Efficiency of Yeast Fermentation with Environmental Variables Eric Dammer Dr. David Brown

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1 Efficiency of Yeast Fermentation with Environmental Variables Eric Dammer Dr. David Brown October 31-November November

2 Dammer, 2 Purpose and Principles Successive trials of contained yeast fermentation are analyzed to understand optimal conditions for fermentation in small containers over short periods of time. These conditions, namely ideal sugar type and temperature for the activated yeast used, are then built into the controlled conditions of subsequent trials. In the case of this group, the later experimental conditions tested are initial presence of known ethyl alcohol concentration and in separate trials, the presence of arsenate ions during the ten minute trial period over which each set of data has been collected. By repeating the same procedure with initial variables as the most basic, iterations of the procedure used can give the optimal conditions to control while varying more exotic variables, such as the presence of any foreign substance such as arsenate, fluoride, sodium ions, etc. By optimizing control conditions to produce the largest possible control output, variations in the output in subsequent experimental groups are more pronounced and statistically significant. However, in varying exotic variables, it is entirely possible that physical phenomena beyond the biological system that may not have been considered can in fact contribute to significant error. Alcohol concentration was varied in the optimized trials to determine whether presence of waste product from the fermentation reaction would have significant effect on the efficiency or capacity of fermentation in the tested yeast. Arsenic acid was added with the knowledge that arsenate ion from the additive can replace phosphate and interfere with particular steps of glycolysis, which, if hampered, would not result in the control efficiency of carbon dioxide production. Results Data collected reflect standard pressure and the temperatures noted, which figure into determination of carbon dioxide gas collected from the tapped flasks into separate beakers under water. The ideal gas law mitigates variation in pressure and temperature in the below efficiency calculations (see the identically calculated data set with a layout of the data set involved in these calculations in table two). Slight variations in carbohydrate and yeast involved in each trial system are considered in this calculation, which is in terms of moles carbon dioxide collected divided by moles carbon present in the carbohydrates commutatively, i.e. in turn, divided by the milligrams yeast used. All reactions were completed in the presence of 5.0 ml water over ten minute intervals. Trials Glucose Fructose Sucrose Mannitol Lactose One 1.39E E E E E-04 Two 1.04E E E E E-04 Three 1.59E E E E E-05* Four n/a 1.71E-04 n/a 4.91E E-04 Average 1.34E E E E E-04 Table 1: Determination of optimal carbohydrate in terms of glycolytic efficiency. (Trials carried out at 42 C.) Based on the data in table 1, sucrose was chosen for subsequent trials. Table 2 indicates the results of the same procedure with variations in temperature.

Dammer, 3 Temp ( C) Pressure (atm) Gas Const. (L atm / mol K) g / mol Sucrose Temp ( C) Efficiency Value 293.15 1.0052632 0.08206 342.3 x10-4 20 0.043 Efficiency 35.1 0.

3 Dammer, 3 Temp ( C) Pressure (atm) Gas Const. (L atm / mol K) g / mol Sucrose Temp ( C) Efficiency Value x Efficiency ML CO2 mol CO2 mg sucrose mg yeast mol C Values average ML CO2 mol CO2 mg sucrose mg yeast mol C Eff. Val average ML CO2 mol CO2 mg sucrose mg yeast mol C Eff. Val average ml CO2 mol CO2 mg sucrose mg yeast mol C Eff. Val average ml CO2 mol CO2 mg sucrose mg yeast mol C Eff. Val average ml CO2 mol CO2 mg sucrose mg yeast mol C Eff. Val average 1.840

4 Dammer, 4 In a third set of trials with identical procedure, both the optimal carbohydrate (sucrose) and temperature (55 C) were controls. Denatured alcohol was introduced as a varying percentage of solution. In a single trial, arsenic acid was added with only water. In both cases, control measurements without sucrose were also run to determine whether measurements of collected gas reflected only carbon dioxide produced as a fermentation product. CO 2 or gas ml CO 2 mol x10-4 Sucrose mg Yeast mg Atomic C mol Efficiency x10-4 Conditions % ethyl alc % ethyl alc % ethyl alc % ethyl alc n/a 40% Control Interpretation E mol Arsenate E-04 0 ~400 0 n/a 0 mol Control 2.68E Estimated Arsenate Table 3: Additional variables tested with verification and/or adjustment of results. It is interesting that trial one gave results so similar for the monosaccharide fructose as for the disaccharide sucrose, which is fifty percent glucose, which clearly is less efficiently fermented (by about 100(0.4/1.75), or 23 percent). This would seem to indicate that the yeast has mechanisms that can make use of energy inherent to the structure of the disaccharide in more efficiently processing the glucose monomer. Because the only unique bond in sucrose is the alpha 1-4 linkage, a possible hypothesis that may be confirmable in other experiments considering the enzymes involved in the fermentation pathway would be that a particular enzyme can involve the energy released by hydrolysis of this bond in processes affecting the fermentation of the glucose monomer just released, or perhaps one already released from an earlier hydrolysis. Fig 1a: Lactose (glucose (α,1-4) galactose). Fig 1b: Sucrose (glucose(α,1-2) fructose). This is, however, speculative, given the focus of subsequent experimental groups in this lab. Moreover, it would seem that the α-linkage in sucrose results in a different unique conformation of the disaccharide.

5 Dammer, 5 The second set of trials showed considerably variable fermentation efficiency with temperature. It is not clear whether activity between C is dampened only to increase once more, but it is possible this is a characteristic of the activated yeast, selected for characteristically better and perhaps also unusual efficiency at higher temperatures. It is possible that more than one enzyme or form of an enzyme exists thus leading to multiple efficiency peaks rather than one, which would be expected for one pathway with a single set of enzymes required for completion to yield carbon dioxide and alcohol. In this vein, the subsequent trial set involving alcohol in the active fermentation environment might have been expected to activate feedback inhibition of fermentation, and reducing measured efficiency. While data initially indicated increased efficiency, this result is highly suspicious, especially in light of the fact that a blank beaker with only forth percent alcohol easily accounts for all vapor collected in the experimental trial with the same concentration of alcohol. It is possible that only fractions of a percent of alcohol may have been sufficient to produce the hypothesized results and a curve for efficiency with respect to alcohol concentration. In addition to procedure not suited to the measured variables, the alcohol used was denatured with as much as three percent solvents, which may have easily compromised the selectively permeable physical properties of the plasma membrane, which in turn may have decreased the ability of the yeast cells to collect and maintain sufficient concentrations of the sugar or waste products of fermentation including ethyl alcohol itself. Finally, a valid result was obtained with the optimized conditions in the presence of arsenate. After adjusting for a small amount of evaporation without any alcohol present, an efficiency of only forty-two percent (100(0.954/1.840)) the average value found for the fermentation under the same conditions occurred. This clearly indicates that arsenate interferes with fermentation, if not glycolysis in particular. Arsenate present would be chemically interchangeable with phosphate groups, especially those involved in phosphorlative oxidation. While such oxidation is coupled with reactions reducing energy carriers such as ATP or coenzymes, arsenate would pose both a more sterically hindered molecule, and a more stable ion, thus preventing phosphorlyation wherever it has been integrated into an intermediate carbohydrate, or ADP itself. Thus, were additional trials carried out with variable time, it would be expected that a curve of efficiency vs. time in the presence of a fixed amount of arsenate would indirectly reflect the rate at which yeast and physical gradients incorporate arsenate into the essential biomolecules of the glycolytic pathway but more directly show the dependence of this particular version of respiration on the energy provided by glycolysis.

Learning Target: Describe characteristics and functions of carbohydrates, lipids, and proteins. Compare and contrast the classes of organic

Learning Target: Describe characteristics and functions of carbohydrates, lipids, and proteins. Compare and contrast the classes of organic compounds. What are inorganic molecules? Molecules that CANNOT