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    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
    (Massey University, 1991) Kanchanatawee, Sunthorn
    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.
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    Tower fermentation of whey permeate and sucrose-enriched whey permeate to ethanol : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University
    (Massey University, 1983) Boontanjai, Chaturong
    Tower fermentation of sulphuric acid whey permeate using Kluyveromyces marxianus Y42 has been investigated. The tower fermenter used was 0.025 m in diameter and 2.69 m high. The straight section of the tower was 2.37 m. The total tower volume was 2.9 litres and the separator section made up 1.6 litres of the total volume. The operating temperature was 30°C. The optimum medium feed rate was observed at a superficial liquid velocity of 0.24 mm/s. It was found that a tower height of only 0.82 m was required, excluding the separator section, and the corresponding residence time was 1 hour. An exit ethanol concentration of 16 g/l was produced at a productivity of 16 g/lh from 45 g/l lactose in the whey permeate feed (94% utilization). This was an ethanol yield of 71% on lactose utilized. If the separator section were considered, the ethanol productivity was 5 g/lh and the exit ethanol concentration was 19 g/l while the overall retention time was 3.7 hours. The cell concentration inside the tower varied between 10 and 100 g/l dried weight (54 and 350 g/l wet weight) being greatest at the bottom of the tower. K. marxianus was found to be inhibited by a high level of ethanol in the growth medium and unable to ferment completely a high concentration of lactose when tested in 10 litre-scale-batch fermentation. Further tests in the presence of sucrose and lactose found that this yeast exhibited diauxic behaviour by utilizing sucrose before lactose. This behaviour generally resulted in incomplete lactose utilization in the tower. In the screening for a flocculent lactose-fermenting yeast, the yeast strain K. marxianus was found to be the only flocculent yeast, but it was only moderately flocculent. Further investigation found that it had good flocculence when grown in media which support good growth, and poor flocculence when grown in acidic media and in media which do not support good growth. A subculture of this yeast strain showed moderate flocculence when grown in whey permeate. Tower fermentation of whey permeate enriched with molasses by mixed culture of Sacoharomyces cerevisiae CFCC39 and K. marxianus Y42 was found to be difficult. The difficulty arose because of incomplete lactose utilization even at a very low feed rate (up to 0.14 mm/s) and incompatible flocculation properties of the two yeast species employed. Blockage of the separator and gas slug formation were caused by the very flocculent yeast mass of S. cerevisiae CFCC39. This caused K. marxianus to be slowly washed out of the tower fermenter. Sucrose was completely utilized at the bottom of the tower fermenter, while lactose utilization was slow and incomplete. The incomplete lactose utilization has been attributed to the diauxic behaviour of K. marxianus, ethanol inhibition and molasses inhibition (probably due to its reaction with whey permeate during autoclaving). Results of tower fermentation of cane molasses have also been given for characterization of the tower fermenter used. Experiments to isolate an ethanol tolerant K. marxianus using a serial subculture in a medium containing increasing ethanol concentrations were performed. The isolate obtained could tolerate up to 50 g/l ethanol. It could ferment lactose in whey permeate to produce ethanol at a faster rate than the parent strain and other lactose-fermenting yeast tested. The isolate was found to be stable. It was not used in the tower fermenter as it was non-flocculent. An attempt was made to isolate a sucrose-negative K. marxianus. This was only partially successful. The mutant did not grow on sucrose agar but reverted to the wild type when grown in liquid medium containing both sucrose and lactose. An experiment to isolate a diauxie-negative K. marxianus strain using D-glucosamine as a glucose analogue was also described. This was unsuccessful because K. marxianus was able to grow on lactose in presence of the analogue.