Executive Summary
As the movement to “go green” progresses around the world there has been
much desire to move away from the use of fossil fuels and towards a bio based fuel to
reduce greenhouse gas emissions. As of now many of the methods used while
technologically possible have been economically infeasible. Recently the US
Department of Energy has partnered with the National Renewable Energy Laboratory
(NREL) to develop a process for producing ethanol from lignocellulosic biomass and a
similar process is presented in this research paper. A feedstock of 2000 metric tons of
corn stover is to be consumed every day. This corn stover is sent through a pretreatment
process to liberate the fermentable sugars from the ligin structure. The fermentable
sugars then go through a saccharification process where they are hydrolyzed by three
cellulase enzymes endo-p-glucanase, exo-p-glucanase and ß-glucosidase to five and six
carbon sugars. These sugars are then sent to the fermentation process where a
genetically modified strain of Saccharomyces cerevisiae is used to ferment the sugars to
ethanol. Finally, the ethanol is recovered by a distillation process, first being passed
through a beer column followed by a rectifying column. An azeotropic mixture of ethanol
and water is removed as the distillate from the rectifying column and is sent to a
molecular sieve adsorption column to purify the ethanol to 99.5% at a production rate of
170,000 gallons of ethanol per day.
Introduction
Ethanol is a bio-based, renewable oxygenated fuel that is currently used as an
oxygenate additive in fuels. If ethanol is to progress further than a fuel additive and
become a player in the liquid fuels market, lignocellulosic feedstocks need to be
utilized.10 Ethanol is an attractive fuel because 77% of the energy contained in the
carbohydrates of the feedstock can be recovered as ethanol. Also, for every gallon of
gasoline replaced by lignocellulosic ethanol, an 86% reduction of greenhouse gas
emissions would occur.5 When considering lignocellulosic feedstock choices and plant
location for bio-ethanol production, feedstock availability and transportation costs must
be considered. Transportation costs are one of the largest costs associated with bioethanol production because ligncellulosic feedstocks have low energy densities which
require large amounts of feedstock to be transported. Corn stover was selected as a
feedstock because of its high availability and carbohydrate content. Using this as a
feedstock, Iowa was determined to be an ideal location for the bio-ethanol plant because
of Iowa’s high density of corn fields. These dense corn fields allow for minimization of
transportation costs. A feed stock rate of 2000 MT/day was selected because this rate
yielded an ideal balance between transportation costs and production revenue.3 In terms
of economics, for a 2010 start-up date to be economically feasible, the ethanol must
have a selling price of $1.07 per gallon.3
Pretreatment
Pretreatment of the feedstock is necessary to break the feedstock down into
usable products for saccharification. Dilute acid hydrolysis was selected for this
process. A diagram of the entire pretreatment process can be seen in the appendix.
The first step in the pretreatment process is to take the corn stover feedstock and put it
though a cleaning process to remove excess dirt and metals that would otherwise
decrease the overall efficiency of the process and cause excess volume. The cleaned
corn stover is then put through a shredding process to increase its reaction area. The
shredded product is then fed to a pre-steaming vessel to further break down the
feedstock. The steamed product is then added to the dilute acid hydrolysis vessel where
sulfuric acid is added until its concentration within the reactor is 1.1% . This hydrolysis
converts hemicellulose carbohydrates into usable sugars. During this reaction, side
reactions occur that create compounds that can inhibit the fermentation process, such as
acetic acid, furfural, and hydroxymethylfurfural (HMF). To remove as much of these
inhibitors as possible, flash and overliming processes were used. The hydrolyzed
product from the hydrolysis reactor is sent to a flash vessel where 7.8% of the acetic
acid and 61% of the furfural and HMF are removed as vapor.3 The slurry that leaves the
flash is then filtered into a solid stream containing cellulose and lignin and a liquid
stream. The liquid stream contains the inhibitors from the hydrolysis and is then treated
in an overliming vessel. In this vessels, lime is added until the pH reaches 10. This
allows for the “overliming reactions” to occur which substantially reduce the amount of
inhibitors in the liquid stream.9 This overlimed stream is then fed to a pH adjustor vessel
where the pH is adjusted to that of the fermentation train and let sit for 4 hours. This
long residence time allows for the gypsum crystals to grow large enough for easy
separation.3 Once the crystals are formed, they are filtered out and the liquid stream is
recombined with the cellulose stream and fed to the saccharification train. Sizes and
residence times for all vessels can be found in table P1.
Vessel
Pre-Steam Vessel
Hydrolysis Vessel
Flash
Overlime Vessel
pH Adjustor Vessel 1
pH Adjustor Vessel 2
Residence Time
Size
Hours
0.33
0.17
0.25
1.0
4.0
4.0
m3
609
2032
2159
324
1396
1396
Table P1: Residence times and sizes of pretreatment vessels.
Cellulase Production
Cellulosic biomass can be converted to fermentable sugars through a mixture of
enzymes known as cellulase. This cellulase enzyme mixture consists of three separate
enzymes each of which are important in the degradation of the cellulose. Endo-pglucanase is the enzyme responsible for random scission of the cellulose chains,
yielding glucose and cello-oligo saccharides. Exo-p-glucanase is responsible for the exoattack on the non-reducing end of cellulose with cellobiose as the primary structure. The
ß-glucosidase enzyme then hydrolyzes the cellobiose to glucose1. In order to produce
this cellulase mixture, a genetically engineered strain of the mold Trichoderma reesei,
ZU-02 was used. This strain was chosen as the ideal choice for the process because the
mold grows particularly well on corn cob residue, which is very similar to the corn stover
feedstock used in this cellulosic bioethanol process.
The process discussed in this paper calls for 2000 metric tons of corn stover per
day. Corn stover is 38% cellulose, which translates to 7.6*108 grams of cellulose per
day. The process requires 12FPU per gram of cellulose, therefore 9.12*109 FPU must be
produced each day. In order to make this process economically feasible, the cellulase
production must be on the order of 500FPU/mL. The cellulase production occurs in a fed
batch process which requires a 4 day fermentation time, this requires a total bioreactor
volume of 4 times that of the daily requirement. In order to meet this demand, 2 36m3
reactors are needed for the production of the cellulase. This process is done by
inoculating a 250mL flask with a 2mL spore suspension of the mold, and growing the
mold in the vessel until it can be split and 2 1.25L reactors can be inoculated. A train of
fed batch bioreactors are placed in the system where each one is fed 10vol% of the total
volume and allowed to grow. The final reactor step jumps from 12.5m3 to the final reactor
volume of 36m3.
The substrate for cell growth in this process is “10 g L−1 glucose, 5.1 g L−1 corn
steep solids (Sigma–Aldrich), 1.4 g L−1 (NH4)2SO4, 2 g L−1 KH2PO4, 0.5 g L−1
CaCl2·2H2O, 0.3 g L−1 MgSO4·7H2O, 0.005 g L−1 FeSO4·7H2O, 0.0016 g L−1 MnSO4·H2O,
0.0014 g L−1 ZnSO4·7H2O” 2 and kept at a temperature of 28-30˚C, a pH of 4.8, with a
substrate concentration of 40g/L, and a C:N ratio of 8:1. For the final fermentor the
aeration rate is 360m3/hr, and the system is agitated at 100rpm to ensure optimal cell
growth. Excluding the flask used for the initial cell growth, the reactors are operated in a
fed batch mode because it is not possible to operate this process in a continuous
fashion. However, the enzyme is produced at a high enough rate that if the bioethanol
process were to be operated in a continuous fashion there would be enough of the
enzyme to make this feasible.
Saccharification
In this plant, saccharification will occur separately from fermentation, although
simultaneous saccharification and fermentation does occur in the fermentors since the
cellulase enzymes are not removed between steps. Arranging the process in this
manner has several advantages. First, separate reactors allow for the use of separate
reaction conditions. Because fermentation requires a whole organism to convert useable
sugars into the product, while saccharification requires only the active enzyme, the
saccharification reactor can operate at more extreme temperatures and product
concentrations. Operating these reactors at higher temperature utilize Arrhenius kinetics
and optimize the rate of hydrolysis without degrading the enzyme3. This reduces both
the holding time of each reactor and the amount of enzyme required. A second
advantage of independent saccharification and fermentation is that smaller reactors are
required than if the processes were to occur simultantously. This is especially
advantageous with process control. If a reactor were to become contaminated, for
example, a large investment would be required to disinfect large reactors necessary for
simultaneous saccharification and fermentation. However, if the two processes are
separated, then potential contaminants would be easier to contain and less down-time
would be required to restart the process. Finally, large reactors that would be required
for simultaneous saccharification and fermentation would introduce problems with mass
transfer. For example, large reactor volumes require more enzyme and substrate to
maintain the same concentrations that allow for optimal reaction rates. They would also
require much larger impellers to agitate the fluid and evenly distribute the substrate. This
is especially problematic since large impellers introduce significant operating costs. All of
these factors greatly outweigh the lower capital costs that result from fewer reactors.
Thus, separate saccharification and fermentation is optimal for this process.
Saccharification will take place in a train of five 500,000 gallon reactors, each
operating at 65°C in fed batch mode. As mentioned before, having multiple reactors is
beneficial because one can control contamination and product output. The holding time
for each reactor will be 36 hours – 24 hours of reaction time and 12 hours of turn-around
time. Tanks are maintained at a constant temperature by continuously pumping reactor
contents through a heat exchanger cooled by cooling water. The cellulase required is
12.0 filter paper units per gram of hydrolyzed sugar3. Assuming pretreatment is capable
of producing 0.75 tons of utilizable sugars per ton of feedstock (the remainder consisting
primarily of lignin), and saccharification conditions allow 71% conversion to
monosaccharides4, then the amount of cellulase required is 12.8 GFPU per day. The
reactors reach concentrations of about 5.1 kFPU/gal cellulase and 0.4 kg/gal sugar. One
problem that may arise, however, is large pumping costs due to Non-Newtonian
behavior of polysaccharide solutions.
To grow up the yeast necessary to ferment the saccharification slurry, 10% of the
product is sent to two trains of five seed fermentors with an inoculum volume of 10%.
The reactors operate at 41°C in fed batch mode with a holding time of 36 hours,
including 12 hours for turn-around time. Each stage scales up by a factor of 10 until the
seed volume is capable of fermenting the slurry. Based on the desired production rate,
fermentor sizes will be 20, 200, 2000, 20,000, and 200,000 gallons in size. As with the
saccharification reactors, the seed fermentors are cooled with cooling water in a heat
exchanger. The yeast will also need nutrients to grow to the desired level. Sufficient
growth occurs with corn steep liquor levels of 0.5% and diammonium phosphate levels
of 2.5 g/gal3. The remaining slurry is then fed to a train of large-scale fermentors, which
are roughly twice the size of the saccharifiers to account for the nutrients that need to be
added.
Fermentation Optimization
Several different microorganisms were considered for the fermentation process.
They needed to be robust and have high productivity while being able to ferment a wide
variety of sugars to ethanol. A high tolerance to ethanol is also a very favorable trait.
Saccharomyces cerevisiae, Zymomnas mobilius and Escherichia coli were all
considered as possibilities but S. cerevisiae was found to have the most favorable
characteristics for this process.
S. cerevisiae has a high ethanol tolerance and can ferment sugars to ethanol
anaerobically at low pH conditions. These low pH conditions minimize the likelihood of
contamination of the fermentation tanks by other microorganisms. Wild types of S.
cerevisiae do not naturally ferment pentoses and as such recombinant DNA
technologies must be used to incorporate sugars such as xylose into the pentose
phosphate pathway of the metabolic flux5. This can be done by incorporating genes from
Pichia stipitis by utilizing inverse metabolic engineering. The necessary genes were
found to be D-xylolukinase, or XYL3, and an open reading frame segment labeled
PsTAL1 which is highly homologous to the native gene ScTAL1. These genes were
found to improve xylose uptake and ethanol production in S. cerevisiae without hindering
its ability to grow on glucose7. Furthermore, S. cerevisiae has natural glucose
transporters that are also capable of transporting xylose across the cell membrane,
however there is a delay as glucose is selectively transported
first5.
To isolate the modified strains of S. cerevisiae that are
capable of growing on and therefore fermenting xylose the
genes were first transfected into wild type strains of the yeast.
They were then grown up on minimal xylose media for several
cycles and then replated onto either glucose or xylose rich agar
plates. The colonies on the glucose rich plates were found to be
of equal size indicating they had the same growth rate, while
those on the xylose rich agar plates ranged in sizes. The larger
colonies had a higher growth rate and therefore increase xylose
assimilation7. Under aerobic conditions, the specific ethanol
Figure 1 - From Watanabe. Fermentation of
productivity of strains with PsTAL1 over expressed was sugars in modified strain of S. cerevisiae. The
increased by 70% from the base strain and a 100% increase in squares and circles are glucose and xylose
respectively, the diamonds are
the specific growth rate of the cells on xylose media7. In a consumption
the ethanol production and the triangles are
similar experiment by Watanabe et. al, the best strain isolated the xylitol accumulation.
was found to have a yield of 0.43 g ethanol per g of total consumed sugars which was
84% of the theoretical yield6. These best growing strains could then be isolated and
grown up in a seed train of continuously increasing bioreactors before being inoculated
into the process.
Under optimal conditions S. cerevisiae can produce
ethanol at a rate of 50 mmol ethanol per hour per gram of cell
protein. During batch fermentation this optimal rate is only
maintained for a brief period followed by a rapid decline as
ethanol accumulates in the broth. Accumulation of ethanol is
known to reduce membrane permeability and can increase
hydrogen ion flux into the yeast cells. This increased proton
flux has been proposed as the cause in the ethanol-induced
reduction of fermentation rates. However, replacement of the
fermentation broth containing ethanol does not immediately
restore the fermentative activity. It was found that ethanol Figure 2 - From Dombek. Fermentation rate as a
concentrations below 12% (vol/vol) do not denature the function of ethanol concentration.
glycolytic enzymes or cause significant irreversible inhibition of
the fermentative activity8.
Ethanol Recovery and Purification
Ethanol vaporizes at 78°C and water vaporizes at 100°C. If distilled an azeotropic
mixture of ethanol and water will form the distillate (95.6% ethanol to 4.4% water). Since
the ethanol-water mixture is not ideal a multi-stage process must be implemented5.
Typically the fermentation mixture will be fed to a beer column. Carbon dioxide
leaves out the top with water coming out the bottom and ethanol is removed from a sidedraw. The carbon dioxide vented from the top of the column is sent to a scrubber to
remove any ethanol which is recycled to the rectifying column. The water contains solids
which are removed by pressurized filtration. The purified water is recycled to the process
while the filters are combusted for energy. The beer column is 32 trays with the feed
entering at tray four. The tray spacing is two feet and the column has a diameter of 14.3
feet. The side draw of ethanol removed at tray eight is 39.4% pure by weight3.
This ethanol is fed to a rectifying column, the product of which is the azeotropic
mixture. This mixture cannot be further purified using distillation. The rectifying column
contains 60 trays and is fed at tray 44 with the side draw from the beer column. The
column diameter is 4 feet below tray 44 and 11.5 feet above tray 44 to accommodate the
increased flow3.
The distillate of the rectifying column is fed to a dehydration unit. Typically a
molecular sieve adsorption unit can be used. The zeolites in the molecular sieve unit
selectively adsorb 95% of the water from vapor mixtures, further purifying the ethanol.
Two adsorption columns are required so that while one is dehydrating the azeotropic
mixture, the other can be regenerated. This results in a 99.5% ethanol pure product
produced at a rate of 170,000 gallons per hour3.
Conclusion
Developing countries like Taiwan will likely struggle to satisfy their energy needs
as industrialization continues to take effect. This is especially true for transportation
fuels, which will increase in demand as more and more people begin to purchase
automobiles. In a time of increasing energy costs and decreasing fossil fuel reserves,
researchers are forced to find an alternative to conventional energy sources. Corn-toethanol plants are a promising step in the transition to renewable energy. However,
before these plants can be implemented feasibly, several improvements need to be
made in the process. Research must be conducted to improve yields of the
saccharification and fermentation reactions. Also, more economical methods must be
developed to make the cellulosic biomass accessible to enzymatic hydrolysis. Finally,
and perhaps most importantly, the price of conventional gasoline will have to increase
significantly before ethanol can become a competitive transportation fuel. Nonetheless,
corn-to-ethanol plants will likely play a large role in the industrialization of developing
countries, especially those which have an abundant supply of crop residues which
otherwise would go to waste.
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