INTRODUCTION:

Sonochemistry refers to the field of chemistry where chemical reactions are induced by sound (hence sono) and are driven by acoustic cavitation.

The conversion of wood or agricultural residues to ethanol and industrial chemicals is an attractive option for utilizing all major components of biomass to produce a liquid automotive fuel and for environmental remediation^1-4. The rising need for ethanol as a universal energy source has stimulated worldwide investigations, not only with respect to high ethanol yielding strains, but also use of cheaper raw materials⁵. Apart from fermentation of carbohydrates, alcohol can also be produced industrially by the hydration of ethylene, which is a product of petroleum industry. Because petroleum is a non-renewable resource, studies have recently been focused on getting alcohol through renewable resources such as agricultural sources^6-8.

To make the fermentation method a cost effective and to meet the great demand for ethanol in the present situation of energy crisis, research has been directed in two areas recently, namely, the production of ethanol from comparatively cheaper source of raw materials and to study the new microorganism or yeast strains efficient for ethanol fermentation⁹.

In this respect, inexpensive raw materials like by-products or wastes, such as, molasses, agricultural wastes, cellulose wastes, fruit wastes, vegetable wastes, municipal and industrial wastes^10-16 have been utilized to produce ethanol cheaply.

Ethanol production by fermentation faces competition with ethanol production from petroleum-based products as feedstock. But with the increasing value of these petrochemical feed stocks, fermentation of ethanol is bound to receive more attention (Ahmeh et al. , 1988). In this regard, use of renewable materials would be more economical, since they are cheaper and easily available.

The aim of this investigation is, therefore, to study the kinetics of ethanol fermentation of grape wastes by Saccharomyces cerevisiae yeast strain isolated from toddy. The use of yeast strain developed from toddy is also significant, as it has shown good fermentation ability for the conversion of sugar to ethanol in this study¹⁷. The effects of various fermentation variables like pH, temperature and amount of fruit waste on the ethanol fermentation have also been investigated

MATERIALS AND METHODS:

Organism and Culture media:

The wild type of Saccharomyces cerevisiae yeast procured from Microbial culture collection Institute, Chandigarh, India was used in the present investigation. Saccharomyces cerevisiae yeast strain developed from toddy was used in this study. The culture was maintained on agar slants containing 1% glucose, 0. 5% peptone, 0. 3% beef extract, 3% malt extract and 2% agar-agar at a pH of 4. 25 and was stored at 4 ⁰C.

Apparatus and procedure:

Fermentation experiments were performed using ultrasonic bath at a frequency of 22. 5 KHz and a nominal power of 120 Watt (ULTRASONICS LABLINE CL 500). The bath had dimensions 15 × 15 ×15 cm³.

Preparation of inocula for fermentation:

The fruit waste was obtained from the farmers, fruit shops and market. For the preparation of yeast inoculum, 100 ml water was added to 100 gm macerated fruit waste, filtered through filter cloth. Further the P^H of the solution was maintained at 4. 25 and was subjected to sterilization in an autoclave for 20 minutes at 20 Psi pressure to kill undesirable microorganisms. The solution was cooled to room temp and then colonies of the yeast were introduced in the broth. After inoculating, the culture was kept for growth in an incubator at 30⁰C with agitator speed of 110 rpm at different time intervals.

Experimental setup and fermentation procedure:

The fruit waste was macerated to small pieces and 100 ml of distilled water was added to each batch. Fermentation experiments were performed in a 1 liter capacity glass reactor equipped with a ultrasound bath, digital P^H meter and temperature control system. The fermenter was cleaned and steam sterilized in an autoclave at 15 psi for 15 minutes. Then the sterilized medium containing fruit waste was transferred to the fermenter and 50 ml of yeast inoculum was added to the broth. The temp of fermentation was maintained within 0. 5ºC by the digital temperature control system. The P^H of the fermentation broth was automatically regulated within 0. 1 P^H units with the help of the digital P^H controller by injecting a fine stream of sulfuric acid or sodium hydroxide through peristaltic pump. The fermentation experiments were carried out under anaerobic conditions. Fermented samples were withdrawn periodically and analyzed for determining the ethanol concentration.

Measurement of ethanol concentration:

The concentration of ethanol was monitored Spectrophotometrically using UV-Vis spectrophotometer, Shimadzu make, and model UV-1650PC, and by measuring optical density at 600 nm.

RESULTS AND DISCUSSION:

In the present study, experiments were carried out at different P^Hvalues, 3. 5, 3. 75, 4 and 4. 5 to evaluate the efficiency of the Saccharomyces cerevisiae yeast strain on ethanol production. The temperature of 35 ⁰C and the amount of fruit waste of 200 gms was kept constant throughout the experiments. The experimental results for the variation of P^Hon ethanol concentration as a function of time are shown in fig. 1

As shown in fig. 2, the corresponding value of ethanol productivity at P^H 4 is 0. 69 g/lh, which is quite higher than the productivity of 0. 58 and 0. 6 g/lh obtained at P^H 3. 5 to 3.75 because the P^H values are too low to activate the enzymes to react in the latter case. However, further increase in P^H did not show any improvement in activity of the biocatalyst.

The experimental results are shown in figs. 3 and 4. From fig. 3, it is observed that ethanol concentration achieved at 30ºC and 35ºC are comparable, the values of which are 6. 5 and 7. 0 V% respectively.

The yield data as shown in fig. 4 indicates that there was no remarkable effect of temperature on ethanol yield. However, the maximum ethanol productivity is 0. 7 g/lh and 0. 4 g/g ethanol yield.

Further, it is important to establish the limit of tolerance of the yeast to ethanol and high sugar concentration. These were tested by conducting fermentation experiments with varying amount of fruit waste of 100, 150, 200, and 250 gms at 35ºC. The P^H of the slurry was adjusted at 4. 0 by adding H₂SO₄. In each batch of fermentation, 100 ml distilled water was added to maintain the different initial concentration and its effect on the ethanol concentration as a function of time is shown in fig. 5

While higher amount of fruit waste (250 gms) containing higher sugar content was fermented, the rate of ethanol production as shown in fig 6. was observed to be quite low requiring more time for complete fermentation, though high ethanol concentration could be achieved.

The ethanol yield for different amount of fruit waste calculated, based on the sugar consumed is shown in fig. 6. No remarkable effect of the amount of fruit waste on the yield of ethanol was observed. The ethanol yield at varying amount of fruit waste was found to be in the range 0.3 to 0.4.

CONCLUSION:

Ethanol can be produced efficiently by slurry fermentation of the fruit waste using SC yeast. The SC yeast strain was found to be efficient to ferment at least 200 gm of fruit waste without any difficulty, producing 7.0% of ethanol with a yield of 0. 4 gm ethanol/gm sugar consumed.

Sonication substantially reduces the fermentation time. Cavitation effect is responsible for improvement in fermentation rate drastically. It seems that the diffusion resistance is overcome due to sonication.

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