tmpC1B4 TMP

ENRICHMENT OF METHANOGENIC MICROCOSMS
ON RECALCITRANT LIGNOCELLULOSIC BIOMASS
by
Michael Lacourt
A thesis submitted in conformity with the requirements

for the degree of Master of Applied Science
Department of Chemical Engineering and Applied Chemistry
University of Toronto
Copyright by W. Michael Lacourt 2011
Abstract
ENRICHMENT OF METHANOGENIC MICROCOSMS

ON RECALCITRANT LIGNOCELLULOSIC BIOMASS
Masters of Applied Science, 2011
William Michael Lacourt
Department of Chemical Engineering and Applied Chemistry
University of Toronto
To improve biogas production from lignocellulosics, methanogenic microbial
enrichments were prepared from moose rumen fluid, beaver droppings, and internal circulation
(IC) reactor granules amended with cellulose, pine needles, lignosulphonate, tannic acid, and
poplar hydrolysate. Tannic acid delayed methanogenesis compared to cellulose only
enrichments, both by reducing initial rates (up to 50% in beaver dropping cultures) and
increasing lag times (up to 50 days in moose rumen cultures). Biogas yields from poplar
hydrolysate were 56 % by beaver droppings, 51% % by IC granules, and 31 % by moose rumen
enrichments. Bacterial community profiles, determined via denaturing gradient gel
electrophoresis (DGGE) showed divergent populations between enrichments. Enrichment on
pine needles or poplar hydrolysate promoted bioconversion of post extraction wash (PEW), and
beaver dropping enrichments fed pine needles equaled the yield (about 23%) from IC granules.
Together, the DGGE and PEW results provide evidence of acclimatization to previously
recalcitrant feeds.
ii
Acknowledgements
To all the great people who helped make this project happen:
My supervisors, Professors Emma Master and Honghi Tran
The Ontario Genomics Institute and the Alternative Fuels for Lime Kilns consortium
My committee members, Professors Elizabeth Edwards and Grant Allen
Collaborators, colleagues, and support staff:

o Ivy Yang
o Liane Catalfo, Hai Tran, Ann Zheng, Angie Tse, Torsten Meyer
o Lyle Biglow, Renee Brunelle, Eric Duschene, Richard Mayhew, and all other
Temiscaming site personnel
o Paul Jowlabar, Mel Duhamel, Line Lomheim, Christina Heidorn, Endang
Susilawati, and Anna Ho
Members of the Master, Edwards, Mahedevan, Allen, Tran, and Biozone labs
My friends and family

o Mom, Dad, and Iain, for all your generosity and support
o Friends too numerous to list: you guys are the best!
Thank you!
iii
Table of Contents
Abstract ........................................................................................................................................... ii
Acknowledgements........................................................................................................................ iii
Table of Contents........................................................................................................................... iv
List of tables.................................................................................................................................. vii
List of figures............................................................................................................................... viii
List of appendices .......................................................................................................................... xi
Abbreviations................................................................................................................................ xii
1.
2.
3.
Introduction............................................................................................................................. 1
1.1.
Background ..................................................................................................................... 1
1.2.
Objectives ....................................................................................................................... 2
Literature survey ..................................................................................................................... 4

2.1.
Lignocellulosic biomass.................................................................................................. 4
2.2.
Anaerobic digestion ........................................................................................................ 5
2.2.1.
Hydrolysis ............................................................................................................... 7
2.2.2.
Fermentation ........................................................................................................... 8
2.2.3.
Methanogenesis....................................................................................................... 9
2.2.4.
Inhibitors of digestion process .............................................................................. 10
2.2.4.1.
Tannic acid.................................................................................................... 10
2.2.4.2.
Lignin............................................................................................................ 13
2.2.4.3.
Pine Needles.................................................................................................. 14
2.3.
Biogas from woody biomass......................................................................................... 18
2.4.
Metagenome studies of digestive system lignocellulose degrading communities........ 19
Materials and Methods.......................................................................................................... 22

3.1.
Inoculum collection and storage ................................................................................... 22
3.1.1.
Moose rumen fluid................................................................................................ 22
3.1.2.
Beaver droppings .................................................................................................. 22
3.1.3.
Internal circulation reactor granules ..................................................................... 22
3.1.4.
Other inoculum sources ........................................................................................ 22
3.1.4.1.
Heritage pile.................................................................................................. 23
3.1.4.2.
Landfill and leachate..................................................................................... 23

iv
3.2.
4.
3.2.1.
Solids analysis....................................................................................................... 24
3.2.2.
Chemical oxidation demand ................................................................................. 24
3.2.3.
Fourier transform infrared spectroscopy (FTIR) .................................................. 25
3.2.4.
Gas volume measurements ................................................................................... 26
3.2.5.
Headspace CH4 content......................................................................................... 27
3.3.
Nutrient medium ........................................................................................................... 29
3.4.
Feedstock preparation ................................................................................................... 29
3.5.
Microcosm setup........................................................................................................... 31
3.6.
Dilution and transfer of cultures ................................................................................... 32
3.7.
Biochemical analysis .................................................................................................... 38
3.7.1.
Deoxyribonucleic acid (DNA) extraction............................................................. 38
3.7.2.
Amplification of 16 S rDNA................................................................................. 38
3.7.3.
Denaturing gradient gel electrophoresis (DGGE)................................................. 40
Results and Discussion ......................................................................................................... 44

4.1.
Setup and transfers........................................................................................................ 44
4.2.
Inoculum Properties ...................................................................................................... 48
4.3.
Substrate Properties ...................................................................................................... 49
4.4.
Gas Production Results ................................................................................................. 53
4.4.1.
Phase I Enrichments.............................................................................................. 54
4.4.2.
Phase II and III Enrichments................................................................................. 60
4.4.2.1.
Time to 20 % COD consumption ................................................................. 61
4.4.2.2.
Biogas yield .................................................................................................. 64
4.4.2.3.
CH4 fraction .................................................................................................. 67
4.4.3.
Phase IV Enrichments........................................................................................... 72
4.4.4.
Phase II to IV: Comparison of Concentrated Additive Degrading Microcosms .. 75
4.4.5.
Phase V Enrichment: Bioconversion of Post Extraction Washer (PEW) Effluent79
4.5.
5.
Measurement of physical and chemical properties....................................................... 24
Analysis of microbial inocula and enrichments by DGGE........................................... 81
4.5.1.
DNA extraction and concentration ....................................................................... 81
4.5.2.
DGGE ................................................................................................................... 83
Scale up................................................................................................................................. 86
5.1.
Reactor design and operating conditions ...................................................................... 86
5.2.
Reactor operation .......................................................................................................... 89

v
6.
Conclusions and Implications ............................................................................................... 91
7.
Recommendations................................................................................................................. 94
8.
References............................................................................................................................. 95
vi
List of tables
Table 2.1. Fermentation reactions................................................................................................... 8

Table 2.2 Free energy of methanogenesis....................................................................................... 9
Table 2.3. Tannin content of North American trees. .................................................................... 11
Table 2.4. Effect of tannins on methanogenesis. .......................................................................... 13
Table 2.5. Effect of lignosulphonates on methanogenesis............................................................ 14
Table 2.6. Extractives reported in Pinus resinosa. ....................................................................... 15
Table 2.7. Toxicity of Resin Acids ............................................................................................... 17
Table 2.8 CH4 yield from woody biomass.................................................................................... 18
Table 2.9. Carbohydrate active enzymes database ....................................................................... 20
Table 3.1. Phase I amendments: initial microcosms..................................................................... 33
Table 3.2. Phase II and III amendments: expanded substrate range enrichments ........................ 34
Table 3.3. Phase IV amendments: best performing enrichment transfer...................................... 36
Table 3.4. DGGE denaturant concentrations ................................................................................ 41
Table 4.1. Microcosm setup and transfer timeline........................................................................ 44
Table 4.2. Substrate COD and VS ................................................................................................ 49
Table 4.3 FTIR peak identification table. ..................................................................................... 50
Table 4.4. Biogas produced in no amendment (NA) reference cultivations................................. 58
Table 4.5 Replicate number from Phase III triplicates used as inoculum for Phase IV enrichments
....................................................................................................................................................... 72
Table 4.6 DNA Concentrations..................................................................................................... 82
Table 4.7 Shared DGGE bands..................................................................................................... 84
Table 5.1 Important parameters for lab scale continuous anaerobic reactors............................... 87
Table A.1 Mineral medium composition ................................................................................ A-103
Table H.1 DGGE bands of microcosm enrichments .............................................................. H-125
vii
List of figures
Figure 2.1. Anaerobic digestion process adapted from Droste [12]. .............................................. 6
Figure 2.2. Schematic representation of a cellulosome. ................................................................. 8
Figure 2.3. Example molecular structure of tannic acid. .............................................................. 12
Figure 2.4. Proposed pathway for the biodegradation of tannic acid as per Nelson et al. [36] .... 12
Figure 2.5. Resin acids identified in pulp and paper wastewaters. ............................................... 16
Figure 2.6. Resin acids of Pinus resinosa needles. A) Manoyl oxide acid, B) Epimanoyl oxide
acid, C) Communic acid, D) Imbricataloic acid. .......................................................................... 17
Figure 3.1. Microcosm composition ............................................................................................. 32
Figure 3.3. Example transfer lineage ............................................................................................ 37
Figure 3.4. DGGE Apparatus........................................................................................................ 41
Figure 4.1 Lineage of A) moose rumen (MR), B) beaver dropping (BD), and C) internal
circulation granules (ICG) enrichment cultures............................................................................ 47
Figure 4.2 COD content in environmental inocula....................................................................... 48
Figure 4.3. FTIR spectra of each substrate ................................................................................... 51
Figure 4.4 Lignocellulose associated FTIR profile peaks. ........................................................... 52
Figure 4.5 Example gas production curve .................................................................................... 54
Figure 4.6 Initial rates of biogas production in Phase I microcosms............................................ 55
Figure 4.7 Biogas yield from Phase I A) moose rumen, B) beaver dropping, C) landfill-leachate,
and D) heritage pile microcosms .................................................................................................. 57
Figure 4.8 CH4 fraction of biogas generated by A) moose rumen, B) beaver dropping, and C)
heritage pile................................................................................................................................... 59
Figure 4.9 Initial rates of biogas production in Phase II and Phase III microcosms .................... 62
Figure 4.10 Initial rates of biogas production in Phase II and Phase III microcosms .................. 63
Figure 4.11 Initial rates of biogas production in Phase II and Phase III microcosms .................. 64
Figure 4.12 Biogas yield from Phase II and Phase III microcosms.............................................. 65
Figure 4.15 CH4 fraction of biogas generated by Phase II and Phase III ..................................... 68
Figure 4.16 CH4 fraction of biogas generated by Phase II and Phase III enrichments................. 69
Figure 4.17 CH4 fraction of biogas generated by Phase II and Phase III enrichments................. 70
viii
Figure 4.18 Initial rates of biogas production in Phase IV microcosms....................................... 73

Figure 4.19 Biogas yield from Phase IV microcosms .................................................................. 74
Figure 4.20 Phase IV CH4 fractions.............................................................................................. 75
Figure 4.21 Initial rates of biogas production from the top performing replicates....................... 76
Figure 4.22 Biogas yields from the top performing replicates. .................................................... 77
Figure 4.23 CH4 fraction from the top performing replicates....................................................... 78
Figure 4.24 Biogas yield from A) moose rumen, B) beaver dropping, and C) internal circulation
granules Phase V microcosms amended with post extraction washer (PEW) wastewater........... 79
Figure 4.25 DGGE profiles of environmental inocula: internal circulation granules (A), beaver
droppings (B), and moose rumen (C). .......................................................................................... 83
Figure 4.26 DGGE profiles of (from left to right) enriched moose rumen, beaver droppings, and
internal circulation granules.......................................................................................................... 85
Figure 5.1 Lab scale anaerobic reactor ......................................................................................... 89
Figure 5.2 Lab scale reactor startup phase gas production ........................................................... 90
Figure D.1 Phase I moose rumen, cellulose and VFA gas production curves........................ D-108
Figure D.2 Phase I moose rumen, cellulose and additives gas production curves. ................ D-108
Figure D.3 Phase I beaver droppings, cellulose and VFA gas production curves.................. D-109
Figure D.4 Phase I beaver droppings, cellulose and additives gas production curves. .......... D-109
Figure D.5 Phase I heritage pile, cellulose and VFA gas production curves.......................... D-110
Figure D.6 Phase I heritage pile, cellulose and additives gas production curves. .................. D-110
Figure D.7 Phase I landfill-leachate, cellulose and VFA gas production curves. .................. D-111
Figure D.8 Phase I landfill-leachate, cellulose and additives gas production curves. ............ D-111
Figure E.1 Phase II, III moose rumen, cellulose and poplar hydrolysate gas production curves. .E112
Figure E.2 Phase II, III moose rumen, cellulose and concentrated additives gas production
curves. ......................................................................................................................................E-112
Figure E.3 Phase II, III moose rumen, cellulose and low concentration additives gas production
curves. ......................................................................................................................................E-113
Figure E.4 Phase II, III moose rumen, poplar hydrolysate additives gas production curves...E-113
Figure E.5 Phase II, III beaver droppings, cellulose and poplar hydrolysate gas production
curves. ......................................................................................................................................E-114
Figure E.6 Phase II, III beaver droppings, cellulose and concentrated additives gas production
curves. ......................................................................................................................................E-114
ix
Figure E.7 Phase II, III beaver droppings, cellulose and low concentration additives gas
production curves.....................................................................................................................E-115
Figure E.8 Phase II, III beaver droppings, poplar hydrolysate additives gas production curves...E115
Figure E.9 Phase II, III internal circulation granules, cellulose and poplar hydrolysate gas
Figure E.10 Phase II, III internal circulation granules, cellulose and concentrated additives gas
Figure E.11 Phase II, III internal circulation granules, cellulose and low concentration additives
gas production curves. .............................................................................................................E-117
Figure E.12 Phase II, III internal circulation granules, poplar hydrolysate additives gas
Figure F.13 Phase IV moose rumen gas production curves.....................................................F-118
Figure F.14 Phase IV beaver droppings gas production curves...............................................F-119
Figure F.15 Phase IV internal circulation granules gas production curves. ............................F-119
List of appendices
Appendix A. Mineral Medium Composition .......................................................................... A-103

Appendix B. CH4 in biogas produced script........................................................................... B-104
Appendix C. Detailed DNA Extraction Protocols .................................................................. C-105
Appendix D. Phase I Gas Production Curves ......................................................................... D-108
Appendix E. Phase II, III Gas Production Curves ...................................................................E-112
Appendix F. Phase IV Gas Production Curves........................................................................F-118
Appendix G. Sample Calculations.......................................................................................... G-120
Appendix H. DGGE bands identified with Image J ............................................................... H-125
Appendix I. Pressure Transducer Calibration...........................................................................I-126
xi
Abbreviations
APHA
American Public Health
IC
Internal circulation
ICG
Internal circulation reactor granules
Association
BCTMP
Bleached
chemithermomechanical pulp
BD
Beaver droppings
MR
Moose rumen
BMP
Biochemical methane potential
VFA & glucose substrate mix
BSA
Bovine serum alvumin
PCR
Polymerase chain reaction
Avicel PH-101 microcrystalline
PEW
Post extraction-washer
cellulose
CBM
Carbohydrate binding module
PH
Poplar hydrolysate
CL
Avicel plus sodium
PH
Steam exploded poplar hydrolysate
lignosulphonate
COD
Chemical oxidation demand
RNA
Ribonucleic acid
CP
Avicel plus ground red pine
SDS
Sodium dodecyl sulphate
needles
CT
Avicel plus tannic acid
SSL
Spent sulphite liquor
CTAB
Cetyltrimethylammonium
TAE
40mM Tris acetate, 1mM EDTA
TCD
Thermal conductivity detector
bromide
DGGE
Denaturing gradient gel

electrophoresis
DNA
Deoxyribonucleic acid
TEMED
Tetramethylenediamine
dNTP
Deoxynucleotide triphosphates
TS
Total solids
EDTA
Ethylenediaminetetraacetic acid
TSS
Total suspended solids
FID
Flame ionization detector
UASB
Upflow anaerobic sludge bed
FTIR
Fourier transform infrared
VS
Volatile solids
VSS
Volatile suspended solids
spectroscopy
GC
Gas chromatograph
HPLC
High performance liquid

chromatography
HW
Poplar hydrolysate
xii
1.
Introduction
1.1. Background
During anaerobic digestion, microorganisms convert complex organic substrates to

biogas in the absence of molecular oxygen. This process mainly involves the following microbial
activities: hydrolysis and fermentation processes catalyzed by bacteria, and conversion of
organic acids, hydrogen (H2) and carbon dioxide (CO2) to methane (CH4) by methanogenic
Archaea. This microbial activity has been employed for decades to treat municipal waste water;
the design of high-rate upflow anaerobic sludge blanket (UASB) reactors in the 1970s was
instrumental to industrial adoption of this technology. The advantages of anaerobic processes
over conventional aerobic treatment systems include the production of CH4 to off-set natural gas
consumption, and up to 90% reduction in secondary sludge production [1].
In recent years there has been renewed interest in anaerobic bioprocesses that generate
biofuels due to increasing fossil fuel costs and a push to reduce greenhouse gas emissions.
Biogas, a mixture containing roughly 50-70 % CH4 and 30-50 % CO2, is one such fuel.
Lignocellulosic biomass, including agricultural and forest industry residuals, has been identified
as an ideal feedstock for biofuel production due to its abundance, renewability, and high energy
content. However, lignocellulosic feedstocks are frequently recalcitrant to high-rate
bioconversion technologies (or difficult to digest anaerobically), while necessary pretreatment
processes are often expensive. Pretreatment processes and various wood species can also release
compounds that are inhibitory to anaerobic processes [2], including tannic acid [3] and resin
acids [4].
Over the last four years, previous graduate thesis projects have investigated i) anaerobic
conversion of evaporator acid condensates and waste streams from BCTMP mills ii) the impact
of pretreatment on the solubilization and anaerobic conversion of pulp mill secondary sludge,
and iii) operating variables affecting the performance of anaerobic reactors using multivariate
data analysis. These studies have identified bioaugmentation as a means of reducing the impact
of inhibitory substances on anaerobic reactor performance and stability, while increasing the
range of process streams that can be converted to biogas. Accordingly, the aim of the current
study is to enrich anaerobic microbial consortia that efficiently transform recalcitrant biomass to
biogas, and to do so in the presence of typical inhibitory chemicals.
1
Bioaugmentation is the technique of supplementing an existing microbial population with

cultures adapted to degrade a certain compound. It is most successful when the newly introduced
culture is able to degrade compounds otherwise toxic to the pre-existing microbial community.
Bioaugmentation has been successfully used in pulp and paper industry UASB reactors to
increase removal rates and decrease toxicity of chlorinated organics [5]. Aerobic treatment
systems can also benefit from this technique: bioaugmentation of a lab scale sequencing batch
reactor treating kraft mill wastewater improved the initial digestion rate, raised the organic
loading limit, sped up sludge settling rate, and reduced water content leading to less expensive
dewatering [6].
Microbial populations used in anaerobic digesters in pulp and paper mills are naturally
enriched with microorganisms that transform wood-derived sugars and volatile fatty acids to
biogas. To extend this anaerobic digestion activity to recalcitrant biomass feedstocks and reduce
the susceptibility of the digesters to inhibitory substances, microbial consortia from several
locations were enriched on cellulosic feedstocks including cellulose plus furfural, tannic acid,
sodium lignosulphonate, resin acid-rich red pine needles, or poplar hydrolysate. To maximize the
chances of finding consortia that efficiently degrade each amendment, samples were obtained
from sites that are chronically subjected to complex lignocellulosic materials. These sites
included landfills containing pulp mill secondary sludge, forest heritage piles, chip piles, and
pulp mill anaerobic digesters. In addition, a microbial community from a moose digestive system
was sampled based on findings from Chiquette et al. [7], who found that these consortia contain
similar CH4 producing microbial communities. Beaver droppings were also sampled based on
their similar diet. From these findings and the ability of moose and beaver to survive by eating
twigs and bark, microbial community samples from these mammalian digestive systems were
collected.
1.2. Objectives
It is hypothesized that environmental samples collected from anaerobic locations

chronically exposed to lignocellulosic material will contain methanogenic microbial
communities able to degrade recalcitrant plant biomass, and that resulting enrichments will
contain higher fractions of microorganisms that transform recalcitrant and/or inhibitory wood-
derived compounds than original environmental samples, and so will represent valuable sources
of industrially relevant microorganisms and enzymes.
Accordingly, the objectives of this study are to:
1. Develop mixed anaerobic cultures with the ability to produce biogas from
o
currently recalcitrant lignocellulosic materials, or
lignocellulosic material in the presence of common inhibitors associated

with this material.
2. Determine the effect of enrichment and acclimatization of these cultures on any one
of their:
o
ability to degrade the target substrates,
ability to degrade an industrial wastewater from a pulp and paper facility,

and
bacterial community structure.
The four main tasks undertaken were:

1. Collect environmental samples and prepare microcosms amended with
lignocellulosic substrates, including poplar hydrolysate, microcrystalline cellulose
(Avicel), and microcrystalline cellulose plus tannic acid, pine needles, and
lignosulphonate.
2. Monitor the biological activity in each microcosm by measuring the biochemical
methane potential of each enrichment.
3. Monitor changes in microbial community composition using DGGE.
4. Assist in the design and construction of a lab-scale anaerobic reactor that supports
microbial granulation.
2.
Literature survey
2.1. Lignocellulosic biomass
Cellulose is the most abundant biopolymer on earth, and is the main component of plant
cell walls. In wood, cellulose typically contains nearly 10,000 glucose units that are linked
through -(1,4) glycosidic bonds. Cellulose is synthesized by the cellulose synthase complex,
which forms a rosette structure in plant cytoplasmic membranes and is composed of 36 cellulose
synthase enzymes. Accordingly, 36 cellulose molecules emerge from each rosette structure, and
then coalesce into microfibrils containing 36 cellulose chains that are associated through
hydrogen bonds [8].
The current model of lignocellulose structure depicts cellulose microfibrils associated with
hemicelluloses through hydrogen bonding, which is facilitated by the similar -(1,4)-linked
conformation of hemicellulose backbone sugars. Hemicellulose have between 500 and 3000
backbone sugars per polysaccharide chain, and include various pentoses (5 carbon sugars),
hexoses, and deoxyhexoses (6 carbon sugars with an H substituted for an OH group);
hemicelluloses also contain branching sugars and can be acetylated. The particular sugar
composition of hemicellulose depends on the plant species and region of the cell wall that it is
extracted from. For instance, the main hemicellulose of primary cell walls in wood is xyloglucan,
while the main hemicellulose in secondary cells walls of hardwoods and softwoods is xylan and
galactoglucomannan, respectively.
The branching sugars of hemicellulose can form covalent ester or ether linkages with
lignin. Lignin is a highly complex polymer, mainly made of phenylpropane units (p-coumaryl
alcohol, coniferyl alcohol, and sinapyl alcohol) linked together by carbon-carbon (C-C) and ether
(C-O-C) linkages. While p-coumaryl alcohol predominates in grasses, coniferyl alcohol is the
main monolignol in softwood, forming G-lignin, and hardwood G/S-lignin contains both sinapyl
and coniferyl monolignols. The lignin content in softwood and hardwood fibre is typically 25-35
% and 18 25 %, respectively [8].
2.2. Anaerobic digestion
Anaerobic digestion is the microbially mediated hydrolysis, fermentation, and

methanogenesis of complex polymeric feedstocks. While it has been employed for centuries to
treat organic wastes and produce CH4-containing biogas fuel, recent research has shown
alternative potential applications of this process in the transformation of low value biomass into
other products including H2 gas, carboxylic acids or liquid alcohol fuels [9], and polymers used
in bioplastics [10]. Although a relatively mature process, anaerobic digestion will likely find new
applications in emerging biorefineries [11] because of its potential to synthesize a range of
products coupled with adaptability to different feedstocks. However, remaining challenges
include inhibition of methanogenesis by extractives and wood polymer derivatives found in
pretreated wood, and the reduction in biogas yield due to the recalcitrance of lignocellulosic
polymers.
Anaerobic digestion is the degradation of organic material by microorganisms in the
absence of molecular oxygen. Often, the end product of this degradation is a gas mixture mostly
composed of CO2 and CH4 called biogas. Anaerobic digestion can be broken down into three
main processes in series: hydrolysis of complex polymeric compounds to simple sugars and
monomers, fermentation of these monomers to simple organic acids and ultimately acetic acid,
and finally methanogenesis of acetic acid to CH4 and CO2. This process is illustrated in Figure
2.1 [12].
Figure 2.1. Anaerobic digestion process adapted from Droste [12].

Three sequential steps, hydrolysis, acetogenesis, and methanogenesis are shown on the left. Feedstocks,
intermediates, and products are shown in boxes, and organisms completing each conversion are shown
adjacent to their respective process. Arrow thickness (and percentage listed) is proportional to the relative
magnitude of each reaction.
The general reaction from an organic polymer to CH4 and CO2 biogas follows this form
[13]:
CcHhOoNnSs + yH2O xCH4 + nNH3 + sH2S + (c-x)CO2
where
x = 1/8 * (4*c + h 20 3*n 2*s)
and
y = 1/4 * (4*c h 20 + 3*n 3*s)
Although different substrates will generally have different expected CH4 fractions based
on this formula, in practice some of the CO2 is absorbed by the water (at neutral pH), either
dissolving in molecular form or forming H2CO3 and subsequently H+ and HCO3-. In a well
buffered system, the pH drop from this dissolution is minimal. Decreased CO2 content can lead
to enriched biogas of around 70 % CH4. The theoretical CH4 content values for each substrate,
following this formula, are listed in Appendix G.
2.2.1. Hydrolysis
A first step in anaerobic bioconversion of carbohydrates, proteins, and lipids to biogas is

the hydrolysis of these polymers to their constituent sugars, amino acids and fatty acids. This can
be a complex process requiring multiple steps. The biological breakdown of cellulose to glucose,
for example, is thought to require three types of enzymes:
Endocellulases: cleave non-terminal -(1,4)-glucosidic bonds internal to the cellulose

substrate, and creates new cellulose chains;
Exocellulases: hydrolyze cellobiose from either the reducing or non-reducing end of

cellulose
-D-glucosides: hydrolyze cellobiose to glucose. [14]
Aerobic bacteria produce these enzymes and secrete them to the surrounding fluid; many
of these enzymes typically contain carbohydrate binding modules (CBMs) to help them adhere to
insoluble cellulose substrates. By contrast, anaerobic microbes accomplish cellulose hydrolysis
either by producing a complex, multiple catalytic-domain cellulase [15] or by producing an
extracellular structure called a cellulosome. Cellulosomes have been identified in many
anaerobic species including the cellulolytic rumen bacteria Ruminococcus albus [16] and
Ruminococcus flaveflaciens [17]. This structure consists of a protein scaffold that attaches to the
bacterial cell membrane and anchors both cellulolytic and hemicellulolytic activities. The
cellulosome structure ensures that hydrolysis products can be quickly recovered by the cell for
energy, rather than be harnessed by surrounding microorganisms. An example of a cellulosome
structure can be seen in Figure 2.2. Cellulosomes are extracellular protrusions made up of
multiple proteins classified by function including:
CBMs, which allow the cellulosome to bind to the substrate;
Scaffoldins, structural proteins that comprise the main branch of the complex;
Cohesins and dockerins, proteins that enable modular attachment of enzymes to

scaffoldin;
Catalytic domains, consisting of enzymes with activity on the substrate [14].
Figure 2.2. Schematic representation of a cellulosome.

(A) Bacterial cell, (B) cell wall anchor, (C) scaffold proteins, (D) multiple enzymes including cellulases and
hemicellulases that are to the scaffold through dockerin domains
The actual composition and arrangement of any cellulosome depends on the

microorganism as well as environmental conditions. Notably, despite very similar sequences,
cohesins are species specific [16].
2.2.2. Fermentation
During anaerobic digestion, following hydrolysis, glucose is fermented primarily to

lactic, butyric, or propionic acid. These are in turn fermented to acetic acid. Co-products released
from these reactions include CO2 and H2 gases. A summary of these reactions is shown in Table
2.1 [18].
Table 2.1. Fermentation reactions
Substrate
Product
Reaction
Lactic Acid
C6H12O6 2CH3CHOHCOOH
Butyric Acid
C6H12O6 2CH3CH2CH2COOH + 2CO2 + 2H2
Acetic Acid
C6H12O6 + 2H2O 2CH3COOH + 2CO2 + 4H2
Propionic Acid
CH3CHOHCOOH + H2 CH3CH2COOH + H2O
Acetic Acid
CH3CHOHCOOH + H2O CH3COOH + CO2 + 2H2
Butyric Acid
Acetic Acid
CH3CH2CH2COOH + 2H2O CH3COOH + 2H2
Propionic Acid
Acetic Acid
CH3CH2COOH + 2H2O CH3COOH + CO2 + 3H2
Glucose
Lactic Acid
The first phase, called acidogenesis, converts the products formed during hydrolysis to
short chained organic acids. This process is conducted by facultative and obligate anaerobic
organisms under anaerobic conditions generated by the consumption of dissolved oxygen by the
facultative bacteria. Once these smaller organic acids are produced, the second stage called
acetogenesis begins. This stage uses the reactions listed in Table 2.1, most of which are
endergonic at standard conditions. Based on these reactions, H2 must be consumed (by
homoacetogenic bacteria and methanogens) at a rate high enough to keep the partial pressure at
very low levels. When the partial pressure of H2 increases beyond tolerable levels, acetogenic
bacteria form butyric, capronic, propionic, and valeric acids plus ethanol [13] instead of acetic
acid. These compounds are not consumed by methanogens and can contribute to a drop in pH,
further inhibiting methane production.
2.2.3. Methanogenesis
The biological production of CH4 is known to occur primarily through either

hydrogenotrophic or acetoclastic methanogenic archaea. About 30% of the CH4 produced is from
CO2 reduction by H2 by the former class of microbe, while 70% is from direct conversion of
acetic acid by the latter. The primary reactions and free energy changes are shown below.
Table 2.2 Free energy of methanogenesis
Type
CO2 reduction
Acetate conversion
Reaction
4H2 +
HCO3-
G (kJ/mol)
+ H CH4 + 3H2O
-135.4
HCO3-
-30.9
CH3COO + H2O CH4 +
Methanogens oxidizing H2 use the metabolite of fermenting bacteria as it is produced,

maintaining a very low partial pressure. Acetoclastic organisms similarly use acetate as it is
produced, leading to low levels of liquid intermediates in the reaction medium. The anaerobic
digestion process is generally stable, but the creation of CH4 is easily limited by inhibitory
conditions: methanogens are strictly anaerobic, function only above pH 6.7, and are susceptible
to many inhibitory compounds organic and otherwise [13].
2.2.4. Inhibitors of digestion process
There are many inhibitory compounds found in pulp and paper mill wastewaters that can
disrupt the digestion process including oxygen, sulphur compounds, nitrate, ammonium and
ammonia, heavy metals, pH below 6.7, tannins, lignins, resin acids, long chain fatty acids, and
halogenated compounds [13], [19]. Since the focus of this work is on the degradation of
recalcitrant organic materials associated with lignocellulosic material, the scope will be limited
to tannins, lignins, and resin acids.
2.2.4.1. Tannic acid
Tannins, a class of polyphenolic compounds associated with bark, leaves, and sometimes
the heartwood of trees, are so named because of their ability to tan or denature and precipitate
proteins. They are divided broadly into condensed and hydrolysable varieties, the former being
derivatives of flavanols and the latter being glucose esters with one or more
trihydroxybenzenecarboxylic acids (pyrogallols) (Figure 2.3) [20]. Hydrolysable tannins,
including tannic acid, are toxic to animals (consumption of oak or other leaves containing about
20 % or more tannin by weight leads to high mortality and morbidity in cattle and sheep [21])
while condensed tannins are not [22]. In North America, tannins have historically been
commercially produced from the bark of oak (Quercus prinus, Q. velutina, Q. alba, Q.
densiflora), eastern hemlock (Tsuga canadenis), or western hemlock (Tsuga heterophylla, t.
mertensiana); sumac leaves; and American chestnut (Castanea dentata) wood [23]. Tannins are
found in varying amounts in wood including some species used in the pulp and paper industry
(Table 2.3). Because tannins are extractable in water, and bark usually has high tannin
concentrations, a correspondingly high concentration of tannin is found in debarking
wastewaters. Bark extracts prepared for an anaerobic inhibition study [24] contained between
895 and 3256 mgCOD/L of total tannins. A literature survey conducted by Field et al. [25] of
debarking wastewaters indicated that about 40 % of the COD (chemical oxidation demand)
content of the water was due to tannins. The tannin yield from wood is substantially increased by
sulphonation and a subsequent increase in solubility much like that of lignin, it is therefore
expected to be present in most sulphur based pulping waste streams [26].
10
Table 2.3. Tannin content of North American trees.
Wood
Softwoods
Hardwoods
Tannin wt %
Reference
Eastern hemlock
Bark
[23]
Western hemlock
Bark
15
[23]
Balsam fir
Bark
3.4 - 4.1
[27]
Red spruce
Bark
13.0
[27]
Sitka spruce
Bark
24.4
[27]
Sumac
Leaves
31
[23]
Lodgepole pine
Wood
.061
[28]
Douglas fir
Wood
0.27
[28]
Western red cedar
Wood
0.2 0.4
[28]
Oak
Bark
[23]
American chestnut
Wood
5 15
[23]
American white oak
Wood
0.2 0.4
[28]
Sugar Maple
Wood
0.1
[28]
White ash
Wood
0.063
[28]
Tannic acid is a hydrolysable tannin mixture containing on average 10, but often between
2 and 12, pyrogallol groups [29]. It is commercially produced from Turkish or Chinese nutgall,
which contains 50-70 % tannic acid by weight, and is used to manufacture dyes and inks, size
paper and silk, clarify beer and wine, and tan leather [20]. Tannic acid is very soluble, where 1 g
dissolves in 0.35 mL water [20].
11
Figure 2.3. Example molecular structure of tannic acid.

Blue indicates the central glucose group; red indicates one of the 10 pyrogallol residues.
Tannic acid has been reported as an inhibitor of methanogens and anaerobic microbes in
general in several anaerobic communities including ruminant digestive systems [22, 30-32], and
anaerobic waste water treatment granules [25, 33-35]. Despite its inhibitory properties, tannic
acid is in fact degradable to lower molecular weight components in ruminant systems [22]. The
lower molecular weight products are themselves also inhibitory to the anaerobic digestion
process [32], and have been found to consist primarily of pyrogallol [36]. Proposed pathways for
the biodegradation of tannic acid show the conversion of gallate to CO2 in addition to pyrogallol,
contributing to the total amount of gas produced (Figure 2.4) [36].
Figure 2.4. Proposed pathway for the biodegradation of tannic acid as per Nelson et al. [36]
Ester linkages (red lines) of a tannic acid molecule (G: pyrogallol groups), are hydrolzed by tannases to gallic
acid (A), then transformed to pyrogallol and CO2 (B).
12
The papers investigating the effect of tannins and tannic acid on ruminant digestive
systems suggest that these compounds could be used to suppress the methanogenic activity of gut
bacteria while maintaining a healthy livestock population. The papers focusing on water
treatment suggest that while tannic acid is inhibitory to methanogenesis, the effluent streams
containing high tannin loadings, debarking in particular, are small and can be diluted or diverted
from the anaerobic treatment process. In bioconversion applications focusing on marginal
biomass utilization, much higher concentrations of tannins may be encountered. Table 2.4 below
shows the effect of tannins, tannin derivatives, or tannin-containing wastewaters on
methanogenesis.
Table 2.4. Effect of tannins on methanogenesis.
Conditions
Tannin Concentration
Tannin as % of
Inhibition
Reference
total COD
4 gCOD/L VFA (25% C2,
700 mg/L
15 %
50 %
[33]
44 64 mg/L
7 11 %
50 %
[35]
Pine
410, 920 mg/L
15, 28 %
Spruce
510, 780 mg/L
18, 25 %
50, 80 %
[25]
Birch
845, 1040 mg/L
26, 31 %
4 gCOD/L VFA
Pine
1256 mgCOD/L
18 %
80 %
(25% C2, 35% C3,
Spruce
1434 mgCOD/L
22 %
97 %
Birch
790 mgCOD/L
14 %
97 %
Pulp
536 mgCOD/L
5.6 %
95 %
35% C3, 41% C4)
+ tannic acid
Pinus radiata, DEDED
bleached, tannin + lignin, 0.6
gCOD/L
4 gCOD/L VFA
(25% C2, 35% C3,
41% C4)a +
debarking
wastewater of:
41% C4)a + lab

prepared hot water
extract of:
a
[24]
Volatile fatty acids containing 2, 3, or 4 carbons (acetic, propionic, and butyric acids respectively)
2.2.4.2. Lignin
Lignin is one of the three major components in wood fibre [37]. During the sulphite
pulping process, lignin is separated from hemicellulose and cellulose through reaction with
13
aqueous SO2 or HSO3. The negatively charged, much smaller lignosulphonate then dissolves in
the pulping liquor and is eventually extracted as a salt with sodium [38]. Sodium
lignosulphonates are used as dispersing agents in concrete, binding agents in animal feed pellets,
and dust suppressants on dirt roads [39]. Lignin, representing about 30% of wood, is found in
most forest industry waste waters. Lignin used in this study is represented by ARBOS01P
sodium lignosulphonate powder supplied by Tembec, a forest products company.
Lignin is recalcitrant because it has a high molecular weight, is chemically
heterogeneous, contains stable aromatic rings, and does not have a regular structure upon which
enzymes can work efficiently [40]. Smaller molecular fractions of lignin or derivatives including
lignosulphonate are thought to be recalcitrant or toxic to methanogenic bacteria [40], [41].
However, the treatment of high strength spent sulphite liquor (SSL) in a lab scale UASB system
has been reported to be feasible [42], as has the treatment of dissolving pulp wastewater
(including a nearly 50 % removal of lignin) [43].
Table 2.5. Effect of lignosulphonates on methanogenesis.
Conditions
Lignosulphonate (Sulphite)
4 g/L VFA (equal C2,C3,C4)
Lignin
Lignin as % of
Concentration
added COD
11 300 mg/L
73 %
Inhibition
None
[40]
3 320 mg/L
45 %
2 g/L acetic acid + lab
13 200 mg/L
93 %
Delay
14.5
produced lignosulphonate
26 700 mg/L
96 %
(days):
17.8
(Sulphite)
40 600 mg/L
97 %
21.1
(MW > 20 000 Da)
54 900 mg/L
98 %
NDb
Sulphonated (Kraft)
4 g/L VFA (equal C2,C3,C4)
Reference
50 %
Volatile fatty acids containing 2, 3, or 4 carbons (acetic, propionic, and butyric acids respectively)
Not determined
[44]
2.2.4.3. Pine Needles
Whole ground pine needles or extractives thereof have been shown to have inhibitory
effects on a variety of wood degrading fungi [45], [46], as well as toxic effects on the larvae of
Neodiprion sertifer [47] and instar gypsy moths (Lymantria dispar L.) [48]. The needles of red
pine (pinus resinosa), a species important to Eastern North American pulp and lumber
14
production [49], contain many different extractable compounds including terpenes and phenolics
(Table 2.6). Pine needles are known to contain resin acids, non-volatile, hydrophobic compounds
known to inhibit anaerobic digestion processes in pulp and paper wastewater treatment [50].
Since resin acids constitute a significant fraction of the total dry mass of green needles (1.57.6%), and the non-volatile compounds are even more prevalent in dried needles (12.7%) [46],
[47], dried ground red pine needles were selected as a compound to enrich microbial
communities on.
Table 2.6. Extractives reported in Pinus resinosa.
Class
Terpenes
Compound
Resin Acids
Notes
Reference
Manoyl oxide acid
50.0% of total resin acids
[49]
Epimanoyl oxide
acid
Communic acid
Imbricataloic acid
-pinene
25% of volatiles
-pinene
50% of volatiles
Other
Phenolics
[45]
Catechin
Total phenolics =
Each
Catechin gallate
25.2 mg tannic acid
flavanoid
Epicatechin gallate
equivalents / g pine
~1 g/mL
needles
Present not quantified
Quercetin 3-O-glucoside
[48]
Quercetin 3-O-rutinoside
6-methylkaempferol-3-glucoside
Resin acids are extractives commonly found in wood having a diterpenoid (containing
four isoprene units plus carboxylic acid [51]), often tricyclic structure [50] (Figure 2.5). Resin
acid solubility is low relative to concentrations that are toxic to methanogenic cultures. At
neutral pH and 20C (i.e. conditions similar to those commonly encountered in digesters), the
solubility of abietic acid is 3 to 5 mg/L. Under these conditions, hydrophobic resin acids tend to
associate with suspended solids, resulting in local concentrations of up to 1500 mg/L [50].
15
Figure 2.5. Resin acids identified in pulp and paper wastewaters.

A) Pimaric acid, B) Palustric acid, C) Neoabeitic acid, D) Sandaracopimaric acid, E) Abietic acid, F)
Levopimaric acid, G) Isopimaric acid, H) Dehydroabietic acid[50].
The major resin acids found in red pine needles are manoyl oxide, epimanoyl oxide,
communic, and imbricataloic acids (Table 2.6; Figure 2.6). The cortical (outer part of the stem or
branch) tissue of red pine contains a different subset of resin acids than the needles, including
primarily levopimaric, palustric, neoabeitic, and communic acids, with no manoyl oxide or
epimanoyl oxide acids observed [49].
Resin acids common to both pulp and paper wastewaters and red pine needles include
sandaracopimaric, levopimaric, palustric, and neoabeitic acids. Since the latter three of these are
known to undergo abiotic isomerization to abietic acid with the addition of mineral acids or high
temperatures common to chemical pulping operations [50], the microbial response to abietic acid
is of particular interest. Methanogenic toxicity of abietic acid, on its own and in combination
with other resin acids, has been well studied in several UASB sludges using several carbon
sources (Table 2.7). It has been posulated that the hydrophobic, lipophillic nature of these
compounds allows them to bioaccumulate within the cell membrane and that subsequent changes
to surface tension and other undescribed chemical reactions may be responsible for their toxicity
[52].
16
Figure 2.6. Resin acids of Pinus resinosa needles. A) Manoyl oxide acid, B) Epimanoyl oxide acid, C)
Communic acid, D) Imbricataloic acid.
Resin acids have been found to be toxic to methanogenic cultures in wastewater

treatment systems [4], [35], [52]. The mechanism of resin acid toxicity is not well understood,
but is possibly related to hydrophobic interaction with cell surfaces. Resin acids with polar
substituents have been found to be less toxic than those that do not [52].
Table 2.7. Toxicity of Resin Acids
Carbon Source
Resin Acid(s) Present
[Resin Acid] (mg/L)
Inhibition
Reference
4 g/L VFA (equal
Abietic acid
89
50%
[52]
Dehydroabietic acid
43
-pinene
105
-pinene
110
Alkaline (pH 12) cortical
46
1178
100%
[53]
Oleic acid
1235
50%
[54]
Abietic acid
114
50%
[25]
6 gCOD/L
Mixture:
20
0%
[4]
methanol
7% Pimaric
40
0%
6% Sandarocopimaric
80
0%
12% Isopimaric
160
40%
21% Levopimaric
320
60%
22% Dehydroabietic
700
60%
parts C2, C3,

C4)a
spruce extract
Acetate
Mixture:
50% Sodium abietate
50% Sodium oleate
3 g/L acetate
Acetate
26% Abietic
6% Neoabietic
a
Volatile fatty acids containing 2, 3, or 4 carbons (acetic, propionic, and butyric acids
respectively)
17
2.3. Biogas from woody biomass
Anaerobic digestion as an industrial process is widely used in wastewater treatment to

convert soluble and dilute suspended organic matter, and has been identified as a method of
generating renewable energy (in the form of biogas) from substantially more solid biomass. A
1997 review [55] of this process summarized the biochemical methane potential of a wide
variety of biomass feedstocks including several woody biomass sources (Table 2.8).
Table 2.8 CH4 yield from woody biomass
Biomass
CH4 Yield (m3/kgVS)
Populus deltoides (cotton wood)
0.220
Populus sp. (hybrid poplar)
0.320
Platanus occidentalus (sycamore)
0.320
Pinus taeda (loblolly pine)
0.063
Eucalyptus sp.
0.014
Black alder
0.240
Red alder
0.280
Abies concolor (white fir)
0.042
Salix sp. (willow 0.8 mm particles)
0.310
Populus sp. (poplar 0.8 mm particles)
0.290
Poplulus sp.
0.003 mm particles
0.330
(poplar)
0.8 mm particles
0.330
8.0 mm particles
0.300
Reference
[56]
[57]
[58], [59]
[59]
Several of these studies found a CH4 yield close to 80% of the 0.417 m3CH4/kgVS
theoretical limit (Appendix G) for pure cellulose. Assuming much of the lignin fraction of these
woody sources is not degraded, this yield is close to the theoretical CH4 expected. Problems with
commercial application of this type of biofuel generation include the availability and transport of
woody biomass, the cost of pretreatment required to improve access to crystalline regions and
remove lignin, and the generation of microbial inhibitors through this pretreatment process [60].
18
2.4. Metagenome studies of digestive system lignocellulose degrading communities
Anaerobic cellulolytic microbial communities are found in the digestive systems of a

variety of higher organisms. This mutualistic symbiosis enables both the higher organism and the
microbes to survive on lignocellulosic materials eaten by the animal and partially digested by the
microbes. Typically, the animal will ingest and partially homogenize plant matter through
mastication, and then swallow the material to an enclosed, anaerobic gut environment.
Hydrololytic and fermentative microbes then degrade carbohydrates to sugars and ferment the
sugars to acetate, which is then absorbed by the host animal. In some cases, food is predigested
to remove easier to digest materials such as starch. Often, the absorption of acetate is not perfect,
and methanogens are able to survive on acetate and H2 produced during fermentation. Many
studies have been completed on these microbial communities, but have had limited success due
to the difficulty of culturing organisms in the lab [61]. To circumvent the cultivation problem,
techniques such as metagenome sequencing have been developed. Increasingly, studies focus on
community DNA extraction, sequencing, and analysis to determine the in vivo community
structure and identify genes (or gene fragments) thought to code for target proteins such as
CAZymes (carbohydrate active enzymes). RNA and protein assays to verify the expression and
activity of these enzymes are also commonly employed. Ecosystems that were previously
selected for metagenome sequencing and identification of lignocellulose-degrading microbes and
enzymes, include the cow rumen [62] , Tammar wallaby gut [63], and the monogastric termite
hindgut [64].
The commercial value of cattle has prompted much scientific interest in the digestive
processes of cows and the associated rumen microbe population, leading to extensive study of
the rumen over the past forty years using techniques from culturing to DNA analysis [65]. The
microbial community found in the rumen is known to contain some of the most active cellulose
degrading bacteria identified so far [66], and is known to be capable of degrading complex
lignocellulosic substrates containing tannic acid [22], [36], solublizing but not necessarily
degrading lignin [67], and partially degrading pine needles [68]. A recently published
metagenome study of the microbial population fixed to switchgrass inside a nylon bag incubated
in a fistulated cow rumen revealed an exceptional 27 755 putative carbohydrate active enzymes.
2 365 of these were endocellulases, 2 360 endohemicellulases, 1 381 debranching enzymes, and
6 591 oligosaccharide degrading enzymes. Over 480 of these genes coded for both a catalytic
19
domain and CBM, while 23 804 and 3 469 genes respectively coded for a catalytic domain or
CBM only (Table 2.9).
Table 2.9. Carbohydrate active enzymes database
Enzyme
CAZy
Enzymes from Cow Rumen Study
-(1,4) endoglucanases
1075
1086
-glucosidases
1199
1477
cellobiohydrolases
251
153
The majority of carbohydrate-active enzymes predicted through the metagenomic analysis of

cow rumen samples were significantly different from those already documented in the CAZy
database. In fact, this single study effectively doubled the number of known carbohydrate active
enzymes.
Termites play an important role in the carbon cycle through their consumption of woody
material. A recent metagenomic analysis [64] of bacterial DNA isolated from the paunch of a
Nasutitermes species from Costa Rica identified a large set of genes coding for cellulose and
xylan hydrolytic enzymes. Samples from about 165 individuals were sampled, pooled, cloned,
and sequenced. Sequence data analysis revealed over 700 glycoside hydrolases in 45 different
carbohydrate active enzyme families (CAZy database). More than 100 of these were relevant to
cellulose hydrolysis, and about 100 were associated with hemicellulose degradation. Several
CBMs were also identified. There was no evidence of methanogenic species either in sequence
data or through 16s rDNA PCR with Archeal primers, nor were any lignin degrading genes
identified.
The Tamar wallaby (Macropus eugenii) also relies on gut microbes to aid the digestion of
lignocellulosics. A metagenomic study of the wallaby intestinal microbe population [69]
indicated a community diversity similar to that of the cow rumen, yet yielded far fewer
carbohydrate active enzymes (557) through genomic sequencing. These genes included 10
cellulases, 28 hemicellulases, 42 debranching enzymes (accessory hemicellulases), and 184
oligosaccharide degrading enzymes.
In addition to identifying many new CAZymes for lignocellulose conversion, significant
gaps in our knowledge were also reveled through these metagenomic analyses. For instance,
genes predicted to encode GH6, GH7 or GH48 -(1,4)-exoglucanases were not found in the
Tammar wallaby or termite gut studies, and only 4 of the 27 755 CAZymes identified in the cow
20
rumen study were predicted to encode these activities [69]. Also, 188 and 80 dockerin and
cohesion genes respectively were found in the cow rumen metagenome while none were found in
the wallaby gut metagenome despite the similarity of the catalytic domain and CBM genes.
Moreover, the wallaby feeds mainly on grasses and forbs, the cow rumen metagenome
was obtained from a cow fed with switchgrass, and termites considerably predigest wood fibre
prior to microbial digestion. Accordingly, the aim of the current project is to enrichment
microbial consortia from digestive systems that directly transform woody biomass that is
typically recalcitrant to biological processes, including bark and pine needles. The resulting data
will allow us to study the particular metabolic processes and community members required to
transform different wood-derived compounds. In addition to improving the conversion of plant
residues to biogas for power generation, it is anticipated that this study will identify many new
industrial enzymes relevant to processing woody-biomass and pretreating wood hydrolysates
prior to fermentation.
21
3.
Materials and Methods
3.1. Inoculum collection and storage

3.1.1. Moose rumen fluid
Moose rumen fluid samples were collected on October 10th, 2009 at a location just west
of Nabakwasi Lake (GPS: 47.5612 N, 81.4504 W), about 100 km north of Sudbury, Ontario.
Twenty 50 mL falcon tubes were filled with unfiltered rumen contents during the moose gutting
process at the site and stored on snow in a sealed plastic bag. The hunters returned to their cabin
and kept the samples in a freezer until they returned to Toronto the following weekend. Samples
were thawed and stored at 4 C one day prior to the microcosm setup.
3.1.2. Beaver droppings
Beaver droppings were collected by a trapper associated with the Ontario Ministry of
Natural Resources in November 2009 from beavers in the area around three different regions in
Ontario: Parry Sound, Mount Albert, and Mississauga. The samples were frozen at -20 C until
one day prior to microcosm setup, when they were thawed and stored at 4 C.
3.1.3. Internal circulation reactor granules
Granules were received on April 21, 2010 from an internal circulation (IC) anaerobic
wastewater treatment reactor installed at a pulp and paper complex in Temiscaming, Quebec.
This reactor is normally fed 15 000 m3/day of mixed wastewater including acid condensate from
the evaporator system and bleached chemithermomechanical pulp (BCTMP) effluent.
3.1.4. Other inoculum sources

Two other sources of microbes were identified and sampled for this project: a wood
heritage pile, and landfill plus leachate from the landfill. Due to the lower absolute performance
22
of these microcosms in the first phase of gas production trials, they were not selected for further
enrichment and analysis.
3.1.4.1. Heritage pile
A former sawmill located near Mattawa, Ontario, disposed of sawdust and waste material
in a landfill on the site. Prior to the creation of this landfill, material including chips and sawdust
was disposed of in a heritage pile. This heritage pile is estimated to be about 20 feet deep, and
is overgrown in most places with bushes and small trees. Samples from several locations on top
of the heritage pile were taken by digging about 0.3 m below the surface and filling several 1 L
mason jars with material. Water collected from a puddle on the site was used to displace air in
the jars, and then they were sealed and stored at room temperature before returning to Toronto
where they were stored at 4 C.
3.1.4.2. Landfill and leachate
A pulp and paper complex located in Temiscaming, Quebec, has a landfill on site that is
used to dispose of various wastes from the pulping process including excess lignocellulosic
biomass, boiler ash, and waste activated sludge from the aerobic wastewater treatment plant.
Landfill solids collected from this landfill were collected by digging roughly 0.3 m below grade
and collecting approximately 2 L of material in mason jars. Leachate from the landfill is
collected through a series of pipes and pumped back to the wastewater treatment plant where it
was collected.
23
3.2. Measurement of physical and chemical properties
3.2.1. Solids analysis

Total suspended solids (TSS) measures the total mass of solids retained on a 1.5 m pore
size glass fibre filter after vacuum filtration, washing with deionized water, and drying in a 103
C oven. After weighing the filter to determine TSS, the remaining solids and the glass filter are
heated to 550 C to ignite volatile suspended solids (VSS). The weight difference between the
sample at 103 C and 550 C is recorded as the VSS, and gives an estimate of the organic
content of the suspended solids. The protocol followed in this study is based on the American
Public Health Association (APHA) Standard Methods for the Examination of Water and
Wastewater [70].
Glass filters and aluminum dishes were fired at 550 C for 2 h and stored in a desiccator
until used. The dish and filter weights were recorded prior to use. A known volume of sample
was passed through the filter under vacuum and washed with 5 mL deionized water, then dried at
103 C overnight. The mass of each dish and filter was again recorded, each sample was fired at
550 C for at least 20 min, and the dish and filter mass recorded again. The difference between
the unused filter and the mass after filtering and drying at 103 C was the TSS, and the
difference between the mass after drying at 103 C and combustion at 550 C was the VSS.
3.2.2. Chemical oxidation demand
Chemical oxidation demand (COD) is a measure of the organic content of a sample

reported as the mass of oxygen required for complete oxidation, allowing an estimation of the
amount of CH4 expected from anaerobic digestion. The protocol followed in this study is based
on the APHA standard method 5220D [70]. An oxidizing chemical (potassium dichromate,
K2Cr2O7) is added to a sample under acidic conditions and heated to complete the reaction.
Sulphuric acid, silver sulphate, and mercuric sulphate are also required to lower the pH, catalyze
the oxidation, and prevent interference from chloride species respectively. At the completion of
the reaction, the chromium is reduced to Cr3+, a species that absorbs light at 600 nm. The extent
of oxidation is measured colourimetrically by measuring absorbance at this wavelength and
24
comparing it to a calibration curve generated with potassium hydrogen phthalate (KC8H5O4) as a

standard.
The stock digestion solution was prepared by dissolving 10.216 g of dried (in a 103 C
oven for 2 h) potassium dichromate in 500 mL of distilled water, then adding 167 mL 98 %
sulphuric acid and 33.3 g mercury(II) sulphate (HgSO4). The mixture was allowed to cool to
room temperature then diluted to 1 L with distilled water. The stock sulphuric acid solution was
prepared by mixing 10.185 g silver sulphate (Ag2SO4) into 1 L of sulphuric acid, then left for a
day for the silver sulphate to completely dissolve.
The COD test was performed in 16 mm Hach test tubes with Teflon lined screw caps.
A 2.5 mL sample was added to each tube, and then 1.5 mL digestion solution and 3.5 mL
sulphuric acid solution were added to make a total volume of 7.5 mL. The Teflon cap was then
secured to the tube, the tube was vortexed, and then placed in a 150 C heat block for 2 h.
Following digestion, the absorbance of each tube was measured at 600 nm. A linear calibration
curve relating known oxygen demand to absorbance at 600 nm was generated using from 0 to
500 gCOD equivalent dried potassium hydrogen phthalate at 50 gCOD intervals.
Spent COD solution and rinse water from tube cleaning was discarded to chemical waste.
Tubes were then washed with hot water and soap, and then rinsed with 25 % sulphuric acid to
eliminate soap residue.
3.2.3. Fourier transform infrared spectroscopy (FTIR)
This technique uses the infrared absorption spectrum of a given material to identify
component organic bonds. The material is finely ground and mixed with potassium bromide then
compressed to form a transparent disc. A broad-spectrum infrared laser is passed through this
disc and the resulting signal is compiled into an absorption profile. Each peak corresponds to a
chemical bond contained in the sample, although the exact position of each maximum may be
shifted depending on the nature of the sample. Peak assignment is accomplished through
literature comparison with samples of similar composition.
Coarse substrates (poplar hydrolysate and Wiley mill ground pine needles) were ground
to a fine powder with an acetone-cleaned mortar and pestle. Approximately 5 mg of each
substrate (poplar hydrolysate, pine needles, sodium lignosulphonate, tannic acid, and
microcrystalline cellulose) was well mixed with about 250 mg of potassium bromide (KBr) and
25
dried in a 103 C oven overnight. Each mixture was compressed using about 15 000 psi pressure
from a hydraulic press into a transparent disc, which was stored at room temperature in a
desiccator until use.
To measure the absorbance spectrum of each sample, a Bruker Tensor 27 infrared
spectrometer attached to a PC running Opus 5.0 data interpretation software was used. It was
calibrated by measuring the background absorbance spectra of air, then measuring the
absorbance of each sample and subtracting the background interference from water and CO2 via
the built in correction method. All results are shown as absorbance values and have been
corrected to a flat baseline using the softwares rubber band correction method.
3.2.4. Gas volume measurements
The volume of gas produced from each microcosm was removed from each bottle at
frequent intervals and discarded to the fume hood. All volume removal techniques are
temperature dependent: to maintain a relatively constant temperature, only eight bottles at a time
were removed from the incubator and measured immediately. Several methods for removing
biogas and measuring its volume were employed. A short description of why each method was
discontinued is outlined below.
The first method used was the lubricated syringe method. A 22.5 gauge needle attached
to a short length of tubing was inserted into the bottle, and pressure inside would force the glass
syringe (fixed horizontally to a retort stand) to open. Once the pressure equalized, the syringe
would no longer move, and volume could be read directly from the syringe. This method was
quickly abandoned once it was clear that there was no easy way to measure quantities of gas
larger than those that fit in the syringe, and that the lubrication of the syringe varied arbitrarily
between samples.
The second method involved a pressure transducer (Omega PX725 Industrial Pressure
Transmitter, Omega DP24-E Process Meter) attached to a short length of tubing and a needle. As
pressure increased at the input to the transducer, a silicon wafer was deformed, proportionally
altering the resistance. A linear calibration curve was generated for the transducer over its full
range (Appendix I) relating excess volume inside the bottle to the reading on the transducer.
Once the reading was taken, the excess pressure was relieved by removing the pressure
transducer tube from the needle and allowing the pressure to drop to about 1 kPa above
26
atmospheric. This method worked well at low gas volumes, but was less accurate if the volume
of liquid varied from bottle to bottle, or if the volume of gas produced was more than the
maximum calibration level.
The third and most commonly used method involved the same pressure transducer and
needle, but instead of simply releasing gas to the fume hood it was drawn out using a 20 mL
syringe until the pressure transducer read the equivalent of 1 kPa above atmospheric. The
volume removed could then simply be read off the syringe. This method was occasionally
modified to use a larger syringe if necessary.
3.2.5. Headspace CH4 content
At the end of each degradation cycle the headspace was sampled and analyzed using a
gas chromatograph (GC) to determine CH4 content. Pressure was equalized by conducting a gas
volume measurement and allowing bottles to reach room temperature immediately prior to
headspace sampling. Headspace samples (300 L) were collected from each bottle using a 500
L gastight glass syringe and injected into a GC for analysis. The Hewlett-Packard 5890 Series
II Gas Chromatograph with a GSQ 30 m x 0.53 I.D. PLOT column from J&W Scientific,
equipped with a flame ionization detector (FID), was used to measure CH4 content. In this case,
the carrier gas was helium at 140 kPa, and the oven and detector temperatures were 200 C and
50 C, respectively. On occasion, a second Hewlett-Packard 5890 Series II GC equipped with a
thermal conductivity detector (TCD) and an Alltech CTR I column was used to detect both CH4
and CO2.
The concentration of CH4 gas in the headspace of each bottle was compared to a
calibration curve generated using known CH4 concentrations. The concentration of CH4 in the
headspace is a function of the total amount of biogas produced, the fraction of CH4 in the biogas,
and the amount of gas removed as part of the volume measurements. Accordingly, an iterative
algorithm was developed to back calculate the average concentration of CH4 in the gas generated
at each sampling interval. At time zero, there is no CH4 present in the headspace of a given
bottle. As the microcosm degrades feedstock, biogas containing a certain percentage of CH4 is
produced. At regular time intervals, a certain amount of gas, now containing CH4, is removed
through sampling. This process is repeated until gas production stops, at which point the
headspace CH4 fraction is measured by GC. The concentration of CH4 in the biogas was then
27
calculated from 1) total volume of gas produced, 2) the CH4 fraction in the final headspace, and
by assuming that 3) the CH4 fraction of the biogas was constant.
With a known volume of gas produced over a given interval (Vprod), and a certain fraction
assumed to be CH4 (Xmeth, prod), the total volume of CH4 in the headspace (Vmeth) can easily be
calculated by adding this generated CH4 to the existing CH4 in the headspace (Vmeth,headspace)
(Equation 3.1.):
Vmeth = Vprod * Xmeth,prod + Vmeth,headspace
Equation 3.1.
Now that the new total volume of CH4 is known, the CH4 fraction of the headspace
(Xmeth,headspace) can be calculated by dividing Vmeth by the total one-atmosphere-pressureequivalent volume (the volume taken up as predicted by the ideal gas law if the headspace was
expanded to atmospheric pressure) of gas in the headspace (Vheadspace + Vprod) (Equation 3.2.)
Xmeth,headspace
= Vmeth / (Vheadspace + Vprod)
Equation 3.2.
Next, a certain amount of gas is removed from the headspace to reduce the pressure back
to one atmosphere. The amount of CH4 remaining in the headspace following this removal is
given by (Equation 3.3.):
Vmeth,headspace = Vmeth (Vprod * Xmeth,headspace)
Equation 3.3.
These three calculations are repeated until all gas produced is accounted for, and a final
Xmeth,headspace value is known. If this value is within 0.001% of the measured headspace CH4
fraction, then the value of Xmeth,prod (representing the CH4 fraction in the biogas) is output for that
bottle. The calculation procedure is then repeated for other bottles. This procedure was
automated using a Matlab script, found in Appendix A.
28
3.3. Nutrient medium
A nutrient medium similar to that developed by Edwards and Gbri-Gali [71] for
methanogenic consortia was created to dilute and maintain adequate nutrients in each culture.
The medium was prepared by adding aliquots of each of four solutions (MM1: 10 mL of
phosphate buffer, MM2: 10 mL salt solution, MM3: 2 mL of trace mineral solution, and MM4: 2
mL of magnesium chloride solution) to about 950 mL of reverse osmosis water, autoclaving at
121 C for 20 min to sterilize, then purging with an 80 % N2, 20 % CO2 gas mix for 45 min to
remove oxygen. The bottle was then sealed and brought into an anaerobic glove box containing
an atmosphere of about 90 % N2 and 10% CO2. Once inside the glove box, pre-sterilized
anaerobic aliquots of three further solutions were added (MM6: 10 mL of saturated bicarbonate
solution, MM7: 10 mL of vitamin solution, and MM8: 5 mL of ferrous sulphide solution). The
medium was buffered to pH 7 by the bicarbonate solution and anaerobic conditions are ensured
by the addition of ferrous sulphide that reacts with residual oxygen to form sulphate. The
composition of the final medium solution is shown in Appendix A.
Nutrient medium was added to each bottle during the initial setup of a microcosm set or
when transferring a set to new bottles. When preparing the initial microcosms in the disposable
glove bag, medium was decanted into a 2 L beaker, leaving FeS sludge in the bottle. This
decanted medium was added to each bottle using a graduated cylinder or 60 mL syringe. In
subsequent transfers, medium was added to bottles using an 18 gauge needle and 60 mL syringe.
3.4. Feedstock preparation
Five carbon sources (microcrystalline cellulose, tannic acid, sodium lignosulphonate, red
pine needles, and poplar hydrolysate) were added to corresponding enrichments at different
times during the course of this project. Given the insolubility and heterogeneity of some of these
carbon sources, a variety of methods were used to add substrates to the microcosms, which were
being cultured in serum bottles that were sealed with a black butyl rubber stopper to maintain a
closed, anaerobic environment . The first method was simply to add the feedstock to a new
bottle, sterilize it, and inject a portion of the culture to the new bottle. Another technique was the
injection of soluble or suspended carbon sources via needle and syringe. Finally, since the
29
particle size of some feeds was too large to fit through a needle, the bottle was simply opened in
the glove box, re-amended, and closed up again.
Avicel PH-101 microcrystalline cellulose (Sigma-Aldrich) is an insoluble powder with
a particle size of about 50 m. It was prepared by weighing out the desired amount into a 250
mL serum bottle then adding 100 mL of reverse osmosis water. The serum bottle was then sealed
with a black butyl rubber stopper, crimped tightly, and autoclaved at 121 C for 20 min. A sterile
22 gauge needle and 0.22 m filter as an inlet, and a second sterile 22 gauge needle as an outlet,
were used to purge each bottle with an 80 % N2, 20 % CO2 gas mix for 10 min to remove
residual oxygen. The bottle was shaken well by hand to suspend the cellulose evenly in the water
and 5 mL of the mixture was drawn through a 22 gauge needle into a 5 mL syringe while the
bottle was still in motion. The needle-tipped syringe was withdrawn from the cellulose stock and
held needle-upright until just before injecting into the culture, when it was tipped over, inserted
through the black butyl rubber stopper on the microcosm bottle, and quickly injected. This
method was used when measuring the chemical oxidation demand of the cellulose, and was
found to contain 1.22 0.12 gCOD/g cellulose, very close to the theoretical value of 1.19
gCOD/g cellulose.
Tannic acid (Sigma-Aldrich) and sodium lignosulphonate (Tembec Industries Inc.) are
soluble powders and were simply dissolved in the concentration appropriate for a 1 mL injection
to each microcosm bottle. The applicable mass of each was added to a 160 mL serum bottle and
sealed with a black butyl rubber stopper, crimped, and autoclaved at 121 C for 20 min. Each
stock solution was then purged with an 80 % N2, 20 % CO2 gas mix for 10 min.
Green needles from a red pine (pinus resinosa) were collected in September 2009, dried
at 103 C, and ground with a Wiley mill at the Faculty of Forestry. Steam exploded poplar
hydrolysate (SunOpta) was obtained in October 2009. The ground pine needle and poplar
hydrolysate particles are both insoluble, do not form a good suspension when dissolved in water,
and are too large to fit through a needle. When creating a new microcosm set or transferring a set
to new bottles, ground pine needle or poplar hydrolysate particles were weighed out in the
appropriate mass and added directly to serum bottles. Then, 5 mL of reverse osmosis water was
added, and each bottle was sealed with a black butyl rubber stopper, crimped, and autoclaved at
121 C.
Post extraction washer (PEW) effluent was collected on October 19, 2010 at a pulp and
paper complex in Temiscaming, QC. Prior to addition to enrichment bottles, it was autoclaved.
Addition proceeded by adding 3 mL of sterilized PEW to each bottle.
30
3.5. Microcosm setup
A set of 160 mL clear glass serum bottles (Wheaton) was prepared by soaking in 10 %
nitric acid overnight, then rinsing with distilled water three times. The bottles were allowed to air
dry, and feedstock was added as described above. The headspace in each sealed bottle was
subsequently purged for 5 min with 80 % N2, 20 % CO2 gas mix, placed in an autoclave bag, and
autoclaved in a polypropylene bin. A second bin, carefully lined with aluminum foil to not allow
any gaps, was filled with several glass stirring rods, two metal measuring spoons (15 and 30 mL
sizes), several scoopulas, extra butyl rubber stoppers, a 2 L beaker, and several polypropylene
funnels. This bin was also autoclaved. The microcosm setup procedure was completed in a glove
bag (medium tape-seal AtmosBag, Aldrich). Most required tools and supplies were placed in the
bag one day before the microcosm setup was to begin: both of the autoclaved bins, aluminum
crimping caps, crimping tool, decrimper, isopropanol wipes, latex gloves, and several 60 mL
syringes. This bag was sealed and filled with N2, unsealed and deflated, refilled and deflated
once more with N2, then finally filled with 80 % N2, 20 % CO2 gas mix. The inflated bag was
left overnight to ensure there were no substantial leaks. Nutrient medium from the glove box and
inoculum from the refrigerator were brought into the glove bag the next day immediately before
setup was to start. Gloves were worn over the hands, and over the glove-bag gloves to reduce the
chance of tearing the bag. All contents of the autoclaved bin containing supplies were removed
and the aluminum foil was carefully removed.
Microcosms of moose rumen, beaver droppings and internal circulation granules were
created to study lignocellulose biodegradation by corresponding microbial consortia. Each
source of inoculum mixed in a sterilized bin, and approximately 15 mL of inoculum was
transferred to a series of sterilized serum bottles using a funnel and glass rod. The nutrient
medium was decanted into a 2 L beaker (to leave behind solid iron sulphide), and a 60 mL
syringe and funnel was used to transfer 40 mL to bottles containing the inocula. A 5 mL volume
of each carbon source was then added, and the remaining 100 mL of headspace contained 80 %
N2 and 20 % CO2 (Figure 3.1). The serum bottles were re-sealed using black butyl rubber
stoppers and new aluminum caps, and then removed from the glove bag and transferred to a 36
C incubator.
31
Figure 3.1. Microcosm composition
Moose rumen microcosms were prepared on October 13, 2009, heritage pile on
November 6, 2009, both landfill-leachate and beaver droppings on February 12, 2010, and
internal circulation granules on August 24, 2010. Inoculum that was not used was transferred to
sterile 50 mL falcon tubes and stored at -80 C.
3.6. Dilution and transfer of cultures
Following the cessation of biogas production from a given set of microcosms, a fraction
of each microcosm was transferred to a new bottle, diluted back to the original volume with
nutrient medium, and fed more lignocellulosic material. As the feedstock degrades, organisms
involved in the decomposition are presumed to grow and multiply, and thereby become enriched
relative to the original microbial population. Therefore, through a regimen of transfer and
feeding, resulting microcosms will mainly comprise microorganisms that are relevant to the
particular bioconversion process. In the current study, the process of transfer and feeding
depended on the carbon source used and stage of enrichment. To date, cultures inoculated with
moose rumen fluid have been transferred four times and also fed four times, cultures inoculated
with beaver droppings have been transferred four times and fed twice, and cultures inoculated
with internal circulation granules have been transferred three times and fed two times. The
composition of carbon sources added to the initial cultures and subsequent transfers are
described in Table 3.1 to Table 3.3, and each round of cultivation is described as Phase I cultures
through Phase V cultures.
Phase I was characterized by the initial setup of microcosms from natural sources (moose
rumen and beaver droppings) and a relatively wide range of substrates at low concentrations to
test the ability of the microcosms to tolerate or degrade each compound without substantial
culture enrichment. About 35 mgCOD equivalent substrate was added to each microcosm in
32
Phase I (Table 3.1). In the case of combined cellulose and additive microcosms, the amount of
cellulose was reduced by 20 % from that of the cellulose only microcosms.
Table 3.1. Phase I amendments: initial microcosms
Feedstock
C per
bottle
C per bottle
a
(mgCOD )
[T,L]
T, L, P,
COD per
Total COD
Th.
(mg/L)
or PH
bottle
(mgCOD)
CH4
per
(mgCOD)
(mg)
(mL
STPb)
bottle
(mg)
Microcrystalline
28.4
33.7
33.7
16.1
22.8
27.0
295
17.7
22.0
49.0
23.4
22.8
27.0
88.3
5.3
8.1
35.1
16.8
22.8
27.0
5.7
7.9
34.9
16.7
28.4
26.6
26.6
12.7
cellulose (C)
C + tannic acid
(CT)
C + sodium
lignosulphonate
(CL)
C + pine needles
(CP)
Poplar
Hydrolysate (PH)
a
milligrams of oxygen required to completely oxidize the added feedstock as measured by the chemical
oxidation demand test.
standard temperature and pressure, 273 K and 101.3 kPa
Following Phase I enrichment, each culture was

divided into three fractions, diluted, and amended with a larger
dose of feedstock. The purpose of subsequent enrichment
phases was to enrich each microcosm for communities able to
subsist off of the supplied amendments. Phase II in particular
was intended to test a range of concentrations of each additive
as well as primary feedstock (cellulose or poplar hydrolysate).
Phase II cultures contained cellulose and poplar hydrolysate
amended with tannic acid, sodium lignosulphonate, or pine
needles in one of two concentrations. A total of 170 mg COD
(five times the total from Phase I), was added to each
microcosm in Phase II (Table 3.2). And as before, the amount
33
Figure 3.2. Microcosm Transfer

Apparatus
of cellulose was reduced by the COD equivalent of the added compound. This amount was
equivalent to about 4 % (low) or 20 % (high) of the total added to each bottle.
To dilute and re-amend Phase I cultures to generate Phase II microcosms, 18 mL of
suspended Phase I culture was transferred to new bottles. The remaining 6 mL was stored at 20C for possible future analyses. Bottles used in this transfer were amended with feedstock as
described in Table 3.2. A needle and syringe could not be used to accurately transfer microcosm
contents to new bottles because of the large particulate sizes, and wide-mouthed pipettes could
not be used because suction was too easily lost (particulates and liquid would fall out). To solve
this problem, an apparatus was designed and built to accurately dispense the correct volume of
mixed solid/liquid to a new bottle (Figure 3.2). This apparatus consisted of a wide mouthed 5 mL
pipette tip fixed to the bottom of a 60 mL syringe and attached to a flexible tube held shut with a
tube clamp. The syringe was calibrated using water. After transferring microcosm contents to
new bottles using the apparatus in a glove box, medium was used to rinse out the old bottle and
dispensed using the same apparatus to effectively transfer as much material as possible.
Following this transfer, the black butyl rubber stopper was replaced and crimped closed. A total
of 37 mL of medium was dispensed to each microcosm for a total volume of 60mL (inoculum,
medium, and feedstock).
Following the cessation of gas production in Phase II enrichment cultures, the contents
of each bottle were transferred to new bottles and diluted with fresh buffer and amendment. The
feeding scheme in Phase III was the same as in Phase II: 18 mL from each Phase II bottle was
transferred to a new bottle pre-amended with feedstock using the apparatus described earlier and
diluted with 37 mL of decanted medium.
Table 3.2. Phase II and III amendments: expanded substrate range enrichments
Feedstock
C or
C or PH
[T,L]
T, L,
T, L, or P
Total COD
Th. CH4
PH per
per bottle
(mg/L)
or P
per bottle
(mgCOD)
(mL
per
(mgCOD)
bottle
(mgCOD )
(mg)
STPb)
bottle
(mg)
Microcrystalline
142.2
168.5
168.5
80.6
113.8
134.9
1495
88.5
109.9
244.8
117.1
cellulose (C)
C + concentrated
tannic acid
(CT - high)
34
Feedstock
C or
C or PH
[T,L]
T, L,
T, L, or P
Total COD
Th. CH4
PH per
per bottle
(mg/L)
or P
per bottle
(mgCOD)
(mL
per
(mgCOD)
bottle
(mgCOD )
(mg)
STPb)
bottle
(mg)
C + dilute tannic
136.5
161.8
295
17.7
22.0
183.8
87.9
113.8
134.9
438.3
26.3
40.5
175.4
83.9
136.5
161.8
87.7
5.3
8.1
169.9
81.3
113.8
134.9
28.3
39.0
173.9
83.2
136.5
161.8
5.7
7.9
169.7
81.2
142.2
132.8
132.8
63.6
136.5
127.5
295
17.7
22.0
149.5
71.5
136.5
127.5
88.3
5.3
8.1
135.6
64.9
136.5
127.5
5.7
7.9
135.4
64.8
acid (CT - low)

C + concentrated
sodium
lignosulphonate
(CL - high)
C + dilute sodium
lignosulphonate
(CL - low)
C + high dose of
pine needles
(CP - high)
C + low dose of
pine needles
(CP - low)
Poplar
Hydrolysate (PH)
PH + dilute tannic
acid (CT PH)
PH + dilute sodium
lignosulphonate
(CL PH)
PH + low dose of
pine needles
(CP PH)
(Table 3.2 continued)
a
Phase IV microcosms followed a different amendment plan (Table 3.3), and was
designed to test the limits of the microcosms by using only the high concentrations of tannic
acid, sodium lignosulphonate, and pine needles. The best performing bottle of each triplicate set
from Phase III (as measured by a large volume of gas produced and a high initial rate of gas
35
production) was chosen as the inoculum for Phase IV enrichments. In contrast with earlier
amendment schemes, cellulose was added in equal amount to each bottle, and then other
amendments were added. This resulted in an unequal COD distribution but would lead to a better
understanding of the kinetics of the system, for example a biphasic gas production curve could
be attributed to degradation of cellulose first then tannic acid later. The transfer of Phase III
microcosms to Phase IV bottles was completed by transferring 18 mL of well mixed microcosm
contents using an 18 gauge needle and 20 mL syringe to a bottle previously loaded with
feedstock. 37 mL of medium was then added via a 60 mL syringe and 22.5 gauge needle.
Table 3.3. Phase IV amendments: best performing enrichment transfer
Feedstock
C or PH per
C or PH
[T,L]
T, L,
T, L, or P
Total
Th. CH4
bottle (mg)
per bottle
(mg/L)
or P
per
COD
(mL
per
bottle
(mgCOD)
STPb)
bottle
(mgCOD)
(mgCOD )
(mg)
Microcrystalline
113.8
134.9
134.9
64.5
113.8
134.9
1495
88.5
109.9
244.8
117.1
113.8
134.9
438.3
26.3
40.5
175.4
83.9
113.8
134.9
28.3
39.0
173.9
83.2
136.5
127.5
132.8
61.0
cellulose (C)
C + concentrated
tannic acid
(CT - high)
C + concentrated
sodium
lignosulphonate
(CL - high)
C + high dose of
pine needles
(CP - high)
Poplar
Hydrolysate (PH)
a
The purpose of the Phase V feeding and degradation cycle was to quantify the differences
in gas production yield from post extraction washer (PEW) effluent in each enrichment culture.
Since, by this point, each microcosm had been enriched to some extent relative to the initial
culture (as indicated by DGGE results), transfer and dilution between Phase IV and Phase V was
36
not performed. Instead, following approximately 30 days of incubation, 3 mL of two out of three
Phase IV cultures (i.e. replicate bottle 2 and 3) was replaced with 3 mL (containing 154
mgCOD equivalent per bottle or 2.57 gCOD/L) of PEW effluent; replicate 1 was not reamended to serve as a control for additional gas production from the original substrate beyond 30
days. Following 30 days of additional incubation, replicate 1 of each microcosm set was reamended with the targeted compound (i.e. 113.8 mg cellulose was added to cellulose control
microcosms, but only tannic acid, sodium lignosulphonate, pine needles, or poplar hydrolysate
were added to those bottles respectively at their Phase IV concentrations). Results from this
amendment were intended to show the degradability of the target compound itself rather than in
conjunction with cellulose.
An example transfer lineage is shown in Figure 3.3. To summarize, each inoculum (e.g.
moose rumen fluid (MR)) was distributed to bottles, which were amended and monitored
through Phase I. The contents of each Phase I bottle was then divided into three portions and
amended as indicated, and then monitored through Phase II. Approximately one-third of the
culture volume was then transferred to a new bottle and reammended, and then monitored
through Phase III. The best performing high concentration bottle, as determined by total gas
produced at the end of Phase III, was then divided into three portions and used as inoculum for
three new bottles in Phase IV. In Phase V, PEW was added to the same bottle as Phase IV, and
subsequently re-amended as noted above.
MR
Inoculum
Phase I
MR CT1
MR CT2
MR CT3
Phase II
MR CT2
Low
MR CT2
High
MR CT2
PH
Phase III
MR CT2
Low
MR CT2
High
MR CT2
PH
Phase IV
MR CT2
High (1)
MR CT2
High (2)
MR CT2
High (3)
Phase V
MR CT2
High (1)
MR CT2
High (2)
MR CT2
PEW (3)
Figure 3.3. Example transfer lineage
37
3.7. Biochemical analysis

3.7.1. Deoxyribonucleic acid (DNA) extraction
Two methods were used to extract metagenomic DNA from samples and obtain a product
that was free of compounds that might inhibit the polymerase chain reaction (PCR). A mixed
chemical, enzymatic, and mechanical lysis and extraction method was employed for raw
inoculum samples based on a method designed to optimize metagenomic DNA yield while
reducing inhibitor concentration [72], while the UltraClean Soil DNA Isolation Kit from Mo
Bio Laboratories Inc. was used to extract DNA from enrichment cultures. The chemical method
yielded larger and higher purity DNA fragments than the kit, but was only employed on the raw
inoculum samples because of the time required and the downstream application of the product.
High purity DNA from the raw inoculum was difficult to isolate in PCR-inhibitor-free form due
to coextraction with impurities potentially including humic acids and other associated
compounds. Since lignocellulosic materials that transferred with environmental inocula were
comparatively dilute in enrichment cultures, the purity of DNA obtained via the UltraClean kit
was sufficient for the PCR reaction. Both DNA extraction protocols can be found in Appendix C.
3.7.2. Amplification of 16 S rDNA
The purpose of the polymerase chain reaction (PCR) is to exponentially increase the
number of copies of a targeted DNA sequence for cloning, sequencing and functional
characterization of gene products. In this case, PCR was used to amplify a 161 base pair length
of the variable V3 region (from position 357 to 518) of 16S rDNA to profile the composition of
microbial communities. A series of guanines (G) and cytosines (C) was added to the end of the
forward PCR primer so that PCR products remained partially annealed when exposed to
denaturant in the DGGE procedure [73]. Each PCR reaction contained:
16.6 L of ultrapure sterilized water,
2 L of DNA template (0.1 - 2 ng/DNA per reaction),
0.4 L of each primer, as per Muyzer et al. [74] (Fermentas):
38
o forward (357F - 40GC clamp, sequence 5CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGC

ACGGGGGGCCTACGGGAGGCAGCAG-3) and
o reverse (R518, sequence 5-ATTACCGCGGCTGCTGG-3)
0.4 L of 25mM deoxynucleotide triphosphates (dNTPs) (Fermantas),
2.5 L of 10x ThermoPol buffer (New England BioLabsTM), containing

o 200 mM Tris-HCl,
o 100 mM (NH4)2SO4,
o 100 mM KCl,
o 20 mM MgSO4,
o 1 % Triton X-100,
2.5 L inhibitor-removing bovine serum albumin (BSA) (Sigma-Aldrich), and
0.2 L of Taq DNA polymerase enzyme (New England BioLabsTM).
All components except template were mixed in a master mix tube and distributed equally to each
PCR tube, and then DNA template was added. The PCR reaction cycle was: 94 C for 6 min,
then the three temperature cycle of 94 C for 30 sec, 55C for 1 min, and 72C for 1 min was
repeated 27 times before a final extension at 72C for 10 min, and hold at 4C.
PCR products were purified using the GeneJETTM kit from Fermentas (Thermo
Scientific), following the manufacturers instructions. Briefly, a 1:1:1 mixture of PCR product,
binding buffer, and isopropyl alcohol was mixed together and added to a GeneJETTM column
containing a silica membrane for DNA binding. Each column was centrifuged for 1 min at 11
000 X g and the flow through discarded. Next, 700 L of wash buffer containing 70 % ethanol
was loaded into each column and again centrifuged for 1 min at 11 000 X g. The flow through
was discarded, and each empty column was again centrifuged under the same conditions to
remove any residual ethanol. Finally, each column was removed and placed in a new tube, 20 L
of elution buffer was added, and was centrifuged for 1 min at 11 000 X g. The specificity of the
PCR reaction and purity of the PCR products was verified by running 1-5 L of each sample
(depending on concentration) through a 1 % (w/v) agarose gel (40 mM Tris acetate and 1 mM
EDTA (1x TAE buffer)); a 100 base pair ladder (Fermentas) was used as a standard to estimate
the size of each PCR product. The concentration of PCR products was estimated by comparing
the intensity of the bands on the agarose gel with those bands of known concentration on the
ladder. PCR products were then stored at 4 C, normally for less than one week, before analysis
by DGGE.
39
3.7.3. Denaturing gradient gel electrophoresis (DGGE)
Purified PCR products were loaded onto a denaturing gradient gel to separate DNA
fragments based on the relative fraction of GC, representative of individual species. Double
stranded DNA molecules are held together with hydrogen bonds between complementary bases:
guanine (G) and cytosine (C) always pair together with three hydrogen bonds (stronger), while
adenine (A) and thymine (T) pair together with two bonds (weaker). This difference in bond
strength can be extrapolated to the whole DNA molecule, with higher percentages of GC content
leading to more stable molecules. To exploit this principle and separate different DNA molecules
of the same size based on nucleotide composition, a linearly increasing concentration of
denaturant is incorporated into a polyacrylamide gel and DNA is run as in an agarose gel
electrophoresis procedure. The gel is then stained using SYBR gold, a nucleic acid gel stain, and
viewed under UV with a gel viewer.
The DGGE procedure requires several solutions to be made in advance. Two gel
solutions are required containing different amounts of urea and formamide denaturants. The 0 %
denaturant solution is prepared by mixing 20 mL of 40 % acrylamide/bisacrylamide (37.5:1)
(Biorad) with 2 mL 50 x TAE buffer (made with ultrapure Tris acetate, EDTA, and water) and
78 mL ultrapure water, then filtering this mixture through a 150 mL 0.22 m filter unit
(Sarstedt). The mixture is then degassed under vaccum for 15 min to remove dissolved air
bubbles. The solution is wrapped in aluminum foil and stored at 4C for up to one month before
use. The 80 % denaturant solution contains the same volume of acrylamide/bisacrylamide and
50x TAE buffer, but also requires 33.6 g ultrapure urea (Fluka-Sigma/Aldrich) and 32 mL
deionized biotechnology grade formamide (BioShop) and is diluted to 100 mL with ultrapure
water. The gel loading dye is prepared with 0.25 mL of 2 % bromophenol blue (w/v), 0.25 mL of
2 % xylene cyanol (w/v), 7 mL of glycerol, and 2.5 mL of ultrapure water. The apparatus
consists of an acetate block mixing chamber designed to combine solutions, containing low and
high concentrations of denaturant as they are pumped by a peristaltic pump to the gap between
two glass plates on a gel casting stand (Figure 3.4). Initially, the chamber on the right is filled
with the solution with a high concentration of denaturant and is pumped first to form the bottom
layer of the gel. As pumping progresses, the level of the more concentrated solution drops and
the less concentrated denaturant solution flows in to equalize the static pressure difference. Each
40
chamber is well mixed, so the solution in the right chamber becomes progressively more dilute
as more liquid is pumped to the casting stand.
Figure 3.4. DGGE Apparatus

Higher concentrations of denaturant in acrylamide solution are placed in the mixing chamber on the right,
lower concentrations on the left (acrylic block depicted on the upper left). The pump removes the higher
denaturant concentration acrylamide solution first, pumping it to the bottom of the casting stand. As the
level drops, lower denaturant concentration acrylamide solution mixes into the right chamber and dilutes the
denaturant, leading to a linear gradient of denaturant in the finished gel.
To determine the community structure and changes within communities after

enrichment, a denaturing gradient gel electrophoresis technique was performed using PCR
amplified 16 S rDNA gene fragments. The apparatus was put together as illustrated in Figure 3.4,
ensuring glass plates were properly clean and dry before assembly. The distance between glass
plates was checked by inserting a comb to determine a proper fit. A line was marked 1cm below
the teeth, and then the comb was removed. To make the two gel solutions, two 50mL falcon
tubes were used. Denaturant acrylamide gel solutions were combined according to the following
table to create the proper concentrations of denaturing agent.
Table 3.4. DGGE denaturant concentrations
% denaturant desired
0 % denaturant solution (mL)
80 % denaturant solution (mL)
30
7.2
4.3
70
1.4
10.1
41
Ammonium persulphate (115L of 10% (w/v)) and then tetramethylenediamine

(TEMED, 11.5L) (BioRad) were sequentially added to the 0 % and 80 % denaturant solutions,
and mixed by inversion, taking care not to introduce bubbles. Together, these two chemicals
catalyze the polymerization of the acrylamide gel. The 30 % denaturing agent gel solution was
then added to the left mixing chamber, ensuring the valve between chambers was closed, and the
70 % solution was added to the right mixing chamber. The magnetic stirrer was then turned on to
ensure complete mixing. At the same time, the valve between high and low denaturant solutions
was opened, and the pump was turned on. The flow into the glass plate sandwich was observed,
making sure the level of liquid was even across the apparatus. Once the solution reached the
marked line on the plate, the pump was turned off and the outflow line removed to a waste
beaker. Deionized water was then used to fill both chambers and the pump turned back on to
rinse the line before the gel polymerized. The gel and glass plates were covered by a piece of
plastic wrap and allowed to polymerize for 1.5 h. Immediately after covering the plates, 7 L of
reverse osmosis water and 150 mL 50 x TAE buffer were added to the immersion tank. The lid
of the tank was replaced, and then the stirrer and heater (set to 60 C) were turned on and
allowed to heat up.
Following polymerization, the gel was unwrapped and the comb was reinserted. A spacer
gel solution was mixed (consisting of 3.75 mL 0 % denaturant acrylamide solution, 1.25 mL 1x
TAE buffer, 45 L 10 % (w/v) ammonium persulphate, and 4.5 L TEMED), then added to the
top of the gel ensuring no air bubbles formed beneath the teeth of the comb. The gel was again
covered with plastic and allowed to polymerize for 30 min. Once set, the comb was slowly
removed, taking care not to break the gel. Excess gel was cut off, and the apparatus was washed
with reverse osmosis water. The plate apparatus was then attached to the core and warm TAE
buffer from the immersion tank was poured onto it to test for leaks. If a leak was detected, the
plate apparatus and core assembly were separated then reassembled and retested until secure.
The core was then inserted into the immersion tank and allowed to heat up for 15 min. During
this time, each PCR product was mixed with 20 L 2x loading dye. Once the temperature of the
immersion chamber reached 60 C, each well was loaded. The lid was replaced, and power
turned on at a constant 80 V for 16h.
Following electrophoresis, the gel apparatus was disassembled, the stacking gel was cut
off, and one corner of the gel was removed to identify the correct orientation. The gel was then
immersed in 250 mL of a 0.01 % (v/v) SYBR gold (Invitrogen) in 1x TAE buffer staining
42
solution and slowly agitated for 30 min. The gel was then transferred to a 1x TAE buffer
destaining solution and agitated for 15 min. The stained gel was then observed under ultraviolet
light on a gel viewer.
43
4.
Results and Discussion
4.1. Setup and transfers
Microcosms were initiated and transferred according to the timeline shown in

Table 4.1.
Table 4.1. Microcosm setup and transfer timeline
Start Date (incubation time in days)

Inoculum
Moose Rumen
Heritage Pile
Phase I
Phase II
Phase III
Phase IV
Phase V
Oct. 13 2009*
Aug. 12 2010
Nov. 17 2010
Jan. 15 2011
Feb. 9
(201)
(62)
(58)
(30)
2011
Nov. 6 2009
Feb. 12 2010
Jul. 23 2010
Sept. 30 2010
Jan. 6 2011
Feb. 9
(124)
(55)
(90)
(30)
2011
Aug. 24 2010
Oct. 23 2010
Jan. 9 2011
Feb. 9
(50)
(76)
(30)
2011
(299)
Landfill-leachate
Feb. 12 2010
(76)
Beaver Droppings
granules
*Moose rumen microcosms were additionally re-ammended with secondary feed (sodium lignosulphonate,
tannic acid, or pine needles) on Dec. 23 2009.
A chart outlining the progression of dilutions and the lineage of each treatment
combination for moose rumen, beaver dropping, and internal circulation granules microcosms
can be seen in Figure 4.1 A), B), and C) respectively. In these diagrams, time progresses from
the centre of the circle to the circumference, from initial inoculum material (ex. MR, or moose
rumen fluid) to subsequent sequential enrichment cultures. The numbers found beside each
acronym, for example C1,2,3, indicate the biological replicates amended in that line.
44
45
46
Figure 4.1 Lineage of A) moose rumen (MR), B) beaver dropping (BD), and C) internal circulation granules
(ICG) enrichment cultures.
C: microcrystalline cellulose alone; CT: cellulose plus tannic acid, CP: cellulose plus pine needles, CL:
cellulose plus lignosulphonate, PH: poplar hydrolysate. Percent transfer refers to the percentage of inoculum
added to the subsequent Phase. Phase I amendments contained about 600 mgCOD/L, approximately 24
mgCOD/L of which was either tannic acid, pine needles, or lignosulphonate. Phase II, III, and IV poplar
hydrolysate and cellulose-containing enrichments, with comparatively low concentration of amendment
(designated by low) contained approximately 3 gCOD/L total, with either tannic acid, pine needles, or
lignosulphonate representing approximately 120 mgCOD/L of the total. The same total amount (3 gCOD/L)
was added to bottles designated by high, these contained a higher amount of either tannic acid, pine
needles, or lignosulphonate (about 600 mgCOD/L). The number of replicate bottles is indicated for each
transfer. Following up to four transfers, two of the three replicate enrichments were amended with 3 mL of
post extraction wash (PEW) from a sulphite mill (equivalent to approximately 2.5 g COD per L).
47
COD content (mg/g)
4.2. Inoculum Properties
20
15
10
5
0
Moose
rumen
Beaver
Landfilldroppings leachate
Heritage
pile
IC
granules
Figure 4.2 COD content in environmental inocula.

n = 3, error bars represent confidence interval at p = 0.05
Despite differences in COD, in all cases, 15 mL of sample was used as inocula in Phase I
enrichments. Notably, heritage pile and landfill-leachate inoculum contained significantly less
soluble COD than moose rumen, beaver droppings, or internal circulation granules cultures. This
agrees with subsequent results showing that unamended microcosms from these latter three were
significantly more active in terms of gas production.
Moose rumen inoculum was very fibrous and contained a significant amount of nondegraded plant material (wood fibres, small twigs). Beaver droppings also contained fibrous
material, but the particle size was smaller. Heritage pile material was primarily soil with a
dispersed fibrous material with a particle size comparable to that observed in beaver droppings.
Landfill inoculum resembled soil with little lignocellulosic material. Internal circulation granules
were was approximately 1-2 mm round black particles, and did not contain visible fibrous
material.
48
4.3. Substrate Properties
Substrates were characterized by COD, volatile solids as a fraction of total solids, and
FTIR absorbance profile (Table 4.2; Figure 4.3).
Table 4.2. Substrate COD and VS
Cellulose
Sodium
Tannic
Pine
Poplar
PEW
lignosulphonate
acid
needles
Hydrolysate
wastewat
er
COD
(gCOD/g)
VS (% of
TS)
a
1.22
0.12
1.54 0.11
1.22 0
95.2 2.3
0.97
0.93 0.30
1.42
100.5 4.9
not
0.10
29.2 3.8
98.9
92.4 0.6
0.3
measured
grams of oxygen required to completely oxidize the added feedstock as measured by the chemical
oxidation demand test, per gram of material tested

b
% volatile solids as a percentage of total solids
n = 3, represents confidence interval at p = 0.05
The theoretical COD for each of the defined substrates (cellulose, sodium
lignosulphonate, and tannic acid) was very close to the respective mean measured value of each.
Volatile solids (VS) values represent the fraction of organic matter in each feedstock; with the
exception of sodium lignosulphonate, close to 100 % of each substrate is volatile organic matter
as expected. Sodium lignosulphonate powder, an industrial product, contains a significant
fraction of ash ranging up to 25 % dry weight [75], [76], explaining some of the discrepancy in
VS content. The remaining portion is perhaps due to insufficient exposure to combustion
temperatures, as a black residue remained on each dish following heating to 550 C.
FTIR profiles can be used to identify compounds within a mixed substance, based on the
chemical bonds contained within the individual compounds. Table 4.3 summarizes FTIR
fingerprints that are typically generated from lignocellulosic substances [77-83].
49
Table 4.3 FTIR peak identification table.
Material Wavenumbers (cm-1)
Bonds
Lignin, carbohydrates
1800-1700
Unconjugated C=O
1700-1550
Conjugated C=O stretch, C=C stretch
Lignin
1515-1490
Aromatic skeletal vibrations
1470-1450
C-H deformation
Lignin
1430-1420
Aromatic skeletal vibrations
Carbohydrates
1380-1370
C-H deformation in carbohydrates
1330-1320
C-H vibrations in cellulose and C1-O vibration in

syringyl derivatives
Lignin
1280-1260
Guiacyl ring breathing, C-O stretch in lignin, C-O

linkage in guiacyl aromatic methoxyl groups
1240-1230
Syringyl ring and C-O stretch
Carbohydrates
1160-1150
C-O-C vibration in cellulose and hemicellulose
1130-1120
Aromatic skeletal and C-O stretch
1065-1040
C-O stretch
898
C-H deformation in cellulose
Cellulose
FTIR was used in the current study to evaluate the main compositional differences in the
amendments used in the various enrichments (Figure 4.5). Distinct peaks in the fingerprint
region (1800 cm-1 to 800 cm-1) were identified for each amendment, and peak assignments were
made using the bonds listed in Table 4.3.
50
Relative Absorbance
Figure 4.3. FTIR spectra of each substrate
While peak intensities do not necessarily reveal the relative abundance of different
components within the same sample, absorbance intensity can be used to compare the abundance
of the same compound between samples. Since the peaks associated with each bond are not
necessarily found at an exact wavenumber, the highest peak within the listed range is reported.
From Figure 4.3, it can be seen that the profiles obtained by FTIR analysis are complex and
difficult to analyze by eye. Notable differences clearly observable include the peaks found at
about 1750 cm-1, normally associated with conjugated or non-conjugated C=O bonds, that can be
seen in all three of tannic acid, pine needles, and poplar hydrolysate but not Avicel or sodium
lignosulphonate. Another peak at about 1600 cm-1, associated with lignin-related bonds, can be
seen in the profile of all compounds except Avicel. To reveal more subtle differences between
amendments, the absorbance at each peak wavenumber was plotted for each substrate (Figure
4.4). Caution should be exercised when interpreting these results for two reasons. Firstly, as
noted earlier, the peaks are not located at a precise location but rather within a range of
wavenumbers, leading to errors in identification. Secondly, the peaks overlap with each other
and would require the application of deconvolution techniques to isolate individual values.
51
Figure 4.4 Lignocellulose associated FTIR profile peaks.

(
- Avicel PH101 cellulose,
SunOpta poplar hydrolysate )
- Tannic acid,
- Sodium lignosulphonate,
- Red pine needles,
Cellulose samples absorbed more strongly than lignin at several carbohydrate associated
peaks, including 900-890 cm-1 and to a lesser extent at 1380-1370 cm-1 and 1160-1150 cm-1.
Lignosulphonate absorbed much more strongly relative to cellulose and tannic acid at aromatic
or lignin associated peaks at 1515-1490 cm-1 (aromatic skeletal vibration), 1280-1260 (guiacyl
ring breathing, carbon-oxygen single bond stretch, carbon-oxygen linkage in guiacyl aromatic
methoxyl groups), and 1430-1420 cm-1 (aromatic skeletal vibration). Tannic acid absorbed
strongly relative to cellulose and lignosulphonate at 1800-1700 cm-1 and 1330-1320 cm-1, which
are absorbed by unconjugated carbon-oxygen double bonds, and carbon-hydrogen vibration or
carbon-oxygen single bonds respectively. One peak reported to correspond to both lignin and
carbohydrate associated bonds, at 1700-1550 cm-1, absorbed much more strongly in lignin
samples than in cellulose. This wavenumber is associated with conjugated (aromatic or carbonyl)
carbon-oxygen double bonds and carbon-carbon double bonds, neither of which is found in
cellulose, but may be found in branched hemicelluloses, and are certainly present in lignins.
From the relative absorbance values of pine needles and hardwood hydrolysate samples
(Figure 4.4), it can be seen that poplar samples absorb more strongly at carbohydrate-associated
wavelengths, while both samples absorb similarly strongly at wavelengths associated with
52
aromatic structures found in lignin and many extractives. At 900-890 cm-1, both cellulose and
tannic acid show much higher peaks than lignosulphonate, while at 1065-1040 cm-1, both
cellulose and lignosulphonate show much higher peaks than tannic acid. The relative difference
between pine needles and poplar hydrolysate is much larger at 1065-1040 than 900-890 cm-1,
possibly indicating a higher concentration of tannic-acid-like compounds in red pine needles.
The relative absorbance between the two samples at 1800-1700 cm-1 is consistent with this
interpretation.
4.4. Gas Production Results
The rate and extent of the methanogenic conversion of lignocellulosic substrates to biogas
was primarily determined by regularly measuring the volume of biogas that had accumulated in
each bottle. At the end of each enrichment phase, the biogas in the headspace was also measured
for CH4 content. Accordingly, three metrics were used to compare the performance of
microcosms:
1. Time required to produce 20 % of the theoretical yield of biogas
2. Yield of biogas;
3. CH4 content of biogas.
The first two parameters were extracted from the gas production data as shown in Figure 4.5.
Sample calculations for each of these metrics are shown in Appendix G. The time required to
produce 20 % of theoretical biogas production is a combination metric encompassing both the
lag time and initial rate of reaction. This was chosen over either of the other two metrics to give a
realistic indication of gas production inhibition. Care should be taken to interpret these results
within a given set of enrichments (encompassing a single inoculum) due to the differences in
biomass loading, since values are not normalized to the amount of bacteria present. A value of 20
% was chosen to best capture this metric because for values lower than this it is difficult to
resolve differences between enrichments (i.e. almost all cultures degraded the first 5 % of COD
added within the first 2 days no finer interval is available due to data collection methods).
Headspace CH4 content was measured directly following the cessation of gas production and
used to calculate the CH4 content of the gas produced via the method described in section 3.2.5.
53
COD Consumed
(mgCOD in gas produced)
mgCOD added
1.0
0.8
Measured gas production
0.6
Yield
0.4
0.2
0.0
Time to 20% COD Removal
Time (days)
Figure 4.5 Example gas production curve
Briefly, each of the moose rumen, beaver droppings, landfill-leachate, and heritage pile sets of
microcosms were monitored for biogas production, which contained a combination of carbon
sources as described in Table 3.1. This period, from setup to the first transfer, is designated
Phase I. In Phase II of the enrichment, moose rumen and beaver dropping consortia were
transferred to new bottles, diluted with fresh medium, and amended with a different combination
of feedstocks. At this stage, microcosms inoculated with internal circulation granules were also
prepared. In Phase III of the enrichments, moose rumen, beaver dropping, and anaerobic granule
consortia were transferred to new bottles, diluted with fresh medium, and amended as in Phase
II. Then, in Phase IV of the enrichment, only of the best performing replicate of each Phase III
enrichment was transferred to new bottles, diluted with fresh medium, and amended with 5-times
more carbon source. Phase V did not involve a transfer; instead, two of the three replicates for
each Phase IV enrichment was directly amended with post extraction washer wastewater from a
sulphite mill.
4.4.1. Phase I Enrichments
Triplicate Phase I cultivations were prepared for moose rumen, beaver droppings,
landfill-leachate, and heritage pile microcosms amended with a soluble volatile fatty acid (VFA)
54
and glucose mix (50 % glucose, 45 % sodium acetate, 5 % sodium propionate on a COD basis)
(P), steam exploded poplar hydrolysate (PH), cellulose (C), and cellulose plus sodium
lignosulphonate (CL), tannic acid (CT), or ground red pine needles (CP). No amendment (NA)
controls for each set of microcosms were prepared to measure biogas that was generated through
the endogenous decay of COD directly from the inoculum. Biogas production curves for each set
of enrichments are reported in Appendix D.
The initial rate of gas production is quantified by the time required by an enrichment to
reach 20 % of the theoretical gas production as calculated by total amount of added COD (Figure
4.6). This metric estimates the inhibitory effects of the amendment on biogas production.
Moose rumen
Beaver droppings
Landfill-leachate
Heritage pile
0
25
50
75
100
125
Time to 20% COD Removal (days)
Figure 4.6 Initial rates of biogas production in Phase I microcosms.

(
- VFAs and glucose,
- SunOpta poplar hydrolysate,
- cellulose and
tannic acid,
- cellulose and sodium lignosulphonate,
- cellulose and red pine needles) Results are
presented as the difference in gas production between unamended controls and each amendment rather
than the total amount of gas produced. Poplar hydrolysate data for moose rumen microcosms is not
available, as the substrate was not available at the time moose rumen cultures were inoculated. n = 3, error
bars represent confidence interval at p = 0.05.
Overall, the landfill-leachate microcosm set reached 20 % of the expected biogas

production the fastest, followed by moose rumen, heritage pile, then finally beaver droppings.
This order is similar to the order of microcosm sets ranked by amount of endogenous COD
content (with the exception of heritage pile and moose rumen, which are switched). Significant
lag times were only observed for beaver droppings, with the addition of cellulose alone, and may
contribute to this result. It is possible that the microbial community initially degraded substrates
present in the inoculum (to which they were already adapted), and only later began to degrade
55
the amended carbon sources. In beaver dropping microcosms amended with cellulose only,
cellulose and pine needles, and poplar hydrolysate; and moose rumen microcosms amended with
cellulose only, the initial rate of biogas production was higher in no amendment controls than in
bottles amended with new carbon sources. However, the significance of this result is unclear
given the high variability between replicate cultures.
The time to 20 % COD removal required on average 23 days for all substrates and
inocula. It was expected that the addition of soluble glucose and VFAs would lead to faster
degradation times because a hydrolysis step is not required, but this was not observed in any of
the microcosms with the exception of beaver droppings. In fact, glucose and VFAs were
degraded significantly more slowly than cellulose in heritage pile microcosms. Poplar
hydrolysate was degraded at a faster initial rate in each of beaver droppings, landfill-leachate,
and heritage pile microcosms (data is not available for moose rumen), perhaps due to easily
accessible compounds in the pretreated fibre. Of all the cellulose-based treatments, only cellulose
plus pine needles in beaver dropping enrichments reached 20% biogas production significantly
more rapidly than enrichments amended with cellulose alone.
Biogas yields were calculated once biogas production was no longer measurable. Not
surprisingly, biogas yield in replicate, Phase I cultivations varied significantly, with beaver
droppings and moose rumen microcosms exhibiting the largest variability between replicates and
heritage pile and landfill-leachate microcosms showing somewhat less variability. Biogas yields
from these cultures are shown in Figure 4.7. Individual replicates are shown to illustrate large
differences between them.
56
Figure 4.7 Biogas yield from Phase I A) moose rumen, B) beaver dropping, C) landfill-leachate, and D)
heritage pile microcosms
- CT:
(
- P: VFAs and glucose,
- PH: SunOpta poplar hydrolysate,
- C: Avicel PH101 cellulose,
cellulose and tannic acid,
- CL: cellulose and sodium lignosulphonate,
- CP: cellulose and red pine
needles) Results are presented as the total amount of gas produced by each microcosm, normalized to the
amount of gas expected based on a COD balance. Values account for background gas production from
degradation of inoculum COD by subtracting the gas produced from unamended microcosms. Poplar
hydrolysate data for moose rumen microcosms is not available, as the substrate was not available at the
time moose rumen cultures were inoculated.
The endogenous biogas production by these cultures (without addition of substrate), and
more importantly the variability in ultimate gas volume, varied widely as can be seen in Table
4.4. Since the total COD added to the average culture in Phase I was expected to result in about
40 mL of biogas, most of the variability in the ultimate gas production can be explained by the
inconsistent biogas volume produced with no amendment.
57
Table 4.4. Biogas produced in no amendment (NA) reference cultivations.

Plus/minus represents confidence interval at p = 0.05, n = 3.
Inoculum
Biogas Yield (mL)
Moose rumen
Beaver droppings
Landfill-leachate
Heritage pile
110 20.3
197 36.0
5.2 6.4
40.1 6.2
Overall, microcosms prepared from landfill-leachate and amended with cellulose alone
produced significantly more biogas than any of the other amendments. In contrast, microcosms
prepared from heritage pile samples, and amended with cellulose alone produced significantly
less biogas than those amended with cellulose plus tannic acid, sodium lignosulphonate, or red
pine needles.
Despite the variance in the data collected for replicate enrichments, several trends were
observed. With the exception of beaver dropping cultures, the average biogas produced by
microcosms amended with VFA and glucose was approximately 90 % of the expected biogas; by
contrast, with the exception of landfill-leachate cultures, cellulose amended bottles typically
produced 50 % of the theoretical biogas yield (Figure 4.7). Notably, the average biogas yield
from microcosms enriched using cellulose plus an additional carbon source, was often higher
than that from microcosms enriched using cellulose alone, where biogas yield was generally
highest in cultures containing cellulose plus sodium lignosulphonate, followed by cellulose plus
tannic acid, and then cellulose plus red pine needles (Figure 4.7).
The CH4 content in biogas from heritage pile microcosms enriched on cellulosic
substrates was generally lower than corresponding enrichments of moose rumen and beaver
droppings (Figure 4.8). In general, the CH4 content in biogas samples was similar between
enrichments of the same inoculum. Exceptions were the comparatively low CH4 content in the
cellulose plus pine needle and cellulose plus tannic acid enrichments of moose rumen, and the
comparatively high CH4 content in the VFA/glucose enrichments of heritage pile samples.
58
Figure 4.8 CH4 fraction of biogas generated by A) moose rumen, B) beaver dropping, and C) heritage pile
(
- NA: unamended microcosms,
- P: VFAs and glucose,
- PH: SunOpta poplar hydrolysate,
- C:
Avicel PH101 cellulose,
- CT: cellulose and tannic acid,
- CL: cellulose and sodium lignosulphonate,
- CP: cellulose and red pine needles) Values were calculated as described in Section 3.2.5. Data from
microcosms prepared using landfill-leachate are not shown due to instrumental inconsistencies experienced
during the data collection.
In summary, Phase I analyses led to the following general conclusions:
Biochemical methane potential of inocula when considering biogas produced by no-
amendment controls: beaver droppings > moose rumen > heritage pile > landfill leachate
Average biogas production rate: landfill leachate > moose rumen > heritage pile >
beaver dropping
Average biogas yield: landfill leachate > beaver dropping > heritage pile > moose
rumen
Average CH4 content: beaver dropping > moose rumen > heritage pile
Time to 20 % COD removal, biogas yield, and CH4 content were not significantly
correlated to each other.
Significant lag times (zero gas production rate) were not observed during phase I.
59
Given the comparative quality of biogas generated by the moose rumen and beaver
dropping microcosms, the total activity measured in the corresponding microcosms (i.e.
including biogas from no-amendment controls), and the comparatively high overall performance
of at least one of three replicate microcosms in both cases, microcosms of moose rumen and
beaver dropping inocula were selected for further enrichment in Phase II cultivations.
4.4.2. Phase II and III Enrichments
Moose rumen and beaver dropping cultures were transferred after 201 and 124 days
respectively from their initial setup to new bottles following the protocol described in Section
3.6. As much of the solid portion as possible was transferred with these microcosms to retain the
microbial community that adhered to lignocellulosic materials. Internal circulation granules were
introduced at this stage of the project to provide a benchmark for bioconversion performance as
well as an additional relevant sample for microbial enrichment. Results from the internal
circulation granules at this stage, particularly Phase II, should not be directly compared to moose
rumen and beaver droppings due to the considerable biogas that was likely produced from COD
retained in the original inoculum.
The objective of Phase II cultivations was to test the effect of the three additives, tannic
acid, sodium lignosulphonate, and ground red pine needles on the degradability of mixed
substrates while avoiding the variability due to endogenous substrate decay characteristic of
Phase I results. Accordingly, five times as much COD was added to each microcosm to ensure
residual endogenous methanogenesis did not significantly contribute to biogas yield or
production rate, at least for the moose rumen and beaver dropping transfers. Further, tannic acid,
lignosulphonate, and ground pine needles were added at either Low or High concentrations.
The Low amendment series contained the same amount of tannic acid, lignosulphonate, or
ground pine needles as used in Phase I cultures (Table 3.2), while the High amendment series
retained the same ratio of cellulose to additive, which meant five times more tannic acid,
lignosulphonate, and ground pine needles were added on a milligram basis. Microcosms
containing poplar hydrolysate plus additive (i.e. tannic acid, lignosulphonate, or ground pine
needles) were also initiated at this stage, where the same amount of additive as listed for
cellulose based cultures was used (Table 3.2). Following the cessation of gas production, onethird of each Phase II microcosm was transferred to a new bottle, and re-amended with
60
feedstock. Phase III enrichments were identical to Phase II cultures, simply diluted and reamended, therefore they are compared here together. Full gas production curves for each of these
inocula and all permutations of substrate are shown in Appendix E.
4.4.2.1. Time to 20 % COD consumption
In most cases, the initial rate of biogas production by microcosms amended with cellulose
only or poplar hydrolysate were not significantly different from each other. In Phase II
experiments, internal circulation granules from a pulp mill were included in the comparative
study, and as expected, were the first to produce 20% of expected biogas, followed by beaver
dropping and then moose rumen enrichments. However, this trend was not observed in Phase III
enrichments (Figure 4.9). As previously noted, these results are likely due to differing amounts
of biomass loading, and should not be used as direct comparison. Biogas production rate was
most stable over time in beaver dropping enrichments, both in terms of time and between
substrates. It is interesting to note that although the concentration of microbes available at the
beginning of Phase III was likely lower than at the same point in Phase II (assuming low growth
rates), the initial rate of gas production did not decrease in most cases, suggesting that the
fraction of active microorganisms had increased, or that the concentration of microbial biomass
was not a limiting factor in degradation.
61
Moose
rumen
IC
Beaver
granules droppings
II
III
II
III
II
III
0
10
20
30
40
50
60
70

Figure 4.9 Initial rates of biogas production in Phase II and Phase III microcosms
(
- Avicel PH101 cellulose). n = 3, error bars represent confidence
interval at p = 0.05. Phase II IC granules were not enriched.
The addition of cellulose plus lignosulphonate and cellulose plus pine needles to Phase II
and Phase III enrichments did not have a significant effect on initial rates of biogas production
(Figure 4.10), an exception was Phase II enrichment of moose rumen samples on cellulose plus
sodium lignosulphonate. In contrast, the addition of cellulose plus 1450 mg/L tannic acid
increased the time to 20 % COD degradation by an average of over 25 days from the cellulose
only case across both Phase II and Phase III and all three microcosm sets. This effect was more
pronounced in moose rumen and beaver droppings, but was also observed (although not
significantly) in internal circulation granules enrichments. Much of this increase in the time to
20% degradation can be attributed to a decrease in the initial rate (as seen in Appendix E),
although not necessarily a complete absence of gas production. Following this lag time (as long
as 50 days, as seen in beaver dropping cultures in Phase III), a sharp increase in gas production
rate was observed, ultimately leading to a gas yield similar to that of other treatments. The time
to 20% degradation for these cultures excluding lag time was similar to that of cellulose only
amendments. The addition of cellulose plus 295 mg/L tannic acid, however, did not significantly
alter the initial rate of degradation from that of cellulose alone.
62
Moose rumen
Beaver droppings
IC granules
II
III
II
III
II
III
10
20
30
40
50
60
70

(
- cellulose and 1450 mg/L tannic acid,
- cellulose and 295 mg/L tannic
acid,
- cellulose and 438 mg/L sodium lignosulphonate,
- cellulose and 88 mg/L sodium
lignosulphonate,
- cellulose and 472 mg/L red pine needles). n = 3, error bars represent confidence
interval at p = 0.05. Phase II IC granules were not enriched.
63
The initial rate of biogas production in moose rumen microcosms was similar in
enrichments amended with poplar hydrolysate only and poplar hydrolate plus tannic acid, pine
needles or lignosulphonate (Figure 4.11). While rates of biogas production generally decreased
in Phase III moose rumen cultivations, biogas production rate by beaver dropping enrichments
was similar in Phase II and Phase III, and comparable to internal circulation granules
IC
Beaver
granules droppings
Moose
rumen
enrichments in Phase III (Figure 4.11).
II
III
II
III
II
III
0
10
20
30
40
50
60
70

(
- SunOpta steam exploded poplar hydrolysate,
- poplar hydrolysate and 295 mg/L tannic acid,
poplar hydrolysate and 88 mg/L sodium lignosulphonate,
- poplar hydrolysate and 95 mg/L red pine
needles). n = 3, error bars represent confidence interval at p = 0.05. Phase II IC granules were not enriched.
4.4.2.2. Biogas yield
On a COD basis, the biogas yield from moose rumen microcosms enriched on cellulose
alone is just over 70 % in both Phase II and III (Figure 4.12). This yield is equivalent to a CH4
yield of about 0.4 L/gVS, and is similar to previously reported CH4 yield from cellulose [58],
[59]. With the exception of Phase II internal circulation granules enrichments and Phase III
64
beaver dropping enrichments, biogas yield was higher in cultivations enriched on cellulose only
COD Consumed
mgCOD added
than on poplar hydrolysate.
3
2.5
2
1.5
1
0.5
0
II
III
Moose rumen
II
III
Beaver droppings
II
III
IC granules
Figure 4.12 Biogas yield from Phase II and Phase III microcosms
(
interval at p = 0.05. IC granule data for phase II represents original inoculum, containing large quantities of
endogenous COD. Phase II IC granules were not enriched.
In both Phase II and Phase III, the yield of biogas in moose rumen cultivations enriched
on cellulose plus 1450 mg/L tannic acid or 472 mg/L red pine needles was significantly lower
than other moose rumen cultivations (Figure 4.13). This suggests that these additives may not be
degradable, or that they are reducing the degradability of cellulose in these cultures. Tannic acid,
and to a lesser extent, ground pine needles, also reduced biogas yield in Phase II enrichments of
beaver droppings (Figure 4.13). Biogas yield by enrichments of moose rumen, beaver droppings
and internal circulation granules were similar in Phase III cultivations.
65
COD Consumed
mgCOD added
3
2.5
2
1.5
1
0.5
0
II
III
Moose rumen
II
III
Beaver droppings
II
III
IC granules

(
lignosulphonate,
- cellulose and 472 mg/L red pine needles,
- cellulose and 95 mg/L red pine needles).
n = 3, error bars represent confidence interval at p = 0.05. IC granule data for phase II represents original inoculum, containing large quantities of endogenous
COD. Phase II IC granules were not enriched.
66
Slightly higher biogas yields in Phase II moose rumen microcosms amended with poplar
hydrolysate plus tannic acid or poplar hydrolysate plus sodium lignosulphonate compared to
cultures amended with poplar hydrolysate alone was statistically significant, however, this
difference was not apparent in Phase III enrichments (Figure 4.14). While beaver droppings
showed slightly lower biogas yields in Phase III enrichments compared to Phase II enrichments,
the most dramatic decrease in biogas yield was observed in enrichments of internal circulation
granules. Similar to the time to 20 % degradation of added COD (Section 4.4.2.1), the presence
COD Consumed
mgCOD added
of additives at the concentrations used, did not have a major, reproducible effect on biogas yield.
3
2.5
2
1.5
1
0.5
0
II
III
II
Moose rumen
III
Beaver
droppings
II
III
IC granules
(
needles). n = 3, error bars represent confidence interval at p = 0.05. IC granule data for phase II represents
original inoculum, containing large quantities of endogenous COD. Phase II IC granules were not enriched.
4.4.2.3. CH4 fraction
The fraction of CH4 in biogas sampled from Phase II moose rumen enrichments was
higher than expected based on a simple mass balance, with about 76 % CH4 in poplar
hydrolysate samples and 65 % in cellulose degrading cultures (Figure 4.15). However, Phase III,
corresponding values decreased to 50 % for both amendments (the theoretical expected value
67
from methanogenesis from either cellulose or hemicellulose (Appendix G). Beaver dropping
enrichments sampled in Phase III showed abnormally low CH4 content. However, due to the
consistently low measurements despite relatively high biogas yield and comparatively high CH4
content in Phase II samples, it is likely that these Phase III results are due to poor calibration of
Methane fraction
the GC column used to analyze the Phase III beaver dropping samples.
1
0.8
0.6
0.4
0.2
0
II
III
Moose rumen
II
III
Beaver droppings
II
III
IC granules
Figure 4.15 CH4 fraction of biogas generated by Phase II and Phase III
(
interval at p = 0.05. Beaver droppings phase III data is low due to instrumental error. Phase II IC granules
were not enriched.
In general, no major reduction in the CH4 content of biogas samples was observed in
cultivations enriched on cellulose plus either concentration of tannic acid (with the exception of
Phase III moose rumen cellulose plus 1450 mg/L tannic acid), or cellulose plus either
concentration of lignosulphonate (excepting Phase II internal circulation granules) (Figure 4.16).
Notably, a small but consistent increase in CH4 was observed in biogas samples from
enrichments on cellulose plus 472 mg/ L and 95 mg/L of red pine needles.
68
Methane fraction
1
0.8
0.6
0.4
0.2
0
II
III
Moose rumen
II
III
Beaver droppings
II
III
IC granules
Figure 4.16 CH4 fraction of biogas generated by Phase II and Phase III enrichments
(
lignosulphonate,
- cellulose and 472 mg/L red pine needles,
- cellulose and 95 mg/L red pine needles).
n = 3, error bars represent confidence interval at p = 0.05. Beaver droppings phase III data is low due to instrumental error. Phase II IC granules were not
enriched.
69
The CH4 content in Phase II moose rumen enrichments were particularly high, and over
80 % in enrichments amended with poplar hydrolysate and 472 mg/L red pine needles (Figure
4.17). Similar to measured CH4 content in Phase III enrichment of beaver droppings on cellulose
alone, poplar hydrolysate alone, and cellulose plus additional carbon sources, the CH4 content in
Phase III enrichments of beaver droppings on poplar hydrolysate plus additional carbon source
Methane fraction
was surprisingly low, and likely due to technical error.
1
0.8
0.6
0.4
0.2
0
II
III
Moose rumen
II
III
Beaver droppings
II
III
IC granules
Figure 4.17 CH4 fraction of biogas generated by Phase II and Phase III enrichments
(
needles). n = 3, error bars represent confidence interval at p = 0.05. Beaver droppings phase III data is low
due to instrumental error. Phase II IC granules were not enriched.
In summary, the main results of Phase II and Phase III enrichments are:
Significant increase in biogas production rate by moose rumen samples enriched on
cellulose only (Figure 4.9) from Phase II to Phase III.
Biogas production rates (Figure 4.10) and biogas yield (Figure 4.13):
o Similar for all moose rumen and beaver dropping Phase III cultivations
enriched on cellulose or cellulose plus lignosulphonate or pine needles
70
o IC granules > Beaver droppings > Moose rumen when comparing results
of tannic acid addition
o Significantly lower time to 20% COD removal and higher gas yield
Lag times (Appendix E) were only consistently observed for tannic acid addition,
although several individual moose rumen cultures amended with any of pine needles,
lignosulphonate, or cellulose only (in addition to tannic acid) in either Phase II or Phase
III were affected. Lag times were observed with greater frequency and duration in Phase
III compared to Phase II.
Similar biogas production rates (Figure 4.11) and biogas yield (Figure 4.14) in Phase
III beaver dropping and internal circulation granules cultivations enriched on poplar
hydrolysate or poplar hydrolysate plus an additional carbon source. Corresponding values
in moose rumen enrichments were comparatively low.
Similar CH4 content (Figure 4.15) in biogas sampled from all Phase III enrichments
of moose rumen, beaver dropping, and internal circulation granules.

While amending enrichments with 295 mg/L tannic acid did not significantly affect
biogas production rate, yield or CH4 content, amending enrichments with 1450 mg/L tannic acid
reduced biogas production rate (or increased the lag time by up to 50 days) and yield in
enrichments of moose rumen and beaver droppings; high concentrations of tannic acid also
reduced the CH4 content in biogas from moose rumen enrichments.
71
4.4.3. Phase IV Enrichments
The objective of Phase IV enrichments was to compare the biological activity of the best
replicate from each set of Phase III enrichments. Accordingly, Phase IV microcosms included
the best (as measured by a combination of biogas yield, time to 20 % of theoretical biogas, and
CH4 content) single bottle of each Phase III moose rumen, beaver dropping, and internal
circulation granules cultivation enriched on poplar hydrolysate, cellulose, and cellulose plus
1495 mg/L tannic acid, 438.3 mg/L lignosulphonate, or 28.3 mg pine needles per bottle (Table
4.5).
Table 4.5 Replicate number from Phase III triplicates used as inoculum for Phase IV enrichments
Cellulose
Cellulose +
Cellulose +
Cellulose +
Poplar
tannic acid
lignosulphonate
pine needles
hydrolysate
Moose rumen
Beaver droppings
granules
The contents of bottles selected for Phase IV experiments were transferred to three new
bottles and made up to 60 mL per bottle total volume with medium and amended as described in
Section 3.6.
The variability in biogas production of replicate cultures was considerably lower in Phase
IV enrichments, likely due to the inoculum having originated from the same Phase III bottle. In
all cases, the presence of tannic acid decreased initial rates of biogas production; lignosulphonate
also affected biogas production rates in moose rumen cultivations (Figure 4.18). Significant lag
times before any gas was produced were observed for both moose rumen and beaver dropping
cultures amended with tannic acid (Appendix F). The lag time for moose rumen cultures was
stagnant at about 50 days, while the lag time for beaver droppings increased from nearly zero to
about 20 days across Phase II to Phase IV. The effect of lignosulphonate on biogas production
rate in moose rumen cultivations was unexpected, since corresponding Phase III cultivations
performed similarly to cellulose only cultivations (Figure 4.10). In fact, biogas production in
Phase IV moose rumen cultures amended with cellulose plus lignosulphonate reached a plateau
before 20 % of the theoretical biogas yield had been reached. This was likely due to acidification
72
of these cultivations, which is known to inhibit methanogenic activity [13]. Indeed, the pH of
liquid samples drawn from these cultures was between 5.5 and 6. To avoid acidification of
cultures in future experiments, culture pH was monitored regularly and 5 M NaOH added in 0.5
mL aliquots when necessary, to maintain neutral pH. This problem is expected to reoccur with
some regularity in the future due to lower concentrations of microbial biomass encountered at
further enrichment and a relatively high amount of feed added to cultures (known to increase the
possibility of acidification during anaerobic digestion of lignocellulosic-containing materials in
batch tests [84]). In future, increasing the inoculum to substrate ratio, or increasing the total
alkalinity of the system through increased sodium bicarbonate addition to the medium may
alleviate this problem.
Moose rumen
Beaver droppings
IC granules
25
50
75
100

Figure 4.18 Initial rates of biogas production in Phase IV microcosms
(
tannic acid,
- cellulose and 1450 mg/L
- cellulose and 472 mg/L red pine
needles). n = 3, error bars represent confidence interval at p = 0.05.
Biogas yield from poplar hydrolysate was similar in Phase IV enrichments of internal
circulation granules and beaver droppings (Figure 4.19). In contrast, biogas production from
cellulose-containing amendments was generally higher in enrichments of internal circulation
granules than enrichments of moose rumen fluid and beaver droppings; this trend was especially
apparent in microcosms amended with cellulose alone (Figure 4.19). Notably, while biogas yield
was highest in cellulose enrichments of internal circulation granules and beaver droppings,
73
moose rumen microcosms enriched on cellulose plus pine needles generated more biogas than
other moose rumen enrichments. The lower total yields of these cultures may also be attributed
COD Consumed
mgCOD added
to lower inoculum to substrate ratios (as cultures are diluted) [84].
1
0.8
0.6
0.4
0.2
0
Moose rumen
Beaver
droppings
IC granules
Figure 4.19 Biogas yield from Phase IV microcosms

(
tannic acid,
The theoretical CH4 fraction of biogas generated through anaerobic conversion of

cellulose is 50 % (Appendix G). By Phase IV enrichment, the majority of COD available for
microbial metabolism is likely derived from the amended carbon source, rather than the original
inoculum. Therefore, if anaerobic digestion proceeded as expected, all Phase IV bottles amended
with cellulose should contain biogas composed of approximately 50% CH4. The CH4 content in
biogas produced by microcosms amended with poplar hydrolysate should be similar to celluloseamended cultivations, assuming lignin is not substantially degraded. Likewise, microcosms
amended with cellulose plus pine needles or sodium lignosulphonate should produce similar
biogas quality as those amended with cellulose alone (Appendix G). Since tannic acid has a
theoretical limit of about 43 % CH4, cellulose plus tannic acid cultures are expected to produce
47 % CH4 (Appendix G). While enrichments of internal circulation granules performed as
expected (Figure 4.20), more deviation was observed within enrichments of both beaver
droppings and moose rumen fluid. In particular, the CH4 fraction of biogas produced by moose
74
rumen and beaver dropping samples enriched on cellulose plus tannic acid was very low, and
was correlated to low biogas yield, suggesting methanogenesis was inhibited in these
cultivations.
Methane fraction
1
0.8
0.6
0.4
0.2
0
Moose rumen
Beaver droppings
IC granules
Figure 4.20 Phase IV CH4 fractions

(
tannic acid,
4.4.4. Phase II to IV: Comparison of Concentrated Additive Degrading Microcosms
To assess the effectiveness of the enrichments performed to date, on improving the

biomass conversion activity of selected environmental samples, the highest performing
microcosm of triplicate bottles prepared during Phase II through Phase IV enrichments were
compared in terms of biogas yield, initial rate of biogas production, and the CH4 fraction in the
biogas produced. Notably, since Phase I moose rumen and beaver dropping enrichments, as well
as Phase II enrichments of internal circulation granules, include degradation of endogenous
COD, these stages are not included in the current comparison.
It is anticipated that with each transfer, resulting microcosms will contain a greater
proportion of microbes capable of degrading the designated substrate, and so the time to 20 %
COD removal should decrease, while lag times for biogas production decrease and initial rates of
biogas production increase. As expected, enrichment of moose rumen fluid on cellulose alone,
75
and cellulose plus pine needles increase biogas production rates; however, similar trends for
other moose rumen microcosms, and enrichments of beaver droppings and internal circulation
granules was not observed (Figure 4.21). In fact, the lag time to detectable biogas production by
moose rumen enrichments and beaver dropping enrichments amended with cellulose plus tannic
acid increased considerably, this was also observed in Phase IV enrichments of moose rumen
samples on cellulose plus lignosulphonate. It is possible that the effective concentration, and
consequent inhibitory effects, of tannic acid and lignosulphonate were comparatively low in
Phase II and Phase III enrichments, due to possible absorption of these compounds onto the
IC granules
Beaver
droppings
Moose
rumen
fibrous material still present from the original inoculum.
C
CT
CL
CP
PH
C
CT
CL
CP
PH
C
CT
CL
CP
PH
0
20
40
60
80

Figure 4.21 Initial rates of biogas production from the top performing replicates.
(
- Phase II,
- Phase III,
- Phase IV) C = cellulose, CT = cellulose + 1450 mg/L tannic acid, CL =
cellulose + 438 mg/L sodium lignosulphonate, CP = cellulose + 472 mg/L pine needles, PH = poplar
hydrolysate.
Microbial enrichment should eventually lead to greater substrate utilization in less time,
although conversion of the substrate to biomass and soluble fermentation products could reduce
biogas yields. While biogas yield from beaver dropping and internal circulation granules
enrichments decreased slightly, biogas yield from moose rumen cultivations enriched on
cellulose plus tannic acid, and cellulose plus lignosulphonate was considerably lower in Phase IV
76
than Phase II enrichments (Figure 4.22). This is potentially due to the lower inoculum to
substrate ratio [84], in some cases leading to acidification, in others possibly due to increased
susceptibility to inhibitors at lower biomass concentrations. This could be resolved through
COD consumed
mgCOD added
increasing the microbial population over time.
1
0.8
0.6
0.4
0.2
0
C CT CL CP PH C CT CL CP PH C CT CL CP PH
Moose rumen
Beaver droppings
IC granules
Figure 4.22 Biogas yields from the top performing replicates.

(
- Phase II,
- Phase III,
hydrolysate.
The expected CH4 fraction in a biogas sample is based on substrate chemistry, with
carbohydrate-containing substrates expected to produce 50 % CH4 with insignificant deviation
for lignosulphonate and pine needle additions. Tannic acid and cellulose cultures were expected
to produce 47 % CH4. With the exception of moose rumen cultivations enriched with cellulose
plus tannic acid, the CH4 content in biogas from Phase II and Phase III enrichments was higher
in moose rumen cultures, compared to corresponding beaver dropping enrichments (Figure 4.23).
The CH4 fraction in biogas samples from Phase III enrichments of beaver droppings was
consistently low; however, this is likely due to poor calibration of the GC column used to
measure CH4. Internal circulation granules declined somewhat in CH4 content but did not exhibit
any drastic drops, eventually reaching CH4 levels very close to theoretical limits.
77
1
Methane fraction
0.8
0.6
0.4
0.2
0
C CT CL CP PH C CT CL CP PH C CT CL CP PH
Moose rumen
Beaver droppings
IC granules
Figure 4.23 CH4 fraction from the top performing replicates.

(
- Phase II,
- Phase III,
hydrolysate.
In summary, the main results of Phase IV enrichments and comparisons to Phase

II and III results are:
Although all microcosms retained at least some activity when amended with
increased amount of tannic acid, sodium lignosulphonate, and red pine needles (Figure
4.21 and Figure 4.22), microbial activity was reduced in all enrichments amended with
1450 mg/L tannic acid. Susceptibility was most apparent in moose rumen enrichments,
followed by enrichments of beaver droppings, and then internal circulation granules.
The performance of internal circulation granules enrichments was generally most
stable between transfers, followed by enrichments of beaver droppings and then moose
rumen samples. This result likely reflects higher microbial density in internal circulation
granules compared to enrichments of beaver dropping and moose rumen samples, which
could be addressed through feeding rather than transfer.
78
4.4.5. Phase V Enrichment: Bioconversion of Post Extraction Washer (PEW) Effluent
Post extraction washer (PEW) effluent is generated at a sulphite mill when washing
wood-derived pulp, and contains dissolved solids extracted from the wood fibre along with small
fibres not retained by process screens. PEW is known to contain resin acids and other
components that are inhibitory to anaerobic bioprocesses. Accordingly, the objective of Phase V
cultivations was to evaluate the potential of enrichments generated in the current project, to
transform an industrially significant pulp mill effluent. In this case, two of the three replicate
enrichments generated in Phase IV were directly fed with 3.0 mL of PEW (equivalent to 2.57
gCOD/L), while the third replicate was not amended and instead was used to track biogas
produced from substrates that remained from Phase IV amendments.
Figure 4.24 Biogas yield from A) moose rumen, B) beaver dropping, and C) internal circulation granules
Phase V microcosms amended with post extraction washer (PEW) wastewater.
(
tannic acid,
needles)
79
Previous studies report 45 % conversion of PEW by internal circulation granules prior to

enrichment [85]. By contrast, a maximum of 23 % of the added COD based on gas production
(accounting for the control) was transformed after 85 days of incubation (Figure 4.24). Notably,
the anaerobic wastewater treatment reactors at the pulp and paper complex were re-seeded with
fresh granules between the previous study and the one performed here; the composition of PEW
used in both studies might also have changed. Still, the previously reported biogas yield (45 %)
assumed that the CH4 fraction in the biogas produced was 70 %. However, if the actual CH4
fraction in biogas generated from internal circulation granules was 50 % (closer to the theoretical
value for carbohydrates and organic acids used in both studies, see Appendix G), then the biogas
yield would have been closer to 32 %.
With the exception of beaver droppings enriched on cellulose plus pine needles,
enrichments of internal circulation granules were able to degrade more PEW effluent than
corresponding enrichments of beaver droppings or moose rumen fluid. This result likely reflects
prior exposure of internal circulation granules to compounds present in PEW effluent.
Interesting, for all sources of inocula (i.e. moose rumen fluid, beaver droppings, and internal
circulation granules), microcosms enriched on poplar hydrolysate or cellulose plus pine needles,
were most able to transform PEW, further suggesting that microbial communities can be
enriched to improve the biotransformation of industrially relevant pulp mill effluents.
80
4.5. Analysis of microbial inocula and enrichments by DGGE
Biomolecules obtained from environmental inocula and enriched cultures can be used to
i) identify microbial community members, ii) monitor shifts in microbial community structure
and complexity, and iii) discover genes that encode enzymes that participate in a bioconversion
pathway. In this work, denaturing gradient gel electrophoresis (DGGE) was used to compare the
microbial community of initial environmental inocula, as well as shifts in microbial community
composition that had occurred during the enrichment process.
4.5.1. DNA extraction and concentration
DNA was extracted from each of moose rumen fluid, beaver dropping, and internal
circulation granules inocula as well as from all Phase III enrichments just prior to being
transferred to Phase IV cultivations (Table 4.5). DNA was extracted from the initial inocula
using a chemical extraction procedure (Section 3.7.1), while DNA from enrichment cultures was
extracted using the Ultraclean soil kit. The purpose of using the chemical extraction procedure
to isolate DNA from moose rumen fluid, beaver dropping, and internal circulation granules
inocula was to generate high-quality DNA that could used to generate Lambda-zap libraries that
could eventually be screened for new enzyme activities.
DNA concentrations were measured spectrophotometrically using a Nanodrop 1000
(Table 4.6). Values representing the amount of DNA per gram of sample processed can be used
to estimate the density of microorganisms present in either the initial inoculum or enrichment
[86]. As predicted in Section 4.4.3, DNA yields reported in Table 4.6 suggest that the density of
microorganisms in original internal circulation granules samples, and resulting enrichments, is
higher than moose rumen and beaver dropping inocula and enrichments. It is possible that the
measurement of eukaryotic and plant DNA in beaver dropping and moose rumen samples may
contribute to significant overestimate of microbial population (perhaps over 90% of the total
amount) [87]. However, much of this DNA was expected to have degraded through microbial
action.
81
Table 4.6 DNA Concentrations
Moose Rumen
Beaver Droppings
Internal Circulation
Granules
Amount
Resulting
Amount
Resulting
Amount
Resulting
extracted
concentration
extracted
concentration
extracted
concentration
(ngDNA /
(ng/L)
(ngDNA /
(ng/L)
(ngDNA /
(ng/L)
g sample)
g sample)
g
sample)
Initial
746.8
186.7
269.6
Enrichment
67.4
5754.4
1438.6
After Phase III
Cellulose
130
6.5
96
4.8
242
12.1
C + Tannic Acid
110
5.5
130
6.5
384
19.2
C+
246
12.3
94
4.7
814
40.7
152
7.6
76
3.8
326
16.3
178
8.9
144
7.2
358
17.9
Lignosulphonate
C + Pine
Needles
Poplar
Hydrolysate
82
4.5.2. DGGE
Denaturing gradient gel electrophoresis (DGGE) uses a linear gradient of DNA

denaturant (urea and formamide) cast in a polyacrylamide gel to separate DNA segments of
similar length by sequence (specifically, GC content). As the double stranded segment passes
through the gel (driven by an 80 V potential), it encounters progressively larger concentrations of
denaturant until a critical threshold is reached, at which point the strands partially separate and
their velocity through the gel is dramatically reduced [73]. Once the run is completed the gel is
stained with a compound that fluoresces under UV light, and then photographed. Bands are
visible within each lane at positions corresponding to an increasing GC content from top to
bottom.
A DGGE analysis of moose rumen, beaver dropping, and internal circulation granules
inocula revealed several bands that were shared between i) all three samples, ii) among two of
the three inocula, and iii) many unique bands within only one inoculum ( Figure 4.25). Further,
this DGGE analysis suggests that the complexity of initial microbial communities was highest in
beaver droppings, followed by internal circulation granules, and finally moose rumen fluid.
Figure 4.25 DGGE profiles of environmental inocula: internal circulation granules (A), beaver droppings (B),
and moose rumen (C).
An image analysis was conducted using Image J (NIH). A straight line was drawn along
the centre down the length of each lane and the relative intensity was measured by the software
at each pixel. Since the background colour between lanes, and along the length of each lane, was
different, the running average of every 60 pixels (1 cm) was subtracted from each data point.
This normalized spectrum still contained noise, so a consistent, arbitrary cutoff level based on
83
the weakest discernible bands in the photograph was used. The analysis identified the shared
bands at the positions indicated in Table 4.7.
Table 4.7 Shared DGGE bands
Beaver droppings
Beaver droppings and
Moose rumen and
and moose rumen
internal circulation
internal circulation
granules
granules
3.45
5.50
4.28
4.89
8.27
6.27
All three inocula
7.15
5.40
Position of shared bands (cm from left side of band)
The number of total bands contained in each lane is much higher than the number of
bands shared between lanes, however it is notable that the analysis identified a larger number of
similar bands between metagenomic bacterial DNA from beaver droppings and moose rumen
than between either beaver droppings or moose rumen when compared with internal circulation
granules. Particularly evident is a band located at about 3.45 cm from the left side of Figure 4.25
that is very prominent in both moose rumen and beaver droppings but absent from internal
circulation granules.
Enriched microcosms were also analyzed by DGGE (Figure 4.26), and full Image J
analysis results are presented in Appendix H.
84
Figure 4.26 DGGE profiles of (from left to right) enriched moose rumen, beaver droppings, and internal
circulation granules
Distance measured in cm from top side of band. Lanes in each band show PCR products of DNA extracted
from (in each image, from left to right) raw inoculum; then enrichment cultures following Phase III amended
with cellulose, cellulose plus 1450 mg/L tannic acid, cellulose plus 438 mg/L sodium lignosulphonate,
cellulose plus 472 mg/L pine needles, and poplar hydrolysate
It can be inferred from these images that different enrichment schemes led to changes in
the relative abundance of bacterial species. However, differences between DNA extracted from
inocula and corresponding enrichments should not be compared directly due to the different
DNA extraction methods employed. The clearest evidence of enrichment is the difference in
band intensity shown within each enrichment lane, indicating relative abundance in that sample.
Internal circulation granules, and to a lesser extent beaver droppings, appear to have retained
much of the complexity of the original inoculum. Moose rumen enrichments appear to contain
many fewer species than were in the inoculum, perhaps due to the relatively longer enrichment
period.
85
5.
Scale up
5.1. Reactor design and operating conditions
Batch biochemical methane potential (BMP) assays such as those described in this
project are advantageous for measuring the interaction between microbial communities and
feedstocks, but are not always good indications of full scale performance. Hydraulic forces and
other conditions unique to continuous operation found in high rate anaerobic reactors can be
simulated using a bench scale reactor allowing for evaluation of performance under more
realistic conditions. In addition to gas and liquid measurements common to both batch and full
scale tests, monitoring of granulation and degranulation processes are also possible in this setup.
A lab scale continuous reactor would also allow bioaugmentation of inhibited granular sludge
with different enriched batch cultures to determine the effectiveness of that strategy.
UASB style reactors operate by continuously feeding wastewater to the bottom of a
cylindrical reactor containing a microbial sludge bed under anaerobic conditions. The microbes
degrade soluble organic material in this influent stream to biogas, reducing COD content of the
stream in the process by about 50%. Insoluble biogas then floats to the top of the reactor where it
is collected and removed. Liquid and suspended granules also flow towards the top of the
reactor, where they enter a region of wider diameter and correspondingly lower upflow rate,
allowing heavy granules to settle back down. Much of the liquid is recirculated back to the
bottom of the reactor, while some is removed as effluent.
It is necessary to consider many parameters when designing a UASB reactor system. The
organic and volumetric loading rates are often predetermined by the needs of the system in a full
scale reactor; these constraints form the basis for choosing the type of reactor required and
determine other parameters. The goal of the reactor system designed in this project was to start
and maintain granulation in as small a volume as possible to minimize the required organic and
hydraulic loading rates. Important operating conditions required for granulation include:
temperature (about 30-35 C for mesophilic sludge),
wastewater composition (VFAs, carbohydrates, low suspended solids concentration,

COD/N and COD/P ratio, trace metal mixture),
gentle mixing (high recirculation rate, slow mechanical agitation),
pH (optimally between 6.7 and 7.5), and

86
organic loading rate (above 0.6 kgCOD/kgVSS/day) [88].
Each of these must be considered during the design stage. Several sets of parameters from lab
scale upflow-type anaerobic reactors are shown in Table 5.1 below, as well as the chosen values
for the reactor design.
Table 5.1 Important parameters for lab scale continuous anaerobic reactors
Type
Substrate
Vol.
H:D
(L)
a
EGSB
Ethanol
2.5
20
Seed
OLR
HRT
sludge
(kg/m3
(hr)
(gVSS/L)
day)
10 30
7 12
0.57
v (m/hr)
Ref.
(C)
2.5 5.5
30
[89]
1.3
AHR
Short chain
16.3
5.5
13
3.5 4.5
48
.056
35
[90]
alcohols
c
Sucrose
1.4
13.4
14 35
5 10
24
30
[91]
UASB
Glucose
3.1
14
2 16
0.72
35
[92]
UASB
0.96
UASB
VFAs
2.3
18.5
70
2 20
24
30
[93]
UASB
Acetate &
3.1
9.3
10.5
30
10
35
This
carbohydrates
a
work
Expanded granular sludge bed reactor
anaerobic hybrid reactor
upflow anaerobic sludge bed reactor
An identical pair of bench-scale sequencing batch reactors (SBRs) previously described

by Tripathi and Allen in [94] were selected for use in this project due to their appropriate volume
and height to diameter ratio. In addition, they are made from and are jacketed with clear glass,
allowing for observation of the granulation process. A schematic of the reactor and support
system is shown in Figure 5.1. The jacketed vessels are 7.5 cm in diameter and 70 cm tall; giving
a height to diameter ratio of 9.3 that allows lower recirculation rates to achieve higher upflow
velocity (and subsequent improvement in mixing). The total volume is 3.1 L including a working
liquid volume of approximately 2.0 L and a 1.1 L headspace. Three sampling ports, normally
sealed with butyl rubber stoppers to maintain anaerobic conditions, are found on the side of each
reactor. A depth-adjustable sludge sampling pipe is found protruding through top of the reactor,
allowing for different sludge bed depths to be sampled.
87
Two feed solutions are continuously fed to the reactor from storage in a refrigerator
below the fume hood, one a phosphate buffer, and the other a solution containing several
nutrients and sodium acetate as a carbon source. A hydraulic retention time of just over 10 hours
was achieved by pumping these two solutions with a Masterflex console drive (77521-50) and a
quad-channel pump head (7535-04), with one phosphate and one acetate line leading to each
reactor. These feeds blend with a headspace gas recirculation line that facilitates sludge mixing
within the reactor using a Masterflex pump motor (7553-70) with two Easy-load heads (751800), through low oxygen diffusion Masterflex Norprene tubing (06402-18). Inside the reactor,
the recirculation line enters a downer pipe leading to the bottom of the reactor and a distributor,
which breaks up the flow and provides mixing. Granules are well mixed in the lower regions of
the reactor both by agitation from the headspace gas flow and by biogas bubbles, but settle prior
to reaching the upper stages. The outlet pipe is located about 3cm below the recirculation draw
pipe; it controls the level in the reactor. Effluent containing treated water and low-density
granules is drawn out this outlet and flows via gravity to a settling beaker, which allows the
granules to settle while effluent flows out the side to the drain. Temperature in the reactors is
maintained via a HAAKE E8 immersion circulator which heats water in a water bath and then
pumps it through the jacket of the reactor.
As gas is produced in the reactor, it flows out a port at the top to a wet tip gas meter
(Archae Press), filled with a 1 M sodium hydroxide solution to remove CO2 and H2S, which
measures the volume of CH4 produced. Once the gas volume is measured, the CH4 is discharged
to the fume hood.
88
Headspace Outlet
Sludge Sample Outlet
Water
Bath
& Pump
Gas Outlet
Recirc Outlet
Wet Tip
Gas
Meter
Water
Jacket
Outlet
Recirc
Pump
Vent
Effluent
Degassing
Effluent
Outlet
Sink
Feed and
Nutrient
Pump
Fume Hood
Feed
solution
Nutrient
solution
Fridge
Liquid
Sample
Ports
Sludge
Bed
Water Jacket Inlet

Jacketed
Reactor
Figure 5.1 Lab scale anaerobic reactor
5.2. Reactor operation
An initial 50 day trial of the reactors was conducted to verify hydraulic, mixing, and gas
collection and measurement operations [95]. The reactors operated under a loading rate of 60
gCOD/day each of sodium acetate feed, an organic loading equivalent to 23.7 L of CH4 per day.
Granular seed sludge obtained from internal circulation reactors at a pulp and paper complex was
added to the reactors at a ratio of 21 gVSS/reactor. Specific operating conditions were set as
indicated in Table 5.1 above. A summary of gas production results from this trial phase is shown
in Figure 5.2 below.
89
Cumulative Gas
Produced (L)
80
60
40
20
0
0
10
20
30
40
50
Time (days)
Figure 5.2 Lab scale reactor startup phase gas production
Gas production over the entire 50 day trial from
- Reactor 1 and
- Reactor 2.
Continuous gas production was observed over the startup time period; however, the
measured gas production rate over this time period was about 6 % of that expected based on the
amount of added organic material. Perhaps due to this low activity, the growth of dispersed
sludge was observed as was some degranulation. After analysis of the results, several problems
were identified with the reactor design. First, the tubing used to transport feed to the reactor from
the refrigerator was repeatedly clogged with a white precipitate, blocking the flow of acetate to
the reactor. Second, a brown precipitate was also observed in the tubing, and found to be caused
by the precipitation of iron-containing micronutrients. Lastly, the recirculation of gas within the
reactor proved insufficient to effectively mix the granules. These problems, to be addressed by
future development, should not prevent the eventual use of this reactor as a tool to measure the
variety of parameters unique to continuous systems.
90
6.
Conclusions and Implications
Anaerobic microcosms, prepared from whole moose rumen fluid, beaver droppings, and
internal circulation granules inocula, were enriched on five lignocellulosic feeds and developed
into cultures with different community structure and ability to tolerate and degrade a recalcitrant
wastewater stream. Several results show evidence of a beneficial enrichment of microbial
communities towards degradation of targeted compounds. These include direct measurement of
initial biogas production rate and ultimate biogas yield across enrichment phases, differences in
biogas yield from a recalcitrant wastewater stream from the pulp and paper industry, and
observation of bacterial community changes between inocula and enrichment cultures. Each of
these results suggests the development of cultures potentially useful for future application in
wastewater treatment or solid phase anaerobic digestion of lignocellulosic materials.
Yield is the most important parameter to consider since it can be easily compared across
different amendments and inocula. Rates and lag time are dependent on other factors that have
not been controlled for across inocula including biomass concentration and external carbon
sources, therefore should only be compared within a single microcosm set. Several conclusions
can be drawn from the results obtained over the course of this study, covering three main areas:
Biogas production measurement and analysis:
The time required to degrade the first 20 % of added COD to biogas in any of the three
sets of enrichments did not, in general, change significantly over time. In many cases, the
time required either decreased somewhat or remained relatively consistent, even though
the cultures had been diluted, suggesting acclimatization to the supplied feedstocks.
The ultimate yield of biogas, in contrast, declined consistently over time. It is believed
that this is the result of increasingly limited biomass within each culture, and that
improvements will be seen following several growth phases.
The addition of red pine needles or sodium lignosulphonate to cellulose degrading

cultures had little effect on either the time to 20 % degradation or yield.
Addition of tannic acid had a significant negative effect on the time required to degrade
20 % of supplied COD to biogas in all three microcosm sets, and led to a substantial
decrease in the average yield of all enrichments.
91
o An increase in the lag time from zero to 20 days was seen for beaver dropping
cultures. However, an improvement in rate was evident as time went on.
o Lag times of about 50 days were consistently observed for moose rumen cultures
with tannic acid, remaining steady over time.
o Tannic acid did not cause a substantial lag time in internal circulation granule
cultures, yet did have a negative effect on the time to 20% degradation.
CH4 fraction declined substantially over time in some moose rumen and beaver dropping
cultures degrading tannic acid, indicating possible selective inhibition of methanogens in
the culture. Future analysis of this effect may focus on the community structure of the
archaea present in each enrichment.
Community structure determination via DGGE:
DGGE profiles of metagenomic DNA from moose rumen and beaver droppings inoculum
were more similar to each other than that of internal circulation granules.
Profiles from enrichments compared to those of raw inocula showed significant changes
in bacterial community (in terms of relative intensity of specific bands) depending on the
amendment that each enrichment was grown on, and this was found to be the case
regardless of the substrate complexity.
The relative complexity (number and position of bands) of beaver dropping and internal
circulation granules enrichments was similar to that of their respective inocula, but the
profile from moose rumen enrichments showed a large drop in the number of bands
present, indicating more enrichment.
Enriched culture response to PEW addition:
Internal circulation granules, exposed to pulp and paper wastewater streams prior to use
in this study, were generally more effective at producing biogas from the recalcitrant
PEW wastewater.
Moose rumen enrichments, possibly due to the loss of complexity shown via DGGE
profiling, were mostly unable to degrade the wastewater to a significant biogas yield.
Enrichments amended with poplar hydrolysate were significantly more able than the
respective cellulose degrading culture from the same set.
92
Beaver dropping enrichments amended with cellulose plus red pine needles were
significantly more active when degrading PEW than any other culture with the exception
of the equivalent internal circulation granules enrichment.
Overall, these results point to the development of unique enrichment cultures
acclimatized to a variety of lignocellulosic biomass feedstocks. The use of biogas as a fuel is

increasingly important, and the ability of these enrichments to degrade solid material to CH4 may
be valuable in improving the production of this fuel from currently recalcitrant solid substrates.
93
7.
Recommendations
Many lignocellulosic derived wastewaters are recalcitrant to anaerobic digestion, and the
development of cultures acclimatized to them or enriched on inhibitor-containing feedstocks
such as those described in this study is a first step on the path to addressing this problem. The
development of a functioning lab-scale anaerobic reactor capable of studying phenomena unique
to continuous operation, such as granulation, is another. The pair of reactors described in this
study will be a useful tool in the continued enrichment of these cultures and could be used for
studies on direct granulation of these enriched cultures, or the bioaugmentation of previously
existing granules (such as the internal circulation granules) to create hybrid communities capable
of degrading currently recalcitrant streams. Due to their observed ability to degrade post
extraction washer wastewater, it is recommended that internal circulation granule derived
cultures enriched on both cellulose plus red pine needles and poplar hydrolysate, as well as
beaver dropping derived cultures enriched on cellulose plus red pine needles, be used as
bioaugmentation inocula and compared to non-enriched internal circulation granules.
Continued feeding of all of the current generation of moose rumen, beaver droppings, and
internal circulation granule cultures, including those fed PEW, is recommended to increase
biomass concentration. Further dilutions are not recommended until biomass concentration
reaches sufficiently high levels. Not only is more biomass necessary for scale up work including
continuous reactor bioaugmentation, but it is also required for studies to confirm the results
obtained over the course of this work. Confirmation of bacterial community changes, as well as
an analysis of archaeal community shifts are both recommended. Further feeding leading to an
increase in biomass concentration will also allow for differences in ability to digest different
waste streams independent of the amount of biomass to be studied.
Finally, sequencing of metagenomic DNA obtained from those cultures previously
indicated as potential targets for bioaugmentation, as well as all three cellulose and tannic acid
enriched cultures, is recommended. This will allow for possible identification of sequences
coding for enzymes possibly responsible for the observed performance of these cultures.
94
8.
References
[1]
B. E. Rittmann and P. L. McCarty, Environmental Biotechnology: Principles and

Applications. New York: McGraw-Hill, 2001.
[2]
N. Mosier et al., Features of promising technologies for pretreatment of lignocellulosic

biomass, Bioresource Technology, vol. 96, pp. 673-686, 2005.
[3]
J. Field and G. Lettinga, The methanogenic toxicity and anaerobic degradability of a

hydrolyzable tannin, Water Research, vol. 21, no. 3, pp. 367-374, Mar. 1987.
[4]
P. Mccarthy, K. Kennedy, and R. Droste, Role of resin acids in the anaerobic toxicity of
chemithermomechanical pulp wastewater, Water Research, vol. 24, no. 11, pp. 14011405, Nov. 1990.
[5]
M. Lanthier, B. Tartakovsky, R. Villemur, G. Deluca, and S. R. Guiot, Microstructure of

Anaerobic Granules Bioaugmented with Desulfitobacterium frappieri PCP-1, Applied
and environmental microbiology, vol. 68, no. 8, pp. 4035-4043, 2002.
[6]
W. Hailei, L. Guosheng, L. Ping, and P. Feng, The effect of bioaugmentation on the

performance of sequencing batch reactor and sludge characteristics in the treatment
process of papermaking wastewater., Bioprocess and biosystems engineering, vol. 29, no.
5-6, pp. 283-9, Dec. 2006.
[7]
J. Chiquette, G. Talbot, F. Markwell, N. Nili, and R. J. Forster, Repeated ruminal dosing

of Ruminococcus flavefaciens NJ along with a probiotic mixture in forage or concentratefed dairy cows: Effect on ruminal fermentation, cellulolytic populations and in sacco
digestibility, Canadian Journal of Animal Science, vol. 87, pp. 237-249, 2007.
[8]
E. Sjstm, Wood Chemistry: Fundamentals and Applications. New York: Academic

Press, 1981.
[9]
M. T. Holtzapple et al., Biomass Conversion to Mixed Alcohol Fuels Using the MixAlco
Process, Applied Biochemistry And Biotechnology, vol. 77, pp. 609-631, 1999.
[10]
G. Du and J. Yu, Green technology for conversion of food scraps to biodegradable

thermoplastic polyhydroxyalkanoates., Environmental science & technology, vol. 36, no.
24, pp. 5511-6, Dec. 2002.
[11]
R. Kleerebezem and M. C. M. van Loosdrecht, Mixed culture biotechnology for

bioenergy production., Current opinion in biotechnology, vol. 18, no. 3, pp. 207-12, Jun.
2007.
[12]
R. L. Droste, Theory and Practice of Water and Wastewater Treatment. New York: John
Wiley & Sons, Inc., 1997.
95
[13]
D. Deublein and A. Steinhauser, Biogas from waste and renewable resources. Weinheim:
Wiley-VCH, 2008.
[14]
W. H. Schwarz, The cellulosome and cellulose degradation by anaerobic bacteria,

Applied Microbiology and Biotechnology, vol. 56, no. 5-6, pp. 634-649, Sep. 2001.
[15]
V. Zverlov, I. S. Mahr, K. Riedel, and K. Bronnenmeier, Properties and gene structure of

a bifunctional cellulolytic enzyme (CelA) from the extreme with separate glycosyl
hydrolase family 9 and 48 catalytic domains, Microbiology, vol. 144, pp. 457-465, 1998.
[16]
R. Lamed, J. Naimark, E. Morgenstern, and E. a Bayer, Specialized cell surface

structures in cellulolytic bacteria., Journal of bacteriology, vol. 169, no. 8, pp. 3792-800,
Aug. 1987.
[17]
J. Kirby, J. Martin, and A. Daniel, Dockerin-like sequences in cellulases and xylanases

from the rumen cellulolytic bacterium Ruminococcus flavefaciens, FEMS microbiology,
vol. 149, no. 2, pp. 213-219, 1997.
[18]
J. T. Kraemer, Effects of Methanogenic Effluent Recycle on Fermentative Hydrogen

Production, University of Toronto, 2004.
[19]
M. Ali and T. R. Sreekrishnan, Aquatic toxicity from pulp and paper mill effluents: a
review, Advances in Environmental Research, vol. 5, pp. 175-196, 2001.
[20]
Merck Index, Tannic Acid, Merck Index. [Online]. Available:

http://themerckindex.cambridgesoft.com.myaccess.library.utoronto.ca/TheMerckIndex/def
ault.asp?formgroup=basenp_form_group&dataaction=db&dbname=TheMerckIndex.
[Accessed: 09-Mar-2010].
[21]
J. D. Reed, Nutritional toxicology of tannins and related polyphenols in forage legumes,

Journal of Animal Science, vol. 73, pp. 1516-1528, 1995.
[22]
B. Singh, T. K. Bhat, and O. P. Sharma, Biodegradation of tannic acid in an in vitro

ruminal system, Livestock Production Science, vol. 68, pp. 259-262, 2001.
[23]
E. H. Fulling, Botanical Aspects of the Paper-Pulp and Tanning Industries in the United
States -- An Economic and Historical Survey, American Journal of Botany, vol. 43, no.
8, pp. 621-634, 1956.
[24]
J. A. Field, G. Lettinga, and L. H. A. Habets, Oxidative detoxification of aqueous bark

extracts. Part I: Autoxidation, Journal of Chemical Technology & Biotechnology, vol. 49,
no. 1, pp. 15-33, Apr. 1990.
[25] J. A. Field, M. J. H. Leyendeckers, R. Sierra-alvarez, G. Lettinga, and L. H. A. Habets,

The methanogenic toxicity of bark tannins and the anaerobic biodegradability of water
soluble bark matter, Water Science and Technology, vol. 20, no. 1, pp. 219-240, 1987.
[26]
J. W. Rowe and A. H. Conner, Extractives in Eastern Hardwoods. Madison: , 1979.

96
[27] E. V. Bakuzis and H. L. Hansen, Balsam Fir: A Monographic Review. Toronto: Copp
Clark Publishing Co. Ltd., 1965, p. 320.
[28] S.-duk Seul, N.-woo Lee, B.-sun Lee, and D. L. Pisaniello, Effects of Environmental
Hormone Characteristics by Toxic Substances in Wood Dust, Korean Chemical
Engineering Meeting. p. 565.
[29]
A. E. Hagerman, Hydrolyzable Tannin Structural Chemistry, in The Tannin Handbook,

2011, pp. 1-8.
[30]
R. Bhatta et al., Difference in the nature of tannins on in vitro ruminal methane and
volatile fatty acid production and on methanogenic archaea and protozoal populations,
Journal of Dairy Science, vol. 92, pp. 5512-5522, 2009.
[31] D. N. Kamra, N. Agarwal, and L. C. Chaudhary, Inhibition of ruminal methanogenesis

by tropical plants containing secondary compounds, International Congress Series, vol.
1293, pp. 156 - 163, 2006.
[32] A. Paterson, I. A. N. M. Morrison, and J. E. Smith, Effects of cell wall phenolics and
fungal metabolites on methane and acetate production under in vitro rumen conditions, J
Gen Appl Microbiology, vol. 41, pp. 475-485, 1995.
[33]
J. A. Field and G. Lettinga, The methanogenic toxicity and anaerobic degradability of a

hydrolyzable tannin, Water Research, vol. 21, no. 3, pp. 367-374, 1987.
[34]
R. Sierra-alvarez, J. A. Field, S. Kortekaas, and G. Lettinga, Overview of the Anaerobic

Toxicity Caused by Organic Forest Industry Waste Water Pollutants, Water Science and
Technology, vol. 29, no. 5-6, pp. 353-363, 1994.
[35]
G. Vidal and M. C. Diez, Influence of feedstock and bleaching technologies on

methanogenic toxicity of kraft mill wastewater, Water Science and Technology, vol. 48,
no. 6, pp. 149-155, 2003.
[36]
K. E. Nelson, A. N. Pell, P. Schofield, and S. Zinder, Isolation and characterization of an

anaerobic ruminal bacterium capable of degrading hydrolyzable tannins., Applied and
environmental microbiology, vol. 61, no. 9, pp. 3293-8, Sep. 1995.
[37]
Merck Index, Lignin, Merck Index. [Online]. Available:

http://themerckindex.cambridgesoft.com/TheMerckIndex/default.asp?formgroup=basenp_
form_group&dataaction=db&dbname=TheMerckIndex.
[38]
H. G. Lawford and J. D. Rousseau, Production of Ethanol from Pulp Mill Hardwood and
Softwood Spent Sulfite Liquors by Genetically Engineered E. Coli, Applied Biochemistry
And Biotechnology, vol. 39/40, pp. 667-685, 1993.
[39]
Arbo, Products. [Online]. Available: http://www.arbo.ca/pages/en/products.html.

[Accessed: 18-Mar-2010].
97
[40]
R. Sierra-alvarez and G. Lettinga, The Methanogenic Toxicity of Wastewater Lignins

and Lignin Related Compounds, Journal of Chemical Technology and Biotechnology,
vol. 50, pp. 443-455, 1991.
[41] H. J. Op Den Camp et al., Effects of lignin on the anaerobic degradation of (ligno)
cellulosic wastes by rumen microorganisms, Applied Microbiology and Biotechnology,
vol. 29, pp. 408-412, 1988.
[42]
T. G. Jantsch, I. Angelidaki, J. E. Schmidt, B. E. Braa de Hvidsten, and B. K. Ahring,

Anaerobic biodegradation of spent sulphite liquor in a UASB reactor., Bioresource
technology, vol. 84, no. 1, pp. 15-20, Aug. 2002.
[43]
S. K. Chakrabarti, P. K. Roychoudhury, and P. K. Bajpai, Biphasic biomethanation of

wood-hydrolysate effluent., Artificial cells, blood substitutes, and immobilization
biotechnology, vol. 27, no. 5-6, pp. 461-7, 1999.
[44]
C. R. Yin, D. I. Seo, M. K. Kim, and S. T. Lee, Inhibitory effect of hardwood lignin on

acetate-utilizing methanogens in anaerobic digester sludge, 2000.
[45]
R. T. Koide, L. Suomi, C. M. Stevens, and L. McCormick, Interactions between needles

of Pinus resinosa and ectomycorrhizal fungi, New Phytologist, vol. 140, no. 3, pp. 539547, Nov. 1998.
[46] B. Berg, K. Hannus, T. Popoff, and O. Theander, Chemical components of Scots pine
needles and needle litter and inhibition of fungal species by extractives, Ecological
Bulletins, no. 32, pp. 391400, 1980.
[47]
S. Larsson, C. Bjrkman, and R. Gref, Responses of Neodiprion sertifer (Hym.,

Diprionidae) larvae to variation in needle resin acid concentration in Scots pine,
Oecologia, vol. 70, no. 1, pp. 7784, 1986.
[48]
C. Beninger and M. M. Abou-Zaid, Flavonol glycosides from four pine species that
inhibit early instar gypsy moth (Lepidoptera: Lymantriidae) development, Biochemical
Systematics and Ecology, vol. 25, no. 6, pp. 505-512, Sep. 1997.
[49]
D. F. Zinkel and W. B. Clarke, Resin Acids of Pinus Resinosa Needles, Phytochemistry,

vol. 24, no. 6, pp. 1267-1271, 1985.
[50]
S. N. Liss, P. a Bicho, and J. N. Saddler, Microbiology and biodegradation of resin acids

in pulp mill effluents: a minireview., Canadian journal of microbiology, vol. 43, no. 7,
pp. 599-611, Jul. 1997.
[51]
E. Breitmaier, Terpenes: flavors, fragrances, pharmaca, pherhemones, 1st ed.

Morlenbach, Germany: Wiley-VCH, 2006.
[52]
R. Sierra-Alvarez and G. Lettinga, The methanogenic toxicity of wood resin

constituents, Biological Wastes, vol. 33, no. 3, pp. 211-226, 1990.
98
[53]
P. Andersson and T. Welander, Wastewater Treatment Method, U.S. Patent

01599721985.
[54]
I. W. Koster and a Cramer, Inhibition of methanogenesis from acetate in granular sludge

by long-chain Fatty acids., Applied and environmental microbiology, vol. 53, no. 2, pp.
403-9, Mar. 1987.
[55]
V. Nallathambigunaseelan, Anaerobic digestion of biomass for methane production: A

review, Biomass and Bioenergy, vol. 13, no. 1-2, pp. 83-114, 1997.
[56]
D. Chynoweth, D. Jerger, and V. Srivastava, Biological gasification of woody biomass,

in Proceedings of the 20th Intersociety Energy Conversion Engineering Conference, 1985,
pp. 573-579.
[57]
X. Tong, Methane fermentation of selected lignocellulosic materials, Biomass, vol. 21,

no. 4, pp. 239-255, 1990.
[58]
C. Turick, Methane fermentation of woody biomass, Bioresource Technology, vol. 37,

no. 2, pp. 141-147, 1991.
[59] D. Chynoweth, C. Turick, J. Owens, D. Jerger, and M. Peck, Biochemical methane

potential of biomass and waste feedstocks, Biomass and Bioenergy, vol. 5, no. 1, pp. 95111, Nov. 1993.
[60]
E. Palmqvist and B. Hahn-hagerdal, Fermentation of lignocellulosic hydrolysates. II:

inhibitors and mechanisms of inhibition, Bioresource Technology, vol. 74, pp. 25-33,
2000.
[61]
R. I. Amann, W. Ludwig, and K. H. Schleifer, Phylogenetic identification and in situ

detection of individual microbial cells without cultivation., Microbiological reviews, vol.
59, no. 1, pp. 143-69, Mar. 1995.
[62]
M. Hess et al., Metagenomic Discovery of Biomass-Degrading Genes and Genomes from

Cow Rumen, Science, vol. 331, no. 6016, pp. 463-467, Jan. 2011.
[63]
L. Tasse et al., Functional metagenomics to mine the human gut microbiome for dietary
fiber catabolic enzymes., Genome research, Sep. 2010.
[64]
F. Warnecke et al., Metagenomic and functional analysis of hindgut microbiota of a

wood-feeding higher termite., Nature, vol. 450, no. 7169, pp. 560-5, Nov. 2007.
[65]
W. Deng, D. Xi, H. Mao, and M. Wanapat, The use of molecular techniques based on
ribosomal RNA and DNA for rumen microbial ecosystem studies: a review, Mol Biol
Rep, vol. 35, pp. 265-274, 2008.
[66]
P. J. Weimer, Why dont ruminal bacteria digest cellulose faster?, Journal of dairy
science, vol. 79, no. 8, pp. 1496-502, Aug. 1996.
99
[67] D. E. Akin and R. Benner, Degradation of polysaccharides and lignin by ruminal bacteria
and fungi., Applied and environmental microbiology, vol. 54, no. 5, pp. 1117-25, May.
1988.
[68] D. C. Adams, J. a Pfister, R. E. Short, R. G. Cates, B. W. Knapp, and R. D. Wiedmeier,
Pine Needle Effects on in vivo and in vitro Digestibility of Crested Wheatgrass, Journal
of Range Management, vol. 45, no. 3, p. 249, May. 1992.
[69]
P. B. Pope et al., Adaptation to herbivory by the Tammar wallaby includes bacterial and
glycoside hydrolase profiles different from other herbivores., Proceedings of the
National Academy of Sciences of the United States of America, Jul. 2010.
[70] APHA, Standard methods for the examination of water and wastewater, 20th ed. 1998.
[71]
E. A. Edwards and D. Grbic-galic, Anaerobic Degradation of Toluene and o-Xylene by a

Methanogenic Consortium, Applied and Environmental Microbiology, vol. 60, no. 1, pp.
313-322, 1994.
[72]
C. Desai and D. Madamwar, Extraction of inhibitor-free metagenomic DNA from

polluted sediments, compatible with molecular diversity analysis using adsorption and
ion-exchange treatments., Bioresource technology, vol. 98, no. 4, pp. 761-8, Mar. 2007.
[73]
G. Muyzer and K. Smalla, Application of denaturing gradient gel electrophoresis

(DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology.,
Antonie van Leeuwenhoek, vol. 73, no. 1, pp. 127-41, Jan. 1998.
[74]
G. Muyzer, E. C. de Waal, and a G. Uitterlinden, Profiling of complex microbial

populations by denaturing gradient gel electrophoresis analysis of polymerase chain
reaction-amplified genes coding for 16S rRNA., Applied and environmental
microbiology, vol. 59, no. 3, pp. 695-700, Mar. 1993.
[75]
Amorphos Chemicals PVT. Ltd., Manufacturers for Sodium Lignosulfonate, 2011.

[Online]. Available: http://amorphos.co.in/sodium-lingo-sulphonate.html. [Accessed: 25May-2011].
[76]
Chemical Trading House (UK) Limited, Sodium Lignosulfonate in Powder, 2008.

[Online]. Available: http://c-t-h.ru/sodium_lignosulphonate_in_powder.html. [Accessed:
25-May-2011].
[77]
K. Pandey, FTIR studies of the changes in wood chemistry following decay by brown-rot
and white-rot fungi, International Biodeterioration & Biodegradation, vol. 52, no. 3, pp.
151-160, Oct. 2003.
[78] K. J. Harrington, H. G. Higgins, and A. J. Michell, Infrared Spectra of Eucalyptus

regnans F. Muell. and Pinus radiata D. Don, Holzforschung, vol. 18, no. 4, pp. 108-113,
Jan. 1964.
100
[79]
H. L. Hergert, Infrared spectra, in Lignins: Occurance, Formation, Structure and

Reactions, K. V. Sarkanen and C. H. Ludwig, Eds. New York: Wiley Interscience, 1971,
pp. 267-297.
[80]
T. P. Schultz and W. G. Glasser, Quantitative structure analysis of lignin by diffuse

reflectance fourier transform infrared spectrometry, Holzforschung, vol. 40, no. 1, pp. 3744, 1986.
[81]
O. Faix, Fourier transform infrared spectroscopy, in Methods in Lignin Chemistry, S. Y.

Lin and C. W. Dence, Eds. Berlin: Springer, 1992, pp. 83-109.
[82]
W. E. Collier, T. P. Schultz, and V. F. Kalasinsky, Infrared Study of Lignin:

Reexamination of Aryl-Alkyl Ether CO Stretching Peak Assignments, Holzforschung,
vol. 46, no. 6, pp. 523-528, Jan. 1992.
[83]
K. Pandey and K. Theagarajan, Analysis of wood surfaces and ground wood by diffuse
reflectance (DRIFT) and photoacoustic (PAS) Fourier transform infrared spectroscopic
techniques, European Journal of Wood and Wood Products, vol. 55, no. 6, pp. 383390,
1997.
[84]
F. Raposo, R. Borja, M. a Martn, a Martn, M. a de la Rubia, and B. Rincn, Influence

of inoculumsubstrate ratio on the anaerobic digestion of sunflower oil cake in batch
mode: Process stability and kinetic evaluation, Chemical Engineering Journal, vol. 149,
no. 1-3, pp. 70-77, Jul. 2009.
[85]
M. (Ivy) Yang, Anaerobic Treatment of In-Mill Wastewaters Collected from a Pulp and
Paper Mill, University of Toronto, 2008.
[86]
J. Taylor, Comparison of microbial numbers and enzymatic activities in surface soils and
subsoils using various techniques, Soil Biology and Biochemistry, vol. 34, no. 3, pp. 387401, Mar. 2002.
[87] E. M. Gabor, E. J. de Vries, and D. B. Janssen, Efficient recovery of environmental DNA

for expression cloning by indirect extraction methods., FEMS microbiology ecology, vol.
44, no. 2, pp. 153-63, May. 2003.
[88]
K.-C. Lin and Z. Yang, Technical review on the UASB process, International Journal
of Environmental Studies, vol. 39, no. 3, pp. 203-222, Dec. 1991.
[89]
M. T. Kato, J. a Field, P. Versteeg, and G. Lettinga, Feasibility of expanded granular

sludge bed reactors for the anaerobic treatment of low-strength soluble wastewaters.,
Biotechnology and bioengineering, vol. 44, no. 4, pp. 469-79, Aug. 1994.
[90]
M. P. Henry, B. a Donlon, P. N. Lens, and E. M. Colleran, Use of Anaerobic Hybrid

Reactors for Treatment of Synthetic Pharmaceutical Wastewaters Containing Organic
Solvents, Journal of Chemical Technology & Biotechnology, vol. 66, no. 3, pp. 251-264,
Jul. 1996.
101
[91] J. Li, B. Hu, P. Zheng, M. Qaisar, and L. Mei, Filamentous granular sludge bulking in a
laboratory scale UASB reactor., Bioresource technology, vol. 99, no. 9, pp. 3431-8, Jun.
2008.
[92] C. Campos and G. Anderson, The effect of the liquid upflow velocity and the substrate
concentration on the start-up and steady-state periods of lab-scale UASB reactors, Water
Science & Technology, vol. 25, no. 7, pp. 4150, 1992.
[93] J. Thaveesri, D. Daffonchio, B. Liessens, and W. Verstraete, Different types of sludge
granules in UASB reactors treating acidified wastewaters., Antonie van Leeuwenhoek,
vol. 68, no. 4, pp. 329-37, Nov. 1995.
[94]
C. S. Tripathi and D. Grant Allen, Comparison of mesophilic and thermophilic aerobic

biological treatment in sequencing batch reactors treating bleached kraft pulp mill
effluent, Water Research, vol. 33, no. 3, pp. 836846, Feb. 1999.
[95]
L. Zheng, Construction and Monitoring of the Anaerobic Reactors Treating Pulp Mill
Effluents, University of Toronto, 2011.
102
Appendix A. Mineral Medium Composition
Table A.1 Mineral medium composition
Stock
MM1
MM2
MM3
Compound
[Stock]
[Medium]
Stock
g/L
mg/L
KH2PO4
21.0
210
MM6
K2HPO4
42.8
428
MM7
NH4Cl
53.5
CaCl26H2O
Compound
[Stock]
[Medium]
g/L
mg/L
NaHCO3
6.9
69
Biotin
0.02
0.2
535
Folic acid
0.02
0.2
7.0
70
Pyridoxine HCl
0.1
1.0
FeCl24H2O
2.0
20
Riboflavin
0.05
0.5
H3BO3
0.3
0.6
Thiamine
0.05
0.5
ZnCl
0.1
0.2
Nicotinic acid
0.05
0.5
Na2MoO42H2O
0.1
0.2
Pantothenic acid
0.05
0.5
NiCl26H2O
0.75
1.5
PABA
0.05
0.5
MnCl24H2O
1.0
2.0
Cyanocobalamin
0.05
0.5
(vitamin B12)
CuCl22H2O
0.1
0.2
Thioctic (lipoic) acid
0.05
0.5
CoCl26H2O
1.5
3.0
Coenzyme M
1.0
10
Na2SeO3
0.02
0.04
(NH4)2Fe(SO4)26H2
39.2
392
MM8
O
MM4
Al2(SO4)318H2O
0.1
0.2
Na2S9H2O
24
240
MgCl26H2O
50.8
101.6
(equivalent FeS)
20
A-103
Appendix B. CH4 in biogas produced script

CH4 Fraction in Gas Produced Matlab Script
function [frac_CH4Out, frac_prod] = meth( vol_prod, frac_CH4_meas)
% CH4 fraction in the gas produced, calculated from headspace methane fraction and gas production volume data.
headspace = 100;
A=size(vol_prod);
imax = A(1);
jmax = A(2);
frac_prod = 0.5*ones(1,jmax);
vol_new = 0;
vol_CH4_prod = 0;
vol_CH4_beforeremoval = 0;
frac_CH4=zeros(imax,jmax);
for j=1:jmax
vol_CH4_afterremoval = zeros(imax,1);
while abs(frac_CH4_meas(1,j)-frac_CH4(imax,j))>0.0001
for i = 1:imax
vol_new = vol_prod(i,j)+headspace;
vol_CH4_prod = vol_prod(i,j)*frac_prod(j);
if i~=1
vol_CH4_beforeremoval = vol_CH4_prod+vol_CH4_afterremoval(i-1,1);
else
vol_CH4_beforeremoval = vol_CH4_prod;
end
%sprintf('frac = %i',frac_CH4(imax,j))
frac_CH4(i,j) = vol_CH4_beforeremoval/vol_new;
vol_CH4_afterremoval(i,1) = vol_CH4_beforeremoval-vol_prod(i,j)*frac_CH4(i,j);
end
if (frac_CH4_meas(1,j)-frac_CH4(imax,j))>0
frac_prod(j)= frac_prod(j) + 0.00001;
else if (frac_CH4_meas(1,j)-frac_CH4(imax,j))<0
frac_prod(j)= frac_prod(j) - 0.00001;
end
end
end
end
frac_CH4Out = frac_CH4(imax,:);
end
B-104
Appendix C. Detailed DNA Extraction Protocols
Chemical Method
DNA was extracted from raw inoculum samples using a technique designed to maximize
the quality of product and reduce PCR inhibitor concentration. The method employed is based on
that published by Desai and Madamwar [71], and is described in detail here. A 5 g sample of
sediment was added to a 50 mL falcon tube containing 5 g of 3 mm glass beads, and 15 mL of
extraction buffer (10% w/v sucrose, 1% w/v cetyltrimethylammonium bromide (CTAB), 1.5M
NaCl, 100mM Tris/HCl at pH 8.0, 100mM ethylenediaminetetraacetic acid (EDTA) at pH 8.0)
and 10 mg lysozyme. This mixture was agitated at 250 rpm on a shaker for 30 min at 30 C, then
50 L of 20 mg/mL proteinase K in 100 mM sodium phosphate buffer at pH 8.0 was added to
denature nucleases, and the mixture was again agitated at 250 rpm for 30 min at 50 C. Next, 2
mL of a 20 % sodium dodecyl sulphate (SDS) solution was added to the mixture, and agitated at
250 rpm for 20 min at 60 C. Following the lysis step, 0.5 g powdered activated carbon (PAC)
was added to the mixture to remove humic acids and other potentially inhibitory organic
compounds. The mixture was agitated slowly overnight at 4C. The next morning, the sample
was centrifuged at 7 000 X g for 30 min, then at 20 000 X g for 10 min. The supernatant (S1)
was collected using a pipette and transferred to a clean 50 mL falcon tube. A 5 mL aliquot of
extraction buffer, 0.33 mg lysozyme, and 15 L of proteinase K solution were added to the
pellet, which was re-extracted by shaking at 250 rpm for 30 min at 30 C, then adding 1 mL of
20 % SDS solution, shaking at 250 rpm for 15 min at 60C, and centrifuging at 7 000 X g for 30
min. The supernatant from the re-extraction procedure (S2) was then combined with S1. One
gram of PAC was added to the mixed supernatant, which was then agitated slowly overnight at 4
C. The following morning, the mixture was centrifuged at 7 000 X g for 30 min and the
supernatant drawn off to a clean 50 mL falcon tube. Na2EDTA was added to the mixture to give
a final concentration of 2.5 mM at pH 8.0. A volume of phenol, chloroform, and isoamyl alcohol
(25:24:1 v/v/v) mixture equal to that of the supernatant was added to the falcon tube, which was
then shaken by hand for 3 min to separate organic soluble impurities. The sample was
centrifuged at 7 000 X g for 15 min to separate the phases, and the upper aqueous phase was
drawn off and added to an equal volume of chloroform and isoamyl alcohol (24:1 v/v) mixture.
The mixture was again shaken for 3 min by hand, then centrifuged at 7 000 X g for 15 min. To
remove heavy metals and other ionic contaminants, the aqueous phase was loaded onto a 20 mL
C-105
glass column containing 10 g of Amberlite IRA 400 ion exchange resin saturated with 1M NaCl
and washed with 100 mM Tris/HCl buffer (pH 8.0) and allowed to drip slowly through the
column to a new 50 mL falcon tube. Isopropanol (0.8 volumes) was added to the flow through
from the ion exchange resin and incubated at room temperature for 30 min to allow DNA to
precipitate, and then the tube was centrifuged at 7000 X g for 15 min. The supernatant was
discarded and the pellet was washed with 1 mL of 70 % ethanol, the alcohol was discarded, and
the pellet was allowed to air dry for 30 min. Once dry, it was resuspended in 20 L of
Tris/EDTA (pH 8.0) buffer. The concentration and purity of extracted DNA was measured
spectrophotometrically using a Nanodrop 1000 and eluted DNA was stored frozen at -20C until
further use.
UltraClean Method
DNA was extracted from enrichment cultures using the UltraClean kit according to the
manufacturers instructions. Briefly, 1 mL of sample was taken from each enrichment culture
and centrifuged at 11 000 X g for 15 min to separate solids from the liquid medium. The medium
was discarded and 500 L of liquid from the provided 2 mL Bead Solution Tubes was pipetted in
to resuspend the solids. This suspension was then transferred back to the tubes, and then the
tubes were vortexed for 5 s. Each tube contained a proprietary buffer solution and was preloaded
with lysis beads to mechanically shear microbial cells. A 60 L aliquot of a proprietary solution
containing sodium dodecyl sulphate (SDS) was then added and the tubes were vortexed for 5 s.
A 200 L aliquot of a provided inhibitor removal solution was then added, and then all tubes
were secured to a high speed vortex and shaken at maximum speed for 10 min. Following lysis,
the bead tubes were centrifuged at 11 000 X g for 1 min to separate the DNA-containing
supernatant from the beads, suspended cell contents, and other debris. The supernatant was
transferred to a new tube and mixed with 250 L of a proprietary protein precipitation solution,
then incubated on ice for 5 min to promote precipitation, and finally centrifuged to again separate
DNA-containing supernatant from solid material. The supernatant was then separated into two
new tubes, and 650 L of a proprietary DNA binding salt solution (DNA binds to silica in the
presence of this solution) was added to each tube. The tubes were vortexed for 5 s to thoroughly
mix the contents. Next, 700 L of this mixture was added to a spin filter and centrifuged at 11
000 X g for 1 min allowing the mixture to pass through the silica membrane. The DNA in the
mixture was retained on the filter and filtrate was discarded. This spin filtration procedure was
repeated using the same spin filter until all of the solution was filtered. A proprietary ethanolC-106
containing wash solution (300 L) was then added to each filter and the tube centrifuged at 11
000 X g for 1 min to remove remaining salt and contaminants. The flow through was discarded,
and the tubes were centrifuged once more at 11 000 X g for 1 min to completely remove residual
ethanol. The spin filter was transferred to a new tube and 20 L of elution buffer provided by the
manufacturer was added to the surface of the filter. The tubes were centrifuged at 10 000 X g for
1 min to remove the solution, now containing relatively pure DNA. The concentration and purity
of this product was measured spectrophotometrically and the DNA in solution was stored at 20C.
C-107
Appendix D. Phase I Gas Production Curves
COD consumed
mgCOD added
1
0.75
0.5
0.25
0
0
20
40
60
80
60
80
Time (days)
Figure D.1 Phase I moose rumen, cellulose and VFA gas production curves.
- glucose, acetate, propionate mixture,
- cellulose,
- poplar hydrolysate )
COD consumed
mgCOD added
1.5
1.25
1
0.75
0.5
0.25
0
0
20
40
Time (days)
Figure D.2 Phase I moose rumen, cellulose and additives gas production curves.
- cellulose,
- cellulose + lignosulphonate,
- cellulose + tannic acid,
D-108
- cellulose + pine needles )
COD consumed
mgCOD added
1
0.75
0.5
0.25
0
0
50
100
150
Time (days)
Figure D.3 Phase I beaver droppings, cellulose and VFA gas production curves.
- cellulose,
COD consumed
mgCOD added
2
1.5
1
0.5
0
0
50
100
150
Time (days)
Figure D.4 Phase I beaver droppings, cellulose and additives gas production curves.
- cellulose,
D-109
COD consumed
mgCOD added
1
0.75
0.5
0.25
0
0
50
100
150
200
Time (days)
Figure D.5 Phase I heritage pile, cellulose and VFA gas production curves.
- cellulose,
COD consumed
mgCOD added
2
1.5
1
0.5
0
0
50
100
150
200
Time (days)
Figure D.6 Phase I heritage pile, cellulose and additives gas production curves.
- cellulose,
D-110
COD consumed
mgCOD added
1
0.75
0.5
0.25
0
0
20
40
60
80
60
80
Time (days)
Figure D.7 Phase I landfill-leachate, cellulose and VFA gas production curves.
- cellulose,
COD consumed
mgCOD added
1.5
1.25
1
0.75
0.5
0.25
0
0
20
40
Time (days)
Figure D.8 Phase I landfill-leachate, cellulose and additives gas production curves.
- cellulose,
D-111
Appendix E. Phase II, III Gas Production Curves
COD consumed
mgCOD added
Moose Rumen
1
0.8
0.6
0.4
0.2
0
0
50
100
150
Time (days)
Figure E.1 Phase II, III moose rumen, cellulose and poplar hydrolysate gas production curves.
- cellulose,
COD consumed
mgCOD added
1
0.8
0.6
0.4
0.2
0
0
50
100
150
Time (days)
Figure E.2 Phase II, III moose rumen, cellulose and concentrated additives gas production curves.
- cellulose (C),
- C + lignosulphonate (high),
- C + tannic acid (high),
E-112
- C + pine needles (high) )
COD consumed
mgCOD added
1
0.8
0.6
0.4
0.2
0
0
50
100
150
Time (days)
Figure E.3 Phase II, III moose rumen, cellulose and low concentration additives gas production curves.
- cellulose (C),
COD consumed
mgCOD added
- C + lignosulphonate (low),
- C + tannic acid (low),
- C + pine needles (low) )
1
0.8
0.6
0.4
0.2
0
0
50
100
150
Time (days)
Figure E.4 Phase II, III moose rumen, poplar hydrolysate additives gas production curves.
- poplar hydrolysate (PH),
- PH + lignosulphonate,
E-113
- PH + tannic acid,
- PH + pine needles )
COD consumed
mgCOD added
Beaver Droppings
1
0.8
0.6
0.4
0.2
0
0
50
100
150
Time (days)
Figure E.5 Phase II, III beaver droppings, cellulose and poplar hydrolysate gas production curves.
- cellulose,
COD consumed
mgCOD added
1
0.8
0.6
0.4
0.2
0
0
50
100
150
Time (days)
Figure E.6 Phase II, III beaver droppings, cellulose and concentrated additives gas production curves.
- cellulose (C),
E-114
COD consumed
mgCOD added
1
0.8
0.6
0.4
0.2
0
0
50
100
150
Time (days)
Figure E.7 Phase II, III beaver droppings, cellulose and low concentration additives gas production curves.
- cellulose (C),
COD consumed
mgCOD added
1
0.8
0.6
0.4
0.2
0
0
50
100
150
Time (days)
Figure E.8 Phase II, III beaver droppings, poplar hydrolysate additives gas production curves.
E-115
- PH + tannic acid,
COD consumed
mgCOD added
Internal circulation granules
2
1.5
1
0.5
0
0
50
100
150
Time (days)
Figure E.9 Phase II, III internal circulation granules, cellulose and poplar hydrolysate gas production curves.
- cellulose,
COD consumed
mgCOD added
2
1.5
1
0.5
0
0
50
100
150
Time (days)
Figure E.10 Phase II, III internal circulation granules, cellulose and concentrated additives gas production
curves.
- cellulose (C),
E-116
COD consumed
mgCOD added
2
1.5
1
0.5
0
0
50
100
150
Time (days)
Figure E.11 Phase II, III internal circulation granules, cellulose and low concentration additives gas
production curves.
- cellulose (C),
COD consumed
mgCOD added
2
1.5
1
0.5
0
0
50
100
150
Time (days)
Figure E.12 Phase II, III internal circulation granules, poplar hydrolysate additives gas production curves.
E-117
- PH + tannic acid,
Appendix F. Phase IV Gas Production Curves
COD consumed
mgCOD added
Moose rumen
1
0.8
0.6
0.4
0.2
0
0
50
Time (days)
100
Figure F.13 Phase IV moose rumen gas production curves.
- poplar hydrolysate,
- cellulose (C),
- C + lignosulphonate,
needles )
F-118
- C + tannic acid,
- C + pine
COD consumed
mgCOD added
Beaver droppings
1
0.8
0.6
0.4
0.2
0
0
Time (days)
50
100
Figure F.14 Phase IV beaver droppings gas production curves.
- cellulose (C),
- C + tannic acid,
- C + pine
needles )
COD consumed
mgCOD added
Internal circulation granules
1
0.8
0.6
0.4
0.2
0
0
Time (days)
50
100
Figure F.15 Phase IV internal circulation granules gas production curves.
- cellulose (C),
needles )
F-119
- C + tannic acid,
- C + pine
Appendix G. Sample Calculations
Electron Equivalents
Organic compounds can be oxidized to form carbon dioxide and water. During this
oxidation process, the net result is a transfer of electrons from the organic compound to oxygen.
For example, the oxidation of acetic acid can be thought of in terms of two half reactions:
CH3COOH + 2H2O
2O2 + 8H+ + 8e-
2CO2 + 8H+ +8e4H2O
(oxidation)
(reduction)
CH3COOH + 2O2
CO2 + 2H2O
(overall)
Electron equivalents are the number of moles of electrons required to fully oxidize one
mole of corresponding organic. In the case above, 8 electron equivalents are required per mole of
acetic acid. Since the net reaction results in the full reduction of molecular oxygen, the electron
equivalents can be determined quickly by writing the complete oxidation reaction, balancing it,
counting the number of oxygen atoms required, and multiplying by 2 (since 2 electrons are
required to fully reduce 1 oxygen atom).
Theoretical Methane Fraction

The theoretical methane fraction in biogas is based on the conservation of electron
equivalents. In the absence of a suitable electron acceptor, are assumed to be conserved, simply
transferring from the substrate organic compound to the product methane. Consider the methane
oxidation reaction:
CH4 + 2O2
CO2 + 2H2O
It can be seen that methane contains 8 electron equivalents. To determine the amount of
methane derived from other organic compounds, first the electron equivalents in the other
organic compound must be determined, for example:
G-120
CH3COOH + 2O2
CO2 + 2H2O
Acetic acid contains 8 electron equivalents as well, indicating a one to one stoichiometric
relationship between acetic acid and methane. The remaining atoms of the organic compound are
assumed to form fully oxidized products:
CH3COOH
CH4 + CO2
From one mole of acetic acid, one mole of methane and one mole of carbon dioxide are
formed. This ratio indicates that 50% methane would be expected in the gaseous product. Similar
calculations can be performed for other organic substances, such as cellulose:
C6H10O5 + 3O2
3CO2 + 5H2O
Cellulose contains 12 electron equivalents, and methane contains 8. Therefore, 3/2 moles
of methane are produced per mole of cellulose.
2C6H10O5
3CH4 + 3CO2
An equation to simplify this process, incorporating nitrogen and sulphur species in a

similar manner is presented below:
CcHhOoNnSs + yH2O xCH4 + nNH3 + sH2S + (c-x)CO2
where x = 1/8 * (4*c + h 20 3*n 2*s)
and
y = 1/4 * (4*c h 20 + 3*n 3*s)
Sample calculations
Gas production was measured periodically. Following the cessation of biogas production,
the total amount of gas produced was divided by the theoretically expected volume of gas to give
a biogas yield. Several factors were accounted for in the calculation of theoretical biogas yield:
Added COD (COD of each substrate was measured and correlated to mass)
G-121
Expected CH4 (COD is a measure of complete oxidation, as such it can be converted to

electron equivalents and ultimately expected CH4)
Expected total gas volume (accounting for CO2)
Temperature, pressure (the ideal gas law, PV = nRT, was used to convert from the
number of moles of CH4 to an expected total volume at the expected temperature and
pressure conditions)
Saturated H2O content (as per Richards et al.)

The total amount of biogas actually produced divided by the theoretical amount of gas
gives the biogas yield. Applying this method to the cumulative gas production totals and
determining at which point the yield exceeded 20 % of the total expected biogas gives the time to
20 % COD conversion. A sample calculation is presented below.
In phase II, 0.142g of cellulose was added to each cellulose-only bottle. From COD
measurements, 1.00g cellulose contains 1.19gCOD. Therefore,
0.142g cellulose * (1.19gCOD / 1.00g cellulose) = 0.169gCOD
Converting from gCOD to moles of CH4:
1 gCOD * (1 mol O2 / 32 gCOD) = 1/32 mol O2
1/32 mol O2 * (4 mol e- / 1 mol O2) = 1/8 mol e1/8 mol e- * (1 mol CH4 / 8 mol e-) = 1/64 mol CH4
0.169 gCOD * (1/64 mol CH4 / 1 gCOD) = 0.00263 mol CH4
Since cellulose theoretically forms half methane and half carbon dioxide, this number is
doubled to account for the total amount of gas, then converted to a volume at standard
temperature and pressure using the ideal gas law, and finally temperature and humidity are
accounted for:
0.00263 mol CH4 * (2 mol biogas / 1 mol CH4) = 0.00526 mol biogas
0.00526 mol biogas * (8.314 J/mol*K * 273.15K / 101.325kPa)
= 0.118 L cold, dry biogas
0.118 L cold, dry biogas / (0.83) = 0.142 L wet, warm biogas
G-122
If the data series below was the set of measured biogas volumes removed from one
cellulose-only bottle over the time listed, then the following calculations would be performed to
obtain a time to 20 % conversion and yield:
Time (days)
0
2
4
6
8
10
50
Volume (mL)
0
15.5
26.2
30.6
35.7
39.0
65.1
The time to 20 % conversion is determined first by finding 20 % of the theoretical

methane value, then finding the corresponding time at which the actual gas production first
exceeded this value:
0.142 L biogas * (0.20) = 0.0285 L biogas
28.5 mL biogas is exceeded at 6 days.
The total yield is determined by dividing the ultimate gas volume (following the
cessation of biogas production) by the theoretical gas volume.
0.0651 L actual biogas / 0.142 L theoretical biogas = 0.458
G-123
Elemental Composition
MW
Na
gCOD/
g substrate
Sodium acetate
82
0.78
Sodium propionate
96
1.17
Glucose
180
12
1.07
Cellulose
162
10
1.19
Furfural
96
1.67
Hemicellulose
96
1.67
Lignin
Sodium
lignosulphonate
150
10
2.24
449
20
26
10
1.55
Tannic acid
1700
76
52
46
1.24
Pine needles
145
1.38
Substrate
G-124
CH4 in
gas
0.88
CO2 in
gas
1.13
CH4
fraction
0.44
1.63
1.38
0.54
3.00
3.00
0.50
3.00
3.00
0.50
2.50
2.50
0.50
2.50
2.50
0.50
5.25
3.75
0.58
10.25
9.75
0.51
33.00
43.00
0.43
3.13
2.88
0.52
Appendix H. DGGE bands identified with Image J

Table H.1 DGGE bands of microcosm enrichments
MR C
0.33
MR CT
MR CL
MR CP
MR PH
0.3
0.36
0.36
0.36
0.23
0.43
0.4
0.56
BD C
BD CT
0.47
BD CL
0.49
BD CP
0.49
0.78
0.89
0.89
0.89
0.93
0.94
2.12
2.15
0.78
0.75
2.11
3.94
TG CL
TG CP
TG PH
0.43
0.5
0.5
0.4
0.4
0.37
0.56
0.6
0.6
0.58
0.53
0.69
0.73
0.76
0.76
0.66
1.93
1.82
0.93
1.24
3.84
1.41
2.45
4.54
4.54
4.74
5.48
2.15
2.24
2.26
2.26
2.16
2.78
2.86
2.88
2.85
2.78
3.71
3.76
3.72
3.64
4.01
3.28
3.94
4.21
TG C
1.69
1.03
2.1
3.74
3.94
TG CT
0.7
0.89
3.6
BD PH
2.4
1.9
3.64
2.48
4.08
4.58
4.67
3.43
3.79
3.9
4.1
4.06
4.57
4.03
4.97
4.12
4.44
5.37
4.09
5.54
4.5
5.66
5.37
5.86
5.6
5.6
5.64
5.84
6.19
5.56
6.06
6.23
6.29
6.32
6.27
6.23
6.59
6.97
7.03
7.58
6.99
6.95
7.6
7.56
7.86
8.29
Position of shared bands (cm from top of gel)
H-125
7.85
8.32
8.35
8.32
8.28
Appendix I. Pressure Transducer Calibration
Pressure Transducer
Reading
2.0
1.5
1.0
y = 0.0641x + 0.0105
2
R = 0.9999
0.5
0.0
0
10
15
20
25
Volume (mL)
Figure I.1 Pressure transducer calibration curve
Test conditions: 25 C, 100 mL headspace, 101.9 kPa atmospheric pressure. The specified volume of air was added to a sealed serum bottle and the
corresponding reading was recorded. The volume of air was then removed, and the process repeated for different volumes.
I-126
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ENRICHMENT OF METHANOGENIC MICROCOSMS

ON RECALCITRANT LIGNOCELLULOSIC BIOMASS

A thesis submitted in conformity with the requirements

Copyright by W. Michael Lacourt 2011

ENRICHMENT OF METHANOGENIC MICROCOSMS

My supervisors, Professors Emma Master and Honghi Tran

My committee members, Professors Elizabeth Edwards and Grant Allen

Collaborators, colleagues, and support staff:

My friends and family

Literature survey ..................................................................................................................... 4

Anaerobic digestion ........................................................................................................ 5

Inhibitors of digestion process .............................................................................. 10

Biogas from woody biomass......................................................................................... 18

Metagenome studies of digestive system lignocellulose degrading communities........ 19

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

Inoculum collection and storage ................................................................................... 22

Moose rumen fluid................................................................................................ 22

Beaver droppings .................................................................................................. 22

Internal circulation reactor granules ..................................................................... 22

Other inoculum sources ........................................................................................ 22

Landfill and leachate..................................................................................... 23

Chemical oxidation demand ................................................................................. 24

Fourier transform infrared spectroscopy (FTIR) .................................................. 25

Gas volume measurements ................................................................................... 26

Headspace CH4 content......................................................................................... 27

Nutrient medium ........................................................................................................... 29

Feedstock preparation ................................................................................................... 29

Dilution and transfer of cultures ................................................................................... 32

Biochemical analysis .................................................................................................... 38

Deoxyribonucleic acid (DNA) extraction............................................................. 38

Denaturing gradient gel electrophoresis (DGGE)................................................. 40

Results and Discussion ......................................................................................................... 44

Setup and transfers........................................................................................................ 44

Inoculum Properties ...................................................................................................... 48

Substrate Properties ...................................................................................................... 49

Gas Production Results ................................................................................................. 53

Phase II and III Enrichments................................................................................. 60

Time to 20 % COD consumption ................................................................. 61

Biogas yield .................................................................................................. 64

CH4 fraction .................................................................................................. 67

Phase II to IV: Comparison of Concentrated Additive Degrading Microcosms .. 75

Phase V Enrichment: Bioconversion of Post Extraction Washer (PEW) Effluent79

Measurement of physical and chemical properties....................................................... 24

Analysis of microbial inocula and enrichments by DGGE........................................... 81

DNA extraction and concentration ....................................................................... 81

Reactor design and operating conditions ...................................................................... 86

Reactor operation .......................................................................................................... 89

Conclusions and Implications ............................................................................................... 91

Table 2.1. Fermentation reactions................................................................................................... 8

Figure 4.18 Initial rates of biogas production in Phase IV microcosms....................................... 73

Appendix A. Mineral Medium Composition .......................................................................... A-103

American Public Health

Internal circulation reactor granules

Biochemical methane potential

VFA & glucose substrate mix

Bovine serum alvumin

Polymerase chain reaction

Avicel PH-101 microcrystalline

Carbohydrate binding module

Avicel plus sodium

Steam exploded poplar hydrolysate

Chemical oxidation demand

x = 1/8 * (4c + h 20 3n 2*s)

y = 1/4 * (4c h 20 + 3n 3*s)