1 The Effect of ph on the Growth (Alcoholic Fermentation) of Yeast Andres Avila, et al School name, City, State April 9, 2015 Abstract We investigated the effect of neutral and extreme ph values on the fermentation of yeast. Using ph 5 as the control we collected data on the growth of yeast in ph values of 3, 7, and 10. Yeast grew best at the neutral ph 7, followed by 5, 10, and 3. There was a marked difference in the growth of yeast in values near the neutral ph values tested when compared with the extreme ph values tested. The study of environmental factors affecting the growth of yeast and the production of ethanol have been important in the past and are important for industry as well as food and drink production today. Introduction Fermentation of yeast is a valued process both in the world of today and the ancient world. Of the products produced during fermentation (CO2 and ethanol), ethanol is valued for its many uses. Ethanol is the primary ingredient in the commercial production of fuel alcohol (Dorta, et al., 2005), and thus is an important commodity in many parts of the world. Additionally, many of us might already be aware of the extensive use of yeast fermentation in the production and preservation of food, a process dating back thousands of years (Belloch, et al., 2007). Yeast is used in the production of beer, wine, and bread; indeed yeast pairs with a particular strain of bacteria to create distinctive sourdough bread. Though the uses for yeast and it products are versatile, its tolerance for extremes of ph might not be. Gänzle et al. (2005) studied the effect of ph on yeast and found that yeast growth was unaffected between the ph ranges tested, 3.5 to 7. It was the aim of our study to investigate the effect of ph levels outside these ranges and resolve the question of whether yeast will grow effectively at extreme ph values. It is our prediction that like most organisms yeast growth will be favorable at ph values near the middle range and less favorable at extreme ph values because organisms generally prefer neutral environments and extremes of ph may be toxic to them.
2 Materials and Methods Equipment and supplies needed: Fermentation tube 10% sucrose solution Dry yeast Millimeter ruler 37 C water bath 1M NaOH 1M HCl Microcent tube Stir rod Beakers Spatula The strain of yeast is unknown, but most likely common commercially packaged yeast, S. cerevisiae. 0.5g yeast was added to 20ml sucrose solution in beaker and hand stirred until yeast was completely dissolved. Solution ph was adjusted to 5 for control group. Solution was added to fermentation tube, filling upright portion, and placed in 37 C water bath. Yeast growth is measured by fermentation. Displacement of fluid by CO2 was measured from top at six 5 minute intervals using ruler. Note: Any foam which developed was measured to the bottom as part of CO2 fermentation. Three test groups with solution ph adjusted to 7, 3, and 10 were each tested over three trials and results recorded. The mean of each interval was calculated and graphed with the results from each ph level tested. A final graph compares the mean of each interval for each ph level tested. Results Trial 1 of control group at ph5 (Fig. 1) produced more CO2 (5mm) at 5 minute interval than all other trials (4mm each), but produced the least CO2 after the 15 minute interval than trials 2 and 3 ending with 63, 72, and 71, respectively. The mean amount of CO2 produced for control group was 68.7. Test group ph 7 trials CO2 production (Fig. 2) were identical at 5 minute interval (3mm), but varied markedly at the 15 minute interval at 15, 15, and 18, respectively. The range of CO2 production total was ±2mm of the mean, 68, 72, and 70, respectively. For test group ph 3 (Fig. 3), the 5 minute resulted in similar CO2 production with trial 1 and 2 at 3mm and trial 3 at 2mm. Similarly, at the 15 minute interval, CO2 production was at 8, 7, and 8mm for trials 1, 2, and 3, respectively. However, the 25 minute interval resulted in a relatively significant difference in CO2 production between each trial with 13mm, 19mm, and 17mm of CO2
3 produced from trials 1, 2, and 3, respectively. Trial 1 produced the least total CO2 with 25mm compared to trial 3 at 36mm. Test group ph 10 (Fig. 4) showed similar results from intervals at 5 minutes through 15, with only a 1mm difference in CO2 production from the next closest amount of CO2 measured; 1 and 2mm, 4 and 5mm, and, 8 and 9mm. However, at 20 minute and 30 minute intervals at least a 2mm gap was observed between the next closest amount of CO2 produced, 20 and 23, and, 54 and 56, respectively. A significant difference in results of CO2 production is revealed when comparing mean rate of alcoholic fermentation (Fig. 5). In total, test ph 7 produced 2.3mm more CO2 than our control ph 5, while ph 3 and 10 produced the least amount of CO2 at 32 and 53.3mm, respectively.
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5 Discussion The central question posed in this experiment was whether extremes of ph affect the rate of yeast growth unfavorably. The data suggests that of the ph ranges tested yeast growth is favorable at ranges closest to neutral (5, and 7) and less favorable at ph levels near the extremes, 3 and 10. The near-neutral and neutral ph ranges 5, and 7 produced the highest amount of fermentation 68.7, and 70 mm, respectively, compared to 32 and 53.3mm for extreme ranges of ph 3 and 10, respectively for a difference of at most 38mm CO2 produced and at least 15.4mm CO2 produced in favor of the neutral ph values. As predicted extremes of ph inhibited the growth of yeast and therefore produced less fermentation at ph values 3 and 10. The large scale usage of ethanol in many industries around the world make the study of environmental factors that can inhibit yeast fermentation important; the implications are farreaching. One unanticipated outcome of the experiment was the amount of fermentation produced at ph 10. It was expected that fermentation would still occur at extreme ph ranges, but not to the extent observed at ph 10. There are a number of limitations which can be identified as part of the experiment. First, in comparing our results to the study by Gänzle, et al. it can be observed that C. milleri yeast was used, possibly a different strain than was used during this experiment, thus a completely accurate comparison of their results cannot be confirmed. Second, in testing extremes of ph I propose the following amendment to the levels tested: For extreme ph levels, test ph 2, and 13; for neutral ph levels test 5, 7, and 9. Though there are no anticipated large deviations in the results from testing ph 2 instead of 3, and 9 instead of 10, we felt it would be a greater representation of so-called extreme ph ranges, likewise with testing the neutral ph 7 range and adjacent ranges. Also, use ph 7, neutral ph as control. Future experiments might test many strains of yeast in the entire ph range in synergy with a combination of environmental factors, similar to the study by Gänzle, et al. Conclusions The following are conclusions that can be derived from the results of this experiment: 1. Neutral ph 7 produced 119% more fermentation than ph 3. 2. Neutral ph 7 produced 31% more fermentation than ph 10.
6 3. Combined, the neutral ph ranges produced 62% more fermentation than the extreme ph ranges, on average. Literature Cited Gänzle, Michael G., Michaela Ehmann, and Walter P. Hammes. 1998. Modeling of Growth in Lactobacillus sanfranciscensis and Candida miller in Response to Process Parameters of Sourdough Fermentation. Applied and Environmental Microbiology. 64: 2616-2623. Dorta, C., P. Oliva-Neto, M.S. de-abreu-neto, N. Nicolau-Junior, and A.I. Nagashima. 2005. Synergism among lactid acid, sulfite, ph and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26). World Journal of Microbiology & Biotechnology. 22: 177-182. Belloch, Carmela, Sani Orlic, Eladio Barrio, Amparo Querol. 2007. Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex. International Journal of Food Microbiology. 122: 188-195.