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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
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
Background
Concentrations more
than 8% (v/v) :
-increasing membrane
fluidity
-interfere on cell
homeostasis and several
metabolic pathways
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
Increasing yeast tolerance ability in the presence of ethanol
is a necessary. Ethanol-tolerant yeast would have
enhanced ethanol productivity and yields.
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
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)
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
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
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
S. cerevisiae NCYC 1195Ethanol-tolerant Mutant
Reseacrh Step
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
• 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
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
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
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
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
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
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
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
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
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
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
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
In the next study, we will determine the optimum
fermentation condition for ethanol production
and investigate the stability of mutants
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore
Future Research
Thank you foryour kind attention
‘International Conference on Advance in Healthcare and Life Sciences’
8-9 November 2014, Singapore