S.Cerevisiae Fermentation in Shake Flask Experiment

1. BIOTECHNOLOGY
ENGINEERING LAB IV
BTE3211
I, 2017/2018
Mohammad Firdaus Bin Abu Bakar 1416451
Mohd Haffizi Bin Hasni 1421995
Abdul Hadi Bin Annuar 1412769
Abdullah Umar Bin Shahrin 1414575
Raja Muhammad Norfarres
bin Raja Nordin 1413989
Osama Abdelhadi 1314409
S.cerevisiae Fermentation in
Shake Flask

2. Introduction
Shake Flask
 Easy to manage
 Preparation of Culture

3. Introduction
Saccharomyces cerevisiae
 fungi (Feldmann, 2010)
 withstand a wide range of pH(Ortiz-Muñiz, 2010)
 withstand ethanol far better (Prasertwasu et al., 2014)

4. Introduction
Fermentation (Moran et al., 2014)
 C6H12O6 + O2  H2O + CO2 + Energy+ biomass
 C6H12O6  CO2+ C2H6O +Energy
 Ethanol as a Secondary metabolite

5. Introduction
Media (Eddleman, 1999)
 20 g/L Peptone
source of nitrogen
 10 g/L yeast extract
Source of nutrient
 50 g/L Glucose

6. Objectives
 To study growth kinetics of baker’s yeast
 To familiarise with batch culturing in flask
 To practice proper aseptic techniques

7. Procedure
Yeast Culture
0.2% w/w
yeast added
into media
Shake
for 90
min
Every 15
min
sample
taken for
analysis

8. Procedure
Analysis I
Optical density
measure at
620nm
Media use as
blank
Graph is plotted

9. Procedure
Analysis II
Boat from
aluminium
foil prepared
Dry
at
80C
Centrifuge
sample (1mL,
10min,
10000rpm)
Supernatant
removed, pellet
resuspended
Content
poured in
boat, dry
overnight at
80C, weight
the boat

10. Results
Boat time
(min)
Absorban
ce
transmittance (%) weight before drying (g) Weight after drying (g) dry cell weight (g)
0a 15 0.552 28.1 0.7152 0.7153 0.0001
0b 15 0.594 28.4 0.8186 0.8194 0.0008
1a 30 0.383 41.4 0.5357 0.5375 0.0018
1b 30 0.384 41.3 0.6624 0.6641 0.0017
2a 45 0.274 53.2 0.4388 0.4405 0.0017
2b 45 0.286 51.7 0.8199 0.8215 0.0016
3a 60 0.469 33.9 0.4119 0.4131 0.0012
3b 60 0.306 49.4 0.5418 0.544 0.0022
4a 75 0.391 40.7 0.53 0.5312 0.0012
4b 75 0.469 33.9 0.8077 0.8112 0.0035
5a 90 0.496 31.9 0.6884 0.6902 0.0018
5b 90 0.717 19.2 0.7512 0.7566 0.0054
6a 105 0.8 15.9 0.7435 0.7456 0.0021
6b 105 1.169 6.78 0.6101 0.6125 0.0024

11. Results
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 20 40 60 80 100 120
Absorbance Vs time
A
B

12. Results
0
0.001
0.002
0.003
0.004
0.005
0.006
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Cell dry weight vs absorbance
a
b

13. Results
0
0.001
0.002
0.003
0.004
0.005
0.006
0 20 40 60 80 100 120
Cell dry weight vs time
a
b

14. DISCUSSION
Hypothesis
 As number of cells increase the absorbance should increase
Error
o Use of the same micropipette tip
o The yeast and media are not entirely homogenized
o Contamination in certain vials due to poor aseptic techniques
o Error in weighing the boats
o Some sample spilled during resuspension
o The wrong cuvette may had been used as a blank

15. CONCLUSION
 Practice the aseptic techniques
 Observe the growth of yeast through optical density
 measure the dry weight of the cell
 precaution steps must be taken