Fungi play important geoactive roles in the biosphere, particularly element biotransformations and biogeochemical cycling, metal and mineral transformations, decomposition, bioweathering, and soil and sediment formation. Fungi can apply various mechanisms to effect changes in metal speciation, toxicity and mobility, mineral formation and/or mineral dissolution. This research has examined fungal roles in bioweathering and bioleaching of zinc sulfide ore, together with an investigation of the role of fungal phosphatases in the bioprecipitation of uranium and lead when utilising an organic phosphorus-containing substrate as the sole phosphorus source. The results obtained revealed that test fungal species showed bioweathering effects on zinc sulfide ore, and clear evidence of biotransformation and bioleaching of zinc sulfide was obtained after growth of A. niger. The formation of zinc oxalate dihydrate resulted from oxalic acid excretion. The formation of uranium- and lead-containing biominerals after growth of yeasts and filamentous fungi with organic phosphorus sources have also been demonstrated and characterized. Test fungi were capable of precipitating uranium phosphate and pyromorphite, and also produced mycogenic lead oxalate during this process. This work is the first demonstration that filamentous fungi are capable of precipitating a variety of uranium- and lead-containing phosphate biominerals when grown with an organic phosphorus source. The role of fungal processes in the bioweathing and bioleaching of mineral ores, and the significance of phosphatases in the formation of uranium and lead secondary minerals has thrown further light on potential fungal roles in metal and mineral biogeochemistry as well as the possible significance of these mechanisms for element biorecovery or bioremediation.

Nitroaromatic explosives have contaminated millions of acres of soil and water across the globe since World War II with known mutagenic, carcinogenic, and ecotoxicological effects. Recently, the U.S. Army initiated a shift away from traditional explosive compounds, such as trinitrotoluene (TNT) and hexahydrotrinitrotriazine (RDX), towards new, insensitive high explosive formulations. The new formulations approved for use include “IMX-101” and “IMX-104,” which contain 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine (NQ). These mixtures are less prone to accidental detonation making storage, transport, and implementation of these formulations safer for soldiers. Furthermore, initial research indicates that these compounds are less toxic than the older analogues. Despite the apparent benefits, the new explosives have higher solubility (approximately 3-300 times) than the compounds they are replacing, and NTO and NQ are fairly recalcitrant to aerobic biodegradation. The refractory nature and high solubility of the compounds raises concerns about leaching and water contamination considering the previous scale of environmental contamination from production and use of legacy explosives, while feasible strategies for cleaning up the new chemicals from soil and water have not been developed. Therefore, there is a critical need for understanding of the mechanisms of biodegradation these compounds will undergo in the environment and in engineered systems. In addition, a number of questions remain about the photochemistry of the compounds and how they may transform in sunlit surface water. Accordingly, this thesis examines biological transformations of DNAN and NTO in vegetative, fungal, and bacterial organisms, as well as photolysis of NTO and NQ in aqueous solution and DNAN in plant leaves. I identified 34 novel biotransformation products of DNAN using stable-isotope labeled DNAN and high resolution mass spectrometry. Most identified biotransformation products were the result of a nitro-group reduction as the first metabolic step. Arabidopsis plants, a Rhizobium bacterium, and a Penicillium fungus all further metabolized DNAN to produce large, conjugated compounds, and no mineralization was observed in the systems studied. All three organisms reduced both para- and ortho-nitro groups of DNAN, with a dramatic preference for ortho reduction. I found that photodegradation of DNAN and its plant metabolites within Arabidopsis leaves could impact the phytoremediation of DNAN and other contaminants. Soil slurries acclimated to nitroaromatic wastewater degraded DNAN with and without carbon and nitrogen amendments and NTO with added carbon. Organisms capable of degrading DNAN and NTO were isolated, and NTO was transformed to urea, amino-triazolone, and hydroxyl-triazolone. Photolysis of NTO sensitized singlet oxygen formation and yielded hydroxyl-triazolone, nitrite, nitrate, and ammonium. The rate of photolysis of NTO increased over the neutral pH range, and natural organic matter quenched the photolysis of NTO. An unknown volatile product accumulated in the headspace of sealed reactors after NTO photolysis. Singlet oxygen degraded NTO and formed nitrite in stoichiometric yield. Photolysis of NQ produced nitrite and nitrate, but at high pH, the reaction occurred much faster than at neutral pH, and the mass balance of inorganic nitrogen was much lower. Further work should be done to investigate the mechanisms of and products from NTO and NQ photolysis.

Selected PCB congeners (PCB 52, 77, and 153) singly and in mixtures were spiked and aged in soil microcosms and subsequently planted with switchgrass (Panicum virgatum) or poplar (Populus deltoids x nigra DN34). The planted reactors showed significantly greater reductions in PCB parent compounds when compared to unplanted systems after 32 weeks, both in single congener exposures and when all three congeners were present in a mixture. There was evidence of reductive dechlorination in both planted and unplanted systems, but higher concentrations of transformation products were observed in the planted systems than the unplanted. Although planted systems resulted in greater biotransformation, this improvement in PCB-reduction was not the result of plant uptake but rather was due to transformations occurring in the root rhizosphere. Parent PCB congeners were transformed by reductive dechlorination resulting in successively less chlorinated PCB congeners. These dechlorination products accounted for approximately all of the molar mass of parent compound lost. Based on the transformation products, reductive dechlorination pathways are proposed for rhizospheric biotransformation of PCB 52, 77, and 153. Results suggest that PCB 52 transformation proceeds through PCBs 18 and 9 down to monochlorinated PCB 1. Biotransformation of PCB 77 occurs through the intermediaries PCB 35 and 37. The pathway for the rhizospheric transformation of PCB 153 is through PCB 101 and PCB 99. This study provides insight into rhizosphere biotransformation pathways for reductive dechlorination in marginally aerobic,intermittently flooded soil as evidenced by a mass balance on transformation products. Despite the marginally aerobic conditions it is likely that highly reduced microzones existed in the soil particles during flooding and provided the opportunity for reductive dechlorination. In these experiments, planted microcosms with fully developed roots and rhizospheres showed significant reductive dechlorination and greater biotransformation than unplanted reactors. In addition, planted systems that were intermittently flooded had greater transformation of the parent PCB compounds than systems that were not. A poplar planted system resulted in the complete removal of 26 of the 29 PCB congeners detected in a commercial garden soil, while the unplanted soil only had 2 congeners completely removed after 96 days. In addition, the most recalcitrant congener, PCB 52, only decreased by 0.1% in the unplanted reactors while declining by 22.3% in the planted system. There was also greater removal of a PCB 77 spike in the planted system when compared to the unplanted system, 17.2% in the planted system versus 2.8% in the unplanted system. The results suggest that phytoremediation may be an effective tool in cleaning commercially available garden soils that are lightly contaminated with PCBs.

The aerobic transformation of TCE and cis-DCE by a tetrabutoxysilane-grown microorganism (Vancheeswaran et al., 1999) led to the investigation of novel substrates, including benzyl alcohol, for promoting cometabolism. The culture grew on carboxylic compounds and alcohols, but did not grow on formate, methanol, methane, propane, butane, ethylene, benzene, toluene, or p-xylene. Cis-DCE transformation was observed when the culture grew on butyrate, glucose, 1-propanol, 1-butanol, ethanol, benzyl alcohol, and phenol, and effectively transformed TCE, cis-DCE, and vinyl chloride when grown on phenol or benzyl alcohol. Several cycles of growth on benzyl alcohol led to increases in TCE transformation rates and transformation capacities. Products of benzyl alcohol degradation shifted from benzaldehyde to 2-hydroxy benzyl alcohol (2HBA) during the several cycles of growth. In resting cells studies, 2HBA production rates were highly correlated with TCE transformation rates. TCE transformation and 2HBA production rates doubled when the culture was grown on phenol and rates of TCE transformation were correlated with 2HBA production rates. Benzyl alcohol- and phenol-grown cells oxidized toluene to o-cresol, which indicated the similarity between benzyl alcohol ortho-monooxygenase, phenol hydroxylase, and toluene ortho-monooxygenase. 2-Butyne and 1-hexyne (but not acetylene) inhibited benzyl alcohol- and phenol-grown cells similarly, indicating the same ortho-monooxygenase was responsible for TCE cometabolism. Resting cell kinetic studies were performed with cells grown on phenol or benzyl alcohol. Benzyl alcohol degradation followed a Monod kinetics while phenol degradation followed a Haldane kinetics. The maximum transformation rates (k[subscript max]) of TCE, cis-DCE, and VC achieved by phenol-grown cells were about a factor of two higher than achieved with benzyl alcohol-grown cells, while the half-saturation constants (K[subscript s]) were in a similar range. Transformation capacities (Tc) for TCE, cis-DCE, and VC were about a factor of two to four higher with phenol-grown cells. The modeling of TCE, cis-DCE, and VC transformation using independently measured k[subscript max] and K[subscript s] values matched well with observed data from batch tests. Benzyl alcohol was shown to be an effective novel substrate for the aerobic cometabolism of TCE, cis-DCE, and vinyl chloride. Being a non-regulated compound, it might have applications for in-situ bioremediation. / Graduation date: 2004

The 1,1-dichloroethene (1,1-DCE) cometabolic transformation abilities of indigenous and bioaugmented microorganisms were compared in microcosms constructed with groundwater and aquifer solids from the Moffett Field site, CA. Microbial community structure in the microcosms and possible community shifts due to 1,1-DCE transformation stress was evaluated by terminal restriction fragment length polymorphism method (T-RFLP). An existing biotransformation model was used to simulate the experimental data using parameter values determined by Kim et al. (2002) and Rungkamol (2001) with small adjustments to the parameter values. The laboratory microcosm studies showed that both indigenous and bioaugmented butane utilizers were capable of transforming 1,1-DCE when fed butane as a primary substrate. A butane-grown enriched culture was bioaugmented into the microcosms and exposed to several repeated additions of butane and/or 1,1-DCE, ranging from 7.1 to 76 ��mol and from 0.17 to 1.99 ��mol, respectively. The bioaugmented butane-utilizers showed a reduced lag period compared to the indigenous butane-utilizers. The greatest ability to transform 1,1-DCE was observed in bioaugmented microcosms, simultaneously exposed to butane and 1,1-DCE. Very little 1,1-DCE was transformed in the bioaugmented microcosms that were not fed butane, presumably due to lack of reductant supply and/or product toxicity of 1,1-DCE transformation. Microbial community analyses revealed similar results for replicate microcosms and differences in the community structure in microcosms subjected to different patterns of substrate addition and 1,1-DCE cometabolism. 1,1-DCE transformation resulted in temporal fluctuations in specific bacterial groups in the bioaugmented microcosms. It could be inferred that microorganisms, correlated with the T-RFL of 183 base pair (bp) were generally predominant in butane-fed bioaugmented microcosms simultaneously exposed to 1,1-DCE. Bioaugmented microcosms that were pre-exposed to 1,1-DCE for 29 days in the absence of growth substrate, followed by the addition of butane showed a significantly different microbial community from bioaugmented microcosms fed butane and 1,1-DCE simultaneously. Microorganisms with T-RFL of 179 or 277.8 bp dominated in these microcosms. These differences were possibly the result of extensive 1,1-DCE transformation product toxicity during the pre-exposure phase of the tests. A model developed by Kim et al. (2002) was used to mathematically describe the rate and extent of butane utilization and the cometabolic transformation of 1,1-DCE in the microcosm tests. Using the kinetic parameter values previously determined by Kim et al. (2002) and Rungkamol (2001), heuristic fits were obtained between the experimental data and model simulations. The model successfully predicted the trend of the butane utilization and 1,1-DCE transformation. The model outputs were statistically quantified for their fit to the experimental data by estimating Standard Error of Estimate (SEE). A reasonable fit between model predictions and experimental observations was achieved. A significant contribution of this study was developing the laboratory methods to evaluate the microbial abilities to cometabolize 1,1-DCE and determining the communities of microorganisms correlated with those biotransformation activities. Furthermore, the model comparison to experimental data indicated that there was a potential in using the existing model to predict and improve bioremediation strategies. The results showed the successful bioaugmentation of a butane-utilizing culture to improve transformation performance. / Graduation date: 2003

Living organisms constantly respond to changing environmental conditions, and some changes can be far from optimal for many organisms. Insects represent the majority of species in many ecosystems and play an important role in bioaccumulation and biotransformation of environmental contaminants such as selenium and arsenic. Some insectivorous predators feeding on these insects are highly sensitive to such elements resulting in reduced growth, reproductive failures and low population numbers. The mechanisms of selenium and arsenic uptake through the food chain are poorly understood. The determination of chemical speciation is a prerequisite for a mechanistic understanding of a contaminants bioavailability and toxicity to an organism. Synchrotron-based X-ray absorption spectroscopy was used to identify the chemical form of selenium and arsenic in insects in both the field and laboratory conditions. Insects living in streams near Hinton, Alberta affected by coal mine activities were examined for selenium speciation. Results showed higher percentages of inorganic selenium in primary consumers, detritivores and filter feeders than in predatory insects. Selenides and diselenides constitute a major fraction of selenium in these insects. In another field setting, speciation of selenium was studied in insects attacking selenium hyperaccumulating plant <i>Astragalus bisulcatus</i>. The effect of selenate and arsenate alone and the combined effects of selenate and arsenate on insects and parasitoids were monitored using a laboratory-reared moth (<i>Mamestra configurata</i>). Hosts receiving selenium biotransformed selenate to organic selenides and diselenides, which were transferred to the parasitoids in the third trophic level. Arsenic fed larvae biotransformed dietary arsenate to yield predominantly trivalent arsenic coordinated with three aliphatic sulfurs. Larvae receiving arsenate used a novel six-coordinated arsenic form as an excretory molecule in fecal matter and cast skin. X-ray absorption spectroscopy imaging with micro X-ray fluorescence imaging on selenate and arsenate fed larvae revealed highly localized selenium and arsenic species, zinc and copper within the gut. The results provide insights into how the insects cope with their toxic cargo, including how selenium and arsenic are biotransformed into other chemical forms and how they can be eliminated from the insects. The implication of selenium and arsenic species in the diet of predators and detritivores is discussed.

Le cadmium (Cd) est un métal lourd auquel la population en générale est exposée par l'alimentation. L'épithélium intestinal accumule beaucoup de Cd ingéré et représente un organe cible. Étant donné le rôle de cet épithélium dans la biotransformation de xénobiotiques ingérés, l'objectif de notre étude était d'évaluer dans quelle mesure une exposition chronique au Cd peut perturber l'expression des enzymes de biotransformation lors de la différenciation des entérocytes. Comme modèle in vitro, nous avons utilisé la lignée cellulaire humaine Caco-2 qui développe spontanément un phénotype entérocytaire. Le Cd étant connu pour troubler certaines voies de signalisation cellulaires, nous avons testé les hypothèses suivantes : 1) ce métal pourrait modifier l'expression (niveaux et/ou profil) des enzymes de biotransformation; 2) il serait susceptible d'altérer le processus de différenciation. Le profil d'expression d'enzymes de biotransformation (CYP1A1, CYP3A4 et GSTP1) a été caractérisé par RT-PCR en fonction du temps de culture : les niveaux d'ARNm de la GSTP1, de la P-gp et du CYP1A1 augmentent durant la différenciation. Parallèlement, nous avons estimé par mesure d'activité MTT (viabilité cellulaire), la LC5, soit la concentration d'exposition menant à 5% de mortalité. Lorsque les cellules sont exposées durant la phase de prolifération, une période de récupération augmente considérablement la viabilité (LC5 = 87 ± 4 vs. 15.2 ± 0.7 µM). Une LC5 de 36 ± 2 µM est obtenue lorsque les cellules sont traitées pendant la phase de différenciation montrant que la période de sensibilité maximale est pendant la prolifération. Étant donné les profils d'expression obtenus selon le temps de culture des quatre enzymes d'intérêt dans les cellules Caco-2 témoins, nous avons choisi la LC5 de 36 ± 2 µM comme concentration d'exposition pendant la phase de différenciation pour tester l'effet du Cd. Une exposition chronique à cette concentration de Cd perturbe le profil d'expression d'enzymes. De plus hauts niveaux d'ARNm de GSTP1 et de la P-gp sont alors observés mais cette induction par le Cd est diminuée en présence de vitamine D3 ou de NAC, toutes deux antioxydantes. Ainsi, l'effet du Cd sur la GSTP1 et la P-gp serait médié par un déséquilibre rédox. Des mesures d'activités enzymatiques de CYP1A1 et de GSTP1 ont été effectuées afin de corréler l'effet du Cd sur les niveaux d'ARNm aux activités. Une activité extrêmement faible d'environ 0,290 pmol/min/mg de protéines microsomales a été mesurée dans les cellules contrôles et traitées au Cd de 21 jours, soit presque 100 fois moins que ce qui est rapporté dans la littérature, mais plusieurs études ont révélé l'effet inhibiteur du Cd sur l'activité EROD. Deux types de réponses sont observées quant à l'effet du Cd sur l'activité de la GST: (1) une légère stimulation, (2) une faible inhibition. Nous avons également mesuré une diminution de la résistance électrique transépithéliale confirmant ainsi que le Cd affecte l'intégrité de l'épithélium intestinal en augmentant la perméabilité paracellulaire. Par ailleurs, nos récents résultats suggèrent que le Cd diminue l'activité de la phosphatase alcaline, un marqueur de différenciation. Néanmoins, les enzymes de biotransformation n'étaient pas toutes affectées de la même façon, l'effet du Cd sur ces enzymes n'est pas le résultat (indirect) d'une action "non spécifique" sur le processus global de différenciation. Nos résultats montrent que l'exposition intestinale au Cd pourrait avoir des répercussions sur le métabolisme de premier passage des médicaments et autres xénobiotiques absorbés oralement. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : cellules Caco-2, épithélium intestinal, cadmium, entérocyte, CYP1A1, CYP3A4, GSTP1

An emplaced source of coal tar creosote within the sandy Borden research aquifer has provided an opportunity to document the long term (5140 days) natural attenuation for this complex mixture. Plumes of dissolved chemicals were produced by the essentially horizontal groundwater flowing at about 9 cm/day. Eleven chemicals were extensively sampled seven times using a monitoring network of ~280 14-point multilevel samplers. A model of source dissolution using Raoult’s Law adequately predicted the dissolution of nine of eleven compounds analysed. Mass transformation has limited the extent of the plumes as groundwater flowed more than 500 m yet the plumes are no longer than 50 m. Phenol and xylenes were removed and naphthalene was attenuated from its maximum extent on day 1357. Some compound plumes reached an apparent steady state and the plumes of other compounds (dibenzofuran and phenanthrene) are expected to continue to expand due to an increasing mass flux and limited degradation potential. Biotransformation is the major process controlling natural attenuation at the site. The greatest organic mass loss is associated with the high solubility compounds. However, the majority of the mass loss for most compounds has occurred in the source zone. Oxygen is the main electron acceptor yet the amount of organics lost cannot be accounted for by aerobic mineralization or partial mineralization alone. After 10 years the source zone was treated with permanganate in situ to reduce the flux of contaminants into the dissolved plume and to permit natural attenuation to further reduce the plume extent. A sufficient mass of permanganate was injected to oxidize ~10% of the residual source. Laboratory experiments demonstrated that eight of ten of the study compounds were readily oxidized by permanganate. Once treated oxidized compounds displayed a reduced plume mass and mass discharge while they migrated through the monitoring network. Once beyond the monitoring network the mass discharge and plume mass of these compounds returned to pre-treatment trends. Non-reactive compounds displayed no significant decrease in mass discharge or plume mass. Overall the partial in situ chemical oxidation of the coal tar creosote source produced no long-term effect on the dissolved plumes emanating from the source.

An emplaced source of coal tar creosote within the sandy Borden research aquifer has provided an opportunity to document the long term (5140 days) natural attenuation for this complex mixture. Plumes of dissolved chemicals were produced by the essentially horizontal groundwater flowing at about 9 cm/day. Eleven chemicals were extensively sampled seven times using a monitoring network of ~280 14-point multilevel samplers. A model of source dissolution using Raoult’s Law adequately predicted the dissolution of nine of eleven compounds analysed. Mass transformation has limited the extent of the plumes as groundwater flowed more than 500 m yet the plumes are no longer than 50 m. Phenol and xylenes were removed and naphthalene was attenuated from its maximum extent on day 1357. Some compound plumes reached an apparent steady state and the plumes of other compounds (dibenzofuran and phenanthrene) are expected to continue to expand due to an increasing mass flux and limited degradation potential. Biotransformation is the major process controlling natural attenuation at the site. The greatest organic mass loss is associated with the high solubility compounds. However, the majority of the mass loss for most compounds has occurred in the source zone. Oxygen is the main electron acceptor yet the amount of organics lost cannot be accounted for by aerobic mineralization or partial mineralization alone. After 10 years the source zone was treated with permanganate in situ to reduce the flux of contaminants into the dissolved plume and to permit natural attenuation to further reduce the plume extent. A sufficient mass of permanganate was injected to oxidize ~10% of the residual source. Laboratory experiments demonstrated that eight of ten of the study compounds were readily oxidized by permanganate. Once treated oxidized compounds displayed a reduced plume mass and mass discharge while they migrated through the monitoring network. Once beyond the monitoring network the mass discharge and plume mass of these compounds returned to pre-treatment trends. Non-reactive compounds displayed no significant decrease in mass discharge or plume mass. Overall the partial in situ chemical oxidation of the coal tar creosote source produced no long-term effect on the dissolved plumes emanating from the source.

Living organisms constantly respond to changing environmental conditions, and some changes can be far from optimal for many organisms. Insects represent the majority of species in many ecosystems and play an important role in bioaccumulation and biotransformation of environmental contaminants such as selenium and arsenic. Some insectivorous predators feeding on these insects are highly sensitive to such elements resulting in reduced growth, reproductive failures and low population numbers. The mechanisms of selenium and arsenic uptake through the food chain are poorly understood. The determination of chemical speciation is a prerequisite for a mechanistic understanding of a contaminants bioavailability and toxicity to an organism. Synchrotron-based X-ray absorption spectroscopy was used to identify the chemical form of selenium and arsenic in insects in both the field and laboratory conditions. Insects living in streams near Hinton, Alberta affected by coal mine activities were examined for selenium speciation. Results showed higher percentages of inorganic selenium in primary consumers, detritivores and filter feeders than in predatory insects. Selenides and diselenides constitute a major fraction of selenium in these insects. In another field setting, speciation of selenium was studied in insects attacking selenium hyperaccumulating plant <i>Astragalus bisulcatus</i>. The effect of selenate and arsenate alone and the combined effects of selenate and arsenate on insects and parasitoids were monitored using a laboratory-reared moth (<i>Mamestra configurata</i>). Hosts receiving selenium biotransformed selenate to organic selenides and diselenides, which were transferred to the parasitoids in the third trophic level. Arsenic fed larvae biotransformed dietary arsenate to yield predominantly trivalent arsenic coordinated with three aliphatic sulfurs. Larvae receiving arsenate used a novel six-coordinated arsenic form as an excretory molecule in fecal matter and cast skin. X-ray absorption spectroscopy imaging with micro X-ray fluorescence imaging on selenate and arsenate fed larvae revealed highly localized selenium and arsenic species, zinc and copper within the gut. The results provide insights into how the insects cope with their toxic cargo, including how selenium and arsenic are biotransformed into other chemical forms and how they can be eliminated from the insects. The implication of selenium and arsenic species in the diet of predators and detritivores is discussed.