North Carolina State University
CHE596-015
Technical Report
Technical Objectives for Fermentation Operations in
Cellulosic Ethanol Production
by xxxxx
Prepared for Assignment
in
Engineering Challenges at the Energy Frontier
North Carolina State University
Department of Chemical Engineering
Raleigh, NC 27606
xxxx, 2011
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Technical Objectives for Fermentation Operations in Cellulosic Ethanol Production
xxxxxxxxx
Department of Chemical Engineering
North Carolina State University
Raleigh, NC 27607, USA.
Background
The 30x30 goal has been set by the United States Department of Energy to replace 30% of the nation’s gasoline consumption with
60 billion gallons of ethanol production per year by the year 2030. Currently, the majority of the production of ethanol in the United
States is made from brewing and distilling yellow feed corn. Ethanol produced from sources that significantly compete with food and
feed is considered a generation one (Gen 1) biofuel. Ethanol is a combustible liquid that can be used in the transportation industry
as an alternative to gasoline. Ethanol has a 30% lower fuel density as compared to gasoline, but when used as an additive in
gasoline it can increase the fuel octane. It is most commonly sold as a gasoline additive blended to 10% ethanol (E10) to replace
the methyl tert-butyl ether (MTBE) additive which has been previously used to oxygenate the fuel. Ethanol can also be blended at
higher levels with gasoline, but requires mechanical modification to the automobiles fuel system as ethanol in high concentrations
can be corrosive. The most common ethanol rich blend of ethanol in the United States is blended to 85% ethanol and 15% gasoline
(E85), but most E85 fueling stations are located around Midwestern ethanol production facilities.
As the demand for ethanol continues to grow, there will be a great amount of stress put on ethanol feedstock suppliers and this will
eventually generate market driving forces to push the growth into generation two (Gen 2) biofuels. Gen 2 ethanol is cellulosic
ethanol produced from lignocellulosic (woody) biomass either through biochemical or thermochemical conversion processes. There
are many unit operations involved in
the biochemical conversion of biomass
to ethanol that must be optimized in
order to make the ethanol produced via
this pathway economically feasible and
for the process to be viable. The three
main areas that control the selling price
of ethanol are feedstock cost and
availability, substrate pretreatment
yield, and the enzyme cost and
enzymatic hydrolysis performance.
Naturally, these areas attract the
majority of the attention and funding for
research in the biofuels area; however,
it is important not to ignore the other
unit operations. Figure 1 shows the
many potential biofuel conversion
pathways, but this technical report will
focus on the current research and
development involving the fermentation
process and the role of fermentation in
creating a successful bioconversion to
Figure 1. Biofuel conversion technologies.1
ethanol process.1
Cellulosic Ethanol Review
Biomass Handling
Cellulose is the most abundant naturally occurring polymer in the world. The ability to utilize this renewable substrate found in many
natural sources is critical for the production of biofuels. There are many feedstocks that contain high contents of cellulose that can
be broken down to form ethanol, and these feedstocks can be separated into different classes including agricultural residue, forestry
residue, grasses, municipal and other wastes, and trees. These sources include, but are not limited to, corn stover, cotton stalks,
straw, switchgrass, bagasse, energy cane, wood chips, sawdust, forest trimmings, paper, plant-derived garbage, spruce, pine, and
fast-growth trees such as poplar, eucalyptus, and willows. An availability and energy content assessment needs to be conducted to
determine which feedstocks provide the greatest process conversion yield.2
Pretreatment
After realizing the multitude of lignocellulosic feedstocks available, it is important to understand how to free the polysaccharides from
the rest of the components of the biomass. The composition of each lignocellulosic feedstock varies but most are comprised of
approximately 50% cellulose, 25% hemicellulose, 20-25% lignin, and 1-5% extractives. To expose and free the cellulose it is
common practice to use a pretreatment. This process is used to solubilize and remove the lignin which can inhibit the enzymatic
hydrolysis and fermentation steps, and to retain as much of the cellulose and hemicellulose as possible. Some common
pretreatments for biochemical cellulosic ethanol conversion are weak alkaline hydrolysis (green liquor, soda, kraft), dilute-acid
hydrolysis, sulfite pretreatment to overcome recalcitrance of lignocellulosics (SPORL), ionic liquid dissolution, ammonia fiber
explosion (AFEX), steam explosion, CO2 explosion, organosolv, and white-rot fungal treatment. Each pretreatment method has its
specific strengths and weaknesses, but all of them have the ability to open up and increase the surface area of the biomass to allow
for increased enzyme to substrate interactions. 2 Recalcitrance is the natural ability of plant cell walls to resist decomposition from
microbes and enzymes and this phenomenon is what makes biomass pretreatment necessary.
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Cellulose Hydrolysis
After pretreatment, the material is charged with an enzyme cocktail to depolymerize the polysaccharides. Enzymes are proteins that
preferentially reduce the activation energy by catalyzing certain reactions. These cocktails generally include cellulase enzymes that
break down cellulose from the end of the chain (exoglucanase), from the middle of the chain (endoglucanase), and between
individual cellulobiose molecules (B-glucosidase) into individual glucose monomers. Hemicellulases, which break down
hemicellulose to pentoses, are often used as well. Enzyme performance is very sensitive to the reaction conditions and can easily
be inhibited by other molecules that decrease the enzyme activity or can be destabilized by changes in temperature, concentration,
etc.2
Fermentation
Once the enzymes have converted all of the biomass into a dissolved sugar solution, this mixture of hexoses and pentoses are sent
to the fermentor to convert the five and six carbon sugars into ethanol. The overall fermentation reaction is as follows:
2 C6H12O6→ 2 CH3CH2OH + 2 CO2
In this bioreactor, the oxygen flow is controlled to optimize ethanol production and to maintain microbe stability. This fermentation
process has been commercialized and is well established for the conversion of corn starch to ethanol, but the mixture of sugars for
lignocellulosic biomass sources remains challenging with many opportunities for improvement.2
Ethanol Recovery
Because most microorganisms are slightly product inhibited, they cannot be exposed to high concentrations of ethanol. This limits
the final ethanol titer, or beer concentration, in the accepts of the fermentation process, requiring the 5-10% ethanol-water solution
to be upgraded to fuel grade ethanol. Because of the azeotrope that exists between ethanol and water at atmospheric conditions,
the ethanol content can only reach 96% after basic distillation.
A molecular sieve, or some other type of separation process,
is required to remove the remaining water from the ethanol. A
stabilizer is generally added to the pure ethanol before storage
and distribution to avoid the absorption of water back to the
azeotrope. In theory, the ideal cellulosic ethanol conversion
process can utilize any number of different feedstocks with a
variable handling and pretreatment system that can
adequately open the substrate for optimum enzymatic
hydrolysis and fermentation and lead to a maximum sugar to
ethanol process yield. A diagram of such process is shown in
Figure 2. Biochemical cellulosic ethanol production process.3
Figure 2.3
Fermentation Introduction
In the biochemical conversion process to produce cellulosic ethanol, fermentation is the operation that uses yeast to anaerobically
consume the monomer sugars in the enzymatic hydrolysate accepts (mainly glucose and xylose) to yield ethanol as the desired
product and carbon dioxide as a by-product. This conversion process is shown in Figure 3.4 It is critical to understand that the
yeast cells are living microorganisms and not just simple nutrients or catalysts that can be added to the process to convert the
sugars to ethanol. In an aerobic environment, with a plentiful supply of oxygen, sugar, and other minerals, the yeast will rapidly
“bud” and produce daughter cells identical to the parent yeast cell. This phenomenon is important for inoculating and growing the
yeast used in industrial settings. The key to producing ethanol is providing the yeast with the sugar solution without any oxygen.
The yeast will use the sugar as energy, breaking the bonds and producing ethanol and carbon dioxide. Without oxygen, yeast is
only able to break one bond and its growth is restricted as the cell excretes the ethanol as waste.5
Figure 3. Fermentation process of glucose.4
There are many microorganisms that can produce ethanol through
the process of fermentation, but only a few have been proven to
have a high conversion yield and be stable enough to be
considered industrially viable.
These include saccharomyces
cerevisiae, pichia stipitus, and zymomonas mobilis. S. cerevisiae
yeast strains are very efficient fermentors of six carbon sugars.
This yeast is commonly known as “baker’s yeast” and has been
used since ancient times in baking as the rising agent for dough
and in many brewing applications, especially as a top-fermenting
yeast for the production of lager.6 Within the saccharomyces
genus, meaning that it too is a budding yeast, the species p. stipitus
is an important strain of yeast because it has the highest know
natural ability to directly ferment five carbon sugars like xylose.7 Z.
mobilis is another microorganism that has shown the ability to be
very promising in the bioethanol production industry. Unlike the
saccharomyces which are classified as fungi, z. mobilis is a
bacterium that converts glucose to the intermediate pyruvate via the
Entner-Doudoroff pathway. This bacterium is generally more
favorable than saccharomyces yeast strains because it has a higher
sugar uptake and ethanol yield, a higher ethanol tolerance, it does
not require controlled addition of oxygen during the fermentation,
and it is very amenable to genetic manipulations. Unfortunately z.
mobilis is limited to the utilization of only glucose as the substrate
and cannot naturally ferment xylose or any of the other sugars
isolated from the decomposition of hemicellulose.8
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Technical Advancements
Simple optimization has been done with naturally occurring microbes, but the process is slow, the final titer has low concentrations,
and the overall conversion and product yields do not reach the high sugar conversion that is desired. Dr. Nancy Ho, a professor at
Purdue University and the founder of Green Tech America, is one of the leaders in ethanol fermentation research. Since 1985 she
has been at the cutting edge of genetic engineering and has successfully developed the technology to allow any Saccharomyces
yeast to co-ferment glucose and xylose sugars to ethanol. This was achieved by cloning three highly modified xylose-metabolizinggenes (XR, XD and XK), cloned on a high-copy-number plasmid, and incorporating the plasmid into the yeast cells that already had
the capability of achieving high glucose to ethanol fermentation. Cloned genes on plasmids, although effective at a lab scale, are
not suitable for large-scale industrial production of ethanol, because they lack the robustness to prevent themselves from being
destabilized during industrial fermentation operations. From 1993 to 1995, Dr. Ho developed the stable engineered yeast with the
cloned genes integrated into the yeast chromosome. After successful development of an industrially stable, recombinant, cofermenting yeast was produced, large-scale screening was conducted for better yeasts with no constraints on converting the mixed
sugars obtained from cellulosic biomass to ethanol. More than ten new yeast strains were tested and integrated with the xylose
fermenting genes, and two strains were manufactured (424A and 259A) that were industrially viable and free of royalties to any
group or foreign company. Since 2002, Dr. Ho’s group has been working on developing strains that will also effectively ferment two
other minor sugars found in the biomass hydrolysate and further improving the yeast to ferment xylose and other sugars 30 to 75%
faster. She has also successfully engineered yeast capable of producing high-value co-products during ethanol production that can
be separated after fermentation.9
Not only have technological advancements been made in improving the actual yeast strain, but research and process design
simplification have also been done to improve the dynamics of the fermentation operation. There are three main scenarios that
have been studied for industrial fermentation in the biochemical cellulosic ethanol conversion process. The first requires a
detoxification step to neutralize and eliminate any inhibitors, such as acetic acid, furfural, hydroxymethylfurfural (HMF), formic acid,
and levulinic acid.10 Following the detoxification, hexose and pentose fermentation can be performed in series, or simultaneously in
the same vessel with either two separate microbes or one genetically modified microbe. The second method involves combining
fermentation with the enzymatic hydrolysis step. This is called simultaneous saccharification and cofermentation (SSCF or SSF).
Combining these two processes would require developing a thermophilic ethanol-producing organism that can tolerate high
temperatures, but has the benefit of significantly reducing the cellulase requirement for the process. Oftentimes the enzymatic
hydrolysis process is product limited by the glucose concentration, so if the process is combined with fermentationand glucose is
consumed continuously by fermentation microorganisms then the process would require a lower cellulase charge. The cellulase
would be produced and charged to the process as a separate step. These new strains would need to be capable of producing
ethanol at 50oC and pH 6.0 which are the optimum conditions for enzymatic hydrolysis. The third method is the ultimate
combination of cellulase production, cellulose hydrolysis, and cofermention of five carbon and six carbon sugars termed
“consolidated bioprocessing” (CBP). This is considered to be the most cost effective configuration for cellulose hydrolysis in both
near-term and long-term contexts.11
Technical Goals
In June 2006, the US Department of Energy generated a research roadmap for breaking the biological barriers associated with
biomass to biofuels production.11 This document outlined the technical milestones for five, ten and fifteen years in the future that
needed to be achieved to develop a successful and competitive sugar to ethanol fermentation process. Table 1 outlines the
technical milestones for the scientific challenges of genetic, metabolic, and evolutionary engineering of microorganisms. Table 2
details the key milestones for process simplification opportunities from separate fermentation to SSCF and eventually CBP. 11
Table 1. Fermentation scientific advancement milestones.11
Within 5 years
■ Mesophilic microbes demonstrated at scales that are capable of full utilization of all lignocellulosic sugars for reduced
commercialization risk. This requires optimization of developed and partially developed strains.
■ Increased strain tolerance to inhibitory hydrolysates and ethanol, with the ability to use all sugars, including mesophile
and thermophile strains.
■ Understanding of multigenic causes of industrial robustness.
■ Candidate microbes such as thermophilic ethanologens compatible with desired cellulase enzyme optima. This allows
process simplification to single-vessel fermentation with efficient use of all biomass-derived sugars
■ Development of coproducts.
Within 10 years
■ Rapid tool adaptation and regulation of genetically engineered strains, including use of minimal media.
■ Ability to engineer ethanol tolerance and robustness into new strains such as thermophiles.
■ Higher-yield microbes via control of growth and energetics.
■ Increased product (ethanol) titer to simplify product recovery and reduce water use
■ Full predictive metabolic pathway systems model for common industrial microbes, including regulation and identification
of unknown genes
Within 15 years
■ Thermophillic microbes demonstrated at scale to enable simultaneous saccharification and fermentation.
■ Further refinement of biofuel process and operation.
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Table 2. Fermentation process milestones.11
Within 5 years
■ Improve hydrolysate-tolerant microbes.
■ Achieve SSCF under desirable conditions (high rates, yield, and titer; solids concentration and industrial media).
■ Functionally express heterologous cellulases in industrial hosts, including secretion at high levels and investigation of cellsurface expression.
■ Conduct lab tests of modified initial CBP microbes.
Within 10 years
■ Eliminate the detoxification step by developing organisms highly tolerant to inhibitors.
■ Have the same response with undefined hydrolysates as with defined hydrolysates.
■ Move to pilot demonstration of CBP.
Within 15 years
■ Develop intrinsically stable cultures that do not require sterilization.
■ Achieve CBP under desirable conditions (high rates, yield, and titer; solids concentration and industrial media), first on
easily hydrolyzed model cellulosic substrates, then on pretreated cellulose.
■ Develop methods to use or recycle all process streams such as inorganic nutrients, protein, biosolids, or coproduct carbon
dioxide
Conclusions
Feedstock availability, biomass pretreatment and enzymatic hydrolysis still require important technical obstacles to be overcome to
make cellulosic ethanol a competitive fuel in the transportation fuels market. However, it is important to not forget about
fermentation. Even though this biological process has been used and studied since ancient times, there is still room for further
microbial and process optimization that can have profound effects on the cellulosic ethanol profit margin. For the fermentation
operation, there needs to be significant advancements made to increase the ethanol conversion rate, maximize the overall ethanol
yield, and allow these microbes to thrive in more extreme conditions. All of these will increase the fermentation efficiency and the
process economics. The focus needs to not only be to demonstrate these advancements at the lab scale, but also to utilize this
technology at the pilot-plant demonstration scale so that industrial cellulosic ethanol plants can begin construction and production
and thereby make an impact on the transportation fuels market as soon as possible. Fermentation yeast costs are negligible when
looking at all of the variables that affect the ethanol production price. This unit operation is a sensitive process, but by creating more
robust microorganisms and making fermentation a higher yield process, benefits can be seen by reducing the required enzyme
charge and by decreasing the feedstock demand, respectively. Contrary to the cost of yeast, enzyme and wood cost, together,
make up over half of the cost associated with ethanol production. Decreasing the amount of enzyme and wood needed per unit of
ethanol can make the cellulosic ethanol production process very lucrative.
References
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http://www.nrel.gov/technologytransfer/pdfs/igf21_gardner.pdf (accessed March 6, 2010).
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Efficiency & Renewable Energy. http://www.afdc.energy.gov/afdc/ethanol/production_cellulosic.html (accessed March 6, 2010).
4. "Fermentation." The McGraw-Hill Companies, inc.
http://www.mhhe.com/biosci/esp/2001_gbio/folder_structure/ce/m5/s6/cem5s6_1.htm (accessed March 6, 2010).
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8. "Zymomonas Mobilis." Wikipedia. http://en.wikipedia.org/wiki/Zymomonas_mobilis (accessed March 6, 2010).
9. Dr. Nancy Ho. "Technology." Green Tech America. http://www.greentechamerica.com/Technology.html (accessed March 6,
2010).
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Biomass Bioenergy 22, no. 2 (2002): 125-138.
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