6 Practical 5: Growth Kinetics of Saccharomyces cerevisiae

Outcomes for Practical 5

1. Understand the typical growth curve
2. Understand the influences on the phases of the growth curve
3. Understand growth kinetics and oxygen demand
4. Able to use sterilisation techniques
5. Able to use the bioreactors for microbial cultivation
6. Able to manipulate and prepare samples
7. Able to perform the following analyses:
a. turbidity
b. dry mass
c. spread plating and colony counts
d. glucose
8. Generate growth curves
9. Find growth kinetics values
Prerequisite: Collection and storage of samples for use in Practical 6

6.1 Background to Microbial Growth Kinetics

Saccharomyces cerevisiae is a yeast used in a variety of industrial applications. There are

two modes of cultivation, aerobic and anaerobic. In aerobic cultivations of S. cerevisiae the
main objective is the production of biomass (eg production of Baker's Yeast). In anaerobic
fermentations of S. cerevisiae conversion of sugars to ethanol is the primary focus of (eg
beer brewing, wine making).
In a typical batch process the number of living cell varies with time, as shown in Figure 6-1.
The shape of the curve is dependent on the microbial species being grown and the
environmental conditions under which they are grown. The growth curve starts off with a
lag phase (A), where no increase in cell numbers is evident. During this time the cells are
adapting to their new environment. The lag phase is followed by the phase of exponential
growth (B), where the cell numbers increase exponentially with time. Naturally in a closed
vessel the cells cannot multiply indefinitely and a stationary phase (C) follows the period of
exponential growth. At this point the population has achieved its maximum size. Eventually
a decline in cell numbers occurs during the death phase. A growth acceleration phase may
be considered to follow the lag phase and precede the exponential phase, and similarly a
growth retardation phase may be considered to occur before the stationary phase.
 cell concentration (logarithmic scale)

A B C

Time

Figure 6-1 Typical growth curve for batch cell cultivations of yeast
showing (A) lag phase, (B) exponential phase, (C) stationary phase, the death phase follows (C) (Bailey and Ollis,
1986).

The length of the lag observed when culture is inoculated into fresh medium depends on
both the changes in nutrient composition experienced by the cells and the age and size of
the inoculum. In general the lag phase can be minimised by obeying the following rules:
 The inoculating culture should consist of actively-growing exponential-phase cells.
 The culture medium used to grow the inoculum should be as close as possible to the
final full scale fermentation composition.
 Use of a large inoculum (5 to 10% of the new medium volume) is recommended
to avoid loss by diffusion of required intermediates or activators.
At the end of the lag phase the population of microorganisms is well adjusted to its new
environment. The cells can multiply rapidly and the cell mass doubles regularly with time at
the maximum specific growth rate. Equation 6-1 describes the increase in cell numbers during
the exponential phase.
𝑑𝑥
= 𝜇max 𝑥 Equation 6-1
𝑑𝑡
Integration of gives:

𝑥 = 𝑥0 𝑒 𝜇max (𝑡−𝑡lag) where 𝑡 > 𝑡lag Equation 6-2

𝑥 = 𝑥0 where 𝑡 = 𝑡lag
The time interval required to double the cell population (𝑡𝑑 ) can be easily calculated from:
ln 2
𝑡𝑑̅ = 𝜇 Equation 6-3
max

As the nutrients for cell growth become depleted in the culture medium and toxins
accumulate, the increase in cells per unit time are no longer given by 𝜇max . A general
equation (Monod equation) that can be applied to describe the rate of growth during the
exponential phase and the period of growth retardation up until the beginning of the
𝑑𝑥
stationary phase is given by = 𝜇𝑥 .
𝑑𝑡
𝑑𝑥
= 𝜇𝑥 Equation 6-4
𝑑𝑡

where
𝑆
𝜇 = 𝜇max 𝑆+𝐾
𝑠

𝐾𝑠 is a constant equal to the substrate concentration at which the specific growth rate is half its
maximum value as shown in Figure 6-2. The latter equation indicates that if the substrate
concentration 𝑆 is large the growth rate tend to 𝜇max . At low concentrations of substrate the
growth rate approaches zero.
Specific Growth Rate (µ)

µmax

Km

Figure 6-2 Idealised representation of limiting substrate concentration on s pecific growth rate (Tuite and Oliver, 1991)

6.2 Methods for Growth Kinetics Analyses

6.2.1 Biomass production

Turbidity
Agitate the sample. Add approximately 3 mL of sample to a plastic cuvette and read the
absorbance on the spectrophotometer against water at a wavelength of 660 nm. If the
absorbance reading is greater than 1.0 dilute the sample with deionised water. If dilution is
required make a note of the dilution factor.

Dry mass
1. Pre-weigh three pre-dried 2 mL Eppendorf tubes from the desiccator. Label and number
these. Agitate the sample and pipette 2.0 mL into each Eppendorf tube. Centrifuge the
tubes for 5 minutes at full speed in the microfuge.
2. Draw off the supernatant from each tube and filter (0.22 µm) the volume into another, labelled,
sterile 2 mL Eppendorf tube for subsequent analyses (ethanol, glucose, VFAs). Store the
filtered sample in the freezer.
Samples in communal spaces (constant temperature rooms, fridges, freezers,
incubators) must ALWAYS be properly labelled including your name.
3. Add 1.0 mL of deionised water to the three numbered tubes containing pellets from 1. Re-
suspend the pellets on a vortex machine. Centrifuge the tubes again for 5 minutes. Decant
the wash water and place the numbered tubes in the oven (80°C) for 48 hours, after which
they must be weighed again.

Spread plating
Using the technique outlined in Practical 2, determine the number of colony forming units
(CFU/mL) at selected sampling points. This is not required for every sample, but observation
of the relationship between OD660 and CFU/mL should be possible.

6.2.2 Glucose analysis

Samples will be analysed by HPLC (see Practical 6) after completion of the cultivation. Total
carbohydrates will be measured at each sampling point using the Phenol-sulphuric acid
method (CeBER Laboratory Methods Manual).

6.2.3 Metabolite production

Ethanol concentration
The ethanol content of the frozen supernatants will be determined by HPLC (see Practical
6).

Volatile Fatty Acid concentration

The VFA analysis will be performed by HPLC (see Practical 6).

6.2.4 Growth kinetics variables

Oxygen Utilisation Rate (OUR) is measured using the following method. Take note of the date
and time when the OUR is measured.
When the culture is actively growing:
1. The operating conditions are as follows: temperature 30°C, agitation rate 500 rpm and
aeration rate 0,5 vvm.
2. Turn the agitation rate as low as possible while still maintaining adequate mixing (about
100rpm). Shut off the air. Log the drop in DO concentration every 10 seconds until the DO
concentration approaches 2 mg/L. Do not allow the DO concentration to fall below 2
mg/L, as oxygen starvation may permanently affect the cells.
 Note that when the oxygen supply to the reactor is switched off, all the oxygen
does not leave the system immediately.
 This causes a rounded curve before the straight-line portion is reached. This initial
part of the plot should be ignored.
3. When the DO concentration approaches 2 mg/L, open the air valve to 0.5 vvm and
increase the agitation to 500 rpm. Record the increase in DO concentration.
Repeat this experiment for different batch growth phases (see Section 6.2). Sample for cell
dry weight and other measurements (see Section 6.2) at each sample point within each growth
phase.
For the steady state method (see Section Error! Reference source not found.), offgas
measurements can be done during the bioreaction using a plastic bag to collect the gas
sample. The gas bag is connected to the offline gas analyser next to the bioreactors. Only
oxygen concentration can be measured and this data can also be used to calculate oxygen
utilisation rate.

6.3 Experimental Tasks for Growth Kinetics

The aim of this experiment is to characterize the growth curve of Saccharomyces cerevisiae (S.
cerevisiae) during a batch cultivation.

6.3.1 Gassing out and change in dissolved oxygen concentration in medium

1. Make up 5 L of the culture medium (YPD medium). The composition of the YPD
medium is (g/L): 20 g glucose, 20 g peptone, 10 g yeast extract.
2. Fill the bioreactor with culture medium
3. Repeat Section Error! Reference source not found. actions 2-6, so that there are 9 runs
each for water and medium. The effect of the medium components on mass transfer can
then be assessed.

6.3.2 Setting up the bioreactors

Pre-inoculum
The medium for the pre-inoculum consists of (g/L): 10 g glucose; 5 g peptone; 3 g yeast
extract; 3 g malt extract. The pre-inoculum is prepared by inoculating 50 mL of medium
in a 250 mL shake flask with some yeast cells from a nutrient agar slant. The pre-inoculum
will be grown for 24 hours at 30°C.

Inoculum
The medium for the inoculum and the reactor consists of (g/L): 20 g glucose, 20 g peptone,
10 g yeast extract. The inoculum is prepared by inoculating 200mI of media in a 1 litre shake
flask with the pre-inoculum to obtain a starting OD660 value of 0.1. The inoculum will be grown
at 30°C for a pre-determined time period (based on the shake flask growth curve already
completed).

Reactors
The aerobic batch cultivation of S. cerevisiae will be carried out in a 7 litre stirred tank
reactor (working volume 5 litres). The reactor will be autoclaved with 4.8 L of medium, with
the remaining volume made up during inoculation. The bioreactor will be inoculated
with the inoculum to obtain a starting OD 6 6 0 value of 0.1. The balance of the
200 mL volume will be made up with sterile medium. The temperature of the
reactors will be maintained at 30°C. The agitation speed will be fixed at 500 rpm and the air
flow rate at 0.5 vvm.

6.3.3 Sampling and analyses

Samples of the cultures will be harvested over a period of two days and analysed in terms
of biomass production (dry weight, turbidity and spread plating), substrate utilization (total
carbohydrates and glucose analysis), metabolite production (ethanol and volatile fatty acid
analysis) and oxygen utilisation.
The class will be divided into groups of two students and sampling will be performed for the
duration of the practical. Each group will do several sampling points for the batch cultivation.
After all the data has been collected, it will be handed out the class for evaluation.
Before taking a sample from the reactor, a note should be made of time of sampling, the pH
and of the dissolved oxygen concentration and agitation speed of t h e cultivation. OUR
measurements are performed at discrete intervals (lag, exponential and stationary phase)
during the cultivations.
Harvest approximately 20 mL of sample from the reactor and perform the following analyses
(see Section Methods for Growth Kinetics Analyses 6.2):
1. Use triplicate samples to measure turbidity
a. Perform serial dilution and spread plating of the culture at discrete intervals
2. Perform Dry Mass analysis in triplicate
a. Freeze supernatants for other analyses (glucose, VFAs, ethanol)
b. Measure the dried eppendorf tubes (containing biomass) again after 48 hours.
Remember to cool in the desiccator.
3. Measure total carbohydrates in centrifuged and filtered samples using the phenol-
sulphuric acid method.
4. Use samples from the frozen supernatants to measure the following in the HPLC practical
(Practical 6):
a. Ethanol
b. Glucose
c. VFAs

6.4 Growth Kinetics Report

6.4.1 Mass transfer in cultivation medium

1. Use the raw data sets for medium to

a. Plot a curve of each raw data set (see Error! Reference source not found.)
b. Plot a curve of the calculated ln(1 – C/C*) vs. t to determine each kLa (see Error!
Reference source not found.)
c. Tabulate and plot kLa for each set (Runs 1-9)
2. From your experimental results, calculate:
a. C* by Henry's Law in the medium system
b. kLa and OTR in the medium system
3. Discuss the differences or similarities in the measurements for water and culture medium
systems.

6.4.2 Evaluation of measurements

4. The Book Chapter by Lawrence Bergman, Growth and maintenance of Yeast, describes
a generic doubling time for S. cerevisiae of 90 min. What was the doubling time for the
yeast cultured within this experiment?
5. Determine the correlation between the dry mass and the turbidity measured at 660 nm.
Does a fixed relationship exist between the dry biomass and the turbidity measurements?
6. Determine the correlation between the turbidity measured at 660 nm and the number of
CFU/mL determined through spread plating. Does a fixed relationship exist between the
two measurements?
7. Based on your findings in this portion of the experimental procedure, why does Lawrence
Bergman recommend diluting the yeast culture to an OD600 <1.0 for accurate measurement
of cell growth?
8. Graphically represent the change with time in dry mass, turbidity, glucose and dissolved
oxygen concentration for the cultivation. Discuss the profiles obtained.

6.4.3 Growth rates and yields

9. Consider the lag phase of the cultivation. Did the culture perform as expected? Explain
why a longer lag phase would result if a glucose-fed inoculum culture were used to
inoculate a lactose medium than if it is used to inoculate a glucose medium?
10. Evaluate the specific growth rate as a function of time. Comment on your findings.
11. Evaluate the maximum specific growth rate (𝜇max ), the K s constant of the Monod
equation and the doubling time (td). Compare with literature values.
12. Calculate the growth yield (Yx/s) and the ethanol product yield (Yp/s). Compare these with
values obtained from the literature.
13. Evaluate the theoretical growth yield for aerobic cultivation using reaction stoichiometry
given a respiratory quotient of 1.1 molCO2/molO2 and a typical yeast elemental composition
of CH1.75N0.15O0.5.
14. Evaluate the maximum possible ethanol yield for the anaerobic fermentation using reaction
stoichiometry.
15. Does the relationship between growth rate and substrate utilization fit Monod theory?
Discuss further the phenomena of catabolite repression and the Crabtree effect, typical
in S. cerevisiae.

6.4.4 Growth kinetics and oxygen demand

16. From your experimental results calculate:

a. C* in the fermentation broth via
i) Henry's Law and
ii) graphically
b. Oxygen demand and specific oxygen demand of the organism in each growth phase
c. KLa and OTR in the fermentation broth.
17. Comment on the following:
a. any variation in the C* values calculated via the two different methods in 16. a.
b. any differences in kLa and OTR values in air-water, medium-water and fermentation
broth systems
18. Comment on the shape of the curve used to determine the OUR and explain any deviation
from the theory.
19. Explain why it is important to maintain a constant temperature during the experimental
determination of kLa and OTR.
6.4.5 Yeast biotechnology

20. Yeast can be grown both aerobically and anaerobically. Briefly discuss the significance
of this for industry. Include commentary on the nature of the products formed during yeast
cultivation under these different growth conditions.

References
Atkinson, B., Mavituna, F., (1983), In Biochemical Engineering and Biotechnology Handbook;
Macmillan, New York.
Bailey, J.E., Ollis, D.F., (1986), In Biochemical Engineering Principles; McGraw-Hill, Singapore.
Doran, P.M., (1999), Bioprocess Engineering Principles, Academic Press, London.
MacDonald, P. N., Ed., (2001), Methods in Molecular Biology, Vol 177, Two-Hybrid Systems:
Methods and Protocols; Humana Press Inc., Totowa, NJ
Salari, R. & Salari, R., (2017), Investigation of the best Saccharomyces cerevisiae growth
condition. Electronic physician. 9(1):3592-3597.
Tuite, M.F., Oliver, S.G., (1991), In Biotechnology handbooks; Saccharomyces. Plenum Press, New
York.
CHE5070Z course notes.