MICROBIAL PRODUCTION OF ETHANOL FROM CELLULOSE

Abstract

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3 strains of fungi & 4 strains of bacteria that showed cellulolytic activity were isolated froftlsoil & Ii strains of ba.cteria were isolated aerobically fran cowdung. 18 strains of bacteria were isolated anaerobically from the rumencontents of cattle. Majorpart of this investigation was carried out with anaerobes isolated fran the rumencontents. The cellulosic substrate used wasmicrocrystalline cellulose (Bee), which is the crystalline portion of cellulose. Dueto its insoluble nature bowever, carboxymethylcellulose (CMC)had to be substituted as the sole carbon source for sane of the growthexperiments. The applicability of most of the growth studies wastested usinq -a strain of Ruminococcusalbus which is knosn to require strict anaerobic conditions for growthand is excellent in cellulose digestibility & the production of ethanol. The cellulolytic ability of the strains isolated fran roil was tested by visual observation of cellulose degradation that included filter paper decay & the formation of cleared zones in agar medium& by the measuranentof total carbohydrate content of t.hemediumby the phenolsulphuric acid colourimetric test. The irolates fran cowdungwere tested by the phenol-sulphuric acid test & chanical analysis of the culture broth. The strains isolated fran the rumencontents were tested by the phenol sulphuric acid test. Althoughisolated fran cultures that showed cellulolytic actvity only two soil isolates showed clearing on agar medium. Four strains showedsignificant cellulolytic activity by the phenol-sulphuric acid test. Three irolates fran cow dung showed filter paper activity while gas production was observed by only one isola te. Of the isolates fran the rumen contents, only nine strains showedsignificant cellulolytic activity by the phenol - sulphuric acid test. For analytical studies, the anaerobic condition of the mediumwas obtained by culturing yeast in the medium, prior to inoculation of the anaerobes. Experiments carried out using R. albus, & twJ isolates indicated that the presence of yeast in the rned.i.urndid not affect growth & cellulose digestibility of the anaerobes. Chemical analysis of the distillate of the culture broth of R. albus indicated positive results for ethanol & sugars. Quantitative analysis of ethanol was done by gas liquid chrcmatcqraphy. Sugars were separated by paper chromatography with isoproPanol-pyridine-acetic acid - water & glucose was quan~itatively analysed by the phenolsulphuric acid test. 9.2 mg. ethanol per 100 ml. broth was produced by R. albus in 1% 0'1: mediumat 10 day incubation at 37 'C. The production of sugars increased upto a maximumof 30 mg. per 10) ml. broth at 6 days & decreased thereafter. Cell growth assessed by optical density measurements of the culture showed a maximumat 6 days. The maximumarnounts of ethanol & sugars produced fran 1% cellulose broth were 1.75 mg. & 15.48 mg. per 100 mi. broth respectively. The effect of sane envirorrnental factors on the product.Lon of ethanol was studied using cultures of R.albus. Of these factors, rraintenance of pH of the broth at 6.8, incubation under CO2 gas & seed cultivation of bacteria in 0.03% cellobiose broth favoured production, while incubation at room temperature with shaking reduced production.A study of growth curves of the isolates sbowed a harvest time of 10 days as suitable for obtaining samples for analysis for ethanol. The pattern of pH variation of each culture studied with the pattern of cell growth, indicates the enzymic activity of the relevant isolate. The negligible arrounts of ethanol produced by the isolates from cellulose have increased under favourable circumstances. Four isolates yielded ethanol fran eM:::: while three others have yielded ethanol from cellulose. Glucose was detected in the culture broth of tv.o isolates only. Cello1!liose was detected in the cultures of four isolates. An unidentified trimer was producer by all isolates except one. The production of ethanol was increased by mixed cultures, carried out using the canbinations of ethanol producers with non-ethanol producers. A further increase in the production was observed using combinations of ethanol producers, coupled with the Cl & ex components. 56 mg. of ethanol Per 100 mI. broth, the highest arrount obtained in this study was produced by the combination of R. albus & an isolate. Al.t.bouqhrrost of the canbinations favoured production one combination did not produce ethanol. However, the cell yields & the production of ethanol & sugars obtained in this study are fairly low1 partly due to the nature of the cellulosic substrate used. Hence, the resul ts obtained are rrore of an acadanic interest rather than of econanic value.