Isolation and Screening of Ethanol Tolerant Yeast, for the Production of
Indigenous Liquor
P. Moopanar
CSIR Supervisor: Sani Gumede
DUT Supervisor: L. Reddy
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Introduction
• Production of traditional beer and its economical impact
Figure 1: Cashew Fruit
Figure 2: Palm tree
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• The focus of this research was to isolate and screen yeasts for the improvement of current production
processes of indigenous liquor.
Aim
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Objectives
• To isolate ethanol tolerant yeasts from traditional juices
• To test ethanol tolerance of the isolated yeast strains
• To prepare and validate a cell bank of the isolated yeasts to minimise process variation
• To determine the growth profile of the yeast isolates.
• Establishing the efficacy of combinations of selected isolates in marula juice fermentation, to produce the highest concentration of ethanol
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Isolation and selection of high ethanol producing
yeasts from Marula, Cashew, & Palm
Isolates were evaluated for ethanol tolerance at a range
of concentrations
Methodology
Shake flask experiments were conducted in
triplicate to determine growth profiles
Purified isolates were cell banked for future
evaluations, the cell bank was subsequently validated
Combinations of the isolates were screened for
ethanol productivity in marula juice
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Fig 1: KM01 Fig 2: KM02
Fig 3: KC01
Results & Discussion
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Cell Growth during ethanol tolerance trial (9%)
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
KM02 KM01 KC01
CFU
/ml
Initial cell counts Cell counts at 9% (v/v) ethanol
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Cell Growth during ethanol tolerance trial (12%)
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
KM02 KM01 KC01
CFU
/ml
Initial cell counts cell counts at 12% (v/v) ethanol
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Cryopreservation
Yeast isolate Average counts before Average counts in cryovials after cryopreservation (cfu.ml-1)
% cell recovery
cryopreservation
KC01 2.47 * 108 1.55 *108 91.50%
KM01 7.205 *107 6.23 *107 97.50%
KM02 1.113*108 1.63*108 95.50%
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Growth curve determination
0
1
2
3
4
5
6
0 5 10 15 20Culture age (hours)
Opt
ical
den
sity
@ 6
60nm
KC01 KM01 KM02
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Ethanol productivity rates
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
ABC AB AC BC
Eth
anol
Pro
duct
ivity
g/l/
h
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Sugar consumption rates
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2su
gar
cons
umpt
ion
rate
g/l/
h
ABC AB AC BC
Glucose consumption rate Fructose consumption rate
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pH profile of yeast combinations
2.8
2.88
2.96
3.04
3.12
3.2
3.28
3.36
3.44
3.52
0.00 10.00 20.00 30.00 40.00 50.00 60.00age (hrs)
pH
ABC AC BC AB
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Conclusion
• Successful isolation of 3 ethanol tolerant yeasts, KM01,
KM02 and KC01
• Combination of three isolates was most productive in
terms of ethanol productivity rates, sugar utilization rates
and the final ethanol concentration obtained
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Proposed Future Work
• Pathogenicity evaluation
• Ethanol productivity of individual isolates
• Lyophilisation
• Impact of starter culture on taste, texture and end product
• Final selection and identification of organism/s
• Productivity of starter culture in large scale application.
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CSIR Biosciences and the Bioprocess development Team, especially Sani Gumede, Zinhle Ngubane and Nodumo Zulu for their intellectual input and all the individuals who aided in the success of the project.
Acknowledgements