KKEK 3171: LABORATORY AND

COMMUNICATION II
SEMESTER 1, SESSION 2014/15

PBL 13: Batch Fermentation

Lecturer:
Dr. Ang Teck Nam
Assoc. Prof. Dr. Ngoh
Group:
Noor Hanisah Abdul Hamid (KEK120038)
Ng Sau Wei
(KEK120037)
Ong Kien Yeap
(KEK120046)

CONTENTS
1.

Introduction to Fermentation

2.

Problem Statement & Objectives

3.

Literature Review

4.

Methodology

5.

Results & Analysis

6.

Conclusion

Introduction to Fermentation

Background

Application

BACKGROUND OF FERMENTATION

Ethanol / 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

Yeast cells

Glucose
NAD+

Pyruvic Acid
NADH

en.wikipedia.org/wiki/Fermentation

CO2 + Ethanol
3

Literature Review

Liquid Medium

LIQUID MEDIUM FOR

FERMENTATION

Factors for Fermentation

Yeast Extract
a form of processed yeast
products
have the same characteristic
of yeast cells

Ammonium Sulphate, (NH4)2SO4

acts as a pH buffer

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

Dipotassium phosphate, K2HPO4

Source of nutrients

Magnesium sulphate, MgSO4

Source of nutrients

Literature Review

Liquid Medium

Factors for Fermentation

FACTORS AFFECTING ALCOHOLIC

FERMENTATION
I) pH
Optimum: 4 < pH < 8
II) Inoculum Dosage
Inoculum
dosage

Rate of
consumption of
glucose

Rate of
fermentation

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

Introduction to Fermentation

Background

Application

APPLICATIONS OF FERMENTATION

Activated sludge process

Alcoholic beverages
Antibiotics and drugs
http://science.jrank.org/pages/2677/Fermentation-Uses.html

Problem Statement

Objectives

PROBLEM STATEMENTS

ACTIVATOR 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.

Problem Statement

Objectives

OBJECTIVES

To 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

Methodology

Negative Control

Actual Experiment

CO2 Test

METHODOLOGY
1.
2.
3.
4.
5.
6.

Negative Control (Problem Statement)

Actual experiment
Chemical test for carbon dioxide
Oxidation test for ethanol
Cell mass analysis
Glucose assay (DNS method)
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Methodology

Negative Control

Actual Experiment

CO2 Test

STUDENTS EXPERIMENT (NEGATIVE

CONTROL)
Yeast extract
Glucose
(NH4)2SO4
MgSO4
K2HPO4

Bakers yeast

10

Methodology

Negative Control

Actual Experiment

CO2 Test

ACTUAL EXPERIMENT
Yeast extract
Glucose
(NH4)2SO4
MgSO4
K2HPO4

Bakers yeast

11

Methodology

Negative Control

Actual Experiment

CHEMICAL TEST FOR CARBON

DIOXIDE

CO2 Test

Acidified saline
(25% NaCl, 3%
H2SO4)

12

Methodology

Oxidation Test for Ethanol

DNS assay

Cell Mass Analysis

OXIDATION TEST FOR ETHANOL

Few drops
of 0.1M
H2SO4

5ml of
K2CrO7

13

Warm

Methodology

Oxidation Test for Ethanol

DNS assay

Cell Mass Analysis

GLUCOSE ASSAY ( DNS METHOD )

Sodium
Metabisulfide
(Rochelle salts)

3mL of DNS
solution

Potassium
Sodium
Tartrate
Tetrahydrate

14

Heat in water bath

Methodology

Oxidation Test for Ethanol

DNS assay

CELL MASS ANALYSIS

1.

2.

3.

4.

Cell Mass Analysis

The 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.

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RESULTS & ANALYSIS

1.
2.
3.
4.
5.
6.

Negative Control (Problem Statement)

Actual experiment
Chemical test for carbon dioxide
Oxidation test for ethanol
Cell mass analysis
Glucose assay (DNS method)
16

Results & Analysis

Physical Observations

CO2 volume

Oxidation test for Ethanol

PHYSICAL OBSERVATIONS
Alcohol scent is detected.
Bubbles floating

Yeast is still growing

17

Results & Analysis

Physical Observations

CO2 volume

Oxidation test for Ethanol

No
significant
level change
in saline
solution!
18

Results & Analysis

Physical Observations

CO2 volume

Oxidation test for Ethanol

Oxidation Test for Ethanol

INITIAL

FINAL

19

Results & Analysis

Physical Observations

CO2 volume

Oxidation test for Ethanol

Actual
experiment
Initial
colour of
acidified
K2Cr2O7
solution

Negative
control

20

Results & Analysis

Physical Observations

CO2 volume

SPECTROMETER RESULT:
ABSORBANCE PLOT

Oxidation test for Ethanol

Run 1

Run 2

Absorbance

Absorbance

0.14

0.12

0.12

f(x) = 0.25x - 0.06

R = 0.98

0.10

0.10
0.08

0.08

f(x) = 0.18x - 0.03

R = 0.96

0.06

0.06
0.04

0.04

0.02

0.02

0.00
0.2

0.3

0.4

0.5

0.6

Glucose concentration (mg/mL)

0.7

0.8

0.00
0.2

0.3

0.4

0.5

0.6

0.7

Glucose concentration (mg/mL) 21

0.8

Results & Analysis

Glucose content

CALCULATION FOR GLUCOSE

CONCENTRATION

Cell mass analysis

Run 2

Run 1
NEGATIVE CONTROL
0.672= 0.2504*x 0.0264
x = 2.9329 mg/mL

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

NEGATIVE 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

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Results & Analysis

Glucose content

Cell mass analysis

Cell Mass Analysis

Initial mass of Bakers yeast: 1 g
Mass of dry yeast at the
end of experiment(g)
Trial 1
Trial 2

Actual

0.9798

Negative

0.3251

Actual

0.9906

Negative

0.1838
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Conclusion

CONCLUSIONS

Sequence of alcoholic fermentation is significant.

->Yeast must be added after autoclaving medium.
The qualitative and quantitative analysis were done and verified.

Presence of alcohol scent

Bubbling in saline solution
K2Cr2O7 turns from orange to green
Glucose is consumed
Increasing final mass of yeast

Fermentation
occurs!
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THANK YOU!
Q & A Session
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