The removal of multiple substrates in a defined mixed culture process was investigated in the treatment of brewery wastewater. The study was conducted using both batch and a semi-continuous reactor system called self-cycling fermentation. Batch experiments were conducted using a synthetic brewery wastewater containing glucose, ethanol and maltose. Activated sludge from a municipal wastewater treatment plant was acclimatized in the synthetic brewery wastewater. The microbes capable of degrading this wastewater were analyzed by a combination of microscopy, spread plating, and Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and identified as Acinetobacter sp., Enterobacter sp. and Candida sp. From the pure culture batch experiments, it was determined that Enterobacter could degrade glucose and maltose but no ethanol, while Acinetobacter and Candida could degrade all three carbon sources. In mixed culture batch experiments, Enterobacter was dominant in degrading the sugar concentrations to levels permissible for Acinetobacter to degrade ethanol. PCR-DGGE was found to be effective in identifying the dominant species but selective carbon source plating was required to determine viability and track the population dynamics. Kinetic experiments were carried out in a semi-continuous, self-cycling fermentation process using the defined mixed culture in media containing glucose and various initial concentrations of ethanol and maltose. The overall rate of substrate removal was attributable to both the suspended culture and the biofilm formed during the process. A rate expression was developed for this system for the range of substrate concentrations tested. The data indicated that substrate removal by the suspended culture was a function of only the biomass concentration. However, substrate removal by the biofilm was found to be limited to the surface cells and determined to be a function of substrate concentration only.

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Self-cycling fermentation (SCF) control was applied to a semi-continuous, aerobic, fixed-film reactor for the treatment of simulated and real brewery wastewater. The simulated wastewater was treated at 25°C. After approximately 3 hrs, 86% to 96% of the soluble BOD (SBOD) was removed, producing an effluent with a SBOD between 169 mg/L and 412 mg/L. The treatment of real brewery wastewater was undertaken at 25°C and 35°C, and was operated under ammonia-nitrogen deficient condition. 83% to 92% of the total BOD (TBOD) was removed after 3 hrs at 25°C, and after 1.5 to 2 hrs at 35°C. The treated effluent produced had a TBOD between 120 and 438 mg/L. The suspended solids in the treated effluent contributed between 63% and 71% of the TBOD. Hence, better treatment efficiency would have been possible if an efficient clarifier was installed, or the suspended solids were removed from the brewery wastewater prior to treatment.

A novel technique was developed to immobilize plant cells. The cells are deposited on a surface of man-made fibrous material which provides for strong binding of the plant tissue biomass growing in the submerged culture. It was shown that the plant cells need to be fully viable for the attachment process to occur. / The scale-up of this technique to laboratory size specifically designed bioreactors was performed successfully. The cell immobilizing matrix was formed into a vertical spirally wound configuration to provide for a high immobilizing area-to-volume ratio (0.8-1.2 cm$ sp{-1}$). A modified airlift (riser-to-downcomer area ratio of 0.03 and vessel height-to-diameter (H/D ratio of 3) and a low H/D ($ sim$1.5) mechanically stirred vessel delivered the optimum bioreactor performance characterized by low foaming of the broth and highly efficient plant cell attachment and retention ($ geq$96%). / The growth of Catharantus roseus plant cells was investigated in these bioreactors. This process was found not to be mass transfer limited above minimal mild mixing and aeration levels ensuring sufficient supply of nutrients, especially oxygen (k$ sb{ rm L}$a $ sim$ 10-15 h$ sp{-1}$) to the immobilized biomass. / The gentle surface immobilization technique developed in this work did not hinder the biosynthesis potential of the SIPC. In fact, it appeared to induce a partial secretion of some valuable compounds into the culture medium. The mildness, easiness, efficiency, mass transfer characteristics, scale-up potential and biomass loading capacity (11-13 g d.w./L) of the surface immobilization technique make it superior to all other immobilization techniques used to culture plant cells. In addition, its bioreactor overall biomass concentration compares favourably to suspended plant cell concentrations attainable in bioreactors (15-20 g d.w./L).

Formate dehydrogenase from Mycobacterium vaccae (MycFDH) was cloned and expressed from various plasmid constructs that incorporate hexahistidine tags onto the N-and C-termini of the protein. The most successful FDH variant, dual-tagged FDH L-S expressed from pET28a+, was batch-purified using ammonium sulphate precipitation and IMAC to achieve 96% homogeneity. A significant proportion of the expressed protein was insoluble, and the expression protocol did not respond to solubility optimization efforts. Expression of an FDH-NusA fusion variant appeared to be vulnerable to proteolytic degradation in the cell. None of the strains expressing tagged-FDH variants produced clarified lysate activity levels that were consistently as high as those from the original pUC 119 vector. However, it is likely that the protein aggregation problems encountered are due to overloading of the protein production machinery or related causes, rather than to the presence of the tags themselves. A bioengineered FDH protein closely related to MycFDH was also investigated. FDH GAV was immobilized in polyacrylamide gel to create gel discs or hollow cylinder mini-reactors. The apparent Km(formate) for this enzyme was 15.6 ± 3.6 mM in the immobilized state, and 17.2 ± 1.9 mM in aqueous solution. The activity of FDH GAV was reversibly inhibited by the presence of acrylamide monomer but was not affected by ammonium persulfate or TEMED (alone or in combination) after incubation for one minute. The activity of the immobilized enzyme system was determined to be at least partially limited by diffusion. FDH GAV was also included in an in vitro analysis of the Methanol Linear Dissimilation Pathway (MLDP), a three enzyme system of NAD+ - dependent dehydrogenases that oxidize methanol sequentially to CO2. Horse liver alcohol dehydrogenase (EC 1.1.1.1) appeared to be the rate-limiting enzyme under the conditions used in these experiments, most likely due to its limited activity on methanol. The applicability of FDH and the MLDP to industry and bioelectronics is also considered.

A common problem observed in large-scale cell cultivation is reduced culture performance compared with small-scale processes due to the existence of concentration gradients caused by poor mixing. Small-scale simulations using microbial cell suspensions have shown that circulation of cells through concentration gradients of oxygen, pH and glucose can result in reduction of cell growth and product formation similar to the effects observed in large-scale bioreactors. This study was aimed at using scale-down studies to investigate poor mixing in large-scale bioreactors used for suspended plant and animal cell culture. Two plant cell suspensions and a hybridoma cell line were used in this work. The range of oxygen transfer coefficients achieved in the hybridoma and plant suspensions were about 50???20 h-1 and 12???6 h-1, respectively. One-vessel simulation was developed to induce fluctuations of dissolved oxygen tension in a 2-L bioreactor using intermittent sparging of air and nitrogen. The effect of dissolved oxygen fluctuations on the cells was examined by comparing the performance of the cultures with those operated at constant dissolved oxygen tension. In the hybridoma suspension culture, only slight effects on cell growth were observed at circulation times above 300 s. No effect on the specific glucose uptake rate or antibody production was observed at the circulation times tested. Analysis of gene expression for selected hypoxia-related genes also suggested that the overall effect was limited. In plant cell suspensions, the specific growth rates and biomass yields on total sugar in the cultures under fluctuating dissolved oxygen tension were essentially the same as those at constant dissolved oxygen tension for both transgenic Nicotiana tabacum and Thalictrum minus. Under fluctuating dissolved oxygen tension, no effect on antibody accumulation was observed in transgenic N. tabacum suspensions, but a decrease in berberine accumulation was observed in T. minus. From the results, it can be concluded that only minimal effects due to the development of concentration gradients would be expected in large-scale bioreactors used for the cultivation of the hybridoma and plant cell suspensions tested in this work.

Macromolecular components, including protein and polysaccharides, are viewed as one type of major foulants in the complex feed membrane filtration systems such as membrane bioreactor (MBR). In this thesis, the mechanisms of macromolecular fouling including protein and polysaccharide in the complex feed solution are explored by using Bovine serum albumin (BSA) and alginate as model solution. During the filtration of BSA and washed yeast with 0.22 ????m PVDF membrane, it was found that the critical flux of mixture solution was controlled by washed yeast concentration while the existence of BSA significantly changed the cake reversibility of much larger particles. The fouling mechanisms of alginate, as a model polysaccharide solution, were investigated both in dead end and crossflow membrane filtration. In the dead end experiments, it was found that the cake model appears to fit the entire range of the ultrafiltration data while the consecutive standard pore blocking model and cake model are more applicable to microfiltration membranes. The alginate was featured with high specific cake resistance and low compressibility despite some variations between different membranes. The specific cake resistance ( c ) is similar to c of BSA and actual extracellular polymer substance (EPS) in MBR systems reported in the literature, and higher than that of many colloidal particles. In a system contained alginate-particles mixture, it was found that the existence of alginate dramatically increased the cake specific resistance and decreased the cake compressibility. The fouling mechanism of alginate was also studied using long term cross flow filtration under subcritical flux. A two-stage TMP profile similar to that typically observed in MBR was obtained, confirming the important role of EPS during membrane fouling in MBR. In addition to adsorption, trace deposition of alginate also contributed to the initial slow TMP increase during the subcritical filtration. TMP increase during the long-term filtration was found not only due to the increase of the amount of deposition, but also the increase of specific cake resistance. A combined standard pore blocking and cake filtration model, using a critical pore size for the transition time determination, was developed and fit the experimental results well.

A common problem observed in large-scale cell cultivation is reduced culture performance compared with small-scale processes due to the existence of concentration gradients caused by poor mixing. Small-scale simulations using microbial cell suspensions have shown that circulation of cells through concentration gradients of oxygen, pH and glucose can result in reduction of cell growth and product formation similar to the effects observed in large-scale bioreactors. This study was aimed at using scale-down studies to investigate poor mixing in large-scale bioreactors used for suspended plant and animal cell culture. Two plant cell suspensions and a hybridoma cell line were used in this work. The range of oxygen transfer coefficients achieved in the hybridoma and plant suspensions were about 50???20 h-1 and 12???6 h-1, respectively. One-vessel simulation was developed to induce fluctuations of dissolved oxygen tension in a 2-L bioreactor using intermittent sparging of air and nitrogen. The effect of dissolved oxygen fluctuations on the cells was examined by comparing the performance of the cultures with those operated at constant dissolved oxygen tension. In the hybridoma suspension culture, only slight effects on cell growth were observed at circulation times above 300 s. No effect on the specific glucose uptake rate or antibody production was observed at the circulation times tested. Analysis of gene expression for selected hypoxia-related genes also suggested that the overall effect was limited. In plant cell suspensions, the specific growth rates and biomass yields on total sugar in the cultures under fluctuating dissolved oxygen tension were essentially the same as those at constant dissolved oxygen tension for both transgenic Nicotiana tabacum and Thalictrum minus. Under fluctuating dissolved oxygen tension, no effect on antibody accumulation was observed in transgenic N. tabacum suspensions, but a decrease in berberine accumulation was observed in T. minus. From the results, it can be concluded that only minimal effects due to the development of concentration gradients would be expected in large-scale bioreactors used for the cultivation of the hybridoma and plant cell suspensions tested in this work.