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Effect of varying temperature on the rate of CO 2 production in baker’s yeast
( Saccharomyces cerevisiae )
Hillary Janssens, Lisa Kim, Icel Lee, Melody Salehzadeh
ABSTRACT We conducted an experiment to find the optimal temperature for cellular respiration of Saccharomyces cerevisiae. Respirometers were incubated in water baths at temperatures of 25°C, 30°C and 35°C. The amount of CO70 minutes, and cell counts were made before and after incubation. The notable trend in our data 2 gas produced was recorded at five minute intervals for was that with increasing temperature, there was less of a lag time before measurable CO 2 appeared, and CO 2 production was more rapid than at lower temperatures. The average CO 2 production rates were 1.66 × 10 -9^ ± 6.95 × 10 -10^ mL/cell, 2.31 × 10 -9^ ± 6.76 × 10 -10^ mL/cell and 3.02 × 10 -9^ ± 6.42 × 10 -10^ mL/cell at 25°C, 30°C and 35°C, respectively. Based on previous research, the lower rate of CO 2 production observed at 25°C is a result of reduced enzyme kinetics and reaction rates at lower temperatures. Additionally, the accelerated rate of CO 2 production seen at 30°C, and even more so at 35°C, can be explained by the increase in enzymekinetics, membrane fluidity and diffusion rates that accompany higher temperatures. Our results suggest that the optimal temperatures for S. cerevisiae growth and metabolism may not be equal. INTRODUCTION
Saccharomyces cerevisiae , more commonly known as baker’s yeast, is a eukaryotic microorganism and a facultative anaerobe. This means that yeast can use sugars to undergo aerobic respiration to produce water and CO 2 gas, or it can undergo fermentation in the absence of oxygen to produce ethanol and CO 2 gas (Berg et al. 2012). Due to its ability to form such by- products, S. cerevisiae has been widely used in multiple areas of scientific research. For example, in health care research, yeast has been used to identify cancer-causing genes in humans (Sloan and Prize 1992). Moreover, the fermentation by-product of ethanol has been used in biofuel manufacturing as an alternative to fossil fuels (Lin et al. 2012). We were interested in determining which temperature S. cerevisiae exhibits the highest metabolic rate while performing aerobic respiration. Literature lists a wide range of optimal growth temperatures for yeast, including 25°C to 30°C (Morano 2012), 30°C to 33°C
(Zakhartsev et al. 2015), as well as 25°C to 35°C (Kuloyo et al. 2014). Our objective was to determine which temperature, 25°C, 30°C, or 35°C, was closest to the optimal temperature for the metabolism in S. cerevisiae. When yeast undergoes aerobic respiration, it produces water, CO 2 gas, and energy in the form of adenosine triphosphate (Berg et al. 2012). As this chemical process constitutes a majority of the cell’s metabolism, the volume of gas produced over time can be used as an indicator of cell activity. This investigation will add to the body of knowledge regarding metabolism in S. cerevisiae , and will enhance the exploitation of yeast by reducing the costs of mass production and increasing the efficiency of manufacturing valuable by-products. Our first null hypothesis was that temperatures of 25°C, 30°C and 35°C have no effect on CO 2 production per S. cerevisiae cell. Our corresponding alternate hypothesis was that temperatures of 25°C, 30°C and 35°C have an effect on CO 2 production per S. cerevisiae cell. Our second null hypothesis was that time has no effect on CO 2 production per S cerevisiae cell. Our second alternate hypothesis was that time has an effect on CO 2 production per S. cerevisiae cell. Lastly, our third null hypothesis was that the effect of time on CO 2 production per S. cerevisiae cell is the same for 25°C, 30°C and 35°C, whereas our last alternate hypothesis was that the effect of time on CO 2 production per S. cerevisiae cell is not the same for 25°C, 30°C and 35°C.
METHODS
We obtained 2.2 L of wild-type yeast stock solution with an approximate concentration of 9.0 × 107 cells/mL as well as 4.0 L of yeast extract peptone dextrose (YPD), a medium that facilitates yeast activity. We determined the rate of cellular metabolism by measuring the volume of CO 2 gas produced in respirometers filled with yeast at temperatures of 25°C, 30°C and 35°C. We set the treatment control to be 30°C, because this was the temperature at which most research found the optimal temperature for growth of S. cerevisiae (Zakhartsev et al. 2015). Each treatment had four replicates (n=4), in addition to four procedural controls that contained only YPD medium. We made marks of 0. mL onto the 4.0 mL test tubes of the respirometers to allow for an accurate and efficient reading of CO 2 volume at each time interval (Figure 2). We first prepared 12 procedural control respirometers containing only YPD medium. After preparing the controls, we concentrated the yeast stock to a final concentration of 4.0 × 10^9 cells/mL by centrifuging and then resuspending the yeast pellets in 200 mL of YPD medium. At this point, we filled the respirometers with the newly suspended yeast and placed four replicates into each water bath of 25°C, 30°C and 35°C. The CO 2 produced by the yeast filled the inside of the inverted respirometer, and we were able to record the volume of CO 2 produced by using the marks we had made on the outside of the tubes.
Figure 2. 0.5mL lines were marked in the 4mL test tube for an accurate reading of CO 2 production.
We collected data every five minutes for a total of 70 minutes. When the yeast solution on the outside of the respirometer obscured the reading of the innermost tube, we pipetted out and discarded the excess liquid for an easier reading (Figure 3). At the conclusion of our experiment, we withdrew 10 μl from each replicate and added 1 μl of fixative so that the cells would stop budding and we could make accurate cell counts. We determined the final cell concentration of each replicate by counting cells using a haemocytometer that was viewed with an Axio microscope. We divided the CO 2 produced at each five minute interval by the number of cells to determine the CO 2 produced per cell at each time. We analyzed the data using the two-way ANOVA with replication, and the p -values were compared with the significance level (α) of 0.05 to determine if there was a significant difference between the treatments with regards to CO 2 production.
RESULTS We analyzed our data by performing a two-way ANOVA test, and we calculated p -values of 3.98 × 10 -25, 2.80 × 10 -49^ and 1.47 × 10 -4, for our first, second and third hypotheses, respectively. We observed increases in the average cell density from 9.57 × 107 cells/mL observed before incubation, to 1.05 × 109 cells/mL, 1.11 × 109 cells/mL and 9.78 × 108 cells/mL observed after incubation at 25°C, 30°C and 35°C, respectively.
Figure 3. Pipetting out excess yeast solution.
Figure 5. 35°C. Error bars represent 95% confidence intervals Average CO 2 production rates (mL/cell/min) of the 4 replicates of (α = 0.05). (n = 4) S. cerevisiae at 25°C, 30°C and
We made several qualitative observations throughout this experiment. When S. cerevisiae was observed with an Axio microscope at a total magnification of 400x, the yeast cells were transparent, round in shape, and surrounded by a thick cell wall which appeared dark (Figure 6). The yeast stock solution that we obtained was a deep amber color, probably due to the brown YPD medium. As we centrifuged the yeast pellets and removed the supernatant, the cells revealed to be a very pale in colour. Once we resuspended the cells in fresh YPD medium, the solution reverted back to
25°C 30°C 35°C TREATMENTS
Figure 6. microscope with a total magnification of 400x. Saccharomyces cerevisiae as seen through an Axio
CO2 PRODUCTION RATE (
-^11 mL/cell/min)
being deep amber. This colour stayed constant throughout the water bath procedure. Moreover, the CO 2 gas that filled each respirometer was clear and rose to the top of the innermost tube.
DISCUSSION
Based on our statistical analysis, we were able to reject all three null hypotheses, and thus lend support to the alternate hypotheses, due to the fact that our p -values were calculated to be less than our significance level of 0.05. Once we obtained a p -value of 3.98 × 10 -25, we rejected our first null hypothesis, which stated that temperatures of 25°C, 30°C, and 35°C have no differing effects on the production of CO 2 gas in S. cerevisiae. We were therefore able to support our alternate hypothesis, which stated that temperature does affect CO 2 production in S. cerevisiae. This finding also corresponds with our prediction. During our experiment, we observed that as temperature was increased, yeast cells produced more CO 2 , with a maximum volume of gas produced at 35°C (Figure 4). Our results are consistent with Arroyo-Lopez et al. (2009) who showed that temperature is the variable with the greatest influence on yeast metabolism. The effect of temperature on the rate of CO 2 production will be further discussed with the analysis of our third hypothesis. After finding the p -value relating to our second hypothesis was 2.80 × 10 -49, we rejected the null hypothesis which stated that time had no effect on the production of CO 2 in S. cerevisiae. We were able to support to our second alternate hypothesis which stated that time does in fact affect CO 2 production in S. cerevisiae. Therefore, our results support our prediction that the rate of CO 2 production varies with time. As the experiment went on, the total amount of CO 2 accumulated. According to Figure 4, it is evident that yeast initially produced CO 2 slowly, and as time passed, the rate of gas production rapidly increased, up to a point where production slowed down. This trend in CO 2 production shown by the yeast follows the model that explains
we observed a slower rate of increase in CO 2 production following this initial lag phase. This can be explained by a decreased rate of enzyme kinetics that slows down reaction rates, and thus cellular processes such as metabolism, at below-optimal temperatures (Tai et al. 2007). Yeast exposed to higher temperatures began to produce CO 2 noticeably sooner after they were placed in the medium compared to those at lower temperatures, and produced gas at a higher rate once the brief lag phase was complete (Figure 5). In our experiment, the 35°C treatment caused yeast to produce CO 2 faster than the supposed optimal growth temperature of 30°C. Zakhartsev et al. (2015) stated that yeast metabolism changes to dissipate more heat when exposed to temperatures that are above optimal, which they defined as being above 31°C. Tai et al. (2007) stated that the molecular mechanisms that allow this heat dissipation to occur include increased diffusion rates and increased fluidity of the cell membrane due to changes in phospholipids. A more fluid membrane enables faster transport and thus higher metabolism at higher temperatures (Tai et al. 2007). These cellular mechanisms enabled yeast at 35°C to undergo cellular respiration and produce CO 2 at a remarkably higher rate than yeast at 30°C. Salvado et el. (2011) found that S. cerevisiae has a maximum growth temperature of 45.4°C, which may allow the strain a competitive advantage over other Saccharomyces species, which are not able to grow as well or as fast at such high temperatures. They also found that at temperatures that are well above the optimal range, metabolism decreases drastically due to enzyme denaturation and consequent loss of function (Berg et al. 2012). This research helps explain why we observed the highest rate of CO 2 production at 35°C, as this is well below the temperature at which enzyme denaturation results in a decline in cellular respiration. However, we made assumptions throughout our experiment which may have affected our results. We assumed that all the yeast cells were at the same point in their life cycle and that cell
counts remained constant throughout the 70 minutes. We were able to assume this because it has been shown that yeast requires 90 minutes to divide in YPD (Sherman 2002). This source of variation could have affected our CO 2 production rate in certain respirometers; if younger cells were present, the amount of CO 2 produced would be less than a respirometer full of mature yeast cells. As we could not determine the age of the yeast when using the haemocytometer and Axio microscope, we have no way of knowing if this factor had an effect on the number of cells, and thus the CO 2 production, in our experiment. As well, we assumed that the small amount of water produced by the yeast during aerobic respiration was negligible. As yeast produced both CO 2 gas and water during this process, the water should have ultimately diluted the cell count at the end of 70 minutes when we calculated the cell concentration of each replicate. Replicates that produced more CO 2 gas should have also produced more water than the others, and should have had their cell counts the most diluted. We assumed that the level of dilution was negligible, and if it was not then we would have recorded a higher rate of gas production than the actual rate as we would have divided the total volume of CO 2 gas by fewer cells to produce a larger rate. As we had three group members measuring the volume of CO 2 produced in each respirometer replicate at each temperature, this may have added error into our data. As we had only marked 0.5 mL differences on each tube, anywhere in between those markings had to be estimated and each group member may have had her own interpretation of the gas levels. This could have caused our data to either be lower or higher than the actual value, depending on the opinion of each group member.
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