiary institution without the prior approval

of The University of Western Australia and where applicable, any partner institution

responsible for the joint-award of this degree.

This thesis does not contain any material previously published or written by another

person, except where due reference has been made in the text and, where relevant, in the

Authorship Declaration that follows.

This thesis does not violate or infringe any copyright, trademark, patent, or other rights

whatsoever of any person.

The work described in this thesis was funded by Australian Research Council under the

ARC Linkage Projects Scheme (Project Number: LP100200137). Financial and other

supports have also been provided by the Australian Commonwealth Government

through the Australia Awards Scholarship.

This thesis contains published work and/or work prepared for publication, some of

which has been co-authored.

Signature:

Date: 06-08-2019

ii

ABSTRACT

The conventional anaerobic digestion (AD) is a mature technology to both manage

putrescible organic waste and generate biogas for energy services. However, the biogas it

produces is often of a low quality and the process is slow and operates over very long

durations. Two phase anaerobic digestion (TPAD) has been conceptualised to improve the

conventional AD. The advantage of the TPAD is the production of hydrogen (H2) from

the first phase to be mixed with the methane (CH4) generated from the second phase to

increase the overall quality of the biogas without sophisticated and expensive gas

processing. The TPAD also produced an increased nutrient availability for organic

fertiliser preparation for agricultural applications. However, improvements to further

enhance biogas production, increasing H2 and CH4 yields, operating TPAD in pilot scale

and adding value to the beneficial utilisation of the effluent are still required. Biochar with

its beneficial characteristics for a wide range of applications has become an innovative

aspect of this study. The present PhD thesis research was aimed to investigate the

utilisation of biochar in (1) the first and second phases of batch TPAD, (2) start-up

performance of a TPAD process demonstration unit (PDU) and (3) preparation of biochar-

loaded organic fertiliser from TPAD effluent.

The specific objectives of this thesis work included a systematic study on the effect of

biochar on the performance of the bench-scale TPAD and an investigation into the

working mechanisms of biochar through experimentation and process optimisation studies

in bench scale TPAD. In addition, a demonstration of the operation of TPAD PDU added

with biochar and evaluation of biochar-loaded organic fertiliser prepared from the effluent

of TPAD PDU and biochar. To accomplish these objectives, an investigation on the

individual effects of biochar addition ratio on the gas yield, gas production rate and

metabolic products in each phase of TPAD was conducted in a batch operation. The

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combined effects of biochar, pH and temperature on the H2 and CH4 production were

systematically studied and optimised using the response surface methodology (RSM). The

start-up performance of the TPAD PDU with the optimum biochar addition suggested

from the batch scale operation of TPAD was then conducted. The biogas production,

biogas composition, pH and metabolic products during the start-up operation were

investigated. Finally, the TPAD effluent was loaded on to biochar with different addition

ratios to prepare organic fertilisers. The biochar-loaded fertilisers were characterised, then

evaluated using seed germination assay.

Bench-scale studies of the first phase of TPAD showed that biochar addition shortened

the lag phase by 21.4 to 35.7 %, increased the maximum H2 production rate by 32.4% and

H2 production potential by 14.2 to 31 %. Biochar addition was shown to substantially

increase H2 yield (YH), especially at lower pH and higher temperatures. The RSM analysis

showed that the maximum YH of 1,331 mL.L-1 and H2 production rate (RH) of 763 mL.L-

1.day-1 were achieved under the optimum conditions of biochar addition ratio 10.1 g.L-1,

initial pH 6.4 and temperature 32C. The biochar initiated the biofilm formation, as

observed with scanning electron microscopy (SEM) images, and provided macro and

nutrients in the culture, enriching the microbial population. Biochar also acted as a pH

buffer of the cultures, preventing the cultures from sharp pH drop caused by acid

accumulation during hydrolysis, enhancing the H2 production.

Bench-scale studies of the second phase of TPAD showed that the biochar addition also

shortened the lag phase, by 41 to 45 %, increased the maximum CH4 production rate by

23.0-41.6% and CH4 production potential by 1.9 to 9.6%. A moderate level of biochar

addition, mesophilic temperature and neutral to alkaline pH were shown to benefit CH4

production in the second phase of TPAD. The effect of biochar addition was more

profound at higher pH. The optimum biochar addition, temperature and initial pH were

found to be 12.5 g.L-1, 36.2oC and 7.8, respectively. Under the optimum condition, the

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CH4 yield (YM) and CH4 production rate (RM) were 1755 mL.L-1 and 500.9 mL.L-1.day-1.

SEM images suggested an establishment of methanogenic biofilm in the pore and surface

of biochar. It was hypothesised that methanogenic biofilm enriched methanogens and

enhanced CH4 production. The alkalinity of biochar, however, was found to be

insignificant in promoting CH4 production in the second phase.

The investigation into the transient performance during the start-up of a TPAD PDU

treating food waste with biochar addition was conducted. A fed-batch followed by

semicontinuous operation strategy was found to be effective in starting up the TPAD PDU.

The fed-batch operation allowed sufficient time for microbial enrichment and adaptation.

Under semi-continuous operation, the peak H2 composition and yields in the first phase

were 49% and 46 L.kg volatile solids (kg VS)-1, respectively. CH4 production with the

composition of up to 59% and yield of 301 L.(kg VS)-1 were attained in the second phase.

The addition of biochar showed a potential to buffer the pH of culture and initiate biofilm

formation, which supported the successful start-up in both the reactors, supported by the

findings observed in bench scale studies of TPAD with biochar addition.

Finally, the biochar loaded organic fertilisers were successfully prepared and subject to

the seed germination assay. It was found that the addition of biochar significantly

increased water holding capacity (WHC) with no significant pH changes. It also increased

essential elements for germination and plant growth, such as potassium (K), calcium (Ca),

magnesium (Mg), sodium (Na), iron (Fe), manganese (Mn), nickel (Ni) and chromium

(Cr), compare to the TPAD effluent alone. The prepared fertilisers were tested using

germination bioassay and compared to the control without fertiliser addition. Fertilisers

with 0-90% biochar addition gave the positive effects on the seed germination, while the

pure biochar significantly reduced germination index (GI). However, despite the low GI,

the pure biochar, and the rest of the fertilisers tested, resulted in the increased sums of root

and shoot length compared to the control. The maximum sums of root and shoot (153.4±8

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and 62.3±5 cm) was achieved with the fertiliser prepared of 10% of TPAD effluent and

90% of biochar. The improved macro and micronutrients in the fertilisers were observed,

contributing to the good seed germination results with the biochar loaded fertilisers.

The outcomes of the current research have contributed new knowledge and useful

experimental data on the utilisation of biochar to enhance H2 and CH4 production in TPAD

operation. The working mechanisms biochar involved in each phase of TPAD have been

proposed. The applications of biochar in pilot scale TPAD and as organic fertiliser for

agriculture applications have also been demonstrated.

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TABLE OF CONTENTS

THESIS DECLARATION ................................................................................................ i

ABSTRACT ..................................................................................................................... ii

TABLE OF CONTENTS ................................................................................................ vi

ACKNOWLEDGMENTS ............................................................................................. xix

AUTHORSHIP DECLARATION: CO-AUTHORED PUBLICATIONS .................. xxii

Chapter 1 Introduction ................................................................................................... 1

1.1 Background and motivation.................................................................. 1

1.2 Scope and aims .................................................................................... 3

1.3 Thesis structure .................................................................................... 3

Chapter 2 Literature Review ......................................................................................... 6

2.1 Introduction .......................................................................................... 6

2.2 Anaerobic digestion (AD) ................................................................... 6

2.2.1 Application of AD for waste treatment and energy utilisation ... 6

2.2.2 Fundamental principles of AD .................................................... 7

2.2.3 Drawbacks of conventional AD ................................................ 10

2.3 Two-phase anaerobic digestion (TPAD) ............................................ 11

2.3.1 Mechanisms and system of TPAD ............................................ 11

2.3.2 Factors influencing TPAD operation ........................................ 14

2.4 Biochar ............................................................................................... 21

2.4.1 Physical characteristics ............................................................. 22

2.4.2 Chemical characteristics ............................................................ 23

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2.4.3 Biological properties ................................................................. 24

2.5 The effect of biochar in the AD system .............................................. 25

2.5.1 Biochar application in biogas production .................................. 25

2.5.2 Biochar application in pilot scale operation of TPAD .............. 32

2.5.3 Organic fertiliser production from AD effluent and biochar .... 36

2.5.4 Biochar application in biogas cleaning ..................................... 38

2.6 Summary of literature review and specific research objecti .............. 43

Chapter 3 Methodology, Approach and Techniques ...................................................... 47

3.1 Overall research strategies .................................................................. 47

3.2 Bench scale experimentation of TPAD .............................................. 48

3.2.1 Materials .................................................................................... 48

3.2.2 Experimental set up ................................................................... 49

3.2.3 Experimental procedure ............................................................ 50

3.2.4 Analysis ..................................................................................... 58

3.3 TPAD Process Demonstration Unit (PDU) ........................................ 62

3.3.1 Principles of TPAD PDU .......................................................... 62


3.3.3 System monitoring and control ................................................. 68

3.4 Preparation, characterisation and evaluation of biochar-

added organic fertiliser ....................................................................... 69


3.4.2 Analysis ..................................................................................... 71

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3.5 Data analysis and modelling ............................................................ 72

3.5.1 Analysis of variant (ANOVA) .................................................. 72

3.5.2 The modified Gompertz Model ................................................. 72

3.5.3 Response surface methodology (RSM) ..................................... 72

Chapter 4 Effect of Biochar Addition on Hydrogen Production .................................... 74

4.1 Introduction ...................................................................................... 74

4.2 Hydrogen production without biochar ............................................. 74

4.3 Hydrogen production with biochar .................................................. 76

4.3.1 Response surface analysis ......................................................... 80

4.3.2 Hydrogen yield .......................................................................... 85

4.3.3 Hydrogen production rate .......................................................... 87

4.4 Effect of biochar on H2 production via anaerobic digestion

as compared to other solid additives: role of acidity ....................... 89

4.5 Mechanisms ..................................................................................... 95

4.6 Summary .......................................................................................... 98

Chapter 5 Effect of Biochar Addition on Methane Production .................................... 100

5.1 Introduction .................................................................................... 100

5.2 Methane production without biochar addition ............................... 100

5.3 Methane production with biochar addition .................................... 101

5.3.1 Response surface analysis ....................................................... 106

5.3.2 Methane yield .......................................................................... 108

5.3.3 Methane production rate .......................................................... 110

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5.4 Methane production in single phase anaerobic digestion ................ 111

5.5 Mechanisms ...................................................................................... 112

5.6 Summary .......................................................................................... 114

Chapter 6 Transient Performance during Start-up of Two-Phase Anaerobic Digestion

Process Demonstration Unit ........................................................................ 116

6.1 Introduction ...................................................................................... 116

6.2 The start-up performance of the first phase ..................................... 116

6.3 The start-up performance of the second phase ................................. 121

6.4 Summary .......................................................................................... 130

Chapter 7 Preparation, Characterisation and Evaluation of Biochar-loaded Organic

Fertiliser ....................................................................................................... 131

7.1 Introduction ...................................................................................... 131

7.2 Characteristics of biochar-loaded organic fertilisers ........................ 131

7.3 Soil less petri dish bioassay .............................................................. 137

7.4 Summary .......................................................................................... 142

Chapter 8 Evaluation and Practical Implications.......................................................... 143

8.1 Introduction ...................................................................................... 143

8.2 Integration of Experimental Findings .............................................. 143

8.3 Evaluation against the Specific Research Objectives ...................... 144

8.4 Evaluation against the Literature ..................................................... 145

8.4.1 Effect of Biochar Addition on Hydrogen Production .......... 145

8.4.2 Effect of Biochar Addition on Methane Production ............ 147

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8.4.3 Mechanisms of biochar in enhancing H2 and CH4 production in

TPAD.................................................................................... 148

8.4.4 Transient Performance during Start-up of TPAD PDU ....... 155

8.4.5 Preparation, Characterisation and Evaluation of Biochar-loaded

Organic Fertiliser .................................................................. 156

8.5 Practical Implications .................................................................... 159

Chapter 9 Conclusions and Recommendations ............................................................ 161

9.1 Introduction .................................................................................... 161

9.2 Conclusions .................................................................................... 161

9.2.1 Effect of Biochar Addition on Hydrogen Production in the

Bench Scale Experiment ...................................................... 161

9.2.2 Effect of Biochar Addition on Methane Production in the Bench

Scale Experiment .................................................................. 162

9.2.3 Transient Performance during Start-up of TPAD PDU ...... 163

9.2.4 Preparation, Characterisation and Evaluation of Biochar-loaded

Organic Fertiliser .................................................................. 163

9.3 Recommendations .......................................................................... 164

xi

LIST OF FIGURES

1.1 Thesis map ........................................................................................... 5

2.1 Steps of anaerobic digestion ................................................................. 8

2.2 Schematic diagrams of SPAD ............................................................ 10

2.3 Schematic diagrams of TPAD ............................................................ 12

2.4 The TPAD utilisation for combined heat and power (CHP)

system ................................................................................................. 14

2.5 The characteristics, possible functions and applications of

biochar ................................................................................................ 23

3.1 Research strategies ............................................................................ 47

3.2 Schematic of the experimental set-up (a) and bench scale

TPAD in the incubator (b) .................................................................. 50

3.3 Schematics of the experimental procedure for TPAD

employed in this study ....................................................................... 51

3.4 Schematics of the experimental procedure for second phase

TPAD ................................................................................................. 56

3.5 Schematic set up of water displacement method for gas

volume measurement ........................................................................ 59

3.6 A typical gas chromatogram of gas collected from the first

phase of TPAD .................................................................................. 60

3.7 Typical standard curves for (a) acetic and (b) butyric acids

analysis .............................................................................................. 61

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3.8 Schematic diagram of TPAD PDU of the Centre for Energy

of University of Western Australia .................................................. 63

3.9 Preparation of organic fertiliser from TPAD effluent and

biochar ............................................................................................ 69

3.10 Experimental set up of soil-less petri dish bioassay ........................ 70

4.1 Cumulative yields and production rates of H2 without

biochar addition ............................................................................... 74

4.2 Cumulative H2 yields at different biochar addition ratios ............... 76

4.3 VFA profiles during H2 production in culture with (a) 0; (b)

8.3; (c) 16.6; (d) 25.1 and (e) 33.3 g.L-1 biochar addition

ratios................................................................................................. 79

4.4 Response surface and contour plots of cumulative H2 yield

(YH) over 8 days of operation as a function of: (a) initial pH

and biochar addition ratio at 32C and (b) temperature and

biochar addition ratio at initial pH 6 ................................................ 86

4.5 Response surface and contour plots of maximum H2

production rate (RH) as function of: (a) initial pH and

biochar addition ratio at 32C; and (b) temperature and

biochar addition ratio at initial pH 6.4 ............................................. 88

4.6 The pH of liquid culture with the addition of the additives

before the pH adjustment ................................................................. 90

4.7 Production rates of H2 from cultures with different types of

additives ...............................................................................................

91

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4.8 Cumulative yields of H2 from cultures with addition of

different additives ............................................................................... 92

4.9 The pH evolution of the cultures with different types of

additives ............................................................................................. 93

4.10 The mechanisms of biochar in promoting H2 and CH4

productions in TPAD from food waste .............................................. 95

4.11 SEM images of (a) biochar and (b) final effluent of H2

production .......................................................................................... 96

5.1 Cumulative yields and production rates of CH4 without

biochar addition ................................................................................ 100

5.2 Cumulative CH4 yields at different biochar addition ratios ............. 102

5.3 VFA profiles during CH4 production in culture with (a) 0;

(b) 8.3; (c) 16.6; (d) 25.1 and (e) 33.3 g.L-1 biochar addition

ratios ................................................................................................. 104

5.4 Response surface and contour plots of CH4 yield (YM) as

function of: (a) biochar addition and temperature at initial

pH 7.8 and (b) biochar addition and initial pH at temperature

36.2ºC ............................................................................................... 109

5.5 Response surface and contour plots of maximum CH4

production rate (RM) as function of: (a) biochar addition and

temperature at initial pH 7.8 and (b) biochar addition and

initial pH at temperature 36.2ºC ....................................................... 110

5.6 SEM images of (a) the fresh biochar and (b) biochar after 40

days of incubation at 35ºC ............................................................... 113

xiv

6.1 (a) Biogas production, (b) biogas composition, and (c) H2

and CH4 production during the start-up of the first phase ............. 117

6.2 VFA of the first phase .................................................................... 118

6.3 pH and temperature during the start-up of the first phase ............. 119

6.4 TS and VS of the first phase .......................................................... 120

6.5 (a) Biogas production, (b) biogas composition, and (c) H2

and CH4 production during the start-up of the second phase ........ 122

6.6 VFA of the second phase ............................................................... 123

6.7 pH and temperature during the start-up of the second phase ......... 125

6.8 TS and VS of the second phase ..................................................... 125

6.9 SEM images of a liquid sample taken from (a) R1 on day 18

and (b) R2 on day 77 of the start-up operation .............................. 127

7.1 Photographs of germination of rocket seed with various

organic fertilisers conducted in the soil-less petri dish

bioassay .......................................................................................... 138

7.2 The effect of various organic fertilisers on root length of

germinated rocket seed conducted in the soil-less petri dish

bioassay .......................................................................................... 140

7.3 The effect of various organic fertilisers on shoot length of

germinated rocket seed conducted in the soil-less petri dish

bioassay .......................................................................................... 141

7.4 The effect of various organic fertilisers on shoot to root ratio

of germinated rocket seed conducted in the soil-less petri

dish bioassay .................................................................................. 141

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8.1 A schematic representation of the mechanisms of the

working of biochar in promoting H2 and CH4 productions in

TPAD from food waste .................................................................... 149

8.2 H2 and CH4 production in pilot scale TPAD using different

feedstock .......................................................................................... 156

8.3 Germination index of petri dish bioassay using different

plants and fertilisers ........................................................................ 160

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LIST OF TABLES

2.1 The studies of the AD application for wastewater treatment

and energy generation ........................................................................ 7

2.2 Major reactions in acidogenesis step ................................................. 8

2.3 Major reactions in acetogenesis ......................................................... 9

2.4 Fuel Properties ................................................................................. 11

2.5 General reactions of H2 and CH4 generation in ............................... 12

2.6 Hydrogenases and methanogens in typical inoculums .................... 18

2.7 Studies on pH effect on anaerobic digestion ................................... 19

2.8 Optimum temperature range for microbial growth .......................... 20

2.9 Existing studies on the biochar utilisation in H2 and CH4

production via AD P ........................................................................ 28

2.10 Studies on the pilot scale operation of TPAD.................................. 35

2.11 Biogas impurities ............................................................................. 39

2.12 Study of biochar and carbon-based materials to remove gas

impurities ......................................................................................... 41

3.1 Characteristics of feedstock, inoculum and biochar ........................ 49

3.2 Central composite design for H2 production.................................... 53

3.3 The characteristics of the additives .................................................. 54

3.4 Parameters and levels of Box Behnken Design ............................... 57

3.5 Box Behnken design for CH4 production ........................................ 57

3.6 Start-up strategy of the first phase of TPAD PDU .......................... 66

xvii

3.7 Start-up strategy of the second phase of TPAD PDU ........................ 67

3.8 Fertiliser composition ......................................................................... 70

4.1 Profiles of the first phase of TPAD without biochar .......................... 75

4.2 One-way ANOVA and post hoc analysis on cumulative H2

production in different biochar addition ratios ................................... 77

4.3 The results of calculation using the modified Gompertz

fitting equation on H2 production with different biochar

addition ratios ..................................................................................... 78

4.4 Central composite design and experimental results for H2

production .......................................................................................... 81

4.5 ANOVA analysis and the fitting model for YH .................................. 82

4.6 ANOVA analysis and the fitting model for RH .................................. 83

4.7 Model validation results ..................................................................... 84


fitting equation on hydrogen production with different

additives ............................................................................................. 93

5.1 Profiles of the second phase of TPAD without biochar ................... 101

5.2 One-way ANOVA and post hoc analysis on cumulative CH4

production in different biochar addition ratios ................................. 102


fitting equation on CH4 production with different biochar

addition ratios ................................................................................... 103

xviii

5.4 Box Behnken design and experimental results for CH4

production ............................................................................................

106

5.5 ANOVA analysis and the fitting model for YM and RM ................ 108

6.1 Studies on the start-up of pilot scale TPAD PDU ......................... 129

7.1 Main characteristics and composition of the organic

fertiliser with different percentages of biochar addition ................ 133

7.2 The concentrations (mg.kg-1) of elements in the organic

fertiliser with different percentage of biochar addition ................. 137

7.3 Germination index (GI) as percentage of germinated seeds

in the assay to the control .............................................................. 139

8.1 A comparison of the current experimental results with the

literature data on the batch H2 production with the addition

of different types of biochar........................................................... 147

8.2 A comparison of the current experimental results with the

literature data on the batch CH4 production with the addition

of different types of biochar........................................................... 148

8.3 Profiles of biochar and proposed mechanisms in H2

production of different studies ...................................................... 153

8.4 Profiles of biochar and proposed mechanisms in CH4

production of different studies ....................................................... 154

8.5 Characteristics of AD effluent and biochar prepared as

organic fertiliser ............................................................................. 158

xix

ACKNOWLEDGMENTS

This study was undertaken at the Centre for Energy, University of Western Australia,

Perth, Western Australia. I wish to acknowledge everyone who was very supportive in my

journey to complete the PhD study.

I would first like to express my sincere gratitude to my principal supervisor, Dongke

Zhang. I sincerely thank him for taking me under his wing and providing as much as

possible facilities and supports for my research. I thank him for being so patient and

considerate during my study. His supervision is not only limited to the academic aspect

but also extended to life philosophy and art of educating. I have and continue to learn from

him as an excellent role model with an intelligent brain, strong character and commitment.

To my co-supervisor, Mingming Zhu, I cannot thank him enough for his patient and

valuable guidance throughout my course of study. He always trains me to think practically,

logically and critically. I will treasure these values and lessons in my heart and apply it in

my future career as a researcher and educator.

I would also acknowledge the financial support for this research provided by ARC

Linkage Projects Scheme (Project Number: LP100200137). I acknowledge Australian

Commonwealth Government through the Australia Awards Scholarship for providing my

PhD scholarship and University of Brawijaya, my home institution for the permission and

support.

xx

I am grateful for wonderful support by Debra Basanovic and Celia Seah, Krystina Haq for

assisting the thesis writing, Peta Clode and Lyn Kirilak (CMCA UWA) for helping with

SEM analysis and interpretations, and Ross Duffield (Woodman Point Wastewater

Treatment Plant) for providing the sludge inoculum.

I also extend my gratitude to Zhezi Zhang for the advice on the technical aspects of my

research, Yii Leng Chan and Carina Wang for always taking care of us. To Zhijian Wan,

Hendrix Setyawan, and Jesse Sheng, I thank them for helping with the technical issues

during the experiments and sharing general PhD experiences, struggle and jokes to help

me survived the journey. Isabelle Jones, for always being so generous with her time and

experience, helping me with the fertiliser characterisation, writing and preventing me from

burning the laboratory.

Special thanks to my fellow PhD candidate, Yusron Sugiarto; TPAD FYP students 2014-

2018, and Tian Zhang, for being very warm and supportive teammates and also for the

time we spent together inside and outside the TPAD PDU. To my labs-mates: Pengfei Liu,

Jorge Preciado, Herry Lesmana, Juan Zhang, Qian Zhang and Chimeika Okoye and all

CFE families, thank you for sharing everything from labs tools to your cultures. I also

thank to Emily, Tine, Juwita and Dina for multiple PhD sharing sessions, it meant a lot.

Also, my office mates in Room 1.96 for being good companies during our long days and

nights. Terima kasih, xiexie, muchas gracias. I also thank Ezmieralda Melissa, Sigit Pria

Perdana, Reliana Lumban Toruan, and other Indonesian students’ cohort 2014, Maroonah,

Warneds, Liqo Rania, Liqo Matilda Bay and AIPSSA for the friendship and always

making me feel at home.

Finally, I would also like to thank my husband, Gilang Agus Setiyono, for being the best

supporter and enjoying the roller coaster of the PhD journey together. It would have been

impossible for me to finish this work without his continuous support and encouragement.

xxi

I thank my mother, Ibu Tri Sayekti, my parents-in-law, my sisters and brothers for their

endless prayer, love and support so I can complete the work. And I dedicate this thesis to

my late Father, Totok Sunyoto.

xxii

AUTHORSHIP DECLARATION: CO-AUTHORED

PUBLICATIONS

This thesis contains work that has been [published and/or prepared for publication].

Details of the work:

Sunyoto, N. M., Zhu, M., Zhang, Z., & Zhang, D. (2016). Effect of biochar addition

on hydrogen and methane production in two-phase anaerobic digestion of aqueous

carbohydrates food waste. Bioresource Technology, 219, 29-36.

Location in thesis: Chapter 4

Student contribution to work:

• The work was supported by and part of ARC Linkage project grant held by Dongke

Zhang

• The candidate designed, conducted and analysed the results of the experiment with

assistance from Zhezi Zhang and Mingming Zhu, and under supervision of Dongke

Zhang

• The candidate drafted the manuscript and worked closely with Dongke Zhang and

Mingming Zhu to critically review it


Sunyoto, N. M., Zhu, M., Zhang, Z., & Zhang, D. (2017). Effect of Biochar Addition

and Initial pH on Hydrogen Production from the First Phase of Two-Phase Anaerobic

Digestion of Carbohydrates Food Waste. Proceedings of the 8th International

Conference on Applied Energy, Beijing.




Zhang



Zhang

xxiii



• The candidate presented the paper at the conference


Sunyoto, N. M., Zhu, M., Zhang, Z., & Zhang, D. (2017). Effect of Biochar Addition

and Initial pH on Hydrogen Production from the First Phase of Two-Phase Anaerobic

Digestion of Carbohydrates Food Waste. Energy Procedia, 105, 379-384.




Zhang



Zhang




Sunyoto, N. M. S., Zhu, M., Zhang, Z., & Zhang, D. (2018). Effect of Biochar Addition

and Temperature on Hydrogen Production From the First Phase of Two-Phase

Anaerobic Digestion of Carbohydrates Food Waste. Proceedings of the 42nd

Clearwater Clean Energy Conference, Florida




Zhang

xxiv



Zhang




Sunyoto, N. M. S., Zhu, M., Zhang, Z., & Zhang, D. (2018). Effect of Biochar Addition

and Temperature on Hydrogen Production From the First Phase of Two-Phase

Anaerobic Digestion of Carbohydrates Food Waste. Journal of Energy Resources

Technology, 140(6), 062204.




Zhang



Zhang




Sunyoto, N., Zhu, M., Sugiarto, Y., & Zhang, D. (2018). Effect of biochar on hydrogen

production via anaerobic digestion as compared to other solid additives: Role of acidity.

Chemeca 2018, 165.




Zhang

xxv



Zhang



• The candidate presented the paper at the conference


Sunyoto, N. M. S., Zhu, M., Sugiarto, Y., & Zhang, D. (2018). Effect of Biochar

Addition, Initial pH and Temperature on Methane Production in Two Phase Anaerobic

Digestion of Carbohydrates Food Waste. Proceedings of the 43rd Clearwater Clean

Energy Conference, Florida.




Zhang



Zhang




Sunyoto, N., Zhu, M., Sugiarto, Y., & Zhang, D. Transient Performance during Start-

up of a Two-Phase Anaerobic Digestion Process Demonstration Unit Treating

Carbohydrate-rich Waste with Biochar Addition. International Journal of Hydrogen

Energy (Submitted – 2018)



xxvi


Zhang

• The candidate planned and designed the experiment under supervision of Dongke

Zhang and Mingming Zhu

• The candidate carried out the experiments, and analysed results with assistance

from Zhezi Zhang and Mingming Zhu

• The manuscript was drafted by the candidate and critically reviewed by Dongke

Zhang and Mingming Zhu

1

Chapter 1 Introduction

1.1 Background and motivation

Anaerobic digestion (AD) is a good means of putrescible organic waste treatment and utilisation

due to its ability both as a system to minimise the environmental effects of waste disposal and

to provide energy service. The AD has been widely applied to convert all types of putrescible

organic waste and wastewater to produce biogas. The AD is preferred over aerobic digestion

process because of its higher effectiveness to reduce high solids and organic materials in

wastewater and convert these materials into the valuable form of energy, the biogas (mainly

CH4), without emissions of harmful volatile organic compounds (VOC). It is also efficient due

to lower energy and smaller space requirement leads to lower total operating costs. It also brings

more economic benefits for its ability to generate biogas [1]. Although AD has been well

applied for some decades, several drawbacks need to be overcome; such as slow conversion

process, long retention time, sub-optimum process condition and low biogas quality [2, 3]. AD

produces typical biogas which has low energy density and less ideal as fuel for gas engines [4].

The UWA Centre for Energy has advocated a new concept of two-phase anaerobic digestion

(TPAD), which is an improvement over the conventional AD. TPAD is a system consisting of

two anaerobic digesters designed to separate hydrogen (H2) production in the first phase from

methane (CH4) production in the second phase. This separation allows the enrichment of

specific microbes and optimisation of operation condition in each reactor [5]. These

improvements increase the effectiveness of anaerobic process by enhancing biogas production

and improving the stability of the system [6, 7]. At the end of the process, a mixture of H2 and

CH4 are produced from TPAD. The presence of H2 in the otherwise low-quality biogas helps

to improve ignition quality thus better for gas engine applications. This system has been trialed

2

in several waste streams including dairy wastewater [8-10], food waste [11-13] and sewage

sludge [14, 15]. To improve the performance, TPAD requires further innovations.

Biochar, a carbon-rich material derived from the thermal decomposition of biomass, has

attracted significant attention due to its potential as an additional feedstock and an ideal packing

material for biofilm formation that enhance the production of biogas. A study conducted by

Mumme et al. (2014) report that biochar addition in single phase AD improves CH4 yield by

32% and prevent the system from mild ammonia inhibition [3]. There are also some studies

investigating the effects of operation conditions on biogas production and composition,

specifically in single-phase anaerobic digestion [16, 17] but not in TPAD. Therefore, it is

essential to study biogas production and composition in TPAD under different biochar addition

and operation conditions.

Typically, impurities such as CO2, H2S and NH3 are also produced during the AD. The

impurities lower the caloric content of biogas and cause utility corrosion. Therefore, attempts

to remove impurities from raw biogas are also needed. One promising method in the removal

of biogas impurities is adsorption using biochar [18]. Different types of biochar such as sewage

sludge [18], shell-derived, camphor-derived, bamboo, rice hull [19, 20] and leaf based [21] have

been used in the study of the removal of single stream biogas impurities such as H2S. However,

up to now, there are limited studies on the removal of biogas impurities especially in NH3 using

biochar. Biogas cleaning using biogas, therefore, is recommended for future studies.

AD generates high nutrient content-effluent as a by-product at the end of the process [22, 23],

thus can be used as an organic fertiliser. It has been studied in a wide range of plants, such as

tomato [24], wheat [25] and grassland [26]. Likewise, studies of biochar as fertiliser have been

conducted in several plants cultivation, for instance, soybean, tea tree, rice, cowpea wheat grass

and oats [27, 28]. However, the results of those studies vary and inconclusive, and only a few

of them examined the “synergistic effect” of the effluent of anaerobic digestion and biochar as

3

fertilisers for agriculture applications [29]. In this study, a method to prepare organic fertiliser

by treating the TPAD effluent with biochar is studied.

1.2 Scope and aims

The present PhD thesis research was aimed to investigate the utilisation of biochar in (1) the

first and second phase of batch TPAD, (2) start-up of TPAD process demonstration unit (PDU),

(3) preparation of biochar loaded organic fertiliser from TPAD effluent. The individual effects

of biochar addition ratio on the gas yield, gas production rate and metabolic products in each

phase of TPAD were investigated in a batch operation. The combined effects of biochar, pH

and temperature on the H2 and CH4 production were further studied and optimised.

The start-up TPAD PDU with the optimum biochar addition suggested from the batch scale

operation of TPAD was conducted. The biogas production, biogas composition, pH and

metabolic product during the start-up operation were investigated. The effluent of TPAD with

different addition percentage of biochar was used to prepare organic fertiliser. The prepared

fertilisers were characterised, and the effects on the seed germination were studied.

1.3 Thesis structure

A schematic map of the thesis is presented in Figure 1.1. There are eleven chapters in this thesis

as outlined below:

Chapter 1 TPAD as an innovation to improve biogas quality is introduced. Biochar

potentials to improve biogas production, purify biogas and prepare organic

fertiliser are identified. The scope of research, overall aims and thesis

structure are defined.

Chapter 2 Fundamental knowledge on AD and TPAD and existing studies of biochar

utilisation in the AD, biogas cleaning, and organic fertiliser preparation are

4

reviewed. Knowledge gaps are identified, and specific objectives of the

thesis are defined.

Chapter 3 To achieve the aims in Chapter 2, methodology, approach and technique

are determined. Experimental set up of bench scale and demonstration

operations of TPAD, biogas cleaning and organic fertiliser preparation are

described.

Chapter 4 The effects of biochar addition, initial pH and temperature on H2

production, composition, and metabolic products in the bench scale

experimentation are presented. The optimisation of each factor on the batch

operation of the first phase is suggested.

Chapter 5 The effects of biochar addition, initial pH and temperature on CH4

production, composition, and metabolic products in the bench scale

experimentation are presented. The optimisation of each factor on the batch

operation of the second phase is suggested.

Chapter 6 The operation of TPAD in a demonstration-scale unit is reported. Practical

considerations of the unit operation are outlined.

Chapter 7 The preparation of organic fertiliser from TPAD effluent and biochar is

described. The effect of each type of fertiliser on the seed germination is

explained.

Chapter 8 The results from the present work are integrated. The findings are evaluated

against the objectives and the literature data. The practical implications are

also identified.

Chapter 9 The new and significant findings are identified. The new knowledge gaps

are recommended.

5

Figure 1.1 Thesis map

6

Chapter 2 Literature Review

2.1 Introduction

Chapter 2 reviews the fundamentals and state of knowledge relating to AD and TPAD.

Specifically, this chapter emphasises on the biochar utilisation as the centre of the study. The

biochar characteristics, utilisation of biochar in the AD and TPAD, including pilot-scale

operation of TPAD, and organic fertiliser preparation are also reviewed. At the end of the

review, the gaps of studies are suggested and specific objectives of this study are appointed.

2.2 Anaerobic digestion (AD)

2.2.1 Application of AD for waste treatment and energy utilisation

Anaerobic digestion (AD) is a series of complex biochemical conversions of organic content in

a material carried out by different groups of microbes in the absence of oxygen [30]. AD serves

as a means to treat waste/wastewater because the process results in reduced solids and organic

content of materials as to achieve an approved quality to be discharged to the environment. AD

also plays a role to generate valuable forms of energy in the form of biogas [31]. Single phase

AD typically produces CH4 (65-77%), CO2 (19-50%), N2 (0-5%) of and trace gases (H2S: 3-

20,000 ppm; NH3:50-100 ppm) [32]. After removal of the CO2 and other trace gases, the biogas

is upgraded to a pipeline quality bio-CH4 and ready to be used as an engine and vehicle fuel

[32]. At the end of the process, the AD also recovers various chemicals and nutrients to produce

useful by-products, such as nitrogen, phosphorus, potassium, alcohol and volatile fatty acids

(VFA) [1, 30, 31, 33]. AD has been applied to a wide variety of putrescible organic feedstocks

for some decades, including lignocellulosic biomass, animal manure, sewage sludge, industrial

and food waste [34, 35]. Table 2.1 shows the report of previous studies using various waste and

wastewater as feedstock in AD and its effectiveness in treating the waste and producing biogas.

7

The listed studies suggest that the AD removes up to 81% of the volatile solids in the waste and

converts each gram volatile solids of the waste into 0.26 – 0.46 litre of CH4.

Table 2.1 The studies of the AD application for wastewater treatment and energy

generation

Feedstock VS removala (%) CH4 yield

(l CH4.gr VS-1)

Ref.

Lignocellulosic biomass (Zea

mays L.)

72 0.30 [36]

Chicken manure 81 0.43 [37]

Sewage sludge 42 0.27 [38]

Dairy industry waste N.A 0.26 [39]

Food waste N.A. 0.46 [40]

a VS removal: volatile solids removal

b COD removal: chemical oxygen demand removal

2.2.2 Fundamental principles of AD

According to the microbial activities and metabolic products generated, the AD consists of 4

major steps namely: hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Figure 2.1)

[41].

Hydrolysis

In the hydrolysis stage, complex materials such as carbohydrate, protein and fats are broken

down into simpler compounds/monomers (short-chain sugars, amino acids and peptides,

glycerine and fatty acids). Wide ranges of hydrolytic bacteria are involved in the hydrolysis

process, including Pseudomonas sp, Clostridia, Micrococci [42]. These hydrolytic bacteria

secrete extracellular hydrolysis enzymes, such as cellulose, amylases, proteases and lipase that

are required during the hydrolysis process. The general reaction on hydrolysis is as follow [42]:

8

C6H10O4 + 2H2O → C6H12O6 + 2H2 (R2.1)

Figure 2.1 Steps of anaerobic digestion [42-45]

Acidogenesis

Table 2.2 Major reactions in acidogenesis step [42]

Product Reaction

Ethanol C6H12O6 2CH3CH2OH + 2CO2 (R2.2)

Propionate C6H12O6 2CH3CH2COOH + 2H2O (R2.3)

Acetic acid C6H12O6 → 3CH3COOH (R2.4)

In acidogenesis stage, different anaerobic bacteria degrade the monomers into short-chain

organic acids, alcohols, H2S, H2 and CO2. The major reactions of acidogenesis are shown in

Table 2.2. The fermentative microorganism, such as Lactobacillus sp; Propionibacterium sp,

contributes a significant role in the reactions [43]. These microbes grow ten times faster than

methanogenic archaea, therefore acidogenesis is the fastest process in an AD. The optimum

value of pH for acidogenesis bacteria is the range of 5.2 and 6.5 [43].

Process Reaction Microbes

Carbohydrate Protein Lipid

Pseudomonas sp,

Clostridia, Micrococci

Hydrolysis

Acidogenesis

Sugars Amino acids Free long fatty

acids and glycerol Lactobacillus sp;

Propionibacterium sp

Volatile fatty

acids, alcohol Acetogenesis

Ammonia

Acetobacter sp;

Syntrobacter wolinii

Methanogenesis

Acetic acid Hydrogen, carbon

dioxide

Methane, carbon

dioxide

a Methanomicrobiales,

a Methanobacteriales

(b) (a)

9

Acetogenesis

In the acetogenesis step, the compounds formed are converted to acetate, formate, CO2 and H2

[30]. Acetogens, bacteria involving in acetogenesis, are slow growing, strictly anaerobic and

work optimally in pH around 6 [42]. Included in this group are Acetobacter sp and Syntrobacter

wolinii [42]. Common reactions in acetogenesis are listed in Table 2.3.

Table 2.3 Major reactions in acetogenesis [42]

Substrate Reaction

Acetate H3CH2COO- + 3H2O CH3COO- + H++ HCO3- + 3H2 (R2.5)

Glucose C6H12O6 + 2H2O 2CH3COOH + 2CO2 + 4H2 (R2.6)

Ethanol CH3CH2OH + 2H2O CH3COO- + 2H2 + H+ (R2.7)

Methanogenesis

In the methanogenesis phase, methanogens transform acetic acid and H2/CO2 to CH4 and CO2.

There are two major pathways for CH4, production namely hydrogenotrophic methanogenesis

and acetoclastic methanogenesis. The first pathway converts CO2 and H2 to CH4 in this

following equation:

CO2 + 4H2 → CH4 + 2H2O (R2.8)

Acetoclastic methanogenesis produces CH4 from acetate (R2.9).

CH3COOH → CH4 + CO2 (R2.9)

In the conventional AD operation, the four steps are conducted in a single reactor (Figure 2.2).

Therefore conventional AD is often referred to as a single phase AD (SPAD)[41]. The SPAD

converts prepared organic materials into methane-rich biogas. The biogas can be used as biofuel

10

after the purification [20, 46]. The remaining volatile fatty acids (VFA) and solids in the final

effluent are also generated [41].

Figure 2.2 Schematic diagrams of SPAD [41]

2.2.3 Drawbacks of conventional AD

Despite its effectivity in treating waste and producing biogas, previous studies report several

drawbacks of SPAD.

a. Sensitive to inhibitions of metabolic products

The overall operation of the AD is “occurred” in a single reactor [41]. Various biochemical

steps requiring different growing conditions are exposed to the same conditions. In the early

stages of SPAD, the accumulation of acid often decreases the pH, while the inhibitory level of

ammonia, long chain fatty acid and sulphide are increased significantly. These factors disrupt

the stability of the SPAD and may lead to reactor failure [47].

b. Slow rate of biological reactions

AD reactions run slowly, especially at a higher organic loading rate [41]. AD requires a longer

period to degrade high organic loading of feedstock. When the treating time is too short, AD

faces an imbalanced process [48]. A conventional AD treating industrial wastewater is reported

to be unfeasible at a loading rate of 2 gr VS.l-1.day-1 or higher and in the hydraulic retention

time 10 days or shorter.

11

c. Low-quality biogas

Table 2.4 Fuel Properties [4]

Fuels Energy density Heating value

(1 atm and 15C) (MJ/kg)

Flame speed (cm/s)

LPG 2.26 45.7 38.25

Natural gas 0.79 50 34

H2 0.08 120 275

Biogas 1.2 17 25

Biogas produced from SPAD has low energy density and the heating value and flame speed are

much lower than liquid petroleum gas (LPG), natural gas, and H2 (Table 2.4) [4]. Therefore, to

optimise the AD and improve biogas quality, innovative alternatives are required.

2.3 Two-phase anaerobic digestion (TPAD)

As an alternative to overcome the drawbacks of the SPAD, TPAD has been developed. TPAD

is a system proposed by Pohland and Ghosh in 1971 to treat high solid organic loading rate and

to produce higher CH4 [49]. TPAD employs separate stages for acidogenesis and

methanogenesis under different operating conditions. This section reviews the mechanism and

operating consideration.

2.3.1 Mechanisms and system of TPAD

Figure 2.3 shows TPAD system. Unlike SPAD, the two main reactions in the AD are separated

into two different vessels. The first phase of TPAD is also called as acidogenesis phase,

accommodates the hydrolysis, acidogenesis and acetogenesis which produce H2, CO2 and VFA.

Meanwhile, the second phase is also known as methanogenesis phase because the primary

reaction is CH4 generation which produces CH4 and CO2. General reactions of H2 and CH4

generation in TPAD are listed in Table 2.5. The Tables 2.2, 2.3 and 2.5 are meant to explain

12

the individual reaction that may occur during AD, although in the reality, the reactions are

complex and occur simultaneously.

Figure 2.3 Schematic diagrams of TPAD [41]

Table 2.5. General reactions of H2 and CH4 generation in TPAD [50]

Phase Reaction

First C6H12O6 + 2 H2O → 4 H2 + 2 CH3COOH +2 CO2 (R2.10)

Second 2 CH3COOH → 2 CH4 + 2 CO2 (R2.11)

TPAD allows the separation of acidogenesis and methanogenesis into two separate reactors,

based on their optimum operating conditions [51]. The independent control of both reactors

optimises the growth and activity of each group for bacteria involved in each step [7, 52].

Typically, the first phase is operated in a relatively shorter period (0.5-1.5 days) than the second

phase (≥ 3 days)[51]. It washes out the CH4-forming bacteria and removes the inhibitory effect

of CH4, thus higher production of H2 is achieved. The first reactor buffers the system by

avoiding a sudden pH drop caused by the accumulation of volatile fatty acid (VFA) which

typically hinders the methanogenesis phase and sometimes leads to reactor failure [49]. The

effluent from the first phase is then used as the feedstock for the second phase with more

suitable characteristics to enhance CH4 production. Therefore, the separation of the phases

improves the stability and resilience of the overall AD system [53].

13

The separation of both phases also improves the efficiency of solubilisation and saccharification

of the feedstock in the first phase of TPAD. The operation time is reduced significantly [52].

Ghosh et al. (1985) [48] proved that TPAD provides “superior performance” over the current

SPAD by comparing the two system in treating wastewater from the soft drink industry. In this

experiment, TPAD outweighs SPAD regarding CH4 production rate (seven times higher),

organic loading rates (OLR) (7.6 times higher) and hydraulic retention time (HRT) (almost a

half-time shorter).

The innovative aspect of the TPAD is the harvest of H2 from the first phase to be mixed with

the CH4 generated from the second phase to increase the overall quality of the biogas [41]. Liu

et al. (2013) state that the H2 present in biogas improves its fuel and heat efficiency by

enhancing flame speed, reducing combustion time and quenching distance, and extending the

flammability of CH4[52]. The H2 also increases the H/C ratio thus decreases greenhouse gas

(GHG) emission [52, 54]. Zhang et al. (2017) report that high efficiency and low emission can

be achieved when the biogas generated from TPAD was used in spark ignition (SI) engine [55].

Lee and Chung (2010) also evaluated that TPAD is more promising in recovering energy than

SPAD [56]. The energy recovery of TPAD outnumbers SPAD by around 18-20% [53, 57]. This

is mainly due to the flexibility in controlling the operation conditions of each reactor [57] and

the increased availability of readily available of VFA that enhances the CH4 production in the

second phase of TPAD [53]. TPAD also reduces ammonia inhibition thus reduces the gas

cleaning requirement [41, 58].

The TPAD principles can be applied in a remote area as an integrated system of TPAD, biogas

cleaning and conditioning (Figure 2.4). The food and agriculture waste are a potential feedstock

for TPAD to produce biogas for household and TPAD operation. The TPAD also produces an

increased nutrient availability in the effluent for organic fertiliser preparation for agricultural

application [41]. This scope of this thesis covers the study on TPAD operation, gas cleaning

unit and fertiliser preparation from TPAD effluent.

14

Figure 2.4 The TPAD utilisation for combined heat and power (CHP) system [41]

2.3.2 Factors influencing TPAD operation

Each phase of TPAD should be operated in appropriate conditions to produce an optimum

amount of biogas. This section elaborates factors affecting the operation of TPAD; including

nutrients, microbial sources, pH and temperature.

Nutrients

According to Weiland (2010) and Wellinger et al. (2013), biogas can be produced from any

biomass which is rich in carbohydrates, proteins, fats, cellulose and hemicellulose [58, 59].

These types of biomass are applicable as feedstock both for SPAD and TPAD. Wide ranges of

feedstock have been used in TPAD, such as food waste [56, 60], industrial wastewater [61-63],

animal manure [64] and agricultural waste [65-67].

The adequacy of nutrients in the feedstock should be evaluated to achieve a good productivity

of the AD. This evaluation of the gas potential of feedstock is suggested to be based on ratio

volatile solids (VS), Chemical Oxygen Demand (COD) or C/N ratio. The organic loading rate

(OLR) of the anaerobic system should be designed between 3.2 and 40 g VS.l-1.day-1[68, 69].

Some researchers suggested that ideal C/N ratio of feedstock is in the range of 15-32 [58, 59,

68]. In TPAD operation, the range of C/N for hydrolysis step is suggested to be 10-45 and for

CH4 production is 20-30 [59, 70].

15

In addition to macronutrients such as C, H, O and N, the micronutrients and trace elements such

as sodium (Na), magnesium (Mg), zinc (Zn), nickel (Ni) and iron (Fe) are also required by

microbes inside TPAD due to their significant role in enzyme synthesis and activity [71-74].

For example, Fe and Ni is reported to be important in hydrogenase synthesis for the hydrolysis

step [71, 74]. The addition of trace elements contained in additives such as granular activated

carbon, biochar and industrial Fe was reported to improve fatty acid consumption and biogas

production in AD [40, 72]. Other studies have claimed that in TPAD, the trace elements give a

more profound effect during methanogenesis than during acidogenesis [71, 75]. However, a

higher concentration of trace elements can cause AD inhibition. For example, it is reported that

the addition of more than 350 mg.L-1 of Na inhibited mesophilic methanogenesis. While the

optimum K addition is less than 400 mg.l-1 at both mesophilic and thermophilic AD [76].

Therefore, a study of dosage optimisation of different additives to enhance AD is crucial.

Microbes

The presence of microorganisms in TPAD is required to degrade feedstock and converts

nutrients into a valuable form of biogas and effluent with reduced solids and organic content.

There are two major groups of microbes involved in TPAD, namely hydrogenanses in the first

phase, and methanogens in the second phase [41]. The separation of the two groups of bacteria

is a central consideration in TPAD [41, 77]. Therefore the understanding of the characteristic

of each group of microorganisms is crucial [41].

The hydrogenases are also known as H2 producing bacteria (HPB). Clostridium and

Enterobacter are a common genera identified in H2 production [50, 78]. Clostridium is

characterised as rod-shaped, strictly anaerobic, and gram-positive. Enterobacter is also rod-

shaped, but facultative anaerobic and gram-negative [78].

Methanogens belong to the Archaea domain, which differs from bacteria by their genomic and

biological signatures [41, 77]. Methanogens are typically rod-shaped or coccoid-rod and strictly

16

anaerobic. Some methane producing microbes are H2 consuming and convert H2 to CH4

(hydrogenotrophic) [44, 77], while others are acetoclastic, producing CH4 from acetate [44, 45].

Ruggeri et al. (2015) suggest the principal difference between the HPB and H2 consuming

archaea [77]:

• HPB can grow in a broader pH range (4.5 - 7), while H2 consuming archaea have a more

limited pH range (7-8).

• The growth kinetics of HPB are faster than H2 consuming archaea.

• HPB can survive extreme conditions such as high temperature and extreme acidity or

alkalinity by forming endospores and germinating back in a favourable environment, while

H2 consuming archaea cannot.

Generally, two types of microbial culture are used in TPAD, namely pure culture and mixed

culture [41]. TPAD with pure culture only employs a single specific strain of microbes [50].

Mixed culture, on the other hand, consists of more than one group of bacteria which can be

found easily in the environment [41]. The mixed culture is typically obtained from AD sludge,

sewage sludge, wastewater treatment plant, compost and soil [41, 78]. The application of mixed

culture is often regarded to as more practical than pure culture as it is easier to operate and less

sensitive to changes on operating conditions. However, since mixed culture consists of H2

producing bacteria (HPB) and H2 consuming archaea, the first phase of TPAD requires selective

enrichment of HPB and the elimination of the H2 consuming archaea [78]. The methods to

selectively enrich the HPB include thermal treatment (heat shock at 80-110ºC for 20-60 mins),

use of chemicals (chloroform), aerobic stress, and kinetic selection (controlling pH, organic

loading rate /OLR and hydraulic retention time/HRT during the operation) [77, 78].

Several studies on TPAD used mixed culture obtained from different sources. The further

microbial studies identified the possible hydrogenases and methanogens involved in the first

17

and second phase of TPAD, respectively. Table 2.6 enlists the corresponding results of the

microbial identification studies.

Table 2.6 suggests the dominance of microbial groups mainly depends on the feedstock and

operating condition of AD. For example, the microbes from genus Lactobacillus are identified

in the first phase of TPAD treating food waste [79, 80]. Thermoanaerobacterium

thermosaccharolyticum, a typical thermophilic bacterium is enriched at the thermophilic first

phase [81]. The knowledge on the microbial profile of each stage of TPAD is useful for a better

process control to achieve optimum biogas production [82].

Several studies highlight low biogas production and process stability may be caused by the

sensitivity of microbes employed in AD [75, 83]. Reports suggest several methods to enrich

microbial growth and activity in AD, including through the molecular engineering of the

HPB[84] and the addition of microbial carrier to facilitate microbial immobilisation [83, 85].

Microbial immobilisation is reported to enhance H2 production rate and retain more microbes

in the reactor[84]. Studies on the suitability of additives for microbial immobilisation,

especially those of lower cost are required [83].

pH

An optimum pH is a crucial factor for AD operation [86] since it is directly correlated to

activities and metabolic pathways of the AD microorganism [41, 86]. During feedstock

degradation, acidic and alkaline compounds are produced. The equilibrium of these compounds

is a function of pH of the digester, therefore, the changes of these compounds will alter the pH

of the system [45].

18

Table 2.6 Hydrogenases and methanogens in typical inoculums

Feedstock Source of inoculum Operating conditions Identified microorganism Ref.

1st phase 2nd phase

Municipal

wastewater

AD sludge treating local

wastewater

Both reactors: Continuous

1st phase: pH 5.5/35ºC

2nd phase: pH 7.0/35ºC

Flavobacteriales, Clostriales Methanobacteriales,

Methanosarcinaceae

[82]

WAS and

OFMSW*)

Wastewater treatment

plant sludge

Both reactors: Semicontinuous

1st phase: pH: NA/55ºC

2nd phase: pH: NA/55ºC

Clostridium sp. Methanosaeta [87]

Food recycling

wastewater

AD sludge Both reactors: Continuous

1st phase: pH: no control/ 35ºC

2nd phase: pH: no control / 35ºC

Lactobacillus acetotolerans-

and Lactobacillus kefiri-like

organisms

Methanosarcina-like

organisms

[79]

Brown water

and food waste

Mesophilic wastewater

treatment plant sludge

Both reactors: Continuous

1st phase: pH: no control/ 35ºC

2nd phase: pH: no control / 35ºC

Firmicutes, Lactobacillus.

amylovorus and Acetobacter

peroxydans

Methanosaeta [80]

Palm oil mill

effluent (90ºC)

AD treating palm oil

waste sludge

Both reactors: batch

1st phase: pH: 5.5/ 55ºC

2nd phase: pH: 7.5 / 28-34ºC

Thermoanaerobacterium

thermosaccharolyticum

Methanoculleus

sp.

[88]

*) WAS and OFMSW: Waste activated sludge and organic fraction of municipal solid waste

19

Generally, pH stability of CH4 phase (6.7-7.4) is narrower than H2 phase (6.5-9.0) [89].

However, Cooney et al. [7] stated that the H2 phase is typically operated at lower pH (5-6). And

although it is common that methanogens are active between pH 6.7 to 7.4, some studies have

reported methanogenic activity at pH 5.8 [82, 90]. The inconsistency of the suggested optimum

pH has led to several studies to understand the effect of pH and determine the optimum pH in

the AD/TPAD using different feedstock (Table 2.7).

Table 2.7 Studies on pH Effect on Anaerobic Digestion

Feedstock AD System pH range Optimum pH Ref.

Glucose SPAD (H2) 4.00-7.00 5.50 [91]

Brewery wastewater SPAD (H2) 5.00-7.00 5.50 [92]

Food waste SPAD (H2) 4.70-7.00 5.30 [93]

Sugarcane bagasse hydrolysate SPAD (H2) 4.50-9.00 6.50 [94]

Swine manure and maize stalk SPAD (CH4) 6.00-8.00 6.81 [86]

Synthetic feed media TPAD 1st: 4.50-6.50

2nd: 6.00-7.70

1st: 5.50

2nd: 6.80

[95]

Kitchen waste TPAD 1st: 5.00-11.00 1st: 7.00 [96]

In conclusion, it seems that the optimum pH for H2 and CH4 production of the previous studies

were feedstock dependant. Moreover, it is also suggested by Ruggeri et al. (2015) that there is

no definite optimum pH value in fermentation due to other factors and environmental conditions

during the process, including the characteristics of feedstock [77]. In order to maintain the pH

of each reactor, several studies have applied methods such as water recirculation and the

addition of the alkaline additives (NaOH, lime, biochar) are often applied [41, 97, 98]. More

studies are required to investigate the efficiency of the methods to adjust the pH of the system

to enhance biogas production with minimum operation cost.

20

Temperature

Table 2.8 Optimum temperature range for microbial growth [45, 99-101]

Phase of AD Temperature

range

Genus Optimal temperature

(°C)

Acidogenesis Psycrophilic Rahnella 20

Mesophilic Bacillus 35

Thermotogales 35

Thermophilic Thermoanaerobacterium 60

Desulfotomaculum 55

Methanogenesis Psychrophilic Methanogenium 20

Mesophilic Methanococcus 35-40

Methanococcoides 30-35

Thermophilic Methanohalobium 50-55

Methanosarcina 50-55

Similar to pH, temperature is also found to be an essential factor in AD operation due to its

effect on the essential enzyme activity of bacteria. Generally, there are three temperature

regimes in fermentation operation, namely psychrophilic (5-25C), mesophilic (30-40C),

thermophilic (50-60C) and hyperthermophiles operation (> 65C). However, the mesophilic

and thermophilic operation is more common in AD operation [45, 77]. Normally, the first phase

is operated at thermophilic condition for enhanced substrate degradation while the second phase

is operated under mesophilic condition for higher CH4 production [41]. Higher temperature

may be beneficial for the reaction kinetics, but it is typically followed by a rapid pH decrease

which inhibits especially H2 production [102]. Appropriate temperature allows optimum

germination, acclimatisation of bacteria to substrate used in the system, carbon consumption

rate and partial pressure of the produced gas [41, 77]. Optimum temperatures for microbial

growth in H2 and CH4 production are listed in Table 2.8.

In TPAD operation, various results on temperature effect on system performance were found.

Using corn straw and pig manure in mesophilic and thermophilic condition, Yang et al. (2014)

21

proposed that mesophilic system is better than thermophilic TPAD because of the less energy

requirement with similar biogas production [103]. On the contrary, Kim et al. (2002) [104]

found that TPAD operation in both mesophilic and thermophilic temperature has no impact in

improving the performance of both systems. An interesting result was reported by Parawira et

al. (2007) [105]. When TPAD operated in mesophilic temperature, higher CH4 yield was

reached, while at the thermophilic conditions, shorter HRT and OLR were achieved. Therefore,

this study suggested operating TPAD according to the final aim of the system. If the higher CH4

yield is the purpose, the mesophilic condition is preferable, while the thermophilic condition is

appropriate for shorter digestion period and higher organic loading rate. The variation of the

optimum temperature may be caused by the origin of the inoculum and the type of feedstock

used [102].

To sum up, it is well known that there is different optimum temperature for H2 producing

bacteria and CH4 producing archaea. However, there are conflicting results on the optimum

temperature of each phase of TPAD treating different feedstock. Therefore, it is suggested to

investigate the effect of temperature on the TPAD operation especially when the study is

conducted using different feedstock.

2.4 Biochar

The existing literature suggests the need to improve the performance and biogas yields of

TPAD. Supplementation of micronutrients and trace elements is required to enhance enzyme

activation and improve microbial activity [106, 107]. The initiation of microbial colonisation

using supportive materials is also thought to enrich and increase the robustness of the microbial

groups under different operating conditions [83]. The addition of neutraliser materials to buffer

the pH of TPAD at the favourable range is needed [98]. One possible solution to address the

above-mentioned requirements is by utilising biochar in TPAD operation.

22

Biochar, also known as charcoal, is a carbon-rich solid residue produced from biomass

pyrolysis. A typical pyrolysis heats organic materials as feedstock at between 300 and 800°C

under very low or preferably no oxygen conditions [108, 109]. The process yields biochar, bio

oil and syngas [110]. In comparison with activated carbon (AC), biochar is more cost effective

[3, 111]. Unlike AC, biochar is neither pyrolysed at a very high temperature nor activated using

steam or carbon dioxide (CO2). Thus energy consumption and operation cost are lower than

that of AC [109]. Also, biochar is generally produced from low-cost and abundant materials

such as agricultural waste, sewage sludge, solid waste and animal droppings and wood chips

[108, 111].

It is well known that the origin and the operating condition of pyrolysis affect the physical,

chemical and biological characteristics of the biochar [27, 108, 112-114] (Figure 2.5).

2.4.1 Physical characteristics

The important physical characteristics of biochar are specific surface area (SSA) and porosity.

These characteristics are affected by different feedstock and operating condition of pyrolysis

[113, 115]. During the pyrolysis, the C mass is removed while the pores in material is created,

influencing the surface area of the biochar [27, 116]. The surface area of the wood increased to

ten to several hundreds of m2.g-1 after the pyrolysis [27]. In general, the structure of feedstock

also plays a role. For example, under the same highest treatment temperature (HTT), the biochar

produced from sewage sludge has lower SSA (38 m2.g-1) than that from sawdust (511 m2.g-1)

[113]. It implies that the lignin-rich biomass is an ideal feedstock to derive the biochar with

high SSA [111, 113].

Larger SSA reflects the higher porosity in the biochar [115]. The pores contribute to the high

adsorptive capacity of biochar for the small molecules such as common solvent and gases [27,

111, 117]. In addition, biochar offers suitable dimension, increased surface area and porosity

as an ideal habitat for bacteria, archaea and fungi [27, 111]. It is also suggested that the porosity

23

and structure of biochar may interact with soil structure therefore directly impacts the soil

structure [27].

Biochar

Physical

- high specific

surface area

- high porosity

- bulk density

CharacteristicsPossible

applications

Anaerobic

digestion

Biogas cleaning

Fertiliser

Possible

functions

Liming agent

Adsorbent

Microbial carrier

Micronutrient

additives

Chemical

- surface

functional groups

- high alkalinity

Biological

- provision of

nutrients

- high water

holding capacity

Soil conditioner

Figure 2.5 The characteristics, possible functions and applications of biochar

A related physical property of biochar is bulk density. Biochar produced from woody biomass

has 0.30 to 0.43 g.m-3 bulk density [27, 118]. It is in an ideal range of bulk density for gas

adsorption, which is suggested in between 0.40 and 0.50 g.cm-3 [27, 119].

2.4.2 Chemical characteristics

Pyrolysis alters the elemental compositions of the raw materials. Generally, after being charred,

the carbon (C) composition increases, while hydrogen (H), nitrogen (N) and oxygen (O)

decrease [111]. Changes in the surface chemical compositions also occur [111]. An abundance

of oxygen-containing functional groups, such as OH, COO and C=O in rice hull biochar

prepared at 400ºC has been reported [19]. At temperature higher than 600ºC, neutral or basic

aromatic groups were formed [109]. The formation of more functional groups (such as

24

carbonyl, carboxylate and hydroxyl) may contribute to the higher cation exchange capacity

(CEC) of biochar [111]. The CEC reflects the total capacity of biochar in adsorbing and

exchanging species with positive charge [114]. Various functional groups and surface charge

of biochar suggest a potential utilisation of biochar as an adsorbent [111]. However, further

study using various sorbates, compounds to be adsorbed, under different operating conditions

are required to understand the sorption behaviour of different types of biochar [27, 111].

The pH of biochar is strongly influenced by the organic functional group and ash content [113,

114]. Biochar produced from slow pyrolysis is alkaline, while most hydrochar produced at a

lower temperature of pyrolysis is slightly acidic [111, 115]. It is believed that the higher HTT

leads to more oxygenated functional groups being consumed and/or deprotonated to the

conjugate bases [114]. The addition of biochar into deionised water increases the pH of the

solution due to the leaching of alkali salts released from feedstock during the pyrolysis [111].

In an AD system, the alkalinity of biochar is believed to buffer the pH of the culture, improve

microbial activity and biogas production [98]. Biochar also adjusts the pH of soil when it is

used for soil amendment [27, 115]. In addition, the alkalinity of biochar also plays a role in gas

adsorption, especially for the removal of acid gases, such as H2S [120]. For example, in a study

conducted by Shang et al (2013), rice hull biochar with the highest pH value (10.6) performed

the highest H2S removal [19]. It is suggested that the removal of H2S is governed by the local

pH in the pore system. Alkaline pH is reported to enhance the dissociation of H2S and its

oxidation to sulphur [19].

2.4.3 Biological properties

Biochar provides an ideal habitat for bacteria, archaea and fungi because of its high surface area

[27]. The high porosity of biochar also allows the biochar to retain moisture. A study by

Pietikainen et al. (2000) suggests that biochar produced from humus and wood has a higher

water holding capacity (WHC) (2.9 ml.gr-1) than AC (1.5 ml.gr-1) and pumice (1 ml.gr-1) [27,

121]. The increased amount of water retained by the biochar and soil may improve the

25

habitability for soil microorganisms. An increase in WHC of biochar improves the overall soil

WHC when it is applied as a soil amendment agent. These characteristics are important for

utilisation of biochar in biological applications, such as fertiliser and as a microbial carrier.

In addition, remaining bio-oils and volatile matters, including water-soluble compounds such

as sugars, alcohols, acids, aldehydes, and ketones, in the biochar surface after pyrolysis may be

utilised by microbes. Producing biochar at the lower temperature is reported to be better for

retaining nutrients for microbes [3, 113]. Some nutrients such as K, P, Na, Mg and Ca also

remained [114]. The provision of the macro and micronutrients in the biochar are expected to

be useful for enhancing the biogas production and supporting plant growth when biochar is

used for organic fertiliser [29, 114, 122].

To sum up, biochar has beneficial physicochemical and biological characteristics that may be

useful for applications in AD, biogas cleaning and soil amendment [20, 28, 108, 123] (Figure

2.5). However, some compounds associated with biochar like polycyclic aromatic hydrocarbon

(PAH), formaldehyde and other toxic compounds may be inhibitive for microorganisms.

Therefore, detailed studies on the utilisation of biochar on the above mentioned applications

are required [111].

2.5 The effect of biochar in the AD system

Biochar with its aforementioned beneficial characteristics for a wide range of applications

therefore becomes an innovative aspect of this study. This thesis investigates the utilisation of

biochar in the (1) TPAD, both bench and demonstration scale, (2) the gas cleaning and (3)

preparation of fertiliser from the TPAD effluent (see Figure 2.3). This section reviews the

existing literature on the utilisation of biochar on these three applications.

2.5.1 Biochar application in biogas production

This section reviews the existing studies on biochar utilisation in AD and the possible

mechanisms by which it enhances biogas production. SSA, nutrient content and pH of biochar

26

has been proposed as useful for H2 production via an AD [3, 113]. It is also reported that biochar

addition in single phase AD prevents the system from mild ammonia inhibition and improves

CH4 yield by 32% [3]. Several studies investigated the effect of biochar on H2 or CH4

production via AD (Table 2.8).

As can be seen from Table 2.9, more studies of the biochar addition were conducted for CH4

production, while only a few studies have investigated the H2 production and TPAD operation.

The existing literature suggests possible mechanisms for the role of biochar in improving AD,

although some studies state that the mechanisms are still unclear. The possible mechanisms by

which biochar in enhances H2 and CH4 are elaborated as follows.

1. Bacterial immobilisation

Bacterial immobilisation is an important strategy in the AD. Bacterial immobilisation is the

attachment of bacteria to the surface of the solid material to establish microbial colonisation. In

the context of bacterial immobilisation, additives are often referred to as supporting material or

microbial carrier. Bacterial immobilisation benefits AD in two ways. Firstly, a microbial carrier

provides an increased available surface for microbial growth. The increased surface allows

more intense cross feeding, co-metabolism and H2 and proton transfer, which then enriches

microbial growth and initiates microbial colonisation [124]. The enriched microbial population

has a greater potential for a higher biogas production. Secondly, microbial immobilisation

increases the robustness of the AD system. With microbes attached to a supporting material,

the AD system has a lower probability of experiencing microbial washout, where the system

cannot retain the microbes when the AD effluent is discharged. The use of a microbial carrier

will better maintain microbes inside the reactor thus improving the robustness of AD. This then

allows the system to operate with shorter hydraulic retention time (HRT) and higher organic

loading rate (OLR) [125].

27

One natural process of bacterial immobilisation is biofilm formation which allows the

colonisation of bacteria, fungi and protozoa on polymerised solid surfaces [123, 126]. The

biofilm formation rate depends on the characteristics of the microbial carrier, including pore

size, SSA, porosity and surface roughness [125, 127].

Luo et al. studied the effect of particle size (75-150 µm, 0.5-1 mm and 2-5 mm) of fruitwoods

biochar on the CH4 production from synthetic wastewater. The result shows that biochar

addition enriched Archea, achieving a higher amount than that of control without biochar.

Biofilm formation is known to depend on the particle size. Biochar selectively enriches the

Archea by tightly binding Methanosarcina in the larger biochar particles, loosely binding

Methanosaeta in all particle sizes and tightly binding it in smaller particle sizes [83].

28

Table 2.9 Existing studies on the biochar utilisation in H2 and CH4 production via AD

System Feedstock Type of biochar Optimum biochar

addition (g.L-1)

Operating

conditions

H2 yield

(mL.g VS-1)

CH4 yield

(mL.g VS-1)

Ref.

SPAD (H2) Glucose Corn bran

residue

0.6 Batch 37ºC

Initial pH: 7

204 mg H2.g

glucose-1)

- [73]

SPAD (H2) OFMSW Woody mass 12.5 Batch 37ºC

Initial pH: 5.5

80 - [128]

SPAD (H2) DAS* and food

waste

Saw dust 10 Batch 35ºC

Initial pH: 5.5

81 - [113]

SPAD (CH4) AD sludge Paper sludge

and wheat husk

20 Batch 42ºC

Initial pH: N.A*

- 158 [3]

SPAD (CH4) Glucose Fruit woods 10 Batch 35ºC

Initial pH: 7

- 13.7 mmol CH4.g

glucose-1

[83]

SPAD (CH4) Glucose + 7 g-

N.L-1

Fruit woods 10 Batch 35ºC

Initial pH: 7

- 16.7 mmol CH4.g

glucose-1

[129]

SPAD (CH4) Citrus peel Coconut shell N.A Batch 35ºC

Initial pH: 7

- 186 [130]

SPAD (CH4) Food waste Fruit woods 5 Batch 35ºC

Initial pH: 7

- 500 [131]

SPAD (CH4) OFMSW Rice straw 5 Batch 35ºC

Initial pH: N.A*

- 167 [132]

SPAD (CH4) Kitchen waste Vermi-compost 50 Batch 35ºC - 175±5 [98]

29

System Feedstock Type of biochar Optimum biochar

addition (g.L-1)

Operating

conditions

H2 yield

(mL.g VS-1)

CH4 yield

(mL.g VS-1)

Ref.

Initial pH: 6.5

SPAD (CH4) Dairy manure Dairy manure 10 Batch 35ºC

Initial pH: 7.7

- 467 [133]

TPAD (CH4) Primary sludge

and WAS

Corn stover 0.25 – 1 g.day-1 Semicontinuous

Batch 55ºC

1st phase: 6.5

2nd phase:

uncontrolled

- 340 [134]

*DAS = dewatered activated sludge

NA = not available

30

Sharma et al (2017) also reported that woody mass-biochar initiated biofilm formation in H2

production from OFMSW using co-culture of Enterobacter aerogenes and Escheria coli [128].

It contributed to the shorter lag phase, waiting period for the system to produce H2, by 25%

compared to control and improved H2 production rate [128].

2. Source of macro- and micro-nutrients

Biochar contains a stable and labile fraction of carbon. Previous studies report that the presence

of a labile fraction of biochar can be used by AD microbes to kick-start the production of biogas

[3]. Especially in the biochar produced at the low temperature (~200°C), often referred to as

hydrochar, there is the readily digestible carbon such as sugar and volatile matters remaining

after the pyrolysis. This portion of carbon is reported to be useful for CH4 production and

improved the production by 32% compared with the control [3]. Jang et al. (2018) reported that

the nutrients in dairy manure-derived biochar (Ca ~ 9.1%; Mg 3.6%; N ~ 1.3% and P ~ 0.14%)

supported the CH4 production of dairy manure at three different temperature regimes tested,

namely psychrophilic, mesophilic and thermophilic condition by 28, 32 and 36% compared to

the control [133].

3. Buffer the pH of the AD system

H2 production is a pH-dependent process. The pH influences the microbial activity and

activation of H2 producing-enzymes. The addition of biochar into the AD system is believed to

buffer the pH of the culture, enhancing the production of biogas. Zhang et al. (2017) reported

the buffering effect of biochar in AD to produce H2. The study used biochar prepared from corn

barn residue (pH 8.92) to produce H2 using glucose as the substrate [73]. In the study, the culture

containing biochar at 0.6 g.L-1 produced higher H2 than the control by 29% with a maintained

higher final pH than the control.

31

Wang et al. (2018) also studied the effects of different feedstock and preparation temperature

of biochar on H2 production. The results suggested that among other factors, the pH buffering

capacity served as the primary factor in improving H2 production to mitigate the pH drop as a

result of VFA accumulation during the process. Biochar contains alkaline compounds (-COOH

and -OH) that may neutralise and act as a buffer when the culture’s pH drops as a result of VFA

accumulation during the AD process [123]. The buffered pH provides a better environment for

microbial growth and activity thus allowing the culture to produce higher H2 [73, 113].

Using a type of biochar derived from vermicompost (VCBC), Wang et al. (2017) investigated

the capacity of biochar in buffering the pH and alleviating the adverse effect of acid

accumulation in the AD system producing CH4 [98]. The results show that the addition of

VCBC improved the buffering capacity to acetic, butyric, propionic and valeric acid. The metal

alkaline content of VCBC (Na and K) and alkaline-earth metals (Ca and Mg) are proposed to

be responsible for the improved buffering capacity, following equation (R2.12).

Ca(Mg)CO3 + CxHyCOOH [CxHyCOO]2Ca(Mg) + H20 + CO2 (R2.12)

The addition of VCBC supported the culture in SPAD with a high loading of chicken manure

(CM) (50 g TS.kg-1) by initiating the biogas production 10 days faster and preventing the system

from rapid pH drop during the operation [98].

On the other hand, Luo et al. (2015) claimed that the role of buffering capacity of biochar is

insignificant in a CH4 production system [83]. Instead, the porosity of biochar which served as

a microbial carrier is thought to be the central role of biochar in CH4 production [83]. Therefore,

it is believed that biochar enhances the H2 and CH4 production through a different mechanism.

Thus, additional experiments using different feedstock, biochar type and operating conditions

are required to investigate the possible mechanisms of biochar effects in AD.

32

4. Adsorption of inhibitors

There is an indication that carbonaceous materials, such as biochar, have a potential to be used

as an adsorbent to remove contaminants [123]. The pores in biochar exhibit an adsorptive

capacity to remove compounds, such as ammonium [128]. Biochar is suggested to adsorb both

ionic and organic compounds by electrostatic forces and Van der Waals forces, respectively.

These mechanisms are similar to those of AC and zeolite, however, biochar is relatively cheaper

than these additives [123, 128].

Sharma et al. (2017) studied the effect of biochar addition on the different addition of

ammonium to investigate the ability of biochar to adsorb ammonium in AD producing H2 from

the organic fraction of municipal solid waste (OFMSW)[128]. At a low concentration, ammonia

is required by microbes for microbial growth; however, it may be harmful at a higher

concentration. The results show that the addition of biochar (2.5 – 35 g.L-1) in the system

mitigated the ammonium inhibition which allowed the culture to produce a higher amount of

H2 than the control by up to four-fold. It is believed that the ability of biochar to adsorb

ammonium ions from matrices which accounts for the improved performance of the culture

with biochar addition [128]. Biochar also has a significant effect in mitigating ammonium

inhibition in a CH4 producing reactor. Mumme et al. (2014) report that the addition of biochar

mitigated the mild ammonia inhibition (up to 500 mg N.kg-1) [3].

2.5.2 Biochar application in pilot scale operation of TPAD

A study on the pilot scale operation of TPAD is necessary to confirm the results of the

bench/laboratory studies [135] and to investigate the technical challenges and the related trouble

shooting on a real full-scale application [56]. An initial study has been done in a demonstration

scale unit developed by the Centre for Energy of the University of Western Australia. The unit

consists of two reactors; the first reactor is an H2 producing reactor with the capacity of 150 L,

while the second reactor accommodates CH4 generation with 500 L of total volume. Prior to

the operation, commissioning and trial runs were conducted [41]. Several studies also focused

33

on the pilot scale operation of co-production of H2 and CH4 via TPAD using different types of

feedstock (Table 2.10).

Most of the studies were conducted in a range of 0.2 – 5 m3 capacity for H2 production and

larger reactor capacity for CH4 production phase (0.76 – 50 m3). Some studies focused on the

sole CH4 production [135-137], while others also took H2 production into account depending

on the scope of the study.

Despite the success in some pilot scale trials, several technical aspects are required to be

considered before and during the operation of the pilot scale TPAD. Cavinato et al. (2012)

reported that the short HRT and high OLR were effective in eliminating the methanogens from

the first phase reactor [138]. However, too high OLR may lead to the reactor shock load and

microbial wash out [85]. In this condition, it is advisable to allow some period for the adaptation

to the increased OLR, achieving a normal H2 production [56]. In some cases, the pH of the

culture of each reactor has to be automatically adjusted at the optimum pH, being at 5.5 and 7.0

-7.5 in the first and second phase, respectively [56]. Effluent recirculation from the second

reactor into the first reactor was also suggested as an alternative to increasing the pH of the first

reactor [138]. However, the methanogenic contamination and ammonium accumulation will be

the consequences [97]. Therefore, other solutions are required. The addition of biochar into the

system is one alternative to overcome the problems.

34

Table 2.10 Studies on the pilot scale operation of TPAD

Feedstock Operating conditions Gas production

Ref. 1st reactor 2nd reactor H2 CH4

Kitchen waste and

fruit/vegetable

waste

• V: 2.0 m3

• pH: N.A.

• T: 35ºC

• OLR: 2 – 10 g (VS)/L/day

• HRT: 10 days

• V: 4.0 m3

• pH: N.A.

• T: 35ºC

• OLR: 1 – 5 g (VS)/L/day

• HRT: 20 days

• Composition (%):

N.A

• Yield:

N.A


65 - 67

• Yield: 0.46 – 0.64 L/g

VS

[135]

Solid municipal

waste

• V: 0.5 m3

• pH: 5.3±0.2

• T: 33±4ºC

• OLR: 12.3 – 71.3 g-

COD/L/h

• HRT: 21 – 66 hours

• V: 2.3 m3

• pH: 7.4±0.3

• T: 36±4ºC

• OLR: 2.7 – 6.4 g

(VS)/L/day

• HRT: 3.9 – 6.4 days


60

• Yield:

1.8 – 2.2 L/L/day


60

• Yield:

4.6 – 5.4 L/L/day

[56]

Biowaste • V: 0.2 m3

• pH: 3.5 – 5.4

• T: 55ºC

• OLR: 16 – 21 g (VS)/L/day

• HRT: 3.3 – 6.6 days

• V: 0.76 m3

• pH: 7.6 – 8.2

• T: 55ºC

• OLR: 4 – 10 g (VS)/L/day

• HRT: 12.6 days


19 - 37

• Yield:

3 - 51 L/kg VS


60 - 65

• Yield:

377 - 410 L/kg VS

[139]

35

Feedstock Operating conditions Gas production

Ref. 1st reactor 2nd reactor H2 CH4

Food waste • V: 0.2 m3

• pH: 5.7±0.3

• T: 55ºC

• OLR: 16.8 g (TVS)/L/day

• HRT: 3.3 days

• V: 0.76 m3

• pH: 7.6 – 8.2

• T: 55ºC

• OLR: 1.3 – 4.8 g

(TVS)/L/day

• HRT: 12.6 days


38.5±9.7

• Yield:

0.067 m3/kg VS


67±3.7

• Yield:

0.48 m3/kg VS

[138]


• pH: 4.6±0.3

• T: 55ºC


• HRT: 20 days

• V: 0.76 m3

• pH: 8.0±0.1

• T: 55ºC


• HRT: 20 days


N.A

• Yield:

N.A


55.2

• Yield:

0.55 m3/kg VS

[137]

Food waste

leachate

• V: 5.5 m3

• pH:

• T: 38.7 – 42.8 ºC

• OLR: 2.18 – 2.45

• HRT: 3 days

• V: 50 m3

• pH: 7.3

• T: 35.6 – 37.7 ºC

• OLR: 2.36 kg VS/m3/day

• HRT: 27 days


N.A

• Yield:

N.A


57 - 65

• Yield:

0.39 – 0.85 Nm3/kg

VSremoved

[136]

36

As explained, alkalinity of biochar contributes in buffering the pH of the system and alleviating

the effect of VFA accumulation as a result of high OLR and short HRT [98, 113]. Also it acts

as a microbial carrier which immobilises the microbes and prevents microbial wash out [83, 85,

140].

Despite growing interest on biochar utilisation in AD, the study on biochar addition in

demonstration or full-scale operation is rare. Cooney et al. (2015) investigated the start-up

period of CH4 production using 1.5 m3 demonstration scale of SPAD with the addition of

biochar. A successful start-up was achieved after 59 days, showing a COD removal of 68% and

CH4 composition of more than 60%. The AD almost achieved the maximum value of theoretical

methane production per kilogram of COD reduced [140]. There has been limited investigation

on the application of biochar in a biogas demonstration scale unit, so more studies are required.

In particular, a study of biochar application in a demonstration scale TPAD has not been

explored to date.

2.5.3 Organic fertiliser production from AD effluent and biochar

At the end of the AD process, a final effluent is generated. AD effluent (also called as digestate,

biogas residues, or biogas slurry) is an attractive material for soil improvement and restoration

due to its organic materials content [141]. This effluent contains nutrients useful for plant

growth [22, 23]. Generally, AD effluent contains 5-7% N, 30-50% P and 70-100% K required

in the first year of the growth of pasture plant [142]. Therefore, AD effluent can be used as an

organic fertiliser to improve soil fertility [143]. Several studies have investigated the utilisation

of AD effluent as fertiliser. Alburquerque et al. (2012) found that two types of AD effluent

produced from (1) combined pig manure and slaughterhouse waste effluent and (2) combined

cattle manure and maize-oat silage effluent positively affected lettuce seed germination. It can

37

be categorised to have a growth stimulant attributes as they exceeded a threshold value of 125%

of germination index (GI) (of the control) [144].

Similarly, biochar also has many beneficial characteristics, for example, high SSA, ability to

improve pH, high WHC, and good affinity for micro and macronutrients for plant growth [29,

122]. These characteristics enable biochar to impact soil fertility by increasing the content of

carbon (C), nitrogen (N) and aggregate stability as well as providing a beneficial environment

for microbes and bioremediation of soil. Also, biochar has a relatively lower cost than other

materials, estimated being 10 times cheaper than AC (2 USD/kg) [141]. Solaiman et al. (2013)

reported a positive effect of adding appropriate biochar on germination and early growth of

several plant seeds. The study used a soil-less petri-dish assay to investigate the potential

toxicity of lignin based-biochar (Oil Mallee, Rice husk, New Jarrah, Old Jarrah, Wheat Chaff)

on wheat, mung bean and subterranean clover seed germination. All biochar addition generally

increased the root length especially in the additional dose of 10, 20 and 50 t/ha. At the 100 t/ha,

biochar addition showed adverse or no significant effect on the root length. It is probably due

to the inhibitive effect of trace elements in the biochar when it is applied above the acceptable

agronomic rate [28].

Another positive effect of biochar on plant growth is also reported by Zhang et al. (2017). In

the study, sulphur-enriched AD-sludge biochar (SulfaChar) generated from biogas cleaning unit

was used. Compared to the control (synthetic S fertiliser), sulphur- enriched biochar achieved

a marked increase of plant biomass ranging from 31 – 49% in Zea mays L after 90-day of

greenhouse study. The study proposed that the SulfaChar may supply the macro (N,

phosphorus/P, potassium/K, calcium/Ca and magnesium/Mg) and micronutrients (zinc/Zn,

manganese/Mn and boron/B) or promoted the uptake of those nutrients [145].

Combining AD effluent and biochar for organic fertiliser may improve their fertiliser properties

and reduce the nutrient leaching from AD effluent [141, 146]. A willow biochar is proven to

38

reduce the P and K leaching from the soil, reduce the toxicity of sewage sludge and simulate

the growth of the tested organisms. The fertilising characteristic is shown by an increased root

growth of grass cress (L. sativum) when the rate of biochar addition increased [147].

On the other hand, Sun et al. (2014) reported that biochar (hickory wood, bagasse and bamboo

prepared at 200, 300, 450 and 600ºC) had an insignificant effect on the germination of brown

top millet seed. The hickory wood hydrochar (prepared at 200ºC) even had a lower germination

rate (45%) compared to the control (78%). It is estimated that the acidic pH of the hydrochar

(pH 5.3) inhibited the seed generation. Note that hydrochar is produced at a lower temperature

of pyrolysis [113]. The biochar dose and plant species were also believed to influence the result

[115]. Cardelli et al. (2018) also reported the fertiliser prepared from AD effluent and biochar

limited the amount and activity of the microbial in the soil, probably because the biochar

reduced the soluble organic compounds (dissolved organic C - DOC and phenols)[148].

In conclusion, the results of studies on the utilisation of AD effluent and biochar for fertiliser

are varied. Therefore, further studies are required.

2.5.4 Biochar application in biogas cleaning

During the AD, trace gases are produced during the H2 and CH4 production. These biogas

impurities, which generally consist of CO2, water vapour, hydrogen sulphide (H2S) and

ammonia (NH3) [149] limit the utilisation of CH4-H2 mixture as biofuel. For example, the water,

H2S and NH3 lead to the corrosion of utilities and CO2 caused a decrease in biogas caloric

content [19, 149, 150]. The types, quantity in biogas and possible effects of each impurity are

listed in Table 2.11.

The removal of H2S from biogas must be undertaken to achieve the H2S standard limit for safety

and fuel application concerns. Safe work Australia suggested 10 and 15 ppm as the 8 hours

average and short-term exposure limits for H2S, respectively. Biogas-fuelled generator engines

39

can tolerate to 200 ppm of H2S, while the H2S concentration limit for biogas boiler is 1000 ppm

[151]. The presence of NH3, which is typically generated in biogas from the nitrogen-containing

feedstock, can vary from a few hundred to 30,000 ppm. The NH3 may provoke NOx emissions

when the biogas is used as fuel in turbines, gas engines and burners [46]. Therefore, before the

utilisation of biogas as a fuel for engine operation requires the gas impurities to be removed.

Table 2.11 Biogas impurities [149, 152, 153]

Component Composition in

Biogas

Possible Effects

CO2 15-60% - Decreases caloric value

- If the gas is wet, it causes corrosion

H2S 0-2% - Provokes corrosion

- Generates SO2 emission in case of imperfect

combustion

- Spoils catalysts

NH3 <1% - Causes corrosion when dissolved in water

- NOx emission

- Increases anti-knock properties of engines

Water vapour 1-10% - Causes corrosion of facilities

- Causes water condensation

- Rises risk of freezing of piping and nozzles

Dust >5 µm - Interfere nozzles

N2 0-5% by volume - Decrease calorific value

- Improve engine’s anti-knock properties

Siloxanes 0-50 mg Nm-3 - Causes engine failure

There are several well-known methods to remove H2S from biogas. Among them are

adsorption, wet scrubbing, absorption with liquids, membrane separation, selective catalytic

oxidation and biological filtration. While for removing NH3, washing using diluted nitric and

sulfuric acid is a common method. Each of the methods has its own merits and limitations.

Chemical processes may generate hazardous compounds while biological processes can be slow

and more sensitive to operating conditions. Therefore, physical treatment such as adsorption is

40

an alternative method. The utilisation of a gas adsorption unit filled with carbon-based materials

is also suggested to remove both H2S and NH3 [21]. The replacement rather than regeneration

of these materials is suggested because of its their low cost and possible utilisation to prepare

sulphur-rich fertiliser [145, 149].

One promising material that can be used in biogas impurities removal is biochar [18]. Biochar

has been extensively used in the study of the removal of single stream biogas impurities such

as H2S both during [3] and after anaerobic digestion [18-20]. Biochar has an excellent adsorbing

ability to remove organic contaminants from soil and is also reported to be a potential gas

adsorbent [19, 20, 145].

Biochar is known to have strong alkalinity buffering ability that is favourable in removing

biogas impurities, particularly H2S [18]. The H2S is an acidic gas, therefore it is easier for H2S

to be adsorbed when it is in contact with an alkaline surface [20]. Xu et al., (2014) investigated

swine manure and sewage sludge-based biochar utilisation to remove H2S. This study proposes

a mechanism of H2S removal by biochar as follow:

H2S(gas) → H2S(ads) → H2S(ads-liq) (R2.13)

H2S(ads-liq) → OH- → HS-(ads) + H2O (R2.14)

HS-(ads) + O2 → S0 (R2.15)

HS-(ads) + O2 + H2O → SO4

2- (R2.16)

It is suggested that the caustic presence in the gas cleaning system catalyses the oxidation of

H2S to the elemental sulphur until the base is exhausted. The results suggest that there was only

a small decrease in pH in the treatment with biochar because of its basic characteristics with

higher quantities of oxygen-containing functional groups. The FTIR analysis confirmed the

presence of some surface structure such as OH, COO, and C=O[18]. On the other hand, in H2S

41

removal using AC having acidic pH, the base was exhausted because of the formation of

sulfuric acid [19].

Another study suggests that a high pH is believed to be the reason for the higher adsorption

capacity performed by biochar derived from rice hull (SR) in removing 10 – 50 ppm of H2S

[19]. The SR was superior to the rest of the materials, namely AC, biochar derived from bamboo

(SB), and camphor (SC), because of its highest pH value [19].

Table 2.12 Study of biochar and carbon-based materials to remove gas impurities

Material Type of

gas

Concentration

(ppm)

Adsorption

capacity

(mg.g-1)

References

Woodchips biochar H2S 1020 273 [153]

Green waste biochar H2S 2000 6.5 [151]

Camphor-derived biochar H2S 50 1.2-121.4 [20]

Activated carbon H2S 10 – 50 35.6 [19]

Rice hull derived biochar H2S 10 – 50 382.7 [19]

Switch grass-derived

biochar

H2S 150 8 [46]

Woody biochar

(Cryptomeria japonica)

NH3 100 RE* = 90% [154]

Switch grass-derived

biochar

NH3 300 8 [46]

*RE = Removal efficiency

Results of previous studies using biochar and carbon-based material to remove gas impurities

are summarised in Table 2.12. Kanjanarong et al. (2016) investigated the biochar made of the

mixture of 80% of woodchip (spruce, pine and fir) and AD residue mixture-based (20%) in

removing low strength of H2S (105, 510 and 1020 ppm) from biogas. The study reports the high

efficiency of H2S removal (up to 98% removal) caused by the high alkalinity of pH (7.98) and

moisture content during operation (80-85). Also, the presence of carboxylic and hydroxide

42

radical groups also contributed to the process [153]. Using a higher H2S concentration of 2000

ppm, Skerman et al. (2017) examined five different materials for H2S removal, namely,

commercial iron-oxide H2S scavenger (cg5), green waste-biochar, granulated steel furnace slag,

red soil, manure-based compost and granulated activated carbon (GAC). Although the biochar

breakthrough time was significantly shorter than the cg5 and red soil, biochar achieved the

second-highest breakthrough capacity (6.5 g S.kg medium-1) [151]. The studies show the

potential of biochar as an ideal H2S adsorbent.

Sahota et al. (2018) used leaf biochar prepared at three different temperatures (200, 300 and

400ºC) to remove 500 – 1300 ppm of H2S. The results suggest that the biochar prepared at

400ºC achieved the higher H2S removal efficiency of 84% owing to its higher pH which allowed

the increased H2S dissociation and elemental sulphur conversion rates. It is also believed that

the higher SSA, carbonisation temperature and surface mineral element of biochar play a role

[21].

While the utilisation of biochar to remove acid gases such as H2S and CO2 are popular, the

studies of biochar use in NH3 cleaning remain scarce. Iyobe et al. (2004) compared woody

biochar and activated carbon (AC) to remove 100 ppm of NH3. At 20ºC, the woody biochar

removed higher NH3 than AC, achieved ca. 90% removal efficiency compared to 15% at 24

hours of operation. It is well known that NH3 is an alkaline and polar gas [154]. It is reported

that biochar was more suitable for this type of gas, while AC performs better in removing

volatile organic carbon (VOC). Also, the presence of acidic functional groups such as carboxyl

and phenolic hydroxyl groups in this woody biochar prepared at 400ºC led to a maximum

absorbability for a base gas like NH3. The study reports that the surface acidity of the biochar

than its specific surface area, was more significantly affected the NH3 removal [154].

A contradictive result was reported by Bhandari et al. (2013) who conducted the study using

biochar and other materials namely activated carbon, acidic surface activated carbon, and mixed

43

metal oxide [46] to remove H2S, NH3 and toluene simultaneously. In the study, biochar was

found to be a moderate material in removing NH3, adsorbing 8 mg NH3.g-1 with 100 min of

breakthrough time. It is thought that the NH3 removal occurred via surface and micro-pore

filling. The study suggested that the mechanism of biochar in removing NH3 via adsorption

follows the following reactions [46]:

2NH3 → N2 + 3H2 (R2.17)

4NH3 + 3O2 → 2N2 + 6H2O (R2.18)

However, the result was significantly lower when compared with the treatment using AC which

adsorbed 0.03 g-NH3.g-1. It is believed that the acidic surface of the AC that sustained the NH3

removal to achieve higher removal efficiency [46].

The existing literature suggests the promising potential of biochar in removing H2S. However,

the results on the utilisation of biochar in NH3 removal are inconsistent, depending on the

feedstock and preparation condition of biochar. Therefore, further investigations are

recommended for future studies.

2.6 Summary of literature review and specific research objectives

The AD is a mature technology to both manage waste and generate energy. However, it

produces a low quality of biogas and operates in a sub-optimum condition. The development

of TPAD is idealised to improve the AD. The innovative aspect of the TPAD is the harvest of

H2 from the first phase to be mixed with the CH4 generated from the second phase to increase

the overall quality of the biogas. The TPAD principles can be applied in a remote area as an

integrated system of TPAD, biogas cleaning and conditioning. The food and agriculture waste

are a potential feedstock for TPAD to produce biogas for household and TPAD operation. The

TPAD also produced an increased nutrient availability for organic fertiliser preparation for the

44

agricultural application. However, optimisation in term of further enhancing biogas production,

improving H2 and CH4 yield, removing impurities from biogas generated from TPAD, and

adding value to the beneficial utilisation of the effluent is still required.

Biochar with its aforementioned beneficial characteristics for a wide range of application,

therefore, become the innovative aspect of this study. The reviews of current literatures suggest

the gaps on the knowledge of the application of biochar on the utilisation of biochar in the (1)

TPAD, (2) the gas cleaning and (3) preparation of fertiliser from the TPAD effluent as follows.

First, to the best of our knowledge, in a TPAD, where phases of the AD are separated into two,

the studies on the effects of biochar addition and operation condition on H2 and CH4 production

are limited. Therefore, it is essential to study the H2 and CH4 generation on TPAD under

different biochar addition. In addition, the majority of the studies of the biochar addition in AD

were conducted under single operating condition. As a result, the information on the interactive

effect of biochar addition and operating conditions in AD are scarce. Therefore, this thesis

investigated the effect of biochar addition under the different operating condition, such as

different initial pH and temperature. This thesis is also aimed to reveal the mechanism of

biochar in enhancing H2 and CH4 production in the separate phases of TPAD.

Second, despite the growing interest on the biochar utilisation in AD, the study on the biochar

addition in demonstration or full-scale operation is rare. To date, the study on the biochar

application in demonstration scale of TPAD has never been explored. The study on the

demonstration scale operation of TPAD is necessary to apply the results of the bench/laboratory

studies and to investigate the practical considerations on the biochar addition in the

demonstration scale application. A commissioning and start up period are important steps

before the continuous operation of demonstration scale TPAD.

45

Furthermore, for further study, it is suggested to investigate the impurities removal using

biochar derived from other sources of waste/feedstock [19]. Moreover, the inconsistent results

from the existing studies of NH3 removal using biochar imply that further studies are important.

Finally, the existing literature suggests that the AD effluent, biochar and the combination of

both materials have shown a possible utilisation both for soil amendment and fertiliser, although

some reports also show their negative effects on plant growth, depending on their

characteristics, application rate and the species of the tested plant [28, 147, 155]. Therefore, the

further characterisation, determination of the appropriate proportion of AD effluent and

biochar, and its fertilising character are in a high necessity to be conducted before the

application of AD effluent as fertiliser or stabiliser [144, 147].

It is expected that this study will fill the gaps in the literature about the biochar application on

the integrated TPAD system. The thesis investigates into the utilisation of biochar in the (1)

TPAD, (2) the gas cleaning and (3) preparation of fertiliser from the TPAD effluent. Overall

aims of this study are to study and reveal the mechanism of the biochar effects on TPAD process

for H2 and CH4 production using laboratory scale bench-top bioreactors, to demonstrate and

optimise performance of TPAD through the PDU with the addition of biochar, as well as to

devise a method to prepare organic fertilisers by treating the TPAD effluent with biochar.

To achieve these overall aims, the following specific objectives have been identified for this

thesis work:

1. To investigate the effect of biochar addition on the gas and metabolic products generation

as a function of time in the first and second phases of the bench-scale TPAD and establish

the working mechanisms of biochar in TPAD;

46

2. To study the effect of operating condition (initial pH and temperature) in the biochar-

added TPAD process on biogas production and composition in the first and second phases

of the bench-scale TPAD;

3. To determine the optimum value of the biochar addition, initial pH and temperature in

the first and second phases of TPAD;

4. To demonstrate the performance of the TPAD Process Demonstration Unit (PDU) using

selected feedstock over a sufficiently long period of operation;

5. To study the effect of biochar addition in the performance in the TPAD PDU operation;

6. To prepare and characterise biochar-loaded fertiliser prepared from TPAD effluent;

7. To assess the fertilising characteristic of the biochar-loaded fertiliser by using

germination assay.

47

Chapter 3 Methodology, Approach and Techniques

The chapter presents the detailed methodology, approach and techniques to achieve the study

objectives. Experimental set up of bench scale and demonstration operations of TPAD, biogas

cleaning and organic fertiliser preparation are described.

3.1 Overall research strategies

The experimental works was focused on the effects of biochar addition and operation in bench

scale TPAD, both first and second phase TPAD to produce H2 and CH4, respectively.

Experiment on the operation of TPAD PDU, fertiliser preparation and biogas cleaning were

also accommodated. Figure 3.1 shows the schematic diagram of the research strategies for the

experimental study.

Experiments

Modelling

Bench scale TPAD

Demonstration scale

TPAD

Preparation of

organic fertiliser

Biogas cleaning

First phase

Second phase

Gompertz model

Research strategies

Figure 3.1 Research strategies

48

3.2 Bench scale experimentation of TPAD

It is known that there is a standardised method to investigate an anaerobic biodegradability of

a given substrate and its ultimate methane potential, namely bio-methane potentials (BMP)

[156-158]. It is also essential for assessing the activity, inhibition and bio stability of an

anaerobic assay [156, 157]. The information then can be used for a feasibility study of design,

economic and full scale of AD operation [157, 158]. A similar method for bio-hydrogen

potential (BHP) is also develop for investigating potential of a certain substrate to produce H2

[159]. A combined assay of BHP and BMP is proposed to simulate a batch TPAD operation

[158, 159]. In this study, the combined BHP and BMP was used to assess the performance of

bench scale TPAD with the addition of biochar under different operating conditions.

3.2.1 Materials

To ensure the consistency in the feedstock quality and characteristics, white bread obtained

from a local supermarket was used to simulate carbohydrate-rich food waste in an aqueous

environment for TPAD. The bread was shredded to ca.1 mm in size before being used in the

TPAD experimentation.

The source of inoculum was a sludge obtained from Woodman Point Wastewater Treatment

Plant, Western Australia. For hydrogen production, the sludge was heated and stirred at the 95

C for 30 minutes to eliminate methanogens. A biochar obtained from pyrolysis of pine sawdust

using an indirectly fired kiln reactor at 650C with a retention time of ca. 20 min. The biochar

was ground and sieved to a size fraction of 3.5-25.9 µm. The biochar sample was dried in an

oven at 105C prior to use.

Elemental (C, H, N and S), total solids (TS) and volatile solids (TS) analysis the food waste,

inoculum and biochar used in the study were conducted according to the ASTM standards

(D3176) and US Environmental Protection Agency (EPA Methods 1694) [160]. Particle size

49

distribution was measured using Cilas 1180 particle size analyser. BET surface area and pore

volume were determined using TriStar II 3020 surface area and porosimeter analyser.

Characteristics of the feedstock, inoculum and biochar are listed in Table 3.1.

Table 3.1 Characteristics of feedstock, inoculum and biochar [161]

Parameter Food waste Inoculum Biochar

Carbon (%) 42.7 ND 77.9

Nitrogen (%) 2.3 ND 0.3

Hydrogen (%) 9.1 ND 3.7

Sulphur (%) 0.3 ND 0.1

Oxygen (%) 45.6 ND 18

C/N ratio 18.7 ND 255.0

pH 4.9 7.3 9.6

Total solids (%) 61.2 2.5 95.2

Volatile solids (%) 59.5 1.8 81.1

Particle size distribution (µm) ND* ND 3.5-25.9

BET surface area (m2.g-1) ND ND 130.0

Pore volume (cm3.g-1) ND ND 0.0138

ND: not determined

3.2.2 Experimental set up

Figure 3.2 shows the experimental set up of bench scale TPAD study. A serum botte with 100

mL total volume was used as the reactor. The working volume was 60 mL, containing sludge,

bread and water. The bottle was airtight sealed with rubber lid and aluminium crimp to ensure

the anaerobic condition and prevent the reactor from gas leaking. The typical bottles were then

placed in incubator at the designed temperature and duration.

50

Gas sample taken for

GC analysis

Liquid sample taken for

VFA analysis

Inoculum

Bread

Water

(a)

(b)

Figure 3.2 Schematic of the experimental set-up (a) and bench scale TPAD in the incubator

(b)

3.2.3 Experimental procedure

3.2.3.1 The first phase of TPAD

The effect of biochar under a fixed initial pH and temperature

The TPAD experimentation was conducted in batch mode. Schematic diagrams showing the

experimental set-up and the typical experimental procedure are shown in Figure 3.3. For H2

production, 8 g VS.L-1 of bread, 10 ml of heated sludge and water were added in a 100 ml serum

bottle with a working volume of 60 ml. The initial pH was adjusted to 5 by adding an

appropriate amount of hydrochloric acid (HCl) or sodium hydroxide (NaOH). Prior to an

experimental run, the reactors were flushed with high purity nitrogen (> 99.99 %) at 10 l.min-1

for one minute and then carefully sealed with rubber plugs and secured with aluminium caps

[156].

51

Figure 3.3 Schematics of the experimental procedure for TPAD employed in this study

The bottle was then placed in an incubator maintained at 35 C until the gas production stopped,

within approximately 8 days. The bottle was shaken once a day prior to sampling the gas for

composition measurement [156]. A sample of accumulated biogas was taken daily from each

reactor using a gas tight syringe and a 1 ml liquid sample was taken on day 2 and 8, respectively,

for further analysis. In order to study the effect of the amount of biochar addition on the

performance of the H2 production process, reactors with different biochar addition ratios of 8.3,

16.6, 25.1 and 33.3 g.l-1 were set up. These experiments were run following the same procedure

Heated sludge Distillate water Bread Biochar

Mix in 100 ml

serum bottle

Bring to initial pH

5±0.1

Purge with nitrogen

(1 minute)

Take initial sample (1 ml)HCl 1 M or NaOH 1 M

Close the serum

bottle with rubber

and alumunium lid

Place in incubator

(35°C) for 8 days

Add unheated

sludge into serum

bottle

Bring to initial pH

7±0.1

Take initial sample (1 ml)HCl 1 M or NaOH 1 M

Close the serum

bottle with rubber

and alumunium lid

Place in incubator

(35°C) for 39 days

Purge with nitrogen

(1 minute)

• Stir the bottles once a day

• Measure gas production

every day

• Take gas sample every day

• Take liquid sample (1 ml)

periodically for VFA analysis

• Stir the bottles once a day

• Measure gas production

every day

• Take gas sample every day

• Take liquid sample (1 ml) on

day 2 and 8 for VFA analysis

Hydrogen

production

Methane

production

52

as detailed above. Each set of experiments was repeated under identical conditions three times,

involving a total of fifteen serum bottle reactors.

The gas volume and composition were analysed every day according to the methods explained

in section 3.2.4.1 and 3.2.4.2, respectively. Liquid samples were taken initially, on day 2 and 8

for VFA analysis (section 3.2.4.3). Initial and final pH values were also recorded.

The effect of biochar under different initial pH and temperature

The experiment was according to the first phase experimental procedure as in section 3.3.1.1.

The batch experimentation of H2 production was designed based on a CCD [162]. A typical

CCD comprises of three different sets of experimental runs, namely a centre, factorial and axial

point runs. The centre point runs combine the medium levels of each factor examined in this

study, namely initial pH, temperature and biochar addition ratio.

The factorial point runs accommodate the upper and lower levels of each factor, and the axial

point runs typically cover slightly upper and lower levels of each factor [163]. Thus a CCD

explores the representative points of the experiments while keeps the number of total test runs

to a minimum.

The three independent factors examined in this study were the initial pH (X1), temperature (X2)

and biochar addition ratio (X3) and the responses were the cumulative H2 yield (YH) and

maximum H2 production rate (RM), respectively. The levels of initial pH were 4, 6 and 8, the

levels of temperature were 25, 35 and 45C and levels of the biochar addition ratio were 5, 10

and 15 g.l-1. The level of biochar addition ratio was determined based on our previous study

[161], in which a biochar addition ratio of 16.6 g.l-1and higher showed no beneficial effect on

H2 production. The total runs of the experiments were 20, consisting of 6 replications of centre

point runs, 8 sets of factorial runs and 6 sets of axial runs (Table 3.2).

53

Table 3.2 Central composite design for H2 production

Run Factor 1

A: Biochar addition (g.L-1)

Factor 2

B: Temperature (oC)

Factor 3

C: initial pH

1 4 25 15

2 8 25 15

3 6 35 10

4 8 25 5

5 6 35 10

6 9.4 35 10

7 4 45 5

8 4 25 5

9 6 35 10

10 6 35 10

11 6 35 18.4

12 8 45 15

13 6 35 10

14 6 51.8 10

15 2.6 35 10

16 8 45 5

17 6 18.2 10

18 6 35 10

19 4 45 15

20 6 35 1.6

Each experimental run was triplicated. The cumulative H2 yield (YH) and maximum daily

volumetric H2 production rate (RM) achieved during the experimentation were examined as the

targeted responses to factors, i.e. the treatment parameters: initial pH, temperature and biochar

54

addition ratio [164]. Additional experiments at biochar addition ratios ranging from 0 – 18.4

g.l-1 at the initial pH 35 and temperature 35C were performed to investigate a typical H2

production under different biochar addition.

The daily volumetric gas and composition were used to calculate the cumulative H2 yield (YH)

and maximum H2 production rate (RH). The YH and RH are the responses of the CCD. The

responses then analysed using response surface methodology explained in section 3.6.3.

Effect of Biochar on H2 Production via Anaerobic Digestion as Compared to Other Solid

Additives: Role of Acidity

The additives with different acidity and surface structure used in the study were pinewood

biochar [161], alumina and zeolite. Glass with a pH of 7 and very small surface area was also

used for a comparison purpose. The additives were crushed to have a particle size less than 50

μm. The pH and BET surface area of the additives are listed in Table 3.3. It is seen that the

biochar is alkaline and alumina is neutral while the zeolite is very acidic. Zeolite has the highest

BET surface area of 538 m2.g-1 followed by biochar (361 m2.g-1) and alumina (153 m2.g-1).

Table 3.3 The characteristics of the additives

Parameter

Additives

Biochar Glass Alumina Zeolite

pH 8.6 7.1 6.9 4.2

BET surface area (m2.g-1) 361 0.26 153 538

The feedstock (1 gram) and 10 mL of heated sludge and 10 g.L-1 of an additive are mixed in a

100 mL serum bottle and distillate water was added to occupy the working volume of 60 mL.

The initial pH was adjusted to 6.4, according to an optimum pH suggested by the optimisation

study, using hydrochloric acid (HCl) and sodium hydroxide (NaOH). The bottles were then

purged with high purity nitrogen and sealed with a rubber lid and aluminium crimp. The bottles

55

were then incubated at 32 °C, as suggested by the optimisation study, for 7 days. A set of control

without any biochar addition was also prepared. Each treatment was conducted in triplicate.

The daily volume and composition of biogas produced were monitored daily. Liquid sampling

was taken periodically for further analysis. A set of identical bottles of each treatment was also

prepared to be opened daily to monitor the pH and VFA changes during the experiment.

3.2.3.2 The second phase of TPAD

The effect of biochar under a fixed initial pH and temperature

This study is the continuation of the preliminary study explained in the section 3.2.3.1. After 8

days, the remaining culture in a reactor was used as the feed for CH4 production in the second

phase (Figure 3.3). The bottle tip was opened and 10 ml of unheated sludge was added to the

bottle to bring the pH to 7. The bottle was then sealed and incubated at 35 C for 39 days. Gas

sampling was conducted daily and liquid samples were taken periodically for further analysis.

The effect of biochar under different initial pH and temperature

The feedstock used in the experiment was the effluent from the first phase of TPAD of food

waste. It was prepared according to optimum biochar addition, initial pH and temperature of

our previous study, which is briefly described as follows. The feedstock was prepared in a one

litre Duran bottle. The heated sludge (100 mL), 10 g volatile solid.L-1 (g VS.L-1) of white bread,

10.1 g.L-1 of biochar and 500 mL of distillate were mixed in one litre bottle and set at optimum

initial pH 6.4. The culture was then incubated at an optimum temperature of 32C until it

stopped producing biogas (7 days). This effluent was used as the feedstock in the second phase

of TPAD to produce methane.

56

Sludge (10 ml) First phase effluent (50 ml) Biochar (as designed)

Mix in 100 ml

serum bottle

Bring to initial pH

(as designed)

Purge with nitrogen

(1 minute)

1M HCl or 1M NaOH

Seal the serum

bottles with rubber

and aluminum lids

Place the bottles in

incubator at

designed

temperature for 35

days

• Stir the bottles once daily

• Measure gas production daily

• Gas sampled and analysed

daily

Figure 3.4 Schematics of the experimental procedure for second phase

The experimental set up is illustrated in Figure 3.4. In a typical experimental run, 50 mL of the

feedstock and 10 mL of the seed sludge were mixed in a 100 mL serum bottle. The amount of

fresh sludge added was determined based on the result of the preliminary study. HCl and NaOH

were added to bring the pH of the liquid to the designed pH. The reactor was then purged with

nitrogen to create an anaerobic environment and closed with a rubber and aluminium lid and

incubated for 30 days. The effect of biochar addition (5, 10, and 15 g.L-1(w/v)), initial pH (5, 7

and 9) and temperature (25, 35 and 45 C) on methane yield (YM) and methane production rate

(RM) was examined in turn using a Box Behnken experimental design method. A control

experiment without biochar addition incubated at an initial pH of 7 and temperature of 35 C

was also conducted.

57

Table 3.4 Parameters and levels of Box Behnken Design

Variable Parameters Level

A Biochar addition (g.l-1) 5 10 15

B Temperature (ºC) 25 35 45

C Initial pH 5 7 9

The batch experimentation for methane production was designed based on a Box Behnken

design (BBD) [165]. The three independent factors examined in this study were the biochar

addition ratio (A), temperature (B) and initial pH (C) and the responses were the cumulative

methane yield (YM) and maximum methane production rate (RM), respectively. The levels of

initial pH, temperature and biochar addition ratio are listed in Table 3.4.

Table 3.5 Box Behnken design for CH4 production

Run Factor 1

A: Biochar addition (g.l-1)

Factor 2

B: Temperature (oC)

Factor 3

C: Initial pH

1 10 35 7

2 10 45 9

3 10 45 5

4 10 25 5

5 15 35 5

6 5 45 7

7 15 35 9

8 10 35 7

9 15 45 7

10 10 35 7

11 5 25 7

58

Run Factor 1

A: Biochar addition (g.l-1)

Factor 2

B: Temperature (oC)

Factor 3

C: Initial pH

12 5 35 9

13 15 25 7

14 10 35 7

15 10 35 7

16 5 35 5

17 10 25 9

The level of biochar addition ratio was determined based on our previous study [161], in which

a biochar addition ratio of 25.5 g.l-1and higher decreased CH4 production. The total runs of the

experiments were 17 with 5 replications of the centre point runs (Table 3.5). A control treatment

without biochar addition at the initial pH 7 and temperature 35C were also performed. Each

experiment was run in triplicate.

The daily volumetric gas and composition were used to calculate the cumulative CH4 yield (YM)

and maximum CH4 production rate (RM). The YM and RM are the responses of the BBD. The

responses then analysed using response surface methodology explained in section 3.6.3.

3.2.4 Analysis

3.2.4.1 Biogas volume

Water displacement is a typical method for volumetric gas measurement (Figure 3.5) [157,

158]. Thus in this current study, daily volumetric biogas production was measured by a water

displacement method [23] and then converted to volumetric biogas production under the STP

condition (273K and 1 atm pressure) according to the ideal gas law [161].

59

Figure 3.5 Schematic set up of water displacement method for gas volume measurement

3.2.4.2 Biogas composition

Studies use gas chromatography (GC) methods with flame ionization (FID) and thermal

conductivity detector (TCD) to analyse the composition of H2, CH4 and CO2 in biogas produced

by the assay [102, 156, 158]. In this study, gas composition was analysed using a Gas

Chromatograph (GC; Agilent 7980) facilitated with a flame ionisation detector (FID) (heater

200 C, hydrogen flow: 30 ml.min-1, air flow: 350 ml.min-1, make up flow: 35.5 ml.min-1), a

back detector thermal conductivity detector (TCD) (heater 250 C, reference flow: 10 ml.min-

1, make up flow: 5 ml.min-1) and a TCD (heater 200 C, reference flow: 7 ml.min-1, make up

flow: 3 ml.min-1) with the oven temperature of 90 ºC. Figure 3.6 shows a typical gas

chromatogram of gas collected from the first phase of TPAD.

60

Figure 3.6 A typical gas chromatogram of gas collected from the first phase of TPAD

The daily volumetric hydrogen or methane production was calculated by multiplying the

volumetric biogas production by the H2 or CH4 percentage as determined from the GC analysis

[156]. The total cumulative H2 or CH4 yield was then obtained by adding daily volumetric H2

or CH4 production ºC. The cumulative H2 yield (YH) and CH4 yield (YM) were the total

volumetric H2 production over the whole duration of an experimental run, calculated according

to the ideal gas law (in STP ml) and normalised to the volume of reactor [161]. The specific

gas production was the cumulative H2 yield relative to the gram VS of the added [53, 156, 161].

3.2.4.3 Volatile fatty acids

Volatile fatty acid (VFA) analysis is important to understand the metabolic products generated

from the assay, for example butyric, acetic and propionic acids [158, 159]. The metabolic

products may give the insight on the possible dominance metabolic pathway of an assay[113].

For the sample preparation, 100 µl of liquid sample was diluted with 300 µl deionised water in

a microcentrifuge tube. As much as 100 µl of 2-ethylbutyric acid (10 mmol.L-1) as internal

standard (IS) was also added. The pH of the sample was reduced by adding 50 µl of 6 N HCl.

To extract the volatile acid, 500 µl of diethyl ether was added. The tubes were then centrifuged

using microcentrifuge (Eppendorf centrifuge 5415D) at 3000 rpm for 10 minutes. After

61

centrifugation, the upper layer containing acid extract in the ether was transferred into GC vials

for VFA analysis. A GC (Agilent 7980) using DB-WAX column (30 m x 250 mm x 0.25 mm),

flame ionisation detector (FID) (heater 250 C, H2 flow: 35 ml.min-1, air flow: 350 ml.min-1,

make up flow 4 ml.min-1, total run time 13 minutes) was used to determine the volatile fatty

acid (VFA) of liquid samples taken from both phases. The calculation of each acid was

determined using the response factor of each acid generated from the standard curve developed

from the VFA analysis using a 10 mmol.L-1 mixed acid standard (Sigma Aldrich). The area of

the Figure 3.7 shows typical standard curves for some selected acids. The acid calculation was

based on the following equation.

𝐶𝑜𝑛𝑐. 𝐻𝑥 =𝐴𝑟𝑒𝑎 𝐻𝑥 𝑥 𝐶𝑜𝑛𝑐. 𝐼𝑆

𝐴𝑟𝑒𝑎 𝐼𝑆 𝑥 𝑅𝐹 (R3.1)

(a) (b)

Figure 3.7 Typical standard curves for (a) acetic and (b) butyric acids analysis

3.2.4.4 pH

A TPS AQUA-pH pH mater (Rowe Scientific) was used to measure the pH of liquid samples.

Prior to the test, two points calibration with pH 4.0 and 7.0 standard buffer solution (Rowe

Scientific) was conducted.

62

3.2.4.5 Scanning Electron Microscopy

In an effort to reveal the working mechanisms of biochar, the morphology of biochar before

and after experimentation was examined using Scanning Electron Microscopy (SEM). Liquid

sample (1 mL) containing biochar particles were taken at the end of the experiment and added

with fixative solution (glutaraldehyde 2.5%). In order to prevent the damage of the samples, the

critical point drying method (CPD) was conducted prior to the SEM analysis. The liquid sample

was washed twice using phosphate buffer solution (PBS) and set at BioWave for 30 second

each. The samples were then dehydrated using gradually increased concentration of ethanol

(50, 70, 90, and 100% in water) and microwaved for 30 second each. [85] The dried samples

were then coated with Au and analysed using SEM Zeiss 55 with SE or InLens detector.

3.3 TPAD Process Demonstration Unit (PDU)

3.3.1 Principles of TPAD PDU

A TPAD process demonstration unit (PDU) operated by the Centre for Energy of the University

of Western Australia consists of two reactors; the first reactor is an H2 producing reactor with

the capacity of 150 L, while the second reactor accommodates methane generation with 500 L

of total volume [41]. Before the operation, the feedstock is milled (where necessary) and diluted

with water to achieve the designed total solids (TS) in storage tank. The feedstock is then

pumped into the first phase of TPAD (R1). R1 1 is operated at low pH (4-6) and mesophilic

temperature to produce H2. Typical hydraulic retention time (HRT) of the R1 is 3 days. The

effluent from the R1 is pumped to the buffer tank to adjust the pH of effluent to around 7. The

effluent is transferred to the second reactor (R2) to produce methane-rich biogas. The HRT of

the R2 is around 7-14 days. The heating pump circulated heated water to double jackets of each

reactor to maintain the temperature. The effluent from the R2 is flown to discharge tank for

further treatment.

63


TPAD PDU system

Figure 3.8 shows the schematic diagram of TPAD PDU of the CFE UWA. The components of

the unit are the process controller, feedstock preparation, digesters, heating system, gas

collection and effluent conditioning.

Figure 3.8 Schematic diagram of TPAD PDU of the Centre for Energy of University of

Western Australia

Process control

TPAD PDU is controlled by a programmable logic controller (PLC), with sensors measuring

pH, temperature and gas flow rate. The instructions for agitation, heating and pumping activities

across the facilities can be automatically executed via the PLC. The data obtained from the

sensors were recorded for further analysis.

64

Feedstock preparation

The unit consists of a mill and a storage tank. The mill is used to mechanically reduce the size

of the feedstock for easier access of microbes into the nutrient contents in the feedstock [41].

The storage tank (volume 200L) is needed for feedstock mixing and dilution, achieving

maximum total solids (TS) of 30% (w/w) in the AD system [166]. Size reduction and dilution

of feedstock also prevents jamming in the valves, pumps and pipes [41]. Both mill and storage

tank were safety guarded that are controlled by micro-switches. The motor of equipment stops

operating when the lid is open.

Digesters

The digester contains two reactors, first for H2 production (150 L) and second for CH4

production (500 L), and one buffer tank (100 L) installed between the two reactors. Both

reactors are continuous stirred tank reactor (CSTR), which comprises of a tank body for the

main reaction, heating jacket for adjusting temperature, an agitator to stir the culture, a stage

inlet and an outlet for effluent discharge. Both reactors are equipped with temperature, pH and

oxidation-reduction potential (ORP) sensors to monitor the operating conditions. The buffer

tank with a pH probe is used for pH adjustment of the effluent from the first phase before being

fed into the second reactor.

Heating system

A 250 L tank is used for water storage. The water is pumped to a 250 L heating tank and the

heated water is circulated by a heating pump and hot water pipe line to the heating jacket of

each reactor. The designed temperature is set and controlled by the PLC. Both the first and

second reactors can be heated up to 50ºC.

65

Gas and effluent collection unit

A wet flowmeter was used to measure daily biogas production from each reactor. A cautious

inspection should be conducted to monitor water level in the flowmeter as an incorrect level of

liquid may cause a flowmeter malfunction. When a low water level is found, additional liquid

should be added into the wet flowmeter and subsequent flow meter calibration needs to be

executed. The biogases produced from the first and second tank are mixed in a 1 m3 gas bag.

Gas can be transferred into a D size gas tank using a gas pressurisation system. The effluent

from the second tank is discharged to the disposal tank for further treatment or utilisation.

Feedstock, inoculum and biochar

White bread from a local supermarket was used as a simulated carbohydrate waste for TPAD

feedstock. Microbial inoculum was effluent from anaerobic digestion at the Woodman Point

Wastewater Treatment Plant, Western Australia. Biochar was prepared by heating pine saw

dust at 650 ºC for ca. 20 minutes in a kiln reactor [167]. Biochar was ground and sieved to a

particle size 3.5-25.9 µm. Elemental (C, H, N and S), total solids (TS) and volatile solids (TS)

analysis of the food waste, inoculum and biochar used in the study were conducted according

to ASTM standards (D3176) and US Environmental Protection Agency (EPA Methods 1694)

[160]. Particle size distribution was measured using Cilas 1180 particle size analyser. BET

surface area and pore volume were determined using TriStar II 3020 surface area and

porosimeter analyser. Characteristics of the feedstock, inoculum and biochar are listed in Table

3.1.

Start-up strategy of the TPAD PDU

First phase: An effective sterilisation and sludge heating were required prior to the start-up to

ensure the elimination of methanogens in the R1. The first tank was thoroughly washed and

sterilised with 70% of ethanol to prevent methanogens contamination. In our bench scale study,

66

the sludge was heated at 95ºC for 30 minutes [161]. However, when the same method was used

in TPAD PDU, the methanogens contamination occurred in R1. Therefore, a longer heating at

95ºC for one hour was conducted, which has been found to be effective in sterilise R1.20 L of

the heated sludge was added once in the beginning of the operation. Prior to the feeding, the

bread was blended with water according to the designed substrate concentration (Table 3.6). A

fed-batch strategy was applied in the start-up period. The feeding with the mixture of the

feedstock, biochar and water was conducted gradually until the working volume of 100 L was

occupied. There were six stages during the start-up of R1, with the HRT of 3 days for each

stage. The fed-batch was conducted during stage 1 – 4 and followed by semi-continuous

operation at stages 5-6 (Table 3.6).

Table 3.6 Start-up strategy of the first phase of TPAD PDU

Stage Sludge

Volume (L)

Water

volume

(L)

Bread Biochar

(g)

HRT

(days) (g) (g VS)

1 20 20 170 100 400 3

2 - 20 340 200 200 3

3 - 20 1000 600 200 3

4 - 20 1350 800 200 3

5 - 20* 1350 800 - 3

6 - 20* 1350 800 - 3

Note: *: used recirculated liquid from tank 1

The biochar was stepwise added with the total biochar addition ratio of 10 g.L-1 of working

volume of the reactor, as suggested by the result of bench scale study on the first phase TPAD

[160]. At stage 4, the designed working volume of the reactor (100 L) was achieved. In stages

5 and 6, 20 litres of the culture was discharged from the reactor and then blended with 1350

67

gram of white bread and recirculated back into the reactor. The first tank was operated under

ambient temperature. 1% (v/v) of 1 M NaOH was added into the reactor at the beginning of

stages 4, 5 and 6 to adjust the pH above 4.

Second phase: R2 was operated under fed-batch conditions by feeding the sludge, white bread,

biochar and diluting water gradually for the first 5 stages until a total working volume of 300 L

was achieved (Table 3.7).

Table 3.7 Start-up strategy of the second phase of TPAD PDU

Stage

Sludge

Volume

(L)

Water

volume

(L)

Bread

Biochar

(g)

HRT

(days) (g) (g VS)

1 15 60 500 300 940 7

2 15 60 1000 600 940 7

3 15 60 1500 900 940 7

4 15 30 2000 1200 375 7

5 - 30 2500 1500 375 7

6 60 - - - 750 21

7 - 20* 2000 1200 - 7

8 - 20* 2000 1200 - 7

9 - 20* 2000 1200 - 7

Note: *: used recirculated liquid from tank 2

In order to enhance CH4 production, 60 L of untreated sludge was added into the reactor at stage

6 [140, 168, 169]. Biochar at with the total addition ratio of 12.5 g.L-1 was applied as suggested

by our previous study [13]. A tiny amount of NaOH (1% v/v) was added into the system in

stages 1 to 5 to adjust pH. NaOH was not required after stage 5 since the pH of the culture

remained above 6. The whole start-up operation was conducted at ambient temperature. In the

68

beginning of start-up, the manhole and sampling ports in both reactors were tightly sealed and

flushed with N2 to check any gas leaking and ensure the anaerobic condition of the tanks.

3.3.3 System monitoring and control

The daily volumetric biogas production from the first and second phase of TPAD was

monitored using a wet gas meter (LMF-1, CCAF Company, Changchun, China), respectively.

The gas samples of each reactor were taken once daily using foil gas bags. The gas composition

of the gas samples were analysed using a gas chromatography (GC) Agilent 7980 (Shanghai,

China) which is equipped with flame ionisation (FID) (H2 flow: 30 ml.min-1, make up flow:

35.5 ml.min-1, air flow: 350 ml.min-1, heater temperature 200ºC), thermal conductivity detector

(TCD) (reference flow: 7 ml.min-1, make up flow: 3 ml.min-1, heater temperature 200ºC) and

back TCD (reference flow: 10 ml.min-1, make up flow: 5 ml.min-1, air flow: 350 ml.min-1, heater

temperature 250ºC) [167]. The daily H2 and CH4 production were calculated by multiplying the

daily biogas production and H2 and CH4 composition, respectively. A liquid sample was also

taken daily from each reactor for measurement of pH and VFA concentrations [161]. A TPS

AQUA-pH pH meter (Rowe Scientific) was used to measure the pH of liquid samples. For VFA

analysis, 2-ethylbutyric acid (10 mmol.L-1) was used as an internal standard (IS) and diethyl

ether was used to extract the volatile acid. A GC (Agilent 7980) using DB-WAX column (30

m x 250 mm x 0.25 mm), flame ionisation detector (FID) (heater 250 C, H2 flow: 35 ml.min-

1, air flow: 350 ml.min-1, make up flow 4 ml.min-1, total run time 13 minutes) was used to

determine the concentrations of VFA of liquid samples taken from both phases [161].

69

3.4 Preparation, characterisation and evaluation of biochar-added organic

fertiliser


TPAD effluent Biochar

Add to container

according to the

designed percentages

Mixed

thourougly

Close the container with

airtight lid

Set at least

24 hours

Figure 3.9 Preparation of organic fertiliser from TPAD effluent and biochar

Figure 3.9 shows the experimental set up of the preparation of organic fertiliser from TPAD

effluent and pinewood biochar. The TPAD effluent was generated form the R2 of TPAD PDU

on the 57th days of the operation. The pinewood biochar prepared at 650ºC was milled to the

particle size c.a. 25 µm.

The organic fertiliser was prepared by mixing TPAD effluent and biochar according to the

designed percentage of each treatment (Table 3.8). Each combination TPAD effluent and

biochar were thoroughly mixed, kept in an airtight container and labelled according to the

sample names in Table 3.8. The organic fertiliser was set for at least 24 hours to reach the WHC

equilibrium of biochar before use [170].

70

Table 3.8 Fertiliser composition

Sample name TPAD effluent (%) Biochar (%)

BC00 100 0

BC10 90 10

BC20 80 20

BC30 70 30

BC40 60 40

BC50 50 50

BC60 40 60

BC70 30 70

BC80 20 80

BC90 10 90

BC100 0 100

Figure 3.10 Experimental set up of soil-less petri dish bioassay

BC90

71

In a petri dish (diameter 8.5 cm), one gram of the organic fertiliser, 50 rocket seeds and 20 ml

of water were added. It is important to make sure that the biochar and the seeds were evenly

distributed across the petri dish. Each type of the fertiliser was tested triplicated. The petri

dishes were then incubated at 25ºC for 72 hours. At the end of the 72 hours, a photograph of

each treatment was taken. The seeds were counted as germinated when the radicle had emerged

[171]. The germinated seed was counted, the root and shoot length were measured, and

root/shoot ratio and germination index (GI) were calculated.

3.4.2 Analysis

Characterisation of the organic fertiliser

The pH analysis of the fertiliser was according to Luo et al. [83]. The analysis of dry matter and

WHC were based on Foster et al. [170]. The macronutrients required for plant growth including

phosphorus (P), potassium (K), sulphur (S), calcium (Ca) and magnesium (Mg), while the

micronutrients are iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), boron (B), chlorine (Cl),

and molybdenum (Mo), and nickel (Ni). P, K and micronutrients analysis was conducted using

ICP-EOS (iCAP 7000 Series). The preparation of the fertiliser samples for the micronutrients

used the modified methods according to USEPA methods 3050B for acid washing the sludge

sample and Enders and Lehman (2012) for biochar [172, 173].

Germination bioassays

The germination, root and shoot measurement were conducted according to Solaiman et al.

(2012) [28]. The germination index (GI) was calculated using the following formula [144].

𝐺𝐼 = 𝐺𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑𝑠 𝑖𝑛 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑡𝑒𝑠𝑡

𝐺𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑𝑠 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑥 100% (E3.1)

Adopting the compost toxicity test, there are three criteria of toxicity based on GI, (1) GI

≥125%: the compost considered to possess plant nutrient and growth stimulants; (2) GI ≥ 80%:

72

the compost free of any toxic and (3) GI ≤ 50%: compost contains strong toxicity [144, 174,

175].

3.5 Data analysis and modelling

3.5.1 Analysis of variant (ANOVA)

To compare the mean of hydrogen and methane yields from each phase of all treatments, the

data were also statistically analysed using Analysis of Varians (ANOVA). Post hoc tests were

carried out using the least squares difference (LSD).

3.5.2 The modified Gompertz Model

In order to obtain the lag phase, maximum production potential and rate of both hydrogen and

methane production in each treatment, the following modified Gompertz [25] model was

employed:

(E3.2)

where G(t) is the cumulative hydrogen or methane production [ml.l-1], t the time [days], P the

maximum hydrogen or methane production potential [ml.l-1] , Rmax the maximum hydrogen or

methane production rate [ml.l-1.per day] and λ is the lag phase [days] defined as a delayed period

of a culture in responding to a new environment and starting to produce hydrogen or methane

[26]. The cumulative hydrogen and methane production results were fitted using the model

3.5.3 Response surface methodology (RSM)

The response surface methodology (RSM) is a popular method to investigate the effects of key

operational factors such as additive concentration, initial pH and temperature on a measured

response, for example biogas yield and biogas production rate [176]. The optimisation of such

( )

+−

−= 1expexp)(

maxt

P

eRPtG

73

factor provides crucial information for the full scale operation [177]. Using RSM, the number

of experiment can be significantly reduced since it only selects representative levels of each

factor involved in the process [178].

To determine the interactions between the factors and the optimum operating conditions, a

quadratic model (Equation 1) was used to fit the experimental data [179].

Y = 𝛼0 + 𝛼1𝑋1 + 𝛼2𝑋2 + 𝛼3𝑋3 + 𝛼12𝑋1 𝑋2 + 𝛼13𝑋1 𝑋3 + 𝛼23𝑋2 𝑋3 + 𝛼11𝑋12 + 𝛼22𝑋2

2+

𝛼33𝑋32 (E3.3)

where X1, X2 and X3 are the actual values of the three factors, Y is the response (YH or RM,

respectively), 0 is a fitting intercept, 1, 2, 3 are linier coefficients, 12, 13 and 23 are

coefficient of interactivity between factors, and 11, 22 and 33 are quadratic coefficients.

The significance of the fitting model, linear, interactive and quadratic terms in the fitting models

was then determined by conducting ANOVA analysis. The optimisation of the model was

conducted using the “Design Expert” software (Design-Expert 10, State-Ease, Inc.,

Minneapolis MN, USA).

74

Chapter 4 Effect of Biochar Addition on Hydrogen Production

4.1 Introduction

Chapter 4 presents the results of bench scale experiments on the H2 production in the first phase

of TPAD. Typical H2 production without biochar addition is reported in Section 4.2. Section

4.3 discussed the effect of biochar addition on H2 production under varying operating conditions

(initial pH and temperature). Investigation into the role of acidity of biochar on H2 production

is presented in Section 4.4, followed by the discussion of possible mechanisms of biochar in

enhancing H2 production (Section 4.5).

4.2 Hydrogen production without biochar

Figure 4.1 Cumulative yields and production rates of H2 without biochar addition

The potential H2 production from AD of food wastes simulated by bread was first examined

under the tested conditions. Figure 4.1 shows the cumulative yields and production rates of H2.

The H2 production started on day 2 and completely stopped on day 8. The highest H2 production

rate was reached on day three with 304±33 mL.L-1.per day. The maximum cumulative H2

75

production was 751±3 mL.L-1. The H2 composition ranged from 49-65 vol% (Table 4.1). The

remaining gas mainly consisted of carbon dioxide (CO2). No CH4 was detected in the first

phase, confirming that the heating of sludge prior to the inoculation to the system was effective

in eliminating methanogens from the culture. The yields and contents of H2 resulted in the

current study were comparative to the data reported in the literature using potato starch, bagasse

fermentation residue and food waste as feedstocks [88, 180].

Table 4.1 Profiles of the first phase of TPAD without biochar

Parameters Unit Value

Cumulative H2 production mL.L-1 751.1±3

Gas composition

H2 % 49-65

CH4 % 0

CO2 % 35-51

Volatile fatty acids (VFA) mg.L-1

Acetic acid mg.L-1 1063± 351

Butyric acid mg.L-1 1172±60

Propionic acid mg.L-1 7±7

Final pH 3.0

During the operation, H2 production was accompanied by the generation of VFAs. The

dominant fatty acids were butyric and acetic acids, as shown in Table 4.1. The accumulation of

2,242 mg.L-1 of VFA occurred at the end of the phase. The pH of the remaining culture dropped

from 5.0, as set at the beginning of the experiment, to 3.0. The accumulation of acid in the end

of H2 production was identified to be the cause of the pH drop.

76

4.3 Hydrogen production with biochar

Figure 4.2 Cumulative H2 yields at different biochar addition ratios

Figure 4.2 shows the cumulative H2 production over 8 days of operation in the first phase with

and without biochar addition. All cultures started to produce H2 after day one. It is clear that all

cultures with biochar addition produced higher cumulative H2 than that without biochar.

However, when the biochar addition was higher than 8.3 g.L-1, the H2 yield started to decrease

slightly. The highest H2 production of 945 ml.L-1 was achieved at 8.3 g.L-1 of biochar addition.

To determine whether the effect of the biochar addition on the H2 yield was statistically

significant, a statistical analysis was conducted by data-add on of Microsoft Excel 2010. The

procedure consisted of a one-way analysis of variance (ANOVA) and post hoc test analysis

[88]. A statistical level of significance was defined by a (p) value less than or equal to 0.05

which indicates that the mean value of at least one of the H2 production was not equal to the

others. Statistical non-significance was defined by (p) value greater than 0.05. The statistical

analysis results are presented in Table 4.2 including the differences in mean values of the final

H2 production of any two compared cultures, standard errors and the associated p value. It can

77

be seen that the differences between mean values of H2 yield of the control system and systems

with biochar additions less than 25.5 g.L-1 were greater than 100 mL with standard deviations

of less than 68.0 and p values of less than 0.05. This suggests that the H2 yields in the culture

with 8.3; 16.6 and 25.5 g.L-1 biochar additions were statistically significantly higher than that

from the control system. However, it is also evident that there was no significant difference

between the H2 yields of the system with the 33.3 g.L-1 biochar addition and the control system.

Table 4.2 One-way ANOVA and post hoc analysis on cumulative H2 production in

different biochar addition ratios

Biochar addition

(g.L-1)

Average cumulative H2

yields (mL.L-1)

Standard deviation P value

0 750.4a 55.5 0.006

8.3 944.5c 38.6 0.006

16.6 859.5b 68.0 0.006

25.1 858.0b 24.3 0.006

33.3 831.0a 32.0 0.006

Note: Different notation indicates a significant difference from post hoc analysis

The experimental data of H2 production in each culture was fitted using the Gompertz model

and the fitted curves were also presented in Figure 4.2. The coefficients of determination (R2)

of all of the fittings ranged from 0.96 to 0.98, suggesting that the model fits the experimental

data very well. The parameters, namely, the lag phase (λ), maximum H2 potential (P), and

maximum H2 production rate (Rmax) against operation time, were derived from the model and

the results are presented in Table 4.3. First of all, the λ changed from 1.4 days for the system

without biochar addition to 0.9 – 1.1 days with biochar additions. The Rmax of all cultures with

biochar (400 mL.L-1.per day) were higher than that of the control (302 mL.L-1.per day).

78

Similarly, the P of all cultures with any biochar addition was also higher than that of the control.

The highest P value was reached by the culture with 8.3 g.L-1 of biochar (981±24 mL.L-1).

Table 4.3 The results of calculation using the modified Gompertz fitting equation on H2

production with different biochar addition ratios

Biochar addition

(g.L-1)

λ (day) Rmax

(mL.L-1.per day)

P

(mL.L-1)

R2

0.0 1.4±0.1 302±32 749±22 0.96

8.3 0.9±0.1 400±37 981±24 0.97

16.6 1.1±0.1 400±41 884±23 0.97

25.1 1.0±0.1 400±31 886±17 0.98

33.3 0.9±0.1 400±36 855±18 0.97

Figure 4.3 shows the yields of VFA during H2 production phase on Days 2 and 8, respectively.

Generally, the cultures with biochar additions generated increasingly more VFA during H2

production than the control, starting from Day 2. However, towards the end of the

experimentation (day 8), the total VFAs showed little differences between the cultures with and

without biochar addition. It can be seen that the dominant VFA in the control and the culture

with 8.3 g.L-1 addition were different from those with higher biochar addition ratios (16.6; 25.1

and 33.3 g.L-1). The culture without biochar and with 8.3 g.L-1 biochar addition produced

butyric and acetic acids as dominant VFAs, while the cultures with higher biochar addition also

produced propionic acid in addition to butyric and acetic acids. This indicates that the dominant

pathway in the control and the culture with 8.3 g.L-1 biochar addition in H2 production was

butyric type of fermentation, while the other cultures, especially the culture added with 33.3

g.L-1 biochar were likely to follow the propionic pathway [181]. It is believed that the shift of

the reaction pathway was due to the organic overloading in the cultures with biochar addition.

At higher biochar additions (16.6; 25.1 and 33.3 g.L-1), the system will achieve a higher rate of

79

acidogenesis that leads to higher nicotinamide adenine dinucleotide (NADH/NAD+/). To

maintain a balanced NADH/NAD+ ratio, propionic acid fermentation was spontaneously

activated since more NAD+ was produced in propiogenesis than in butyrogenesis [180].

(a)

(b)

(c)

(d)

(e)

Figure 4.3 VFA profiles during H2 production in culture with (a) 0; (b) 8.3; (c) 16.6; (d)

25.1 and (e) 33.3 g.L-1 biochar addition ratios

Sharma et al. observed similar finding that the predominant acids were butyrate and acetate at

biochar addition ratios less than 12.5 g.L-1. The higher addition ratios of biochar (12.5 - 35 g.L-

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1) was reported to increase propionic acid while decrease H2 production [128]. It is suggested

that the propionic acid follows H2 consuming pathway thus decrease H2 [128, 182].

The previous sections suggest that the addition of biochar enhanced H2 and VFA generation,

depending on the addition ratio. Other crucial factors affecting H2 production in the first phase

of TPAD are temperature and pH, because of their major influences on the microbial

metabolism [183, 184]. Understanding the interactions between biochar and operation

conditions including temperature and pH, therefore, is essential to optimise the AD process

[177, 178].

Extending the investigation reported in the previous section, this section reports systematic

examination of the effect of biochar addition on H2 production the first phase of the TPAD

under various initial pH and temperature conditions. The experiments were designed using the

central composite design (CCD) and response surface methodology (RSM) in order to obtain

the optimised condition [185, 186]. Scanning Electron Microscopy (SEM) analysis is used to

observe the morphological changes in the biochar before and after the experiment to reveal the

possible mechanism of biochar addition in enhancing H2 production and reported in Section

4.5.

4.3.1 Response surface analysis

The experimental data as listed in Table 4.4 were fitted using the quadric model as shown in

Equation 3.3 (Chapter 3) leading to the following correlations, respectively:

𝑌𝐻 = −4665.4 + 1138.9𝐴 + 119.3𝐵 + 92.2𝐶– 3.7𝐴𝐵– 1.3𝐴𝐶– 0.00625𝐵𝐶– 78.9𝐴2– 1.5𝐵2– 4.1𝐶2

(4.1)

81

𝑅𝐻 = −3649.3 + 563.9𝐴 + 121.3𝐵 + 94.2𝐶– 2.8𝐴𝐵 – 1.5𝐴𝐶 + 0.007𝐵𝐶– 31.8𝐴2– 1.6𝐵2– 4.2𝐶2

(4.2)

Table 4.4 Central composite design and experimental results for H2 production

Run Initial

pH

Temperature

(°C)

Biochar addition ratio

(g.L-1)

YH (mL.L-1) RH (mL.L-1.day-1)

1 4 25 15 800 334

2 8 25 15 950 582

3 6 35 10 1363 700

4 8 25 5 1060 662

5 6 35 10 1344 755

6 9.4 35 10 709 679

7 4 45 5 681 212

8 4 25 5 794 294

9 6 35 10 1310 690

10 6 35 10 1300 751

11 6 35 18.4 1094 459

12 8 45 15 539 278

13 6 35 10 1118 653

14 6 51.8 10 881 263

15 2.6 35 10 81 27

16 8 45 5 588 297

17 6 18.2 10 846 265

18 6 35 10 1424 858

19 4 45 15 624 195

82

Run Initial

pH

Temperature

(°C)

Biochar addition ratio

(g.L-1)

YH (mL.L-1) RH (mL.L-1.day-1)

20 6 35 1.6 901 371

Table 4.5 ANOVA analysis and the fitting model for YH

Source

Degree of

Freedom

Regression

coefficient

F

Value

p-value

Model 9 8.84 0.0011

Lack of fit 5 3.58 0.0938

R2 =0.89

A-pH 1 137.79 4.99 0.0494

B-temperature 1 -65.37 3.70 0.0834

C-biochar 1 5.30 0.039 0.8469

AB 1 -56.13 1.80 0.2093

AC 1 -15.24 0.059 0.8131

BC 1 0.36 3.174E-005 0.9956

A2 1 -127.17 58.43 < 0.0001

B2 1 -158.65 13.20 0.0046

C2 1 -105.25 6.18 0.0322

The ANOVA analysis was performed to evaluate the adequacy of the quadratic models and the

results are presented in Table 4.5 for YH and Table 4.6 for RH. As can be seen, the p-values of

both YH (p= 0.0010) and RH (p= 0.0006) are less than 0.05, indicating Equation 4.1 and

Equation 4.2 represent the experimental data very well [177]. The “lack of fit” describes the

fitness between the model predictions and the experimental data. The p-values of “lack of fit”

lower than 0.05 show a significant “lack of fit” of the model. Thus, a value > 0.05 is desired. In

this study, the p-values for both YH (p=0.09) and RH (p=0.11) are higher than 0.05 [187],

83

showing the “lack of fit” of the models were not significant. The R2 (squared regression

statistics) values of both fittings of YH and RH are 0.89 and 0.90, respectively. These values are

relatively high [179], showing that the quadratic models are sufficiently good to predict YH and

RH.

Table 4.6 ANOVA analysis and the fitting model for RH

Source

Degree of

Freedom

Regression

coefficient

F

Value

p-value

Model 9 10.16 0.0006

Lack of fit 5 3.16 0.1164

R2 = 0.90

A-pH 1 94.87 24.16 0.0006

B-temperature 1 -81.62 5.44 0.0419

C-biochar 1 8.41 0.036 0.8538

AB 1 -74.43 2.35 0.1563

AC 1 -13.47 0.17 0.6861

BC 1 -0.31 9.528E-005 0.9924

A2 1 -315.91 21.72 0.0009

B2 1 -150.17 33.80 0.0002

C2 1 -102.76 14.88 0.0032

Equations 4.1 and 4.2 derived from the quadratic model were further validated against the

experimental data. The experiments were performed under random combinations of the three

operation factors. The experimental conditions and the results of YH and RH are presented in

Table 4.7. YH and RM were also predicted using Equations 4.1 and 4.2 and the predicted results

are listed in Table 4.7. As seen from Table 4.7, the deviations between the experimental data

84

and the calculated results are less than 8% for YH while less than 10% for RM. This proves that

the quadratic model is adequately capable of predicting the H2 production.

The optimum biochar addition, initial pH and temperature were obtained using the numerical

optimisation function of Design Expert Software ver. 11 [188]. According to the optimisation

analyses, the optimum conditions with 0.93 desirability was found to be at the biochar addition

ratio 10.1 g.L-1, initial pH 6.4 and temperature 32C, reaching the maximum values of 1,330.7

mL.L-1 for YH and of 762.5 mL.L-1.day-1 for RH. The optimum addition of biochar at the range

tested was 10 g.L-1, which is at range of optimum biochar suggested by other studies. The

optimum biochar addition at the range of 10 -12.5 g.L-1 was reported to enhance H2 production

in several studies [113, 128]. Lin and Lay reported that in their study, H2 production was

occurred between pH 4 and 7, however, the maximum H2 was found to be at pH value between

6 and 7 [189], as suggested in this current study (pH 6.4).

Table 4.7 Model validation results

pH

Tempe-

rature

(°C)

Biochar

(g.L-1)

Cumulative YH (mL.L-1) RH (mL.L-1.day-1)

Predicted Experiment

Deviation

(%)

Predicted Experiment

Deviation

(%)

4 35 10 898 910 1 468 499 7

4 45 15 659 678 3 216 195 10

6 35 18.4 1033 1094 6 445 459 3

6 35 10 1309 1300 1 733 751 2

6.4 32 10.1 1331 1225 8 763 706 8

Normally, the first phase is operated at thermophilic condition for enhanced substrate

degradation [41]. Higher temperature may be beneficial for the reaction kinetics, but it is

typically followed by a rapid pH decrease which inhibits especially H2 production [102].

Appropriate temperature allows optimum germination, acclimatisation of bacteria to substrate

85

used in the system, carbon consumption rate and partial pressure of the produced gas [41, 77].

Other study suggested that at operating temperature range of 25-45C, the maximum H2

production was achieved at 35C. The reactor produced the minimum production at 25C and

inhibited at 45C [77]. In this current study, the similar optimum temperature was suggested

(32C).

4.3.2 Hydrogen yield

The response surface plots from the RSM analysis showing the influence of interactions

between variable on the YH are presented in Figure 4.4. Figure 4.4(a) illustrates the effect of

biochar addition ratio on YH at various pH values at 32C. The two-dimensional contour curves

beneath the 3D surface show a clear elongated running diagonally shape. This indicates a

significant interaction between biochar and initial pH [179]. It is evident that, at each pH, YH

increased first and then decreased with increasing biochar addition ratio from 1.6 to 18.4 g.L-1,

reaching a maximum value at biochar addition ratio 10g.L-1. The maximum YH increased with

increasing initial pH, peaking at pH 6 and then decreased as the initial pH was further increased

to 9.4.

As can be seen from Figure 4.4(a), the effect of biochar in enhancing YH was more profound at

a lower pH. For instance, at pH 4 and temperature 35C, the 10 g.L-1 biochar addition increased

YH by 57% compared to biochar addition 1.6%, while at pH 6, the increase was 30%. Starting

at low initial pH, the culture experienced delay in H2 production caused by longer adaptation

period [160, 190, 191]. Also, it is reported that at the pH below 4, the hydrogenase activity

declined [183]. The biochar used in the study, contains 10wt% of volatile matter and

micronutrients (Na, K, Mg, Fe) which was suspected to enriched the microbial growth and

initiated H2 production. In addition, our previous study showed that biochar addition prevented

a sharp pH decrease caused by accumulation of the acids produced in the first phase of TPAD

[98, 131, 161].

86

Figure 4.4 Response surface and contour plots of cumulative H2 yield (YH) over 8 days of

operation as a function of: (a) initial pH and biochar addition ratio at 32C and

(b) temperature and biochar addition ratio at initial pH 6.4

Figure 4.4 (b) shows the effect of biochar addition on YH at different temperatures while at a

constant initial pH 6.4. It is clear that, at each temperature, YH increased first with increasing

biochar addition ratio, reached the maximum at the biochar addition ratio 10 g.L-1, and then

decreased as biochar addition ratio rose to 18.4 g.L-1. As the temperature changed from 18 to

52 C, the maximum YH increased first and then decreased, reaching a peak value at temperature

(b)

(a)

87

35 C. It is noted that the influence of biochar addition ratios on YH at various temperatures was

similar, suggesting that the biochar effect on YH was not sensitive to changes in temperature. It

is estimated that the addition of biochar from 1.6 to 10 g.L-1 increased the YH around 30-39%

at temperature range 25 – 35%. Temperature was found to more significantly affected RH as

discussed in Section 4.3.3.

4.3.3 Hydrogen production rate

Figure 4.5 shows the 3D surface responses and the contour plots of combined effects of (a)

biochar addition and initial pH (b) biochar addition and temperature on RH. As seen from Figure

4.5 (a), it is clear that the effect of biochar on RM was significantly affected by initial pH of the

cultures. At each initial pH, RH reached a maximum value at the 10g.L-1 biochar addition ratio.

However, the maximum RH changed significantly at different initial pH. For instance, with the

addition of 10 g.L-1, RH increased drastically from 27 mL.L-1 at initial pH 2.6 to 733 mL.L-1 at

initial pH 6, remained almost constant at pH 8 (744mL.L-1), and decreased gradually at pH 9.4

(600 mL.L-1).

This implies that the effect of biochar in enhancing RH was more profound at lower pH. For

example, at initial pH 4, as the biochar addition ratio increased from 1.6 to 10 g.L-1, RH

increased by almost three folds, whereas at initial pH 6, RH only increased by 83%. At higher

pH, it has been reported that RH of the cultures plummeted three folds as the initial pH increased

from 6 to 7 [91] due to the poor ability for the cultures to degrade glucose used in their study.

However, at the optimum biochar addition ratio 10 g.L-1, as observed in this study, RH remained

almost stable while pH was increased from 6 to 8. This indicates that biochar could help stabilise

the activity of the cultures in degrading feedstock to acids and converting it to H2, at higher

initial pH.

88

Figure 4.5 Response surface and contour plots of maximum H2 production rate (RH) as

function of: (a) initial pH and biochar addition ratio at 32C; and (b) temperature

and biochar addition ratio at initial pH 6.4

Figure 4.5(b) shows the effect of biochar addition ratios on RH at different temperatures at initial

constant pH 6.4. At each temperature tested, the RH peaked at 10 g.L-1 addition of biochar.

However, the value of maximum RH varied significantly, increasing from 395 mL.L-1.day-1 at

temperature 18C to 733 mL.L-1.day-1 at the temperature 35C, and decreased to 176 mL.L-

1.day-1 at 52C. The microbes in cultures were more sensitive to higher temperatures. This is

consistent with literature reports that too high temperatures could deactivate and denature

(a)

(b)

89

enzyme and related proteins [192]. It is clear that with the biochar addition, RM substantially

increased regardless of temperature. However, such enhanced behaviour due to biochar addition

was more remarkable at higher temperatures. For example, the RH increased by 150% when the

biochar addition ratio increased from 1.6 to 10 g.L-1 at 45C, compared to only 72% at 35C

with the same addition ratio of biochar. The possible mechanisms of biochar promoting H2

production will be elaborated in Section 4.4 and 4.5.

4.4 Effect of biochar on H2 production via anaerobic digestion as

compared to other solid additives: role of acidity

In the previous sections, it has found that biochar addition significantly increased the H2 yield

by 31% in the first phase of a two-phase anaerobic digestion [161]. It was hypothesised that the

acidity and surface structure of the biochar play an important role. The high accumulation of

volatile fatty acid (VFA) in AD would reduce the pH of the culture supressing H2 production.

Alkaline additives have been proposed to add into AD to maintain or increase the alkalinity of

the system [193]. Several alkaline additives such as lime mud from paper making and calcined-

red mud showed positive effects on the H2 production [73, 194, 195]. Biochar can be alkaline

depending on the source of biomass and the pyrolysis temperature [193]. This section reports a

systematic investigation into the possible working mechanisms of biochar in enhancing H2

production in the first phase of TPAD. In order to achieve this objective, the effect of biochar

and several other solid additives with different acidity as references, namely alumina, glass and

zeolite, were tested and compared. The effect of these additives on the cumulative H2

production (YH), H2 production rate (RH) and pH during the operation were monitored.

90

Figure 4.6 The pH of liquid culture with the addition of the additives before the pH

adjustment

When the additives were mixed with the feedstock and sludge, they affected the pH of the

culture significantly. Figure 4.6 shows the pH of the culture with the addition of the additives

before it was adjusted to initial pH 6.4. The pH of the control and the culture with glass addition

were similar at 6.7. It is clear that the addition of glass particles into the cultures did not alter

the initial pH because the pH of the glass is neutral. The addition of alumina and biochar brought

the pH of the culture higher than that of the control. The pH of the culture dropped significantly

to 6.3 with the zeolite addition.

Figure 4.7 shows the daily H2 production rate (RH) of the cultures with addition of different

additives. The culture started to produce H2 after 12 hours of incubation. The culture with

alumina addition produced the highest rate of H2 on the first day, followed by the cultures with

additions of biochar. The glass bead addition did not change the H2 production rate, as expected.

The zeolite addition slowed down the H2 production. Most of the cultures reached the peak

production rate on the first day except the culture with zeolite addition, which experienced a

significant delay of reaching the peak value on day 2. The H2 production of all cultures with

91

and without the addition of the additives dropped on the day 2.5 and stopped producing H2 on

day 3.

Figure 4.7 Production rates of H2 from cultures with different types of additives

Figure 4.8 shows the comparison of cumulative H2 yields (YH) of the cultures with addition of

different additives during 7 days of incubation. The cultures with biochar and alumina addition

produced similar YH, which was much higher than the other cultures. The glass particle addition

did not affect YH while the zeolite addition showed slightly higher YH compared to the control.

It is clear that the cultures with addition of biochar and alumina had shorter lag phase than the

rest of the treatment, while the culture with zeolite addition experienced a delay in H2

production.

92

Figure 4.8 Cumulative yields of H2 from cultures with addition of different additives

The experimental data was then fitted using Gompertz model to obtain the maximum H2

production potential (P), maximum H2 production rate (Rmax) and lag phase () [196]. Table

4.8 shows a good agreement of the experimental data with the model fitting with the R2 of 0.97-

0.99. The fitting curves are also shown in Figure 4.8. The P values of the cultures with biochar

and alumina additions were predicted to be 1,234±14 and 1,216±14 mL H2.L-1, being higher

than the control by ca. 10%. However, the addition of glass particle and zeolite did not change

the P. The addition of different additives also significantly affected the Rmax of the culture. The

Rmax of the cultures with biochar and alumina additions were projected to be 1,148±98 and

1,076±120 ml H2.l-1.day-1, which was 32 and 24% higher than that of the control, respectively.

The calculated suggests that the zeolite addition would delay the hydrogen production, which

is consistent with the experimental observations.

93

Table 4.8 The results of calculation using the modified Gompertz fitting equation on

hydrogen production with different additives

Parameter Control Biochar Alumina Glass Zeolite

R2 0.99 0.99 0.97 0.99 0.99

P (ml H2.l-1) 1118±12 1234±14 1216±20 1127±8 1144±12

Rmax (ml H2.l-1.day-1) 870±60 1148±98 1076±120 969±54 957±67

λ (days) 0.59±0.05 0.59±0.05 0.51±0.07 0.62±0.03 0.65±0.05

Figure 4.9 The pH evolution of the cultures with different types of additives

The changes of pH of each treatment during the whole period of incubation are shown in Figure

4.9. It is clearly seen that the addition of biochar and alumina brought the initial pH of the

culture from 6.7 to 7.0 and 7.5, respectively, while the zeolite addition reduced the pH of the

culture. Prior to the incubation, the pH of all cultures was adjusted to 6.4. During the incubation,

the pH of all the cultures dropped significantly on day 1 and further decreased on day 2 but

slightly increased on the final day. However, the pH of the cultures with biochar and alumina

additions had higher pH than that of the control except on day 1. The culture with the zeolite

94

addition also showed a slightly higher pH than the control although zeolite itself had a very low

pH value.

The results suggest that the alumina and biochar addition in the culture generally increased both

RM and YH compared to the control while the zeolite addition suppressed H2 production. The

possible working mechanisms of biochar are discussed as follows. The biochar used in current

study is a pine wood biochar with a pH of 8.6. It is known that high pH promoted H2 production

in the AD [190, 197]. The biochar and alumina addition significantly increased the pH value of

the culture. Therefore, in the beginning of the incubation, the cultures with biochar and alumina

addition produced significantly higher amount of H2 compared to the control. The rapid H2

production usually results in quick accumulation of acids, decreasing the pH of the culture [14].

This explains why all the cultures showed a pH drop and the cultures with biochar and alumina

addition experienced even a greater pH drop compared to the control on day 1 (Figure 4.9).

However, as the incubation time increased, the cultures with biochar and alumina additions

produced more H2 but also exhibited higher pH in comparison with the control. This suggests

that the biochar and alumina acted as a pH buffer of the cultures. The biochar contains some

alkaline groups that may act as neutralizer for certain acids [73]. Due to the high acidity, the

addition of zeolite reduced the pH of the culture significantly (Figure 4.9 - initial). This explains

why the culture with the zeolite addition produced a very low amount of H2 on the first day and

it took a longer time for the culture to adapt the environment (Figure 3). Due to the slow H2

production rate, the amount of acids generated during the incubation process was low, which

was inadequate to cause dramatic pH drop as experienced by the rest of the treatment as

observed in Figure 4.9.

95

4.5 Mechanisms

The preceding discussion has shown that biochar was beneficial in enhancing H2 in TPAD. A

mechanism of the working of biochar in enhancing H2 production in TPAD has been proposed,

as shown in Figure 4.10, and is discussed as follows.

Figure 4.10 The mechanisms of biochar in promoting H2 and CH4 productions in TPAD from

food waste

The results show that the biochar addition in the culture promoted H2 production at all initial

pH and temperature tested. In the H2 production process, it is believed that biochar enhanced

biofilm formation [3], provided temporary nutrients for microbial growth and buffering pH of

the culture. A possible mechanism of biochar in enhancing H2 is that the biochar provides

surface area to initiate biofilm formation to immobilise microbes. Biochar has high surface area

(130 m2.g-1), facilitating microbial colonisation in the anaerobic digestion process [27]. Figure

4.10 shows the SEM images of the biochar samples before and after anaerobic digestion of

culture with 10 g.L-1 addition, at initial pH 6 and 35ºC. It is observed that there was the clear

development of biofilm on the surface of the biochar and the rod shape of bacteria on the surface

of biofilm was also evident.

The length of bacteria ranges from 2-5 µm, a typical characteristic of H2 producing bacteria

(HPB) as reported by Yin et al [198]. The development of biofilm in the biochar is explained

as follow. The biochar used in this study has a high specific surface area (130 m2g-1) which

provides the surface area for the initial attachment of the microbes. It is known that the microbes

First phase

▪ Promoting biofilm formation

▪ Providing temporary nutrients

▪ Buffering pH

Biochar

Results:

▪ Shorter lag phase

▪ Faster VFA generation

▪ Higher H2 production rate

▪ Higher cumulative H2 production

96

could produce extracellular polymeric substances (EPS), which consist of polysaccharides,

proteins, nucleic acids and lipids [85]. EPS is responsible for the nutrient supply, stability of

the bacteria and act as polymer bridges between microbes [85, 126, 199]. Once the microbes

were attached on the surface of the biochar, the microbial division, growth and colonisation

happened. The enriched microbial population in the microbial carrier has reported to enhance

biogas production [124, 199], which was confirmed by the results of the current study.

Figure 4.11 SEM images of (a) biochar and (b) final effluent of H2 production

HPB

Biofilm

Biochar

(a)

(b)

97

In addition, as Lehmann [27] stated that biochar produced from low temperature pyrolysis

contains temporary substrate to support microbial metabolism and growth [27]. Our previous

study showed that the biochar used in this study contained 10 wt% of volatile matter that would

serve as temporary nutrients in the beginning of H2 production [200]. It also contains some

beneficial temporary micronutrients that may be useful for microbial growth and enzyme

activation. Our previous study has found that the biochar leached sodium (Na) (183 mg.kg

biochar-1) and potassium(K) (1639 mg.kg biochar-1) at a leaching temperature of 30ºC for 24

hours [110]. Light metals such as Na+ and K+ are necessary for microbial growth [123, 201].

The Na+, especially, is useful for cofactor of bacterial enzyme, transport processes, the

formation of adenosine phosphate (ADH) and dehydrogenase [123, 201]. Lin et al reported that

Na, together with magnesium (Mg), zinc (Zn) and iron (Fe) were important trace elements in

H2 production from synthetic substrate using anaerobic sewage sludge as the inoculum and

incubated at 35ºC [201, 202]. It is also suggested that at moderate concentration (< 400 mg.l-1),

K stimulates AD process, promoting H2 production [41, 125, 203]. The provision of temporary

nutrients and trace element supported microbial growth and activity in the H2 production. It is

evident that biochar addition shortened the lag phase and produced higher VFA in cultures.

Finally, it is also worth noticing that at the end of the AD experiment (Section 4.1), the pH of

the control was 3.0 while the pH of the cultures with 16.6; 25.1 and 33.3 g.L-1 biochar additions

was 3.5; 3.7 and 3.7, respectively. This pH stability prevented the culture from significant

decrease in the pH caused by VFA accumulation, which is known to inhibit anaerobic digestion

[204]. This allowed the systems with biochar addition to achieve a higher H2 production rate

and accumulation.

Also, the biochar addition increased the YH in all pH tested in bench scale experiment under

different operating conditions, particularly at the lower pH. At the lower pH, the culture usually

produced lower amount of H2 since it prevents a further use of feedstock for H2 production due

to rapid acid inhibition and pH drop [190, 205]. The biochar used in this study is an alkaline

98

additive (pH 8.6) which help alleviate the negative effect of pH drop due to the accumulation

of volatile fatty acid (VFA) [205]. Biochar also contains some alkaline groups that may act as

neutraliser for certain acids [73], thus supports the culture to produce higher H2 production.

Further experiment suggests that biochar behaves similarly with the alumina as a reference. The

biochar and alumina addition significantly increased the pH value of the culture. Therefore, the

cultures with biochar and alumina addition produced significantly higher amount of H2 and a

greater pH drop compared to the control, in the beginning of the incubation. However, the

cultures produced more H2 but also exhibited higher pH in comparison with the control during

the rest of the operation. This suggests that the biochar and alumina acted as a pH buffer of the

cultures, enhancing the H2 production.

4.6 Summary

The effect of biochar addition on the H2 and CH4 production in laboratory scale TPAD of

simulated carbohydrate food waste was studied systematically. Biochar addition was shown to

shorten the lag phase by 21.4 to 35.7 %, increased the maximum production rate by 32.4% and

H2 production potential by 14.2 to 31 % of H2.

Further investigation on the effect of biochar addition under different initial pH and temperature

on the cumulative H2 yield (YH) and maximum daily production rate (RH) in the first phase of

TPAD was conducted and optimised using the response surface methodology (RSM). The RSM

analysis showed that the maximum YH of 1,331 mL.L-1 and RH of 763 mL.L-1.day-1 could be

achieved under the optimum conditions of biochar addition ratio 10.1 g.L-1, initial pH 6.4 and

temperature 32C. Biochar addition was shown to substantially increase YH, especially at lower

pH and higher temperatures.

It is observed that the biochar initiates the biofilm formation and provides temporary nutrients

in the culture, enriching the microbial population. Also, the addition of biochar was observed

99

to bring the condition of the culture from acidic to alkaline at the beginning of operation and

prevented significant pH drop during incubation. As a result, the cultures with biochar additions

generated more H2 at faster rates.

100

Chapter 5 Effect of Biochar Addition on Methane Production

5.1 Introduction

Chapter 5 reports the experimental study on the bench scale of the CH4 production. Section 5.2

discusses the CH4 production without biochar addition. Section 5.3 presents the results on the

effect of biochar addition under different initial pH and temperature according to Box Behnken

Design (BBD) and response surface methodology (RSM). The result on additional experiment

on the single phase anaerobic is reported in Section 5.4. Possible mechanisms of the biochar in

enhancing CH4 in the second phase of TPAD are proposed in Section 5.5.

5.2 Methane production without biochar addition

Figure 5.1 Cumulative yields and production rates of CH4 without biochar addition

As shown in Figure 5.1, in the second phase, the culture started to produce a substantial amount

of CH4 on day 9. The CH4 production continued and achieved the peak production rate on day

15 with 139 mL.L-1 of CH4 followed by a significant drop on day 19. The CH4 production

completely stopped on day 39. The maximum cumulative CH4 production in the end of

101

operation was 1070±3 mL.L-1. CH4 composition in the biogas produced from the system at the

conclusion of an experimental run was found to be in the range of 55-78% (Table 5.1). The H2

production was not observed in the second phase. The fresh sludge containing methanogenesis

added into the second phase converted the acetic acids produced in the first phase to CH4.

Therefore, there was no H2 was detected in the second phase. Low amount of VFA remained in

the final effluent of the second phase. The acetic acid decreased from 1063 to 78 mL.g-1 at the

end of the first phase, while butyric decreased from 1172 to 44 mL.g-1.

Table 5.1 Profiles of the second phase of TPAD without biochar

Parameters Unit Value

Cumulative CH4 production mL.L-1 1070±3

Gas composition

H2 % 0

CH4 % 55-78

CO2 % 22-45

VFA mg.L-1

Acetic acid mg.L-1 78±34

Butyric acid mg.L-1 44±15

Propionic acid mg.L-1 119±0

Final pH 6.7±0.1

5.3 Methane production with biochar addition

The cumulative CH4 production of the cultures with and without biochar is shown in Figure

5.2. In the beginning, all cultures with biochar addition produced higher CH4 yields than the

control. However, from day 29 until the end of the experiment, the cultures with 25.1 and 33.3

g.L-1 biochar additions started to show less CH4 yields and production rate than the control. At

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the end of the experiment, the highest cumulative CH4 production was achieved by the culture

with 8.3 g.L-1 biochar addition, while the culture with 33.3 g.L-1 biochar addition exhibited the

lowest cumulative CH4 production, even lower than that of the control.

Figure 5.2 Cumulative CH4 yields at different biochar addition ratios

Table 5.2 One-way ANOVA and post hoc analysis on cumulative CH4 production in

different biochar addition ratios

Biochar addition

(g.L-1)

Average cumulative CH4

yields (mL.L-1)

Standard deviation P value

0 1070.0b 5.1 0.002

8.3 1136.6b 7.0 0.002

16.6 1057.3b 8.7 0.002

25.1 956.2a 4.9 0.002

33.3 931.7a 1.8 0.002

ANOVA analysis on the cumulative CH4 production data shows a significant difference in

methane production among the various treatments as listed in Table 5.2. However, the LSD

103

post hoc test suggested that the cumulative CH4 yields of cultures with 0, 8.3 and 16.6 g.L-1

biochar addition were not significantly different between each other but they were significantly

higher than that of cultures with 25.1 and 33.33 g.L-1 biochar addition according to a post hoc

analysis.

Table 5.3 The results of calculation using the modified Gompertz fitting equation on CH4

production with different biochar addition ratios

Biochar

addition (g.L-1)

λ (day)

Rmax

P (mL.L-1) R2

(mL.L-1.per day)

0 10.0±0.2 113±5 1027±9 0.98

8.3 5.9±0.2 156±7 1126±7 0.98

16.6 5.7±0.1 160±3 1046±3 0.99

25.1 5.5±0.1 145±3 949±2 0.99

33.3 5.7±0.2 138±8 918±6 0.98

The experimental data of cumulative methane production was also fitted using the Gompertz

model and the fitted curves were presented in Figure 5.2. The R2 of all of the fittings ranged

from 0.98 to 0.99, suggesting a good fitting between experimental data and the modelling

results, thus the feasibility of using the Gompertz model for the calculation of λ, P and Rmax.

The λ, P, and Rmax against operation time derived from the model are presented in Table 5.3.

As shown in Table 5.3, the λ of the cultures with various biochar additions were in the range of

5.5-5.9 days, while the lag phase of the control was 10.0 days. It is also clear that the Rmax of

the cultures with biochar additions were higher than that of the control. However, the Rmax

increased as the biochar addition increased from 0 to 16.6 g.L-1 biochar and then decreased as

the biochar addition was further increased to 33.3 g.L-1.The P of the cultures with 8.3 and 16.6

g.L-1 additions were higher than that of the control, whereas, the cultures with higher biochar

104

additions showed lower P. The highest P was achieved for the culture with 8.3 g.L-1 biochar

addition. A proposed detailed mechanism of biochar effects on CH4 production are explained

in section 5.2.3.

(a)

(b)

(c)

(d)

(e)

Figure 5.3 VFA profiles during CH4 production in culture with (a) 0; (b) 8.3; (c) 16.6; (d)

25.1 and (e) 33.3 g.L-1 biochar addition ratios

The VFA profiles of the cultures on Day 7, 14, 21, and at the end of the experiment were

illustrated in Figure 5.3. The results confirm that the dominant VFA were acetic, acetate and

105

propionic acids [180]in all cultures. All cultures with biochar addition degraded VFA faster

than without biochar during the first 14 days of the experiment. VFA of all cultures significantly

degraded by Day 21 and propionic acid accumulated in all cultures. The propionic acid content

became higher with increasing biochar addition. In the end of the experiment, the cultures with

25.1 and 33.3 g.L-1 biochar addition had more propionic acid accumulation. This might be the

cause of lower CH4 production of these two cultures.

In addition, as shown in Figure 5.3, final cultures with 25.1 and 33.3 g.L-1 biochar remained in

the reactors, which were used as influent for methane production, had lower quantities of VFA

and higher amounts of propionic acid. According to Wang et al (2009) and Amani et al (2011),

the energy to anaerobically oxidize propionic acid to acetate (+76.1 kJmol-1) is almost doubled

as compared to that for butyrate (+48.1 kJ.mol-1). Therefore, the accumulation of propionic acid

in the culture resulted in a slower acetogenic rate of propionic acid [180, 206], as a consequence,

a lower CH4 production rate. The accumulation of propionic acid also led to low pH. When the

pH is reduced to below 7.0 methanogenic activity is often inhibited [58].

The pH and temperature are the two important factors affecting CH4 production in the second

phase of TPAD due to their influences on microbial growth and activity and metabolic products

generated [86]. It is well known that the optimal conditions for CH4 production are in the pH

range from 6.7 to 7.4 [207] and more stable under mesophilic conditions [2, 105]. However,

the behaviour of the cultures with biochar addition under different initial pH and temperature

regimes is still not clear. Therefore, the present chapter reports the examination of the effect of

biochar addition on CH4 production in the second phase of TPAD, utilising the pre-digested

feedstock from the first phase of TPAD (as prepared in Section 3.2.3.2), under different initial

pH and temperature conditions.

106

5.3.1 Response surface analysis

The experimental data as listed in Table 5.4 were fitted using the quadric model as shown in

Equation 3.3 resulting in the following equations:

𝑌𝑀 = −7047.15 − 54.13𝐴 + 290.33𝐵 + 863.49𝐶 − 1.58𝐴𝐵 + 7.60𝐴𝐶 + 11.51𝐵𝐶 +

3.54𝐴2 − 4.97𝐵2 − 83.70𝐶2

(5.1)

𝑅𝑀 = −1837.63 − 12.75𝐴 + 45.08𝐵 + 323.08𝐶 − 0.33𝐴𝐵 + 0.59𝐴𝐶 + 5.31𝐵𝐶 +

0.83𝐴2 − 0.97𝐵2 − 31.61𝐶2

(5.2)

Table 5.4 Box Behnken design and experimental results for CH4 production

Run Factor 1

A: Biochar

addition (g.L-1)

Factor 2

B: Temperature

(oC)

Factor 3

C: initial

pH

Response

1 YM

(mL.L-1)

Response 2 RM

(mL.L-1. day-1)

1 10 35 7 1532.8 388.4

2 10 45 9 1392.2 525.1

3 10 45 5 0.3 0.2

4 10 25 5 582.2 137.134

5 15 35 5 1183.8 212.5

6 5 45 7 1457.9 616.3

7 15 35 9 1770.6 492.1

8 10 35 7 1771.2 508

9 15 45 7 1325.4 494.6

10 10 35 7 1655.2 474.3

11 5 25 7 877.4 216.6

107

Run Factor 1

A: Biochar

addition (g.L-1)

Factor 2

B: Temperature

(oC)

Factor 3

C: initial

pH

Response

1 YM

(mL.L-1)

Response 2 RM

(mL.L-1. day-1)

12 5 35 9 1348.3 460.7

13 15 25 7 1060.3 160.3

14 10 35 7 1490.1 442

15 10 35 7 1492.1 429

16 5 35 5 1065.6 205

17 10 25 9 1052.7 237

Table 5.5 lists the results of ANOVA analysis of the fitting models of both YM and RM. The

probability (p value) of the quadratic models for both YM and RM were less than 0.05, indicating

the significance of the quadratic model in fitting and representing the experimental data. The

R2 (squared regression statistics) values of both fittings of YM and RM are 0.89 and 0.90,

respectively. These values are relatively high [179], showing that the quadratic models are

sufficiently good to predict YM and RM.

Using Design Expert ver. 11, the optimum biochar addition, temperature and initial pH were

then optimised. The calculation suggested the optimum biochar addition; temperature and

initial pH were 12.5 g.L-1, 36.2oC and 7.8, respectively. In the optimum condition, the YM and

RM were estimated to be 1755.3 mL.L-1 and 500.9 mL.L-1.day-1. The optimum addition of

biochar at the range tested was 12.5 g.L-1, which is at range of optimum biochar suggested by

other studies.

Literature suggest that the optimum biochar was at 5- 70 g.L-1 depending on the AD feedstock

and the type of biochar [3, 131]. It is well known that the optimum pH range for CH4 production

is 6.7 – 7.4, which is slightly lower than the optimum pH suggested by the current study (7.8).

108

Normally, the second phase is operated under mesophilic condition for higher CH4 production

[41]. Optimum temperatures for microbial growth in typical mesophilic CH4 production are

listed at range 30 – 40C, depending on the source of inoculum and microbial genus, which also

reported in this study (36.2C) [45, 99-101].

Table 5.5 ANOVA analysis and the fitting model for YM and RM

Source

YM RM

Degree of

freedom

Regression

coefficient

p-

value

Degree of

freedom

Regression

coefficient

p-

value

Model 9 0.0126 9 0.0153

R2 0.8885 0.8814

A-biochar

addition

1 -54.13 0.3883 1 -12.75 0.6045

B-temperature 1 +290.33 0.3790 1 +45.08 0.0107

C-pH 1 +863.39 0.0038 1 +323.08 0.0027

AB 1 -1.58 0.5099 1 -0.33 0.7291

AC 1 +7.60 0.5247 1 +0.60 0.8989

BC 1 +11.52 0.0821 1 +5.31 0.0516

A2 1 +3.54 0.4497 1 +0.83 0.6544

B2 1 -4.97 0.0028 1 -0.97 0.0641

C2 1 -83.70 0.0193 1 -31.61 0.0243

5.3.2 Methane yield

The response surface plots from the RSM analysis showing the influence of interactions

between variable on the YM are presented in Figure 5.4. Figure 5.4 (a) shows the effect of

109

biochar on the YM under different temperatures at optimum pH 7.8. Regardless of the

temperature, the YM increased slightly with the increase of biochar from 5 to 15 g.L-1. It is

evident that the temperature affected the YM significantly. The value of YM increased as the

temperature increased, reaching its peak at 35 ºC then slightly decreased as the temperature was

further increased to 45ºC. The YM peaked at temperature 35 ºC regardless of biochar addition.

(a)

(b)

Figure 5.4 Response surface and contour plots of CH4 yield (YM) as function of: (a) biochar

addition and temperature at initial pH 7.8 and (b) biochar addition and initial pH

at temperature 36.2ºC

110

Figure 5.4 (b) illustrates the effect of biochar on the YM at varying initial pH at temperature

36.2 ºC. It is clear that at low initial pH, biochar at various addition ratios gave similar YM. At

high initial pH, a higher biochar addition ratio resulted in higher YM. It is also noted that initial

pH greatly affected the YM. The YM was similar at initial pH 7 and pH 9, while the YM at pH 5

was very low.

5.3.3 Methane production rate

Figure 5.5 Response surface and contour plots of maximum CH4 production rate (RM) as

function of: (a) biochar addition and temperature at initial pH 7.8 and (b) biochar

addition and initial pH at temperature 36.2ºC

111

Figure 5.5 illustrates the response surface plots of RM as a function of various factors. Figure

5.5 (a) shows that the increase in temperature significantly increased the RM. Regardless of the

biochar addition ratio, the highest value of RM was always achieved under the thermophilic

conditions.

It is noted from Figure 5.5 (b) that higher initial pH led to greater RM regardless of biochar

addition ratio. This suggests that alkaline conditions support CH4 production in the culture. It

has been reported [208] that alkaline conditions promote activity of methanogens and a

shortened lag phase.

Comparing the YM and RM among cultures, the higher temperature led to the higher RM of the

cultures. However, while the higher initial pH and temperature led to the highest RM, it is noted

that at the end, they produced a similar amount of YM to the cultures set at neutral pH and

mesophilic temperature. Parawira et al [105] reported in their study of TPAD of potato waste,

thermophilic condition is to be preferred to shorten the operation duration, while mesophilic

condition supports the system to achieve the higher CH4 yield.

5.4 Methane production in single phase anaerobic digestion

A single-phase AD simulation was conducted using the same feedstock concentration and

operating conditions as the experiment in Section 5.3.2. The single phase of AD was added with

10 (g VS). L-1 of fresh bread, 10 g.L-1 of biochar set at initial pH 7 and incubated at temperature

35C.

The result shows that with the addition of 10 (g VS). L-1 of white bread, the culture produced

H2 instead of CH4. The reactor started producing H2 on the first day, achieving the peak of 730

mL.L-1. The H2 production then gradually decreased and stopped on day 4. The total cumulative

H2 production was 1079 mL.L-1. The experiment was extended until 14 days, however the CH4

production was not observed until the end of experiment. The result suggests that the SPAD

112

simulated in the study may suffer from feedstock overloading. In the early stages of SPAD, the

accumulation of acid often decreases the pH (4.2), while the inhibitory level of VFA is increased

significantly. These factors disrupt the stability of the SPAD thus led reactor to a failure [47].

A conventional AD treating industrial wastewater is reported to be unfeasible at a loading rate

of 2 gr VS.l-1.day-1 or higher [48]. The high feedstock concentration in this study disrupted the

culture, thus it failed producing CH4.

The culture treating the effluent from the first phase TPAD showed CH4 production was 1533

mL.L-1 (Table 5.4). The separation of both phases allowed the hydrolysis and acidogenesis to

occur in the first phase, therefore the second reactor was prevented from the acid accumulation.

The VFA produced in the first reactor was then converted into CH4 in the second phase. The

result of this experiment suggests that the separation of the acidogenesis and methanogenesis

allowed the TPAD to produce CH4 particularly in the high concentration of feeding.

5.5 Mechanisms

In the CH4 production process, it is believed that biochar mostly acted as a good microbial

carrier in enhancing methane production. According to recent studies of the effect of biochar

addition on CH4 production in conventional single phase AD [3, 83], the porous structure of

biochar acted as a microbial carrier and favoured biofilm development, accommodating a wide

range of microbial population including acido, aceto and methanogens [140].

Figure 5.6 shows the SEM images of the biochar samples before and after 40 days of anaerobic

digestion at initial pH 7 and temperature 35 ºC. It is clear that there was biofilm on the surface

of the biochar. The rod (bacillus) shape of bacteria on the surface of biofilm was also observed

as shown on Figure 5.6 (b). Biofilm allows the colonisation of bacteria, fungi and protozoa

[123, 126], which benefits CH4 production in two ways. Firstly, it provides more surface area

available for microbial growth. The biofilm allows more intense cross feeding, co-metabolism

113

and H2 and proton transfer, which then enrich microbial growth and initiate the microbial

colonisation [124]. Secondly, it immobilises microbes, increasing the robustness of the AD

system. With a microbe attached to the biofilm, it has a lower probability of loss of microbes.

The promoted formation of biofilm is assumed to enhance microbial activity, leading to a

shorter lag phase, faster VFA degradation and CH4 production in the cultures with biochar.

Figure 5.6 SEM images of (a) the fresh biochar and (b) biochar after 40 days of incubation

at 35ºC

(b)

(a)

114

Note that pH values of the control (6.72) and cultures with biochar additions (6.96) were similar

(experiment Section 5.1). Therefore, it is assumed that unlike in the first phase, the pH buffering

capacity of biochar did not directly affect the pH condition of the cultures in the second phase

of TPAD. In other words, the pH buffering capacity of biochar is not a critical factor in

promoting CH4 production in the second phase.

5.6 Summary

A preliminary study on the effect of biochar addition on the CH4 production in laboratory scale

TPAD of simulated carbohydrate food waste was conducted. The biochar addition shortened

the lag phase by 41 to 45 %, increased the maximum production rate by 23.0-41.6% and CH4

production potential by 1.9 to 9.6% of CH4. Furthermore, the effect of biochar addition on CH4

production in the second phase of TPAD under varying initial pH and temperature conditions

has been studied. A moderate level of biochar addition, mesophilic temperature and neutral to

alkaline initial pH were shown to benefit CH4 production in the second phase of TPAD. In a

typical CH4 production process at initial pH 7 and 35 C, 10 g.L-1 of biochar addition increased

YM by 14% relative to cultures without biochar addition.

The effect of biochar addition was more profound at higher pH. The optimum biochar addition;

temperature and initial pH were found to be 12.5 g.L-1, 36.2oC and 7.8, respectively. Under the

optimum condition, the YM and RM were 1755 mL.L-1 and 500.9 mL.L-1.day-1. The SEM

analysis revealed the development of biofilm and the accumulation of the bacillus rods of

bacteria on the surface of biochar. The promoted methanogenic biofilm formation in the second

phase of TPAD may contribute to the enriched methanogens and enhanced CH4 production

An additional experiment of single-phase AD using fresh food waste was conducted. The result

suggests that at a high concentration of feedstock, the single-phase AD suffered from acid

accumulation thus failed to produce CH4, while the second phase TPAD successfully produced

115

substantial amount of CH4. It is due to the separation of acidogenesis and methanogenesis,

which favour CH4 production in the second phase AD.

116

Chapter 6 Transient Performance during Start-up of Two-Phase

Anaerobic Digestion Process Demonstration Unit

6.1 Introduction

Although there is a growing interest in biochar utilisation in AD, there are very limited reports

on the effect of biochar addition in a demonstration or pilot scale operation [140]. The studies

on the pilot scale operation of TPAD are necessary to confirm the results of bench/laboratory

studies and to investigate the technical challenges and the related troubleshooting required for

a real full-scale application [56]. Chapter 6 reports strategy and transient performance of the

TPAD PDU during the start-up period with biochar addition since this is a key step to achieve

a successful operation. During the start-up, rate, amount and composition of both H2 and CH4

and the production of metabolic products were studied [209]. The outcomes of this work are

expected to provide technical advices on the strategy of the start-up of TPAD PDU and

evidences on the beneficial effect of biochar in enhancing H2 and CH4 production in

demonstration scale [209, 210].

6.2 The start-up performance of the first phase

Figure 6.1 shows the gas production, composition and H2 yield of the first reactor during the

start-up period. The reactor experienced a delay for three days in producing biogas during stage

1. At the stages 2 and 3, the reactor started to produce biogas but the percentage of H2 was

negligible (Figure 6.1(b)). H2 was produced at the end of stage 3. Both the biogas production

and H2 composition continued to increase during stages 4 and 5, reaching up to gas production

of 63 L.day-1 of and H2 composition of 49% at the end of stage 5. At the stage 6, the gas

production decreased but the H2 composition in the biogas continued to increase. It is noted that

there was no trace of CH4 in the reactor during the whole start-up period, showing the

effectiveness of the sterilisation and sludge heating.

117

Figure 6.1 (a) Biogas production, (b) biogas composition, and (c) H2 and CH4 production

during the start-up of the first phase

(a)

(b)

(c)

118

Figure 6.1(c) shows the H2 yield during each stage. The H2 yield was negligible at the stages 1

to 2, reaching ca. 2 L H2 per gram VS of bread added at the end of stage 3. The H2 yield increased

significantly during stage 4 and fluctuated at a range between 22 - 46 L.kg VS-1 afterwards. The

decline in H2 yield in stage 6 of the first phase was believed to have been caused by VFA

accumulation, as discussed below [113, 161, 190].

The H2 production is typically accompanied with the generation of metabolic products, such as

VFA [190, 211]. The yield of H2 is significantly affected by the metabolic products and

microbial metabolism pathway [212, 213]. The investigation into VFA during the start-up gives

an insight on the possible metabolism pathway carried out by the culture in producing H2 [212].

A theoretical 4 moles of H2 is produced when the final product is acetic acid (HAc) and 2 moles

of H2 when butyric acid (HBu) is produced [214]. Thus molar ratio of acetate to butyrate

(HAc/HBu) has been used as an indicator of the efficiency of H2 production [213].

Figure 6.2 VFA of the first phase

Figure 6.2 presents the VFA profile during the start-up period of the first reactor. The culture

did not produce VFA at stage 1 and started to produce a low amount of acetic and butyric acid

at stage 2. The VFA production increased during stages 3 and 4 and reached the highest at stage

5 followed by a slight decrease at stage 6. This follows a similar trend to the H2 production as

119

shown in Figure 6.1(c). For instance, an increased H2 production was observed at stage 4 when

the culture produced higher VFA than the previous stages. At stage 5, the culture achieved the

highest H2 production and yields when the VFA production was also the highest. The HAc/HBu

during stages 5 – 6 was 1.3 – 1.6, which indicates a relatively high H2 production efficiency

compared to the literature reports [27]. In this study, the predominant VFAs were acetic and

butyric acids with very low propionic acid (HPr), indicating that the culture underwent a

favourable pathway for H2 production [213]. Propionic acid (HPr) is unfavourable for H2

production, thus a low amount of HPr in this study suggested an efficient H2 production [213].

Figure 6.3 pH and temperature during the start-up of the first phase

The temperature profile and evolution of pH during the start-up is shown in Figure 6.3. It is

evident that the temperature remained almost constant during the whole start-up period. It

should be mentioned that the temperature was slightly low in the first two stages due to the cold

weather, which might have delayed the biogas production at the beginning of the trial. The pH

changed significantly during the start-up. At stage 1, the pH remained at around 8 because the

culture was in an adaptation period with no VFA production. At stages 2 and 3, the culture

started producing VFA thus the pH decreased gradually. At the end of stage 3, the pH was 5.0.

At the beginning of stages 4, 5 and 6, the pH was fixed at around 6.0. This is due to the addition

120

of a base solution (4M NaOH, 1% (v/v)) in order to provide a favourable pH for the H2

producing bacteria. This suggests that in a typical pilot scale operation of H2 producing reactor,

an automatic addition of base solution, such as NaHCO3 for pH adjustment is needed [211]. In

the present study, it is observed that the pH was still in a favourable range for H2 production

during stages 1-3 and only a very small amount of base solution was required in the beginning

of stages 4-6. One possible reason may be due to the beneficial effect of biochar. The addition

of biochar has shown a potential to buffer the pH of the culture in the reactor [113, 161].

Figure 6.4 TS and VS of the first phase

The TS of the culture in R1 was 2.5% in the beginning of the trials and increased to 4.6% at the

end of operation. In addition, the VS (as a percentage of the TS) also slightly increased from

81.5 to 83.6% (Figure 6.4). The increase of TS and VS may be due to the accumulation of TS

and VS of the feedstock when the recirculation water was used for feeding in stage 5 and 6. The

increased TS and VS at stages 4 – 6 may also contribute to the increased H2 and VFA production

at those stages.

121

6.3 The start-up performance of the second phase

Figure 6.5 shows the biogas production during the start-up of the second reactor. The reactor

started to produce biogas at the end of stage 1. During stages 2 to 5, the biogas production

achieved the highest at the beginning and then dropped significantly towards the end at each

stage. This is believed to be caused by feedstock overloading (0.65 (g VS).L-1.day-1) and high

accumulation of VFA (up to 8200 mg.L-1) as discussed below.

The biogas production almost stopped at the end of stage 5 but resumed at stage 6 after 60 L of

sludge and 12.5 g.L-1 of biochar were added into the reactor. The biogas production fluctuated

at the range of 55 – 207 L.day-1 at stages 7 to 9. The CH4 production was negligible during

stages 1 to 5 and small amount of H2 was produced. The CH4 production increased while H2

composition decreased during the recovery period (stage 6). CH4 composition fluctuated during

stages 7-9 at the range between 35 – 59% with trace amount of H2 in the biogas. After the

recovery period of stage 6, the CH4 yield increased from 222 at stage 7 to 301 L.kgVS-1 at stage

9. There was no H2 data after stage 6 because H2 production potential had exhausted after stage

6 during this start-up run. In stage 6, the feeding was temporarily stopped, leading to the absence

of hydrolysis and thus H2 production ceased. After stage 6 (stages 7-9), when the feeding was

resumed, H2 produced during the hydrolysis was converted to CH4 quickly. This indicates the

effect of the addition of untreated sludge which is rich in methanogens in supporting CH4

production by converting the VFA accumulated in the beginning of stage 6 and the fresh

feedstock added at the beginning of stages 7-9 [215].

122

Figure 6.5 (a) Biogas production, (b) biogas composition, and (c) H2 and CH4 production

during the start-up of the second phase

(c)

(b)

(a)

123

Figure 6.6 VFA of the second phase

Figure 6.6 presents the VFA production during the start-up. At stage 1, the dominant VFA were

HBu and HAc. HPr was produced at stage 2. During stages 3 to 6, production of all acids

significantly increased. The accumulation of the VFA reached the highest at the end of stage 5,

with the concentration of each VFA following this order: HAc>HBu>HPr. The VFA

concentration was then decreased during the recovery period (stage 6). At the end of each stage

after recovery (stages 7 to 9), the VFA concentration was slightly increased, but showed much

lower than the peak observed at the end of stage 5. It is also observed that the concentration of

HPr was higher than HBu and HAc at stage 9.

During stages 1-5, the very low production of biogas and CH4 was mainly due to feedstock

overloading and the high accumulation of VFA. At the stage 5, the concentration of HAc and

HPr were 3310 and 1553 mg.L-1 which were much higher than their inhibitive concentration

which reported to be 2500 and 800, respectively [216, 217]. At the recovery stage (stage 6), the

VFA decreased for two reasons. First, feeding was stopped to prevent further accumulation of

the VFA. Second, untreated sludge, which is believed to contain abundant HAc consuming

microbes, was added into the tank [140, 168, 169]. At the end of the stage 6, HAc concentration

124

reduced by around 50% (Figure 6.6) and consequently the CH4 production increased from 0.38

to 33 L.day-1 (Figure 6(c)). HAc and HBu are the main precursors for CH4 production. Around

65-95% of CH4 was converted from HAc [218]. It is evident that HAc and HBu were

significantly decreased at stages 7- 9 along with a high production of CH4. The high percentage

of HPr at the stages 7-9 may be due to its inhibited degradation. The degradation of HPr was

inhibited when its concentration more than 800 mg.L-1 with pH 7 and required a longer time

compared to the degradation of HAc and HBu at the same concentrations [216]. Clearly, the

stepwise addition of unheated sludge during the operation was required. It is necessary to

convert the accumulated VFA to CH4. Some studies suggested the stepwise addition of acetate

metabolising microbes during the start-up period [140, 168, 169]. This strategy is also found to

be effective in the current study.

Figure 6.7 describes the changes of pH and temperature during start-up of R2. Similar to the

R1, an adaptation period occurred during the stage 1 of the trial. During stages 1 to 3, the pH

was at the range of 5 – 7.5. At stages 4 and 5, extreme pH drops occurred caused by a high VFA

accumulation (Figure 6.6), leading to significant drop in the production of biogas and CH4.

Although, 4M of NaOH (1% (v/v)) was added to increase the pH at the beginning of stage 5,

the biogas production remained very low. The possible reason was the inhibition caused by

VFA (acetic, butyric, propionic acids) accumulation [216]. After the addition of 750 gr of

biochar and 60 L of unheated sludge in the beginning of stage 6, the pH of the reactor remained

stable at 6.8 – 7.1. The system seemed to experience a recovery period with the stable pH

condition and a gradual increase of biogas production at stage 6. The pH decreased at the

beginning of stage 7, 8, and 9, but it then increased towards the end of each stage. The

experiment was conducted under ambient temperature without a control of temperature. The

temperature during the trial fluctuated at a range between 13 – 29ºC. The slight decrease in

temperature at the end of the start-up was due to the natural variations in the ambient

125

temperature. However, such a decrease in the temperature did not influenced CH4 production

significantly as evident on Figure 6.5.

Figure 6.7 pH and temperature during the start-up of the second phase

Figure 6.8 TS and VS of the second phase

Figure 6.8 shows the changes in TS and VS during the operation. The increase of TS and VS

occurred during stage 1 to 5, and then decreased after the recovery stage until the end of the

start-up. Both TS and VS increased by 25% compared to the initial values at stages 1 to 5. This

is similar to the observation of R1. When the reactor was under fed-batch operation, the TS and

VS increased. At the end of stage 9, the TS and VS decreased by 25 and 35%, respectively,

126

compared to the TS and VS on the stage 5. At the recovery stage 6 when the unheated sludge

and biochar were added, the TS and VS decreased. It seems that the sludge addition not only

increased the CH4 production but also decreased the TS and VS of the culture.

The strategies applied in the current study led to a successful start-up of TPAD PDU. The H2

composition in this study is higher than the results of several pilot scale TPAD studies treating

bio and food wastes which were in the range of 19-39% [138, 139, 211]. The H2 yield (1.6 – 46

L H2.kg VS-1) was comparable to similar studies (3 - 67 L H2.kg VS-1) [138, 139]. The

composition of CH4 was at the range of the typical studies on pilot scale TPAD, which varies

from 55 – 67% [135, 137-139]. The CH4 yield, however, was slightly lower than similar studies.

A longer operation and of the second tank may lead to the higher CH4 yields which achieved

by other studies (377 – 550 L.kgVS-1) (Table 6.1) [138, 139].

127

Figure 6.9 SEM images of a liquid sample taken from (a) R1 on day 18 and (b) R2 on day

77 of the start-up operation

(a)

(b)

128

Table 6.1 Studies on the start-up of pilot scale TPAD PDU

Feedstock Operating conditions Gas production Ref.

1st reactor 2nd reactor H2 CH4

Biowaste • V: 0.2 m3

• pH: 3.5 – 5.4

• T: 55ºC

• OLR: 16 – 21 g

(VS)/L/day

• HRT: 3.3 – 6.6 days

• V: 0.76 m3

• pH: 7.6 – 8.2

• T: 55ºC

• OLR: 4 – 10 g (VS)/L/day

• HRT: 12.6 days

• Composition: 19 – 37

%

• Yield: 3 - 51 L/kg VS

• Composition : 60 – 65%

• Yield: 377 - 410 L/kg

VS

[139]


• pH: 5.7±0.3

• T: 55ºC

• OLR: 16.8 g

(TVS)/L/day

• HRT: 3.3 days

• V: 0.76 m3

• pH: 7.6 – 8.2

• T: 55ºC

• OLR: 1.3 – 4.8 g

(TVS)/L/day

• HRT: 12.6 days

• Composition: 39 %

• Yield: 67 L/kg VS

• Composition: 67%


[138]

Food waste • V: 0.2 m3 • V: 0.76 m3 • Composition : N.A • Composition: 55.2% [137]

129

• pH: 4.6±0.3

• T: 55ºC


• HRT: 20 days

• pH: 8.0±0.1

• T: 55ºC


• HRT: 20 days

• Yield: N.A



• pH: > 4

• T: ambient

• OLR:

• HRT: 3 days

• V: 0.5 m3

• pH: > 6

• T: ambient

• OLR:

• HRT: 7 – 21 days

• Composition :

10 – 49%

• Yield: 24 – 46

L.kg VS-1

• Composition :

35 – 59%

• Yield: 222 – 301

L.kg VS-1

This

study

130

6.4 Summary

Transient performance during the start-up of a TPAD PDU treating food waste with biochar

addition was investigated. A fed-batch followed by semicontinuous operation strategy was

found to be effective in starting up the TPAD PDU. The fed-batch operation allowed sufficient

time for microbial enrichment and adaptation. Under semi-continuous operation, the peak H2

composition and yields in the first phase were 49% and 46 L.(kg VS)-1, respectively. CH4

production with composition of up to 59% and yield of 301 L.(kg VS)-1 were attained in the

second phase. The addition of biochar has shown a potential to buffer the pH of culture and

initiate biofilm formation, which supported the successful start-up in both the reactors. This

provides evidences on the beneficial effect of biochar in enhancing H2 and CH4 production in

demonstration scale.

131

Chapter 7 Preparation, Characterisation and Evaluation of

Biochar-loaded Organic Fertiliser

7.1 Introduction

At the end of the operation of TPAD PDU, the final effluent containing biochar was generated,

which can be utilised as a fertiliser for agriculture applications. The aim of this Chapter is to

prepare organic fertilisers from TPAD effluent from the TPAD PDU (reported in Chapter 6)

with and without addition of biochar and evaluate their potential for rocket seeds germination.

The TPAD PDU effluent was collected first and mixed with biochar with the biochar ratio

ranging from 10 to 100 wt% to prepare the biochar loaded-organic fertilisers. The fertilisers

prepared were then characterised with the results reported and discussed in Section 7.2. The

effect of the organic fertilisers on the rocket seed germination using the soil-less petri dish

bioassay experimentation was evaluated and the results were presented in Section 7.3.

7.2 Characteristics of biochar-loaded organic fertilisers

Table 7.1 shows the characteristics and nutrient composition of TPAD effluent and biochar

loaded fertilisers. It is evident that TPAD effluent (BC00) is alkaline, which is probably due to

the consumption of VFA and the addition of biochar, which is alkaline, into the reactor. It is

also reported that ammonia produced during the second phase of TPAD also brought the

effluent into alkaline condition [144]. Unfortunately, the ammonia production was not

monitored during this study. With 20% of moisture (dry matter = 80%), the water holding

capacity (WHC) of the TPAD effluent was low (0.2±0.02 mL.g-1).

The major macronutrients were P and Ca, followed by S, K and Mg. In terms of micronutrients,

Na was the most abundant, followed by Fe and others. The values of the macronutrients were

considered lower compared to AD effluent used by other studies [142, 144]. The AD effluent

132

cannot be considered as a balanced fertiliser, therefore in the application, it is suggested to be

complemented with other materials [144].

Table 7.1 Main characteristics and composition of the organic fertiliser with different

percentages of biochar addition

Treatment

Biochar

content

(%)

pH

(H2O)

Dry

matter

(%)

WHC1

(mL.g-1)

P2 K3 S4 Ca5 Mg6

(mg.kg-1)

BC00 0 8.59±0.1 80±1 0.2±0.02 447.3 152.4 207.2 420.7 118.9

BC10 10 8.58±0.0 84±0 0.2±0.00 344.8 332.7 180.2 638.1 182.2

BC20 20 8.58±0.1 85±0 0.2±0.00 309.8 650.2 228.5 1191.3 345.7

BC30 30 8.60±0.0 87±1 0.8±0.07 302.0 1033.1 285.0 2254.8 670.5

BC40 40 8.49±0.0 89±1 0.8±0.00 303.0 1423.6 289.6 2425.3 706.1

BC50 50 8.57±0.0 90±0 0.9±0.02 322.0 1517.3 254.8 2366.6 682.8

BC60 60 8.55±0.1 95±4 0.9±0.08 228.6 1687.7 267.0 2639.5 778.4

BC70 70 8.60±0.1 94±0 1.1±0.08 214.3 1417.6 203.7 2558.7 737.9

BC80 80 8.52±0.0 97±0 1.2±0.02 150.5 1806.2 189.9 2436.3 690.2

BC90 90 8.48±0.0 97±0 1.4±0.08 211.1 2592 256.5 4450.9 1160.8

BC100 100 8.54±0.0 98±0 1.3±0.01 211.3 2702 207.9 4645.5 1211.8

Note: 1 WHC: water holding capacity; 2 P: Phosphorus; 3 K: Potassium; 4 S: Sulphur; 5Ca:

Calcium; 6 Mg: Magnesium; * trace: under detection limit

Both TPAD effluent (BC00) and biochar (BC100) are alkaline. Therefore, the mixtures between

the two materials are also characterised as alkaline. There are no significant pH differences

among the fertiliser, ranging at pH 8.48 – 8.60. The TPAD effluent is in a slurry form, with the

dry matter (DM) 80%, while the pure biochar is solid with DM of 98%. The DM of the fertilisers

prepared from the TPAD effluent and biochar mixtures range from 80 – 98%. The DM

133

increased with the higher percentage of biochar addition. The fertilisers with 0-30% of biochar

formed watery fertilisers, fertilisers with 40-60% were sludge-like while the fertilisers with 70-

100% of biochar were solid with little trace of water.

According to Table 7.1, generally, the higher percentage of biochar addition increased the

WHC. The WHC of BC00 - BC20 were significantly lower than others. At the biochar addition

of 30 – 60% (BC30-BC60), the WHC increased to 0.8 – 0.9 mL. g-1. WHCs of BC70-BC100

were more than 1 mL.g-1. The addition of biochar into the mixture seems to provide an increased

porosity to retain higher moisture. The WHC of BC90 was 1.4 mL.g-1, comparable to the WHC

of biochar used by other studies (Wheat Chaff (1.4 mL.g-1), New Jarrah (1.6 mL.g-1)), activated

carbon (AC) (1.5 mL.g-1) and pumice (1 mL.g-1) [27, 28, 121]. The increased amount of water

retained by the biochar and soil may improve the habitability for soil microorganisms. An

increase in WHC of biochar improves the overall soil WHC when it is applied for soil

amendment [27].

The macronutrients in the prepared fertilisers, such as P, K, Ca and Mg are shown in Table 7.1

(standard deviation = 0.04 – 4%). The P concentration of the TPAD effluent was significantly

higher (447.3 mg.kg-1) than the biochar (211.3 mg.kg-1). Therefore, the P content of the fertiliser

decreased as higher percentage of biochar was added. However, the P value was still

comparable with other studies using AD effluent and pine/woody biochar [144, 219]. It is

reported that the P content reduced during pyrolysis [219].

On the contrary, the K content of the pure biochar was significantly higher than the TPAD

effluent. The addition of 10% biochar in BC10 doubled the K content compared to that of BC00.

The K kept increasing with the higher percentage of biochar addition, reaching the highest at

100% biochar addition (BC100). The highest K value of the fertiliser was higher than the K

content of pine chips-biochar prepared at 500±70ºC (1,450 – 2,354 mg.kg-1) [219, 220]. K

134

activates enzymes required by plant metabolisms and growth. Also, it contributes to the cell

division, especially the turgor pressure driven [221, 222].

Low sulphur contents range at 180 - 290 mg.kg-1were detected in the TPAD effluent and the

fertiliser. Although lower than other macronutrients, the S contents was higher than that Gaskin

et al which found very low S content on the biochar produced from pine-chips (60 mg.kg-1)

[219]. The low content of S may be caused by the S loss during the pyrolysis. As much as 35 –

50% of S is released during the pyrolysis at 400-500ºC [27]. The biochar in this study was

prepared at 650ºC, therefore the higher loss of S might be occurred.

Ca was found to be one of the highest constituents of both TPAD effluent and pure biochar.

The addition of biochar significantly improved the Ca content of the fertiliser. Ca content in the

TPAD effluent was 421 mg.kg-1, while that in the pure biochar was 4646 mg.kg-1. At 90%

biochar addition (BC90), the Ca content was ten times higher (4451 mg.kg-1) than that of BC00.

Ca content of biochar derived from the same materials were varied, being closer to the Ca

content of biochar derived from willow and demolition wood [27, 219]. Ca plays a vital role in

cell expansion and enhances seed germination and growth, thus Ca deficiency may inhibits root

growth [221, 223].

Clearly, the content of Mg also extremely increased with the addition of biochar (Table 7.1).

The Mg contents improved from 182 to 1161 mg.kg-1 with the increased addition of biochar

from 10 – 90% (BC10 to BC90). Typical woody biochars contain Mg at 360 – 2,107 mg.kg-1

[27, 219, 224]. Mg acts as enzyme activators and osmoregulator and supports the root and

shoots growth [222, 223].

Table 7.2 enlists the micronutrients contents, including Na, Fe, Mn, and Cu, and heavy metals

(Cr, Ni) in the fertilisers. Generally, all micronutrients increased with the higher percentage of

biochar addition, except that of Na. Although the Na of the pure biochar is lower than that of

TPAD effluent, it is still in the range of typical study [27, 219].

135

Fe was a most abundant micronutrient in the pure biochar. The high content of Fe in biochar

significantly increased the Fe content of fertilisers with biochar addition. In woody biochar, the

Fe content range from 350 – 10,000 mg.kg-1[27]. Mn and Cu concentrations were much lower

than other micronutrients.

Heavy metals such as Ni and Cr were also observed in low concentrations. Generally, Ni (1.7

– 31 mg.kg-1) and Cr (3.3 – 60 mg.kg-1) concentrations also increased with the higher percentage

of biochar addition. It is interesting that the concentration of both Ni and Cr peaked at the

addition of 90% of biochar then decreased at 100% biochar. The value of Ni and Cu of the

fertiliser investigated in the current study are lower than the limits allowed for the utilisation of

AD effluent [144].

136

Table 7.2 The concentrations (mg.kg-1) of elements in the organic fertiliser with different percentage of biochar addition

Parameter

Biochar (%)

0 10 20 30 40 50 60 70 80 90 100

Na (mg.kg-1) 1850.12 1491.44 1638.75 1562.29 1822.58 1457.44 1290.91 773.05 804.74 994.56 818.52

Fe (mg.kg-1) 570.38 1133.71 2224.71 4551.88 5077.93 5211.22 6453.77 6191.43 6342.63 10718.19 10229.94

Mn (mg.kg-1) 5.99 14.54 30.99 68.86 75.18 79.15 95.22 87.92 88.19 156.12 151.49

Cu (mg.kg-1) 8.50 52.71 88.56 216.97 190.88 135.33 102.20 104.98 86.73 158.69 162.93

Ni (mg.kg-1) 1.73 3.23 5.77 12.91 21.89 10.82 17.55 12.54 13.22 30.65 12.68

Cr (mg.kg-1) 3.26 4.82 9.70 17.77 30.78 20.42 33.32 19.94 18.43 59.60 27.98

137

7.3 Soil less petri dish bioassay

Figure 7.1 Photographs of germination of rocket seed with various organic fertilisers conducted in the soil-less petri dish bioassay

40%

70% 80% 90%

BC40 BC10 BC00 Control BC20 BC30

BC50 BC60 BC70 BC80 BC90 BC100

138

Figure 7.1 shows the photographs of the germination of rocket seeds with the applications of

various organic fertilisers. Visual observation suggests that the rocket seeds were succesfuly

germinated after 72 hours of incubation under 25 ºC and dark condition. The length of root and

shoot, however, varied among the treatments.

Table 7.3 Germination index (GI) as percentage of germinated seeds in the assay to the

control

Treatment GI (%) S.D.

Control 100 0

BC00 102 11

BC10 99 12

BC20 97 4

BC30 99 3

BC40 87 2

BC50 91 6

BC60 103 5

BC70 99 2

BC80 87 16

BC90 100 10

BC100 68 4

Typical indicators of the assay are percent of germination (and/or germiantion index), lengths

of radicle (root) and shoot [28, 225]. Table 7.4 shows the germination index (GI) calculated to

compare the seed germination of each treatment to the control (as percentage). The GIs of

treatment BC00 to BC 90 ranged from 87±16 to 103±5% of the control. The treatment of BC100

appeared to decrease the seed germination. It is noted that the GIs of almost all tested treatment

were > 80% of the control, except the BC100 which GI was 68%.

139

The nutrient micro and micronutrients content of the TPAD PDU is reported to have a strong

contribution to the seed germination. The P, as the major macronutrient in the effluent, has

reported to be an important element required during germination and seedling growth [226].

The P holds an important role in cellular metabolism and enhances root growth [223, 226]. The

lowest GI of the BC100 indicates that it was toxic that reduced the GI. The high amount of K,

Cu in BC100 may contribute to the toxicity. Other elements such as Cl and Zn which may also

contribute to the reduced GI, shoot and root length [144].

Figure 7.2 The effect of various organic fertilisers on root length of germinated rocket seed

conducted in the soil-less petri dish bioassay

The entire structure of plants is represented by the root and shoots. The sum of the root length

of each treatment was shown in Figure 7.3. It is clear that the sum of root length increased with

the percentage of biochar in the fertilisers increased, but then decreased when the pure biochar

(BC100) was used. However, it is worth mentioning that the sum of root length of BC100 was

still higher than the control. The highest sum of root length was achieved with the treatment

with BC90 (153±8 cm), being three times higher than the control (52±15 cm).

140

Figure 7.3 The effect of various organic fertilisers on shoot length of germinated rocket

seed conducted in the soil-less petri dish bioassay

Figure 7.4 The effect of various organic fertilisers on shoot to root ratio of germinated

rocket seed conducted in the soil-less petri dish bioassay

Similar trend was observed in the sum of shoot length regarding the effect of the biochar-loaded

organic fertiliser, as shown in Figure 7.3. The sum of shoot length increased with the higher

biochar percentage in the fertilisers achieving the highest with the BC90 treatment, attaining

141

63±5 cm. The shoot length became smaller with the BC100 treatment compared with the BC90

treatment. The ratios of shoot to root ratio calculated and presented in Figure 7.4. It is clear that

the shoot to root ratio tended to increase with the higher biochar percentage in the fertilisers.

The higher sum of root and shoot in the treatment with higher biochar percentage in the

fertilisers may attribute to higher nutrient contents. As suggested in Section 7.2, the addition of

biochar in the TPAD PDU significantly increased the macro and micronutrients required for

seed germination and plant growth, such as K, Ca, Mg and Fe. It is known that K, Mg and Fe

are cofactor of enzymes required by plant metabolisms and growth. Also, K contributes to the

cell division, especially the turgor pressure driven [221, 222]. Ca plays a vital role in cell

expansion and enhances seed germination [221, 223]. Mg also acts as enzyme activators and

osmoregulator, and supports the root and shoot growth [222, 223].

Root to shoot ratio indicates the allocation of the nutrient to the part of the plants [227]. Figure

7.4 suggests that the nutrients contained in the fertiliser enhanced root growth than shoot. The

abundance of Fe and Ca in biochar, may contribute to the enhance root growth [221-223].

It is worth noticing that the BC100 performed better than the control in term of shoot and root

length, despite the decrease in GI [171]. Similar result is reported in a study of germination

assay of watercress using biochar derived from compost-like output (CLO) mixed with sewage

sludge [171]. It was observed that although the GI reduced, the nutrient availability in the

biochar promoted the root and shoot once the seed germinated [171, 225]. The similar results

were found by Rogovska et al., (2012) and Stephenson et al., (1997). This suggests that using

GI alone as an indicator to evaluate the potential of biochar-loaded fertilisers appeared to be

insufficient. It is proposed to use lengths of root and shoot, together with GI, as indicators for

the assay [225, 228].

142

7.4 Summary

The TPAD PDU effluent was loaded onto biochar with different biochar loading ratios to

prepare the organic fertilisers. The biochar-loaded fertilisers were characterised and evaluated

using seed germination assay. The results suggest that the addition of biochar significantly

increased essential elements for germination and plant growth, such as K, Ca, Mg, Na, Fe, Mn,

Ni and Cr, compare to the treatment with the TPAD effluent alone. Biochar addition also

increased WHC of the fertiliser with no significant pH change. Fertilisers with 0-90 wt%

biochar loading gave the positive effects on the seed germination, while the pure biochar

reduced germination index (GI). However, despite the low GI, the pure biochar, and the rest of

the fertilisers tested, resulted in the increased sums of root and shoot length compared to the

control without the fertiliser. The maximum sums of root and shoot (153.4±8 and 62.3±5 cm)

was achieved with the fertiliser prepared of 10% of TPAD effluent and 90% of biochar. The

improved macro and micronutrients in the biochar-loaded fertilisers contributed to the good

seed germination results.

143

Chapter 8 Evaluation and Practical Implications

8.1 Introduction

In Chapter 8, the findings from the entire thesis work as presented in the previous four chapters

are integrated and evaluated against the specific objectives and literature data, respectively. In

addition, the practical implications are identified.

8.2 Integration of Experimental Findings

The study has provided strong, convincing and systematic evidence that the biochar addition

improves H2 and CH4 production both in the first and second phases of TPAD. It shortens the

lag phase, increases the maximum H2/CH4 production rate, and H2/CH4 production potential. It

was further observed and verified that the volatile fatty acids (VFA) generation and conversion

are also enhanced in the first and second phase, respectively. The beneficial characteristics of

biochar observed to contribute to the enhanced H2 include the high surface area for the initiation

of biofilm formation and provision of additional nutrients, enriching the microbial growth and

population. The pH buffering ability of biochar was also found to be essential in enhancing H2

production. In the second phase, biochar plays a significant role in biofilm formation which

increased microbial population and activity. The buffering ability of biochar, however, was an

insignificant factor.

A fed-batch followed by semicontinuous operation strategy was found to be effective in starting

up both phases of the TPAD process demonstration unit (PDU) at a pilot scale. Confirming the

findings in the bench scale study, biochar addition shows potential buffering capacity and

microbial immobilising ability, which contributed to the successful start-up of the TPAD PDU.

Finally, the biochar-loaded organic fertilisers were successfully prepared, characterised and

evaluated. The biochar addition significantly increased water holding capacity (WHC) and

144

micro and macronutrients with no significant pH changes compared to the TPAD effluent alone,

thus enhanced the seed germination.

8.3 Evaluation against the Specific Research Objectives

The objectives stated in Chapter 2 were achieved through a systematical and thorough study

according to the methodology explained in Chapter 3 then reported in Chapters 4, 5, 6 and 7.

The first three objectives were achieved by investigating the effect of biochar in bench scale

TPAD operating the two separate phases individually. Biochar was found to exert profound

beneficial effects on both the first and second phase of TPAD (Chapters 4 and 5). The biochar

used in the study, however, was only limited to one type of biochar, which was the pine sawdust

biochar, prepared at 650C. Considering the different characteristics which may be possessed

by different types of biochar prepared under different operating conditions, their effects and

mechanisms in TPAD may be different. There could well be some biochars that may not show

the same or similar beneficial effects as the pine biochar studied in the present work does and

it would be scientifically interesting and practically useful to clearly define the key

characteristics of biochar and relate them to the biochar effects in enhancing H2 and CH4

production in TPAD operations. Therefore, further studies using different types of biochar in

TPAD are recommended. In addition, in the current study, the presence of typical AD microbes

and the establishment of biofilm on the pore and surface of biochar were observed as evidenced

in the SEM images. However, a further study on the bacterial and biofilm identification using

molecular biological tools is necessary to give a better understanding of the interaction between

biochar, TPAD microbes and operating conditions.

The investigations into the effect of biochar on H2 and CH4 production were focused on the

batch operation in a bench scale AD. Very limited studies examined the biochar addition in

long term continuous operations on large scales. An attempt to achieve the fourth and fifth

objectives of the current study, demonstrating the operation of H2 and CH4 production with the

145

biochar addition in a larger scale was conducted, as reported in Chapter 6. The operation of

TPAD PDU with the addition of biochar was successfully conducted. However, it was limited

to the start-up period under fed-batch and semicontinuous operation. Further study on the

biochar addition in long term continuous operation of pilot scale TPAD is suggested.

The final specific objectives namely the preparation, characterisation and evaluation of biochar

and TPAD effluent as fertiliser were successfully achieved as detailed in Chapter 7. The biochar

addition improved the characteristics of TPAD effluent alone. The results also suggest the

potential utilisation of combined biochar and effluent as fertiliser as shown by seed germination

assay results. Further studies on the utilisation of the fertilisers in pot trials and using different

application ratio were necessary to provide confidence before the field application.

8.4 Evaluation against the Literature

8.4.1 Effect of Biochar Addition on Hydrogen Production

The current study was systematically conducted to examine the beneficial effect of biochar on

H2 production. The results show that the biochar addition significantly increased H2 production.

The results from the present study are compared with the literature data. Similar studies report

the beneficial effects of biochar on H2 production (Table 8.1); although some studies report its

negative effects. While some types of biochar are reported to have adverse effects on H2

production [113], in the present study, the pine sawdust biochar at all ratios tested gives positive

effects in H2 production. It was found that the biochar also enhances VFA generation [73, 128].

In addition, it has been found that the effect of biochar addition in H2 production is dependent

on the type and addition ratio of biochar, the feedstock of AD and operating conditions. The

present work was able to explain the wide variations in the optimal biochar addition ratio

ranging from 0.6 to 12.5 g. L-1 (Table 8.1). It implies the difficulty to achieve agreement on the

146

quantitative data among the literature data since different types and addition of biochar,

feedstock and operating conditions were used in each study [128].

Table 8.1 A comparison of the current experimental results with the literature data on the

batch H2 production with the addition of different types of biochar

Feedstock

of AD

Feedstock

of biochar

Biochar addition

(g.L-1) Operating

conditions

H2 yields

increase

compared to

control

Ref. Studied

range

Optimal

OFMSW1 Woody

mass

2.5 – 35 12.5 Batch 37ºC

Initial pH: 5.5

1.4 - 3.3

times

[128]

Glucose Corn bran

residue

0.2 – 0.8 0.6 Batch 37ºC

Initial pH: 7

11- 29% [73]

DAS2 and

food

waste

Sawdust 10 10 Batch 35ºC

Initial pH: 5.5

0-13% [113]

DAS2 and

food

waste

Wheat bran 10 10 Batch 35ºC

Initial pH: 5.5

6 – 13% [113]

DAS2 and

food

waste

Sewage

sludge

10 10 Batch 35ºC

Initial pH: 5.5

(-10) - 6% [113]

DAS2 and

food

waste

Peanut

shell

10 10 Batch 35ºC

Initial pH: 5.5

(-44) - (-

10)%

[113]

Current

study

Pine

sawdust

8.3 – 33.3 10 Batch 35ºC

Initial pH: 5.0

14– 31% [161]

Note: 1: OFMSW: organic fraction of municipal solid waste

2: DAS: dewatered activated sludge

Finally, while most of the literature focuses on the study of type and addition ratio of biochar

under a fixed set of operating conditions, the current study also examined the effect of biochar

addition under various operating conditions of initial pH values and temperatures. The

147

beneficial effect of biochar on H2 production was found to be more profound at lower pH and

higher temperatures.

8.4.2 Effect of Biochar Addition on Methane Production

Table 8.2 A comparison of the current experimental results with the literature data on the

batch CH4 production with the addition of different types of biochar

Feedstock

of AD

Feedstock

of biochar

Biochar addition

(g.L-1) Operating

conditions

CH4 yields

improvement

compared to

control (%)

Ref. Studied

range

Optimal

AD sludge Paper

sludge and

wheat husk

20 20 Batch 42ºC

Initial pH: N.A

31 [3]

Glucose Fruit

woods

10 10 Batch 35ºC

Initial pH: 7

21 [83]

Glucose Fruit

woods

10 10 Batch 35ºC

Initial pH: 7

11.5 [129]

Citrus

peel

Coconut

shell

9.7 9.7 Batch 35ºC

Initial pH: 7

13 [130]

Food

waste

Fruit

woods

2 - 10 2 Batch 35ºC

Initial pH: 7

39 - 44 [131]

Dairy

manure

Dairy

manure

1 - 10 10 Batch 35ºC

Initial pH: 7.7

5 – 24.5 [133]

Food

waste and

DAS

Sawdust 2 - 15 6 Batch 35C

Initial pH: NA

(-2) - 4 [229]

Current

study

Pine

sawdust

8.3 –

33.3

8.3 Batch 35C

Initial pH: 7

(-10) – 10 [161]

*DAS = dewatered activated sludge

NA = not available

The present work into the second phase of TPAD suggested strong evidence of the enhancement

of CH4 production with the addition of biochar addition, in good agreement with other studies

148

in the literature using various wastes as the feedstock (Table 8.2). The results of the current

study are within the range of the findings of other studies. At the appropriate ratios of biochar

additions, the CH4 yields increased by 4 – 44% compared to the control, depending on the types

and addition ratio of biochar, the feedstock of AD and operating conditions [3, 83, 129-131,

133, 229]. Similar to the results in H2 productions, the optimal biochar additions are varied in

the existing studies, which range from 2 to 20 g.L-1 (Table 8.2).

In addition, the current study observed that at the addition ratio of 25.5 – 33.3 g.L-1, the CH4

yields were lower than control due to the biochar overdose. However, the CH4 production rate

increased (by 23 – 42%). Similar results were reported by Wang et al. that the CH4 yield

decreased with the higher addition of biochar addition, while the CH4 production rate increased

at all biochar addition ratios tested [229]. Finally, further study on the biochar addition under

various operating condition (initial pH and temperature) suggested that the effect of biochar

additions was greater at the higher pH.

8.4.3 Mechanisms of biochar in enhancing H2 and CH4 production in TPAD

Figure 8.1 A schematic representation of the mechanisms of the working of biochar in

promoting H2 and CH4 productions in TPAD from food waste

First phase


▪ Buffering pH

▪ Providing temporary nutrients

Biochar

Results:


▪ Faster VFA generation

▪ Higher H2 production rate

▪ Higher cumulative H2

production

Second phase


Results:


▪ Faster VFA degradation

▪ Higher CH4 production rate

149

The possible mechanisms of the biochar effects in H2 and CH4 production have been proposed

in the current study (Figure 8.1). It is observed that in the first phase, biochar promoted biofilm

formation, buffered the pH and provided temporary nutrients. Meanwhile, in the second study,

the biofilm formation initiated by biochar was observed to be the main mechanism of biochar

in enhancing CH4. The proposed mechanisms were supported by other studies (Table 8.3 and

8.4) and discussed as follow.

Previous studies suggested that different types of biochar affected H2 production with different

mechanisms. First, Sharma et al. and Shang et al. found that biochar enhanced H2 production

due to the high surface area of biochar which facilitated microbial immobilisation by biofilm

formation, although there was no direct evidence of the establishment of biochar presented in

their studies [73, 128]. In the current study, a clear establishment of biofilm in biochar was

observed in the SEM images (Figure 4.11). The microbial immobilisation is reported to play a

critical role in increasing the microbial density in the culture compared to the suspended culture

without microbial immobilisation [85, 230]. The increased density of microbes favours H2

production [85, 230].

In addition, biochar was also shown to be an efficient pH buffer in this study as supported by

the findings in the study comparing biochar with other additives with different acidity as

references (Section 4.4). This finding is supported by other studies [73, 113]. Zhang et al. and

Wang et al. [113] reported an increased H2 with the increased in final pH which corresponds to

the higher addition of biochar, which was also observed in this study (Section 4.4 and 4.5).

Yet another mechanism was proposed that the provision of additional nutrients due to biochar

addition may be useful for microbial activity in enhancing H2. The biochar used in this study

contains 10 wt% of volatile matter that was available as a temporary microbial nutrient [27].

This mechanism is also reported in CH4 production using biochar produced at the lower

production temperature, where the remaining nutrient was readily available [3].

150

In addition, biochar used in this study contained micronutrients, such as Fe (10,230 mg.kg-1)

(Table 7.2). Zhang et al. found that the addition of 50 – 300 mg.L-1 of Fe2+ increased the H2 by

17 - 38 % compared to the control. H2 production increased from 158 to 212 mg.g glucose-1

with the addition of 0 – 100 mg.L-1 then gradually decreased to 184 mg.g glucose-1 with further

Fe addition to 300 mg.L-1[73]. Similar results were found in the current study, using the biochar

ranged at 8.3 – 33.3 g.L-1, which corresponded to 85 – 341 mg. L-1 of Fe. The H2 production

increased from 749 to 981 mL.L-1 with the addition of 8.3 g.L-1, and then decreased to 855

mL.L-1 when the biochar was increased to 33.3 g.L-1. This agreement shows that the nutrient

contained in biochar is essential for H2 production. Particularly Fe, it is required by the H2

producing bacteria (HPB) for hydrogenase protein redox, which directly correlates to microbial

growth and biogas release, however toxic when it is overdosed [73, 231, 232].

In the second phase of TPAD, the possible mechanisms of biochar effects in CH4 production

proposed in the current study were supported by other studies and discussed as follows (Table

8.4). A significantly shorter lag phase than control (41 – 45% shorter) and higher maximum

production rate (23 – 42%) was observed in the current study (Table 5.3). These observations

imply that the addition of biochar enhanced the microbial growth and activity. Thus the culture

started the CH4 production faster. It was observed that the microbial immobilisation was

facilitated with biochar addition (Figure 5.6). The promoted formation of biofilm has been

reported to enhance microbial activity, leading to a shorter lag phase, faster VFA degradation

and CH4 production in the cultures with biochar.

Some literature reported the beneficial effect of biochar in buffering the pH of the culture [133,

229]. The role of biochar as pH buffer, however, was found to be insignificant in the current

study (Section 5.5). The separation of acidogenesis from the methanogenesis was observed to

be effective in preventing the culture from extreme pH drop and high accumulation of VFA, as

151

reported in Section 5.4. Therefore, the pH drop was no longer an issue in the second phase.

Thus the pH buffer capability of biochar insignificantly affected the CH4 production.

152

Table 8.3 Profiles of biochar and proposed mechanisms in H2 production of different studies

References

Biochar

Proposed mechanisms

Feedstock pH SSA (m2.g-1)

Sharma et al. [128] Woody biomass N.A 125 - Biochar facilitated biofilm formation

- Biochar mitigated ammonia inhibition

Zhang et al. [73] Corn-bran residue 8.92 58 - Biochar acted as a microbial support carrier

- Biochar acted as pH buffers

Wang et al. [113] Sawdust 7.27 – 10.07 15.3 – 511.3 Biochar acted as pH buffers

Wang et al. [113] Wheat bran 7.37 – 10.33 4.2 – 45.9 Biochar acted as pH buffers

153

Table 8.4 Profiles of biochar and proposed mechanisms in CH4 production of different studies

References

Biochar

Proposed mechanisms


Mumme et al. [3] Paper sludge and

wheat husks

9.3 N.A - Hydrochar provides anaerobically degradable carbon

- Biochar prevents ammonia inhibition

Luo et al. [83] Fruitwoods 8.6 N.A - Biochar selectively enriched methanogens

- Biochar improved the resistance of the microbes to highly

acidic conditions

Lu et al. [129] Fruitwoods 8.6 N.A - Fine biochar enhanced acid generation which can be useful

for H2 production

- Biochar mitigated ammonia and acid stress

- Biochar initiates microbial colonisation

Fagbohungbe et al.

[130]

Coconut shell 8.3 NA - Biochar adsorbed an inhibitive compound in the AD

feedstock

- Biochar immobilises methanogens

154

References

Biochar

Proposed mechanisms


Cai et al. [131] Fruit woods 8.6 N.A - Biochar was a favourable material during easily degradable

feedstock

Jang et al. [133] Dairy manure N.A 6.3 - Biochar served as a pH buffer

Wang et al. [229] Sawdust 9.2 248.6 - Biochar provides excellent pH buffering capacity

- Biochar enriched specific methanogens for direct

interspecies electron transfer

155

8.4.4 Transient Performance during Start-up of TPAD PDU

The results of the TPAD PDU study are compared with the literature data of pilot scale TPAD

operation. Figure 8.2 shows studies on pilot scale TPAD using different feedstock, reactors and

operating conditions. All studies presented in Figure 8.2 were conducted without biochar

addition, except for the current study. The volume H2 and CH4 producing reactors were 150 –

200 L and 42 – 1,300 L, respectively. Some literature report both H2 and CH4 production, while

the others only considered the CH4 production. The results of the current study were comparable

to the results from other studies, where the H2 yield range at 51 to 68 L.(kg VS)-1 and the range

of CH4 yield are at 179 – 550 L.(kg VS)-1. A longer and continuous operation of the TPAD

PDU in the future study is expected to improve the H2 and CH4 production.

Figure 8.2 H2 and CH4 production in pilot scale TPAD using different feedstock

There is limited research work on the biochar addition on the pilot scale AD. To the best of

author’s knowledge, to date, there is only one study reporting biochar addition of biochar in

pilot scale single AD producing CH4 (reactor volume 900 – 1500 L) [140]. The study reports

the correlation between the maturity of biofilm with the pH changes during the operation. It

was observed that towards the end of the 59 days operation, the culture approached pH 7 more

156

rapidly, which implied the enhanced biofilm maturation [140]. Similar trends were also

observed in the current study (Figure 6.7), suggesting a mature establishment of methanogenic

biofilm as also suggested SEM images (Figure 6.9).

8.4.5 Preparation, Characterisation and Evaluation of Biochar-loaded Organic

Fertiliser

The characterisation of TPAD effluent and biochar in this study was conducted and evaluated

against literature data (Table 8.5). The pH of the TPAD effluent and biochar are consistent with

those of effluent from other studies. The macro and micronutrient depend on the feedstock of

the AD and biochar. Compared to other AD effluents, the nutrients of TPAD were relatively

lower, except for the Fe and Na. Therefore, the supplementation of biochar is aimed to increase

the essential macro and micronutrients for agricultural applications.

Figure 8.3 shows the comparison of the minimum and maximum germination index (GI) of the

current study and other studies. The GI presents the germination of the tested treatment relative

to the control without the addition of the tested fertilisers [144]. The GI of rocket seed used in

the current study ranged at 68 – 103 %, which is at the range of other studies. It is comparable

with the GI of those experiments using (1) pig slurry added with crop waste and (2) New Jarrah

biochar. Also, it is significantly higher than that of AD effluent (cattle slurry) added with

glycerol. The GI of the assay with AD effluent (pig slurry) added with animal waste and AD

effluent (cattle slurry) added with agriculture waste are higher than that of others. It is likely

that the significant content of P and K, that are higher than that of our study, play an essential

role in increasing the GI of cress used in the study by Albuquerque et al. [144].

157

Table 8.5 Characteristics of AD effluent and biochar prepared as organic fertiliser

Parameter

Feedstock of AD/TPAD effluent Feedstock of biochar

Food

waste

Pig slurry and animal by-

product

Cattle slurry and

agriculture waste

Pinewood sawdust Pine chips

New Jarrah

pH 8.59 7.95 7.50 8.54 8.30 9.52

P (mg.kg-1) 447.3 500 800 211.3 140 N.A

K (mg.kg-1) 152.4 2200 3100 2702 1450 N.A

S (mg.kg-1) 207.2 219 457 207.9 60 N.A

Ca (mg.kg-1) 420.7 799 4026 4645.5 1850 N.A

Mg (mg.kg-1) 118.9 324 698 1211.8 590 N.A

Fe (mg.kg-1) 570.4 51 301 10229.94 50 N.A

Na (mg.kg-1) 1850 696 746 818.52 13 N.A

Mn (mg.kg-1) 5.99 11.4 27.5 151.49 258 N.A

Cu (mg.kg-1) 8.50 14.3 10.8 162.93 9 N.A

Ni (mg.kg-1) 1.73 N.A. N.A. 12.68 3 N.A

Cr (mg.kg-1) 3.26 N.A. N.A. 27.98 18 N.A

158

Parameter

Feedstock of AD/TPAD effluent Feedstock of biochar

Food

waste

Pig slurry and animal by-

product

Cattle slurry and

agriculture waste

Pinewood sawdust Pine chips

New Jarrah

Reference This

study

[144] [144] This study [219] N.A

Note: N.A; Not available

159

Figure 8.3 Germination index of petri dish bioassay using different plants and fertilisers

The results from this current study and literature suggest strong evidence that the TPAD effluent

and biochar are potential materials for soil amendment for agricultural application. Studies

using germination assay are required for the TPAD effluent and biochar utilisation derived from

the different feedstock.

8.5 Practical Implications

The results and new findings from the present research significantly contribute to the practical

applications of TPAD in processing wastes for biogas production with the use of biochar in

enhancing H2 and CH4. Although TPAD is more promising in recovering energy than single

phase AD [56], it is still challenging to achieve stable operation and high production rates and

yields of both H2 and CH4.

The findings of the study provided strong evidence that biochar addition enhanced H2 and CH4

production. First, the enhanced H2 and CH4 production rates lead to the shorter time of

operation. Thus more waste/wastewater can be treated. Furthermore, with higher production of

H2 and CH4, the unit required less addition of buffering solutions due to the buffering ability of

160

biochar. Also, the strong establishment of biofilm on biochar can prevent the microbial wash

out in the real application of continuous operation of pilot scale AD [85]. Finally, the

experimental results from TPAD PUD provide valuable data and reference for the design,

operation and economic evaluation of TPAD in commercial scale with the use of biochar.

The findings suggest a very promising solution to the management of AD effluent, which can

sometimes be challenging. The TPAD effluent containing biochar with and without further

biochar addition provides nutrients for plants growth. The TPAD effluent loaded with biochar

has shown positive effects in the germination of the tested seed. The biochar-loaded organic

fertilisers can be used for agriculture, industry and commercial applications.

As a whole, the thesis has proposed an approach to the practical implication of integrated

pyrolysis and TPAD operation to achieve a sustainable bioenergy generation and bio-wastes

management, particularly in remote areas. Woody biomass or agricultural wastes are potential

feedstock for biochar production via pyrolysis. The biochar produced is an ideal material for

enhancing H2 and CH4 production via TPAD treating animal manure, food and agricultural

wastes. The effluent from the TPAD can be utilised for the agricultural applications.

161

Chapter 9 Conclusions and Recommendations

9.1 Introduction

The outcomes of the present thesis research have contributed new knowledge and useful

experimental data on the utilisation of biochar to enhance H2 and CH4 production in bench and

demonstration scale of TPAD operation, as well as the utilisation of the AD effluent and biochar

as fertilisers. The conclusions and evaluation of the current research as elaborated in Chapter 8

can naturally lead to recommendations for future studies. This Chapter summarises the key

conclusions and recommendations as follows.

9.2 Conclusions

9.2.1 Effect of Biochar Addition on Hydrogen Production in the Bench Scale Experiment

• The effect of biochar addition on the H2 production in laboratory scale TPAD of simulated

carbohydrate food waste was studied systematically. Biochar addition was shown to

shorten the lag phase by 21.4 to 35.7 %, increased the maximum production rate by 32.4%

and H2 production potential by 14.2 to 31 % of H2.

• Further investigation of the effect of biochar addition under different initial pH and

temperature on the cumulative H2 yield (YH) and maximum daily production rate (RH) in

the first phase of TPAD was conducted and optimised using the response surface

methodology (RSM). Biochar addition was shown to substantially increase YH, especially

at lower pH and higher temperatures.

• The RSM analysis showed that the maximum YH of 1,331 mL.L-1 and RH of 763 mL.L-

1.day-1 could be achieved under the optimum conditions of biochar addition ratio 10.1 g.L-

1, initial pH 6.4 and temperature 32C.

162

• The working mechanisms of biochar in enhancing H2 production were proposed. It is

observed that the biochar initiates the biofilm formation and provides temporary nutrients

in the culture, enriching the microbial population. Also, the addition of biochar was

observed to bring the condition of the culture from acidic to alkaline at the beginning of

the operation and prevented significant pH drop during incubation. As a result, the cultures

with biochar additions generated more H2 at faster rates.

9.2.2 Effect of Biochar Addition on Methane Production in the Bench Scale Experiment

• A preliminary study on the effect of biochar addition on the CH4 production in laboratory

scale TPAD of simulated carbohydrate food waste was conducted. The biochar addition

shortened the lag phase by 41 to 45 %, increased the maximum production rate by 23.0-

41.6% and CH4 production potential by 1.9 to 9.6% of CH4.

• Furthermore, the effect of biochar addition on CH4 production in the second phase of

TPAD under varying initial pH and temperature conditions has been studied. A moderate

level of biochar addition, mesophilic temperature and neutral to alkaline initial pH were

shown to benefit CH4 production in the second phase of TPAD. The effect of biochar

addition was more profound at higher pH.

• The optimum biochar addition; temperature and initial pH were found to be 12.5 g.L-1,

36.2oC and 7.8, respectively. Under the optimum condition, the YM and RM were 1755

mL.L-1 and 500.9 mL.L-1.day-1.

• The possible working mechanism of biochar in enhancing CH4 is related to the high surface

area of biochar, which initiates biofilm formation. The SEM analysis revealed the

development of biofilm and the accumulation of the bacillus rods of bacteria on the surface

of biochar. The promoted methanogenic biofilm formation in the second phase of TPAD

may contribute to the enriched methanogens and enhanced CH4 production. The pH

buffering capacity of biochar, however, was found to be insignificant.

163

• An additional experiment of single-phase AD using fresh food waste was conducted. The

result suggests that at a high concentration of feedstock, the single phase AD suffered from

acid accumulation thus failed to produce CH4, while the second phase TPAD successfully

produced a substantial amount of CH4. It is due to the separation of acidogenesis and

methanogenesis, which favour CH4 production in the second phase AD.

9.2.3 Transient Performance during Start-up of TPAD PDU

• Transient performance during the start-up of a TPAD PDU treating food waste with biochar

addition was investigated. A fed-batch followed by semicontinuous operation strategy was

found to be effective in starting up the TPAD PDU. The fed-batch operation allowed

sufficient time for microbial enrichment and adaptation.

• Under semi-continuous operation, the peak H2 composition and yields in the first phase

were 49% and 46 L.(kg VS)-1, respectively. CH4 production with the composition of up to

59% and yield of 301 L.(kg VS)-1 were attained in the second phase.

• The addition of biochar has shown a potential to buffer the pH of culture and initiate biofilm

formation, which supported the successful start-up in both the reactors. This provides

evidence on the beneficial effect of biochar in enhancing H2 and CH4 production in

demonstration scale

9.2.4 Preparation, Characterisation and Evaluation of Biochar-loaded Organic

Fertiliser

• The TPAD PDU effluent was loaded onto biochar with different biochar loading ratios to

prepare the organic fertilisers. The biochar-loaded fertilisers were characterised and

evaluated using seed germination assay. The results suggest that the addition of biochar

significantly increased essential elements for germination and plant growth, such as K, Ca,

Mg, Na, Fe, Mn, Ni and Cr, compare to the treatment with the TPAD effluent alone.

Biochar addition also increased WHC of the fertiliser with no significant pH change.

164

• Fertilisers with 0-90 wt% biochar loading gave the positive effects on the seed germination,

while the pure biochar reduced germination index (GI). However, despite the low GI, the

pure biochar, and the rest of the fertilisers tested, resulted in the increased sums of root and

shoot length compared to the control without the fertiliser.

• The maximum sums of root and shoot (153.4±8 and 62.3±5 cm) were achieved with the

fertiliser prepared of 10% of TPAD effluent and 90% of biochar. The improved macro and

micronutrients in the biochar-loaded fertilisers contributed to the good seed germination

results.

9.3 Recommendations

The overall objectives of the present study have been achieved. However, various new gaps

have also been identified during the evaluation of the findings of the current research. The new

gaps of knowledge are suggested in this Recommendations section.

• The biochar used in the study, however, was only limited to one type of biochar, which

was the pine sawdust biochar (prepared at 650C) and one type of feedstock (carbohydrate

food waste). Considering the different characteristics, which may be possessed by different

types of biochar and feedstock, their different effects and mechanisms may be found in the

AD. Therefore, further studies using different types of biochar and other feedstock in

TPAD are recommended.

• In the current study, the presence of typical AD microbes and the establishment of biofilm

on the pore and surface of biochar were observed in the SEM images. However, a further

study on the bacterial and biofilm identification using molecular biological tools is

necessary to give a better understanding of the interaction between biochar TPAD, H2 and

CH4 microbes and operating conditions.

• The operation of TPAD PDU with the addition of biochar was successfully conducted.

However, it was limited to the start-up period under fed-batch and semicontinuous

165

operation under ambient temperature and uncontrolled pH. Therefore, further study on the

biochar addition in long term continuous operation of pilot scale TPAD with controlled

operating conditions is suggested.

• The results also suggest the potential utilisation of combined biochar and effluent as

fertiliser as shown by seed germination assay results. Further studies on the utilisation of

the fertilisers in pot trials and using different application ratio were necessary to provide

confidence before the field application.

166

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