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D-Xylose Fermentation to Ethanol by Encapsulated D-Xylose
Isomerase and Saccharomyces cerevisiae
Honors Thesis Proposal
Author: Brian Frederick
Advisor: Dr. Phillip Christiansen
Co-Advisor: Dr. Silvana Andreescu
Objectives:
The objective of this study is to develop a procedure for encapsulating
Saccharomyces cerevisiae, brewers’ yeast, with D-xylose isomerase to efficiently
ferment both six- and five-carbon monosaccharides to ethanol. If time allows, this
procedure will further be modified to improve ethanol yield and fermentation efficiency
in dilute-acid hydrolyzates.
Overview:
With rising concerns of global warming and future supplies of oil dwindling,
alternative renewable energy sources are increasingly being sought. One such potential
source is ethanol, derived from the fermentation of waste plant matter such as corn husks.
This plant matter can be hydrolyzed by dilute acid or enzymatically digested into
monosaccharide sugars that can then be used by yeast to produce ethanol. The ethanol
can be mixed with gasoline supplies, increasing the octane rating, or can be used straight
in modern combustion engines. Some areas of the world, most notably Brazil, already
offer ethanol as an alternative to gasoline at the gas pump and run planes purely on
ethanol. It is currently comparable in price to gasoline to sell, is carbon neutral since the
amount of carbon dioxide released by its combustion is equivalent to the amount
removed from the atmosphere by the growth of the plant matter for fermentation, and is
entirely renewable. Furthermore, there are several strains of yeasts readily available that
are highly efficient at ethanol production and have been used by humans for thousands of
years. One set back, however, is that these strains are limited to six-carbon
monosaccharide (hexose sugar) fermentation, such as glucose. Though this is typically
the most abundant type of monosaccharides found in plant matter, five-carbon
monosaccharides (pentose sugars), primarily D-xylose, comprise a significant
proportion13. To increase efficiency and yield of ethanol, yeast capable of fermenting
pentose sugars are being sought.
Several approaches have been explored for developing pentose fermentors. One
such approach is to genetically modify hexose fermentors to introduce the enzymes
necessary to ferment five-carbon monosaccharides. This increases the utility of the yeast
strain but has many complications including the need to ensure NADH (an electron
carrier vital to many processes in the cell) usage in the synthetic pathway is equivalent to
its re-synthesis to ensure no imbalance occurs14. Furthermore, genetically modified
organisms are tightly regulated and their use will both be limited and expensive.
Alternatively, several strains of yeast, including Pichia stipitis, have been discovered
that, given the right conditions, will ferment pentose sugars to ethanol. However, these
yeast are inefficient at this form of fermentation and have further complications from low
ethanol resistance7. Forced adaptation attempts have been made to selectively enhance
certain traits of these yeast, such as increasing their resistance to ethanol and low pH,
however this is a slow process with limited results6. Attempts have also been made to
take these pentose fermentors and hybridize them to efficient hexose fermentors, most
notably Saccharomyces cerevisiae, in the hopes of creating a strain of yeast that is
efficient at both five- and six-carbon fermentation with a higher resistance to ethanol.
Such hybrids have been shown to be possible but tend to be unstable4. Further
exploration of S. cerevisiae, brewers’ yeast, has shown that while these yeast cannot
ferment most pentose sugars they can produce ethanol from D-xylulose5. D-xylulose can
be produced from D-xylose, the primary pentose sugar of plant matter, using the enzyme
D-xylose isomerase. When S. cerevisiae are grown under anaerobic conditions with Dxylose and a high concentration of D-xylose isomerase, ethanol is produced3.
Unfortunately, the high concentration of enzyme needed makes this procedure
impractical.
Encapsulation of particles with an artificial, permeable membrane has been
carried out for many reasons. By encapsulating certain enzymes in a membrane
permeable to substrates but not to product molecules, with subsequent recovery of the
capsules, economically desirable products can be easily isolated and environmentally
harmful substances can be removed. Controlled release of encapsulated drugs1 and
bioartificial liver assist devices acting like dialysis tubes8 are other applications. The
capsules allow the researcher to select what can enter and leave the interior12, potentially
protecting the encapsulated substance from harmful substances. Yeast have been
encapsulated for this reason. As previously mentioned, the plant matter to be used for
fermentation is hydrolyzed by a weak acid to yield monosaccharides. This produces
many substances in addition to the monosaccharides, some of which have been shown to
inhibit yeast activity or are toxic to the cells. Encapsulating the yeast has been shown to
protect the cells from some of these toxic substances while still giving the cells access to
monosaccharides and nutrients11. Overall, this increases the efficiency and life-span of
the yeast. An additional advantage is that the capsules can be reused, reducing the
amount of waste per fermentation batch.
A further advantage, hitherto unexplored, of encapsulation is that one can
encapsulate more than just yeast. As previously outlined, one approach for using hexose
fermentors to produce ethanol from pentose sugars is to use S. cerevisiae and D-xylose
isomerase. The disadvantage to this approach is that the enzyme concentration must be
significantly high. By encapsulating S. cerevisiae with D-xylose isomerase, we will
create a very high localized concentration of the enzyme that is in very close proximity to
the yeast cells. This will allow the D-xylulose produced by the enzyme to be efficiently
taken up by the yeast cells, giving them the ability to ferment pentose sugars to ethanol.
Since S. cerevisiae is one of the most efficient yeast fermentors, with high resistances to
ethanol, the result should be a very effective six- and five-carbon fermentor.
Furthermore, use of this procedure would not be restricted, as in the case of genetically
modified yeast, and should be more stable than yeast hybrids. Economically, it will
employ inexpensive materials, efficiently ferment nearly all monosaccharides in plant
material, and will allow the yeast to be reusable. Another advantage is that, since the
yeast are encapsulated and not freely suspended, the capsules can be packed into a
column with fermentation material continuously flowing through. This increases the
flow-through rate of material and increases the production of ethanol compared to
traditional batch fermentation.
Methods:
The first phase of this study will consist of encapsulating Saccharomyces
cerevisiae in an artificial membrane of sodium alignate, carboxymethylcellulose, and
Tween 20 as in reference 11. Adjustments to the viscosity of the capsule interior and the
porosity of the membrane will be made to optimize yeast ethanol fermentation from
glucose. In the next phase, S. cerevisiae will be encapsulated with D-xylose isomerase
and adjustments to the capsules will be made to optimize ethanol fermentation from D-
xylose. The last phase will be conducting the ethanol fermentation on dilute-acid
hydrolyzates, which will consist of a mixture of pentose and hexose sugars. Capsules
will be adjusted to reduce toxicity and optimize fermentation. In all phases, anaerobic
fermentation will be conducted as previously outlined9,10 and the amount of ethanol
produced will be quantified spectrophotometrically2.
Timeline:

May 2007: Successfully encapsulate Saccharomyces cerevisiae, optimizing
ethanol fermentation on glucose.

October 2007: Successfully encapsulate S. cerevisiae with D-xylose isomerase,
optimizing ethanol fermentation on D-xylose.

December 2007: Adapt procedure to optimize fermentation of capsules on diluteacid hydrolyzate.
References:
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Klebsiella planticola. Appl. Environ. Microbiol. 53: 2039-2044.