Auswahl der wissenschaftlichen Literatur zum Thema „Pseudomonas putida mt2“

Biofilm-forming bacteria can protect mild (unalloyed) steel from corrosion. Mild steel coupons incubated with Rhodoccocus sp. strain C125 and Pseudomonas putida mt2 in an aerobic phosphate-buffered medium containing benzoate as carbon and energy source, underwent a surface reaction leading to the formation of a corrosion-inhibiting vivianite layer [Fe3(PO4)2]. Electrochemical potential (E) measurements allowed us to follow the buildup of the vivianite cover. The presence of sufficient metabolically active bacteria at the steel surface resulted in an E decrease to -510 mV, the potential of free iron, and a continuous release of ferrous iron. Part of the dissolved iron precipitated as vivianite in a compact layer of two to three microns in thickness. This layer prevented corrosion of mild steel for over two weeks, even in a highly corrosive medium. A concentration of 20 mM phosphate in the medium was found to be a prerequisite for the formation of the vivianite layer.

ABSTRACT Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by Pseudomonas putida mt2 and the transconjugant rhizosphere bacterium P. putidaWCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three meta-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer o-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.

ABSTRACT Mild (unalloyed) steel electrodes were incubated in phosphate-buffered cultures of aerobic, biofilm-formingRhodococcus sp. strain C125 and Pseudomonas putida mt2. A resulting surface reaction leading to the formation of a corrosion-inhibiting vivianite layer was accompanied by a characteristic electrochemical potential (E) curve. First, E increased slightly due to the interaction of phosphate with the iron oxides covering the steel surface. Subsequently, E decreased rapidly and after 1 day reached −510 mV, the potential of free iron, indicating the removal of the iron oxides. At this point, only scattered patches of bacteria covered the surface. A surface reaction, in which iron was released and vivianite precipitated, started. E remained at −510 mV for about 2 days, during which the vivianite layer grew steadily. Thereafter, E increased markedly to the initial value, and the release of iron stopped. Changes in E and formation of vivianite were results of bacterial activity, with oxygen consumption by the biofilm being the driving force. These findings indicate that biofilms may protect steel surfaces and might be used as an alternative method to combat corrosion.

L’impact de l’Homme sur l’environnement et les écosystèmes constitue une problématique de premier plan de ce 21ème siècle. La pollution de l’air, des sols et des eaux endommage des écosystèmes déjà fortement affaiblis par les différents fléaux de l’activité humaine (réchauffement climatique, surexploitation, destruction des habitats naturels menant à la sixième extinction de masse, etc…). La pollution des eaux est particulièrement problématique à cause de la large diffusion possible des polluants et à l’importance de l’eau pour toute forme de vie. Cette pollution impacte ainsi la santé des êtres humains directement et indirectement à travers les réseaux trophiques. Ces polluants peuvent être naturellement dégradés – dégradation physique, chimique ou biologique (à travers la bioremédiation) – cependant la persistance de certains de ces contaminants nécessite une intervention humaine en vue de la restauration des écosystèmes. La bioaugmentation est une stratégie prometteuse consistant en l’introduction d’organismes exogènes (provenant d’un autre milieu) choisis pour leur capacité à dégrader spécifiquement les polluants persistants. Ces organismes exogènes peuvent être des plantes, des algues, des champignons ou encore des bactéries. Cette introduction d’organismes exogènes soulève une double question : celle d’un possible déséquilibre de l’écosystème, déjà perturbé, mais aussi de la sensibilité des organismes introduits aux conditions du milieu qui peuvent affecter leur viabilité et efficacité de dépollution. Dans ce contexte, l’utilisation de cellules encapsulées – plutôt qu’en suspension – a été rapportée dans la littérature1. Cette approche d’emprisonnement des microorganismes dans des matrices adaptées permet de limiter leur diffusion et leurs impacts potentiellement néfastes sur l’écosystème, tout en leur conférant une protection contre les conditions physico-chimiques du milieu. L’efficacité de la dépollution d’une telle approche réside dans la capacité des microorganismes à dépolluer les molécules ciblées mais aussi dans le choix de la matrice d’encapsulation. Les matériaux utilisés ainsi que la structure de la matrice jouent un rôle crucial pour permettre une dépollution par les microorganismes. L’utilisation de biopolymères comme matrice d’encapsulation a été largement étudiée dans la littérature2,3 en raison de leur cyto-comptabilité. La structure, quant à elle, est préférée macroporeuse en vue de la diffusion des polluants à travers le substrat. Dans cette thèse nous présenterons les résultats et réflexions sur l’encapsulation de bactéries dans des matrices d’alginate par la technique de congélation directionnelle, pour la biodégradation de colorants azoïques. La congélation directionnelle est une technique facile à mettre en œuvre, peu chère et applicable à une large gamme de biopolymères. Elle repose sur le contrôle de la croissance de cristaux de glace dont le négatif permet l’obtention de pores fortement anisotropes ainsi que l’encapsulation de cellules au sein des parois des matrices4. Nous avons alors étudié le contrôle de la structure macroporeuse et de la viabilité à travers différents leviers tels que la vitesse de croissance des cristaux (appelée vitesse du front de glace) et la concentration des microorganismes. La compréhension de l’évolution du microenvironnement autour des bactéries lors de leur congélation nous a aussi particulièrement intéressé. Malgré le fort potentiel de cette technique pour former des matrices cellularisées, anisotropes et macroporeuses, les nombreuses étapes de stabilisation nécessaires pour une utilisation en milieu aqueux diminuent drastiquement la viabilité des bactéries encapsulées. Afin de maximiser la viabilité de P. putida dans ces matrices d'alginate – ce qui permettrait une biodégradation plus efficace – nous avons conçu une nouvelle approche de traitement basée sur la dépression cryoscopique de la glace induite par des agents de réticulation ioniques. Cette approche [...]
Bioremediation is expected to play a central role in the future of water treatment. In this context, the use of encapsulated cells – rather than in suspension – to clean-up effluents has been widely reported1. To cope with the need to fabricate bacteria-laden materials, numerous strategies emerged: from bead bioreactors to nanoencapsulation2. Here we present our recent results in the entrapment of bacteria inside biopolymer matrix using the freeze casting process – for the bioremediation of Polycyclic Aromatic Hydrocarbons. This process is easy to implement, cost effective and applicable to a wide range of water-soluble encapsulation matrices3. It relies on ice crystal growth to create an anisotropic porous matrix and permit the encapsulation of various cells in the matrix’s biopolymer walls4,5. Despite the technique’s attractiveness to build cell laden materials, the numerous stressing steps of the process led to poor bacteria survival. To maximise viability of P. putida in alginate matrices – which should translate to higher biodegradation efficiency – we designed a new processing approach based on the cryoscopic depression of ice induced by the biopolymers’ cross-linking ions, Ca2+. This approach that we coined topotactic cross-linking – and are currently extending to other biopolymer matrices6,7 – enables a one step process, eliminating the need for a drying step. The resulting macroporous materials host metabolically active bacteria and the aligned channels promote efficient liquid transport, suggesting interesting performance in flow biodegradation. We will discuss the implication of each processing steps (freezing, drying, topotactic crosslinking) on the local environment surrounding bacterial cells and their impact on viability. Finally, using a dye degrading strain (DSM: 3931), we show that freeze-casting coupled with topotactic cross-linking leads to a functional bioremediation system

An exceptionally important stress response of Pseudomonas putida strains to toxic chemicals is the induction of efflux pumps that extrude solvents, as well as other toxicants, into the surrounding medium. However, the bacterial tolerance mechanisms are still not fully understood, thus in this thesis metabolomic approaches were used to detect and identify metabolites involved in P. putida DOT-T1E tolerance to abiotic stresses, in particular focussing on the role of efflux pumps. To elucidate any metabolome alterations several strains of P. putida, including the wild type DOT-T1E, and the efflux pump knockouts DOT-T1E-PS28 and DOT-T1E-18, were challenged with different levels of propranolol. Fourier-transform infrared (FT-IR) spectroscopy, which provided a rapid, high-throughput metabolic fingerprint of P. putida strains, was used to investigate any phenotypic changes resulting from exposure to propranolol. FT-IR data illustrated phenotypic changes associated with the presence of propranolol within the cell that could be assigned to the bacterial protein components. To complement this phenotypic fingerprinting approach metabolic profiling on the same samples was performed using gas chromatography mass spectrometry (GC-MS) to identify metabolites of interest during growth of bacteria following this toxic perturbation with propranolol. GC-MS revealed significant changes in ornithine levels which can be directly linked to bacterial tolerance mechanisms, and alterations in the levels of several other metabolites which were also modified in response to propranolol exposure. Moreover, the effect of the organic solvent toluene was also investigated using the same approach. Examination of FT-IR data indicated that protein and fatty acids were the most affected components of P. putida strains due to the presence of toluene within the cell. Moreover, application of GC-MS allowed for the identification and quantification of several metabolites which were differentially produced or consumed in the presence of toluene. To investigate the role of efflux pumps in P. putida DOT-T1E, several analytical techniques were employed including Raman spectroscopy, gas and liquid chromatography to identify and quantify the level of propranolol or toluene in P. putida cells. These analyses showed that propranolol and toluene accumulated in the mutant P. putida DOT-T1E-18 (lacking the TtgABC pump) at higher levels in comparison with the levels found in the wild-type DOT-T1E and the mutant DOT-T1E-PS28 (lacking the TtgGHI pump), indicating the key role of efflux pumps in solvent tolerance. Furthermore, the effect of Mg2+ and Ca2+ on the stabilisation of the toluene tolerance of P. putida DOT-T1E strains was examined in order to elucidate whether divalent cations interact with efflux pumps or other resistant mechanisms to improve solvent tolerance. FT-IR analysis suggested that the influence of divalent cations on the stabilisation of the toluene tolerance could be due to the contribution of metal ions towards other tolerance mechanisms such as lipopolysaccharide (LPS) instead of enhancing the activity of efflux pumps. In conclusion, this thesis presents evidence that phenotypic fingerprinting and metabolic profiling approaches in combination with chemometric methods can generate valuable information on phenotypic responses occurring within microbial cultures subjected to abiotic stress.

碩士
元智大學
化學工程與材料科學學系
97
Azo dyes which are considered to be the most recalcitrant and persistent among all groups of dye were biodegraded by various kinds of bacteria. In this study, the decolorization of Methyl Orange was determined under different conditions by non-immobilized and immobilized Pseudomonas putida mt2. For non-immobilized cell system, it was further confirmed that decolorization was much more favorable under anoxic condition since no dye degradation was obtained with 200 rpm shaking, whereas 100% dye was removed in static condition after 3 incubation days. Temperature and pH dependences were evaluated based on the specific decolorization rate and the equilibrium conversion values to investigate the highest capability of Pseudomonas putida mt2 for decolorization in static condition. The optimal temperature range is quite narrow (33oC to 35oC) and the decolorization seems not to be suitable in acidic medium since the optimal pH for methyl orange decolorization occurred at pH 7.0 and significantly decreased at pH 5.0. The Michalis Menten equation was utilized to establish the dependence of the specific decolorization rate on the concentration of dye. The kinetic parameters of Vmax and Km were predicted up to 7.5 mg g-1 h-1 and 283 mg L-1, respectively. External diffusion coefficient kL was evaluated 7.5 x 10-6 cm s-1. The Ca-alginate immobilized cells can not improve aerobic degradation when the dye color decreased insignificantly but completely disappeared under anaerobic condition. The optimal range of pH and temperature were obtained at 7 to 9 and 35oC to 37oC, respectively. The effects of initial biomass and initial dye concentration were also determined to confirm the predominance of immobilized cells on dye treatment when comparing with free suspended cells. Kinetics parameters were also determined to give the values of Vmax of 6.3 mg g-1 h-1 and Km of 257 mg L-1. The internal diffusion coefficient inside the beads was also investigated to give the value of 6.2 x 10-5 cm2 s-1. Paint-PVA biofilm was created to immobilize cells instead of Ca-alginate beads when the integrity of beads was not fully maintained. Since the degradation took very long duration, the procedure of biofilm preparation was tried to recover cell’s activity. Better expression was obtained with twice adaptation. Paint-PVA immobilized cells showed the best performance on biodegradation at 35oC to 37oC, the identical optimal temperature rang with Ca-alginate immobilized cells, but the favorable range of pH is quite large from 5 to 9. In this system, the kinetics was also done to obtain the values of Vmax of 2.66 mg g-1 h-1 and Km of 161 mg L-1. The slow biodegradation rate was explained by small value of diffusion coefficient of 2.12 x 10-7 cm s-1.