Bioreactor Bioprocessing

PRACTICAL 1
Kinetics Study Batch Fermentation of Baker’s Yeast

Objective
To study the growth kinetic of Baker’s yeast in batch production

Introduction
Batch fermentation can be considered to be a “closed system”. At the time of zero, the
sterilized nutrient solution in the fermenter is inoculated with microorganism & incubation
is allowed to proceed under optimal physiological conditions. In the course of the entire
fermentation, nothing is added except oxygen (in a form of air), an antifoam, and acid/base
to control the pH. Composition of the culture medium, biomass, and metabolite
concentration change constantly as a result of the cell metabolism. The growth of a
microbial culture may be divided into a number of stages and already discussed by Monod
(1949) and Stanbury and Whitaker (1984). After the lag phase where there are no increase
in cell number, one period of faster growth cell was occur (cell increase with time
exponentially) or called log phase. After substrate was exhausted the growth ceases and this
is called stationary phase. The exhausted of substrate for maintenance and the presence of
toxic cause the cell death and this was called death phase. Many fermentation industries
still used batch fermentation even the increase of productivity for many types of
fermentation can be achieved using continuous and fed-batch fermentation (Whitaker,
1980)

Yeast has been used 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, supporting multibillion dollar alcoholic beverages industry, and single cell protein
industry (animal food supplement).

Methodology
Microorganism
The Baker’s yeast, Saccharomyces cerevisiae, was used in all experiment. This is hybrid yeast
produced for commercial baker.

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. The well
formulated defined medium was used in this experiment.

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Stock Culture Medium

Compound Concentration (g/L)
Glucose 50
Yeast extract 5
KH2PO4 2
MgCl2 1
NH4Cl2H2O 1
Technical Agar 15
pH 5.5

Pre-seed Culture and Batch Fermentation Medium

Concentration (g/L)
Compound
Pre-seed Batch fermentation
Glucose 50 100
Yeast extract 5 10
KH2PO4 2 4
MgCl2 1 2
NH4Cl2H2O 1 2
pH 5.5 5.5

Batch Fermentation
Batch fermentation was carried out in fermenter with 1L culture volume. The inoculum was
prepared as instructed.

A single colony of pure culture from a petri dish, or a slant tube (containing solid gel) was
inoculated into the medium flask (250mL) containing 100mL pre-seed medium. Medium was
sterilized by autoclaving at 121°C, 15psi for 20 min prior to inoculation. The inoculated flask
was constantly agitated in a temperature control flask shaker for 12-14 hours to reach
exponential growth phase, or log phase. The S.cerevisiae culture was then inoculated into
sterile fermenter containing 900mL batch culture medium to start the fermentation. During
the fermentation, samples were withdrawn at time intervals.

In normal practice for preparation of inoculums, subculturing was done repeatedly for
several times to ensure that the culture is acclimated before employed in fermentation.

Sample Analysis
Measuring dry weight
1. 1.5mL of sample was inserted into the eppendorf tube.
2. Step (1) was repeated for another four samples from different time interval.
3. Samples were centrifuged at 4000rpm for 20 min.
4. The cell pellet was dried in the oven at 100°C for approximately 30 minutes and dry
weight of the sample was calculated.

Measuring pH value and optical density at 610nm

1. Spectrophotometer was reset. Wavelength was calibrated at 610nm.
2. Sample was taken and inserted into up to 2/3 of the cuvette.

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3. Cuvette was placed on the sample holder and the cover was closed.
4. Machine was started. Absorbance value was determined.
5. pH value was determined by using electronic pH detector.
6. Step 1-5 was repeated for another four samples from different time interval.

Specific Growth Rate

µ = 1/x (dx/dt)

Results
Cell biomass concentration against time

Cell Biomass Concentration, X vs Time

18.000 IV
III
16.000
14.000
II
12.000
10.000 I
X (g/L)

8.000
6.000
4.000
2.000
0.000
1 2 3 4 5
Time (day)

Legend
I Lag phase
II Log phase
III Stationary phase
IV Death phase

Specific Growth Rate

t (day) X (g/L) 1/X dx dt µ

1 0.6667 1.4999 0 1 0
2 6.7333 0.1485 6.0666 2 0.0490
3 12.7333 0.0785 12.0666 3 0.3157
4 15.5333 0.0644 14.8666 4 0.2394
5 12.5333 0.0798 11.8666 5 0.1894

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X, cell biomass concentration:

[(dry cell (g) + dry filter paper (g)) – (dry filter paper (g))] x 1000
1.5(ml)

Dry cell + Difference Divide with

t (day) Filter paper Difference
filter paper X 1000 1.5 (ml) ≈ X
1 0.8507g 0.0010g 1 0.6667
2 0.8598g 0.0101g 10.1 6.7333
3 0.8688g 0.8497g 0.0191g 19.1 12.7333
4 0.8700g 0.0203g 20.3 15.5333
5 0.8685g 0.0188g 18.8 12.5333

µ, specific growth rate:

dx/dt = µX
µ = 1/X (dx/dt)

t (day) 1/X dx dt Calculation (see formula) µ

1 1.4999 0 1 (1.4999)(0/1) 0
2 0.1485 6.0666 2 (0.1485)(6.0666/2) 0.0490
3 0.0785 12.0666 3 (0.0785)(12.0666/3) 0.3157
4 0.0644 14.8666 4 (0.0644)(14.8666/4) 0.2394
5 0.0798 11.8666 5 (0.0798)(11.8666/5) 0.1894

Value of pH of culture and optical density at 610nm

t (day) pH value Optical Density (A)

1 6.30 0.045
2 6.23 0.093
3 6.21 0.106
4 6.19 0.146
5 6.18 0.158

Discussion
Spectrophotometer and dry weight analysis
 Spectrophotometer is an instrument which measures the amount of light of a specified
wavelength which passes through a medium. According to Beer's law, the amount of
light absorbed by a medium is proportional to the concentration of the absorbing
material or solute present.
 Monitoring cell growth using spectrophotometer (based on absorbance value) has some
advantages: it is vary rapid means of monitoring growth with very minimum disruption
to the culture.
 However, this method does not directly indicate the cell concentration in the culture
(especially when different medium type, condition, or cell strain was analyzed). In this
experiment, error of spectrophotometer reading can result from cell shape and medium
colour. Spectrophotometer does not able to distinguish between the cell and large
particles of debris and viable cells or not.

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 Dry weight measurements of biomass are independent of cell size and directly
proportional to the absorbance (absorbance is a measure of cell mass rather than cell
number).
 Biomass concentration is one of the most critically needed measurements in
fermentation studies. It is also one of the most difficult and unreliable ones. Dry/wet
weight methods fail completely if the broth contains other insoluble particulate matter,
which is often the case in a practical fermentor. These methods cannot distinguish the
viable cells from the dead ones, too.

Specific Growth Rate

µ represent the intrinsic rate of growth of the organism under the condition used
(temperature, culture medium, etc). If µ is zero, then the number of organism is not
decreasing and increasing. If µ is more than zero, then the number of organism will increase
exponentially and if µ is less than zero, then the number of organism will decrease.

pH value
Value of pH becomes more acidic as growth proceeds as a result of metabolic by-product.

Conclusion
Through this experiment, I become aware and understood about growth kinetic of Baker’s
yeast in batch production.

References
Robb, F.T. 1995. Archaea: A Laboratory Manual. CHSL Press, New York. 1037p.

Panikov, N.S. 1995. Microbial Growth Kinetics. Chapman & Hall, London. 378p.

Trun, N.J. and Trempy, J.E. 2003. Fundamental Bacterial Genetics. Blackwell Publishing,
Oxford. 287p

Mendes-Ferreira, A., Mendes-Faia, A., and Lea, C. 2004. Growth and Fermentation Patterns
of Saccharomyces cerevisiae Under Different Ammonium Concentrations and its
Implications in Winemaking Industry. Journal of Applied Microbiology. 97(7): 540-
545.

http://www.engr.umd.edu/~nsw/ench485/lab9c.htm (accessed on 150209)