PowerPoint Presentation

KKEK 3171: Laboratory and Communication IISemester 1, Session 2014/15Lecturer: Dr. Ang Teck NamAssoc. Prof. Dr. Ngoh

Group:Noor Hanisah Abdul Hamid (KEK120038)Ng Sau Wei (KEK120037)Ong Kien Yeap (KEK120046)PBL 13: Batch Fermentation

CONTENTSIntroduction to Fermentation

Problem Statement & Objectives

Literature Review

Methodology

Results & Analysis

Conclusion

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3Background of FermentationEthanol / Lactic acid fermentation

Metabolic process that converts sugar into acids or alcohol

Anaerobic process carried out by microorganisms

Liquid medium contains of specific nutrients for growth

GlucoseCO2 + EthanolPyruvic Acid

NAD+NADHYeast cellsIntroduction to FermentationBackgroundApplication

en.wikipedia.org/wiki/Fermentation

Liquid medium for fermentationYeast Extract a form of processed yeast products have the same characteristic of yeast cells

Glucose serves as a source of energy for growth under anaerobic condition

4Ammonium Sulphate, (NH4)2SO4 acts as a pH buffer

Magnesium sulphate, MgSO4Source of nutrients

Dipotassium phosphate, K2HPO4Source of nutrientsLiterature ReviewLiquid MediumFactors for Fermentation

Factors affecting alcoholic fermentationI) pHOptimum: 4 < pH < 8II) Inoculum Dosage

III) TemperatureOptimum: 30oC < T < 38oCBeyond 45oC: yeast cells are destroyed.Low temperature: dormant stage -> slow fermentation rate

5Literature ReviewLiquid MediumFactors for Fermentation

Applications of Fermentation6Introduction to FermentationBackgroundApplication

http://science.jrank.org/pages/2677/Fermentation-Uses.html

Alcoholic beverages

Antibiotics and drugs

Activated sludge process

PROBLEM STATEMENTSACTIVATOR OF FERMENTATION: Yeast -> convert glucose in liquid medium to ethanol.

PROBLEM: Yeast suspension added to medium PRIOR to autoclaving.

OBSERVATION: No yeast growth and alcohol production.

HYPOTHESIS: The high temperature & high pressure autoclave treatment destroyed the yeast cells.7Problem StatementObjectives

ObjectivesTo investigate the correct sequence of alcoholic fermentation by Bakers yeast.

To verify the process of alcoholic fermentation by conducting a series of qualitative and quantitative analyses

8Problem StatementObjectives

METHODOLOGYNegative Control (Problem Statement)Actual experimentChemical test for carbon dioxide Oxidation test for ethanolCell mass analysisGlucose assay (DNS method)9MethodologyNegative ControlActual ExperimentCO2 Test

Students experiment (negative control)10MethodologyNegative ControlActual ExperimentCO2 Test

Yeast extractGlucose(NH4)2SO4 MgSO4K2HPO4

Bakers yeast

11MethodologyNegative ControlActual ExperimentCO2 Test

Yeast extractGlucose(NH4)2SO4 MgSO4K2HPO4

Bakers yeast

Actual Experiment

Chemical test for carbon dioxide12MethodologyNegative ControlActual ExperimentCO2 Test

Acidified saline(25% NaCl, 3% H2SO4)

5ml of K2CrO7Few drops of 0.1M H2SO4

Warm13MethodologyOxidation Test for EthanolDNS assayCell Mass Analysis

Oxidation Test for ethanol

Heat in water bath

3mL of DNS solutionSodium Metabisulfide(Rochelle salts)Potassium Sodium Tartrate Tetrahydrate14MethodologyOxidation Test for EthanolDNS assayCell Mass Analysis

Glucose Assay ( DNS Method )

Cell Mass AnalysisThe dried filter paper is weighed.

The fermented mixture is filtered using Buchner funnel.

After filtration, the filtrate is dried together with the filter paper for 24hours.

The final weight is measured.15

MethodologyOxidation Test for EthanolDNS assayCell Mass Analysis

16RESULTS & ANALYSISNegative Control (Problem Statement)Actual experimentChemical test for carbon dioxide Oxidation test for ethanolCell mass analysisGlucose assay (DNS method)

Physical ObservationsAlcohol scent is detected.Bubbles floating Yeast is still growing 17Results & AnalysisPhysical ObservationsCO2 volumeOxidation test for Ethanol

18No significant level change in saline solution!Results & AnalysisPhysical ObservationsCO2 volumeOxidation test for Ethanol

Oxidation Test for EthanolINITIALFINAL19Results & AnalysisPhysical ObservationsCO2 volumeOxidation test for Ethanol

Initial colour of acidified K2Cr2O7solution20Negative controlActual experimentResults & AnalysisPhysical ObservationsCO2 volumeOxidation test for Ethanol

Spectrometer result: Absorbance plot 21Run 1Run 2Results & AnalysisPhysical ObservationsCO2 volumeOxidation test for Ethanol

Calculation for glucose concentration22NEGATIVE CONTROL0.672= 0.2504*x 0.0264 x = 2.9329 mg/mL

ACTUAL EXPERIMENT 0.604 = 0.2504*x 0.0264 x = 2.6613 mg/mL

Results & AnalysisGlucose contentCell mass analysis

Run 1NEGATIVE CONTROL 0.462=0.1752*x-0.0346 x = 2.8345 mg/mL x(negative)= 10* 2.8345 mg/mL = 28.345 mg/mL

ACTUAL EXPERIMENT 0.022=0.1752*x-0.0346 x = 0.3231 mg/mL

x(actual)= 10*0.3231 mg/mL = 3.231 mg/mL

Run 2

Mass of dry yeast at the end of experiment(g)Trial 1Actual0.9798Negative0.3251Trial 2Actual0.9906Negative0.1838

Initial mass of Bakers yeast: 1 g23Cell Mass AnalysisResults & AnalysisGlucose contentCell mass analysis

CONCLUSIONSSequence of alcoholic fermentation is significant.->Yeast must be added after autoclaving medium.

The qualitative and quantitative analysis were done and verified. Presence of alcohol scentBubbling in saline solutionK2Cr2O7 turns from orange to greenGlucose is consumedIncreasing final mass of yeast 24

Fermentation occurs!Conclusion

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25Q & A SessionTHANK YOU!