Wetlands account for approximately 20% of annual global methane emissions. Many wetlands receive inputs of organic matter, nutrients, metals and various toxic compounds from adjacent agricultural and industrial areas. The present study aimed to investigate the microbial composition of a natural methanogenic consortium. A consortium-based molecular approach to study diversity of methanogenic microbial communities in a natural wetland at the primary inflow was used. Key microorganisms of a nethane producing consortium were identified. Extracted high molecular mss DNA ws analysed by PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rDNA. This study was also aimed to identify syntrophic microorganisms in the wetland system. The data obtained suggest a well established syntrophic relationship within the wetland.

Wetlands account for approximately 20% of annual global methane emissions. Many wetlands receive inputs of organic matter, nutrients, metals and various toxic compounds from adjacent agricultural and industrial areas. The present study aimed to investigate the microbial composition of a natural methanogenic consortium. A consortium-based molecular approach to study diversity of methanogenic microbial communities in a natural wetland at the primary inflow was used. Key microorganisms of a nethane producing consortium were identified. Extracted high molecular mss DNA ws analysed by PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rDNA. This study was also aimed to identify syntrophic microorganisms in the wetland system. The data obtained suggest a well established syntrophic relationship within the wetland.

Magister Scientiae - MSc / Wetlands account for approximately 20% of annual global methane emissions. Many wetlands receive inputs of organic matter, nutrients, metals and various toxic compounds from adjacent agricultural and industrial areas. The present study aimed to investigate the microbial composition of a natural methanogenic consortium. A consortium-based molecular approach to study diversity of methanogenic microbial communities in a natural wetland at the primary inflow was used. Key microorganisms of a nethane producing consortium were identified. Extracted high molecular mss DNA ws analysed by PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rDNA. This study was also aimed to identify syntrophic microorganisms in the wetland system. The data obtained suggest a well established syntrophic relationship within the wetland. / South Africa

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 117-157).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 132-152).

No description available.

No description available.

Doctor of Philosophy / Dental caries continues to be a significant public health problem affecting mankind in many parts of the world. Microbial activities include the progressive localised destruction of teeth that without treatment, would eventually result in infection of the dental pulp and surrounding periapical tissues. Although the bacteria responsible for caries initiation and early caries progression have been extensively studied, the microbiology of dentine caries is reported to show considerable diversity and has not yet been fully identified. Few studies have analysed the microbiology of deep caries or examined the relationship between the microflora and the histopathy of chronic pulpits in symptomatic teeth. Matched carious dentine samples and dental pulps were obtained from teeth without evidence of periodontal disease but with coronal caries and symptoms of pulpits. Bacteria were cultured from the carious dentine samples under both anaerobic and microaerophilic conditions. Real-time polymerase chain reaction (PCR) technology was also used to identify and enumerate the bacteria. Development of the techniques for the efficient extraction of bacterial DNA from both Gram-negative and Gram-positive bacteria found in carious dentine was an essential prerequisite for molecular analysis. In addition, the dental pulps were processed and categorised into one of four groups on the basis of dominant pathology of the tissue (minimal inflammation, soft tissue degeneration, hard tissue degeneration, inflammatory degeneration). Analysis of the culture data indicated a predominance of Gram-positive bacteria, particularly lactobacilli, while Gram-negative bacteria were also present in significant numbers with Prevotella species the most numerous anaerobic group cultured. Real-time PCR indicated a greater anaerobic microbial load than that determined by colony counting. The total number of anaerobes detected by PCR was 41-fold greater, while Prevotella spp. and Fusobacterium ssp. were 82-fold and 2.4-fold greater respectively. PCR also identified the presence of Micromonas micros, Porphyromonas endodontalis and Porphyromonas gingivalis in 71%, 60% and 52% of carious dentine samples, respectively. Correlation matrices from the real-time PCR data revealed significant multiple associations involving Fusobacterium spp. in combination with P. endodontalis, M. micros and/or Prevotella in the tissue response categories of minimal inflammation, soft and hard disuse degeneration. A positive correlation was also observed between M. micros and P. endodontalis for the category of inflammatory degeneration of the dental pulp. These anaerobes have been strongly implicated in the endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of threshold levels of these bacteria in carious dentine may be indicative of irreversible pulpitis. Knowledge of the microbial predictors associated with irreversible pulpitis creates potential for the development of a diagnostic tool, and for restorative materials with antimicrobial properties.

Abstract not available

2,4,6-trinitrotoluene (TNT) is a toxic and recalcitrant pollutant contaminating soils and groundwater. Therefore, characterization of microbial populations of TNT-contaminated soils and isolation of bacteria degrading this pollutant are of primordial importance. Comparison of hybridizations of 16S rRNA derived from uncontaminated and TNT-contaminated soil samples required the development of a functional ANOVA model. Specifically, a statistical tool was necessary to compare dissociation curves obtained from thermal dissociation analysis of RNA hybridizations to DNA microarrays, and to determine if the dissociation curves significantly differed. To test and validate the model, we used dissociation curves from in vitro transcribed 16S rRNA amplified from two environmental samples hybridized to a phylogenetic microarray. Detection and rejection of outlier curves was important for appropriate discrimination between curves. The identification of significantly different curves was more efficient with the model than approaches relying on measurements at a single temperature. This functional ANOVA analysis was used to improve discrimination between hybridizations of two soil microbial communities. Following hybridization of in vitro transcribed 16S rRNA derived from an uncontaminated and a TNT-contaminated soil sample to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and mismatch (MM) variants, thermal dissociation was used to analyze the nucleic acid bound to each PM-MM probe set. Functional ANOVA of the dissociation curves generally discriminated PM-MM probe sets when values of Td (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greater than Td. Comparison of signal intensities measured prior to dissociation analysis from hybridizations of the two soil samples revealed significant differences in domain-, group-, and species-specific probes. Functional ANOVA showed significantly different dissociation curves for 11 PM probes when hybridizations from the two soil samples were compared, even though initial signal intensities for 3 of the 11 did not vary. These differences in hybridizations between the two soil samples were likely the result from the presence of TNT. The effect of TNT on soil microbial communities was further investigated with additional uncontaminated and TNT-contaminated soil samples using 16S rRNA PCR-DGGE and cultivation-dependent techniques. In all contaminated soil samples, the amount of DNA extracted was lower than in the uncontaminated ones. Analysis of bacterial diversity by DGGE showed a predominance of Pseudomonadaceae and Xanthomonadaceae in the TNT-contaminated soil samples compared to the uncontaminated ones. Caulobacteraceae were also present in several contaminated soil samples. The culturable microflora of these soils was studied by plate counts on agar supplemented with dilute nutrient broth. The number of CFUs was lower in a TNT-contaminated soil inoculum than in an uncontaminated one. In the former, most of the CFUs belonged to Pseudomonadaceae, and to a lesser extent, to Caulobacteraceae. In addition to the above contaminated soil samples, a pristine soil was artificially contaminated with different concentrations of TNT and incubated for 4 months. The amount of DNA extracted decreased in the highly contaminated soil samples (1.4 and 28.5 g TNT/kg soil). After 7 days of incubation of these soil samples, there was a clear shift of their original flora to a population dominated by Pseudomonadaceae, Xanthomonadaceae, Comamonadaceae and Caulobacteraceae. When the TNT concentration was lower (140 mg TNT/kg soil), a moderate shift in the bacterial population was observed. These results indicate that TNT affects soil bacterial diversity and richness by selecting for a narrow range of bacterial species that belong mostly to Pseudomonadaceae and Xanthomonadaceae. TNT-contaminated soil samples probably contained TNT-degrading bacteria. In order to isolate bacteria that can denitrate TNT, enrichment cultures were carried out with TNT as sole nitrogen source and in the absence of oxygen. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum, in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. Concomitantly, TNT-reduced compounds were detected as well as unidentified polar metabolites. The concentration of nitrite released from TNT was proportional to the concentration of ferrihydrite in the medium. The release of nitrite was lower when the concentration of initially spiked TNT was reduced by one order of magnitude. Under these conditions, the concentration of nitrite peaked and then its concentration slowly decreased and production of ferrous ions was detected. A decrease of nitrite concentration and production of ferrous ion were observed when TNT was omitted and nitrite and ferrihydrite were provided. These results suggest that nitrite-reducing conditions were initially achieved, followed by iron-reducing conditions. When grown aerobically on a chemically defined medium, P. aeruginosa strain ESA-5 produced a greenish extracellular compound. This product was identified as phenazine-1-carboxylic acid (PCA). When purified PCA was incubated with TNT in the presence of NADH, nitrite was released. The concentration of nitrite released was dependent on the concentration of NADH and PCA. Denitration also occurred with two TNT-related molecules, 2,4,6-trinitrobenzaldehyde and 2,4,6-trinitrobenzyl alcohol. The release of nitrite was coupled with the formation of two polar metabolites and mass spectrometry analyses indicated that each of these compounds had lost two nitro groups from the trinitroaromatic parent molecule. The results obtained with the PCA mediated denitration of TNT in the presence of inhibitors of oxygen reactive species suggested the involvement of superoxide (O2.-). When exogenous PCA was added to a P. aeruginosa ESA-5 liquid culture containing TNT as sole nitrogen source, bacterial growth was significantly enhanced compared to cultures containing TNT without PCA.