Isolation and Screening of Ethanol Tolerant Yeast, for the Production of

Indigenous Liquor

P. Moopanar

CSIR Supervisor: Sani Gumede

DUT Supervisor: L. Reddy

Slide 2 © CSIR 2006 www.csir.co.za

Introduction

• Production of traditional beer and its economical impact

Figure 1: Cashew Fruit

Figure 2: Palm tree

Slide 3 © CSIR 2006 www.csir.co.za

Slide 4 © CSIR 2006 www.csir.co.za

• The focus of this research was to isolate and screen yeasts for the improvement of current production

processes of indigenous liquor.

Aim

Slide 5 © CSIR 2006 www.csir.co.za

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

Slide 6 © CSIR 2006 www.csir.co.za

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

Slide 7 © CSIR 2006 www.csir.co.za

Fig 1: KM01 Fig 2: KM02

Fig 3: KC01

Results & Discussion

Slide 8 © CSIR 2006 www.csir.co.za

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

Slide 9 © CSIR 2006 www.csir.co.za

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

Slide 10 © CSIR 2006 www.csir.co.za

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%

Slide 11 © CSIR 2006 www.csir.co.za

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

Slide 12 © CSIR 2006 www.csir.co.za

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

Slide 13 © CSIR 2006 www.csir.co.za

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

Slide 14 © CSIR 2006 www.csir.co.za

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

Slide 15 © CSIR 2006 www.csir.co.za

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

Slide 16 © CSIR 2006 www.csir.co.za

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.

Slide 17 © CSIR 2006 www.csir.co.za

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