The main objective of this study was to improve our understanding of biological filtration (biofilm type) treatment for manganese (Mn) removal in drinking water. Biological filtration treatment involves biofilms of Mn(II)-oxidizing microorganisms attached to solid filter material that remove and immobilize dissolved Mn(II) in raw water by conversion to black MnO2(s) precipitates. Mn-biological filtration is an emerging green technology that can serve as an alternative to conventional physicochemical treatments but its full potential is hindered by various factors. These include lack of understanding the: (1) optimal removal conditions for Mn, (2) mechanisms for Mn releases of the accumulated Mn in the biofilter, and (3) effects of recalcitrant natural organic matter (NOM) on biofiltration. Confounding these issues is the unknown identity of the diverse microbial communities which occupy the biofilms attached to the filter media. To investigate these issues, biological Mn removal was studied in laboratory bench scale reactors using a new Mn(II)-oxidizing bacterium isolate, Pseudomonas Putida EC112. The main research hypothesis formulated that the transition metal catalyst, MnO2(s), can increase the bioavailable carbon and energy from recalcitrant NOM (e.g., humic acids (HA)) to biological filters. Mn and HA can be found in most natural waters, including groundwaters, lakes and streams. To test the hypothesis, the potential for strain EC112 growth and Mn(II) oxidation utilizing the organic substrate products from the oxidation reaction between HA and MnO2(s) was assessed. Biological Mn(II)-oxidation kinetics were investigated in batch (suspended cell) and continuous flow (biofilm) bioreactors at optimal pH and temperature conditions for strain EC112. Batch kinetics was successfully characterized with the Monod model. Continuous flow steady-state kinetics was modeled with a single, zero-order kinetic parameter. Enhanced Mn(II) removal capacity was observed for strain EC112 in batch and continuous flow reactors in the presence of HA and MnO2. The effect of MnO2(s) on HA biodegradability was studied and optimal conditions for biodegradation were identified. Biofilter Mn(II) releases were observed during the continuous flow bioreactor experiments. Release conditions were identified and releases modeled using pseudo first-order kinetics. Changes in HA structure induced by MnO2(s) oxidation were studied with Fourier transform infrared (FT-IR) and proton nuclear magnetic spectroscopy (1H-NMR).

TOL plasmids of Pseudomonas putida encode enzymes for the degradation of toluene and related aromatics. These genes are organized into two operons regulated by the Xy1R and Xy1S transcriptional activators. Previous analysis of the TOL pDK1 catechol-2,3-dioxygenase gene (xy1E) and a comparison of this gene to xy1E from the related TOL plasmid pWW0, revealed the existance of a substantial level of sequence homology (82%).

The TOL plasmids of Pseudomonas putida encode enzymes required for the oxidation of toluene and other related aromatic compounds. These genes are organized into two operons, the xylUWCMABN operon (upper), and the xylXYZLTEGFJQKIH operon (lower). Here we report the nucleotide sequence of a 7107 bp segment of the TOL pDK1 plasmid encoding the region just upstream of the "upper" operon through the genes encoding xylUWCMA. Sequence analysis, comparison of base-usage patterns, codon-usage patterns, and intergenic distances between genes help support the idea that the "upper" and "lower" operons have evolved independently in different genetic backgrounds and have only more recently been brought together in TOL and related catabolic plasmids.

Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.

"Bacteria during the course of their life undergo a lot of developments on their surface. The changes that occur inside a cell result in the production of a variety of biopolymers on the cell surface. These polysaccharides have been found to play a major role in deciding the adhesive or repulsive nature of a bacterial cell. Based on the application the adhesive nature of a cell sometimes needs to be manipulated such that bacteria are required to have higher adhesions for bioremediation applications and in the case of bioreactors bacteria must not stick to walls to avoid fouling. In order to control adhesions of a cell to a variety of substrates, knowledge of the polysaccharides present on its surface is needed. Therefore the goal of the present study is to detect the sugars present on the surface of Pseudomonas putida KT2442 using Atomic force microscopy and to relate properties of the polysaccharides to bacterial adhesion. Previous experiments suggested that cellulose and other sugars were produced by Pseudomonas putida KT2442. Thus the cells were grown to late exponential phase and treated with cellulase to degrade any cellulose, if present, on the surface of the cells. Control experiments were done on untreated cells and cells that were not treated with cellulase but were centrifuged, since centrifugation is a part of the cellulase treatment and may also affect the bacterial surface. An appropriate (Steric) fitting model for the atomic force microscope (AFM) approach curves was applied to calculate the height and density of the polymer brush layer present on the cell surface. There was a decrease in the density of the polymer brush and increase in the height of the brush upon treatment with cellulase. Centrifugation alone did not affect the approach curves. From looking at the retraction curves it verified the results got from the approach curves and indicated stretching out of the polymer brush to greater distances after the treatment with cellulase. Another batch of cells was treated with dextranase to check for the presence of dextran on the cell surface. Dextranase treated cells behaved identical to the control cells, suggesting that dextran is not one of the polysaccharides present on the bacterial surface. No change was observed in retraction curves data for dextranase treated and untreated cells."

Microorganisms are capable of producing advanced biofuels that can be used as ‘drop-in’ alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms for improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. First, using genomic DNA from Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. Additionally, we used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP. putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP. putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP. putida, while others provide tolerance across a wide range of rpoDP. putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries. This dissertation describes a method of effectively screening genomic DNA from multiple organisms for genes to mitigate biofuel stress and shows how tolerance genes can improve bacterial growth in the presence of toxic biofuel compounds. These identified genes can be targeted in future studies as candidates for use in biofuel production strains to increase biofuel yields.

Biocatalysis involves the use of enzymes to perform stereo- and enantio-specific reactions. One of the reactions where biocatalysis is a valuable technology is oxidation of naphthalene to 1-naphthol using Toluene ortho-Monooxygenase (TOM) variant TmoA3 V106A, also known as TOM-Green. Whole-cell biocatalysis in a water-organic solvent biphasic system was used to minimize naphthalene and 1-naphthol toxicity, and to increase substrate loading. Recombinant Escherichia coli TG1 cells expressing TOM-Green were used for biphasic biocatalysis and lauryl acetate gave best results among the solvents tested. On a constant volume basis, 8 - fold improvement in 1-naphthol production was achieved using biphasic systems compared to biotransformation in aqueous medium. The organic phase was optimized by studying the effects of organic phase ratio and naphthalene concentration in the organic phase. The efficiency of biocatalysis was further improved by application of a solvent tolerant strain Pseudomonas putida S12. P. putida S12 is solvent tolerant owing to its two adaptive mechanisms: outer membrane modification and solvent extrusion using solvent resistant pump srpABC. P. putida S12, in addition to its tolerance to various organic solvents, showed better tolerance to naphthalene compared to E. coli TG1 strain expressing TOM-Green. Application of solvent tolerant P. putida S12 further improved 1-naphthol productivity by approximately 42%. Solvent tolerance of P. putida S12 was further analyzed by transferring its tolerance to a solvent sensitive E. coli strain by transfer of solvent resistant pump srpABC genes. Engineered E. coli strain bearing srpABC genes either in low-copy number plasmid or high-copy number plasmid grew in the presence of a saturated toluene concentration. Engineered E. coli strains were also more tolerant to toxic solvents, e. g., decanol and hexane, compared to the control E. coli strain without srpABC genes. The expression of solvent resistant pump genes was confirmed by Reverse Transcriptase PCR analysis. The main drawbacks of biocatalysis for production of chemicals were addressed and approaches to minimize the drawbacks have been presented. The production of 1-naphthol was significantly improved using biocatalysis in liquid-liquid biphasic systems.

Résumé Certaines bactéries de la rhizosphère (PGPR, rhizobactéries promotrices de la croissance des plantes) exercent un effet bénéfique sur la croissance des plantes en stimulant des mécanismes de défense inductibles chez lhôte, rendant celle-ci moins susceptible vis-à-vis dune infection ultérieure par un agent pathogène. Ce phénomène appelé résistance systémique induite (ISR) a été mis en évidence chez plusieurs plantes pour lutter contre une gamme relativement large de pathogènes fongiques, bactériens ou viraux. Cependant, les bases moléculaires des mécanismes de défense proprement dits stimulés lors de lISR restent assez méconnues malgré les nombreux travaux réalisés cette dernière décennie. Dans ce contexte, des souches de PGPR (Bacillus subtilis et Pseudomonas putida) capables de protéger certaines plantes via linduction de lISR sont étudiées depuis plusieurs années au laboratoire. Dans le cadre de cette thèse de doctorat, nous avons étudié l´effet protecteur de ces bactéries dans deux pathosystèmes différents, tomate/Botrytis cinerea et concombre/Colletotrichum lagenarium. Nos résultats ont montré la capacité de P. putida BTP1 à induire l´ISR chez la tomate et le concombre sur base de la réduction des symptômes de maladie observée et sur base de la séparation spatiale des deux agents, bénéfique au niveau racinaire et pathogène au niveau foliaire. Cette résistance a été clairement associée avec la stimulation de la voie des oxylipines chez la tomate. Linduction de cette voie métabolique a dabord été mise en évidence biochimiquement par une augmentation des activités lipoxygénase et lipide hydroperoxydase dans les feuilles des plantes traitées avec Pseudomonas. De plus, au niveau moléculaire, les analyses par northern blot nous ont permis d´identifier un nouvel isoforme de gène Lox chez la tomate qui est exprimé différentiellement chez les plants traités par la bactérie. Ce gène dénommé LoxF montre une homologie de 82% avec un des cinq isoformes connus chez cette plante, LoxC. LoxF est essentiellement surexprimé dans les feuilles de tomates pré-inoculées avec BTP1 suite à l´infection par le pathogène. Il en est de même pour lactivité enzymatique correspondante de la lipoxygénase qui naugmente significativement chez les plants traités par rapport aux témoins quaprès infection par Botrytis. Ces résultats sous-entendent un phénomène de « priming » ou « mise en alerte » étroitement associé avec linduction de résistance systémique chez les plantes. La reconnaissance de la bactérie au niveau racinaire met en alerte la plante sans que des bouleversements majeurs au niveau métabolique ou génétique ne soient observés. Lhôte réagit alors plus fortement et plus rapidement pour mettre en uvre ses mécanismes de défense une fois le pathogène perçu. Par biotest sur TLC et analyses HPLC, CPG et LC-MS, nous avons mis en évidence laccumulation dune molécule dans les feuilles de tomate prétraitées avec BTP1. Cette molécule semble être de nature apolaire mais non phénolique et ne correspond pas aux phytoalexines connues de la tomate. Des études complémentaires doivent être réalisées pour lidentifier chimiquement mais sa cinétique daccumulation dans les tissus de la plante est étroitement associée à celle de la stimulation de la lipoxygénase. Elle pourrait donc dériver de cette voie métabolique. Des réactions de défense similaires ont été observées suite au traitement avec Bacillus subtilis chez la tomate et ces essais ont permis de mettre en évidence le rôle des lipopeptides produits en tant quéliciteurs impliqués dans linduction du phénomène de lISR par cette souche. Par contre, bien que les deux souches soient capables dinduire la résistance chez le concombre, aucune accumulation claire de phytoalexines ni de stimulation significative de la voie de la lipoxygénase nont pu y être associées chez cette plante. Globalement, nos résultats suggèrent que les voies métaboliques activées dans le cadre de lISR varient en fonction de lespèce végétale même si le microorganisme inducteur est identique. Abstract Some plant growth promoting rhizobacteria (PGPR) are able to stimulate inducible defence mechanisms that render the host plant less susceptible to a subsequent pathogen attack. This phenomenon called induced systemic resistance (ISR) can occur in several plant species against a wide range of bacterial, viral and fungal pathogens. Despite extensive work carried out this last decade, many aspects of the molecular basis underlying this rhizobacteria-mediated ISR remain unclear. In this context, Bacillus subtilis and Pseudomonas putida strains able to protect plants via induction of ISR have been studied for several years in our laboratory. In this thesis, we first aimed at evaluating the protective effect of these strains in different pathosystems and second at identifying the associated defence mechanisms. Our results showed the capacity of P. putida BTP1 to induce ISR in the tomato/Botrytis cinerea and cucumber/Colletotrichum lagenarium pathosystems on the basis of reduction of disease symptoms and on the basis of the spatial separation of the two agents, beneficial at the root level and pathogenic at the leaf level. This resistance was clearly associated the stimulation of the oxylipin pathway in tomato. The induction of this metabolic pathway was evidenced biochemically by an increase in the lipoxygenase and lipid hydroperoxydase activities in the leaves of plants treated with Pseudomonas compared to controls. Moreover, at the molecular level, northern blot analyses enabled us to identify a new isoforme of Lox gene in tomato which is expressed differentially in plants treated with the bacterium. This gene called LoxF shows a homology of 82% with one of the five isoformes known in this plant, LoxC. LoxF is mainly expressed in tomato leaves pre-inoculated with BTP1 after infection by pathogen. The same applies for the lipoxygenase enzyme activity which increases significantly in treated plants only after infection by Botrytis. These results imply a phenomenon of "priming" closely associated with the systemic resistance induction in plants. The recognition of the bacterium at the root level primes the plant but in most cases major changes at the metabolic or genetic level are not observed. The host then reacts more strongly and more quickly to express defence mechanisms once the pathogen is perceived. In addition, by combining the use of TLC-based biotests and analytical methods such as HPLC, CPG and LC-MS analyses, we highlighted the accumulation of a molecule in BTP1-pretreated tomato leaves. This molecule seems to be of a non-polar nature but not phenolic and does not correspond to any phytoalexin known in tomato. Complementary studies must be carried out to identify its structure but its kinetic of accumulation in the plant tissues is closely associated with the stimulation of the lipoxygenase enzyme. It could thus derive from this metabolic pathway. Similar defence reactions were observed in tomato following treatment with Bacillus subtilis and through these tests, we also highlighted the role of some lipopeptides produced by this strain as elicitors responsible for the induction of the ISR phenomenon. On the other hand, although the two strains are able to induce resistance in cucumber, no clear accumulation of phytoalexins nor of significant stimulation of the lipoxygenase pathway could be associated with disease reduction in this plant. All together, our results suggest that the metabolic pathways activated during ISR vary in function of the plant and pathogen species even if the inducing micro-organism is identical.

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.

Bacteria living in soil and aquatic habitats rapidly adapt to changes in physico-chemical parameters that influence their energy status and thus their ability to proliferate and survive. One immediate survival strategy is to relocate to more metabolically optimal environments. To aid their movement through gradients (a process called taxis), many bacteria use whip like flagella organelles. Soil-dwelling Pseudomonas putida possesses a polar bundle of flagella that propel the bacterium forward in directed swimming motility. P. putida strains are generally fast growing, have a broad metabolic capacity, and are resistant to many harmful substances – qualities that make them interesting for an array of industrial and biotechnological application. This thesis identifies some of the factors that are involved in controlling the flagella driven motility of P. putida. In the first part of the thesis, I present evidence that P. putida displays energy-taxis towards metabolisable substrates and that the surface located Aer2 receptor (named after its similarities to the Escherichia coli Aer receptor) is responsible for detecting the changes in energy-status and oxygen-gradients that underlie this response. Aer2 is expressed simultaneously with the flagella needed for taxis responses and its expression is ensured during nutrient scares conditions through the global transcriptional regulators ppGpp and DksA. In addition to Aer2, P. putida possesses two more Aer-like receptors (Aer1 and Aer3) that are differentially expressed. Like Aer2, Aer1 and Aer3 co-localize to one cell pole. Although the signals to which Aer1 and Aer3 respond are unknown, analysis of Aer1 uncovered a role in motility control for a protein encoded within the same operon. This protein, called PP2258, instigated the work described in the second part of my thesis on the involvement of the second messenger c-di-GMP in regulation of P. putida motility. Genetic dissection of the catalytic activities of PP2258 revealed that it has the unusual capacity to both synthesize and degrade c-di-GMP. Coupling of the c-di-GMP signal originating from PP2258 to motility control was traced to the c-di-GMP binding properties of the protein PP4397. In the last part of the thesis, I present possible mechanisms for how these different components might interact to create a signal transduction cascade – from the surface located Aer1 receptor to PP2258 and the c-di-GMP responsive PP4397, and from there to the flagella motors – to ultimately determine flagella performance and the motility status of P. putida.