An in-depth study of the biodeterioration of the Hulett Aluminium hot roll mill emulsion, Prosol, was conducted. Samples of the emulsion in use at the hot roll mill were taken from various areas of the emulsion reticulation system in order to identify regions of highest microbial contamination. Standard plate count techniques and diagnostic procedures were employed to quantify and identify the microorganisms in these samples. In some of the highly contaminated areas of the emulsion reticulation system, microorganisms exceeded lxlO'CFUml'1 emulsion. A range of bacteria was identified which included members of the genera: Bacillus; Pseudomonas; Escherichia; Enterobacter; Sporosarcina; Micrococcus; Aeromonas; Chromobacterium and Desulfovibrio. Various fungi, including several yeasts, were also isolated and some of the filamentous spore-forming types were identified zsAspergillus spp.; Penicillium spp. and a Cladosporium sp. A visual scale was developed to assess the growth rate of the isolated microorganisms on a range of specific media containing various emulsion components as carbon and energy source. Although the results obtained by using this scale were not conclusive, a few biodegradable components were nonetheless identified. It was found that mixed cultures of the above microorganisms had a greater biodeteriorative effect on the emulsion than did any of the pure cultures when applied separately. This suggested complex microbial interactions were involved in the breakdown of the emulsion. A laboratory-scale model system representative of the Hulett Aluminium hot roll mill was designed and constructed to carry out a series of tests on unprotected and biocide-treated emulsions. A range of biocide concentrations were tested from which the minimum biocide inhibitory concentration was calculated. It was shown that microorganisms exposed to sublethal doses of the biocide Busan (active component glutaraldehyde) over a prolonged period of time, exhibited greater levels of tolerance and resistance to the biocide than did those microorganisms not previously exposed. It was deduced that less frequent, shock doses of biocide are more effective in the control and eradication of emulsion degrading microorganisms than are frequent, low level doses of the same biocide. In addition to the biocide studies, three imported so-called 'biostable' emulsions were evaluated as possible replacements for the susceptible Prosol. Of these three imported emulsions, two viz. HRF3 and Houghton Biostable were shown to be more resistant than Prosol to biodeterioration. After assessing the current hot roll mill management practices, a number of recommendations were made, including: the improvement of plant hygiene; education of the mill workers; improvement of emulsion monitoring; improvement of down-time management and improvement of biocide dosing regimes. Recommendations are also made for minimizing potential microbial growth in the new hot roll mill currently under construction at the Hulett Aluminium processing plant at Pietermaritzburg, South Africa. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

1,2-Dichloroethane (1,2-DCA), a highly toxic and recalcitrant compound, is produced anthropogenically in larger quantities than any other chlorinated compound. It is regarded as a mutagen and carcinogen, thus making it a priority target molecule for biological degradation. In addition, the intermediates of 1,2-DCA degradation are highly reactive and toxic, due to the electrophilic nature of the carbonyl groups in these compounds. Aerobic biodegradation of 1,2-DCA, resulting in complete mineralization, has previously been reported in Xanthobacter autotrophicus GJ10 and some Ancylobacter aquaticus strains. X. autotrophicus GJ10 has been found to possess chloroacetaldehyde (CAA) dehydrogenase and haloacid (HA) dehalogenase enzymes, both of which play a crucial role in 1,2-DCA degradation. Five strains of Ancylobacter aquaticus capable of utilizing 1,2-DCA as a sole carbon and energy source have recently been isolated in our laboratory. The degradation potential and specific dehalogenase activities of these bacterial isolates against 1,2-DCA and other halogenated compounds as a carbon source were investigated and compared to previously characterized organisms, viz., X. autotrophicus GJ10 and Ancylobacter aquaticus strains AD25 and AD27. Furthermore, this study proposed to detect the presence of the CAA dehydrogenase (aldB) and HA dehalogenase (dhlB) encoding genes in these isolates. Growth of all strains in the presence of 1,2-DCA as a carbon source was monitored over an 84 h period, in minimal medium supplemented with either vitamins or yeast extract. Dehalogenase activities were measured colorimetrically by monitoring halide release by crude cell extracts of the isolates. In order to detect the presence of dhlB and aldB genes, genomic DNA of the isolates was digested with individual restriction endonucleases, viz., EcoRI, PstI, HindIII and BamHI, and then subjected to Southern hybridization experiments. All isolates demonstrated significant growth rates in both vitamin and yeast extract supplemented media, with the former having a greater overall growth effect. Ancylobacter aquaticus DH5 demonstrated the highest growth rate of 0.147.h-1 in the presence of vitamins while Ancylobacter aquaticus DH12 displayed the highest growth rate of 0.118.h-1 with yeast extract. Optimum haloalkane dehalogenase activities of these bacterial isolates were confirmed at pH 8, similar to the activity in X. autotrophicus GJ10, while haloaciddehalogenase activity had a broader pH range. Hydrolytic dehalogenase activity of the bacterial isolates using a range of halogenated aliphatic compounds was also determined. Results demonstrated a wide substrate range with activity being observed on 1,3- dibromopropane, 1,2-dibromoethane and 1,3-dichoropropene, for all isolates. Southern Hybridization experiments confirmed the presence of both aldB and dhlB genes in X. autotrophicus GJ10. The dhlB probe produced a positive signal for an EcoRI fragment in Ancylobacter aquaticus DH12 while the aldB probe hybridized and produced a single positive signal on similar sized PstI fragments for all organisms except A. aquaticus AD25 which produced two positive signals. The results in this study demonstrate the potential application of the newly isolated strains of Ancylobacter aquaticus. in future bioremediation strategies. The detection of the genes involved in 1,2-DCA degradation further support the use of these isolates and/or their enzymes for the degradation of 1,2- DCA as well as other halogenated compounds. Future work need to determine sequence similarity of these genes detected in A. aquaticus strains to the genes in Xanthobacter autotrophicus GJ10 and other previously reported genes. It may also be important to investigate the activity of the enzymes under various environmental conditions and to determine enzyme structure and the catalytic sites, so as to gain knowledge of their degradation potential on site. Characterization of enzymes at both the molecular and protein levels may be necessary and beneficial for implementation in strategies involving bioremediation for the biological degradation of a wide range of halogenated aliphatic hydrocarbons. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Present within the biodiverse hypersaline environment are a wide variety of halotolerant filamentous fungi. Many of these phytopathogens are capable of hydrolysing plant cell wall polysaccharides such as hemicellulose which are comprised of mannans and heteromannans which are polymers of the mannose sugars. Endoacting hydrolytic enzymes such as endo-β-1,4-mannanases are secreted into the extracellular environment and are involved in the catalysis of the random hydrolysis of β-1,4-mannosidic linkages within the backbone of mannan, galactomannan, glucomannan, and galactoglucomannan. Poultry are monogastric animals that are unable to efficiently digest high-fibre and mannan rich feeds such as soybean meals and this results in decreased or depressed animal performance. The use of feeds supplemented with β-mannanases has been shown to enhance the feeding value of mannan-based meals. In the current study, the degradation of β-mannan polysaccharides present in poultry feed by halotolerant Scopulariopsis candida LMK008 β-mannanase was investigated. SDS-PAGE, Native-PAGE in conjunction with zymogram analysis was used to assess the molecular weight of the endomannanases. At least three isozymes were detected: two of 56 kDa (pI 3.5 and 6.7) and one of 28 kDa. Anion exchange chromatography was used to purify the 28 kDa isozyme. Three mannan-based substrates, viz., locust bean gum, guar gum and soybean flour, were used to evaluate the hydrolysis capability of the crude as well as the purified β-mannanase via the release of reducing sugars and was detected using the DNS assay. The β-mannanase exhibited low activity with pure guar gum but high activity with locust bean gum galactomannan and soybean flour mannan. The hydrolysis activities of the crude and purified enzyme were then tested further on mannan-based soybean meals. In general it was found that more reducing sugars were released from the grower feed than the starter and layer feeds. Another common hydrolysis pattern observed in all feed types was that after prolonged incubation of 24 h there was a decrease in the amount of reducing sugars released which could be attributed to the presence of naturally-occurring microorganisms in the feed sample which metabolised the simple sugars resulting from the enzymatic hydrolysis of the mannan components in the feed samples. This was confirmed by standard plate count assays. The results obtained are encouraging and the purified β-mannanase could be applied as an industrial feed additive within the animal feed industry, however, further testing of the enzyme in situ is needed in order to prove its applicability. The cloning of the endomannanase has to date proven unsuccessful despite numerous techniques being employed and further research is also needed to accomplish this task. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Previous studies of fungal decay have mainly examined long term effects of wood decay. In contrast, the present work, was designed to quantify fungal degradation of wood during incipient decay. Three facultatively anaerobic, dimorphic fungi were isolated from the rumen of sheep. These fungi were identified as Mucor racemosus, Candida tropicalis and Geotrichum capitatum. Scanning electron microscopy showed that these fungi colonised Pinus patula and Eucalyptus grandis extensively but did not appear to degrade the wood. The obligate anaerobe Neocallimastix frontalis colonised wood very sparsely, whereas the white rot bas id iomycetes Cori 01 us versicolor, and Phanaerochaete chrysosporium, and the brown rotters Coniophora puteana and Lentinus lepideus, colonised wood under both aerobic and anaerobic conditions. The extents of colonisation were greater under aerobic conditions. The work then quantified the effects of the basidiomycetes C. versicolor, P. chrysosporium, C. puteana and L .lepideus, and the non-decay mould, M. racemosus in individual and coculture experiments. Wood colonisation was quantified by Kjeldahl nitrogen determinations converted to biomass assays, and degradation was quantified by weight losses, and Klason lignin determinations. Furthermore, the degraded wood samples were also analysed by HPLC analysis of hydrolysates and their sugar contents were determined to establish whether the glucose of cellulose and xylose + mannose of hemicellulose had been utilised by the respective fungi. The extent and nature of sugar utilisation by monocultures and cocultures in wood were then compared with the biomass and degradation data. statistical analyses of' these comparisons correlated the extents of colonisation, degradation, and the patterns of wood sugars predominantly utilised by each fungus. The results of the corresponding glucose, xylose and 'lignin analyses confirmed the brown rot physiological capacity of C.puteana in both'woods. The white rot fungi behaved as simultaneous rotters and,<M·~<.racemosus was shown to be ligninolytic in P .patula. The white rot physiological capacity of C.versicolor was confirmed in 'E.grandis and that of P.chrysosporium in P.patula. Antagonism and synergism in wood was detected between individuals 'within cocultures during incipient decay. The significance of these findings becomes apparent when decayed wood of unknown history is analysed as described here. Such findings may be interpreted to provide valuable information describing the physiological nature of the responsible fungi, even if these are no longer viable or culturable. / Thesis (M.Sc.)-University of Durban-Westville, 1992

No abstract available. / Thesis (M. Sc.)-University of Durban-Westville, 1994.

The proper organization and differentiation of the anterior segment is pivotal for normal eye development. Neural crest-derived POM cells are key contributors to correct anterior segment formation, differentiating to form the monolayered corneal endothelium. Mice with homozygous null mutations in the forkhead transcription factor gene, Foxc1, fail to develop a proper corneal endothelium stabilized by adherens junctions, with the endothelium adhering to the lens, preventing anterior chamber separation. The aim of this study was to evaluate the interaction between Foxc1 and the adherens junction protein, N-cadherin, as well as an associated gene, Msx1, during key stages in corneal endothelium development. Foxc1 was over-expressed in E12.5 and E13.5 POM cells and qPCR was carried out to determine the effect of Foxc1 on N-cadherin and Msx1 gene expression. Data showed over-expression of Foxc1 in wildtype E12.5 and E13.5 POM cells to cause significant fluctuations in N-cadherin and Msx1 expression (p < 0.05). POM cells were then transfected with a Foxc1 knock-down plasmid or the Foxc1 overexpression plasmid to evaluate the effect of Foxc1 on N-cadherin protein expression by Western blot analysis, however, these results were inconsistent with the gene expression analyses with no significant differences in N-cadherin expression detected. N-cadherin protein expression and localization was then further assessed by means of immunocytochemistry (ICC) and confocal microscopy in monolayer and hanging-drop POM cell cultures. Both qPCR and confocal microscopy data showed consistency, indicating increased amounts of N-cadherin in E12.5 cells relative to E13.5 cells, with membrane-bound N-cadherin showing a clear lattice-work pattern in hanging drop culture. Foxc1 over-expression/knock-down studies on E12.5 and E13.5 POM cells together suggest that N-cadherin is transcriptionally regulated by Foxc1 and that Foxc1 has a threshold level at which it is able to exert control over N-cadherin in POM cells. Foxc1 expression is therefore essential in establishing N-cadherin adhesion junctions in the corneal endothelium. Preliminary data also suggests that Msx1 may directly interact with Foxc1 in POM cells, however, further studies must be undertaken to verify and establish the effects of Foxc1/N-cadherin/ Msx1 interaction in the development of a cohesive, integrated corneal endothelium and functional anterior segment. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

In recent years there has been widespread interest in rearing aquatic organisms of nutritional and commercial value (Calaprice, 1976). The most hopeful prospect for marine prawn culture in the United Kingdom (Wickins, 1976), the Americas (Hanson & Goodwin, 1977) and South Africa probably lies in intensive culture under controlled conditions. A closed system approach, in which a captive body of water is circulated, provides the scope for water quality management which results ~n maximum water utilization and minimal discharge. On the other hand, direct utilization of sea-water in open systems presents problems for aquaculture since this water is subjected to diurnal and seasonal fluctuations in temperature, salinity and turbidity, as well as contamination from industrial, agricultural and maritime sources. Furthermore, large mariculture farms release enormous amounts of organic wastes which result in eutrophication and could lead to environmental deterioration of coastal waters (Gerhardt, 1978). It is well established that circulated sea-water develops an unusual ~on~c composition as a result of the metabolic activity of the prawns and of the nitrifying bacteria in the biological filter. The changes include elevated levels of ammonia, nitrite and nitrate and reduced pH. The presence of even sublethal levels of these nitrogenous compounds ~n closed systems have been found to affect growth of penaeid spec~es (Wickins, 1976). Ammonia and nitrite, which rapidly accumulate in the water, are usually maintained at nontoxic levels by nitrification in the biological filters (Spotte, 1974; Johnson & Sieburth, 1974). The chemolithotrophic bacteria responsible for nitrification are presently classified by their · cellular morphology and by the oxidation of either ammonia and nitrite (Watson, 1974). The predominant ammonia- and nitrite-oxidizing bacteria isolated from natural environments are Nitrosomonas europaea and Nitrobacter winogradskyi, respectively (Watson et aZ., 1981). Direct observation of nitrifying bacteria in natural environments, however, has been limited to studies involving light microscopy with immunofluorescent techniques (Fliermans et aZ., 1974; Fliermans & Schmidt, 1975). The electron microscopic observation of nitrifying bacteria ~sdifficult in natural microcosms with low levels of nitrification and with the presence of sunlight and anaerobic conditions conducive to the enrichment of other bacteria with a similar ultrastructure. However, in closed systems with extremely active nitrification but poor light conditions, the occurrence of morphologically similar forms in numbers that could be easily detected by electron microscopy is unlikely (Johnsort & Sieburth, 1976). Furthermore, the cyst-like colonies of the nitrifiers are unique and are not found with the methane-oxidizing bacteria with a similar ultrastructure (Davies & Whittenbury, 1970; Smith & Ribbons, 1970), whereas the thick cell wall of the cyanobacteria (Carr & Whitton, 1973) and the distinctive cell morphologies of the purple sulphur and purple nonsulphur bacteria (Pfennig, 1967) separate them from the nitrifiers. Therefore, closed systems with active nitrification provide the ideal environment to study the activities of nitrifiers in conjunction with their relative abundance, nature and diversity. In spite of the opportunity offered by closed systems, previous studies (Kawai et aZ., 1965; Wickins, 1976; Gerhardt, 1978; Mevel & Chamroux, 1981) on nitrification have been primarily indirect observations on rates of ammonia and nitrite oxidation to nitrate (Johnson & Sieburth, 1976). Studies on the enumeration and identification of nitrifiers ~n closed systems have been seriously neglected. Kawai et aZ. (1964) included the enumeration of nitrifiers in their study on nitrification while,in a qualitative study, an attempt to identify the in situ nitrifiers 1n closed systems (Johnson & Sieburth, 1976) was not very successful. This study was undertaken to investigate the three basic aspects of nitrification necessary for the understanding of such a process in closed systems, viz., the oxidation of ammonia and nitrite to nitrate, and the enumeration and identification of the nitrifying bacteria. Prior to determining the concentrations of the nitrogenous compounds in the culture water, various methods were evaluated for their accuracy and reproducibility with both sea-water and culture water samples. This approach is necessary in order to gauge the accuracy of results obtained by such methods. Enumeration of nitrifying bacteria was preceded by an investigation on the effect of incubation time on the maximum most probable number , estimate. Such an investigation was necessary because of the inconsistent approach to the enumeration of nitrifiers in previous studies (Wilson, 1927; Walker et al., 1937; Lewis & Pramer, 1958; Molina & Rovira, 1964; Meiklejohn, 1965; Smith et al., 1968). Incubation periods appear to have been chosen arbitrarily in previous investigations. Identifi~ation of nitrifying bacteria necessitates the isolation and purification of these organisms. Isolation of nitrifiers 1S a difficult and time-consuming task (Watson et al., 1981) and could be the main reason for not being included in previous studies on nitrification. Since the success of this study depended upon the isolation and purification of these chemolithotrophs, this aspect is de~lt with in detail. The changes most likely to be associated with nitrification in a closed system were also monitored 1n the culture water. These included pH, dissolved oxygen and biochemical oxygen demand. Apart from a biological sand filter, no other form of culture water treatment was effected during the investigation. The effect of growing the "sugpo" or jumbo tiger prawn, Penaeus monodon (Kinne, 1977) for 22 weeks in a captive body of sea-water was evaluated by comparing the survival and wet mass with those reported by other workers. This study differs greatly from previous reports on nitrification in closed systems because both the "causes" and "symptoms" of this important detoxifying process are investigated. It is intended that the findings of such a study would aid culturists in exploiting the nitrifying potential of closed systems to its utmost. / Thesis (M.Sc.)-University of Durban-Westville, 1984.

Ratoon stunting disease (RSD) of sugarcane was first recognized in 1944 in Queensland, Australia (Steindl, 1961). The disease occurs worldwide and causes significant yield losses, especially during drought. RSD produces no external symptoms except a non-specific stunting (Steindl, 1961). RSD, which was first recorded 1n South Africa in 1953 (Anon., 1960), causes a greater overall loss in yield than any other sugarcane disease in South Africa. Yields of sugarcane are reduced by 20% to 40% and the harvest of affected fields declines progressively with successive ratoons (Anon., 1980b). A virus was originally thought to cause RSD, but in 1973, a coryneform bacterium was implicated as the causal agent (Gillaspie et al., 1973; Teakle et al., 1973). In 1980, our laboratory reported the successful isolation and culture of a coryneform bacterium associated with RSD of sugarcane and was indicated to be the causal agent (Nayiager et al., 1980). The lack of a rapid diagnostic technique applicable to mass screening of sugarcane has hindered progress in the control of the disease. There are two types of commonly used diagnostic tests. One test depends on the evaluation of internal stalk symptoms which may require from two to twenty six weeks to develop (Gillaspie et al., 1966; Matsuoka, 1971; Schexnayder, 1960; Singh, 1969). However, these symptoms are not always present in RSD affected plants and similar symptoms can sometimes result from other causes (Steindl, 1961). The other test involves establishing the presence of the coryneform bacterium associated with diseased plants. The bacterium is visible under high magnification by phase-contrast microscopy (Gillaspie et al., 1973) or by electron microscopy (Teakle et al., 1973). Although identification by the latter methods requires little time, the technology involved severely limits the number of samples that can be examined. Recently, serological techniques have been used (Brlansky et al., 1982; Damann et al., 1977; Davis et al., 1980; Gillaspie, 1978b; Gillaspie et al., 1979; Harris and Gillaspie, 1978) but their success has been limited. Besides problems with diagnosis of the disease, the precise morphology and taxonomy of the causal organism is unclarified. The objectives of this research programme were, firstly, to characterize the cultured intact bacterium and its constituent membranes both ultrastructurally and immunologically, and secondly, to evaluate various immunological methods for detection of the bacterium. This study should contribute to enhancing the taxonomic status of the bacterium and to the use of a rapid diagnostic technique applicable to mass screening of sugarcane. Such a technique should eventually contribute to effective control of RSD. / Thesis (M.Sc.)-University of Durban-Westville, 1984.

Corneal endothelial development is an intricate process driven by finely tuned gene expression. Its formation is necessary for the continued normal development of the anterior segment of the eye. The presence of an inductive lens able to secrete factors such as TGFβ2 as well as the expression of Foxc1 and Pitx2 is essential to corneal endothelial development, as in the absence of any of these; the corneal endothelium fails to form. Corneal endothelial development begins as peri-ocular mesenchyme (POM) cells migrate into the space between the lens and surface ectoderm at E11.5. From E12.5, these cells begin to transition from a mesenchymal to an epithelial/endothelial (MET) phenotype, differentiating into a monolayered endothelium by E15 characterised by inter-cellular junctions. To study the initial process of development, immortalised POM cell lines from E12.5 and E13.5 embryos were used. Expression of the key genes, the transcription factors, Foxc1 and Pitx2 and two genes involved in EMT/MET, Slug and Tsc22, were analysed at these stages to establish the developmental norm. The effect of the lens on these expression levels was then determined. To establish whether TGFβ2 is the lens secreted signal responsible for gene expression changes, cells were subjected to TGFβ2 treatment. In all these experiments, the role of Foxc1 in regulating gene expression was determined by Foxc1 overexpression and knockdown. The effect of the lens on cellular proliferation and on the expression and cellular arrangement of N-cadherin, a junction protein was also determined. The results showed that, at E12.5, the lens downregulates Foxc1 and Pitx2 expression, is a potent inducer of Tsc22 expression and is required for maintaining Slug levels. TGFβ2 was shown to play a role in Foxc1 and Pitx2 downregulation. Analysis suggests that Tsc22 expression is responsive to lens signals, but that TGFβ2 is not the signal responsible for its downregulation between E12.5 and E13.5. The lens has no effect on Slug expression in the presence of Foxc1, but when Foxc1 is silenced, Slug is induced. Thus, Foxc1 plays a crucial regulatory role in Slug expression. At E13.5, as differentiation is initiated, Foxc1 expression remains responsive to the lens and to TGFβ2. Pitx2 expression is still induced by the lens but, at this stage, TGFβ2 does not play a part in Pitx2 regulation suggesting involvement of other unknown lens secreted signals. Other lens secreted signal/s were also shown to downregulate Tsc22 and Slug at this stage. The lens was implicated in MET as it was shown to have an effect on N-cadherin localisation in 3-dimensional culture. E12.5 Spheroids exposed to E6 lenses formed a distinct lattice arrangement of N-cadherin compared to the uniform distribution in control cells. Although the 13.5 control cell aggregates also showed a lattice framework, it was more pronounced in the lens treated cells. The transcriptional role of Foxc1 was determined by overexpression and knockdown experiments where Foxc1 overexpression and knockdown upregulated Tsc22 and downregulated Pitx2 and Slug at E12.5. At E13.5, Pitx2 was downregulated and Slug was upregulated in response to aberrant expression of Foxc1. This was illustrative of the sensitivity these genes have to Foxc1 expression during development. It is known that the presence of a functioning lens and Foxc1 are essential for proper development of the corneal endothelium, which in turn is necessary for normal eye development. The understanding of the precise molecular mechanisms required for corneal endothelial development and the processes requisite for cell proliferation and differentiation has important consequences for providing further insight into the pathophysiology of anterior segment dysgenesis and glaucoma. Previous studies suggest that stem-cell like qualities are conferred in cells undergoing EMT. Such an investigation may lead to application in regenerative medicine such as the bioengineering of corneal tissue. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

No abstract available. / Thesis (M.Sc.)-University of Durban-Westville, 1989.