Browsing by Author "Wallis, Frederick Michael."

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Application of image analysis in microecophysiology research : methodology development.
(1998) Dudley, B. T.; Wallis, Frederick Michael.
Rehabilitation of landfill sites is important for successful land utilization. Revegetation is one key element of the process since it can overcome aesthetic problems. The inimical challenges of landfill leachate and gas are largely responsible for the difficulties associated with the revegetation of completed sites. Many components of landfill leachate can be catabolized by microbial associations thereby reducing their impacts on the environment. The importance of research on interactions between pollutants, microorganisms and soil is its applicability in environmental risk assessment and impact studies of organic pollutants which enter the soil either accidentally or intentionally. The application of image analysis with microscopy techniques to landfill soil-pollution interactions provides a means to study surface microbiology directly and to investigate microbial cells under highly controlled conditions. This research focused on the development of a method to study the real time processes of attachment, establishment, growth and division of microbial cells/associations in site covering soils. Image analysis provides a powerful tool for differential quantification of microbial number, identification of morphotypes and their respective responses to microenvironment changes. This minimal disturbance technique of examining visually complex images utilizes the spatial distributions and metabolic sensitivities of microbial species. It was, therefore, used to examine hexanoic acid catabolizing species, both free-living and in a biofilm, with respect to obviating the threat of hexanoic acid to reclamation strategies. The three sources of inoculum (soil cover, soil from the landfill base liner and municipal refuse) were compared for their ability to provide associations which catabolized the substrate rapidly. During the enrichment programme the inocula were challenged with different concentrations of hexanoic acid, a common landfill intermediate. From the rates at which the substrate was catabolized conclusions were drawn on which concentration of hexanoate facilitated the fastest enrichment. The results of initial batch culture enrichments confirmed that the soil used contained microbial associations capable of catabolizing hexanoic acid at concentrations < 50mM, a key leachate component. Exposing the landfill top soil microorganisms to a progressive increase in hexanoic acid concentration ensured that catabolic populations developed which, in situ, should reduce the phytotoxic threat to plants subsequently grown on the landfill cover. The analysis of surface colonization was simplified by examining the initial growth on newly-exposed surfaces. The microbial associations generated complex images which were visually difficult to quantify. Nevertheless, the dimensional and morphological exclusions which were incorporated in the image analysis software permitted the quantification of selected components of the associations although morphology alone was inadequate to confirm identification. The effects of increasing the dilution rate and substrate concentration on the growth of surface-attached associations in Continuous Culture Microscopy Units (CCMUs) were examined. Of the five dilution rates examined the most extensive biofilm development (9.88 jum2) during the selected time period (72h) resulted at a dilution rate of 0.5h' (at 10mM hexanoic acid). The highest growth (608 microorganisms.field"1) was recorded in the presence of 50mM hexanoic acid (D = 0.5h"1). To ensure that the different morphotypes of the associations were able to multiply under the defined conditions a detailed investigation of the component morphotypes was made. Numerically, after 60h of open culture cultivation in the presence of 50mM hexanoic acid, rods were the predominant bacterial morphotypes (43.74 field'1) in the biofilms. Both rods and cocci were distributed throughout the CCMUs whereas the less numerous fungal hyphae (0.25 field'1) were concentrated near the effluent port. The specific growth rates of the surface-attached associations and the component morphotypes were determined by area (//m2) colonized and number of microorganisms.field"' and compared to aerobic planktonic landfill associations. From area determinations ( > 0.16 h'1) and the number of microorganisms.field"1 10mM hexanoic acid was found to support the highest specific growth rate ( > 0.05 h"1) of the surfaceattached association isolated from municipal refuse. With optical density determinations, the highest specific growth rate (0.01 h'1) was recorded with 25mM hexanoic acid. The surface-attached microbial associations component species determinations by area and number showed that the hyphae had the highest specific growth rate ( > 0.11 h"1). The surface-attached microbial association specific growth rate determinations from the discriminated phase (0.023 h'1), area colonized (0.023 h"1) and number of microorganisms (0.027 h"1) calculated from the results of the component species rather than the association should give more accurate results. The specific growth rate obtained differed depending on the method of determination. Any one of these may be the "correct" answer under the cultivation conditions. Depending on the state (thickness) of the association (free-living, monolayer or thick biofilm) the different monitoring methods may be employed to determine the growth. As a consequence of the results of this study, the kinetics of microbial colonization of surfaces in situ may be subjected to the same degree of mathematical analysis as the kinetics of homogeneous cultures. This type of analysis is needed if quantitative studies of microbial growth are to be extended to surfaces in various natural and artificial environments.
Biodeterioration of aluminium hot roll mill emulsions.
(1998) Ramsden, Peter John.; Wallis, Frederick Michael.
An in-depth study of the biodeterioration of the Hulett Aluminium hot roll mill emulsion, Prosol, was conducted. Samples of the emulsion in use at the hot roll mill were taken from various areas of the emulsion reticulation system in order to identify regions of highest microbial contamination. Standard plate count techniques and diagnostic procedures were employed to quantify and identify the microorganisms in these samples. In some of the highly contaminated areas of the emulsion reticulation system, microorganisms exceeded lxlO'CFUml'1 emulsion. A range of bacteria was identified which included members of the genera: Bacillus; Pseudomonas; Escherichia; Enterobacter; Sporosarcina; Micrococcus; Aeromonas; Chromobacterium and Desulfovibrio. Various fungi, including several yeasts, were also isolated and some of the filamentous spore-forming types were identified zsAspergillus spp.; Penicillium spp. and a Cladosporium sp. A visual scale was developed to assess the growth rate of the isolated microorganisms on a range of specific media containing various emulsion components as carbon and energy source. Although the results obtained by using this scale were not conclusive, a few biodegradable components were nonetheless identified. It was found that mixed cultures of the above microorganisms had a greater biodeteriorative effect on the emulsion than did any of the pure cultures when applied separately. This suggested complex microbial interactions were involved in the breakdown of the emulsion. A laboratory-scale model system representative of the Hulett Aluminium hot roll mill was designed and constructed to carry out a series of tests on unprotected and biocide-treated emulsions. A range of biocide concentrations were tested from which the minimum biocide inhibitory concentration was calculated. It was shown that microorganisms exposed to sublethal doses of the biocide Busan (active component glutaraldehyde) over a prolonged period of time, exhibited greater levels of tolerance and resistance to the biocide than did those microorganisms not previously exposed. It was deduced that less frequent, shock doses of biocide are more effective in the control and eradication of emulsion degrading microorganisms than are frequent, low level doses of the same biocide. In addition to the biocide studies, three imported so-called 'biostable' emulsions were evaluated as possible replacements for the susceptible Prosol. Of these three imported emulsions, two viz. HRF3 and Houghton Biostable were shown to be more resistant than Prosol to biodeterioration. After assessing the current hot roll mill management practices, a number of recommendations were made, including: the improvement of plant hygiene; education of the mill workers; improvement of emulsion monitoring; improvement of down-time management and improvement of biocide dosing regimes. Recommendations are also made for minimizing potential microbial growth in the new hot roll mill currently under construction at the Hulett Aluminium processing plant at Pietermaritzburg, South Africa.
Bioremediation of arsenic contaminated groundwater.
(2008) Teclu, Daniel Ghebreyohannes.; Wallis, Frederick Michael.; Tivchev, George V.; Laing, Mark Delmege.
Sulphate-reducing bacteria (SRB) mediate the reduction of metals/metalloids directly or indirectly. Bioremediation of arsenic contaminated water could be a cost-effective process provided a cheap carbon source is used. To this end, molasses was tested as a possible source of carbon for the growth of sulphate-reducing bacteria (SRB). Its chemical composition and the tolerance of SRB toward different arsenic species [As (III) and As (V)] were also investigated. Batch culture studies were carried out to assess 1, 2.5 and 5 g l-1 molasses as suitable concentrations for SRB growth. The results indicate that molasses does support SRB growth, the level of response being dependent on the concentration; however, growth on molasses was not as good as that obtained when lactate, the usual carbon source for SRB, was used. The molasses used in this study contained several metals including Al, As, Cu, Fe, Mn and Zn in concentrations ranging from 0.54-19.7 ìg g-1, but these levels were not toxic to the SRB. Arsenic tolerance, growth response and sulphate-reducing activity of the SRB were investigated using arsenite and arsenate solutions at final concentrations of 1, 5 and 20 mg l-1 for each species. The results revealed that very little SRB growth occurred at concentrations of 20 mg l-1 As (III) or As (V). At lower concentrations, the SRB grew better in As (V) than in As (III). Batch cultures of sulphate-reducing bacteria (SRB) in flasks containing pine bark, sand and polystyrene as support matrices and Postgate medium B were used to study formation of biofilms. The effects of the support matrices on the growth of the organisms were evaluated on the basis of pH and redox potential change and the levels of sulphide production and sulphate reduction. Characterisation of the matrix surfaces was done by means of environmental scanning electron microscopy (ESEM). A consortium of SRB growing on polystyrene caused a 49% of original sulphate reduction whereas on sand a 36% reduction occurred. Polystyrene was further examined for its durability as a long-term support material for the growing of SRB in the presence of As(III) and/or As(V) at concentrations of 1, 5 and 20 mg l-1. Both sulphate reduction and sulphide production were greater in this immobilised system than in the matrix-free control cultures. With pine bark as support matrix no significant sulphate reduction was observed. The kinetics of sulphate reduction by the immobilised cells were compared with those of planktonic SRB and found to be superior. The leaching of organic compounds, particularly phenolic substances, from the pine bark had a detrimental effect on the growth of the SRB. Different proportions of pine bark extract were used to prepare media to investigate this problem. Growth of SRB was totally inhibited when 100% pine bark extract was used. Analysis of these extracts showed the concentration of phenolics increased from 0.33 mg l-1 to 7.36 mg l-1 over the extraction interval of 15 min to 5 days. Digested samples of pine bark also showed the presence of heavy metals. The effects of nitrate, iron and sulphate and combinations thereof were investigated on the growth of a mixed culture of sulphate-reducing bacteria (SRB). The addition of 30 mg l-1 nitrate does not inhibit the production of sulphide by SRB when either 50 or 150 mg l-1 sulphate was present. The redox potential was decreased from 204 to -239 mV at the end of the 14 day batch experiment in the presence of 150 mg l-1 sulphate and 30 mg l-1 nitrate. The sulphate reduction activity of the SRB in the presence of 30 mg l-1 nitrate and 100 mg l-1 iron was about 42% of original sulphate, while if no iron was added, the reduction was only 34%. In the presence of 20 mg l-1 either As(III) or As(V), but particularly the former, growth of the SRB was inhibited when the cells were cultured in modified Postgate medium in the presence of 30 mg l-1 nitrate. The bioremoval of arsenic species [As(III) or As(V)] in the presence of mixed cultures of sulphate-reducing bacteria was investigated. During growth of a mixed SRB culture adapted to 0.1 mg l-1 arsenic species through repeated sub-culturing, 1 mg l-1 of either As(III) or As(V) was reduced to 0.3 and 0.13 mg l-1, respectively. Sorption experiments on the precipitate produced by batch cultured sulphate-reducing bacteria (SRB-PP) indicated a removal of about 77% and 55% of As(V) and As(III) respectively under the following conditions: pH 6.9; biomass (2 g l-1); 24 h contact time; initial arsenic concentration,1 mg l-1 of either species. These results were compared with synthetic iron sulphide as adsorbent. The adsorption data were fitted to Langmuir and Freundlich isotherms. Energy dispersive x-ray (EDX) analysis showed the SRB-PP contained elements such as sulphur, iron, calcium and phosphorus. Biosorption studies indicated that SRB cell pellets removed about 6.6% of the As(III) and 10.5% of the As(V) from water containing an initial concentration of 1 mg l-1 of either arsenic species after 24 h contact. Arsenic species were precipitated out of synthetic arsenic-contaminated groundwater by reacting it with the gaseous biogenic hydrogen sulphide generated during the growth of SRB. The percentage removal of arsenic species was dependent on the initial arsenic concentration present. Lastly, laboratory scale bioreactors were used to investigate the treatment of arsenic species contaminated synthetic groundwater. A mixed culture of SRB with molasses as a carbon source was immobilised on a polystyrene support matrix. The synthetic groundwater contained either As(III) or As(V) at concentrations of 20, 10, 5, 1 or 0.1 mg l-1 as well as 0.1 mg l-1 of a mixture with As(III) accounting for 20, 30, 40, 60 and 80% of the total. More that 90% and 60% of the As(V) and As(III) respectively were removed by the end of the 14-day experiment. At an initial concentration of 0.1 mg l-1 total arsenic had been reduced to below the WHO acceptable level of 10 ìg l-1 when the proportion of As(III) was 20 and 30%, while at 40% As(III) this level was reached only when the treatment time was increased to 21 days. The efficiency of As(III) removal was increased by first oxidising it to As(V) using MnO2.
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 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.
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.
The effect of various crop residue management practices under sugarcane production on soil quality.
(2003) Graham, Martha Helena.; Haynes, Richard John.; Wallis, Frederick Michael.
This study examined the influence of different management practices under sugarcane production on soil chemical, biological and physical properties on a long-term (59yr) field experiment in KwaZulu-Natal. These management practices included conventional post-harvest burning of crop residues, with the tops either left on the soil surface or with tops removed, and green cane harvesting with the retention of crop residues on the soil surface as a trash blanket. Each of these treatments were either fertilized or did not receive fertilizer. The data collected was used to evaluate the effect of crop residue management on soil quality under sugarcane production. Soil organic matter content increased from 39 g C kg¯¹ soil, under conventional burning to 55 g C kg¯¹ soil in the surface 10 cm under green cane harvesting where crop residues are returned to the soil. It also resulted in greater recycling of nutrients and increases in exchangeable K and Ca and extractable P. Fertilizer application resulted in a build-up of soil nutrients in combination with trash retention. Fertilizer application induced increases in exchangeable P and also some accumulation in soil organic P. Exchangeable and non-exchangeable K concentrations were also higher in fertilized than non-fertilized treatments. However, nitrogen fertilizer application and, to a lesser degree, organic matter mineralization, resulted in soil acidification to a depth of 30 cm. Acidification in the fertilized treatments resulted in a concomitant increase in exchangeable acidity and exchangeable Al, due to the increase in H⁺ ions and solubilized Al species on exchange sites. Base cations moved into soil solution and were leaching to lower soil layers. The decrease in soil pH resulted in the surface charge conferred on the variable charge surfaces on soil colloids becoming less negative and as a result there was a decrease in ECEC. Acidification in fertilized treatment not only increased exchangeable Al but also the buffering reserve of non-exchangeable al; both that complexed with soil organic matter (CuCl₂- extractable) and that present as hydroxy - Al associated with mineral colloids (ammonium acetate - extractable). The increased organic matter content under trash retention resulted in an increase in ECEC. This enabled the soil to retain greater amounts of Ca²⁺, Mg²⁺ and K⁺ which were returned to the soil in the trash. Both residue retention and fertilizer application had a positive effect on the microbial biomass C and N and the microbial quotient increased from 0.39% to 0.86% as organic C increased from 39 g C kg¯¹ soil under burnt treatments to 55 g C kg¯¹ soil under trashed, fertilized treatments. This increase was associated with increased concentrations of labile organic material (K₂S0₄₋extractable) present as well as increased amounts of nutrients being cycled through the plant-soil system. The light fraction organic matter also increased with increasing returns of organic residues. However, the large active microbial biomass under the trashed, fertilized treatment resulted in an increased turnover rate of this fraction and consequently resulted in lower LF dry matter, C and N than in the unfertilized treatment. Aggregate stability increased with increasing amounts of organic material returned due to trash retention. Nevertheless, fertilized treatments induced a lower aggregate stability than unfertilized ones, despite the tendancy for the latter to have higher organic C and microbial biomass values. This was attributed to an increase in the proportion of exchangeable cations present in monovalent form (due to application of fertilizer K and leaching of Ca and Mg) favouring dispersion and a decline in aggregate stability. Green cane harvesting resulted in an increase in microbial activity (basal respiration, FDA hydrolytic activity, arginine ammonification rate and dehydrogenase activity) and in the activity of specific soil enzymes involved in turnover of C, N, P and S to a depth of 30 cm. Increased activities of these enzymes reflect a higher rate of turnover of C, N, P and S. The metabolic quotient decreased with increasing residue return, indicating a more metabolically efficient microbial community. Fertilizer application resulted in a variable effect on enzyme activity. Long-term fertilizer application resulted in an increase in invertase and acid phosphatase, a decrease in L-histidase and arylsulphatase and had little effect on protease and alkaline phosphatase. These variable effects were explained in terms of an interaction between fertilizer - induced increases in Corg and soil nutrient status and fertilizer - induced soil acidification. The size and activity of the soil microbial biomass was studied in the plant row and in the inter-row of a sugarcane field under burning or green cane harvesting. Soils were sampled to 30 cm depth in (i) the centre of the plant row, (ii) 30 cm out from the row centre and (iii) 60 cm out from the row centre (i.e. the middle of the inter-row area). Under burning, the only substantial input of organic matter to the soil was from root turnover in the row area where the root biomass was concentrated. As a consequence, the size (microbial biomass C) and activity (basal respiration of the soil microbial community were concentrated in the row. However, under green cane harvesting there was a large input of organic matter in the inter-row area in the form of the trash blanket itself and through turnover of crop roots that were concentrated in the surface 10 cm of the soil below the blanket. As a result, soil microbial activity was considerably higher in the interrow area under green cane harvesting than under burning. Phospholipids are essential membrane components of microorganisms and a good correlation was found between the total PLFA's extracted from soils and the microbial biomass C, indicating that phospholipids are an accurate measurement of living biomass. Mutivariate statistical analysis (PCA) was used to separate different PLFA profiles under burning versus trash retention and under different land uses (sugarcane, maize, annual and perennial pasture and undisturbed veld). Soil organic matter content contributed the greatest variance in the data along the first axis. That is increasing soil organic matter return not only increased the size of the microbial biomass, but also affected the composition of the microbial community. There was a shift in the different sub-fractions under different management practices. MUFA's are general biomarkers of Gram negative bacteria and were found to be a sensitive indicator of higher substrate availability (i.e they increased under green cane harvesting). Fungal biomarkers indicated an increased fungal biomass associated with surface application of residues. Soil physical conditions were considered to be a contributing factor to the shift in microbial community structure. Increased organic matter content improved soil physical conditions and preferentially stimulated the growth of aerobic microorganisms. In addition to this, the proportion of SATFA (gram positive bacteria) was found to increase in response to burning. This increase was attributed to the survival mechanisms of these microorganisms (i.e. endospore formation). It was found that the conversion from burning to trash management changes the composition of the soil microbial community. The effect of management practices on soil functional diversity was also evaluated using two methods (i.e. Biolog plates and substrate induced respiration (SIR)). Biolog plates are a selective technique that stimulate growth of a small proportion of the soil microbial community whereas the SIR technique measures the activity of the metabolically active microbial community in situ. As a result the SIR method separated treatments more effectively than Biolog plates (i.e. annually tilled treatments, permanent grassland sites and fertilized and unfertilized treatments). The quantity and the quality of organic C supply influenced the catabolic diversity. Conversion from burning to green cane harvesting greatly increased catabolic evenness and richness and therefore presumably also tended to increase the resilience of the soil to stress and disturbance particularly in relation to decomposition functions. It was concluded that conversion from preharvest burning to green cane harvesting results in an increase in soil organic matter content, an improvement in soil structure and soil nutrient status, an increase in the size, activity, taxonomic and functional diversity of the soil microbial community. The practice should therefore be promoted to the South African sugar industry.
Effects of management practices on soil organic matter content, soil microbial activity and diversity in the KwaZulu-Natal midlands.
(2002) Nsabimana, Donat.; Wallis, Frederick Michael.
The objective of this study was to investigate the effects of land use and management practice on the soil organic matter content and the size, activity and diversity of the microbial biomass. These effects were investigated using samples taken from the top (0-10 cm) layer of the soils from long-term agricultural managements including natural grassland, maize under conventional (maize CT), maize under zero tillage (maize ZT), annual ryegrass, Eucalyptus, Pinus, and permanent kikuyu pasture. The natural grassland was used as a control since records indicated that no agricultural activity had ever been exerted on the soil. The measurements used to investigate these effects included soil organic C, total N, soil pH, microbial biomass C, basal respiration rate, microbial quotient, metabolic quotient, dehydrogenase activity, fluorescein diacetate (FDA) hydrolysis, arginine ammonification rate, arylsulphatase activity and acid and alkaline phosphatase activities. The microbial functional diversity was measured using the Biolog Ecoplate and catabolic response profiles methods. Soil organic Cand total Nwere lowest under maize CT, followed by maize ZT and annual ryegrass and were higher under natural grassland, Eucalyptus and Pinus plantations while permanent kikuyu pasture had the highest values. The other analyses, namely microbial biomass C, basal respiration rate, FDA hydrolysis, arginine ammonification rate and arylsulphatase activity also followed the same pattern. Annual cultivation was responsible for a decrease in microbial biomass C, basal respiration rate and enzyme activity, principally because there was an appreciable decrease in soil organic matter content. Conversely, permanent pasture, Eucalyptus and Pinus plantations increased appreciably the amount of organic C and consequently, promoted the size and activity of the microbial biomass in the soils. The principle component scores showed that management practices affected the microbial functional diversity because different treatments were found in separate zones of the principle component spaces. The regression analysis showed that the variation in the PC1 and PC2 scores was correlated with the variation in soil organic C, exchangeable acidity, extractable P and exchangeable K and Mg. In addition, richness, evenness, Shannon, and Simpson diversity indices showed that any management practice affects the dynamics of soil microbial diversity.
The epidemiology and control of crucifer chocolate spot.
(1998) Machaba, Kulani Patrick.; Laing, Mark Delmege.; Wallis, Frederick Michael.
In the 1991/92 season, a leaf spotting disease of crucifer seedlings appeared at a number of nurseries in Kwazulu-Natal (KZN) Province. Towards the end of 1996, the disease had also been detected in Western and Northern Provinces, and even in Zambia. The disease appeared in nurseries during warm humid conditions. It initially started as small, water-soaked spots on leaves, which later expanded and coalesced, forming halo regions surrounded by a dark brown to black ring. Due to the typical dark brown spots, the disease was named crucifer chocolate spot (CCS). From the studies conducted by Qhobela & Laing (1994), the causal agent of CCS was found to be a Xanthomonas campestris pathovar that was in symbiotic association with a unique bacteriophage. Koch's postulates were conducted. The causal agent of CCS was inoculated on cabbage seedlings (6-8 weeks old), and the seedlings subjected to post-inoculation periods of approximately 48 hours at 28-30°C, and relative humidity (RH) of ≥70%. The best method of inoculation was achieved when the leaves were pricked with pins prior to application of the inoculum (≥10(7)cfu/ml). The latent period of CCS varied from 2-6 days, depending on RH, temperature, nutritional status of the host and other factors within the crop pathosystem. Artificial inoculation studies showed the host range of CCS X. campestris to be similar to X. campestris pv. armoraciae and X. campestris pv. campestris. All the pathogens were able to infect cabbage, cauliflower, broccoli and lettuce plants during artificial inoculation. A hypersensitive response was elicited on pepper, tomato, and tobacco plants. Electron microscopic studies showed epiphytic colonization by CCS X. campestris, and also the colonization of xylem, phloem and parenchyma tissues. Although xylem tissues were also colonized, the V-shaped lesions and vein blackening characteristics typical of X. campestris pv. campestris were not observed. Profuse multiplication of the pathogen resulted in rupturing of cell organelles and cell membranes. Different crucifer cultivars from two seed companies were found to have varying levels of susceptibility to CCS disease during artificial inoculation, with levels of leaf area infected ranging from 0% to 34%. Disease severity was also affected by seasonal variations. None of the seed-lots tested in this research were found to be infected by CCS X. campestris, although some were infected by other xanthomonads. Chemical seed treatment (soaking in bactericide solutions at 30°C for two hours) with biocides; e.g., Kocide 101, copper oxychloride, and copper ammonium carbonate was more effective than hot water treatment (50°C for 30 min). All seed treatments lowered pathogen levels, however, none resulted in complete control of seedborne inoculum. Percentage germination of seed was low for chemical treatment compared to the hot water treatment. Disease levels were reduced with increased rates of Ocean 3.1.3 (38) fertilizer applied, with significant differences in the percentage leaf area infected at obtained at application levels of 400 ppm. Increased levels of N, P, K, Zn and Cu in leaf tissues were associated with low levels of disease severity. Copper bactericides; e.g., Kocide 101, Copper Count N, Copper Ammonium Carbonate B and Copper oxychloride were more effective than quaternary ammonium compounds in reducing disease levels (≤30%). The combination of copper bactericides and mancozeb improved the performance of the copper compounds. Application of copper bactericides up to four times the recommended dosage did not result in increased efficacy. CCS was found in 80% of KZN nurseries surveyed between December 1995 and December 1996. During this period, it was not detected from Mpumalanga and Northern Provinces. Factors that contributed to CCS disease outbreaks include favourable climatic conditions (≥ 40% RH, ≥24°C), wounding of seedlings by pests, and nutrient stress.
A histopathological study on selected bacterial vascular diseases with emphasis on ultrastructure.
(1975) Wallis, Frederick Michael.; Truter, Susarah J.
No abstract available.
Humic acid pretreatment for enhancing microbial removal of metals from a synthetic 'wastewater'.
(2004) Desta, Tsegazeab Goje.; Wallis, Frederick Michael.
The presence of heavy metal ions in waste streams is one of the most pervasive environmental issues of present times. A rotating biological contactor (RBC) was used to investigate the potential capacity of microbial biofilms in remediation of the metal ion species from a mixed metal contaminated effluent solution containing Cr+3 , Pb+2 and Cu+2 , each at a concentration of 200 mg r1 • In the first part of this study the effectiveness of various support materials for the development of microbial biofilms capable of removing heavy metals from a synthetic effluent was investigated. EDX analysis showed that none of the support matrices investigated, viz. gravel, polyester batting and sand, adsorbed metal ions on their surfaces; hence, metal adsorption was due purely to microbial activities. The biofilms attached more firmly and uniformly to polyester batting than to gravel and sand. The characteristics of polyester batting which made it a superior support matrix were its surface roughness and porous hydrophilic nature, which provided a larger surface area for the adhesion of microorganisms and attraction of nutrients during the biofilm development process. The selective accumulation of metal ion specIes by various microbial populations grown as biofilm using polyester batting as support matrix in separate compartments of a single-stage RBC bioreactor was examined. Lead ions were readily accumulated by almost all the microbial biofilms tested. Fungus-dominated biofilms selectively accumulated chromium ions whereas biofilms comprising mainly bacteria more readily accumulated copper ions from the mixed metal contaminated effluent solution. However, where interactions between the bacterial and fungal components were encouraged the mechanical stability of the biofilms was enhanced so that large amounts of all three metal ion species were removed by this biofilm. The combined effect of a series of bench-scale columns containing liquid humic acid and a three stage RBC bioreactor on the removal of metal ion species from a mixed metal contaminated effluent was investigated. After seven days of treatment the combined system had removed approximately 99% of the Cr+3, 98% of the Pb+2 and 90% of the Cu+2 ions from the mixed metal contaminated synthetic effluent. Complexation of the metal ions with humic acid was the predominant factor accounting for approximately 68-86% Cr+3 , 70-86% Pb+2 and 53-73% Cu+2 removal levels within the columns. A large proportion of the remaining Cr+3 and Pb+2, but not of the Cu+2, was removed in compartment 1 of the RBC. This suggested that the presence of the former two metals in solution might have reduced the removal of the Cu+2 ions from the system. The removal of substantially large amounts of the competing ions chromium and lead during the initial stages of the treatment process meant that copper was successfully taken up in the second and third RBC compartments. Hence, the economy of the treatment process was improved as larger quantities of the metal ions were removed in a shorter period of time than was possible when using the individual treatments (humic acid-metal complexation and biofilm adsorption) separately. More than 75%,92% and 86% of the adsorbed Cr+3 , Pb+2 and Cu+2 ions, respectively, were recovered from the three RBC bioreactor compartments following repeated washing of the biofilms with 0.1 M HCI. This relatively easy desorption suggested that the metal ions were simply adsorbed onto the surfaces of the biofilm cells rather than being taken into the cytoplasm of the cells.
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.
Isolation and identification of antibiotic producing microorganisms from natural habitats in the KwaZulu-Natal midlands.
(2001) Okudoh, Vincent Ifeanyi.; Wallis, Frederick Michael.
The search for new antibiotics continues in a rather overlooked hunting ground. In the course of screening for new antibiotic-producing microorganisms, seventy-nine isolates showing antimicrobial activity were isolated from soil samples from various habitats in the KwaZulu-Natal midlands, South Africa. Existing methods of screening for antibiotic producers together with some novel procedures were reviewed. Both modified agar-streak and agar-plug methods were used in the primary screens. The use of selective isolation media, with or without antibiotic incorporation and/or heat pretreatment, enhanced the development of certain actinomycete colonies on the isolation plates. Winogradsky's nitrite medium (Winogradsky, 1949), M3 agar (Rowbotham and Cross, 1977), and Kosmachev's medium (Kosmachev, 1960), were found to be selective for actinomycetes. Statistical analysis showed highly significant interactions between isolates, assay media and the test organisms. The diameters of inhibition zones were found to be larger on Iso-sensitest agar (ISTA)[Oxoid, England] than in nutrient agar plates. Of the 79 isolates that showed antimicrobial activity, 44 isolates were selected for confirmatory screening. Of these, 13 were selected for secondary screening. Criteria for selection were based on significant inhibition of at least two test organisms and/or the inhibition of the specifically targeted organisms, Pseudomonas and Xanthomonas species. Following secondary screening eight isolates were considered for further investigation. The isolates were tentatively identified . on the basis of morphological features, using both light microscopy and scanning electron microscopy(SEM); their ability to utilize various carbon sources; and selected physiological and staining tests. Suspected actinomycetes were further characterized on the basis of selected chemical properties using thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) techniques. High pressure liquid chromatography analysis (Beckman 6300 analyzer) detected the presence of diaminopimelic acid (DAP) in whole-cell hydrolysates of six of the isolates while TLC analysis confirmed the type ofDAP present. The isolates N2, N12, N16, N19 and N35 were tentatively identified as Thermomonospora, Saccharopolyspora, Nocardiodes, Corynebacterium and Promicromonospora, respectively. Isolate N30 was identified as belonging to the coryneform group ofbacteria, possibly an Arthrobacter species. Isolate, N8, tentatively identified as Actinosynnema, was unique among the isolates tested as it showed good antimicrobial activity against all the Gram- positive and Gram-negative bacteria, and yeasts used as test organisms in the present investigation.
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.
Screening of aerobic endospore-forming bacterial isolates as candidate biocontrol agents against rhizoctonia solani.
(2016) Hunter, Charles Haig.; Schmidt, Stefan.; Laing, Mark Delmege.; Wallis, Frederick Michael.
Bacterial-based biocontrol of soil-borne phytopathogens has gained prominence as a promising technology for developing sustainable agricultural pest control practices. Aerobic endospore-forming bacteria are seen as potential candidates for biocontrol applications due to various ecological and physiological traits which have been shown to influence plant health and disease control. Their ability to produce endospores also provides a major commercial advantage over non spore-forming bacteria. Appropriate screening methods are central to the discovery of successful biocontrol agents and should ideally be both ecologically relevant and able to evaluate a large number of isolates. A study was therefore undertaken with the aim of establishing screening methods that facilitate the selection of aerobic endospore-forming bacteria as candidate biocontrol agents against Rhizoctonia solani, an economically important fungal pathogen exhibiting a wide host range. Aerobic endospore-forming bacteria were isolated from rhizosphere material of five crop types grown in composted pine bark medium and screened for R. solani antagonism using traditional in vitro dual-culture bioassays. Isolates exhibiting antifungal activity were then evaluated in vivo for biocontrol activity against R. solani in cucumber seedling trials. Selected isolates were evaluated further using several screening approaches including: genomic fingerprinting; characterization of, and PCR-based screening for genes involved in the biosynthesis of bioactive lipopeptide compounds; and, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of rapidly screening bacterial isolates. Approximately 6% of the bacterial isolates (n=400) showed antagonism towards R. solani in vitro. Dual-culture bioassays against R. solani, Fusarium oxysporum, Botrytis cinerea and Pythium arrhenomanes revealed that the antagonistic activity amongst isolates varied considerably and was influenced by the duration of the assay. From these assays it was possible to rank isolates based on the extent and stability of the inhibitory response in vitro as well as by the spectrum of antifungal activity observed. Twenty-four isolates were selected for in vivo screening for biocontrol activity against R. solani, using susceptible cucumber plants grown under greenhouse conditions. In preliminary experiments the pathogen loading rates were shown to have a marked influence on disease severity. In experiments where R. solani was seeded in the form of colonized agar plugs, significant differences between treatments and controls were recorded and several potential biocontrol candidates were identified. A general observation was that isolates that achieved high rankings in vitro performed better in the in vivo trial than those with lesser rankings; although some exceptions were noted. These findings support the notion that fungal antagonism is an important determinant of biocontrol potential that can be used in preliminary biocontrol screening programmes. Internal-transcribed spacer region (ITS) PCR and randomly amplified polymorphic DNA (RAPD) PCR were evaluated as methods to differentiate isolates exhibiting antifungal activity in vitro. ITS-PCR distinguished three major groupings, but proved to be limited in its ability to detect inter- and intra-specific variation amongst closely related organisms. Based on 16S rRNA gene sequence analysis, two of the groups were identified as members of the “Bacillus subtilis” and “Bacillus cereus” clusters; while, the third group consisted of a single isolate identified as a strain of Brevibacillus laterosporus. RAPD-PCR revealed further levels of genetic diversity within each ITS grouping. The “Bacillus subtilis” cluster was distinguished further into four distinct groups, which based on gyrA gene fragment sequence analysis, were identified as strains of B. amyloliquefaciens subsp. plantarum and B. subtilis respectively. Sequence matches were consistent with the RAPD-PCR groupings, indicating that this method was appropriate for differentiating related isolates at the strain and possibly the sub-species level. Clonal similarities were evident for a number of strains isolated from different plant species suggesting that these may reflect populations of rhizosphere competent strains and/or plant adapted ecotypes. Strains of B. amyloliquefaciens subsp. plantarum and B. subtilis were amongst the best performers in the in vivo biocontrol seedling trial and generally performed better than the “Bacillus cereus” group of isolates. RAPD-PCR of the “Bacillus cereus” isolates showed that they exhibited greater levels of genetic heterogeneity and that the groupings detected were not consistent when different primer sets were evaluated. Genomic fingerprinting was found to provide an insight into the prevalence, distribution and possible rhizosphere competency of related strains. Liquid chromatography was used in conjunction with electrospray-ionization time-of-flight (ESI-TOF) mass spectrometry (MS) to characterize bioactive lipopeptides purified from culture supernatants of selected strains that ranked highly in the in vitro/in vivo assays. Phylogenetically related strains produced very similar lipopeptide profiles. Bacillus subtilis strains were found to produce isoforms of surfactin and fengycin. In addition to these lipopeptides, B. amyloliquefaciens subsp. plantarum strains were also found to produce isoforms of bacillomycin D or iturin A. Bacillomycin/iturin and fengycin fractions exhibited antifungal activity in vitro, whereas surfactin fractions did not. Isolates that ranked the highest in the R. solani dual-culture bioassays all produced either isoforms of bacillomycin D or iturin A. Bacillomycin D producing isolates were amongst the best performers in the in vivo biocontrol trials. Gene markers targeting the biosynthetic apparatus of the detected lipopeptide classes were then assessed for screening purposes using PCR. BACC1F/1R primers targeting the bacillomycin D synthetase C (bmyC) gene correlated well with the ESI-TOF MS findings, whereas ITUD1F/1R primers targeting the malonyl-CoA-transacylase (ituD) gene linked to iturin A biosynthesis were unable to distinguish between isolates that produced iturin or bacillomycin in culture. Disparities between some of the PCR and ESI-TOF MS results suggested that primers targeting srfA (surfactin) and fenD (fengycin) biosynthetic genes showed limited specificity amongst the strains screened. Phylogenetic comparisons of srfD and fenD gene sequences from selected strains of B. amyloliquefaciens subsp. plantarum and B. subtilis revealed that these genes clustered according to species with marked heterogeneity between clusters being evident. Using fenD gene sequence data from B. amyloliquefaciens subsp. plantarum FZB42, primers (FENG1F/1R) targeting fengycin synthetase genes of strains of B. amyloliquefaciens subsp. plantarum isolated in this study were successfully established. MALDI-TOF MS was assessed as a means of identifying isolates antagonistic to R. solani in vitro and determining their associated lipopeptide profiles. Mass spectra were obtained in the m/z range 2000 to 20000 for identification and grouping purposes and in the m/z 750 to 2500 range in order to profile lipopeptide production. The available Bruker BDal spectral library allowed for the identification of isolates to the genus level but proved to be limited for identifying environmental isolates to the species level. Extension of the library using “inhouse” mass spectra generated from isolates identified in this study significantly improved the level of isolate identification in subsequent identification runs. Cluster analysis of mass spectra allowed for the relationships between isolates to be established and provided a means of grouping closely related isolates. Strains of B. subtilis and B. amyloliquefaciens were clearly distinguished from one another and the potential for differentiating strains at the subspecies level was also shown. MALDI-TOF MS also provided a convenient means of detecting bioactive lipopeptides directly from whole cell preparations, cell extracts and crude culture filtrates. Lipopeptide profiles varied depending on taxonomic groupings. Results for isolates within the “Bacillus subtilis” group supported the earlier ESI-TOF MS findings and were found to be more reliable than PCR screening for lipopeptide synthesis genes. “Bacillus cereus” group isolates produced distinct spectral profiles with peaks that were consistent with biomarkers previously described in the literature as isoforms of the kurstakin class of lipoheptapeptides. Brevibacillus laterosporus CC-R4 yielded a unique spectral profile in the m/z 750-2000 range with mass fragments which were similar to antimicrobial compounds recently reported in the literature. Overall, MALDI-TOF MS was found to fulfil the requirement for a practical yet robust technique suitable for processing large numbers of aerobic endospore-forming bacteria for biocontrol screening. This study has shown that genomic fingerprinting and MALDI-TOF MS characterization of bacterial isolates are worthwhile additions to preliminary in vitro screening practices. They provide a level of isolate differentiation and characterization that is beneficial for selecting candidate biocontrol agents, which is not possible with traditional screening practices. Effectively, they allow traditional biocontrol screening to move away from empirically based approaches to ones which are “knowledge” based, allowing for representative groups of bacteria with specific traits to be selected for further evaluation.
Studies on Cercospora zeae-maydis, the cause of grey leaf spot of maize in KwaZulu-Natal.
(2000) Caldwell, Patricia May.; Laing, Mark Delmege.; Wallis, Frederick Michael.; Rijkenberg, Fredericus Hermanus Johannes.
In 1983, Latterell and Rossi described grey leaf spot (GLS) of maize (Cercospora zeae-maydis Tehon and Daniels) as "a disease on the move". This pathogen has more than lived up to its reputation. It is estimated to be spreading at a rate of 80-160 km each year, and is recognized as one of the most grain yield-limiting diseases of maize worldwide. The occurrence of the pathogen in the Province of KwaZulu-Natal (KZN), Republic of South Africa (RSA), in 1988, was its first official report from the African Continent. It has since become pandemic, causing grain yield losses of up to 60%. It has spread to other provinces in RSA as well as other African countries, namely Cameroon, Kenya, Malawi, Mozambique, Nigeria, Swaziland, Tanzania, Uganda, Zaire, Zambia and Zimbabwe. It has also been reported to occur in Brazil, China, Columbia, Costa Rica, Mexico, Peru, Trinidad, and Venezuela. The use of soil macro- and micronutrients in the management of fungal plant pathogens is widely documented in the literature. Specific nutrients are known to increase or decrease disease resistance in plants. However, each host-pathogen interaction must be considered on an individual disease basis, together with environmental and soil variables. Although few diseases can be eliminated by a corrective fertilizer regime, the severity of a disease can be reduced by specific nutrients, particularly when used in conjunction with other cultural practices. However, the economic implications, and not grain yield alone, of different control measures should be considered; i.e., farmers must compare the expected added gross margin ha -1 (added income minus added costs) with the potential variability in expected added gross margin ha -1 (upper and lower limits) of each treatment when deciding on which fertilizer applications and/or fungicide treatments to use. Literature reviews were undertaken on both GLS and the use of soil nutrients to control fungal plant pathogens to provide the necessary background technical information in order to conduct research under local conditions, and to assist in interpretation of results of experiments. Nutrient trials to control GLS were conducted at two sites in KZN, i.e., Cedara (1995/96, 1996/97 and 1997/98) and Ahrens (1995/96). Research at Cedara showed that with increased applications of nitrogen (N) at 0, 60 and 120 kg N ha -1 and potassium (K) at 0, 25, 50 and 150 kg K ha -1, leaf blighting occurred earlier, and final percentage leaf blighting and the standardized area under disease progress curve were higher. The Ahrens trial also showed that with increased applications of N (0, 60, 120 and 180 kg N ha -1) and K (0, 50, 100 and 150 kg K ha -1), there were also increases in final percentage leaf blighting. Increasing phosphorus levels of 0, 30, 60 and 120 kg P ha -1 did not have any effect on final percentage leaf blighting. The application of systemic fungicides to GLS-susceptible maize was highly effective in controlling GLS and increasing grain yields substantially with increased N and K applications. In the non-fungicide treated plots, grain yields did not increase with increased applications of K in all three years of the trial. This was probably because grain yield response, which should have occurred at higher K applications, was reduced by increased GLS severity. Similarly, grain yields did not increase significantly with N application in 2 of the 3 years of the trial. At Cedara, non-fungicide treated maize produced a financial loss of -R165 and -R48 with 25 and 50 kg K ha -1 respectively, relative to 0 kg K ha -1. However, increasing N applications resulted in increasing grain yields, and added gross margins of R714 ha -1 and R536 ha -1 with applications of 60 and 120 kg N ha -1, respectively. The drop in added gross margin at 120 kg N ha -1 was probably because of increased GLS levels at higher fertiliser rates, resulting in reduced grain yields. In fungicide treated maize, added gross margin relative to 0 kg K ha -1 increased from R851 to R1212 ha -1. However, there was a loss of -R133 ha -1 in added gross margin relative to 0 kg N ha -1 at 60 kg N ha -1 as increased grain yields did not offset the added cost of N fertilizer and fungicide applications. At 120 kg N ha -1 added gross margin relative to NO was R423 ha -1. Highest grain yields and gross margins in fungicide treated maize were obtained with 120 kg N ha -1 and 150 kg K ha -1, as expected. However, in non-fungicide treated maize, highest grain yields and gross margins were obtained using 60 kg N ha -1 and 50 kg K ha -1. This was because of higher GLS severity at the higher N and K application rates. Yields of wheat grown in soils with residual fertilizers after non-fungicide treated maize were higher (4.21 ha -1) compared to yields (3.61 ha -1) grown on residual fertilizers after maize that had been sprayed to control GLS. This was probably as a result of GLS reducing the photosynthetic area of maize leaves, causing premature death with a concomitant reduced uptake of nutrients by roots. This resulted in higher residual levels of fertilizers in soils where fungicide applications were not used to control GLS on maize compared to soils planted with maize where GLS was controlled through the application of fungicides. In KZN there are approximately 350,000 small-scale farmers. The same diseases that affect commercial agricultural production also affect the small-scale farmer, the major difference being in the methods of disease control employed. At the commercial level, most farmers rely on the use of agro-chemicals, which are often not available to the small-scale farmer due to the relatively high cost of agro-chemicals, application methods, and the non-availability of products in the rural areas. The level of illiteracy of the small-scale farmer may also inhibit the use of agro-chemicals. In many African countries, the per capita consumption of maize may be as high as 100 kg per year. Production of cereals in Africa has fallen in the past 25 years. This, together with yield reductions of maize caused by GLS, is likely to contribute to an even greater food deficit in many African countries. At present, low soil fertility and pH levels are a problem among small-scale farmers both in the RSA and other parts of Africa. In the RSA, government policy is to increase maize production by small-scale farmers through improved agronomic methods, including increased fertilizer application. Appropriate and affordable rotations and other improved agronomic practices need to be developed and promoted to ensure food security and sustainable systems for smallscale farmers. The results from the nutrient trials presented in this thesis have practical applications for the small-scale farmer who does not have the option of controlling GLS through the use of agrochemicals. The small-scale farmer will be able to attain a maximum gross margin from his maize crop by applying 60 kg N ha -1 and 50 kg K ha -1, if no fungicides are applied. However, comparative analyses of manure showed that a small-scale farmer would have to apply 1-3 tonnes of manure in order to achieve similar nutrient levels - a procedure that would be impractical. Comparative financial analyses of aerial and knapsack fungicide applications showed that it would be uneconomical for the small-scale farmer to apply fungicides using a knapsack sprayer. A simple spreadsheet has been created to help farmers make the best choice of N (0, 60 or 120 kg N ha -1) and K (0, 25, 50 or 150 kg K ha -1) and the number of fungicide application (O, 1, 2 or 3). This will eliminate the guesswork needed for farmers to maximize gross margins, based on a specific amount of money available. The resistance expressed by different hybrids on conidial germination of C. zeae-maydis at varying temperatures, desiccation periods and interrupted dew periods was investigated using the susceptible ZS 206 and the less susceptible SC 625 maize cultivars. Germination of conidia was maximized at 28°C on both cultivars by 48 hr with ZS 206 showing 100% germination, in contrast to only 63% germination in SC 625. As the number of days (1-5) of desiccation increased following inoculation, germination decreased from 100 to 47% in ZS 206 and from 62 to 0% in SC 625, respectively. The observation that C. zeae-maydis is able to tolerate unfavourable conditions and resume germ tube growth when favourable conditions return was confirmed in interrupted dew period studies. There was no change in percentage germination after 48 hrs., when plants were subjected to interrupted dew periods of 2-36 hrs, following a 6 hr period at 95-100% RH at 28 °C in a dew chamber. However, germination was lower (64%) on SC 625 than ZS 206 (90%). The wider range of temperature conditions favourable for conidial germination of ZS 206, and the fact that it was less affected by desiccation and interrupted dew periods than SC 625, could account for the different susceptibility levels of these two hybrids to GLS. Peak daily conidial catches were found to be between 1200 and 1400 hrs when temperatures and vapour pressure deficits were highest and leaf wetness lowest. Multiple regression analyses identified high evaporation over a 24 hr period, low temperatures over a 48 hr period and wind over a 72 hr period as the weather variables most strongly associated with high conidial releases. Rain, high vapour pressure deficit values and temperatures between 20-30 °C with leaf wetness over a 72-day period, together with prolonged high evaporation over a 48 hr period were identified as limiting factors in conidial release. These results indicate that temperatures (< 20 °C) and moisture 24-48 hrs prior to release is required for production of conidia. However, dry air and leaf surfaces are required for conidia to break off conidiophores at the point of attachment, i.e., a hygroscopic process is involved in release of conidia in C. zeae-maydis. In general, the process of conidiogenesis in C. zeae-maydis is similar to that observed on C. beticola. Successive formation of conidia on the same conidiophore are in accord with previous observations on C. zeae-maydis. Conidial measurements are also similar to other taxonomic descriptions of C. zeae-maydis. Hyphae aggregate in the substomatal cavity and give rise to fascicles of 1-2 septate conidiophore initials which emerge through the stoma. A single, aseptate conidium develops from the conidiogenous cell of the conidiophore initial. Extension growth of the conidiogenous cell from the base and one side of the terminal conidium, leads to the lateral displacement of the conidium on the conidiophore. After conidial secession, the conidiophore continues to grow, producing a second conidium from the conidiogenous cell at the apex of the extended conidiophore. This sympodial and successive proliferation of the fertile conidiogenous cell results in the formation of a characteristic 1-3 geniculate, occasionally 4, conidiophore, bearing a single conidium at each apex. This body of research has added information that was previously missing in the lifecycle of C. zeae-maydis. However, this additional information has, in turn, led to other yet unanswered questions which need to be addressed in the future, particularly under southern African conditions. A thorough knowledge and understanding of the epidemiology of this pathogen can result in more effective control strategies with increased yields for both commercial and small-scale farmers in KZN.
The transformation of South African soya bean cultivars with a synthetic Basta resistance gene.
(1995) Van Huyssteen, Tracy.; Wallis, Frederick Michael.
The development of a genetic engineering system for soya bean (Glycine max L.) is described in this thesis. Routine tissue culture regeneration systems were developed for South African cultivars of soya bean despite the recalcitrant nature of this plant to in vitro manipulation. Regeneration of shoots was obtained when cotyledons were excised from seeds germinated for two days and cultured on B5 BA 20 medium containing 2 mg/I BA. The important problems of in vitro shoot elongation and rooting were overcome by culturing cotyledons in the dark for four weeks to produce shoots with unusually long stems. This was followed by one week of culture under conditions of high light intensity to obtain healthy green shoots which could be rooted , either in sorbarods or on solid Y2MS 30 medium. The use of a mist bed for the hardening off of rooted soya bean regenerants was essential for the recovery of fertile soya bean plants. Molecular techniques for the cloning of foreign genes into binary vectors suitable for plant genetic engineering were also studied and are described in the thesis. The Basta herbicide resistance gene, pat, was successfully cloned into the binary vector pBI121 which contains the [beta]-glucuronidase (GUS) reporter gene, uidA. The new construct, pB1121/Ac, was conjugated into various disarmed Agrobacterium tumefaciens strains and these strains, along with other binary vector-containing strains, were used to transform soya bean plant material. Although a protocol for the routine transformation of soya bean was not developed, transgenic soya bean material resistant to kanamycin and showing GUS activity was obtained. Transformation of wound sites on cotyledons was obtained in several experiments and transgenic shoots were regenerated from inoculated cotyledons. Only the A. tumefaciens strain C58C1 (pGV2260)(pJIT119) was able to transform cotyledonary cells of soya bean and, therefore, only kanamycin resistant soya bean shoots were produced. Transgenic soya bean plants resistant to the herbicide Basta were not produced due to the recalcitrant nature of the crop to genetic engineering. Transformation of the non-recalcitrant plant, tobacco, which is a model system for plant genetic engineering was achieved. The binary pat gene containing vector constructed in th is study, as well as vectors obtained from AgrEvo, were tested. The transgenic Basta resistant tobacco plants obtained were used to optimize assay systems for the analysis of transformed plant material containing the pat gene. These assay systems included the use of the polymerase chain reaction as well as digoxigenin-Iabelling of a DNA probe suitable for detection of the pat gene.
Utilization of Bacillus spp. as plant probiotics.
(2000) Yobo, Kwasi Sackey.; Laing, Mark Delmege.; Wallis, Frederick Michael.
Numerous microorganisms produce beneficial effects on plant developmentwhen applied to crop seeds or incorporated into soil. Research efforts worldwide over the past two decades have renewed commercial interest in plant growth promoting rhizobacteria (PGPR).With successes being recorded in PGPR research, it is expected that within the next few years, more commercial PGPR products will be available on the market. In particular, commercial PGPR could be advantageous to plant nurseries if they enabled earlier sale of plants, more rapid turnover of seedlings and further crop production cycles. Trials were carried out to evaluate the growth stimulation and biological control abilities of Biostart™, a Bacillus-based plant probiotic comprising seven Bacillus spp. and Biostart® 2000 (a combination of three of the Bacillus spp.). The first priority was to determine the survival pattern of six Biostart™ Bacillus spp., namely B. chitinosporus, B. uniflagellatus, B. laterosporus, B. pumilus, B. subtilis and B. licheniformis in potting soil in the presence or absence of a crop plant, i.e., cucumber, with or without NutriStart-AC. Bacterial numbers in pots in the absence of cucumber seedlings, with or without NutriStart-AC, declined slowly but steadily. Population sizes in pots without NutriStart-AC decreased steadily from Day 1 to Day 14 for all six Bacillus spp. and thereafter remained constant between 6.19 and 6.15 log cfu g -1 of wet soil for all six Bacillus spp. up to termination of the experiment on Day 35. A similar effect was observed in pots supplemented with one gram of NutriStart-AC. In the presence of cucumber seedlings, population sizes in pots without NutriStart-AC supplement declined faster until Day 14 than those in the NutriStart-AC supplemented pots. Populations remained stable after Day 14 for all six Bacillus spp. in the NutriStart-AC unsupplemented pots, while there was a variation in population sizes among Bacillus spp. in pots supplemented with NutriStart-AC. Growth stimulation trials in tunnels were carried out using four crops, i.e., lettuce, tomato, sorghum and beans. Seed treatment and seed treatment plus drenching with or without NutriStart-AC were evaluated. All Bacillus spp. used stimulated plant growth. Growth stimulation was more pronounced with a 4% NutriStart-AC supplement. Growth stimulation was best in lettuce, with Biostart® 2000. There was an increase of 466% compared to the dry biomass of the water control lettuce seedlings. The lowest responses were recorded in sorghum and beans. Three tomato cultivars, i.e., Roma, Floradade and Rodade and a pepper cultivar Thai were evaluated for growth stimulation by applying Biostart™ as seed treatment and seedling drench. The highest growth stimulation, 96%, was obtained using B. licheniformis on Roma as a seedling drench. Growth response was better in Roma and Floradade cultivars than in the Rodade cultivar. Pepper plants drenched with Biostart™ Bacillus spp., and supplemented weekly with a 4% NutriStart-AC suspension, showed increased fruit yield. Using B. subtilis, a 533% increase in fruit yield was recorded when seedlings were supplemented weekly with a 4% NutriStart-AC suspension. Similar results were recorded using an unidentified Bacillus strain CM-33 (433%) and B. licheniformis (333%). In a nematode control trial, no galls were found on the roots of treated and untreated control seedlings inoculated with Meloidogyne spp. Early inoculation of seedlings might have failed because there were no roots for the nematodes to attack at the time of inoculation. In a biological control trial, Biostart™ Bacillus spp. were applied by seed treatment and seedling drench to control Rhizoctonia causing damping-off of marigold, cabbage and eucalyptus seedlings. Biostart™ was ineffective under the conditions of this trial. Most seedlings died seven days after pathogen inoculation and by Day 21 about 90% of the seedlings were dead. The results presented in this thesis have some practical applications to seedling growers in South Africa, especially in growth promotion. Applying Biostart™ probiotic Bacillus spp. may increase the turnover of seedlings in nurseries. More trials are needed if the growth promotion and biological control potentials of Biostart™ probiotic Bacillus spp. are to be fully exploited.