NAME : NOLUBABALO

SURNAME :NKOSAYIDLI

STUDENT NUMBER : 202108442

COURSE CODE : BCH 223 PRACTICAL 2

GROUP NO. : 3

TITTLE B:THE PRODUCTION OF PYRUVATE AND

ACETALDEHYDE

DURING THE FERMENTATION OF GLUCOSE BY YEAST.

TITTLE: The production of pyruvate and acetaldehyde during the fermentation of
glucose by yeast.

INTRODUCTION:

Glycolysis is the metabolic pathway that converts glucose ( C 6 H 12 O6 ), into pyruvate (

C H 3 COCO 2 H ). The free energy released in this process is used to form the high-
energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine
dinucleotide (NADH). Glycolysis is a determination sequence of ten enzyme
catalyzed reactions. However, the products of glycolysis are sometimes disposal of
the using atmospheric oxygen. when molecular oxygen is used in the disposal of the
products of glycolysis the process is usually referred to as aerobic, whereas if the
disposal uses no oxygen the process is said to be anaerobic. Glycolysis can occur
with or without oxygen. In the presence of oxygen glycolysis is the first stage of
cellular respiration. In the absence of oxygen glycolysis allows cells to make small
amounts of ATP through a process of fermentation. Glycolysis takes place in the
cytosol of the cell’s cytoplasm. The 10 steps of glycolysis are organized by the order
in which specific enzymes act upon the system. (Dennis, 1995).

Step 1: the enzyme hexokinase phosphorylates or adds a phosphate group to

glucose in a cell’s cytoplasm. In the process, a phosphate group from ATP is
transferred to glucose producing glucose 6-phosphate or G6P. one molecule of ATP
is consumed during this phase. Step2: the enzyme phosphoglucomutase isomerize
glucose 6-phosphate(G6P) into its isomer fructose 6-phosphate or F6P. isomers
have the same molecular formula as each other but different atomic arrangements.
Step 3: the kinase phosphofructokinase uses another ATP molecule to transfer a
phosphate group to F6P to form fructose 1.6-bisphosphate or FBP. Two ATP
molecules have been used so far. Step 4: the enzyme triose-phosphate isomerase
rapidly converts DHAP into GAP whilst these isomers can inter-convert. GAP is the
substrate needed for the next step of glycolysis. Step6: the enzyme glyceraldehyde
3-phosphate dehydrogenase (GAPDH) serves two functions in this reaction. First, it
dehydrogenates GAP by transferring one of its hydrogen molecules to the oxidizing
agent nicotinamide adenine dinucleotide (NAD) to form NADH and hydrogen. Step7:
the enzyme phosphoglycerokinase transfers a phosphate from BPG to a molecule of
ADP to form ATP. This happens to each molecule of BPG. This reaction yields two-
phosphoglycerate (3 PGA) molecules and two ATP molecules. Step 8: the enzyme
phosphoglyceromutase relocates the P of the two 3PGA molecules from the third to
the second carbon to form two 2-phosphoglycerate (2 PGA) molecules. Step9: the
enzyme enolase removes a molecule of water from 2-phosphoglycerate to form
phosphoenolpyruvate (PEP). This happens for each molecule of 2 PGA from step 8.
Step 10: the enzyme pyruvate kinase transfers a P from PEP to ADP to form
pyruvate and ATP.

Fermentation refers to the metabolic process by which organic molecules normally

glucose is converted into acids, gases, or alcohol in the presence of oxygen or any
electron transport chain. Fermentation pathways regenerate the coenzyme
nicotinamide adenine dinucleotide (NAD), which is used in glycolysis to release
energy in the form of adenosine triphosphate (ATP). Fermentation only yields a net
of 2 ATP per glucose molecule through glycolysis, while aerobic respiration yields as
many as 32 molecules of ATP per glucose molecule with the aid of electron transport
chain. (Mayo,1994).

The study of fermentation and its practical uses is named zymology and originated in
1856 when French chemist Louis Pasteur demonstrated that fermentation was
caused by yeast. Fermentation occurs in certain types of bacteria and fungi that
require an oxygen- free environment to live which is known as obligate anaerobes, in
facultative anaerobes such as yeast, and in muscle cells when oxygen is in short
supply, such as in strenuous exercise. The process of fermentation is valuable to the
food and beverage industries, with the conversion of sugars into ethanol used to
produce alcoholic beverages, the release of carbon dioxide (CO2) by yeast used in
leavening of bread and with the production of organic acids to preserve and flavor
vegetables and dairy products. (Chojnacka.2006).

After pyruvate is produced from glycolysis, it enters the mitochondria to begin

aerobic respiration. Aerobic respiration begins with the conversion of pyruvate to
acetyl CoA. This conversion takes place in three steps: decarboxylation, the
reduction of NAD+, and the attachment of coenzyme A. Pyruvate is oxidatively
decarboxylated to acetyl-coenzyme A by the metalloenzyme pyruvate ferredoxin
oxidoreductase (POR) and pyruvate formate lyase. An acetyl-CoA is then converted
to acetaldehyde by a CoA-dependent- acetylating acetaldehyde dehydrogenase
(AcDH). (Rumble,2018).

AIM: Is to form pyruvate and acetaldehyde from glucose and use nitroprusside to
test the present of pyruvate.

METHODS AND MATERIALS

FORMATION OF PYRUVATE FROM GLUCOSE:

All the apparatus was rinse with distilled water. Five milliliter of glucose solution were
pipetted into two boiling tubes A and B. Five milliliter of yeast suspension was added
in the slightly alkaline solution of NA2HPO4 to tube A. Test tubes were placed in a
water bath at 37℃ for 1 hour. Test tubes were observed during the incubation period
and their appearance was noted. At the end of the incubation period 2ml of
trichloroacetic acid solution was added to each tube, then mixed thoroughly, and
centrifuged for 20 minutes on a benchtop centrifuge. Supernatant was removed and
the presence of pyruvate was tested.

SODIUM NITROPRUSSIDE TEST FOR PYRUVATE.

An amount of 2ml of boiled supernatant was added to 1 cm of solid ammonium

sulphate in a test tube. Two drops of freshly prepared sodium nitroprusside solution
of 50g/L were added, then mixed thoroughly, and concentrated ammonia was
carefully running down the side of the tube to form two layers.

FORMATION OF ACETALDEHYDE FROM GLUCOSE

An amount of 5ml of glucose solution were pipetted into two tubes C and D. Five
milliliter of yeast suspension were added in water to both tubes, and 0.5g of sodium
sulphate to tube D. Then mixed thoroughly, two test tubes were incubated at 37 ℃
for 1 hour. Test tubes were observed during the incubation period and their
appearance were noted. At the end of the incubation period, the test tubes were
centrifuged then the supernatant were removed, and 0.5 ml of freshly prepared
sodium nitroprusside was added to 2 ml of the supernatant followed by 2ml of
aqueous piperidine and were mixed. Whether the acetaldehyde was present then a
blue color was observed.
RESULTS:

Table 1: the observation of all test tubes during the 60 minutes

Time Test tube A Test tube B Test tube C Test tube D

10 Two layers formed Two layers formed Little foam No layers appeared
but there was no and the colour formed but there here but foam was
foam changed to light was no layers there and the
brown and little bit of colour changed to
colour change coffee brown
20 No colour changes Lot of foam Two layers of 1 little layer of foam
and no foam appeared and the foam formed appeared and the
formed colour was light and dark brown colour remained
brown colour change coffee brown
30 No layers formed More foam was One layer of 1 thin layer
and there was lot formed, the colour foam, the colour formed, more
of residues at the remained more was coffee residues at the
bottom light brown, two brown, no bottom and the
layers formed, residues at the colour were still
layer at the bottom bottom. coffee brown.
was stronger than
the one above and
it was lighter
45 No layer formed No residues at the A thin layer of No layer just few
even after bottom, a thick every white bubles residues at
incubation, light layer formed of foam appeared, the bottom and the
brown color was white foam, and coffee brown colour were
there and residues the colour was still colour and no caramel like.
at the bottom light brown residues
 60 No layer formed No residue at the A thin layer of No layers just few
even after bottom, a thick very white foam bubbles residues at
incubation, light layer formed of appeared, the bottom and the
brown colour was white foam, and coffee brown colour were
there and residues the colour was still colour and no caramel like.
at the bottom light brown residues.
\

FIGURE 1: Incubation of test tubes after 30 minutes

FIGURE 2: incubation of test tubes after 60 minutes

FIGURE 3: pyruvate and acetaldehyde test color formation after four tubes were
incubated for the length of 1 hour at 37℃ .
Discussion:

In the absence of oxygen yeast cells convert glucose to alcohol and carbon dioxide
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. 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
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 37◦C which is the reason why the test tubes were
incubated in a water bath at 37◦C, 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 ammonium sulphite. (Banowetz,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,2002).

The fermentation productivity differs in every test tube. Test tube D after incubation,
there was no changes, this means that there was no productivity. In test tube C, the
foam was also formed later than B. there were changes during the incubation of the
incubation of the formation of pyruvate from glucose and in acetaldehyde formation.
The reason for the changes is because of the closed tubes not getting any oxygen
so fermentation was taking place.

Fermentation does not require oxygen. If oxygen is present, some species of yeast
will oxidize pyruvate completely to carbon dioxide and water in a process called
cellular respiration, hence these species of yeast will produce ethanol only in an
anaerobic environment. The acetaldehyde was present, a blue colour was seen in
both test tubes but it was faint in test tube C and pyruvate was observed. Pyruvate
decarboxylase is inactive in slightly alkaline solution, so pyruvate accumulates, and
its presence is demonstrated by the reaction with sodium nitroprusside. So, we can
conclude that yeast can produce pyruvate and acetaldehyde based on this
experiment. ( Martinek, 1969).
REFERENCES:

Banowetz.S.(1996). Biochemistry and molecular

Chojnacka.K.(2006). Fermentation products. Chemical engineering and chemical

process technology,12.

Dennis.R.(1995). Principles of biochemistry. 4th edition. page.89-92.

Martinek.R.(1968). Practical Clinical Enzymology: J.Am.Med.,31-162.

Mayo.I.(1994). Chemist, biotechnology, and statesman. Journal of chemical

education.71(3), (209).

Romano.A.H.(1996). Bacterial physiology and metabolism.3 rd edition.47(28):16-48.