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PROJECT FINAL REPORT COVER PAGE

GROUP NUMBER____R1

PROJECT TITLE___Anaerobic and Aerobic Yeast Growth

DATE SUBMITTED______4/12/00

ROLE ASSIGNMENTS

ROLE GROUP MEMBER

FACILITATOR………………………..Jocelyn Poruthur

TIME & TASK KEEPER……………..Grant Foy (did not participate in report)

SCRIBE……………………………….Danielle Antalffy

PRESENTER…………………………Keyur Shah

SUMMARY OF PROJECT CONCLUSIONS

A comparative study on the growth of aerobic and anaerobic growth of anaerobically prepared baker’s yeast, Saccharomyces cerevisiae, at different temperatures showed no significant difference between doubling times at anaerobic and aerobic conditions at 32(C and 37(C. The doubling time at 32(C was 234.01(5.61 min at anaerobic conditions and 247.55(6.60 min at aerobic conditions; at 37(C the doubling time was 212.56(5.46 at anaerobic conditions and 203.87(3.35 at aerobic conditions; since the values overlap within in their confidence intervals, which were 212.56±11.74 and 203.87±7.62 minutes at 37 degrees and 234.01±11.86 and 247±12.73 minutes at 32 degrees, no significant difference can be seen between aerobic and anaerobic conditions at the two specified temperatures. However, anaerobic data taken at 25(C suggests no growth at the specified conditions due to the length of the lag time, which was 111.423±10.93 minutes the large standard error of 9.81% of the mean in the lag time and 27.1% of the mean in the doubling time. The lag times compared at aerobic and anaerobic conditions for 32(C and 37(C do not show a significant difference, once again due to an overlap in the confidence intervals. Also, experimental results show that anaerobic growth causes a decrease in pH over time due to alcohol production and accumulation in the PENNCELL apparatus.

OBJECTIVES

The experimental aim of this project is to determine at which conditions the growth of Saccharomyces cerevisiae is most rapid. We will be comparing aerobic and anaerobic growth at 25, 32, and 37(C. Also the effect of anaerobic growth on pH is an aim of this project. Anaerobic data will be compared to previously compiled aerobic data obtained from fellow lab groups. Manufacturer correspondence stated that Fleischmann’s yeast is anaerobically prepared. Thus, lag phases are expected to be shorter under anaerobic conditions when compared to aerobic conditions at a specific temperature since literature states that yeast synthesize enzymes essential for nutrient metabolism during lag phase (Hohmann 15). Therefore, anaerobically prepared yeast should require less time to synthesize additional enzymes when cultured anaerobically as opposed to being cultured aerobically, in which enzymes required for the Krebs cycle and the electron transport chain would need to be made. Also, pH is expected to decrease over time under anaerobic conditions due to alcohol production. In addition, growth rates are expected to be greater at higher temperatures over both aerobic and anaerobic conditions because of the laws of enzyme kinetics, which state that enzyme activity increases as a function of temperature (Castellan 433). Also, the growth rate is expected to be greater under aerobic conditions, since more ATP is produced via cellular respiration under aerobic conditions (Campbell 147). Doubling time is a more practical application of the growth constant; this value is inversely related to the growth constant, hence it decreases as the growth constant increases. Thus, we expect to see shorter doubling times for aerobic conditions and higher temperatures.

BACKGROUND

According to Hohmann, S. cerevisiae is a facultative anaerobic yeast which ferments hexose sugars under aerobic and anaerobic conditions (Hohmann 10). The yeast will be grown in glucose-rich medium where it will utilize different modes to harvest energy depending upon the specific growth conditions.

According to Campbell, organisms employ cellular respiration to harvest energy in the form of ATP in the presence of oxygen (Campbell 150). Enzymatic activity is the driving force behind the three main stages of cellular respiration – glycolysis, the Krebs cycle and the electron transport chain. Without oxygen, organisms undergo fermentation which only involves the first stage of cellular respiration, glycolysis, and subsequently requires less enzymes (Campbell 161). Cellular respiration yields 38 ATP molecules while fermentation only produces 2 ATP molecules (Campbell 162). Organisms rely on ATP to drive their cellular processes and promote growth. According to Hohmann, very little growth occurs during lag phase where the yeast acclimate themselves to their environment and synthesize enzymes necessary to catabolize nutrients (Hohmann 12). According to Mills, the yeast will synthesize fewer enzymes in anaerobic environment than in an aerobic environment since fermentation requires fewer enzymes than cellular respiration (Mills 54). According to Miller, it is possible that yeast growth will be inhibited under anaerobic conditions since fermentation produces alcohol, which also decreases pH over time and kill the yeast (Miller 60). Consequently, the partial pressure of oxygen is a key factor affecting yeast growth in addition to temperature and pH. The optimum temperature growth range of yeast is between 25(C and 37(C (Miller 64).

METHODS OF CALCULATION

Absorbances where converted to cell concentrations using the conversion ratio of 1 unit Absorbance = 3 x 108 cells/ml (Ausbel 13.2.1). The growth constant, doubling time and lag time were determined from the concentration vs. time graphs. These values were obtained once concentration vs. time graphs were converted to semi-log plots to linearize the data. We visually inspected the log growth phase and chose the corresponding array of points. Linear regressions were performed on different sets of consecutive points in the observed log phase until we obtained the highest Pearson’s R2 value and lowest standard error, which denoted the log phase.

Exponential regressions were fitted to the log phase on the original graph. The resulting equation was in the form y=Coerx , where y is the concentration at time x, Co is the mathematically determined initial concentration, and r is the growth constant in min-1. Statistical error analysis provided standard errors and 95 % interval of the growth constant. The growth constant was then used to determine both the doubling time and lag phase. Using the formula (ln 2) / r = (, the doubling time,(, was found. The lag phase for the anaerobic growth was determined by using the formula ln(Ci/Co)/r = t(lag), where Ci is the concentration at time zero. This method did not work well for the aerobic data since the initial aerobic data was taken 1 hour after growth whereas we began taking measurements 10 minutes after growth.

Also, a normalized pH vs. time plot was constructed to show the overall pH change. Since the pH meter was calibrated at 25(C the plots of 32(C and 37(C were normalized to the results of 25(C, because the initial pH should have been within the precision range of the pH (i.e., 6.5(0.1) of the growth medium determined by the manufacturer regardless of temperature. The normalization method involved using the formula, (pH (25)i-pHi) + pH = pH(norm), where pH(25)i is the initial pH at 25 degrees, pHi is the initial pH at either 32(C or 37(C, and pH is the pH being normalized.

Apparatus

• PENNCELL Culture Apparatus

• Milton-Roy Spectronic 20D Spectrophotometer

• All American Electric Pressure Steam Sterilizer Model 25X

• Assorted glassware and plastic ware (2 cuvettes, 2 syringes)

• Sterile Water

• Thermometer

• Fisher Scientific Accumet Model 625 pH meter with electrode

• Oxygen Monitor with Electrode

• Electrode Membrane

• Nitrogen Tank

Materials

• 8.0 ( 0.1g Saccharomyces cerevisiae (Fleischmann’s Active dry yeast)

• 200.0 ( 0.1g YPD Media Powder (Difco Bacto YPD broth #0428-17-5)

• pH 7.0 and pH 4.0 Buffer Solutions

• 1.5M KCl solution

Procedure

The basic experimental procedure followed can be found in the BE 210 lab manual. The yeast were grown under anaerobic conditions at three temperatures: 25, 32 and 37(C. Protocol modification involved the addition of a period of nitrogen treatment to obtain an anaerobic environment. The first method employed involved constantly monitoring the O2 concentration of the PENNCELL via an oxygen monitor and maintaining 0.75 L/min flow of N2. This attempt revealed the sensitivity of the oxygen monitor to the presence of yeast manifested through the fluctuations in the O2 readings. Therefore, the protocol was modified to a 10 minute N2 treatment at 0.75 L/min N2. The length of the treatment was determined by calibrating the O2 monitor in both sterile water and YPD media. The lowest O2 concentration of 3ppm was obtained for both calibrations and occurred within approximately 10 minutes. After the N2 treatment, a baseline absorbance reading and pH were taken and subsequent readings were taken in 10-minute intervals until the plot of absorbance versus time reached the stationary phase observed by a plateau in the graph.

Results

[pic]

Figure 1: A Plot of the Logarithmic Phase of Anaerobic Growth at 25, 32 and 37 Degrees Celsius:

Figure 1 shows the plot of the logarithmic growth phase of anaerobic yeast at three varying temperatures: 25(C shown in blue, 32(C shown in red, and 37(C shown in green. From the exponential regression plotted to each of the data sets both the growth rate and mathematical initial concentrations were found. The growth rate constants for the three temperatures were .001, .0029 and .0032 min-1, at 25, 32, and 37(C respectively. The mathematical initial concentrations were also determined using the equations, which yielded values of 2.3348, 2.138, and 2.0027 *108 cells/ml, respectively.

[pic]

Figure 2: Plot of the Normalized pH Values Versus Time:

Figure 2 shows the plot of normalized pH versus time with a fitted linear regression line. To normalize the pH at 32(C, .273 pH units were added to all the pH values, and to normalize the pH at 37(C, .355 pH units were added to the recorded pH values. The overall change in pH for the three temperatures were .973, .951, and 1.089 pH units at 25, 32 and 37(C respectively. Also, the y-intercept of the regression lines were fixed at 6.38 pH units because this was the baseline value at 25(C to which the other two temperatures were normalized.

|  |Temperature |Growth Constant |95% Interval |Standard Error |Doubling Time |95 % Interval |Standard Error |

|Anaerobic |25 |1.07E-03 |2.90E-04 |1.05E-04 | | 176.23| |

| | | | | |650.23 | |63.81 |

|  |32 |2.96E-03 |1.50E-04 |7.10E-05 | | | |

| | | | | |234.01 |11.86 |5.61 |

|  |37 |3.26E-03 |1.80E-04 |8.38E-05 | | | |

| | | | | |212.56 |11.74 |5.46 |

|Aerobic |25 |2.20E-03 |1.70E-04 |7.87E-05 | | | |

| | | | | |330.07 |25.51 |11.81 |

|  |32 |2.80E-03 |1.44E-04 |7.46E-05 | | | |

| | | | | |247.55 |12.73 |6.60 |

|  |37 |3.40E-03 |1.27E-04 |5.58E-05 | | | |

| | | | | |203.87 |7.62 |3.35 |

Table 1: Chart of Growth Constants and Doubling Times with 95% confidence Interval and Standard Error:

|  |Temperature |Lag Time |95 % Interval |Standard Error |

|Anaerobic |25 | | | |

| | |111.423 |30.20 |10.93 |

|  |32 | | | |

| | |48.370 |2.45 |1.16 |

|  |37 | | | |

| | |57.850 |3.19 |1.49 |

|Aerobic |25 | | | |

| | |50.000 |3.86 |1.79 |

|  |32 | | | |

| | |60.000 |3.09 |1.60 |

|  |37 | | | |

| | |60.000 |2.24 |0.98 |

Table 2: Chart of the Lag Times with 95 % Confidence Intervals plus Standard Error:

Table 1 and 2 are the tabulated results of the growth constant, doubling time and lag time with their respective 95% intervals and standard errors for both the anaerobic data and previously recorded aerobic data (see appendix) for the three temperatures. As mentioned before, the lag times for the aerobic data were not calculated using the formula in the calculations section, but was estimated through visual inspection of the graph.

DISCUSSION

Overall trends in the data show that temperature and O2 concentration have major influences in growth rate, doubling time, and lag phases. For conceptual ease doubling time will be discussed instead of growth rates. Since the two are inversely related an increase in one would correspond to a decrease in the other. At 25(C the doubling time for the anaerobic yeast was 650.23 ( 63.81 minutes compared to 330.07 ( 11.81 minutes for aerobic yeast. At 32(C the doubling time for yeast grown anaerobically was 234.01 ( 5.61 minutes, whereas the doubling time for aerobically grown yeast was 247.55 ( 6.60 minutes. Finally, at a temperature of 37(C the doubling time for anaerobic yeast was found to be 212.56 ( 5.46 minutes in comparison to 203.87 ( 3.35 minutes.

At 32(C and 37(C there was no significant difference observed between anaerobic and aerobic conditions. However, because aerobic conditions produce more ATP, which drives cellular processes, greater growth is expected under aerobic conditions when compared to anaerobic conditions, but the data collected failed to support this hypothesis. Also, for both aerobic and anaerobic growth, the doubling times were smaller at higher temperatures as hypothesized following the laws of enzyme kinetics (Castellan 433). Since the values at 32(C and 37(C have overlapping 95% confidence intervals, the upper bound of the anaerobic and the lower bound of the aerobic, it can be said that at 32(C and 37(C the growth rate is similar under aerobic and anaerobic conditions.

The overall precision of the data recorded was very good, ranging from 1.67 % - 3.55 % of the mean, with the exception of the data recorded at 25(C for the anaerobically grown yeast. At this condition the standard error was 9.81% of the mean value of 650.23 minutes, suggesting very low precision in the measurements. Because of the huge error and the large 95 % confidence interval it is possible that the yeast did not grow at 25(C, as demonstrated by the long doubling time and the very short growth phase. This might signify that at 25(C, in the absence of oxygen, yeast cannot grow efficiently.

From the normalized pH vs. time graphs, the rate of decrease was faster at higher temperatures. Since pH decreases with alcohol production (Miller 60) our data shows greater alcohol production at higher temperatures.

The method employed to determine lag times for the anaerobic data did not prove to be an effective method when applied to the aerobic data. Thus, the lengths of the lag phases were taken until the point where the log phase was determined to begin. Different experimental procedures were employed for the aerobic and anaerobic conditions where the first time-point was taken 1 hour after growth for the aerobic data and the first time-point was taken 10 minutes after growth for the anaerobic data. The discrepancy between the baseline readings prevents the use of the same method in determining the lag phases under both conditions. No significant conclusions could be drawn from a comparison of the lag times under aerobic and anaerobic conditions at 32(C and 37(C since they are similar within their confidence intervals. However, at 25(C no conclusions can be drawn because of the long lag time of the anaerobic data, indicating that there was possibly no growth. It was expected that when yeast prepared anaerobically is placed in an anaerobic environment, the yeast should not exhaust much time to acclimate since they were grown in an anaerobic environment and would not need to synthesize the additional enzymes that would be required for catabolizing nutrients in an aerobic environment. Subsequently, when anaerobically prepared yeast is placed in an aerobic environment, new enzymes would need to be synthesized in order to catabolize nutrients in the presence of oxygen.

Many improvements on the protocol and design of this experiment can lead to more accurate results and conclusive data. First, a more accurate spectrophotometer could have been used. As the absorbance values increased, the fluctuations in the reading increased 5 fold from .001 to .005 absorbance units. This large increase in fluctuation increases both the standard error and 95% intervals. A more accurate spectrophotometer would decrease these fluctuations decreasing the standard error and allowing for more accurate measurements.

Due to the limitations of the present protocol, which allows for the presence of too many variables such as varying pH and partial pressure of oxygen, conclusive data could not be drawn. Improvements include monitoring a constant pH by placing a pH meter within the apparatus and maintaining this pH with NaOH and HCl solution. Additionally, the use of an oxygen monitor that is not sensitive to the presence of yeast would allow for a constant partial pressure of oxygen to be maintained and adjusted with the nitrogen tank. Also, the present protocol inhibited the complete removal of oxygen and an accurate measurement of the concentration of oxygen in the apparatus; thus, the environment was not truly anaerobic.

A more air tight apparatus could have been used, which would allow for the creation of a better anaerobic environment. The PENNCELL apparatus used in this experiment allowed us to reach a near anaerobic environment (3 ppm of oxygen). Consequently, the data suggests that yeast is able to use this small fraction of oxygen to grow at an equivalent rate of yeast growing at atmospheric conditions (21% oxygen) as seen in the overlap in the confidence intervals of the doubling times. Also, it would be interesting to observe the affect of aerobic growth at 21% oxygen on pH; alcohol production might even occur at atmospheric oxygen since our experimental setup did not provide a totally oxygen-free environment but still caused the pH to decrease over time.

CONCLUSION

1. The most significant conclusion drawn from this experiment was that the data obtained for anaerobically grown yeast at 25(C was inconclusive. Due to time limitations, it was possible that growth was never achieved at these conditions as the data revealed an extremely long lag phase with a large standard error (9.81 % of the mean) and large confidence interval (27.1% of the mean).

2. From the protocol employed in this experiment there is no significant difference in the doubling times and lag phases at both 32(C and 37(C between anaerobic and aerobic conditions, due to overlapping values within the calculated confidence intervals.

3. Finally, as temperature increases from 25(C to 37(C, the doubling time decreases at both environmental conditions, which follows the laws of enzyme kinetics. At the anaerobic conditions, as the temperature decreased from 25(C to 37(C the doubling time also decreases from 650.23 to 212.56 minutes. The Aerobic conditions also displayed this decrease in doubling time as the temperature increased, decreasing from 330.07 to 203.87 minutes.

REFERENCES

1. Ausbel, Frederick M. Current Protocols in Molecular Biology. Volume 2. New York: Wiley Insterscience, 1998.

2. Campbell, Neil A. Biology. 5th Edition. Menlo Park: Addison Wesley, 1996.

3. Castellan, Stephan. Physical Chemistry. 3rd Edition. New York: Addison Wesley, 1983.

4. Hohmann, Stefan. Yeast Stress Response. Austin: R.G Landes Company, 1997.

5. Miller, Alex K. Micro-organisms and fermentation. 3rd edition. London:

Macmillan, 1900.

6. Mills, A.K. Aspects of Yeast Metabolism. Oxford: Blackwell Science Publications,

1967.

7. BE 210 Bioengineering Laboratory II Laboratory Manual Spring 2000.

APPENDIX

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Figure 3: Aerobic Data collected for 25, 32 and 3 Degrees C

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