Experiment 2, 3 & 4

Production of Bakers yeast by fermentation

1.0 Theory
Yeast has been in use since the beginning of human civilization. It is still a very versatile
microorganism widely used in a wide range of fermentation industries. For example, the
carbon dioxide released as a result of carbohydrate metabolism is used to raise dough in
baking; the ethanol produced supports a multi-billion dollar alcoholic beverage industry;
single cell protein is used to supplement animal feed. It is also currently the most widely
used microorganism for ethanol production as the altemative energy source,
Zymomonas mobilis being the other potentially dominant one. Although fermentation
was practiced even before recorded history, the fact that microorganisms were
responsible for the leavening and brewing actions was not realized until the last century.
Yeast biomass is produced not only for the baking industry but also for pharmaceutical
and microbiological purposes and is used for animal feed in the farming industry.

The Baker's yeast, Saccharomyces cerevisae, is used in all experiments. This is hybrid
yeast produced for commercial Baker's, which is developed for their capacity to quickly
ball on the dough with gas, especially when primmed with sugar and milk. Baker's yeast
die in an oven before they have chance to digest much of the actual starch, producing a
loaf that inevitably tastes of them and raw flavour-something the Baker's attempt to
mitigate by sweetening the bread with malted barley. However, when yeast is given time
to digest the starch, the result is a more nutritious loaf (additional protein and B vitamin)
and one with superior texture and richer, sweet flavour.

1.1 Media
Most practical industrial fermentation processes are based on complex media
because of the cost and the choice of the nutrients and the ease of nutrient preparation.
For example, complex media for yeast fermentation can be easily prepared in a lab by

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following the same recipe as that used in the YPG agar, minus the agar: 5 g/L yeast
extract, 10 g/L peptone, and 5 g/L glucose. However, the use of complex media is
discouraged in the fundamental studies of fermentation kinetics because of the
possibility of variations in the nutrient composition from run to run. For example, the
exact content of a yeast extract preparation is not known, and its nutritional quality may
vary from batch to batch. On the other hand, a defined medium can be reproduced time
after time to ensure the reproducibility of biochemical experiments. The disadvantage of
a defined medium is that there always the possibility of missing some important growth
factors. The formulation of a defined medium is often a tedious process of trial and
error.

However, a well formulated defined medium can support the healthy growth and
maintenance of cells as effectively as, or sometimes superior to, a complex one. A very
simple defined medium will be used in this experiment.
1.2 FERMENTATION
Two modes of fermentation, batch and continuous, are carried out for Baker's yeast
production.
1.2.1 Batch Fermentation
Batch fermentation is carried out in the bioreactor with 1 L culture volume. The inoculum
is prepared as follows. A single colony of pure culture from a Petri dish, or a slant tube
(containing solid gel) is inoculated into the inoculum flask (250 mL) containing 100 mL
pre-seed medium. The inoculated flask is constantly agitated in a temperature controlled
flask shaker for 12-14 hr to reach the exponential growth phase, or log phase. The
S.cerevisae culture is then inoculated into sterile fermenter containing sterile 900 mL
batch culture medium to start the fermentation.
In normal practice for preparation of inoculum, sub-culturing is done repeatedly for
several times to ensure that the culture is acclimated before it is employed in
fermentation. A similar process of repeated inoculation is carried out in the fermentation
industry to build up enough inoculum needed to seed a larger fermenter. To reduce the
shock resulting from a drastic change in the growth environment, the composition of the

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media used in preparing the inoculum should optimally be identical as that used in the
main process. When working with a pure culture, one must operate under the
assumption that contaminating microorganisms are present everywhere in the open
environment, a fact demonstrated in our previous experiment. It is important to know
intuitively when sterile tools or glassware must be used and when sterilization is not
necessary. This requires the ability to distinguish clearly the sterile side from the non
sterile side.
In this experiment, the interior of the shaker flask or the fermenter is the sterile portion of
the system. Anything that is part of the system and anything that ever comes in direct
contact with that part of the system must be sterile. Thus, the nutrient in the shaker flask
before inoculation must be sterile, which in turn requires that the reservoir storing the
filtered nutrient is sterile and that the entire process of dispensing the nutrient from the
medium jar to the shaker flask is carried out aseptically. In addition, items that enter the
shaker flask such as the cotton plug, inoculation loop, sampling pipets, and even air
must all be sterile. This will be demonstrate an practice during the experiment
Specific growth rate:
dX
X
dt

eq. 1

dX
dt
X

eq. 2

Xt

X
0

ln

t1 t 0
max S
KS S

eq. 3

eq. 4

Growth yield:
The growth yield Y X

is defined as the increase in biomass (dX) as a result of the

utilization of substrate (dS)

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YX

dX
dS

eq.5

The growth yield is assumed constant and reproducible when taken over the phase of
growth-associated fermentation

YX

Xm X0

Sf

eq.6

Substrate consumption rate:

The utilization of substrate in a culture during an infinitely small interval of item given by,
DS q S Xdt

Eq. 7

where qs is termed the "specifc substrate uptake rate"

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

3.0 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 30C. The exponential phase will last from 2 to
24 hours after inoculation. The cell count should be around 5 x 106 cells/ml.

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Pre-seed culture and batch fermentation medium

Compound

Concentration(g/L)

Glucose
Yeast extract
KH2PO4
MgCl2
NH4Cl2.H2O
pH

45
5
2
1
1
6

4.0 PROCEDURES
4.1

Microbial Growth Determination

1. Dissolve all the composition of inoculation (pre-seed) medium except glucose in
150mL 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 X mL of distilled water and
sterilize separately at 121C for 20 minutes. After the sterilization, blend both
parts of the medium aseptically. (Total volume of the medium is 250 ml, X is
calculated after considering 5% (v/v) inoculum)
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 3 days, 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

(refer APPENDIX).
6. Plot the calibration curve, substrate consumption and finally the graph for
microbial growth and pH tolerant from the data obtained.

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4.2 Production Fermentation

4.2.1 Seed culture
1. Perform step 1 until step 2 from the above section 4.1, 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.
4.2.2 Production Fermenter
1. Follow the procedure for bioreactor set up below (Section 4.3).
2. Prepare 2L bioreactor that consists of 1350 ml of medium. Separate the glucose
solution. Autoclave at 121oC for 15 minutes. (1350 ml is total volume)
3. Transfer aseptically 10% (v/v) of inoculum into the sterile medium.
4. Sample out 20 ml for analysis purposes (t=0).
5. Run the bioreactor for 3 days, 30oC, at 300 rpm, 1vvm. Take samples at 4 hrs
interval.
6. Perform the analysis to the samples collected (refer APPENDIX).
7. 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.

4.3 BIOREACTOR
A 2 L stirred tank bioreactor is used for all experiments (Biostat B, B.Braun,Melsungen).
Two six-bladed turbine impellers mounted on the agitator are used for agitation. For
aeration, sterile air is sparged through the air sparger placed just below the impeller. The
fermenter is equipped with temperature, dissolved oxygen and pH controllers. During all
fermentations, agitation speed is fixed at 300 rpm and the temperature within the
fermenter was controlled at 30C.
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4.3.1 SETTING UP BIOREACTOR

All sensors, except for temperature sensor, are sterilizable and would be sterilized right
inside the reactor. The pH sensor must be calibrated before the sterilization in a
subprogram of the main menu CALIBRATION.
Calibration of the pH sensor: use an arrow on the keyboard and set the cursor to
second row and switch the regime from auto to man. Switching is performed using
button ALTER and the choice is confirmed by button ENTER. The cursor relocates to
temperature; manually enter desired temperature. Press ENTER. Immerse the pH
sensor into pH 7 buffer and press ENTER. When the pH value stabilizes, the biostat
would record the value and it will automatically relocate the cursor to following row.
Immerse the electrode into pH 4 buffer and press ENTER. When the pH value stabilizes,
the regime is automatically switched to auto and the calibration is successfully finished.
At sterilization, it is important that the ends of all hoses and all of metallic parts would be
covered by aluminum foil; the outlet of out-gassing from reactor must not be closed. After
the sterilization and cooling, place the reactor into inoculation box and pour inside the
glucose solution under aseptic conditions; insert the temperature probe and connect
sterilized air filters to inlet and outlet of air.
Transport the reactor back to biostat; connect all sensors, cooling water, air supply. Turn
on the cooling water from central supply and turn on the flow of air, also from central
supply. Carefully and in parallel, turn on the air supply on the biostat and simultaneously
open the squeezer on the air inlet line. Regulate the flowrate of air using a rotameter,
which is an accessory of the biostat. Set the value to 1.0 vvm.
After stabilization of temperature, adjust pH to value of 6. Turn on the temperature
control. In the subprogram CONTROL LOOPS on the first page, change the regime from
Off to Auto. Set the cursor to respective row, press ALTER and then ENTER.
Navigate between rows using the arrows in the main keyboard of the biostat. Turn on the
stirring control: Change the regime from Off to Auto in the same way.

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Calibration of the oxygen sensor: switch air supply to supply of nitrogen. Navigate to
second page in subprogram CALIBRATION (navigate between pages using button
ENTER). Set cursor to second row using the arrows on the keyboard and switch regime
from man (manual) to auto. Switching between regimes is performed using button
ALTER; confirm the choice by button ENTER. The cursor relocates to temperature;
manually enter desired temperature. Press ENTER. Navigate the cursor to NITR and
press ENTER. When the value of concentration of dissolved nitrogen stabilizes at value
of approximately 0%, navigate the cursor to NITR and press ENTER. Biostat records a
stable value and the cursor automatically relocates to AIR. Switch the supply of nitrogen
to supply of air and wait until the value of concentration of dissolved oxygen grows to
value of approximately 100%. Press ENTER to record a stable value corresponding to
saturation state. The cursor relocates to row TEMP and the regime switches
automatically to Auto. The calibration is successfully finished.
After calibration of oxygen sensor, disconnect the reactor and place it into inoculation
box. Transfer 5%v/v of seed culture (inoculums) under sterile conditions into separator
connected to bioreactor. Reconnect the reactor to biostat. Turn on the inlet of cooling
water and air. Turn on the temperature control and stirring control and, after checking the
completeness of the device and connections, inoculate the reactor.

6.0 RECOVERY AND PURIFICATION

At the end of the growth phase, the yeast will be removed from fermentation broth by
using centrifugal separator run at a minimum speed of 5000rev/min. Harvested cells are
then washed several times with water, chilled to 2-4C and finally dried to around 7075% (w/w) moisture using vacuum filter dehydrators. Alternatively, it may be dried further
to 7-10% (w/w) moisture to form dried yeast at 50-60C, which can be stored for long
periods without refrigeration. Weigh the biomass obtained and calculate the yield for 2L
bioreactor.

Reports: Please prepare three reports from this experiment. First report from the
shake flask fermentation, second report from 2L bioreactor fermentation and third
report on the recovery and purification.
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APPENDIX: ANALYTICAL METHODS

A1 Sample processing:
Determine the absorbance (refer section A2) of the culture (600nm), retain 2 x 1.5mL for
cell dry weight (CDW) determination (section A3). The supernatant from the CDW
determination can be used to estimate glucose and ethanol.

A2 Absorbance
The growth of microbial cells can be determined by measuring the absorbance and
relating this value to a calibration curve of absorbance against cell dry weight. Generally
600nm is used for yeasts, whereas 400-450nm may be used for bacteria (Fig. 1). One of
the major sources of error in such measurements is the nonlinearity of the absorbance
measurements at high cell densities. If the absorbance is above 0.3, carefully and
accurately dilute the culture until a suitable value has been obtained. Measure the
absorbance against a medium blank.

Figure 1: The relationship between absorbance of yeast cultures against cell dry
weights.
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A3 Dry weight determination

This measurement forms the basis for the determination of most of the important growth
parameters such as productivities and yields, since the concentration of parameters can
be directly related to a constant state of reference that is the cell composition. Thus,
microbial cells are like all living systems, composed of ~80% water. The actual water
content within the cells can vary considerably, depending on their physiological state,
whilst the intracellular water content will depend on the method used to separate the
cells from the medium, as well as the nature of the medium itself and the organism.
Dry weight methods aim to completely remove both intra- and intercellular water
completely, so that the non viable cell material remaining is composed of carbohydrates,
fats, proteins and nucleic acids.
One method is as follows:
1. Carefully and precisely weigh two dry clean microcentrifuge tubes (1.5mL volume
size).
2. Carefully mix the culture sample and accurately pipette 1.5mL into each tube.
3. Centrifuge the tubes at 3000rpm for 10 minutes.
4. Carefully decant the supernatant and resuspend cell pellet in approximately
1.5mL saline (0.9% NaCl) and re-centrifuge.
5. Decant supernatant and place tubes in 90C oven for 20 hours.
6. Remove tubes from oven and immediately place in a dessicator containing a
drying agent until cool.
7. Reweigh tubes.
8. Calculate CDW (X):

The correlation between absorbance and CDW can now being developed. This standard
curve will be used in the latter experiments.
A4. Determination of Total Reducing sugars (GLUCOSE)

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The dinitrosalicylic acid (DNS) method as proposed by Miller et al. (1959) was employed
for total reducing sugar determination. In this method, 1 ml of supernatant with 1 ml DNS
reagent was mixed, and the tubes were placed in a boiling water bath for exactly 5 min.
The tubes were then cooled under running tap water to room temperature. A 10 ml
distilled water was added to a reaction mixture and shaked well. Finally, the tubes were
allowed to stand for at least 20 minutes and the absorbance was read using
spectrophotometer (Shimadzu, Model UV-1601 PC) at 540 nm. The absorbance (after
subtraction of the reagent blank) was then translated into glucose equivalent using a
standard graph obtained by plotting glucose against absorbance.

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