The State of Florida ranks fifth in the world's production of phosphate. The phosphate industry relies on surface mining to withdraw the phosphate ore, and this process can devastate the natural environment. One of the most impacted natural resources is wetlands. Federal laws permit the legal destruction of wetlands providing their loss is compensated by the mitigation (i.e., the restoration, creation, or enhancement) of other wetlands, but the complexity of wetland ecosystems makes the mitigation process difficult. One of the goals of this thesis is to review the established Federal, State and non-regulatory guidelines for the development and maintenance of mitigated wetlands, evaluate their efficacy and present some underlying reasons for successful versus unsuccessful mitigation projects.The environmental repercussions of phosphate mining are not only pertinent to Florida or the United States. Wetland mitigation has become a global issue. Laws and programs that facilitate specific countries do not benefit wetland ecosystems on a landscape level. It is important to remain cognizant of the ramifications of wetland destruction and avoid piecemeal solutions to a wide-spread problem. Thus, my second objective is to investigate the progress and status of international wetland preservation. I will examine how different countries and international organizations are addressing the environmental impacts of mining, and underscore the relevant methods and protocols. I will also supplement this review by proposing the use of soil microbial communities as bioindicators of wetland development and sustainability. I will describe the laboratory and field procedures necessary to evaluate the various biological and physical aspects of mitigated wetlands, thereby offering mangers an effective monitoring technique. My intention is to confirm that microorganism development and preservation are critical to wetland health and longevity. My final objective is to document the relevant literature on environmental policy, and provide current scientific and policy review for researchers, managers and legislators. This thesis will synthesize the diverse and often contradictory theories, and suggest possible methodologies to bridge the science-policy gap.Overall, I intend to supply researchers, managers, and government agencies with a source of publications that can assist in evaluating, managing and monitoring wetland mitigation projects.

No description available.

A standardized method to examine soil microbial health incorporating biomass, activity, and diversity measurements is currently lacking, limiting the use of this ecologically relevant endpoint in ecological risk assessments. The soil microbial health of a petroleum hydrocarbon-contaminated boreal forest soil, relative to a reference soil, was examined using a suite of tests. Microbial health impacts in the contaminated soil were observed using nitrification, organic matter decomposition, bait lamina, denaturing gradient gel electrophoresis, community level physiological profiling, and most enzyme assays. Results of heterotrophic plate count and respiration tests indicated higher culturable numbers and activity in the contaminated soil. A data integration technique was developed to incorporate the results from individual tests into an overall conclusion, indicating that soil contamination at the site moderately to severely impacted microbial health. The research presented lays the foundation for the development of a soil microbial health standardized method.

Most natural products and xenobiotic molecules, irrespective of their molecular or structural complexity, are degradable by some microbial species/associations within particular environments. Atrazine- and selected petroleum hydrocarbon (benzene, toluen~ and 0-, m- and p-xylene (BTX))-degrading associations were enriched and isolated"trom atrazine- and petroleum hydrocarbon (PHC)-contaminated KwaZuluNatal loamy and sandy soils, respectively. In total, eight pesticide- and forty BTXcatabolizing associations were isolated. Electron microscopy revealed that, numerically, rods constituted the majority of the populations responsible for both atrazine and PHC catabolism. Cocci and, possibly, spores or fungal reproductive bodies were observed also. For the BTX-catabolizing associations, the population profiles appeared to be dependent on the enrichment pH and the molecule concentration. After combining selected associations, to ensure that all the isolated species were present, batch cultures were made to determine the optimum pH and temperature for growth; With an atrazine concentration of 30 mgr1, the highest specific growth rates, as determined by biomass (OD) changes, were recorded at 30DC and pH 4 although the rate§ at 25DC and pH 5 were comparable. For the BTX (50 mgr1)-catabolizing associations, the highest growth rates were recorded at pH 4 for the four temperatures (15, 20, 25 and 30DC) examined. The sole exception was p-xylene with the highest specific growth rate recorded at pH 5 and 30De. Batch and continuous (retentostat) cultivations in the presence/absence of methanol and under C- and N-limited conditions were used to investigate the impacts of the solvent and the catabolic potentials of a combined atrazine-catabolizing culture (KRA30). In general, different degradation rates were recorded for the culture in response to element limitation. Addition of citrate as the primary carbon source / effected atrazine (100 mg!"l) degradation rates comparable to that of Pseudomonas sp. strain ADP while succinate addition effected herbicide co-metabolism. Carbon supplementation may, therefore, be considered for site amelioration practices. To complement conventional culture-based microbiological procedures, molecular techniques were employed to explore the diversities and analyze the structures of the microbial communities. In parallel, anaerobic microbial associations which targeted atrazine were also characterized. The soil DNA isolation/characterization protocol adopted consisted of a clean-up step followed by the polymerase chain reaction (peR) and 16S rDNA fingerprinting by denaturing-gradient gel electrophoresis (DGGE). The preliminary results suggested that despite different, but chemically similar, petroleum hydrocarbon molecules, the common selection pressures of the primary enrichments effected the isolation of similar and complex aerobic microbial associations. Some similar numerically-dominant bands characterized the aerobic and anaerobic atrazine-catabolizing associations although distinct differences were also recorded on the basis of the enrichment/isolation pH value and the concentration of the herbicide. Cloning and sequencing were then used to identify some of the numerically-dominant and non-dominant association members. Community-level physiological profiling (CLPP) for physiological fingerprinting was made with Biolog EcoPlates and highlighted the differences in the isolated aerobic atrazine-catabolizing associations depending on the enrichment pH and molecule concentration. Logarithmic-phase cultures of the combined atrazine- and BTX-catabolizing associations were used to explore the association profiles following pH and temperaiure optImIzation. Although some common numerically-dominant components were maintained, differences in numerical and, possibly, activity dominance were observed in the 16S rDNA profiles in response to changes in pH and temperature. This indicated that environmental parameter optimization and characterization of catabolic association structure must precede bioaugmentation so that control of key variables will facilitate maintenance of the dominant site-specific species. Following KRA30 cultivation in the presence/absence of methanol and under carbon and nitrogen-limited conditions, the population fingerprints showed that the presence of methanol effected shifts in species numerical dominance and, possibly, changes in atrazine catabolic capacity. Also, Coulter counter results, optical density readings and 16S rDNA characterization by DGGE indicated that degradation rate changes were accompanied by shifts in species numerical/activity dominance within the association. Although N-limitation effected the highest rates of herbicide catabolism, a potential versatility of the combined association for bioaugmented and/or biosupplemented remediation with acceptable rates regardless of any elemental limitation was recorded. To determine if the contaminated and pristine source soils contained comparable catabolic populations and, thus, offered potential for intrinsic bioremediation, PCRDGGE was used to characterize the populations in comparison with the enriched/isolated associations. Some similar dominant bands characterized the contaminated soils and the enriched/isolated associations. The significance of this, in relation to a possible correlation between numerical and activity dominance in the component species, is discussed with respect to the use of PCR-DGGE to identify natural attenuation potential and monitor sustained intrinsic and enhanced (bioaugmented and biosupplemented) bioremediation. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2001.

The environmental impact of anthropogenic disturbances such as agriculture, on the soil ecosystem, and particularly on soil microbial structural and functional diversity, is of great importance to soil health, conservation and remediation. Therefore, this study assessed the effects of various land use and management practices on both the structural (genetic) and functional (catabolic) diversity of the soil bacterial and fungal communities, at two long-term sites in KwaZulu-Natal. The first site is situated at Baynesfield Estate, and the second at Mount Edgecombe Sugarcane Research Institute. At site 1, the land uses investigated included soils under pre-harvest burnt sugarcane (Saccharum officinarum, Linn.) (SC); maize (Zea mays, Linn.) under conventional tillage (M); permanent kikuyu (Pennisetum clandestinum, Chiov) pasture (KIK); pine (Pinus patula, Schiede) plantation (PF); and wattle (Acacia mearnsii, De Wild) plantation (W), all fertilized; and undisturbed native grassland (NAT) that had never been cultivated or fertilized. At site 2, a sugarcane (Saccharum officinarum × S. spontaneum var. N27) pre-harvest burning and crop residue retention trial was investigated. The treatments studied included conventional pre-harvest burning of sugarcane with the tops removed (Bto), and green cane harvesting with retention of crop residues on the soil surface as a trash blanket (T). Each of these treatments was either fertilized (F) or unfertilized (Fo). The polymerase chain reaction (PCR), followed by denaturing gradient gel electrophoresis (DGGE) were used to determine the structural diversity, and community level physiological profiling (CLPP) using BIOLOG plates, the catabolic diversity. In addition, the soils were analysed with respect to selected physicochemical variables, and the effects of these on the soil microbial communities were determined. Replicate soil samples (0–5 cm) were randomly collected from three independent locations within each land use and management, at both sites. Soil suspensions for the CLPP analyses were prepared from fresh soil subsamples (within 24 h of collection) for the bacterial community analyses, and from 8-day-old soil subsamples (incubated at 4°C to allow for spore germination) for the fungal community analyses. BIOLOG EcoPlates™ were used for the bacterial CLPP study and SF-N2 MicroPlates™ for the fungal analysis, the protocols being adapted and optimized for local conditions. This data was log [X+1]-transformed and analysed by principal component analysis (PCA) and redundancy analysis (RDA). For PCRDGGE, total genomic DNA was isolated directly from each soil subsample, and purified using the MO BIO UltraClean™ soil DNA Isolation kit. Protocols were developed and optimized, and fragments of 16S rDNA from soil bacterial communities were PCR-amplified, using the universal bacterial primer pair 341fGC/534r. Different size 18S rDNA sequences were amplified from soil fungal communities, using the universal fungus-specific primer pairs NS1/FR1GC and FF390/FR1GC. Amplicons from both the bacterial and fungal communities were fingerprinted by DGGE, and bands in the fungal DGGE gels were excised and sequenced. The DGGE profiles were analysed by Bio-Rad Quantity One™ Image analysis software, with respect to band number, position, and relative intensity. Statistical analyses of this data then followed. Soil properties [organic C; pH (KCl); exchangeable acidity; total cations (ECEC); exchangeable K, Ca and Mg; and extractable P] were determined by PCA and were shown to have affected the structural and catabolic diversity of the resident microbial communities. At Baynesfield, canonical correspondence analysis (CCA) relating the selected soil variables to bacterial community structural diversity, indicated that ECEC, K, P and acidity were correlated with CCA1, accounting for 33.3% of the variance, whereas Mg and organic C were correlated with CCA2 and accounted for 22.9% of the variance. In the fungal structural diversity study, pH was correlated with CCA1, accounting for 43.8% of the variance, whereas P, ECEC and organic C were correlated with CCA2, and accounted for 30.4% of the variance. The RDA of the catabolic diversity data showed that the same soil variables affecting fungal structural diversity (organic C, P, ECEC and pH) had influenced both the bacterial and fungal catabolic diversity. In both the bacterial and fungal RDAs, organic C, P and ECEC were aligned with RDA1, and pH with RDA2. However in the bacterial analysis, RDA1 accounted for 46.0%, and RDA2 for 27.5% of the variance, whereas in the fungal RDA, RDA1 accounted for only 21.7%, and RDA2 for only 15.0% of the variance. The higher extractable P and exchangeable K concentrations under SC and M, were important in differentiating the structural diversity of these soil bacterial and fungal communities from those under the other land uses. High P concentrations under M were also associated with bacterial catabolic diversity and to a lesser extent with that of the soil fungal communities under M. Similarly, the higher organic C and exchangeable Mg concentrations under KIK and NAT, possibly contributed to the differentiation of these soil bacterial and fungal communities from those under the other land uses, whereas under PF, the high exchangeable acidity and low pH were possibly influencing factors. Under W, low concentrations of P and K were noted. Other factors, such as the presence/absence and frequency of tillage and irrigation, and the diversity of organic inputs due to the diversity of the above-ground plant community, (in NAT, for example) were considered potentially important influences on the nature and diversity of the various land use bacterial and fungal communities. At Mount Edgecombe, CCA showed that organic C and Mg had a significant effect on soil bacterial structural diversity. Organic C was closely correlated with CCA1, accounting for 58.7% of the variance, whereas Mg was associated with CCA2, and accounted for 41.3% of the variance. In the fungal structural diversity study, ECEC and pH were strongly correlated with CCA1 and accounted for 49.1% of the variance, while organic C was associated with CCA2, accounting for 29.6% of the variance. In the functional diversity studies, RDA showed that both bacterial and fungal community catabolic diversity was influenced by soil organic C, pH, and ECEC. In the bacterial analysis, RDA1 was associated with organic C and pH, and accounted for 43.1% of the variance, whereas ECEC was correlated with RDA2, accounting for 36.9% of the variance. In the fungal analysis, RDA1 was correlated with ECEC and accounted for 47.1% of the variance, while RDA2 was associated with pH and organic C, accounting for 35.8% of the variance. The retention of sugarcane harvest residues on the soil surface in the trashed treatments caused an accumulation of organic matter in the surface soil, which did not occur in the pre-harvest burnt sugarcane. This difference in organic C content was a factor in differentiating both bacterial and fungal communities between the trashed and the burnt treatments. Soil acidification under long-term N fertilizer applications caused an increase in exchangeable acidity and a loss of exchangeable Mg and Ca. Thus, as shown by CCA, a considerably lower exchangeable Mg concentration under F compared to Fo plots resulted, which was influential in differentiating the bacterial and fungal communities under these two treatments. In the structural diversity study at Baynesfield, differences were found in bacterial community species richness and diversity but not in evenness, whereas in the fungal analysis, differences in community species richness, evenness and diversity were shown. The soil bacterial and fungal communities associated with each land use were clearly differentiated. Trends for bacterial and fungal diversity followed the same order, namely: M < SC < KIK < NAT < PF < W. At Mount Edgecombe, no significant difference (p > 0.05) in bacterial structural diversity was found with oneway analysis of variance (ANOVA), but two-way ANOVA showed a slight significant difference in bacterial community species richness (p = 0.05), as an effect of fertilizer applications. A significant difference in fungal species richness (p = 0.02) as a result of management effects was detected, with the highest values recorded for the burnt/fertilized plots and the lowest for the burnt/unfertilized treatments. No significant difference was shown in species evenness, or diversity (p > 0.05), in either the bacterial or the fungal communities. In the catabolic diversity study at site 1, the non-parametric Kruskal-Wallis ANOVA showed that land use had not affected bacterial catabolic richness, evenness, or diversity. In contrast, while fungal catabolic richness had not been affected by land use, the soil fungal community catabolic evenness and diversity had. At site 2, the land treatments had a significant effect on soil bacterial community catabolic richness (p = 0.046), but not on evenness (p = 0.74) or diversity (p = 0.135). In the fungal study, land management had no significant effect on the catabolic richness (p = 0.706), evenness (p = 0.536) or diversity (p = 0.826). It was concluded, that the microbial communities under the different land use and trash management regimes had been successfully differentiated, using the optimized protocols for the PCR-DGGE of 16S rDNA (bacteria) and 18S rDNA (fungi). Sequencing bands produced in the 18S rDNA DGGE, enabled some of the soil fungal communities to be identified. CLPP of the soil microbial communities using BIOLOG plates showed that, on the basis of C substrate utilization, the soil bacterial and fungal communities’ catabolic profiles differed markedly. Thus, it was shown that the different land use and management practices had indeed influenced the structural and catabolic diversity of both the bacterial and fungal populations in the soil. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Water treatment residue (WTR), a by-product of the water treatment process, consists primarily of precipitated hydroxides of the coagulants used in the water treatment process, along with sand, silt, clay, humic compounds, and dissolved organic matter. It is usually disposed of by landfill, a technology with numerous problems that include dwindling landfill capacity, extensive dewatering requirements for the WTRs, high costs of transportation, and potential liability for landfill clean-up. Therefore, land disposal (or land treatment) presents a popular alternative disposal method based on the principle that the physical, chemical, and microbial properties of the soil can be used to assimilate applied waste without inducing any negative effects on soil quality. The objective of this study was to investigate the effects of land disposal of the WTR generated by Umgeni Water, a local water treatment authority, on soil quality. These effects were investigated using depth samples from soil profiles of Westleigh and Hutton soil forms at field trials located at Ukulinga Research Farm, near Pietermartizburg and Brookdale Farm, Howick, KwaZulu-Natal, South Africa, respectively. Four rates of WTR (0, 80, 320, and 1280Mg ha-1 incorporated into the soil) were investigated at both trials, in addition to mulched treatments at rates of 320 and 1280Mg ha-1 at Brookdale only. Sampling of plots was carried out in September 2001 and May 2002, and all treatments were investigated under fallow and grassed cultivation. Laboratory measurements used to assess soil quality included pH, electrical conductivity (EC), organic carbon (QC), and microbial activity using f1uorescein diacetate (FDA) hydrolysis. At both trials in September 2001 WTR-amended plots displayed higher pH in the 0-200mm soil in comparison to the controls, whereas by May 2002 pH had returned to the condition of the controls. Addition of WTR at Ukulinga resulted in higher QC in September 2001, but in May 2002 this was similar to the controls. However, at Brookdale QC was unaffected by WTR. At Ukulinga and Brookdale the effect of WTR on EC was variable, and microbial activity in the soil profile was unaffected by WTR addition. Observations at Ukulinga and Brookdale reflected long term changes (3 and 5 years, respectively) to soil quality following WTR addition. To examine the initial changes in soil quality a laboratory experiment was set up using the field trial soils. Research objectives were also extended to include WTRs from Rand Water (Johannesburg), Midvaal Water Company (Stilfontein), Amatola Water (East London), and two samples from the Faure Water Treatment Plant (near Cape Town). The second Faure sample (Faure2 ) was collected when blue green algal problems were experienced at the plant. The measurements used to investigate these short term effects on soil quality were soil pH, EC, and microbial activity as indicated by respiration rate. Each of the WTRs added to the Hutton and Westleigh soils increased soil pH by varying increments, and the higher the WTR application rate, the higher was the pH recorded. With the exception of the Rand and Umgeni WTRs that clearly increased soil EC, the effect of the otherWTRs on EC was variable. The Faure1 and Amatola WTRs appeared to have no effect on microbial activity, whereas the Umgeni, Rand, Midvaal, and Faure2 WTRs stimulated microbial activity by Day 2 following the addition of WTR, but this had declined by Day 14. As for pH, higher microbial activity was recorded at higher WTR application rates. Changes in microbial community structure of the Hutton soil only, following the addition of WTR were examined using denaturing gradient gel electrophoresis (DGGE) analysis. Community profiles of the different WTRs proved to be markedly different. However, WTR-amended soil retained banding patterns consistent with the control soil indicating that dominant populations in the Hutton soil had been retained. The field trials indicated that long term effects of land disposal of WTR were not detrimental to the measured indicators of soil quality namely, pH, EC, QC, and microbial activity. The laboratory assessments of the short term response of the Hutton and Westleigh soil forms to WTR addition suggested that the tested variables were altered by WTR, but not significantly changed to the detriment of soil quality. Microbial community analysis indicated that the community structure of the Hutton soil was not significantly altered by WTR amendments. Present findings provide no evidence to suggest that land disposal of WTR is detrimental to soil quality. It is therefore regarded as a feasible disposal option although there are some aspects that should be investigated further. These include investigations into rhizosphere/microbial interactions and the feasibility of growing cash crops. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

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. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

A novel approach for screening metagenomic libraries by merging both metagenomic and microarray platforms was developed and optimized. This high-throughput screening strategy termed "metagenomic microarrays" involved the construction of two Arctic soil large-insert libraries and the high density arraying of the clone plasmid DNA (~50 kb) onto glass slides. A standard alkaline lysis technique used for the purification of plasmid DNA was adapted and optimized to function efficiently in a 96-well format, providing an economically viable means of producing sufficient high-quality plasmid DNA for direct printing onto microarrays. The amounts of printed material and probe, required for maximal clone detection, were optimized. To examine catabolic clone detection libraries were first screened by PCR for catabolic genes of interest. Two PCR-positive clones were printed onto microarrays, and detection of these specific clones in the printed libraries was achieved using labeled probes produced from PCR fragments of known sequence. Also, hybridizations were performed using labeled PCR fragments derived from the amplification of a catabolic gene from the total community DNA. The ability of selected probes to specifically target clones of interest was demonstrated. This merger of metagenomics and microarray technologies has shown great promise as a tool for screening the natural microbial community for catabolic potential and could also be used to profile microbial diversity in different environments.

Graduation date: 1993

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 140-151).