Media Modification to Control Yeast Growth

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 are is components of dead yeast cells which are 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-glucose[2], 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 to grow cells producing the pharmaceuticals 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 industry[4]. 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.

Aims & Hypotheses

We aimedOur 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.

We 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).

We It was further hypothesized that lowering the peptone concentration to 10 g/L will 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 will bewas devoted to making the media for week 2. YPD and Low Y, P,D media will bewere made, sterilized, and stored for use the next week.

In the second week, we compared 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 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, we compared 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 by distilled water, seal the mouth with paraffin tape and repeatedly invert repeatedly till complete dissolution.
3. After all powder dissolved completely, add more distilled water to 250ml mark.
4. Transfer dissolved medium solution to a polypropylene bottle.
5. Loosen the bottle lid and place the bottle in the autoclave at 121-125 C for 15 minutes.
6. Cool down for 30 minutes, close the lid, label, store for a week.
7. For Low Y, P,D medium, repeat steps 1 to 6 except add only 1.25g of Yeast Extract.
8. 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 tubeit 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 will bewere 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 it 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.

1. Calculate economic cost of each media type using the function, Cost = $80.70(Y/500) + $65.15(P/500) + $27.15(D/500).
2. Estimate savings by percentage of saved cost to Commercial Medium cost.
3. 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 an 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 indicatesd a very strong linear fits, but does 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%[LC1].

The growth rate constant for the Low Y, P,D groups from the twoeach weeks wasere 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 Hemisulfate[5]. 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. 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 – Tables and Figures

Figure 1 shows the growth curves for the second week of experimentation. The growth rate constant, , is the slope of this curve.

Table 1 shows descriptive statistics for the control and experimental groups from the second week of experimentation.

Figure 2 shows the growth curves for the third week of experimentation. The growth rate constant, , is the slope of this curve.

Table 2 shows descriptive statistics for the control and experimental groups from the third week of experimentation.

[1] “Yeast Treatise – Biology of Yeast Cells” accessed 3.28.04

[2] “Dextrose” accessed 4.23.04

[3] “Yeast Kits & Media” Qbiogene 2003 Catalogue, Midwest Scientific, St. Louis, MO.

“Yeast Kits & Media” Qbiogene 2003 Catalogue, Midwest Scientific, St. Louis, MO.

[4] “Brewer’s Yeast” Thompson/PDR. accessed 4.23.04

[5] accessed 4.26.04

[LC1]I like the emphasis, you guys are welcome to disagree