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Production and analysis of the products of the acetone-butanol-ethanol fermentation of beet extract by butyl clostridia

Thompson, Roger Neil
Date
1988
Type
Thesis
Fields of Research
ANZSRC::1003 Industrial Biotechnology
Abstract
Rises in the price of oil during the 1970's stimulated interest in the production of fuels and chemicals from biomass. Although fuel ethanol has received a great deal of attention, the physical properties of butanol make the longer chain alcohol more desirable as a fuel, and this has been born out by vehicle tests. Previous work at Lincoln College had shown that sugar beet was a suitable substrate for producing ethanol by fermentation. This thesis describes work that was carried out to evaluate sugar beet as a substrate for the production of acetone-butanol-ethanol by fermentation with butyl clostridia. Investigations on the method of culturing and propagating such bacteria and the quantitative determination of fermentation residues and end products are also described. Four media were tested for their ability to support the growth and fermentation of butyl clostridia over a series of subcultures and it was found that an undefined medium incorporating freshly harvested potato gave the most reliable results. It was necessary to heat shock clostridial spores for 1-3 min in order to initiate germination of healthy solvent producing bacteria. Eleven strains of butyl clostridia were evaluated for the fermentation of sugars to ABE and it was found the Cl. acetobutylicum strains NRCC 2490. ATCC 27022 and P262 produced the greatest yields of butanol. Strain 2490 was selected for further investigation, and it was found that the fermentation of beet juice produced optimum yields of solvents when yeast extract was added. NH₄⁺ and phosphate did not improve solvent yields. Beet juice containing up to 115.5 g/l sugars required 1-2 g/l CaC0₃ to ensure adequate pH buffering against acetic and butyric acid metabolic intermediates. At sugar concentrations from 115.5 g/l to 221.8 g/l no CaCO₃ buffering was required. Increasing amounts of sugar in the juice resulted in decreasing fermentation efficiencies but solvent yields were maintained. Up to 17.5 g/l of butanol were produced, and a minimum of 60-75 g sugars was required to achieve this. In extracts containing more than these sugar levels residual carbohydrate was found at the end of fermentation. Fermentations of Cl. acetobutylicum in media containing more than 10% sugar have not been reported as yet, and so it is suggested that strain 2490. which grew in juice containing as much as 221.8 g/l sugar has considerable potential in fermentation systems where continuous product removal is incorporated. Gas chromatographic analysis of neutral solvents and acids was investigated and the best column was found to be a Chromosorb W-AW (80/100)/10% Carbownx 20M-TPA + 0.1 % MPA column. Other commonly used columns gave poor precision and accuracy, especially with acetic and butyric acids. The study revealed a need for the development of inert packing materials that permitted baseline separation of the products of the ABE fermentation. Of several columns evaluated for the quantitative measurement of sugars by HPLC, an ion exchange system was found to be the most suitable. Ca²⁺ was a better counter-ion than Pb²⁺ and an anionic guard column was necessary to remove acetate and butyrate which interfered with sugar peaks.
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