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Item The acetone butanol ethanol fermentation : preliminary studies on some practical aspects : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology(Massey University, 1982) Gapes, James RichardThe dilute nature of solvents at the end of fermentation and slow overall rate of fermentation are major economic burdens on a commercial plant producing acetone, butanol, and ethanol. A preliminary feasibility costing of such a plant showed the cost of fermenters represents almost 50% of the total purchased equipment cost, and emphasised the need for improvements in the fermentation. Experiments were performed in 10-litre and 30-litre pressure vessels, a 1.5-litre vessel at atmospheric pressure, and trial runs in 100 ml bottles. Good correlations were found for the different fermentation headspace pressures (100 to 250 kPa abs.) and minimum observed pH's (pH 4.2 to pH 4.65) with final butanol yields (0.92 to 11.6 g/1); increases in both parameters correlating with increased butanol concentration. Ethanol was found to be correlated with pressure only, and acetone with neither parameter directly. Other chemical species present in the broth were also correlated with each other. It was found that a tree diagram drawn using the strongest correlations resembled closely the known metabolism of the organism in terms of the metabolic pathways, specification of active forms of the metabolites, and effect of external influences. Use of multiple linear regression in this manner was named The Factor Correlation Method, and is potentially useful for research on metabolism and similar investigation on a much broader basis. Application of this technique showed that the pressure effect was possibly due to more than a single metabolic cause, and further experiments also emphasised the complex nature of the pressure effect. The experimental work also highlighted the potential hazard of culture degeneration leading to substandard fermentation yields and eventual nonviability. Discussion on the experimental results and of the literature suggests the phenomenon is due to infection by lysogenic phage rather than spontaneous mutation, and an approximate model based on simultaneous partial differential equations parallels some observed characteristics of the phenomenon. Other topics include theoretical exercises with laboratory work on the water tolerance of methanol-petrol mixtures, the error associated with cell enumeration using a haemocytometer, and evaluation of growth and solvent production characteristics and some relevant parameters.Item Intensification of the acetone, butanol, ethanol fermentation using whey permeate and Clostridium acetobutylicum : a preliminary study : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University(Massey University, 1987) Ennis, Brett MillsThe use of whey permeate as the fermentation substrate for the production of acetone:butanol:ethanol (solvents), using C. acetobutylicum P262 was studied. Initial experiments were conducted in a batch mode using sulphuric acid casein whey permeate medium, in an attempt to optimize the culture conditions for maximal extent of lactose utilization and solvents production. A high initial lactose concentration (65-75 g/l) in combination with a culture pH maintained in the region pH 5.4 to 5.6 were the most favourable conditions for solvent production. An inverse relationship between the lactose utilization rate and solvents yield was observed. Solvent productivities were only 60% however, of that achievable with this strain of organism on an industrial scale using a molasses medium, but comparable productivities were obtained using a semi-synthetic medium containing glucose. Hydrolysed-lactose sulphuric acid casein whey permeate medium was investigated as a medium for solvent production. Glucose and galactose were utilized simultaneously, although glucose was used preferentially. Only a small increase in solvents productivity was obtained compared with that obtained using non-hydrolysed permeate. Experiments were performed in continuous culture using cheese whey permeate medium and alginate-immobilized cells. Significantly greater solvent productivities were obtained, compared with those achieved using free cells in batch culture. Fermentations were operated for over 650 hours with no detectable loss in fermentation performance. The extent of lactose utilization was low, however (less than 40%), and attempts to increase this by the use of pH regulation or a two-stage process were unsuccessful. This fermentation process was described as a biomass volume process (volumetric fraction of alginate beads in the reactor), where the lactose utilization and hence the solvents production, was defined by an inhibitory concentration of butanol, approximately 5 g/l. An alternative continuous fermentation process using free cells and cheese whey permeate medium was investigated. External cell recycle using cross-flow microfiltration (CFM) membrane plant to continuously separate cells from the fermentation culture and recycle them back to the fermenter was utilized. Biomass was continuously removed from the fermenter in order to achieve a stable biomass concentration. Stable solvents production was not achieved under the range of culture conditions investigated; culture degeneration was attributed to the complex interactive morphological cyclic behaviour of the organism. A tubular CFM unit which could be periodically backflushed to maintain the filtrate flux, was found to be the most suitable of those tested. The integration of in-situ or in-line solvents recovery with batch culture using free cells, and continuous fermentation using cells immobilized by adsorption to bonechar, was investigated in order to remove toxic solvents and so increase the extent of lactose utilization and solvents productivity. A novel process using gas-stripping with an inert gas, and solvents recovery from the vapour phase by condensation using a cold trap, was described. An increase in lactose utilization and solvents productivity was achieved in both fermentation modes compared with control fermentations. The use of adsorbent resins and a molecular sieve for integrated fermentation solvents recovery was also demonstrated. However, the adsorption of medium components may mitigate against the usefulness of such a process option. The batch refermentation of batch fermentation effluent treated by gas-stripping to remove solvents was investigated. However, solvent production was favoured only when lactose and nutrients were supplemented to concentrations similar to those present originally. Conversely, fermentation medium treated by gas-stripping to remove solvents could be readily refermented to produce solvents when an existing cell population was used, suggesting that this option of an integrated continuous fermentation-product recovery process may be promising for whey permeate solvent production.