Fermentation Analyses: Literature Reports Below are abstracts from various papers reporting findings of fermentation analyses using S. cerevisiae and other species of microorganisms. Keep in mind that you do NOT need to understand all the details of the analyses. Focus on picking out things that could be helpful for developing your hypothesis and/or methods. If there’s something that confuses you, ask your lab TA. You are welcome to do some research on your own, however remember that you need to develop your hypothesis and generate a detailed experimental protocol to test your hypothesis before you leave lab.

Lin, Yan, et. al. (2012) “Factors affecting ethanol fermentation using Saccharomyces cerevisiae BY4742.” Biomass and Bioenergy. 47: 395-401. Fermentation of sugar by Saccharomyces cerevisiae BY4742, for production of ethanol in a batch experiment was conducted to improve the performance of the fermentation process. The thermotolerant ability of S. cerevisiae to grow and ferment glucose at elevated temperatures similar to the optima for saccharification was investigated. The influences of temperature, substrate concentration and pH on ethanol fermentation were observed. The yield for ethanol production and changes in the fermentation pathway were compared under different conditions. When the temperature was increased to 45°C, the system still showed high cell growth and ethanol production rates, while it was inhibited at 50°C. The maximum specific growth rate and the maximum specific ethanol production rate were observed between 30 and 45°C with different initial glucose concentrations. The maximum sugar conversion at 30°C after 72 h incubation was 48.0%, 59.9%, 28.3%, 13.7% and 3.7% for 20, 40, 80, 160 and 300 kg m-3 of glucose concentrations respectively. Increased substrate supply did not improve the specific ethanol production rate when the pH value was not controlled. pH 4.0‒5.0 was the optimal range for the ethanol production process. The highest specific ethanol production rate for all the batch experiments was achieved at pH 5.0 which is 410 g kg-1 h-1 of suspended solids (SS) which gave an ethanol conversion efficiency of 61.93%. The highest specific ethanol production rate at 4.0 was 310 g kg-1 h-1 of SS. A change in the main fermentation pathway was observed with various pH ranges. Formation of acetic acid was increased when the pH was below 4.0, while butyric acid was produced when the pH was higher than 5.0. In the presence of oxygen, the ethanol could be utilized by the yeast as the carbon source after other nutrients became depleted, this could not occur however under anaerobic conditions.

D’Amore, Tony, et. al. (1989) “Sugar utilization by yeast during fermentation.” Journal of Industrial Microbiology. 4: 315-324. When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer’s wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in the Saccharomyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake in

Saccharomyces cerevisiae revealed a Km of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.

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Casey, Elizabeth, et. al. (2013) “Effect of salts on the Co-fermentation of glucose and xylose by a genetically engineered strain of Saccharomyces cerevisiae.” 6: 83. Background: A challenge currently facing the cellulosic biofuel industry is the efficient fermentation of both C5 (five carbons) and C6 (six carbons) sugars in the presence of inhibitors. To overcome this challenge, microorganisms that are capable of mixed-sugar fermentation need to be further developed for increased inhibitor tolerance. However, this requires an understanding of the physiological impact of inhibitors on the microorganism. This paper investigates the effect of salts on Saccharomyces cerevisiae 424A(LNH-ST), a yeast strain capable of effectively co-fermenting glucose and xylose. Results: In this study, we show that salts can be significant inhibitors of S. cerevisiae. All 6 pairs of anions (chloride and sulfate) and cations (sodium, potassium, and ammonium) tested resulted in reduced cell growth rate, glucose consumption rate, and ethanol production rate. In addition, the data showed that the xylose consumption is more strongly affected by salts than glucose consumption at all concentrations. At a NaCl concentration of 0.5 M, the xylose consumption rate was reduced by 64.5% compared to the control. A metabolomics study found a shift in metabolism to increased glycerol production during xylose fermentation when salt was present, which was confirmed by an increase in extracellular glycerol titers by 4-fold. There were significant differences between the different cations. The salts with potassium cations were the least inhibitory. Surprisingly, although salts of sulfate produced twice the concentration of cations as compared to salts of chloride, the degree of inhibition was the same with one exception. Potassium salts of sulfate were less inhibitory than potassium paired with chloride, suggesting that chloride is more inhibitory than sulfate. Conclusions: When developing microorganisms and processes for cellulosic ethanol production, it is important to consider salt concentrations as it has a significant negative impact on yeast performance, especially with regards to xylose fermentation.

Osman, Yehia and Ingram, Lonnie. (1985) “Mechanism of Ethanol Inhibition of Fermentation in Zymomonas mobilis CP4.” Journal of Bacteriology. 164: 173-180. Accumulation of alcohol during fermentation is accompanied by a progressive decrease in the rate of sugar conversion to ethanol. In this study, we provided evidence that inhibition of fermentation by ethanol can be attributed to an indirect effect of ethanol on the enzymes of glycolysis involving the plasma membrane. Ethanol decreased the effectiveness of the plasma membrane as a semipermeable barrier, allowing leakage of essential cofactors and coenzymes. This leakage of cofactors and coenzymes, coupled with possible additional leakage of intermediary metabolites en route to ethanol formation, is sufficient to explain the inhibitory effects of ethanol on fermentation in Zymomonas mobilis.

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