A Pseudomonas strain, AI08, which was capable of detoxifying the herbicide acetochlor (2- chloro-N-ethoxymethyl-6'-ethylacet-o-toluide) was isolated from soils. The microbe was isolated using a combination of batch culture enrichment techniques, phenotypic agar plate based assays and a qualitative bioassay for detecting acetochlor detoxification. With the aid of a bioassay developed specifically for the quantification of acetochlor concentrations, it was determined that over a 21 day period Al 08 was capable of detoxifying 20 % of the acetochlor present in a medium containing no other organic carbon and 53 % of the herbicide in a medium containing glucose and yeast extract at concentrations of 0.02 g.l-l and 0.005 g.l-l respectively. A fragment of A108 DNA was cloned in Escherichia coli which produced recombinant cells with both elevated acetochlor resistance and the ability to detoxify 15 % of the acetochlor present in a minimal nutrient medium (containing 0.02 g.l-l glucose and 0.005 g.l-l yeast extract) over a 21 day period. Partial sequencing of the cloned A108 DNA revealed that it encoded an amino acid sequence with significant homology with the dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complexes of Azotobacter vinlandii, E. coli and Alcaligenes eutrophus. Theories are proposed as to the possible biochemical mechanisms whereby expression of the dihydrolipoyltransacetylase gene of Al 08 in recombinant E. coli cells may function in the detoxification of acetochlor. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

Dyes are often recalcitrant organic molecules that produce a colour change and contribute to the organic load and toxicity of textile industrial wastewater. Untreated effluent from such sources is harmful to aquatic life in the rivers and lakes due to reduced light penetration and the presence of highly toxic metal complex dyes. The use of alum as flocculant/coagulant in wastewater treatment is not encouraged as it induces Alzheimer’s disease in humans and results in the production of large amounts of sludge. Therefore, the development of safe and biodegradable flocculating agents that will minimize environmental and health risks may be considered as an important issue in wastewater treatment. Bioflocculants are extracellular polymers synthesized by living cells. In this study, bacterial bioflocculants were assessed for their ability to remove dyes from textile wastewater as well as reducing the microbial load in untreated river water. The bacteria were isolated from a wastewater treatment plant and identified using standard biochemical tests as well as the analysis of their 16S rDNA gene sequences. Six bacterial isolates were identified viz. Staphylococcus aureus, Pseudomonas plecoglossicida, Pseudomonas pseudoalcaligenes, Exiguobacterium acetylicum, Bacillus subtilis, and Klebsiella terrigena. The flocculating activities of the bioflocculants produced by these isolates were characterized. The effect of temperature, pH, cations and bioflocculant concentration on the removal of dyes, kaolin clay and microbial load was also determined. The amount of bioflocculants produced by the bacterial isolates ranged between 5 and 27.66 g/l. According to the findings of the present study, bacterial bioflocculants were composed of carbohydrates, proteins, uronic acid, and hexosamine in varying quantities. The bioflocculants were effective to varying degrees in removing the dyes in aqueous solution, in particular whale dye, medi-blue, fawn dye and mixed dyes, with a decolourization efficiency ranging between 20-99.9%. Decolourization efficiency was influenced by the bioflocculant concentration, pH, temperature, and cations. The bacterial bioflocculants were also capable of reducing both the kaolin clay and the microbial load from river water. The flocculating activity ranged between 2.395–3.709 OD-1 while up to 70.84% of kaolin clay and 99% of the microbial load from the river water was removed. The efficiency of kaolin clay flocculation increased with higher concentration of bacterial bioflocculants. The optimum pH for the flocculating activity was observed between 6 and 9. The best flocculating activity was observed at 28oC. Divalent cations such as Mg2+ and Mn2+ improved the flocculation while salts such as K2HPO4, CH2COONa, and Na2CO3 did not. The findings of this study strongly suggest that microbial bioflocculants could provide a promising alternative to replace or supplement the physical and chemical treatment processes of river water and textile industry effluent. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2008.

The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

The objective of this study was to investigate the effects of land use and management practice on the soil organic matter content and the size, activity and diversity of the microbial biomass. These effects were investigated using samples taken from the top (0-10 cm) layer of the soils from long-term agricultural managements including natural grassland, maize under conventional (maize CT), maize under zero tillage (maize ZT), annual ryegrass, Eucalyptus, Pinus, and permanent kikuyu pasture. The natural grassland was used as a control since records indicated that no agricultural activity had ever been exerted on the soil. The measurements used to investigate these effects included soil organic C, total N, soil pH, microbial biomass C, basal respiration rate, microbial quotient, metabolic quotient, dehydrogenase activity, fluorescein diacetate (FDA) hydrolysis, arginine ammonification rate, arylsulphatase activity and acid and alkaline phosphatase activities. The microbial functional diversity was measured using the Biolog Ecoplate and catabolic response profiles methods. Soil organic Cand total Nwere lowest under maize CT, followed by maize ZT and annual ryegrass and were higher under natural grassland, Eucalyptus and Pinus plantations while permanent kikuyu pasture had the highest values. The other analyses, namely microbial biomass C, basal respiration rate, FDA hydrolysis, arginine ammonification rate and arylsulphatase activity also followed the same pattern. Annual cultivation was responsible for a decrease in microbial biomass C, basal respiration rate and enzyme activity, principally because there was an appreciable decrease in soil organic matter content. Conversely, permanent pasture, Eucalyptus and Pinus plantations increased appreciably the amount of organic C and consequently, promoted the size and activity of the microbial biomass in the soils. The principle component scores showed that management practices affected the microbial functional diversity because different treatments were found in separate zones of the principle component spaces. The regression analysis showed that the variation in the PC1 and PC2 scores was correlated with the variation in soil organic C, exchangeable acidity, extractable P and exchangeable K and Mg. In addition, richness, evenness, Shannon, and Simpson diversity indices showed that any management practice affects the dynamics of soil microbial diversity. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2002.

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

Microbial leaching plays a significant role in the natural weathering of silicate containing ores such as diamond-bearing kimberlite. Harnessing microbial leaching processes to pre-treat mined kimberlite ores has been proposed as a means of improving diamond recovery efficiencies. The biomineralization of kimberlite is rarely studied. Therefore, this study investigated the feasibility of exploiting both chemolithotrophic and heterotrophic leaching processes to accelerate the weathering of kimberlite. Preliminary investigations using mixed chemolithotrophic leaching cultures were performed on four finely ground kimberlite samples (<100μm) sourced from different mines in South Africa and Canada. Mixed chemolithotrophic cultures were grown in shake flasks containing kimberlite and inorganic basal media supplemented either with iron (Fe2+, 15g/l) or elemental sulfur (10g/l) as energy sources. Weathering due to dissolution was monitored by Inductive Coupled Plasma (ICP) analyses of Si, Fe, K, Mg and Ca in the leach solutions at known pH. Structural alterations of kimberlite after specified treatment times were analyzed by X-ray Powder Diffraction (XRD). The results of the preliminary investigation showed that weathering can be accelerated in the presence of microbial leaching agents but the degree of susceptibility and mineralogical transformation varied between different kimberlite types with different mineralogical characteristics. In general, the results showed that the kimberlite sample from Victor Mine was most prone to weathering while the sample from Gahcho Kue was the most resistant. It was therefore deduced that kimberlite with swelling clays as their major mineral component weathered relatively more easily when compared to kimberlite that consisted of serpentine and phlogopite as their major minerals. Gypsum precipitates were also distinguished indicating that a partial alteration in the kimberlite mineralogical structure occurred. Both energy sources positively influenced the dissolution process, with sulfur producing superior results. This was attributed to the generation of sulfuric acid which promotes cation dissolution and mineral weathering. Success in the preliminary investigations led to further experimental testing performed to determine the effect of particle size and varying energy source concentrations on the biotransformation of kimberlite. It was observed that although weathering rates of the larger kimberlite particles (>2mm<5mm) were lower than that of the finer particles, slight changes in their mineralogical structures represented by the XRD analyses were seen. Optimisation studies of energy source concentration concluded that although the highest concentration of elemental sulfur (20% w/w) and ferrous iron (35% w/w) produced the most pronounced changes for each energy source tested, the leaching efficiency at these concentrations were not drastically greater than the leaching efficiency of the lower concentrations, as expected. Following the success of batch culture shake flasks weathering tests, the effect of continuous chemolithotrophic cultures on the biotransformation of larger kimberlite particles (>5mm<6.7mm) was investigated. A continuous plug-flow bioleach column was used to model the behaviour of chemolithotrophic consortia in a dump- or heap leaching system. Two sequential columns were setup, in which the first consisted of kimberlite mixed with sulfur and the second purely kimberlite. Inorganic growth medium was pumped to the first column at a fixed dilution rate of 0.25h-1 and the leachate from the first column dripped into the second. After an 8 week investigation period, the ICP and XRD data showed that weathering did occur. However, the pH results showed that the leaching process is governed by the amount of acid produced by the growth-rate independent chemolithotrophic consortia. Data from pH analyses also showed that the leaching bacteria reached ‘steady state’ conditions from day 45 onwards. The pH also remained higher in the second column than in the first column highlighting the alkaline nature of the kimberlite ores and its ability to act as a buffering agent and resist weathering. This important factor, as well as further optimisation studies in process operating conditions and efficiency, needs to be considered when establishing heap-leaching technology for these kimberlite ores. In the preliminary heterotrophic investigation, Aspergillus niger was used to produce organic metabolites to enhance kimberlite mineralization. The results demonstrated that the organic acid metabolites generated caused partial solubilization of the kimberlite minerals. However, it was deduced that for more significant changes to be observed higher amounts of organic acids need to be produced and maintained. The results obtained in this study also showed that the type of kimberlite presents a different susceptibility to the dissolution process and the presence of the fungal cells may improve the leaching efficiency. The results in this study provided an optimistic base for the use of microbial leaching processes in accelerating the weathering of kimberlite. These findings may also serve to supply data to formulate recommendations for further and future column microbial leach tests as well as validation and simulation purposes. / Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2008.

The sources of gentamicin resistant pseudomonads and enterobacteria were studied in detail. A total of 1703 gentamicin resistant gram negative bacilli (GRGNB) isolated from patients, staff and their immediate environment were studied over a 6 month period . Of these 954 were isolated from clinical specimens obtained from patients and 540 from their immediate environment. A furthur 209 stains were isolated from the staff members who were responsible for the care of these patients. Pseudomonas aeruginosa; pyocin type 1 phage type F7 and .serotype 11 was the commonest isolate. It constituted 24,9% of all isolates in this study. This organism was distributed in all the wards investigated and was isolated throughout the 6 month study period. This strain, therefore, appears to be part of the "resident'' flora of King Edward Vlll Hospital for it was found on patients, staff and their immediate environment. Among the Enterobacteriaceae, Klebsiella pneumoniae was the commonest isolate and made up 13,6 % of all isolates. All the isolates obtained in this study were resistant to five of more antibiotics tested (gentamicin, tobramycin, kanamycin, streptomycin, carberricillin, polymyxin B amikacin and sisomicin). Of 310 staff members screened 25,2% harboured GRGNB on their hands. Among patients the commonest source of GRGNB was stool which yielded 141 (14,8 %) of the clinical isolates. Of the environmental sources studied, sinks harboured 87 (14%) GRGNB. The isolates from the environment and staff members were identical to patient strains. The significance of these findings is discussed. / Thesis (Fellowship of the Society of Medical Laboratory Technologists of South Africa)-University of Natal, Durban, 1985.

Members of the genus Bacillus are mainly Gram positive, aerobic rod shaped, endospore-forming bacteria that are increasingly being recognised for their ability to promote plant growth and antagonise fungal pathogens. From a biological control perspective, Bacillus spp. strains that produce antifungal compounds are of particular interest. In this study, aerobic endospore-formers were isolated from an undisturbed indigenous grassland soil and screened for antifungal activity and other plant growth promoting traits. Endospore-formers were also isolated from rhizosphere soil associated with the roots of maize, wheat and kale grown in pots containing soil from the same grassland site. Microbial diversity amongst isolates showing antifungal activity was investigated using different molecular fingerprinting methods, namely, intergenic transcribed spacer–PCR (ITS-PCR), random amplified polymorphic DNA-PCR (RAPD-PCR) and 16S rRNA gene amplification and sequencing. Characterization of the active antimicrobial compound(s) associated with selected isolates was also attempted. Prior to isolating from bulk and rhizosphere soils, samples were pre-heated to eliminate heat sensitive vegetative cells. Mean endospore counts were; wheat rhizosphere, Log 6.03 c.f.u g-1 soil; maize rhizosphere, Log 5.88 c.f.u g-1 soil; kale rhizosphere Log 5.90 c.f.u g-1 soil; and bulk soil Log 5.67 c.f.u g-1soil. A total of three hundred and eighty-four isolates were screened for antagonism towards Rhizoctonia solani using dual-culture plate bioassays. Thirty four of the isolates (~9%) mostly isolated from the bulk soil inhibited R. solani at varying degrees. Differences in antimicrobial interactions were apparent in in vitro bioassay; supposedly due to different concentrations and/or types of antimicrobial compounds. Biochemical tests for amylase, cellulase, chitinase, and proteinase activity, siderophore production and inorganic phosphate solubilisation were conducted. None of the isolates possessed all of these attributes and only a few showed multiple traits. Ninety-one percent of the isolates exhibited proteinase activity, 76% were able to hydrolyze starch whereas only four displayed cellulase activity. Only four isolates from the bulk-soil were capable of solubilising inorganic phosphate. ITS-PCR and 16S rRNA gene sequence analysis showed high levels of genetic homology amongst isolates and the majority were closely associated with representatives of the B. cereus group. Isolate C76 was the exception, being closely matched with B. subtilis. ITS-PCR banding profile was useful for distinguishing between species but did not distinguish within species. RAPD-PCR distinguished finer levels of genetic diversity between and within sample sets, with primer OPG-11 showing the greatest levels of heterogeneity. DNA extraction methods and the influence of template DNA dilution were investigated to determine their influence on RAPD-PCR analysis reproducibility. Prominent bands were comparable for crude template- and kit-extracted DNA but slight changes in band intensity and in some instances, additional faint bands were observed. At the highest DNA concentrations tested (7 μg/ml), further bands with molecular weights above 2.5 kbp were apparent. Strict standardization of PCR conditions greatly reduced variability of the RAPD-PCR analysis. Isolates from the different sample sets were screened for the presence of genetic markers associated with the biosynthesis of zwittermicin A, an aminopolyol antibiotic produced by some members of the B. cereus group. In an initial screen only one isolate, W96, yielded PCR amplicons consistent with those previously reported in the literature for the zwittermicin A genes. Later a further sixteen isolates grouped with W96 on the basis of the RAPD-PCR fingerprinting profiles, were screened for the presence of these genes. Of these, only six showed PCR amplification products similar to W96. Sequence homology testing against the GenBank database confirmed the presence of the zwittermicin A genes in these isolates. Isolate W96 was selected for further extraction and characterization of its antifungal compound(s). However, after culturing in various broth media cell free supernatants of W96 failed to show antifungal activity in vitro even when the supernatants were concentrated 20-fold. These findings provide a general overview of the diversity of aerobic endospore-forming bacteria present in an undisturbed indigenous grassland soil that exhibited antifungal activity in vitro and the limited influence tested crop rhizospheres have on this diversity. Combined use of ITS-PCR, 16S rRNA sequencing and RAPD-PCR techniques served as a rapid and effective means of grouping isolates for further investigations of their potential use as biocontrol agents and plant growth promoting rhizobacteria. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

The incidences of diarrhoeal episodes in infants and children have mostly been associated with the consumption of contaminated weaning foods. This is especially true in developing countries where factors such as the lack of sanitation systems and electricity have been found to contribute to an increase in the incidence of microbiologically contaminated weaning foods. The process of fermentation has been found to reduce the amount of microbiological contamination in such foods as a result of the production of antimicrobial compounds such as organic acids, peroxides, carbon dioxide and bacteriocins. In this study, microbiological surveys were conducted on sorghum powder samples and their corresponding fermented and cooked fermented porridge samples collected from an informal settlement of the Gauteng Province of South Africa. The process of fermentation was found to result in significant decreases (P>0.05) in Gram-negative counts and spore counts, while aerobic plate counts decreased slightly. Lactic acid bacteria counts, however, increased significantly (P>0.05). The cooking process was found to result in further significant decreases (P>0.05) in all counts. Sorghum powder samples and fermented porridge samples were found to be contaminated with potential foodborne pathogens, including Bacillus cereus, Clostridium perfringens and Escherichia coli, however, none of the pathogens tested for were detected in any of the cooked fermented porridge samples. SDS-PAGE and phenotypic analysis of 180 lactic acid bacteria isolated from sorghum powder samples and their corresponding fermented and cooked fermented porridge samples showed that a majority of the isolates were lactobacilli and leuconostocs, however, some isolates were identified as pediococci and lactococci. These results demonstrated the heterogeneity of the lactic acid bacteria isolates that were associated with fermentation processes in this study. Of the lactic acid bacteria identified, Lactobacillus plantarum and Leuconostoc mesenteroides strains were found to have the highest distribution frequencies, being distributed in 87% and 73% of the households, respectively. Analysis of Lactobacillus plantarum (58) and Leuconostoc mesenteroides (46) strains isolated from sorghum powder samples and corresponding fermented and cooked fermented porridge samples by AFLP fingerprinting showed that they originated from a common source, which was sorghum powder. There was, however, evidence of strains that may have been introduced at household level. Antimicrobial activity of selected lactic acid bacteria was found to be mainly due to a decrease in pH in fermented and cooked fermented porridge samples. None of the lactic acid bacteria tested seemed to produce bacteriocins.

With the exceptions of mixing and heating mechanisms, and the recycling of settled solids, no radical changes or improvements have been made to conventional anaerobic digesters treating municipal sewage. These digesters usually function with a hydraulic retention time of 30 to 60 days and at a total solids concentration of 2.6 %(m/v). Volumetric loading is limited since high loadings effect the displacement of the slow growing methanogens. Thus, the hydraulic retention time is coupled to the solids retention time. A crossflow microfiltration unit has been constructed at Northern Waste Water Treatment Works, Durban, to concentrate sludge from a conventional anaerobic digester and, thus, facilitate operation with a higher solids concentration. In addition, this process should result in the retention of the active biomass which would otherwise be lost as a waste product of the treatment process. The solids retention time is, thus, decoupled from the hydraulic retention time. The net result could be higher volumetric loadings, increased microbial activity and increased volatile solids destruction and, hence, improvement in the efficiency of anaerobic digestion of sewage sludge. To test these, different experiments were conducted to specifically determine the effect of higher solids loads. Preliminary experiments were undertaken to determine the biodegradability of primary sludge from the Northern Waste Water Treatment Works. Results showed that primary sludge of 76% VS could be reduced to approximately 48 to 50% VS during an experimental period of 85 days. Reduction of the first 20% VS was rapid if conditions were optimum but subsequent reduction from 55 to 50% VS was slow. It was calculated that approximately 0.88 l gas was produced for every g volatile solids catabolised. Further experiments were conducted to investigate the effects of different solids concentrations on microbial activity. The results showed that the volume of gas produced increased as the solids concentration increased from 2 to 6%(m/v). Digesters with solids concentrations of 6 to 13%(m/v) produced similar volumes of gas. Digesters with solids concentrations of 6 to 13%(m/v) TS produced approximately 300 ml more gas than the control during the 20 days experimental period. The rate of gas production also increased as the solids concentration increased. However, digesters containing 11%(m/v) and 13%(m/v) TS produced similar rates. These results indicate that the introduction of concentrated sludge into the digester improves digestion efficiency. Finally, a semi-continuous digester was operated at a 30 days retention time and at optimum temperature to investigate the efficacy of digesters with increased solids concentrations. The results showed that the rate of gas production increased as the solids concentration increased from 2%(m/v)(control) to 3.8%(m/v). However, the digester operated with 4.7%(m/v) TS produced gas at a rate lower that the digester with 3.8%(m/v) TS. The volatile solids concentrations of all four digesters were similar, indicating neither favourable nor unfavourable effects from increased solids concentrations. The digesters operated with 3.8%(m/v) and 4.7%(m/v) TS produced higher concentrations of volatile acids than the control. The alkalinity concentrations (>_4000 mg t-1 ) were similar for all four digesters. / Thesis (M.Sc.)-University of Natal, Durban, 1995.