characterization of ligninolytic microbial consortia...

Tawaf Ali Shah

2018

Department of Biotechnology

Pakistan Institute of Engineering and Applied Sciences

Nilore-45650, Islamabad, Pakistan

Characterization of Ligninolytic Microbial

Consortia and Analysis of Recalcitrant

Structural Properties of Pretreated

Lignocellulosic Biomass

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Characterization of Ligninolytic Microbial

Consortia and Analysis of Recalcitrant

Structural Properties of Pretreated

Lignocellulosic Biomass

Tawaf Ali Shah

Submitted in partial fulfillment of the requirements

for the degree of Ph.D.

2018

Department of Biotechnology

Pakistan Institute of Engineering and Applied Sciences

Nilore-45650, Islamabad, Pakistan

iii

Copyrights Statement

The entire contents of this thesis entitled Characterization of Ligninolytic

Microbial Consortia and Analysis of Recalcitrant Structural Properties of

Pretreated Lignocellulosic Biomass by Tawaf Ali Shah are an intellectual property

of Pakistan Institute of Engineering & Applied Sciences (PIEAS). No portion of the

thesis should be reproduced without obtaining explicit permission from PIEAS.

iv

Dedicated

To

My Grand father,

My Family and to All the good

people of my life.

Acknowledgements

First and foremost, I would like to thank the creator for giving me a functioning body

and mind to live life and learn. It is my immense pleasure to acknowledge the role of

several people, who have been instrumental for completion of my PhD.

I would like to pay deepest gratitude to my supervisor Prof. Dr. Romana

Tabassum, DCS Head TSD, who expertly taught me not only fermentation and

bioenergy production but has also been the source of inspiration in every walk of life.

Her determined passion for biofuels production kept me constantly engaged with my

research. Mam, I am really thankful and honored to have you as my mentor. I hope

you will guide me throughout my professional carreer. I am also very thankful to

director NIBGE, Dr. Shahid Mansoor, SI for providing the means to fulfil my

scientific explorations.

I would also like to acknowledge the valuable inputs of our collaborator, Prof.

Dr. Charles Lee, USDA-ARS, 800 Buchanan St. Albany, CA 9471, USA. His

scientific guidance and critical analysis in manuscript writing helped me a lot to grow

as a research scientist and achieve my goals with in time. Thank you for providing me

the facility of DNA Sequencing, FT-IR and SEM analysis in your lab.

I am very thankful to all my lab fellows both past and present, whose

assistance and cooperation were essential not only for completion of my field work

but also for bench work. I would like to thank , Dr. Saira Bashir, Aimen, Ahmad,

Khalid, Amjad, Naveed, and averyone who was there whenever I needed. Sincere

and special gratitude goes to Asifa Afzal and Shahbaz Ali for their company and

support. I would also like to pay gratitude to all my friends at NIBGE.

I would specially like to thank Dr. Raheem Ullah, Dr. Zafar Ali and Dr.

Tariq for their generous support and valuable suggestions specially for reviewing my

thesis efficiently. I am glad that I have friends like you.

I would like to express an eternal love and gratitude for my parents and family

members, who have always been there for me no matter where I am and regardless of

vi

the situation. The unconditional support and patience of my caring Late Grand

Father and My Uncle throughout my educational carreer is the key factor in my

success and achievements. My loving father Dada, from whom I learn philosophy of

life and guidance that helped me get through the difficult times, I faced in different

phases of my life. I am also thankful to Ali Imran, M. Asif and M. Iqbal from

university cell at NIBGE for all their help and coordination during my stay at NIBGE.

Last but not the least I am also obliged to Higher Education Commission,

(HEC) Pakistan, and EMMA-WEST 2013 project selection committee for awarding

me a scholarship for Doctorate of 10 months at University of Padova, Italy.

.

Tawaf Ali Shah

vii

Table of Contents

Declaration of Originality ......................................... Error! Bookmark not defined.

Copyrights Statement ................................................................................................ iii

Dedicated…...................................…………………………………………………...iv

Acknowledgments ........................................................................................................ v

Table of Contents ....................................................................................................... vii

List of Figures ............................................................................................................. xii

List of Tables ……………………………………………………………………….xiv

List of Abbreviations and Symbols .......................................................................... xv

List of Publications .................................................................................................. xvii

Abstract………………… .......................................................................................... xix

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

1.1 General Introduction ........................................................................... 1

1.2 Lignocellulosic Waste Biomasses ........................................................ 2

1.2.1 Recalcitrant Structure of Biomasses ...................................................... 3

1.2.2 Composition ........................................................................................... 4

1.2.3 Cellulose ................................................................................................ 4

1.2.4 Hemicellulose ........................................................................................ 5

1.2.5 Lignin ..................................................................................................... 5

1.3 Functional Groups in Lignocellulose Waste Biomass (LWB) ......... 7

1.4 Pretreatment Methods ......................................................................... 8

1.4.1 Mechanical Pre-Treatment ..................................................................... 8

1.4.2 Thermal Pre-Treatment .......................................................................... 8

1.4.3 Effect of Autoclave, Water Bath, and Microwave Heating ................... 9

1.4.4 Chemical Pre-Treatment ........................................................................ 9

1.4.5 Biological Pre-Treatment ..................................................................... 10

1.4.6 Enzymes for Lignin Degradation ......................................................... 12

1.4.7 Lignin Peroxidase ................................................................................ 13

1.4.8 Manganese Peroxidase ......................................................................... 14

1.4.9 Laccase ................................................................................................. 14

1.5 Finding of New Lignin-Degrading Species ...................................... 15

1.6 Techniques for Analysis of Pretreated and Untreated biomass .... 16

viii

1.6.1 Fourier Transform Infrared (FT-IR) Spectra Analsysis ....................... 16

1.6.2 Scanning Electron Microscope (SEM) ................................................ 16

1.6.3 X-ray Diffraction XRD ........................................................................ 17

1.7 Biohydrogen and Biomethane Production....................................... 17

1.7.1 Hydrolysis ............................................................................................ 18

1.7.2 Acidogenesis ........................................................................................ 18

1.7.3 Acetogenesis ........................................................................................ 19

1.7.4 Methanogenesis.................................................................................... 19

1.8 Objective of the Study ....................................................................... 21

2 Materials and Methods ........................................................................................... 22

2.1 Chemical Treatment of Agribiomass ............................................... 22

2.1.1 Reagents and Materials ........................................................................ 22

2.1.2 Conditions of Alkali Treatment ........................................................... 22

2.1.3 Delignification Method ........................................................................ 22

2.1.4 FTIR Analysis ...................................................................................... 23

2.1.5 Scanning Electron Microscopy ............................................................ 23

2.1.6 High-performance Liquid Chromatography of Carbohydrates ............ 23

2.1.7 Anaerobic Digestion Experiment ......................................................... 24

2.1.8 Statistical Analysis ............................................................................... 25

2.2 Ca(OH)2 Soaking ................................................................................ 25

2.2.1 Scanning Electron Microscopy (SEM) ................................................ 26

2.2.2 Biomethane Potential Test (BMP) ....................................................... 26

2.3 Bilogical Treatment Method ............................................................. 26

2.3.1 Isolation of Aerobic Ligninolytic Bacteria .......................................... 26

2.3.2 16S rRNA analysis ............................................................................... 27

2.3.3 Lignin and Dyes Decolorization Assays .............................................. 27

2.3.4 Growth on Alkali Lignin and Azure B dye .......................................... 28

2.3.5 Screening for Extracellular Hydrolytic Activities ............................... 28

2.3.6 Assessment of Lignin Degradation Efficiency .................................... 28

2.3.7 Lignin-Degrading Enzyme Activity Assays ........................................ 29

2.3.8 Rice Straw Pretreatment ...................................................................... 30


2.3.10 Biomethane Potential Assay ................................................................ 31

2.4 Methodlogy of Biohydrogen and Biomethane Production ............. 32

2.4.1 Reagents and Chemicals ...................................................................... 32

2.4.2 Experimental Design ............................................................................ 32

2.4.3 Isolation of Ligninolytic Bacteria ........................................................ 32

ix

2.4.4 Preliminary Screening of Ligninolytic Bacteria .................................. 33

2.4.5 Biohydrogen Fermentation Batch Assay ............................................. 34

2.4.6 Biomethane Potential Test (BMP) for the Wheat Straw ...................... 35

2.4.7 Analytical Method ............................................................................... 36

2.5 Pretreatment Method with Neurospora Crassa F5 Strain .............. 37

2.5.1 Waste Biomass ..................................................................................... 37

2.5.2 Alkali Pretreatment of Wheat straw ..................................................... 37

2.5.3 Recombinant N. Crassa Treatment ...................................................... 37

2.5.4 Enzyme Activity .................................................................................. 37


2.5.6 FT-IR and X-ray Diffraction (XRD) Analysis..................................... 38

2.5.7 The Automatic Methane Potential Test System (AMPTS) ................. 39

2.5.8 Analytical Method ............................................................................... 39

2.6 Biohydrgoen Production from OFMSW ......................................... 40

2.6.1 Microbial Strains .................................................................................. 40

2.6.2 Screening for the Production of Extracellular Hydrolytic Enzymes ... 40

2.6.3 Cellulase Activity (CelA) .................................................................... 41

2.6.4 Lipolytic Activity (LipA) ..................................................................... 41

2.6.5 Pectinolytic Activity (PecA) ................................................................ 41

2.6.6 Proteolytic Activity (PrA) .................................................................... 41

2.6.7 Starch-Degrading Activity (StA) ......................................................... 41

2.6.8 Xylan-Degrading Activity (XylA) ....................................................... 42

2.6.9 Amylolytic Enzymes Characterization ................................................ 42

2.6.10 Batch Test for Hydrogen Production from Glucose ............................ 42

2.6.11 Batch Test for Hydrogen Production from OFMSW ........................... 43

2.6.12 Analytical Methods and Calculations .................................................. 44

3 Results……. ............................................................................................................. 47

3.1 Alkali Treatment ................................................................................ 47

3.1.1 Analysis for Biomass Composition ..................................................... 48

3.1.2 Alkali Treatment to Remove Lignin from Lignocellulosic Biomass... 49

3.1.3 Substrate Base Lignin Removal ........................................................... 52

3.1.4 SEM for the Surface Degradation of Biomass .................................... 55

3.1.5 Fourier Transformed-Infrared Spectroscopy ....................................... 57

3.1.6 Biogas Potential of Pre-Treated and Untreated Substrates .................. 60

3.1.7 Conclusion ........................................................................................... 62

3.2 Enhancing Biogas from Lime Soaked Corn Cob Residue ............. 64

3.2.1 Composition and Ca(OH)2 Soaking Effect .......................................... 65

x

3.3 Scanning Electron Microscopy ......................................................... 65

3.4 Anaerobic Digestion and Biomethane Potential.............................. 66

3.5 Biological Pretreatment of Rice Straw ............................................. 71

3.5.1 Isolation and Characterisation of Lignin-Degrading Bacterial Strains 72

3.5.2 Growth on Lignin and Azure B ........................................................... 74

3.5.3 Decolorization of Lignin and Azure B ................................................. 74

3.5.4 Lignin Degradation Efficiency ............................................................ 75

3.5.5 Biological Pretreatment of Rice Straw ................................................ 76

3.5.6 Scanning Electron Microscopy of Rice Straw ..................................... 77

3.5.7 Fourier Transformed-Infrared Spectroscopy of Rice Straw ................ 78

3.5.8 Biogas and Methane Yield from Pretreated Rice Straw ...................... 81

3.5.9 Conclusion ........................................................................................... 83

3.6 Two Separate Biohydrogen and Biomethane Fermentation .......... 83

3.6.1 Selection of Ligninolytic Bacterial Strains .......................................... 85

3.6.2 Identification of Ligninolytic Bacterial Strains ................................... 87

3.6.3 Evaluation for Hydrogen Production from Cellulose and Xylose ....... 88

3.6.4 Hydrogen Production from Wheat Straw ............................................ 90

3.6.5 Biogas Potential from the Bio-Pretreated Wheat Straw……….. ........ 92

3.6.6 Application and advantages of two-phase batch fermentation ............ 94

3.6.7 Conclusion ........................................................................................... 95

3.7 Pretreatment of Wheat Straw using N. Crassa Strain ................... 96

3.7.1 Chemical Composition of Wheat Straw .............................................. 98

3.7.2 Cellulase Production of N. Crassa F5 and Hydrolysis of Wheat Straw

.............................................................................................................. 98

3.7.3 Scanning Electron Microscopy (SEM) for Analysis of Wheat Straw

Surface Degradation............................................................................. 99

3.7.4 Fourier Transformed-Infrared Spectroscopy (FT-IR) for Wheat Straw

Fiber ................................................................................................... 100

3.7.5 N. Crassa F5 Pretreatment Improved Biogas Production .................. 103

3.7.6 Changes in Organics Composition during BMP Process .................. 106

3.7.7 Conclusion ......................................................................................... 109

3.8 Bacillus sp. Strains to Produce Bio-Hydrogen ............................. 109

3.8.1 Screening for Extracellular Enzymatic Activities .............................. 111

3.8.2 Hydrogen Potential from Glucose by Selected Microbial Strains ..... 113

3.8.3 Characterization of Amylolytic Enzymes by F2.5 and F2.8 .............. 116

3.8.4 Hydrogen Production by F2.5 And F2.8 ............................................ 117

3.8.5 Hydrogen Potential from OFMSW .................................................... 120

3.8.6 VFAS Profiles from Fermentations ................................................... 123

xi

3.8.7 Conclusion ......................................................................................... 123

4 Discussion............................................................................................................... 124

4.1 Influence of Alkalies Treatment on Lignocellulosic biomass ....... 125

4.2 Biological Pretreatment using Ligninolytic Bacterial Culture .... 128

4.3 Biohydrogen and Biomethane Batch Fermentation ..................... 131

4.4 Preatment with Recombinant Neurospora. Crassa F5 strain ....... 133

4.5 Bio-Hydrogen from the OFMSW ................................................... 134

4.6 Summary and Future Scenarios ..................................................... 136

References… .................................................................................................. ……140

xii

List of Figures

Figure 1-1 The report of total energy supply according to the European

Environment Agency and International Energy Agency (IEA) . ........... 2

Figure 1-2 Lignocellulosic waste biomass and pre-treatment effect. ...................... 6

Figure 1-3 Catalytic reaction of Lignin peroxidase ............................................... 13

Figure 1-4 Catalytic reaction of Manganese Peroxidase ....................................... 14

Figure 1-5 Catalytic reaction of Manganese Peroxidase ....................................... 15

Figure 1-6 Pathways and processing steps involved in the biodegradation of waste

biomass for biogas production [15]. .................................................... 20

Figure 1-7 The figure show process of two-separate phases of anaerobic digestion

for bioenergy production...................................................................... 20

Figure 3-1 Box plot demonstration for NaOH base comparative delignification, 51

Figure 3-2 Box plot demonstration for KOH base comparative delignification, .. 51

Figure 3-3 Effect of alkali on hemicellulose and cellulose degradation. .............. 52

Figure 3-4 Comparative delignification of NaOH for microwave (MW), autoclave

(Auto), and water bath (WT), .............................................................. 53

Figure 3-5 Comparative delignification of KOH for microwave (MW), autoclave

(Auto), and water bath (WT), .............................................................. 54

Figure 3-6 Comparative delignification of Ca(OH)2 for microwave (MW),

autoclave (Auto), and water bath (WT), .............................................. 55

Figure 3-7 SEM micrographs for the untreated and pre-treated wheat straw, ....... 56

Figure 3-8 SEM micrographs for the untreated and pre-treated Kallar grass, ....... 57

Figure 3-9 FT-IR spectrum of wheat straw for microwave sample,...................... 59

Figure 3-10 Comparision of FTIR peak variation of wheat straw, .......................... 60

Figure 3-11 Daily volumetric biogas of NaOH and KOH pretreated wheat straw. 62

Figure 3-12 Daily volumetric biogas of NaOH and KOH pretreated almond shell.63

Figure 3-13 Cumulative biogas of NaOH (A) and KOH (B) pretreated wheat straw.

.............................................................................................................. 63

Figure 3-14 Cumulative biogas of NaOH (A) and KOH (B) pretreated almond

shell. ..................................................................................................... 64

Figure 3-15 Morphology of the corn cob before and after Ca(OH)2 soaking, ........ 66

Figure 3-16 Daily biogas of untreated, 7, 15, and 30 days Ca(OH)2 soaked corn

cob, ....................................................................................................... 68

xiii

Figure 3-17 Daily methane of untreated, 7, 15, and 30 days Ca(OH)2 soaked corn

cob, ....................................................................................................... 69

Figure 3-18 Cumulative biogas of untreated, 7, 15, and 30 days Ca(OH)2 soaked

corn cob, ............................................................................................... 70

Figure 3-19 Cumulative methane of untreated, 7, 15 , and 30 days Ca(OH)2 soaked

corn cob, ............................................................................................... 71

Figure 3-20 Isolation of bacterial isolates on lignin and selection based on Azure B

decolorization on (A) solid medium and (B) liquid medium. .............. 73

Figure 3-21 Phylogenetic analysis with related species strains based on 16S rRNA

of the bacterial isolates......................................................................... 74

Figure 3-22 Efficiency of alkali lignin degradation with various substrate

concentrations at pH 5 (A) and pH 7 (B). ............................................ 76

Figure 3-23 Effect of pretreatment by individual isolates (TL4, TL6, TL8, TL26,

TL27, TL33) or co-culture on the composition of rice straw, Error bar

= standard deviation. ............................................................................ 77

Figure 3-24 Surface morphology of rice straw pretreated with bacterial cultures, . 79

Figure 3-25 FT-IR spectrum of untreated rice straw sample (C) compared to treated

sample of TL4,TL6,TL8,TL24,TL26,TL27, and TL33 isoaltes. ......... 80

Figure 3-26 Daily biogas from rice straw of untreated, pretreated with individual

isoalte (TL4, TL6, TL26) and co-culture, ............................................ 82

Figure 3-27 Cumulative biogas from rice straw of untreated and pretreated with

individual isoalte (TL4, TL6, TL26) and co-culture,........................... 82

Figure 3-28 Dye degradation potential of isolates, Azure B dye (A) and Lignin (B)

in evaluation with control and inoculated conditions respectively. ..... 86

Figure 3-29 Evolutionary relationships and phylogenetic tree with related species

strains based on 16S rRNA of the bacterial isolates. ........................... 88

Figure 3-30 Cumulative hydrogen productions from xylose (A) and cellulose (B)90

Figure 3-31. Cumulative H2 productions from wheat straw in first batch

fermentation assay, .............................................................................. 91

Figure 3-32 Cumulative biogas from wheat straw in second batch, ........................ 93

Figure 3-33 Cellulase production of N. crassa F5 on wheat straw, ........................ 99

Figure 3-34 SEM images of wheat straw after hydrolysis, ................................... 100

Figure 3-35 FT-IR spectra of wheat straw after hydrolysis, .................................. 102

Figure 3-36 XRD analysis of wheat straw after hydrolysis, ................................. 103

Figure 3-37 Optimization experiment for biogas potential for N. crassa F5

treatement, .......................................................................................... 105

Figure 3-38 Cumulative biogas of pretreated and untreated wheat straw, ............ 105

Figure 3-39 The effect of pH (a) and incubation temperature (b) on the amylase

activity of Bacillus sp. F2.5 ( ) and Bacillus sp. F2.8 ( ) grown for 72

h in SPM containing 20 g/L soluble starch. ....................................... 117

Figure 3-40 Cumulative hydrogen productions of Bacillus sp. F2.5 (a) and Bacillus

sp. F2.8 (b) ......................................................................................... 118

xiv

List of Tables

Table 1-1 Lignocellulosic biomass composition. ................................................... 6

Table 1-2 Different functional groups in lignocellulose biomass molecules ......... 7

Table 1-3 Enzymes and mechanism of associated reaction ................................. 12

Table 2-1 Results from manual sorting procedure of the OFMSW……………. 44

Table 3-1 Estimated percentage composition of different waste biomasses ........ 48

Table 3-2 One-way ANOVA for NaOH delignification ...................................... 49

Table 3-3 One-way ANOVA for KOH delignification ........................................ 50

Table 3-4 Methane yield (NmL/gVS) of the NaOH and KOH treated biomass .. 64

Table 3-5 Effect of Ca(OH)2 soaking on composition of corn cob ...................... 65

Table 3-6 Growth, decolorization potential of lignin and Azure B dye………...75

Table 3-7 Methane content (%) and cumulative methane yield. ......................... 83

Table 3-8 Decolorization potential of lignin and Azure B ................................. 86

Table 3-9 Volatile fatty acid (VFA) production ………………...……………..92

Table 3-10 Comparison of the biohydrogen and methane yield ............................ 95

Table 3-11 Wheat straw composition ..................................................................... 98

Table 3-12 Crystallinity of untreated wheat straw and treated wheat straw ........ 102

Table 3-13 The COD removal efficiency of pretreated wheat straw ................... 107

Table 3-14 The VS removal efficiency of pretreated wheat straw ....................... 108

Table 3-15 VFA production in anaerobic digestion of pretreated wheat straw ... 108

Table 3-16 The accumulation of ammonia in anaerobic digestion…….………..109

Table 3-17 Extracellular enzymatic activity of microbial strains. ....................... 113

Table 3-18 Comparison of hydrogen production potential of Bacillus sp. .......... 115

Table 3-19 VFAs profiles of the biogas produced on different substrates.. ......... 120

Table 3-20 Comparison of hydrogen production from OFMSW. ........................ 122

xv

List of Abbreviations and Symbols

NmL/gVS

LWB

AD

OBA

MC

TS

VS

CH4

H2

FT-IR

SEM

XRD

NaOH

KOH

MV

Auto

WT

LB

mL

U/mL

Ca(OH)2

Lip

Lac

Mnp

%

S

°C

LB

Lig

Normalized biogas in mL per gram volatile solid

Lignocellulosic waste biomass

Anaerobic digestion

Online Biogas App

Moisture content

Total solid

Volatile solid

Methane

Hydrogen

Fourier Transform Infrared Spectroscopy

Scanning Electron Microscopy

X-rays differaction

Sodium hydroxide

Potassium hydroxide

Microwave

Autoclave

Water bath

Lignocellulosic biomass

Mili litter

Units/ mL

Calcium hydroxide

Lignin peroxidase

Laccase

Manganase peroxidase

Percent

Second

Degree Celsius

Lignocellulosic biomass

Lignin

xvi

MV

N. CRASSA

WS

RS

Microwave

Neuraspora Crassa

Wheat straw

Rice straw

xvii

List of Publications

Journal Publications

Tawaf Ali Shah, Shehbaz Ali, Asifa Afzal, Romana Tabassum ‘A review on

biohydrogen as a prospective renewable energy’ International Journal of

Biosciences Vol.11.P.106-130.2017.

Tawaf Ali Shah, Romana Tabassum "Enhancing biogas production from Lime

soaked corn cob residue". International Journal of Renewable Energy Research

(IJRER, Vol 8, No 2, June 2018, IF 1.25).

Tawaf Ali Shah , L. Favaro, L. Alibardi, L. Cagnin, A. Sandon, R. Cossu , S.

Casella, M. Basaglia ‘’Bacillus sp. strains to produce bio-hydrogen from the

organic fraction of municipal solid waste’’ Journal of Applied Energy 176

2016, 116–124, IF 7.5).

Tawaf Ali Shah, Shehbaz Ali, Asifa Afzal, Romana Tabassum’’Effect of

Alkalis pretreatment on Lignocellulosic Waste Biomass for Biogas

Production’’ International Journal of Renewable Energy Research

(IJRER).Vol-8,No3, 2018 (IF 1.3).

Tawaf Ali Shah, Charles C. Lee, William J. Orts and Romana Tabassum ‘’Biological pretreatment of rice straw by ligninolytic Bacillus sp. strains for

enhancing biogas production’’ Environmental Progress & Sustainable Energy

.2018. (IF 2.01)

Tawaf Ali Shah, Shehbaz Ali, Asifa Afzal, Romana Tabassum.‘’Two separate

biohydrogen and biomethane batch fermentation by ligninolytic Bacillus sp.

strains using wheat straw as a substrate’’ Journal of Bioenergy Research, pp1-

15,31-2018. (IF 2.5).

Tawaf Ali Shah, Romana Tabassum, Ruihong Zhang, Takao Kasuga,

Zhiliang Fan’’Pretreatment of wheat straw using a recombinant Neurospora

crassa strain for improved biogas production’’ (Manuscript).

Shehbaz Ali, Tawaf Ali Shah, Asifa Afzal, Romana Tabassum , 2018.

Exploring lignocellulosic biomass for bio-methane potential by anaerobic

digestion and its economic feasibility. Energy & Environment 0(0) 1–10 DOI:

10.1177/0958305X18759009

Shehbaz Ali, Tawaf Ali Shah, Asifa Afzal, Romana Tabassum 2017.

Evaluating the co- digestion effects on chicken manure and rotten potatoes in

batch experiments. International Journal of Biosciences 2017; 10: 150-159.

Shehbaz Ali, Tawaf Ali Shah, Asifa Afzal, Romana Tabassum, 2018. Analysis

of agricultural substrates for nutritive values and bio-methane potential; Journal

of Current Science, PP115(2).2018.

Shehbaz Ali, Tawaf Ali Shah, Asifa Afzal, Romana Tabassum, 2018. A

concise review of methane production from lignocellulosic biomass; Biomass

xviii

characteristics; Anaerobic digestion and microbial ecology of digestion media.

In review; International Journal of Renewable Energy Research.

Oral and poster Research Presentations

Tawaf Ali Shah, Favaro L, Alibardi L, Cagnin L, Cossu R, Casella S, Basaglia

M,Exploring microbial diversity of a brewery full scale anaerobic digester to

look for robust and efficient H2- producing microbes, National student

conference in Biotechnology 11 October 2015at NIBGE.

Lorenzo Favaro, Tawaf Ali Shah, Shehbaz Ali, Lorenzo Cagnin, Annalisa

Sandon Raffaello Cossu, Sergio Casella, Marina Basaglia ‘’Mining full scale

anaerobic digester as source of robust microbes for bio-hydrogen production

from organic waste’’ ISME -16 meeting (http://www.isme-

microbes.org/isme16 16th International Symposium on Microbial Ecology)

held 21-26 August 2016.

Tawaf Ali Shah “Evaluation of different pretreatment of waste biomass for

biogas production’’ 1st International Conference on Biotechnology (ICB),

Challenges and opportunity in Pakistan (Oct 11-12-2017).

Tawaf Ali Shah, L. Favaro, S. Casella, M. Basaglia; Bacillus sp. strains to

produce bio-hydrogen from the organic fraction of municipal solid waste

(OFMSW). First National Conference on Emerging Trends in Bioinformatics

and Biological Sciences at Department of Bioinformatics, Hazara University

Mansehra, Pakistan.20-22 July 2017.

L. Favaro, Tawaf Ali Shah, L. Alibardi, L. Cagnin, R. Cossu, S. Casella, M.

Basaglia ; Full scale anaerobic digester as promising source of robust microbes

for H2 production from organic waste ECO Bio 2016_0515 conference.

http://www.isme-microbes.org/isme16

http://www.isme-microbes.org/isme16

xix

Abstract

The aims of this dissertation was to evaluate chemical and biological treatment

methods to remove and degrade lignin from agriculture waste biomass for increasing

the yield of biogas and biohydrogen. In chemical treatment approach, three alkali

reagents at various dosages: NaOH (1-5%), KOH (1-5%), and Ca(OH)2 (0.5%) and

three different heating processes, water bath, autoclave and short time microwave

were tested for 10 different agriculture substrate. The Scanning Electron Microscopy

(SEM) images showed visible degradation on the alkalies treated surface of biomass

as compared to the untreated biomass. Additionally, disapperance and emergence of

new peaks were observed in treated substrates using Fourier Transform Infrared

spectroscopy (FT-IR). Microwave heating with 2% NaOH treated substrates showed

more total biogas yield as compared to other treatment conditions. The Ca(OH)2

(0.5%) soaking of corn cob for 7, 15, and 30 days incubation was tested. The highest

cumulative biogas was 360.5 NmL/gVS, 3-times higher than the cumulative biogas

produced from the untreated corn cob 115.1 NmL/gVS. For biological treatment of

waste material, 27 ligninolytic bacteria were isolated from soil, wood compost, and

waste sludge. Seven of the most active strains were selected. The optimum yields of

lignin peroxidase and laccase were achieved at pH 3-5. The co-cultures demonstrated

2.5 times more rice straw lignin degradation than using single culture. Likewise, the

greatest enhancements of cumulative methane yield (70-76%) occurred from co-

cultures treated rice straw as compared to individual culture. To produce biohydrogen

and biomethane separatly in batch fermentation, 20 ligninolytic Bacillus sp. strains

were isolated from granular sludge of full scale anaerobic digester. Among them, four

ligninolytic Bacillus sp. strains were selected based on their lignin and Azure B

degradation. Brevibacillus agri AN-3 exhibited the highest decrease in COD (88.4%)

of lignin and (78.1%) of Azure B. Brevibacillus agri AN-3 showed hydrogen (H2)

yield of 1.34 and 2.9 mol-H2/mol from xylose and cellulose respectively. In two-

phase wheat straw batch fermentation, Brevibacillus agri AN-3 produced 72.5 and

125.5 NmL/gVS cumulative H2 and methane (CH4) respectively. It was perceived that

using ligninolytic Bacillus sp. strains, 48.6% more methane yield could be obtained

xx

from the wheat straw than using the untreated wheat straw in batch fermentation. A

consolidating bioprocessing of recombinant Neurospora crassa F5 strain was used for

saccharification of wheat straw (WS) to increase the biogas production. The WS was

pretreated with 2% NaOH followed by 2,4, and 6 days hydrolysis with N. crassa F5

strain at 28±1℃ and 200 rpm using 0.5 g/L glucose in Vogel media. Scanning

Electron Microscopy (SEM) analysis showed a visible change on the surface structure

of the pretreated WS as compared to the untreated WS. The 2,4 and 6 days N. crassa

F5 saccharified WS was used for biomethane potential (BMP) analysis using

automatic methane potential testing system (AMPTS). A maximum cumulative biogas

of 700.8 mL/gVS was obtained from 2% NaOH pretreated WS followed by 2 days N.

crassa F5 treatment. The recombinant N. crassa F5 treated WS produced daily biogas

which was 6-fold higher per day and 339.3% more in cumulative volume than the

untreated WS sample. Finally, a single culture was tested for the potential of

biohydrogen from Organic Fraction of Municipal Solid Waste (OFMSW). One

hundred and twenty bacterial strains were isolated from heat-treated granular sludge

of a full scale anaerobic digester. The best hydrolytic strains were assessed for H2

production from glucose and soluble starch. Two Bacillus sp. strains, namely F2.5 and

F2.8, exhibited high H2 yields and were used as pure culture to convert OFMSW into

hydrogen. The strains produced up to 61 mL of H2 per grams of volatile solids and

could be considered as good candidates towards the development of industrially

relevant H2-producing inoculants. This was the first successful application of pure

microbial cultures in bio-hydrogen production from OFMSW.

1 Introduction

1.1 General Introduction

Production of bioenergy from lignocellulosic waste biomass (LWB) sources needs

uncompromising considerations to supersede the dependence on fossil fuel. The key

obstacle is an effective waste management and pretreatment method in the conversion

of solid waste biomass into a valuable product of liquid and gaseous biofuels. Fossil

fuel sources (coal, oil and natural gas) are depleting and release an extreme quantity

of greenhouse gases. Greenhouse gases polluting our environment, increasing urban

smog and damaging biodiversity [1]. The continuous depletion of fossil fuel further

generating global warming. Therefore, an effective waste management method is the

application of the anaerobic digestion (AD) technology for the production of different

kinds of environmentally harmless biofuels. Biogas is one of the alternative energy

generated through anaerobic digestion process from LWB [1].

Looking into the realities of the future every country is investing in the

production of renewable energy from waste biomass source. According to the data

recorded by U.S. Energy Information Administration (EIA), the European

Environment Agency and International Energy Agency (IEA), the energy production

in 2013 was 28.9% from coal, 31.1% (oil), 21.4% (natural gas), 4.8% (nuclear), 2.4%

(hydro), 10.2% (biofuels and waste), and 1.5% from other sources as shown in the

(Figure 1.1) [2]. The IEA reports further proved that the world is now thinking of

energy production from renewable sources instead of depending on non-renewable

sources. The 2013 reported value indicates that biofuels production are not considered

as important as it keeps value. However, due to the large demand for energy and

depletion of fossil fuel, bioenergy production from waste biomass sources is a

sustainable approach in the future. The problems of global warming, energy crises,

pollution and economy can be solved by using the suitable pretreatment methods and

advance technolgy for anaerobic digestion process from LWB.

https://en.wikipedia.org/wiki/Energy_Information_Administration

https://en.wikipedia.org/wiki/European_Environment_Agency

https://en.wikipedia.org/wiki/European_Environment_Agency

https://en.wikipedia.org/wiki/International_Energy_Agency

Chapter 1: Introduction

2

Figure 1-1 The report of total energy supply according to the European

Environment Agency and International Energy Agency (IEA) .

1.2 Lignocellulosic Waste Biomasses

The most favored input sources in anaerobic digestion technology are LWB due to

high energy yield and sufficient availability. But, presently agriculture biomass adds

around 7–10% to the world’s energy stream [3]. A 9–14% to the total energy supply

is reported in industrialized countries, while in developing countries, the energy

contribution is much higher from biomasses up to one‐fifth [4]. Whereas, in

developing countries much of the agricultural waste products are utilized for cooking

in wood‐stoves or burn in the field. Existing energy production percentage from waste

biomass is low and could be increase considering the huge quantities of waste

biomass being produced [3].

The relatively high abundance of energy crops and LWB (soft wood,

hardwood, grasses, forestry and agricultural residues) needs to be utilized in suitable

method. Agriculture waste biomass contained high water content and useful for

production of energy carriers such as biogas, biodiesel, biohydrogen and bioethanol.

Amongst, these fuel sources, anaerobic digestion using waste biomass for biogas

being the most commonly used approach. So bioenergy could be given the first

priority to replace fossil fuel production.


3

1.2.1 Recalcitrant Structure of Biomasses

For the development of efficient anaerobic technology, the selection of crop residue

and composition of biomass is particularly important. Amount of water and lignin

value is secondary vital property in choosing substrates for the biochemical process.

While the total organic composition is the most crucial consideration in the hydrolysis

of the biomass [5]. The productivity of the microbial transformation of the substrate

in anaerobic digestion process is associated to the biodegradability of LWB. The

organic constituents in agricultural biomass have a recalcitrant structured cell wall

which is called “lignocellulose” [5].

The recalcitrant structure of the LWB is the main obstacle. Hence pre-

treatment is essential, with an aim to reduce the recalcitrant property and expose the

cellulose to enzymes hydrolysis of LWB for increasing efficiency of biofuels

production. The recalcitrance of LWB is due to several factors. They contain lignin

carbohydrate complexes (LCCs), structure and content of lignin, acetylated

hemicelluloses, cellulose crystallinity and degree of polymerization (DP) [6]. Lignin

performs a function as a physical barrier to stop enzyme access to the carbohydrate

fraction of LWB. The mechanism of inhibition and acting as a barrier of lignin against

enzymatic hydrolysis is still unclear. Although cross-linkages between lignin and

carbohydrate are believed to be a significant contributing factor [6]. Additionally,

enzymes loss their activities by irreversibly binding to lignin through hydrophobic

interactions during biomass hydrolysis [7]. Unbranched hemicelluloses covalently

bond to lignin and generate enzyme impermeable cross-links. Acetyl groups of

hemicelluloses make a sheath around cellulose microfibrils and hinder enzyme attack.

The cross-links between lignin-hemicellulose and cellulose are also called LCCs,

which are major barriers to enzyme access to cellulose [8].

The LWB in general consists of 40-50% cellulose, 25-30% hemicellulose and

20% lignin and other extractable components [9]. However, the composition of the

substrate can be considerably varied for hemicellulose, lignin, cellulose, and other

content depending on its source whether its derive from soft,hardwood or grasses

[10]. LWB is being commonly generated through agriculture waste processing

practices and also from agriculture industries with a comparative amount of domestic

use, however, unfortunately, most of these LWB is regularly predisposed by burning.


4

Therefore, the enormous quantities of LWB theoretically supposed to be converted

into different bio-fuels are vanished [11].

1.2.2 Composition

Higher plants, hardwood, and softwood are also known as LWB as it mainly

composed of the main three components, hemicellulose, lignin and cellulose as shown

in (Table 1-1). A maximum portion of the cell wall is made of cellulose which

provides both mechanical and chemical stability to the plants. The second copolymer

in the plant composition is hemicellulose which is composed of various C5 and C6

sugars polymers. Aromatic compounds (Lignin) is the third and important protective

layer of plant cell wall produce by the biosynthetic process in the plant system.

Besides the three main components in the plant cell wall, water, proteins, minerals

and other components are also present in small quantities. Plant materials and

agricultural waste biomass need a pretreatment process to hydrolysed hemicellulose,

degrade lignin and open the chains of cellulose as shown in (Figure 1-2).

1.2.3 Cellulose

Cellulose is made of a basic unit of cellubiose, 4-O-β-D-glucopyranosyl-D-glucose

and is more usually thought as a polymer of glucose because cellobiose comprises of

two molecules of glucose. (C6H10O5)n is the chemical formula of one chain of the

polymer in cellulose. Most of the properties of cellulose is related to the degree of

polymerization (number of glucose units in one polymer) and common number of DP

of cellulose is between 800-10000 units. The bond between β-1,4 glucosidic glucose

molecules is flexible and allow each molecule to form a long straight chain. Hydrogen

bonds are present between the molecules which produced several parallel chains in

the compound [12]. Cellulose is present in both non-crystalline and crystalline

structure. The combination of numerous polymer chains produces microfibrils and

finally united to form fibers to complete its full crystalline structure. Figure 1-2

illustrates structure as well as the placement of cellulose in the cell wall. At normal

atmospheric conditions cellulose can absorb water up to 8-15% and swells out as it is

not soluble in water, similarly, cellulose is not soluble at low temperature in dilute

acid. The cellulose hydrolysis is directly related to its solubility and it is soluble at

high temperature and in a high concentration of acids solutions, however, alkali


5

treatment swells crystalline structure of the cellulose and reduce their degree of

polymerization [11].

1.2.4 Hemicellulose

The second rich heterogeneous polymer is hemicellulose which collectively

comprises of different sugar molecules i.e glucomannan, glucuronoxylan, galactans

and small quantities of other polysaccharides. They are classified with sugar as a

backbone, i.e., mannans, xylans, and glucans, are common and lower amount of

galactans and arabinans are present in the hemicellulose molecule, however, they are

different in b-1,4 linked backbone structure. Xylan is the most common plymer in

hemicellulose which involves 1-4 linkages of xylopyranosyl units, these units

attached anhydroxylose units with α-(4-O)-methyl-D-glucuronopyranosyl produce a

branched polymer chain of six carbon sugar monomers (glucose) and five carbon

sugar monomers (xylose) [11]. Importantly hemicellulose insoluble in water at low

temperature, and presence of acids in water highly improves their solubility.

Hemicellulose is not crystalline in nature due to the presence of acetyl groups in the

polymer, also have a wide range of shape, size and mass characteristics with a low

degree of polyemrisation. To make a strong backbone hemicellulose are bound tightly

through non-covalent bonds to the surface of each cellulose micro-fibril [11].

1.2.5 Lignin

The most complex fraction of plant cell wall is lignin which is a long chain of

aromatic heterogeneous polymer comprise of phenyl-propane units and methoxy

groups in poly phenolic element connected by ether bonds. The core function of

lignin is to tightly bind both hemicellulose and cellulose molecules thus act like a glue

to fill every gap between and around making lignocellulosic biomass a harder

structure for the degradation, it is therefore known as barrier in biofuels production

from lignocellulosic substrates. Coniferyl alcohol, p-coumaryl alcohol, and sinapyl

alcohol are the three major phenolic components of lignin which make them

hydrophobic that resist during hydrolysis process. The distribution of lignin varies

noticeably among different plants cell wall [13]. Lignin is soluble in different solvents

acetone, pyridine, dioxane, dimethyl sulfoxide and in acidic or alkaline solutions at

high temperature [14].


6

Waste biomass

Pretreatment effect

Figure 1-2 Lignocellulosic waste biomass and pre-treatment effect.

Table 1-1 Lignocellulosic biomass composition adopted from Sun and Ghimire

[10, 15] et al.

Substrate Cellulose (%) Hemicellulose (%) Lignin (%)

Paper 40-55 25-35 15-20

Wheat straw 35-40 30-35 15-16

Corn straw 38-45 26 17-20

Corn cob 40-50 20-25 15-17

Paragrass 35-45 25-30 15-20

Grasses 30-40 20-30 20-25

Peanut 40-45 15-17 20-30

Rice straw 38 19 13

Barley straw 37 24 16

Corn stalk 36 26 16

Cornstalk 34 27 21

Lawn grass 30 43 3-5

Sugarcane bagasse 33 23 5

Sweet sorghum 38 21 17


7

1.3 Chemical Properties of Functional Groups in

Lignocellulose Waste Biomass (LWB)

LWB composed of different functional groups, among them the interesting groups are

those which are involved in hydrolysis (furfural), partial depolymerization of lignin

(phenolic compounds). Different functional groups are listed in the (Table 1-2), where

hydrogen bond is considering a functional group due to the properties of changing the

solubility of a molecule and helps in degradation of biomass compounds.

Table 1-2 Different functional groups in lignocellulose biomass molecules

Group Cellulose Hemicellulose Lignin

Hydroxyl group X X √

Aromatic ring X X √

Ether (glucosidic linkage) √ √ √

Hydrogen bond √ √ X

Ester bond X √ X

Carbon to carbon linkage X X √

Among the three components, lignin polymer holds maximum different

functional groups which bring its degradation and depolymerization to water-soluble

byproducts. Cellulose contains only hydrogen and ether glucosidic bond and can

easily produce simple sugar monomers. These functional groups are utilizing in the

substitution and transformation reactions. Kirk-Otmer [16] described in details about

the transformation and substitutions in functional groups. According to Kirk-Otmer,

lignin polymer aromatic ring are substituted with chlorine or nitro groups as a result

of chlorination and nitration reactions. Similarly, hydroxyl group is transformed to

aryl or allylic ether with acidic solutions renders the lignin polymer water soluble.

The most important bond is ether among the functional groups, as it holds (glucosidic

linkage) in glucose monomers polymer and is the principal bond of the lignin

polymer, hence termination of ether bond can bring separation of lignin from the

lignocellulosic biomass. Solvolytic reactions cleave ether bond, whereas alkaline

conditions separate aromatic rings and acidic conditions convert ether bond into

hydroxyl follow by carbonyl or carboxyl before it closely broke into C3 or C2

molecules. In hemicellulose, the acetyl group builds ester bond with a hydroxyl chain

of the polysaccharides and hydrolysis convert the ester bond carboxyl and hydroxyl

groups but the position of ester bond is uncertain between lignin,-hemicellulose and


8

lignin –cellulose [12]. Hydrogen bonds is present in the hydrogen atom of glucose

monomer between an oxygen atom and a hydroxyl group of another glucose

monomer in cellulose and hemicellulose polymer chains makes the parallel chain of

both the molecules insoluble in water. High temperatures break hydrogen bonding by

introducing newly high energy hydrogen or by altering the structure of the polymer

with a low hydrogen bond between the molecules [17].

1.4 Pretreatment Methods

Pre-treatment are used to disrupting cellulose, lignin crystallinity, and hemicellulose

content. A wide range of pre-treatment methods has been tested, including physical,

hydrothermal, chemical and biological. The best pre-treatment method must have

properties like (a) require less chemical (b) maximum carbohydrate recovery (c) very

limited amount of by-products (d) cost-effective for large-scale application (e)

applicable for different kinds of biomass feedstock’s and (f) reduce the amount of

enzymes required for substrate hydrolysis [18]. Generally, the above mentioned

properties are the key limitations of most pre-treatment methods, in viewing to these

points, here a brief story of only those pretreatment methods are described which are

considered in experimental research of this thesis.

1.4.1 Mechanical Pre-Treatment

Mechanical pre-treatment makes solid particles of the feedstock coarse, increasing the

specific surface area and break them down into small size particles with low water,

easy implementation and a moderate energy consumption in initial pre-treatment

process. A variety of mechanical pre-treatment methods -i.e piston press, high-

pressure Homogenizer, bead mill, sonication, and grinding are reported.

1.4.2 Thermal Pre-Treatment

Amongst the pre-treatment methods, thermal treatment is extensively used at

industrial scale for pathogen removal. This treatment let down the viscosity of the

digestate and solubilized organic compounds. Thermal treatment includes

temperatures ranges from 100-160 ᵒC with shorter or longer treatment time depending

on limiting loss of volatile organics, solubilisation of proteins and carbohydrates.


9

1.4.3 Effect of Autoclave, Water Bath, and Microwave Heating

Generally, providing heating combined with acid or alkali treatment is to break down

bonds in lignin, increase the surface area and decrease the crystallinity of cellulose

[2]. Hence a proper pre-treatment is necessary to remove lignin and expose the

cellulose for hydrolysis in further process. Different heating devices are practiced for

the pretreatment of agriculture biomass. Commonly, for the supply of conventional

heating hot plate, water bath and burners etc is studied. These devices provide heat

slowly and gradually within the sample. Autoclave heating uses moist heat under

pressure for supplying heating for lignocellulosic biomass hydrolysis. However,

ultimately, the supply of heating results in a substantial breakdown of the lignin in

biomass and improved the digestibility of substrate [19].

Microwaves heating using electromagnetic and infrared radiation with a

frequency range of 300 MHz to 300 GHz for energy and wave transformation [20].

Beside of electromagnetic spectrum it also produces heating of materials by induced

molecular vibration as a result of dipole rotation or ionic polarization [21]. The

heating process is different than the conventional heating system based on volumetric

heat generation and inward to outward heating system. Microwave is also used for

commercial and household purposes include blanching, pasteurization, sterilization,

selective heating and enhanced reaction kinetics etc [21]. Importantly. It is also used

for the pretreatment of lignocellulosic biomass and is proved to 36 times more

efficient pretreatment method than the conventional heating [22]. In this regards,

Microwave radiation has been applied breaking down lignin to improve digestibility.

Zhu et al. [23] reported three different microwave pretreatment alkali, combine acid-

alkali, and acid-alkali-H2O2 using rice straw as a substrate. They reported the highest

rate of rice straw hydrolysis and releasing of glucose in the hydrolysate. Pretreatment

with lower microwave power for a longer period of time and pretreatment with higher

microwave power for less processing time has an equally similar effect on the lignin

removal, weight loss, and composition of waste biomasses [22].

1.4.4 Chemical Pre-Treatment

Chemical pretreatments have been extensively used to improve the biodegradability

of cellulose by removing lignin and hemicellulose. Concentrated and diluted acids are

involved to break the rigid structure of the lignocellulosic material. The most


10

commonly used acid is dilute sulphuric acid (0.5-1% w/v) hydrolyzing the

hemicellulose portion of lignocellulosic biomass to simple sugars minimizing the

production of fermentation inhibitors during anaerobic digestion [24]. Alkali

treatment removes the lignin and leftover relatively pure cellulose from agricultural

waste biomass. The most extensively used alkaline pretreatment bases are potassium

hydroxide, sodium hydroxide and calcium hydroxide [25] to degrade the lignin of the

feedstock and to maximize the accessibility of enzymes to cellulose [26]. It is tough to

categorize the most suitable pre-treatment method for all types of lignocellulosic

biomass [27]. However, the appropriate pre-treatment method should increase

porosity of the substrate and minimize the development of inhibitors [27]. Literature

study and review on lignocellulosic chemistry [28] makes it clear that alkaline pre-

treatment can be performed at low temperatures so that very little of the saccharide

fractions of cellulose is solubilized which is a necessary benefit for bioenergy

production from energy-rich components of lignocellulose. Sodium hydroxide,

potassium hydroxide, and calcium hydroxide are the most extensively studied alkalies

which degrades lignocellulose principally in the same manner; however, sodium

hydroxide has a higher reaction rate [29] compared to calcium hydroxide, but the

main problem is high expenses on largescale implementation. Among the alkaline

bases, calcium hydroxide is one of the inexpensive alkali for pretreatment of

lignocellulosic feedstock [30]. Therefore, the researchers need to use raw materials as

less expensive substrate and cost-effective chemical pre-treatment method for

increasing yield of biofuels production from solid waste biomass through anaerobic

fermentation.

1.4.5 Biological Pre-Treatment

Biological pre-treatment with microbial consortia or addition of specific enzymes

such as laccases, xylanases, lignin peroxidases, peptidases, endoglucanases and other

hydrolases to the lignocellulosic substrate. Saccharification process is an alternative

cheap method to chemical pre-treatment. Biological pre-treatment can be performed

by enzymatic Saccharification, composting or micro-aeration to obtain a maximum

hydrolysis of complex substrates prior to biofuel production. A wide range of fungal

strains Pleurotus ostreatus, Trametes versicolor, Ceriporiopsis subvermispora,

Phanerochaete chrysosporium and a number of bacterial spp are reported for

oxidative biodegradation of lignin from agricultural wastes to increases the enzymatic


11

hydrolysis efficacy. Biological pre-treatment consumed less energy, environmentally

non-toxic and require no chemicals treatments. However, slow treatment rate, growth

conditions and requirement of bulky space are the only limitations of the biological

process [31]. Thermophile Anaerocellum thermophilum and other clostridiums spp

can degrade lignin [32, 33]. Brown-rot fungi principally hydrolysed cellulose, while

white fungi degrade both lignin and cellulose [34]. These microbes utilize their

ligninolytic enzymes such as manganese peroxidase, lignin peroxidase, laccase and

versatile peroxidase [35]. Stereum hirsutum (white-rot fungi) is reported with 14.5 %,

and 7.8 % degradation of the lignin from the wood [35]. In a 20 days of incubation,

Coniochaet ligniaria fungus degrades 75 % of pepper plant residues. Similarly,

Pleurotus Florida degrade 45 % lignin during 60 days incubation from corn straw

[36].

A number of studies reported production of hydrolytic and cellulolytic

enzymes (cellulases, cellobiohydrolase, and endoglucanase) from fungal culture on

agricultural biomasses [37]. The single enzyme pretreatment is not efficient in term of

pretreatment and prolonged the pretreatment time, however, the purified enzymes

cocktails can speed up the hydrolysis but the treatment become expensive and

uneconomic [38]. Similarly, the fungal culture takes weeks to months, this further

cause possible contamination in the culture and consumed some of the holocellulose

[37]. Consolidating bioprocessing (CBP) is a new approach to use microbial

hydrolysis, enzyme production, and fermentation simultaneously for the

deconstruction of different agriculture feedstock [39]. However, all of these studies

proposed that biological treatment is economical pre-treatment method amongst the

all for inexpensive biofuels productions.

Ligninolytic Microbial Consortia

Lignin is one of the leading aromatic component and is harder to degrade as

compared to cellulose and hemicellulose in plant biomass composition. It is natural

that plant leftover residue in the soil is degraded by soil harboring ligninolytic strains.

Therefore, the idea of isolating indigenous ligninolytic bacteria and application of mix

microbial consortia is an effective way of pretreatment. Pretreatment of waste

biomass with individual culture does not occures at the same rate as that of mix

microbial consortia. Mostly different fungi are reported for lignin degradation and


12

enzymatic study, however, bacteria are the simple tool that grows faster and can better

anticipate ligninolytic enzymes production. Verities of enzymes are reported from

both bacterial and fungal strains, these are mainly lignin peroxidase, laccase, and

manganase peroxidase [40, 41]. The majority of studies focus fungi for lignin-

degrading enzymes [41-43]; however, some enzymes have been characterized from

bacteria, such as Streptomyces viridosporus T7A [44], actinomycetes [45],

Rhodococcus jostii RHA 1 and P. putida [41, 42], Pseudomonas sp. LD002 [43] and

Bacillus sp. [20-23].

Lignin creates problems in waste biomass degradation for biofuel production.

The application of lignin-degrading microbes and enzymes in degradation of waste

material is of core interest nowadays. To achieve this goal the isolation of robust

lignin degrading bacterial strains and characterization of their enzymes can be a

valuable study.

1.4.6 Enzymes for Lignin Degradation

It is obvious that lignin has a complex structure, therefore a set of different enzymes

may degrade lignin by slicing different parts of the lignin structure. There are many

enzymes but here we describe function and mediators of only three main enzymes

involved in lignin degradation (Table 1-3).

Table 1-3 Enzymes and mechanism of associated reaction

Enzyme

Activity

Substrate Mediator Reaction

Lignin

peroxidase

veratry alcohol H2O

2 Oxidized aromatic

ring to cation radical

Manganese

peroxidase

Mn, organic acid as

chelator

H2O

2 Oxidized to Mn(II)

Mn(III); chelated

Mn(III) oxidizes

phenolic compounds

to phenoxyl radicals

Laccase ABTS O2 Phenol are oxidized

to phenoxyl radicals;

otherreactions in the

presence of

mediators.


13

1.4.7 Lignin Peroxidase

Lignin peroxidase (LiP) was discovered in 1983 and was produced as a family of

isoenzymes by various plant degrading fungi [16]. Recently LiP is also reported in

different bacterial phyla particularly Bacillus sp. [46]. Lignin peroxidase are heme-

dependent proteins and are approximately 37,000 Daltons in size. The enzyme utilizes

hydrogen peroxide and organic peroxides as a cofactor for oxidation. Lignin

peroxidase directly cleaves Cβ-Cβ linkages opening the phenolic rings and

mineralized the recalcitrant structure of lignin analogous aromatic compounds. Lignin

peroxidase has a broad range of substrate specificity. It can oxidize both phenolic and

nonphenolic aromatic compounds. LiP oxidize polycyclic aromatic compounds but

particularly lignin monomers, dimers and trimers [47]

The mechanism of Lip (Figure 1-3) involved mainly one oxidation and two reduction

steps. i.e.

1) Oxidation of (LiP-Fe(III)) with two electron charges by H2O2, to Compound-I

oxo-ferryl intermediate Fe (IV)

2) In the second step, Compound-I is reduced by gaining one electron from the

non-phenolic aromatic substrate (A)

3) Lastly, the substrate (A) passes the electron to Compound II and return them

to the resting ferric state.

H2O2 H2O

(LiP-Fe(III)) (LiP+ )-Fe (IV)

Lip-1 (compound I)

A+ A

(LiP )-Fe (IV)

A Lip1 (compound II) A+

Figure 1-3 Catalytic reaction of Lignin peroxidase


14

1.4.8 Manganese Peroxidase

Manganese Peroxidase (MnP), needs Manganese (Mn) ions for the induction and

lignin degradation. The Manganese Peroxidase (MnP) has more lignin degradation

potential compared to laccase, due to high redox potential. The mechanism of action

of MnP is similar to that of LiP (Figure 1-4). Both are heme-containing glycoproteins

which need H2O2 as an oxidant. In MnP reaction manganese serve as a mediator and

oxidizes Mn2+ to Mn3+. In addition, malonate and lactate can chelate Mn3+ ion and

make a complex of Mn3+ —organic acid that oxidizes phenolic compounds [48]. The

addition of Mn can provoke MnP enzymatic activity and increase the lignin

degradation [47].

H2O2 H2O

(MnP-Fe(III)) (MnP+ )-Fe (IV)

MnP-1 (compound I)

Mn2+ A Mn2+ A

(MnP )-Fe (IV)

Mn3+ A+ MnP II (compound II) Mn3+ A+

Figure 1-4 Catalytic reaction of Manganese Peroxidase

1.4.9 Laccase

Bacterial Laccase (Lac) is a copper-containing oxidase enzyme uses O2 as a mediator

for oxidation of organic and inorganic substances [40, 49]. In a large number of

studies reported laccase activity from different fungi but particularly from white rot

fungus [41-43]. However, some bacterial strains are identified with laccase enzyme

activity. The catalytic activity of laccase involved a mediator as a conveyor in the

reaction process from Lac to substrate. The oxidized lac mediator further oxidizes

more substrate and subsequently reduced by the substrate to its original state. Finally,

the H2O2 are formed by transferring an electron to O2 from Lac enzyme (Figure 1-5).

The laccase has broad substrate specificity and can oxidized compounds like

diphenols, polyphenols, aromatic amines and benzenethiol [47].


15

O2 Laccase Oxidized Mediator Substrate

H2O2

Laccase Mediator oxidized

oxidized Substrate

Direct oxidation of substrate without mediator

Figure 1-5 Catalytic reaction of Manganese Peroxidase

1.5 Finding of New Lignin-Degrading Species

To extend the knowledge of microbial diversity, it’s better to study detailed

information about lignin degradation. The ideal way is to find out new bacterial

strains which are more actively involved in lignin degradation than earlier reported

ones. The new species might have developed a new catalytic mechanism for lignin

degradation, in case the species origin is from extreme habitat, selective plant dump

or wood-feeding insects and an anaerobic lignin degradation environment. In this

regard, bacterial strains have some advantages relatively than fungi because the

bacterial genetic modification is easier. The genes from bacterial species can be

transferred to another strain quickly. The metabolic pathways can be modified to

enhance the production of lignin-degrading enzymes. The introduction of new aerobic

and anaerobic lignin degrading bacterial species might increase the biosynthetic

capability of methanogenic microorganisms and might improve the anaerobic

digestion process [41, 47].

The lignin-degrading enzymes of bacterial species are unique as they erupt

certain Cα-oxidation and Cβ-Cβ bonds of lignin structure which fungal lignin

peroxidases cannot [50]. The pre-treatment of waste biomass with this microflora,

reduce the recalcitrant structure of biomass by partial degradation of lignin,

disassemble the matrix of lignocellulosic biomass and exposed the structure of

hemicelluloses and cellulose for cellulolytic enzymes [51]. The advantages of this

treatment are minimum inhibitors and minimum loss of carbohydrate in pre-treatment

process [10]. The application of co-culture of microbial consortia could be a useful


16

tool for evaluating biofuel production. The synergistic reaction of culture accomplish

hydrolysis without any thermochemical pre-treatment and the main problem i.e lignin

is degraded leaving behind the cellulose and hemicellulose for biomethane production

[52, 53].

1.6 Techniques for Analysis of Pretreated and Untreated

biomass

1.6.1 Fourier Transform Infrared (FT-IR) Spectra Analsysis

To see the vibrational changes induced in solid waste biomass at the molecular level

FT-IR technique is used that further confirm the compositional alterations. The FT-IR

analysis does not required any tidous procedure for sample preparations, need a little

amounts of substrate and yield numerical data at very quickly. This data is evaluated

for confirmational changes as result of treatment compared to untreated sample [54].

The single band of the hemicellulose, lignin and cellulose frame are agred upon the

associated functional groups to some what related waste biomass subrtates [55]. The

conjectural analysis of the spectra generated through FT-IR is diverse because the

agriculture biomass contains various structure units and functional groups due to

presence of heteropolymer chains in hemicellulose, cellulose, and lignin skeleton.

Hence, the groups are given based on expected spectral data of the compounds from

the published studies on analogous susbtrate [55, 56].

Commonly in the lignocellulosic feedstock the band in the range of 1500–

1650 cm-1 and 1200–1300 cm-1 is occupied by aromatics (lignin), while the band in

the range of 1000–1100 cm-1 and 1800–1900 cm-1 is occupied by the hemicellulose

groups and the band in the range of 1300–1450 cm-1 and 2700–2900 cm-1 is occupied

by the cellulose contents [56]. These possibilities of assigning many bands to the

samples spectra based on common features [55].

1.6.2 Scanning Electron Microscope (SEM)

A scanning electron microscope (SEM) is a kind of electron microscope that uses a

beam of electrons during scanning of a sample to produce an image. SEM images

reflect information’s about the sample's shape and surface topography. The electron

beam can be spot with the detected signal for image production. The sample must fit

specimen chamber and must be of an appropriate size. The structural modifications


17

induced after treatment can be examined with the help of scanning electron

microscope techniques. Before analysis each sample can be coated with Kbr and

labeled according to the experimental code. The magnification range of sample can be

set from 200-4000 um to visualized the fibrous structure of sample [25].

1.6.3 X-ray Diffraction XRD

X-ray diffraction XRD is used to detect the crystallinity of a sample. The sample

needs to be grounded, homogenized and a fine powder can be used for defining

changes in bulk composition and crystallinity. The pocket of X-ray diffractometer

system is packed with dried powder and pressed into a compact material for the

analysis [57]. The XRD instrument is set at 45 kV, using radiation of Cu Ka (λ D 1.54

Alpha 1) with a range between 10- 38° and a step size of 0.1° [57]. The time per step

are set as 8 with a scanning rate of 1.33 degree/minute. The Crystallinity (%) is

calculated as shown in Equation 1-1.

Crystallinity (%) = cryst amor

cryst

I I

I

……….. 1-1

Where Icryst= represents the crystalline region, and Iamor= represents the

amorphous region.

1.7 Biohydrogen and Biomethane Production

Today anaerobic digestion (AD) is considered a reliable and established technology

for the management of various organic wastes and animal manure for biofuels

production [58]. Anaerobic digestion could be either single stage anaerobic digestion

or two-stage anaerobic process. The single stage anaerobic digestion is normally

simple dark fermentation, while the two-stage anaerobic is divided into (a)

hydrolysis/acidogenesis and (b) acetogenesis/methanogenesis process operated in

separate bioreactors. Beside traditional biogas process for CH4 production the two-

stage anaerobic digestion could yield both H2 and CH4 simultaneously, in this process

organic-rich substrate is converted to H2, CO2 and fatty acids, which is then converted

to CH4 by methanogens (Figure 1-6) [59]. These biogases can be produced from a

varity of raw lignocellulosic and other waste organic biomass and can be recycled for

different energy purpose such as power, heat or electricity and as a vehicle fuel and

could substitute about 20-30% of the natural gas reduction [60]. Biogas usually


18

contains above 50% methane and other gases in comparatively low quantities namely,

H2, CO2, N2 and O2. The mixture of these gases is flammable if the methane content is

more than 40-50% [61]. Anaerobic digestion is temperature dependent; some of the

anaerobic bacteria have a temperature range of 32-35 °C, while other bacteria require

50-55 °C for optimal degradation [62]. To acquire profitable output bioenergy

(biohydrogen and Biomethane), the dark fermentation must be coupled to the second

phase of the anaerobic digestion, this process could be ideally separated as

microorganisms of both stages has significant differences in terms of nutritional

needs, physiology, pH sensitivity and sensitivity to others environmental conditions.

A methane gas of 270 mL/h and 119 mL/h hydrogen is reported using a two-stage

anaerobic digestion system [63]. In future, the two-stage anaerobic fermentation

process could be considered more feasible than others processes for bioH2 and CH4

production as shown in the (Figure 1-7). The AD process consisting of the following

steps.

1.7.1 Hydrolysis

During hydrolysis biopolymers proteins, carbohydrates and fats are degraded by

extracellular enzymes and broken down to their respective monomers. These new

molecules are used by another group of microorganisms in AD and transformed into

subsequent products [64]. In this step lignin polymer resist degradation, therefore pre-

treatment is performed to enhanced hydrolysis [65]. Hydrolysis covert complex

matrixes to simple to make it easy for enzymatic attack. During hydrolysis of AD,

glucose and different products are formed (Equation 1-2).

Biomass cellulose + glucose organic acids + hydrogen + carbon dioxide

…………. 1-2

1.7.2 Acidogenesis

Acidogenic bacteria breakdown less complex material to a mixture of volatile fatty

acids (VFAs), alcohols and other compounds. This step is sometimes referred to as

fermentation. Production of a higher amount of hydrogen accompanied by carbon

dioxide is a hallmark of acidogenesis. Acids formed in this step are short-chain

organic acids such as acetic acid, propionic acid, acetate and butyric acid. Production

of VFAs in this phase is important for methanogens. But the higher amount of VFAs

in digester induce microbial stress due to low pH and eventually lower down AD.


19

Therefore, for ideal AD process, the concentration of VFAs plays a key role.

Efficiency of AD process and their optimum conditions are often measured by

examining VFA. Acidogens grow well at pH 5-6; however, fluctuation of pH allows

them to live in hostile conditions of AD process. Organic acids accumulation rapidly

decreases pH and inhibit digestion process if transformed to other product in the next

step of fermentation [64]. Overall during this step simple sugars, amino acids and

fatty acids are converted into short-chain organic acids and alcohols [66].

1.7.3 Acetogenesis

In this step, acetogenic bacteria transform VFAs and others metabolites of

acidogenesis step into acetic acid, hydrogen and carbon dioxide [64]. The

acidogenesis and acetogenesis steps work together, if a significant hydrogen pressure

is present then acetate production stops due to inhibition of specific bacterial activity

[67]. It is known that acetate (equation 1-3) contributes almost seventy percent

towards methane production, the final product of anaerobic digestion [68].

Acidogenesis and acetogenesis reaction:Volatile fatty acid acetate or butyrate +

carbon dioxide+ hydrogen …………… 1-3

1.7.4 Methanogenesis

Two important groups of methanogens are involved in biomethane production

depending upon on the nature of carbon source as a substrate. One group consumes

acetic acid and yield methane and the other group transform hydrogen and carbon

dioxide to methane. The first group is of acetoclastic microbes and have the ability to

generate up to 70% methane concentration in biogas composition. Methanogens

belong to acetoclastic group are slow growing i.e. their doubling time consist of

several days. They show a sensitive behavior towards lack of nutrients, pH, certain

compounds [64]. The other group use oxidation of hydrogen and reduction of carbon

dioxide to methane, a distinctive mechanism of metabolic pathway of methanogens.

Most of the methanogens can be used H2 with CO2, H2 with acetate and methanol

with formate as a substrate for methane production [67].


20

Waste biomasses

Anaerobic digestion

Carbohydrate, lipid, proteins

1st step

Hydrolysis

2rd step

AcidogenesisProduction of VFA (propionic acid, butyric

acid, valaric acid etc)

3rd step

AcetogenesisProduction of acetate, butyric acid H2 production

4th step

MethanogenesisVFA, CO2, H2 CH4 production

Figure 1-6 Pathways and processing steps involved in the biodegradation of

waste biomass for biogas production adopted from Ghimire et al. [15].

Biomass

Two-phase Anaerobic digestion system

1-Hydrolysis

3-Acetogenesis

2- Acidogenesis4-Methanogenesis

CH4

CO2

H2

VFA

Figure 1-7 The figure show process of two-separate phases of anaerobic

digestion for bioenergy production.


21

1.8 Objective of the Study

The main objective of the study was to investigate different chemical and biological

pretreatment stratagies for lignin removal/degradation of agriculture waste biomass to

increase bioenergy production.

Sub-objective

To study different chemical pretreatment methods for lignin removal of

agriculture waste biomass

Analysis of structural properties of pretreated substrate by Scanning Electron

Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR)

Isolation of ligninolytic bacteria for biological pretreatment of lignocellulosic

waste biomass and perceiving its effect on substarte and biogas production

To study bio-hydrogen potential of lignin degrading bacteria and combined

effect of chemical and biological treatment on wheat straw to improve biogas

production

To explore single bacterial strain with potential of bio-hydrogen from the

organic fraction of municipal solid waste

http://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CCgQFjAB&url=http%3A%2F%2Fen.wikipedia.org%2Fwiki%2FFourier_transform_infrared_spectroscopy&ei=D8G1U6vpKO6V7AblyoDQCg&usg=AFQjCNGjtZiMOS4ehEw2Lkiz2JCTOTimaQ&bvm=bv.70138588,d.bGE

2 Materials and Methods

2.1 Chemical Treatment of Agribiomass

2.1.1 Reagents and Materials

Chemicals and materials were purchased from Merck group of Chemical company

and Fisher Scientific company. A total of 10 different substrates, wheat straw, kallar

grass, para grass, paper wastes, almond shell, peanut shell, corn cob, rice straw,

bagasse, and pulses peel were collected at National institute for Biotechnology and

Genetic Engineering (NIBGE) Pakistan. The substrates were ground to 20 mm mesh

size, and stored at room temperature in polyethylene bags. Volatile solid (VS), total

solid (TS), ash, moisture, hemicellulose, lignin, and cellulose were measured

according to the standard NREL laboratory analytical procedure [69]. The

degradation effect on biomass contents before and after treatment was calculated.

2.1.2 Conditions of Alkali Treatment

The lignin removal was carried out from all tested substrates with 1, 2, 3 and 5%

concentrations of NaOH and KOH; however, a single low concentration of 0.5%

Ca(OH)2 was used. The water bath (WT), microwave (MW), and the autoclave (Auto)

were used as heating containers. The conditions of heating were 121°C for 20 minutes

in autoclave, 80°C for 60 minutes in water bath, and 3 time soakings for 1-3 minutes

with intervals in a general purpose laboratory microwave (C.R.S117 Dawlence) as

described previously [70]. The control samples were treated with distilled water under

the same conditions.

2.1.3 Delignification Method

Five grams of each substrate were immersed in 100 mL of alkaline solution of

different concentrations: 1, 2, 3 and 5% (w/w) of sodium hydroxide and potassium

hydroxide and 0.5% of Ca(OH)2 in a 250 ml flask [71]. These alkali-immersed

samples were then subjected to autoclave, water bath, or microwave heating. The

Chapter 2: Materials and Methods

23

resulting leftover solid residue was washed thrice with water until the pH reached 7.0.

The composition of all the substrates in the given biomasses prior to alkaline

delignification was calculated using National Renewable Energy Laboratory (NREL)

procedure as described earlier [69]. These values were set as the actual value for

measuring the extent of delignification. The amount of lignin in the substrate was

determined before and after the treatment and the extent of delignification was

calculated by the equation 2-1 using TAPPI T222 om-02 method.

Lignin extraction % = weight of lignin extracted × 100 ……….. 2-1

acid insoluble lignin in biomass

The neutralized solid residue was used for SEM, FTIR, and AD process.

2.1.4 FTIR Analysis

The samples of treated and untreated LB solid residue were embedded in KBr pellets

prior to Fourier Transform Infrared (FT-IR) spectral analysis. The spectra were

collected in the range of 500-4000 cm-1 with 32 scans per sample with a resolution of

4 nm in absorption band mode as described previously [72]. Spekwin 32-7.1 FTIR

software was used for data normalization, and changes in the peak positions of pre-

treated samples compared to untreated residue.

2.1.5 Scanning Electron Microscopy

The morphological changes due to pre-treatment could be analysed with scanning

electron microscopy (SEM) techniques. The surface morphology of untreated and pre-

treated substrates were imaged using a vacuum-desiccated SEM (S-3700 microscope

Hitachi) with the magnification ranges of 1K, 2K and 3K to visualize the broken and

distorted fibrous structure of feedstock.

2.1.6 High-performance Liquid Chromatography (HPLC) Analysis

of Carbohydrates

The hemicellulose and cellulose sugars were analysed by HPLC for filtrate samples

extracted from different feedstocks with a cation exchanger column (Perkin Elmer,

USA). All samples were filtered (0.45 um) before being subjected to HPLC analysis.

De-ionized water was used as the mobile phase at a flow rate of 10 ml/minute. The

injection volume was 10 ml/minute, and the column temperature was maintained at


24

80°C. The eluted products were detected by the software Turbochrome work station

version 6.3.1.

2.1.7 Anaerobic Digestion Experiment

The biomethane potential (BMP) of 1, 2, 3 and 5% NaOH and KOH microwave

pretreated biomass was assessed by AD process. The culture volume in each serum

bottle was 100 mL. The 100 mL contains, 8 mL of K2HPO4 (2.5g/L), 8 mL of 3.5 g/L

sodium bicarbonates solution, and 2 mL from stock solutions of vitamin solutions

folic acid (0.02 g/L), thymine (0.01 g/L), riboflavin (0.05 g/L), vitamin B12 (0.001

g/L), panthonic acid (0.05 g/L), lipoic acid (0.05 g/L). The serum bottles were airtight

with rubber cork and aluminum crimp caps. The initial pH of all serum bottles was

7.5. Control samples of inoculum without substrate, water without the substrate, and a

substrate without inoculum were included. The untreated and pretreated substrates

were run parallel to compare the biogas and methane production. The serum bottles

were flushed with N2 gas for 4 min and were incubated at static condition. Volume of

biogas was determined by the water displacement method for 40 days at regular

intervals. The CH4 content of the biogas was analyzed using GFM series gas analyzer.

Biogas Calculation

Daily volume of biogas, composition of methane (CH4), and composition of carbon

dioxide (CO2) were determined from each bottle. Cumulative biogas from raw data

was summarized using OBA (https://biotransformers.shinyapps.io/oba1/). This biogas

software (R package) calculated cumulative volume of methane, cumulative volume

of biogas, volumetric rate of biogas, and volumetric rate of methane. The R package

used (Equation 2-2) to calculate the daily biogas for each bottle based on displaced

volume and % CH4 at each previous and current reading [73].

Vb = P × Vh × C ……… 2-2

R × T

Where Vb = is the volume of daily produce gas (mL), P = is the absolute

pressure difference (kPa), Vh = is the volume of head space (mL), T = is absolute

temperature in (K), C = gas molar volume (22.14 L mol-1) at 273.15 K and 101.325

kPa, R = 8.314 L kPa k-1 mol-1 is universal gas constant.


25

Kinetics Model

The solver function in Microsoft Office Excel 2016 was used for modified Gompertz

equation (Equation 2-3) the minimum methane production (mL/gVS) was calculated

of each untreated and pretreated biomass sample by minimizing the least square

difference between predicted and experimental values as described previously [74].

1

emaxR

M t P exp expp t

……… 2-3

Where Mt is cumulative methane production (mL) during the incubation time,

t (hours), P is the methane production (mL), Rmax is the maximum production rate

(mL/h), and Δ (delta) is the lag phase duration (hours) and e is equal to 2.718282.

2.1.8 Statistical Analysis

All experiments in the present study were conducted in triplicates. Mean values and

standard deviation (SD) among each triplicate were calculated through Microsoft

Office Excel 2016. One-way ANOVA and box plot scheme were used to present the

data.

2.2 Ca(OH)2 Soaking

Corn cob residue (50 g/L) was soaked in 100 mL of 0.5% of Ca(OH)2 and was kept

for 7, 15 and 30 days incubation time. The control sample of corn cob residue was

immersed in 100 mL of distilled water without Ca(OH)2. The experiment was run in

the triplicate. After soaking time, the corn cob residue was collected and was washed

with tap water several times with cheese cloth. The solid residue was dried at room

temprature. Change in lignin content was calculated using National Renewable

Energy Laboratory (NREL) procedure [69]. Reduction in total weight of the samples

were calculated at the end of each soaking time. Weight loss was measured in

percentage using (equation 2-4), the final weight of corn cob was minus from the

initial weight as such,

Dry weight loss (%) = 1 2 100

1

C C

C

…….. 2-4

C1= initial weight, C2= final weight


26

2.2.1 Scanning Electron Microscopy (SEM)

The untreated corn cob, 7, 15, and 30 days of 0.5% Ca(OH)2 soaked samples were

subjected for Scanning Electron Microscopy (SEM) (JSM-7800F PRIME, JEOL's

USA). Images of the untreated corn cob, 7, 15, and 30 days of 0.5% Ca(OH)2 soaked

were fixed on a black carbon tape and sputter with gold palladium and analysis of

SEM was done using magnification range of 1k, 2k and 3k um to show the effect of

pretreatment from samples morphology as described earlier [72].

2.2.2 Biomethane Potential Test (BMP)

The BMP was carried out by mixing 2-gram substrate as a starting material. Untreated

corn cob and 7, 15, and 30 days 0.5% Ca(OH)2 soaked treated corn cob samples were

prepared. The 20 mL of active inoculum of anaerobic digester plant was used as

inoculum. A total working volume of media was 50 mL in each serum bottle. The

rest of procedure was same for BMP experiment and biogas calculation as described

in section 2.1.7.

2.3 Biological Treatment Method

2.3.1 Isolation of Aerobic Ligninolytic Bacteria

Microorganisms were collected from three different environments: soil surface, wood

compost, and waste sludge. The samples were moistened in 9 g/L (w/v) sterile NaCl

for 30 minutes before inoculation into mineral salt media (MSM) (1.0 g/L KH2PO4,

1.0 g/L MgSO4, 1.0 g/L NaCl, 0.5 g/L CaCl2, 0.4 g/L CuSO4 and 0.002 g/L MnSO4)

supplemented with 1 g/L alkali lignin. The lignin was prepared from rice straw by

suspending the substrate in 20 g/L NaOH and autoclaving at 121ᵒC for 20 min. The

brown liquor was then filtered, and lignin was precipitated with 1 M H2SO4 at pH 3

and dried at room temperature [75].

The microorganisms (NaCl suspension) were inoculated into MSM

supplemented with lignin and cultured at 37°C and 150 rpm for 72 hours. Fresh

cultures were serially inoculated twice more under the same growth conditions to

enrich for lignin-degrading microbes [76, 77]. Aliquots of the liquid culture were then

spread onto plates (30 g/L agar) containing the MSM supplemented with lignin to

isolate individual colonies capable of growth on lignin as the sole carbon substrate.

The colonies were characterized based on visualization of colony morphology, light


27

microscopy, and gram staining. All isolates were then maintained in Luria broth (LB)

containing 10 g/L tryptone, 5 g/L NaCl, and 5 g/L yeast extract at pH 7.

2.3.2 16S rRNA analysis

Genomic DNA was extracted from bacterial cells with 500 μL lysis buffer (10 mM

Tris Cl pH 8 , 1 mM EDTA, 1 M NaCl), 70 μL 10% SDS, and 40 μL of proteinase K

(20 mg/mL) using an ethanol precipitation method [78]. The 16S rRNA genes were

amplified from the genomic DNA by PCR using the following primers [79].

FD1: 5’-AGAGTTTGATCCTGGCTCAG-3′

RD1: 5’-AAGGAGGTGATCCAGCC-3’

The genomic DNA was combined with the above primers and amplified with

Taq DNA polymerase (Fermentas, USA), using 10x PCR reaction mixture. The PCR

reaction was conducted with 1 cycle at 94°C for 3 min and 30 cycles at 94°C for 1

min, 72°C for 1 min, 55°C for 1 min. The PCR products were cleaned with QIAquick

PCR Purification Kit (Qiagen). The resulting PCR products were sequenced and

studied by Basic Local Alignment Search Tool (BLAST) analysis. The phylogenetic

tree was constructed using the Mega 6.06 software (Molecular Evolutionary Genetic

Analysis).

2.3.3 Lignin and Dyes Decolorization Assays

The bacterial strains were assessed for their ability to decolorize Azure B dye

(structural analogue of lignin) in agar media. The Azure B dyes were used at 0.0025

g/L, respectively. The autoclaved agar media was applied on sterile agar plate, and 10

μL of bacterial cell suspension (in 9 g/L NaCl) were slanted onto each agar plate

containing the dye. The plates were incubated at 37°C and monitored regularly for

growth and development of decolorization zones over a period of 120 hours.

The extent of dye decolorization by selected bacterial isolates was quantified

by culturing the strains in liquid media containing either lignin or Azure B dye. The

bacteria were first grown in LB broth for 24 hours. These cultures were then used to

inoculate 50 mL MSM (supplemented with 5 g/L peptone and 5 g/L yeast extract)

containing either 10 g/L lignin or 1 g/L Azure B dye to an initial optical density at

600 nm (OD600) of 0.5. The cultures were incubated at 150 rpm at 37ᵒC on a shaker


28

for one week. Two mL samples were periodically collected and centrifuged at 10000

rpm for 5 min. The supernatant was collected, and the absorbance was measured at

465 nm or 651 nm to determine the extent of lignin and Azure B dye decolorization,

respectively [77]. The percent decolorization of lignin and Azure B dye for each

isolate was calculated as shown in equation 2-5:

% decolorization = Ai – Af x 100 ………… 2-5

Ai

Where Ai = initial absorbance at first day, Af = final absorbance at last day

2.3.4 Growth on Alkali Lignin and Azure B dye

The pure bacterial isolates were inoculated in MSM supplemented with either 1 g/L

lignin or 1 g/L Azure B dye as the sole carbon source as described previously [77,

80]. The flasks were incubated at 150 rpm at 37ᵒC for one week. The growth of each

isolate was monitored daily at 620 nm with a UV-visible spectrophotometer.

2.3.5 Screening for Extracellular Hydrolytic Activities

Bacterial cultures were grown in LB broth at 37°C at 150 rpm for 18 h. The cells were

resuspended in 9 g/L NaCl, and 10 μL samples of cell suspension were spotted onto

MSM agar plates supplemented with 0.5 g/L carboxymethylcellulose (CMC), 1 g/L

tributyrin (Trb), 10 g/L skim milk, 20 g/L starch, 10 g/L xylan, and 5 g/L

polygalacturonic acid (PG) to assay for cellulase, lipase, protease, amylase, xylanase,

or pectinase activity, respectively. Maltose, sucrose, and cellobiose were used for

carbohydrate fermentation on MSM agar plates supplemented with 10 g/L of each

carbohydrate. Phenol red solution was used to check the change in color of the

medium from red to yellow to detect the fermentation ability of bacterial strains of

tested sugars. After aerobic incubation at 37°C for 3 days, the agar plates were

checked for the presence of enzyme activity as described previously [81].

2.3.6 Assessment of Lignin Degradation Efficiency

The efficiency of lignin degradation in the presence of various concentrations of

substrate was determined in 50 ml MSM supplemented with peptone (5 g/L) and yeast

extract (5 g/L) at pH 5 and pH 7 as previously described [46]. The concentrations of

alkali lignin tested were 0.5, 1, 3, 6, 8, 10, 20, 30, and 40 g/L. All the bacterial isolates


29

were first grown in LB broth for 24 h. These cultures were used to inoculate the

lignin-containing media with a starting OD600 of 0.5. The cultures were incubated at

150 rpm at 37ᵒC for one week. Control media (uninoculated) were used in all cases.

Two ml samples were collected from the culture at relevant time points and

centrifuged at 10000 rpm for 5 min. The supernatant was measured at 280 nm to

assess the level of lignin degradation on a UV-visible spectrophotometer. The percent

degradation of lignin for each isolate was calculated using equation 2-6.

% degradation = Ai – Af x 100 ………… 2-6

Ai


2.3.7 Lignin-Degrading Enzyme Activity Assays

Bacterial isolates were assayed at pH 3-7 and temperatures 30-70ºC for the presence

of lignin peroxidase (LiP) and laccase (Lac) enzyme activities. The culture was grown

on MSM supplemented with lignin (1%), peptone (0.5%), and yeast extract (0.5%),

and incubated at 150 rpm at 37ᵒC. Bacterial cells were spin down at 10000 rpm for 10

min and supernatant containing excreted proteins were used for enzyme assay. Crude

supernatant were assayed at pH 3-7 and temperatures 30-70ºC for the presence of

lignin peroxidase (LiP) and laccase (Lac) enzyme activities.

Lignin peroxidase (LiP)

Two hundred μL of crude enzyme were added to 200 μL of 2 mM veratryl alcohol

and 1 mL of 0.1 M sodium citrate/sodium phosphate buffer (pH 3-7), and the reaction

was initiated with 100 μL of 2 μM H2O2. The reactions were incubated at 30-70ºC for

1 hour. The oxidation of veratryl alcohol was measured by absorbance at 310 nm. A

unit (U) of enzyme activity is defined as amount of enzyme that converts 1 µmole

substrate per minute. The activity was calculated as: (U/ml) = [OD* total assay

volume] ÷ [Є veratryl alcohol* time*enzyme volume]. The molar coefficient of

veratryl alcohol is ε310=9300 M-1cm-1.

Laccase (Lac)

The reaction mixtures containing 200 μL of crude enzymes, 200 μL of 2 mM guaicol,

and 500 μL of 0.1 M sodium citrate/sodium phosphate buffer (pH 3-7) were incubated

at 30-70ºC for 1 h, and the absorbance was read at a wavelength of 450 nm. A unit


30

(U) of enzyme activity is defined as amount of enzyme that converts 1 µmole

substrate per minute. The activity was calculated as: (U/ml) = [OD* total assay

volume] ÷ [Є guaicol* time*enzyme volume]. The molar coefficient of guaicol is

65000 M-1cm-1 at a wavelength of ε450 nm.

2.3.8 Rice Straw Pretreatment

Rice straw (RS) residue was collected and dried at room temperature, ground to

approximately 20 mesh size, and stored in polyethylene plastic bags at room

temperature. The ground rice straw (RS) (5 g) was suspended in 100 mL of mineral

salt media (1.0 g/L KH2PO4, 1.0 g/L MgSO4, 1.0 g/L NaCl, 0.5 g/L CaCl2, 0.4 g/L

CuSO4 and 0.002 g/L MgSO4). RS was pretreated using either pure bacterial culture

or co-culture of all the bacteria.

For the bacterial culture experiments, both individual Bacillus sp. strain as

well as a mixture of all seven strains were tested. For pretreatment by the individual

strain, the individual bacterial culture was inoculated into the flask to reach an OD600

= 0.5. For pretreatment by the co-culture of bacteria, a mixture of all the strains were

added such that each strain contributed an OD600 = 0.5. The culture was incubated at

37±1°C, pH 7, 150 rpm for 7 days.

Samples were taken at the beginning and end of the pretreatment for

determination of weight loss. Non-inoculated RS served as a control. The weight loss

of RS was calculated according to a previously described method [82]. The weight

loss was determined from the final weight of rice straw minus initial weight using the

equation 2-7.

Dry weight loss (%) = 1 2

1

w w

w

x100 …………. 2-7

1w = initial weight , 2w = final weight

The composition of RS was determined using a National Renewable Energy

Laboratory (NREL) procedure [69]. The reductions in cellulose, hemicellulose, and

lignin were each calculated. The RS samples were kept for SEM analysis and for

anaerobic digestion assessment.


31


The morphological changes of RS caused by bacterial and enzymatic pretreatments

were analyzed using a scanning electron microscope (SEM) (JSM-7800F PRIME,

JEOL's USA). The dried RS samples were fixed on black carbon tape and sputter-

coated with gold palladium [72]. Images of the RS were taken using SEM with

magnification ranges of 1000, 2000 and 3000 µm to visualize the broken and distorted

fibrous structure of feedstock.

2.3.10 Biomethane Potential Assay

The biomethane potential assay (BMP) was carried out using anaerobic serum bottles.

Each bottle (100 mL capacity) contained 8 ml of K2HPO4 (2.5 g/L), 8 mL of

bicarbonates solution, 2 mL of vitamin solution, and 1 g of one of the following RS

samples: untreated or bacterial treated RS. A control inoculum without RS was

included. A 0.28 (S/I ratio) for BMP experiment were prepared based on the volatile

solid (VS) content as food to microorganisms ration (F/M ratio). The serum bottles

were flushed with N2 gas for 4 min and incubated 37±1°C at pH 7.5. The bottles were

mixed manually on a daily basis. The volume of biogas was determined by the water

displacement method at regular intervals. The CH4 and CO2 content of the biogas

were analyzed using a GFM406 – multichannel portable gas analyzer. The raw data

was analyzed using OBA (https://biotransformers.shinyapps.io/oba1/). This biogas

software package (R package) calculated cumulative volume of methane, cumulative

volume of biogas, volumetric rate of biogas, and volumetric rate of methane. The R

package used (Equation 2-2) to calculate the daily biogas for each bottle based on

displaced volume and % CH4 at each reading [73].

The solver function in MS Excel 2016 was used for the modified Gompertz

equation (equation 2-3) to study the experimental cumulative methane yield

(mL/gVS) to define the minimum methane production potential (mL/gVS). The

minimum methane production (mL/gVS) was calculated for each sample by

minimizing the least square difference between predicted and experimental values as

described previously [74].


32

2.4 Methodology of Biohydrogen and Biomethane

Production by Ligninolytic Bacteria Culture

2.4.1 Reagents and Chemicals

All chemicals of analytical grade were obtained from Sigma-Aldrich (Chemie GmbH,

Germany). The chemicals and reagents used in experiments were alkali lignin, Azure

B, hydrogen peroxide, guaiacol, veratryl alcohol, magnesium sulphate, calcium

chloride, sodium chloride, amonium sulphate, copper sulphate, potassium dihydrogen

phosphate, peptone, yeast extract and bacteriological agar.

2.4.2 Experimental Design

The culture enrichment technique was used to isolate the robust ligninolytic bacteria

to evaluate their potential of lignin degradation and enhancing biogas production.

The following experiment were performed

o Bacterial strains were isolated using mineral salt media (MSM) supplemented

with alkali lignin

o Strains were selected based on high decolorization of Azure B dye

o Selected strains were screened for extracellular enzymatic hydrolytic activity

o Xylose and cellulose were tested in fermentation batch assay for H2

production

o Two un-couple batch tests were conducted: a fermentation test for H2 and CH4

using wheat straw as feeding material

Each experiment was conducted in triplicates and average data was presented.

2.4.3 Isolation of Ligninolytic Bacteria

Sample was collected from granular sludge of full scale anaerobic digester. 50 gram

of sample in triplicates was moistened in 100 mL sterile 9 g/L sodium chloride for 60

minutes. Alkali lignin was prepared from wheat straw treated with 20 g/L NaOH and

autoclaved at 121ᵒC for 20 min. The brown liquor was then filtered, and alkali lignin

was precipitated with 1 M H2SO4 at 50ᵒC, pH 3 followed by drying at room

temperature [75]. A mineral salt media 80 mL (MSM) 1.0 g/L potassium dihydrogen

phosphate, 1.0 g/L magnesium sulphate, 1.0 g/L sodium chloride, 0.5 g/L calcium


33

chloride, 0.4 g/L copper sulphate, 0.002 g/L amonium sulphate) supplemented with 5

g/L peptone and 10 g/L alkali lignin was used. Firstly, the mineral salt media (MSM)

in 250 mL of serum bottle was autoclaved at 121°C for 20 minutes. Then, 10 mL

sample of sodium chloride suspension from each triplicates sample was inoculated

using a syringe into sterile 80 mL mineral salt media (MSM) in 250 mL of serum

bottle. The isolation procedure was done as reported previously [83]. The serum bottle

was incubated at 37 °C and 150 rpm for 72 hours. The culture enrichment processes

from the serum bottle were repeated successively two more times under the same

growth conditions.

The Aliquots of 100 uL of culture were then spread onto agar plates

containing the mineral salt media (MSM) supplemented with 10 g/L alkali lignin. The

culture plates were incubated at 37 °C temperature, and growth was observed after 48

hours. Single colony of pure culture was streaked in to fresh Luria broth (LB) agar

plates based on physical and morphological differences. The isolates were then

maintained in LB agar media plates and stored in Luria broth (LB) containing 10 g/L

tryptone, 5 g/L sodium chloride and 5 g/L yeast extract at -80°C.

2.4.4 Preliminary Screening of Ligninolytic Bacteria

The isolates were evaluated for their ability to decolorize Azure B dye ‘structural

analog of lignin’ in liquid MSM media (pH 7). The bacteria were first grown in LB

broth for 24 hours. The optical density of 0.5 at 600 nm was used to inoculate 250 mL

serum bottles containing 100 mL MSM supplemented with 2 g/L peptone and 0.0025

g/L Azure B dye. MSM along with 2 g/L peptone and 0.0025 g/L Azure B dye

without culture was used as a control. MSM with 2 g/L peptone inoculated with

culture without Azure B dye was used to check bacterial growth. Sterile glucose

solution equivalent to 5 g/L was added in to each bottle. The serum bottles were

sterilized at 121°C for 20 minutes. The cultures were then incubated at 150 rpm and

37 ᵒC on a shaker for one week. Two mL samples were collected at initial and final

time point of the batch assay and centrifuged at 10000 rpm for 5 min. The supernatant

was collected, and the absorbance was measured at 651 nm to determine the extent of

Azure B dye decolorization [77]. The percent decolorization of Azure B dye for each

isolate was calculated as shown in equation 2-8.

% decolorization = Ai – Af x 100 Equation 2-8


34

Ai


Control media and assay samples were carried out in triplicates.

Similarly, in a parallel experiment for the COD reduction of lignin and Azure

B dye was carried out in 250 mL serum bottles containing with 2 g/L peptone in 100

mL of liquid MSM media (pH 7). 0.0025 g/L Azure B dye and 10 g/L alkali lignin

was added in to separate serum bottles. Sterile glucose solution equivalent to 5 g/L

was added in to each bottle. The serum bottles were sterilized at 121°C for 20

minutes. One milliliter of the inoculum of each pure isolate with an initial OD (600

nm) of 0.5 was inoculated in each serum flask. The serum bottles were incubated in a

shaking incubator of 150 rpm at 37 ºC for 7 days. Control MSM media without Azure

B dye and alkali lignin was added. Control media and assay samples were carried out

in triplicates. The initial and final value for the COD reduction was measured with

COD kit (Hach company, Germany) using DRB200 thermostat.

The enzyme assay for lignin peroxidase and laccase and molecular

identification were done as described in methodology of section (2.2.2 to 2.2.7).

2.4.5 Biohydrogen Fermentation Batch Assay

Four of the best and most active hydrolytic strains were then evaluated for H2

potential from cellulose, xylose and wheat straw. 250 mL serum bottles were filled

with 100 mL of liquid MSM media (pH 7) supplemented with or without 10 g/L

cellulose, xylose, and wheat straw separately. These serum bottles were autoclaved at

121 °C for 20 min, then five milliliters of the pre-grown overnight inoculum of each

pure isolate with an initial OD (600 nm) of 0.5 was inoculated in each serum bottle.

After inoculation in a sterile condition, the serum bottles were capped with a butyl

rubber stopper and flushed with N2 for 5 min. Followed by incubation at batch

condition without shaking at 37°C. Water displacement method was used to record

the amount of daily biogas. Gas chromatography was used to measure the biogas

composition of hydrogen, carbon dioxide, and methane. Volatile fatty acids (VFAs)

concentration was analyzed at the end of batch experiment fermentation. All the

experiments were conducted in triplicate and average results presented.


35

The data of hydrogen productions was calculated using the equation 2-9 below.

Equation 2-9

Where:

2,H tV = represent the volume of hydrogen produced in the interval between t

and t-1; 2,H tC , 2, 1H tC = hydrogen concentrations measured at times t and t-1;VBG, t =

volume of biogas produced between time t and t-1; VHS =volume of the headspace of

reactors.

Cumulative hydrogen production (VH2cum) was calculated as sum of hydrogen

productions between each measurement (VH2, t) during dark fermentation batch tests,

according to the following equation 2-10.

2, 2,1

n

H cum H tt

V V

Equation 2-10

Hydrogen yields, expressed as NmL H2/g VS and mol H2/mol substrate, were

calculated according to the following equation 2-11.

2,

2 /H

Hydrogen yield NmLV cu

H gm

Wsub Equation 2-11

Where:

VH2cum: cumulative hydrogen production at the end of the dark fermentation

test; W sub: weight of added VS.

The cumulative hydrogen volume was calculated using modified Gompertz

equation (equation 2-3) by solver function in Microsoft Office Excel 2016 with a

Newtonian algorithm for each batch.

2.4.6 Biomethane Potential Test (BMP) for the Biopretreated Wheat

Straw

After biohydrogen batch fermentation assay of the wheat straw, the serum bottles of

the four strains were subjected to BMP assay. Briefly, a 20 mL media was drained off

cautiously from each serum bottle and fresh 20 mL sludge was added to each bottle of

the experiment. The 20 mL sludge was an active inoculum of anaerobic digester plant

2, 2 . 2, 2, 1 , , .( )H t H t BG t SH H t H tV C V V C C


36

fed with organic food waste. A total working volume was 100 mL in each serum

bottles. A 10 g/L untreated wheat straw along with 20 mL sludge in 80 mL of liquid

MSM media (pH 7) and control sludge of 20 mL in 80 mL of liquid MSM media (pH

7) serum bottle were used as a control to BMP assay. The pH was adjusted to 7.5 and

then all the bottles were airtight with a butyl rubber stopper. The batch assay was

started 37±1°C incubation temperature after N2 gas flushing for 5 min. All the bottles

were manually mixed and daily biogas volume was determined by water displacement

method for 25 days. The CH4 content of the biogas was evaluated using gas

chromatography.

The raw data of daily biogas volume and CH4 composition obtained from each

bottle was analyzed using OBA (https://biotransformers.shinyapps.io/oba1/). This

biogas package software (R package) calculated cumulative methane yield,

cumulative biogas and daily rate of methane using a standard Equation 2-3 [73].

2.4.7 Analytical Method

The analysis of wheat straw for cellulose, hemicellulose, lignin, total solid (TS) and

volatile solid (VS) was estimated through National Renewable Energy Laboratory

(NREL) procedure [69]. COD for each sample was determined using COD kit (Hach

company, Germany). The pH values of each bottles were measured using a pH meter.

The samples of wheat straw after batch fermentation for hydrogen production were

filtered with 0.45 µm filters for volatile fatty acids (VFA). The samples were acidified

with phosphoric acid (H3PO4) before start of analysis. Concentrations of VFA

(methanol, ethanol, n-butyric, iso-butyric, acetic, propionic, n-valeric, and iso-valeric)

in the samples were determined using gas chromatograph (Hewlett Packard 5890A,

Agilent Technologies, USA) equipped an HP-FFAP column (Agilent Technologies,

HP 6890, CA USA) and automatic headspace sampler (Perkin Elmer). Nitrogen was

the carrier gas with a flow rate of 6 mL/min. The chromatographic conditions were as

follows: injector temperature 120 °C; detector temperature, 80 °C and oven

temperature program cooling temperature of 40 °C was used. A standard solution of

VFA, water, and triplicates of each sample was run, and average value was calculated.


37

2.5 Pretreatment Method with Recombinant Neurospora

crassa F5 Strain

2.5.1 Waste Biomass

The Wheat straw (WS) residue was collected in University of California, UC-Davis

(City), USA. The WS residue was dried at room temperature and then grinded with a

machine to approximately 20 mesh size. The coursed samples were then packed in

polyethylene plastic bags and placed in shelter at room temperature.

2.5.2 Alkali Pretreatment of Wheat straw

Dried 10 g of WS in triplicate was first pretreated with 2% NaOH in a 1:20 substrate

to volume ratio, autoclaved at 121°C for 20 min. The suspension was drained using

Nylon cheese cloth and solid residue of WS was washed thrice with water and then

oven dried at 105°C.

2.5.3 Recombinant N. crassa Treatment

A 50 mL 1x Vogel’s media containing 2% NaOH treated or untreated WS in 250 mL

Erlenmeyer flasks were inoculated by 10-14 day old conidia suspensions of the

recombinant N. crassa F5 strain at a final OD420 of 0.05 [84]. Equalient of 0.5 g/L or

3 g/L glucose was added to initiate conidia germination. Flasks were incubated at 28

ºC and shaken at 200 rpm with constant light. Samples were taken at various time

intervals for enzyme activity analysis and compositional analysis. The fermentation of

N. crassa F5 strain on WS was carried out for 2 to 6 days.

2.5.4 Enzyme Activity

The enzyme activity was carried out using 2% NaOH-WS and untreated WS. A final

OD420 of 0.1 was inoculated of the recombinant N. crassa F5 in 100 mL Vogel media

(1X solution) in to 250 mL flasks. A glucose solution was autoclaved separately and

a concentration of 0.5 g/L and 3 g/L was added to the 50 mL medium during

inoculation. The recombinant N. crassa F5 was inoculated to 2% NaOH-WS and

untreated WS. Both 2% NaOH-WS and untreated WS supplemented with 0.5 g/L and

3 g/L glucose solution serve as a control. The fermentation of N. crassa F5 were

carried out in shake flasks using 200 rpm at 28°C for one week. A 10 mL of culture

supernatant was centrifuged daily for 20 minutes at 10,000 rpm and cellulase assay

was performed as described previously [85]. A 96 well plate was used for absorbance


38

reading. The enzyme assay was used with 500 μL enzyme, 1000 μL of 50 mM sodium

citrate buffer (pH 5.0) and strip of filter paper was incubated at 50°C for 60 min. The

reaction was stopped by adding 30 μL of 3,5-dinitrosalicylic acid (DNS) reagent to

180 μL of each sample. The reaction mixture was heated to 95°C for 5 min, followed

by 1 min at 4°C in PCR plates. Glucose standard and samples absorbance was

measured at 540 nm as described previously [86].


The untreated WS, autoclaved at 121°C for 20 min with distilled water WS, 2%

NaOH pretreated WS, and N. crassa F5 strain treated WS samples were prepared for

Scanning electron microscopy (SEM). The morphological variations after pre-

treatment was analyzed with the help of scanning electron microscope techniques

(SEM) (JSM-7800F PRIME, JEOL's USA). SEM analysis was carried out at

different magnification range of 1000, 2000 and 3000 um. Images of the samples

were taken using the dried treated and untreated samples, fixed on a black carbon

tape and sputter with gold palladium as described previously [57].

2.5.6 FT-IR and X-ray Diffraction (XRD) Analysis

The dried samples of treated and untreated WS were embedded in KBr pellets prior

Fourier Transform Infrared (FT-IR) spectra analysis (Thermo Nicolet iS10 FT-IR

spectrometer with an ATR attachment, USA). The spectra were collected in the range

500-4000 cm-1 with 32 scans per sample with a resolution of 4 cm-1 in absorption

band mode as described previously [72]. Spekwin 32-7.1 FT-IR software were used

for data normalization, graphing and determination of changes in the peak position of

treated samples compare to untreated WS.

The X-ray diffraction XRD profiles of WS fibers before and after N. crassa F5

pretreatment were scanned for the crystallinity of each samples. The samples were

grounded, homogenized and a fine powder was used for defining changes in bulk

composition and crystallinity. The pocket was packed with dried powder and pressed

into a compact material for the analysis. The XRD instrument was set at 45 kV,

radiation was Cu Ka (λ D 1.54 Alpha 1) with a range between 10- 38° with a step size

of 0.1°. The time per step was 8 with a scanning rate of 1.33 degree/minute. The

Crystallinity (%) was calculated as shown in equation 2-12.


39

Crystallinity (%) = cryst amor

cryst

I I

I

Equation 2-12

Where Icryst= represents the crystalline region, and Iamor= represents the

amorphous region.

The samples were subjected in amorphous to Panalytical X'PERT, X-ray

diffractometer system CA, USA as described previously [57].

2.5.7 The Automatic Methane Potential Test System (AMPTS)

The biomethane potential assay (BMP) was carried out using anaerobic bottles

containing untreated WS, N. crassa F5 treated WS, N. crassa F5 + 2% NaOH

pretreated WS and 2% NaOH pretreated WS samples. A stable inoculum of anaerobic

REED digester plant (USA) was used. The inoculum had average of TS and TS/VS

value of 2.2 and 66.6% respectively. The anaerobic digestion experiment was started

with a substrate loading of 3 gVS/L, and inoculum loading of 2.25% using

food/microbe (S/I) of 0.28 in to each anaerobic digestion (AD) bottle. The final

volume was adjusted to 400 mL using the same Vogel media (1 X). The initial pH of

each AD bottle was between 7.8-8.0. Inoculum in Vogel media bottles without

substrate sample were included as a control at the same conditions. The untreated and

pre-treated substrates were run parallel to compare the biogas and methane

production. The AD bottles were airtight with rubber septa and a screw cap. All the

bottles were flushed with N2 gas for 4 min and were incubated at thermophilic

50±2°C condition. All the bottles were incubated for a period of 3 weeks. The daily

volume of biogas was measured continuously by AMPTS-II system version 1.6

(Bioprocess Control Sweden, AB) set at thermophilic 50±2°C condition. A biogas

samples was collected periodically from each triplicate bottles and were analyzed for

CH4 and CO2 content of the biogas using gas chromatography (Hewlett Packard

5890A Agilent GC, USA). The GC system used helium as a carrier, column head

pressure of 350 kPa, 100, 120 and 120°C temperatures of oven, injector port and

thermal conductivity detector respectively.

2.5.8 Analytical Method

The analysis of structural carbohydrate of wheat straw before pretreatment and for

each pretreated sample of wheat straw was carried out through standard laboratory


40

analytical procedure (LAP). Total solid (TS), volatile solid (VS), ash contents were

determined. The National Renewable Energy Laboratory (NREL)’s method of two

step acid hydrolysis analytical method was used for lignin content, quantification and

detection of sugar monomers (xylose, glucose, cellubiose, mannose and arabinose) as

described previously [87]. High Performance Liquid Chromatography (Shimadzu,

SPD-MZ0A, CA, USA) equipped with a Refractive Index Detector C. Column was

used. Methanol and water were used as an eluent at a flow rate of 0.6 mL/min and

85°C to detect sugar monomers. Cellulose and hemicellulose were quantified from

these monomers. COD and ammonia of initial time point and final time point of AD

for each samples were determined using COD, ammonia kit (Hach company,

Germany). The pH values of each bottles were measured using a pH meter. The

samples of AMPTS before BMP and post-BMP were filtered with 0.45µm filters for

VFA. The samples were acidified with phosphoric acid (H3PO4) before start of

analysis. Concentrations of VFA (methanol, ethanol, n-butyric, iso-butyric, acetic,

propionic, n-valeric, and iso-valeric) in initial and final samples from the AMPTS-II

system were determined using gas chromatograph (Hewlett Packard 5890A, Agilent

Technologies, USA) equipped an HP-FFAP column (Agilent Technologies, HP 6890,

CA USA) and automatic headspace sampler (Perkin Elmer). Nitrogen was the carrier

gas with a flow rate of 6 mL/min. The chromatographic conditions were as follows:

injector temperature 120°C; detector temperature, 80°C oven temperature program

cooling temperature of 40°C was used. A standard solution of VFA, water, and

triplicates of each sample was run and the average value was reported.

2.6 Biohydrgoen Production using Pure Bacillus sp. strains

from OFMSW

2.6.1 Microbial Strains

One hundred and twenty microbial strains were previously isolated from granular

sludge samples heat-treated (100°C) with increasing residence times in order to

inhibit indigenous methanogenic bacteria. All the strains were identified by 16S

rDNA sequencing [88].

2.6.2 Screening for the Production of Extracellular Hydrolytic

Enzymes

Calibrated suspensions (A600 = 0.9, corresponding to an average concentration of 106


41

cells per mL) of bacterial cells, grown for 24 h at 37 °C in NB broth at 100 rpm, were

used to inoculate plates containing the appropriate media described below and

purified agar (Sigma, Italy). Petri dishes were checked for the presence of enzymatic

activity described below, after aerobic incubation at 37 °C for 3 days. No discrepant

results were recorded in repeated experiments.

2.6.3 Cellulase Activity (CelA)

Cellulase production was detected on Hankin and Anagnostakis Medium containing 5

g/L carboxymethyl-cellulose (CMC). After cell growth, the presence of cellulolytic

activity (CelA) was detected by Congo red method [89].

2.6.4 Lipolytic Activity (LipA)

Strains were tested on tributyrin agar medium containing (g/L): peptone, 5; yeast

extract, 3; tributyrin, 10; agar, 15; pH 6.0. Lipase activity (LipA) of the strains were

indicated by a clear halo around the colony in an otherwise opaque medium as

previously described [90].

2.6.5 Pectinolytic Activity (PecA)

The secretion of extracellular pectin enzymes was tested on polygalacturonic acid

medium (g/L): yeast nitrogen base, 6.7; glucose, 5; polygalacturonic acid (Fluka,

Italy), 7.5; pH 7.0 [91]. The screening was performed using polygalacturonic acid

medium with or without glucose (10 g/L). After cell growth, plates were flooded with

a solution of 6 N HCl. The appearance of a degradation halo around bacterial colony

was considered an indication of the polygalacturonic acid hydrolysis [92].

2.6.6 Proteolytic Activity (PrA)

Extracellular protease production was determined on protein medium with skim milk

(Difco, Italy), pH 6.5. A clear zone around the colony indicated protease activity

(PrA) as described in literature [91, 93].

2.6.7 Starch-Degrading Activity (StA)

Microbial strains were screened for the ability to hydrolyse soluble potato starch

(Sigma, Italy) on Wollum medium containing (g/L): Yeast Extract (Difco), 1;

Na2NO3, 1; KCl, 0.5; MgSO4, 0.5; starch, 10; agar, 17 [92]. After incubation, Petri


42

dishes were flooded with iodine solution. A pale yellow zone around colonies in a

blue medium indicated starch degrading activity (StA) [94, 95].

2.6.8 Xylan-Degrading Activity (XylA)

Cultures were screened for xylan degrading activity by growth on modified Hankin

and Anagnostakis Medium containing 0.5% xylan from oat-spelt (Fluka, Italy).

Colonies showing xylan-degrading activity (XylA) were identified by a clear

hydrolysis zone around the colony after treatment with Congo Red.

2.6.9 Amylolytic Enzymes Characterization

The starch degrading strains were tested for their amylolytic activity once cultivated

in NB with 20 g/L soluble starch or Starch Production Medium (SPM) supplemented

with (g/L): peptone, 5; soluble starch, 20; Na2HPO4, 2; KH2PO4, 1. The pH was set to

7.0 for both media. The strains were aerobically grown at 37 °C for up to 168 h. Ten

mL samples were withdrawn at 24 h intervals and, after centrifugation (10 min, 5,500

x g), the supernatant was used for enzymatic assays.

Total amylase activity was determined in liquid assays using the reducing

sugar method with glucose as standard [96]. The optimal enzyme pH was assessed at

50 °C with 50 μL of the supernatant and 450 μL of the substrate (0.1% soluble potato

starch) suspended in 0.05 M citrate-phosphate or sodium-phosphate buffer at pH

values ranging from 5.5 to 8.0. The optimal assay temperature was determined at pH

6.0 and 7.0 using temperatures ranging from 30 to 60°C. The enzymatic reactions

were conducted for 10 min and terminated by boiling in a water bath for 15 min.

Enzymatic activities were expressed as unit (U) per mL of supernatant, which

is defined as the amount of enzyme which releases 1 μmole of reducing end groups

per min. All experiments were carried out in triplicate.

2.6.10 Batch Test for Hydrogen Production from Glucose

To evaluate the H2 potential from glucose of the twenty strains with the most

promising hydrolytic phenotype, 100 mL Pyrex vessels, were filled with 50 mL of

Nutrient Broth (NB, Oxoid, pH 6.0) with or without glucose (5 g/L) and sterilized by

autoclave (121° C, 20 min). Each strain was pre-grown overnight in NB and

inoculated into the batch reactors at an optical density (600 nm) value of 0.2. After


43

inoculation, the reactors were hermetically closed using a silicon plug. Once flushed

with N2 gas for 3 min, the vessels were incubated without stirring in a thermostatic

chamber at 37° C.

The amount of biogas produced was recorded daily, using the water

displacement method [88]: the biogas accumulated in reactors headspace is released

in a second bottle filled with an acidified (pH < 3) and saline (NaCl 25%) solution,

which avoids the dissolution of gas into the liquid. The biogas moves an equivalent

volume of liquid that was subsequently measured with a graduated cylinder. Biogas

composition in terms of hydrogen, carbon dioxide and methane were measured by gas

chromatography as indicated in the “Analytical methods and calculations” paragraph.

At the end of fermentation, liquid samples were kept at -20 °C to analyse the

volatile fatty acids (VFAs) concentration and the amount of residual glucose or starch

as described below in the “Analytical methods and calculations” paragraph.

All experiments were carried out in triplicate and the results averaged.

2.6.11 Batch Test for Hydrogen Production from Soluble Starch and

OFMSW

The most promising starch-hydrolyzing strains were evaluated for their ability to

convert soluble starch into H2. The strains were grown in SPM for 72 h and then used

to inoculate 50 mL fresh SPM into Pyrex bottles as described above. Sodium

phosphate buffer (pH 6.0 and 7.0) was used.

In the case of H2 production from OFMSW, each vessel was supplemented with 10 g

VS/L (which corresponds to 150 g/L of fresh weight), instead of soluble starch.

OFMSW was sterilized by autoclave (121° C, 20 min) to suppress the indigenous

microbes [97]. The experiments were monitored until biogas production stopped. At

the end of H2 fermentation, liquid samples were withdrawn and kept at -20 °C for

further analysis. All the experiments were carried out in triplicate and the results

averaged.

The sample of OFMSW used for batch tests was obtained in May 2015 from

separate collection of MSW in Padova (Italy). Approximately 200 kg of organic waste

was manually sieved, sorted and divided into the following fractions: fruits (F),

vegetables (V), meat–fish–cheese (MFC), bread-pasta-rice (BPC), undersieve 20 mm


44

(U) and rejected materials. Undersieve 20 mm was composed of materials smaller

than 20 mm. The rejected materials were shoppers, plastics, metals, glass, bones,

paper and cardboard, shells and fruit kernels. Results of manual sorting procedure are

reported in Table 2-1.

Table 2-1 Results from manual sorting procedure of the OFMSW used in this

study

Fraction Weight (Kg) Percentage (%)

Fruit 52.01 25.9

Vegetable 42.21 21.0

Meat-Fish-

Cheese

8.95 4.5

Bread-Pasta-

Rice

44.44 22.1

Rejected

materials

33.52 16.7

Undersieve 20

mm

19.67 9.8

Total 200.80 100

Using the sorted fractions, a sample of organic waste was prepared

maintaining the same proportion of the single fractions without the rejected materials.

The prepared sample of OFMSW was ground in a kitchen mill prior to be used as

substrate for H2 production. The shredded OFMSW had total solid (TS) concentration

of 146±11 g TS/L and volatile solid (VS) and total organic carbon (TOC)

concentration of 93±1 % and 45±1 %, respectively, referred to dry weight. Total

Kjeldahl nitrogen (TKN), ammonium and total phosphorus concentration was

2861±113 mg N/L, 408±35 mg N/L and 375±18 mg P/L, respectively. Concentrations

(of dry weight) of lipids, proteins, cellulose, hemicellulose, lignin, starch and pectin in

OFMSW sample were also detected as follow: 18±1, 17±1, 5.0±0.6, 6.0±0.5, 2.0±0.2,

19±1, 8.0±0.7, respectively.

2.6.12 Analytical Methods and Calculations

TS, VS, TKN, ammonium and total phosphorous concentrations were analysed

according to standard methods [98]. TOC values were obtained by difference between

Total carbon (TC) and inorganic carbon (IC). TC and IC were analysed by a TOC

analyser (TOC-V CSN, Shimadzu). Concentration of lipids, proteins, pectin, lignin,

cellulose, hemicellulose and starch were analysed according to official methods [99].


45

VFAs concentrations (acetic, propionic, iso and n-butyric, iso and n-valeric,

iso and n-caproic and heptanoic acids) were analysed by a gas chromatograph (Varian

3900) equipped with a CP-WAX 58 WCOT fused silica column (25 m x 0.53 mm ID,

Varian) and a Flame Ionization Detector (FID). Nitrogen was used as carrier gas at a

flow of 4 mL/min in column. The oven temperature programme was initially set at 80

°C for a min, then increased at a rate of 10 °C/min to 180 °C (finally maintained for 2

min). Injector and detector temperatures were both set to 250 °C.

Residual glucose and soluble starch in the NB or SPM broths were measured

using the peroxidase-glucose oxidase method with the D-glucose and starch assay kit,

respectively (Boehringer Mannheim). Biogas composition in the headspace of

reactors, in terms of hydrogen (H2), carbon dioxide (CO2) and methane (CH4)

concentrations, was analysed by gas chromatography using a micro-GC (Varian 490-

GC) equipped with i) a 10-meter MS5A column (to analyse H2 and CH4) ii) a 10-

meter PPU column (to analyse CO2) and iii) two Thermal Conductivity Detectors

(TCDs). Helium was used as carrier gas at a pressure of 150 kPa in columns. Injector

and column temperatures were both set to 80 °C.

Data on biogas and hydrogen productions was expressed at a temperature of 0

°C and pressure of 1 atm. Hydrogen volumes produced in the time interval between

each measurement [t - (t-1)] during dark fermentation batch tests, were calculated

using a model considering i)the hydrogen gas concentration at times t and t-1,

together with the total volume of biogas produced at time t, ii) the concentration of the

specific gas at times t and t-1, and iii) the volume of the head space of reactors [100].

The equations 2-4, 2-5, 2-6 was applied for hydrogen volume calculation, cumulative

hydrogen production (VH2cum), hydrogen yields, expressed as NmL H2/g VS and mol

H2/mol glucose. The hydrogen yield was calcualted using Equation 2-13.

Equation 2-13

Where:

VH2cum: cumulative hydrogen production at the end of the dark fermentation

test; 22.414 L/mol: volume occupied by 1 mole of ideal gas at 1 atm pressure and 0°


46

C; Wglucose: weight of glucose equivalent added at the beginning of the batch test; 180

g/mol: weight of 1 mole of glucose equivalent.

The volumetric productivity (Q) was based on as NmL H2/g VS per litre of

culture medium per day (NmL H2/L/d) and the maximum volumetric productivity

(Qmax) was compared as the highest volumetric productivity displayed by the strains.

3 Results

3.1 Alkali Treatment

Fossil fuels are continuously depleting and their consumption releases greenhouse gases

that raise environmental problems. Therefore, interest is developed in looking for

alternative energy. Biogas production from agriculture waste residue through anaerobic

digestion (AD) is focused on sustainable bioenergy production process [101]. The

agriculture waste residue is composed of lignin, hemicellulose, cellulose, and some

extractable components. The percentage of these components varies among crop residue,

but in general, hardwood biomass contains 40-50% cellulose, 15-25% lignin, 22-35%

hemi-cellulose, and 2-7% extractives, whereas softwood biomass contains 20-31% lignin,

24-32% hemicellulose, 40-45% cellulose, and 1-7% extractives [102, 103]. Cellulose is

homopolysaccharide chains of glucose units. Hemicellulose is heteropolymers of pentose

sugars that is the outer surface layer of the biomass cell wall [40]. Lignin is the most

complex hydrocarbon polymer and contains multiple phenylpropane units, crosslinking

of these phenylpropane units and hydrophobic nature of lignin make the LB structure

more resistant to microbial and enzyme degradation [104]. The degradation resistance of

lignin significantly decreases the yield of biogas in AD process [28]. One of the initial

rate-limiting steps is to choose a suitable pretreatment method to remove lignin and make

the cellulose accessible to hydrolytic enzymes [104, 105]. The second concern is

contingent on testing of high concentration of chemicals in biomass pretreatment. Several

methods, steam explosion, hydrothermal process, acid treatment, alkalies treatment,

ammonia fiber explosion etc, have been reported for biomass pretreatment with low and

high solid concentration [105, 106].

For lignin removal among all of the listed methods and many others, the preferred

selection is alkaline pretreatment method. The lignin can be released by different alkalies,

especially, KOH, Ca(OH)2 and NaOH [107]. Treatment of waste biomass with these

alkalies decreased the degree of polymerization, remove lignin, and make cellulose more

Chapter 3: Results

48

accessible to enzymatic and microbial degradation. However, the high concentration of

alkalies increase the cost of treatment than it produces energy and generate volatile fatty

acids (VFA) that inhibit digestion process [108]. Therefore, selection of optimum

pretreatment method is the crucial step for improving anaerobic digestion from waste

biomass for increasing biogas production [109, 110]. Although there are wide-range of

reports on the treatment methods, none of the studies has compared the different heating

conditions and alkali reagents on various substrates for an optimum and effective method,

the current study emphasized to test different concentration of alkalies coupled with

thermal heating for delignification and evaluate the effect on the biogas and methane

yield through anaerobic digestion of the pretreated and untreated biomass.

3.1.1 Analysis for Biomass Composition

Composition of each biomass was determined as described previously [69]. The lignin

composition obtained was 10% in peanut, 15% in raw paper, 23% in almond, 26% in

kallar grass, and 18-22% in wheat straw (Table 3-1). Similarly, the amounts of

hemicellulose and cellulose contents was high in wheat straw, waste paper and low in

almond shell and peanut shell compared to other tested substrate (Table 3-1).

Table 3-1 Estimated percentage composition of different waste biomasses

S.No Substrate Cellulose Hemicellulose Lignin

T.S V.S

1 Waste Paper 50 25 15 90 80.5

2 Wheat straw 40 26 18 81.5 65.5

3 Rice straw 38 24 17 85.3 80

4 Para grass 29 34 22 65.5 80

5 Kallar grass 38 23 25 67 74

6 Bagasse 28 32 19 72 76

7 Pulses peel 29 24 18 80 65

8 Peanut shell 31 23 8-10 89 72

9 Corn cob 42 29 18.5 89 70.5

10 Almond shell 28 31 23 94.5 90.3 T.S = total solid, V.S = volatile solid, W.E = water extractive

Chapter 3: Results

49

3.1.2 Alkali Treatment to Remove Lignin from Lignocellulosic Biomass

An alkali treatment method was used for the measurement of lignin removal percentage.

After the composition analysis, 1,2,3, and 5% NaOH, 1,2,3, and 5% KOH, and 0.5% of

Ca(OH)2 solutions were used to remove lignin from the substrates.

To find a significant difference among each alkali treatment and heating process a

one-way ANOVA was used to find statistical difference in lignin removal. All the tested

substrates using one alkali at different concentration were compared, the average result of

one factor ANOVA showed p-values of 0.05 and 0.001, indicating that the reactions were

significant for most of the conditions. A clear significant difference between increasing

value can be seen in (Tables 3-2 and 3-3). The difference in percentage of lignin removal

was high in case of 1-2% alkali dosage and was less between 3-5% alkali concentration.

Among the heating conditions and different concentration of alkalies, the

optimum results were observed using 2% of NaOH and KOH. For 2% NaOH, water bath

heating treatment displayed an average value of 56.3% lignin removal, whereas autoclave

heating 61.6% and the highest lignin removal percentage was 67.9% with microwaving

from all the tested substrates (Table 3-2). Similarly, the 2% KOH removed an average of

40, 47.7 and 55.7 % lignin with water bath heating, autoclave heating and microwave

treatment respectively from all the tested substrates (Table 3-3).

Table 3-2 One-way ANOVA for NaOH delignification of all tested substrates

Groups Count Sum Average Variance

1%NaOH (WT) 10 322.2 32.2 19.6

1%NaOH (AUTO) 10 390.5 39.0 37.7

1%NaOH (MV) 10 458.5 45.8 33.1

2%NaOH (WT) 10 563.1 56.3 26.7

2%NaOH (AUTO) 10 616.3 61.6 18.9

2%NaOH (MV) 10 679.2 67.9 11.9

3%NaOH (WT) 10 683.7 68.3 60.9

3%NaOH (AUTO) 10 735.6 73.5 47.1

3%NaOH (MV) 10 818.3 81.8 21.1

5%NaOH (WT) 10 704 70.4 56.9

5%NaOH (AUTO) 10 764 76.4 37.8

5%NaOH (MV) 10 858 85.8 24.1

ANOVA

Chapter 3: Results

50

Source of Variation SS df MS F P-value F crit

Between Groups 25810.03 10 2581.003 76.26397 0.05 1.927679

Within Groups 3350.459 99 33.84302

Total 29160.49 109

MV= microwave, WT= water bath. Auto= autoclave

Table 3-3 One-way ANOVA for KOH delignification of all tested substrates

Groups Count Sum Average Variance

1%KOH (WT) 10 216.0 21.6 16.7

1%KOH (AUTO) 10 254.0 25.4 27.0

1%KOH (MV) 10 307.0 30.7 29.2

2%KaOH (WT) 10 407.4 40.7 19.7

2%KaOH (AUTO) 10 470.4 47.0 26.5

2%KaOH (MV) 10 557.2 55.7 25.2

3%KaOH (WT) 10 505.1 50.5 36.6

3%KaOH (AUTO) 10 583.6 58.3 42.6

3%KaOH (MV) 10 650.4 65.0 29.0

5%KOH (WT) 10 525 52.5 32.0

5%KOH (AUTO) 10 609 60.9 29.2

5%KOH (MV) 10 678 67.8 27.0

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 26028.3 11 2366.209

83.1977317

6 0.001 1.878388

Within Groups 3071.605 108 28.44079

Total 29099.9 119

MV= microwave, WT= water bath. Auto= autoclave

From the temperature effect, the microwave heating was more effective in lignin

removal than autoclave and water bath. In addition, it was observed that, the potential of

lignin removal was higher using NaOH in all the heating conditions than KOH and

Ca(OH)2 treatments. The box plot results elucidate that delignification was maximum

using microwave compared to the autoclave and water bath heating for each of the 1,2,3,

and 5% NaOH and KOH conditions. Increase in lignin removal was observed with

increasing concentrations of NaOH and KOH as shown in the box plot (Figure 3-1 and 3-

2). The results for alkali treatment concluded that alkali treatment significantly reduced

lignin from all the biomass tested. In control, untreated samples (treated with distilled

water without alkali), no lignin was removed, however a negligible amount of weight loss

was observed from all the substrates at the same heating conditions.

Chapter 3: Results

51

0

10

20

30

40

50

60

70

80

90

100

1%

WT

1%

Au

to

1%

MV

2%

WT

2%

Au

to

2%

MV

3%

WT

3%

Au

to

3%

MV

5%

WT

5%

Au

to

5%

MV

Del

ign

ific

atio

n (%

)

Figure 3-1 Box plot demonstration for NaOH base comparative delignification,

WT= water bath, Auto= autoclaving, MW= microwave.

0

10

20

30

40

50

60

70

80

90

1%

WT

1%

Auto

1%

MV

2%

WT

2%

Au

to

2%

MV

3%

WT

3%

Au

to

3%

MV

5%

WT

5%

Au

to

5%

MV

Del

ignif

icat

ion

(%)

Figure 3-2 Box plot demonstration for KOH base comparative delignification, WT =

water bath, Auto= autoclaving, MW= microwave.

The results of alkalies treatment verified maximum delignification as the alkalies

concentration was increased, however, an opposing consequence of higher concentration

of alkalies was observed on holocellulose. The highest tested dosage of alkalies beside

Chapter 3: Results

52

lignin removal, also decreased hemicellulose and cellulose composition of the tested

biomass. In (Figure 3-3), the effect of 1,2,3 and 5% NaOH on holocellulose is shown, the

reduction of hemicellulose was less in case of 1-2% NaOH dosage and significantly

higher reduction was observed at maximum dosage of 5% NaOH tested. Similarly, the

cellulose content also decreased as the concentration of NaOH increased from 1 to 5%

NaOH. A maximum level of 40-50% hemicellulose reduction was observed from pulses

peel, corn cob and almond shell and lower percentage in the wheat straw, rice straw and

paper waste when 5% NaOH is tested. Similarly, a maximum of 30-40% reduction in

cellulose was observed with 5% NaOH from wheat straw, rice straw and paper waste and

lower 25-32% from pulses peel, corn cob and almond shell (Figure 3-3).

Figure 3-3 Effect of alkali on hemicellulose and cellulose degradation, HM=

hemicellulose, CL=cellulose, PW =paper waste, WS= wheat straw, RS=rice straw,

PG=para grass, KG= kalar grass, BG=bagasse, PP=pulse peel, PN = peanut,

CC=corn cob, AL=almond.

3.1.3 Substrate Base Lignin Removal

In (Figure 3-4 , 3-5, 3-6 ) a comparative delignification of 2% NaOH, 2% KOH, and

0.5% Ca(OH)2 from each tested substrate is shown. Base on the substrate, a different

potential of lignin removal was observed. The highest lignin removal was obtained from

Chapter 3: Results

53

rice straw, wheat straw, kallar grass, and bagasse respectively with 2% NaOH using the

microwave heating as shown in (Figure 3-4). It was observed that, the delignification was

lower in case of almond, corn cob and paper pulp. A similar pattern of lignin removal

was observed for 2% KOH and 0.5% Ca(OH)2 from all the substrates treated by the

microwave, autoclave, or water bath, although the lignin reduction was comparatively

lower than 2% NaOH treated substrates (Figure 3-5 and 3-6).

The results of the current and previously reported study proved that Ca(OH)2 has

less ability for lignin removal as compared to NaOH and KOH, this could be due to

solubility reason of Ca(OH)2, it is hardly soluble beyond this concentration and,

secondly, it makes a calcium–lignin complex, as calcium ions with two positive charges

are inclined to crosslink with negatively charged lignin molecules under alkaline

conditions that result in functional group ionization, thus preventing higher amounts of

lignin degradation [111, 112].

Figure 3-4 Comparative delignification of NaOH for microwave (MW), autoclave

(Auto), and water bath (WT), P =paper, WS= wheat straw, RS=rice straw, PG=para

grass, KG= kalar grass, BG=bagasse, PP=pulse peel, PN = peanut, CC=corn cob,

AL=almond.

Chapter 3: Results

54

Figure 3-5 Comparative delignification of KOH for microwave (MW), autoclave



AL=almond.

Chapter 3: Results

55

Figure 3-6 Comparative delignification of Ca(OH)2 for microwave (MW), autoclave



AL=almond.

3.1.4 Scanning Electron Microscopy for the Surface Degradation of

Biomass

The surface structures of the untreated and 1,2,3, and 5% NaOH pre-treated wheat straw

biomass were compared by analysing scanning electron microscope (SEM) micrographs.

The untreated wheat straw was highly compact with a clear and smooth structure Figure

3-7 (A). After alkaline pre-treatment, wheat straw was missing its dense structure, and

distortion were observed on the surface. Similar observation of degradation after alkali

treatment on the surface of straw is also reported earlier [113]. The SEM micrographs

demonstrated the impact of pre-treatment removal of lignin and hemicellulose Figure 3-7

(B), (C), (D), and (E). The severity of the treatment of the surfaces increased with the

concentration of NaOH indicating that the pre-treatment promoted the degradation. The

images clearly showed ruptures in the silicon waxy structure, broken fibrils, and

disrupted wall bundles in the lignin and hemicellulose complex in each sample. The

morphologies of the wheat straw and other biomass from the SEM micrographs were

very similar in term of the alkaline pre-treatment process. Kallar grass SEM images

Chapter 3: Results

56

under the same heating and alkali treamtent further proved simillar effect on substrate

surface as shown in (Figure 3-8 ).

A B

C ED

Figure 3-7 SEM micrographs for the untreated and pre-treated wheat straw, (A)

untreated (B) 1% NaOH treated (C) 2% NaOH treated (D) 3% NaOH (E) 5%

NaOH treated wheat straw with 1000X magnification.

Chapter 3: Results

57

A B

C ED

Figure 3-8 SEM micrographs for the untreated and pre-treated Kallar grass, (A)

Untreated (B) 1% NaOH treated (C) 2% NaOH treated (D) 3% NaOH treated and

(E) 5% NaOH treated Kallar grass SEM images at 1000X magnification.

3.1.5 Fourier Transformed-Infrared Spectroscopy

FT-IR technique is used for changes and compositional transformations of lignocellulosic

biomass at the molecular level. FT-IR spectroscopy was used for the chemical group

breakdown to see the corporal changes tempted using alkalies pre-treatment. The

conjectural analysis of the spectra generated through FT-IR is diverse because the

agriculture biomass contains various structure units and functional groups due to

presence of heteropolymer chains in hemicellulose, cellulose, and lignin skeleton. Hence,

the groups are given based on expected spectral data of the compounds from the

Chapter 3: Results

58

published studies on analogous susbtrate [55, 56]. Generally, in agriculture waste

biomass, the band in the range of 1499–1640 cm-1 and 1199–1310 cm-1 is occupied by

aromatics (lignin), while the band in the range of 990–1150 cm-1 and 1799–1890 cm-1 is

occupied by the hemicellulose groups, and the band in the range of 1320–1470 cm-1 and

2690–3000 cm-1 is occupied by the cellulose contents [56]. As an example, FT-IR

spectra for 1-5% NaOH treated and untreated wheat straw residue is shown. A severe

changes in the spike and peak positions of treated biomass as compared to untreated

wereobserved. The FT-IR spectra of wheat straw treated with NaOH showed obvious

changes in the banding pattern and peak position (Figure 3-9). The band at 1665 cm-1

representing unconjugated carbonyl stretching was more prominent in control WS,

however missing from 1,2, 3 and 5% NaOH, as can be seen in (Figure 3-9). The bands

from 1300-1550 cm-1 are attributed to aromatic rings, these bands are degraded and

distorted in the 1,2,3 and 5% NaOH treated WS as compare to untreated WS. The bands

1250 cm-1 specify existence of syringyl in lignin's structure of wheat straw. This bands

and peak position was also detached due to pre-treatment as shown in (Figure 3-9).

Further the region of 1000-1600 nm was focused to see more clear shift in peak positions,

and it was noticed that untreated and pre-treated substrate with same alkali act differently

using different heating conditions but the action of alkali is target specific to particular

group as shown in (Figure 3-10).

Chapter 3: Results

59

1250

1511

C-O absorption in

guaycyle ring in lignin

C-O-C

stretch

(diaryl)

1665

C=O stretch 1760–

1665 (carbonyls)

1355-1315

(aromatics)

C-H aldehyde stretch

(~2850 & ~2750

C-H stretch (Alkane)

(2950-2800)

5%NaOHl (blue)

2% NaOH(aqua)

1% NaOH(dark)

3% NaOH (green)

Control (purple)

Wavenumber

Ab

so

rbance

Figure 3-9 FT-IR spectrum of wheat straw for microwave sample, untreated control

(purple), 1% NaOH (black) 2% NaOH (aqua), 3% (green) and 5% (blue).

Chapter 3: Results

60

Ab

so

rb

an

ce

Wavenumber

0

0.2

0.4

0.6

0.8

1

05001000150020002500300035004000

Control

1% MV-WS

1% Auto-WS

1% Wt-WS

Figure 3-10 Comparision of FTIR peak variation of wheat straw, untreated control

(red), 1% NaOH microwave (blue) 1% NaOH autocalave (green), 1%NaOH water

bath (black) treated sample.

3.1.6 Biogas Potential of Pre-Treated and Untreated Substrates

The biogas potential is often defined as the volume of biogas produced per gram volatile

solid (VS) added for the specific substrate. The accumulated final methane production is

regarded as the methane potential of the particular substrate. In the current study,

inoculum of a full-scale anaerobic digester with a moisture content (MC) of 87.9%, 4.2%

TS, and 2.8% VS was used. For the comparison of anaerobic digestion, the 1,2,3, and 5%

NaOH and KOH treated samples of almond shell and wheat straw were tested. The

selection of substrate was based on lower delignification (almond shell) and relatively

higher delignification (wheat straw) among the tested substrates. The highest methane

(CH4) concentration of the biogas was 65-66% for pre-treated wheat straw and 50-55%

for pre-treated almond shell respectively. However, 30-40% methane (CH4) content was

observed from the same untreated substrates.

The average highest daily volumetric biogas rate was between 50.4-70.5 mL from

KOH and NaOH treated wheat straw (Figure 3-11). Similarly, the maximum daily

volumetric biogas rate was between 30-45 mL from NaOH and KOH treated almond

Chapter 3: Results

61

shell (Figure 3-12). Correspondingly, the average daily methane yield was 10-12 and 5-7

mL/gVS from wheat straw and almond shell treated with NaOH and KOH respectively.

It was observed that; the daily volume of biogas and methane rate was 2-2.6 times

higher in case of pretreated almond shell and wheat straw comparatively to their

untreated substrates. Similarly, the biogas yield of NaOH treated samples was more than

the daily biogas and methane yield from KOH treated samples. This could be related to

the high lignin removal of NaOH treatment than KOH.

In addition; the daily biogas was high in the first 10 days of anaerobic digestion

from 3-5% KOH and NaOH batch assay than 1- 2% treated substrates. This results

support that; high delignified substrate yield more biogas at the start of anaerobic

digestion process than less delignified substrate.

The cumulative biogas obtained from the alkali treated substrate was 2-times

higher than the untreated substrates. The highest cumulative biogas was 560.6 NmL/gVS

from 2% NaOH treated wheat straw (Figure 3-13 AB). Similarly, the highest cumulative

biogas was 310.2 NmL/gVS from 2% NaOH treated almond shell (Figure 3-14 AB). The

total cumulative biogas and methane yield was minimum in case of 3 and 5% batch assay

than 1 and 2% NaOH and KOH samples. This could be related to the hemicellulose and

cellulose degradation from 3 and 5% delignified substrates (Figure 3-3). The total biogas

yield was less in case of KOH treated samples than the NaOH treated substrate. This

observation is also supported from the Gompert kinetic calculation for cumulative

methane yield, a comparison of 2% NaOH and KOH is shown in (Table 3-4).

The results of the current study proved that, pretreatment is necessary to include

for increasing biogas production from agriculture biomass. Our data from different

pretreatment conditions unfold that low alkali dosage for short time heating process can

decrease lignin up to more than 50% without degrading useful carbon sources

(hemicellulose and cellulose). Beside this, the short heating process with low dosage of

alkali has shown significant results in term of cumulative biogas yield.

Chapter 3: Results

62

3.1.7 Conclusion

Three alkali reagents at different concentrations were compared at three heating

conditions to evaluate the impact of treatment on agriculture waste biomass.

Delignification from the waste residue of agribiomass increased as the concentration of

alkali reagent was increased. However, the high dosage of alkali has shown adverse

effect on holocellulose, removing hemicellulose and cellulose, which showed a negative

effect on net biogas yield during BMP batch assay. The results of lignin removal and

anaerobic digestion batch experiments shown that, the optimum results were displayed by

2% NaOH treatment that removed a significant level of lignin from all the tested

substrates. Similarly, the biogas obtained with 2% NaOH has also shown more total

biogas yield than 2% KOH and even from the concentrated alkali dosage treated samples.

Among the heating processes, short time microwave heating was the most effective

treatment for lignin reduction. Notably, wheat straw batch samples displayed highest

cumulative biogas production compare to almond shell.

Incubation time (d)

0 10 20 30 40 50

Dai

ly b

iogas

(m

L)

0

20

40

60

80

Untreated

1% KOH

1% NaOH

2% KOH

2% NaOH

3% KOH

3% NaOH

5% KOH

5% NaOH

Fermentation time (d)

Daily

bio

gas (m

L)

Figure 3-11 Daily volumetric biogas of NaOH and KOH pretreated wheat straw.

Chapter 3: Results

63

Incubation time (d)

0 10 20 30 40 50

Dai

ly b

iogas

(m

L)

0

10

20

30

40

50

60

Untreated

1% KOH

1% NaOH

2% KOH

2% NaOH

3% KOH

3% NaOH

5% KOH

5% NaOH

Daily

bio

gas (m

L)


Figure 3-12 Daily volumetric biogas of NaOH and KOH pretreated almond shell.

Cum

ula

tive

bio

gas (N

mL

/gV

S)

Cum

ula

tive

bio

gas (N

mL

/gV

S)

Fermentation time (d) Fermentation time (d)

Figure 3-13 Cumulative biogas of NaOH (A) and KOH (B) pretreated wheat straw.

Chapter 3: Results

64

Cum

ula

tive

bio

gas (N

mL

/gV

S)

Cum

ula

tive

bio

gas (N

mL

/gV

S)

Fermentation time (d)Fermentation time (d)

Figure 3-14 Cumulative biogas of NaOH (A) and KOH (B) pretreated almond shell.

Table 3-4 Methane yield (NmL/gVS) of the NaOH and KOH treated biomass

Untreated

WS

Untreated

AL

2% NaOH

WS

2% NaOH

AL

2% KOH

WS

2% KOH

AL

(P) 131.00 108.00 430.60 275.50 308.10 220.90

Rmax 35 30 64.5 49.9 40.3 30.5

(λ) 94.6 96.5 48.1 51.2 49.2 52.5

R2 96.1 97.3 98.2 98.2 97.5 97.5 (P)= Methane production, Rmax= maximum production rate, (λ)=lag phase, WS=wheat straw, AL=almond

shell.

3.2 Enhancing Biogas Production from Lime Soaked Corn

Cob Residue

The advantage of lime (Ca(OH)2) pretreatment is the low-cost of Ca(OH)2 as compared to

other alkalies. Secondly, it does not generate much inhibitors and can be easily recovered

from hydrolysate by reaction with CO2 during treatment process[114]. Soaking with

Ca(OH)2 did not need any thermal heating, so it is also beneficial for large scale

development and energy output yield. The corn cob is a waste biomass of maize an

Chapter 3: Results

65

important cereal crop grown in Pakistan. Thus, corn cob could be assessed as substrates

for biogas production. Being an inexpensive pretreatment method, the current study

focused to test the consequences of lime (Ca(OH)2) pretreatment on structural properties

of corn cob using scanning electron microscopy (SEM). Additionally, the effect of

Ca(OH)2 soaking before and after treatment was compared for composition, biogas and

methane yields of the corn cob.

3.2.1 Composition and Ca(OH)2 Soaking Effect

The effect on the compositions of corn cob before lime treatment (initial value) and after

treatment is listed in the (Table 3-5). Corn cobs are a lignocellulosic biomass composed

of hemicellulose, cellulose and lignin. The percent composition of each constituent may

be vary depending upon the variety, growth and analysis parameters. Corn cob was

composed of 19% lignin, 30% hemicellulose, and 42% cellulose determined by using the

protocol and methodology as described by Sluiter et al. [69]. The compositions of

soaking pretreatment method with 0.5% of Ca(OH)2 was assessed for the measurement of

lignin removal percentage and dry weight loss. The highest removal of lignin was

57.8±1% and maximum loss of dry weight was 33±1% after 30 days of soaking

incubation as shown in the (Table 3-5).

Table 3-5 Effect of Ca(OH)2 soaking on composition of corn cob

Soaking days Lignin content Lignin loss (%) Total weight loss (%)

Initial 19±1 0 0

7 days 16±1 15.7±2 12±1

15 days 12±1 36.8±1 23±2

30 days 8±1 57.8±1 33±1


The effect of incubation time on corn cob were compared to see the surface degradation

and morphological changes. The Ca(OH)2 remove lignin from the substrates and make it

swollen to increase it digestibility. This degradation effect can be seen in the images of

scanning electron microscopy. The Ca(OH)2 soaked solid residues of corn cob clearly

displayed degradation and visual distortion on their surfaces as can be seen in (Figure 3-

Chapter 3: Results

66

15). The SEM micrographs confirmed the impact of Ca(OH)2 soaking in Figure 3-15

(B), (C), (D), as compare to control (A). The intensity of the Ca(OH)2 soaking treatment

on corn cob surfaces increased with the incubation period. Figure 3-15 (B) is 7 days, Fig.

1 (C) is 15 days, and Figure 3-15 (D) is 30 days Ca(OH)2 soaked sample. The prolong

incubation time clearly indicating effect and severity of degradation. The images visibly

displayed ruptures in the waxy structure in each sample.

2.0kv 18.6mmx1.00kSE(M) 6/5/17 14:50 200 um

2.Okv 18.6 mmx1.00k SE(M) 6/5/17 15:10 200 um

2.Okv 18.6mmx1.00k SE (M) 6/5/17 14:35 200 um

2.Okv 18.6mmx1.00k SE(M) 6/5/17 15:25 200um

A B

DC

Figure 3-15 Morphology of the corn cob before and after Ca(OH)2 soaking, (A)

untreated (B) 7 days treated (C) 15 days treated (D) 30 days Ca(OH)2 soaked image

with 1000X magnification.

3.2.3 Anaerobic Digestion and Biomethane Potential

Anaerobic digestion process is known for production of biogas from organic waste

biomass. The volume of biogas is reported as the volume of biogas produced per gram

Chapter 3: Results

67

volatile solid (VS) added for the specific substrate. The methane yield is calculated from

the volatile solid (VS) of the substrate. The initial total solid of the corn cob was 85.5%

and VS was 71.3%.

In this study, inoculum sample of a full-scale anaerobic digester was used. The

inoculum was containing moisture content (MC) of 80.2%, 5.4% TS, and 3.2% VS. The

highest methane (CH4) concentration of the biogas was 50-55% for pre-treated corn cob

and 35% for untreated corn cob respectively.

The 0.5% Ca(OH)2 soaked samples for 7 days, 15 days, and 30 days were

evaluated for biogas production compared to untreated corn cob. The highest daily

biogas was 35 and 50 NmL/gVS from 15 days, and 30 days treated corn cob (Figure 3-

16). However, daily biogas was 27 and 15 NmL/gVS from 7 days treated and untreated

corn (Figure 3-16). Similarly, the highest daily volumetric methane was 18 and 20

NmL/gVS from 15 days, and 30 days treated corn cob (Figure 3-16). However, daily

volumetric methane was was 10 and 7 NmL/gVS from 7 days treated and untreated corn

cob (Figure 3-17). Although the maximum daily volumetric methane rate was 10.4 and

13.5 mL/gVS from 15 days, and 30 days treated corn cob while, 5.5 and 2.2 mL/gVS

from 7 days treated and untreated corn cob.

It was observed that; the daily volume of biogas and methane rate was 2 times

higher in case of pretreated corn cob as compared to untreated corn cob. In addition, lag

phase of the daily volume of biogas and methane was minimum up to 48 hr of incubation

and was longer in the case of untreated corn cob up to 7 days. The daily biogas and

methane yield was maximum between 7-17 days of anaerobic digestion process.

Routinely, the yield of biogas and methane slowly get down after 20 days of anaerobic

fermentation process.

Chapter 3: Results

68

Untreated

7 days treatment

15 days treatment

30 days treatment


Daily

bio

gas (m

L)

Figure 3-16 Daily biogas of untreated, 7, 15, and 30 days Ca(OH)2 soaked corn cob,

error bar indicates standard deviation among the triplicates samples.

Chapter 3: Results

69

Untreated

7 days treatment

15 days treatment

30 days treatment


Vo

lum

etr

ic m

eth

ane

rate

(m

L)

Figure 3-17 Daily methane of untreated, 7, 15, and 30 days Ca(OH)2 soaked corn

cob, error bar indicates standard deviation among the triplicates samples.

The cumulative biogas obtained from 0.5% Ca(OH)2 soaked samples for 7 days,

15 days, and 30 days was higher as compared to untreated corn cob obtained. The best

result was observed after 30 days of 0.5% Ca(OH)2 soaking with a cumulative biogas

volume of 356.5 NmL/gVS. Similarly, a cumulative biogas volume of 218 and 305

NmL/gVS was obtained from 7 and 15 days 0.5% Ca(OH)2 soaked samples. A lower

cumulative biogas of 115.5 NmL/gVS was obtained from untreated corn cob (Figure 3-

18).

The highest cumulative volume of methane for 0.5% Ca(OH)2 soaked samples for

30 days was 136.8 NmL/gVS. Similarly, a cumulative volume of methane of 44 and 104

NmL/gVS was obtained from 7 and 15 days 0.5% Ca(OH)2 soaked samples. A lower

cumulative methane of 27.5 NmL/gVS was obtained from untreated corn cob (Figure 3-

19). The cumulative methane obtained from the treated corn cob was increased as the

time of soaking was increased from 7 to 30 days compare to untreated corn cob.

Chapter 3: Results

70

Untreated

7 days treatment

15 days treatment

30 days treatment


Cum

ula

tive

bio

gas (N

mL

/gV

S)

Figure 3-18 Cumulative biogas of untreated, 7, 15, and 30 days Ca(OH)2 soaked

corn cob, error bar indicates standard deviation among the triplicates samples.

Chapter 3: Results

71

Untreated

7 days treatment

15 days treatment

30 days treatment


Cum

ula

tive

me

thane

(N

mL

/gV

S)

Figure 3-19 Cumulative methane of untreated, 7, 15 , and 30 days Ca(OH)2 soaked

corn cob, error bar indicates standard deviation among the triplicates samples.

3.3 Biological Pretreatment of Rice Straw by Ligninolytic

Bacillus Sp. Strains for Enhancing Biogas Production

In many countries, waste crop residues are burned in the open field. This practice leads to

serious environmental pollution and results in the loss of a valuable commodity that

could be used to supplement farm income. Crop residues represent an inexpensive,

renewable resource for biofuels production. In Asia, about 670 million tons of rice straw

are produced annually, and this material could be harnessed for bioenergy production

[115].

Rice straw is composed primarily of carbohydrate (67%) and lignin (28%) [116].

The high amounts of carbohydrate in rice straw can be utilized as a carbon source for

microbial biogas production [117]. However, the lignin serves as a barrier to prevent

access to the carbohydrate fraction. Lignin is the most complex hydrocarbon polymer

and contains three phenylpropane units (guaiacyl, p-hydroxyphenyl, and sinapyl) linked

by C–C bonds or aryl-ether bonds. The hydrophobic nature of lignin and the crosslinking

of the phenylpropane units make the rice straw more resistant to microbial and enzymatic

Chapter 3: Results

72

degradation. Therefore, this lignin must be removed through a pretreatment process. The

rice straw can be subjected to a chemical pretreatment with alkali [113] or a biological

pretreatment with fungal or bacterial strains [118]. The advantages of biological over

chemical pretreatments are that they are less costly and do not generate high levels of

toxic byproducts that could inhibit downstream biological conversions [119].

Biological pretreatments with naturally-occurring microbial strains and enzymes

have been explored [120]. Two enzymes that are critical to lignin degradation are lignin

peroxidase and laccase. Most examples of these enzymes are from fungal strains;

however, some bacteria, such as Bacillus sp., are also known to produce lignin

peroxidase and laccase [121]. In such bacterial strains, both of these enzymes are actively

involved in degradation of phenols, aromatic amines, diamines, and many other

xenobiotic molecules [118]. Bacterial ligninases are unique in that they can cleave certain

Cα-oxidation and Cβ-Cβ bonds of the lignin structure which are resistant to the fungal

lignin peroxidases [50]. Some bacteria can degrade plant residue by either tunneling into

the interior cell walls or making stripy erosions in the microfibrils of cellulose [122].

Recently, microbial consortia of Bacillus sp. have been shown to have extensive

interactions for lignin degradation [123]. Such ligninolytic microbial consortia may help

in the process of anaerobic digestion to increase biogas production [124].

In this study, microbial populations were collected at various environmental sites,

and individual isolates were selected based on high activity against lignin substrates. The

seven most active isolates were identified as Bacillus sp. strains and were used for rice

straw pretreatments. The treated rice straw was analyzed for compositional and

morphological changes after the pretreatment process. In addition, the effect of

biologically pretreating the rice straw upon biogas production from anaerobic

fermentation was measured.

3.3.1 Isolation and Characterisation of Lignin-Degrading Bacterial

Strains

The current study was focused on isolating ligninolytic microorganisms that could be

used to pretreat agricultural biomass to increase renewable fuel production. Samples

containing mixed microorganisms were collected from three different environments: soil

Chapter 3: Results

73

surface, wood compost, waste sludge, and populations of lignin-degrading microbes were

enriched by repeated culturing in liquid media containing lignin as the sole carbon

source. The enriched cultures were then spread onto solid media plates, and 27 colonies

(12, 7, and 8 from soil, wood compost, and waste sludge samples, respectively) were

isolated. The isolates were screened both in agar and liquid medium for their ability to

decolorize Azure B, a synthetic dye substrate with a structure similar to lignin (Figure 3-

20). Of the 27 specimens, the seven strains (TL4, TL6, TL8, TL24, TL26, TL27, and

TL33) that showed the highest levels of synthetic dye decolorization were chosen for

further analysis. These seven isolates were gram positive with white colonies, jelly

smooth coats, and long rods. In addition, these isolates all tested positive for extracellular

enzymatic activities against cellulase, lipase, protease, amylase, xylanase, and pectinase

substrates.

Lignin

inoculatedLignin

control

Azure B

inoculatedAzure B

control

A B

Figure 3-20 Isolation of bacterial isolates on lignin and selection based on Azure B

decolorization on (A) solid medium and (B) liquid medium.

Genomic DNA was isolated from the 7 strains, and for the identification the 16S

rRNA genes were isolated by PCR, sequenced, and analyzed by BLAST analysis. All the

isolates were identified as different Bacillus sp. displayed 99% identity to the 16S rRNA

gene of Bacillus sp. strain Phylogenetic analysis was done using Mega 6.06 software

(Figure 3-21). The gene sequences were deposited and the sequences are available under

accession no (GenBank accession KY744570-KY744576).

Chapter 3: Results

74

Bacillus mojavensis. strain (KY608825)Bacillus sp. strain TL24 (KY744573)

Bacillus axarquiensis. strain (KY608836)

Bacillus subtilis subsp. Inaquosorum. strain (KT720239)Paenibacillus polymyxa. strain (MF001284)

Bacillus licheniformis (LT599743)

Bacillus tequilensi.s strain (KY910154)Bacillus sp. TL26 (KY744574)

Bacillus sp. strain TL8 (KY744572)Bacillus subtilis. strain L7 (KU179326)

Bacillus licheniformis. strain (KT985636)Bacillus tequilensis. strain (KT982221)

Bacillus subtilis. strain (KJ870191)Bacillus methylotrophicus. strain MER. (KT719451)Bacillus tequilensis. strain (HQ844469)Bacillus amyloliquefaciens. strain (FJ657666)Bacillus pumilus .strain (EF178456)

Bacterium Te22R (AY587809)Bacillus subtilis. strain EJH-1 (DQ402043)

Paenibacillus polymyxa. strain PP17 (MF001284)Bacillus sp. AB242d (FR821122)

Bacillus cereus. strain NCIM (KU218526)Bacillus mojavensis strain HEP6B11 (KY608825)Bacillus siamensis. strain EN23 (KU512904)Bacillus subtilis. strain YS51 (KU551250)

Bacillus subtilis. strain L7 (KU556326)Bacillus amyloliquefaciens. strain AZB42 (KU738862)Bacillus velezensis. strain EC2 (KX242452)Bacillus sp. TL27 (KY744575)

Bacillus sp. TL33 (KY744576)Bacillus velezensis. strain GA3 (KY978399)Bacillus methylotrophicus. (LT223629)

Bacillus amyloliquefaciens strain SB1 (MF171193)Bacillus amyloliquefaciens. strain B39 (KC441735)

Bacillus sp. strain TL4 (KY744570)Bacillus sp. strain TL6 (KY744571)

7130

64

95

73

60

0.01

Figure 3-21 Phylogenetic analysis with related species strains based on 16S rRNA of

the bacterial isolates.

3.3.2 Growth on Lignin and Azure B

The growth potential of the isolates on lignin and Azure B dye at 5th day of incubation

strains TL4, TL6, and TL26 had the highest growth, and strains TL8 and TL24 had the

lowest growth. Overall, there was lower growth and a longer lag phase on the Azure B

substrate compared to lignin (Table 3-6 ).

3.3.3 Decolorization of Lignin and Azure B

When the decolorization potential of the isolates was tested. Bacillus sp. strains TL4 TL6

and Bacillus sp. strains TL26 were of the most prominent strains observed. Strain TL6

exhibited the highest decolorization of 92% of lignin and 97% of Azure B. The details

decolorization after 5 days is shown in (Table 3-6).

Chapter 3: Results

75

The most important types of enzymes that have been implicated in biological lignin

degradation are lignin peroxidase (LiP) and laccase (Lac). The seven Bacillus sp. strains

were analyzed for both the presence of these enzyme activities as well as the optimal

expression time and activity condition. Crude cell filtrate from the culture media of each

strain was collected daily and tested for the two enzyme activities at a variety of

temperatures (30-70ᵒC) and pH (3-7). Table 3-6 shows the highest activity achieved at

the optimum expression and activity condition for each of the two enzyme activities. All

the strains demonstrated LiP and Lac activities with optimal performance at 50˚C. The

optimum LiP expression was observed at 48 h growth, and optimal activity was achieved

at pH 3. The highest LiP activities were 2.99 U/mL, 2.81 U/mL, and 2.74 U/mL for TL6,

TL4, and TL26, respectively. The optimal expression of Lac activity was at 72 h growth,

and the highest activity was seen at pH 5. The strains TL6, TL4, and TL26 had the

highest Lac activities of 2.46 U/mL, 2.39 U/mL, and 2.33 U/mL, respectively. The

activity of lignin peroxidase and laccase was optimum at acidic pH 3-5.

Table 3-6 The highest growth, decolorization potential of lignin and Azure B dye

and enzyme activities at optimum conditions of bacterial isolates.

Isolates Growth

(OD)

Lignin

decolorization

(%)

Azure B dye

decolorization

(%)

Lip (U/mL)

(50˚C,pH 3)

Lac (U/mL)

(50˚C,pH 5)

Bacillus sp. strain TL4 1.8 81.2 91.6 2.81 2.39







3.3.4 Lignin Degradation Efficiency

As illustrated in the current study, the most notable observation was the maximum lignin

degradation efficiencies at pH 5 compared to pH 7. This degradation potential can be

associated to the optimum activity of the LiP and Laccase activities at pH 5 among all the

Bacillus sp. strains. At pH 5, the isolates TL4, TL26, and TL6 displayed the maximum

Chapter 3: Results

76

lignin degradation efficiencies of 79.8, 82.2, and 85.7%, respectively than other Bacillus

sp. strains of the current study at pH 5 (Figure 3-22 A). However, as the concentration of

lignin was increased, the degradation efficiency decreased. At pH 7, all the bacterial

strains had similar degradation efficiencies in contrast to their performance at pH 5;

however, overall the lignin degradation efficiencies were lower at pH 7 compared to pH 5

(Figure 3-22 B). The isolates TL4, TL26, and TL6 showed maximum lignin degradation

efficiencies of 56.1, 57.6, and 57.4%, respectively, at 0.5 g/L at pH 7. The strains all

expressed lignin peroxidase and laccase, which are important enzymes implicated in the

degradation of lignin. Maximum lignin degradation was obtained at 5 days’ growth at pH

5 which coincided with the maximum production of laccase. High lignin degradation

rates by these strains indicate their potential utility in pretreatment of waste biomass

hydrolysis for bioenergy production.

Concentration of Lignin (g/L)

0 10 20 30 40 50

% D

egradati

on

0

10

20

30

40

50

60

70

80

TL-4

TL-6

TL-8

TL-24

TL-26

TL-27

TL-33

BA

Concentration of Lignin (g/L)

0 10 20 30 40 50

% D

egradati

on

0

20

40

60

80

100

TL-4

TL-6

TL-8

TL-24

TL-26

TL-27

TL-33

Figure 3-22 Efficiency of alkali lignin degradation with various substrate

concentrations at pH 5 (A) and pH 7 (B).

3.3.5 Biological Pretreatment of Rice Straw

Rice straw lignin degradation was achieved by either culturing with individual Bacillus

sp. strains or co-culturing with a mixture of the seven Bacillus sp. strains. In the co-

culture pretreatment, a decrease of 53.1% lignin was detected which was 2.5 times more

Chapter 3: Results

77

than that of any of the individual Bacillus sp. strain pretreatments (Figure 3-23). A 22.4%

total dry weight reduction was the maximum value obtained in the case of pretreatment

by the individual culture; however, the co-culture treatment reduced the total dry weight

up to 61.3% from rice straw residue. Notably, the loss of the hemicellulose and cellulose

fractions were relatively low.

Figure 3-23 Effect of pretreatment by individual isolates (TL4, TL6, TL8, TL26,

TL27, TL33) or co-culture on the composition of rice straw, Error bar = standard

deviation.

3.3.6 Scanning Electron Microscopy of Rice Straw

The surface structures of the untreated and pretreated rice straw were compared using

scanning electron microscope (SEM) microscopy (Figure 3-24). The untreated rice straw

had a highly compact, smooth, and homogeneous structure that was degraded after

bacterial culture pretreatments. The highly dense structure was lost, and the surface was

Chapter 3: Results

78

clearly degraded and distorted. The images show broken fibrils and disrupted bundles in

the cell wall complex of each pretreated sample.

3.3.7 Fourier Transformed-Infrared Spectroscopy (FT-IR) Analysis of

Rice Straw

The effect of individual strains treatment on rice straw of all active seven isolates were

assessed compared to untreated rice straw using FT-IR analysis. Commonly in the

lignocellulosic feedstock the band at around 1500–1650 cm-1 and 1200–1300 cm-1 is

occupied by aromatics (lignin), while the band at around 1000–1100 cm-1 and 1800–1900

cm-1 is occupied by the hemicellulose groups and the band at around 1300–1450 cm-1

and 2700–2900 cm-1 is occupied by the cellulose contents [56, 72]. The peak position

between 1000-1700 cm-1 are attributed to lignin and aromatic group of solid agriculture

biomass contents [56, 72]. We observed a mark variation in the absorbance and peak

positions in all the tested treated samples. Particularly a peak position of 1448.8 cm-1

was present in untreated RS and was missing in the pretreated samples. We also observed

emergence of some new peaks 1539.9, 1558.7, and 1596.8 cm-1 in the treated samples.

These variation and changes suggest the severity and action of ligninolytic microbial

system of Bacillus sp. strains is different in each isolate. The disappearance and emerging

of new peak proved a hydrolytic effect and degradation of lignin from rice straw as

shown in (Figure 3-25).

Chapter 3: Results

79

Figure 3-24 Surface morphology of rice straw pretreated with bacterial cultures, (C)

= untreated RS; (TL4, TL6, TL8,TL24,TL26, TL27, and TL33) are treated images

of bacterial cultures.

Chapter 3: Results

80

CTL4

TL6TL26

Tra

nsm

itta

nce %

Tra

nsm

itta

nce %

Wavenumber cm-1 Wavenumber cm-1

Tra

nsm

itta

nce %

Wavenumber cm-1

Wavenumber cm-1

Tra

nsm

itta

nce %

Wavenumber cm-1

TL27

TL24TL8

Tra

nsm

itta

nce %

TL33

Wavenumber cm-1

Tra

nsm

itta

nce %

Wavenumber cm-1

Tra

nsm

itta

nce

%

Tra

nsm

itta

nce %

Wavenumber cm-1

Wavenumber cm-1

TL27

Figure 3-25 FT-IR spectrum of untreated rice straw sample (C) compared to treated

sample of TL4,TL6,TL8,TL24,TL26,TL27, and TL33 isolates.

Chapter 3: Results

81

3.3.8 Biogas and Methane Yield from Pretreated Rice Straw

The biogas potential is often defined as the volume of biogas produced per gram volatile

solid (VS) added for the specific substrate. The accumulated final methane production is

regarded as the methane potential of the particular substrate. In the current study, the

methane (CH4) content of the biogas was 32% for untreated rice straw and 41% and 67%

for substrate pretreated with individual cultures and the co-culture, respectively (Table 3-

7).

The daily biogas volume, daily volumetric methane rate, cumulative biogas, and

cumulative methane yield were measured from the anaerobic fermentation of untreated

rice straw and substrates pretreated with cultures (individual or combined) (Figure 3-26).

The highest daily biogas volume using culture-pretreated rice straw was 28-30 and 60

mL/day from individual isolates (TL-4, TL-6, TL-26) and a co-culture of all seven

Bacillus sp. strains, respectively. The maximum daily volumetric methane rate using

culture-pretreated rice straw was 6-8.5 and 17.3 mL/gVS from individual isolates and a

co-culture of all seven Bacillus sp. strains, respectively.

The cumulative maximum biogas using culture-pretreated rice straw was 270-280

and 528.9 NmL/gVS from individual isolates (TL4, TL6, TL26) and a co-culture of all

seven Bacillus sp. strains, respectively (Figure 3-27).

The minimum cumulative methane yields from rice straw subjected to the various

biological pretreatment are shown in Table 3-7. The minimum cumulative methane yield

calculated through Gompertz (Equation 2) was in the range of 252-254 ml/gVS from

substrates pretreated with individual cultures. However, the minimum cumulative

methane yields were greatly increased (444 mL/gVS) when substrates were pretreated

with a co-culture of all seven Bacillus sp. strains were used.

There was no substantial difference in cumulative methane yield between

untreated rice straw and individual bacterial isolates. However, a significant increase of

76.1% was obtained from cumulative methane yield with co-culture of all seven Bacillus

sp. strains.

Chapter 3: Results

82

Figure 3-26 Daily biogas from rice straw of untreated, pretreated with individual

isolate (TL4, TL6, TL26) and co-culture, (d) = days. Error bar = standard deviation.

Figure 3-27 Cumulative biogas from rice straw of untreated and pretreated with

individual isolate (TL4, TL6, TL26) and co-culture,

(d) = days, Error bar = standard deviation.

Chapter 3: Results

83

Table 3-7 Methane content (%) and cumulative methane yield based on Gompertz

equation obtained from anaerobic fermentation of rice straw pretreated with

individual culture, co-culture compare to untreated rice straw.

Sample CH4 (%) P Rm Delta (Δ) R2

Untreated RS 32 229.9 0.29 112.8 0.96

TL4 42 252.4 0.31 72.5 0.98

TL6 40.5 255.5 0.32 72.5 0.95

TL26 41.2 254.5 0.32 72.5 0.98

Co-culture 66.6 444.4 0.46 40 0.98 P = cumulative methane yield (mL), Rmax= maximum production rate, (Δ) =lag phase duration

(hours).

3.3.9 Conclusion

Seven robust ligninolytic Bacillus sp. strains were collected and identified from

environmental samples. Pretreatment by a co-culture of all seven Bacillus sp. strains

resulted in a significant reduction of lignin from rice straw in comparison to untreated

rice straw residue. This decrease in lignin corresponded to increased fermentation yields

of renewable biogas when using the pretreated rice straw as substrate. Thus, the

application of these Bacillus sp. strains might be a promising strategy for maximizing

biogas yield from waste crop residues.

3.4 Two Separate Biohydrogen and Biomethane Batch

Fermentation by Ligninolytic Bacillus Sp. Strains using

Wheat Straw as a Substrate

In biofuels production from lignocellulosic waste biomass (LWB), the removal of lignin

from these substrates is a major impediment and rate limiting reaction. Therefore,

production of any type of biofuel depends on the efficiency of the pretreatment method.

The LWB, is an important feedstock because of its abundance and it does not compete

with food source [125, 126]. However, generally these waste biomasses are burnt in the

field and leading to the disappearance of a key source. The LWB contains cellulose,

hemicellulose, and lignin as main constituents. The hemicellulose and cellulose of the

LWB can be used as substrate for biofuels production, however lignin inhibits biomass

hydrolysis [127]. Unfortunately, to convert LWB into biofuel, pretreatment is required to

Chapter 3: Results

84

improve digestibility of biomass prior anaerobic digestion [128, 129]. For the lignin

removal chemical treatments are also tested [28, 130]. However, the chemical treatments

are not suitable for large-scale application due to carbohydrates degradation, production

of toxic products and increasing the cost of anaerobic fermentation process [131, 132].

An alternative to chemical treatment for lignin removal, use of natural

microorganism for biodelignification could be a promising strategy and a suitable

alternative to chemical treatment [133]. The hydrolysis of LWB biomass by

microorganisms can consolidate also to the anaerobic fermentation process [134]. But the

modification of anaerobic fermentation process from single phase anaerobic digestion to

the two-phase anaerobic digestion process has several advantages [135]. In the two-phase

anaerobic fermentation, hydrolysis and acidogenesis can be conducted separately then

acetogenesis and methanogenesis process in separate bioreactors. The significance of

separating single anaerobic fermentation process to two-phase is that the biomass is

degraded into simple carbohydrates polymer producing H2, CO2, organic acid in the first

phase as intermediate metabolites followed by rapid fermentation into CH4 [136].Thus,

two kinds of biogas fuels H2 and CH4 biogas can be produced [15, 137]. The separation

of the anaerobic fermentation process is important because microorganisms of each phase

has significant changes in terms of physiology, pH sensitivity, nutritional needs, and

sensitivity to other environmental conditions. Hence in comparison to single stage, the

two-stage anaerobic fermentation is considered more practicable for H2 and CH4

production [138, 139].

Therefore, the current study is focused to isolate indigenous ligninolytic bacterial

strains as a biocatalyst for lignin degradation bypassing the needs of chemical treatment.

The inherit ability of the indigenous ligninolytic microorganism to degrade lignin

network through the hydrolytic enzymes can be used for lignin degradation of LWB. In

addition, the biohydrogen potential of the lignin degrading bacteria was explored.

Microbial communities both fungal and bacterial strains are reported for lignin

degradation and production of hydrolytic enzymes i.e laccase, lignin peroxidase,

manganese peroxidase, cellulase, and hemicellulases. But the fungal treatment needs

longer incubation time for biomass hydrolysis and not often increases the final energy

Chapter 3: Results

85

yield [140]. In contrast, to the fungal treatment, bacterial pretreatment is more attractive

as bacterial species have rapid growth and can degrade lignin efficiently [122]. Bacterial

strains are known with hydrolytic enzymes [50] however, the Bacillus sp. strains have

been widely reported for lignin degradation and production of ligninolytic enzymes [141,

142]. It is also notable, that Bacillus sp. strains are capable of growing in aerobic and

anaerobic conditions effectively and degrade aromatics compounds [83].

Although, the lignin degradation potential and production of extracellular

enzymes from ligninolytic bacterial strains are reported, but, none of the studies

identified biohydrogen potential of indigenous ligninolytic bacterial strains. Therefore,

the current study was aimed to isolate and select the most active ligninolytic bacterial

strains to assess in hydrolysis of wheat straw as a substrate. Also, the anaerobic batch

fermentation process was modified to tow-phase batch fermentation experiment to

evaluate the potential of biohydrogen (H2) production of ligninolytic bacterial strains.

The first batch fermentation was followed by the second batch fermentation for (CH4)

production from the biotreated wheat straw samples of first batch fermentation.

3.4.1 Selection of Ligninolytic Bacterial Strains

Twenty bacterial strains were isolated from the granular sludge using culture enrichment

technique. The mixed microorganisms culture from granular sludge of full-scale

anaerobic digester was screened in MSM media supplemented with alkali lignin for

lignin degrading bacterial strain. The culturing process was repeated to enrich the desired

microorganisms from the sample source. A preliminary estimation of Azure-B dye

decolorization was used as criteria in the selection of strains for further investigations. Of

the 20 isolates, the four: 4, 9, 13 and 18 strains were named as AN-1, AN-2, AN-3, and

AN-4 showed best performance. A visible decolorization of Azure B dye was obtained

after 4 days of incubation as shown in (Figure 3-28). Among the four strains the AN-2

and AN-3 showed the highest level of 87 and 96.9 % Azure B dye decolorization (Table

3-8).

The percent decrease of COD after 7 days is shown in Table 3-8. Bacillus

altitudinis AN-2 showed the maximum reduction 84.5% of lignin and 76.3% of Azure B.

Chapter 3: Results

86

Whereas, Brevibacillus agri AN-3 exhibited the highest decrease COD of 88.4% of lignin

and 78.1% of Azure B. In a batch assay of 7 days, a remarkable visible bacterial growth

was observed during the first 4 days of incubation exhibiting a significant color reduction

of both lignin and Azure B dye reaching a maximum level after the 7th day. Microscopic

study revealed that all the four isolates were rod and gram positive bacteria.

A B

Figure 3-28 Dye degradation potential of isolates, Azure B dye (A) and Lignin (B) in

evaluation with control and inoculated conditions respectively.

Table 3-8 Decolorization potential of lignin and Azure B of the selected bacterial

strains.

Strains Azure-B

dye decolorization (%)

Lignin

COD reduction

(%)

Azure B dye

COD reduction (%)

Bacillus tequilensis AN-1 67.6 82.6 70.2

Bacillus altitudinis AN-2 83.1 84.5 76.3

Brevibacillus agri AN-3 90.9 88.4 78.1

Bacillus pumilus AN-4 66.5 76.5 64.5

Chapter 3: Results

87

3.4.2 Identification of Ligninolytic Bacterial Strains

Genomic DNA was isolated from these strains, and the 16S rRNA genes (Gene Bank

accession MG736065-MG736068) were isolated by PCR, sequenced, and analyzed by

BLAST analysis. All the isolates displayed 99% identity to the 16S rRNA gene and were

identified as Bacillus tequilensis AN-1, Bacillus altitudinis AN-2, Brevibacillus agri AN-

3, and Bacillus pumilus AN-4 (Figure 3-29). The evolutionary history was created using

the Neighbor-Joining method. The tree was drawn to scale with branch lengths in the

same units as those of the evolutionary distances used to infer the phylogenetic tree. The

evolutionary distances were computed using the Maximum Composite Likelihood

method. The analysis was conducted in MEGA 6.06 for a total of 45 nucleotide

sequences.

Chapter 3: Results

88

Bacillus stratosphericus strain (MG561355.1)Bacillus altitudinis strain (MG645240.1)Bacillus sp strain C1 (MG461669.1)Bacillus aerophilus strain (KY038792.1)Bacillus pumilus strain (KX832897.1)Bacillus subtilis strain BS05 (KX783558.1)Bacillus stratosphericus strain (KY886238.1)Bacillus xiamenensis strain M28 (KY595443.1)

Bacillus altitudinis AN-2 (MG736066)

Bacillus sp AECSB02 (AB748939.1)Bacillus pumilus strain (KM226929.1)

Bacillus sp strain JDMASP34 (KX817950.1)

Bacillus sp strain (MF996674.1)Bacillus safensis strain (MF078473.1)Bacillus methylotrophicus strain (KR140184.1)Bacillus cereus strain UBT4 (KR709243.1)Bacillus sp (LN849695.1)

Bacillus pumilus AN-4 (MG736068)Bacillus pumilus strain IPB (KM357837.1)

Bacillus tequilensis AN-1 (MG736065)Paenibacillus polymyxa strain (MF185647.1)Bacillus subtilis strain (KX708698.1)Bacillus velezensis strain X-14 (MF988731.1)Bacillus sp strain LCSB-11 (MG062754.1)Bacillus licheniformis strain SJ32 (MG396981.1)Bacillus vallismortis strain CA6B (MG397007.1)Bacillus subtilis strain KBL40 (MG576193.1)Bacillus tequilensis strain CICR-H3 (MG645241.1)

Bacillus sp strain FJAT (KY949518.1)

Paenibacillus polymyxa strain PP17 (MF001284.1)Brevibacillus sp Bac165R (KP795828.1)Brevibacillus sp enrichment culture (JQ956516.1)

Brevibacillus agri AN-3 (MG736067)Aneurinibacillus aneurinilyticus (JN663805.1)Brevibacillus sp DMS (KR709235.1)Bacillus sp SH22 (EU374144.1)Brevibacillus agri (HE993879.1)Streptomyces sp (AB731746.1)Brevibacillus sp AMBR2 (KM403208.1)Brevibacillus agri strain EB54 (KC352740.1)

Uncultured bacterium clone (KY962971.1)45

96

62

100

100

65

99

100

98

0.005

Figure 3-29 Evolutionary relationships and phylogenetic tree with related species

strains based on 16S rRNA of the bacterial isolates.

3.4.3 Evaluation for Hydrogen Production from Cellulose and Xylose

The ligninolytic abilities, the decolorization of Azure B dye, and the presence of different

extracellular enzymatic activities were considered promising capabilities of the four

isolates. Once the phylogenetic analysis confirmed it a distinct group of Bacillus species,

and the hydrolytic potential of these strains developed an interest to screen them for H2

production from complex substrates.

Chapter 3: Results

89

As per composition, wheat straw contains cellulose and hemicellulose. Therefore,

the individual culture of Bacillus tequilensis AN-1, Bacillus altitudinis AN-2,

Brevibacillus agri AN-3, and Bacillus pumilus AN-4 were first evaluated for H2 potential

were provided with cellulose and xylose as a feeding material. The strains produced H2

with different yields from xylose and cellulose. The hydrogen concentration was

observed after 72 hours of incubation. H2 yield was comparatively higher from cellulose

than that of xylose for all the tested strains. A lower H2 yield of 0.8 and high H2 yield of

1.34 mol-H2/mol of consumed xylose was obtained from Bacillus pumilus AN-4 and

Brevibacillus agri AN-3 respectively. The potential of hydrogen production from xylose

is important, because the composition of hemicellulose constitutes 20-30% xylose which

is a pentose sugar of the wheat straw and other agribiomass.

Whereas, the H2 yield of Bacillus tequilensis AN-1 and Bacillus pumilus AN-4

was 1.75 and 1.41 mol-H2/mol of cellulose used respectively. Similarly, the H2 yield of

Bacillus altitudinis AN-2 and Brevibacillus agri AN-3 was 2.78 and 2.9 mol-H2/mol of

cellulose respectively. In comparisons of H2 conversion potential of the strains, higher H2

yield was shown by Bacillus altitudinis AN-2 and Brevibacillus agri AN-3 from both

cellulose and xylose. Similarly, the H2 volumetric rate production was similar from

xylose and cellulose. The highest H2 volumetric rate was 50 mL H2/d from Brevibacillus

agri AN-3 and 40.5 mL H2/d from Bacillus altitudinis AN-2. The other two strains

Bacillus tequilensis AN-1 and Bacillus pumilus AN-4 produced H2 volumetric rate of

35.4 and 32.9 mL H2/d respectively.

The (Figure 3-30 A) depicts the cumulative H2 production from xylose. The

maximum cumulative H2 of 64 and 68 mL was obtained from Bacillus altitudinis AN-2

and Brevibacillus agri AN-3 respectively. Correspondingly, in (Figure 3-30 B)

cumulative H2 production from cellulose is shown. The highest cumulative H2 of 160 and

147.5 mL was obtained by Brevibacillus agri AN-3 and Bacillus altitudinis AN-2 at the

end of fermentation batch assay. However, a lower cumulative H2 of 130.5 and 120.2 mL

was observed by Bacillus tequilensis AN-1 and Bacillus pumilus AN-4 at the end of

fermentation batch assay. From the xylose and cellulose fermentation assay, no methane

Chapter 3: Results

90

gas was detected in the whole fermentation experiment. The relative H2 concentrations

were 45-55% for the tested Bacillus sp. strains.

Figure 3-30 Cumulative hydrogen productions from xylose (A) and cellulose (B),

AN-1= Bacillus tequilensis , AN-2 = Bacillus altitudinis, AN-3= Brevibacillus agri =

AN-4 grew in MSM supplemented with 10 g/L of xylose and cellulose respectively.

3.4.4 Hydrogen Production from Wheat Straw

In the current study, biopretreatment of wheat straw was started with the selected

ligninolytic Bacillus sp. strains to explore H2 potential. As the strains were capable of

hydrogen production from the xylose and cellulose, therefore, wheat straw was batch

incubated with individual anaerobic Bacillus sp. strains for 25 days’ retention time. The

wheat straw was composed of 42.5, 27.11 and 22.51 % cellulose, hemicellulose and

lignin respectively. The total solid (TS) and volatile solid (VS) of wheat straw was 92.5

and 85.5% respectively. In batch fermentation experiment on wheat straw, initially, a 4-5

days of lag phase was observed. At the end of batch assay, Bacillus altitudinis AN-2 and

Brevibacillus agri AN-3 were found to be the most efficient hydrolyzing strains, leading

to the highest amount of 81.1 and 88.3 mL/gVS of H2 yield (Figure 3-31). Similar results

with lower cumulative hydrogen yield were obtained for the Bacillus tequilensis AN-1

and Bacillus pumilus AN-4 (Figure 3-31). The concentration of H2 reached to 35-45 % after

14 days of batch fermentation for all the tested isolates. Moreover, the results of wheat straw

Chapter 3: Results

91

batch fermentation for H2 yield were lower than the H2 yield obtained from the xylose and

cellulose for all the tested strains. The range of H2 yield was 0.6-1.1 mol-H2/g of wheat straw.

Individually, the H2 yield of Bacillus tequilensis AN-1 and Bacillus pumilus AN-4 was 0.61 and

0.75 mol-H2/g of wheat straw respectively. Similarly, the H2 yield of Bacillus altitudinis AN-2

and Brevibacillus agri AN-3 was 0.9 and 1.1 mol-H2/g of wheat straw respectively.


0 5 10 15 20 25 30

Cu

mu

lati

ve

H2 y

ield

(m

L/g

VS

)

0

20

40

60

80

100

AN-1

AN-2

AN-3

AN-4

Figure 3-31. Cumulative H2 productions from wheat straw in first batch

fermentation assay, AN-1= Bacillus tequilensis , AN-2 = Bacillus altitudinis, AN-3=

Brevibacillus agri = AN-4.

Generally, H2 yield is related to the production level of volatile fatty acids (VFA)

during fermentation system. The concentration and the quantity of the produced VFAs

show performance of batch assay and illustrate the H2 production pathways. The high

VFAs concentrations were observed at the end of bio-pretreatment of the wheat straw

batch assay, suggesting the optimum activity and the growth of all the tested strains

(Table 3-9). Among the list of tested VFA standard, only 4 kinds of organic metabolites

Chapter 3: Results

92

namely i.e. acetate, butyrate, iso-butyrate, and propionate were produced. In all batch

experiments, the butyrate was the major metabolite followed by acetate.

Table 3-9 Volatile fatty acid (VFA) production during batch fermentation assay of

H2 from wheat straw, data is mean value of replicates.

Strains Aa

mg/L Pa mg/L Ba mg/L

Ia

mg/L

TVFA

mg/L

Bacillus tequilensis AN-1 723±45 547±28 1523 ±54 337±21 3130

Bacillus altitudinis AN-2 588±28 444±18 2045± 48 409±14 3486

Brevibacillus agri AN-3 566±24 550±25 2223± 30 345±18 3684

Bacillus pumilus AN-4 677±30 491±19 1487 ± 45 289±15 2944

Acetic acid,Pa= Propionic acid,Ba= Butyric acid, Ia= Iso-butyric, TVFA, total volatile fatty acid

3.4.5 Biogas Potential from the Bio-Pretreated Fermented Wheat Straw

The batch assay for biomethane was initiated from the finished batch fermentation assay

conducted for hydrogen production as a second phase of the anaerobic digestion process.

A 20 mL of culture supernatant was drained off from the serum bottles and stored at -4°C

for VFA analysis, and 20 mL of inoculum sludge from anaerobic digester with TS (4.5%)

and 62.5% VS characteristics was added. The biogas potential was calculated as the

volume of biogas produced per gram of the wheat straw added. In the current study, the

methane (CH4) content of the biogas was 30% for untreated wheat straw and 50-60% for

all the biotreated wheat straw sample. When the second batch of anaerobic digestion was

started, no latency phase was observed. The highest daily biogas volume of 41 and 56.4

mL/d was obtained after 24 h of the batch assay from the Bacillus altitudinis AN-2 and

Brevibacillus agri AN-3 respectively. Similarly, the maximum daily biogas volume of 35

and 22.6 mL/d was obtained after 24 h of the batch assay from the Bacillus tequilensis

AN-1 and Bacillus pumilus AN-4 respectively. The daily methane yield was 6.08, 9.2,

9.7, and 5.2 mL/d from AN-1- AN-4 Bacillus sp. strains respectively. However, the daily

methane yield was 1.2 mL/d from the untreated wheat straw batch sample.

Chapter 3: Results

93

The cumulative biogas was 105 NmL/gVS from the untreated wheat straw batch

fermented sample. Whereas a 70.8% maximum cumulative biogas of 360 NmL/gVS was

obtained from Brevibacillus agri AN-3 followed by 335.3 NmL/gVS, (68.6%) more

cumulative biogas for Bacillus altitudinis AN-2. However, the cumulative biogas was

305 and 282.6 NmL/gVS for Bacillus tequilensis AN-1 and Bacillus pumilus AN-4

respectively (Figure 3-32). Overall, the biotreated wheat straw samples exhibited 63-71%

increase in biogas yield than the untreated wheat straw sample.


0 5 10 15 20 25 30

Cum

ula

tive B

iogas

(Nm

L/g

VS

)

0

100

200

300

400

Sludge

Untreated WS

AN-1

AN-2

AN-3

AN-4

Figure 3-32 Cumulative biogas from wheat straw in second batch, sludge, untreated

WS , AN-1= Bacillus tequilensis , AN-2 = Bacillus altitudinis, AN-3= Brevibacillus

agri = AN-4.

Chapter 3: Results

94

3.4.6 Application and Advantages of Two-phase Batch Fermentation

In Table 3-10, cumulative methane is calculated using online biogas packages tools OBA

(https://biotransformers.shinyapps.io/oba1/). In addition to that, the Gompertz equation

was also used for hydrogen yield calculated manually through Excel spreadsheets of

Microsoft 2016. In this study, the highest and the best performance in the term of total

yield was shown by Bacillus altitudinis AN-2 and Brevibacillus agri AN-3. Bacillus

altitudinis AN-2 exhibited 190.7 NmL/gVS and Brevibacillus agri AN-3 shown 198

NmL/gVS combine H2 and CH4 yield from wheat straw in two-stage batch fermentation

experiment. The untreated wheat straw showed 64.5 NmL/gVS CH4 yield. These results

are important because only few studies reported the production of biohydrogen from a

pure bacterial culture using simple sugar as a substrate. In comparison to reported

literature, the efficiency of ligninolytic Bacillus sp. strains for biohydrogen using wheat

straw as a substrate is more interesting. Numerous studies have been conducted and

reported on the single stage batch fermentation process either for biohydrogen or

biomethane production from wheat straw as shown in Table 3-10. The earlier reported

studies practiced mixed cultures and single batch fermentation either for hydrogen or

methane yield. According to the Table 3-10, the H2 yield from wheat straw is very low

when no pretreatment is performed. Although the H2 yields were improved when wheat

straw was treated either with acid, alkali or biological treatment. However, the H2 yields

observed in this study are higher than the H2 yield from acid and Ca(OH)2 treated wheat

straw as reported [143, 144], even though they used mix culture of the sludge. The

hydrogen yield of C.Saccharolyticus was comparable to the hydrogen yield of Bacillus

pumilus AN-4 and Bacillus tequilensis AN-1 of the present study, however, the wheat

straw hydrolysate was prepared by heating up to 130 °C for 30 min [145]. The maximum

hydrogen yield of 1.67-1.92 mol/mol glucose is reported from different Bacillus sp.

strains [146] which is almost equivalent to the hydrogen yield from xylose but lower than

the hydrogen yield from the cellulose of this study. These results proved that ligninolytic

Bacillus sp. strains can bypass the needs of costly chemical pretreatment of agriculture

waste biomass.

https://biotransformers.shinyapps.io/oba1/

Chapter 3: Results

95

In the current study, from the two-stage anaerobic batch fermentation of wheat

straw a 48.6% more methane yield was obtained as compared to untreated wheat straw

batch sample.

Table 3-10 Comparison of the biohydrogen and methane yield from wheat straw as

substrate with other pretreatment conditions.

Pretreatment H2 yield

(mol/mol sub)

Cumulative CH4

(mL/gVS)

Cumulative H2 (mL/gVS)

Inoculum Ref

No treatment - - 6.4 Mix culture [143]

2% H2SO4 - - 41.9 Mix culture of seed sludge [143]

No treatment - - 1 Cow dung [147]

HCl - - 68.1 Cow dung [147]

7.4% Ca(OH)2 - - 58.78 Granular sludge [144]

Acid steam - 280 - Mix culture [148]

NaOH - 165.9 - Mix culture [149]

130 °C, 30 min - - 44.7 C.Saccharolyticus [145]

180 °C, 15 min - 297 178 Mix culture [150]

Biotreated - 214 - Trichoderma reesei [151]

Biotreated - - 178 Thermophilic mix culture [152]

- 1.67 a - 215 B. thuringiensis EGU45 [152]

- 1.92 a - 240 B. cereus EGU44 [152]

- 0.96 a - 125 Bacillus cereus EGU3 [152]

- 1.12 a - 145 Bacillus cereus 43 [152]

Biotreated 0.9 b,1.75 c 108.6 39.5 Bacillus tequilensis AN-1 This study

// 1,2 b.78 c 121.3 69.4 Bacillus altitudinis AN-2 This study

// 1.34 b,2.9 c 125.5 72.5 Brevibacillus agri AN-3 This study

// 0.8 b,1.41 c 104.4 44.5 Bacillus pumilus AN-4 This study

Untreated - 105 --- Digester sludge This study

a= mol/mol glucose, b= xylose, c= cellulose

3.4.7 Conclusion

In the present study, four Bacillus sp. strains that produce lignin-degrading enzymes were

selected. Bacillus sp. strains shown a maximum of 78.1 and 88.4% COD reduction of

Chapter 3: Results

96

lignin and Azure B dye. All the strains expressed lignin peroxidase, laccase, xylanase,

and cellulase which are important enzymes implicated in the degradation of

lignocellulosic biomass. In addition, these strains exhibited H2 potential from xylose,

cellulose and wheat straw. The isolates exhibited 48.6% greater biomethane yield when

used in the anaerobic fermentation of wheat straw. Thus, the application of these

ligninolytic Bacillus sp. strains represents a promising approach for maximizing biogas

yield from waste agribiomass. Further, optimization of culture conditions will help in

developing a ligninolytic Bacillus sp. strains inoculum for hydrogen production.

Assessment of these strains can play a vital role as a biotreatment agent for effective

anaerobic technology development.

3.5 Pretreatment of Wheat Straw using a Recombinant

Neurospora crassa Strain for Improved Biogas Production

The expectable shortage of fossil fuels is bringing main structural changes in the

worldwide economy. The oil, gas, and coal reserve are depleting continuously [153].

Beside this, other major concern is increasing of world population and consumption rate

of energy [154]. Collectively, these complications generate a substantial challenge around

the globe. To address the challenge, novel industrial bioprocesses are essential to explore

renewable energy production from sustainable resources. This challenge needs to be

accomplished before a shortage incites social, economic, and global conflicts. Waste

lignocellulosic biomasses are a low-cost source for renewable biofuels production and

can play a critical role in overcoming the demand for energy production in future [155].

About 60-70% of lignocellulosic biomass (LB) is waste residue, this huge amount of

waste biomass can be an economical source for LB biogas production. The LB residue

composed of 35-50% cellulose, 20-35% hemicellulose, and 10-25% lignin [43]. The

hemicellulose and cellulose are used easily as a carbon source, however, lignin resistance

is the rate-limiting step in biomass hydrolysis [43, 156, 157]. To convert LB biomass to

its monomeric components, a pretreatment process that is efficient in hydrolysis of LB to

a set of desired components is a scientific challenge [158]. Usually, chemicals

pretreatment are used to remove lignin from LB biomasses, the free and swollen residue

(cellulose) are hydrolyzed with enzymes to convert it into glucose [159]. The glucose

Chapter 3: Results

97

monomers are then converted into preferred bioenergy. However, chemical pretreatments

methods are expensive [160] and generate a lot of toxic by-products that inhibit

downstream biological conversions [119].

The primary objective must be to overcome the pretreatment cost and make

cellulosic biorefineries sustainable [3,4,5]. Biological pretreatment with lignocellulolytic

fungi has significant potential of direct enzymatic hydrolysis. Fungal treatment is simple,

mild reaction, no need for extra energy, and is environmentally safe [4,6]. Several,

studies reported different fungal strains such as white and brown fungi or cellulolytic

bacteria for delignification of LB to enhance its digestibility, White-rot fungi and other

strains are reported with agriculture straw degradation [6,7,8,9]. The fungal strains

breakdown waste biomass with three major enzymes, laccase, manganese peroxidase and

lignin peroxidase. They oxidized lignin and similar aromatic compounds [161]. However,

some fungi strains do not produce all of these enzymes, they either produce one or two of

these enzymes [162]. A number of studies reported production of hydrolytic and

cellulolytic enzymes (cellulases, cellobiohydrolase, and endoglucanase) from fungal

culture on agricultural biomasses [37]. The single enzyme pretreatment is not efficient in

term of pretreatment and prolonged the pretreatment time, however, the purified enzymes

cocktails can speed up the hydrolysis but the treatment becomes expensive and

uneconomic [38]. Similarly, the fungal culture takes weeks to months, this further cause

possible contamination in the culture and consumed some of the holocellulose [37].

Consolidating bioprocessing (CBP) is a new approach to use microbial hydrolysis,

enzyme production and fermentation simultaneously for the deconstruction of different

agriculture feedstock [39].

In the present study, a recombinant Neurospora crassa F5 strains was used for

wheat straw hydrolysis. The N. crassa F5 was a Knocking out strains of six of the seven

bgl genes. The carbon catabolite repression (CCR) (cre-1 gene), and a cellulase repressor

(ace-1 gene) was knock out to improve cellulase production [163]. This recombinant N.

crassa F5 strain and dilute alkali (NaOH) treatment combination was used for wheat

straw hydrolysis. A short hydrolytic and saccharification pretreatment time was used. The

N. crassa F5 saccharified wheat straw samples were consolidated directly to the

Chapter 3: Results

98

anaerobic digestion process. The Automatic Methane Potential Test System (AMPTS-II)

was used for biogas potential of treated and untreated wheat straw samples.

3.5.1 Chemical Composition of Wheat Straw

Chemical and biological pretreatment induced changes in the physical characteristics,

chemical structures and chemical compositions of WS. The WS constitute 43.45, 26.5,

and 23.1 % cellulose, hemicellulose, and lignin respectively. The total solid (TS), volatile

solid (VS), and moister content were 94.4, 87.3 and 5.59% respectively as shown in

(Table 3-11).

Table 3-11 Wheat straw composition

Parameters value

Total solid (TS %) 94.40 ±1

Volatile solid (VS %) 87.30 ±1

Moister (%) 5.59 ±0.5

Ash (%) 8.56 ±0.5

Cellulose 43.45 ±3

Hemicellulose 26.5 ±2

Acid soluble Lignin 23.1 ±1

Acid insoluble lignin 7.7 ±1

3.5.2 Cellulase Production of N. crassa F5 and Hydrolysis of Wheat

Straw

This study combined the process of direct hydrolysis and fermentation of straw coupled

with anaerobic digestion. N. crassa F5 was screened for cellulase production before BMP

process of WS. N. crassa F5 was showing maximum enzyme production with 0.5 g/L and

3 g/L glucose added to NaOH treated WS Vogel media. However, a lower enzyme

production was obtained with 0.5 g/L and 3 g/L glucose added to untreated WS in Vogel

media. The cellulase activity was 1.35 units/mL at day 5 for 0.5 g/L glucose added to

NaOH treated WS Vogel media. Similarly, the cellulase activity was 1.17 units/mL at day

Chapter 3: Results

99

5 for 3g/L glucose added to NaOH treated WS Vogel media. The cellulase activities were

not detected from untreated WS in Vogel media supplemented with 0.5 g/L and 3 g/L

glucose as shown in the (Figure 3-33). The results suggested that 0.5 g/L glucose and 2%

NaOH diligninified wheat straw induced production of cellulase.

Figure 3-33 Cellulase production of N. crassa F5 on wheat straw

3+2%NaOH-WS= 3g/L glucose added to N. crassa F5 and NaOH treated wheat straw

sample,

0.5+2%NaOH-WS = 0.5g/L glucose added, N. crassa F5 and NaOH treated wheat straw

sample,

3F5-WS= 3g/L glucose added, N. crassa F5 and NaOH treated wheat straw sample,

3F5-WS= 3g/L glucose added, N. crassa F5 only treated wheat straw sample,

0.5F5-WS= 0.5g/L glucose added, N. crassa F5 only treated wheat straw sample.

3.5.3 Scanning Electron Microscopy (SEM) for Analysis of Wheat

Straw Surface Degradation

The surface structures of the untreated WS, distilled water autoclaved WS, 0.5 g/L

glucose added N. crassa F5 pretreated WS and 3 g/L glucose added N. crassa F5

pretreated WS, and 2% NaOH pretreated WS samples were compared by analyzing

scanning electron microscope (SEM) micrographs. The untreated wheat straw was highly

compact with a clear and smooth structure. After alkaline pre-treatment, and fungal

hydrolysis the dense structure of wheat straw was destructed. The degradation can be

seen on the WS surface. The SEM micrographs (Figure 3-34) verified the effect of pre-

Chapter 3: Results

100

treatment, suggesting the lignin and hemicellulose component might have been removed

from the WS. The degradation effect was punitive in case of 2% NaOH pretreated WS

samples and N. crassa F5 pretreated WS than other treatment conditions. The images

clearly showed ruptures in the silicon waxy structure, surface destruction and

morphologies changes of the wheat straw as results of fungal hydrolysis further

suggesting the cellulase production and facilitation of fermentation process.

D-H20 WS

0.5-F5 WS

Untreated WS

3-F5 WS 2%NaOH WS

Figure 3-34 SEM images of wheat straw after hydrolysis

(A) was untreated WS, (B) was distilled water autoclaved WS, (C) was 0.5g/L glucose

added N. crassa F5 pretreated WS,(D) was 3g/L glucose added N. crassa F5 pretreated

WS, (E) was 2% NaOH pretreated WS.

3.5.4 Fourier Transformed-Infrared Spectroscopy (FT-IR) for Wheat

Straw Fiber

FT-IR was used to study the change in peak position as a result of pre-treatment. In FT-

IR spectroscopy the bands and peaks represent a particular group of molecules in the

feedstock. Commonly in lignocellulosic biomass, the band in the range of 1500–1650 cm-

1 and 1200–1300 cm-1 is occupied by aromatics (lignin), while the band in the range of

Chapter 3: Results

101

1000–1100 cm-1 and 1800–1900 cm-1 is occupied by the hemicellulose groups, and the

band in the range of 1300–1450 cm-1 and 2700–3100 cm-1 is occupied by the cellulose

contents [56]. The FTIR spectra for untreated WS, distilled water autoclaved WS, was

same and no major changes in the peaks and bands were observed. A significant change

was observed in the 2% NaOH treated WS band between 1400-1600 cm-1. The band

1420.4 cm-1 was present in untreated WS and disappeared from 2% NaOH treated WS.

This band corresponds to aromatic rings (lignin) of wheat straw. In the N. crassa F5

pretreated WS a strong intensities differences were generated between 1100-1200 cm-1,

interestingly a significant change in peak intensities between 2800-3340.1 cm-1 was

observed. A peak intensity at 3330.1 cm- expressed hydroxyl group (–OH) representing

(cellulose) shifting and stretching in N. crassa F5 pretreated WS as shown in (Figure 3-

35). Generally, FT-IR analysis confirmed partial changes in lignin and hemicellulose

component of wheat straw samples.

XRD profiles of wheat straw fibers before and after N. crassa F5 pretreatment

were examined for the crystallinity of each sample. Based on the XRD results, the N.

crassa F5 pretreated WS samples decreased the crystallinity of the WS to 42-45%.

Simialrly, the crystallinity of 2%NaOH reduced up to 49.3, however, the D-H20

autoclave and untreated WS sample crystallinity was closed to each other 54.0, and

55.5% respectively as shown in Table 3-12. There was no major difference in term of

crystallinity, however, interestingly a significant variation in peak intensities between the

N. crassa F5 pretreated WS and untreated sample were observed as shown in (Figure 3-

36). After the fungal treatment, the cellulose is easy to convert into free sugar, become

more accessible and hydrolysable. The SEM, FT-IR, and XRD results revealed visible

changes and degradation on the amorphous structure of WS after N. crassa F5 pretreated

WS as compared to untreated WS sample.

Chapter 3: Results

102

Untreated WS

D-H2O autoclaved WS

2% NaOH treated WS

0.5 FN treated WS

3 FN treated WS

Ab

so

rbance

Wavelength (nm)

Figure 3-35 FT-IR spectra of wheat straw after hydrolysis, untreated wheat straw

(black), D-H20 autoclaved WS= distill water autoclave WS (red), 2% NaOH treated

WS (blue), 0.5FN= 0.5g/L glucose added N. crassa F5 treated WS (light yellow),

3FN=3g/L glucose added N. crassa F5 treated WS (dark yellow).

Table 3-12 Crystallinity of untreated wheat straw and treated wheat straw

Samples Crystallinity (%)

Untreated WS 55.5

D-H2O WS 54.0

2%NaOH treated WS 49.3

0.5-F5 treated WS 42.2

3-F5 treated WS 45.8

Chapter 3: Results

103

10 15 20 25 30 35

Inte

nsity

(Arb

itra

ry U

nits)

2-theta (degrees)

XRD Overlay View

3-F5WS

Untreated WS

2% NaOH WS

D-H20 WS

0.5 F5WS

Figure 3-36 XRD analysis of wheat straw after hydrolysis, untreated wheat straw

(red), distill water autoclave WS (brown red), 2% NaOH treated WS (blue),

O.5F5WS= 0.5g/L glucose added N. crassa F5 treated WS (light pink), 3F5WS=

3g/L glucose added N. crassa F5 treated WS (black).

3.5.5 N. crassa F5 Pretreatment Improved Biogas Production

The aerobic recombinant N. crassa F5 consolidated hydrolyzed wheat straw was

subjected directly to anaerobic digestion to evaluate biogas potential. In the (Figure 3-

37), the untreated WS bottle supplemented with 3 g/L glucose produced more biogas

compare to untreated WS bottle supplemented with 0.5 g/L glucose after the hydrolytic

retention time of 14 days. The 2 days treated N. crassa F5 WS bottle supplemented with

3 g/L glucose produced less biogas than the 2 days treated N. crassa F5 WS bottle

supplemented with 0.5 g/L glucose. The 4 days treatment and 6 days treated WS

supplemented with either 0.5 or 3 g/L glucose did not improve biogas production as

compared to 2 days treatment and untreated as shown in the (Figure 3-37). The starting

methane (CH4) concentration of biogas was 15.5% and reached up to 40.2% for un-

treated WS. However, the starting methane (CH4) concentration of biogas was 32.5% and

reached up to 70-71% after the hydrolytic retention time of 14 days for N. crassa F5 WS.

Chapter 3: Results

104

In the (Figure 3-37) results, it was concluded that 2 days N. crassa F5 treatment

and an addition of 0.5g/L glucose to WS bottle is optimum for biogas production

compared to 4 and 6 days treatment supplemented either 0.5 or 3 g/L glucose.

In the (Figure 3-38) 0.5 g/L glucose, 2 days treatment, and 2% NaOH treatment

was used as an optimum condition of the previous experiment. A considerably high 740.8

mL/gVS volume of biogas was obtained from 2% NaOH + 2 days N. crassa F5

saccharified WS bottle after the hydrolytic retention time of 25 days. Correspondingly,

maximum daily volumetric biogas of 130-150 mL was obtained from 2% NaOH+ N.

crassa F5 treated WS bottle, this was 6-fold higher than the untreated WS bottle. Further,

the 2% NaOH + N. crassa F5 treated WS sample minimized the lag phase of AD process.

However, in case 2% NaOH pretreated WS bottle a 635.8 mL/gVS volume of biogas was

produced. A maximum daily volumetric biogas of 70-80 mL was obtained from 2%

NaOH treated WS bottle, this was 3-fold higher than the untreated WS bottle. The 2 days

N. crassa F5 treated WS bottle produced only 325.7 mL/gVS volume of biogas and even

lower volume of biogas was obtained from the untreated WS bottle after the hydrolytic

retention time of 25 days. The cumulative biogas produced from the 2% NaOH+N.

crassa F5 treated WS was significantly high 339.3% more than the untreated WS bottle.

However, an improvement of 50.4% cumulative biogas was observed from the N. crassa

F5 treated WS bottle than the untreated WS.

Chapter 3: Results

105


Cum

ula

tive

bio

ga

s (

Nm

L/g

VS

)

Figure 3-37 Optimization experiment for biogas potential for N. crassa F5

treatement, 3 g/L= 3 g/L glucose added, 0.5 g/L= 0.5 g/L glucose added, 2,4 and 6

DT= 2,4 and 6 days treated samples.


Cum

ula

tive

bio

ga

s (

Nm

L/g

VS

)

Figure 3-38 Cumulative biogas of pretreated and untreated wheat straw,

F5+2%NaOH= N. crassa F5 and NaOH treated wheat straw, F5= N. crassa F5 and

NaOH treated wheat straw, 2%NaOH= NaOH treated wheat straw, control=

untreated wheat straw.

Chapter 3: Results

106

3.5.6 Changes in Organics Composition during BMP Process

The results of AD process illustrate different rates of substrate degradation in terms of

total COD (TCOD), soluble COD (SCOD), total solid (TS), volatile solid (VS),

accumulation of ammonia and VFA during anaerobic digestion. The COD removal

efficiencies presented in (Table 3-13), showed that a maximum of 20.1 and 22.6% SCOD

and TCOD was removed in case of 2% NaOH followed by N. crassa F5 treated WS

bottles respectively. Similarly, a high 17.2 and 20.1% SCOD and TCOD was removed in

case of 2% NaOH treated WS samples. However, a lower COD removal efficiency of

8.6, 10.8, 8.1 and 11.8% SCOD and TCOD was observed from N. crassa F5 treated WS

and untreated WS bottles respectively. The higher COD removal efficiencies demonstrate

the maximum production of biogas of the samples.

In the fungal pretreated as well as the untreated show slight improvement in VS

removal efficiency. The highest VS and cellulose removal ability occurred of 21.7 and

52.4% respectively in case of 2% NaOH followed by N. crassa F5 treated WS bottles.

Similarly, 17.3 and 35.5% VS and cellulose respectively were removed in case of 2%

NaOH treated WS bottles. However, in conditions of N. crassa F5 treated WS bottles and

untreated WS, a lower VS and cellulose removal efficiency of 10.7, 24.4 and 9.1,17.7%

was observed respectively. In this study the TCOD, SCOD removal ability was high but

VS removal was slightly lower (Table 3-14).

The (Table 3-15) shows the concentration of individual volatile fatty acids (VFA),

namely, acetic acid, propionic acid, butyric acid, isobutyric acid and isovaleric acid. The

methanol, ethanol and valeric acid were tested in the standard but we did not found any

of them in an anaerobic digestion processed samples. Before the start of BMP process, in

the initial VFA analysis acetic acid and propionic acid was the major acid detected in all

the reaction AD bottles. The initial concentration of acetic acid and propionic acid was

8900, 1700 mg/L respectively in case of 2% NaOH followed by N. crassa F5 treated WS

bottles. However, the initial concentration of acetic acid and propionic acid was 9900,

1800 mg/L respectively in case of N. crassa F5 treated WS bottles. A high diversity in

the final VFA profile was observed at the end of the anaerobic digestion process. A

variety of acids, namely acetic acid, propionic acid, butyric acid, isobutyric acid and

Chapter 3: Results

107

isovaleric acid were detected in each sample. The production of new VFA and increase in

the concentration of VFA clearly specify the production and conversion into methane. In

the (Table 3-15) highest concentration of acetic acid, propionic acid, isobutyric acid and

isovaleric acid was 14300, 23900, 2800, and 700 mg/L respectively in case of 2% NaOH

followed by N. crassa F5 treated WS bottles. However, a lower concentration of acetic

acid, propionic acid, butyric acid, isobutyric acid and isovaleric acid was 11300, 9300,

2100, 500 and 400 mg/L respectively in case of 2% NaOH followed by N. crassa F5

treated WS bottles. The initial and final VFA of the control sample (sludge+ water) was

low in both case and generate a negligible volume of biogas.

The results of the ammonia estimation (Table 3-16) showed that 38.2% ammonia level

was increased at the end of anaerobic digestion experiment of 2% NaOH followed by N.

crassa F5 treated WS bottles. Similarly, that 27.1% ammonia level was increased in 2%

NaOH treated WS bottles. However, a lower level of ammonia 15.5 and 19.8% were

observed N. crassa F5 treated WS and untreated WS bottles respectively. The relation of

increasing VFA and ammonia is correlated, the highest level of acetic acid, propionic

acid production might be due to the accumulation of ammonia (NH+3).

Table 3-13 The COD removal efficiency of pretreated wheat straw

Sample SCOD

(I)(mg/L)

SCOD

(F)(mg/L)

Reduction

(%)

TCOD

(I)(mg/mL)

TCOD

(F)(mg/mL)

Reduction

(%)

NaOH+ F5 treated WS 11150 8900 20.1 22500 17400.5 22.6

NaOH treated WS 10150 8400 17.2 17900.5 14300 20.1

F5 treated WS 11000 10050 8.6 19400 17300 10.8

Untreated WS 11550 10600 8.2 19400.5 17100.5 11.8

Sludge 7850 6300 24.6 19300.5 14700.5 31.1

(I)=initial, (F)= final, S=soluble, T=total

Chapter 3: Results

108

Table 3-14 The VS removal efficiency of pretreated wheat straw

Sample Initial

VS

(%)

Final VS

(%)

VS Reduction (%) Cell (i) Hemicell

(i)

Cell

(F)

Hemicell

(F)

NaOH+F5 treated WS 66.4 52.33 21.7 43.07 17.4 20.5 12.7

NaOH treated WS 68 56.2 17.3 42.39 15.8 27.3 13.05

F5 treated WS 65.34 58.2 10.1 45.8 17.09 34.5 14.29

Untreated WS 71.3 64.2 9.7 46.1 16.7 37.9 14.29

Sludge 2.64 2.15 18.5 20.5 7.1 6.8 6.27

(I)=initial, (F)= final, Cell= cellulose, Hemicell= hemicellulose

Table 3-15 VFA production in anaerobic digestion of pretreated wheat straw

Initial VFA Ac mg/L Pa mg/L Ia mg/L Ba mg/L Isov mg/L

NaOH+ F5 treated WS 8900 1700 0 0 5

NaOH treated WS 8400 1600 0 0 0

F5 treated WS 9900 1800 0 0 0

Untreated WS 9700 1800 0 0 0

Sludge 2000 1500 0 0 0

Final VFA Ac mg/L Pa mg/L Ia mg/L Ba mg/L Isov mg/L

NaOH+ F5 treated WS 14300 23900 2800 0 700

NaOH treated WS 8600 19900 6900 400 600

F5 treated WS 11340 9300 2100 500 400

Untreated WS 7860 8700 1000 2100 520

Sludge 1140 1210 0 1400 0

Aa=Acetic acid, Pa= Propionic acid, Ia= Isobutyric acid, Ba= Butyric acid, Isov= Isovalaric acid

Chapter 3: Results

109

Table 3-16 The accumulation of ammonia in anaerobic digestion of pretreated

wheat straw

Sample Initial NH+3 (mg/mL)

Final NH+3 (mg/mL)

Increase (%)

NaOH+ F5 treated WS 1155 1870 38.2

NaOH treated WS 1720 2360 27.1

F5 treated WS 1605 1900 15.5

Untreated WS 1295 1615 19.8

Sludge 2040 1620 -0.259

3.5.7 Conclusion

In this study, we earned the speedy saccharification of wheat straw using recombinant N.

crassa F5 strain. A partial transformation of wheat straw residue was achieved in 48

hours of batch time. A significant degradation on the surface of wheat straw was

observed after pretreatment of NaOH, recombinant N. crassa F5 treatment as compared

to untreated wheat straw. Consolidated bioprocessing of wheat straw with recombinant N.

crassa F5 strain significantly introduced shorter hydrolytic retention time in the

anaerobic digestion system. Recombinant N. crassa F5 strain degraded wheat straw

exhibited higher biogas production compared to untreated wheat straw. This study shows

that novel treatment is vital for effective and economical anaerobic technology

development.

3.6 Bacillus sp. Strains to Produce Bio-Hydrogen from the

Organic Fraction of Municipal Solid Waste

Biological hydrogen production from organic waste represents both an energy production

process and a first stage of stabilization for organic biomass since it degrades complex

substrates to readily biodegradable compounds or to metabolites of commercial interest

(i.e. organics acids and solvents) [164-166].Organic waste and low-cost organic by-

products of food-processing industry have been already investigated as promising

renewable materials to be converted into hydrogen and other fuels, polymers, enzymes

and bulk chemicals [97, 167-175]. However, to guarantee the economical sustainability

Chapter 3: Results

110

of the organic waste-to-hydrogen route, one of the main requirements is linked to the

availability of efficient H2 producing microbes with proper robustness to be used at

industrial scale [164]. In order to obtain suitable inoculants, methanogens and hydrogen-

consuming bacteria should be inhibited. To this purpose, several methods for pre-

treatment of inocula have been proposed, including heat-treatment, aeration, irradiation,

freezing, addition of chemical inhibitors such as acid, alkali, chloroform, etc., as

extensively reviewed in [15, 100, 176-178].

The Organic Fraction of Municipal Solid Waste (OFMSW), characterized by high

moisture and high biodegradability due to a large content of food waste, kitchen waste

and leftovers from residences, cafeterias and markets, has been previously evaluated for

H2 production through the addition of heat-treated inocula [97, 179-181]. Although heat

shock pre-treatment contributed to good H2 performances in short lab scale operations,

increasing evidences show that a stable H2 production and methanogens repression is not

possible for long-term continuous mode [15, 164, 182]. Further research is also needed to

establish whether the additional technical complexity of heat-treating the inoculum at

industrial scale is cost-effective. Pragmatically thinking, heat shock of inocula is

technologically more difficult during scale-up as compared to other pre-treatments [176].

Moreover, the use of exogenous inocula does not allow to guide properly the

fermentation process [15, 97]. To address this issue, recent research advances have been

reported indicating that OFMSW itself could produce, without any external inoculum

supplementation, high H2 yields [97]. Natural decomposition occurs to food waste when

left for few days at room temperature due to the presence of indigenous microorganisms.

In case of no or very low oxygen concentration, fermentation of organic matter takes

place and methane production may also occur with time. Therefore, some species of

indigenous microbial population of organic waste may have good characteristics for the

hydrolysis of complex substrates and for an efficient conversion into H2. As a result, food

waste could serve both as substrate and source for H2 production and H2-producing

bacteria, respectively [97, 183]. This novel approach paves the way for the development

of inoculants to produce H2 from OFMSW relying on the indigenous microbes.

Chapter 3: Results

111

Another recent research strategy is the use of selected microbe(s) for the

conversion of organic waste into H2 [181, 184]. The main advantages of using pure

cultures over mixed microflora are that metabolic changes are easier to detect/tune and

more information on the conditions that promote H2 production can be disclosed [177,

178, 185]. Furthermore, even in non-sterile environments, pure cultures may be useful in

bioaugmentation to achieve higher gas outputs [176, 178, 183, 186]. The possibility to

select strain(s) for their hydrolytic and fermenting abilities according to the main

complex substrates available in the food waste makes this avenue very effective.

However, it remains still unexplored as pure cultures have been so far mostly applied for

H2 production from simple sugars (i.e., glucose, sucrose and xylose) or laboratory-grade

soluble starch [15, 177, 187]. Thus, more researches using pure cultures for H2 production

from organic waste are recommended [177, 178, 185].

In this paper, to look for microbes with both high hydrogen production potential

and proper robustness, granular sludge from a brewery full scale Up Flow Anaerobic

Sludge Blanket (UASB) digester was selected as promising source because of processing

complex substrates at industrial scale. One hundred and twenty bacterial strains,

previously isolated from heat-treated granular sludge and selected for their high H2

production [88], were screened for extracellular hydrolytic profile on cellulose,

hemicellulose, starch, pectin, lipids, protein. The isolates exhibited a broad range of

hydrolytic activities and the most interesting strains were assessed for their H2 production

from glucose. The top H2-performing microbes were evaluated using starch as main

carbon source. Two Bacillus sp. strains showed high H2 levels and were evaluated also on

OFMSW, mainly composed by starch, lipids and protein. The microbes gave promising

H2 yields and could be considered as good candidates towards the future development of

industrially relevant microbes for the processing of organic waste into H2. This is the first

successful application of pure microbial cultures in bio-hydrogen production from

OFMSW.

3.6.1 Screening for Extracellular Enzymatic Activities

One hundred and twenty microbial strains were previously isolated and identified from

samples of heat-treated granular sludge used to perform hydrogen production batch tests

Chapter 3: Results

112

[88]. The heat-treatment (100°C for increasing residence times of 0.5, 1, 2 and 4 hours)

strongly affected the microbial viability in the sludge and the heat-treated sludge

produced high and variable hydrogen yields from glucose, with the microbial consortia

surviving after 2 and 4 hour boiling times having the most promise [88]. All isolates were

screened for the production of industrially relevant extracellular enzymes and exhibited a

broad range of hydrolytic activities (Table 3-17).

Fifty-seven strains were found proteolytic with a great majority of positive

isolates belonging to Bacillus genus. A high number of pectinolytic strains has been also

detected: the fact that only four out of 34 strains confirmed their potential once grown in

the presence of both glucose and polygalacturonic acid (PecA+glucose) clearly indicates

that, in the screened microbial collection, the production of pectinolytic enzymes is

mainly not constitutive. Twenty-seven microbes gave positive results for starch-

degrading activities. As reported in Table 3-17, three strains produced active xylanases

meanwhile only a B. licheniformis isolate was found to be cellulolytic. No lipolytic

microbes were recovered.

The majority of the catalytic activities were found to be protease, amylase and

pectinase. This outcome could be explained considering that the strains have been

isolated from an anaerobic digester of a brewery whose fed by-products are usually rich

in starch, pectin and protein. Overall, the isolates belonging to Bacillus sp. genus

displayed the highest number of hydrolytic activities. They are attractive species for the

industry as they are rarely pathogenic, grow fast and secrete high amounts of proteins.

These properties make bacilli very useful in industrial applications where they contribute

up to 50% of the enzyme market.

Chapter 3: Results

113

Table 3-17 Extracellular enzymatic activity of 120 microbial strains isolated from

samples of heat-treated granular sludge.

Strains No. of

strains CelA LipA PecA

PecA

+ glu PrA StA XylA

Bacillus sp. 31 - - 8 - 16 6 1

Bacillus badius 20 - - 7 - 11 5 -

Bacillus berjingensis 6 - - 3 - 2 - -

Bacillus farraginis 8 - - - - - - -

Bacillus flexus 1 - - - - - 1 -

Bacillus licheniformis 3 1 - 2 1 1 3 1

Bacillus megaterium 3 - - 3 - 3 3 -

Bacillus subtilis 3 - - 3 - 1 3 -

Bacillus tequilensis 4 - - 2 3 1 4 1

Brevibacillus sp. 3 - - - - - - -

Brevibacillus agri 3 - - - - 1 - -

Brevibacillus brevis 2 - - - - 1 - -

Brevibacillus

parabrevis 1 - - - - - - -

Enterobacter sp. 2 - - - - - - -

Enterobacter cloacae 1 - - - - - - -

Lysinibacillus sp. 16 - - 5 - 5 3 -

Paenibacillus sp. 6 - - 2 - - - -

Paenibacillus cookii 3 - - - - - 1 -

Sporosarcina sp. 4 - - - - - - 1

Total n. of strains 12

0

Total n. of positive

strains 1 - 34 4 57 27 3

(CelA: cellulolytic activity; LipA: lipolytic activity; PecA: pectinolytic activity; PecA + glucose:

pectinolytic activity screened in the medium supplemented also with glucose; PrA: proteolytic activity;

StA: starch-degrading activity; XylA: xylan-degrading activity).

3.6.2 Hydrogen Potential from Glucose by Selected Microbial Strains

The presence of different extracellular enzymatic activities in many screened isolates was

Chapter 3: Results

114

considered promising towards the definition of a proper inoculum for the conversion of

complex organic waste into hydrogen. In literature, indeed, Bacillus species are known as

strong candidates for biological H2 production because (i) they can survive under harsh

conditions, hence could compete with other microbes (ii) they have large and versatile

enzymatic activities, therefore a diverse range of bio-waste could be used as substrate for

bio-hydrogen production, (iii) they do not require light for H2 production, (iv) Bacillus

sp. spores are being used as probiotics in humans and animals; thus, they may not pose

environmental health concerns [188, 189].

Twenty strains belonging to Bacillus sp. and Brevibacillus sp. were selected for

their hydrolytic activities and evaluated for H2 potential. Firstly, the microbes were

screened in NB supplemented with 5 g/L glucose and compared in terms of hydrogen

yield and glucose consumption after 48 hours of incubation. The microbes produced H2

with variable yields (0.16-1.53 mol of H2 per mol of consumed glucose). The most

proficient microbes are reported in Table 3-18 together with other H2-performances

recently described for Bacillus sp. grown on the same amount of glucose.

Chapter 3: Results

115

Table 3-18 Comparison of hydrogen production potential of Bacillus sp. and

Brevibacillus sp. strains from glucose (5 g/L) as carbon source.

Strain Enzymatic profile H2 yield

(mol/mol

glucose)

Residual

glucose (%) Reference

Bacillus sp. F2.5 StA 1.53 nd This study

Bacillus sp. F2.7 PrA, StA 0.88 2.9 This study

Bacillus sp. F2.8 PrA, StA 1.47 nd This study

B. farraginis F4.10 PrA, StA 0.31 nd This study

B. megaterium F1.22 PectA, PrA, StA 0.57 nd This study

B. tequilensis F2.16 PectA, StA, XylA 0.36 2.5 This study

Brevibacillus sp. F4.12 PectA, PrA 0.75 nd This study

Brevibacillus sp. F4.16 PrA 0.69 nd This study

Bacillus sp. EGU444 PrA 0.35 na [190]

B. thuringiensis EGU378 LipA, StA 0.26 na [190]

B. megaterium ATCC15374 StA 0.60 1.0 [191]

B. thuringiensis EGU45 nd 1.67 24.0 [146]

B. cereus EGU44 nd 1.92 23.2 [146]

B. cereus EGU43 PrA 1.12 21.6 [146]

B. cereus EGU3 nd 0.96 22.4 [146]

Bacillus sp. FS2011 nd 2.04 0.5 [192]

na: not available; nd: not detectable

Interestingly, the glucose-to-H2 conversion efficiencies of the newly isolated

bacteria were comparable to those of the literature and the highest yields were exhibited

by two Bacillus sp. strains (namely F2.5 and F2.8) with 1.53 and 1.47 mol of H2 per mol

of used glucose, respectively. The majority of the microbes investigated in this study

completely utilize the glucose available in the system meanwhile other Bacillus sp.

strains, although exhibiting high H2 yields, did not convert all the substrate. This finding

Chapter 3: Results

116

is of great interest since a microbial strain should have both high substrate utilization and

H2 yield for being implemented in the industrial bio-hydrogen technology.

As reported in Table 3-18, the strains selected in this study showed one to three

hydrolytic capabilities whereas only few Bacillus sp. microbes with high H2 potential

were described in literature also for enzymatic activities. The most efficient strains,

Bacillus sp. F2.5 and F2.8, were selected for further studies. Their amylolytic enzymes

could be very useful for the H2-conversion of food waste, where starch can account up to

30% of the TS.

3.6.3 Characterization of Amylolytic Enzymes Secreted by Bacillus Sp.

F2.5 and F2.8

To study the degrading activity of Bacillus sp. F2.5 and F2.8, the strains were grown both

in NB and SPM supplemented with 20 g/L soluble starch. The highest enzymatic

activities were detected in SPM broth after 72 h of incubation at 37°C (data not shown),

thus this medium was selected to deeply investigate their amylolytic abilities. The

activity of both microbes after 72 h incubation in SPM was firstly assessed at 50°C using

different pH values (Figure 3-39 a). The two strains displayed comparable amylase

activities: Bacillus sp. F2.8 showed the most promise with the highest enzymatic

activities (67.8 U/mL) detected at pH 7.0 meanwhile the uppermost catalytic ability of

Bacillus sp. F2.5 was found at pH 6.0 (62.5 U/mL). pH greatly influenced the enzymes of

both strains: the total amylase activity of Bacillus sp. F2.5 at higher pH progressively

dropped to 25.1 U/mL at pH 8.0, which stand for almost 40% of the highest value. The

amylase activity of Bacillus sp. F2.8 was high in the pH range of 6.0-8.0.

The amylolytic enzymes were assayed at temperatures from 30 to 60 °C at the

optimal pH for each strain, namely pH 6.0 and 7.0 for Bacillus sp. F2.5 and F2.8,

respectively. Enzyme activity increased with temperature up to 50 °C, which was found

to be the optimum for the two microbes (Figure 3-39 b).

Chapter 3: Results

117

Figure 3-39 The effect of pH (a) and incubation temperature (b) on the amylase

activity of Bacillus sp. F2.5 ( ) and Bacillus sp. F2.8 ( ) grown for 72 h in SPM

containing 20 g/L soluble starch.

At 60 °C, the enzymatic values were lower, 57 and 67% of the highest activity

detected at 50 °C for Bacillus sp. F2.5 and F2.8, respectively. Both microbes had high

relative activity at 30 and 40 °C (on average 64 and 74%, respectively). Overall, Bacillus

sp. F2.5 and F2.8 produced amylase with high potential with enzymatic activities.

Moreover, the high enzymatic activities registered at thermal levels near to those optimal

for growth (37 °C) could be beneficial for the saccharification of starchy substrates into

glucose during the starch-to-H2 fermentation.

3.6.4 Hydrogen Production from Glucose and Soluble Starch by

Bacillus Sp. F2.5 And F2.8

Considering that OFMSW is usually quite rich in starch, with the final aim of assessing

their ability to convert OFMSW into H2, Bacillus sp. F2.5 and F2.8 were firstly evaluated

for their H2 potential from soluble starch (20 g/L) at pH 6.0 and 7.0, selected as the

optimal values for the amylase secreted by each strain (Figure 3-39a). The microbes were

also cultivated in the presence of the equivalent amount of glucose (22 g/L), as reference

medium.

No methane was detected throughout the experiments whereas the strains were

able to produce H2 from glucose and soluble starch (Figure 3-40 a, b).

Chapter 3: Results

118

Figure 3-40 Cumulative hydrogen productions of Bacillus sp. F2.5 (a) and Bacillus

sp. F2.8 (b)

grown in SPM supplemented with 22 g/L of glucose ( ), 20 g/L soluble starch (▲) or 10

g VS/L of OFMSW ( ). Filled and empty symbols report values obtained at pH 6.0 and

7.0, respectively. Data shown are the mean values of three replicates and standard

deviations are included.

The two microbes completely utilized glucose within five days yielding high

levels of hydrogen. Bacillus sp. F2.5 obtained the uppermost H2 concentrations both at

pH 7.0 and 6.0, with 114 and 101 mL of H2, respectively, whereas Bacillus sp. F2.8

produced lower volumes: 101 and 85 mL at pH 7.0 and 6.0, respectively. As a result, the

top fermenting abilities were achieved at pH 7.0, with the H2 yield of 0.91 and 0.81 mol

per mol of consumed sugar for Bacillus sp. F2.5 and F2.8, respectively. Lowering the pH

resulted in a reduced efficiency, mostly for Bacillus sp. F2.8 whose yield was 0.69 mol

per mol of consumed sugar meanwhile the other strain produced 0.82 mol of H2 per mol

of used glucose. Bacillus sp. F2.8 displayed the most efficient fermenting profile with the

highest H2 productivity attained at pH 7.0 (26.8 mL of H2 per day), which was 1.12-fold

that of Bacillus sp. F2.5 (24.0 mL of H2 per day). Relative H2 concentration was found to

be similar (about 45%) for the two strains (Table 3-19).

Chapter 3: Results

119

In the presence of soluble starch, Bacillus sp. F2.5 and F2.8 produced high H2

levels, too (Figure 3-40), consuming all the available polysaccharide. At pH 7.0, Bacillus

sp. F2.8 confirmed the most efficient hydrolyzing ability, obtaining the highest amount of

H2 (51.8 per gram of consumed starch) in a shorter timeframe (Figure 3-40 b). Similar

performances but with lower productivity were detected for Bacillus sp. F2.5 (Figure 3-

40 a): in the first days, higher amounts of hydrogen were produced at pH 6.0 while, at the

end of incubation, pH 7.0 supported slightly better the H2 potential of Bacillus sp. F2.5.

This finding could be explained considering that, for this strain, pH 6.0 and 7.0 were

found to be optimal for amylases and H2 yield, respectively.

The relative concentration of H2 was similar for the two microbes (Table 3-19):

44 and 45 % for Bacillus sp. F2.8 and F2.5, respectively, and the highest H2 efficiencies

were found at pH 7.0: 0.42 and 0.41 mol of H2 per mol of consumed starch for Bacillus

sp. F2.8 and F2.5, respectively. Their yields from soluble starch were 51 (0.81/0.42) and

44% (0.91/0.41), respectively, of those above presented in the same broth from glucose.

Interestingly, although the two strains had similar starch-to-H2 efficiency, Bacillus sp.

F2.8 showed H2 potential from glucose lower than Bacillus sp. F2.5 (Tables 3-19, Figure

3-40). This could be associated with the most efficient starch-degrading activity

described for Bacillus sp. F2.8 at pH 7.0 (Figure 3-39 a). On the other hand, as reported

in (Table 3-19), both Bacillus sp. strains described in the present work showed

productivity (about 4 mL of H2 per day) lower than those found in other studies on H2

production from starch. However, their limited H2 production rate, which could be

mainly influenced by their low inoculum size and static incubation, are likely to be

improved by optimizing the growth conditions and other environmental factors such as

micronutrients availability, buffers and temperature which were reported as key

parameters to boost H2 productivity.

Chapter 3: Results

120

Table 3-19 VFAs profiles of the biogas produced on different substrates, (mg/L

and % TVFA, Total Volatile Fatty Acid), maximum volumetric H2 productivity (Qmax),

(NmL/L/d), and relative H2 concentration (%) Data shown are the mean values of three

replicates and and standard deviations are included.

Substrate Strains p

H

H2

%

Qmax

mL/L/

d

TVFA

mg/L

Acetate

mg/L %

Propionate

mg/L %

Butyrate

mg/L %

Glucose F2.5 6 45 23.9 1774 973±85 55 247±48 14 554 ± 60 31

7 45 26.8 2115 1134±99 54 367±26 17 613 ± 62 29

F2.8 6 45 19.6 1548 873±68 56 182±18 12 493± 48 32

7 45 24.0 1861 1087±99 58 205±16 11 569 ± 55 31

Starch F2.5 6 45 3.7 896 490±69 55 123±25 14 282 ± 30 31

7 45 3.9 1058 568±99 54 183±40 17 307 ± 51 29

F2.8 6 45 3.8 774 437±45 57 91±19 12 247 ± 45 31

7 44 4.1 1106 637±29 58 123±27 11 345 ± 43 31

OFMSW F2.5 6 38 4.2 1117 625±71 56 158±20 14 334 ± 39 30

7 38 3.9 1131 601±55 53 199±17 18 331 ± 28 29

F2.8 6 39 4.0 945 527±49 56 122±15 13 296 ± 30 31

7 39 5.0 1277 737±58 58 144±13 11 396 ± 35 31

3.6.5 Hydrogen Potential from OFMSW

The fractions analysis of the OFMSW obtained from manual sorting procedure (Table 2-

1) revealed a composition similar to those of other OFMSW. Fruit, vegetable and bread-

pasta-rice were the most abundant shares on wet weight basis, meanwhile, as reported in

Materials and Methods (section 2.1), starch, protein and lipids were found to be the main

components of TS, with 19, 18 and 17% of TS, respectively. From OFMSW, no methane

was detected whereas H2 production was found to be feasible with both strains: H2

concentrations were slightly higher for Bacillus sp. F2.8 (Figure 3-40 b), which produced

almost 61 mL of H2 per g VS, at pH 7.0. At pH 6.0, the strain achieved slightly lower H2

Chapter 3: Results

121

levels and productivity. On the other hand, Bacillus sp. F2.5 exhibited fermenting

abilities comparable for both tested pH values and H2 production was found 55 and 53

mL per g VS for pH 6.0 and 7.0, respectively (Figure 3-40 a).

Bacillus sp. F2.8 confirmed the most efficient productivity already described from

soluble starch. At pH 7.0, the strain produced 5.0 mL of H2 per day whereas 4.0 mL of H2

were daily produced at lower pH (Table 3-19). Bacillus sp. F2.5 had similar H2

productivity at pH 6.0 (4.3 mL of biogas and H2) while, at pH 7.0, its productivity was

lower resulting in 3.9 mL of H2 (Table 3-19). Both strains produced comparable relative

H2 concentrations (nearly 38%) which were inferior than those above reported from

soluble starch and glucose (Table 3-19).

Hydrogen levels produced in this study were consistent with those previously

described for batch H2 fermentation of OFMSW or food waste by using pure or mixed

cultures (Table 3-20). Further, in the present study, inoculum pre-treatment inoculum was

not required. Moreover, as described in Table 3-20, this is one of the earliest accounts on

a single microbe capable of converting organic waste into H2 with a high rate and yield.

Only recently, Marone and colleagues described few Enterobacteriaceae strains, isolated

by the bioaugmentation of vegetable waste (Rahnella sp. 10, Buttiauxella sp. 4 and

Raoultella sp. 47), for their promise in producing H2 from vegetable kitchen waste

collected from a cafeteria. However, this is the first successful application of pure

microbial cultures in bio-hydrogen production from OFMSW. Furthermore, both Bacillus

sp. strains exhibited high starch-degrading activities meanwhile the above reported

microbes did not produce any relevant hydrolytic enzymes. Their spore-forming ability

and their being isolated from granular sludge of a full scale UASB anaerobic digester are

two additional noteworthy benefits which count for the potential development of Bacillus

sp. F2.5 and F2.8 as efficient and robust inoculants.

Chapter 3: Results

122

Table 3-20 Comparison of hydrogen production from OFMSW achieved in this

study and other performances previously reported from OFMSW and food waste.

Feedstock Inoculum Pre-treatment

inoculum

Pre-

treatment

feedstock

Temp(°C)

Yield

(mL

H2/g VS)

Reference

OFMSW Bacillus sp. F2.5 NO Sterilized 35 61 This

study

OFMSW Bacillus sp. F2.8

NO Sterilized 35 55

This

study

OFMSW

pre-adapted H2-

producing

bacteria

NO NO 37 180 [181]

OFMSW pre-treated

digested sludge 100 °C 15 min NO 37 140 [181]

OFMSW NO NO NO 35 42 [97]

OFMSW granular sludge 100°C 4 h NO 35 70 [97]

OFMSW granular sludge 100°C 4 h Sterilized 35 57 [97]

OFMSW granular sludge 100°C 4 h NO 35 25-85 [180]

Food waste anaerobic sludge NO NO 35 39 [193]

Food waste anaerobic sludge NO NO 50 57 [193]

Food waste grass compost 180°C 3 h NO 35 77 [194]

Food waste NO NO NO 35 4 [195]

Food waste Food waste 90 °C 20 min 60-90 °C

20 min 35 26-149 [195]

Vegetable waste Vegetal waste NO NO 28 22 [184]

Vegetable

waste and potato

peels

Vegetable

waste and potato

peels

NO NO 28 18 [184]

Vegetable waste Rahnella sp. 10 NO NO 28 47 [184]

Vegetable waste Buttiauxella sp. NO NO 28 71 [184]

Vegetable waste Raoultella sp. 47 NO NO 28 70 [184]

Chapter 3: Results

123

3.6.6 VFAS Profiles from Glucose, Soluble Starch and OFMSW

Fermentations

H2 production is coupled with production of VFAs and/or solvents. The composition of

VFAs generated is a useful indicator for monitoring the H2 production pathways. The

high VFAs concentrations achieved in this study indicate that favourable conditions for

the growth and the activity of both strains were established during the course of the

experiments (Table 3-19). The detected soluble metabolites were acetate, butyrate and

propionate. In all batch experiments the acetate was the major component (53-58%) with

butyrate as the second most abundant acid (29-32%). This finding proved that similar

metabolic pathways were involved and the acetate-butyrate was the predominant

fermentation mode, which was reviewed as favouring H2 production. As a result,

supplementing different substrates significantly changed only the VFAs quantity rather

than their shares: the highest amount of Total VFA (TVFA) was obtained from glucose

meanwhile starch and OFMSW supported similar TFVA values. The higher the level of

VFA accumulation (Table 3-19), the higher H2 production was achieved (Figure 3-40 a,b)

3.6.7 Conclusion

This study demonstrated for the first time the effective conversion of OFMSW into H2 by

using pure cultures of Bacillus sp. strains properly selected for both their proficient

enzymatic activities and their high fermenting abilities from glucose and starch. Future

studies will further increase their H2 performances and techno-economical evaluations

will determine the actual feasibility of the whole process. Taken together, the results of

this work gave advances in knowledge towards the development of microbial inoculants

for the industrial processing of organic waste in H2.

4 Discussion

In this Chapter, an assessment between the different pretreatment approaches conducted

in the research study is presented with relation to previously reported studies. A

comparison is considered to see the effects of chemical and biological pretreatment for

changes in composition of substrates and methane production. The additional results

conducted for supporting the correlation with biogas improvement is also discussed. The

consequence of the pretreatment on chemical composition and biogas production from

agribiomass varied among different pretreatment categories. The effects of the both

chemical and biological processes were considered under batch mode in order to define

the optimum pretreatment approach for a future scale-up of the anaerobic digestion

technology.

Anaerobic digestion process is widely reported, although most anaerobic digesters

are not adjusted for high energy production. Present and upcoming research in anaerobic

technology is dedicated on effective pretreatment methods and bioprocess control,

bioreactor design and digestate processing. This thesis only examines the effective

pretreatment methods aspect for biomethane potential from different agriculture waste

biomass as a feeding material.

Biomass-based energy are converging attention for the reduction of human-

induced climate change and solid waste management. The application of waste biomass

in biomass-based energy is considering important and relevant as bioeconomic

feedstock. Bioenergy and biofuel production from waste lignocellulosic biomass (LB) is

a sustainable approach. In addition, crop-based bioenergy is feasible to be not only a

substitute, sustainable and inexpensive, but also not to damage the food security. For the

crop-based bioenergy production, agriculture wastes are abundant throughout the world.

A proper management is necessary to utilize them for production of value added

products. This is important because fossil fuels are rapidly depleting, raising

environmental pollution by releasing greenhouse gases. To overcome these challenges

Chapter 4: Discussion

125

anaerobic digestion (AD) has developed serious consideration [196]. Bioenergy

production depends upon on the composition of the evaluating substrates. Substrate with

high carbohydrates, fats, proteins, hemicelluloses and cellulose yield maximum volume

of biogas compared to substrate with low quantity of these components [197]. The

expected theoretical and practical biogas yield also depended on individual carbon

composition of each substrate [198]. Biogas production potential is related to the amount

and composition of percentage of total solid, volatile solid and total oraganic content in

any biomass [199]. The volatile solid (VS) and total solid (TS) content of biomasses

effects AD performance, especially efficiency of biogas yield. Substrate with less

hemicellulose and high content of VS has maximum methane production [197]. Any

material that contains inhibitory elements needs to be identified and must be reduced later

to not effect bacterial growth or microbial population [200].

4.1 Influence of Alkalies Treatment on Lignocellulosic Waste Biomass

As observed in chapter 3 in Table 3-1 of the current thesis, the different biomasses were

composed of cellulose, hemicellulose and lignin as main constituents. The composition of

these constituents were similar to the plant waste biomass of previously reported studies,

40-50% cellulose, 15-25% lignin and 22-35% hemi-cellulose [102, 103]. However, the

compositions among selected substrates were different from each other, although some of

the tested substrates has closely similar composition. The estimation of the hemicellulose

and cellulose of the substrate is important as the output of biogas is correlated to carbon

content in each biomass. Whereas, the lignin is the non-biodegradable component and

made of aromatic complex hydrocarbon polymer [201]. Lignin cross link with

hemicellulose and cellulose sugars make the biomass structure more difficult to microbial

degradation [202]. This is the rate-limiting reaction and it lower down the process of

biomass degradation and reduce the biogas yield in AD [28].

As presented in the (Figures 3.1,3-2 and Tables 3-2 and 3-3) summarized the

effect of alkali treatment and heating process on lignocellulosic biomasses. Among the

alkalis tested (NaOH, KOH, Ca(OH)2 for lignin removal, NaOH was observed the most

appropriate alkali for lignin removal from agriculture waste biomasses. Among the

heating process microwave is an effective heating process for lignin removal from all of


126

the biomass type. These observations are correlated to previously reported studies, where

among the alkalis, NaOH has shown higher affinity for lignin due to strong hydroxyl ions

bonding to remove effectively lignin from each waste biomass [203]. Ca(OH)2 is reported

with lowest potential in term of lignin reduction in all of the heating processes. The low

lignin removal capability of Ca(OH)2 is due to solubility reason of Ca(OH)2 , it is barely

soluble beyond this concentration, it makes a calcium–lignin complex under alkaline

conditions and stopping higher amounts of lignin removal [111, 112].

The second observation in the box plot (Figure 3-1 and 3-2) showing increasing

of lignin removal with increasing the concentration of alkali, This is also consisted with

a previous report on lignin reduction [204]. However, the increasing of the alkali

concentration also remove some of the hemicellulose and cellulose from the treated

substrates (Figure 3-3), this is an accordance with a an earlier study where high alkalies

concentrated is reported a negative effect on the sugar recovery, degrade useful carbon

sources and effect the overall pre-treatment process [205].

The scanning electron microscope (SEM) images of the untreated and alkalies

treated samples clearly showed visible degradation on the surface of each substrate. The

surface wall was broken and disrupted in each pretreated sample compared to the

untreated. The degradation was prominent in samples treated with high dosage alkali

reagent. Similar observation of degradation after alkali treatment on the surface of

biomass is also reported earlier [113]. The FTIR spectra of waste biomass treated with

alkalies further showed obvious changes in the banding pattern and peak position in the

sample compared to untreated substrate. Which proved that pretreatment has done some

molecular changes in the waste biomass [56].

For the comparison of anaerobic digestion of 1,2,3, and 5% NaOH and KOH

treated samples of almond shell and wheat straw were tested. It was observed that; the

daily volume of biogas and methane rate was 2-2.6 times higher in case of pretreated

almond shell and wheat straw comparatively to their untreated substrates. Similarly, the

biogas yield of NaOH treated samples was more than the daily biogas and methane yield

from KOH treated samples. This could be related to the high lignin removal of NaOH

treatment than KOH. In addition; the daily biogas was high in the first 10 days of


127

anaerobic digestion from 3-5% KOH and NaOH batch assay than 1- 2% treated

substrates. This results support that; high delignified substrate yield more biogas at the

start of the anaerobic digestion process than less delignified substrate. The cumulative

biogas obtained from the alkali treated substrate was 2-times higher than the untreated

substrates. The results of lignin removal and anaerobic digestion batch experiments

shown that, the optimum results were shown by 2% NaOH treatment that removed a

substantial level of lignin from all the tested substrates. Similarly, the biogas obtained

with 2% NaOH has also shown more total biogas yield than 2% KOH and even from the

concentrated alkali dosage treated samples.

In conclusion to the chemical treatment methods, microwave was found more

effective heating process because microwave radiation is rapid, efficient, selective,

precise, controllable, and produces hot spots on the subjected biomass, therefore, it

solubilized more hemicellulose and lignin than conventional alkali pre-treatment [23].

Additionally, the results of this study verifying that pre-treatment with alkali is vital for

lignin reduction and improving biogas production from agriculture waste biomass [206,

207]. Application of conventional heating or microwave using alkali combined pre-

treatments might be a significant process for increasing delignification and biogas

production, however it must be study for the economically value for commercial

applications [208, 209].

In the alkalies treatment process, heating and high concentration of alkali may

increase the overall cost of the treatment process [210]. So lime (Ca(OH)2) soaking was

tested with the objective of being less expensive treatment as compared to the above

tested conditions. Among the alkalies Ca(OH)2 is cheaper and it does not generate

inhibitors during treatment process[114]. Additionally, soaking with Ca(OH)2 did not

need any thermal heating, so it is also beneficial for large scale development and energy

output yield. Dilute lime (Ca(OH)2) soaking pretreatment shown a considerable

percentage of lignin removal from corn cob and improved the biogas yeild. These

observations suggested that lime soaking can be considered to move forward from lab

research to industrial scale process for less expensive biogas production. Further

adjustment to an anaerobic process by co-substrate strategy can be proved as vital [211,


128

212]. The suitable design of anaerobic digester, generator, and optimization of conditions

for anaerobic digestion process is also crucial for industrial-scale development [213,

214].

4.2 Biological Pretreatment using Ligninolytic Bacterial Culture

In this thesis, an approache of biological preatment of agriculture waste biomass was also

considered. Lignin was used as a sole carbon source for isolation and screening of

ligninolytic strains, and were considered for lignin degradation of rice straw to to

improve biogas production [215]. Of the 27 isoaltes, 7 robust different Bacillus sp.

strains were selected base on high decolorization. The Bacillus sp. strains were positive

for different extracellular enzymatic activities. Bacillus sp. strains are reported in

disticntion for lignin and dye degradation among different bacterial phyla proteobacteria,

Actinobacteria, Firmicutes and archaea [216]. In addition to an enzymatic repertoire of

cellulase, lipase, protease, amylase, xylanase, and pectinase, Bacillus strains actively

grow on lignin and dyes and typically displaying highest decolorization and degradation

of lignin and synthetic dyes from other species [41, 42, 141, 142]. As illustrated in the

current study, the most notable observation was the lignin degradation efficiencies. The

Bacillus sp. strains displayed maximum lignin degradation efficiencies at pH 5 rather

than at pH 7 (Figure 3-22). This degradation potential can be associated to the optimum

activity of the LiP and Laccase activities at pH 5 among all the Bacillus sp. strains.

These results are consistent with those of a previous study focused on the Azotobacter sp.

HM121 isolated from a soil sample [76]. The activity of lignin peroxidase and laccase

was optimum at acidic pH 3-5. Similar to our result a previous study also reported

optimum LiP activity at pH 3 and 65˚C for Bacillus sp. strain VUS [217] and Bacillus

subtilis at pH 3 and at 30˚C [218]. The strains all expressed lignin peroxidase and

laccase, which are important enzymes implicated in the degradation of lignin.

These seven Bacillus sp. strains were used either pure culture, co-cultures of all

the seven strains to degrade lignin of rice straw. Only in co-culture pretreatment

displayed high rate of lignin degradation which was 2.5 times more than that of any of

the individual Bacillus sp. These observations are consistent with an earlier study where a

lignocellulolytic microbial consortium in a mixed microbial population enhanced total


129

weight reduction and consumed some quantities of hemicellulose and cellulose using rice

straw as a substrate [124, 219]. The scanning electron microscope (SEM) images show

broken fibrils and disrupted bundles in the cell wall complex of each pretreated sample.

Our results are consistent with a previous SEM study of rice straw treated with

ligninolytic microorganisms and ligninolytic enzymes in which similar degradation and

distortions were observed [72, 220]. In another study of rice straw degradation by a

microbial consortium, the SEM micrographs showed penetration of the outer structure

and disruption of the substrate [221].

Finally, in batch mode experiment for biogas production of the (TL-4, TL-6, TL-

26) treated samples, an increase of 76.1% cumulative methane with co-culture of all

seven Bacillus sp. strains were obsereved. Like our observations, in a previous study of

biological pretreatment of lignocellulosic straw, it was reported that the microbial activity

structurally modified the cellulose and increased accessibility up to six-fold within the

first few days of the treatment [222]. Furthermore, it has been proved that biological

pretreatment of rice straw results in enhancement of cumulative biogas yields [223].

However, optimization studies are necessary regarding nutrients, and synergistic growth

of mix culture for further increasing of lignin degradation of waste biomass for biofuel

production.

Biological pretreatment of lignocellulosic biomass is consider an inexpensive

hydrolysis method for lignin degradation of agriculture feedstock to yield low-cost

biofuel production [215]. The application of microbes for enzymatic hydrolysis of lignin

in waste biomass offers advantages over chemical pretreatment strategies to decrease

energy and equipment costs [224]. Co-inoculation of bacterial strains and application of

enzymes mixture consolidating pretreatment process could be an active method for

increasing biogas production [225]. Ligninolytic bacterial strain and fungal strains are

particularly important microorganisms involve in lignin degradation. These microbes

normally have lignin-degrading enzymes, laccase (Lac), lignin peroxidase (LiP), and

manganese peroxidase (MnP) [226, 227]. These enzymes involve in the oxidation of

phenolic rings [40, 49]. Common fungal strains are known for dyes and lignin-

degradation using ligninolytic enzymes [228, 229]. However, bacterial strains such as


130

Streptomyces viridosporus T7A [44], actinomycetes [45], Rhodococcus jostii RHA 1,

Pseudomonas sp. [230], and Bacillus sp. also contains lignin-degrading enzymes [46].

Lignin can be used as a sole carbon source for isolation and screening of

ligninolytic strains, that can be considered in future for lignin degradation of agriculture

feedstock to yield inexpensive biofuel production [215]. The Soil, wood compost and

waste sludge environments are preeminent source of microorganisms capable of

degrading heterogeneous and polymeric aromatic compounds. Bacterial strains of

different phyla including proteobacteria, Actinobacteria, Firmicutes, and archaea are

reported for lignin and aromatic dyes degradation [216]. However, Bacillus are one of the

high diverse genera in the class bacilli. They are consistently inhibited in many extreme

niches. It includes rod-shaped, gram-positive, aerobic and facultative anaerobic.

Moreover, Bacillus sp. strains are reported for a wide range of applications

predominantly for the production of industrial compounds [231]. Bacillus sp. strains

displaying an enzymatic repertoire of cellulase, lipase, protease, amylase, xylanase, and

pectinase which access them to grow on complex organic compounds [81]. These

Bacillus sp. strains predominantly decolorize and degrade lignin and synthetic dyes [41,

42, 141, 142].

Lignin degradation is associated with the optimum pH, the maximum lignin

degradation efficiencies can be associated to the optimum activity of the lignin degrading

enzyme activities and can be further improve by addition of growth supplements [232].

The enzymes of these Bacillus sp. strains showing maximum activity of lignin peroxidase

and laccase at acidic pH 3-5 [217]. Biological degradation of agriculture biomass with

naturally degrading bacterial strains are recognized pretreatment process [120]. It is

beneficial to do the treatment process of waste biomass through biological treatment,

because it strip the matrix of hemicelluloses and lignin, lessen the loss of carbohydrate

and generation of inhibitory compounds [10]. Both aerobic and anaerobic lignin

degrading microbial consortia and enzymes mixture pretreatment is known a useful tool

for biomass hydrolysis [233]. Screening of robust hydrolytic microbial consortium could

be assess to degrade lignin during anaerobic digestion for biomethane production [53].


131

Several aerobic Bacillus sp. strains namely Bacillus subtilis, Bacillus megatarium,

Bacillus ligniniphilus L1 showing lignin degradation [121, 141, 142, 234].

The combine reaction of co-inoculation of Bacillus sp. strains contribute more to

degrade RS residue particularly lignin and increase biogas production [49]. Bacterial

strains showing active lignin degradation from straw and helps in anaerobic digestion

process [219]. The improvement in biomass hydrolysis needs assessment of advance

technology, robust isolates, and synergistic use of hydrolytic enzymes for production of

biofuels from lignocellulosic biomass [235]. Beside of alkali pretreatment, bacterial

strains also proving in biomass hydrolysis [236]. The hydrolyzed and easily accessible

carbohydrate component (cellulose) is then rapidly transform in to biogas [223]. High

lignin degradation rates of Bacillus sp. strains indicate their potential utility in

pretreatment of waste biomass hydrolysis for bioenergy production. Further optimization

studies are necessary regarding nutrients, metabolism of aromatic compounds, and co-

inoculation of these strains to improve lignin degradation. The current study suggest that

combine bioprocessing of ligninolytic Bacillus sp. strains co-inoculation might be a cost-

effective pretreatment method to remove lignin from waste biomass for biogas

production.

4.3 Biohydrogen and Biomethane Batch Fermentation by Ligninolytic

Bacillus Sp. Strains

Indigenous ligninolytic bacterial strains are present in a natural enviromental sources,

however the isolation and selection of robust strains as a biocatalyst for lignin

degradation needs to considered. Fungal treatment took more time period in hydrolysis of

biomass, whereas bacterial pretreatment are effective because of fast growth rate and

potential of lignin degradation [122]. In the current thesis (section 3.6), out of 20 isoaltes,

the four selcted strains were Bacillus sp. strains. These strains were lignin degrader and a

source of extracellular enzymatic activities against various substrates. Bacillus sp. strains

are well known for biohydrogen production from glucose, starch and organic fraction of

municipal solid waste [81]. Bacillus sp. strains has been also reported for growth and

degradation of aromatics compounds under aerobic and anaerobic condition [83] and

proved as one of the most predominant species demonstrating decolorization of lignin


132

and synthetic dyes [42]. Bacillus sp. strains of the current study displayed lignin and

Azure B dye decolorization up to 80-90%. Similar to our observation, lignin and dye

decolorization are also reported with different bacterial strains. A bacterial strain i.e

Acetoanaerobium sp. WJDL-Y2 have been reported with 25% COD lignin

decolorization, however the authors used a higher lignin concentration in the medium

[237]. Simialr to our results of Bacillus sp. strains lignin and Azure B dye decolirization,

an 83% decolorization of black liquor of paper industry is reported by bacterial strains,

Citrobacter sp., Serratia marcescens and Klebsiella pneumoniae [238]. Likewise, three

aerobic bacterial strains, Aneurinibacillus aneurinilyticu, Paenibacillus sp. and Bacillus

sp. supplemented with glucose and peptone as extra carbon and nitrogen sources have

been reported with a lower 30-37% lignin degradation and COD reduction after one week

of incubation time [141, 239].

The four Bacillus sp. strains of the present study were positive for lignin

peroxidase (LiP) and laccase (Lac), both are important types of enzymes involved in

lignin degradation. The enzyme activity and decolorization potential of Bacillus sp.

strains are proved in also previous studies with minor changes in conditions [217, 218].

Bacillus species having multipurpose enzymatic activities are identified for H2 production

as an individual culture [81]. The four Bacillus sp. strains of the present study produced

H2 with different yields from xylose and cellulose. The potential of hydrogen production

from xylose was important, because the composition of hemicellulose constitutes 20-30%

xylose which is a pentose sugar of the wheat straw and other agribiomass. H2 production

from xylose fermentation is reported with a final product either acetate or butyrate using

synthetic cell free enzyme cascade [240]. It is also reported, that xylose as a substrate

produce fewer inhibitory compounds because xylose can be consumed and metabolized

by other H2 producing microorganism in fermentation system [241].

The results of the current study are comparable with the H2 yield of earlier

reported Bacillus sp. [188]. Instead of difference in H2 volumetric rate and H2 yield from

cellulose and xylose, a previous study concluded glucose, sucrose, maltose, lactose, and

cellulose as an ideal substrate for the hydrogen production [242]. In an early reported

study, the performance of cumulative H2 yield was comparable to cumulative hydrogen


133

yield of 68.1 ml H2/g TVS, however the author used HCl pretreated wheat straw and cow

dung in batch fermentation experiment [147]. The cumulative H2 yield of the current

study was higher than the monoculture of C. thermocellum reported with cumulative 61.4

mL/g of H2 from untreated cornstalk waste, however, the authors obtained a hydrogen

yield of 75 mL of H2/g when co-culture of C. thermocellum and

C.thermosaccharolyticum were used [243, 244].

The average H2 yield from the untreated agribiomass of 1.41 mmol H2/g have

been reported [241]. Although a similar study shown a higher 1.5 mol-H2/g from wheat

grain using co-culture of Bacillus licheniformis JK1 and Enterobacter [245]. The high

percentage of VFA production is consistent with the earlier reported study, proved that

H2 producing microorganisms has similar metabolic pathways [246, 247]. Beside of

chemical treatment, biological treatment of wheat straw different basidiomycetes have

been reported as lignin degrading and pretreatment agent for delignification and

increasing of biogas yield [248, 249]. Similarly, mixed bacterial culture enriched in

minimal medium under semi-anaerobic mesophilic conditions has also shown substantial

effect on methane yield from wheat straw [117]. Whereas, several studies have isolated

and screened lignin degrading bacterial sp. strains [41, 42, 45], however none of the study

reported biohydrogen potential and improvement of biomethane from wheat straw using

anaerobic lignin degrading bacteria. Recently, in a similar study, addition of 4% enriched

inoculum of cellulolytic rumen microbiota has shown only 27 % increase of methane

yield from wheat straw [250], which is less than the combine yield percentage of H2 and

CH4 yield from wheat straw of the current study. Similarly, 37% increase in methane

yield was obtained from ligninolytic fungal pretreated wheat straw compare to untreated

wheat straw, with a longer time of pretreatment incubation 4-10 weeks [251].

4.4 Preatment with Recombinant Neurospora crassa F5 strain

To extend the objective of selecting an inexpensive pretreatment process and hydrolyzed

the cellulosic material before fermentation reaction [3,4,5]. Additionally, in biological

pretreatment the induction of recombinant fungi has significant potential in direct

enzymatic hydrolysis because fungal enzyme machinery is more powerfull than bacterial

strains [4,6]. So far the most reported studied about delignification from waste biomasses


134

is reported with white-rot and brown fungi [6,7,8,9]. Fungal strains can utilized their

enzmyes mechanism consist of laccase, manganese peroxidase, lignin peroxidase,

hydrolytic, and cellulolytic enzymes (cellulases, cellobiohydrolase, and endoglucanase)

to degrade lignin and similar aromatic compounds during waste biomass breakdown

[161]. But the limitation in fungal treatment is that, the fungal culture takes several weeks

for growth and hydrolysis [37]. In this thesis the consolidating bioprocessing (CBP)

pretreatment of recombinant Neurospora crassa F5 strian was tested for wheat straw

hydrolysis [39]. Recombinant N. crassa F5 strain combined pretreatment hydrolysis and

anaerobic digestion of waste biomass using waste residue of wheat straw [252].

In this study, using recombinant N. crassa F5 strain degradation of the wheat

straw was also confirmed using SEM, FT-IR and XRD analysis. But interstingly, the

wheat straw samples that was treated for 2 days has shown more biogas yeild than 4 and

6 days treated wheat straw sample. The total biogas yeild of wheat straw sample that was

treated with 2% NaOH and followed by N. crassa F5 hydrolysis for 2 days significantly

increased 339.3% biogas yeild than the untreated wheat straw sample. So the

consolidated bioprocessing of wheat straw with recombinant N. crassa F5 strain

significantly introduced shorter hydrolytic retention time in the hydrolysis process.

Recombinant N. crassa F5 strain results demonstrat that innovative treatment is

necessory for effective and economical anaerobic technology development [251].

4.5 Bio-Hydrogen from the Organic Fraction of Municipal Solid Waste

One hundred and twenty microbial strains were previously isolated and identified from

samples of heat-treated granular sludge used to perform hydrogen production batch tests

[88]. All isolates were positive for extracellular enzymes and exhibited a broad range of

hydrolytic activities. This outcome could be explained considering that the strains have

been isolated from an anaerobic digester of a brewery whose fed by-products are usually

rich in starch, pectin and protein [253]. In literature, indeed, Bacillus species are known

as strong candidates for biological H2 production because (i) they can survive under harsh

conditions, hence could compete with other microbes (ii) they have large and versatile

enzymatic activities, therefore a diverse range of bio-waste could be used as substrate for

bio-hydrogen production, (iii) they do not require light for H2 production, (iv) Bacillus


135

sp. spores are being used as probiotics in humans and animals; thus, they may not pose

environmental health concerns [188, 189].

Out of 120 strains, the most efficient strains, Bacillus sp. F2.5 and F2.8, were

hydrogen producer using glucose as substrate, also these strains were capable of amylase

production and starch hydrolysis and could be very useful for the H2-conversion of food

waste, where starch can account up to 30% of the TS [180, 254, 255]. The optimum

catalytic ability of Bacillus sp. F2.5 was found at pH 6.0 whereas, the highest amylase

activity of Bacillus sp. F2.8 was at the pH 7.0-8.0. These findings are in accordance with

those described in literature regarding Bacillus sp. amylases, where the optimal pH values

were reported to be within the broad range of 3.5-12 and the pH was found to deeply

affect their catalytic activity on starch [256-258]. Both strains had high relative activity at

50°C and their optimal temperature values were lower than those usually reported for

other Bacillus sp. amylases (60-70 °C) [257, 259-261]. Overall, Bacillus sp. F2.5 and

F2.8 produced amylase with high potential with enzymatic activities comparable to those

recently reported by efficient amylolytic Bacillus sp. strains [188, 257].

. Interestingly, although the two strains had similar starch-to-H2 efficiency, Bacillus

sp. F2.8 showed H2 potential from glucose lower than Bacillus sp. F2.5 (Table 3-18).

This could be associated with the most efficient starch-degrading activity described for

Bacillus sp. F2.8 at pH 7.0 (Figure 3-39 a). Nevertheless, both strains exhibited

promising H2 yields which were found to be comparable with those described in literature

mainly by mixed consortia [262, 263]. The highest H2 yield from starch reported so far

by a strain belonging to the Bacillus genus was recently disclosed as 0.70 mol H2 per mol

of reducing sugar [264]. On the other hand, as reported in (Table 3-19), both Bacillus sp.

strains described in the present work showed productivity (about 4 mL of H2 per day)

lower than those found in other studies on H2 production from starch. However, their

limited H2 production rate, which could be mainly influenced by their low inoculum size

and static incubation, are likely to be improved by optimizing the growth conditions and

other environmental factors such as micronutrients availability, buffers and temperature

which were reported as key parameters to boost H2 productivity [262, 263, 265].

Hydrogen levels produced in this study were consistent with those previously


136

described for batch H2 fermentation of OFMSW or food waste by using pure or mixed

cultures (Table 3-20). Further, in the present study, inoculum pre-treatment inoculum was

not required. Moreover, as described in Table 5, this is one of the earliest accounts on a

single microbe capable of converting organic waste into H2 with a high rate and yield.

Only recently, Marone and colleagues described few Enterobacteriaceae strains, isolated

by the bioaugmentation of vegetable waste (Rahnella sp. 10, Buttiauxella sp. 4 and

Raoultella sp. 47), for their promise in producing H2 from vegetable kitchen waste

collected from a cafeteria [183]. However, this is the first successful application of pure

microbial cultures in bio-hydrogen production from OFMSW. Furthermore, both Bacillus

sp. strains exhibited high starch-degrading activities meanwhile the above reported

microbes did not produce any relevant hydrolytic enzymes [183].

Their spore-forming ability and their being isolated from granular sludge of a full

scale UASB anaerobic digester are two additional noteworthy benefits which count for

the potential development of Bacillus sp. F2.5 and F2.8 as efficient and robust inoculants.

4.6 Summary

The aim of thesis was to study different approaches consisting of chemical and biological

treatment to increase bioenergy production using agriculture waste biomass. In each case

anaerobic digestion processes were the final estimation system to conclude the efficiency

of tested approach. The overall theme of the thesis is the production of biomethane and

biohydrogen from organic waste biomass through anaerobic digestion processes. The

scientific works and previous literature undertaken for the digestion of agriculture waste

biomass using chemical and biological treatment highlighting the importance of the

research work in this thesis for renewable energy production. The thesis comprised of the

journal paper and academic thesis model of the Pakistan Institute of Engineering and

Applied Sciences (PIEAS), that is a sequence of submitted or published journal papers

that can be read for full understanding.

The results (chapter 3, section 3.1-3.1.7) of the thesis is emphases on the

importance of alkalies treatment (NaOH, KOH and Ca(OH)2) among the other chemicals

treatment approaches for lignin degradation and biomethane yield. NaOH with short time


137

microwave heating process had the highest delignification of 70-86% as compared to

KOH, Ca(OH)2 and autoclave and water bath heating processes. Scanning Electron

Microscophy (SEM) and Fourier Transform Infrared Spectroscophy (FT-IR) results

showed a visible degradation effect of alkalies on biomass surface. These akalies

treatment increased 2-times the output of cumulative biogas from pretreated substrate

compared to the cumulative biogas produced from the untreated substrate. It is

summarised that alkalies treatment of agriculture waste biomass is necessory with

thermal heating for increasing potential of biogas.

Lime (Ca(OH)2) soaking treatment (chapter 3, section 3.2-3.2.3) on corn cob for

different time 7, 15, and 30 days of incubation was tested. In the SEM micrograph a clear

destruction in the morphology of treated corn cob was noticed compared to untreated

corn cob. Further, the best cumulative biogas and methane yield was obtained for 30 days

soaking incubation time. This study summarised that soaking with Ca(OH)2 for longer

time is effective for increasing digestibility of agriculture waste biomass and improving

biogas production.

Biological pretreatment of rice straw (chapter 3, section 3.3-3.3.9), seven

ligninolytic Bacillus sp. strains out of 27 isoaltes were selected based on their robustness

for lignin degradation. These strains were producing ligninolytic enzymes and were able

to degrade lignin from rice straw. It was observed that mixed combinations of these

seven ligninolytic Bacillus sp. strains was more effective than using individual strain.

Overall, the study demonstrates the potential of using these Bacillus sp. strains as robust

biocatalysts for processing lignocellulosic waste biomass.

Biodegradation of wheat straw to biohydrogen and biomethane was explored

using two separate biohydrogen and biomethane batch fermentation by ligninolytic

Bacillus sp. strains. Four ligninolytic Bacillus sp. strains were selected out of 20 isolates

from granular sludge of full scale anaerobic digester based on their lignin and Azure B

degradation. These strains were capable of lignin and Azure B dye COD reduction. The

strains were also found to show hydrogen (H2) potential from xylose and cellulose. In

two-phase wheat straw batch fermentation, it was perceived that using ligninolytic

Bacillus sp. strains, 48.6% more methane yield could be obtained from the wheat straw


138

than using than the untreated wheat straw in batch fermentation. This is the first study

establishing not only the hydrogen potential of ligninolytic Bacillus sp. strains but also

indicates a vital role of these species for developing standard inoculum and a biocatalyst

for processing agribiomass.

A consolidating bioprocessing of recombinant Neurospora crassa F5 strain was

tested for saccharification of wheat straw (WS) to increase biogas production. The 2,4,

and 6 days treated samples of WS with recombinant N. crassa F5 strain, the 2 days

treatment increase 6-time daily biogas volume and 339.3% more in cumulative volume

than the untreated WS samples as compared to 4 and 6 days treatment. This treatment

introduced that shorter hydrolysis time is optimum for increasing of biogas production

from waste biomasses.

Bio-hydrogen, obtained by fermentation of organic residues, is considered a

promising source of renewable energy. However, the industrial scale H2 production from

organic waste is far to be realized as technical and economical limitations have still to be

solved. Low H2 yields and lack of industrially robust microbes are the major limiting

factors. To look for bacteria with both interesting hydrogen fermentative traits and proper

robustness, granular sludge from a brewery full scale Upflow Anaerobic Sludge Blanket

(UASB) digester was selected as trove of microbes processing complex substrates.

Bacterial strains isolated from heat-treated granular sludge were containing a reportiar of

multiple extracellular hydrolytic enzymes. Two Bacillus sp. strains, namely F2.5 and

F2.8 were the most active hydrolytic strains showed H2 potential from glucose, soluble

starch and Organic Fraction of Municipal Solid Waste (OFMSW). The strains could be

apply as a respectable inoculants comercially for H2 production. This is the first

successful application of pure microbial cultures in bio-hydrogen production from

OFMSW.

4.7 Future Prospect

This dissertation proved two very important strategies to be consider in future application

of anaerobic technology. The alkalies especially of dilute NaOH treatment method needs

to be tested at commercial scale to evaluate its economical feasibility for processing of


139

lignocellulosic waste biomass. The natural lignin degrading bacteria can also be applied

as as biocatalyst tool for processing of agriculture waste biomass at large scale. However,

still the microorganisms of full scale-scale digester is not fully explored, as the inoculum

in each digester is different plus the variations in physiology and nutrition can change the

community of micorflora [59]. Thus, it is important to isolate even more robust and

intersting microorganism for biohydrogen and biogas production [266]. Secondly,

consolidated biological pretreatment of ligninolytic bacteria follow by simultaneous

operation of the two-stage AD process needs optimisation process in every aspect so that

it can be practicable as an economical method for biohydrogen and methane production

[138, 139]. In future, one of the key requirment is also to build a standard inoculum of

robust microflora to convert organic waste into biogas for industrial scale to avoid

continuous dependence on animal dung [164]. Finally, the selection of robust culture

needs more information about the conditions, carbon/nitrogen source and identification of

isolation source in future research advancement [177, 178, 185].

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