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Direct microbiological conversion of cellulosic biomass to fuel ethanol by a simultaneous saccharification/fermentation process using thermophilic anaerobic bacteria

Kirumira, Abdullah K. (1989) Direct microbiological conversion of cellulosic biomass to fuel ethanol by a simultaneous saccharification/fermentation process using thermophilic anaerobic bacteria. PhD thesis, Murdoch University.

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The major objective of this thesis was to investigate and improve a direct microbial fermentation process, using the thermophilic anaerobic bacteria Clostridium thermocellum and Clostridium thermohvdrosulfuricum for the production of fuel ethanol in economically significant concentrations, from cellulose and hemicellulose contained in renewable biomass. Two model substrates representative of readily available lignocellulosic materials were selected for this study. These were wheat straw, which is the largest single biomass resource available in Australia, and waste paper, which is a major component of municipal solid wastes.

At the onset of the research, proximate analyses of the composition of the two substrates were carried out to assess the potential yield of ethanol. Based on the total carbohydrate (hexose as well as pentose sugars) content of the materials, a theoretical potential yield of ethanol over 500 litres per dry tonne of biomass was estimated for both substrates. The feasibility of effecting biomass conversion to ethanol by direct fermentation of the substrates was then examined.

Fermentation characteristics of 9 strains of the potent cellulolytic anaerobe, C. thermocelIum and 5 strains of the saccharolytic ethanoloaen. C. thermohvdrosulfuricum. were investigated on a range of sugars. Cellulose hydrolysis and fermentation by C. thermocellum strains were also studied with alpha-cellulose and the two model substrates. Variations in growth characteristics, extent and rates of substrate utilization, as well as in the stoichiometry of product formation were noted, not only between the two species, but also among the different strains. A stable coculture comprising the most potent strains of the two species could be established, and this culture efficiently fermented crystalline and native cellulosic substrates to produce ethanol at substantially higher yields than could be achieved with cultures of C. thermocellum alone. At 1% (w/v) concentration of wheat straw and newspaper, ethanol yield amounting to 70% of theoretical was obtained with the coculture, compared to 25% of theoretical yield exhibited bv C. thermocellum. The metabolic basis for the enhanced fermentation effectiveness of the coculture system has been discussed.

The feasibility of attaining higher ethanol concentrations in the fermentations was investigated next by employing increased substrate concentrations in batch as well as fed-batch mode of operation. The bioconversion efficiency was observed to systematically decrease with increased substrate concentration, and a limiting ethanol concentration for the cocultures appeared to be around 10-12g/l. At the highest substrate loadings used, the yield of ethanol was only 25% of theoretical. Lignaceous components of biomass and inhibition of bacterial growth by products of fermentation, as well as the physical nature of the substrates were determined to be the major factors limiting the effectiveness of the fermentation.

The above observations led to further studies involving a comparative evaluation of range of substrate delignification treatments and a systematic program of strain improvement with respect to increased ethanol tolerance and end product selectivity. A selective solvent extraction procedure using an alkaline ethanol solvent yielded the best delignification performance of all the alternatives examined. Up to 70% lignin removal with a loss of less than 10% of the available carbohydrates was obtained with this method. Coculture fermentation of wheatstraw and newspaper pretreated by this procedure showed a four-fold increase in the maximum volumetric degradation rate as well as nearly 100% increase in the overall extent of substrate utilization, compared to untreated material.

Studies aimed at improving the fermentation efficacy were undertaken on both species of organisms. Improved ethanol tolerance was achieved through progressive adaptation of parent strains to higher ethanol concentrations in the growth medium. The strains isolated in this work however tended to have a significantly higher yield of the acid products concomitant with their enhanced ethanol productivity. A separate program of mutation and selective isolation of low acid producing cultures eventually resulted in strains which in coculture, fed batch fermentations were able to produce ethanol at concentrations of up to 30g/l at a net ethanol yield exceeding 60% of theoretical, when grown on pretreated wheat straw and newspaper.

A relatively reduced yield of ethanol however, was noted on real biomass compared to similar fermentations using pure substrates. This coincided with increased production of acetate with the crude substrates. An analysis of fermentation kinetics for the various experiments revealed that the ethanol/acetate ratio for deregulated strains of C. thermocellum and C. thermohvdrosulfuricum was strongly dependent on the specific growth rate the organisms achieve during fermentation, which, in turn is determined by the substrate hydrolysis and/or consumption rates. The implications of this to future process improvement studies has been briefly discussed.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Venkitachalam, Hari
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