Nutrient requirements for acetone-butanol-ethanol production from whey permeate by Clostridium acetobutylicum P262 in a range of bioreactors : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand

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The acetone-butanol-ethanol (ABE) fermentation process, using Clostridium acetobutylicum P262, was studied. Experiments were conducted in a traditional batch fermentation using freely-suspended cells in a defined medium with lactose as carbon source. Solvent production did occur under conditions of nitrogen-, phosphate- or iron-limitation. However, the optimum conditions were observed when all nutrients were present slightly in excess of growth requirements. A greater excess of nutrients caused the fermentation to be acidogenic rather than solventogenic. Sulphuric acid casein whey permeate, without nutrient supplementation, proved to be a poor substrate for growth and solvent production by this organism. However, the addition of yeast extract (5 g/1) led to strong solventogenesis. The deficiency in the whey permeate was shown to be iron rather than assimilable nitrogen, phosphate or vitamins. Experiments were also performed in a defined medium in continuous culture using freely-suspended cells in a CSTR. An inverse relationship was observed between the biomass concentration and the specific butanol productivity. It is suggested that this was due to the cell population not being homogeneous, and that a change in the nutrient balance led to a change in the relative proportions of acidogenic, solventogenic and inert cells (spores). The addition of supplementary yeast extract during continuous solvent production from whey permeate using free cells in a CSTR showed that yeast extract supplementation of less than 1 g/1 favoured acid production rather than solvent production. It is unlikely that high solvent productivities can be attained with this substrate in a single stage continuous culture system with freely-suspended cells. A maximum solvent productivity of 0.05 g/l.h was observed in continuous culture compared to 0.06 g/l.h in batch culture. A continuous fermentation process was investigated using cells immobilized by adsorption onto bonechar and operated in a packed bed reactor. Three nutrients (i.e. nitrogen, phosphate and iron) were selected for this investigation. Solvent production was favoured by high concentrations of these nutrients in the influent medium. It was not possible to restrict the supply of phosphate or iron in the reactor due to leaching of the bonechar. However, conditions where biomass growth was restricted by a restriction in the nitrogen supply were not conducive to solvent production. This reactor was also operated using whey permeate as the substrate. The biomass build-up was controlled by minimizing the supplementary yeast extract concentration of the feed, but this had deleterious effects on solvent production. Hence, when producing solvent from whey permeate in this type of reactor a compromise must be made between strong solventogenesis and reactor longevity.
Content removed due to copyright restriction: Appendix 1 Kanchanatawee, S. S., & Madox, I. S. (1970). Effect of phosphate and ammonium ion concentrations on solvent production in defined medium by Clostridium acetobutylicum. Journal of Industrial Microbiology, (5), 277. Appendix 2 Kanchanatawee, S. S., & Madox, I. S. (1990). The nutritional status of whey permeate as a substrate for the abe fermentation. In P-L, Yu (ed.),Fermentation Technologies: Industrial Applications (pp. 173-177). London, UK: Elsevier Applied Science. Appendix 3 Kanchanatawee, S. S., & Madox, I. S., (1990). The effect of selected nutrients on solvent production by clostridium acetobutylicum. Proceedings of the 9th Australian Biotechnology Conference, 24-27 September 1990, Surfers Paradise, Queensland, Australia: University of Queensland. Appendix 4 Kanchanatawee, S. S., & Maddox, I. S. (1991). Effect of biomass concentration on the specific solvent productivity of Clostridium acetobutylicum in chemostat culture. Journal of Industrial Microbiology, (2), 151.
Clostridium acetobutylicum, Fermentation, Whey, Whey permeate