The g-proteobacterium Shewanella oneidensis MR-1 reduces a wide range of terminal electron acceptors, including solid Fe(III) oxides. Pathways for Fe(III) oxide reduction by S. oneidensis include non-reductive (organic ligand-promoted) solubilization reactions, and either direct enzymatic, or indirect electron shuttling pathways. Results of the present study expand the spectrum of electron acceptors reduced by S. oneidensis to include the naturally occurring disulfide compounds cystine, oxidized glutathione, dithiodiglycolate, dithoidiproponiate and cystamine. Subsequent electron shuttling experiments demonstrated that S. oneidensis employs the reduced (thiol) form of the disulfide compounds (cysteine, reduced glutathione, mercaptoacetate, mercaptopropionate, and 2-nitro-5-thiobenzoate, cystamine) as electron shuttles to transfer electrons to extracellular Fe(III) oxides. The results of the present study indicate that microbial disulfide reduction may represent an important electron-shuttling pathway for electron transfer to Fe(III) oxides in anaerobic marine and freshwater environments.

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

Outer membrane cytochromes OmcB and OmcS of Geobacter sulfurreducens are two important components of the respiratory chain for extracellular Fe(III) reduction. OmcS is a loosely bound cell surface protein involved in the reduction of insoluble Fe(III). OmcB is an outer membrane protein and required for insoluble and soluble Fe(III) reduction. The objective of this study was to understand better the mechanism of dissimilatory Fe(III) reduction, focusing on the cell surface proteins by further localization, identification of protein-protein interactions, and biochemical characterization of OmcB and OmcS. OmcB was found to be surface-exposed but embedded in the outer membrane because mild protease treatment of cells resulted in partial degradation of OmcB. Removal of surface-exposed proteins inhibited Fe(III) reduction, which is at least partially due to the degradation of OmcB. Co-immunoprecipitation studies with outer surface proteins using an antibody against OmcS revealed that OmcS interacts with several proteins, of which some are implicated in Fe(III) reduction, such as PilA, OmpJ, and OmpB, and in electricity production, such as OmcZ. Other OmcS-associated proteins, which have not been studied, include a cytochrome (GSU2887), a hypothetical and a conserved hypothetical protein, and a putative protease with a PDZ domain. The results suggest that co-immunoprecipitation with other antibodies would help to identify more elements of electron transport pathways related to extracellular Fe(III) reduction. OmcB was purified via preparative sodium dodecylsulfate polyacrylamide gel electrophoresis (SDSPAGE) and anion-exchange chromatography. The molecular mass was determined as 82 kDa, and 11.5 hemes per molecule were found. OmcB was able to transfer electrons to either soluble or insoluble Fe(III). OmcS was purified by detergent extraction. The molecular mass was 47 kDa and it contains 6 heme groups. UV-visible, EPR, and NMR spectroscopies determined that all hemes are bis-histidyl hexacoordinated and low-spin in both oxidized and reduced forms. OmcS has a –212 mV midpoint redox potential, and donates electrons to soluble and insoluble metals and quinones. Transient state kinetics showed that OmcS reduces anthroquinone-2, 6-disulfonate 10 times faster than it reduces Fe(III) citrate. This study revealed valuable further details about the mechanism of Fe(III) reduction by G. sulfurreducens by identifying the localization, protein-protein interactions and biochemical characteristics of the components of extracellular electron transport.

Microorganisms control many processes pertinent to the stability of radwaste inventories in nuclear storage and disposal facilities. Furthermore, numerous subsurface bacteria, such as Shewanella spp. have the ability to couple the oxidation of organic matter to the reduction of a range of metals, anions and radionuclides, thus providing the potential for the use of such versatile species in the bioremediation of radionuclide contaminated land. However, the organisms promoting these processes will likely be subject to significant radiation doses. Hence, the impact of acute doses of ionizing radiation on the physiological status of a key Fe(III)-reducing organism, Shewanella oneidensis, was assessed. FT-IR spectroscopy and MALDI-TOF-MS suggested that the metabolic response to radiation is underpinned by alterations to proteins and lipids. Multivariate statistical analysis indicated that the phenotypic response was somewhat predictable although dependent upon radiation dose and stage of recovery. In addition to the cellular environment, the impact of radiation on the extracellular environment was also assessed. Gamma radiation activated ferrihydrite and the usually recalcitrant hematite for reduction by S. oneidensis. TEM, SAED and Mössbauer spectroscopy revealed that this was a result of radiation induced changes to crystallinity. Despite these observations, environments exposed to radiation fluxes will be much more complex, with a range of electron acceptors, electron donors and a diverse microbial community. In addition, environmental dose rates will be much lower than those used in previous experiments. Sediment microcosms irradiated over a two month period at chronic dose rates exhibited enhanced Fe(III)-reduction despite receiving potentially lethal doses. The microbial ecology was probed throughout irradiations using pyrosequencing to reveal significant shifts in the microbial communities, dependent on dose and availability of organic electron donors. The radiation tolerance of an algal contaminant of a spent nuclear fuel pond was also assessed. FT-IR spectroscopy revealed a resistant phenotype of Haematococcus pluvialis, whose metabolism may be protected by the radiation induced production of an astaxanthin carotenoid. The experiments of this thesis provide evidence for a range of impacts of ionizing radiation on microorganisms, including the potential for radiation to provide the basis for novel ecosystems. These results have important implications to the long-term storage of nuclear waste and the geomicrobiology of nuclear environments.

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Thesis (Ph. D.)--Miami University, Dept. of Geology, 2007. / Title from second page of PDF document. Includes bibliographical references.

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