Media Modification to Control Yeast Growth
Group W4: Chang, Kuang, Litcofsky, Muhammad, Siew
Background
Saccaharyomyces cerevisiae, or baker’s yeast, is a common unicellular organism
with wide applications to scientific research in both academic and commercial
biotechnology. Naturally occurring in almost all locations on earth, these eukaryotic
fungi are favored in academia and industry for their relatively simple biochemistry and
their improved compatibility with eukaryotic genes over bacteria, a characteristic
important for genetic engineering. Under favorable conditions, S. cerevisiae reproduces
through a process known as cell separation, or “budding,” in which a daughter cell
protrudes from a mother cell, develops while still attached, and then splits from the
mother cell. This process repeats as long as growth conditions are favorable. When
growth conditions become harsh, yeast cells form sturdy spores that can survive for a
long time and germinate into actively growing yeast again when conditions improve.1
The common media used for S. cerevisiae growth is YPD Broth, a media
comprised of yeast extract (Y), peptone (P), and dextrose (D). Yeast extract is
components of dead yeast cells which primarily aids in the lag phase of yeast growth by
providing ready-made yeast components for the new cells, which are then not restricted
to producing the components from scratch. Peptone, a protein derivative, is the primary
source of amino acid building blocks for new proteins synthesized in a growing yeast
population. Dextrose, also known as corn sugar, grape sugar, or D-glucose2, is a naturally
occurring form of glucose and provides the essential nutrients and energy needed for
yeast reproduction and growth. The most common YPD Broth available commercially is
composed of 10g/L Yeast Extract, 20 g/L Peptone, and 20 g/L Dextrose.3 The quoted cost
of YPD growth media provided in bioengineering lab is $5.31/L.
The optimization of growth media with regard to cost and growth has many
important applications in industry. The pharmaceutical/biotechnology industry, for
example, must pay close attention to the growth media used in large bioreactors. The
large scale of those processes requires that the media be optimized for a particular
application, which can save drug companies significant amounts of funds that can be used
in more research in drug discovery. S. cerevisiae is also used extensively in the brewery
and alcoholic beverages industry4. In addition to its scientific value, detailed knowledge
of both methodology and past research data on growth characteristics of the yeast can aid
in quality control and cost reduction in this $150 billion industry. Data on S. cerevisiae
response to varying YPD concentrations can also serve as a gauging basis for future tests
of addition nutrient’s contribution to yeast growth.
1
2
“Yeast Treatise – Biology of Yeast Cells” http://www.theartisan.net/biology_of_yeast_cells_simplified.htm accessed 3.28.04
“Dextrose” http://www.webref.org/chemistry/d/dextrose.htm accessed 4.23.04
“Yeast Kits & Media” Qbiogene 2003 Catalogue, Midwest Scientific, St. Louis, MO.
http://www.midsci.com/docs/qbio/
3
“Yeast Kits & Media” Qbiogene 2003 Catalogue, Midwest Scientific, St. Louis, MO.
http://www.midsci.com/docs/qbio/
4
“Brewer’s Yeast” Thompson/PDR. http://www.pdrhealth.com/drug_info/nmdrugprofiles/herbaldrugs/100410.shtml accessed 4.23.04
Aims & Hypotheses
Our aim, through a series of experiments and analysis completed over three weeks,
was to lower the cost of yeast growth media by reducing the concentrations of media
constituents without compromising growth rates.
It was hypothesized that yeast grown in media containing a lowered concentration
of yeast extract (5g/L) and concentrations of peptone and dextrose nutrients unadjusted
from market YPD growth media would not have a statistically different growth rate
constant, , than a control group of yeast grown using broth of market YPD media
concentration (10 g/L Y, 20 g/L P, 20 g/L D).
It was further hypothesized that lowering the peptone concentration to 10 g/L
would yield a growth rate constant not statistically different than the growth rate constant
of a control group grown in broth containing lowered yeast extract concentration (5 g/L)
and concentrations of peptone and dextrose consistent with commercially available
growth media.
General Protocol
The first week of experimentation was devoted to making the media for week 2.
YPD and Low Y, P,D media were made, sterilized, and stored for use the next week.
In the second week, the difference in growth rate constants was compared using
Y,P,D (10g/L yeast extract, 20g/L peptone, 20g/L dextrose) and Low Y,P,D (5g/L yeast
extract, 20g/L peptone, 20g/L dextrose) media. Each group had a sample size of 4. After
allowing sufficient time to ensure the start of the log phase, the absorbance of the growth
cultures was measured approximately every 15 minutes for about three hours. In order to
ensure that the experiment could be completed by the end of class, the inoculation culture
was started one hour prior to the start of class. From the absorbance readings, a plot of
Ln(A/A0) vs. Time was constructed from which the growth rat constants could be
obtained. A T -test was performed to determine if the two groups' growth rate constants
were statistically different. In addition to the comparison, we made the media used for
the following week.
In the third week, Low Y, Low P, D (5g/L yeast extract, 10g/L peptone, 20g/L
dextrose) media was compared to Low Y, P, D (5g/L yeast extract, 20g/L peptone, 20g/L
dextrose) media, which was shown to have the same growth rate constant as the
commercial YPD media from the previous week. The only change from the previous
week was the media used. Similarly, a T-test (significance at α = 0.05) was performed to
determine if the two group’s growth rate constants were statistically different.
Specific Methods
Preparation of Growth Medium
1. For Commercial YPD Medium, mix a powder of 2.5g Yeast Extract, 5g Peptone and 5g
Dextrose in a 250 mL Volumetric flask.
2. Wash inner wall of the flask with distilled water, seal the mouth with paraffin tape and
invert repeatedly till complete dissolution.
3. After all powder dissolved completely, add distilled water to 250ml mark.
4.
5.
6.
7.
8.
Transfer dissolved medium solution to a polypropylene bottle.
Loosen the bottle lid and place the bottle in the autoclave at 121-125 C for 15 minutes.
Cool down for 30 minutes, close the lid, label, store for a week.
For Low Y, P,D medium, repeat steps 1 to 6 except add only 1.25g of Yeast Extract.
For Low Y, Low P, D medium repeat steps 1 to 6 except add only 2.5g of Peptone.
Preparation of Yeast Inoculation Culture
1. Weigh 150 mg of dried yeast into 50ml centrifuge tube and add 15 ml of sterile
Growth Media for a final concentration of 10mg/ml.
2. Screw cap and place tube on the rocker for 15 minutes at room temperature.
Starting Growth Culture Tubes
1. Fill 50ml sterile centrifuge tubes with 38 mL sterile Growth Medium.
2. Add 2ml of 10mg/ml Inoculation Culture to give a final yeast concentration of
0.5mg/ml in the tube.
3. Seal tubes tightly and place on the rocker.
4. Repeat steps 1 - 3 three more times for each Growth Medium, for n=4.
5. Start timer at the moment that the inoculation culture is added to the first growth tube
in each group. (each group is on a separate timer)
6. For each week, there were 2 groups of 4 tubes (due to maximum number of tubes that
can be placed onto the rocker).
Running the Test
1. Set Spec-20 to read at 550nm.
2. At some T ~ 150 min, remove 3 ml from each 8 tubes into clean cuvettes with 1mL
micropipette.
3. Zero Spec-20 with growth medium used in the growth tube about to be measured
4. Read Absorbance of sample, Record on Excel spreadsheet.
5. Repeat steps 3,4 for each of 8 growth tubes.
6. Discard the culture and rinse the cuvettes with distilled water. Dry with KimWipe.
7. Record room temperature.
8. At ~15 min intervals repeat steps 1 to 4 until the growth tube is empty (approx. 11
times).
9. Plot Absorbance vs Time.
10. Plot ln(A/Ao) vs. Time and determine growth rate, μ from the slope.
Analyze Result/Statistical Methods
1. Perform t-test on average μ of two different groups of medium at each week with
95% confidence level.
2. Calculate economic cost of each media type using the function, Cost = $80.70(Y/500)
+ $65.15(P/500) + $27.15(D/500).
3. Estimate savings by percentage of saved cost to Commercial Medium cost.
4. Compare the results from the Low Y, P, D group from each week.
Results
The results of the second week of experimentation show that a reduction of the
yeast extract component of the growth media by half does not cause a change in the
growth rate of the yeast. The growth rate constant, , for the test group did not deviate
significantly from the control’s. The  values in Table 1 were generated from the slopes
of the curves in Figure 1, the graph of Ln(A/A0) vs. Time. The linear slopes are
believable because the R2 values for all eight of the curves are greater than 0.995,
indicating excellent fits. A Student’s T-test (significance at α = 0.05) indicated that there
was no significant difference between the test and control groups. Because of these
results, we can conclude that the Low Y, P, D growth medium, can replace the YPD
medium without a change in growth rate.
Based on the results of the second week, The Low Y, P, D medium served as the
control for the week 3 comparison to Low Y, Low P, D medium. A T-test again showed
no significant difference between the growth rate constants for the media. The  values
were generated in the same manner as in the prior week’s analysis. The curves in Figure
1 all had R2 values greater than 0.971, which indicated very strong linear fits, but also
show more variability than the data from the first week. This variability can be
recognized from a comparison of the curves in Figures 1 and 2. The lack of difference
between the two groups means that the Low Y, Low P, D medium can be used to replace
the control, Low Y, P, D, which was already shown to be equivalent to YPD medium in
terms of the growth rate constant.
The two tests showed that a medium with half the concentration of yeast extract
and peptone compared to commercial YPD medium produces a similar growth rate
constant as the YPD medium. The reduction in concentration of media components
decreased the cost of the medium. The cost of the new, Low Y, Low P, D medium is
$3.20 per liter compared to $5.31 per liter of YPD media. These costs were generated
from the costs of the individual components in the following formula: Cost =
$80.70(Y/500) + $65.15(P/500) + $27.15(D/500) where Y,P, and D are the
concentrations of yeast extract, peptone, and dextrose respectively. The new formulation
represents savings of $2.11, or 39.7%.
The growth rate constant for the Low Y, P,D groups from the two weeks were
also compared to see if there was any change in conditions that affects our experiment. A
T-test (significance at α = 0.05) showed that there was no significant differences between
the groups from each week. This indicates that laboratory conditions likely remained
constant from week 2 to week 3.
Discussion
YPD growth media with commercial concentrations of 10g/L for yeast cell extract,
and 20g/L for dextrose and peptone, is a non-strain specific growth media that supports
yeast cell growth of all strains. However, each strain of yeast, due to mutations or other
factors, has its own specific set of growth requirements. Therefore, it is possible to tailor
a growth media that would better suit the specific requirements of our strain of yeast.
This would lead to a more cost efficient growth media that would reduce the total cost of
the media constituents while still yielding the same growth rate as with YPD. This may
be achieved either by reducing the concentration of media constituents and keeping the
others unchanged, or by increasing the concentration of one constituent while decreasing
the concentration of other(s). Only the former method was used because the latter method
would require more experimentation than time permits for this project.
Due to time and equipment constraints, and for our results to have statistical
significance (4 of the 8 growth tubes had to be used as controls), we realized that we
could only test 2 of the media constituents. We felt that dextrose, being the main energy
source, would have the greatest effect on the growth rate constant. We therefore decided
to keep dextrose concentration constant and vary the other 2 media constituent
concentrations instead. In addition, since we could only run 8 growth tubes on our rocker
each week, and 4 tubes were used as controls, only 4 tubes were left and for our results to
have statistical significance, only 1 new concentration for 1 media constituent could be
tested each week.
Although this satisfies our goal of obtaining a cheaper medium, our final
optimized concentrations are very likely not at the optimal point since it’s likely that the
concentrations of both yeast cell extract and peptone could be reduced further without
changing the growth rate constant. If there was more time, however, we would have
tested other lower concentrations of both yeast cell extract and peptone to explore further
cost reduction. Dextrose would be tested last not only because it’ll have the largest effect
on the growth rate constant, but also because it’s the cheapest of the three media
constituents.
Although we have achieved our goal of developing a more cost efficient growth
media for our yeast, it is possible that other aspects of the experiment that weren’t
measured might have been affected. For example, it is possible that the length of the lag
phase might be different with our optimized media. During the lag phase, the yeast cells
are adapting to their new environment by synthesizing the required proteins and enzymes.
Peptone might be one of the proteins synthesized or could be a source of protein that
could be broken down by the yeast cells to synthesize the required proteins and enzymes.
Yeast cell extract, on the other hand, would likely contain proteins and enzymes
necessary for yeast cell growth and proliferation and would also function as a source of
protein and minerals. Therefore, reducing the concentration of both peptone and yeast
cell extract would possibly increase the length of the lag phase by a large amount. This
fact should be taken into consideration in future experiments that use media constituent
concentrations determined from this experiment. In commercial labs and businesses like
beer breweries, time can be a large cost. Therefore, it’s likely that cost savings with our
optimized media would not be able to justify the increased opportunity cost of time.
In addition, YPD is only one of the commercially available types of yeast growth
media. Other types of commercial growth media include additional constituents such as
L-tryptophan and Adenine Hemisulfate5. Adding in L-tryptophan and other amino acids
would provide the precursors for synthesizing proteins and enzymes, decreasing the yeast
cell’s need to break down protein to obtain these amino acids. Adenine Hemisulfae would
be a good source of adenine base and sulfate for DNA/RNA and ATP synthesis.
DNA/RNA are necessary for cells to proliferate and ATP is a necessary store of energy,
which is needed for various cell processes such as synthesizing proteins and enzymes.
5
http://www.teknova.com/liquidcul-main.html?liquid/yeast-liq/YPD-broths/ypdbroths.htm. accessed 4.26.04
Including these might significantly increase the growth rate constant and shorten the lag
phase. If the cost of including these amino acids and adenine hemisulfate is low, it might
be possible to reduce the concentration of peptone, yeast cell extract or dextrose so as to
yield a more cost effective media. Also, if the strain of yeast in question is deficient in
one of these amino acids, purines, or pyrimidines, including them would have a dramatic
increase in the growth rate constant while also reducing the lag phase.
Appendix
Figure 1 - Growth Curves Comparing Low Y to YPD media
0.7
0.6
YPD 1
YPD 2
YPS 3
0.4
YPD 4
LowY 1
)
LN(A/A0)
0.5
0.3
LowY 2
LowY 3
0.2
LowY 4
0.1
0
0
50
100
150
200
250
300
350
Time
Figure 1 shows the growth curves for the second week of experimentation. The growth
rate constant, , is the slope of this curve.
Table 1 - Week 2 Growth
Rate Constants
YPD
Low Y
mean 0.00364 0.00354
sd
0.00011 0.00016
Table 1 shows descriptive statistics for the control and experimental groups from the
second week of experimentation.
Figure 2 - Growth Curves Comparing Low Y, Low P to Low Y
0.8
0.7
LowY 1
0.6
LowY 3
LowY 4
0.4
LowY, Low P 1
)
LN(A/A0)
LowY 2
0.5
LowY, LowP 2
0.3
LowY, LowP 3
0.2
LowY, LowP 4
0.1
0
0
100
200
300
400
Time
Figure 2 shows the growth curves for the third week of experimentation. The growth
rate constant, , is the slope of this curve.
Table 2 - Week 3 Growth
Rate Constants
Low Y,
Low Y
Low P
mean 0.00352 0.00334
sd 0.00003 0.00007
Table 2 shows descriptive statistics for the control and experimental groups from the
third week of experimentation.