The effect of salts on yeast fermentation Goes, A. & Milani, P.

Colégio Bandeirantes, São Paulo, Brazil

Received May 2010 Summary

Nowadays, ethanol is one of the viable alternatives as a renewable source of energy and it can be obtained from the anaerobic fermentation of sugars performed by Saccharomyces cerevisiae. Considering this, questions about ways of improving the fermentation process to optimize the production of ethanol arise. In order to find answers to them, five different salts were added to the fermentation (MgCl2, NaCl, NaNO3, CaCl2, and Na2HPO4) and the volume of CO2 produced was measured. The experiments showed an increase in the reaction rate with the addition of Na2HPO4 and a reduction with the addition of NaCl. Such results answered some questions, but raised others, like why these salts influenced the fermentation.

Fermentation, yeast, salts, Na2HPO4

Introduction Ethanol has many uses; as fuel,

it will be important to replace oil, which contributes to global warming and can run short. The main process used in industrial scale to produce ethanol is the fermentation of glucose, performed by yeasts to obtain energy: C6H12O6 2C2H5OH + 2CO2

Most countries use starch as source of glucose for fermentation. However, in Brazil, considered one of the leading countries in the biofuel industry, sucrose from sugarcane is the raw material for the production of ethanol. Therefore, the polysaccharides and disaccharides must suffer hydrolysis prior to fermentation. The hydrolysis of sucrose produces glucose and fructose, two isomers that can be fermented by yeast. Such reaction is catalyzed by the enzyme invertase, found in yeast: C12H22O11 + H2O C6H12O6 + C6H12O6

(sucrose) (glucose) (fructose) Yeasts are eukaryote and

heterotrophic beings that belong to the Kingdom of Fungi. One of the species used in fermentation is the Saccharomyces cerevisiae, also known as baker’s yeast. The S. cerevisiae is considered a facultative anaerobe1, because it can grow in either the

presence or absence of oxygen. If there is oxygen, the yeast might perform other reactions in order to obtain energy2, like the oxidations of glucose and ethanol. So, it is possible to conclude that, to optimize the yield of ethanol, the reaction must occur in an oxygen free environment, to maximize the fermentation and minimize the oxidations. Another way to improve the production of ethanol is adjusting temperature and pH to optimal conditions, which previous researches indicate to be around 35° C3 and 54, respectively.

The alcoholic fermentation is not a single reaction, but instead consists of a complex group of reactions, which are catalyzed by a number of yeast enzymes1. Researches appear to indicate that some ions serve as cofactors for such enzymes1. We also believe that the S. cerevisiae needs some elements as part of its nutrition, so it could theoretically grow better in their presence. This raises the question: Can the addition of certain salts increase the fermentation rate?

Our hypothesis is that some salts will slightly increase the fermentation rate, due to the two reasons explained before.

Experimental procedure Kitasatos were filled with 35 mL

of acetate buffer (pH = 5.6) and completed with more 5 mL of buffer (control experiment) or 5 mL of a 0.1 mol/L salt solution in buffer (the salts used were: MgCl2, NaCl, NaNO3, CaCl2 and Na2HPO4). After this, 5g of S. cerevisiae (Itaiquara fresh biological yeast) were dissolved in each solution and then 10 mL of a 15% (m/m) sucrose (União sugar) solution in buffer were added to the flasks. The kitasatos were immediately closed and the CO2 produced was collected in graduated cylinders, as shown in figure 1. All tests were repeated three times each, always at room temperature (around 26.5o C) and under constant stirring. The fermentation was kept running for 25 minutes and the volume of gas produced was recorded every 5 minutes. In the end, the pH of the solutions was measured once again and the values

obtained guaranteed that the buffer solution had worked. Figure 1: Photo of the experimental set-up. The piece of equipment under the kitasato is a magnetic stirrer. Results

The results obtained are shown in the tables and graph below:

Table 1: CO2 collected in control, MgCl2 and NaCl tests and respective averages.

Table 2: CO2 collected in NaNO3, CaCl2 and Na2HPO4 tests and respective averages.

Time (min)

Control (mL) MgCl2 (mL) NaCl (mL)

1 2 3 Avg 1 2 3 Avg 1 2 3 Avg 0 0 0 0 0 0 0 0 0 0 0 0 0 5 2 5 2 3 4 2 5 3.7 0 1 1 0.7

10 16 18 20 18 18 18 17 17.7 7 15 12 11.3 15 37 40 44 40.3 41 42 38 40.3 26 38 31 31.7 20 61 68 70 66.3 69 67 63 66.3 47 61 55 54.3 25 85 94 98 92.3 95 93 85 91 74 84 80 79.3

Time (min)

NaNO3 (mL) CaCl2 (mL) Na2HPO4 (mL)

1 2 3 Avg 1 2 3 Avg 1 2 3 Avg 0 0 0 0 0 0 0 0 0 0 0 0 0 5 2 1 2 1.7 3 2 4 3 2 2 5 3

10 16 15 22 17.7 16 17 20 17.7 30 19 26 25 15 36 34 48 39.3 35 36 42 37.7 62 42 58 54 20 62 60 78 66.7 60 63 68 63.7 95 72 96 87.7 25 89 85 103 92.3 88 92 92 90.7 135 110 132 125.7

Figure 2: Graph of average CO2 collected versus time for all tests. Discussion and Conclusion Based on the results, the five salts can be divided into three groups: the ones that do not alter the fermentation rate, the ones that slow it down and the ones that speed it up. In the first group, there are MgCl2, NaNO3 and CaCl2, because the fermentation rate was very similar between them and the control. They probably do not interfere with the yeast metabolism and do not act as cofactors nor as inhibitors. The second group has the NaCl, or common salt, since a 14% reduction in the reaction yield was observed after 25 minutes. It may interfere negatively on S. Cerevisiae’s metabolism or may act as an inhibitor for some reactions in the fermentation. The third group consists of Na2HPO4, because it made the fermentation produce 36% more CO2 in 25 minutes. This salt probably provides important nutrients for the yeast or act as a cofactor in one of the reactions of the fermentation process. The results could also be interpreted from another point of view. It is known that in a reaction with multiple stages, like fermentation, the slowest stage is the one that determines the rate of the overall reaction. So, a

possible explanation for the effect of the first and third group of salts is that the MgCl2, NaNO3 and CaCl2 might actually be cofactors for some stages of the fermentation, but they speed up parts which are already fast, without affecting the rate of the reaction as a whole; the Na2HPO4, on the contrary, might speed up some of the slowest stages of the fermentation, therefore producing a perceptible increase in its rate.

To conclude, the inquiry question was answered, since the experiments showed not only one salt capable of increasing the fermentation rate (Na2HPO4), but also one capable of reducing it (NaCl) and three that do not influence it (MgCl2, NaNO3 and CaCl2).

Evaluation

On evaluating the experimental set-up, we believe that most of the control variables, like temperature, pH, stirring and amounts used were managed well. Nonetheless, in some cases, one value of CO2 produced differed considerably from the other two, which indicates that we might have overlooked something and that each test should have been repeated more than three times. One

more thing that could be improved was the time the experiment was kept running: when we stopped recording the values, 25 minutes after the beginning of the fermentation, the reaction was far from over. It would be interesting to know what would have happened if the experiment had lasted longer. Finally, further inquiries could test the effect of salts that were not used or even the mixture of more than one salt. Another approach to this idea could be to test the effect of those same salts on the fermentation of starch and compare the results with the ones we got. Since starch fermentation is much slower than the sucrose one, it might be easier to verify the effects of the salts on this process.

Bibliography 1- Dr. Susan Petro’s Paper. “Fermentation

in the Yeast Saccharomyces cerevisiae”, available at http://phobos.ramapo.edu/~spetro/lab_pdf/Fermlab.pdf (at 05/23/2010)

2- Loureiro, V. & Malfeito-Ferreira, M. (2003). Spoilage yeasts in the wine industry. International Journal of Food Microbiology, 86, 23-50

3- Slaa et al. (2009). Yeast and Fermentation: the optimal temperature. Journal of Organic Chemistry: Chem. Dut. Aspects, 134

4- Laboratory exercise developed by the University of Colorado, available at http://spot.colorado.edu/~kompala/lab2.html (at 05/23/2010)