Graduation date: 1993 / Figures in original are black and white photocopies. Best scan available.

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains x, 282 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)(Plant Production)--University of Pretoria, 2001. / Includes bibliographical references).

No description available.

Although the physiological nitrogen demand of the soil microbial biomass is a major determinant of N mineralization in forest soils, the exact nature of the relationship is unclear. This study investigated the relationships between a respiration-based indicator of microbial physiological N demand (NIR) and N availability in forest soils. NIR was found to correlate significantly with net mineralized N in the field and annual foliar litterfall N fluxes. In a laboratory incubation, NIR was shown to be sensitive to changes in soil available C and N pools. These results demonstrated that microbial physiological N demand is determined by relative availabilities of labile C and N, and that it is significantly related to N cycling in forest soils. Results from a seasonal study of a forested watershed suggest that nutrient availability determined tree production and soil C availability, which in turn determined microbial physiological N demand and nitrogen dynamics in the forest.

A polyphasic approach was used to examine the impact of contamination on soil microbial community structure. Two systems were examined using a combined biochemical and molecular biological approach. Petroleum hydrocarbon contaminated soils from two Northern Canadian sites, representing long-term contamination, were examined using Biolog GN plates and PCR-denaturing gradient gel electrophoresis (DGGE) analysis of total community 16S rDNA. Results obtained using both methods demonstrated a positive correlation between samples that was based on the geographical origin of the samples, not on contamination level. In the second system, non-contaminated soil was contaminated with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to monitor the effect of short- to medium-term contamination. Changes in the soil microbial community were examined using PCR-DGGE of total community 16S rDNA combined with RDX mineralization and chemical analysis of intermediates. The non-contaminated loam soil had an inherent RDX degradative capability and contamination of soil columns with 1000 mg RDX/kg soil did not significantly change the 16S rDNA bacterial community profile. The bacterial diversity remained high as estimated by the number of bands present in the DGGE and by NQ-78704 statistical rarefaction analysis of 16S rDNA clone RFLPs. The same soil, used in 10% soil slurries (w/v), demonstrated two apparently different RDX degradation mechanisms based on mineralization and chemical analysis. The differences were based on aerobic versus anaerobic conditions and the presence/absence of Na3 citrate. PCR-DGGE performed on 16S rDNA from aerobic slurries amended with Na3-citrate detected the stimulation of 3 operational taxonomic units, identified as Stenotrophomonas sp., Sphingomonas sp. and a member of the Alcaligenaceae. The results from the two systems examined (short- to medium-term and long-term contamination) demonstrated the utility of a polyphasic approach in the examina

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. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Many studies have been undertaken to determine the effects of soil and soil properties on migrating metal pollutants. Organic pollutants, however, in addition to their interactions with soil components , are also susceptible to degradation (catabolism) by microorganisms. Soil-microorganism-pollutant interactions have, traditionally, been studied in soil columns (microcosms). One of the shortcomings of column and in situ studies is that the identity and specific effect(s) of the soil component(s) affecting or influencing attenuation are not known and cannot readily be determined. Attenuation effects of the soil components are, therefore, difficult to interpret. ("Attenuation" in this context is the combined effects of both soil adsorption and microbial catabolism). Attenuation studies often only consider the physical conditions such as aeration, permeability, flow rate, temperature, etc. This approach assumes the soil to be a homogeneous matrix with no specific physico-chemical properties attributable to different components within the matrix. Soil physical factors suspected of influencing pollutant attenuation could be misleading without consideration of the physico-chemical interactions between soil components, microorganisms and pollutants. Adhesion of pollutants and microorganisms seems to be most important in this regard. The initial phase of this study was undertaken to examine the effects of three different soil materials on attenuation of key landfill leachate molecules. Examination of the effects of soil surface type on attenuation focused on adsorption / desorption of the pollutant molecules and microorganisms. These experiments sought to investigate the physico-chemical effects of soil, microorganism, pollutant interactions and were done as batch slurry experiments as well as in soil columns. Two soil horizons from the Inanda soil form (humic A and red apedal B) and the topsoil (vertic A) from a Rensburg soil form were used. The Inanda topsoil had a high organic matter content and both the topsoil and subsoil had a kaolinitic clay mineralogy; the Rensburg topsoil clay mineralogy was predominantly smectitic with a relatively low organic matter content. From the batch experiments, the adsorption of a hydrophobic molecule (naphthalene) and a heavy metal (cadmium) were found to be influenced to a significant extent by soil characteristics. Adsorption of naphthalene was due to the soil organic matter (SOM) content whereas cadmium adsorption was due to the cation exchange capacity (CEC) of the soil. Soil characteristics did not seem to have a significant influence on the adsorption of a water soluble compound such as phenol at the concentrations used. Attenuation of naphthalene was found to be affected by adsorption of the pollutant molecule (related to SOM) as well as the CEC of the soil. The attenuation of hydrophobic molecules can possibly be ascribed to the influence of CEC on the microbial population responsible for attenuation. This would seem to indicate interaction between the soil surfaces and the catabolizing microbial population. Desorption of the pollutant (and possibly also of the microbial population) was achieved by the addition of acetonitrile and methanol both of which reduced the polarity of the water. These solvents were also found to be toxic to the catabolizing microbial population at high concentrations. The toxicity thresholds of both solvents for catabolizing microorganisms differed significantly between soil- (> 15 %, v/v) and soil free (< 5 %, v/v) treatments. This discrepancy cannot be accounted for by adsorption and is ascribed to physico-chemical interaction between microorganisms and the soil surfaces. This interaction probably affords protection from, otherwise, toxic concentrations of solvents or metals. The important effects of soil surfaces on attenuation processes were thought to be due to the strong adsorption of naphthalene. Surface attachment of microorganisms was, however, also inferred from results obtained with phenol. This seemed to indicate that microbial attachment to soil surfaces was an important aspect in attenuation and did not occur only because of pollutant adsorption. Soil column experiments were made with both naphthalene and phenol. The naphthalene, which was adsorbed to the soil, did not leach from the columns to any appreciable extent. This was despite the addition of acetonitrile to some columns. This was probably due to greater microbial catabolism caused by desorption and, subsequent, increased soluble concentrations of the molecule. After extraction from the soil at the end of the experiment it was clear that the sterile controls held much higher concentrations of naphthalene than the experimental columns. The soil type and treatments showed little difference in the naphthalen concentration extracted from the soil columns. This did not reflect the differences found between soil materials in the batch experiments and was probably due to the masking effect of the soil physical factors on attenuation processes. Unlike naphthalene, phenol, because of its high solubility, was detected in the column leachates at relatively high concentrations. The phenol concentrations were much higher for the Inanda subsoil (approximately 4 mM) than the Inanda topsoil (approximately 2 mM) and Rensburg topsoil (< 1 mM). The Rensburg topsoil produced the lowest phenol concentrations in the leachate and this can probably be ascribed to the larger quantity of micropores in this soil. Thus, it seems that the soil physical features had a pronounced influence on attenuation. Whether this effect was directly on the studied molecule or indirectly, because of the effects on the microbial population, is not known. Inoculation of the columns with a phenol catabolizing population had only a slight increased effect on leachate phenol concentrations from all columns. This increased effect was, however, only prolonged in the case of the Inanda subsoil. The flow rate through the columns affected leachate phenol concentration which was lower with a slower flow rate and, thus, longer retention time. From the column experiments soil physical parameters were suspected of influencing, and possibly overriding, the soil surface effects on microbial activity (capacity to catabolize a organic molecule of interest). Soil porosity, as caused by different soil materials, was suspected of being the most important soil physical parameter influencing microbial activity. To investigate the potential effect of soil porosity, relatively homogeneous porous media i.e. chromatography packing material and acid washed sand were used. These materials had more defined and distinct porosities and were considered to be suitable for investigating the fundamental influence of porosity on microbial activity. Saturated continuous flow columns were used and three types of packing configurations were tested: chromatography packing (CHROM) material (porous particles); acid washed sand (non-porous) (AWS); and a 1: 1 (w/w) mixture of chromatography packing and acid washed sand (MIX). Only a single water soluble molecule, phenol, was used in this phase of the investigation. Bacterial filtration ("filtration" as a component of "attenuation'') was found to be highest for the CHROM and lowest for the AWS materials. This difference in microbial retention affected the phenol catabolism in response to increased column dilution rates. The CHROM and MIX materials had distinctly different porosities than that of the AWS, due to the internal porosity of the chromatography packing. This greater pore size distribution in the MIX and CHROM packing materials created pores with different effective pore dilution rates within the microcosms at similar overall flow rates. The greater pore size distribution in the MIX and CHROM packing materials facilitated pore colonization since some pores did not participate, or conduct, mass flow as occurred in macropores. This led to different microcolonization effects in the macro- vs micropores. Since the MIX and CHROM packing materials had more micropore colonization sites these packing materials showed a greater range of substrate affinities (i.e. Ks values) for the phenol substrate. The extent to which micropore colonization occurred could be detected by the effect it had on phenol breakthrough curves. In the MIX and CHROM materials, microbial colonization caused blocking of micropores with a subsequent effect on the phenol breakthrough curves. The AWS material, however, which had a low inherent microporosity, showed microbially induced microporosity probably due to biofilm development. The fact that the MIX and CHROM packing materials facilitated micropore colonization was also responsible for the greater resistance to, and the recovery from , potentially inhibitory cadmium concentrations. This effect was also apparent in the presence of acetonitrile, although this effect was not identical to that observed with cadmium. Finally, column pressure build up as a function of pore clogging was determined and was found to occur in the order AWS > MIX > CHROM. This was most likely due to fewer potential liquid flow paths with a higher blocking potential in the AWS. Extrapolation of the fundamentals of the above findings led to the conclusion that soil surface- and soil porosity effects are extremely important factors in determining the behavior of soils as bioreactors. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1995.

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

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