University of Akureyri
Faculty of Natural Resources
33
Metabolic pathways and biofuel production from
lignocellulosic biomass by thermophilic anaerobes isolated
from Icelandic hot springs
Sara Lind Jónsdóttir
Faculty of Natural Resources
School of Business and Natural Science
Sara Lind Jónsdóttir | BSc Biotechnology
University of Akureyri
2014
i
University of Akureyri
Faculty of Natural Resources
Metabolic pathways and biofuel production from
lignocellulosic biomass by thermophilic anaerobes isolated
from Icelandic hot springs
Sara Lind Jónsdóttir
A 12 ECTS Bachelor‘s thesis in B.Sc. Biotechnology
Supervisor
Dr. Jóhann Örlygsson
Professor and Dean of the Faculty of Biotechnology
Faculty of Natural Resources
School of Business and Natural Science
University of Akureyri
Akureyri, April 2014
Sara Lind Jónsdóttir | BSc Biotechnology
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University of Akureyri
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I hereby attest that I am the author of the dissertation herein and that it is a product of my own
academic research from the autumn of 2013 and the spring of 2014.
_____
Sara Lind Jónsdóttir____________
Signature, date and place
I hereby certify that the dissertation successfully fulfills the requirements to complete
the Bachelor of Science in Biotechnology at the University of Akureyri.
______________Jóhann Örlygsson_____________
Signature, date and place
Sara Lind Jónsdóttir | BSc Biotechnology
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Faculty of Natural Resources
Metabolic Pathways and Biofuel Production from Lignocellulosic Biomass by Thermophilic
Anaerobes isolated from Icelandic hotsprings
Metabolic Pathways and Biofuel Production from Lignocellulose by Thermophiles
12 ECTS thesis for Baccalaureaus Scientiae in Biotechnology
Copyright © 2014 Sara Lind Jónsdóttir
All rights reserved
School of Business and Science
Faculty of Natural Resource Sciences
University of Akureyri
Sólborg, Norðurslóð 2
600 Akureyri
Tel: +354 460 8000
Registration information:
Sara Lind Jónsdóttir, 2014
Metabolic Pathways and Biofuel Production from Lignocellulosic Biomass by Thermophilic
Anaerobes isolated from Icelandic hot springs
Bachelor thesis, School of Business and Science
Faculty of Natural Resource Sciences
University of Akureyri, 61 pp.
Printed by Háskólaprent
Reykjavík, April 2014
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Abstract
The present investigation is, firstly, a literature review of most current studies on biofuel
production (e.g. ethanol, hydrogen and methane) from lignocellulosic biomass and the use of
the isolated thermophilic anaerobic strains from Icelandic hot springs in the process. Various
factors affect the production of biofuels from lignocellulosic biomass including partial
pressure of hydrogen, initial concentration of lignocellulosic hydrolysates, acidity and
alkalinity of pretreatment, inhibitory effects of end-product formation and enzymes used.
Good ethanol and hydrogen yields depend greatly on optimising these factors and the use of
the suitable lignocellulosic hydrolysate. Thermoanaerobacterium strain AK17 has the highest
ethanol yield of 5.5 mM g sugar-1 from 2.5 g L-1 grass hydrolysate, followed by
Thermoanerobacter strain AK5 with ethanol yield of 4.4 mM gsugar-1 from grass hydrolysates.
Thermoanaerobacter strain GHL15 has the highest hydrogen yield of 7.6 mM g sugar-1 from
grass hydrolysates followed by Clostridium strain AK14 with 6.23 mM g sugar-1 also from
grass hydrolysates. Fermentative performance of the methane-producing strains isolated in
Iceland is yet to be studied and published.
Secondly, biofuel production processes are reviewed in the paper. Several challenges
concerning the upscaling and the high cost of pretreatment are presented.
Keywords:
Thermophilic anaerobic bacteria, biofuel, lignocellulosic biomass, renewable, Iceland
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University of Akureyri
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Acknowledgments
I would like to thank my professor and supervisor Dr. Jóhann Örlygsson for the opportunity to
work on this project. My family for endless support and compassion. And David Glaser for
the unsurmountable encouragement, inspiration and love.
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University of Akureyri
Faculty of Natural Resources
Útdráttur
Verkefnið er fyrst og fremst heimildarritgerð á nýjustu rannsóknum á lífeldsneytisframleiðslu
(etanól, vetni og metan) úr flóknum lífmassa og notkun einangraðra hitakæra baktería úr
íslenskum
hverum
í
framleiðsluferlinu.
Mismunandi
þættir
hafa
áhrif
á
lífeldsneytisframleiðslu úr flóknum lífmassa s.s. hlutþrýstingur vetnis, upphafsstyrkur flókins
lífmassa, formeðhöndlun með sýru- og basa, hrindrunaráhrif lokaafurða, og ensím sem eru.
Góð etanól og vetnisnýting er háð því að hámarka þessa mismunandi þætti og hvaða lífmassi
er notaður. Thermoanaerobacterium stofn AK17 hefur hæstu etanól nýtingu 5.5 mM g sugar-1
við 2.5 g L-1 upphafsstyrks úr gras hydrolysati. Næstur er Thermoanerobacter stofn AK5 með
etanól gildi 4.4 mM g sugar-1 úr grashydrolysati. Thermoanaerobacter stofn GHL15 er með
hæstu vetnisnýtingu, 7.6 mM g sugar-1 úr grashydrolysati. Síðan er Clostridium stofn AK14
með 6.23 mM g sugar-1 einnig úr gras hydrolysati. Metanframleiðsla hjá metanframleiðandi
hitakærum baktería sem einangraðar eru á Íslandi hafa ekki verið rannsakaðar og birtar.
Einnig er farið í mismunandi framleiðsluferla til að búa til lífeldsneyti í ritgerðinni,
sérstaklega m.t.t. hugsanlega erfiðleika við uppskölun og kostnað af formeðhöndlun.
Lykilorð:
hitakærar bakteríur, lífeldsneyti, loftfirrtar bakteríur, einangraðar á Íslandi, flókinn lífmassi
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Table of Contents
Background and research objectives .......................................................................................... 1
Introduction ................................................................................................................................ 3
Iceland and renewable energy ................................................................................................ 4
Biomass ...................................................................................................................................... 5
Biofuels .................................................................................................................................... 12
Metabolic Pathways: Fermentation .......................................................................................... 14
Thermophilic Anaerobes – the background ............................................................................. 17
Thermophilic Anaerobes from Icelandic Hot Springs ............................................................. 22
Bioprospecting Icelandic Hot Springs .................................................................................. 23
Process of Isolation .................................................................................................................. 28
Ethanol-Producing strains isolated in Iceland .......................................................................... 29
Hydrogen-producing strains isolated in Iceland ....................................................................... 32
Methane-producing strains isolated in Iceland ......................................................................... 35
Effects of Different Factors on the yield .................................................................................. 37
Upscaling .................................................................................................................................. 41
Biofuel Plants of the future ...................................................................................................... 42
Improvement strategies......................................................................................................... 44
Pros and cons of using thermophiles for biofuel production ................................................... 45
Genetic engineering of thermophiles ....................................................................................... 46
Discussion ................................................................................................................................ 47
Concluding Remarks ................................................................................................................ 49
Bibliography ............................................................................................................................. 50
Appendix I ................................................................................................................................ 60
Appendix II .............................................................................................................................. 61
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List of Figures
Figure 1. Renewable energy share of global final energy consumption, 2009........................................ 3
Figure 2. Primary energy use in Iceland .................................................................................................. 4
Figure 3. Major components of plant cells: lignin, hemicellulose, cellulose .......................................... 5
Figure 4. Cellulose: glucose monomers linked by β-1,4 glycosidic bonds within units and hydrogen
bonds between. ........................................................................................................................................ 6
Figure 5. Chemical structure of xylan. .................................................................................................... 7
Figure 6. Lignin precursors: (a) p-coumaryl alcohol, (b) coniferyl alcohol and (c) sinapyl alcohol ...... 8
Figure 7. A simplified scheme of glucose degradation to various end-products................................... 14
Figure 8. Stage I and II in glycolysis – Preparation and Oxidation.......................................................15
Figure 9. Stage III – Reduction. Ethanol, hydrogen, carbon dioxide and organic acids formation ......15
Figure 10. Anaerobic digestion: hydrolysis, fermentation, methanogenesis.........................................16
Figure 11. A map of geothermal fields in Iceland and the isolation location of biofuel-producing
thermophilic anaerobes .........................................................................................................................22
Figure 12. Upscaling .............................................................................................................................41
Figure 13. Cellulosic ethanol production model....................................................................................42
Figure 14. Diagram of ethanol production from lignocellulosic biomass .............................................43
List of Tables
Table 1. Ethanol-producing thermophilic strains isolated by researchers at the University of Akureyri
............................................................................................................................................................... 25
Table 2. A summary of best biofuel-producing substrates with each isolated thermophilic anaerobe . 40
Appendices
Appendix I. Profiles for anaerobic digestors.........................................................................................60
Appendix II. Minimum inhibitory concentrations of ethanol for seven thermophilic bacterial
strains.....................................................................................................................................................61
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Background and research objectives
The research question of this paper is: Are thermophilic anaerobes isolated from Icelandic hot
springs ideal candidates for pretreatment for the biofuel production from lignocellulosic
biomass?
The objective of this research is therefore twofold.

Firstly, to do a literature study of most current research on biofuel production (e.g.
ethanol, hydrogen and methane)
from lignocellulosic biomass with thermophilic
bacteria isolated from hot springs in Iceland. As a result of the investigation, an
overview of Icelandic thermophilic anaerobic strains that are promising candidates for
biofuel production is provided.

Secondly, to investigate and analyse the process of biofuel production with
thermophilic anaerobes. This will be done by investigating the pilot scale processes,
challenges of upscaling, and its potential for the future.
Sara Lind Jónsdóttir | BSc Biotechnology
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University of Akureyri
Faculty of Natural Resources
“Progress means getting nearer the place you want to be. And if you take a wrong turning,
then to go forward does not get you any nearer. If you are on the wrong road, progress means
doing an about face and walking back to the right road, and in that case the man who turns
back the soonest is the most progressive man.“ (C S Lewis in “Mere Christianity“)
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Introduction
Global greenhouse emissions as a result of industrialisation since the 19th century and the
consequential climate change are amongst mankind‘s biggest challenges. According to the
United Nations Framework Convention on Climate Change (‘Climate Convention‘), its
ultimate objective is “ to [stabilise] greenhouse gas concentrations in the atmosphere at a level
that would prevent dangerous anthropogenic interference with the climate system.“ Global
greenhouse gas emissions amount to 50.1 gigatonnes of carbon dioxide equivalent (GtCO2e)
per year (range 45.6-54.6 GtCO2e per year) as of 2010 (UNEP, 2013).
The power sector produces the highest emission of greenhouse gases in the atmosphere and
therefore have the highest emission reduction potential in 2020 of 2.2-3.9 GtCO2e per year.
The manufacturing industry has the second highest emission reduction potential or 1.5-4.6
GtCO2e per year, followed by transportation with 1.7-2.5 GtCO2e per year. Waste has around
0.8 GtCOse per year emission reduction potential (UNEP, 2013).
Renewable energy source is an expanding energy sector that is of vital importance in
addressing the challenge of greenhouse gas emission reduction (Figure 1). An estimated 19%
of global energy consumption is accounted for renewable energy source. Renewables
represent rapid growth of total energy supply, including heat and transport, in countries such
as the United States (10.9%), China (9%), Germany (11%), Denmark (22%), Portugal (21%),
Spain (15.4%), and Ireland (10.1%) (REN21, 2013).
Figure 1. Renewable energy share of global final energy consumption, 2011 (REN21, 2013)
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Iceland and renewable energy
Iceland is an island situated in the North Atlantic just south of the Arctic Circle. It is located
on a geological hotspot in the Mid-Atlantic Ridge making the country volcanically active with
an average eruption every five years. As a northern country, Iceland‘s landmass is covered by
glaciers which form powerful rivers from the ice caps. These geographical characteristics of
the country made Iceland uniquely abundant with both geothermal resource and hydropower
(NEA, 2013).
Geothermal and hydropower account for more than 70% of Iceland‘s primary energy
consumption. The period between 1940 and 2010 shows a dramatic increase in national
energy consumption. Most of the energy in 1940 was obtained from coal (Figure 2). In the
year 2006, in contrast, 60% of the primary energy consumption is geothermal and 15% is
hydropower. The rest is mainly oil for transportation of vehicles and for the fishing fleet
(Steingrimsson et al., 2007). Even more dramatic increase in energy consumption as well as
the ratio between different sources is seen in the years between 2006 and 2010 (Figure 2).
Figure 2. Primary energy use in Iceland (Icelandic National Energy Authority (NEA), 2013)
The transportation sector and the fishing fleet in Iceland still depend on fossil fuel imports
whose cost is about one tenth of the cost of the country‘s entire imports. Hence it should be in
the nation‘s priority to find locally-produced alternatives to fossil fuels. Electricity-based
alternatives, such as battery vehicles, hydrogen fuel cells; biomass; and biofuels are promising
candidates in addressing the challenge to alter the uneconomical import of fossil fuels (NEA,
2012).
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Biomass
Biomass is a promising renewable energy resource. With the world‘s increasing energy
resource problem, as well as the challenge of reducing greenhouse gas emissions, biomass
gives a whole new alternative – it is abundant and renders environment-friendly end-products.
Biomass, in a biotechnological context, means “all organic matter that grows by the
photosynthetic conversion of solar energy” (Glazer and Nikaido, 2007). Biofuel is any fuel
that is derived from biomass – either from plant material or animal waste.
There has been a significant increase in annual ethanol production in recent years. More than
95% of ethanol production today is a first-generation biofuel, i.e. produced from simple
biomass such as starch and mono- or disaccharides (Renewable Fuels Association, 2011).
This source of biomass is thus a subject of public scrutiny as it requires extensive use of land
which can otherwise be used for food crops (Sanchez and Cordona, 2008). Lignocellulosic
biomass is hence a better choice of source for biofuel production as it is abundant in nature
and is largely regarded as waste in agricultural activities
(Hamelinck et al., 2005). Hydrogen and methane, along with
ethanol are the compounds that are produced as a result of
fermenting lignocellulosic biomass.
The main challenge of harvesting these compounds from
lignocellulosic biomass lies in the pretreatment. The problem
to-date
is
hydrolysing
the
complex
biomass
before
fermentation takes place, as well as the continuity of the
process despite the formation of inhibitory by-products.
Relatively
new
investigation
shows
that
thermophilic
anaerobes are the most promising candidates to address the
pretreatment challenge (Sanchez and Cordona, 2008).
Lignocellulosic Biomass
Major components of plant biomass. Cell walls of the vascular
tissues of higher land plants consist of cellulose fibrils that are
embedded in an amorphous matrix of lignin and hemicelluloses
(Figure 3). These three major components, i.e. cellulose,
lignin and hemicellulose, are bound strongly by both
Figure 3. Major components of plant cells:
lignin, hemicellulose, cellulose (US. DOE,
2014)
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noncovalent forces and covalent cross-links, which overall form what is called lignocellulose.
This composite material forms about 90% of a plant‘s cell dry weight. Percentage of each of
the three lignocellulosic component varies from each plant species to another. For instance,
cellulose content is higher in hardwoods (45-55%) than grasses (25-40%), whereas lignin
content is relatively higher in softwoods( 25-35%) than hardwoods (18-25%) and grasses (
10-30%) (Glazer and Nikaido, 2007).
An estimated annual production of lignocellulose ranges from 2 to 5 x 1012 metric tons
(Glazer and Nikaido, 2007) and hence, a contender for both sustainable and renewable energy
source.
Energy tapping from waste biomass creates additional value to the society, as fossil fuel
consumption is replaced by this alternative source which would otherwise go to waste
(Worldwatch Institute, 2006).
Cellulose
A worldwide production between 1010 and 1011 tons is accounted for cellulose alone, making
it the most abundant organic compound on earth (Lavoine et al., 2012). Interestingly, urban
waste is a promising source of biomass. An estimated 40% of urban waste consists of
cellulose (Gray et al., 2006). A cellulose polymer consists of linear chain of glucose
molecules linked by β-(1:4)-glycosidic bonds (Figure 4).
HO
O
HO
OH
O
O
O
OH
HO
HO
HO
OH
O
O
O
HO
H
HO
OH
O
HO
Figure 4. Cellulose: glucose monomers linked by β-1,4 glycosidic bonds within units and hydrogen bonds
between.
Cellulose is an immensely tough substance whose primary role is to provide plants protection
from physical strains. A number of bacteria and fungi contain substances that can break down
cellulose by hydrolysing its chemical bonds. These are called cellulases or enzymes that break
down cellulose. They are divided into three subgroups: endoglucanases, cellobiohydrolases
and β-glucosidases (Lynd et al., 2002). Cellulases play an important role on the enzymatic
hydrolysis of lignocellulose which is a crucial step before fermentation of this complex
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biomass takes place. Relatively few bacteria and fungi, however, have the complete enzyme
system that is capable of breaking down crystalline cellulose. A rare example of this type of
enzyme is the cellulosome Clostridium thermocellum which was discovered in the early
1980‘s (Bayer and Lamed, 2006). It is a multifunctional protein complex that has a
scaffolding unit including a series of nine highly similar binding modules called domains that
interact together in the complete breakdown of crystalline cellulose (Shimon et al., 1997;
Bayer et al., 1998).
Hemicellulose
Hemicellulose units are defined as those polysaccharides that are non-covalently bonded with
cellulose (Figure 3). They consist of 1,4-linked β-D-pyranosyl units which are structurally
analogous to cellulose. Hemicelluloses are highly branched, and mostly noncrystalline
heteropolysaccharides. Sugar units that are generally found in hemicellulose are pentoses ( Dxylose, L-arabinose), hexoses (D-galactose, L-galactose, D-mannose, L-rhamnose, L-fucose),
and uronic acids (D-glucorinic acid) (Glazer and Nikaido, 2007). Hemicellulose generally
comprises 15-35% of plant biomass. Xylan and glucomannan are amongst the most relevant
hemicellulose units. Xylan (Figure 5) is a major structural polysaccharide in plant cells and
the second most abundant polysaccharide in nature. Xylan is found at the interface between
lignin and cellulose and is believed to be accountable for fibre cohesion and over-all plant cell
wall stability (Collins et al., 2005).
Figure 5. Chemical structure of xylan.
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Lignin
Lignin is the most abundant aromatic polymer on Earth. It is found in the cell walls of
gymnosperms angiosperms, ferns and club mosses. Lignin consists phenylpropare (C9) units.
Direct precursors of lignin are three alcohols – the lignols, which are derived from phydroxycinnamic acid: coniferyl, sinapyl, and p-coumaryl alcohols (Figure 6). Lignin
molecules increase the mechanical strength and durability of plants. Huge trees that are
several metres tall, for instance, remain upright because of what is called lignification – the
process wherein lignin molecules fill up the spaces between the preformed cellulose fibrils
and hemicellulose chains of the cell wall (Glazer and Nikaido, 2007).
Figure 6. Lignin precursors: (a) p-coumaryl alcohol, (b) coniferyl alcohol and (c) sinapyl alcohol
Lignin is a non-fermentable part of lignocellulose and protects cell wall polysaccharides from
microbial degradation. Hence, lignin is an inhibiting factor in plant biomass conversion to
biofuels. Lignin degradation is a costly process which requires the use of variety of enzymes:
lignin peroxidase, Mn-peroxidase, and laccase. Environmental conditions such as moisture,
temperature, pH, and levels of nitrogen and oxygen are critical factors for successful
degradation of lignin molecules (Glazer and Nikado, 2007; Tuor et al., 1995).
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Pretreatment of Biomass
Pretreatment is a significant stage of biofuel production from lignocellulosic biomass. This
stage incorporates modification of the complex structure of lignocellulose into a more
accessible form for enzymes that convert the polymeric carbohydrates into sugars before
fermentation. The processes involved in pretreatment to-date are financially demanding
thereby making biofuel production from lignocellulosic biomass considerably less
competitive to fossil fuels (Mosier et al., 2005; Zhu et al., 2010). There are several methods of
pretreatment as described below. A worldwide challenge remains on developing processes
with minimal energy consumption while maximising sugar and biofuel yield.
Physical Pretreatment
Physical pretreatment of lignocellulosic biomass involves mechanical processing such as
milling, chipping or grinding. This is mainly done to reduce cellulose crystallinity and thus
improve hydrolysis. Physical pretreatment is often used for woody biomass or hardwoods
(Zhu et al., 2010; Mosier et al., 2005). Raising the temperature of the biomass up to more than
300°C – in a process called pyrolysis – is also accounted as physical pretreatment, which
causes a rapid hydrolysis of cellulose (Sun and Cheng, 2002). Handling of lignocellulosic
biomass using physical pretreatment methods may prove to be impractical as the energy cost
is likely to be higher than the theoretical energy content available in the biomass involved
(Menon and Rao, 2012; Sanchez and Cordona, 2008).
Chemical Pretreatment
Chemical pretreatment of biomass
involves varying the pH. Both acidic and alkaline
treatments methods are widely used in the pretreatment of lignocellulosic biomass.
Concentrated and diluted inorganic acids such as hydrocholoric acid (HCl) and sulphuric acid
(H2SO4) are powerful agents in cellulose hydrolysis therefore they have been found useful in
acidic pretreatment of lignocellulosic biomass (Guo et al., 2008). Alkaline methods, on the
other hand, involve sodium-, calcium- and ammonium hydroxide which are especially
powerful in lignin separation (Yang and Whyman, 2007).
Physio-chemical pretreatment
Physio-chemical pretreatment includes both physical and chemical processes in preparing
lignocellulosic biomass for enzymatic degradation (Sveinsdottir, 2012). The most commonly
used physio-chemical method is steam explosion which uses high pressure (0.69 to 4.83 Mpa)
and saturated steam (160-260°C) to treat the biomass (Talebnia et al, 2010). The process is
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considered to be more environmental-friendly than some other pretreatment methods due to
less hazardous chemicals being produced. (Datar et al, 2007).
Ammonia fiber explosion (AFEX) method is similar to steam explosion except that liquid
ammonia is used at high temperature and pressure followed by a rapid pressure reduction.
This method considerably improves saccharification rates and has been used for various types
of lignocellulosic biomass, except that it is not as efficient for biomass with high lignin
content (Vlasenko et al., 1997; Mosier et al., 2005).
Liquid-hot water method is used to solubilise hemicellulose making cellulose more
accessible. It uses increased pressure for the water to stay in liquid state at elevated
temperatures. The pH is required to be between 4 and 7 (Hendriks and Zeeman, 2009; Mosier
et al., 2005).
Another physio-chemical method is SO2-added steam explosion which improves enzymatic
hydrolysis of the solid fraction of biomass and increases the recovery of sugars from
hemicellulose. CO2 explosion, on the other hand, uses an explosive release of CO2 pressure to
increase the accessible surface area of the substrate (Chiaramonti et al., 2012). Lastly,
microwave-chemical pretreatment has been shown to enhance enzymatic digestibility of the
biomass and is more effective than conventional heating (Ma et al., 2009).
Enzymatic Hydrolysis
Enzymatic hydrolysis is a critical component of the conversion process of lignocellulosic
biomass. The aforementioned pretreatment processes generally separate the biomass into its
three major components: lignin, cellulose and hemicellulose. Enzymatic hydrolysis of the two
latter fractions is needed to completely hydrolyse them into their building blocks which will
then be used for fermentattion (Sveinsdottir, et al., 2012).
Cellulases and hemicellulases are specifically used in enzymatic hydrolysis. The three types
of cellulases work together to completely degrade cellulose: endo-glucanase hydrolyses the
middle of a low crystalline cellulose chain as exo-glucanase attacks the end to create a
cellobiose disaccharide, which is hydrolysed by β-glucosidase to form two free glucose
molecules (Bayer et al., 1998). Hemicellulases, on the other hand, degrade xylan and other
parts of hemicellulose. More complex structure of hemicellulose requires synergistic action of
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various types of hemicellulases for efficient degradation (Shallom and Shoham, 2003).
Hemicellulases are either glycoside hydrolases, which cleaves glycosidic bonds, or
carbohydrate esterases, which act on the ester bonds between carbohydrate molecules. Main
enzyme groups of hemicellulases are: xylanase, β-mannanase, α-L-arabinofuranosidase, α-Dglucuronidase, β-xylosidase, and hemicellulytic esterase (Shallon and Shoham, 2003).
Biological Pretreatment
Fungal strains are still predominantly used in cellulose degradation: Trichoderma, Penicillium
and Aspergillus (Galbe and Zacchi, 2002). This method, however, shows poor results as
regards to competing with acid hydrolysis therefore more efficient degradation at higher
temperature is needed. In order to surmount this challenge, relevant enzymes have been
found in thermophiles and hyperthermophiles (Turner, 2007). The problem with bacterial
cellulases is that they are relatively larger and more complex enzymes and that they are
generally part of a cellulosome with many different activities. Research today that has been
addressing this problem generally aims on metabolically engineering presently-used
fermentation strains (Turner, 2007).
An advantage of biological pretreatment is the lack of potentially hazardous chemicals
involved in the process making it an environmentally-friendly method. The energy input is
very low but it is time consuming and requires a lot of space and control of growth conditions
(Menon and Rao, 2012).
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Biofuels
Biothanol
An estimated 83.1 billion litres of fuel ethanol was produced globally in 2012, with the United
States and Brazil being the leading producers which account for 61% and 26% of the global
production respectively. China, Canada and France are other leading producers of ethanol
(REN21, 2013).
Major ethanol production to-date is mainly by first-generation production1 which is subject to
much controversy because it competes with food production (Sanchez and Cordona, 2008).
Several countries are therefore aiming to produce biofuel from lignocellulosic feedstock.
Production of advanced biofuels in U.S., i.e. biofuels produced from lignocellulosic biomass,
has reached 2 million litres in 2012. China has produced 3 million litres of ethanol from
lignocellulosic feedstock in 2012. Europe has several pilot plants but has only produced small
volumes to date (REN21, 2013).
Biohydrogen
Hydrogen is a good candidate as an alternative to fossil fuels. An ideal energy carrier for the
future (Bockris, 2002; Turner, 2004), hydrogen can be produced from renewable energy
sources by thermo-photo-, and electro-chemical, radiolytic and biological processes (Dincer,
2002; Ni et al., 2006). Hydrogen can be produced biologically through the process of
biophotolysis, through photosynthesis (Licht et al., 1997; Hansel and Lindblad, 1998) and by
fermentation (Yokoi et al., 1997). Dark fermentation is seen as the most practical of all the
biological processes because of the highest production rates (Levin et al., 2004). With dark
fermentation there is also a potential of producing hydrogen simultaneously with waste
treatment (Kapdan and Kargi, 2006).
Biomethane
Biomethane is already widely used as a vehicle fuel in different parts of the world. For
instance, 10% of the natural gas vehicles in Germany uses compressed biomethane; Sweden‘s
capital, Stockholm, uses biomethane to fuel 50% of its public transportation fleet as of
October 2012 (REN21, 2013); and Iceland has utilised methane collected from a landfill since
2003 and has since started using that to fuel cars (NEA, 2013).
1
First-generation production – ethanol production from simple sugars such as starch and corn
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Waste is an invaluable source of biomethane. Anaerobic digestion is an efficient means of
treating waste and is already widely used. This process converts organic waste into biogas in
the form of methane (and carbon dioxide), which is a renewable energy source (Speece,
1983). The main drawback of the process is its rather slow rate of degradation, and the
susceptibility of the process to variations in operating conditions (Chen et al., 2008),
especially in a larger-scale production. The challenges that biomethane processing technology
is facing have thus made researchers look into utilising thermophilic anaerobes for the
digestion process (Kelly et al., 1993).
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Metabolic Pathways: Fermentation
Ethanol and Hydrogen production
Thermophilic bacteria facilitate carbohydrate degradation whose end-products include e.g.
ethanol and hydrogen. There are two possible pathways in which microorganisms degrade
glucose and similar carbohydrates: Embden-Meyerhof pathway (EMP) and Entner-Douderoff
pathway (ED). Figure 7 shows the carbon flow from glucose by fermentation thorough
Embden-Meyerhof pathway. This generates two NADH and two pyruvate molecules, together
with two ATP molecules. Degradation through the ED-pathway only generates 1 mol of ATP
(Siebers and Schönheit, 2005).
Figure 7. A simplified scheme of glucose degradation to various end-products (Embden-Meyerhof
pathway). Enzyme abbreviations: ACDH, acetaldehyde dehydrogenase; ADH, alcohol dehydrogenase; AK,
acetate kinase; Fer:NAD(P), ferredoxin:NAD(P) oxidoreductase; H 2-ase, hydrogenase; LDH, lactate
dehydrogenase; PFOR, pyruvate:ferredoxin oxidoreductase; PTA, phosphotransacetylase (Sveinsdottir,
2012).
The end-product of glycolysis by strict anaerobic bacteria is pyruvate, which can then be
converted to fermentation products like hydrogen, ethanol, and more (Figure 7). Glycolysis is
a two-step process where the first phase is a preparation stage and does not involve oxidation
or reduction. Molecular rearrangements in this stage require an energy input consisting of two
ATP molecules. The second stage involves six reactions and is the oxidation part of the
glycolytic pathway yielding 2 moles of pyruvate, 2 NADH’s and 4 ATP’s (Figure 8).
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Figure 8. Stage I and II in glycolysis – Preparation and Oxidation (Madigan et al., 2003)
The pyruvate molecule can then be converted to several end-products such as organic acids,
alcohols, carbon dioxide and hydrogen (Figure 9). The driving force behind the reduction of
pyruvate is the spontaneous oxidation of NADH to NAD+. Thus, fermentation,yields 2 ATP
molecules, whereas aerobic respiration yields 38 ATP molecules (Madigan et al., 2003;
Nelson and Cox, 2005).
Figure 9. Stage III – Reduction. Ethanol, hydrogen, carbon dioxide and organic acids formation
(Madigan et al., 2003).
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Several reductive pathways are possible for producing the aformentioned end-products from
pyruvate, as seen in Figure 9. Ethanol and lactate fermentational pathways generally result in
no hydrogen production (Thauer et al., 1977). The most feasible pathway to produce
hydrogen from carbohydrate fermentation is towards acetate and butyrate with 4 and 2 moles
of hydrogen, respectively:
C6H12O6 + 2H2O  2CH3COOH + 4H2 +2CO2
[acetate pathway]
C6H12O6  CH3CH2CH2COOH + 2H2 +2CO2
[butyrate pathway]
Methane production
Thermophilic anaerobic digestion is comprised of four steps: hydrolysis, fermentation,
acetogenesis, and methanogenesis, with different microorganisms that are active in each step
(Suryawanshi et al., 2010). Figure 8 shows a simplified scheme of methane production from
lignocellulosic biomass which include the three respective steps of anaerobic digestion.
The first steps of anaerobic digestion leading to
methane production are essentially the same as
ethanol and hydrogen fermentation leading to
production of hydrogen, carbon dioxide and
acetate. Methane is then formed either by
acetocalastic methane formation from acetate or
by
hydrogenotrophic
methanogenesis
from
hydrogen and carbon dioxide (Figure 10).
Figure 10. Anaerobic digestion: hydrolysis, fermentation,
methanogenesis
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Thermophilic Anaerobes – the background
Bacteria and Archaea whose natural habitat involves high environmental temperature and do
not require oxygen for growth are described as thermophilic anaerobes (Stetter and Zillig,
1985; Wagner and Wiegel, 2008). Thermophilic anaerobes represent ancient adaptation to
heat (Blöchl et al., 1997), and can be divided into several categories: moderate thermophiles
(Topt between 45 and 55°C), true thermophiles (Topt between 55 and 75°C), and extremophiles
(Topt above 75°C) (Brock, 1986).
Due to thermophiles‘ characteristic heat tolerance, they have been of interest for further
developments in biotechnological processes (Huber and Stetter, 1998). Thermophilic
anaerobes are promising candidates as biofuel-producing organisms – ethanol, hydrogen and
methane production.
Thermophilic anaerobes are able to thrive in harsh environment. Geothermal areas are indeed
favourable habitats for these organisms as these areas offer stability in heat (Brock, 1986;
Kristjansson and Alfredsson, 1986). Such environments are characterised by volcanic
solfatares and hot springs high in sulphur and toxic metals, as well as hydrothermal vents that
are both high in pressure and temperature (Stetter, 1999; Stetter, 2006a). The physiological
characteristics of thermophilic anaerobes that enable them to thrive in such environments are
of significant interest amongst researchers as these involve thermostable enzymes which have
a great potential in designing biotechnological processes – including the degradation of
lignocellulosic biomass (Vieille and Zeikus, 2001; Unsworth et al., 2007).
Early Life on Earth
Oldest biogenic signatures have been dated to 3.85 to 3.8 billion years ago (Ga) and complex
microfossil communities are dated to 3.5-3.4 Ga, where as oxygen accumulation happened
later, approximately 2.1-2.3 Ga (Baross, 1998; Kasting, 1993). Therefore, it is widely
accepted that the earliest forms of life on Earth had lived in an atmosphere with significantly
less oxygen, or devoid of it altogether (Wagner and Wiegler, 2008). Also, it is conjectured
that life began at elevated temperatures, around 80°C on clay or iron-sulphur mineral surfaces
in shallow pools (Wagner and Wiegler, 2008; Russell et al., 1998).
Natural thermal environments such as geothermal areas are similar to those postulated
environments in which life began (Wagner and Wiegler, 2008). Remoteness of the
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environment from the atmosphere, low solubility of oxygen in water at elevated temperatures,
hypersalinity, inputs of reducing gasses such as H2 and H2S contribute to the anaerobic feature
of these thermal enviroments (Brock, 1970; Stetter, 1996). Natural thermobiotic environments
are of terrestrial, marine or subsurface nature. Terrestrial geothermally-heated features include
hot springs, geysers, solfatares, mud pools, and some solar-heated environments. Examples of
these are found in Iceland, the Hawaiian volcanoes, Japan, New Zealand, Pacific islands that
are located at the Ring of Fire, and the Kamchatka Peninsula in the Russian Far East (Stetter,
2006b).
There are several possible reasons why thermophilic anaerobes thrive in environments where
they would otherwise not survive, considering their physiological properties. One reason, as
proposed by Wagner and Wiegel (2008) is that they survive by taking advantage of temporary
thermal microniches that become available when proteinaceous biomass is degraded. Other
reasons include that there is no growth amongst these organisms but mere existence, and that
they have transient habitation from one thermal environment to the next (Engle et al., 1996).
Diversity of Thermophilic Anaerobes
Worldwide grip on the isolated strains of these bacteria
More than 300 species of thermophilic anaerobes have been described (Wagner and Wiegler,
2008).
The
majority
of
isolated
taxa
of
thermophilic
anaerobes
are
chemoorganoheterotrophic. Thermophilic anaerobes are metabolically diverse (Wagner and
Wiegler, 2008). Below the main environmental factors prevailing in these types of ecosystems
are dicussed.
Temperature Relationship
The etymology of the term thermophiles suggests that these organisms „love“ heat. The base
of the phylogenic tree with inferred 16S rRNA gene sequence contains organisms of
extremophilic taxa. This supports the hypothesis that life emerged on Earth when the average
temperature significantly higher than today (Wagner and Wiegel, 2008). Temperature-tolerant
thermophiles are considered to grow over a 35-40°C temperature span (Wiegel, 1990). The
record widest temperature growth range is 22-75°C by a Methanothermobacter
thermautotrophicus-like strain isolated from river sediment (J. Wiegel, unpublished results).
However, some thermophilic anaerobes have especially narrow temperature growth ranges,
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such as 42-55°C for Anaerolinea thermolimosa (Yamada et al., 2006) and 50-60°C for
Anaerolinea thermophila (Sekiguchi et al., 2003). Isolates that have optimal growth above
100°C are generally found at deep-sea vents (Adams, 1994), or deep in terrestrial hot spring
channels and sediments. Increased pressure allowing water to remain in liquid form at
temperatures above 100°C is ascribed as to how these isolated strains of thermophilic
anaerobes are often found at a certain depth (Wagner and Wiegel, 2008).
Oxygen Relationship
Many thermobiotic environments are either anaerobic or low in oxygen. Not surprisingly,
thermophiles that have been observed to-date are predominantly anaerobic (Wagner and
Wiegel, 2008). Most axenic thermophiles are either anaerobes or facultative aerobes (Brock,
1970; Stetter, 2006a ). From a biochemical point, anaerobes are unable to use oxygen as the
terminal electron acceptor, even with oxygen present (i.e., an oxygen-tolerant anaerobe),
whereas facultative aerobes are able to use oxygen as a terminal electron acceptor. Some
obligately anaerobic thermophiles can survive exposure to atmospheres with oxygen,
especially when they are metabolically inactive – at suboptimal temperatures, or lack of
metabolisable substrates. Respiring anaerobic or facultative aerobic thermophiles can use,
through electron transport phosphorylation, various compounds as electron acceptors,
including: CO2, CO, NO3-, NO2-, NO, N2O, SO42-, SO32-, S2O32-, S0, Fe(III), Mn(IV), and
Mo(VI), in the absence of oxygen (Amend and Shock, 2001; Slobodkin, 2005). The energy
generated from these respiratory pathways together with the energy produced anaerobically
through substrate-level phosphorylation represent the over-all energy production of these
organisms (Wagner and Wiegel, 2008).
pH Relationship
The isolated thermophilic anaerobes to-date are generally neutrophilic, i.e. the corresponding
acidity or basicity of the environment with which the strain grows optimally (pHopt) is about
7.0. None of the known strains grow below pH 3.0 (Wiegel, 1998) whereas only a few known
strains of Archaea grow optimally at high pH (Wagner and Wiegel, 2008).
However, there are thermophilic anaerobes with wide pH growth ranges; Thermococcus
hydrothermalis, for instance, has a pH range of 3.5-9.5 (Godfroy et al., 1997). Interestingly,
Thermoanaerobacter ethanolicus strain JW200 has a broad and flat pH optimum from pH 5.5
to 8.5. There are also isolated strains of thermophilic anaerobes with growth range of less than
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1 pH unit: Pelotomaculul thermopropionicum with a pH range of 6.8 and 7.5 (Imachi et al.,
2002) and Thermodesulfobacterium hydrogeniphilium ranging from pH 6.3 and 6.8 (Jeanthon
et al., 2002).
Metabolic Diversity of Thermophilic Anaerobes
Most thermophilic anaerobic Archaea and Bacteria are generally chemoorganoheterotrophic,
i.e. they use organic compounds for carbon and energy. Other strains of thermoanaerobes
have also been found to have metabolic strategies such as chemolithoautotrophy,
chemolithoheterotrophy, photoheterotrophy, and photoautotrophy (Amend and Shock, 2001).
Unique Physiological Adaptation
Further categorisation of chemoorganoheterotrophic metabolism are as follows: proteolytic,
glycolytic, cellulolytic, lipolytic, and peptidoglytic metabolisms (Wagner and Wiegel, 2008).
The Emden-Meyerhof and Entner-Doudoroff pathways are generally utilised by glycolytic
thermophilic anaerobes, although a variety of modifications of these pathways have been
discovered, particularly within the Archaea (Siebers & Schonheit, 2005). The main
fermentation products of glycolytic thermophilic anaerobes include acetate, lactate, ethanol,
CO2, and H2. Although it is known that chemoorganoheterotrophic metabolisms are amongst
the most common metabolic strategies for the axenic thermophile anaerobes studied in the
lab, the natural substrates for these microorganisms remain largely unknown (Amend and
Shock, 2001).
Among chemolithotrophic pathways, the methanogenic reaction, 4H2 + CO2  CH4 + 2H2O
has
been
studied
extensively
and
is
used
by
thermophilic
taxa
within
the
Methanobacteriaceae, Methanothermaceae, Methanocaldococcaceae, and Methanococcaceae
(Amend and Shock, 2001). Another chemolitotrophic metabolism of anaerobic thermophiles
utilises CO, a normal component of escaping volcanic gas of terrestrial and deep-sea
hydrothermal origin (Sokolova et al., 2004), thus performing the metabolic reaction CO +
H2O  CO2 + H2.
Chemolithoheterotrophs produce energy chemolithotrophically and assimilate carbon
heterotrophically. Archaeoglobus profundus (Burggraf et al., 1990) and Stetteria
hydrogenophila (Jochimsen et al., 1997) within Archaea; Desulfotomaculum alkaliphilum
(Pikuta et al., 2000), Desulfotomaculum carboxydivorans (Parshina et al., 2001), and
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T.carboxydiphila (Sokolova et al., 2005) within Bacteria are examples of thermophilic
anaerobes that use chemolithoheterotrophic metabolism.
Two other known metabolism mechanism of thermophilic anaerobes are photoheterotrophy
and photoautotrophy – requiring collection and conversion of light energy into chemical
energy depending on photochemical reaction centres containing (bacterio)-chlorophyll
(Bryant & Frigaard, 2006). The few currently known thermophilic anaerobic phototrophs are
found within the phyla Proteobacteria, Chloroflexi and Firmicutes. Thermochromatium
tepidum, from phylum Proteobacteria, is one of the very few thermophiles that utilises
photoautotrophic metabolism (Imhoff et al., 1998; Madigan, 1986).
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Thermophilic Anaerobes from Icelandic Hot Springs
Typical Icelandic geological areas feature volcanoes, hot springs, geysers, mud pots and
fumaroles. About 0,5% of Iceland‘s total land area (more than 500 km2) is classified as
geothermal fields, which is then further categorised as either high or low temperature fields
(Hreggivdsson and Kristjansson, 2003).
Several strains of thermophilic anaerobes capable of producing ethanol, hydrogen and
methane from lignocellulosic biomass have been isolated in different areas in Iceland. The
distribution of high- and low temperature fields in Iceland along with the isolation location of
each strain are shown in Figure 11.
Figure 11. A map of geothermal fields in Iceland (Orkustofnun, 2012) and the isolation location of biofuelproducing thermophilic anaerobes (Orlygsson and Baldursson, 2007; Jessen and Orlygsson, 2012;
Brynjarsdottir et al., 2012; Stetter et al., 1981; Burrgraf et al., 1990; Kurr et al., 1991; Bellack et al.,
2011).
The geothermal areas in Iceland have various physical and chemical compositions providing
unique environments for microbes to thrive. Low temperature alkaline hot springs (45-50°C)
provide the environment for phototrophic microbial biomats that are dominated by
cyanobacteria Mastigocladus laminosus or Phormidium laminosum. Anaerobic bacteria are
found underneath the photosynthetic layer of the biomat fermenting the decaying remains
(Skirnisdottir et al., 2000; Kristjansson & Alfredsson, 1986). In comparison, higher
temperature hot springs (60-80°C) are dominated by species belonging to the family
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Aquificales. Also, some hydrogen-oxidising, suphate-reducing and methane-producing
bacteria have been isolated from high temperature areas (Kristjansson & Alfredsson, 1986;
Marteinsson et al., 2004; Vesteinsdottir et al., 2011a)
Figure 11 shows the locations where ethanol-, hydrogen- and methane-producing strains of
thermophilic anaerobic bacteria have been isolated: Viti, Krafla geothermal area, Grensdalur,
Hengill geothermal area, Hveradalir, Kerlingarfjöll, and Kolbeinsey Ridge
Bioprospecting Icelandic Hot Springs
Hot springs are invaluable source of thermophilic biofuel-fermenting microorganisms.
Thorough batch screening of samples taken from Icelandic hot springs had been shown to
indicate promising enrichments for methane, hydrogen and ethanol production (Prieur et al.,
1994; Koskinen et al., 2008; Suryawanshi et al., 2010).
Ethanol Producing Strains
Thermophilic microorganisms hold a great potential in facilitating ethanol production from
lignocellulosic biomass (Taylor et al., 2009). The main reason for using thermophiles for
ethanol production is because of their broad substrate spectra and tolerance to various extreme
environmental factors. Up to date, most studies to date have been on Gram positive, strictly
anaerobic bacteria that belong to the phylum Firmicutes. Many of the isolated strains are of
geothermal origin with optimal growth temperature above 60°C. Mostly they belong to the
genera Clostridium, Thermoanaerobacter, and Thermoanaerobacterium (Lamed and Zeikus,
1980; Ben Bassat et al., 1981; Wiegel and Ljungdahl, 1981; Fardeau et al., 1996; Sveinsdottir
et al., 2009).
One disadvantage about using thermophiles for ethanol production is their relatively low
ethanol tolerance to S. cerevisiae and the well-known mesophilic bacterium Zymomonas
mobilis (Lynd, 1989). However, the advantages of these microorganisms such as lower risk of
contamination, increased bioconversion rates and product recovery, are sufficient
encouragement in advancing the technology of using thermophilic anaerobes for ethanol
production
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Thermoanaerobacter mathranii is the earliest recorded ethanol-producing strain isolated
from Icelandic hot springs (Ahring et al., 1996). The optimal growth range of the bacterium
is between 70 and 75°C at pH 7.0. It was isolated from sediment in a slightly alkaline hot
spring in Grensdalur area. The bacterial cells are Gram-variable, straight, rod-shaped and
spore-forming. T. mathranii utilises various energy sources and its end-products from
fermentation are ethanol, lactate, acetate, CO2 and H2 (Larsen et al., 1997). Several studies
have been conducted on the ethanol-producing capacity of T. mathranii since it was isolated
in 1997. One study investigated ethanol production of T. mathranii (strain A3M1) from wet
oxidised wheat straw hydrolysates. Optimisation of different factors including oxygen
pressure, sodium carbohydrate concentration, acid treatment (H2SO4), and use of enzymes
(Celluclast®) was done. The highest ethanol yield achieved was 0.8 g/L as higher acid
concentration during pretreatment led to lower yield (Ahring et al., 1999). Another research
focused on the effects of potential inhibitory compounds on T. mathranii (strain A3M4) from
wet oxidised wheat straw. The study showed that various inhibitory compounds are present at
low concentrations in the hydrolysates, but only severely affect ethanol yield at increased
concentration of hydrolysates (Klinke et al., 2001).
Various genetic modifications of T. mathranii have been performed. The first one being was
the deletion of lactate dehydrogenase gene to eliminate NADH oxidation pathway (strain
BG1L1). In addition to that was the insertion of heterologous gene (gldA) coding for NAD+dependent glycerol dehydrogenase. Both modifications facilitate NADH regeneration
resulting in a recombinant strain BG1G1. Increased ethanol yields in the presence of glycerol
with xylose as a substrate were observed. Acetate production decreased as a result of restoring
the redox balance, thus shifting the yields towards more ethanol production (Yao &
Mikkelsen, 2010b). Another type of genetic modification involving overexpression of one of
four alcohol dehydrogenases present in T. mathranii led to the development of strain BG1E1.
This strain is capable of producing increased ethanol yields from xylose. Analysis showed
that NAD(H)-dependent bifunctional aldehyde-alcohol dehydrogenase (AdhE) has a
significant role in ethanol production due to its importance in acetyl-coA reduction to
acetaldehyde in the ethanol-formation pathway (Yao & Mikkelsen, 2010a).
Interestingly, further studies on BG1L1 showed that the strain displays a high tolerance of
exogenously added ethanol compared to other thermophilic anaerobic bacteria, while still
maintaining a relatively high and stable ethanol production. BG1L1 has ethanol tolerance of
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8.3% (Georgieva et al., 2007). Moreover, the strain shows promising ethanol-producing
capacity from lignocellulosic biomass such as corn stover and wheat straw hydrolysates.
Ethanol yield of T. mathranii was 2.61 mM g sugar-1 from wheat straw hydrolysates (Ahring
et al., 1999) and later found to be 5.30 mM g sugar-1 (Klinke et al., 2001) from the same
substrate. A significant resistance of hydrolysates occured when concentrations were
increased (Georgieva & Ahring, 2007; Georgiva et al., 2008).
Thermophiles isolated by researchers from the University of Akureyri
Bioprospecting Icelandic hotsprings for thermophilic anaerobes that have the potential of
aiding biofuel production from lignocellulosic biomass has been an important research area at
the University of Akureyri since 2005. Table 1 shows the most efficient ethanol-producing
strains that have been isolated, site of isolation, optimal growth conditions, end-product
formation, and phylogenetic relationship.
Table 1. EtOH producing thermophilic strains isolated by researchers at the University of Akureyri
(Sveinsdottir, 2012)
Strain
AK1
Sampling
Isolation
Sites
Year
Grensdalur
2005
Topt
pHopt
Fermentation
Genus
end-products
45°C
6.5
EtOH, acetate,
Clostridium
H2, CO2
AK5
Grensdalur
2009
65°C
7.0
EtOH, acetate,
Thermoanaerobacter
H2, CO2
AK15
Viti
2005
60°C
7.0
EtOH, acetate,
Thermoanaerobacter
lactate, H2
AK17
Viti
2005
58-60°C
6.0
EtOH, acetate,
Thermoanaerobacterium
H2, CO2
AK54
Grensdalur
2007
65°C
5.0-6.0
EtOH, acetate,
Thermoanaerobacterium
lactate, H2,
CO2
J1
Grensdalur
2009
65°C
7.0
EtOH, acetate,
Thermoanaerobacter
H2, CO2
GHL15
H2, acetate
Thermoanaerobacter
A bioprospecting survey on both ethanol and hydrogen producing bacteria showed that
thermophiles that come from moderately high temperatures (50-60°C) were good ethanol
producers (Thermoanaerobacterium, Clostridium and Caloramator), whereas isolates from
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higher temperatures (70 and 75°C) (Thermoanaerobacter and Caldicellulosiraptor) were
better hydrogen (and acetate) producers (Örlygsson & Bakken, 2010). Other good ethanol
producing strains isolated at the University of Akureyri include Thermoanaerobacter strain
AK5 (Brynjarsdottir et al., 2012) Thermoanaerobacterium AK17 (Almarsdottir et al., 2012),
Thermoanaerobacter strain J1 (Jessen & Orlygsson, 2012) and Clostridium strain AK1
(Örlygsson, 2012).
Hydrogen-producing strains
Hydrogen production from carbohydrates is mainly produced through acetate and butyrate
production. Fermentation leading up to acetate and butyrate end-products are most feasible in
hydrogen production because these compounds are not reduced and electron-scavengers like
ethanol and lactate (Thauer et al., 1977; Wiegel, 1980). The highest recorded yield for
hydrogen production was observed from the extreme thermophile Caldicellulosiruptor
saccharolyticus under gas sparging (de Vrije et al., 2007) and Thermoanaerobacter
tengcongensis which produces up to 4 moles of H2 from one mole of glucose under nitrogen
flushed fermentor systems (Soboh et al., 2004).
Hot springs are an ideal source for hydrogen-producing thermophiles (Wiegel and Ljungdahl,
1981). Icelandic hot springs are no exception. Several strains that belong to the genus
Caldicellulosiruptor have been isolated from hot springs in Iceland, e.g. Caldicellulosiruptor
kristjanssonii, Caldicellulosiruptor lactoacedicus and Caldicellulosiruptor acetigenus
(Mladenovska et al., 1995; Bredholt et al., 1999; Nielsen et al., 1993). Species within the
genus are generally known to be good hydrogen producers (van Niel et al., 2003) but these
three above mentioned species have been neglected concerning hydrogen production .
Researchers at the University of Akureyri have isolated some hydrogen-producing strains.
Most of these strains are combined ethanol and hydrogen producers (Sveinsdottir et al., 2011;
Sigurbjornsdottir & Orlygsson, 2011). The strain AK54, member of the genus
Thermoanaerobacterium, has gained considerable attention in recent years as a producer of
both ethanol and hydrogen (Sigurbjornsdottir and Orlygsson, 2012). AK14, a moderate
thermophile from genus Clostridium, has also shown promising hydrogen yields from
lignocellulosic biomass (Almarsdottir et al., 2010). And finally, the strain GHL15, which
belongs to the genus Thermoanaerobacter produces good hydrogen yields from
lignocellulosic hydrolysates (Brynjarsdottir et al., 2013).
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Methane-producing strains
Methane-producing thermophilic archaea have unique properties (e.g. resistance to antibiotics
and presence of F250) making them relatively easier to isolate and identify than other
microbial groups. They carry out reactions in extreme anaerobic habitats and also account for
a significant fraction of the carbon flow in them. Thermophilic methanogens are found in
various habitats/regimes such as (a) mud from cattle pasture, (b) submarine hydrothermal
vents in different oceans, (c) at depths of 100-2600 m in different oceans, (d) adapted to
nuclear power plant/oil field/municipal sewege/paper mill waste water environments. There
are a total of 25 species of thermophilic methanogens in 12 genera that have been isolated to
date (Suryawanshi et al., 2010). Five of these were isolated in Iceland and will be discussed in
later sections.
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Process of Isolation
The process of isolation of hydrogen and ethanol producing strains are described by
Orlygsson and Baldursson (2007). The process includes details on samplings sites, medium,
determination of optimum pH and temperature for growth, preparation of the hydrolysates
from complex biomass substrates, physiological experiments, strain identification by 16S r
RNA analysis and analysis of ethanol, acetate and hydrogen yields by gas chromatograph
(Orlygsson & Baldursson, 2007) .
The isolation process of methanogens is variously described in their respective research
papers (Stetter et al., 1981; Burggrad et al., 1990; Laurer et al., 1986; Bellack et al., 2011;
Kurr et al., 1991).
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Ethanol-Producing strains isolated in Iceland
Clostridium strain AK1
This strain was isolated from a hot spring (47.8°C; pH 7.6) from Grensdalur. The strain has an
ethanol (EtOH) yield of 1.5 mol EtOH mol glucose-1, or 8.22 mM g-1 glucose (Orlygsson,
2012). The carbon balance is almost complete at 94% (± 1.5%) showing the following
stoichiometry for glucose degradation:
1.0 Glucose  1.50 EtOH+ 0.38 Acetate + 0.55 H2 + 1.88 CO2 (Orlygsson, 2012)
The strain degraded xylose from the three pentoses (not ribose, arabinose) studied while all of
the hexoses (glucose, fructose, galactose and mannose) were degraded as well as the
disaccharides sucrose and lactose. Additionally, starch, xylan and pectin were degraded but
not the rest of the substrates (see footnote 2) (Orlygsson, 2012).
Results from 16s rRNA analysis of strain AK1 shows that is it most likely related to species
within Clostridium Cluster IV. The temperature growth range is only about 15°C (40 to 55°C)
(Orlygsson and Baldursson, 2007).
Strain AK1’s main drawback is its relatively low tolerance to low initial glucose concentration
(Orlygsson, 2012). Growth on lignocellulosic biomass hydrolysates showed that the strain
produced up to 3.1 and 3.5 mM ethanol g-1 on chemically pretreated grass and hemp stem,
respectively (Orlygsson, 2012).
Thermoanaerobacterium strain AK17
The strain was isolated from a hot spring (70°C; pH 6.5) at Víti, in North East Iceland
(Örlygsson & Baldursson, 2007). The strain produces up to 1.5 mol EtOH mol glucose-1 and
1.1 mol EtOH mol xylose-1 as well as good yields from lignocellulosic biomass (Sveinsdottir
et al., 2009; Almarsdottir et al., 2012). The strain also produces acetate, hydrogen, and carbon
dioxide during fermentation. The stoichiometry of glucose and xylose degradation of AK17
has been found to be as follows (Sveinsdottir et al., 2009):
20 mM Glucose  29,9 mM EtOH + 7.5 mM Acetate + 13.8 mM H2 + 37.4 mM CO2
2
Cellulose, formate, succinate, malate, pyruvate, oxalate, crotonate, glycerol, inositol, sorbitol, casamino acids,
peptone, beef extract, tryptone, alanine, aspartate, glycine, glutamate, serine, threonine, histidine, and
cysteine (Orlygsson, 2012).
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20 mM Xylose  21.3 mM EtOH + 8.3 mM Acetate + 14.3 mM H2 + 29.6 mM CO2
16s rRNA analysis of strain AK17 has showed that it belongs to the genus
Thermoanaerobacterium (Lee et al., 1993; Sveinsdottir et al., 2009) with a 99.1% homology
to T. aciditolerans making the strain its closest relative (Kublanov et al., 2007). Optimum
temperature and pH for AK17 were found to be 60°C and 6.0, respectively, with generation
time of 1.0 h (Almarsdottir et al., 2012).
Ethanol production by AK17 from most hexoses was between 1.0 and 1.5 mol EtOH mol
hexose-1 with 0.75 and 1.0 mol EtOH mol pentose-1 and disaccharides-1 from pentoses and
disaccharides. Acetate and hydrogen production by AK17 is considerably less compared to its
ethanol production (Almarsdottir et al., 2012).
The strain produces more than 40 mM of ethanol from hydrolysates made from 5 g/L of
cellulose hydrolysates. Without any chemical pretreatment of complex biomass, hemp stems
give highest ethanol yields (2.1 mM g-1 dw) whereas hemp leaves give the lowest ethanol
yield (0.4 mM g-1 dw). Acid and alkali chemical pretreatment however substantially increase
the yields on all lignocellulosic biomass (grass, hemp and straw) tested, with grass
hydrolysate wielding the highest ethanol yield at 3.6 mM g-1 dw. Most ethanol production was
observed on grass hydrolysates (5.5 mM g-1 dw) obtained at very low concentration of
hydrolysate (2.5 g L-1) (Almarsdottir et al., 2012).
Thermoanaerobacter strain AK5
Strain AK5 was isolated from a hot spring (64°C, pH 6.7) in Grensdalur in southwest Iceland.
The strain belongs to the genus Thermoanaerobacter, with close homology to T.
thermohydrosulfuricus (99.1%) and T. wiegelii (98.9%), and is most closely related to T.
ethanolicus (98.7%), T. siderophilus (98.4%), and T. acetoethylicus (98.0%) (Brynjarsdottir et
al., 2012).
The strain has a growth range of 55.0-75.0°C with optimal temperature of 65°C. The pH
range of growth is pH 4.0-8.0 with optimal pH of 7.0.
Glucose degradation of AK5 yields 1.70 mol ethanol per mol glucose. The strain is highly
flexible for either ethanol or acetate production, depending on the culture conditions.
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Cellulose hydrosylates produces up to 7.7 mM ethanol g-1 cellulose with strain AK5. Without
chemical pretreatment, hemp stem gives the highest ethanol yield of 3.0 mM g-1 dw whereas
straw gives the lowest 0.9 mM g-1 dw. Addition of either alkali or acid increased the yield
substantially on most of the lignocellulosic biomass tested. The highest increase was seen on
straw hydrolysates pretreated with alkali (by a factor of 2.73) and acid (by a factor of 2.36).
The highest ethanol yields was from grass hydrosylate (4.4 mM g-1 dw) pretreated with acid
(Brynjarsdottir et al., 2012).
Thermoanaerobacter strain J1
Thermoanaerobacter strain J1 is a thermophilic anaerobe isolated from a hot spring (69°C, pH
7.5) in Grensdalur, southwest of Iceland. Phylogenetic analysis shows that the strain is a
member of the genus Thermoanaerobacter whose closest neighbours are T. uzonensis (97.7%
homology) and T. sulfurigenes (95.5%). The strain grows between 55.0°C and 75.0°C with
optimal temperature being 65.0°C. The pH range of growth is pH 4.0 and 9.0, with optimal
pH being 7.0 (Jessen and Orlygsson, 2012).
Strain J1 is a powerful ethanol-producer with the following stoichiometry of glucose
degradation:
1.0 Glucose  1.70 EtOH + 0.15 Acetate + 0.30 H2 + 1.85 CO2 (Jessen and Orlygsson,
2012)
The substrate range of J1 is impressively broad, showing good capacity in degrading pentoses
(xylose, arabinose), hexoses (glucose, mannose, galactose, fructose, and rhamnose),
disaccharides (maltose, cellobiose, lactose, trehalose, and sucrose), the trisaccharide raffinose,
starch, pyruvate, and serine. Ethanol is the major end-product in all substrates with the
exception of serine and pyruvate whose major end-product is acetate. Highest ethanol yields
were produced from the trisaccharide raffinose (75.2 mM) (Jessen and Orlygsson, 2012).
Highest ethanol yields on more complex biomass types without enzymatic nor chemical
pretreatment were observed on hemp (2.6 mM g-1 dw) and lowest on straw (0.8 mM g-1 dw).
Chemical pretreatment by adding either acid or alkali substantially increased yields on most
of the lignocellulosic biomass substrates tested. Most profound increase was observed on
hydrolysates from straw pretreated with alkali. Highest ethanol yields was observed on hemp
(4.3 mM g-1 dw) (Jessen and Orlygsson, 2012).
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Hydrogen-producing strains isolated in Iceland
Caldicellulosiruptor kristjanssonii
It is known that some of the best hydrogen-producing microorganisms are found within the
genus Caldicellulosiruptor. Caldicellulosiruptor saccharolyticus, for instance, shows good
hydrogen yields when grown on pretreated lignocellulosic biomass (Ivanova et al., 2009). C.
kristjanssonii, isolated in Iceland, was found to have similar fermenting performance as C.
saccharolyticus. More interestingly, the two strains, C. kristjanssonii and C. saccharolyticus,
obtain a maximum theoretical hydrogen yield of 3.8 mol H2 / mol C6 sugar equivalent, which
utilise glucose and xylose simultaneously. This is a higher yield than when each strain is used
individually, suggesting that a possible synergetic mechanism occurs when these different
strains are used together for fermentation (Zeidan and Niel, 2009).
Clostridium strain AK14
Strain AK14 was isolated from a hot spring (51°C and pH 7.6) at Hengill geothermal area in
Grændalur in SW Iceland (Orlygsson & Baldursson, 2007). The strain is a powerful
hydrogen- producer isolated from the same area as AK1 and has a similar optimum growth
range as the latter strain (Orlygsson and Baldursson, 2007; Almarsdottir et al., 2010). The
strain belongs to genus Clostridium using 16S rRNA gene sequence analysis, having
Anaerobacter polyendosporus as its closest relative with 95.1% homology. Glucose
degradation using strain AK5 has the stoichiometry of:
20 mM Glucose  2.8 mM EtOH + 7.5 mM Acetete + 10.7 mM Butyrate + 30.5 mM H2 +
31.7 mM CO2
Hydrogen and carbon dioxide as well as acetate and butyrate are the main products of the
strain‘s degradation of glucose, alongwith ethanol as a minor product (Almarsdottir et al.,
2010).
From three pentoses tested by Almarsdottir et al. (2010), AK14 only degraded xylose but all
four hexoses tested (glucose, fructose, galactose and mannose) were degraded as well as the
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disaccharide but not lactose. The strain also degraded xylan and starch. All the other
substrates (see footnote 3 were not degraded (Almarsdottir et al., 2010).
The strain’s highest yield of hydrogen (8.5 mol H2 g-1 VS) was from cellulose hydrolysate.
However, paper and lignocellulosic biomass substrates only yielded between 0.26 to 3.60 mol
H2 g-1 substrate using the strain. Acid and base pretreatment significantly enhances hydrogen
yield with the highest production from grass (6.23 mol H2 g-1 VS) (Almarsdottir et al., 2010).
Thermoanaerobacterium strain AK54
The strain Thermoanaerobacterium AK54 was isolated from an Icelandic hot spring (66°C, pH
5.3) in Grensdalur, southwest of Iceland. 16s rRNA phylogenetic analysis shows that the
strain is a member of the genus Thermoanaerobacterium, with T. aciditolerans its closest
relative of 99.0% homology. AK54 has a relatively narrow temperature growth range of 55.0
to 70.0°C, with optimal temperature of 65.0°C. The strain’s pH range of optimum growth is
between 5.0 and 6.0 (Sigurbjornsdottir and Orlygsson, 2011).
Glucose degradation of the strain has been shown to be affected by the partial pressure of
hydrogen; ethanol and acetate production decreases with increasing media volume compared
to gas head space as well as hydrogen yields (Sigurbjornsdottir and Orlygsson, 2011).
Thereby making low liquid-gas volume ratio more practical in both ethanol and hydrogen
production. The following stoichiometric relation of glucose degradation is found by
Sigurbjornsdottir and Orlygsson (2011):
1.0 Glucose  1.25 EtOH + 0.66 Acetate + 0.09 Lactate + 1.80 H2 + 1.91 CO2
From the biomass substrates tested by Sigurbjornsdottir and Orlygsson (2011), highest
ethanol and hydrogen yields (24.2 mM) were observed from cellulose, even when the glucose
released by enzymatic and chemical pretreatments was not fully degraded. Ethanol production
from grass hydrolysate ( Phleum pratense) without pretreatment was only 68% of the ethanol
produced from cellulose hydrolysates.
Pretreatment of barley straw, hemp and grass
increased ethanol and hydrogen yields, but not from cellulose or newspaper . The highest
increase after chemical pretreatment (both acidic and basic) was observed for barley straw
3
Xylose, ribose, arabinose, glucose, fructose, galactose, mannose, sucros, lactose, lactate, formate, succinate,
malate, pyruvate, oxalate, crotonate, glycerol, inositol, starch, cellulose, xylan, sorbitol, pectin, casamino acids,
peptone, beef extract, tryptone, alanine, aspartete, glycine, glutamate, serine, threonine, histidine, and
cysteine (Almarsdottir et al., 2010).
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(Hordeum vulgare) with more than 100%increase in ethanol yield, whereas paper
hydrolysates did not give as high increase after either chemical pretreatment. Cellulose
hydrolysates gave the most yield of hydrogen (6.7 mol H2 g-1 TS) pretreated with alkali;
whereas from lignocellulosic biomass, the highest yields were from grass (4.9 mol H2 g-1 TS)
also pretreated with base (Sigurbjornsdottir and Orlygsson, 2011).
Thermoanaerobacter strain GHL15
The strain Thermoanaerobacter GHL15 was isolated from a geothermal area (70.1°C, pH 7.0)
in Grensdalur, southwest of Iceland. Its primary end-products are hydrogen and acetate.
Brynjarsdottir et al. (2013) investigated the fermentative properties of GHL15 on various
carbon substrates as well as with different lignocellulosic biomass including cellulose, hemp,
newspaper, barley straw, and Timothy grass (Brynjarsdottir et al., 2013).
Phylogenetic analysis of GHL15 shows that it is a member of the genus Thermoanaerobacter
and its most closely related species are Thermoanaerobacter yonsiensis (98.9% homology),
Thermoanaerobacter keratinophilus (98.8%), Caldanaerobacter subterraneus (98.5%), and
Thermoanaerobacter tengcongensis (98.0%).
Growth temperature of GHL15 ranges from 55.0-75.0°C, with optimum growth temperature at
65.0°C. The range of pH for the strain’s lies between pH 3.0 and 9.0, with optimum growth at
pH 6.5. Generation time of GHL15 is about 10 h (Brynjarsdottir et al., 2013).
GHL15 has versatile fermentative characteristics as it degrades most of the carbohydrates (see
footnote 4) tested to the same end-products as for glucose (acetate, ethanol, and hydrogen). The
dominant end-products of GHL15 are acetate and hydrogen, mostly in the ratio of 1.0:1.15.
Ethanol production using GHL15 is relatively low, with yield of mostly of 5 mM or less. The
strain degraded xylose, most of the hexoses and disaccharides, starch, pyruvate, and the amino
acides serine and threonine. The other remaining substrates (arabinose, rhamnose, raffinose,
glycerol, xylan, and cellulose substrates) wielded no results (Brynjarsdottir et al., 2013).
The stoichiometry of glucose (20 mM) fermentation by strain GHL15 is as follows:
4
Xylose, arabinose, glucose, mannose, galactose, fructose, rhamnose, maltose, cellobiose, sucrose, lactose,
trehallose, raffinose, starch, cellulose, CMC, avicel, xylan, glycerol, pyruvate, serine, and threonine
(Brynjarsdottir et al., 2013).
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1.0 C6H12O6  1.60 CH3COOH + 0.05 CH3CH2OH + 3.00 H2 +1.65 CO2 (Brynjarsdottir et
al., 2013)
Hydrogen production from lignocellulosic biomass substrates (cellulose, hemp leaves and
stem fibres, newspaper, barley straw, and grass) show promising results. Grass hydrolysate
pretreated with 0.5% sulphuric acid gave the highest hydrogen yield with 7.6 mmol H2 g-1
grass. The major drawback of GHL15 is its extreme sensitivity towards initial substrate
concentration and acetate accumulation (Brynjarsdottir et al., 2013).
Methane-producing strains isolated in Iceland
The following list shows the methane-producing strains isolated in Iceland. However, there
are no accessible data on methane-producing performance of the isolated thermophilic
anaerobic methanogens described in this paper.
Methanothermus fervidus
This strain was isolated in 1981 from a hot spring (85°C, pH 6.5) in the Hveradalir solfataric
field in Kerlingarfjöll in Iceland (Stetter et al., 1981). The bacterium does not grow below
60°C but optimal growth is at 83°C with pH 6.5. The upper temperature limit it 97°C. Ths
stein is a hydrogenotrophic methanogen, producing methane from from H2 + CO2.
Mb. thermoautotrophicum seems to be mainly involved in the thermophilic decomposition of
organic matter in sewage digestors and waste piles, and of decaying blue green algal mats in
hot springs (Zeikus et al., 1980). Species within the genus Methanothermus, on the other
hand, appears to depend on geothermal hydrogen and CO2 as algal mats can not grow at
temperatures around 90°C.
From the survey done by Stetter et al. (1981) in Icelandic
solfaratas, M. fervidus was only found once indicating its extreme oxygen sensitivity and may
occur more frequently in the depth of volcanic areas (Stetter et al., 1981). It is therefore
speculated (Stetter et al., 1981) that Methanothermus fervidus and other unknown members of
the Methanothermaceae may also be responsible for bacterial methane production in
geothermally heated deep sediments, e.g. methane found in pliocene sediments situated in
3,000 m depth with temperature around 90°C in the area of Porto Corsini in northern Italy
(Schoell, 1980).
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Methanopyrus kandleri
The strain Methanopyrus kandleri belongs to a novel group of hyperthermophilic rod-shaped
motile methanogens. It was isolated from a shallow marine hydrothermal system (Kolbeinsey
ridge, Iceland) and from a hydrothermally heated deep sea sediment (Guaymas Basin, Gulf of
California). The growth range of the strain is between 84 and 110°C, with optimal growth at
98°C. The isolates are obligate chemolithoautotrophes that use hydrogen and carbon dioxide
as energy and carbon sources. Methanopyrus kandleri is also able to produce hydrogen
sulphide (H2S) in the presence of sulphur, and its cells tend to lyse (Kurr et al., 1991).
Methanocaldococcus villosus
Methanocaldococcus villosus was isolated from a sumbarine hydrothermal system at the
Kolbeinsey Ridge, north of Iceland (Bellack et al., 2011). It is a novel chemolithoautotrophic
and hyperthermophilic methanogen that belongs to the order Methanococcales, with 95%
gene sequence homology to Methanocaldococcus jannaschii, making the latter-mentioned
strain its closest relative. The isolate grows at 55-90°C, with optimum growth at 80°C and pH
6.5. The cells of Methanocaldococcus villosus gain their energy exclusively by reduction of
CO2 with H2. Selenate, tungstate and yeast extract stimulate the growth of the strain
significantly (Bellack et al., 2011).
Other methanogens isolated in Iceland with limited amount of information are Methanotorris
igneus
and
Methanothermus
sociabilis.
Methanotorris
igneus
(named
before
as
Methanococcus igneus) is a novel hyperthermophilic strictly chemolithoautotrophic member
of the genus Methanotorris. It was isolated from a submarine vent system, with depth of 106
m, at the Kolbeinsey ridge, Iceland. The isolate has a growth range between 45°C and 91°C
with optimal growth at 88°C (Suryawanshi et al., 2010; Burggraf et al., 1990).
Methanothermus sociabilis is a motile rod-shaped microorganism. Optimum growth is
observed at pH 6.5 and temperature of 88°C (Suryawanshi et al., 2010; Laurer et al., 1986).
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Effects of Different Factors on the yield
Several factors have been shown to affect the end-product yield of each biofuel-producing
thermophile. Initial substrate concentration may have a significant effect in growth and endproduct formation rates and yields. This has been investigated for different thermophilic
bacteria such as Clostridium and
Thermoanaerobacterium (Lacis and Lawford, 1988;
Sommer et al., 2004; Almarsdottir et al., 2012). Concentration of the end-products may also
have an effect on fermentative metabolism. For instance, end-product inhibition by hydrogen
or organic acids and alcohols. The partial pressure of hydrogen (ρH2) may especially have a
significant effect on fermentation – as ρH2 increases, some thermophilic microorganisms may
change their metabolism from acetate to lactate or ethanol production which results in
decrease of H2 yields and production rates (van Niel et al., 2003). Below, various factors
concerning yields of biofuel production are discussed.
Initial glucose concentration. Almarsdottir et al. (2012) examined the effects of various
factors on lignocellulosic biomass degradation of strain AK17. It was found that increased
glucose loadings led to lower pH, increased acetate formation and a lower degree of glucose
utilisation in cultures. An increase of initial glucose concentration from 5 to 10 mM led to a
twofold increase in end-product formation and 98% degradation of glucose. Further increase
in glucose concentration did not, however, have a positive correlation with end-product
formation. Analysis of the results indicate that the inhibition was more likely cased by low pH
rather than the high substrate loadings. Similar phenonmenon was also shown to be present
for most of the strains investigated for ethanol and hydrogen production. The only striking
exception was Thermoanarobacter strain J1 where ethanol production was not inhibited at up
to 200 mM glucose concentrations (Jessen & Orlygsson, 2012).
Acid/Alkali. Lignocellulosic biomass consists of cellulose, hemicellulose and lignin. Various
pretreatment methods have been used for releasing the sugars from such biomass. For the
studies done on hydrogen and ethanol producing strains isolated from Icelandic hot springs,
chemical pretreatement by either alkali and acids, as well as heating (autoclaving) has been
done. Almarsdottir et al. (2012) showed that the effects of chemical pretreatment involving
acid/alkali addition with low concentrations (0.25%) of acid or alkali increased ethanol yields
to 3.2 and 3.5 mM g-1, respectively. Further increase in acid concentration slightly increased
the yield, whereas addition of more alkali did not increase ethanol yields (Almarsdottir et al.,
Sara Lind Jónsdóttir | BSc Biotechnology
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2012). Similar spectra was also showed from other strains isolated from Icelandic hot springs;
namely acid pretreatment was more effective on grass and hemp whereas alkali pretreatement
was more effective from straw hydrolysates (Jessen & Orlygsson, 2012; Brynjarsdottir et al.,
2012; Sigurbjornsdottir & Orlygsson, 2011).
Enzyme concentration. One of the major challenge for large scale ethanol production from
lignocellulosic biomass is the high enzyme cost. Almarsdottir et al. (2012) showed that no
end-products are produced from cellulose hydrolysates without adding enzymes5. However,
adding 0.01 mL g-1 dw of the enzyme, which is one-tenth of the „usual amount“, a dramatic
increase of ethanol yield was observed (from 0 to 3.5 mM g-1EtOH). Twice the amount of the
usual enzyme concentration (0.2 mL g-1 dw) further increased the ethanol yield up to 10.2
mM g-1 EtOH. Grass hydrolysates, on the other hand, produce ethanol yields of up to 0.9 mM
g-1 without enzyme addition; although it was increased to 1.9 mM g-1 with adding 0.1 mL g-1
enzyme. Interestingly, acetate formation from grass hydrosylates does not increase with
increased enzyme concentration, as opposed to ethanol formation (Almarsdottir et al., 2012).
Effect of furan derivatives from pentoses and hexoses. Lignocellulosic biomass consists of
different compounds which when degraded may affect the formation of the desired endproducts. Furfural and hydroxymethylfurfural (HMF) are examples of these. Furfural and
HMF are furan derivatives from pentoses and hexoses, respectively, which have been reported
to act as strong inhibitors of glycolytic enzymes (Banarjee et al., 1981; Palmqvist and HahnHagerdal, 2000). The study by Almarsdottir et al. (2012) shows the inhibitory effects of the
two compounds. At 0.5 g L-1 of furfural or HMF, the decrease in ethanol yield was 17% and
15%, respectively. A decrease of approximately 50% was observed at 2 g L-1 concentration of
furfural, and no growth was observed at higher concentrations. Slightly less decrease in
ethanol yield was observed for HMF. Interestingly, acetate concentration is positively
correlated to HMF concentration (Almarsdottir et al., 2012).
Partial pressure of hydrogen. Partial pressure of hydrogen as a great influence on hydrogen
yield (van Niel et al., 2002). It had been observed that for the strain AK15 the theoretical yield
of hydrogen increased from 10% to 58% by changing the liquid/gas (L-G) phase ratio of 0.75
to 0.043 (Orlygsson and Baldursson, 2007).
5
The enzymes Celluclast® and Novozyme 188 were used in the study by Almarsdottir et al., 2012.
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For hydrogen producers, electron scavenging or low L-G ratios enhance hydrogen production
(Sigurbjornsdottir & Orlygsson, 2011) whereas for ethanol producers high L-G ratios shifts
end product formation more towards ethanol (Jessen & Orlygsson, 2012).
Ethanol tolerance
Minimum inhibition concentration (MIC) determinantion was performed in order to
determine the maximum ethanol tolerance of each strain. Sveinsdottir et al. (2009) determined
maximum ethanol tolerance for seven thermophilic anaerobic strains using MIC
determination. The effect of various ethanol concentrations ranging from 0% to 8% on seven
of the isolated thermophilic anaerobes from Icelandic hot spring. The results are found in
Appendix II. Four of the Thermoanaerobacterium species and the Paenibacillus strain
showed ethanol tolerance up to 3.2% (v/v) whereas the two Thermoanaerobacter species had
lower ethanol tolerance (1.6% v/v).
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The following table shows a summary of the best biofuel-producing substrate with each of the
isolated thermophilic anaerobe described above.
Table 2. A summary of best biofuel-producing substrates with each isolated thermophilic anaerobe
Thermophilic anaerobe
Biomass
Substrate
Ethanol
Hydrogen
Pre
Conc. (g
Yields
Yields
treatment
(mM g
(mM g
-1
L )
-1
sugar )
Genus
Strain
Clostridium
AK1
hemp
Reference
sugar-1)
5,0
3,5
acid/base
5,0
3,1
acid/base
Orlygsson (2012)
stem
grass
Thermoanaerobacter
AK5
grass
4,4
Brynjarsdottir et al.
(2012)
Thermoanaerobacterium
AK17
Grass
5,0
3,6
Almarsdottir et al.
(2012)
Grass
Thermoanaerobacter
J1
2,5
Hemp
5,5
4,3
acid/base
Jessen and Orlygsson
(2012)
Clostridium
AK14
Grass
6,23
acid/base
Almarsdottir et al.
(2010)
Thermoanaerobacterium
AK54
Grass
4,9
base
Sigurbjornsdottir and
Orlygsson (2012)
Thermoanaerobacter
GHL15
Grass
4,5
7,6
acid
Brynjarsdottir et al.
(2013)
The highest ethanol yield (5.5 mM g sugar-1) is produced from grass hydrolysate by the strain
AK17, with a relatively lower substrate concentration of 2.5 g L-1, followed by strain AK5
with a yield of 4.4 mM g sugar-1 from grass hydrolysate. Highest hydrogen yields are
observed from the strain GHL15 (7.6 mM g sugar-1) from grass hydrolysate.
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Upscaling
The thermophilic, fermentative process of producing the desired biofuel – ethanol, hydrogen and
methane – is merely the first step, albeit a crucial one, towards effectively harnessing
sustainable lignocellulosic biomass energy source. The next step is to increase the magnitute
of the whole process, or more technically called upscaling.
Upscaling of processes that involve
microorganisms is a big challenge as
working conditions inevitably change. A
slight variation in any of the
environmental conditons such as
tempereture, pH, oxygen exposure, etc.,
can affect the processing pathway as a
whole. Generally, the optimal
Figure 12. Upscaling
conditions change with every 10 times
increase of the process scale. Upscaling is thus a tedious task that requires a serious deal of
simultaneous research and immediate execution of research results.
There is a wide variation in the morphological, biochemical and nutritional characteristics of
methanogens that have been isolated to-date, thus requiring specific conditions for growth and
sustenance (Suryawanshi et al., 2010). Similarly, the isolated ethanol and hydrogen-yielding
thermophilic anaerobes require specialised growing conditions, it is a complicated task to
assign a process pathway that is suitable for all microorganisms. Each factory therefore has to
be explicitly designed and optimised based on individual prerequisites of survival of the
thermophilic anaerobes being used.
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Biofuel Plants of the future
ethanol, hydrogen and methane
Large-scale biofuel production from lignocellulosic biomass has yet to be accomplished. A
simplified, graphical image of an ideal ethanol production plant is shown in Figure 13.
Lignocellulosic biomass is collected and brought to an ethanol plant where it undergoes
physical treatment including size reduction, chemical pretreatment and heating. This leades to
various polymeric compoungs (cellulose, hemicellulose, oligosaccharides) which are then
used as substrates for enzyme-catalysed conversion to sugars finally fermented by microbes
to ethanol. Here,
thermophilic anaerobes pose a great potential in fermenting not only
hexoses but also the various pentoses formed. In addition to this, thermophilic anaerobes give
the possibility of increasing the temperature at this stage due to its naturally high heat
tolerance. This increase the kinetics of the fermentation reaction, thereby producing more
yield. The ethanol that is produced from the fermentation is then distilled from possible
contamination in the process and is then prepared for distribution (U.S. DOE, 2007)
Figure 13. Cellulosic ethanol production model (US-DOE, 2007).
A more detailed process of ethanol production from lignocellulosic biomass is shown in the
following page.
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Figure 14. Diagram of ethanol production from lignocellulosic biomass (US-DOE, 2007)
Figure 14 shows more detailed representation of the process of ethanol production.
Lignocellulosic biomass is first reduced in size before it undergoes physical and/or chemical
pretreatment. Toxins and contaminants which might be present in pretreatment are then
removed through the process of detoxification and neutralisation. After solid-liquid phase
separation, the process goes to one of the three steps: fermentation of hemicellulose sugars,
enzymatic hydrolysis and enzyme production. The substrates that go through enzymatic
hydrolysis either goes to enzyme production or towards fermentation of cellulosic sugars,
which together with the fermentation of hemicellulosic sugars move to the phase of product
recovery. The final products either ethanol and hydrogen are then harvested, as the residues
go to processing which may lead to production of coproducts (US-DOE, 2007).
The same theory of process is applicable for hydrogen production from lignocellulosic
biomass. Methane production, on the other hand, is slightly different, especially when the
targeted source of lignocellulosic biomass is oftem more complex, e.g. municipal solid waste.
Suryawanshi et al.
summarised 16 case studies of anaerobic digestion of waste using
methanogens in their study (2010) in which 13 pertain to lab scale studies, 1 pilot scale study
and 2 large scale studies all over the world (See Appendix I). The majority of cases had
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reactors as small as 0.2-0.4 L capacity with a maximum of 1-8 L capacity. Various reactors
were used in the case studies: glass reactors, continuous stirred tank reactors (CSTR), and
anaerobic sequencing batch reactors (ASBR). The sources of lignocellulosic biomass in the
case studies include cattle or cow manure, industrial waste from dairy, fruit and vegetable,
and paper industries. The amount of biogas and the percentage of methane content are all
summarised in Appendix I (Zinder et al., 1984; Nielsen et al., 2004; Hamed et al., 2004; ManChang et al., 2006; Kaparaju et al., 2007; Perez et al., 2007; Bouallagui et al., 2008; Yilmaz et
al., 2008).
Improvement strategies
Strategies to improve and maximise net energy production from anaerobic digestion of
lignocellulosic biomass put emphasis on improving (i) the steps involved in feedstock
hydrolysis and (ii) factors affecting these steps – particularly temperature (Suryawanshi et al.,
2010).
The use of thermophiles involves raising the temperature of the whole biomass digestion
process. Complementary use of renewable energy source such as sunlight has therefore been
suggested to improve the energy cost of heating up the system, hence increasing the net
energy production.
The mixing of biomass substrate is an important aspect in the process as it enables heat
transfer, improves the homogeneity of pH, encourages interaction between microbial
consortium and substrates in the medium, reduces the particle sizes to physically facilitate
digestion, and helps release the biogass generated from the generator content (Meynell, 1976).
There have been conflicting results, however, on what mode of mixing optimises biogas
production. Nevertheless, the consensus amongst researchers was that in order to minimise
killing of microbial bimass, due to shearing forces of the mixer, intermediate mixing appeared
to be optimal for substrate conversion (Dague et al., 1970; Smith et al., 1996).
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Pros and cons of using thermophiles for biofuel production
Thermophilic anaerobes offer a great potential in treating lignocellulosic biomass for biofuel
production. There are several advantages in utilising these microorganisms as well as
challenges that are yet to be met. The advantages of anaerobic digestion under thermophilic
conditions include the following:
Broad substrate range. The naturally wide substrate range of thermophilic anaerobes makes
them ideal fermentative microorganisms for biofuel production from lignocellulosic biomass.
Various compounds that are present in lignocellulosic biomass are more likely to be degraded
by microorganisms with a broad range of substrates compared to specialised degradation of
enzymes and other fermentative methods.
Higher kinetic advantage and higher specific growth rate. The kinetics and stoichiometry of
biofuel-fermenting reactions are generally favoured by high temperature, making
thermopihilic anaerobes the ideal candidates for this. The rates of chemical and enzymatic
reactions are positively correlated with increasing temperature. Increased reaction rate as a
result of relatively higher growth rate of thermophilic microorganisms, in spite of their
inherently slow doubling time, will significantly contribute to increased biogas production
(Suryawanshi et al., 2010; van Groenestijn et al., 2002). For instance, the thermodynamics of
H2-producing metabolism become more favourable with increasing temperature (Stams,
1994), and less affected by the partial pressure of hydrogen in the liquid phase (Levin et al.,
2004; van Groenestijn et al., 2002). This helps lengthen the culture‘s survival rate throughout
the process of biofuel production.
Eradication of pathogens. Non-desired microorganisms are more likely to be eradicated with
elevated temperature conditions (van Groenestijn et al., 2002). Larsen et al. (1994) concluded
that thermophilic as well as mesophilic anaerobic digestion together with thermophilic pretreatment sufficiently reduce vegetative pathogens (e.g. E.coli and Enterococci) and intestinal
parasites that are normally found in animal wastes. Salmonella and Mycobacterium
paratuberculosis are inactivated within 24 h at thermophilic conditions (Sahlstrom, 2003).
There are still plenty of drawbacks that remain to be solved in this thermophilic anaerobic
digestion of biomass. Higher energy input for operation is needed in anaerobic digestion
under thermophilic condition compared to its mesophilic counterpart (Suryawanshi et al.,
2010). Net energy production is significantly reduced as the energy requirement for operation,
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especially in higher altitude and cold regions, may be greater than the actual energy produced
(Mackie and Bryant, 1995). Also, lower growth yields of thermophilic microorganisms result
in longer start-up times. This increases the susceptibility of the process to toxicity,
fluctuations in temperature and other environmental conditions (Ong et al., 2000, Hamed et
al., 2004). According to Ahring et al. (2001) imbalance in microbial consortium is apparent
when operational temperature is increased from 55°C to 65°C. This entails imbalance
between the fermenting, acid-producing and acid-consuming microbial consortia (Ahring et
al., 2001).
Another challenge with regards to thermophilic anaerobic digestion for methane production is
the interaction between temperature and ammonia – when ammonia concentration is in the
inhibitory range, the effect of temperature cannot be independently examined (Gerardi, 2003).
Also, the cell density achieved that is produced by thermophilic H2 fermentation processes
has still been relatively low (Hallenbeck, 2005). Further development of cell retainment
methods is needed.
Genetic engineering of thermophiles
Thermophilic anaerobes may seem to hold the potential in biofuel production from
lignocellulosic biomass, however, there are certain criteria that need to be met in order to
fulfill the increasingly sought-after functionality. Genetic engineering holds the key to making
this a reality.
Low tolerance to ethanol and the production of other end products like acetate and lactate are
the main hindrance of utilising thermophilic bacteria for biofuel production. Efforts on
enhancing ethanol tolerance through mutations and adaptations have been made (Lovitt et al.,
1984, 1988; Georgiva et al., 1988) and mentioned in the previous chapters.
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Discussion
Energy production from lignocellulosic biomass offers an ideal and promising solution on
how to alleviate the alarming environmental situation of our world today. Lignocellulosic
biomass is a complex form of biomass. It is abundant and exist in many forms which are
typically regarded as waste, such as crop residues and urban waste. The use of lignocellulosic
biomass for biofuel production is thus more preferable than the more simple type – starch and
sucrose derived from corn and sugarcane – which requires growing these crops specially for
energy production. Simple biomass, however, has been used for ethanol production for
decades, as the conversion from the crop to biofuel is relatively easier than producing biofuels
from lignocellulosic biomass.
The main challenge of converting lignocellulosic biomass into biofuels such as ethanol,
hydrogen and methane is the degradation of its chemical constituents – cellulose,
hemicellulose and lignin – into more simple types of carbohydrates in order for fermentation
occur. Thermophilic anaerobes that have been isolated in Icelandic hot springs give a
promising answer to this challenge. These microorganisms thrive in hot and hostile
environments of the geothermal areas in Iceland. Their heat tolerance is of great advantage for
the conversion process from complex biomass to ethanol, hydrogen and methane, as it gives
the possibility of raising the process temperature, and hence increasing the rate of reaction of
each fermentational stage. Moreover, thermophilic anaerobes have broad spectra of substrate
degradation including various hexoses, pentoses, disaccharides and polymeric substrates,
which is a great advantage when degrading complex biomass source such as urban waste and
crop residues.
Several thermophilic anaerobes isolated in Iceland that are capable of producing ethanol,
hydrogen and methane are discussed in this paper. Strain AK17 which belongs to genus
Thermoanaerobacterium has the best ethanol yield of 5.5 mM g sugar-1 from grass
hydrolysates (g L-1). From the isolated hydrogen-producing strains, GHL15, from genus
Thermoanaerobacter has the most yield of 7.6 mM g sugar-1 from grass hydrolysates (g L-1).
Various factors affect the biofuel yields of thermophilic anaerobes from lignocellulosic
substrates. Pretreatment with either acid/alkali with appropriate concentration significantly
improves the yield of a given strain. Degradation of grass hydrolysate with AK17 increased its
ethanol yield from 1.9 mM g-1,without chemical pretreatment, to 3.2 and 3.5 mM g-1, with
acid and alkali pretreatment respectively. Further increase in acid concentration of the
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pretreatment slightly increased the yield whereas increase in alkali concentration did not have
any effect (Almarsdottir et al., 2012). This suggests that degradation of lignocellulosic
biomass has optimal pH pretreatment condition wherein it gives the most yield from a given
substrate source, making this aspect an important criterion on optimising biofuel yields.
Interestingly, the yields of these newly isolated thermophilic anaerobic strains by the
researchers at the University of Akureyri are only slightly better than that of T. mathranii
(2.61- 5.30 mM g-1 from wheat straw hydrolysates) which was the first ethanol-producing
thermophilic anaerobic strain isolated in Iceland. This may suggest that more thermophilic
anaerobic strains which have even better fermenting capacities may still remain unidentified
therefore it is vital to continue the isolation of such strains.
Factors such as initial glucose concentration, enzyme concentration, partial pressure of
hydrogen, inhibitory effects of furan derivatives, and tolerance to end-products such as
ethanol are amongst the crucial factors that have been shown to have important effects on the
yield of lignocellulosic degradation of thermophilic anaerobes. Each strain and type of
substrate source may have various optimal conditions of fermentation therefore it is of vital
consideration to study these factors in designing an optimal process of biofuel production
from lignocellulose.
Various advantages have been acknowledged in the use of thermophilic anaerobes in biofuel
production such as broad substrate spectrum, higher rate of reaction and eradication of
pathogens whereas disadvantages including the high cost of production, lower growth yield of
thermophilic anaerobes and the challenges of upscaling continue to slow down the full
utilisation of lignocellulosic biomass as a worldwide source of fuels.
The research question is thus addressed – thermophilic anaerobes isolated from Icelandic hot
springs are indeed good candidates for biofuel production from lignocellulosic biomass due to
their aforementioned advantages. Apparent challenges are an inevitable part of the
development of the technology.
More research and international cooperation need to be done to address the challenges that are
involved in using lignocellulosic biomass as biofuel feedstock. Previous genetic modifications
done on T.mathranii serves as a stepping stone for further modifications on the other isolated
thermophilic anaerobes described in this paper. Studies on how genetic modifications could
improve growth yield and tolerance to inhibitory compounds can be made in the future. Also,
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the high energy cost of biofuel production from lignocellulosic biomass can be addressed by
using other renewable energy sources such as solar, wind, geothermal or hydroelectric energy,
to further encourage the overall use of renewable energy sources. Iceland is teeming with
geothermal and hydroelectric power therefore it is an ideal place to carry out further research
and upscaling projects of biofuel production from lignocellulosic biomass.
Concluding Remarks
Thermophilic anaerobes isolated in Iceland provide a great potential in producing biofuels
such as ethanol, hydrogen and methane from lignocellulosic biomass. Several challenges still
remain to be addressed for full-scale utilisation of the technology. Broad substrate spectra,
higher rate of reaction and eradication of pathogens due to natural heat tolerance are of
considerable advantage in developing the process of harnessing energy from lignocellulose as
well as the amount of other renewable energy source in Iceland. Further research needs to be
done on optimising the yields of biofuel products through variation of different environmental
factors as well as genetic modification of the isolated strains and continuous search of more
thermophilic anaerobic strains.
As Earth‘s oil reserves continue to deplete while every societal framework depends greatly on
the availability of energy, it is of vital importance to develop an increasingly important
technology such as the biofuel production from lignocellulosic biomass. As studied in this
paper, the utilisation of an ubiquitous energy source – lignocellulosic biomass – poses a
victorious situation wherein energy, which is an absolute necessity today, produced from an
abundant source which otherwise would not be used at all is a lot more economical, practical
and a sustainable solution compared to pursuing an extremely limited energy production from
oil reserves.
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Appendix I
Appendix I. Profies of anaerobic digestors as a function of digester type, substrate, biogas and % methane
Source: Suryawanshi et al. (2010)
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Appendix II
Appendix II. Minimum inhibitory concentrations of ethanol for seven thermophilic bacterial strains. The final
optical density (OD) was used as the indicator of growth; ++++ = OD > 1.0; +++ = OD between 0.7 and 1.0; ++
= OD between 0.3 and 0.7; + = OD below 0.3 but above control
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