grds international conference on health and life (13)

SCREENING OF FLOCCULANT

SACCHAROMYCES CEREVISIAE

(NCYC-1195) FOR HIGH TOLERANCE

OF ETHANOL CONCENTRATION

Harsojo

National Nuclear Technology Agency (BATAN)

Jakarta - Indonesia

Edwin Yulian E.

Brawijaya University

Malang - Indonesia

Agustin K. Wardani

Brawijaya University

Malang - Indonesia

‘International Conference on Advance in Healthcare and Life Sciences’

8-9 November 2014, Singapore

Background



Background

Concentrations more

than 8% (v/v) :

-increasing membrane

fluidity

-interfere on cell

homeostasis and several

metabolic pathways



Increasing yeast tolerance ability in the presence of ethanol

is a necessary. Ethanol-tolerant yeast would have

enhanced ethanol productivity and yields.



Background

Mutagenesis research by Mehdikhani, P. et al. (2011) showed

that gamma ray 60Co can increase yeast tolerance on high

ethanol concentration and high temperature during

fermentation

Increasing the ethanol tolerance of yeast can be done by

mutation technique. One of the mutation technique is

random mutagenesis by gamma ray 60Co.

Its have ability to do self-floculating → easy separation →

reduce cost production.

Flocculant S. cerevisiae (NCYC 1195)



Aim of The Study

Increase the tolerance ability of

flocculant Saccharomyces cerevisiae

(NCYC 1195) to high concentration of

ethanol

Determine gamma ray 60Co dose that

give highest cell lethality to flocculant

Saccharomyces cerevisiae (NCYC

1195)

Methodology



S. cerevisiae NCYC 1195Ethanol-tolerant Mutant

Reseacrh Step



• Gamma ray treatment

• Isolation survival coloniesRadiation

• Ethanol-tolerant screening

• Isolation ethanol-tolerant mutantScreening

• Determine the viable cell count

• Determine the sugar consumption

Growth Profile test



Radiation

3 x 108 cell/ml

0 KGy

0,6 KGy

1,4 KGy

0,8 KGy

1 KGy

1,2 KGy

Incubation 24

hours at 30OC

Ethanol-tolerant Screening



Day 1 Day 2 Day 3 Day 4

0.1 ml 108 cell/ml

5 ml YPG broth

2.5 % ethanol (v/v)

Day 5



Growth Profile Test

100 ml YPG Broth medium :

10 % glucose

2 % peptone

1% yeast extract

30 OC and 120 rpm

At shaker waterbath for 72 hours

5 % inoculum



Results

Dose (KGy) Survival Colonies (CFU/ml)

0 44 x 106

0.6 26

0.8 2

1 0

1.2 0

1.4 0

Dose

(KGy)

Number of Screened

Colonies

Mutant

0.6 26 4

0.8 2 2

Table 1. Number of survival colonies

by different doses of gamma ray

26 survival colonies obtained after gamma

ray treatments at 0.6 KGy dose

2 survival colonies obtained after gamma ray

treatments at 0.8 KGy dose

No survival colonies obtained excess

1 KGy dose

1.

2.

Table 2. Number of screened colonies and

ethanol-tolerance 4 colonies (Y6a, Y6b, Y6c and Y6d) from

0.6 KGy dose that could survive in the

presence of 2.5 % (v/v) ethanol

concentration2 colonies (Y7 and Y8) from 0.8 KGy dose

that could survive in the presence of 5 %

(v/v) ethanol concentration

Mutant Y7 and Y8 show higher

tolerance of ethanol concentration than

the WT.

Mutant Y7 and Y8 = 5% (v/v) ethanol

concentration

WT = 2.5% (v/v) ethanol concentration



Growth Profile

-20

0

20

40

60

80

100

120

140

0 12 24 36 48 60 72

0

2

4

6

8

10

12

Cells (

10

6C

FU

/ml)

Time (h)

To

tal R

esid

ual

Su

gars

(g

/100m

l)

▬▲▬ Viable cell growth of Mutant Y8 ▬■▬ Viable cell growth of WT

▲ Sugar consumption of Mutant Y8 ■ Sugar consumption of Mutant Y8

The present study shows that :

1. Flocculant Saccharomyces cerevisiae (NCYC 1195) have highest

cell lethality by gamma irradiation treatments at 0.6-0.8 KGy.

2. Two mutants, Y7 and Y8, have higher ethanol tolerance (5 %

ethanol concentration (v/v)) than the wild type (2.5 % ethanol

concentration (v/v)).

3. Mutant Y8 display 15.9% higher numbers of viable cell than the

wild type.

Conclusion



In the next study, we will determine the optimum

fermentation condition for ethanol production

and investigate the stability of mutants



Future Research

Thank you foryour kind attention



grds international conference on health and life (13)

Health & Medicine

singapore ethanol

ethanol fermentation

ethanol tolerance of

ethanol tolerance ability

life sciences8

presence of ethanol

ethanol productivity

high ethanol concentration