No description available.

Petroleum spills causes contamination of drinking water with carcinogenic aromatic compounds including benzene and cresol. Current knowledge of anaerobic benzene and cresol degradation is extremely limited and it makes bioremediation challenging. Geobacter daltonii strain FRC-32 is a metal-reducing bacterium isolated from radionuclides and hydrocarbon-contaminated subsurface sediments. It is notable for its anaerobic oxidation of benzene and its unique ability to metabolize p-, m-, or o-cresol as a sole carbon source. Location of genes involved in aromatic compound degradation and genes unique to G. daltonii were elucidated by genomic analysis using BLAST. Genes predicted to play a role in aromatic degradation cluster into an aromatic island near the start of the genome. Of particular note, G. daltonii has two copies of the bss genes, which are responsible for the first step in anaerobic toluene oxidation. This bacterium is unique among the family Geobacteraceae and other toluene degraders in this aspect. The a subunits have 74% identity to one another. The remaining genes in each operon are not identical. BssA was upregulated when G. daltonii was grown on benzene and toluene while the grlA was upregulated during growth on m-cresol. Toluene was accumulated during degradation of benzene by cell lysate. Cells grown with benzene and toluene exhibited a similar protein profile compared to cells grown with benzoate. These results indicate that benzene is converted to toluene and further degraded via the toluene pathway. Both the bss and grl operons were predicted to have sigma54-dependent promoters. This was confirmed using 5' RACE and sequence analysis. E. coli transformed with the bss operon were able to grow in the presence of toluene but lost this capability when sigma 54 was knocked out. Growth was restored with complementation of sigma 54. The sigma 54-dependent signaling system bamVW was upregulated in the presence of all aromatic compounds tested. These results suggest that the bss operon is regulated via sigma 54-dependent mechanisms. This study significantly contributes to anaerobic aromatic gene regulation which is crucial in effective oil spill bioremediation.

Universiẗat, Diss., 2003--Konstanz.

An air cathode, membrane-less microbial fuel cell (MFC) containing a co-culture of Geobacter sulfurreducens and Escherichia coli was constructed and compared to pure culture MFCs of both organisms. The E. coli containing MFCs were unsparged and relied on E. coli for oxygen removal. The pure G. sulfurreducens MFC had a power output of 128 mW/m2, compared to 63 mW/m2 for the co-culture at an early stage and 56 mW/m2 for the late stage co-culture. The limiting current density is 404 mA/m2 for the pure G. sulfurreducens culture, 184 mA/m2 for the early co-culture, and 282 mA/m2 for the late co-culture, despite an increase in internal resistance between the early and late co-culture cells. Analysis of metabolites has shown that succinate production is likely to have negatively affected current production by G. sulfurreducens, and the removal of succinate is responsible for the increased current density in the late co-culture cell.

An air cathode, membrane-less microbial fuel cell (MFC) containing a co-culture of Geobacter sulfurreducens and Escherichia coli was constructed and compared to pure culture MFCs of both organisms. The E. coli containing MFCs were unsparged and relied on E. coli for oxygen removal. The pure G. sulfurreducens MFC had a power output of 128 mW/m2, compared to 63 mW/m2 for the co-culture at an early stage and 56 mW/m2 for the late stage co-culture. The limiting current density is 404 mA/m2 for the pure G. sulfurreducens culture, 184 mA/m2 for the early co-culture, and 282 mA/m2 for the late co-culture, despite an increase in internal resistance between the early and late co-culture cells. Analysis of metabolites has shown that succinate production is likely to have negatively affected current production by G. sulfurreducens, and the removal of succinate is responsible for the increased current density in the late co-culture cell.

The findings of this study demonstrate that Dehalococcoides species are intimately involved in complete reductive detoxification of chlorinated ethenes and are widely distributed in anoxic sediments and aquifers, including non-contaminated (pristine) environments. Careful examination of enrichment culture dechlorination kinetics, 16S rRNA gene based analyses, and reductive dehalogenase gene targeted PCR approaches revealed that complete reductive dechlorination is carried out by multiple dechlorinators. Two new dechlorinating species were isolated from contaminated and non-contaminated site materials. The first new isolate, designated strain SZ, was isolated from PCE-to-ethene dechlorinating microcosms established with creek sediment. 16S rRNA gene sequence of the strain SZ indicates that the new isolate is affiliated with the genus Geobacter most closely related to G. thiogenes. Strain SZ is capable of stepwise dechlorination of PCE to cis-DCE, while the closest relatives were not able to dechlorinate PCE or TCE. Dechlorination of PCE or TCE by strain SZ was supported by acetate, hydrogen or pyruvate as electron donor. Chloroethene-dechlorinating populations have been shown to have distinct electron donor requirements. However, none of previously described chlorinated ethene degrading population can use both, acetate and hydrogen, as electron donors. PCE dechlorination by strain SZ uses both acetate and hydrogen as electron donors suggesting that the ability to versatile electron donor utilization may increase the efficiency of bioremediation approaches. Importantly, strain SZ reduced two environmental priority pollutants, PCE and U(VI) concomitantly and detected from both bio-stimulated chloroethene and uranium contaminated sites, strongly suggesting that strain SZ play a important roles in in-situ bioremediation of chloroethene and U(VI) contaminated sites. The second, a new Dehalococcoides species designated strain GT, was isolated from contaminated site materials. Strain GT uses trichloroethene (TCE), cis-DCE, 1,1-dichloroethene (1,1-DCE), and the human carcinogen vinyl chloride (VC) as growth supporting electron acceptors producing products ethene and inorganic chloride. The new isolate shares common traits of Dehalococcoides such as ampicillin resistance, strict hydrogen-dependent metabolism, and a low hydrogen consumption threshold concentration. Culture-dependent and independent, 16S rRNA gene and reductive dehalogenase gene targeted PCR approaches suggested culture purity.

Geobacter sulfurreducens possesses type IV pili that are considered to be conductive nanowires and a crucial structural element in biofilm formation, enabling electron transfer to insoluble metal oxides in anaerobic sediments and to graphite anodes in microbial fuel cells. The molecular mechanism by which electrons are transferred through the nanowires to the electron acceptor is not fully understood. Prior to the work described in this thesis, the gene (pilA) encoding the structural pilus subunit had been identified, but little was known about the functional translation start codon, the length of the mature secreted protein, or what renders the pili conductive. Using mass spectrometry, I found that a tyrosine residue (Y32) near the carboxyl terminus of the mature PilA protein is posttranslationally modified by attachment of glycerophosphate. I studied the significance of Y32 for biofilm formation on various surfaces and for growth of G. sulfurreducens with insoluble electron acceptors. A mutant in which Y32 was replaced by phenylalanine lacked the glycerophosphate; biofilm formation on graphite surfaces was severely diminished and current production in microbial fuel cells was initiated only after a long lag phase. Moreover, cells with Y32F mutation in the pilA gene exhibited growth deficiency when Fe(III) oxide was the sole electron acceptor. My data confirm the role of G. sulfurreducens pili in biofilm formation and electron transfer to Fe(III) oxide and identify an amino acid in the PilA protein that is essential for these two processes. I also confirmed the existence of two functional translation start codons for the pilA gene and identified two isoforms (short and long) of the PilA preprotein by series of genetic complementation experiments. The short PilA isoform is found predominantly in an intracellular fraction, and seems to stabilize the long isoform and influence the secretion of several outer surface c-type cytochromes. The long PilA isoform, on the other hand, is required for secretion of PilA to the outer surface of the cell, a process that requires co-expression of pilA and the nine genes on its 3’ side. The long isoform is essential for biofilm formation on various surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxide. This study provides new insight concerning the function and biogenesis of Geobacter type IV PilA, as well as a foundation for further research that will be conducted on microbial nanowires.

Arsenic sorbs strongly to the surfaces of Fe(III) (hydr)oxides. Under aerobic conditions, oxygen acts as the terminal electron acceptor in microbial respiration and Fe(III) (hydr)oxides are highly insoluble, thus arsenic remains associated with Fe(III) (hydr)oxide phases. However, under anaerobic conditions Fe(III)-reducing microorganisms can couple the reduction of solid phase Fe(III) (hydr)oxides with the oxidation of organic carbon. When ferric iron is reduced to ferrous iron, arsenic is mobilized into groundwater. Although this process has been documented in a variety of pristine and contaminated environments, minimal information exists on the mechanisms causing this arsenic mobilization. Arsenic mobilization was studied by conducting controlled microcosm experiments containing an arsenic-bearing ferrihydrite and an Fe(III)-reducing microorganism, Geobacter metallireducens. Results show that arsenic mobility is strongly controlled by microbially-mediated disaggregation of arsenic-bearing iron nanoparticles. The most likely controlling mechanism of this disaggregation of iron oxide nanoparticles is a change in mineral phase from ferrihydrite to magnetite, a mixed Fe(III) and Fe(II) mineral, due to the microbially-mediated reduction of Fe(III). Although arsenic remained associated with the iron oxide nanoparticles and was not released as a hydrated oxyanion, the arsenic-bearing nanoparticles could be readily mobilized in aquifers. These results have significant implications for understanding arsenic behavior in aquifers with Fe(III) reducing conditions, and may aid in improving remediation of arsenic-contaminated waters. / Master of Science

L'industrie pétrolière et gazière subie d’importantes pertes économiques en raison de problèmes liés à la corrosion. Parmi ces problèmes, la corrosion induite par les micro-organismes (biocorrosion) fait toujours l’objet de recherche, le mécanisme le plus souvent évoqué et documenté étant lié aux bactéries sulfato-réductrices (BSR). Cependant certaines études ont montré que la biocorrosion pouvait se produire même en absence de BSR dans l'environnement corrosif ; le principal objectif de la thèse était donc de fournir un nouvel éclairage sur la corrosion anaérobie de l'acier au carbone en proposant des mécanismes différents de ceux impliquant les BSR. En premier lieu, l’influence d'une souche électro-active, G. sulfurreducens, sur la protection/corrosion de l'acier C1145 a été étudiée. Lorsque des espèces phosphate sont présentes dans le milieu, la bactérie favorise la formation d’une couche de Fer/Phosphate qui ensuite protège le matériau. En présence d’ammonium, les vitesses de corrosion sont plus élevées mais les bactéries réduisent la dissolution du métal. En deuxième partie, des échantillons de terrain issus des opérations de nettoyage des pipelines des systèmes d’injection ont été analysés d’un point de vue microbiologique et électrochimique. L’analyse moléculaire et l’identification de la communauté bactérienne montre la présence d'espèces sulfurogènes autre que les BSR. Ces bactéries peuvent stimuler la corrosion des métaux par la production d'acides organiques, de CO2 et de différentes espèces soufrées telles que H2S. De surcroît, il a été prouvé que le consortium contenu dans les échantillons de terrain accélérait la corrosion de l'acier au carbone, principalement par la production d'espèces sulfures. / The oil and gas industry is impacted by important economic losses due to corrosion problem. As part of this problem, microbially influenced corrosion (MIC) is still a subject of research. The most often evoked and well acknowledge MIC mechanism is linked to sulphate reducing bacteria (SRB). However, some studies have shown that MIC can occur even when SRB is not present in the corroding environment; in this framework, the main objective of the thesis is to provide new insights on corrosion of carbon steel caused by other mechanisms different to those described with SRB. First, the influence of an electroactive strain, G. sulfurreducens (an iron reducing bacteria, IRB) on the corrosion/protection of steel C1145 was studied. When phosphate species are present in the medium, bacteria promote the formation of an iron phosphate layer (vivianite) that afterwards protects the material. In presence of NH4+, corrosion rates are higher but bacteria decrease the dissolution of the material. In the second part, field samples from pigging operations performed in water injection pipelines were analysed from microbiological and electrochemical corrosion points of view. Molecular analysis and identification of the biofilm community show the presence of sulfidogenic species besides SRB. These bacteria can stimulate metal corrosion through production of organic acids, CO2 and different sulphur species such as H2S. Moreover, it was proved that the consortium contained in field samples accelerated corrosion of carbon steel mainly by production of sulphide species.

De multiples micro-organismes ont la capacité de catalyser l’oxydation électrochimique de matières organiques en s’organisant en biofilm à la surface d’anodes. Ce processus est à la base de procédés électro-microbiens très innovants tels que les piles à combustible microbiennes ou les électrolyseurs microbiens. L’interface biofilm/électrode a été l’objet de nombreuses étudesdont les conclusions restent difficiles à démêler en partie du fait de la diversité des paramètres interfaciaux mis en jeu. L’objet de ce travail de thèse est d’exploiter les technologies microsystèmes pour focaliser l’impact de la topographie de surface des électrodes sur le développement du biofilm et sur ses performances électro-catalytiques. La formation de biofilmsélectroactifs de Geobacter sulfurreducens a été étudiée sur des électrodes d’or présentant des topographies bien contrôlées, sous la forme de rugosité, porosité, réseau de piliers, à des échellesallant du nanomètre à quelques centaines de micromètres. La présence de microrugosité a permis d’accroitre les densités de courant d’un facteur 8 par rapport à une surface lisse et son effet a étéquantifié à l’aide du paramètre Sa. Nous avons tenté de distinguer les effets des différentes échelles de rugosité sur le développement du biofilm et la vitesse des transferts électroniques.L’intérêt de la microporosité a été discuté. L’accroissement de surface active par la présence de micro-piliers s’est avéré très efficace et une approche théorique a donné des clés de compréhension et d’optimisation. Les connaissances acquises dans les conditions de culture pure ont finalement été confrontées avec la mise en oeuvre de biofilms multi-espèces issus d’un inoculum complexe provenant de sédiments marins.