Chapter 7
Science of Alternative Feedstocks
Hans P. Blaschek and Thaddeus C. Ezeji21
Introduction
Biomass, which represents both cellulosic and non-cellulosic materials, contains the most
abundant source of fermentable carbohydrates that can be fermented into fuels and chemicals.
According to the Department of Energy (DOE) “Roadmap for Biomass Technology in the United
States,” biobased transportation fuels are projected to increase from the 0.5% of U.S. consumption in
2001 to 4% in 2010, 10% in 2020, and further to 20-30% in 2030, or about 60 billion gallons of
gasoline equivalent per year. The production of ethanol from low-cost lignocellulosic biomass that
does not compete with food crops may be the key to meeting the DOE target. This approach would
be consistent with ethanol as an economically viable and sustainable energy source.
As a result, interest in the cultivation of lignocellulosic crops such as switchgrass and
Miscanthus for subsequent conversion into fermentable sugars is receiving considerable attention.
In addition, industrial and agricultural co-products such as corn fiber, corn stover, dried distillers’
grains with solubles (DDGS), wheat straw, rice straw, soybean residues, as well as various types of
agricultural and industrial wastes are presently considered as potential feedstocks for the production
of fermentable sugars. The depolymerization of these renewable and abundant resources to
fermentable sugars represents a challenge for microbiologists and chemical engineers due to their
recalcitrant nature. One of the initial steps in the lignocellulosic biomass-to-fermentable sugars
conversion process is pretreatment. The purpose of pretreatment is to alter the biomass macroscopic
and microscopic structure as well as its sub-microscopic chemical composition, thereby allowing
cellulase enzymes to access the cellulose with greater ease in order to obtain a greater yield of sugars
[Mosier et al., 2005]. Depending on the ethanol production route, pretreatment may or may not be a
factor in the conversion of lignocellulosic biomass to ethanol. Pretreatment adds approximately
30% to the cost of processing of the biomass. Various strategies have been developed to utilize
alternative substrates for conventional and unconventional ethanol fermentations.
This report reviews recent approaches for utilization of alternative feedstocks for ethanol
production, including different ethanol production routes, as well as for selecting and manipulating
the fermenting microorganisms in order to achieve better product specificity and yield.
21
Professor and Research Assistant, respectively, Department of Food Science and Human Nutrition, University of
Illinois, Urbana-Champaign.
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Terminology
To facilitate the understanding of some of the terms used in this chapter, below are a few
key definitions:
•
Butanol is a four carbon alcohol. It can be produced via clostridial fermentation.
•
Cellulose is a polymer of glucose. Unlike starch, the glucose monomers of cellulose are
linked together through β-1-4 glycosidic bonds by condensation resulting in tightly
packed and highly crystalline structures that are resistant to hydrolysis.
•
Distillation is a method of separating chemical compounds based on their differences in
volatility; and volatility is a measure of the speed at which a chemical compound
evaporates.
•
Esterification is a general name for a chemical reaction between alcohols and acids
(carboxylic acids, mineral acids, and acid chlorides) to form compounds called esters.
•
Fermentation is microbial conversion of carbohydrates into alcohols or acids by
microorganisms. The most common type of fermentation involves product production in
the absence of oxygen.
•
Gasification involves a group of processes that turn biomass into combustible gas by
breaking apart the biomass using heat and pressure to produce a combustible gas,
volatiles, char, and ash. The gases can then be used as a fuel or feedstock chemical.
•
Glucan is the anhydrous form of D-glucose as found within a polysaccharide such as
starch or cellulose that has 1 molecule of water (18 g/mol) less mass due to a
condensation reaction forming the polymer,C6H10O5
•
Hemicellulose is a highly branched and substituted polymer comprised mainly of xylose
and arabinose, with minor amounts of galactose and glucose. In the plant cell walls,
hemicellulose holds crystalline microfibers of cellulose in place.
•
Hydrogenolysis is the process of cleaving a molecule or compound with the addition of
hydrogen atoms.
•
Hydrolysis is the breaking of a glycosidic bond (formed through a condensation reaction)
within a polysaccharide chain through the addition of water.
•
Saccharification is the process of hydrolyzing a complex carbohydrate into a simple
soluble fermentable sugar. Starch or oligosaccharides can be saccharified to produce
glucose using glucoamylase enzyme.
•
The solubles in DDGS contain residual oligosaccharides, organic acids, and non-volatile
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metabolic by-products of the yeast-based ethanol fermentation.
Biomass Composition and Depolymerization
Typically, lignocellulosic biomass contains 56-72% fermentable carbohydrates (cellulose
and hemicellulose) by dry weight (Figure 1; Table 1). Due to the nature of lignocellulosic biomass, it
can be found virtually everywhere in our environment ranging from plants to municipal wastes.
Given the right conditions, these different biomass sources can be fermented to ethanol and other
liquid fuels (Figure 2). Cellulose, the major constituent of lignocellulosic biomass, is a polymer of
glucose. Unlike starch, the glucose monomers of cellulose are linked together through β-1-4
glycosidic bonds resulting in tightly packed and highly crystalline structures that are resistant to
hydrolysis. Cellulose fibers are embedded in a lignin-hemicellulose matrix and this property
contributes to the recalcitrance of lignocellulosic biomass to hydrolysis. Therefore, pretreatment of
lignocellulosic biomass before enzymatic hydrolysis is a vital step.
Lignin
Hemicellulose
22-30%
15-27%
Cellulose
35-48%
Figure 1. Typical Lignocellulosic Biomass Composition
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Table 1. Composition of Representative Potential Lignocellulosic Raw Materials For Ethanol Production
% Dry weight basis
Corn
Corn
fiber
Corn
stover
DDGS
Wheat
straw
Sugarcane
bagasse
Switchgras
s
Poplar
Hexan
Glucan
2.4a
36.5
36.1
22
36.6
38.1
32.2
39.8
Galactan
2.9
2.5
0.3
2.4
1.1
0
0
Mannan
NA
1.8
NA
0.8
NA
0.4
2.4
Total
71.7b 39.4
40.4
22.3
39.8
39.2
32.6
42.2
Pentan
5.5c
Xylan
18.4
21.4
9.5
19.2
23.3
20.3
14.8
Arabinan
13.3
3.5
5.5
2.4
2.5
3.7
1.2
Total
5.5
31.7
24.9
15
21.6
25.8
24
16
Total fermentable 79.6
71.1
65.3
37.3
61.4
65
56.6
58.2
Lignin
0.2
6.9
17.2
3.1
14.5
18.4
23.2
29.1
NA: not available. Data on switchgrass and poplar from [Chung et al., 2005]. Data on wheat straw and sugarcane
bagasse from [Lee, 1997]. Data on corn stover from [Laureano-Perez et al., 2005]. Data on corn fiber and dried distillers
grains and solubles (DDGS) from author’s laboratory [unpublished]. Data on corn from [Gulati et al., 1996]. a:
cellulose; b: starch; c: xylan + arabinan.
Types of Biomass
Sugar-based: sugarcane, sugar beets
Starch-based: corn, potatoes, barley,
wheat, etc
Lignocellulosic: Switchgrass,
Miscanthus, corn stover,
corn fiber, DDGS, etc
Animal waste: cow, swine, and
poultry
Food waste
Municipal waste
Liquid biofuel
Methanol CH 3OH
(Bio)conversion
Ethanol C2H5OH
Butanol C 4H9OH
Feedstock chemical
e.g. acetic acid
Industrial (especially food industry)
waste
Figure 2. Biomass Conversion to Liquid Biofuels and Feedstock Chemicals
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Hemicellulose, the second major constituent of lignocellulosic biomass, contributes
significantly to the total fermentable sugars of the lignocellulosic biomass (Table 1 & Figure 1).
Unlike cellulose, hemicellulose is chemically heterogeneous and easily hydrolyzed to its constituent
monosaccharides. Depending on the plant source, these monosaccharides may include hexoses
(glucose, galactose, mannose, rhamnose) and pentoses (xylose, arabinose). Acetate and uronic acids
(glucuronic and galacturonic acids) are also constituents of the hemicellulosic component of
lignocellulosic biomass. These compounds have been found to be a potential source of microbial
inhibitors in lignocellulosic hydrolysates (Ezeji et al., 2007).
Lignin is a condensate of products obtained from lignin monomers such as p-coumaryl
alcohol, coniferyl alcohol, and sinapyl alcohol (Klinke et al., 2004). While cellulose and
hemicellulose contribute to the amount of fermentable sugars for ethanol production, products of
lignin degradation are recognized as a potential source of microbial inhibitors (Ezeji et al., 2007). It
was proposed over two decades ago that the lignin fraction of biomass be used as an ash-free solid
fuel for the generation of energy. To date, there is no known study on the feasibility and scalability
of this approach. But can we harness the energy content of lignin through an indirect ethanol
production route. This will be discussed later in the chapter.
Ethanol Production from Alternative Feedstocks
Feedstock costs make up a significant portion of the ethanol production costs and the
selection of readily available and cheap feedstock is vital to the economics of the ethanol
fermentation. Extensive research is currently being carried out on the various processes that can be
used to produce ethanol from heterogeneous feedstock such as lignocellulosic biomass. We
therefore contend that the choice of feedstock will determine the ethanol production route (whether
conventional or unconventional). In addition to the conventional ethanol production route, we have
identified two alternative ethanol production routes using alternative feedstocks which we believe
have great potential for scale-up and commercialization.
Conventional Ethanol Production
In conventional ethanol production, starches and sugars are converted into ethanol in a few
steps involving a series of enzymes. Thermostable α-amylase is used in the presence of water and
heat to liquefy the starch, followed by glucoamylase which saccharifies the liquefied starch to
sugars. Subsequently, a biocatalyst is added in the form of yeast that ferments the sugars to ethanol.
This is followed by distillation of the beer to produce pure ethanol (Figure 3). The overall reaction
shows the conversion of glucose to ethanol and CO2, which can be represented stoichiometrically as
follows:
Starch
+
Amylolytic
enzymes
C6H12O6
+
Yeast
2C2H5OH + 2CO2 + ATP
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This process is a mature technology that is carried out industrially. When lignocellulose is
the substrate for ethanol production, there may be some drawbacks, however. These drawbacks
relate to the complexity of the lignocellulosic cell wall components that make up the biomass.
Furthermore, hydrolysis of the hemicellulose portion of the lignocellulosic biomass generates
significant amounts of pentose sugars and potential microbial inhibitors such as uronic, ferulic and
acetic acids. The presence of these compounds in the hydrolysates exerts undue stress on the
fermenting microorganism, leading to poor cell growth and low ethanol titers and productivity.
Unconventional Ethanol Production
Gasification and Fermentation
One unconventional ethanol production route involves gasification of the biomass, such as
switch grass, Miscanthus, crop residues, etc. to generate producer gas. The producer gas, composed
primarily of CO, CO2, H2, and N2, is fermented by a biocatalyst to ethanol and acids. Recent work
by Datar et al., 2004 and Ahmed & Lewis, 2007, demonstrated the integration of a fluidized-bed
gasifier with a bioreactor to utilize biomass-syngas for producing ethanol. The process is illustrated
in Figure 4. The combined technologies of gasification and fermentation have the potential to
produce ethanol from recalcitrant lignocellulosic biomass. One major advantage of this process is
that the carbon-rich lignin fraction (which contains 50% more carbon than the fermentable
carbohydrates) is converted to producer gas and then to ethanol. Pretreatment and hydrolysis of
lignocellulosic biomass to produce fermentation sugars is totally eliminated when ethanol is
produced via the gasification and fermentation route.
Fermentation Esterification Hydrogenolysis Process
A second unconventional ethanol production route is a radically new approach for producing
fuel ethanol from biomass. The process involves fermentation, esterification, and hydrogenation.
During the first step, monosaccharides are fermented to acetic acid at near 100% carbon yield by
homoacetogenic microorganisms. In the second step, the acetic acid is esterified in the presence of
an alcohol to produce an ester, while in the third step; the ester undergoes hydrogenolysis to produce
ethanol (ZeaChem, Inc.). The process is stoichiometrically illustrated in the following reactions:
Fermentation
Esterification
Fermentation: C6H12O6
Hydrogenolysis
Ethanol
O
3CH3COH [Acetic acid]
O
Esterification: 3CH3COH +3ROH
3CH3COR [Acetate ester]+ 3H2O
Hydrogenolysis: 3CH3COR + 6H2
3C2H5OH + 3ROH [alcohol]
Net:
C6H12O6 + 6H2
3CH3CH2OH + 3H2O
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In this process, 3 moles of ethanol are produced from 1 mole of glucose resulting in a 50%
improvement over the conventional route where 2 moles of ethanol are produced from 1 mole of
glucose. The energy for the third mole of ethanol is supplied by hydrogen (ZeaChem, Inc.), which
can be generated by the gasification of the lignocellulosic biomass. We therefore suggest that in
order to accelerate the development of an efficient ethanol production process using alternative
feedstocks, research in this area should be diversified to include unconventional ethanol production
routes.
Alternative Feedstocks
The substrate commonly used in the United States today for fuel ethanol production is starch
from agricultural crops, primarily corn. In order to meet the DOE projected bioenergy target in the
year 2030, the use of alternative feedstocks for fuel ethanol production must increase dramatically.
A joint study by the USDA and the U.S. Department of Energy concluded that at least 1 billion tons
of biomass in the form of corn stover, wheat straw, wood wastes, etc. could be collected and
processed in the U.S. each year which would be independent of the food supply. These feedstocks
represent an equivalent of over 65 billion gallons of ethanol, with a potential for replacing 30% of
the gasoline consumption in the U.S. (U.S. Department of Energy Biofuels: 30% by 2030 Website).
Of course there are significant regional differences in the availability of lignocellulosic feedstocks
which must be considered. While corn stover is abundant in the Midwestern States of U.S., rice
straw will be an important feedstock source in California and Texas and soft woods will dominate in
the Southeastern U.S.
Dried Distillers’ Grain and Solubles (DDGS) and Corn Fiber
Dried distillers’ grain with solubles (DDGS) are residues (proteins, fiber, and oils) obtained
following the yeast fermentation of saccharified whole corn grain. DDGS is produced by blending
corn distillers’ liquid solubles with wet corn distillers’ grains and the mixture is dried. The number
of dry grind ethanol plants is growing rapidly in the United States. The boom in construction of drymill based ethanol plants is evidence of the biobased opportunities in this area. For every bushel of
corn being converted into ethanol, 18 lb of DDGS is generated, which can be further converted to
approximately 6.2 lb of fermentable sugars. In a concerted effort undertaken by the Midwest
Consortium for Sustainable Biobased Products and Energy to address the proliferation of low value
DDGS, a team of scientists from Purdue University, Michigan State University, University of
Illinois, Iowa State University, Ames Laboratory, and USDA NCAUR is conducting research to
further process DDGS into fermentable sugars for the production of ethanol and butanol (a secondgeneration liquid fuel), while leaving a solid that is reduced in weight and rich in protein.
Furthermore, corn fiber, a co-product from the corn wet milling industry represents another
readily available alternative feedstock for fuel ethanol production. Approximately 13.9 billion
pounds of corn fiber is produced annually in the U.S. Corn fiber contains about 70% fermentable
sugars, of which approximately 20, 14, and 35% come from starch, cellulose, and hemicellulose,
respectively. Currently, corn fiber is marketed as a low cost animal feed ingredient. The further
utilization of corn fiber and DDGS to produce value-added products such as ethanol and butanol is
consistent with the National Renewable Energy Laboratory (NREL) sugar-platform biorefinery
model for future dry-grind plants. Such an integrated approach is expected to contribute to biofuel
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profitability and efficiency.
Corn Stover
Corn stover represents about 50% of the corn plant. Approximately one ton of corn stover is
generated from one ton of corn grain produced. Corn stover is made up of about 50% stalks, 23%
leaves, 15% cob, and 12% husks. Corn stover contains about 66% fermentable sugars, of which
approximately 38 and 28% come from cellulose, and hemicellulose, respectively. Producing ethanol
from corn stover hydrolysates will increase the possibility of ethanol production on a larger scale.
How much of the corn stover that can be collected without detrimental effect on the soil is a
complex question whose answer is beyond the scope of this report. However, one school of thought
suggests that about 85% of corn stover residue rots on the ground which releases CO2 into the
atmosphere. The remaining 15% is incorporated in soil as organic matter. Another estimate suggests
that 50% of the corn stover left after harvest can be collected without negative impact on the soil
carbon. Other critical factors such as erosion control, soil moisture retention and regional climate
must also be considered when harvesting corn stover.
Switchgrass and Miscanthus
Interest in the cultivation of lignocellulosic crops such as switchgrass and Miscanthus for
subsequent conversion into fermentable sugars is receiving considerable attention. Switchgrass
contains about 56.6% fermentable sugars and 23.2% lignin (Table 1). On the other hand,
Miscanthus contains about 68% fermentable sugars, of which approximately 44 and 24% come from
cellulose, and hemicellulose, respectively (http://bioenergy.ornl.gov/papers/misc/biochar_
factsheet.html). In addition, Miscanthus contains about 17% lignin. By applying the appropriate
processing conditions, these fermentable sugars and the lignin should be convertible to ethanol.
Miscellaneous (Wheat and Rice Straws, Sugarcane Bagasse, Wood, Etc.)
For decades, farmers in the rice-growing region north of Sacramento, California have
collected left over rice straw into heaps and burned them after harvest. However, in the early
1990s, California lawmakers passed a law and put in place a program for rice farmers to
gradually discontinue routine burning in order to reduce the associated production of smoke and
its impact on urban areas. As a result, rice farmers have been looking for alternative ways to
utilize or dispose of rice straw. Table 2 shows different lignocellulosic biomass materials which
have potential for ethanol fermentation. If these feedstocks are broken down into sugar
monomers, the released sugars can be used to make ethanol. This lignocellulosic ethanol can
also be purified using the same technology as corn-based ethanol production (Figure 3).
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Table 2. Percentage of Cellulose, Hemicellulose and Lignin Content in Common
Agricultural Residues and Wastes
Agricultural residue
Cellulose
Hemicellulose
Lignin
Hardwood
40–50
24–40
18–25
Softwood
45–50
25–35
25–35
Corn cobs
45
35
15
Grasses
25–40
35–50
10–30
Wheat straw
33–40
20–25
15–20
Rice straw
40
18
5.5
Source: Prasad et al. (2007), Kaur et al. (1998), and McKendry (2002)
Figure 3. Process of Making Ethanol From Whole Corn
(Source: Genencor, with Modification)
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Lignocellulosic Biomass Pretreatment For Ethanol Production
About 98% of the ethanol produced in the United States today is produced by dry or wet
milling processes using corn. These processes are relatively simple: corn grinding (and starch
separation in the case of wet milling), liquefaction, saccharification, fermentation, distillation,
and dehydration (Figure 3). Alternatively, biomass is composed of lignin, cellulose, and
hemicellulose. One of the primary functions of lignin is to provide structural support for the
plant; and unfortunately from the standpoint of biofuel production, it also encloses the cellulose
and hemicellulose molecules. In addition to the structural characteristics of biomass feedstock,
the encapsulation of cellulose by lignin makes the lignocellulosic biomass nearly inaccessible to
hydrolytic enzymes, and as a result more difficult to hydrolyze than more traditional starchy
materials. Therefore, one of the key steps in the lignocellulosic biomass-to-fermentable sugars
conversion is pretreatment.
The purpose of pretreatment (Figure 5) is to break the lignin-hemicellulose matrix in
order to facilitate cellulase enzymes access to the cellulosic portion of the biomass for
subsequent hydrolysis to glucose. The common methods that have been employed to make
lignocellulosic biomass more accessible to hydrolysis are dilute acid, alkaline, hot water, and
ammonia pretreatments. Due to the recalcitrant nature of these lignocellulosic feedstocks, their
pretreatment often requires a combination of physical, chemical, and heat treatments to disrupt
the structure and convert it into a more hydrolysable form. The complete depolymerization of
these renewable feedstocks in a cost-effective manner with minimal formation of degradation
products represents a significant challenge for microbiologists and chemical engineers.
Figure 5. Schematic of Goals of Pretreatment on Lignocellulosic Material (Adapted From
Hsu et al., 1980).
Dilute sulfuric acid pretreatment can be applied to agricultural residues to bring about
hydrolysis. This is the oldest technology for converting lignocellulosic feedstock to fermentable
sugars with subsequent fermentation to ethanol. Unfortunately, during acid hydrolysis, a
complex mixture of microbial inhibitors is generated. Lignins are oxidized or degraded to form
phenolic compounds and parts of the sugars that are released during hydrolysis are also degraded
into products that inhibit cell growth and fermentation. Examples of the inhibitory compounds
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that may be produced include furfural, hydroxymethyl furfural (HMF), and acetic, ferulic,
glucuronic, ρ-coumaric acids (Zaldivar et al., 1999; Ezeji et al., 2007a). These inhibitors can be
divided into three groups based on their origin: (1) compounds released from the hemicellulose
structure, e.g. acetic, ferulic, glucuronic, ρ-coumaric acids, etc; (2) lignin degradation products,
e.g. syringaldehyde; and (3) sugar degradation products, e.g. furfural and hydroxymethylfurfural.
Using a single feedstock (corn stover), common analytical protocols, and consistent data
interpretation, five research teams documented the technical and economical feasibility of
selected pretreatment techniques (Wyman et al., 2005; Eggeman and Elander, 2005). They
found that, among the dilute acid (Lloyd and Wyman, 2005), hot water (Mosier et al., 2005b),
ammonia fiber expansion (AFEX) (Teymouri et al., 2005), ammonia recycle percolation (ARP)
(Kim and Lee, 2005), and lime (Kim and Holtzapple, 2005) pretreatments, low cost pretreatment
reactors are often counterbalanced by the higher costs associated with either pretreatment
catalyst recovery or down-stream processing. A summary of the pretreatment methods and their
limitations is given in Table 3.
Table 3. Summary of Biomass Pretreatment Methods For Ethanol Production
Pretreatment
1
Increases
Decrystalizes
Removes
Removes
surface
cellulose
hemicellulose
lignin
area
Dilute acid
Yes
Yes
Hot water2
Yes
Yes
AFEX3
Yes
Yes
Yes
4
Yes
Yes
Yes
Lime5
Yes
ND
Yes
ARP
a
Limitations
Corrosion, neutralization,
formation of inhibitors, and
disposal of neutralization salts
High temperature, need to add
alkaline to control pH, and
relatively high cost in product
recovery
Cost of ammonia
Cost of ammonia and relatively
low conversion of xylose
Long treatment time, relatively
low conversion of xylose, and
relatively low solids
Cumulative soluble sugars as (oligomers + monomers)/monomers. Single number = just monomers. ND: Not
determined. be-hydrolysis = enzymatic hydrolysis. 1 Lloyd and Wyman (2005); 2Mosier et al. (2005b); 3Teymouri et
al. (2005); 4Kim and Lee (2005); 5Kim and Holtzapple (2005).
The complete hydrolysis of hemicellulose requires xylanase, β-xylosidase, and several
other complimentary enzymes such as acetylxylan esterase, α-arabinofuranosidase, αglucuronidase, α-galactosidase, ferulic and/or p-coumaric acid esterase (Ezeji et al., 2007b). The
activities of these enzymes, in addition to the activities of cellulases on the cellulose component
of the biomass, result in the generation of complex mixture of acids (ferulic, p-coumaric, acetic,
glucuronic) in addition to monomeric sugars such as glucose, galactose, xylose, and arabinose in
the biomass hydrolysates. Acids such as ferulic and p-coumaric have been found to be inhibitory
to the solventogenic clostridia (used for producing butanol and acetone) at concentrations as low
as 0.3 g/L [Ezeji et al., 2007a]. Therefore, for complete depolymerization of lignocellulosic
122
biomass, it is difficult to totally avoid the generation of inhibitory compounds irrespective of the
pre-treatment and hydrolysis method utilized.
A closer look at Table 3 reveals that the acidic pretreatment methods (including hot
water) are effective in removing hemicellulose while the alkali methods (AFEX, ARP, and lime)
function to remove lignin and decrystallize the cellulose. Both acidic and alkaline pretreatments
are known to improve enzymatic digestibility. In addition, economic analysis of the
pretreatment methods has shown that the relatively high costs associated with ethanol production
from lignocellulosic biomass arise mainly from three factors: a) harsh pretreatment conditions
(high temperature, use of acids or bases, etc.), b) use of costly enzymes, and c) recovery of end
products (Eggeman and Elander, 2005). Technologies that lead to improvement in any of the
above areas will help to improve the profitability of a biofuel production operation (Dr. Hao
Feng, University of Illinois Urbana-Champaign, personal communication).
Strain Selection and Improvement For Alternative Feedstock Utilization
The fermentation of lignocellulosic biomass to ethanol is both timely and challenging. The
ability to utilize all the sugars present in the lignocellulosic feedstock is necessary for efficient
production of ethanol. It is not clear which of the three major microbial platforms (yeast,
anaerobic Gram-positive bacteria such as clostridia, or Gram-negative microbes such as E. coli,
Zymomonas mobilis) for biofuel production will be the primary production microbes of the
future.
Yeast remains the traditional microorganism of choice for ethanol production due to its
high tolerance for ethanol during fermentation. The use of yeast involves fermentation of
glucose and sucrose to ethanol. Efficient fermentation of lignocellulosics to ethanol by yeast is
difficult due to the heterogeneity (pentoses and hexoses) of the feedstock. Metabolic
engineering research is being carried out to develop new industrial yeast strains with the ability
to efficiently convert lignocellulosic hydrolysates to ethanol. However, engineering
Saccharomyces cerevisiae to co-ferment hexose and pentose sugars is constrained by the
stoichiometric feasibility of the enzymatic activities of the introduced genes and the physiology
of the yeast. Recombinant S. cerevisiae developed in several laboratories have used xylose
oxidatively as opposed to fermentative utilization of glucose for ethanol production (Jin and
Jeffries, 2004). Since xylose is a major constituent of lignocellulosic hydrolysate, ethanol
production from xylose (with comparable ethanol yield from glucose) is essential for successful
utilization of this feedstock for ethanol production.
Dr. Lee Lynd has examined simultaneous hydrolysis and fermentation using the
anaerobic bacterium Clostridium thermocellum to make ethanol from cellulosic feedstocks
(Lynd, 1989; Lynd, 1996; Lynd et al., 1991), but there is a problem. C. thermocellum is capable
of hydrolyzing cellulose and xylan to glucose and xylose, respectively. However, this
microorganism can only utilize hexoses (and not the pentose sugars) generated from cellulose
and hemicellulose (Demain et al., 2005). On the other hand C. thermosaccharolyticum (Saddler
et al. 1984, Venkateswaren& Demain, 1986), C. thermohydrosulfuricum (Germain et al., 1986),
and Thermoanaerobacter ethanolicus (Wiegel & Ljungdahl, 1981) are capable of utilizing
hexose sugars as well as pentose sugars. As a result, the use of mixed culture systems where
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cellulose is broken down by the cellulase complex of C. thermocellum to sugars and the
fermentation of these sugars by this microorganism in combination with pentose utilizing
microorganisms to ethanol is of great interest (Demain et al., 2005). One such mixed culture
system involved C. thermocellum and C. thermosaccharolyticum for ethanol production when
using cellulosic feedstock as a carbon source (Duong et al., 1983). However, the formation of
co-products such as acetate and lactate that decrease the yield of ethanol and can act as weak
uncouplers and inhibit cell growth (Herrero et al. 1985) have been a problem. More work is
needed to address this problem. One potential solution is the elimination of metabolic pathways
for co-products formation in order to make ethanol as the sole or major fermentation product,
thereby enhancing ethanol yield. One obvious approach is to knock out the genes (encoding
acetate kinase and/or phosphotransacetylase and lactate dehydrogenase) that are responsible for
the branched metabolic pathways (Demain et al., 2005). The effect of this approach on the
physiology of the microorganisms needs to be investigated.
The question of which microbial platform will dominate as biocatalyst in the quest for
alternative feedstock utilization for ethanol production will depend on which area (yeast, Grampositive and Gram-negative bacteria) of research is most successful. When strains that can
efficiently utilize mixed pentose and hexose sugars for ethanol production are developed, the
process will require that these microbes also have a high tolerance for degradation products such
as acetic acid, furfural, HMF, ferulic acid, etc.
Conclusions
The rationale behind seeking alternative feedstocks for ethanol production is the
continued increase in energy demand worldwide, notably in China, India, United States, and the
United Kingdom. The use of alternative feedstocks such as lignocellulosic biomass for ethanol
production holds great promise due to its widespread availability and abundance. This feedstock
is available in the form of agricultural and forestry residues, and industrial and municipal wastes.
Due to the recalcitrant nature of lignocellulosic biomass to enzymatic hydrolysis, several
investigations have been carried out by agronomists and plant geneticists to improve biomass
characteristics. These include development of plants that self-produce cellulases, plants with
low lignin content, high polysaccharide content, and higher overall plant biomass yield.
Significant progress has been made in this area, but fermentation of lignocellulosic biomass to
ethanol still remains a challenge due to the necessity of converting both hexose and pentose
sugars (constituents of the hydrolysates) obtained from this feedstock to ethanol in a single
fermentation step. In addition, the generation of degradation and microbial inhibitory products
during pretreatment and hydrolysis of biomass results in poor growth and ethanol productivity
by the fermentation microorganisms. To circumvent these problems, more research effort needs
to be directed toward unconventional methods for ethanol production. For instance, lignin and
other non-fermentable materials account for nearly 40% of the energy content of the
lignocellulosic biomass. This portion can be harnessed and utilized for ethanol production via
the simultaneous gasification and fermentation process (Figure 4).
124
Biomass: Energy crops, Crop
residues, Animal waste, Food
waste, etc
Bioreactor: Fermentation of
producer gas to ethanol (and
other value-added products)
Gasifier: Conversion of
biomass to producer gas
(H2, CO2, CH4, CO, N2)
Clostridium ljungdahlii, C.
autoethanogenum, C.
carboxidivorans P7T, etc
Ethanol
Figure 4. Process For Ethanol Production By Simultaneous Gasification and Fermentation
125
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