Doctoral Degrees (Microbiology)

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Alkalic salt-based pretreatment strategies for enhancing sugar recovery from corn cobs and process development for simultaneous saccharification and bioethanol production.
(2018) Sewsynker-Sukai, Yeshona.; Gueguim Kana, Evariste Bosco .
Abstract available in PDF file
Bacterivory of three freshwater ciliates isolated from Blackbarough Spruit in KwaZulu-Natal, South Africa.
(2021) Bulannga, Rendani Bridghette.; Schmidt, Stefan.
Abstract available in PDF.
Beta-lactamase mediated resistance in Salmonella spp. at a tertiary hospital in KwaZulu-Natal.
(2008) Govinden, Usha.; Essack, Sabiha Yusuf.; Sturm, Adriaan Willem.; Moodley, Prashini.
Extended spectrum (3-lactamases (ESBLs) were characterized in Salmonella spp. isolates from a pediatric ward of a hospital in Durban. Forty one Salmonella spp. were subjected to serotyping, antibiotic susceptibility testing, E-Tests for ESBL detection, iso-electric focusing, polymerase chain reaction for detection of genes and sequencing. Isolates were screened for the presence of WaTEM, WaSHV, WaCTX-M, WaOXA , WaCMY, WaDHA and WaACC genes. The most common serotype was Salmonella Typhimurium. Isolates were multi-drug resistant with 100% susceptibility only to meropenem and ciprofloxacin. Tazobactam was the most effective inhibitor. Forty-one percent of the isolates were resistant to ceftriaxone, thus limiting therapeutic options for Salmonella infections.TEM-1 was the most predominant (3-lactamase found in 51% of isolates while SHV-12 found in 39 % was the most common ESBL. TEM-63 was evident in 29 %, TEM-116 in 10 % and TEM-131 was found in one isolate. The high ceftazidime MICs of isolates expressing only TEM-63 were indicative of R164S substitution which widens the binding cavity to accommodate the bulky side chains of oxyiminoaminothiazolyl cephalosporins. The identification of TEM-131 which differs from TEM-63 by 1 amino acid reiterates the evolutionary potential of the TEM-type plactamase. Other ESBLs identified included SHV-2, CTX-M-3, CTX-M-15 and CTX-M-37. CMY-2 and the OXA-1 p-lactamase were also detected. This is the first report of TEM-116, CTX-M-3, -15 and -37 in Salmonella spp. in South Africa. All isolates with nalidixic acid MICs > 48 ug/ml had the mutation D87N, or D87G in the QRDR of the gyrA gene. This study showed that Salmonella spp. may be multi-drug resistant with the propensity to harbour p-lactamases in unique combinations. The diversity of ESBLs and the co-expression of quinolone resistance suggests that their incidence in salmonellae needs to be monitored.
Biodegradation of pentachlorophenol by Bacillus tropicus isolated from activated sludge of a wastewater treatment plant in Durban, South Africa.
(2021) Aregebesola, Oladipupo Abiodun.; Olaniran, Ademola Olufolahan.; Mokoena, Mduduzi Paulos.
Abstract available in PDF.
Bioremediation of chemically contaminated soil : extraction/analysis methodology development.
(2002) Khan, Fatima.; Wallis, Frederick Michael.
The efficacies of soil extraction methods, namely, Soxhlet, sonication, agitation, alkaline digestion and the ethyl acetate micro-method, for monitoring soil bioremediation were evaluated using three soil types, Swartland, Rensburg and Hutton, encompassing the mineralogical range prevalent in Kwa Zulu Natal. Phenol, atrazine and the BTEX component of petrol were the molecules used in this study and were extracted under different spiking concentrations, after prolonged ageing times up to 21 days and after changing the composition of the spiking solution. It was concluded that extraction methods must be validated for the specific conditions under which they would be used, taking into consideration, soil type, spiking solutions, moisture content, weathering times and the analyte(s) in question. A preliminary appraisal of atrazine degradation in a Hutton soil was then made under the conditions of sterilized, fertilized/non-fertilized and non-sterilized, fertilized/nonfertilized soils. The predominant pathway of atrazine degradation was deemed to be chemically/abiotically mediated due to the soil pH and the presence of iron and aluminium oxides as well as the high levels of manganese in the soil. The results obtained prompted further study into atrazinecatabolism using soil-slurry reactors, under the conditions of carbon-limitation, nitrogen limitation, carbon/nitrogen non-limitation and carbon/nitrogen limitation. A comparison was made between inoculated and non-inoculated bioreactors. The ability of the indigenous microbial population to return the Hutton soil to its original pristine state was confirmed. The expense of inoculation and culture maintenance could be avoided since carbon and nitrogen supplementation would be as equally effective as inoculation.
Bioremediation of creosote-contaminated soil by microbial intervention..
(2002) Atagana, Harrison Ifeanyichukwu.; Haynes, Richard John.
No abstract available.
Bioremediation of heavy metal polluted waters.
(1995) Meyer, Angela.; Wallis, Frederick Michael.
Microorganisms have the potential to remove heavy metals from polluted waters and effluents and may be used in clean-up processes. Microbial associations were enriched for and adapted to grow in nutrient solutions containing various concentrations of different metals. As immobilised cells are known to be more stable and more efficient in metal uptake than are corresponding planktonic or free-living cells the attachment of the microbial associations was investigated using a model stream and it was found that biofilm development was better on rough surfaces such as ground glass and polystyrene than on smooth surfaces such as unetched glass plates and glass beads. When comparing metal uptake by planktonic and attached microorganisms, attached populations were found to have a greater metal-uptake capacity. The uptake of individual metals from various metal combinations was tested with various proportions of pregrown metal-adapted microbial populations as inoculum and it was found that a particular metal was taken up more readily by microbial associations which had previously been exposed to that metal. Lead (Pb2+) appeared to be taken up more readily than copper (Cu2+) or cadmium (Cd2+) while Cd2+ was more actively removed than Cu2+ from solution. pH also affected metal uptake and the optimum range for Cu2+ uptake by the Cu2+ -adapted microbial association was found to be between 5.8 and 7.0. Dead microbial biomass was investigated and found to have efficient metal uptake capacity. Living mycelium from an isolated Aspergillus species showed poor uptake of Cu2+ initially, but when this fungus was pregrown and subsequently killed by moist heat treatment the non-living mycelium was efficient in removal of Pb2+ and Cu2+ ions. The optimum mycelial biomass concentration for metal uptake was also determined. The mechanism of metal uptake by this Aspergillus species was determined, using electron microscopy and EDX techniques, to be metabolism-independent biosorption onto the hyphal surface. Thus the microbial associations and fungal cultures used in this study were shown to have the potential for use in the removal of heavy metals from polluted waters.
Bioremediation of oil-contaminated soil : a South African case study.
(1996) Lees, Zoë Marie.; Senior, Eric.; Hughes, Jeffrey Colin.
In 1990, an oil recycling plant situated in Hammarsdale, South Africa, was decommissioned and a decision was taken by management to rehabilitate the site in preparation for resale. The heavily impacted area covered over two hecatares and oil contamination penetrated soil to depths in excess of three metres, making excavation and removal of the soil very expensive. The options for remediation of the site were limited. No facility for incineration of contaminated soil exists in South Africa, and landfilling was not permitted. The emphasis in developing a remediation strategy, therefore, focussed upon the possibility of in situ remediation with minimal excavation of soil. This study, the first of its kind in South Africa, was subsequently initiated to assess the feasibility of this approach, the results of which would underpin a full-scale cleanup programme. The development of such a strategy involved four key stages of work : (1) a comprehensive site investigation to evaluate and fully understand the particular problems at the site; (2) treatability studies to determine the potential for biological treatment of the contaminated soil and the optimisation of such treatments, particularly in terms of time and cost; (3) the testing of some of the more effective treatments on a pilot-scale; and (4) recommendations for full-scale bioremediation of the contaminated site. various conditions unique to South Africa had to be considered at each stage viz. the lack of funds and remediation experience, which created numerous problems and emphasised the requirement for a simple, "low-tech" approach. Site investigations revealed that in situ remediation may be possible due to the high permeability of the sandy soils and low concentrations of heavy metals. Laboratory experiments also showed that a mixed association of indigenous microorganisms was present which, once stimulated by nutrient supplementation at C:N:P, ratios of between 10:1:1 and 20:1:1, was capable of degrading total petroleum hydrocarbons at an average rate of 11% week -1. Further experimentation, aimed at reducing the cost of remediation and improving the soil quality, focussed on the efficacy of oil solubilisers, a soil ameliorant (composted pine-bark), indigenous fungi and higher plants in the remedial process. Three commercial surfactants (Arkopal N-050, N-060 and E2491) and one natural solubiliser (soybean lecithin) were tested for their biotoxicity, solubilisation and biodegradability at various concentrations (0.01 - 1.0%). Formulation E2491 was able to support a microbial population and was selected as the preferred commercial surfactant if soil washing was to be recommended; however, lecithin was considered to be more useful in situ because of its localised solubilising effect, biological origin and nutritional contribution. The use of fungi was of particular interest in addressing the persistent organic compounds, such as the heavy fractions of oil, for which bacterial remediation methods have been slow or ineffective. While it was not possible, however, to demonstrate in the laboratory that the indigenous fungi contributed significantly towards the degradation of the contaminating oil, the basic trends revealed that the fungal component of the indigenous microbial population was readily stimulated by the addition of nutrient supplements. The bulking-up process was also a success and additional exploratory work was proposed in the form of a larger scale composting design. Finally, the potential for using higher plants and 20% (v / v) composted pinebark (in addition to nutrients) to increase the microbial degradation of the contamination was investigated in both greenhouse and field plot studies. Greenhouse investigations employed soybeans which were postulated to have soil quality and cost benefits. However, although the soybeans were found to significantly enhance the remedial process, the complex soil-contaminant- plant interactions gave rise to strange nutritional effects and, in some cases, severe stunting. In contrast, the field studies employed grasses that had previously established on the site and which ultimately demonstrated a better tolerance for the contaminated conditions. Scanning electron microscopy revealed that there were considerable differences between the root tips of soybean plants which had been grown in contaminated soil and those which had been grown in uncontaminated soil. It was concluded that toxicity symptoms, which are readily observed in the root, could be used as an early indicator for determining the suitability of vegetation for remediation purposes. In both instances, despite the differences, the addition of composted pine-bark and nutrients (nitrogen and phosphorus) resulted in total petroleum hydrocarbon reductions of >85%, illustrating the benefits of plant establishment and oxygen availability. The need to link results from laboratory or pilot-scale experiments to achieve reliable predictions of field-scale behaviour was an essential component of this research. The results of the field study provided evidence, similar to that found in the pot trial, of the accelerated disappearance of organic compounds in the rhizosphere. All experiments incorporated parallel measurements of hydrocarbon residues, microbial activity and pH changes in the contaminated soil, the results of which strongly supported the argument that biodegradation was the dominant component of the remediation process. Thus, after consideration of the significant interactions which dominated the study (time-contaminant-nutrient; time-contaminant-pine-bark; and time-contaminant- pine-bark-plant), it was clear that, aside from these limiting factors, little should preclude the in situ bioremediation of the impacted soil.
Characterisation of chlorinated-hydrocarbon-degrading genes of bacteria.
(2009) Govender, Algasan.
1,2-dichloroethane (DCA) is one of the most widely used and produced chemicals of the modern world. It is used as a metal degreaser, solvent, chemical intermediate as well as a fuel additive. This carcinogen is toxic to both terrestrial and aquatic ecosystems and accidental spills and poor handling has resulted in contamination of the environment. Thus far several bacteria in the Northern hemisphere have been identified that are capable of utilizing this compound as a sole carbon and energy source. This report focuses on the isolation and characterization of bacterial isolates from the Southern hemisphere that are capable of degrading DCA as well as the global distribution of the DCA catabolic route. Samples obtained from waste water treatment plants were batch cultured in minimal medium containing DCA and repeatedly sub-cultured every five days over a 25 day period. A halogen release assay was performed in order to determine whether individual isolates possessed dehalogenase activity. Confirmation of DCA utilization by bacterial isolates positive for dehalogenase activity was done by sub-culturing back into minimal medium containing DCA. Enzyme activities were confirmed with cell free extracts using all of the intermediates in the proposed DCA degradative pathway and compared to a known DCA degrading microorganism. Biochemical tests and 16SrDNA sequencing indicated that all the South African isolates belonged to the genus Ancylobacter and were different from each other. Based on enzyme activities, it was found that the South African isolates may possess a similar degradative route as other DCA degrading microorganisms. Primers based on genes involved in DCA degradation were synthesized and PCR analysis was performed. It was found that all isolates possessed an identical hydrolytic dehalogenase gene whereas the other genes in the pathway could not be PCR amplified. Southern hybridization using probes based on known genes indicated that some of the isolates had homologous genes. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis indicated that the five South African isolates of Ancylobacter aquaticus are distinguishable from each other. This study is the first report indicating that microbes from different geographical locations use similar metabolic routes for DCA degradation. The first gene of the pathway (dhlA) has undergone global distribution which may be due to widespread environmental contamination.
Chlorine dioxide and ozone facilitated disinfection of selected bacteria in aqueous systems.
(2018) Ofori, Isaac.; Jonnalagadda, Sreekantha Babu.; Lin, Johnson.
Chlorination is the most commonly used disinfection technology for the control of pathogenic microorganisms in drinking water or wastewater treatment. However, the reactions of chlorine with natural organic matter in water have been found to produce harmful by-products including trihalomethanes, haloacetic acids, and haloacetonitriles. Regulations on these deleterious disinfection by-products keep increasing and have consequently focussed considerable attention on the use of alternative chemical disinfectants. The design and operation of an efficient disinfection system at a water treatment facility aim at providing adequate control of microbial threats and simultaneously satisfying regulatory requirements on by-products. Achieving these require an in-depth understanding of the inactivation kinetics of the disinfectant on potential target organisms. In this study, the microbial inactivation kinetics of chlorine dioxide (ClO2) as an alternative chemical disinfectant for water treatment was monitored on two Gram-negative bacterial species: Escherichia coli (ATCC 35218) and Pseudomonas aeruginosa (ATCC 27853) and a gram-positive: Staphylococcus aureus (ATCC29313) under varied conditions of disinfectant concentration, pH, temperature and bacterial density in an oxidant demand free water. Further studies were conducted to investigate the effect of ClO2 on bacterial outer cell membrane permeability, the cytoplasmic membrane integrity, inhibition of intracellular enzyme activity and changes in cell morphology by TEM to elucidate the bactericidal mechanism of action of ClO2. In addition, autochnous bacteria from urban wastewater were exposed to chlorine dioxide and the susceptibilities monitored and compared by a culture-dependent heterotrophic plate count technique and culture-independent 16S rRNA gene-directed polymerase chain reaction (PCR) based denaturing gradient gel electrophoresis (DGGE). Furthermore, the influence of four organic solvents commonly discharged from industrial lines into wastewater systems, namely, ethanol, methanol, ethyl acetate and dimethyl sulfoxide (DMSO) on ozone absorption, stability and consequent inactivation of Escherichia coli (ATCC 25218) and Staphylococcus aureus (29213) in water were also examined. Chlorine dioxide showed strong and rapid disinfection capabilities at relatively lower dosages with significant influences by pH and temperature. However, the efficiency generally appeared unaffected by changes in bacterial density. The PCR- DGGE technique showed that 1.0 mg/L was sufficient to inactivate three predominant bacterial species from an urban wastewateridentified as Arcobacter suis F41, Pseudomonas sp strain QBA5 and Pseudomonas sp B-AS- 44, whereas a significant population of other species such as Pseudomonas sp CCI2E was observed to presumably remain viable to 5.0 mg/L chlorine dioxide whilst the heterotrophic plate count method indicated complete elimination of bacteria at 3.0 mg/L. ClO2 was not found to inactivate bacteria by inflicting gross morphological damages to the cell wall, but instead, increases the permeability of the outer cell membrane, disrupts the integrity of the inner cytoplasmic membrane which leads to the efflux of intracellular contents of the cell and hence, resulting in the overall cell death. The presence of ethyl acetate and DMSO were observed to significantly enhance ozone absorption and stability in water with a consequent increase in bacteria inactivation efficiency whilst methanol-containing water rather accelerated the decomposition of ozone. The findings herein provide further knowledge to enhance the disinfection operations at a water treatment plant when ClO2 or O3 is applied.
Clinical strains of mycobacterium tuberculosis induce strain-specific patterns of cytokine production, gene expression and pathway changes in pulmonary alveolar epithelial cells.
(2016) Mvubu, Nontobeko Eunice.; Pillay, Manormoney.; Pillay, Balakrishna.
The epidemiological success of M. tuberculosis strains, dominant in different geographic regions globally, may be ascribed to a subversion of the host‟s protective immune response. The increasing prevalence of F15/LAM4/KZN, Beijing, F11 and F28 Mycobacterium tuberculosis strain families, coupled with rapidly evolving drug resistance within the KwaZulu-Natal province of South Africa population has resulted in a need to characterize host response associated with infection by these strains. Therefore, in this study, cytokine/chemokine production and host transcriptomics were investigated in A549 pulmonary epithelial cells infected with the F15/LAM4/KZN, Beijing, F28, F11, Unique and H37Rv strains. Cytokines/chemokines were quantified using the Bio-Plex Pro Human Cytokine 27-Plex assay at 0, 24, 48 and 72 hr post-infection. Changes in host gene expression were determined by whole genome RNA Sequencing (RNA-Seq) using the Illumina HiSeq 2000 platform. The 50 bp reads were mapped to the human genome (hg19) using Tophat (2.0.10). Differential expression was quantified using Cufflinks (2.1.0) with false discovery rate (FDR) of 0.05 and a log fold change cutoff of ≥2. R commands (Bioconductor), MeV and Ingenuity Pathway Analysis (IPA) were used to generate heat maps, network and pathways analysis. Twenty-three out of 27 analytes were detected. All strains, except the F28 strain induced an increased production of 18, and a decrease in 5 cytokines/chemokines at 24, 48 and 72 hr post-infection, compared to the uninfected control. Increased production of all 23 analytes by the F28 strain occurred at 48 and 72 hr. Among the 23 cytokines/chemokines that were detected, anti-inflammatory and pro-inflammatory cytokines, as well as chemokines were produced at the different time intervals. Compared to the other strains, high cytokine levels were induced by the F28 strain at 48 hr and F15/LAM4/KZN strain at 72 hr for most analytes. A lower cytokine production was induced by the Beijing and Unique strains at all time intervals. In the case of the laboratory strain H37Rv either a higher or lower cytokine/chemokine production was observed, compared to the clinical strains. RNA-Seq revealed differential gene expression that varied among the strains with respect to both up- and down-regulated genes: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A total of 292 genes were commonly induced by all strains, of which 52 were down-regulated and 240 were up-regulated. Different strain combinations induced different genes that were involved in a variety of pathways, including immune response and apoptosis pathways. Furthermore, strain specific genes were activated by each strain as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (185) and H37Rv (125). The F15/LAM4/KZN, Unique and H37Rv were the only strains that had molecular signatures with overlapping functional Kegg and Reactome pathways for their specific genes. IPA analysis revealed canonical pathways that differed among the strains, with the interferon signalling and hepatic fibrosis/hepatic stellate cell activation pathways being among the top 5 pathways in all the strains. Cholesterol biosynthesis and immune related pathway enrichment was similar in the Beijing and Unique strains whilst the F15/LAM4/KZN strain showed closer relatedness to the F11 strain, and the F28 strain closely clustered to the H37Rv strain. The Beijing and Unique strains highly enriched cholesterol biosynthesis pathways compared to other clinical and laboratory H37Rv strain. The top scoring networks induced by these clinical strains varied among the strains with the associated functions. These gene networks were involved in antimicrobial response, developmental disorder, organismal injury, infectious disease and cellular development. Among the transcriptional factors, only EHL, IRF7, PML, STAT1, STAT2 and VDR were induced by all clinical strains, while other factors were strain specific. In conclusion, low cytokine/chemokine production and activation of immune associated pathways by the Beijing and Unique strains suggest a higher virulence for these strains compared to the F15/LAM4/KZN, F11 and F28 strains. These characteristics may explain the high transmissibility and prevalence of the Beijing strains. A similar pattern exhibited by the less prevalent, non-clustering Unique strain, may suggest some virulence attributes in common with the Beijing strain. Findings in this study have the potential to reveal useful biomarkers that can be used as targets for alternative TB therapeutics including immunomodulators that take into consideration network regulations and strain-specific pathways and molecular signatures.
The culture-independent analysis of fungal endophytes of wheat grown in KwaZulu-Natal, South Africa.
(2016) Burgdorf, Richard Jörn.; Jamal-Ally, Sumaiya Faizal.; Laing, Mark Delmege.
Fungal endophytes are of interest due to their diverse taxonomy and biological functions. A range of definitions exists based on their identity, morphology, location and relationship with their host. Fungal endophytes belong to a wide range of taxa and they are categorized by a variety of characteristics. The detection and identification of these fungal endophytes can be performed using culture-dependent and culture-independent methods. These organisms have a range of application in pharmaceutical discovery and agriculture. Agricultural applications include the exploitation of the growth promoting and protective properties of fungal endophytes in crops such as wheat. This important crop is grown in South Africa where biotic and environmental stresses pose a challenge to its cultivation. Fungal endophytes have demonstrated potential to ameliorate these challenges. Future research will reveal how they can be harnessed to fight food insecurity brought about by stress factors such as climate change. Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under Variable Pressure Scanning Electron Microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies. Denaturing gel electrophoresis (DGE) can be used in culture-independent studies of microbial community composition and the technique has several variants. This work compared two of these variants, namely denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient electrophoresis (TTGE), to establish their relative performance in terms of resolution and detection, as well as cost and preparation time. Per gel reagent and material costs and preparation times were recorded for comparison. Conversion formulae were developed to standardize denaturing conditions for comparison of DGGE and TTGE gels. For all gel samples, band numbers, positions, peak height and base width were recorded. Samples run on DGGE gels tended to be clearer and more distinct from each other and DGGE tended to provide higher band numbers and better resolution. However, TTGE was quicker and cheaper to prepare. The TTGE and DGGE gel data were strongly correlated but DGGE provided more accurate dendrograms for comparisons of pure fungal isolates. Non-metric multidimensional scaling showed that TTGE data profiles were more heterogeneous, while DGGE produced tighter clustering of replicate samples. Although TTGE could be an acceptable technique for resolving DNA sequences in certain applications, DGGE is preferable for fungal wheat endophyte studies. Fungal endophyte community composition can be affected by various factors, such as the host genome. Research into the host genome effects on fungal endophyte composition can assist in harnessing the potential benefits of such relationships in agro-ecosystems. Several culture-based studies have investigated the presence of a cultivar effect on endophyte composition. However, a culture-based approach can only detect organisms that can be isolated and grown. Culture-independent methods can detect both culturable and non-culturable fungal endophytes for comparisons of fungal endophyte community composition (ECC) between wheat cultivars. Denaturing gradient gel electrophoresis (DGGE), high-resolution melt (HRM) analysis of community profiles, quantitative PCR, and sequence analysis were used to analyse and compare the fungal ECC of four wheat cultivars grown under field conditions. A significant organ and cultivar x organ interaction effects on fungal biomass were observed. A chytrid, namely Olpidium brassicae formed a significant component of the fungal endophyte community across all tissues in wheat. This finding highlighted the utility of the culture-independent in revealing cryptic interactions and endophytes, and raised questions about the factors that influence the organisms that reside within field-grown wheat. Systemic fungicides used in wheat production are pathogenic to many plant-inhabiting fungi such as fungal endophytes. The aim of the study was to reveal the effect of tebuconazole on the eukaryotic endophytes of wheat flag leaves using next generation sequencing (NGS). Treated and untreated leaves were surface sterilized prior to metagenomic DNA (mDNA) extraction. NGS was performed on DNA amplified using universal ITS primers. SCATA analysis was used for operational taxonomic unit (OTU) assignment of sequences, which were identified against CBS, UNITE and Genbank databases. A maximum likelihood (ML) tree was developed for taxonomic assignment of key genera. OTU mean read numbers and OTU richness were compared. The treatment effects were analysed using Principal Component Analysis (PCA), permutational multivariate ANOVA (PERMANOVA), distance-based test for homogeneity of multivariate dispersions (PERMDISP) and similarity percentage analysis (SIMPER). With one exception, non-wheat OTUs belonged to the Dikarya. Puccinia read numbers differed significantly (p = 0.01) between treatments and fungicide treatment tended to reduce total OTU read numbers and OTU richness. The variability of most key OTUs correlated positively with unsprayed samples. Treatment influenced OTU composition. Treated samples had the greatest homogeneity in endophyte composition and Puccinia made the greatest contribution to variation, with low contribution from the other OTUs. Dikarya were the dominant wheat flag leaf endophytes, and while the fungicide suppressed Puccinia and reduced fungal endophyte abundance, it did not significantly alter the community assemblage.
Design, optimisation and costing of a novel forced-upflow bioreactor for bioremediation of leachates from selected landfill sites in KwaZulu-Natal.
(2011) Vaughan, Halina.; Tivchev, George V.; Wallis, Frederick Michael.; Laing, Mark Delmege.
Most waste generated in South Africa is sent to landfills for disposal, and although it is confined in specific areas, it can potentially affect both above and below ground water resources, impacting environmental and public health. This is particularly relevant in a country where water supplies are limited and groundwater resources are prone to pollution. The primary objective of this study was to assess the performance of an upflow packed-bed bioreactor purposedesigned for the treatment of leachates produced by landfills in the Durban Metropolitan Area (DMA). The effect of parameters such as the nature of the biofilm support matrix, aeration rate and recycle rate on the efficacy of the system were investigated. Another major aim of the project was to develop a low maintenance technology that could, nonetheless, bioremediate leachate effectively at minimum cost. This aspect of process design is a crucial factor in areas where there is a shortage of both funds and skilled labour. The glass 132 l packed-bed upflow bioreactor was evaluated by measuring its efficiency in terms of chemical oxygen demand (COD) and biological oxygen demand (BOD) reduction and ammonia removal. The bioreactor could be configured as a batch-type system, which was useful for comparing operating conditions; or as a continuous cascade system, which was used to assess its overall performance. Different biofilm support matrices viz. various grades of pine bark, plastic bioballs and ceramic noodles were evaluated in 22 l batch-type reactors. Leachates from five landfill sites were remediated during the course of the study, and only the leachate from Shongweni landfill, which had a remarkably low BOD:COD ratio (0.05), was intractable and could not be successfully treated; even in flask trials designed to test strategies such as augmentation of microflora and biostimulation. The other leachates investigated were from the Umlazi, Marianhill, Bisarsar Road (all general sites) and Bul-Bul Drive (a semi-hazardous site) landfills, all of which were remediated to some degree. Originally, leachate from the Umlazi landfill site was used, but it became unavailable when the site closed enforcing the use of other leachates for the remainder of the investigation. Leachates from Marianhill, Bisarsar Road and Bul-Bul Drive were treated simultaneously in duplicate operating the six-chambered bioreactor in the batchtype configuration. The highest COD removal efficiency (49 %) was obtained in the chambers treating the Bul-Bul Drive leachate, which was therefore used for further investigations. This leachate had the highest BOD:COD ratio and was therefore expected to be the most suited to biological remediation. The bioreactor performed best when plastic bioballs were used as biofilm support matrix with a relatively low level of aeration, although the uncomposted form of pine bark was used initially as the support matrix because it is inexpensive and readily available in South Africa. However, although satisfactory COD reduction (30 – 61 %) and ammonia removal (87 – 98 %) was achieved when the Umlazi leachate was treated, the possibility of compounds leaching out of the bark and affecting the quality of the treated leachate was a concern. Also, pine bark would be prone to mechanical degradation in a full scale operation. Of the other solid support matrices tested using the Bul-Bul leachate, COD removal efficiencies were superior with plastic bioballs (60 %) than with pine bark chips (29 %). The former therefore became the preferred biofilm support matrix. Aeration level did influence bioremediation of the Umlazi landfill leachate since those chambers aerated with an aquarium pump (0.05 – 0.1 litres air/litre leachate/min; 60 % COD removal) performed better than those aerated with a blower (0.6 -0.7 litres air/litre leachate/min; 42 % COD removal) and those that remained unaerated (44 % COD removal). Recycle rate did not significantly affect bioremediation, but the performance of the system was higher when operated in batch mode (up to 60 % influent COD removal), rather than in continuous flow-through (cascade) mode when only 37 % of the influent COD in the Bul-Bul leachate was removed. Under the latter conditions, most of the reduction occurred in the first four chambers and very little biodegradation occurred in the final two chambers. The cascade-mode will require some refinement to enhance the COD removal efficiencies achieved. However, it did eliminate 89 % of the BOD present in the raw leachate, producing a treated effluent with a consistent BOD:COD ratio of 0.05. The COD removal efficiencies achieved covered a wide range from a minimum of 23 % with Marianhill leachate to a maximum of 63 % with leachate from Bul-Bul Drive. These results are comparable with many of those reported by other authors treating landfill leachate. Up to 98 % of the ammonia was removed when the Umlazi leachate was treated. However, ammonia removal from the other leachates tested was erratic. Although the treated leachate from this system could not be released into the environment without further remediation, the reduction in concentration of pollutants would allow its return to the local water supply via a wastewater treatment plant. This was achieved without temperature and pH regulation or addition of extraneous nutrient sources. A cost-effective, low maintenance technology such as this one would be a useful tool for the treatment of effluents such as landfill leachate in countries like South Africa where although water conservation is urgently required, resources for highly sophisticated effluent remediation are often not readily available.
Development of a bioreactor system using a pine bark matrix for the removal of metal ions from synthetic aqueous solutions.
(2013) Van Zuydam, Jason Peter.; Wallis, Frederick Michael.
Many industries use, or produce, metal-containing solutions which must be treated for reuse or discharge to sewer. One such treatment is biological and both living and dead materials have been investigated for the abstraction of metal ions from solution. Studies on systems containing only a single biosorbent are well documented, and mostly involve optimisation of biosorption capacities and metal uptake rates through modification of Biological Support Particle (BSP) size and surface characteristics. Literature on dual biosorbent studies is sparse. The commercial application of biosorption technology in wastewater treatment remains largely unexplored and unexploited. The primary objective here was to assess the potential of forced-upflow packed-bed bioreactors, containing dual biological sorbents, for treating a synthetic wastewater containing copper, zinc and cadmium, at both laboratory- and pilotscale. Pine bark was selected as BSP since it is an abundant, relatively cheap, agricultural waste product in South Africa, and is known to sorb metal ions. Initial experiments aimed to optimise biofilm development on the pine bark surfaces, since microbial biomass is also known to sequester metal ions. Systems comprising either one, or both, these biosorbents were compared for their efficiency in metal removal. The effects of type, size, and state of decomposition, of the pine bark, the addition of supplementary nutrients (Voermolas) and the mixing conditions, on the metal biosorption capacity and reaction kinetics of the systems were also studied. All experiments were conducted at an initial metal concentration of 100mg.ℓ⁻¹with both composted and uncomposted pine bark as BSP. The former supported microbial colonisation and resisted biofilm sloughing, but degraded rapidly causing engineering difficulties. Uncomposted pine bark showed the same ability, but was also physically more robust. Organic compounds leached from the pine bark did not hinder microbial colonisation of the BSP; rather they served as additional nutrients. Literature studies suggest that these compounds would not significantly compromise the COD or increase the toxicity of the final effluent. Biofilms developed without supplementary nutrients, but Cd²⁺ and Zn²⁺ were sorbed more effectively in bioreactors containing Voermolas (39% and 38% Cd²⁺ removal, 36% and 32% Zn²⁺ removal, in 0.2% and 0.1% Voermolas solutions respectively) than in unsupplemented systems (25% Cd²⁺ removal and 20% Zn²⁺ removal). Conversely, Cu²⁺ was removed most efficiently in the absence of supplementary nutrients. Based on biosorption of the target metal ions, 0.1% (v/v) Voermolas was the most effective concentration of supplementary nutrients. Raw, un-colonised pine bark nuggets (16-24mm), and plastic bioballs (commercially available, bespoke BSP), were compared in laboratory-scale bioreactors by measuring the decrease in residual metal ion concentrations over time, and changes in the solution pH. These experiments showed that the two BSPs did not differ significantly in their performance as a support matrix, or as a metal sorbent (30.6% and 32.6% of metal ion remained in solution when using bioballs and pine bark respectively). However, the presence of a biofilm on both these BSPs, improved the overall performance of the bioreactors significantly (for the bioball BSP, residual metal ion levels decreased from 30.6%, in the absence of a biofilm, to 11.0% with a biofilm present. Similarly, for the pine bark BSP, residual metal ion levels decreased from 32.6%, in the absence of a biofilm, to 7.3% with a biofilm present). A cost comparison of the two BSPs showed that raw pine bark nuggets were available at less than 0.1% of the cost of the bioballs. At pilot-scale, modelled kinetic data compared poorly with experimentally determined results, but minimum residual metal concentrations for Cu (1.7mg.ℓ⁻¹) and Zn (4.2 mg.ℓ⁻¹) were below South African (eThekwini Municipality) regulatory limits for discharge to sewer (5mg,ℓ⁻¹ for both), and sea outfall (3mg.ℓ⁻¹ Cu and 20mg.ℓ⁻¹ Zn). However, for Cd the final residual metal concentration (5.6mg.ℓ⁻¹) was above the regulatory discharge threshold for any receiving system. Although some of the effluents from the system investigated could not be legally released into the municipal sewer system without further remediation, the study showed that a system combining living and dead biomass in a single reactor is capable of significantly reducing dissolved metal concentrations in synthetic wastewaters without temperature or pH adjustment. Furthermore, such a system can operate at pilot-scale, where a pine bark matrix represents a significant cost saving over conventional plastic BSPs.
Development of a code of practice for co-disposal to obviate inimical environmental impacts of generated gases and leachates.
(1996) Daneel, Richard A.; Senior, Eric.
Despite its phasing out in numerous countries, such as Germany and the U.S.A., co-disposal of hazardous waste with municipal solid waste continues to be widely practised in South Africa. Co-disposal utilises properties and microbial activities in the refuse to attenuate the hazardous waste and thus obviate its environmental impact potential. All landfill operations require careful planning in not only site selection criteria but also the type and amount of various wastes accepted for disposal. It is clear, however, that the practice of co-disposal requires special precautions and management as the methods employed in the landfill operation determine to a large extent the environmental effects and, thus, the public acceptability of the operations. Although co-disposal is not suitable for all industrial wastes the results of recent research efforts, conducted mainly in the U.K., have indicated that, when properly managed, co-disposal can be regarded as a safe and efficient disposal option for many hazardous wastes. Environmental awareness in many European countries ensures that numerous hazardous compounds are either recycled or recovered. Unfortunately, in South Africa the lack of similar concern has resulted in increased concentrations of toxic compounds being co-disposed on a regular basis. Since fundamental studies of this technology, pertaining to South African conditions, have been lacking laboratory models/microcosms were built to address this paucity. Model. To effect the separation of species habitat domains of component species of growth rate-dependent interacting microbial associations responsible for terminal catabolic processes of the refuse fermentation, with retention of overlapping activity domains, and so facilitate examination of species in isolation without violating the integrity of each association, multi-stage models were constructed. The accidental overgassing of the culture with liquid petroleum gas (LPG) effected interesting fermentation balance changes which also emphasised the need for an Anaerobic Bioassay Test to assess the impacts of specific perturbants. Evidence of differential susceptibility of the component species to phenol was demonstrated in this study. Microcosm. A total of 42 refuse packed single-stage glass column bioreactors were commissioned and subjected to phenol and/or anaerobically digested sewage sludge codisposal. The effects of four different operational modes: leachate discard (single elution); leachate recycle; batch; and simulated rain on the co-disposals as well as refuse catabolism per se were examined. The results of these studies indicated that protracted periods of adaption to phenol (1000 and 2000 mg l -1) could have resulted from nutrient (elemental) limitation. Circumstantial evidence was also gained which indicated that the nitrate- and sulphate-reducing bacteria (SRB) were particularly sensitive to the added xenobiotic. Further, without the effective participation of the nitrate- and SRB the active and total fermentation of both the phenol and refuse components were depressed. It was also determined that the operating regime employed was a key factor in refuse degradation although with time, and especially following the phenol resupplementations, the operating conditions played a less significant role. In general, the single elution operated columns demonstrated increased phenol removal rates which were, unfortunately, coincident with low pH values and increased leachate residual phenol concentrations. Leachate recycle, on the other hand, unlike the batch operated columns, facilitated increased pH values and methane evolutions. The simulated rain columns were characterised by rapid washout of the added phenol as well as methanogenic precursors. The sewage sludge co-disposal experiments, likewise, demonstrated that, depending on the sludge:refuse ratio, the operating regime was extremely important in optimising the refuse degradation processes although, in general, leachate recycle appeared to be the most favoured method of operation.
Diversity of quorum sensing pherotypes amongst ecotypes of plant-associated Bacillus subtilis sensu lato isolates.
(2021) Tredgold, Heather Rayne.; Hewer, Raymond.; Hunter, Charles Haig.
Ecologically-adapted populations, or ecotypes, are species forms that are functionally adapted to particular habitat niches. Bacterial ecotypes are challenging the significance and implications of ecological adaptation in terms of prokaryote taxonomy, community ecology, and biocontrol applications. Plant-associated members of the Bacillus subtilis sensu lato group—in particular B. subtilis and B. velezensis—are ecologically specialised to perform numerous functions that are beneficial to plant and soil health. Underpinning these beneficial activities is the ability to colonise plant surfaces by biofilm formation. Biofilms are a result of co-ordinated social behaviour amongst microorganisms. In the B. subtilis sensu lato this sociality is governed by the ComQXPA quorum-sensing cassette, which uses the ComX pheromone for intercellular communication leading social behaviours like biofilm formation. This peptide pheromone contains a post-translational modification on a conserved tryptophan residue. This modification is highly variable between populations, resulting in discrete ComX variants which form communication groupings known as pherotypes. The limitation of communication to within a pherotype may constitute a form of ecological adaptation designed to protect the products of co-operative behaviour and restrict their benefit to the producing population. The present study aimed to explore ecotypes and pherotypes amongst a subset of plantassociated B. subtilis and B. velezensis isolates. These isolates originated from phylloplane and rhizosphere samples from seven crop species grown across the KwaZulu-Natal province, South Africa, and had demonstrated biocontrol potential in previous studies. An exploratory study set out to apply in silico approaches to determine gene-sequence-based variation amongst representative strains of the B. subtilis sensu lato available in the GenBank database. Nine housekeeping gene targets (viz., 16S rRNA, cheA, dnaJ, groEL, gyrA, gyrB, polC, purH, and rpoB) were evaluated for suitability to resolve clustering of closely-related B. subtilis sensu lato. Four of these gene sequences (viz., 16S rRNA, dnaJ, gyrA, and rpoB) were identified as candidates for a Multilocus Sequence Analysis (MLSA) scheme to distinguish between members of the B. subtilis sensu lato group. Putative pherotype variation amongst these reference strains was explored in silico using comQXP gene sequence data. The suitability of a comQXP PCR-RFLP protocol with potential for rapid pherotyping amongst B. subtilis sensu lato was evaluated in silico using simulated comQXP amplicons. This necessitated the design of a PCR primer set targeting the quorum-sensing gene region of B. velezensis. Four restriction enzyme candidates namely, BtsCI, Fnu4HI, Cac8I, and Hpy166II, were identified for further study. Ecotyping amongst the B. subtilis and B. velezensis isolates was carried out using a four-gene (viz., 16S rRNA, dnaJ, gyrA, and rpoB) MLSA. This concatenated sequence dataset was applied to ecotype simulation (ES) analysis to corroborate putative ecotype sub-clusters in the MLSA phylogeny. Two DNA fingerprinting approaches—Repetitive Element Palindromic PCR (Rep-PCR) and Random Amplified Polymorphic DNA PCR (RAPD-PCR)—were also evaluated for their potential to identify putative ecotypes within the isolate subset. This investigation of pherotypes amongst the putative ecotype groupings examined isolate comQ sequence data as well as the comQXP PCR-RFLP, and also applied a srfA-LacZ reporter gene assay to examine isolate stimulation of seven known pherotype tester strains (viz., 168, RO-B- 2, RO-C-2, RO-E-2, RO-FF-1, RO-H-1, and RS-D-2). The MLSA of isolate gene sequences presented distinct sequence clusters suggestive of ecotype populations amongst the two Bacillus species which were corroborated by ES analysis. The MLSA and ES determined two putative ecotypes within the B. subtilis isolates, and six within the B. velezensis isolates. Ecotype groupings were found to contain isolates from different crop species and locations, and four B. velezensis ecotypes were distinct from included B. subtilis sensu lato reference strains. PCR fingerprinting identified strain-level variances amongst the isolates, and were able to differentiate plant-associated B. velezensis from closelyrelated B. amyloliquefaciens, but did not define groupings entirely consistent with the ES and MLSA phylotypes. The MLSA, ES, and PCR fingerprinting delineated a group of five isolates (viz., B81, CT-R67, bnd136, bng221, and sqo271) to be grouped with reference strain Bacillus sp. JS in the MLSA. This grouping is of interest as Bacillus sp. JS is a strain demonstrating biocontrol capability and formed a clade distinct to B. subtilis sensu stricto in gene sequence phylogenies. This Bacillus sp. JS grouping was further confirmed by ES to constitute a single ecotype, and both Rep-PCR and RAPD-PCR OPG-16 distinguished this grouping based on fingerprint profiles. Investigation of reference strain variation in the comQ gene sequence determined significant levels of sequence variation amongst reference strains evaluated in silico, and showed divergence in some strains from known pherotype counterparts. Furthermore, sequence clusters were resolved in B. velezensis that suggested unique pherotype variants amongst reference strains, and showed comQ similarity amongst the five isolates related to Bacillus sp. JS. This trend was observed for the comQ phylogeny applying isolate and pherotype sequences, which resolved two distinct clusters each amongst the B. velezensis and B. subtilis isolates, with only two isolates (viz., bnd134 and bnd156) grouping with known pherotypes. The B. velezensis clades demonstrated significant comQ sequence variance from the pherotype reference strains based on clustering distances in the phylogeny. Of the four enzyme candidates identified for potential use in a comQXP PCR-RFLP protocol, two were found to be applicable: BtsCI resolved the grouping of the Bacillus sp. JS clade, while Fnu4HI was found to distinguish between more closely-related isolates in B. velezensis. The profile groupings for both of these enzymes demonstrated most of the isolates to be distinct from known reference pherotype strains, and the technique proved able to corroborate groupings in the comQ phylogeny. The pherotyping srfA-LacZ reporter gene assay failed to support the pherotype groupings defined by the comQ phylogeny, but demonstrated that the environmental isolates were capable of eliciting substantial responses in the pherotype tester strains, particularly amongst the B. velezensis isolates. Eight isolates did not elicit significant responses in any of the tester strains, while most matched to multiple pherotypes on the basis of tester strain response to conditioned medium from isolates. These findings demonstrated that putative ecotype variation was present amongst the B. subtilis and B. velezensis isolates that were resolvable by MLSA, and that these ecotype groups comprised isolates from different crop types and location sites. Some of these ecotypes bore only distant relation to B. subtilis sensu lato reference strain counterparts. The investigation of PCR fingerprinting methods for ecotyping purposes found that the primer sets applied were not able to consistently corroborate putative ecotype groupings defined by MLSA and ES. Pherotyping investigations demonstrated that there was evidence of gene-sequence-based pherotype variation amongst the isolates within these putative ecotype groupings. The pherotype clades resolved with the comQ phylogeny and with comQXP PCR-RFLP were not corroborated by srfA-LacZ reporter gene pherotyping assays, but the data suggested that assay optimisation to suit environmental strains is warranted in future studies. This study presents the first instance of ecotyping and pherotyping amongst plant-beneficial B. velezensis. The B. velezensis isolates were largely distinct in ecotype and pherotype from the plant-associated model strain FZB42, which further promotes that these isolates demonstrate localised adaptation. The Bacillus sp. JS-related isolate grouping is of interest as these isolates comprised a single putative ecotype resolvable by MLSA, ES, and Rep-PCR and RAPD-PCR OPG-16. This grouping was distantly-related to B. subtilis sensu stricto, and this is interesting from a taxonomic standpoint, as is the fact that that Bacillus sp. JS is reported to have plantbeneficial capabilities. The existence of uniquely South African plant-associated B. subtilis sensu lato ecotypes is valuable in agricultural management approaches targeting beneficial microbes, as these organisms likely possess adaptations allowing them to compete with extant bacterial communities and thrive within the South African agricultural climate.
Evaluation of the larvicidal potential of Bacillus velezensis strain PHP1601 as a viable biological control agent against selected fly species.
(2024) Ramesar, Danvir Rajesh.; Hunter, Charles Haig.
Flies are one of the most abundant and prevalent insect pests posing a growing threat to various sectors of the economy. In response to this, a study was undertaken to evaluate Bacillus spp. strain PHP1601 as a candidate biocontrol agent against Lucilia cuprina larvae as a proxy for fly species of biocontrol significance. The identity of PHP1601 was confirmed as B. velezensis using MLSA and species-specific PCR. Bioassays demonstrated a larvicidal effect of cell, endospore (102 – 1010 cells/endospores g -1 ) and cell-free supernatant (1 – 30% v w -1 ) treatments on second instar larvae of L. cuprina. Studies were directed to the larvicidal effect of extracellular compounds, namely lipopeptides. Crude lipopeptide extract (CLP) was acquired using organic extraction from Landy broth. Bioassays with CLP extract (5 – 1000 μg g -1 ) resulted in a dose-dependent larvicidal response. Lipopeptides in the CLP extract were purified by TLC and characterised using UPLC ESI-TOF MS. This indicated the presence of iturin, fengycin and surfactin homologues of which, the purified surfactin fraction (Rf 0.91) was the most larvicidal. Bioassays were repeated with commercial surfactin, confirming its larvicidal potency, exhibiting an LC50 of 9.87 μg g -1 at 240 h. Larvae scent choice tests using TSB and MG bioassay medium fermented by PHP1601 showed that resulting VOCs were attractive to fly larvae, which was considered a viable trait of a fly biocontrol agent. CG-MS of the VOCs produced indicated that ketones were the dominant VOC class and, presumably, the major contributor to this larvae attraction effect. Field performance evaluation using pig manure trials demonstrated successful inhibition of several fly species of agricultural and veterinary importance using endospore treatments (105 and 1010 endospores g -1 ) of PHP1601. qPCR and REP-PCR fingerprinting confirmed that PHP1601 could grow in the manure slurries and was amiable to recovery and monitoring. Zebrafish embryo toxicity bioassays of the CLP produced by PHP1601 indicated that they achieved an LC50 of 22.77 µg ml-1, which characterised these metabolites as slightly toxic. Genome mining detected no genes associated with pathogenicity or virulence and presented no apparent pathogenic threat. The investigation demonstrated that B. velezensis PHP1601 is a viable fly biocontrol candidate and constitutes the first report of a B. velezensis antagonist of Brachycera flies.
Genetic characterisation of an indigenous plasmid in xanthomonas albilineans.
(1999) Permaul, Kugenthiren.; Pillay, Balakrishna.; Pillay, D.
Abstract available in PDF file.
Genetic manipulation of saccharomyces cerevisiae for improved ethanol production from d-xylose.
(1999) Govinden, Roshini.
No abstract available.
Heterologous expression and application of lipolytic enzymes for mitigation of lipophilic compounds in eucalyptus species.
(2017) Ramnath, Lucretia.; Govinden, Roshini.; Sithole, Bishop Bruce.
Abstract available in PDF file.

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