2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
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B. Microorganism and medium
The strain used for bio-ethanol production was sake
brewing strain,
Saccharomyces cerevisiae
Kyokai no.7
(ATCC 26422) obtained from American Type Culture
Collection and baker’s yeast
Saccharomyces cerevisiae
obtained from Mauri Sdn Bhd. The strains were
maintained at 4
o
C and sub-cultured every month on
YPD medium agar (g/L): glucose (dextrose) 10, peptone
5, yeast extracts 5, agar 20.
C. Inoculums preparation
Stock cultures were prepared by transferring
S.
cerevisiae
Kyokai no.7 or baker’s yeast
S. cerevisiae
into 500 mL Erlenmeyer flask containing 250 mL of
medium (NaCl 5 g/L, peptone 5 g/L, beef extract 1.5
g/L and yeast extracts 1.5 g/L). Strain was incubated for
12- 18 h at 30
o
C and 180 rpm until reached standard
initial concentration 0.34-0.35 g/L (turbidimetry by
OD= 3.6
–
3.7) determined at 600 nm.
D. Diffferent strains fermentation
After the initial both strains have been standardized,
25 ml of inoculums (10% v/v) was transferred into a
500 ml Erlenmeyer flask containing 225 ml of oil palm
trunks sap. Fermentation of sap was run for 48 hours at
conditions 30
o
C and 180 rpm. Samples were collected
and analyzed at 0, 24 and 48 h.
E. Sterilization test
Sterilized medium was prepared by autoclaving sap
at 121
o
C for 15 min and let it cooled down before use.
For non-sterilized medium, sap was directly used after
filtration with 9.0 μm filter. Fermentations were carried
out by transferring 10% v/v of yeast (use the best strain
based on result from previous experiment)
into a 500 ml
Erlenmeyer flask containing 225 ml of palm oil trunks
sap. All flasks were run at conditions 30
o
C and 180 rpm
for 48 hours. Fermentation samples were collected and
analyzed at 0, 24 and 48 h.
F. Analysis
The fermentation broth was centrifuged at 10,000
rpm for 5 minutes to separate supernatant from pellet.
The supernatant was removed and stored at -20
o
C for
ethanol and sugar analysis. Pellet were rinsed twice and
then dissolved into distilled water and read at 600 nm
(UV-Visible spectrophotometer). Distilled water was
used as blank. Absorbance values were converted into
biomass concentration by using standard curve obtained
from absorbance versus cell dry weight calibration y =
10.582x
–
0.0058, where Y is biomass (g-dry
biomass/L) and X is absorbance at 600 nm. For cell dry
weight determination, pellets were dried at 60
o
C until
constant weight.
Ethanol concentrations were determined by a gas
chromatograph (HP Agilent) equipped with a flame
ionization detector (FID) and Innowax column (30 m x
12 mm ID x 2 μm film thickness). Temperature of
injector and detector were set at 150
o
C and 200
o
C,
respectively. The carrier gas was nitrogen at a flow rate
of 15 ml/min. Ethanol and n-propanol is used as
external and internal standard for quantitative analysis.
Total sugars were determined by DNS method
(absorbance at 540 nm) and HPLC.
III. R
ESULTS AND DISCUSSION
A. Effects of different strains on bio-ethanol production
The total initial sugar (expressed as total glucose
concentration) in the sap was 14.6- 25.0 g/L. Analysis
by High Performance Liquid Chromatography (HPLC)
showed that sap contained sucrose as the main sugar
and also modest amount of glucose and fructose. Since
fructose content was not detected by HPLC after 48 h of
fermentation, we can conclude that it is fully consumed
by yeast.
Both strain performances were compared based on
maximum ethanol concentration and percentage yields
obtained since initial concentrations of sugar have been
fixed. Fig. 1 showed the fermentation profile of
S.
cerevisiae
Kyokai no.7 and baker’s yeast
S. cerevisiae
in
the sap for 48 h.
Fig. 1. Fermentation Profile of
S. cerevisiae
Kyokai no.7 and
baker’s yeast
S.
cerevisiae in 48 hours. Blue lines represent result for
S. cerevisiae
Kyokai no.7 and b
lack lines represent for baker’s yeast
S.
cerevisiae
.
It was clearly shown that
S. cerevisiae
Kyokai no.7
was able to produce more ethanol compared to
baker’s
yeast
S. cerevisiae
and has higher productivity (Fig 1
and Fig 2).
S. cerevisiae
Kyokai no.7 able to convert
88.9-98.9% of sap sugar to ethanol compared to only
58.5-
63.8% for baker’s yeast
Saccharomyces cerevisiae.
This might due to the ability of
S. cerevisiae
Kyokai
no.7 to adapt and growth fast in sap.
