Fermentation (from Lu)
Microorganisms used for fermentation must be robust and have a high productivity. Also,
the microorganism selected must be able to convert a mixture of different sugars to ethanol.
Other favorable traits include high tolerance to ethanol or other inhibitors and resistance to
contamination by undesired microorganisms. Three main microorganisms will be considered as
possibilities for this fermentation reaction
Saccharomyces cerevisiae – S. cerevisaiae has been considered the workhorse for
ethanol fermentation for years. This microorganism has high ethanol tolerance and operates at
low-pH fermentation conditions. Low pH conditions can be utilized to minimize the potential for
contamination. Wild type strains can ferment glucose, mannose and fructose as well as
disaccharides like sucrose and maltose, but they cannot ferment pentoses which make up 40%
of total biomass carbohydrates. Recombinant DNA technologies have been applied to increase
this function. Major enzymes required for xylose fermentation must incorporate xylose flux into
the pentose phosphate pathway, these enzymes are xylose reductase and xylitlo
dehydrogenase which can be found in yeasts like P. stipitis and Candida shehatae. Additionally,
xylose must be transported across the cell membrane. It is believed that this is carried out by
glucose transporters. These transporters have a higher affinity for glucose which delays the
transport of xylose until the majority of the glucose has been consumed which decreases the
effectiveness of the co-fermentation process. This delay results in an increase the time required
for fermentation as well as increased capital costs.
Zymomnas mobilius – The Entner-Doudoroff pathway is utilized to anaerobically produce
ethanol from sugars. Since only one mole ATP is generated per mole sugar consumed, glucose
metabolism must be high to compensate for low energy yield which results in high ethanol
productivity. Compared to traditional yeast, a 5-10% increase in yield and a 5 fold higher
volumetric productivity is observed. Z. mobilius must be metabolically engineered to ferment
xylose in addition to hexoses. Some success has been achieved. Major hurdles include
industrial adaptation of the recombinant type, increased likelihood of contamination due to
operation at neutral pH and less robust cells.
Escherichia coli – Wild type E. Coli can ferment a mixture of sugars to acids with ethanol
as a minor by-product. Only half the theoretical yield of ethanol can be achieved. By overexpressing pyruvate decarboxylase and alcohol dehydrogenase the ethanol production and
tolerance can be increased.
Based on this information it seems that using S. cerevisiae will be the best option for this
process. It is the most robust microorganism and being able to operate over a wide range of
conditions will allow us to optimize the productivity of the fermentation reaction.
S. cerevisiae Preparation (from Yong-Su Jin)
Inverse metabolic engineering can be utilized to create strains of S. cerevisiae that can
ferment xylose in addition to glucose. The recent advances in genome sequencing are taken
advantage of using this method. A library of strains is screened for the desired phenotype and
then the genetic modifications correlated to that phenotype are determined using sequencing.
This approach identify strains that will likely yield the desired phenotype opposed to the older
approach of random mutation which are more difficult to trace.
Genes were borrowed from Pichia stipitis in order to construct a metabolic pathway
through which xylose could be assimilated. Xylose reductase and xylitol dehydrogenase, or
XYL1 and XYL2 respectively, were initially chosen to be expressed in S. cerevisiae by Jin et al.
These strains had inefficient xylose assimilation and the accumulation of xylitol bounded the
ethanol production. With further study, the XYL3 and PsTAL1 genes were found to both improve
xylose uptake and ethanol production in S. cerevisiae without inhibiting the yeast’s growth ability
on glucose. It was found that strains with these two genes overexpressed were able of rapidly
ferment of glucose-xylose mixtures.
 Strains and Plasmids
 Media and culture conditions
 Construction of Pichia stipitis genomic library
 Yeast transformation
 Serial transfer and screening of fast-growing transformants
 Insert identification and sequence analysis
 Enzymes, primers and chemicals
 Plasmid construction
 Preparation of crude extract and enzyme assay
 Analytical methods
 Nucleotide sequence accession number
 ****Ask Professor Hu how much detail he wants on the above bullet points?
The genes of interest were transferred to recombinant strains of S. cerevisiae that
contained copies of XYL1 and XYL2. These strains were grown up on minimal xylose media for
10 transfers and then plated onto either a glucose or xylose agar plate. The colonies on the
glucose plate were of similar sizes, while those on the xylose plate varied greatly indicating that
those with the higher growth rate had increase xylose assimilation. (Graphic?)
Of 16 plasmids isolated, 10 contained PsXYL3 and one contained an inserted highly
homologus to the sequence of ScTAL1. This confirmed the hypothesis that increased
xyluokinase activity by either XYL3 or XKS1 would increase the efficiency by which xylose was
utilized by S. cerevisiae. It was found that over expression of PsTAL1 would increase the xylose
assimiliation while have no effect to the yeasts ability to grow on glucose rich media, while over
expression of ScTAL1 inhibited the yeast cell’s ability to grow on glucose as a carbon source. In
general over expression of PsTAL1 dramatically improved xylose uptake, cell growth and
ethanol production. Under aerobic conditions, the specific ethanol productivity of strains with
PsTAL1 over expressed was increased by 70% from the base strain and a 100% increase in the
specific growth rate of the cells on xylose media. (Graphic?)
The best strain created by Watanabe et al was found to produce 5.94 g ethanol per liter
with a yield of 0.43 g ethanol per g of total consumed sugars which was 84% of the theoretical
yield.
Fermentation Activity (from Dombek)
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. Molecular
oxygen and magnesium have been identified as critical factors for maintaining high fermentative
rates, but do not completely prevent the rate decrease. (Graphic?)
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 concentrations below 12% (vol/vol) do not denature the glycolytic
enzymes or cause significant irreversible inhibition of the fermentative activity.
Reactor Design and System
Train of fed batch reactors
Aerobic/Anaerobic?
Separator to recycle yeast cells and send product ethanol on to distillation sequence
Ethanol Recovery and Purification (from Lu and Aden)
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.
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. 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 effluent is therefore fed to a dehydration unit.
Typically a molecular sieve adsorption unit can be used. The zeolites in the molecular sieve unit
selectively adsorb water from vapor mixtures, further purifying the ethanol.
The energy input of the distillation/dehydration unit must be minimized to make the
process economically feasible. Pressure swing adsorption can be implemented to remove the
water from the azeotropic mixture, this gives a further optimization consideration for the
process.
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.