Experiment 2, 3 & 4

Production of Baker’s yeast by fermentation

1.0 Objectives
1. To perform an aerobic cultivation of baker’s yeast in a shake flask and 30L
stirred tank bioreactor.
2. To monitor development of concentration of substrate, biomass, pH and oxygen
during the process.
3. To calculate average yield factors based on these measurements.
4. To recover and purify the product obtained.

2.0 PROCEDURES

2.1 DETERMINATION OF MICROBIAL GROWTH

2.1 .1 Preparation of Inoculum

1. Dip one loop of Saccharomyces cerevisae isolated colony on YM broth. Swirl the
loop in the solution to dislodge the selected culture from the loop.
2. Place the broth bottle in incubator at 30◦C. The exponential phase will last from 2 to
24 hours after inoculation.
Pre-seed culture and batch fermentation medium
Compound Concentration(g/L)
Glucose 45
Yeast extract 5
KH2PO4 2
MgCl2 1
NH4Cl2.H2O 1
pH 6

2.1.2 Batch Cultivation in shake flask

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 1. Dissolve all the composition of inoculation (pre-seed) medium except glucose in
100mL distilled water in a 500 ml Erlenmeyer flask. Adjust pH to value of 5.8
using 1M NaOH or 1M HCl. Dissolve the glucose in 100 mL of distilled water and
sterilize separately at 121°C for 20 minutes. After the sterilization, blend both
parts of the medium aseptically.
2. Transfer aseptically 5% (v/v) Saccharomyces cerevisae suspension into the
sterile medium.
3. Sample out 10 ml for analysis purposes.
4. Incubate in the incubator shaker for 48 hrs, 30oC, at 200 rpm. Take the samples
at 4 hours interval.
5. Analyze the samples collected for
a. pH
b. cell dry weight
c. optical density
d. glucose concentration
6. Plot the calibration curve, substrate consumption and finally the graph for
microbial growth and pH tolerant from the data obtained.

2.2 Production Fermentation

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2.2. 1 Seed culture

1. Perform step 1 until step 2 from the above section 2.1.2, but reduce the volume
to 150 ml.
2. Incubate the medium in incubator shaker at 30oC and 200 rpm. Stop the
incubation when it reaches log phase.

2.2.2 Production Fermenter

1. Prepare 2L bioreactor that consists of 1350 ml of medium. Separate the glucose

solution. Autoclave at 121oC for 15 minutes.
2. Transfer aseptically 10% (v/v) of inoculum into the sterile medium.
3. Sample out 20 ml for analysis purposes (t=0).
4. Run the bioreactor for 3 days, 30oC, at 300 rpm, 1vvm. Take samples at 4 hrs
interval.
5. Perform the analysis to the samples collected
6. The batch culture data of S.cerevisae are presented in the form of graphs of cell
concentration, glucose concentration, culture pH and DOT against fermentation
time. In addition, the data are analyzed in terms of the various kinetic
parameters, specific growth rate, growth yield and specific glucose uptake rate.

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