Methylosinus trichosporium OB3b is a wild type, obligate methanotroph that grows only on one-carbon compounds and, in the absence of copper, produces high levels of soluble methane monooxygenase (sMMO) to metabolize methane to methanol. SMMO has gained a great deal of attention in the bioremediation and chemical industries because of its low substrate specificity and its ability to oxidize chlorinated hydrocarbons. Much literature exists on cultivating this organism on methane, however no one has achieved dry cell weight densities exceeding 18 g/L. Biomass growth is limited due to mass transfer of methane to cells. This study investigated the growth of M. trichosporium on the water soluble carbon source, methanol while retaining sMMO activity. Methanol was found to completely inhibit growth at 40 g/L. For online methanol measurements during fed-batch cultivation, an in situ probe was constructed from autoclavable materials and equipped with a Figaro TGS822 vapor sensor. The probe was designed to prevent the sensor coming in contact with water aerosols which affect its performance. The probe was an essential component of a feedback methanol control system. The cumulative CO2 production (CCP) strategy was used to feed methanol in fed-batch experiments. In an initial bioreactor study, growth nutrients were fed in excess. The yields of biomass to nutrients were determined and the growth medium modified accordingly. A biomass density of 19 g/L (growth rate of 0.013-0.065 h-1) was achieved with sMMO activity of 300 to 500 [µmol naphthol][g of biomass]-1[h]-1. The subsequent bioreactor study involved feeding of nutrients based on their yields in relation to methanol, a biomass density of 62 g/L (growth rate of 0.034- 0.08 h-1) was achieved. The inoculum cultures utilized in the bioreactor studies were maintained on Noble agar plates containing nitrogen minimal salts medium and methane. After 6 months of subsequent plate transfers, M. trichosporium lost the ability to produce high levels of sMMO. The enzyme activity in methanol grown cells was recovered by subculturing in liquid NMS medium with methane as the sole carbon source, the activity increased from 8 to 600 [µmol naphthol][g of biomass]-1[h]-1. It is recommended that further studies be carried out on stimulating sMMO activity during cultivation on methanol. / Thesis (Master, Chemical Engineering) -- Queen's University, 2008-08-21 15:14:42.475

Ce travail de thèse a pour objectif le développement et l'optimisation d'une nouvelle configuration de bioréacteur à membranes (BRM) pour l'hydroxylation efficace et sûre du méthane par la bactérie Methylosinus trichosporium OB3b. Ce BRM couple un bioréacteur à deux contacteurs membranaires gaz/liquide macroporeux qui alimentent en continu chaque substrat gazeux (méthane et air) sans générer de bulle dans la suspension bactérienne, évitant ainsi la formation d'un mélange gazeux méthane/air explosif. Dans un premier temps, la faisabilité et la reproductibilité de ce nouveau bioprocédé de conversion du méthane en méthanol ont été démontrées. D'une part, la productivité moyenne obtenue dans ce BRM (75 ± 25 mg méthanol.(g cellules sèches)-1.h-1) est près de deux fois meilleure que celle obtenue dans un réacteur fermé conduit dans les mêmes conditions que le BRM, traduisant un transfert de masse gaz-liquide accru dans le BRM. D'autre part, la productivité obtenue dans ce nouveau BRM est similaire aux meilleures productivités reportées dans la littérature pour des réacteurs alimentés avec un distributeur de gaz à bulles et près de 35 fois meilleure que celle reportée pour le seul autre BRM (à membranes denses) présent dans la littérature. Dans un second temps, le suivi cinétique de l'activité intrinsèque hydroxylante du biocatalyseur a permis de vérifier que l'arrêt de production du méthanol qui est observé après 14 h de réaction correspond à une perte quasi-totale de l'activité du biocatalyseur. Plusieurs essais ont été réalisés pour appréhender les facteurs pouvant avoir une influence sur l'activité hydroxylante de la bactérie, en vue de trouver le moyen d'augmenter le temps de production. Ces essais ont mis en évidence que l'arrêt de production est dû à la fin de vie du biocatalyseur. En parallèle à ces études, dans l'objectif de régénérer le cofacteur NAD nécessaire à la réaction d'hydroxylation de manière économique et in situ, des essais ont été conduits avec un système bio-électrochimique innovant (biocathode) visant à remplacer les électrons d'un donneur d'électrons (formiate) par ceux d'un métal faiblement polarisé. Ces essais ont montré l'incapacité de ces bactéries à utiliser les électrons d'une électrode dans les conditions de la réaction. / This work aimed to develop and optimize a new configuration of membrane bioreactor (MBR) for an efficient and safe methane hydroxylation by the Methylosinus trichosporium OB3b bacterium. This BRM couples a bioreactor with two gas/liquid macroporous membrane contactors supplying continuously each gaseous substrate (methane and air) without generating any bubble in the bacterial suspension, avoiding thus the formation of an explosive methane/air gas mixture. In the first step, the feasibility and the reproducibility of this new bioprocess for the conversion of methane into methanol was demonstrated. In the one hand, the average productivity achieved in the MBR (75 ± 25 mg methanol.(g dry cell)-1.h-1) is twice higher than that obtained in a batch reactor operated with the same conditions, highlighting an increased mass transfer in the MBR. In the other hand, productivity obtained in this MBR is similar to the best productivities reported in the literature for reactors (either fed-batch or continuous) using gas bubble spargers and about 35-times better than the one reported for the only other MBR (with dense membranes) present in the literature. Secondly, a kinetic monitoring of the intrinsic hydroxylating activity of the biocatalyst confirmed that the methanol production stop observed after 14 hours of reaction matched a quasi-total loss of the biocatalyst activity. Several trials were conducted to understand the factors which may influence the bacterial hydroxylating activity, in order to find a way to increase the production time. These trials put in evidence that the production stop is caused by the end of life of the biocatalyst. In parallel to these studies, aiming to regenerate the NAD cofactor required for the reaction by a cheap and in situ way, several tests were conducted with an innovative bio-electrochemical device (biocathode) to replace the electrons from an electron donor (formate) by those from a weakly polarized metal. These trials showed the inability of this bacterium strain to use electrons from an electrode in the conditions of the reaction.