M. Brooke Hooper M. Brooke Hooper Microbiology Undergraduate Microbiology Undergraduate

student student Biology department Biology department

Tennessee Technological Tennessee Technological Univerisity Univerisity

Cookeville, TNCookeville, TN

3850538505

Introduction IIntroduction I TitleTitle: : Affect of disaccharide concentration on the Affect of disaccharide concentration on the

fermentation of saccharomyces cerevisiaefermentation of saccharomyces cerevisiae ObjectiveObjective: To relate affects of disaccharide : To relate affects of disaccharide

concentration on the alcohol by volume percentage of concentration on the alcohol by volume percentage of wine from the fermentation of saccharomyces wine from the fermentation of saccharomyces cerevisiae cerevisiae

Hypothesis:Hypothesis: A stronger disaccharide concentration will A stronger disaccharide concentration will yield a higher alcohol by volume percentage yield a higher alcohol by volume percentage

Null hypothesisNull hypothesis: a stronger disaccharide : a stronger disaccharide concentration will yield a lower alcohol by volume concentration will yield a lower alcohol by volume percentage percentage

Introduction IIIntroduction II

Saccaromyces cerevisiaeSaccaromyces cerevisiae is the yeast used in the is the yeast used in the fermentation of wine.fermentation of wine.

Bio-ethanol production from Bio-ethanol production from S. cerevisiaeS. cerevisiae is presently is presently the largest fermentation process in industrial ethanol the largest fermentation process in industrial ethanol production (Medina 2010).production (Medina 2010).

During anaerobic growth of During anaerobic growth of S. cerevisiaeS. cerevisiae, sugar , sugar dissimilation occurs via alcoholic fermentation (Lin dissimilation occurs via alcoholic fermentation (Lin 2002).2002).

Belonging to the kingdom Fungi,S. cerevisiaeBelonging to the kingdom Fungi,S. cerevisiae feed off feed off of disaccharides for energy and fermentation as they of disaccharides for energy and fermentation as they acquire carbon from their immediate surroundings acquire carbon from their immediate surroundings (Truckses 2004).(Truckses 2004).

Introduction IIIIntroduction III

Yeast cells divide asymmetrically by budding Yeast cells divide asymmetrically by budding (Kaeberlein 2008).(Kaeberlein 2008).

Ethanol fermentation from S. cerevisiae takes the Ethanol fermentation from S. cerevisiae takes the following three steps (Devi 2000):following three steps (Devi 2000): Development of a solution of soluble sugarsDevelopment of a solution of soluble sugars Fermentation of these sugars to alcoholFermentation of these sugars to alcohol Purification of the ethanol Purification of the ethanol

Materials & Methods

Six 0.35L bottles prepared with: 60ml Concentrated Grape Juice Sugar solutions of 0g, 10g, 20g, 25g, 35g, or 45g. Red Star Montrachet brand Saccharomyces cerevisiae

Each solution allowed to ferment in darkness for six weeks

Methods followed by:http://www.instructables.com/id/Make-your-own-wine/

http://www.homebrewzone.com/

Materials & Methods Materials & Methods

ContainerContainer DisaccharideDisaccharide JuiceJuice WaterWater

A (control)A (control) nonenone 60ml60ml 240ml240ml

BB 10g10g 60ml60ml 240ml240ml

CC 20g20g 60ml60ml 240ml240ml

DD 25g25g 60ml60ml 240ml240ml

EE 35g35g 60ml60ml 240ml240ml

FF 45g45g 60ml60ml 240ml240ml

Home Wine Brewing Home Wine Brewing

http://www.leftofme.com/how-to-brew-cheap-wine/

Materials & Methods Materials & Methods

Data CollectionData Collection: Specific gravities measured : Specific gravities measured using a hydrometer before and after fermentation.using a hydrometer before and after fermentation.

EquationEquation: (specific gravity1- specific gravity2) x : (specific gravity1- specific gravity2) x 125= ALCOHOL PERCENT BY VOLUME 125= ALCOHOL PERCENT BY VOLUME

StatisticsStatistics: T-tests using : T-tests using Dr. Machida’s statistic tools

Saccharomyces cerevisiaeSaccharomyces cerevisiae

Budding yeast cellhttp://www.ianchadwick.com/tequila/

Fermenting yeast cellhttp://microbewiki.kenyon.edu/

ResultsResultsAffect of disaccharide concentration on the fermentation of saccharomyces cerevisiae.

0

5

10

15

20

25

30

35

40

45

50

1 2 3 4 5 6 7

Wine Speciment (let 1=A, 2=B, 3=C, etc).

Series1Series2Series3

-Series 2=%

Alcohol

-Series 3 = g of sugar

•A positive correlation is noted between the increase in sugar and the increase of alcohol content.

Results A negative correlation noted between increase of sugar

concentration and the decrease of specific gravity.

A positive correlation noted between percent of alcohol and change in specific gravity

Specific gravities of each wine solution decreased with time Ethanol Specific gravity=0.78 & Water Specific

gravity=1.0 Null hypothesis rejected

DiscussionDiscussion

Benefits: amplification of understanding the ideal sugar amount Benefits: amplification of understanding the ideal sugar amount required for optimal ethanol production.required for optimal ethanol production.

Future Experiment 1: Study of other genus varieties. Future Experiment 1: Study of other genus varieties. Species S. diastaticus is distinguished from S. cerevisiae primarily by its ability Species S. diastaticus is distinguished from S. cerevisiae primarily by its ability

to ferment starch (Innis 1985)to ferment starch (Innis 1985)

Future Experiment 2: Study of External factorsFuture Experiment 2: Study of External factors Glycerol protects the yeast cells in high osmolarity (Medina 2009). Glycerol protects the yeast cells in high osmolarity (Medina 2009).

Future Experiment 3: Future Experiment 3: Study over max. lifespan/fermentation.Study over max. lifespan/fermentation. Cerevisiae become dormant after the ethanol % reaches a maximum or the Cerevisiae become dormant after the ethanol % reaches a maximum or the

yeast consume all of the sugar need for fermentation (Uptain 2002).yeast consume all of the sugar need for fermentation (Uptain 2002).

Future Experiment 4: Genetic study of genusFuture Experiment 4: Genetic study of genus Gene redundancy: thought to be the consequence of whole-genome Gene redundancy: thought to be the consequence of whole-genome

duplications in the species (Langki 2003).duplications in the species (Langki 2003).

Conclusions

A stronger disaccharide concentration will yield a higher alcohol by volume percentage.

A change in specific gravity connects with the percent alcohol by volume in a positive correlation.

A negative correlation is noted between increase of sugar concentration and the decrease of specific gravity as fermentation takes place.

Further InformationFurther Information

http://mbhooper21.tripod.com/

http://www.homebrewzone.com/hydrometer.htmhttp://www.homebrewzone.com/hydrometer.htm

http://www.instructables.com/id/Make-your-own-http://www.instructables.com/id/Make-your-own-wine/wine/

Works CitedWorks Cited

CitationsCitations Devi, R. N., and S. Sumitra. 2010. Bioconversion of cellulose into fermentable sugars by Devi, R. N., and S. Sumitra. 2010. Bioconversion of cellulose into fermentable sugars by

Saccharomyces cerevisiae cells for the production of ethanol using cellulolytic fungi isolated from Saccharomyces cerevisiae cells for the production of ethanol using cellulolytic fungi isolated from soil. soil. The Internet Journal of MicrobiologyThe Internet Journal of Microbiology 77: not applicable. : not applicable.

Home Brew Zone.“Using your hydrometer.” http://www.homebrewzone.com/hydrometer.htm Home Brew Zone.“Using your hydrometer.” http://www.homebrewzone.com/hydrometer.htm (March 17, 2010).(March 17, 2010).

Innis, M.A., et al. 1985. Expression, glycosylation, and secretion of an Aspergillus Glucoamylase Innis, M.A., et al. 1985. Expression, glycosylation, and secretion of an Aspergillus Glucoamylase by Saccharomyces cerevisiae. by Saccharomyces cerevisiae. ScienceScience 228: 228: 21-36.21-36.

Instructables. “How to make wine.” http://www.instructables.com/id/Make-your-own-wine/ Instructables. “How to make wine.” http://www.instructables.com/id/Make-your-own-wine/ (January 28, 2010).(January 28, 2010).

Kaeberlein, M. 2008. A molecular age barrier: a mother's instinct is to protect her children at any Kaeberlein, M. 2008. A molecular age barrier: a mother's instinct is to protect her children at any cost. In the budding yeast Saccharomyces cerevisiae this 'maternal instinct' comes at a high cost. In the budding yeast Saccharomyces cerevisiae this 'maternal instinct' comes at a high price--accelerated ageing and premature death. price--accelerated ageing and premature death. NatureNature 454454: 709-719.: 709-719.

Langkjaer, R. B., et al. 2003. Yeast genome duplication was followed by asynchronous Langkjaer, R. B., et al. 2003. Yeast genome duplication was followed by asynchronous differentiation of duplicated genes. differentiation of duplicated genes. NatureNature 421421: 848-860.: 848-860.

Lin, S. J., et al. 2002. Calorie restriction extends Saccharomyces cerevisiae lifespan by Lin, S. J., et al. 2002. Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. increasing respiration. NatureNature 418418: 344-356.: 344-356.

Medina, V. G., et al. 2010. Elimination of glycerol production in Anaerobic cultures of a Medina, V. G., et al. 2010. Elimination of glycerol production in Anaerobic cultures of a Saccaromyces cerevisiae Saccaromyces cerevisiae strain engineered to use acetic acid as an electron acceptor. strain engineered to use acetic acid as an electron acceptor. Applied Applied and Environmental Microbiologyand Environmental Microbiology 7676: 190-195.: 190-195.

Truckses, D. M., Lindsay S. G., and J. Thorner. 2004. Jekyll and Hyde in the microbial world. Truckses, D. M., Lindsay S. G., and J. Thorner. 2004. Jekyll and Hyde in the microbial world. ScienceScience 306306: 1509-1520.: 1509-1520.

Uptain, S. M., and S. Lindquist. 2002. Prions as protein-based genetic elements. Uptain, S. M., and S. Lindquist. 2002. Prions as protein-based genetic elements. Annual Review Annual Review of Microbiologyof Microbiology 5656: 703-741: 703-741