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Biochemical and microbial changes that occur during
fermentation of Caryota urens phloem sap were studied with
a view to upgrading the local fermentation industry and to
developing new biotechnological process by recognizing
yeasts suitable to local conditions.
First sample of sap which was taken after keeping the
sterilized pot for 10 hours, was apparently unfermented.
Paper chromatographic studies done with unfermented sap
revealed that the main type of sugar was sucrose, while
glucose and fructose were in trace amounts. The content of
reducing sugar of unfermented measured by Lane and r
sap,
Eyon method, was very low (0.14 '*0, w/v) . However this
started to increase after 24 hours and reached a maximum
value (4.1 '*0, w/v) after 24 hours. The alcohol
concentration, measured by ebulliometer, increased only
after appreciable quantities of reducing sugar was formed.
Maximum alcohol concentration of 7 %, v/v was obtained on
the fifth day. The unfermented sap had apH around 7. This
decreased substantially and reached a constant value around
4 after the fourth day of fermentation. Decrease in pH
coincided well with increase of acidity. Microbial analysis of fermenting sap revealed that the type
of organisms present in the sap changed with time. At the
beginning of fermentation the sap contained mainly bacteria
with a colony count in the order of 107
cells/ml. The yeast
cell number increased after 24 hours and reached a maximum
of 8 x 107
cells/ml after third day. During the later
stages of fermentation, there was a drop in the viable
yeast cell count, while the bacteria count increased again
to 106
cells/ml.
A total of eleven yeast strains were isolated from
fermenting phloem sap,some of these were identified as
species of Candida, and species of Saccharomyces. A strain
which fermented inulin , isolated from fermenting sap and
it's identification was not possible. This may be a new
strain that has not been reported earlier.
These yeast were examined to determine their suitabili ty
for high temperature ethanol fermentation and for single
cell protein production.
When the yeast isolates were screened for their ability to
ferment sucrose to ethanol at 40°C only five strains gave
substantial yields, (ie more than 50 % of the theoretical
maximum). Out of these, three strains were selected for
further studies. For the purpose of selecting the best
strain for alcohol fermentation at 40°C , certain parameters of these strains were checked. These strains had
different growth rates at 40°C when checked using a
complete synthetic medium. Strain S 2-5 had the highest
growth (2.2 x 106
cells/ml/h) while Sll F-3 had the
lowest growth rate (1 x 106
cells/ml/h) . Lower growth rate
would favour high ethanol production and hence, Sll F-3
seems to be better in this aspect. When fermentation tests
done with these three strains (S 2-5, Sll F-3, S5 MB-29)
at different temperatures, 30, 35 and 40°C in complete
synthetic medium with 20 % sucrose, the highest alcohol
concentration of 14.2 %, v/v was obtained with strain
Sll F-3 at 40°C. At 35 °c, all three strains gave more than
9 %, v/v alcohol. At 40°C strain Sll F-3 performed better,
giving an alcohol yield of around 9.2 %, vivo With increase
of temperature from ,30 -c to 40°C, the ethanol yield of
strain Sll F-3 decreased by about 35 % while the strain S5
MB-29 and S 2-5 decreased about 36.5 % and 40 %
respectively. When the temperature was increased from 35°C
to 40 °c the decreased in ethanol yield was only about 10
% in the case of strain Sll F-3. With the molasses medium,
at 40°C, Sll F-3 gave highest alcohol yield of 8.4 %. This
value is very much higher than the value obtained with
Baker's yeast when used under the same conditions ( 4.7 %)
Maximum CO2 productivity (g/h), final ethanol concentration
(%,v/v), yield efficiency [(g ethanol/g glucose) / 0.511]
anu~axi~umcell growth rate (No.of cells/ml/h) were used as selection criteria of thermotolerant yeast. The strain
Sll F-3 which gave highest alcohol yield gave higher values
with all the above parameters except growth rate.
Four yeast strains which were unable to give high alcohol
yields in fermentation experiments were tested for their
nitrogen contents and higher growth rates were selected for
further studies (85 M-15 and 88 M-15). Their optimum growth
temperature was around 35°C. The moisture contents were in
the range of 6.5-7.3 %, while the ash contents were around
7 % on dry weight basis. Two strains showed considerably
good growth in molasses medium and produced about 4 g cell
dry weight/lOa ml of molasses. Amino acid composition of
two strains were determined using HPLC method (Pico-Tag)
and strain 88 M-15 consisted of 82 % of essential amino
acids, while strain (85 M-15 consisted of 70 % essential
amino acids from the total amino acid content of each of
the strains. Thiamine and riboflavin contents were analyzed
by HPLC method (Hagg) and two strains contained about 0.2
mg riboflavin and 0.1 mg thiamine per gram of dried cells.
The results suggest that changes during natural
fermentation of Caryota urens phloem sap is very much
similar to coconut sap fermentation and among the isolated
strains, strain 811 F-3 could be regarded as the best
strain for production of alcohol at higher temperature
while strains 85 M-15 and 88 M-15 which could be grown more
