Doctoral Degrees (Microbiology)

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An ultrastructural study on rusts with special reference to Puccinia sorghi on Oxalis corniculata.
(1973) Rijkenberg, Fredericus Hermanus Johannes.; Truter, Susarah J.
No abstract available.
A histopathological study on selected bacterial vascular diseases with emphasis on ultrastructure.
(1975) Wallis, Frederick Michael.; Truter, Susarah J.
No abstract available.
Purification and electron microscopy of the tomato spotted wilt virus.
(1976) Martin, Michael Menne.; Thung, T. H.; Noordam, D.
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.
Microbiological investigations into granular sludge from two anaerobic digesters differing in design and industrial effluent purified.
(1995) Howgrave-Graham, Alan R.; Wallis, Frederick Michael.
Due to a combination of selection criteria, sludges from upflow anaerobic digesters treating industrial waste waters consist primarily of well-settling, dense agglomerates called granules. Quantification of the component mixed microbial populations of these granules has been severely restricted by the inability of researchers to disrupt them without concomitantly destroying numerous cells. In situ quantification using light and electron microscopy is complicated by the high cell numbers and bacterial diversity; the small cell size; and the destructive nature of electron microscopy preparative techniques preventing the viewing of more than a small percentage of the population at a time. For these reasons, in this investigation, standardization of qualitative electron microscopic techniques was performed prior to their application to granules. Isolation and electron and light microscopic techniques were applied to granules from a fullscale clarigester treating effluent from a maize-processing factory. In addition, a method using montaged transmission electron micrographs (TEMs) taken along a granule radius, and image analysis, was developed for bacterial quantification within granules. This method, together with antibody probe quantification, was applied to granules from an upflow anaerobic sludge blanket (UASB) digester treating a brewery effluent. The clarigester granules contained a metabolically and morphologically diverse population of which many members were not isolated or identified. By contrast, the UASB digester granules consisted primarily of morphotypes resembling Methanothrix, Methanobacterium and Desulfobulbus, in order of predominance. However, only about one-third of the population reacted with antibody probes specific to strains of bacterial species expected to occur within these granules. According to the antibody probe library used, the Methanobacterium-like cells observed in TEMs were probably Methanobrevibacter arboriphilus. From this study it is apparent that different anaerobic digester designs, operational parameters, and the chemical composition of the waste water purified, are factors which influence the formation and maintenance of granules differing with respect to their microbial populations. Until the difficulties associated with quantification are overcome, the processes governing granule formation and/or population selection will remain obscure.
The influence of soil particle surfaces and soil porosity on the biodegradation of key refuse leachate organic molecules.
(1995) Du Plessis, Chris Andre.; Senior, Eric.; Hughes, Jeffrey Colin.
Many studies have been undertaken to determine the effects of soil and soil properties on migrating metal pollutants. Organic pollutants, however, in addition to their interactions with soil components , are also susceptible to degradation (catabolism) by microorganisms. Soil-microorganism-pollutant interactions have, traditionally, been studied in soil columns (microcosms). One of the shortcomings of column and in situ studies is that the identity and specific effect(s) of the soil component(s) affecting or influencing attenuation are not known and cannot readily be determined. Attenuation effects of the soil components are, therefore, difficult to interpret. ("Attenuation" in this context is the combined effects of both soil adsorption and microbial catabolism). Attenuation studies often only consider the physical conditions such as aeration, permeability, flow rate, temperature, etc. This approach assumes the soil to be a homogeneous matrix with no specific physico-chemical properties attributable to different components within the matrix. Soil physical factors suspected of influencing pollutant attenuation could be misleading without consideration of the physico-chemical interactions between soil components, microorganisms and pollutants. Adhesion of pollutants and microorganisms seems to be most important in this regard. The initial phase of this study was undertaken to examine the effects of three different soil materials on attenuation of key landfill leachate molecules. Examination of the effects of soil surface type on attenuation focused on adsorption / desorption of the pollutant molecules and microorganisms. These experiments sought to investigate the physico-chemical effects of soil, microorganism, pollutant interactions and were done as batch slurry experiments as well as in soil columns. Two soil horizons from the Inanda soil form (humic A and red apedal B) and the topsoil (vertic A) from a Rensburg soil form were used. The Inanda topsoil had a high organic matter content and both the topsoil and subsoil had a kaolinitic clay mineralogy; the Rensburg topsoil clay mineralogy was predominantly smectitic with a relatively low organic matter content. From the batch experiments, the adsorption of a hydrophobic molecule (naphthalene) and a heavy metal (cadmium) were found to be influenced to a significant extent by soil characteristics. Adsorption of naphthalene was due to the soil organic matter (SOM) content whereas cadmium adsorption was due to the cation exchange capacity (CEC) of the soil. Soil characteristics did not seem to have a significant influence on the adsorption of a water soluble compound such as phenol at the concentrations used. Attenuation of naphthalene was found to be affected by adsorption of the pollutant molecule (related to SOM) as well as the CEC of the soil. The attenuation of hydrophobic molecules can possibly be ascribed to the influence of CEC on the microbial population responsible for attenuation. This would seem to indicate interaction between the soil surfaces and the catabolizing microbial population. Desorption of the pollutant (and possibly also of the microbial population) was achieved by the addition of acetonitrile and methanol both of which reduced the polarity of the water. These solvents were also found to be toxic to the catabolizing microbial population at high concentrations. The toxicity thresholds of both solvents for catabolizing microorganisms differed significantly between soil- (> 15 %, v/v) and soil free (< 5 %, v/v) treatments. This discrepancy cannot be accounted for by adsorption and is ascribed to physico-chemical interaction between microorganisms and the soil surfaces. This interaction probably affords protection from, otherwise, toxic concentrations of solvents or metals. The important effects of soil surfaces on attenuation processes were thought to be due to the strong adsorption of naphthalene. Surface attachment of microorganisms was, however, also inferred from results obtained with phenol. This seemed to indicate that microbial attachment to soil surfaces was an important aspect in attenuation and did not occur only because of pollutant adsorption. Soil column experiments were made with both naphthalene and phenol. The naphthalene, which was adsorbed to the soil, did not leach from the columns to any appreciable extent. This was despite the addition of acetonitrile to some columns. This was probably due to greater microbial catabolism caused by desorption and, subsequent, increased soluble concentrations of the molecule. After extraction from the soil at the end of the experiment it was clear that the sterile controls held much higher concentrations of naphthalene than the experimental columns. The soil type and treatments showed little difference in the naphthalen concentration extracted from the soil columns. This did not reflect the differences found between soil materials in the batch experiments and was probably due to the masking effect of the soil physical factors on attenuation processes. Unlike naphthalene, phenol, because of its high solubility, was detected in the column leachates at relatively high concentrations. The phenol concentrations were much higher for the Inanda subsoil (approximately 4 mM) than the Inanda topsoil (approximately 2 mM) and Rensburg topsoil (< 1 mM). The Rensburg topsoil produced the lowest phenol concentrations in the leachate and this can probably be ascribed to the larger quantity of micropores in this soil. Thus, it seems that the soil physical features had a pronounced influence on attenuation. Whether this effect was directly on the studied molecule or indirectly, because of the effects on the microbial population, is not known. Inoculation of the columns with a phenol catabolizing population had only a slight increased effect on leachate phenol concentrations from all columns. This increased effect was, however, only prolonged in the case of the Inanda subsoil. The flow rate through the columns affected leachate phenol concentration which was lower with a slower flow rate and, thus, longer retention time. From the column experiments soil physical parameters were suspected of influencing, and possibly overriding, the soil surface effects on microbial activity (capacity to catabolize a organic molecule of interest). Soil porosity, as caused by different soil materials, was suspected of being the most important soil physical parameter influencing microbial activity. To investigate the potential effect of soil porosity, relatively homogeneous porous media i.e. chromatography packing material and acid washed sand were used. These materials had more defined and distinct porosities and were considered to be suitable for investigating the fundamental influence of porosity on microbial activity. Saturated continuous flow columns were used and three types of packing configurations were tested: chromatography packing (CHROM) material (porous particles); acid washed sand (non-porous) (AWS); and a 1: 1 (w/w) mixture of chromatography packing and acid washed sand (MIX). Only a single water soluble molecule, phenol, was used in this phase of the investigation. Bacterial filtration ("filtration" as a component of "attenuation'') was found to be highest for the CHROM and lowest for the AWS materials. This difference in microbial retention affected the phenol catabolism in response to increased column dilution rates. The CHROM and MIX materials had distinctly different porosities than that of the AWS, due to the internal porosity of the chromatography packing. This greater pore size distribution in the MIX and CHROM packing materials created pores with different effective pore dilution rates within the microcosms at similar overall flow rates. The greater pore size distribution in the MIX and CHROM packing materials facilitated pore colonization since some pores did not participate, or conduct, mass flow as occurred in macropores. This led to different microcolonization effects in the macro- vs micropores. Since the MIX and CHROM packing materials had more micropore colonization sites these packing materials showed a greater range of substrate affinities (i.e. Ks values) for the phenol substrate. The extent to which micropore colonization occurred could be detected by the effect it had on phenol breakthrough curves. In the MIX and CHROM materials, microbial colonization caused blocking of micropores with a subsequent effect on the phenol breakthrough curves. The AWS material, however, which had a low inherent microporosity, showed microbially induced microporosity probably due to biofilm development. The fact that the MIX and CHROM packing materials facilitated micropore colonization was also responsible for the greater resistance to, and the recovery from , potentially inhibitory cadmium concentrations. This effect was also apparent in the presence of acetonitrile, although this effect was not identical to that observed with cadmium. Finally, column pressure build up as a function of pore clogging was determined and was found to occur in the order AWS > MIX > CHROM. This was most likely due to fewer potential liquid flow paths with a higher blocking potential in the AWS. Extrapolation of the fundamentals of the above findings led to the conclusion that soil surface- and soil porosity effects are extremely important factors in determining the behavior of soils as bioreactors.
The microbiology of ex situ bioremediation of petroleum hydrocarbon-contaminated soil.
(1996) Snyman, Heidi Gertruida.; Senior, Eric.; Oellermann, Rolf Alfred.
Bioremediation is the process whereby the degradation of organic polluting compounds occurs as a result of biochemical activity of macro- and microorganisms. Bioremediation of hydrocarbon contaminated soils can be practised in situ or ex situ by either stimulating the indigenous microorganisms (biostimulation) or introducing adapted microorganisms which specifically degrade a contaminant (bioaugmentation). This investigation focused on ex situ remediation processes with special attention to the processes and microbiology of landfarming and thermal bioventing. Landfarming was investigated at pilot-scale and full-scale, and thermal bioventing at laboratory and pilot-scale. This study indicated that pilot-scale bioremediation by landfarming was capable of effecting a total petroleum hydrocarbon concentration (TPHC) reduction of 94% (m1m) from an initial concentration of 320 gkg-I soil to 18 gkg-I soil over a period of 10 weeks. Reactors receiving biosupplements showed greater rates of bioremediation than those receiving nutrients. Promotion of TPHC catabolism by addition of a commercial or a site-specific microbial biosupplement was similar. Seedling experiments proved that bioremediation did not necessarily leave the soil in an optimal condition for plant growth. The full-scale landfarming operation reduced the TPHC concentrations from 5 260 - 23 000 mgkg- I to 820 - 2335 mgkg- I soil over a period of 169 days. At full-scale, the larger fraction of more recalcitrant and weathered petroleums. and the less intensive treatment resulted in a slower rate of TPHC reduction than was found in the pilot-scale study. Three distinct decreases in the TPHC were observed during the full-scale treatment. These presented an ideal opportunity to investigate the microbiology of the soil undergoing treatment. The dominant culturable microorganisms were isolated and identified. The bioremediation process was dominated by Bacillus and Pseudomonas species. The method used to study the population was, however, biased to culturable, fast growing microorganisms which represent a small portion of the total microbial population. For this reason, a method to study the total eubacterial population in situ with rRNA targeted oligonucleotide probes was adapted and found to be a valuable technique. Soil microorganisms respiratory activity was investigated at different times in the full-scale treatment. A clear correlation between activity and degradation was recorded. The effect of a supplement. anaerobically digested sludge, was also assessed by this method. Thermal bioventing was investigated as an ex situ in-vessel treatment technology for small volumes of highly contaminated soils. This proved to be a viable technique for the bioremediation of petroleum hydrocarbons at laboratory-scale. Volatilisation contributed to at least 40% of the reduction. Of the two supplements evaluated. dried sludge promoted degradation to a greater extent than chicken manure. The pilot-scale study proved that a chemical contaminant reduction of at least 50% could be achieved in 13 weeks by thermal bioventing. Of the supplemented reactors. the presence of dried sludge and commercial biosupplement etfected the largest contaminant decrease. As a possible supplement to increase the rate of bioremediation. dried anaerobically digested sludge was more effective than chicken manure. A parallel laboratory-scale experiment gave similar results. Gravimetric analyses were found to be conservative indications of the remediation process. The results of this study shed some light on our. still. limited understanding of bioremediation. The gap between the technology in the laboratory and field was narrowed and a better understanding of the soil microbiology was achieved. Due to the limited control of environmental parameters in the case of landfarming. thermal bioventing was investigated and proved to be an effective alternative. The latter technology is novel in Southern Africa.
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.
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.
Genetic manipulation of saccharomyces cerevisiae for improved ethanol production from d-xylose.
(1999) Govinden, Roshini.
No abstract available.
Genetic characterisation of an indigenous plasmid in xanthomonas albilineans.
(1999) Permaul, Kugenthiren.; Pillay, Balakrishna.; Pillay, D.
Abstract available in PDF file.
P15 trypanosome microtubule associated protein : structure/function analysis and vaccine development for the prevention of African sleeping sickness.
(2001) Rasooly, Reuven.; Balaban, Naomi.
Trypanosomes are hemoflagellated protozoan parasites causing chagas disease in South America, Leishmaniasis throughout the world, and African sleeping sickness in humans and nagana in animals in Africa. About 55 million people and 25 million cattle have been estimated to be at risk of contracting African sleeping sickness or nagana respectively. Once injected into the blood stream via the bite of a tsetse fly, the parasite evades the host's immune response by repeatedly changing its surface antigens, thus making the development of a vaccine seem impossible. Furthermore, chemotherapy existing today can be toxic, suggesting that novel methods to prevent diseases caused by trypanosomes are essential. All parasites of the Trypanosomatidae family contain unique microtubular structures called the subpellicular microtubules. Microtubules are made of tubulin and of microtubule associated proteins (MAPs). Unlike other microtubules, the subpellicular microtubules are crosslinked to one another and to the plasma membrane. The unique structure of the subpellicular microtubules has been attributed to unique trypanosome subpellicular MAPs which stabilize the microtubule polymers and crosslink them to one another. Three unique types of subpellicular MAPs have been identified: MARP, which is a high molecular mass MAP that stabilizes microtubules, p52 that is a 52kDa MAP which crosslinks microtubules, and pI5, which is a I5kDa protein which bundles microtubules. Because trypanosome MAPs have been shown to be unique to these parasites, these molecules could serve as useful target sites for therapy. In this study pI5 was cloned and sequenced and shown to contain highly organized, nearly identical tandem repeats with a periodicity of 10 amino acids, rich in positively charged and in hydrophobic amino acids. It was shown that pI5 can also bind phospholipids, suggesting that it may not only bundle the microtubule polymer through its positively charged amino acids but may also crosslink the microtubules to the plasma membrane through its hydrophobic regions, thus contributing to the stable structure of the subpellicular microtubules. To test for the efficiency of pI5 as a vaccine candidate, the recombinant pI5 was cloned into an adenovirus, which was used as a vaccine delivery system for pI5. Mice were vaccinated with the native purified pI5, with the expressed recombinant pI5 and with the adenovirus containing the recombinant pI5 gene (Ad-pI5). The results indicated that pI5 protected 100% of the animals vaccinated with the recombinant molecule (8/8), and 87% of the animals vaccinated with the native protein (7/S), while none of the control animals were protected. Animals that were vaccinated with the Ad-pI5 were protected but so were the control animals vaccinated with an adenovirus containing the lacZ gene. We have shown that vaccination with the adenovirus is associated with an elevated CDS+ T cell response which is known to be trypanostatic (S6), suggesting that animals vaccinated with Ad-pIS may have been protected not only by the specific anti-plS response but also by non specific immunity that was induced by the adenovirus itself. The source of the native and recombinant pI5 was from a different strain of T. brucei that was used for challenge. Since the subpellicular microtubules are common to all members of the Trypanosomatidae family, pI5 may ultimately serve as a common target for therapy to all types of diseases caused by trypanosomes.
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.
The syndromic management of sexually transmitted diseases : clinical microbiological response in relation to aetiology, susceptibility patterns and co-infection with HIV-1 [electronic resource].
(2002) Moodley, Prashini.; Sturm, Adriaan Willem.
HIV-1 is the most prevalent and notorious sexually transmitted pathogen locally, and constantly challenges our foundation of knowledge regarding the classical STIs. The ultimate objective of the syndromic management strategy was to reduce the load of sexually transmitted infections, and hence HIV transmission. This strategy is multifaceted and not only includes the recognition of symptoms by the patient and an effective treatment regime that comprehensively covers the possible aetiological agents for a defined syndrome, but also appropriate health seeking behaviour of infected individuals, recognition of syndromes by the health care worker, partner management (notification and treatment), behavioural counselling and condom promotion. Understanding the complexity of sexual networking and transmission dynamics is part of such a strategy. So, although the rationale and design of syndromic case management appears simplistic, it is by no means easy to implement
Bioremediation of creosote-contaminated soil by microbial intervention..
(2002) Atagana, Harrison Ifeanyichukwu.; Haynes, Richard John.
No abstract available.
Molecular characterisation of Peronospora parasitica infecting Brassica species.
(2002) Jugmohan, Maneshree.; Lucas, John Alexander.; Achar, Premila. N.
Peronospora parasitica (Pers. ex. Fr.) Fr., pathotype, single spore isolate, Brassica spp., downy mildew, internally transcribed spacers, ITSI , 5.8S, ITS2 sequence, radial phylogram, phylogeny, peR, micro satellite markers, SSRs, DNA library, microsatellite primer sequences, allele, geographic and pathotype diversity, polyploidy The downy mildew disease caused by Peronospora parasitica (Pers. ex. Fr.) Fr. infects approximately lOO plant species in the family Cruciferae. It is primarily a foliage blight which causes 60-70% seedling losses to nurserymen and rural subsistence farmers in Kwazulu-Natal, South Africa who depend on this crop to maintain a healthy cash flow not provided by other products. The disease is also frequent in India, France, Portugal and in the UK. The pathogen is especially debilitating in winter when low temperatures and high humidity favour infection and spread of the disease. Control of the disease with the use of fungicides is inefficient since P. parasitica has developed resistance to metalaxyl-based fungicides. There is a need to establish patterns of genetic diversity in order to address the ambiguities surrounding species definition of P. parasitica. The present study focused on clarifying the relationships between different pathotypes of P. parasitica found on various Brassica species viz. B. oieracea, B. juncea, B. napus, B. rapa, as well as the Arabidopsis thaiiana pathotype, a wild host of P. parasitica. Co-evolution with plant hosts over long periods was suggested to have lead to the divergent forms of this pathogen adapted to different host taxa. Genetic analysis of host specificity was investigated, based on sequence analysis of intergenic spacer regions of ribosomal DNA and on micro satellite markers. The current study provides the first comprehensive ITS-based phylogeny of pathotypes of P. parasitica. Based on ITSl, 5.8S and ITS2 sequences all pathotypes of P. parasitica from Brassica species (viz. Brassica oieracea, Brassica napus, Brassica juncea and Brassica rapa) were monophyletic. Based on ITSl sequences, the pathotype of P. parasitica from the wild host, Arabidopsis thaliana, was found to be significantly different from the Brassica pathotypes (i.e. cultivated host pathotypes). Furthermore the genetic distance between the genus Peronospora and Phytophthora was closer compared to other taxa such as Pythium, supporting the observation that the downy mildews have derived from a Phytophthora ancestor. rDNA sequence analysis was unable to differentiate Brassica pathotypes of P. parasitica. The development of a powerful class of genetic markers known as microsatellites (SSRs) in the present study provided greater insight into the relationships between Brassica pathotypes of P. parasitica. A genomic DNA library highly enriched for various micro satellites was prepared and a large number of potential SSRs consisting primarily of dinuceotide repeats (CA)n and (CT)n were obtained. Sequence analysis of 351 clones yielded 120 clones containing SSR loci, and 29 (24%) potentially useful SSRs from which primers could be designed were identified. PCR amplification with radiolabelled probes at 8 loci yielded useful polymorphisms across 27 isolates representing four pathotypes of P. parasitica. The alleles showed pathotype specific diversity since isolates of the same pathotype could be grouped together. The value of micro satellites as a strong discriminatory tool for intraspecies variation was further demonstrated by the ability to significantly separate isolates of the Brassica oleracea pathotype by their geographic origin and even field population in some instances. Several monoconidial lines from the same field population were grouped together and in some instances were found to be alike. The large number of alleles observed per genotype in the present study suggests that P. parasitica is polyploid and more complex than previously known. This parallels similar observations in other downy mildews such as Phytophthora, the closest relative of Peronospora. Future work involving sequencing at microsatellite loci would provide more insight into the exact ploidy, as well as the mapping of characterised single nucleotide polymorphisms (SNPs) which may be useful in specific identification of pathotypes. The primers developed for P. parasitica were able to amplify genomic DNA of Bremia lactucae indicating the value of the library developed in this study in comparative diversity studies with other Oomycetes. ITS-PCR and micro satellites markers provide useful tools for improved classification and diagnosis of downy mildew diseases. Furthermore, knowledge on the genetic diversity in P. parasitica together with the wealth of information now available about RPP genes of P. parasitica in Arabidopsis thaliana provides many exciting avenues for a complete understanding of the genetics of host specificity.
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.
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.
Isolation and characterization of antibiotic(s) produced by bacteria from KwaZulu-Natal soils.
(2010) Okudoh, Vincent Ifeanyi.; Wallis, Frederick Michael.
This work reports the continued search for new antibiotics in the relatively under investigated region of KwaZulu-Natal, South Africa. A soil bacterium designated strain N8 with antibacterial activity against both Gram-positive and Gram-negative bacteria was isolated from a poultry farm in Pietermaritzburg, South Africa. The organism was one of approximately 2600 strains isolated from various habitats in the KwaZulu-Natal midlands, South Africa during an actinomycete screening programme. The highest number of antimicrobially-active isolates came from a forest soil site whereas the lowest number was present in a riparian soil. Morphological, physiological and cultural characteristics indicated that strain N8 belonged in the genus Intrasporangium. In the literature, members of this actinomycete genus have not been associated previously with antibiotic production. Studies on the influence of different nutritional compounds on antibiotic production showed that the highest antibacterial activities were obtained when glycerol at 1% (w/v) was used as sole carbon source in the presence of mineral trace elements. Using solvent extraction and various chromatographic techniques, the antibiotic produced by strain N8 was recovered from the fermentation broth. The use of a three-solvent system, petroleum ether: acetone: ethyl acetate enhanced the separation of the antibiotic complex in broth. Bioassay results established that the antibacterial agent was in the ethyl acetate fraction (EAF) and chromatographic methods were used in its purification. The chromatographic methods used were: flash column chromatography (FCC), thin-layer chromatography (TLC), and Harrison research chromatotron (HRC). Further purification was carried out by reverse phase high performance liquid chromatography (HPLC). Most of the inactive, coloured material was removed from the antibiotic extract by FCC, while TLC chromatograms run using a range of the most polar to the least polar solvent systems [SS1 (most polar) – SS5 (least polar)] showed best separation of EAF with SS2. TLC chromatograms using SS2 usually showed 3 bands. Bioautograms of SS2-separated EAF revealed that the antibiotic activity was located in the region with an Rf value of 0.56 – 0.64. The Harrison research chromatotron technique also gave good separation of the EAF sample. Preparative HPLC was used as the final purification step for most of the EAF samples. Although, a number of peaks were observed during isocratic-HPLC (IHPLC) runs, they were not as clearly separated as those obtained with gradient-HPLC (GHPLC). Three major peaks PI, PII and PIII with elution times of 3.56 min, 4.53 min and 23.06 min respectively were revealed under GHPLC runs with decreasing concentrations (100% – 50%) of methanol in water. Methanol concentrations between 50% and 70% in water were considered the optimum GHPLC mobile phases. Since these chromatographic methods were all time consuming, required large volumes of solvents, and resulted in low yields of the antibiotic, an alternative procedure producing better results was sought. This led to the development of a procedure combining a three-solvent extraction system with a pH precipitation process which efficiently recovered the antibiotic in solid/crystal form. Using this procedure, sufficient quantities of the antibiotic were recovered from the fermentation broth to permit a degree of structural elucidation. Two types of crystals (brown and pink-yellow in colour) were obtained and their chemical natures established by means of 1H- and GCOSY- nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS). On further LC-MS analysis, the brown crystals appeared to be a protein and since it did not show inhibitory activity against any of the test organisms, no further studies were carried out on it. The pink-yellow crystals when suspended in a minimal volume of methanol showed inhibitory activity against S. marcescens confirming that the antibiotic activity resided therein. The LC-MS spectrum of these crystals showed a prominent/base peak at 304.2724 [mass to charge ratio (m/z) in positive mode]. The elemental composition of this compound suggests a molecular formula close to C16H36N2O3 with a molar mass of 304.4686 g/mol. No existing name could be assigned to it from the database of known natural compounds. Hence, the possibility that it is a novel antimicrobial compound cannot be excluded. Characterisation of the antimicrobial substance using GC-MS revealed that it contained at least seven components (A – G). These components were then subjected to mass spectrum analysis and their retention indices compared to computer database listings of known compounds. Components A and B were regarded as representing one compound (possibly isomers) since they have the same molecular weight and formula. Their different retention indices strongly suggest they are indeed isomers. Thus a total of six different compounds were detected in the extract by GC-MS and the molecular formulae assigned to them include: C6H10O (A and B); C6H12O2 (C); C9H14O (D); C8H7N (E); C21H44 (F); and C12H14N2O (G). Since only low probability matches were obtained for A – F and as the sample could not be recovered from the analyser, they were not studied further. The closest match (71% probability) with substances listed in the computer database of natural compounds was for compound G (C12H14N2O) which was thus provisionally identified as N-acetyltryptamine. A structurally related compound known as melatonin is attributed with the ability to inhibit tumour growth in vivo and in vitro. Attempts were made to assign a chemical structure to the antibiotic produced by strain N8 using all the data available. The indications are that it is a tryptamine, the chemical structure of which is postulated to be: In order to monitor the antimicrobial activity of the antibiotic produced by strain N8, bioassays were conducted after all major steps during the isolation and characterization processes. The antimicrobial activity of the pink-yellow crystals was confirmed on the test organisms used during the primary screening phase, namely, Escherichia coli, Pseudomonas fluorescens, Serratia marcescens, Staphylococcus aureus, Enterococcus faecalis and Xanthomonas campestris pv. campestris, and the yeast Candida utilis, indicating that the crude substance had maintained its inhibitory activity against Gram-positive and Gram-negative bacteria, and the yeast tested. The study was extended to include investigations into the use of combinations of the GHPLC separated peaks of the antibiotic (PI, PII and PIII) to improve the efficacy of growth inhibition of the test pathogens for possible use in chemotherapy. Data from these studies showed that PI inhibited the growth of E. coli and X. campestris pv. campestris while PII and PIII inhibited the growth of the latter organism and also that of S. marcescens. Individually, the peaks showed no growth inhibition on Pseudomonas fluorescens but the combination PI+PII+PIII was antimicrobially effective. In all cases, the use of combinations was significantly more effective than the use of any single component alone. For example, the combination of GHPLC PI and PII had a greater growth inhibitory effect (synergic action) against Serratia marcescens than did either alone; the inhibition-zone diameter being double (30mm) that caused by the single peaks (15mm) against S. marcescens. Likewise mixing PI and PIII resulted in a much improved action against X. campestris pv. campestris. These findings may meet the current call by many scientists that all infectious diseases should be treated with a combination of two antibiotics with different mechanisms of action in order to counter the serious problem of emerging bacterial resistance. Since the antibiotic isolated during this study showed activity against both mammalian and plant pathogenic bacteria it is hoped that this work will encourage further investigation in this field in South Africa. The results obtained should impact on the pharmaceutical industry as well as agriculture and will, hopefully, help curb both plant and human infectious diseases in our African communities. This study also confirmed that KwaZulu-Natal soils do harbour rare actinomycetes that produce novel antimicrobial compounds.
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.

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