Optimization of fermentation parameters

were set up by taking 50ml of YEPD. These were inoculated with 5×10 8 cell/ml,

6×10 8 cells/ml, 7×10 8 cells/ml, 8×10 8 cells/ml and 9×10 8 cells/ml. For glucose

concentration, the above medium was modified by taking 5, 10, 15, 20 and 25% of

dextrose for RPDPMP whereas 5, 10, 15, 20, 25 and 30% of dextrose was used for

R3DSC5 with an initial pH 5 (Ekunsanmi and Odunfa, 1990). For studies on the

effect of initial pH, the above medium having 20% dextrose and initial pH values

3, 5, 7 and 9 were used for both the strains. For temperature studies, the above

medium having 20% dextrose for R3DPMP strain and 30% glucose for R3DSC5

strain with initial pH 5 was taken into separate flasks and incubated at 30, 35, 40

and 45°C temperatures. Ethanol concentration in the samples was determined by

Gas Chromatography (Agilent make GC equipped with flame ionization detector).

The fermented samples were diluted with Dimethyl Sulfoxide (DMSO) at a

concentration of 40mg/ml and analyzed using Agilent DB-624 column (length

30mts, film thickness 3µm, and diameter 0.53). The sample was introduced in to

the column through head space technique. The oven was programmed to hold at

40°C for 12 min followed by gradual and uniform increase from 40°C to 220°C

at the rate of 30°C per minute and hold for 5 min. Injector temperature was

maintained at 180°C and detector temperature at 240°C. The carrier gas (Helium)

was swept through the column with a flow rate of 3ml/min. Glucose concentration

was estimated by DNS method (Miller, 1959).

Results and Discussion

Data on ethanol production and maximum growth noticed for different parameters

are presented in Table 1. Significant variations were noticed with respect to

glucose concentration and temperature and therefore, graphs (Figs. 1-4) for these

two parameters are included for both the strains.

Effect of inoculum density

For R3DPMP, maximum growth was observed at 24 hours and a maximum cell

number of 532.2×10 5 cells/ml was observed with the highest inoculum density of

158.8×10 5 cells/ml. Ethanol production was found to increase proportionately with

inoculum level reaching peak ethanol concentration at 158×10 5 cells/ml inoculum.

Peak accumulation of ethanol was also observed in less time with increase in

inoculum densities. Therefore, it was planned to carry out ethanol production with

higher inoculum densities and the results indicated that in general, increasing the

initial inoculum density resulted in increased growth and ethanol production for

both the strains. Simultaneously, for R3DPMP, glucose levels decreased to less than

1% at 9 hours in flasks with 7, 8 and 9× 10 8 cells/ml of inocula and for R3DSC5,

glucose was almost exhausted at all inoculum densities by 48 hours. Moreover,

unutilized glucose in all the flasks was less than 0.5% at the end of fermentation

and this is important because an efficient strain should be able to utilize most of the

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