Several problems concerning the use of conventional synthetic flocculants has necessitated the need for alternative cost effective, safe and efficient bioflocculants from microorganisms inhabiting many environments, particularly those from unusual environments. Hence, this study assessed bioflocculant production by three marine bacteria isolated from the bottom sediment of Algoa Bay in the Eastern Cape Province of South Africa. Analysis of the 16S rDNA sequences led to their identification as Halobacillus sp. Mvuyo, Virgibacillus sp. Rob and Oceanobacillus sp. Pinky. Several factors affecting the production and activity of the bioflocculant(s) were studied. Halobacillus sp. Mvuyo produced bioflocculant optimally with glucose (76%) and ammonium chloride (93%) as sole carbon and nitrogen sources, respectively and at neutral pH and in the presence of Ca2+. On the other hand, Virgibacillus sp. Rob preferred glucose (70.4 %) and iron sulphate (74%) as carbon and nitrogen source respectively; an alkaline pH (12.0) and Fe2+. Oceanobacillus sp. Pinky produced bioflocculant optimally when sucrose (80%) and peptone (72.4 %) were used as carbon and nitrogen source respectively, at neutral pH, and in the presence of Ca2+ cation. The chemical analyses of the partially purified bioflocculants revealed that the bioflocculants produced by Halobacillus sp. Mvuyo and Oceanobacillus sp. Pinky were glycoproteins, while that produced by Virgibacillus sp. Rob was a polysaccharide. We thus conclude that Halobacillus sp. Mvuyo, Virgibacillus sp. Rob and Oceanobacillus sp. Pinky hold promise as producers of new and efficient bioflocculant(s). We recommended development of process conditions for large scale production of the bioflocculants followed by their detailed characterization, as well as pilot scale assessment of the applicability of the purified bioflocculant in water/wastewater treatment and other industrial uses

Submitted in complete fulfillment for the degree of Doctor of Philosophy (Biotechnology), Durban University of Technology, Durban, South Africa, 2015. / Anaerobic digestion, a proven and highly efficient biological process for treating industrial wastewater and biogas generation is an underutilized technology in South Africa. Some of the industries that have on-site anaerobic reactors tend to face problems in operating these reactors due to poor understanding of the process and implementation of the technology. This has resulted in high pollutant loads in their final effluents and low energy recovery. In this study, an on-site full–scale upflow anaerobic sludge blanket (UASB) reactor treating brewery wastewater was extensively monitored in order to evaluate the efficiency in terms of effluent quality, biogas production and microbial structure. Furthermore, developed and adopted kinetic models were used to optimize the performance of the full–scale UASB reactor using a combined Pareto differential evolution (CPMDE) algorithm. A preliminary analysis of the raw wastewater has shown that the wastewater produced from the brewery industry was high in organic matter with a total chemical oxygen demand (COD) between 1096.41 to 8926.08 mg/L. The average removal efficiency of COD from the UASB reactor after treatment was 79% with a methane (CH4) production of 60-69% at temperature ranges of 28-32˚C and hydraulic retention time (HRT) of 12 h within the optimal pH range for anaerobic bacteria (6.6 and 7.3) under various organic loading rates. However, the results also showed an increase in total suspended solids (TSS), nitrogen (N2), ammonia (NH3) and orthophosphate concentrations when comparing the influent to the effluent, which indicated the necessity for further optimization of the reactor condition in order to reduce these effluent parameters to acceptable standards and to increase CH4 production. In order to optimize the process, a thorough understanding of microbial interaction was essential. A combination of different molecular techniques viz., fluorescence in–situ hybridization (FISH), polymerase chain reaction (PCR) and quantitative real-time PCR (QPCR) were employed to understand the microbial community structure of the granular sludge samples using species specific primers and probes. The results revealed that the dominance of diverse groups of eubacteria belonging to phyla Proteobacteria, Firmicutes and Chloroflexi and an uncultured candidate division WS6 with four different orders of methanogenic Archaea viz., Methanomicrobiales, Methanococcales, Methanobacteriales and Methanosarcinales belonging to hydrogenotrophic and aceticlastic methanogens were within the reactor samples. Quantification of the 16S rDNA copies of eubacteria and methanogenic Archaea using species-specific primers further confirmed the spatial distribution of these microorganisms within the different compartments of the reactor where, the upper compartments were dominated by eubacteria and the lower compartments by methanogenic Archaea. The concentration of Archaea per nanogram of DNA was much higher (96.28%) than eubacteria (3.78%) in lower compartments, while, the eubacteria concentration increased to 98.34% in upper compartments with a decrease in Archaea quantity (1.66%). A modified kinetic methane generation model (MMGM) was developed on the basis of mass balance principles with respect to substrate (COD) degradation and the endogenous decay rate to predict CH4 production efficiency of the reactor. Furthermore, a Stover–Kincannon kinetic model was adopted with the aim of predicting the final effluent quality in terms of COD concentration and model coefficients were determined using the data collected from the full–scale reactor. Thereafter, a model-based multi-objective optimization was carried out using the CPMDE algorithm with three–objective functions namely; maximization of volumetric CH4 production rate; minimization of effluent substrate concentration and minimization of biomass washout, in order to increase the overall efficiency of the UASB reactor. Important decision variables and constraints related to the process were set for the optimization. A set of non-dominated solutions with high CH4 production rates of between 2.78 and 5.06 L CH4/g COD/day at low biomass washout concentrations were obtained at almost constant solution for the effluent COD concentration. A high COD removal efficiency (85-87%) at ~30-31˚C and 8-9 h HRT was obtained for the multi-objective optimization problem formulated. This study could significantly contribute towards optimization of a full–scale UASB reactor treating brewery wastewater for better effluent quality and biogas production. Knowledge on the activity and performance of microbial community present in the granular sludge taken from the full–scale UASB reactor would contribute significantly to future optimization strategies of the reactor. In addition, optimization using an evolutionary algorithm under different operational conditions would help to save both time and resources wasted in operating anaerobic bioreactors.

Bacillus cereus is a group of ubiquitous facultative anaerobic spore forming Gram-positive rods commonly found in soil. It has been detected and implicated in several contaminated food products and raw milk in dairy farms and it causes foodborne gastroenteritis by producing several toxins. This study is aimed at characterizing virulence determinants of B. cereus from cow‟s raw milk. A total of 400 raw milk samples were collected in Fort Hare Dairy Trust and Middledrift Dairy Farm; and cultured on Polymyxin pyruvate Egg-Yolk Mannitol Bromothymol Agar (PEMBA) for 48 hours at 37°C. DNA was isolated from the isolates and 16S rDNA was amplified and sent to Central Analytical Laboratory for sequencing. The gyrB gene of B. cereus was also used to confirm the identity of the isolates. Antibiotic susceptibility profiles of the isolates together with virulence genes were investigated. Multilocus Sequence typing was used to investigate the genetic relatedness of some selected isolates. Furthermore, spores of the isolates were produced, harvested and their concentrations determined. All (100%) of the isolates were identified as having a 96-99% similarity to B. cereus, B. thuringiensis and B. anthracis using sequencing; while gyrB gene was observed in all (100%) of the isolates. Three virulence genes nheA, nheB, nheC encoding for non haemolysin enterotoxin were amplified in all (100%) the isolates. All (100%) of the isolates were susceptible to doxycycline, gentamycin, tetracycline, ciprofloxacin, chloramphenicol and streptomycin. Resistance to rifampicin and penicillin G was predominant with equal rate of 100%, while susceptibility to erythromycin, clindamycin and doxycycline ranged from 60% to 100%. The selected isolates were related and are descendants of the same ancestor. All (100%) the isolates produced spores. The B. cereus isolates contain virulence genes, has multiple antibiotic drug resistance and produce spores, which poses a health risk to the public and cannot be used as probiotics.

MSc (Microbiology) / Department of Microbiology

MSc (Microbiology) / Department of Microbiology / Background: Escherichia coli is a facultative anaerobic bacterium that forms part of the gut microbiota. It is used as an indicator that confirms recent faecal contamination. E. coli have been identified amongst the pathogens that are mostly responsible for moderate to severe diarrheal outbreaks in the low and middle-income countries. With South Africa facing an issue in water scarcity, issues concern poor sanitation and hygiene practices results in serious public health problems and allows E. coli to be transmitted from infected human or animal faeces to a new susceptible host using environmental reservoirs such as soil, water, hands as the transmission pathway. Objective: The primary objective of the study was to characterize E. coli strains from rural communities of Vhembe district, Limpopo, South Africa. Methodology: Households of 7 villages in the Vhembe district were randomly selected. A total of 81 households (HHs) were part of the study. In each household, a structured questionnaire was used to background information on WASH practices. Samples taken from each HH included toilet seat swabs, floor swabs, child and mother handwash samples, stored water samples and running tap water samples. A total of 399 samples were analysed using Colilert® Quanti-trays®/2000 method to detect the presence of Escherichia coli. Positive E. coli samples were further identified using multiplex polymerase chain reaction (m-PCR) to determine the pathogenic strains of E. coli. Transmission pathways were established using identified strains. Results: Data from the structured questionnaires showed common problems of availability of running tap water; lack of provision of sanitation; open practice on defaecation and very little hand hygiene practices. A total of 91 (22.81%) samples tested positive for E. coli with the Colilert® Quanti-trays®/2000 method. The mothers’ handwash samples had the most E. coli prevalence followed by stored water samples. The most prevalent E. coli pathotype was EPEC with the virulence gene eae. Atypical EPEC (60.44%) outnumbered the typical EPEC (5.49%). The pathotype ETEC was detected in 41.76% samples and EHEC in 9.89% samples. Transmission pathway was observed from the different households; with eae gene (aEPEC) being the most detected from samples looking at the LT gene (ETEC). v | P a g e Discussion: All 7 villages are facing common issues such as lacking running water, poor sanitation and improper hand hygiene practices. The mothers were the most contaminated and it was observed that its due to the daily activities that they perform around the house. It is of importance for them to practice proper hand hygiene to prevent transmission of pathogenic E. coli to the children via direct or indirect transmission route. The pathogenic E. coli was detected from all different samples collected from the households including the floor and toilet seat samples. EPEC was detected the most, and studies have shown that this strain is known to cause diarrheal infections in young children from developing countries. Conclusion: The members of the study village households were aware of the WASH services and its importance. However, proper implementation into their day-to-day life is lacking due to the high number of TC and E. coli detected from handwash samples and stored water samples from the households. Recommendation: Appropriate WASH strategies should be established to ensure good health at the village households. Further studies should be done to check possible transmission pathways from more village households. / NRF

MSCAGR / Department of Animal Science

This study focuses on the isolation and characterization of bacteria from lignocellulosic biomass obtained from the sediments of the Tyume River in Alice, Eastern Cape and to determine those bacterial isolates with good potential for modification and decomposition of lignocellulosic biomass for industrial application. Several bacterial isolates were recovered and screened for ability to degrade various lignocellulosic materials. Nine of the isolates were positive for lignocellulolytic activity. Four isolates were cellulase positive and six were xylanase positive. Moreover, one isolate (SB1) was positive for both xylanase and cellulase activities and showed the best hydrolysis zone on solid media. This isolate was then chosen as the best and identified molecularly. The 16S rDNA sequence analysis indicated that SB1 was a Bacillus cereus species. Factors affecting the cellulose and xylanase enzyme production by the organisms were studied. The organisms produced the enzymes maximally at earlier hours of incubation (12-30 hr) and optimally at acidic pH (3-5) and at moderate temperatures (35-45ºC). SB1 appears to hold promise in the decomposition of lignocellulosic wastes.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

MSc (Microbiology) / Department of Microbiology / Background: The high on-going incidences of infectious diseases, specifically those caused by multi-drug resistant bacteria in the last decade has made it a necessity to investigate a variety of antimicrobial drug sources, such as plants. Medicinal plants have played a significant role in drug discovery for western pharmaceuticals recently and have also been used successfully by traditional healers and herbalists to treat various infectious diseases for centuries. Currently, a few medicinal plants are commercialized, reason being most medicinal plants phytochemicals have not been studied yet, although they have been traditionally used by healers. Due to the constant development of multi-drug resistance of bacteria to antibiotics, S. africana extracts can provide an opportunity to finding new antibacterial compounds that can be used as the foundation for formulating new antimicrobial drugs. Objectives: The aim of this study was to screen antibacterial activity of the crude extract and fractions of S. africana against multi-drug resistant bacteria and to also evaluate other biological properties. Methods: Preliminary screening of phytochemical constituents of S. africana and fractions was done using standard qualitative and quantitative methods. Antibacterial activity of the extracts was evaluated using the agar well diffusion method and the microdilution assay against MDR bacterial strains. Antioxidant activities of the MCE and its fractions were measured by DPPH and reducing power assays, and the toxicity of the MCE and its fractions was tested on Vero cells using Cell-based high content screening assay. Results: Phytochemical analysis of the MCE and fractions obtained in this study showed the presence of phenolics, flavonoids, alkaloids, steroids, saponins, cardiac glycosides and terpenoids in most of S. african’s test samples. Fraction F1 and F2 both lacked alkaloids and saponins. The micro-plate dilution assay demonstrated that the MCE and all its fractions can inhibit the growth of all selected MDR bacterial strains tested against at different concentrations (0.1mg/ml to >12.5mg/ml), wherein the lowest MIC averages were obtained from fractions F3 and F6, with 0.59 mg/ml and 0.71 mg/ml MIC averages respectively. Contrary to the micro-plate dilution assay, the well diffusion assay demonstrated that MCE and all its fractions were not active against all the selected MDR bacterial strains tested against, as no inhibition was shown against the growth of K. pneumonia by any of S. african’s test samples. For DPPH assay, the IC50 of S. african’s test samples ranged between 0.01 ±0.34 mg/ml to 0.62 ± 0.05 mg/ml, whiles for the reducing power assay, EC50 measured ranged between 0.61 ± 0.01 mg/ml and 11.30 ± 0.04 mg/ml. The MCE and fraction F2 exhibited the highest toxicity to Vero cells. Conclusion: The MCE and fractions of the plant S. africana have antibacterial activity against MDR bacterial strains, beneficial biological properties and contains potential antibacterial compounds that may be valuable in the discovery of new potential drugs for treatment of infectious diseases / NRF