Executive Summary (Dana Olson)
As the movement to “go green” progresses around the world there has been much
desired to move away from the use of fossil fuels and towards a biobased fuel to reduce
greenhouse gas emissions. As yet 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 to develop a process for producing ethanol from
lignocellulosic biomass and a similar process is presented in this research paper. (Too
introductory??) 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 ßglucosidase to five and six carbon sugars. Then these sugars are 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 and second 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.
Fermentation Optimization (Dana Olson)
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.
Include discussion of E. coli and Z. mobilius??
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 flux
[Lu]. 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 glucose [Jin]. 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 first [Lu].
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
1 - From
Watanabe.
Fermentationofof
and therefore increase xylose assimilation [Jin]. Under Figure
Figure
##. From
???. Fermentation
sugars
in modified
strain
of S.
The
sugars
in
modified
strain
of cerevisiae.
S. cerevisiae,
aerobic conditions, the specific ethanol productivity of strains squares and circles are glucose
and xylose
squares
and
circles
are
glucose
and
with PsTAL1 over expressed was increased by 70% from the consumption respectively, the diamonds are
xylose consumption respectively,
the ethanol production and the triangles are
diamonds
are ethanol production and
the xylitol accumulation.
triangles are xylitol accumulation
base strain and a 100% increase in the specific growth rate
of the cells on xylose media [Jin]. In a similar experiment by
Watanabe et. al, the best strain isolated was found to have
a yield of 0.43 g ethanol per g of total consumed sugars
which was 84% of the theoretical yield [Watanabe]. These
best growing strains could then be isolated and grown up in Figure 2 - From Dombek. Fermentation rate as a
a seed train of continuously increasing bioreactors before function of ethanol concentration.
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 ethanolinduced reduction of fermentation rates. However, replacement of the fermentation broth
containing ethanol does not immediately restore the fermentative activity. It was found that
ethanol concentrations below 12% (vol/vol) do not denature the glycolytic enzymes or cause
significant irreversible inhibition of the fermentative activity [Dombek].
Ethanol Recovery and Purification (Dana Olson)
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 implemented [Lu].
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 side-draw. 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 weight [Aden].
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 flow [Aden].
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 hour
[Aden].
Resources
1. Aden, A., M. Ruth, K. Ibsen, J. Jechura, K. Neeves, J. Sheehan, B. Wallace, L.
Montague, A. Slayton, and J. Lukas. Lignocellulosic Biomass to Ethanol Process Design
and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis
for Corn Stover. National Renewable Energy Laboratory, 2002. Print.
2. Dombek, K. M., and L. O. Ingram. "Ethanol Production during Batch Fermentation with
Saccharomyces cerevisiae: Changes in Glycolytic Enzymes and Internal pH." Applied
and Environmental Microbiology 53.6 (1987): 1286-291. Department of Microbiology and
Cell Science, University of Florida, Gainesville. Web. 6 Dec. 2009.
3. Jin, Yong-Su, Hal Alper, Yea-Tyng Yang, and Gregory Stephanopoulos. "Improvement
of Xylose Uptake and Ethanol Production in Recombinant Saccharomyces cerevisiae
through an Inverse Metabolic Engineering Approach." Applied and Environmental
Microbiology 71.21 (2005): 8249-256. Department of Chemical Engineering,
Massachusetts Institute of Technology. Web. 4 Dec. 2009.
4. Lu, Yulin, and Nathan S. Mosier. "Current Technologies for Fuel Ethanol Production from
Lignocellulosic Plant Biomass." Genetic Improvement of Bioenergy Crops. Ed. Wilfred
Vermerris. Gainesville, FL: Springer, 2008. 161-77. Print.
5. Watanabe, Seiya, Ahmed Abu Saleh, Seung Pil Pack, Narayana Annaluru, Tsutomu
Kodaki, and Keisuke Makino. "Ethanol Production from xylose by recombinant
Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose
reductase from Pichia stipitis." Microbiology 153 (2007): 3044-054. Kyoto University.
Web. 6 Dec. 2009.