<p><p>I investigated the usefulness of phytoplankton for the removal of surface water contaminants. Three experiments, consisting of nine large mesocosms (92.2 m³) were suspended in the flooded DJX uranium pit at Cluff Lake (Saskatchewan, Canada), and filled with contaminated mine water. During the summer of 2003, each mesocosm was fertilized with a different amount of phosphorus throughout the 35 day experiment to stimulate phytoplankton growth, and to create a range in phosphorus load (g) to examine how contaminants may be affected by different nutrient regimes. Algal growth was rapid in fertilized mesocosms as demonstrated by chlorophyll a profiles. As phosphorus loads increased there were significant declines in the surface water concentrations of As, Co, Cu, Mn, Ni, and Zn. This decline was near significant for uranium. The surface water concentrations of Ra²²⁶, Mo, and Se showed no relationship to phosphorus load. Contaminant concentrations in sediment traps suspended at the bottom of each mesocosm generally showed the opposite trend to that observed in the surface water, with most contaminants (As, Co, Cu, Mn, Ni, Ra²²⁶, U, and Zn) exhibiting a significant positive relationship (<i>P</i> < 0.05) with phosphorus load. Sediment trap concentration of Se and Mo did not respond to nutrient treatments.</p> <p>Similar experiments were repeated during the mid- and late-summer of 2004, with 5 mesocosms being fertilized with phosphorus, and another 4 with both phosphorus and ammonium to create different nutrient gradients. Results from these experiments were much more variable than those seen in the experiment conducted in 2003, and small samples (<i>n</i> = 5 for phosphorus treatments and <i>n</i> = 4 for both phosphorus and ammonium treatments) yielded insufficient statistical power to effectively determine statistically significant trends. However, contaminant sedimentation tended to respond to phosphorus treatments in a similar manner as results from 2003; phosphorus-with-ammonium treatments had little positive effect on contaminant sedimentation rates.</p> <p>My results suggest that phytoremediation has the potential to lower many surface water contaminants through the sedimentation of phytoplankton. Based on our results from 2003, we estimate that the Saskatchewan Surface Water Quality Objectives (SSWQO) for the DJX pit would be met in approximately 45 weeks for Co, 65 weeks for Ni, 15 weeks for U, and 5 weeks for Zn if treated using phytoremediation.</p><p>Note:</p><p>Appendix A content (pages 92-95) contains copyrighted material which has been removed. It can be viewed in the original thesis upon request.</p>

Nitrogen is known to be a limiting factor in polluted environments, however many studies overlook the potential role for nitrogen to significantly influence the removal rate and efficiency with which microorganisms can degrade aromatic hydrocarbons. In this study, inoculated and uninoculated aerated soil microcosms containing different s-triazines were examined for their ability to efficiently and rapidly treat contaminated soils containing naphthalene, nitrobenzene, and toluene (NNT), via a microbial consortium consisting of Pseudomonas, Rhodococcus, and Aeromonas. After an experimental period of 14 days, greater than 90% degradation of NNT supplemented with different s-triazines, at concentrations of 1000-3000 ppm was observed. A difference in the degradation of NNT was seen in inoculated box reactors supplemented with cyanuric acid, melamine, and atrazine in comparison to uninoculated box reactors. Combined usage of 16s rDNA and 16s rRNA analysis was then applied to study the bacterial communities, and determine the abundance and survival of inoculated strains within box reactors contaminated with NNT. The bacterial diversity within clone libraries obtained illustrated a dominance of proteobacteria and gram positive bacteria. Analysis from clone libraries also showed that inoculated strains did survive within each condition, but were not the most predominant members present in the communities. This research shows that significant removal of NNT can be achieved in two weeks with the supplementation of one of the s-triazines. However, differences in degradation and the microbial populations present within contaminated communities will be seen depending on which nitrogen sources are used and whether or not environments are bioaugmented or not.

Phytoremediation systems for petroleum hydrocarbons rely on a synergistic relationship between plants and their root-associated microbial communities. Plants exude organic compounds through their roots, which increase the density, diversity and activity of plant-associated microorganisms, which in turn degrade hydrocarbons. Understanding the mechanisms driving this relationship poses one of the more intriguing challenges in phytoremediation research. This study was designed to address that challenge. Plant-microbe interactions in a weathered-hydrocarbon contaminated soil were examined under controlled growth chamber, and field conditions. In both environments single-species grass treatments initially facilitated greater total petroleum hydrocarbon (TPH) degradation than <i> Medicago sativa </i> (alfalfa), mixed species, or control treatments. In growth chamber studies increased degradation was linked to increased aliphatic-hydrocarbon degrader populations within the rhizosphere. Under field conditions, specific recruitment of endophytic aliphatic-hydrocarbon degraders in response to high TPH levels may have facilitated increased degradation by the grass <i> Elymus angustus</i>(Altai wild rye, AWR). AWR stably maintained these communities during times of local drought, enabling them to act as subsequent source populations for rhizosphere communities. The broad phylogenetic diversity of AWR endophytes, compared to the <i> Pseudomonas</i>-dominated communities of other plants, contributed to the observed stability. The relative composition of exudates released by plants also impacted both degradation activity and potential. Alfalfa released higher concentrations of malonate, which hindered degradation by decreasing metabolic activity and concomitantly inhibiting catabolic plasmid transfer. In contrast, AWR exudates contained high levels of succinate, which was linked to increased catabolic gene expression and plasmid transfer. A reciprocal relationship between exudation patterns and endophytic community structure likely exists, and both parameters have a specific influence on rhizosphere degradation capacity. In this study, grasses were more successful in maintaining the specific balance of all parameters required for the transfer, preservation, and stimulation of hydrocarbon catabolic competency.

This thesis presents the potential of integrating chemical and biological treatment technologies for the removal of high concentrations of phenol in a bioremediation medium. High concentrations of phenol in wastewater are difficult to remove by purely biological methods. Chemical oxidation is one way to treat high concentrations of phenol but complete oxidation is not always possible or will make the treatment process uneconomical. An experimental design approach, based on central composite rotatable design (CCRD) was used to evaluate the effects of process parameters on phenol oxidation by Fentons reagent and chlorine dioxide. Performance of the chemical oxidation was evaluated by determining the percentage of phenol oxidized at equilibrium. The reaction mechanism for the oxidation of phenol by Fentons reagent was proposed based on identification of the intermediate compounds.<p> The effects of H₂O₂ concentration (2000 to 5000 mg L^-1) and FeSO₄.7H₂O concentration (500 to 2000 mg L^-1) were investigated on phenol oxidation and optimal concentrations of H₂O₂ and FeSO₄.7H₂O for complete oxidation of 2000 mg L^-1 phenol in medium were found to be 4340 mg L^-1 and 1616 mg L^-1, respectively, at 25°C and pH 3. The main oxidation products were identified as catechol, hydroquinone and maleic acid.<p> In the case of phenol oxidation by chlorine dioxide, the effects of chlorine dioxide concentration (500 to 2000 mg L^-1), temperature (10 to 40°C) and pH (3 to 7) on the oxidation of 2000 mg L^-1 of phenol were determined. The optimal concentration of chlorine dioxide to completely oxidize 2000 mg L^-1 of phenol was 2000 mg L^-1. The other parameters did not significantly affect the oxidation over the ranges studied. The main oxidation products were identified as 1,4-benzoquinone and 2-chloro-1,4-benzoquinone.<p> Finally, the biodegradation of 1,4-benzoquinone, the main oxidation product of phenol oxidation by chlorine dioxide, was studied in batch and continuous systems using Pseudomonas putida 17484 in two dose McKinneys medium. The effects of 1,4-benzoquinone concentration and temperature were studied on biodegradation of 1,4-benzoquinone in batch reactors. Under optimal conditions, it was found that 150 mg L^-1 1,4-benzoquinone could be successfully biodegraded at 15°C. In a continuous reactor operating at 15°C the highest removal rate with 500 mg L^-1 of 1,4-benzoquinone was found to be 246 mg L^-1 h^-1. The values of µmax, Ks and yield were also determined as 0.74±0.03 h^-1 and 14.17±3.21 mg L^-1 and 2x10¹³ cell mg^-1, respectively.

Cyanide is acutely toxic to the environment. However, this simple nitrile is used in several industrial applications especially the mining industry. Due to its high affinity to metals, cyanide has been used for years to extract gold and other precious metals from the ore. Cyanide nitrilases are considered for the detoxification of the industrial wastewaters contaminated with cyanide. Their application in cyanide remediation promises cheaper and safer processes compared to chemical detoxification. However, application of these enzymes in industry requires improving their characteristics. The goal of this dissertation is to better understand cyanide nitrilases, in particular the cyanide dihydratase from of Bacillus pumilus and Pseudomonas stutzeri and to improve their activity and stability. The lack of any high resolution structure of these enzymes calls for isolating or screening for mutants showing enhancement in enzyme properties. Described first is a simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification. This method is useful for generating large pools of randomly mutagenized clones after error-prone PCR mutagenesis. Several parameters were investigated to optimize this technique; length of homology region, vector treatment, induction time and ratio of fragment to vector. Using error-prone PCR for random mutagenesis, several CynDpum mutants were isolated for higher catalysis at pH 7.7. Three point mutations, K93R, D172N and E327K increased the enzyme’s thermostability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 suggesting an effect on the active site. However, the A202T mutation located in the dimerization or the A surface rendered the enzyme inactive by destabilizing it. No significant effect on activity at alkaline pH was observed for any of the purified mutants. Lastly, an important region for CynDstut activity was identified in the C-terminus. This same region increased the stability of CynDpum compared to the wild-type enzyme. Also, CynDpum-stut hybrid was found to be highly more stable than CynDpum. This same hybrid exhibited 100% activity at pH9, a pH where the parent enzyme is inactive, and retained 40% of its activity at pH 9.5 making it a true pH tolerant mutant.

Contamination of soil with Polycyclic Aromatic Hydrocarbons (PAHs) is a serious environmental issue because some PAHs are toxic, carcinogenic and mutagenic. Bioremediation is a promising option to completely remove PAHs from the environment or convert them to less harmful compounds. One of the main challenges in bioremediation of PAHs in a conventional roller bioreactor is the limitation on mass transfer due to the strong hydrophobicity and low water solubility of these compounds. To address this challenge, a novel bead mill bioreactor (BMB) was developed by Riess et al. (2005) which demonstrated a significant improvement in the rates of mass transfer and biodegradation of PAHs. <p> In this study, to further improve mass transfer rates, baffles have been installed in both the conventional and bead mill bioreactors. Mass transfer rates of 1000 mg L-1 suspended naphthalene, 2-methylnaphthalene and 1,5-dimethylnaphthalene, three model compounds of PAHs, have been investigated in four bioreactors: conventional (control), baffled, BMB and baffled bead mill bioreactors. The baffled bioreactor provided mass transfer coefficients (KLa) that were up to 7 times higher than those of the control bioreactor. <p> Bioremediation of suspended naphthalene or 2-methylnaphthalene as a single substrate and their mixtures was studied using the bacterium <i>Pseudomonas putida </i>ATCC 17484. Both baffled and bead mill bioreactors provided maximum bioremediation rates that were 2 times higher than the control bioreactor. The maximum bioremediation rates of 2-methylnaphthalene were further increased in the presence of naphthalene by a factor of 1.5 to 2 compared to the single substrate. <p> Another rate-limiting step for bioremediation of PAH-contaminated soil is the strong sorption between the contaminant and soil. To find out the effect of sorption on the bioavailability of naphthalene, the appropriate sorption isotherms for three types of soils (sand, silt and clay) have been determined. It was observed that the sorption capacity of soils for naphthalene was proportional to the organic carbon content of the soils. The mass transfer of soil-bound naphthalene from the artificially prepared contaminated soils with short contamination history to the aqueous phase was studied in both the control and bead mill bioreactors. It was observed that the mass transfer was unexpectedly fast due to the increased interfacial surface area of naphthalene particles and the weak sorption between naphthalene and soils. It was concluded that artificially, naphthalene contaminated soils would likely not be any more difficult to bioremediate than pure naphthalene particles.

<p><p>I investigated the usefulness of phytoplankton for the removal of surface water contaminants. Three experiments, consisting of nine large mesocosms (92.2 m³) were suspended in the flooded DJX uranium pit at Cluff Lake (Saskatchewan, Canada), and filled with contaminated mine water. During the summer of 2003, each mesocosm was fertilized with a different amount of phosphorus throughout the 35 day experiment to stimulate phytoplankton growth, and to create a range in phosphorus load (g) to examine how contaminants may be affected by different nutrient regimes. Algal growth was rapid in fertilized mesocosms as demonstrated by chlorophyll a profiles. As phosphorus loads increased there were significant declines in the surface water concentrations of As, Co, Cu, Mn, Ni, and Zn. This decline was near significant for uranium. The surface water concentrations of Ra²²⁶, Mo, and Se showed no relationship to phosphorus load. Contaminant concentrations in sediment traps suspended at the bottom of each mesocosm generally showed the opposite trend to that observed in the surface water, with most contaminants (As, Co, Cu, Mn, Ni, Ra²²⁶, U, and Zn) exhibiting a significant positive relationship (<i>P</i> < 0.05) with phosphorus load. Sediment trap concentration of Se and Mo did not respond to nutrient treatments.</p> <p>Similar experiments were repeated during the mid- and late-summer of 2004, with 5 mesocosms being fertilized with phosphorus, and another 4 with both phosphorus and ammonium to create different nutrient gradients. Results from these experiments were much more variable than those seen in the experiment conducted in 2003, and small samples (<i>n</i> = 5 for phosphorus treatments and <i>n</i> = 4 for both phosphorus and ammonium treatments) yielded insufficient statistical power to effectively determine statistically significant trends. However, contaminant sedimentation tended to respond to phosphorus treatments in a similar manner as results from 2003; phosphorus-with-ammonium treatments had little positive effect on contaminant sedimentation rates.</p> <p>My results suggest that phytoremediation has the potential to lower many surface water contaminants through the sedimentation of phytoplankton. Based on our results from 2003, we estimate that the Saskatchewan Surface Water Quality Objectives (SSWQO) for the DJX pit would be met in approximately 45 weeks for Co, 65 weeks for Ni, 15 weeks for U, and 5 weeks for Zn if treated using phytoremediation.</p><p>Note:</p><p>Appendix A content (pages 92-95) contains copyrighted material which has been removed. It can be viewed in the original thesis upon request.</p>

Contamination of soil with Polycyclic Aromatic Hydrocarbons (PAHs) is a serious environmental issue because some PAHs are toxic, carcinogenic and mutagenic. Bioremediation is a promising option to completely remove PAHs from the environment or convert them to less harmful compounds. One of the main challenges in bioremediation of PAHs in a conventional roller bioreactor is the limitation on mass transfer due to the strong hydrophobicity and low water solubility of these compounds. To address this challenge, a novel bead mill bioreactor (BMB) was developed by Riess et al. (2005) which demonstrated a significant improvement in the rates of mass transfer and biodegradation of PAHs. <p> In this study, to further improve mass transfer rates, baffles have been installed in both the conventional and bead mill bioreactors. Mass transfer rates of 1000 mg L-1 suspended naphthalene, 2-methylnaphthalene and 1,5-dimethylnaphthalene, three model compounds of PAHs, have been investigated in four bioreactors: conventional (control), baffled, BMB and baffled bead mill bioreactors. The baffled bioreactor provided mass transfer coefficients (KLa) that were up to 7 times higher than those of the control bioreactor. <p> Bioremediation of suspended naphthalene or 2-methylnaphthalene as a single substrate and their mixtures was studied using the bacterium <i>Pseudomonas putida </i>ATCC 17484. Both baffled and bead mill bioreactors provided maximum bioremediation rates that were 2 times higher than the control bioreactor. The maximum bioremediation rates of 2-methylnaphthalene were further increased in the presence of naphthalene by a factor of 1.5 to 2 compared to the single substrate. <p> Another rate-limiting step for bioremediation of PAH-contaminated soil is the strong sorption between the contaminant and soil. To find out the effect of sorption on the bioavailability of naphthalene, the appropriate sorption isotherms for three types of soils (sand, silt and clay) have been determined. It was observed that the sorption capacity of soils for naphthalene was proportional to the organic carbon content of the soils. The mass transfer of soil-bound naphthalene from the artificially prepared contaminated soils with short contamination history to the aqueous phase was studied in both the control and bead mill bioreactors. It was observed that the mass transfer was unexpectedly fast due to the increased interfacial surface area of naphthalene particles and the weak sorption between naphthalene and soils. It was concluded that artificially, naphthalene contaminated soils would likely not be any more difficult to bioremediate than pure naphthalene particles.

An External Loop Airlift Bioreactor with a small amount (99% porosity) of stainless steel mesh packing inserted in the riser section was used for bioremediation of a phenol polluted air stream. The packing enhanced VOC and oxygen mass transfer rates and provided a large surface area for cell immobilization. Using a pure strain of Pseudomonas putida, fed-batch and continuous runs at three different dilution rates were completed with phenol in the polluted air as the only source of growth substrate. 100% phenol removal was achieved at phenol loading rates up to 33120 mg/h.m3 using only one third of the column, superior to any previously reported biodegradation rates of phenol polluted air with 100% efficiency. A mathematical model has been developed and is shown to accurately predict the transient and steady state data.

Phytoremediation systems for petroleum hydrocarbons rely on a synergistic relationship between plants and their root-associated microbial communities. Plants exude organic compounds through their roots, which increase the density, diversity and activity of plant-associated microorganisms, which in turn degrade hydrocarbons. Understanding the mechanisms driving this relationship poses one of the more intriguing challenges in phytoremediation research. This study was designed to address that challenge. Plant-microbe interactions in a weathered-hydrocarbon contaminated soil were examined under controlled growth chamber, and field conditions. In both environments single-species grass treatments initially facilitated greater total petroleum hydrocarbon (TPH) degradation than <i> Medicago sativa </i> (alfalfa), mixed species, or control treatments. In growth chamber studies increased degradation was linked to increased aliphatic-hydrocarbon degrader populations within the rhizosphere. Under field conditions, specific recruitment of endophytic aliphatic-hydrocarbon degraders in response to high TPH levels may have facilitated increased degradation by the grass <i> Elymus angustus</i>(Altai wild rye, AWR). AWR stably maintained these communities during times of local drought, enabling them to act as subsequent source populations for rhizosphere communities. The broad phylogenetic diversity of AWR endophytes, compared to the <i> Pseudomonas</i>-dominated communities of other plants, contributed to the observed stability. The relative composition of exudates released by plants also impacted both degradation activity and potential. Alfalfa released higher concentrations of malonate, which hindered degradation by decreasing metabolic activity and concomitantly inhibiting catabolic plasmid transfer. In contrast, AWR exudates contained high levels of succinate, which was linked to increased catabolic gene expression and plasmid transfer. A reciprocal relationship between exudation patterns and endophytic community structure likely exists, and both parameters have a specific influence on rhizosphere degradation capacity. In this study, grasses were more successful in maintaining the specific balance of all parameters required for the transfer, preservation, and stimulation of hydrocarbon catabolic competency.