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Biofuels and Value-added Products from Extremophiles
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Chapter 2
Biofuels and Value-added
Products from Extremophiles
Mohit Bibra, Jia Wang, Phillip Squillace, Rebecca Pinkelman, Sam Papendick,
Steven Schneiderman, Vanessa Wood, Vinod Amar, Sudhir Kumar#,
David Salem, and Rajesh K. Sani*
Department of Chemical and Biological Engineering, South Dakota School of Mines and
Technology, 501 East St. Joseph Street, Rapid City, SD 57701-3995
#
Present address: Department of Biotechnology and Bioinformatics, Jaypee University
of Information Technology, Waknaghat, Solan 172234, Himachal Pradesh, India
ABSTRACT
It has now been established that extremophiles and their enzymes will likely play important
roles in many kinds of bioprocessing e.g., in conversion of nonfood biomass into biofuels,
including bioethanol, biobutanol, and biogas; bioremediation of contaminated aquifers; and
pharmaceuticals. Extremophiles, which can grow under extreme conditions of temperature,
acidity, alkalinity, or salinity, have been reported to produce biofuels and valued added
products of unique properties. The processes employing extremophilic microorganisms to
produce biofuels and value added products are cost effective, more economical, having
low risk of unwanted microbial contamination, increased reaction kinetics, higher yields of
products, and minimal environmental hazards. This chapter presents state-of-the-art review
and performs in-depth analysis of the biofuels (e.g., bioethanol, biobutanol, biodiesel,
biogas) and value added products (e.g., polyhydroxy alkanoates, exopolysaccharides)
production using extremophiles. In addition, existing technologies of biofuel production
and their limitations and strategies to overcome those limitations are discussed.
Keywords: Bioenergy, Butanol, Ethanol, Hydrogen, Methane, Polyhydroxyal-kanoates, and

Exopolysaccharides
2.1 INTRODUCTION
The depletion of non-renewable energy sources is a serious concern and extensive
research has been carried out to develop new, alternative energy sources. Many
countries are developing new energy resources that can address the increasing
imbalance between energy demand and supply, and this global search for new
energy sources has motivated extensive research on biofuels. When research on
biofuel began, the source for their production was mainly staple food sources like
corn, sugarcane, and sugar beet. However, given the increasing global demand
*Corresponding Author: E-mail: Rajesh.Sani@sdsmt.edu

#
The first eight authors have made equal contributions to this review article.
18 Advances in Biotechnology
for staple food, using these sources for biofuel production is now considered
a defective strategy. The realization of this problem has prompted researchers
to look for alternative, nonfood sources for biofuel production.
Nonfood lignocellulosic feedstocks are among the Earth’s most abundant
resources for biofuel production. Regional sources of lignocellulosic feedstocks
will be important in reducing costs associated with transportation of raw
materials to biorefineries. For example, in South Dakota, pinewood, prairie cord
grass (PCG), and corn stover (CS) are major cellulosic renewable materials.
In a joint report, the USDA and Department of Energy (2005) estimated the
annual supply of forest residues is 1,207 thousand dry tons. Nationally, in the
same report it was estimated that the US could sustainably produce a total
of 368 million dry tons of forest biomass, with residues contributing roughly
86% of this total (145 million dry tons from wood processing mills and
pulp and paper mills, 47 million dry tons of urban wood residues including
construction and demolition debris, 64 million dry tons from logging and site
clearing operations, and 60 million dry tons of biomass from fuel treatment
operations to reduce fire hazards). Lignocellulosic feedstocks vary widely
in-terms of type and composition. A comparison of cellulosic feedstock
compositions currently being employed for biofuel production is shown in
Fig. 2.1 [Karunanithy & Muthukumarappan (2010), Raspolli Galletti & Antonetti
(2011), Zhang et al. (2012)].
Attempts have been made to convert lignocellulosic biomass into alternative
biofuels using certain chemical, physical, physiochemical, and biological
processes. With concerns about potential environmental hazards related to several
biofuel production processes, current focus is on using biological processes, since
these are safe, operationally simple, and environment-friendly. Extremophiles and
their enzymes will likely play important roles in many kinds of bioprocessing,
including conversion of nonfood biomass into biofuels. Extremophiles grow
under extreme conditions of temperature, acidity, alkalinity, or salinity and have
been reported to produce not only biofuels but also value-added products with
unique properties. The processes employing extremophilic microorganisms to
produce biofuels and value-added products can be cost effective as they have (i)
low risk of unwanted microbial contamination, (ii) increased reaction kinetics,
(iii) higher yields of products, and (iv) minimal environmental hazards.
This chapter presents a review and in-depth analysis of the state of the
art of biofuels (e.g., bioethanol, biobutanol, biodiesel, and biogas) and value-
added products (e.g., polyhydroxyalkanoates and exopolysaccharides) generated
through extremophiles. In addition, existing technologies of biofuel production
and their limitations are discussed in relation to strategies that overcome those
limitations using extremophiles.
Extremophilic Bioprocessing for Energy 19
Fig. 2.1 Composition of various cellulosic wastes currently being

used for biofuel production.
2.2 PRETREATMENT
A typical lignocellulosic biofuel technology can be broken down into four parts:
(i) pretreatment (removal of lignin barrier from biomass), (ii) saccharification
(conversion of carbohydrate polymers to monomeric sugars), (iii) fermentation
(production of biofuel), and (iv) biofuel recovery. One of the obstacles in
converting lignocellulosic feedstocks to biofuel is the recalcitrant nature of such
feedstocks [Bhalla et al. (2013), Somerville (2006)]. Production of biofuels from
these lignocellulosic feedstocks requires energy intensive, enzyme intensive,
or time intensive techniques for the delamination of the plant cell wall and
conversion of its components into fermentable sugars. Several biological,
chemical, and physical pretreatment methods are currently used to disrupt
the recalcitrant structure of lignocellulosic biomass to make cellulose and
hemicellulose susceptible to an enzymatic hydrolysis [De Frias (2013)].
Physical pretreatments (i.e. chipping, grinding, and milling) are primarily

used to reduce particle size and disrupt the crystallinity of lignocellulosic
biomass. Chemical pretreatments mainly involve acid or alkali additions and,
depending on the nature of the biomass, can produce inhibitory compounds
such as furfural (FF), hydroxymethylfurfural (HMF), formic acid, and levulinic
acid. Ammonia fiber expansion (AFEX) is a chemical pretreatment method
that reduces lignin content and decrystallizes celluslose [Balan et al. (2009)].
Biological pretreatment is an ecofriendly approach which utilizes microorganisms
and their enzymes. All the above pretreatments have several limitations;
biological pretreatments involve long incubation time and consume sugar;
mechanical pretreatments require high energy (e.g., high voltage extruders); and
chemical pretreatments shift the pH to high alkalinity or acidity. In addition,
alkali and AFEX pretreatments are expensive, and recovery of the bases is
critical for the viability of the process.
Combinations of the above-mentioned physicochemical pretreatments
have been reported for efficient hydrolysis of lignocellulosics [Brodeur et al.
(2011)]. Either single or combined pretreatment makes the process complex
and expensive due to the need to remove inhibitory compounds and recover
solvents. For efficient microbial fermentation of sugars, inhibitory compounds
generated during pretreatment should be removed. Even in the case of enzymatic
hydrolysis, the pH of the pretreated biomass needs to be adjusted prior to
hydrolysis. All these factors make the process of lignocellulose conversion into
biofuels less economical. Lignocellulosic biomass is inexpensive (US $2-4/GJ at
a cost of US $39-60/dry ton biomass) but its pretreatment cost (US $15-25/GJ),
along with the high cost of commercially available lignocellulose-hydrolyzing
enzymes, dramatically reduces the overall cost-efficiency of the process [Raj
et al. (2012)]. A cost-effective pretreatment method is required for the biofuel-
based industry and recent interests have been looking at a single step process
using extremophiles and their enzymes [Bhalla et al. (2013)].
2.3 LIQUID AND GASEOUS BIOFUELS

2.3.1 Bioethanol
Ethanol, a renewable substitute for current hydrocarbon fuels, is readily
produced through the fermentation of sugars. Ethanol production is dominated
by first generation feedstocks, e.g. corn, sugar cane, sugar beet. Saccharomyces
cerevisiae-based fermentation is industrially proven and well developed for the
production of ethanol. One of the major constituents of lignocellulosic biomass
is pentose (C5) sugars, but lack of C5 catabolism by S. cerevisiae reduces the
efficiency of the conversion process. This issue has been addressed by xylose
and arabinose utilizing recombinant strains of S. cerevisiae and Zymomonas
mobilis [Barnard et al. (2010)].
On the other hand, several wild-type thermophilic microorganisms are

able to ferment C5 as well as C6 sugars. They also typically have a higher
pH tolerance and maintain their activities through environmental fluctuations.
Thermophilic Clostridia sp. operating at 60-65°C has cellulosomes, containing
cellulases, that are adept at digesting crystalline cellulose. C. thermocellum
JW20 has broad substrate specificity, and can grow on cellulose, cellobiose,
xylooligomers, glucose, fructose, and xylose. Elevated temperatures also reduce
the risk of contaminations, as there are fewer indigenous bacteria that are
competitive at higher temperatures [Taylor et al. (2008)]. Currently one of the
leading contaminants is Lactobacilli, which competes for carbohydrate substrates
and produces inhibitors. Antibiotics will control contamination, but with an
additional cost. Lactobacilli do not grow above 50°C and thus cannot compete
with a thermophilic fermentative organism. In addition, elevated temperatures
are good for distillation and ethanol recovery; however, the two major hurdles
of poor ethanol yield and low ethanol tolerance in thermophiles remain.
Novel organisms from extreme conditions provide a good industrial source
of genes for enzymes because of their ability to deal with harsh industrial
conditions [Blumer-Schuette et al. (2008)]. This has led to the harvest of novel
organisms from a variety of places and conditions including the subterranean,
industrial waste streams, and thermal vents [Blumer-Schuette et al. (2008),
Bai et al. (2010), Rastogi et al. (2009), Rastogi et al. (2010), Zambare et al.
(2011)]. The sustained activity of the extremophilic enzymes is of interest in
ethanol production because it allows for more variation in the pretreatment and
hydrolysis conditions while maintaining normal rates of saccharification.
Saccharolytic fermentative thermophiles can produce lignocellulose-
deconstruction enzymes. They ferment pentose and hexose and are desirable for
efficient bioethanol production and cost-effective operation. In fermentation, the
focus needs to remain on increasing ethanol tolerance and yields. Adaptations
or mutations that lead to higher ethanol tolerance and higher yields would
make these organisms comparable to the current yeast-dominated process.
Extremophiles have been engineered for higher ethanol tolerance, and co-
fermentation of the major pentose and hexose sugars has also been demonstrated
Thermoanaerobacterium saccharolyticum ALK2 produced 33 g/L ethanol by
fermentation of mixed sugars (xylose, glucose, mannose, and galactose) [Barnard
et al. (2010)]. Some extremophile fermenters have shown cellulase activity that
can help promote simultaneous saccharification and fermentation.
In summary, novel microorganisms need to be compared directly with
industrial organisms (e.g., Saccharomyces and Zymomonas spp.) for accurate
assessment. For an ideal microbial strain (e.g., a thermophile) for bioethanol,
there should be high yield of ethanol (>20%, v/v), no side fermentation
byproducts, low inhibitor sensitivity, and high ethanol tolerance comparable
to Saccharomyces and Zymomonas spp. These thermophiles should have fast
growth rates and ferment both pentose and hexose sugars at high temperatures
(e.g. >80°C, for easier recovery of ethanol). These microbial strains should also
have high resistance to microbial contaminants. For the purpose of developing
various metabolic engineering approaches to improve ethanol yield and tolerance,
genome sequencing of thermophiles is desirable. Finally, if extremophiles could
produce thermostable lignocellulosic-deconstruction-enzymes, hydrolyze the
untreated biomass, and ferment the sugars in a single reactor, then this could
add a new dimension to the available methods of replacing fossil fuels with
biofuels. Such extremophiles may produce new engineering solutions to biofuels
production, which are more efficient and more economical.
2.3.2 Biobutanol
Biobutanol was first discovered in Clostridium sp. It has a higher energy density
(29.2 MJ/L) than ethanol (19.6 MJ/L), and can easily be blended with gasoline
and even diesel fuel without any modifications to existing vehicles [Berezina
et al. (2009)]. It has a low volatility and does not increase the Reid vapor
pressure (RVP) of gasoline. Butanol also offers the potential to be upgraded
to aviation jet fuel, a product generally not associated with biofuels (www.
greenbiologics.com). Butanol fermentation can use the same feedstocks as
bioethanol and can be transported through existing infrastructure. One of the
biggest problems faced by the industry is the low butanol tolerance exhibited by
most microorganisms. While ethanol-producing organisms such as S. cerevisiae
can tolerate ethanol concentrations close to 20% v/v, butanol producers are
generally limited to 2-3% v/v [Knoshaug & Zhang (2009)]. This leads to
increased downstream separation costs and larger production equipment.
Using extremophiles or their genes in the production of butanol could
mitigate these problems. Organisms of the genera Bacillus and Pseudomonas
spp. have been shown to be able to tolerate butanol concentrations in the 6-7%
range. Although these organisms are not extremophiles in the traditional sense,
their ability to withstand the harsh conditions of high solvent concentrations
could be considered an “extreme” behavior.
Another problem with using Clostridium sp. for butanol production is
mixed solvent production (30% acetone, 60% butanol and 10% ethanol, also
known as ABE fermentation). An approach to improve biobutanol productivity
is taking desirable genes from mesophiles or extremophiles with specificity
to butanol production and/or increased solvent tolerance and inserting them
into a host organism to create a recombinant strain. Often the host organism
of choice is Escherichia coli because its genome is well understood and the
tools necessary for genetic engineering are readily available. An example of
a novel way of engineering E. coli to produce butanol can be seen through
work of Shen and Liao (2008), in which the native keto-acid pathway was
modified to produce butanol in E. coli. The transfer of mesophilic genes from
Clostridium acetobutylicum has also been used in E. coli to produce butanol
[Atsumi et al. (2008)]. Thermophiles (and thermophilic genes) have also been
investigated as a means for efficient biobutanol fermentation. For example, the
CimA enzyme from Methanococcus jannaschii was used in E. coli to produce
1-butanol and propanol (Atsumi and Liao, 2008). The biofuels company Gevo
(www.gevo.com) has also been involved in using an engineered microbial strain
to produce butanol (Gevo’s proprietary strain).
In summary, butanol is an attractive biofuel because of its higher energy
density and compatibility with existing fossil fuels. There has been organism
development with proprietary strains, but low butanol tolerance is still the
most challenging aspect of economical production. Genes from extremophiles
or mesophiles like Bacillus and Pseudomonas spp. allowing for increased
butanol tolerance and efficient production of butanol should be investigated. For
cost-effective production of butanol, a microbial strain (extremophile) should
provide higher butanol yield; this could be achieved using the recovery of
butanol during fermentation, blocking the pathways for ethanol and acetone by
metabolic engineering, or using a consortium of microorganisms. The microbial
strain should also have high tolerance to butanol, which could be achieved
using genetic manipulations (e.g., mutagenesis or genetic engineering). Finally
the microbial strain needs to have a high butanol production rate (comparable
to ethanol production by yeast) to be competitive with other biofuels.
2.3.3 Biodiesel
Biodiesel, a clean-burning renewable energy source, is a fatty acid ester
compound produced from the transesterification of triglycerides and fatty acids
from vegetable oils, waste oils, animal fats, or microalgae with alcohol usually
methanol or ethanol. It contains long-chain alkyl (methyl, ethyl, or propyl)
esters and has higher energy content (about 35 MJ/L) compared to ethanol or
butanol. It increases fuel lubricity, is not detrimental to engine performance, and
is cost comparable with current diesel. More importantly, it is biodegradable,
non-toxic, and free of sulfur and aromatics [Yang et al. (2009)]. Third generation
biodiesel, in particular using microalgae as the oil source, is a viable option as
a biofuel. Biodiesel is currently produced from oil crops such as soybeans and
rapeseed using a base catalyst for the transesterification process. As the focus is
switching from second generation to third generation biofuels, microalgae are
an excellent source of oil compared to crops such as soybean. They are able
to produce 15- to 300-times more oil than conventional oil crops, are grown
on non-arable land, do not conflict with the food versus fuel issue, and are
continually harvested [Dragone et al. (2010)]. Extremophilic algae are being
discovered and reviewed as an alternative oil source along with an extremophilic
source of lipase enzyme as a biocatalyst in the transesterification process.
This section will focus on extremophiles for biodiesel production, mainly the
lipid (algal oil) source and extremophilic sources of lipase enzymes for the
transesterification process as shown in Fig. 2.2.
Fig. 2.2 Production of biodiesel. Possible roles of extremophiles

are highlighted by green.
2.3.3.1 Algae (Lipid Source)

Biodiesel from algal oil is considered to be more sustainable than from oil
crops. Algae produce more oil and do not have to be grown on arable land.
Algae can be mass cultivated by two different methods, in an open air pond
system or closed system such as a photobioreactor. To prevent contamination in
an open air system, such as a raceway pond, extremophilic strains of algae are
preferred that can grow at high temperatures, either low or high pH values, and/
or high salinity. Spirulina, Dunaliella, and Chlorella spp. are different types of
algae that have been shown to grow well in highly alkaline, halophilic, nutrient-
rich media environments, respectively, and are good options for an open air
system. In a closed photobioreactor system, there is no limitation on the type
of strain since contamination is not an issue, and higher cell densities can be
achieved [Dragone et al. (2010)]. In addition, after extraction of the lipids and
oils for transesterification, the algal biomass can be used as an animal feed,
biofertilizer, or for human nutrition. Value-added products may also be produced
simultaneously with the oil, such as polyunsaturated fatty acids, polysaccharides,
antioxidants, colors, food-coloring products, toxins, and stable isotopes.
Another extremophilic source for oil is the psychrophilic yeast Rhodotorula
glacialis DBVPG 4785. This yeast was isolated from an alpine glacial
environment and can grow at zero and sub-zero temperatures but has optimum
growth at 20°C. It was shown that under nutrient limitation with carbon in
excess, larger amounts of lipids are produced by the yeast cell, up to 68%
(lipids/biomass). The majority of the fatty acids produced is in the range of C14
to C18 with traces of C12 and includes saturated, mono-unsaturated, and poly-
unsaturated forms. The high glucose concentration increases mono-unsaturated
forms at the expense of the poly-unsaturated forms. This lipid content is similar
to soybean oil and rapeseed oil, and thus has high potential as a biodiesel
precursor [Amaretti et al. (2010)].
2.3.3.2 Lipases for Transesterification for Biodiesel Production

Lipases, part of the α/β hydrolase superfamily (E.C.3.1.1.3), are carboxyl ester
hydrolases that catalyze hydrolysis and synthesis of long chain ester compounds.
These enzymes perform transesterification of triglycerides to biodiesel, instead
of using a typical chemical transesterification. In a chemical transesterification,
either an acid or base catalyst is used such as sulfuric acid, sodium hydroxide,
or potassium hydroxide. These chemicals are corrosive and have large negative
impact on the purification process after transesterification but have a high yield of
biodiesel with a low reaction time. As a biocatalyst in transesterification, lipases
are advantageous due to their synthesis of specific alkyl esters, easy recovery of
the biodiesel and glycerol by-product, and transesterification of solutions with a
high free fatty acid composition, whereas chemical transesterification has high-
energy consumption and difficulty in converting triglycerides with a high free
fatty acid content. Lipases typically have low yields over extended periods of
time and are intolerant to the organic solvents typically used in transesterification
[Bajaj et al. (2010), Lam et al. (2010)]. The desired characteristics of lipases for
biodiesel production include high alcohol (ethanol or methanol) tolerance, high
organic solvent tolerance, high yield using mono, di, and triglycerides along
with free fatty acids, low reaction time, temperature resistance, low product
inhibition, and reusability of free or immobilized enzyme [Bajaj et al. (2010),
Yang et al. (2009a)]. Extremophilic sources of lipases such as psychrophiles,
thermophiles, and organic solvent tolerant microorganisms may have more
desirable characteristics for the transesterification of triglycerides and free fatty
acids into biodiesel.
Bacillus sp. was isolated from a soil sample near a furnace in the metallurgical
industry and secretes an extracellular lipase that has been shown to be thermo-
and solvent-tolerant. It has a temperature optimum at 55°C and pH optimum at
7. The activity of the lipase in polar solvents, such as methanol or ethanol, was
low since they disrupt the protective water layer around the enzyme’s active
site. In low water reactions, the polar organic solvents do not have this strong
effect on the active site of the lipase enzyme. Non-polar organic solvents such
as n-hexane and t-butanol also do not have this effect, and the enzyme remains
stable. This lipase is also activated by Ca2+, Mg2+, and K+ but inhibited by
ethylenediaminetetraacetic acid (EDTA), Fe2+, and Zn2+ [Sivaramakrishnan &
Muthukumar (2012)]. Bacillus pumilus B106 also produces a thermo-tolerant
lipase that has an optimum temperature of 50°C, optimum pH of 8.0, and salinity
tolerance of 0-150% psu (practical salinity units). B. pumilus B106 lipase is
active at low concentrations of organic solvents i.e. it is activated at methanol
concentrations from 10-20%, then is inhibited at higher concentrations [Zhang
et al. (2009)]. Psychrophilic microorganisms are another unique source for lipase
production for a less energy intensive biodiesel production process. Psuedomonas
fluorescens B68 expresses an extracellular lipase with a temperature optimum
of 20°C and pH optimum of 8.0. Photobacterium lipolyticum M37 produces a

psychrophilic lipase at a slightly higher temperature at 25°C and optimum pH
of 9.0. This lipase is active at low concentrations of organic solvents such as
dimethyl sulfoxide (DMSO), ethyl acetate, and acetonitrile but decreases slowly
in activity as the concentration of solvent is increased, whereas in methanol or
ethanol the enzyme is active in a concentration of solvent up to 30%, then is
inhibited at higher concentrations [Yang et al. (2009a)].
The thermo- and organo-tolerant lipase from Bacillus sp. can hydrolyze
a wide variety of oils effectively including olive oil, palm oil, sunflower oil,
castor oil, ground nut oil, and coconut oil. The yield of fatty acid methyl
esters (FAMES) was 76% by Oedogonium sp. oil after 40 hours. The stability
of the lipase in t-butanol allowed the higher yield due to the reduction of
inhibitory effects of methanol and glycerol on the enzyme [Sivaramakrishnan
& Muthukumar (2012)]. The psychrophilic lipase from P. fluorescens B68 most
efficiently hydrolyzed pNp-caprate (C10 acyl group) and had a yield of 92%
of biodiesel after 12 hours on soybean oil at 20°C. As the temperature was
increased, the activity of the lipase decreased. Using olive oil as the substrate for
biodiesel, the P. lipolyticum M37 lipase had 90, 90, and 70% yield after 48 hours
using a 3-step, 2-step, and 1-step methanol feed, respectively, in comparison
to the commercial CalB which had 90, 60, and 0% yield, respectively, under
the same conditions. Using waste oil as the substrate, biodiesel was produced
with the free fatty acids and the higher water content (22.6%) did not have an
effect on production [Yang et al. (2009a)].
Some of the drawbacks of using lipases instead of the traditional chemical
transesterification are the lower reaction rates, low yield, and sensitivity to the
methanol co-reactant. Methanol can be used as both the solvent and reactant
in the transesterification process such as for the lipase from P. lipolyticum M37
and B. pumilus B106. A 1:3 ratio of oil to methanol is commonly used for the
transesterification reaction, which is determined to be a methanol concentration
of 10% in the reaction, which activates the P. lipolyticum M37 and B. pumilus
B106 lipases [Yang et al. (2009a), Zhang et al. (2009)]. The psychrophilic lipase
enzymes have higher yields and reaction rates using industrial substrates for
biodiesel production in comparison to the thermo- and organo-tolerant lipases
[Sivaramakrishnan & Muthukumar (2012), Yang et al. (2009a), Zhang et al.
(2009)]. A psychrophilic lipase would lower the energy consumption needed in
the transesterification process. This activity at low temperatures is due to the
flexibility of the enzyme structure to enzyme-substrate complexes.
In summary, extremophiles can play a major role in the production of
biodiesel. Extremophilic sources of algal oils are only necessary if an open air
cultivation system is used. Higher biomass yields, thus larger lipid yields, can
be achieved in a photobioreactor but the costs of building and maintaining a
photobioreactor are higher than a simple raceway pond design. Biodiesel known
as ‘Soladiesel’ produced by Solazyme (www.solazyme.com) is well established
using algae under dark fermentation conditions. Another option is using a
psychrophilic yeast such as R. glacialis DBVPG 4785 which can produce up
to 68% (lipids/biomass) under optimum C:N ratios, whose oil is very similar
to soybean and rapeseed oil. Extremophilic sources of lipase enzymes for the
transesterification of the oil to biodiesel have a much greater potential for use in
biodiesel production. A lipase that is methanol tolerant and has high yields and
reaction rates at lower temperatures would replace the need for a catalyst in a
chemical transesterification process. Lipases have broader substrate specificities,
have lower energy requirements, and are more environmentally friendly than
traditional catalysts.
2.3.4 Biohydrogen
In contrast to fossil fuels and liquid biofuels, hydrogen (H2) has the ability to be
converted to electrical energy in fuel cells. It is therefore a clean energy carrier
with a high energy density (143 MJ/kg), thus a promising alternative fuel of the
future [Kumar et al. (2012), Raj et al. (2012)]. It is estimated that current global
demand for H2 exceeds 45 million tons per annum. A vast array of physical,
chemical, physiochemical, and biological processes are currently being employed
for H2 production [Rittman & Herwig (2012)], including water electrolysis,
steam reformation, catalytic steam gasification of biomass, biomass pyrolysis,
supercritical water gasification, photolysis of water, and fermentation. To date,
however, 96% of the current H2 supply comes from fossil fuels (49%-natural
gas, 29%-crude oil, and 18%-coal) through steam reforming, and 4% H2 is
produced by electrolysis. Fossil fuel reforming generates greenhouse gasses
(e.g. CO, CH4, CO2) and is not renewable. On the other hand, the biohydrogen
(BioH2) production process is eco-friendly (non-polluting in nature), generates
no greenhouse gasses, and is renewable (as it can be produced from biomass).
Generating H2 from microbial origins can meet the requirements of a viable
biofuel prospect, providing a cost-effective, pollution-free, and energy-saving
alternative to current production practices. Several options for the biological
production of H2 are being investigated such as bio-photolysis of water through
algae and cyanobacteria, the use of photosynthetic bacteria for the photo-
fermentation of organic substances, and dark fermentation of organic substances
by anaerobic organisms. The last approach, dark fermentation, is generally
preferred because it does not rely on the availability of light sources. The major
advantages of dark fermentation are: (i) its simple reactor design; (ii) process
operation; (iii) its wide variety of feedstocks utilization; and (iv) higher H2
production rates compared to other biological methods of H2 production [Saripan
& Reungsang (2013)].
A variety of anthropogenic and natural sources such as kitchen wastes,

fruits and vegetables wastes, municipal solid wastes (MSW), and lignocellulosic
materials can serve as substrates for the BioH2 production. Lignocellulosic
feedstock, especially from agricultural and forest origins is regarded as
an abundant renewable resource and may be ideal for H2 production. The
recalcitrant nature of the lignin in the lignocellulosic biomass makes it very
difficult to use in its natural form [Yang et al. (2009a)]. A variety of physical,
chemical, physiochemical, and biological processes are currently being used
for the pretreatment that not only increase the cost of production but also pose
several environmental hazards [Talluri et al. (2013)]. Lignocellulosic feedstocks
and their pretreatment are discussed above under sections 1 and 2.
Production of H2 is currently being carried out using mesophiles and
thermophiles with pretreated (acid, alkaline, and/or enzymatic using commercial
enzyme cocktails) lignocellulosic substrates [Cui et al. (2010), Raj et al.
(2012), Saripan & Reungsang (2013)]. For example, poplar leaves were first
pretreated using acid and alkali as well as enzymes and then fermented using
anaerobic mixed culture of bacteria for H2 production. Thermoananerobactrium
thermosaccharolyticum KKU-ED1 using acid treated sugarcane bagasse
hydrolysate produces only 1.12 mole-H2/mol sugar. Clostridium thermocellum
can utilize hexose sugars and has been shown to produce H2 from delignified
wood fibers [Levin et al. (2006)]. Caldicellulosiruptor saccharolyticus has been
shown to achieve the theoretical Thauer limit of 4 moles of H2/mole of glucose
used [Willquist (2010)]; however, it generated only 11.2 mmol H2/g switchgrass
[Talluri et al. (2013)]. Thermoananerobactrium thermosaccharolyticum PSU-2
produces 12.12 mole H2/L/h using starch [Thong et al. (2008)]. Using filter
paper as a substrate, 0.96 mmol H2/g was obtained from the anaerobic digester
for treating sewage sludge at 55°C using a consortium [Ho et al. (2012)].
The use of thermophilic microorganisms such as Caldicellulosiruptor
saccharolyticus and Thermotogo maritima has shown promising results [Ivanova
et al. (2009), Wilquist et al. (2010)]. For example, thermophilic H2 fermentations
have higher H2 yields than mesophilic ones due to the suppression of H2-
consuming bacteria such as methanogens and sulfate-reducing bacteria
(Hallenbeck 2012, Ren et al. 2010). In addition, several thermophiles
are able to produce H2 from both C5 and C6 sugars. The thermophile, C.
saccharolyticus produces H2 from pentose sugars, while Thermoanaerobacterium
thermosaccharolyticum W16 (50°C) was shown to ferment a biomass hydrolysate
containing a mixture of glucose and xylose to H2 [Ren et al. (2008)].
Microbial production of H2 has been studied for the past few years, but it
has not yet been developed to an economically viable status. A challenge to
fully realize the potential of dark fermentation for H2 production depends at
least upon the presence of several suitable microbial strains and the nature and
availability of feedstocks. For example, most of the microorganisms including
thermophiles produce higher yields of BioH2 only when pretreated lignocellulosic

biomasses are used. Literature suggests that conversion of lignocellulose
from specific energy crops, agricultural residues, or forest products to H2 will
require (i) various physiochemical pretreatments; (ii) enzymatic pretreatment
(enzyme cocktails to hydrolyze cellulose and hemicellulose into to fermentable
sugars usually under conditions of high temperature and extreme pH); and
(iii) robust fermentative microbes. Pretreatments employed for delignification
of lignocellulosic feedstocks and the high current costs of lignocellulose-
deconstructing commercial enzymes used in the breakdown of lignocellulose
to fermentable sugars make dark fermentation more challenging. These factors
warrant the development of a cost-effective H2 production route.
A viable option to lower the costs of feedstock and lignocellulolytic
commercial enzymes costs is to screen for and identify H 2-producing
microorganisms capable of utilizing cellulose and hemicellulose directly from
the biomass without pretreatment at higher temperatures (≥60°C). This would
eliminate the need for the separate steps of pretreatment, lignocellulolytic
thermostable enzyme production, enzymatic hydrolysis of biomass, and
would improve the process economics. However, information on such
microorganisms which produce H2 from untreated lignocellulosic biomass is
relatively scarce. Table 2.1 summarizes the recent studies of H2 production
from untreated lignocellulosic biomass. All the reports mentioned in Table 2.1
have used different parameters for H2 yield calculations. Therefore, it was not
possible to compare H2 yields. Some of these reports did utilize pretreatments
[Chen et al. (2012), Mars et al. (2010)] and also none of these reports (Table 2.1)
elucidated the deconstruction mechanisms of untreated lignocellulosic materials.
In summary, existing technologies on BioH2 production from complex wastes,
such as cellulosic wastes, utilize several steps including (i) physiochemical
pretreatments (acid, alkali, hydrothermal, or ammonia); (ii) enzymatic hydrolysis
(enzymes cocktails to hydrolyze cellulose and hemicellulose into fermentable
sugars); and (iii) fermentation. The inclusion of several steps reduces the overall
cost-efficiency of the process. For example, cellulosic biomass is inexpensive
(US $2-4/GJ at a cost of US $39-60/dry ton biomass) but its pretreatment
cost (US $15-25/GJ) along with the high cost of commercially available
cellulosic waste-hydrolyzing enzymes reduces the overall cost-efficiency of the
process. An alternative to the use of various steps in hydrogen production is
the development of an efficient and cost-effective single step process utilizing
untreated-lignocellulose-degrading and -fermentative thermophiles. Therefore,
bioprospecting such microorganisms would lead to a more complete utilization
of biomass. This would eliminate the need for separate steps of pretreatment,
lignocellulolytic enzymes production, enzymatic hydrolysis of biomass, and
fermentation and thus, will improve the process economics. In addition, the use
of elevated temperatures offer several potential advantages such as (i) improved
hydrolysis of cellulosic substrates; (ii) higher mass transfer rates leading to

better substrate solubility; and (iii) lowered risk of potential contamination,
thus improving the overall economics of the process. This will likely impact
ongoing multiple-step conversion processes of complex wastes to biofuels by
providing a safe, more efficient, and economical process.
Table 2.1 H2 production from untreated lignocellulosic materials

Feedstock Bacterial Strain Pretreatment Temp (°C) Reference
Rice straw Heat treated sludge Physical 55 Chen et al., 2012
pulverization
Potato stem Thermotoga Treatment with 75 Mars et al., 2010
peels neapolitana alpha-amylase
(Novozymes)
Potato stem Caldicellulosiruptor Treatment with 72 Mars et al., 2010
peels saccharolyticus alpha-amylase
(Novozymes)
Maize leaves Caldicellulosiruptor None 70 Ivanova et al.,
saccharolyticus 2009
Poplar, Anaerocellum None 70 Yang et al., 2009b
Switchgrass, thermophilum DSM
Napier and 6725
Bermuda
grasses
Dried distillers Clostridium None 60 Magnusson et al.,
grain, Barley thermocellum 2008
hulls, Fusarium ATCC 27405
head blight
contaminated
barley hulls
Lignocellulosic Enriched sludge None 30* Ho et al., 2012
feedstock
* Mesophilic conditions
2.3.5 Biogas
Industrial and municipal wastes are the inevitable byproduct of an ever-
increasing global economy. Anaerobic digestion (AD) is a promising tool for
treating the biodegradable organic wastes while gaining fuel (i.e. methane) as
a value-added product. Although methane (CH4) itself may not be a profitable
product, integration of AD to produce gaseous biofuel can offset some required
energy input. The AD fundamentals have been established for treating various
wastes including wastewater sludge [Tyagi et al. (2011)], municipal solid wastes
[Karagiannidis et al. (2009)], whole/thin stillage [Alkan-Ozkaynak et al. (2011)],
and algae [Sialve et al. (2009)]. There are still, however, opportunities to optimize
the AD process using extremophiles and their associated operating conditions.
A general mechanism for the AD of complex organics is shown in Fig. 2.3.

The process begins with the hydrolysis and fermentation of complex organic
matter through various hydrolytic and fermentative bacteria. Subsequently,
secondary fermentative H2-producing acetogens and H2-utilizing homoacetogens
work together to produce substrates (H2, CO2, acetate, methanol, methylamine)
for methanogens. In this sequence, hydrolysis is accepted as a rate limiting, thus
process limiting, step [Charles et al. (2009), Shahriari et al. (2012)]. In current
biofuel processing, waste streams are produced in conditions that are almost
uninhabitable. Therefore, this section discusses several benefits of integrating
AD in various bioprocessing units by implementing extremophiles.
Fig. 2.3 Basics of anaerobic digestion.
2.3.5.1 Connecting Pretreatment and Anaerobic Digestion in

Bioprocessing
Municipal waste has extremely high biological oxygen demand (BOD) and
contains harmful pathogens. Anaerobic digestion is becoming an integral part
in treating municipal waste. Collecting biogas from only one quarter of the
total biodegradable organic fraction was shown to make the process energy
sufficient [Walker et al. (2009)]. Mesophilic AD is commonly used but takes at
least two weeks for startup, requires extended hydraulic retention times (HRT),
and leaves pathogens intact. Various pretreatments are employed to increase
organic fraction of municipal solid waste (OFMSW) solubility and alleviate the
hydrolysis rate limitations. By microwaving OFMSW to 145°C, the whole and
liquid fractions subsequently yielded 7% and 26% more biogas, respectively
[Shahriari et al. (2012)]. The feed must then be cooled for mesophilic AD,
whereas integrating thermophilic AD would save energy costs of cooling. Pre-
aeration and composting is also used to raise reactor temperatures. The microbial
activity associated with composting consumes high loading concentrations
of volatile fatty acids (VFA) and raises reactor pH to be more favorable for
methanogens. Pre-aeration and composting may be obsolete when handling a
large feed with high VFA concentrations. Since methanogens are sensitive to low
pH, seeding the reactor with an acidophilic consortium would be an adaptive
solution. Additionally, the VFA normally consumed aerobically would now be
available for methanogenesis, increasing final CH4 yields.
Similar concepts are applied to waste water treatment plants (WWTP) where
the AD feed is waste activated sludge (WAS) and the biodegradable material
is enclosed within cellular walls or extracellular polymeric matrices [Gabriel
Coelho et al. (2011)]. It has been shown that pretreatment reduces digester
HRT, increases biomass production, and enhances dewatering properties. WAS
pretreatment methods have been extensively reviewed [Carrere et al. (2010),
Tyagi et al. (2011)]. Several pretreatment categories include: biological, thermal,
chemical, mechanical, and combinations of two or more.
Biological pretreatment uses hydrolytic enzymes (e.g., proteases, cellulases,
xylanases, amylases, lipases, etc.) to initiate hydrolysis but has the downfall
of high enzyme costs. One of the more promising methods is thermophilic
hydrolysis with thermostable enzymes, which allows a lower HRT before the
main AD. With the AD of primary sludge running at 55°C, methane yields
and rates increase 48% and 115% when the samples were pretreated with
thermostable enzymes [Lu et al. (2008)]. Various biological pretreatment
methods for AD are summarized elsewhere [Carrere et al. (2010)], but the
general outcome is higher methane yields. To alleviate the costs associated with
adding pre-concocted enzymes, adding thermophilic bacteria, e.g., Geobacillusor
Bacillus spp. that produce their own hydrolytic thermostable enzymes may be the
key. Some organisms are already inherent to the sludge itself (e.g. Geobacillus
stearothermophilus) and some are being engineered and introduced separately
e.g., Bacillus stearothermophillus [Carrere et al. (2010)]. Finding a heat-resistant,
robust, and substrate-flexible microbe will undoubtedly benefit this pretreatment
technique.
Thermal pretreatment of WAS is accepted as an effective means to reduce
recalcitrance and enhance methanogenesis. In this method, WAS is heated up
to 175°C using either conventional means (e.g. heat exchangers or steam) or
microwaves to disintegrate biomass and increase organic matter solubility.
Thermal pretreatment also destroys pathogenic organisms and increases sludge

viscosity [Tyagi et al. (2011)]. In brief, thermal pretreatment can increase biogas
yields up to 100% depending on the pretreatment temperature, sludge source,
and AD reactor configuration [Carrere et al. (2010)]. Running reactor vessels
at 175°C expends significant energy and reduces the economy of the process.
Alternatively, extended pretreatment times at moderate temperatures (90°C) can
still solubilize organic and inorganic materials and has been shown to increase
methane yields 11-fold [Appels et al. (2010)]. By using a novel and robust
thermophilic consortium in AD, a lower operating cost will be associated with
cooling pretreated WAS, and initial capital and space will be saved due to
lower HRT.
Combining alkali and moderate thermal pretreatment is also an effective
means to minimize high temperature heating requirements. Base addition
supplements cellular lyses in the sludge, but elevates Na+ or K+ concentrations
that cause viability problems in the downstream AD [Carrere et al. (2010)].
Halophilic mixed cultures should be implemented in the AD. Alternatively,
acid pretreatment also supplements hydrolysis; at a pretreatment pH of 2.0,
methane production rates almost doubled in batch experiments and increased
14.3% in methane yield during semi-continuous digestion [Devlin et al. (2011)].
Acidophilic mixed cultures are an obvious choice for the AD in this case and
would reduce the cost of neutralizing acidic feeds.
2.3.5.2 Integrating Anaerobic Digestion into Other Biofuel

Processes
Thin stillage is a byproduct from the bioethanol process and contains high
chemical oxygen demand (COD) for AD [Eskicioglu et al. (2011)]. Treating
stillage with AD has great potential to offset natural gas used for distillation.
A 43-59% reduction in off-site natural gas consumption was achieved when
a thermophilic continuously stirred tank reactor at a 20 day HRT was used
[Schaefer et al. (2008)]. AD can also minimize water footprints. After tuning
inoculum-to-substrate ratios (e.g. 2), the AD effluent can contain dissolved
yeast nutrients with relatively low inhibitors. Recirculating effluent will have
the combined effect of returning growth factors to fermenting yeast and reusing
water [Alkan-Ozkaynak et al. (2011)]. Thermophilic AD in this case again
produces higher reaction kinetics (decreasing capital costs) and can be coupled
to a high temperature feeds (less cooling costs).
Combining AD with microalgae was originally proposed for waste
water treatment plants, but the more recent processing of algal biodiesel has
regenerated interest in utilizing methanogenesis for lipid-extracted algal biomass.
Algae require large amounts of N and P for growth and thus, for lipid synthesis.
Similar to the nutrient recirculation concept in ethanol fermentation, AD can
re-mineralize lipid-extracted algal biomass and recirculate the nutrients to the
algae. From an additional biogas processing perspective, algae are not sensitive
to CH4 toxicity and can scrub 90% of CO2 generated in AD [Sialve et al.
(2009)]. Three main issues can be highlighted: (i) the recalcitrance of algae cell
walls; (ii) ammonium toxicity generated from algae with high protein content;
and (iii) high salinity requirements for marine algae. The concepts behind
pretreatment were described above in MSW and WAS but can also be applied
to algae pretreatment and subsequent thermophilic AD. Regarding the high
salinity required by marine algae, using a halophilic methanogenic consortium
would be a viable option.
2.3.5.3 General Considerations for Extremophilic Anaerobic

Digestion
The carbon cycle occurs in most corners of the globe, and methanogenesis is
a critical step providing a plethora of extremophilic consortia to choose from.
Salinity gradients across the Illinois Basin shale rocks have adapted methanogenic
consortia up to 2.2 M Cl– [Waldron et al. (2007)], which could be integrated
into algal processing. An extensive review covers thermophilic methanogens and
their various uses in AD [Suryawanshi et al. (2010)]. In brief, methanogenic
growth temperatures span as high as 110°C, which would accommodate
most high temperature pretreatment feeds. Whether a technology uses these
extremophilic microbes directly or favorable genes from the population, waste
handling and biofuel generation will need to integrate extremophiles into their
processes. Instead of wasting energy to force process streams to environmentally
friendly conditions, extremophilic AD may provide the means to treat these
“uninhabitable” conditions in a controlled and beneficial manner.
2.4 VALUE-ADDED PRODUCTS

2.4.1 Polyhydroxyalkanoates
Polyhydroxyalkanoates (PHAs), or endocellular PHAs, are biodegradable
polyesters of various hydroxyalkanoates. As a type of polyester, they are
composed of hydroxyl fatty acids and are biosynthesized and stored as lipid
inclusions. PHA was first discovered in 1901, but only after 30 years was more
detailed research completed after Maurice Lemoigne reported it for the first
time in a Gram-positive bacterium Bacillus megaterium and named PHB as and
lipides b-hydroxybutyriques in 1925 [Kaplan (1998) p. 220]. PHAs are stored
by the cell for future use, being surrounded by a single-layered phospholipid
membrane and deposited as water-insoluble cytoplasmic nano-sized inclusions.
The membranes can be varied in composition, and some of them are made of
proteins while others contain lipids.
It has been shown that under nutritional limiting conditions of N, P, S, O, or
Mg and in the presence of an excess carbon source, several bacteria accumulate
these PHAs as carbon and energy storage materials [Poli et al. (2011)].
When carbon and energy are required, PHA is normally depolymerized to
hydroxybutyric acid and then metabolized to acetoacetate and acetoacetyl-CoA.

In addition to being biodegradable, PHAs possess several valuable properties
such as thermoplasticity, biocompatiblity, piezoelectricity, and brittle to elastic
deformation behavior. PHAs are non-linear, optically active, usually hydrophobic,
and not permeable to gas. Some of the monomers from PHA are chiral, and
they can be molecularly designed to attain some desired properties. PHAs can
be applied in the material industry, fuel industry, medical industry, cosmetic
industry, etc. [Chen et al. (2010)].
According to the number of carbon atoms constituting monomer units,
microbial PHAs can be classified into two groups: short-chain-length (SCL,
PHAs consisted of 3-5 carbon atoms) and medium-chain-length (MCL, PHAs
consisted of 6-14 carbon atoms). The monomers of PHAs frequently found are
the 3-hydroxyalkanoates (3-HAs) of 3-14 carbon atoms or 4-HAs and 5-HAs
of 3-5 carbon atoms. These monomers may be saturated or unsaturated and
straight or branched chains containing aliphatic or aromatic side groups. The
physical properties of PHAs are highly dependent upon their monomer units.
Incorporating different monomer units in PHAs can make different biodegradable
polymers having a wide range of properties.
Rhodobacter sphaeroides strain 14 produces PHA when cultivated in
modified glutamate-malate (GM) medium under two-stage aerobic dark
fermentation [Lorrungruang et al. (2009)]. Sangkharak and Prasertsan (2008)
used three halo-tolerant bacterial strains namely, R. spaeroides ES 16 and two
mutants, N20 and U7, obtained from mutation of strain E16 by using N-methyl-
N¢-nitro-N-nitrosoguanidine and ultraviolet light, respectively. It is observed that
among these three strains, R. sphaeroides N20 gave the highest values (44.4%
w/v) for polyhydroxybutyrate (PHB). Halo-tolerant photosynthetic bacteria have
an advantage over the other microorganisms as they possess the ability to adjust
themselves both in the presence and absence of light and also can withstand
high saline conditions.
Alkalohalophiles with optimal growth at pH values ≥ and 9.0, can produce
novel substances like polyhydroxyalkanoic acid under unbalanced nutritional
conditions. For example, Halomonas campisalis from Lonar Lake in India
produced PHA with a yield of 75g/100g of dry cell weight within 24 h of
incubation. Naheed et al. (2012) screened for potential PHA producing bacterial
strains which showed resistance to various heavy metals. The PHA producing
strain, Pseudomonas guezennei RA26, isolated from ‘kopara’ mat located in
Rangiroa was able to produce medium-chain-length PHAs [Collin et al. (2008)].
Another bacterium, Halomonas boliviesis, is capable of utilizing a wide range of
carbohydrates and can grow in a wide range of temperatures (0-45°C), producing
PHB. These organisms tend to grow at environments requiring a much lower
NaCl concentration when compared to extremely halophilic archaea, thereby
reducing the production costs and preventing corrosion in reactors [Quillaguaman
et al. (2005)].
Haloferax mediterranei was used to produce PHA under hypersaline

conditions in which only a few microorganisms can survive [Don et al.
(2006)]. The growth of these organisms in such a hypersaline environment is
useful in avoiding the contamination problem thereby reducing the sterility
requirements and thus production costs. It is also easy to recover a PHA
pellet from haloarchaea, which can be easily lysed in distilled water, compared
to other microorganisms. Don et al. (2006) reported the chemical structure
of PHBV (poly-b-hydroxybutyrate-co-3-hydroxyvalerate) produced by the
H. mediterranei. They also indicated that the fed-batch fermentation using
glucose as carbon source gave a maximum PHA content of 48.6 wt. % by 48.6
wt% and a volumetric production of PHA of 0.36 g L−1 h−1. Due to properties
like biodegradability, thermoplasticity, and biocompatibility, PHAs are widely
used in industrial packaging e.g., medicine, pharmacy, agriculture, and food, or
as raw materials for the synthesis of enantiomerically pure chemicals and the
production of paints. PHB is the most widely known PHA; and its co-polymer
can be used as biodegradable plastic that can reduce the current problems with
depleting fossil resources and environmental impact caused by plastic garbage.
However, their production is limited by the high price when compared to
synthetic plastic.
PHA production using extremophilic organisms still needs to be
commercialized. The main objectives to be considered for efficient production
are the utilization of inexpensive carbon sources, higher PHA yields and
productivity, and tailoring the PHA to have the desired properties. Metabolic
engineering can be used to develop recombinant microbial strains to produce
PHAs. Combination of metabolically engineered strains, which are capable of
utilizing inexpensive carbon sources and efficient fermentation strategies, could
possibly increase the efficiency of PHA production. There is a need to improve
the growth media for PHA production, and more information about the effect
of conditions on enhancing PHA production is also required. Further research
needs to be conducted in developing unusual PHAs and understanding their
importance in replacing synthetic polymers for future use.
2.4.2 Exopolysaccharides
Microorganisms synthesize a wide variety of polysaccharides that serve a broad
and diverse range of functions. The diverse functionality of these polysaccharides
parallels the vast variance seen in the microbial community itself. These
polysaccharides can be grouped into three general categories: intracellular,
structural, and extracellular polysaccharides (or exopolysaccharides, EPSs).
EPSs are defined as high-molecular-weight polymers that are composed of sugar
residues that are secreted by microorganisms into the surrounding environment
[Nicolaus et al. (2010)]. Sutherland and colleagues first coined the term
extracellular polysaccharide to describe a high molecular weight carbohydrate
polymer excreted by marine bacteria. Since then, the term EPS has evolved
considerably, is no longer limited to marine bacteria, and now applies to

polysaccharides secreted extracellularly by any microorganism. The purpose
of this section is to examine the current knowledge on known EPSs, such as
their origin, function, and potential commercial and industrial applications,
with particular attention paid to EPSs that are produced by microbes living in
extreme environments.
In nature, the physiological functions of these EPSs are as diverse as the
microbes that produce them. In general EPSs are produced in response to
environmental stresses, including extreme temperature, pressure, pH, salinity or
light. EPSs are usually a kind of ‘slime’ that encapsulates the cell without an
obvious association to individual cells. Most of these slimes are polyanionic,
while some are neutral or polycationic and are composed of extracellular DNA,
polysaccharides, and proteins. EPSs are important in several biological activities,
such as intercellular signal transmission, molecular recognition, immunity against
attacks, construction of a comfortable extracellular environments, etc. The
properties and functions of these natural polymers in their native environments
have resulted in their use in several industrial applications. For example, EPSs
are employed as gelling agents, thickeners, and stabilizers in food products.
They have also been utilized as depollution agents for bioremediation of waste
in a variety of applications, as medical and pharmaceutical agents, and in food
applications as antitumor, antioxidant, and prebiotic agents [Donot et al. (2012),
Liu et al. (2010)]. Until now numerous microbial EPSs have been proposed for
use in various industrial applications, but only a handful have been successfully
implemented in large-scale commercial applications. This situation can be
primarily attributed to the challenges with their economic viability associated
with the cost of their production.
In order to survive in extreme conditions, extremophiles have adapted
to these hostile environments in unique manners. One such adaptation is the
secretion of EPSs. Extremophilic EPSs exhibit properties that are fascinating
from a fundamental science perspective, and they have unique properties that
may be exploited and employed in a variety of applications. For example,
high temperatures generally increase the speed of chemical reactions and can
improve product yield for a set time frame. In addition, highly acidic or basic
conditions typically minimize environment contamination from microbial growth
thus reducing operational maintenance cost while improving overall efficiency.
Hence, EPSs of extremophilic origin are promising types of polymer materials
for industrial process improvements as they can survive and even thrive in
extreme operating conditions that are known to improve operational efficiency
in terms of yield or economic cost.
2.4.2.1 Prokaryotic Producers of EPSs

EPS producing extremophiles are seen in both Bacteria and Archaea.
Thermophiles, psychrophiles, halophiles, alkaliphiles, and acidophiles have been
shown to excrete EPSs in order to enhance their survival in extreme conditions.

Considerably less is known about eukaryotic organisms from extreme niches
with regards to EPS production, but in a few cases, EPS production has also
been identified [Pavlova et al. (2011), Poli et al. (2010)]. Marine hot springs,
terrestrial hot springs, and deep-sea thermal vents have been demonstrated as
the primary habitats that promote thermophilic microbial organisms. These
environments are typically characterized by their high temperature, high pressure,
and toxicity due to a high concentration of inorganic molecules or metals. The
majority of EPSs produced by thermophiles have been located in these types of
environments. For example, Thermotoga maritima [VanFossen et al. (2008)] is
one such microbe that produces a significant amount of EPS in biofilm. Another
biofilm producing extremophile from such environments is the hydrogen-
consuming methanogen Methanococcus jannaschii [Muralidharan et al. (1997)].
These types of organisms can survive extreme temperatures, and their EPS
production has been proposed as an explanation of their adaptation mechanism.
Geobacillus tepidamans from a terrestrial hot spring produces a slime that
is highly thermostable. This particular EPS does not begin to decompose until
temperatures are approaching 300°C [Kambourova et al. (2009)]. Another
thermophilic strain isolated from sea sand can produce three different kinds of
EPSs, and it is classified in the genus Geobacillus. The thermophilic aerobic
spore-forming bacteria, which were isolated from hydrothermal springs in
Bulgaria, were able to produce EPSs. One strain, Aeribacillus pallidus,
produced two different kinds of EPSs, consisting of an unusually high variety
of sugars [Radchenkova et al. (2013)]. A thermophilic Bacillus strain, isolated
from a shallow hydrothermal vent, generated three different kinds of EPSs
[Spanò et al. (2013)]. Although these thermophilic polymer excretions have been
proposed for commercial and industrial applications, to date only laboratory
scale implementation has been examined.
Several archaeal halophiles have been shown to produce copious quantities
of EPSs, and hence their commercial exploitation has been considered. Haloferax
mediterranei excretes a large volume of anionic EPS, which have a high
viscosity at moderate salt concentrations and excellent rheological properties
for resistance to extreme pH and temperature. H. mediterranei has also been
proposed as a potential avenue to enhance oil recovery from oil wells. As an
additional benefit in this application, the archaeal membrane lipids are believed
to improve the oil carrying capacity and acts as a surfactant. Halomonas maura
and H. eurihalina show promise as well, producing large volumes of polyanionic
EPSs that are considered strong emulsifying agents with pseudoplastic behavior.
EPS from H. maura has also demonstrated its strong performance as an
immunomodulator. Aphanothece halophytica, a unicellular cyanobacterium,
found in solar salterns of the benthic cyanobacterial lakes and other high salt
concentration lakes are well known to produce high quantities of EPSs. In the
biomedical community, this salt loving bacteria has gained special interest. It
has been shown that after oral administration in mice, the EPS significantly
inhibited pneumonia induced by the influenza virus H1N1 [Zheng et al. (2006)].
However, the advantageous implementation of EPSs produced from halophilic
microbes faces several challenges. For instance, aqueous mixtures with high
salt concentrations propose a significant hurdle with respect to corrosion in
bioreactor vessels. Although corrosion resistant bioreactors are possible to build,
their cost is significantly higher than conventional reactors, which can make a
process less economical [Oren (2010)].
Psychrophiles are usually found in Antarctic, Arctic, or deep-sea sediment.
EPSs from psychrophiles are usually composed of neutral sugars, and they
also contain sulfate as well as high levels of uronic acids, such as galacturonic
acid, which is similar to some of the halophilic EPSs. Most of the EPSs
from marine microbes are acidic with a net negative charge, which gives the
molecule a ‘sticky’ quality [Qin et al. (2007)]. The period for the growth of
psychrophiles is much longer than other extremophiles, and this phenomenon
is partly ascribed to the fact that at a relatively low temperature, the activities
of enzymes in microorganisms is expected to be lower than at more normal
ambient temperatures. So far, fewer psychrophiles have been reported compared
to thermophiles or halophiles. It is, however, necessary to study the role EPSs
play in the ability of psychrophiles to survive in extremely cold environments.
EPS secretion by acidophiles and alkaliphiles was identified recently,
but there are few reports on the structure, properties, and functions of the
EPSs secreted by them. Acidophilic bacteria like Leptospirillum ferroxidans,
Sulphobacilli, and Acidithiobacillus thioxidans, currently implanted in
bioleaching processes, produce EPSs that are believed to play a role in the
attachment of microorganisms to sulfide surfaces in metals [Sand & Gehrke
(2006)]. The alkaliphilic bacterium, Cronobacter sakazakii, has been isolated
from oil contaminated wastewater, and its characterization has illuminated the
production of a biosurfactant EPS [Jain et al. (2012)]. Biosurfactants have a
low toxicity, are biodegradable, and are effective in a wide range of operating
conditions (e.g., extreme temperature, pH, and salinity when compared to
chemical surfactants) while remaining ecologically acceptable. In multiple
studies, the biosurfactant EPS produced by C. sakazakii was reported to
have pseudoplastic rheology and an anionic property that is promising for
bioremediation as cations (seen in polyaromatic compounds) can readily bind
to the pseudoplastic natural polymer. Additionally, this EPS dissolved rapidly
in water in a broad range of temperatures and pH, furthering its candidacy as
a promising biosurfactant for biotechnological and industrial applications [Jain
et al. (2012)]. EPSs produced by thermophiles, halophiles, and psychrophiles
are summarized in Table 2.2.
In summary, the EPSs secreted by extremophiles can be essential for
them to survive under extreme environments. It is now widely accepted that
Table 2.2 EPSs produced by thermophiles, halophiles, and psychrophiles
40
Strains Source environment Extreme Chemical composition of Special Reference

conditions for EPS characteristics of
growth EPS
Thermophiles
Bacillus thermoantarcticus Close to the crater of 65°C EPS1 Not available Manca et al.,
Mount Melbourne, Mannose:glucose = 1:0.7 1996
Antarctica EPS2
Advances in Biotechnology
Mannose:glucose = 1:trace Nicolaus et al.,

2004
Geobacillus tepidamans Velingrad hot spring, 60°C Glucose:galactose:fucose: Degradation at Kambourova

Bulgaria fructose = 1:0.07:0.04:0.02 280°C. et al., 2009
Inhibition of
cytotoxic effects
produced by
avarol in brine
shrimp bioassay
Geobacillus sp. 4004 Sea sand at Maronti, 60°C EPS1 In 1% CaCl2 Moriello et al.,
near Sant’ Angelo Mannose:glucose:galactose and 1% NaCl 2003
(Ischia) = 0.5:1.0:0.3 water solution,
EPS2 with increasing
concentrations of
Mannose:glucose:galactose
EPS3, there was
= 1.0:0.3:trace
an increase in
EPS3 viscosity in all
the solutions.
Brevibacillus thermoruber a hot spring close 55°C Glucose:galactose:mannose: The EPS behaved Yildiz et al., 2013
strain 423 to the village galactosamine:mannosamine like a typical
Gradechnitsa (N = Newtonian fluid
41·683°; E 23·183°), 57.7:16.3:9.2:14.2:2.4 and viscosity of
Blagoevgrad region, the EPS solution
South-West Bulgaria increased with
increasing Ca2+
ion concentration.
Halophiles
Halomonas sp. AAD6 (JCM CamltiSaltern area in 137.2 g/L NaCl Fructose Degradation Poli et al., 2009
15723) strain Turkey temperature
(Td):253°C
Aphanothecehalophytica Guangraosaltworks 1 M NaCl EPS1 Gelling properties Li et al., 2001
GR02 in the Shandong Arabinose:rhamnose:fucose: and strong affinity Morris et al.,
province of P.R. mannose:glucose:galactose: for metal ions 2001
China glucuronic acid =
tr:0.06:0.05:0.08:1:0.75:3.58
EPS2
Arabinose:fucose:mannose:
glucose:glucuronic acid =
1:2.08:1.57:2.87:15.78
Halomonas ventosaeA112T Saline soils in Jaén, 7.5% (w/v) Glucose:mannose:galactose: Emulsifying Mata et al.,
southeastern Spain marine salts xylose: arabinose: activity, heavy 2006
galacturonic acid = metal binding
1.75:4:1:tr:tr:tr capacity
HalomonasMaura Saline soil bordering 7.5% (w/v) Mannose:galactose:glucose: Heavy- Arias et al.,
a solar saltern in marine salts glucuronic acid = metal uptake, 2003
Asilah, Morocco 34.8:14:29.3:21.9 viscosifying
potential
42
Psychrophiles
Pseudoalteromonas sp. 1855 m deep-sea 15°C 6-Glucose, terminal EPS can Qin et al., 2007
SM9913 sediment arabinofuranosyl, terminal obviously enhance
glucopyranosyl, terminal the thermostability
galactose, 4-xylose, ofon the proteases
4-glucose and 3,6-galactose secreted by the
(61.8/11.0/11.2/3.1/3.9/5.0 same strain.
/4.0)
Metal-binding,
stabilizing and
Advances in Biotechnology
flocculation
properties
Flavobacteriumfrigidarium Antarctic marine 20°C Arabinose:mannose:galact Cryoprotectant for Nichols et al.,
CAM005 ose:glucose:glucuronicaci microorganisms in 2005
d:N-acetyl-glucosamine = Antarctic marine
5:74:3:8:8:1 environment
MyroidesodoratusCAM030 Antarctic marine 20°C Arabinose:rhamnose:xylose: Cryoprotectant for Nichols et al.,
mannose:galactose:glucose:g microorganisms in 2005
alacuronicacid:glucuronicaci Antarctic marine
d:N-acetyl-galactosamine:N- environment
acetyl-glucosamine =
6:1:2:48:4:9:2:10:10:8
Shewanella Antarctic marine 20°C Arabinose:rhamnose:xy Cryoprotectant for Nichols et al.,
livingstonensisCAM090 lose:mannose:galactose microorganisms in 2005
:glucose:glucuronicacid Antarctic marine
:N-acetyl-galactosamine = environment
13:2:2:41:5:10:20:7
extremophilic microorganisms will provide a valuable resource not only for

liquid or gaseous biofuels but also for value-added products such as PHAs
and EPSs. These products will have promising commercial applications and
will likely be produced at industrial scale in the near future. The EPSs from
extremophiles, as compared to other microbial isolates that thrive at “normal”
temperature, salinity, and pH values in the biosphere, can offer unique
physicochemical properties essential for various industrial applications. By
testing the structure and chemical-physical characteristics of extremophilic EPSs,
it is possible to gain insight into their structure-function relationships and the
mechanisms of their modulation in the biosynthesis process of EPSs. With the
continuous improvement of methods and equipment for analyzing and detecting
EPSs, more and more extremophilic EPSs will be discovered, characterized,
and employed in various industries.
CONCLUSION
The chapter has discussed the exploitation of extremophiles to develop improved
biofuel or value-added product generation processes using nonfood waste
materials including lignocellulosics. Liquid and gaseous biofuels and value-
added products produced by extremophiles could successfully substitute or
supplement their traditional counterparts. For example, it is well known that
bioethanol can be blended with gasoline. Biobutanol and biodiesel have bright
prospects for being used as aviation fuels. However, although biofuels like
bioethanol, biodiesel, and biobutanol can be used as additives or substitutes for
existing transportation fuels, they do not provide a solution to the problem of
environmental pollution. On the other hand, clean energy sources like BioH2
is environmentally friendly and can be used for transportation and even for
household purposes such as electricity generation.
As discussed, existing technologies on biofuel production from complex
wastes, e.g., cellulosic wastes, utilize several steps including physiochemical
pretreatments, enzymatic hydrolysis, fermentation, and recovery of biofuels.
An alternative to the use of these multiple steps in biofuel production is the
development of a more efficient and cost-effective single step process using
saccharolytic fermentative extremophiles. Assuming the efficient utilization
of all fermentable sugars from lignocellulosic feedstocks, the waste products
of cellulosic biofuel (e.g. H2) production will be the leftover lignocelluloses
(unhydrolyzed fraction), lignin, organic acids produced by microbes, and
microbial cells. The bioconversion of this leftover organic waste to bioenergy
(e.g., value-added products, biogas) using other extremophiles can increase the
overall energy output of the process. A three-stage process can be developed
involving the fermentation of the lignocellulosic or solid waste feedstocks to
H2, CH4, and value-added products, as shown in Fig. 2.4. The organic waste
stream generated in the first stage reactor #1 can be used to generate value-
added products (e.g., EPS, PHA, or Proteins) in a second stage reactor #2. Also
left over lignocelluloses, lignin, and microbial cells of reactors #1 and 2 can
be transfer to reactor #3 for thermophilic anaerobic digestion.
Fig. 2.4 Integrated processes for BioH2 and value-added products production
using extremophiles.
In addition, isolation of new microorganisms capable of using the

lignocellulosic biomass without any pretreatment is a prerequisite for
bioprocessing. The recalcitrant nature of the lignocellulosic biomass is the
main factor that limits achievable yields and commercial profitability. Tools like
genetic engineering, metabolic engineering, mutagenesis etc. can help to improve
the strain with enhanced yields of biofuels and value added products. Using the
extremophiles in their native form or producing genetically modified organisms
(GMOs) using genes from extremophiles will certainly add a new dimension in
the biofuel bioprocessing area. Better yields and increased commercial revenues
necessitate development of such improved strains to minimize the gap between
achievable and achieved products.
In summary, the use of extremophiles in biofuel processes can make these
processes more economically feasible, environmental friendly, and easily
operable. In addition, high value polymer products with unique properties can
be derived from these microbes. Whereas further development of extremophilic
bioprocessing is a key factor in the successful commercial exploitation of
extremophiles in the applications discussed, other factors such as genetic
manipulations, and the discovery of new isolates can also play a major role in
moving the technology forward.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support provided by the
National Science Foundation – Industry/University Cooperative Research Center
(NSF-I/UCRC). The support from the Department of Chemical and Biological
Engineering at the South Dakota School of Mines and Technology is also
gratefully acknowledged.
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Biofuels and Value-added Products from Extremophiles

Chapter · January 2015

Mohit Bibra Jia Wang

SEE PROFILE SEE PROFILE

Rebecca J. Pinkelman Samuel Papendick

SEE PROFILE SEE PROFILE

B.Tech. Undergraduate - Research Project, June 2016 View project

Renewable Energy - Hydrogen Production View project

The user has requested enhancement of the downloaded file.

Keywords: Bioenergy, Butanol, Ethanol, Hydrogen, Methane, Polyhydroxyal-kanoates, and

*Corresponding Author: E-mail: Rajesh.Sani@sdsmt.edu

Fig. 2.1 Composition of various cellulosic wastes currently being

Physical pretreatments (i.e. chipping, grinding, and milling) are primarily

2.3 LIQUID AND GASEOUS BIOFUELS

On the other hand, several wild-type thermophilic microorganisms are

Fig. 2.2 Production of biodiesel. Possible roles of extremophiles

2.3.3.1 Algae (Lipid Source)

2.3.3.2 Lipases for Transesterification for Biodiesel Production

of 20°C and pH optimum of 8.0. Photobacterium lipolyticum M37 produces a

A variety of anthropogenic and natural sources such as kitchen wastes,

thermophiles produce higher yields of BioH2 only when pretreated lignocellulosic

hydrolysis of cellulosic substrates; (ii) higher mass transfer rates leading to

Table 2.1 H2 production from untreated lignocellulosic materials

A general mechanism for the AD of complex organics is shown in Fig. 2.3.

Fig. 2.3 Basics of anaerobic digestion.

2.3.5.1 Connecting Pretreatment and Anaerobic Digestion in

Thermal pretreatment also destroys pathogenic organisms and increases sludge

2.3.5.2 Integrating Anaerobic Digestion into Other Biofuel

2.3.5.3 General Considerations for Extremophilic Anaerobic

2.4 VALUE-ADDED PRODUCTS

hydroxybutyric acid and then metabolized to acetoacetate and acetoacetyl-CoA.

Haloferax mediterranei was used to produce PHA under hypersaline

considerably, is no longer limited to marine bacteria, and now applies to

2.4.2.1 Prokaryotic Producers of EPSs

shown to excrete EPSs in order to enhance their survival in extreme conditions.

Strains Source environment Extreme Chemical composition of Special Reference

Mannose:glucose = 1:trace Nicolaus et al.,

Geobacillus tepidamans Velingrad hot spring, 60°C Glucose:galactose:fucose: Degradation at Kambourova

extremophilic microorganisms will provide a valuable resource not only for

In addition, isolation of new microorganisms capable of using the

2010, Pretreatment methods to improve sludge anaerobic degradability: A review,

feedstock via one-step process, International Journal of Hydrogen Energy, 37,

Manca MC, Lama L, Improta R, Esposito E, Gambacorta A & Nicolaus B, 1996,

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