Hyperthermophilic proteins are of great interest in both the academic and industrial world in understanding how these proteins are capable of retaining their biological activity under such harsh environmental conditions. This thesis studies a tetrabrachion stalk domain from Staphylothermus marinus, know as Right Handed Coiled Coil (RHCC). This protein is of interest due to its extreme thermostability and its affinity for heavy metals. We aim to better understand the reason for the extreme thermal stability of the protein and to take advantage of the proteins affinity for heavy metals with a view to developing a novel approach to bioremediate Hg2+, a major environmental pollutant. Our results clearly indicated that the protein is more thermostable in alkaline conditions in comparison to acidic conditions. This observation can be explained by careful inspection of the high resolution structure. Our data also clearly show that RHCC is able to bind ionic mercury compounds such as mercury nitrate and dipotassium mercury iodide.

KB-1 is an anaerobic Dehalococcoides-containing microbial culture used industrially to bioremediate sites impacted with chlorinated solvents. The culture is typically grown at pH 7. However, lower pH is often encountered and therefore the effect of pH was investigated. Both sudden and stepwise decreases in pH from 7 to 6 and 5.5 were investigated over a period of 450 days. An electron balance was also calculated to look at the flow of electrons for dechlorination. More than 95% of the reducing equivalents went towards methanogenesis and acetogenesis. Select microorganisms were compared by quantitative Polymerase Chain Reaction. It was found that lower rates of dechlorination correspond to low Dehalococcoides numbers and that different methanogens were enriched on different electron donors.

Bioremediation of chlorinated contaminants in groundwater can be achieved by reductive dechlorination by anaerobic dechlorinating cultures. However, at sites impacted with multiple different chlorinated contaminants, reductive dechlorination is often inhibited by co-contaminants. The inhibitory effects of trichloroethene, cis-dichloroethene and vinyl chloride on chloroform dechlorination were studied using a Dehalobacter-containing chloroform dechlorinating mixed culture (ACT-3). The inhibitory effect of chloroform on chlorinated ethene dechlorination was studied in a Dehalococcoides-containing trichloroethene dechlorinating mixed culture (KB-1). Vinyl chloride was found to be the strongest inhibitor of chloroform in ACT-3. Chloroform exerted a complex and strong inhibitory effect on chlorinated ethene dechlorination in KB-1. The potential for microbial reductive defluorination was also examined in the enrichment cultures. Quantifying the substrates and substrate interactions in dechlorinating cultures is necessary to most efficiently use these cultures to remediate contaminated sites.

The purpose of this research was to investigate the microorganisms, genetics and biochemistry of anaerobic dechlorination of chlorinated ethanes, which are common groundwater contaminants. Specifically, this project used mixed microbial cultures to study the dechlorination of 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,1-trichloroethane (1,1,1-TCA). A mixed microbial culture enriched from a contaminated multilayered aquifer in West Louisiana dechlorinated 1,2-DCA, 1,1,2-TCA, tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride (VC) to non-toxic ethene when amended with ethanol as the electron donor. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organisms Dehalobacter and Dehalococcoides spp. Denaturing gradient gel electrophoresis analysis and quantitative PCR (qPCR) with species-specific primers demonstrated that both organisms grew during the dichloroelimination of 1,2-DCA to ethene. Conversely, during the dichloroelimination of 1,1,2-TCA to VC only Dehalobacter grew, while during the reductive dechlorination of VC to ethene only Dehalococcoides grew. Further enrichment with 1,2-DCA, H2 and acetate yielded a co-culture of Dehalobacter and Acetobacterium spp. that did not dechlorinate other chlorinated ethanes or ethenes. Dehalobacter grew in the presence but not in the absence of 1,2-DCA, while Acetobacterium growth was not affected by 1,2-DCA. A novel putative Dehalobacter-associated 1,2-DCA reductive dehalogenase gene was identified and was shown to be transcribed only in the presence of 1,2-DCA. An enrichment microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States was also studied. This culture, referred to as MS, reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) when amended with methanol, ethanol, acetate and lactate. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organism Dehalobacter sp., whose growth during 1,1,1-TCA and 1,1-DCA dechlorination was confirmed by qPCR. In the presence of chlorinated ethenes, dechlorination 1,1,1-TCA by the culture MS was slowed, while dechlorination of 1,1-DCA was completely inhibited. Experiments with cell-free extracts and whole cell suspensions of culture MS suggested that chlorinated ethenes have direct inhibitory effects on 1,1,1-TCA reductive dehalogenase(s), while the inhibition of 1,1-DCA dechlorination may be due to effects on non-dehalogenase components of Dehalobacter sp. cells. Additionally, two novel reductive dehalogenase genes associated with 1,1,1-TCA reductive dechlorination were identified.

KB-1 is an anaerobic Dehalococcoides-containing microbial culture used industrially to bioremediate sites impacted with chlorinated solvents. The culture is typically grown at pH 7. However, lower pH is often encountered and therefore the effect of pH was investigated. Both sudden and stepwise decreases in pH from 7 to 6 and 5.5 were investigated over a period of 450 days. An electron balance was also calculated to look at the flow of electrons for dechlorination. More than 95% of the reducing equivalents went towards methanogenesis and acetogenesis. Select microorganisms were compared by quantitative Polymerase Chain Reaction. It was found that lower rates of dechlorination correspond to low Dehalococcoides numbers and that different methanogens were enriched on different electron donors.

Bioremediation of chlorinated contaminants in groundwater can be achieved by reductive dechlorination by anaerobic dechlorinating cultures. However, at sites impacted with multiple different chlorinated contaminants, reductive dechlorination is often inhibited by co-contaminants. The inhibitory effects of trichloroethene, cis-dichloroethene and vinyl chloride on chloroform dechlorination were studied using a Dehalobacter-containing chloroform dechlorinating mixed culture (ACT-3). The inhibitory effect of chloroform on chlorinated ethene dechlorination was studied in a Dehalococcoides-containing trichloroethene dechlorinating mixed culture (KB-1). Vinyl chloride was found to be the strongest inhibitor of chloroform in ACT-3. Chloroform exerted a complex and strong inhibitory effect on chlorinated ethene dechlorination in KB-1. The potential for microbial reductive defluorination was also examined in the enrichment cultures. Quantifying the substrates and substrate interactions in dechlorinating cultures is necessary to most efficiently use these cultures to remediate contaminated sites.

Alternative strategies involving in situ remediation technologies have been developed to assist with property clean up, however, cost-effectiveness and discrepancies in success rates and timeliness continue. The objective of my research was to critically demonstrate the application and usefulness of an in situ remediation technology at a petroleum hydrocarbon impacted site. This project was proposed as part of the research programs: Groundwater Plume Formation and Remediation of Modern Gasoline Fuels in the Subsurface and Enhancing In Situ Bioremediation at Brownfield Sites funded by the Ontario Centres of Excellence for Earth and Environmental Technologies as part of the multiphase project entitled “Enhancing in situ Bioremediation at Brownfield Sites”. This research focused on the demonstration of nitrate-enhanced in situ bioremediation at a decommissioned service station. Petroleum hydrocarbon impacted soil and groundwater is a common occurrence at gasoline distribution facilities, where toxicological effects are known for gasoline constituents of interest such as benzene, toluene, ethylbenzene and total xylenes (BTEX). These chemicals are volatile, readily soluble, and persistent in groundwater. In particular, residual contaminants present in the saturated zone were targeted for remediation as they serve as a long term source of contamination and contribute to mobile vapour phase and dissolved phase plumes. Site investigations characterized the complex hydrogeological conditions and contaminant distribution present in order to effectively design an in situ bioremediation treatment system. The addition of nitrate as a terminal electron acceptor (TEA) to an aquifer enhances in situ biodegradation of petroleum hydrocarbons, by providing the microbes with a sustainable energy source to promote cell maintenance and growth of the microbial population. The remediation strategy involved pulsed injections of remedial solution amended with a conservative bromide (200 mg/L Br-) and reactive nitrate (90 to 265 mg/L NO3-) tracers with the purpose of providing a continuous supply of TEA available to the indigenous microbial populations. Nitrate was selected as an alternative electron acceptor over the thermodynamically favoured O2 because of typical challenges encountered using O2 in bioremediation applications in addition to the existing anaerobic environment. In situ anaerobic degradation of BTEX compound using TEA amendments has been well documented; however benzene is often recalcitrant under denitrification conditions. The results of the Br- tracer breakthrough curves indicate that different preferential flow pathways were established under the transient saturated conditions present at the Site, although the behaviour of the injected remedial slug was generally consistent between the different units and the test solution was ultimately delivered to the target zone. The delivery of the remedial test solution was greatly influenced by the hydrogeological conditions present at the time of injection. The injectate was preferentially transported in the high permeability zone of sandy gravel aquifer Unit 3 under high saturated condition and background hydraulic gradients. However the seasonal decline in groundwater levels and hydraulic gradients resulted in the lower portion of Unit 4 comprised of higher permeable materials being able to transmit the test solution more effectively. Given the variable hydrogeological conditions present at the Site influenced by seasonal effects, the delivery of the remedial solution to target zones containing petroleum hydrocarbons at residual saturation is more effective under reduced saturated conditions. The delivery of TEA amended water to enhance the in situ biodegradation of petroleum contaminants is more effective when the treatment water has an increased residence time in the target remedial zone, attributed to low gradients and groundwater transport velocities at the Site. Longer residence periods enable the indigenous microbes to have increased contact time with the TEA which will be preferentially utilized to degrade the contaminants.   A reducing zone enriched with TEA in the anaerobic aquifer was established following consecutive injections of remedial test solution. A cumulative mass of 4 kg of NO3- was added to the target aquifer during the course of the remedial injections. Evidence demonstrating NO3- utilized as a terminal electron acceptor in the bioremediation of the petroleum-contaminated aquifer include: laboratory microcosm study confirming local indigenous microbial population’s ability to degrade hydrocarbons using NO3- as the TEA in addition to observed decrease in NO3- relative to a conservative Br- tracer and generation of nitrite, an intermediate product in denitrification in the pilot-scale operation. Contaminant mass removal likely occurred as Br- tracer evidence indicates that NO3- was utilized in the study area based on the inference of denitrification rates. Post-injection groundwater sampling indicate declining concentrations of toluene, however long term monitoring is recommended in order to evaluate the success of the remediation activity and assess the potential for rebound. Post-injection soil core results are unable to demonstrate the reduction in individual toluene, let alone BTEXTMB hydrocarbon levels, as a result of insufficient quantities of nitrate delivered to the target zone relative to the significant but heterogeneously distributed residual mass in the subsurface.

Olive Mill Wastewater (OMW), by-product of oil industry, is a dark liquid with a characteristic fetid smell, bitter taste and bright appearance / having a high pollution potential, creating serious problems in countries producing olive oil. Azotobacter chroococcum as a Nitrogen-fixing bacteria can bioremediate OMW, by degrading its toxic constituents. With the help of this detoxification process OMW can be used as biofertilizer. In this study, the dynamics of growth and nitrogen fixation at different physiological conditions and nutrient requirements of A. chroococcum in chemically defined N-free medium was determined. These parameters were cultivation conditions such as pH, temperature and aeration and some additives such as inorganic salts, boric acid and nitrogen. Consequently, the maximum cell concentration were obtained when A. chroococcum was grown at neutral pH, 35&amp / #61616 / C, 150 rpm and in medium supplemented with manganese salt at 0.01% concentration. The maximum nitrogen fixation products were attained when A. chroococcum was grown under the same conditions except at pH 8. Further, bioremediation of OMW by A. chroococcum was examined. When A. chroococcum was cultivated in OMW containing basal medium at 10% OMW concentration, a cell density 12 times higher than in the OMW free medium was achieved. Also, it was found to have maximum increase in extracellular protein concentration (112 mg/l) at 10% OMW containing medium and maximum increase in ammonia concentration (9.05 mg/l) at 5% OMW containing medium.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M.Sc.). / Written for the Dept. of Natural Resource Sciences. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.