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Yeast and fermentation: the optimal pH level. Joosten, M. & Peeters, M. Philips van Horne sg. Weert, The Netherlands April 14th 2010 Summary of the inquiry Yeast is an eukaryotic organism which is an important factor in the producing of ethanol. It can ferment glucose into ethanol and carbon dioxide. This fact stimulated interest in the producing of ethanol, and the optimum pH level this process requires. For this inquiry, yeast cells of the species saccaromyces cerevisiae, ordinary baker’s yeas
   Yeast and fermentation: the optimal pH level.  Joosten, M. & Peeters, M. Philips van Horne sg. Weert, The Netherlands  April 14 th 2010 Summary of the inquiry Yeast is an eukaryotic organism which is an important factor in the producing of ethanol. Itcan ferment glucose into ethanol and carbon dioxide. This fact stimulated interest in theproducing of ethanol, and the optimum pH level this process requires. For this inquiry, yeastcells of the species saccaromyces cerevisiae, ordinary baker’s yeast, were used. Thefermentation process was followed at pH levels varying between 2,8 and 8,3, measuring therelease of carbon dioxide gas. This resulted in an optimum pH for the fermentation processbetween 4.8 at 6.0. This inquiry also raised further questions, like if the pH level can changeduring the fermentation process. Introduction Yeasts are eukaryotic micro-organismsclassified in the kingdom Fungi, with about1,500 species currently described (1) Aneukaryote is an organism whose cellscontain complex structures inside themembranes. The defining membrane-bound structure that sets eukaryotic cellsapart from prokaryotic cells is the nucleus,or nuclear envelope, within which thegenetic material (DNA) is carried (2) Figure 1:  A Yeast cell (3) The yeast species Saccharomycescerevisiae has been used in baking andfermenting alcoholic beverages for thousands of years. It is also extremelyimportant as a model organism in moderncell biology research, and is one of themost thoroughly researched eukaryoticmicroorganisms(4) Ethanol fermentation isa biological process in which sugars suchas glucose, fructose, and sucrose areconverted into cellular energy and therebyproduce ethanol and carbon dioxide asmetabolic waste products. Because yeastcells perform this process in the absenceof oxygen, ethanol fermentation isclassified as anaerobic.(5) During thefermentation, one glucose molecule isconverted into two ethanol molecules andtwo carbon dioxide molecules : (6) Figure 2: The fermentation of sugar Figure 3 : glucose molecule.(7) In research, it is often found that sucrose(table sugar) ferments better than glucose(8) Figure 4: Sucrose molecule (8) When a fermentation of ethanol withoxygen takes place, vinegar is made.Vinegar is an acidic liquid processed formthe fermentation of ethanol by the aceticacid bacteria. This means that when all theglucose is fermented into ethanol andcarbon dioxide, and there is oxygen, theethanol can ferment into vinegar.(9) Figure 5: acetic acid structure, importantcomponent of vinegar(10)   C 6 H 12 O 6    2 CO 2 + 2 C 2 H 5 OH  During our research we considered thefact that alcoholic beverages can be madefrom different fruits. This arouse thequestion whether a fermentation processhas an optimum pH level, and what thatlevel might be. Our hypothesis is that theoptimum pH level will be around 7, sinceit’s a neutral environment in which mostorganisms can live well. Experimental procedure and approach Some citric acid buffers were madeaccording to Mac Ilvain’s method.(10)Then, 10 grams of table sugar (sucrose)were added to each buffer solution, andCO 2 gas was immersed in the solution.This was to make sure the solution wasfully repleted, so none of the produced gaswould dissolve in the solution. After that, 5grams of  Saccharomyces cerevisiae ,baker’s yeast, were added. The flaskswere closed with a cork, connected to agas cylinder, and were placed in a water bath with a constant temperature of 37°C.The solutions were allowed to ferment for 19½ minutes, before the amount of produced gas was determined. Everybuffer solution was made twice. Theamounts of gas produced were graphicallypresented. Figure 6 shows the experimental set-up. Results  After the yeast was added to the buffer solution and the flask was put into thewater bath, a ‘foam’ layer started to formon top of the solution. And, from thatmoment on, the gas cylinder started to fillup. We noticed that a solution with a lowpH level begins to form gas quickly, butslows down after some time, while thesolution with a higher pH level (thoughunder 8) starts to form gas more slowly,but goes faster after some time. Table 1presents the measured amount of gas, inmL, trapped in the gas cylinder. Table 1: measured amount of gas, in mL,trapped in the gas cylinder. pH levelQuantity of produced gas [mL]2.88485,04.26795.04.883110.15.483108.76.063111.66.45599.07.05985.57.1471027.654104.67.66290.58.34425.0Figure 7 shows the measured amount of gas, in mL, trapped in the gas cylinder atdifferent pH levels. Figure 7: Measured amount of gas, in mL,trapped in the gas cylinder at different pHlevels. Data analysis.  As presented in table 1, the quantity of gasproduced is highest between a pH level of 4.883 and 6.063. Interpreting the graph infigure 7 , it can be said that that yeast cellsof  Saccharomyces cerevisiae are moreactive in an acid environment, than in analkaline environment, since the produce of CO 2 gas drastically decreases from an pHof 8 onwards. Conclusion, discussion & evaluation  As can be seen in both table 1, and figure7, CO 2 gas is produced at every measuredpH level. However, it works best between  a pH level of 4.883 and 6.063. Our conclusion is that yeast cells of  saccharomyces cerevisiae work best at aslightly acid pH level.Looking critically at our experimentalprocedure and approach we that weconsidered the same independent anddependent variables, and kept the samevariables constant. There is a possibilitythat there were some errors in our measurements. It is probable that the pHlevel changed during fermenting becausevinegar could’ve formed out of ethanol andoxygen. After some time CO 2 gas canevaporate from the solution. It is possiblethat in the 19½ minutes we measured,CO 2 gas has escaped from the solution.This would lead to a too high amount of measured gas.This raises further inquiry questions; for example: Does the pH level indeedchange during fermenting? Bibliography. 1. http://www.ars.usda.gov/research/ publications/publications.htm?SEQ_NO_115= 176765 2. http://en.wikipedia.org/wiki/Eukaryote 3. http://www.biocourseware.com/ iphone/cell/img/yeast_cell.jpg 4. http://mmbr.asm.org/cgi/content/full /64/1/34?view=long&pmid=10704473 5. http://www.rurdev.usda.gov/rbs/pub/ sep06/ethanol.htm 6. http://www.chemie.uni-regensburg.de/Organische_Chemie/Didaktik/Keusch/D-fermentation_sugar-e.htm 7. http://www.columbia.edu/itc/biology/ chasin/lecture8/lec8_00.htm 8. http://www.chemheritage.org/ EducationalServices/FACES/glossary/sucrose.htm 9. http://www.fda.gov/ICECI/ComplianceManuals/CompliancePolicyGuidanceManual/ucm 074550.htm 10. http://www.fda.gov/ICECI/ComplianceManuals/CompliancePolicyGuidanceManual/ucm0 74471.htm 11. Van der Pluijm, J.E., Ter Braak, A.H.M., Hallmann, P.P.H.,Houwen, P.J.W., Marquenie,J.G.M.,Van Ree, W. & Schraag,J.A. (1978). Citroenzuurbuffer. Biothema 2, 128.
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