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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
ii
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
2.3.9 Scanning Electron Microscopy ............................................................ 31
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
2.5.5 Scanning Electron Microscopy ............................................................ 38
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.
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.
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.
Chapter 1: Introduction
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.
Chapter 1: Introduction
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
Chapter 1: Introduction
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].
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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.
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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.
Chapter 1: Introduction
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
Chapter 1: Introduction
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].
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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
Chapter 1: Introduction
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.
Chapter 1: Introduction
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].
Chapter 1: Introduction
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.
Chapter 1: Introduction
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
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
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Where Ai = initial absorbance at first day, Af = final absorbance at last day
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
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
31
2.3.9 Scanning Electron Microscopy
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].
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
34
Ai
Where Ai = initial absorbance at first day, Af = final absorbance at last day
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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].
2.5.5 Scanning Electron Microscopy
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.
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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
Chapter 2: Materials and Methods
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].
Chapter 2: Materials and Methods
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°
Chapter 2: Materials and Methods
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
(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
55
Figure 3-6 Comparative delignification of Ca(OH)2 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.
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)
Fermentation time (d)
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
3.2.2 Scanning Electron Microscopy
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
Fermentation time (d)
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
Fermentation time (d)
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
Fermentation time (d)
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
Fermentation time (d)
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
Bacillus sp. strain TL6 1.7 92.0 96.5 2.99 2.46
Bacillus sp. strain TL8 0.7 65.0 66.9 1.11 1.12
Bacillus sp. strain TL24 0.7 62.0 65.5 1.10 1.16
Bacillus sp. strain TL26 1.6 80.8 87.6 2.74 2.33
Bacillus sp. strain TL27 0.8 65.6 62.6 1.13 1.14
Bacillus sp. strain TL33 0.8 67.8 68.0 1.12 1.38
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
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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
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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.
Fermentation time (d)
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
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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.
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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.
Fermentation time (d)
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.
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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.
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
Fermentation time (d)
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.
Fermentation time (d)
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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,
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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].
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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
Chapter 4: Discussion
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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