Experiment:

Aim: Alcohol fermentation by using Baker’s yeast and its quantification by dichromate method
Principle:
Baker's yeast is the common name for the strains of yeast commonly used in baking bread and other
bakery products. Baker's yeast is of the species saccharomyces cerevisiae
Fermentation is generally defined as the conversion of carbohydrates to acids or alcohols. The
conversion of corn sugar (glucose) to ethanol by yeast under anaerobic conditions is the process used
to make the renewable transportation fuel, bioethanol.
A fermentor is operated by inoculating a complex sugar medium with a microorganism. This
microorganism is generally allowed to reproduce under aerobic conditions before the fermentor is
switched to anaerobic conditions to produce secondary metabolites such as ethanol.
Carbon sources such as glucose (C6H12O6) serve two purposes: material building blocks for
biosynthesis and for energy. In this experiment, yeast uses the glucose in three ways:

Fermentation will be aerobic for 3-4 hours until noon, when air will be turned off and fermentation
will be allowed to continue under anaerobic conditions for another 4 hours. One mole of glucose is
converted into two moles of ethanol and two moles of carbon dioxide:
C12H22O11 +H2O + invertase →2 C6H12O6 (1)
C6H12O6 + Zymase → 2C2H5OH + 2CO2 (2)
The second stage is the fermentation process to convert glucose into ethanol and CO 2. Fermentation
ethanol is a change of a mole of glucose into 2 moles of ethanol and 2 moles of CO2.
The yeasts will mainly metabolize glucose and fructose to form pyruvic acid through the stages of the
reaction pathway Embden-Meyerhof-Parnas, nevertheless pyruvic acid generated would be
decarboxylated to acetaldehyde which then experiences dehydrogenation to ethanol.
Requirement for production and harvesting of alcohol
• YEPD broth (yeast extract: peptone: dextrose - 1:2:2)
• Conical flasks
• Refrigerated culture of Saccharomyces cerevisiae/Baker’s yeast powder
• Digital weighing machine, spatula
• Laminar air flow
• Autoclave
• Micropipette
• Rotary shaker incubator, Parafilm, cotton, tissue paper
Requirement for estimation of harvested alcohol by potassium dichromate method
• Potassium dichromate reagent
• Test tubes, test tube stand, micropipette
• Distilled water
• Ethanol
• Sample containing produced ethanol under aerobic and anaerobic cond.
• Heating element
• Spectrophotometer, cuvettes, tissue paper
1. Overview of the procedure:

1
 • Reactivation of culture of Saccharomyces cerevisiae in YEPD broth
• Preparation and sterilization of YEPD broth (Production media)
• Transfer of reactivated culture into production medium (Inoculation)
• Maintaining inoculated flask in an incubator under aerobic and anaerobic conditions
• Harvesting of cell free crude extract containing alcohol
• Estimation of harvested alcohol by potassium dichromate method
1.1. Reactivation of culture of Saccharomyces cerevisiae
• 100 ml of the YEPD broth was prepared in conical flask, autoclaved and inoculated with 1 ml
of the refrigerated culture of provided Saccharomyces cerevisiae, incubated for 2 days under
aerobic condition
Or
• The dry yeast S. cerevisiae was used, 50ml of sterile distilled water and 5g of sugar were added
to a beaker; it was mixed and heated to a temperature of 37ºC, then 1g of it was added, mixing
constantly until homogenized. It was lowered and allowed to cool to room temperature.
1.2. Preparation and sterilization of production medium (YEPD broth)
• 100 ml of YEPD broth in 4 separate conical flask was prepared, labelled them as anaerobic
(24h, 48h, 72h, 96h) and autoclaved
1.3. Transfer of reactivated S. cerevisiae culture into production medium (Inoculation)
• 1 ml of the reactivated culture of S. cerevisiae was transferred aseptically in 100 ml of the
YEPD broth contained in 250 ml conical flasks.
• In this way all 4 conical flasks were inoculated separately with 1 ml of the reactivated culture
of S. cerevisiae
1.4. How anaerobic conditions were created in inoculated flasks
• Multiple layers of Parafilm were covered over the cotton plug which did not allow diffusion of
air and create partial or completely anaerobic conditions
1.5. Maintaining inoculated flask in an incubator under aerobic and anaerobic conditions
• 4 inoculated flasks labelled as anaerobic were placed in the incubator and maintained under
anaerobic conditions in shaking for 24-96 h.
1.6. Harvesting of cell free crude extract containing alcohol
• Take out the flask after end of the incubation period (24h and 96h).
• Filter the fermentation broth through Whatmans No.1 filter paper in order to remove yeast
cells.
• Do the centrifugation of the filtrate at 8000 rpm 10 min.
• Discard the pellet and use the supernatant for ethanol assay
2. Estimation of harvested alcohol by potassium dichromate method
2.1. Plotting of standard curve for ethanol
Principle: Most of the chemical oxidation methods are based on the complete oxidation of ethanol by
dichromate in the presence of sulphuric acid with the formation of acetic acid. This reaction is highly
preferred because potassium dichromate is easily available in high purity and the solution is
indefinitely stable in air. The reaction that occurs between alcohol and potassium dichromate is:
2Cr2O7-- + 3C2H5OH + 16H+ -----> 4Cr+++ + 3CH3COOH + 11H2O
Dichromate (Cr2O7--, Cr(VI)) is yellowish in color and the reduced chromic product (Cr +++, Cr(III)) is
intensely green. Because the absorption spectra of dichromate and chromic ions overlap significantly,
Beer’s law is not obeyed. Instead, the spectra of the solution of interest must be analyzed at multiple
wavelengths to calculate the individual concentrations of dichromate and chromic ions in a mixture
subject to the material balance that the total number of chromium atoms must be conserved.

2
 Proper concentration of sulphuric acid in the surrounding solution will direct the oxidation of ethanol
toward acetic acid instead of acetaldehyde.
Reagent preparation:
Potassium dichromate: The dichromate reagent was prepared by dissolving 10 g of potassium
dichromate in 100 ml of 5 M sulphuric acid solution.
5M sulphuric acid solution: Take 27 ml of concentrated sulphuric acid and add 73 ml of distilled
water to it.
Procedure:
1. Pipette 20-180µl of ethanol solution (50%) in different test tubes and diluted upto 3 ml with distill
water.
2. To avoid the loss of liquid due to evaporation, cover the test tube with a piece of paraffin film ifs a
plain test tube is used.
3. Add 5ml potassium dichromate reagent to all test tubes
4. Heat the mixture at 90ºC for 15 minutes to develop the red-brown colour.
5. After cooling to room temperature in a cold water bath, add 9 ml of distilled water. This will give 4
times dilution.
6. Record the absorbance with a spectrophotometer at 600 nm
Table: Standard curve of ethanol
S.N Volume Volume Amount Volume of Mix all the content Transfer Add OD
. of of of potassium and place tubes on 0.2ml from distille (600
alcohol distilled alcohol dichromate boiling water bath each test d water nm)
(95%) water (ml) for 15 min and cool tube
(µl) it. separately in
The color intensity new set of
will be extremely tubes
1 0 (Blank) 3000 µl 0 5 ml higher. Therefore, 0.2ml 3.8ml 0.000
transfer 0.2ml from
2 20µl 2980µl 0.63% 5 ml 0.2ml 3.8ml 0.055
each test tube
3 40µl 2960µl 1.26% 5 ml separately in new 0.2ml 3.8ml 0.070
4 60µl 2940µl 1.89% 5 ml set of tubes. This 0.2ml 3.8ml 0.127
5 80µl 2920µl 2.53% 5 ml will give 20 times 0.2ml 3.8ml 0.414
6 100µl 2900µl 3.16% 5 ml dilution. This 20 0.2ml 3.8ml 0.178
timed dilution will
7 120µl 2880µl 3.79% 5 ml 0.2ml 3.8ml 0.178
be used in
8 140µl 2860µl 4.43% 5 ml calculation 0.2ml 3.8ml 0.179
9 160µl 2840µl 5.06% 5 ml 0.2ml 3.8ml 0.222
10 180µl 2820µl 5.69% 5 ml 0.2ml 3.8ml 0.388

N1V1 = N2V2
95% X 0.02 = N2 (%) X 3.00ml, N2 = 0.63%
95% X 0.04 = N2 (%) X 3.00ml, N2 = 1.26%
95% X 0.06 = N2 (%) X 3.00ml, N2 = 1.89%
95% X 0.08 = N2 (%) X 3.00ml, N2 = 2.53%
95% X 0.10 = N2 (%) X 3.00ml, N2 = 3.16%
95% X 0.12 = N2 (%) X 3.00ml, N2 = 3.79%
95% X 0.14 = N2 (%) X 3.00ml, N2 = 4.43%

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95% X 0.16 = N2 (%) X 3.00ml, N2 = 5.06%
95% X 0.18 = N2 (%) X 3.00ml, N2 = 5.69%
Standard curve of alcohol
0.45
0.4
Absorbance at 600nm

0.35
0.3 f(x) = 5.94443244685801 x
0.25 R² = 0.82032312482252
0.2
0.15
0.1
0.05
0
0.00% 0.50% 1.00% 1.50% 2.00% 2.50% 3.00% 3.50% 4.00% 4.50% 5.00% 5.50% 6.00%
Percentage of alcohol

Table: Ethanol assay

S.N Volume of Volume of Volume of Mix all the Volume of OD
culture distilled potassium content and distilled (600 nm)
supernatant water (µl) dichromate place tubes water (ml)
(sample) (ml) on boiling
water bath
1 0 (Blank) 3000 µl 5 ml for 15 min 9 ml 0.000
and cool it.
2 120µl 2880µl 5 ml 9 ml
(Anaerobic 24h)
3 120µl 2880µl 5 ml 9 ml
(Anaerobic 48h)
4 120µl 2880µl 5 ml 9 ml
(Anaerobic 72h)
5 120µl 2880µl 5 ml 9 ml
(Anaerobic 96h)
Trial Type of Duration of O.D. Ethanol concentration %
fermentation fermentation per ml of cell free
supernatant
1 Anaerobic 24h

48h

2 Anaerobic 72h

96h

Calculation:
Y=------------------- (From standard curve)
4
X = -------------------
X = --------------------
Ethanol concentration (%) in unknown sample = % from standard curve x dilution factor (4)
(3 ml of solution is diluted to 12 ml with distilled water it gives 4 times dilution therefore dilution
factor is 4)
Ethanol concentration (%) in unknown sample=---- x 4 = --- %
------- ml of unknown sample has ------alcohol
Then 1 ml of unknown sample has ----/---- %= -----%
Results
The amount of Ethanol secreted after 24 h incubation under anaerobic cond is=
The amount of Ethanol secreted after 48 h incubation under anaerobic cond is=
The amount of Ethanol secreted after 72 h incubation under anaerobic cond is=
The amount of Ethanol secreted after 96 h incubation under anaerobic cond is=
Precautions:
1. Wear hand gloves properly
2. Handle sulphuric acid carefully
3. Autoclaved media properly
4. Use the capped test tubes for estimation of ethanol