Métodos de Microbiologia de anaeróbios estritos e de Biologia Molecular foram empregados para se conhecer a diversidade de microrganismos relacionados à degradação de ascarel em reatores anaeróbios metanogênicos. A avaliação de potencial metanogênico foi realizada para a escolha da melhor condição nutricional, bem como, para a seleção de material suporte e solvente adequado a solubilização do ascarel. Nos ensaios em batelada, a produção de metano foi maior nos reatores contendo etanol (média de 0,22 - 0,46 molCH4/gSTV, 46h). Remoção de 85,6% (86,7 mg/L de PCB em Aroclor 1016 e 1260) foi obtida na condição com espuma de poliuretano, etanol (46g/L) e formiato (680 mg/L). Diferentes solventes e surfactantes, tais como, DMSO, dioxano, ácido acético, ácido fórmico, n-hexano, acetona, etano, metanol, Tween 80, SDS (10%) e Triton X-100 foram avaliados para a solubilização de ascarel. Dentre esses, metanol, Triton X-100 e ácido fórmico foram eleitos para a realização de ensaio em reatores em batelada contendo espuma de poliuretano, com o propósito de avaliar o potencial metanogênico na degradação de PCB. Os valores de produção de metano foram muito semelhantes (0,21 0,38 molCH4/mLgSTV, 45h) nas diferentes condições, no entanto, a remoção de PCB foi maior nos reatores com metanol 790 mg/L (86,6%), ácido Fórmico 600 mg/L (84,5%) e Triton X-100 1% (72,1%). Portanto, a melhor condição foi contemplada para a operação do reator anaeróbio horizontal de leito fixo (RAHLF) no tratamento do ascarel, ou seja, células imobilizadas em espuma de poliuretano, etanol e formiato (como fonte de carbono), Triton X-100 (0,1%) e metanol (como solvente). No RAHLF, a remoção média de matéria orgânica (DQO) foi de 91% para concentração afluente média de 1270 mg/L. A presença de morfologias semelhantes à Methanosarcina e bacilos fluorescentes foi confirmada em exames microscópicos. Na análise filogenética, por meio de PCR/DGGE e seqüenciamento das bandas recortadas, os grupos encontrados foram relacionados aos Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi e Chloroflexi, sendo que neste último estão incluídos representantes relacionados a degradação de PCBs. Dentre as arquéias metanogênicas verificou-se similaridade de 99% e 97% com Methanosaeta sp. e Methanolinea sp., relacionadas com a metanogenese acetoclástica e hidrogenotrófica, respectivamente / Molecular biology and microbiology methods were used to study the microbial communities related to degradation of ascarel at methanogenic conditions in an anaerobic reactor. The methanogenic potential was evaluated to choose the better nutritional condition as well as to select the better support material and the most suitable solvent to favor the solubilization of ascarel. The methane production was higher (0.22 0.46 molCH4/mLgSTV, 46h) in batch reactors containing ethanol (46 g/L) and formate (680 mg/L), the PCB elimination attaining 85.6% (86.7 mg/L de PCB as Aroclor 1016 and 1260) when Polyuretane foam was used as support material. Different solvents, namely DMSO, dioxane, n-hexane, acetic acid, formic acid, acetone, ethane, methanol, and surfactants, such as 10% SDS,, Triton X-100, were evaluated aiming o determine the better condition to solubilize ascarel. According to the results of such experiments, methanol, formic acid and Triton X-100 were selected for carrying out the batch experiments in reactors containing polyurethane foam to evaluate the methane production during the PCBs degradation. Regardless of the operation conditions the methane production rates were similar (0.21 0.38 molCH4/gSTV, 45h), however the elimination of PCB was higher in the reactors containing methanol (790 mg/L), formic acid (600 mg/L) and Triton X-100 (1%). Therefore, the better condition for treating ascarel-containing residues in a bench-scale horizontal-flow immobilized biomass (HAIB) was attained with cells immobilized in polyurethane foam when ethanol and formate were used as carbon sources, and in presence of Triton X-100 and methanol, the average elimination of organic material attaining 91% for affluent concentration of 1270 mg/L. The presence of Methanosarcina and fluorescent rods was confirmed by microscopy analysis. According to the filogenetics analysis, which was carried out by PCR/DGGE and band-sequencing, the Bacteria domain are related to the Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi and Chloroflexi, this latter being directly related to the degradation of PCB. Among the methanogenic Archea, a similiraty of 99% and 97% was observed to Methanosaeta sp. and Methanolinea sp. related to acetoclastic and hydrogenthrophic methanogenesis, respectively

A integração de diferentes processos de tratamento na degradação de efluente têxtil foi avaliada utilizando-se a técnica de precipitação, processos oxidativos avançados (POA) e tratamento biológico aeróbio. Os POAs (Ozônio/UV e reagente de Fenton/UV) foram avaliados com experimentos em bateladas de acordo com um planejamento fatorial L18 (Método Tagushi) em função da vazão de ozônio, concentração de reagente Fenton, radiação UV, pH e temperatura. No tratamento microbiológico por processo contínuo, foi utilizado um reator de leito fluidizado com células imobilizadas de zeólitas e avaliados parâmetros como pré-tratamento com POAs, vazão de aeração, taxa de diluição. Concluiu-se que com o pré-tratamento físico-químico obteve-se resultados bastante satisfatórios na redução da DQO e COT, porém gerou uma quantidade desnecessária de lodo. A integração lodo ativado/Reagente Fenton mostrou-se bastante adequada, principalmente na remoção da cor e turbidez; a variedade e freqüência dos microrganismos observados durante o monitoramento biológico foram condizentes com os sistemas de lodo ativado operando eficientemente e o emprego de zeólitas como suporte mostrou-se significativo, alcançando 63,3 % de imobilização do microrganismo no suporte. / The integration of different treatment processes in the degradation of textile wastewater was evaluated using precipitation technique, advanced oxidation processes (AOP) and aerobic biological treatment. AOPs (Ozone/UV and reagent of Fenton/UV) were evaluated in batch experiments according to a factorial planning L18 (Tagushi Method) in function of the flow of ozone, concentration of reagent Fenton, UV radiation, pH and temperature. In the microbiological treatment for continuous process, a reactor of bed fluidized was used with immobilized cells of zeolites some parameters were evaluated such as pre-treatment with AOPs, aeration flow, diluition rate. Satisfactory results were obtained with the physical-chemistry pre-treatment in the reduction of COD and TOC, however it generated an unnecessary amount of sludge. The integration activated sludge/Reagent Fenton was shown quite appropriate, mainly in the removal of the color and turbidity; the variety and frequency of the microorganisms observed during the biological monitorament were suitable with the systems of activated sludge operating efficiently and the use of zeolites as support was shown significant, reaching 63,3 % of immobilization of the microorganism in the support.

by Sze Kwok Fung Calvin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 118-130). / Acknowledgement --- p.i / Abstract --- p.ii / Content --- p.iv / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Literature review --- p.1 / Chapter 1.1.1 --- Heavy metals in the environment --- p.1 / Chapter 1.1.2 --- Heavy metal pollution in Hong Kong --- p.2 / Chapter 1.1.3 --- Electroplating industry in Hong Kong --- p.6 / Chapter 1.1.4 --- Chemistry and toxicity of copper in the environment --- p.7 / Chapter 1.1.5 --- Methods of removal of heavy metal from industrial effluent --- p.9 / Chapter A. --- Physico-chemical methods --- p.9 / Chapter B. --- Biological methods --- p.9 / Chapter 1.1.6 --- Methods of recovery of heavy metal from metal-loaded biosorbent --- p.17 / Chapter 1.1.7 --- The physico-chemical properties of magnetites --- p.18 / Chapter 1.1.8 --- Magnetites for water and wastewater treatment --- p.19 / Chapter 1.1.9 --- Immobilized cell technology --- p.24 / Chapter 1.1.10 --- Stirrer-tank bioreactor --- p.26 / Chapter 1.2 --- Objectives of the present study --- p.28 / Chapter 2. --- Materials and Methods --- p.30 / Chapter 2.1 --- Selection of copper-resistant bacteria --- p.30 / Chapter 2.2 --- Culture media and chemicals --- p.30 / Chapter 2.3 --- Growth of the bacterial cells --- p.32 / Chapter 2.4 --- Immobilization of the bacterial cells on magnetites --- p.32 / Chapter 2.4.1 --- Effects of physical and chemical factors on the immobilization of the bacterial cells on magnetites --- p.34 / Chapter 2.4.2 --- Effects of pH on the desorption of bacterial cells from magnetites --- p.34 / Chapter 2.5 --- Copper ion uptake experiments --- p.35 / Chapter 2.6 --- Effects of physico-chemical and operational factors on the Cu2+ removal capacity of the magnetite-immobilized bacterial cells --- p.35 / Chapter 2.7 --- Transmission electron micrograph and scanning electron micrograph of Pseudomonas putida 5-X loaded with Cu2+ --- p.36 / Chapter 2.7.1 --- Transmission electron micrograph --- p.36 / Chapter 2.7.2 --- Scanning electron micrograph --- p.37 / Chapter 2.8 --- Copper ion adsorption isotherm of the magnetite-immobilized cells of Pseudomonas putida 5-X --- p.37 / Chapter 2.9 --- Recovery of adsorbed Cu2+ from the magnetite-immobilized cells of Pseudomonas putida 5-X --- p.38 / Chapter 2.9.1 --- Effects of eluents on the Cu2+ removal and recovery capacity of the magnetite-immobilized cells --- p.38 / Chapter 2.9.2 --- Batch type multiple adsorption-desorption cycles of Cu2+ using ethylenediaminetetra-acetic acid (EDTA) --- p.39 / Chapter 2.10 --- Removal and recovery of Cu2+ from the electroplating effluent by a bioreactor --- p.39 / Chapter 2.10.1 --- Batch type multiple adsorption-desorption cycles using the copper solution and electroplating effluent --- p.39 / Chapter 2.10.2 --- Continuous type bioreactor to remove and recover Cu2+ from copper solution and electroplating effluent --- p.40 / Chapter 2.11 --- Statistical analysis of data --- p.43 / Chapter 3. --- Results --- p.44 / Chapter 3.1 --- Effects of physical and chemical factors on the immobilization of the bacterial cells on magnetites --- p.44 / Chapter 3.1.1 --- Effects of cells to magnetites ratio --- p.44 / Chapter 3.1.2 --- Effects of pH --- p.44 / Chapter 3.1.3 --- Effects of temperature --- p.44 / Chapter 3.2 --- Effects of pH on the desorption of bacterial cells from magnetites --- p.49 / Chapter 3.3 --- Copper ion uptake experiments --- p.49 / Chapter 3.4 --- Effects of physico-chemical and operational factors on the Cu2+ removal capacity of the magnetite-immobilized bacterial cells --- p.49 / Chapter 3.4.1 --- Effects of pH --- p.49 / Chapter 3.4.2 --- Effects of temperature --- p.53 / Chapter 3.4.3 --- Effects of retention time --- p.53 / Chapter 3.4.4 --- Effects of cations --- p.53 / Chapter 3.4.5 --- Effects of anions --- p.57 / Chapter 3.5 --- Transmission electron micrograph of Pseudomonas putida 5-X loaded with Cu2+ --- p.62 / Chapter 3.6 --- Scanning electron micrograph of Pseudomonas putida 5-X loaded with Cu2+ --- p.62 / Chapter 3.7 --- Copper ion adsorption isotherm of the magnetite-immobilized cells of Pseudomonas putida 5-X --- p.68 / Chapter 3.8 --- Recovery of adsorbed Cu2+ from the magnetite-immobilized cells of Pseudomonas putida 5-X --- p.68 / Chapter 3.8.1 --- Effects of eluents on the Cu2+ removal and recovery capacity of the magnetite-immobilized cells --- p.68 / Chapter 3.8.2 --- Batch type multiple adsorption-desorption cycles of Cu2+ using ethylenediaminetetra-acetic acid (EDTA) --- p.74 / Chapter 3.9 --- Removal and recovery of Cu2+ from the electroplating effluent by a bioreactor --- p.74 / Chapter 3.9.1 --- Batch type multiple adsorption-desorption cycles using the copper solution and electroplating effluent --- p.74 / Chapter 3.9.2 --- Continuous type bioreactor to remove and recover Cu2+ from copper solution and electroplating effluent --- p.81 / Chapter 4. --- Discussion --- p.89 / Chapter 4.1 --- Selection of copper-resistant bacteria --- p.89 / Chapter 4.2 --- Effects of physical and chemical factors on the immobilization of the bacterial cells on magnetites --- p.89 / Chapter 4.2.1 --- Effects of cells to magnetites ratio --- p.89 / Chapter 4.2.2 --- Effects of pH --- p.90 / Chapter 4.2.3 --- Effects of temperature --- p.91 / Chapter 4.2.4 --- Effects of pH on the desorption of bacterial cells from magnetites --- p.92 / Chapter 4.3 --- Copper ion uptake experiments --- p.93 / Chapter 4.4 --- Effects of physico-chemical and operational factors on the Cu2+ removal capacity of the magnetite-immobilized bacterial cells --- p.94 / Chapter 4.4.1 --- Effects of pH --- p.95 / Chapter 4.4.2 --- Effects of temperature --- p.96 / Chapter 4.4.3 --- Effects of retention time --- p.97 / Chapter 4.4.4 --- Effects of cations --- p.98 / Chapter 4.4.5 --- Effects of anions --- p.101 / Chapter 4.5 --- Transmission electron micrograph of Pseudomonas putida 5-X loaded with Cu2+ --- p.101 / Chapter 4.6 --- Scanning electron micrograph of Pseudomonas putida 5-X loaded with Cu2+ --- p.102 / Chapter 4.7 --- Copper ion adsorption isotherm of the magnetite-immobilized cells of Pseudomonas putida 5-X --- p.103 / Chapter 4.8 --- Recovery of adsorbed Cu2+ from the magnetite-immobilized cells of Pseudomonas putida 5-X --- p.104 / Chapter 4.8.1 --- Effects of eluents on the Cu2+ removal and recovery capacity of the magnetite-immobilized cells --- p.104 / Chapter 4.8.2 --- Batch type multiple adsorption-desorption cycles of Cu2+ using ethylenediaminetetra-acetic acid (EDTA) --- p.105 / Chapter 4.9 --- Removal and recovery of Cu2+ from the electroplating effluent by a bioreactor --- p.107 / Chapter 4.9.1 --- Batch type multiple adsorption-desorption cycles using the copper solution and electroplating effluent --- p.107 / Chapter 4.9.2 --- Continuous type bioreactor to remove and recover Cu2+ from copper solution and electroplating effluent --- p.108 / Chapter 5. --- Conclusion --- p.110 / Chapter 6. --- Summary --- p.112 / Chapter 7. --- References --- p.115

Réalisé en codirection avec Karen C. Waldron et Dominic Rochefort. / L’objectif principal de ce projet de recherche est d’étudier l’efficacité de la microencapsulation, technique d’immobilisation d’enzymes utilisée pour la réalisation des nouveaux biocapteurs électrochimiques. Généralement, l’analyte d’intérêt produit ou consomme des électrons, et la réponse électrochimique est mesurée, afin d’identifier ou quantifier l’analyte. Dans le développement d’un biocapteur, il est désirable de quantifier la conversion du substrat (analyte) et/ou la formation de produit de réaction enzymatique. Les similarités structurales entre le substrat et le produit de réaction dans les réactions redox demandent que la technique utilisée pour les identifier soit très sélective. Le haut pouvoir de résolution de l’électrophorèse capillaire (EC) pour des séparations rapides de produits similaires en fait une méthode de choix, spécialement quand le substrat et le produit peuvent être suivis pendant et après la réaction catalysée par l’enzyme immobilisée. Un choix judicieux du substrat, compte tenu de son comportement en EC peut fournir des informations autant sur l’activité de l’enzyme que sur l’efficacité de la microencapsulation. Pour cette raison, nous avons choisi le substrat o-phenylènediamine qui est oxydé par la laccase, pour former le produit 2,3-diaminophenazine, tout en réduisant l’oxygène en eau. Pour commencer, nous avons préparé les microcapsules et évalué l’impact de la microencapsulation sur le comportement de l’enzyme. Ensuite, nous avons développé une méthode de séparation en EC afin de quantifier la conversion de l’OPD en DAP par la laccase libre. La même méthode d’analyse a été utilisée pour caractériser la laccase immobilisée dans les microcapsules. Par la suite, afin de suivre la réaction enzymatique, un microréacteur à base d’enzyme microencapsulée a été couplé hors ligne au système d’EC. Finalement, nous avons essayé l’implémentation du système en ligne et les résultats préliminaires seront présentés. / The principal objective of this research project is to study the efficiency of microencapsulation, technique used for enzyme immobilization in order to create new types of electrochemical biosensors. Generally, the target analyte involved either produces or consumes electrons and the electrochemical response is measured to identify or quantify the analyte. In the development of a biosensor, it is desirable to quantify the conversion of substrate (analyte) and/or the formation of product of the enzymatic reaction. The structural similarity between substrate and product in redox reactions means that the technique used to determine these species must be very selective. The high resolving power of capillary electrophoresis (CE) for rapidly separating similar compounds is thus an attractive method, particularly if substrate and product can both be monitored during or following the reaction catalyzed by microencapsulated enzyme. A judicious choice of substrate with respect to its behaviour in CE separations can help provide information on enzyme activity as well as microencapsulation efficiency. To achieve this, we chose the substrate o-phenylenediamine (OPD), which is oxidized by laccase to form the product 2,3-diaminophenazine (DAP) concomitant with the reduction of molecular oxygen to water. We firstly prepared the microcapsules and evaluate the impact of microencapsulation on the behaviour of the enzyme. After that, we developed a CE based separation method to quantify the conversion of OPD to DAP by free laccase. We also used the CE method to characterize laccase immobilized in microcapsules. Subsequent, the microencapsulated laccase was packed into a microreactor format permitting its off-line coupling with CE as a means to follow the enzymatic reaction. Finally, we tried to implement the on-line system and the preliminaries results are presented.

A conversão enzimática da sacarose pela ação sucessiva da invertase e da glicose oxidase (GOD), permite obter produtos de maior valor agregado, a saber, frutose e o ácido glicônico, dois produtos de amplo uso na indústria farmacêutica, alimentícia e química. Foi estudada a aplicação da invertase imobilizada em resinas aniônicas do tipo Dowex® (um copolímero de poliestireno-divinilbenzeno) sobre a hidrólise da sacarose bem como a oxidação da glicose pela glicose oxidase solúvel ou imobilizada no mesmo suporte em separado (sistema bifásico), utilizando-se um reator de membrana acoplado à membrana de ultrafiltração (100kDa) ou de microfiltração (5µm). Posteriormente, avaliou-se o desempenho de ambas as formas de enzimas, solúveis ou imobilizadas num sistema monofásico empregando o mesmo reator. A bioconversão executada em sistema bifásico permitiu a obtenção de xarope de frutose da ordem de 70% através da separação de glicose e frutose utilizando-se a resina catiônica 50W:8-100. O rendimento de 96,6% e 67,4% para as formas solúveis e imobilizadas respectivamente foram obtidas em sistema monofásico. O não desprendimento das enzimas dos suportes viabilizou o uso da membrana de microfiltração, trazendo vantagens à operação de biorreator com membrana. / The enzymatic conversion of sucrose through a successive action of invertase and glucose oxidase (GOO) allows the obtainment of products with higher commercial value, fructose and gluconic acid, which are widely used in pharmaceutical, food and chemical industries. Invertase and GOO immobilized on Dowex® anionic resin (a polystyrene divinylbenzene copolymer) as well as soluble GOD were used in a membrane bioreactor (MS) for sucrose hydrolysis and glucose oxidation. The MB was coupled with a UF-membrane (100kDa) or a MF-membrane (5µm). The bioconversion was conducted in two steps (biphasic system) as well as in one step (monophasic system). The bioconversion operated in a biphasic system permitted obtaining a fructose syrup with a concentration of about 70% through a separation of glucose and fructose using a cationic resin, 50W:8-100. As for the monophasic system, the yield of 96.6% and 67.4% for soluble and immobilized forms were attained respectively. No leakage of the enzymes from the support allowed the use of a microfiltration membrane, adding advantages to the membrane bioreactor operation.

Métodos de Microbiologia de anaeróbios estritos e de Biologia Molecular foram empregados para se conhecer a diversidade de microrganismos relacionados à degradação de ascarel em reatores anaeróbios metanogênicos. A avaliação de potencial metanogênico foi realizada para a escolha da melhor condição nutricional, bem como, para a seleção de material suporte e solvente adequado a solubilização do ascarel. Nos ensaios em batelada, a produção de metano foi maior nos reatores contendo etanol (média de 0,22 - 0,46 molCH4/gSTV, 46h). Remoção de 85,6% (86,7 mg/L de PCB em Aroclor 1016 e 1260) foi obtida na condição com espuma de poliuretano, etanol (46g/L) e formiato (680 mg/L). Diferentes solventes e surfactantes, tais como, DMSO, dioxano, ácido acético, ácido fórmico, n-hexano, acetona, etano, metanol, Tween 80, SDS (10%) e Triton X-100 foram avaliados para a solubilização de ascarel. Dentre esses, metanol, Triton X-100 e ácido fórmico foram eleitos para a realização de ensaio em reatores em batelada contendo espuma de poliuretano, com o propósito de avaliar o potencial metanogênico na degradação de PCB. Os valores de produção de metano foram muito semelhantes (0,21 0,38 molCH4/mLgSTV, 45h) nas diferentes condições, no entanto, a remoção de PCB foi maior nos reatores com metanol 790 mg/L (86,6%), ácido Fórmico 600 mg/L (84,5%) e Triton X-100 1% (72,1%). Portanto, a melhor condição foi contemplada para a operação do reator anaeróbio horizontal de leito fixo (RAHLF) no tratamento do ascarel, ou seja, células imobilizadas em espuma de poliuretano, etanol e formiato (como fonte de carbono), Triton X-100 (0,1%) e metanol (como solvente). No RAHLF, a remoção média de matéria orgânica (DQO) foi de 91% para concentração afluente média de 1270 mg/L. A presença de morfologias semelhantes à Methanosarcina e bacilos fluorescentes foi confirmada em exames microscópicos. Na análise filogenética, por meio de PCR/DGGE e seqüenciamento das bandas recortadas, os grupos encontrados foram relacionados aos Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi e Chloroflexi, sendo que neste último estão incluídos representantes relacionados a degradação de PCBs. Dentre as arquéias metanogênicas verificou-se similaridade de 99% e 97% com Methanosaeta sp. e Methanolinea sp., relacionadas com a metanogenese acetoclástica e hidrogenotrófica, respectivamente / Molecular biology and microbiology methods were used to study the microbial communities related to degradation of ascarel at methanogenic conditions in an anaerobic reactor. The methanogenic potential was evaluated to choose the better nutritional condition as well as to select the better support material and the most suitable solvent to favor the solubilization of ascarel. The methane production was higher (0.22 0.46 molCH4/mLgSTV, 46h) in batch reactors containing ethanol (46 g/L) and formate (680 mg/L), the PCB elimination attaining 85.6% (86.7 mg/L de PCB as Aroclor 1016 and 1260) when Polyuretane foam was used as support material. Different solvents, namely DMSO, dioxane, n-hexane, acetic acid, formic acid, acetone, ethane, methanol, and surfactants, such as 10% SDS,, Triton X-100, were evaluated aiming o determine the better condition to solubilize ascarel. According to the results of such experiments, methanol, formic acid and Triton X-100 were selected for carrying out the batch experiments in reactors containing polyurethane foam to evaluate the methane production during the PCBs degradation. Regardless of the operation conditions the methane production rates were similar (0.21 0.38 molCH4/gSTV, 45h), however the elimination of PCB was higher in the reactors containing methanol (790 mg/L), formic acid (600 mg/L) and Triton X-100 (1%). Therefore, the better condition for treating ascarel-containing residues in a bench-scale horizontal-flow immobilized biomass (HAIB) was attained with cells immobilized in polyurethane foam when ethanol and formate were used as carbon sources, and in presence of Triton X-100 and methanol, the average elimination of organic material attaining 91% for affluent concentration of 1270 mg/L. The presence of Methanosarcina and fluorescent rods was confirmed by microscopy analysis. According to the filogenetics analysis, which was carried out by PCR/DGGE and band-sequencing, the Bacteria domain are related to the Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi and Chloroflexi, this latter being directly related to the degradation of PCB. Among the methanogenic Archea, a similiraty of 99% and 97% was observed to Methanosaeta sp. and Methanolinea sp. related to acetoclastic and hydrogenthrophic methanogenesis, respectively

<p>The main aim of this thesis was to characterize membrane-solute interactions using artificial membranes in immobilized liposome chromatography or capillary electrophoresis. The partitioning of a solute into a cell membrane is an essential step in diffusion across the membrane. It is a valid parameter in drug research and can be linked to the permeability as well as the absorption of drugs. Immobilized liposome chromatography was also used to study partitioning of lignin precursor models. Lignin precursors are synthesized within plant cells and need to pass the membrane to be incorporated into lignin in the cell wall.</p><p>In immobilized liposome chromatography, liposomes or lipid bilayer disks were immobilized in gel beads and the partitioning of solutes was determined. Capillary electrophoresis using disks as a pseudostationary phase was introduced as a new approach in drug partitioning studies. In addition, octanol/water partitioning was used to determine the hydrophobicity of the lignin precursor models.</p><p>Electrostatic interactions occurred between bilayers and charged drugs, whereas neutral drugs were less affected. However, neutral lignin precursor models exhibited polar interactions. Moreover, upon changing the buffer ionic strength or the buffer ions, the interactions between charged drugs and neutral liposomes were affected. Hydrophobic interactions were also revealed by including a fatty acid or a neutral detergent into the bilayer or by using a buffer with a high salt concentration. The bilayer manipulation had only a moderate effect on drug partitioning, but the high salt concentration had a large impact on partitioning of lignin precursor models.</p><p>Upon comparing the partitioning into liposomes and disks, the latter showed a more pronounced partitioning due to the larger fraction of lipids readily available for interaction. Finally, bilayer disk capillary electrophoresis was successfully introduced for partitioning studies of charged drugs. This application will be evaluated further as an analytical partitioning method and separation technique.</p>

Se ha desarrollado un nuevo método de inmovilización de tirosinasa de champiñón, basado en la adsorción de esta enzima a un éster cinámico de un carbohidrato fotoentrecruzado, con muy buenos resultados. Se han obtenido inmovilizados de tirosinasa directamente a partir de extractos de champiñón. Se han estudiado los parámetros que afectan a la inmovilización (pH, tiempo y concentración de enzima), las características de tirosinasa inmovilizada (cantidad de enzima inmovilizada, actividad enzimática, pH y temperatura óptimos de reacción, estabilidad térmica, operacional y al almacenado), las propiedades cinéticas (constante de Michaelis, velocidad máxima y constante catalítica) de tirosinasa inmovilizada sobre distintos soportes al actuar sobre diversos sustratos y la estereoespecificidad de tirosinasa inmovilizada sobre un soporte quiral y otro no quiral respecto a los isómeros L y D y racémicos. Los conocimientos adquiridos se han aplicado con éxito a la producción de diversos o-difenoles como L-dopa, a partir de sus correspondientes monofenoles. / A new mushroom tyrosinase immobilization method consisting of tyrosinase adsorption on a photocrosslinked cinnamic carbohydrate ester has been developed with very good results. Immobilized tyrosinase was obtained directly from a mushroom extract. We studied the parameters affecting the immobilization process (pH, time and enzyme concentration), the properties of immobilized tyrosinase (quantity of immobilized enzyme, enzymatic activity, optimum pH and reaction temperature; thermal, operational and storage stability), kinetic properties (Michaelis constant, maximum steady-state rate and catalytic constant) of tyrosinase immobilized on several supports and acting over several substrates, and tyrosinase stereospecificity immobilized on a chiral and a nonchiral support acting on L and D isomers and racemics. The knowledge acquired was used to successfully produce several o-diphenols, such as L-dopa, from the corresponding monophenols.

The main aim of this thesis was to characterize membrane-solute interactions using artificial membranes in immobilized liposome chromatography or capillary electrophoresis. The partitioning of a solute into a cell membrane is an essential step in diffusion across the membrane. It is a valid parameter in drug research and can be linked to the permeability as well as the absorption of drugs. Immobilized liposome chromatography was also used to study partitioning of lignin precursor models. Lignin precursors are synthesized within plant cells and need to pass the membrane to be incorporated into lignin in the cell wall. In immobilized liposome chromatography, liposomes or lipid bilayer disks were immobilized in gel beads and the partitioning of solutes was determined. Capillary electrophoresis using disks as a pseudostationary phase was introduced as a new approach in drug partitioning studies. In addition, octanol/water partitioning was used to determine the hydrophobicity of the lignin precursor models. Electrostatic interactions occurred between bilayers and charged drugs, whereas neutral drugs were less affected. However, neutral lignin precursor models exhibited polar interactions. Moreover, upon changing the buffer ionic strength or the buffer ions, the interactions between charged drugs and neutral liposomes were affected. Hydrophobic interactions were also revealed by including a fatty acid or a neutral detergent into the bilayer or by using a buffer with a high salt concentration. The bilayer manipulation had only a moderate effect on drug partitioning, but the high salt concentration had a large impact on partitioning of lignin precursor models. Upon comparing the partitioning into liposomes and disks, the latter showed a more pronounced partitioning due to the larger fraction of lipids readily available for interaction. Finally, bilayer disk capillary electrophoresis was successfully introduced for partitioning studies of charged drugs. This application will be evaluated further as an analytical partitioning method and separation technique.

L’objectif principal de ce projet de recherche est d’étudier l’efficacité de la microencapsulation, technique d’immobilisation d’enzymes utilisée pour la réalisation des nouveaux biocapteurs électrochimiques. Généralement, l’analyte d’intérêt produit ou consomme des électrons, et la réponse électrochimique est mesurée, afin d’identifier ou quantifier l’analyte. Dans le développement d’un biocapteur, il est désirable de quantifier la conversion du substrat (analyte) et/ou la formation de produit de réaction enzymatique. Les similarités structurales entre le substrat et le produit de réaction dans les réactions redox demandent que la technique utilisée pour les identifier soit très sélective. Le haut pouvoir de résolution de l’électrophorèse capillaire (EC) pour des séparations rapides de produits similaires en fait une méthode de choix, spécialement quand le substrat et le produit peuvent être suivis pendant et après la réaction catalysée par l’enzyme immobilisée. Un choix judicieux du substrat, compte tenu de son comportement en EC peut fournir des informations autant sur l’activité de l’enzyme que sur l’efficacité de la microencapsulation. Pour cette raison, nous avons choisi le substrat o-phenylènediamine qui est oxydé par la laccase, pour former le produit 2,3-diaminophenazine, tout en réduisant l’oxygène en eau. Pour commencer, nous avons préparé les microcapsules et évalué l’impact de la microencapsulation sur le comportement de l’enzyme. Ensuite, nous avons développé une méthode de séparation en EC afin de quantifier la conversion de l’OPD en DAP par la laccase libre. La même méthode d’analyse a été utilisée pour caractériser la laccase immobilisée dans les microcapsules. Par la suite, afin de suivre la réaction enzymatique, un microréacteur à base d’enzyme microencapsulée a été couplé hors ligne au système d’EC. Finalement, nous avons essayé l’implémentation du système en ligne et les résultats préliminaires seront présentés. / The principal objective of this research project is to study the efficiency of microencapsulation, technique used for enzyme immobilization in order to create new types of electrochemical biosensors. Generally, the target analyte involved either produces or consumes electrons and the electrochemical response is measured to identify or quantify the analyte. In the development of a biosensor, it is desirable to quantify the conversion of substrate (analyte) and/or the formation of product of the enzymatic reaction. The structural similarity between substrate and product in redox reactions means that the technique used to determine these species must be very selective. The high resolving power of capillary electrophoresis (CE) for rapidly separating similar compounds is thus an attractive method, particularly if substrate and product can both be monitored during or following the reaction catalyzed by microencapsulated enzyme. A judicious choice of substrate with respect to its behaviour in CE separations can help provide information on enzyme activity as well as microencapsulation efficiency. To achieve this, we chose the substrate o-phenylenediamine (OPD), which is oxidized by laccase to form the product 2,3-diaminophenazine (DAP) concomitant with the reduction of molecular oxygen to water. We firstly prepared the microcapsules and evaluate the impact of microencapsulation on the behaviour of the enzyme. After that, we developed a CE based separation method to quantify the conversion of OPD to DAP by free laccase. We also used the CE method to characterize laccase immobilized in microcapsules. Subsequent, the microencapsulated laccase was packed into a microreactor format permitting its off-line coupling with CE as a means to follow the enzymatic reaction. Finally, we tried to implement the on-line system and the preliminaries results are presented. / Réalisé en codirection avec Karen C. Waldron et Dominic Rochefort.