Title: Production of pyruvate and acetaldehyde during the fermentation of glucose by yeast

Introduction
Not all organisms rely on oxygen and aerobic respiration to metabolize glucose. Some
prokaryotic organisms survive solely through anaerobic respiration pathways, never using
oxygen to produce ATP. Two types of anaerobic respiration pathways exist, complete
anaerobic respiration and fermentation, Fermentation is an extremely common anaerobic
respiration pathway utilized by many different prokaryotic and eukaryotic cells. In
fermentation, one or a few chemical reactions follow glycolysis to convert pyruvate into a
waste molecule such as lactic acid or ethanol. Without these reactions, pyruvate would
accumulate in the cytoplasm and inhibit the entire pathway of glycolysis. In addition to
removing excess pyruvate, the fermentation reactions also serve to recycle NADH molecules
produced during glycolysis. Because cells have a limited supply of NAD + coenzymes, the
NADH molecules produced during glycolysis must be oxidized back to NAD + for continued
glycolysis reactions to occur.( Ashworth et al.,1974).

Although many different types of fermentation exist, these reactions share one significant
characteristic. All fermentation pathways are significantly less efficient than aerobic
respiration, producing only two ATP molecules (during glycolysis) for each glucose molecule
degraded. The vast majority of the potential energy originally stored in glucose remains in
the organic molecule waste product at the end of fermentation. Although much less efficient,
fermentation is faster than aerobic respiration because the reactions occur completely in the
cytoplasm and do not require multiple phases in sequence.( Dennis et al.,1995)

Aim
To produce pyruvate and acetaldehyde from glucose in a limited amount of oxygen, then test
the presence of these two intermediates by making use of sodium nitroprusside and piperidine
in which the colour changes can observed confirming their presence or absence.

Materials and methods

Formation of pyruvate from glucose
A pipette was used to obtain 5ml of glucose solution, in which 5ml of glucose solution was
placed in separate boiling tubes A and B, followed by addition of small amount of disodium
hydrogen phosphate to tube A, also small amount of potassium dihydrogen phosphate was
added to tube B and then finally 5ml of yeast suspension was added to both tubes. Both test
tubes were placed in a water bath at 37C for about an hour and observations were made and
recorded. After the incubation period 2ml of trichloracetic acid solution was added to each
tube and the mixture was mixed thoroughly, then centrifuged for 20 minutes on a benchtop
centrifuge .the supernatant was removed and the test for the presence of pyruvate was made
by using sodium nitroprusside when 2ml of boiled supernatant was added to 1cm of solid
ammonium sulphate in a test tube. About two drops of freshly prepared sodium nitroprusside
solution were added to the test tubes and concentrated ammonia was added carefully at the
wall side of the tubes so as to form two layers .where the presence of pyruvate would be
noted by the formation of green or blue rings at the junction of the two liquids in which a
transient pink ring may appear before the characteristic blue or green colour given by
pyruvate.
Formation of acetaldehyde from glucose
About 5ml of glucose solution was pipetted into two different test tubes C and D and 5 ml of
yeast suspension was added to both tubes followed by the addition of 0.5g of sodium sulphite
to tube D, the mixture was mixed thoroughly. Both tubes were incubated at 37C for about an
hour and observations were made and recorded.at the end of the incubation period, both tubes
were centrifuged and the supernatant was removed after centrifugation. About 0.5ml of
freshly prepared sodium nitroprusside was added to 2ml of the supernatant followed by the
addition of 2ml of aqueous piperidine then the mixture was mixed. The presence of
acetaldehyde was noted by a blue colour observed.

Results
Table 1 : a tabular representation of observations made during the fermentation process when
four test tubes were incubated at 37C at 10 minutes interval for 1 hour.
Time
After 10
minutes

Tube A
No change in
appearance the
solution
remained brown
colour
Two distinct
layers were
formed, the top
layer was light
brown in colour
and dark brown
at the bottom
two distinct
layers of the
supernatant
were still
present and a
dark brown
precipitate at
the bottom

Tube B
A thick layer of
froth was
formed on top
of the solution

Tube C
Few bubbles
like were
forming

Tube D
A thin layer of
froth was also
formed

A pellet was
forming and the
froth was
gradually
increasing

No change
observed ,just
few bubbles at
the top

White pellet at
the bottom was
observed and
thin layer of
froth was still
present

Froth forms
almost one
quarter of the
test tube on top
of the solution

A thin layer of
froth was still
forming. Two
layers of the
supernatant also
formed with the
upper layer
light brown and
bottom layer
dark brown

In between 4050 minutes

Froth formed ,
and two distinct
layers of the
solution were
still present

A homogeneous
mixture with no
layers

In between 5060 minutes

Froth layer
thickens but the
colour of the
solution
remained brown

Large bubbles
were observed
and no change
in appearance

A thin layer of
froth was
beginning to
form. Two
layers of the
supernatant also
formed with the
upper layer
light brown and
bottom layer
dark brown
A uniform
colour solution
with no
partitions ,froth
was present was
thicker than in
A and D
Uniform colour
and very thick
layer of froth

After 20
minutes

After 30
minutes

Two layers of
the solution
were still
present and a
dark brown
precipitate
White pellet at
the bottom and
thin layer of
froth. Three
layers were
observed top
layer was a
clear
solution ,mid
layer was light
brown and

bottom layer
was like a dark
brown
precipitate
Sodium nitroprusside test results for pyruvate:
After adding sodium nitroprusside solution and concentrated ammonia solution to both tubes
A and B, no colour change was observed in tube B ,however two distinct layers of liquids
were formed in tube A in which a transient pink ring was formed at the junction of these
liquids and this was due to the presence of thiol group. After this temporary colour a green
ring was formed which indicates a positive result of pyruvate.

Test results for acetaldehyde formation

After adding sodium nitroprusside solution, aqueous piperidine a positive result for the
presence of acetaldehyde was observed in tube D in which a blue colour was observed and in
tube C no colour change was observed the solution remained brown in colour

Discussion
In the absence of oxygen yeast cells convert glucose to alcohol and carbondioxide in the
process called fermentation. This process usually involves several intermediates in which
glucose is converted to pyruvate then to acetaldehyde and finally alcohol. In this practical we
performed our goal was to test for the presence of these intermediates that is pyruvate and
acetaldehyde, since the end product of this reaction is an alcohol several methods were
applied in order to slow down the rate of the enzyme that catalyses the oxidative
decarboxylation of pyruvate to alcohol and this was done by adding an inhibitor and changing
the physiological pH. In order for us to be able to detect the presence of pyruvate we added
alkaline solution of disodium hydrogen phosphate to our test tube before adding the yeast
solution this was done so as to slow down the enzymatic activity of pyruvate decarboxylase
which is an enzyme responsible for the conversion of pyruvate to acetaldehyde, this process
was done because this enzyme is inactive in slightly alkaline solution.( Gumport ,et al.,1989)

For fermentation reaction to occur it has to be promoted by certain conditions such as a

temperature of approximately 37C which is the reason why the test tubes were incubated in a
water bath at 37C, the other condition is the presence of yeast cells which contain enzymes
for catalyses the reaction and also exclusion of air which provides low concentration of
oxygen which is the reason why we closed our test tubes with aluminium foils during the
incubation process. Fermentation does not require oxygen and if oxygen is present, some
species of yeast for example Kluyveromyces lactis will oxidize pyruvate completely to
carbon dioxide and water with no formation of acetaldehyde and this process is called cellular
respiration, however these species of yeast will only produce our intermediate only in an
anaerobic environment. In the second experiment of the formation of acetaldehyde, further
reaction to the formation of alcohol was reduced its rate by inhibiting the enzyme with
sodium sulphite.( Banowetz, et al.,1996)

In this practical, positive result for the presence of pyruvate was observed only in test tube A
and test tube B gave the negative result for this test the reason for this might be as a result of
disodium hydrogen phosphate which was added only in test tube A, this alkaline solution
acted as an inhibitor in slowing down the reaction for the formation of acetaldehyde from
pyruvate, since the enzyme which catalyses that reaction will be inactive in alkaline
conditions.in test tube B potassium dihydrogen phosphate that was added acts as buffer in
chemical reactions, so pyruvate was not observed which suggests that the reaction was very
fast such that the intermediate could not be detected. The same reason applies in experiment
two where we tested for the presence of acetaldehyde, sodium sulphite was added only to test
tube D, and this sodium sulphite traps acetaldehyde that is the reason why test tube D gave
the positive result confirming the presence of acetaldehyde. .( Alicia , et al.,2002)

Conclusion
Fermentation is a metabolic process that converts glucose to an alcohol, during this process
some intermediates such as pyruvate and acetaldehyde are formed, in this practical these
intermediates were able to be tested their presence by inhibiting enzymes that are responsible
for further reactions.

References

Alicia N, Norman R, Jones F, 2002, principles and techniques of biochemistry, 4th

edition, page 96.
Ashworth B, Brewer M , Pesce J , 1974 , experimental techniques in biochemistry,
page 76-78.
Banowetz S, Jodi K and Hancock L, 1996, biochemistry and molecular biology, 5th
edition, page 89-92.
Dennis V , Geoffrey Z , William P , 1995 , principles of biochemistry , page 501.
Gumport R, Jonas A and Mintel R, 1989, students companion to stryers biochemistry,
volume 1, page 345-345.