Chem*4570 Applied Biochemistry

Lecture 2 - Production of ethanol by yeast

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Ethanol production represents an effective method for conversion of biomass into liquid fuel, as a way to
replace or supplement our reliance on fossil fuels.
Ethanol is readily produced from a glucose or sucrose rich medium in brewers yeast
(Saccharomyces cerevisae). This closely resembles the natural environment of yeasts (fermenting
fruit), something that was observed and adapted very early in human history - there is evidence of
brewing and winemaking in early archeological records.
What is the biochemistry of ethanol production?
How is it that yeast can be so efficient in this process?
Glycolysis is the normal pathway for breakdown of glucose in most organisms. Under normal
conditions, the endpoint is pyruvate, which is then broken down to CO2 via the TCA cycle.

Glycolysis itself results in a net gain of 2 ATP and 2 NADH per glucose consumed; this plus the
oxidation of the two pyruvates yields a total of 30-32 ATP per glucose. This combination makes
glucose a good energy source to support cell growth.
However the process shown above is strictly dependent on availability of oxygen, which is required
so that NADH can be recycled by oxidative phosphorylation.
Oxidative phosphorylation generates most of the ATP produced.

NADH must be recycled to NAD +

Whereas glucose can be regarded as a consumable energy source, and can be imported from the
medium as the cellular pool is used up, NAD is only present in the cell in limited amounts, and
cant be imported from the medium. In the absence of O2 , NADH accumulates and NAD+ is depleted,
to the point where NAD+ dependent reactions such as glyceraldehyde-3-phosphate dehydrogenase
approach equilibrium or even reverse direction:
high NAD+ d
glyceraldehyde-3-phosphate + NAD+ + Pi 1,3-bisphosphoglycerate + NADH + H+
b high NADH
To circumvent this, many organisms use lactate dehydrogenase to balance NADH production by
glycolysis.

As pyruvate is converted to lactate, an equivalent number of NAD+ are produced, maintaining a balance
with glyceraldehyde-3-P dehydrodenase in glycolysis. However, only two ATP can be produced per
glucose, so high glucose consumption is necessary to keep up with energy demands.
An additional problem is the production of H+ so that anaerobic glucose consumption results in
acidification. As a result animals can only maintain anaerobic glycolysis for a short time. Some acid
tolerant bacteria can maintain growth by secreting lactate to the medium, as happens when milk goes
sour.

Yeast provides a better solution to the problem of recycling NADH, through the enzyme pyruvate
decarboxylase (A). This converts pyruvate to acetaldehyde by releasing CO2 , which is a non-acidic
product. Acetaldehyde can then be reduced to ethanol by alcohol dehydrogenase (B), which
consumes NADH and releases NAD+ so that glycolysis can continue.

A major advantage is that the final products are neutral, so that acidification is no longer a problem.
Since yeast lacks a central nervous system to fall under the adverse influence, significant amounts of
ethanol can accumulate while maintaining good growth rates. Also since ethanol can permeate the
cell membrane, ethanol can accumulate in the medium and not just in cell cytoplasm.
Pyruvate decarboxylase acts by reaction of pyruvate with thiamine pyrophosphate, displacing CO2 .
This is very similar to the action of subunit E1 of pyruvate dehydrogenase. The difference is that
pyruvate decarboxylase releases the acetaldhyde product whereas pyruvate dehydrogenase continues
reaction by an oxidative attack of lipoamide so that acetyl-CoA is produced.

How is it that yeast can be so efficient?

Yeast grows rapidly in a glucose- or sucrose-rich medium, with a doubling time of about 60 minutes.
High growth rates result in O2 depletion, and the neutral reaction products allow growth to be sustained
anaerobic mode. There are strains of yeast able to tolerate as much as 20% ethanol, although strains
producing 12-15% may actually grow better.
Key points
ethanol is the end point of the only metabolic pathway that yeast can use for ATP
production.
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ATP is produced at a low rate, 2 ATP per mole of glucose and per 2 moles of ethanol
produced so that substrate is rapidly converted to product to provide the energy needs of cell
growth.

Ethanol can escape into the medium rather than accumulating in the cell. This makes
recovery of the product easier.

Pure ethanol has to be recovered by distillation, which could represent the most expensive component
of the production process. In tropical countries, solar distillation is possible making ethanol production
economically viable.
Yield considerations.
1 tonne of glucose can be converted into a bit less than 0.5 tonne of ethanol. The reason for
this is very simple. The molar mass of glucose is 180 g mol-1, ethanol is 46 g mol-1 and CO2 is 44 g
mol-1.
glucose d 2 ethanol + 2 CO2
C6 H12 O6
2 C2 H6 O
180 g
92 g
88 g