B33C-0432
Comparison of emission from the water column and wetland plants at the Berry's Creek estuary, Hackensack Meadowlands, New Jersey.
Berry's Creek is a tidal tributary to the Hackensack River, and was historically subjected to discharges of mercury from the Ventron-Velsicol mercury processing site. The emission of mercury from this site to the atmosphere can follow three pathways: 1) emission from the water column, 2) transpiration through plants, and 3) emission from exposed wetland sediments. In this poster, we present a comparison of the first two emission pathways which have been studied at this site to date. Emission of mercury from the water column mercury to the atmosphere results from complex biogeochemical reactions between photoreactive dissolved organic carbon, ultraviolet light, and dissolved aqueous mercury. Emission rates measured using a dynamic flux chamber ranged from -0.64 to 34 ng/m2-h. Solar radiation and DOC spectral slope appear to exert the strongest control on mercury emission, with solar radiation alone accounting for up to 98% of the diel changes in mercury emission. Emission of mercury from plants appears to be a complex process that includes emission of mercury along with water vapor during transpiration as well as deposition to plant surfaces. Emission rates from Phragmites australis leaves ranged from -0.64 to 0.17 ng/m2-h. Annual and diel cycles are considered in an estimation of the magnitude of total mercury emitted through each pathway over the duration of 1 year.
B33C-0433
Mercury Emission From Phragmites in a Contaminated Wetland
Characterizing the role of vegetation has been an elusive component to a complete understanding of the mercury cycle. Defining this contribution is of ecological and environmental significance as it pertains to contaminated industrial sites. Various studies have demonstrated that foliar exchange of gaseous mercury is bi-directional and may depend on atmospheric concentrations of mercury as well as other environmental parameters. In particular emergent aquatic vegetation such as Typha, Cladium, and Phragmites, in areas of elevated mercury soil concentrations have been shown to generate relatively high daytime fluxes of ~30ng/m2/hr, ~20ng/m2/hr, and in one case 90ng/m2/hr, respectively. For this research mercury fluxes were measured from foliar surfaces of Phragmites australis in a highly contaminated portion of the New Jersey Hackensack Meadowlands using a dynamic flux chamber. The chamber is constructed from UV transparent acrylic sheets sized to average Phragmites leaves and employs a sheath-like design so that it may be easily slid over foliage with minimal interference. The design also circumvents the use of foams or silicone as sealant which in the past have been shown to emit or absorb mercury. Laboratory and field tests have shown good agreement between ambient air and chamber blank mercury levels. During field excursions generally 5-7 adjacent plants would be sampled for 20-30 min each.Over one 6-hour sampling period in late summer 2008 mean Phragmites flux was - 0.12ng/m2/hr±0.25 with a maximum negative flux of -0.64ng/m2/hr. Another sampling period showed a positive average of 0.07ng/m2/hr±0.07 with a maximum of 0.11ng/m2/hr. These values, as well as those observed in earlier literature, are likely the result of significant environmental parameters operating on the mechanism by which foliar flux is produced. Such parameters include, incoming solar radiation, wind velocity, air temperature, air quality, humidity, sediment pore water mercury concentrations, as well as internal leaf properties such as transpiration and relative humidity. It is the ongoing goal of this study to relate the magnitude of mercury flux with said parameters in order to better understand the controls by which emission is enhanced or diminished.
B33C-0434
Biomolecular Aspects of Mercury Transformations
Bacteria participate significantly in mercury transformation in natural and industrial environments. Previous studies have shown that bacterial mercury resistance is mediated by the mer operon, typically located on transposons or plasmids. It encodes specific genes that facilitate uptake of mercury species, cleavage of organomercurials, and reduction of Hg(II) to Hg(0). Expression of mer operon genes is regulated by MerR, a metal-responsive regulator protein on the level of transcription. In vitro studies have shown that MerR forms a non-transcribing pre-initiation complex with RNA polymerase and the promoter DNA. Binding of Hg(II) induces conformational changes in MerR and other components of the complex resulting in the transcription of mer operon genes. As part of ongoing investigations on allosteric conformational changes induced by Hg(II) in dimeric MerR, and the implications on the binding of RNA polymerase to the promoter of the mer operon, we applied small angle scattering to study the regulatory mechanism of MerR in the presence and absence of Hg(II). Our results show that in the presence of Hg(II) the MerR dimer undergoes a significant reorientation from a compact state to a conformation revealing two distinct domains. Bacterial reduction of Hg(II) can also occur at concentrations too low to induce mer operon functions. Dissimilatory metal reducing bacteria, such as Shewanella and Geobacter are able to reduce Hg(II) in the presence of mineral oxides. This process has been linked to the activity of outer membrane multiheme cytochromes. We isolated and purified a decaheme outer membrane cytochrome OmcA from Shewanella oneidensis MR-1 and characterized its envelope shape in solution by small angle x-ray scattering. Structural features were identified and compared to homology models. These results show that OmcA is an elongated macromolecule consisting of separate modules, which may be connected by flexible linkers.
B33C-0435
The role of photochemical reactions of suspended particulate matter on mercury cycling in aquatic systems
Suspended particulate matter (SPM) provides a large amount of surface area for chemical reactions that are important for trace metal cycling. In many settings, particulate-bound mercury (Hg) dominates the total Hg pool. Coatings of iron (Fe) oxides or organic material (OM) on suspended inorganic constituents have long been recognized as important surfaces for reactions with trace metals in aquatic systems. Both Fe and OM are photoreactive and have been well studied in the dissolved phase, and extensive data has been published regarding photochemical influences on mercury cycling. Photoreactions involving particulates have been little studied, but could affect the stability of mercury-SPM complexes in natural and contaminated systems. The purpose of this experiment was to assess the importance of photochemical reactions of SPM for controlling mercury partitioning and emission. The influence of particle coating type (Fe vs. OM vs. no coating) was also evaluated. A pilot experiment was conducted using filtered (F) and unfiltered (UF) waters collected from a contaminated estuary (Berry's Creek, NJ, USA) to evaluate the effect of particle presence and absence on Hg emission rates. Four treatments (F-Light, F-Dark,UF-Light, UF-Dark) in flux chambers were exposed to sunlight for approximately 6 hours. Light was able to penetrate the flux chambers from the sides as well as the top throughout the experiment, and Hg emission was continuously measured using atomic absorption. Results of the pilot study indicated significantly different Hg emissions from the 4 treatments, with emissions increasing in the following order: F-Dark, UF-Dark, F-Light, and UF-Light. Additional experiments were then conducted to better characterize the role of particulates, especially particle type, on Hg partitioning and emission rates. Ground quartz (5-20 um) was cleaned and then coated with iron or organic matter, or left uncoated. Deionized water was amended with mercury to achieve a final concentration of approximately 30 ng/L and then equilibrated with each particle type (approx. 50 mg SPM/L) prior to experiments. Light exposures and dark controls are compared for each particle type as well as particle-free controls. The effect of Fe and OM coatings on Hg binding to SPM, as well as Hg partitioning to the dissolved phase and emission from the water surface to the atmosphere will be evaluated.
B33C-0436
Molecular Scale Dissolved Organic Matter Interactions Impact Mercury Bioavailability for Uptake and Methylation by Sulfate-Reducing Bacteria
Biogeochemical factors such as dissolved natural organic matter (DOM) type and abundance may play a major role in governing the bioavailability of aqueous Hg(II) for uptake and methylation by sulfate-reducing bacteria (SRB). MeHg production correlates in some cases with predicted dominance of hydrophobic, neutrally-charged, aqueous HgS. This species is thought to interact strongly with DOM via hydrophobic attractions. Field and laboratory observations suggest that DOM promotes methylation. We therefore hypothesized that DOM isolates of differing (well-characterized) functional compositions (e.g., hydrophobic versus hydrophilic) could variably enhance bacterial methylation. Methylation assays using Desulfobulbus propionicus 1pr3 in fermentative growth were performed using a mercury isotope tracer applied at concentrations of roughly 100 ng/L. The tracer was pre-equilibrated with 5-10 uM aqueous sulfide and approximately 40 mg/L of either hydrophobic or hydrophilic DOM prior to inoculation. Results showed roughly 1-3% tracer methylation in both hydrophobic DOM+ and DOM- cultures. However, a similar amount of non- tracer (background) mercury associated with the hydrophobic DOM fraction was also methylated. Preliminary results suggested that pre-equilibration of the isotope tracer for up to one month with hydrophobic-fraction humic acids resulted in a roughly 2-3X increase in the quantity and rate of methylation, indicating an important role for aging on DOM in Hg bioavailability. Mercury-sulfide-DOM equilibration products were investigated with synchrotron-based x-ray fluorescence spectroscopy (EXAFS) at liquid nitrogen temperatures. Hg L(III)-edge spectra from resin-concentrated Hg-S-DOM equilibration products exhibited high similarity to a metacinnabar-like conformation. Culturing and EXAFS results, taken together, suggest that nanophase metacinnabar, "packaged" in DOM, could have been the bioavailable form of Hg(II) in culturing experiments. Further experiments involving other DOM fractions, and new EXAFS investigations are ongoing, to explore further the mechanism of bacterial mercury methylation.
B33C-0437
Mercury Retention and Accumulation by Plants at the Abandoned New Idria Mine Site - a Preliminary micro-XRF and micro-XRD Study
Due to its high toxicity and increasing levels in ecosystems, Hg pollution has become a serious global problem. A lot of research has been conducted with regard to Hg biogeochemical cycles in aquatic systems. Much less is known about terrestrial Hg-cycles in general and in plants specifically. Plants play an important role in these cycles; they are known to be an important sink for both atmospheric and soil Hg, the vegetative cover significantly influences soil erosion and migration of contaminants into aquatic systems. However, the processes involved in the interactions of Hg with plants and plants products are poorly studied. Information concerning the interaction of Hg in plants at the molecular level is sparse. The present study is intended to provide new information on Hg retention, translocation, and accumulation in plants associated with mercury mine wastes in central California. We present here preliminary results of Hg distribution in root and leave samples, taken from different plant species, which have adapted to the hostile environment at the New Idria site. Samples were taken at two locations that differ in water acidity and flooding regime. The distribution of Hg appears to be affected by plant species, growing conditions, and development stage. Micro-XRF images of root sections show that Hg is mainly associated with Fe plaque at the outer surfaces and epidermis, but is distributed differently in roots of the two plants. Micro-XRD showed evidence for mineralogical changes in the plaque through the sections. Mercury in leaves was found to be highly diffuse in its distribution, and is not associated with Fe-rich particles attached to the outer surface of the leaf. This finding implies that Hg is assimilated in the leaf tissue. Further examination of Fe plaque characteristics and associated Hg, as well as Hg speciation in the different organs of these plants, is being conducted in our lab.
B33C-0438
Roles of Dissolved Organic Matter in Aqueous Speciation of Hg(II) and CH3Hg+ in East Fork Poplar Creek, Oak Ridge, Tennessee
The aqueous speciation of mercury [Hg(II)] and methylmercury (CH3Hg+) in contaminated East Fork Poplar Creek (EFPC) at Oak Ridge, Tennessee, is not well understood despite recognition of the important roles played by dissolved organic matter (DOM) in controlling the cycling, transport and bioavailability of Hg in the system. We determined Hg species distributions in the EFPC based on EFPC water chemistry and a selected database of reaction constants of Hg(II) and CH3Hg+ with various organic and inorganic ligands, using the "PHREEQC" geochemical speciation computer code. Results show that DOM affects both Hg(II) and CH3Hg+ speciation by forming strong Hg(II)-DOM and CH3Hg-DOM complexes through reactive sulfur groups or thiol-like functional groups in DOM in EFPC surface waters under conditions of pH = 7.5 to 8.1 and DOM about 3 mg/L. The neutral complexes of Hg(OH)2 and Hg(OH)Cl, and CH3HgCl and CH3HgOH were found to be of secondary importance in EFPC surface water. The competitive effects of co-existing metal ions (i.e., Ca2+, Mg2+, Zn2+, Cu2+, Ni2+, Cd2+, Pb2+, Fe3+ and UO22+) on the Hg(II)-DOM and CH3Hg-DOM complexation in the EFPC surface water were evaluated using analogous model organic compounds (i.e., cysteine and glutathione), because reliable binding constants of these co- existing metal ions with thiol-like functional groups in DOM are scarce in the literature. Results show that only Zn2+ (present at relatively high concentrations in EFPC) competes with Hg(II) for the binding sites (thiol) in DOM and therefore contributes to the decreased distribution of Hg(II)-DOM. The effect of other metal ions on the distribution of Hg(II)-DOM and CH3Hg-DOM complexes are estimated to be minimal. These results provided further understanding of mercury speciation, bioavailability and transformation in the EFPC stream water. A literature review of the conditional formation constants (log K) of Hg(II) and CH3Hg+ with reactive sulfur groups (RS) in DOM gives a broad spread of values of log K from 21 to 40 for Hg(II)-RS complexes and from 10 to 17 for CH3Hg-RS complexes, suggesting that the quantitative characterization of reactive binding sites in DOM and their interactions with Hg(II), CH3Hg+, H+ and other metal ions in environments, remain to be developed. Methodologies to develop these areas of understanding are essential for more accurate modeling of Hg(II) and CH3Hg+ speciation in aquatic ecosystems.
B33C-0439
Stabilization of Nanoparticulate HgS by Thiols and Humic Substances During HgS Precipitation
In the aquatic environment mercury has a strong affinity for reduced sulfur-containing ligands such as inorganic sulfides and thiolate functional groups in natural organic matter (NOM). Complexation of aqueous Hg(II) is particularly important because coordination to inorganic sulfide and humic compounds governs Hg(II) speciation (and subsequent bioavailability and mobility) in contaminated water and sediment. The purpose of this study was to explore the potential for NOM-coated HgS nanoparticles in the aquatic environment. HgS precipitation experiments were conducted in the presence of natural organic acids that are prevalent in surface water and sediment porewater. Dynamic light scattering was used to the monitor the size of HgS particles precipitating over time. The results indicated that humic substances decreased growth rates of precipitating HgS particles and stabilized particles with aggregate diameters smaller than 0.2 μm for at least 8 hours. Thiol-containing low molecular weight acids such as cysteine and thioglycolate also decreased growth of HgS particles whereas the hydroxyl-containing acids (serine and glycolate) did not affect particle growth rates. As the humic and thiol concentration increased in solution, growth rates of HgS particles decreased. Growth rates of the aggregates increased in solutions with greater ionic strength. Nanoparticles of HgS would be possible in aquatic environments where HgS precipitation is possible. We conducted equilibrium speciation calculations to determine HgS(s) saturation indices under conditions typical for sediment porewater. The calculations indicated that the metacinnabar saturation index was 1 to 3 orders of magnitude above or below saturation, depending on Hg-(bi)sulfide and Hg-NOM binding constants, which vary by orders of magnitude. These insights suggest that HgS nanoparticles may exist in surface waters and porewater of contaminated sediments as a result of kinetically-hindered mineralization reactions. Hg(II) uptake rates by methylating organisms may be governed by rate-limited mineralization reactions, rather than equilibrium Hg(II) speciation in porewater. However, further studies are needed to directly quantify the bioavailability of nanoparticulate Hg to microbes.
B33C-0440
An Evaluation of Microbial Community Structure and Function in Mercury Contaminated Stream Sediments
Although, there has been extensive work on the presence of mercury resistance genes in mercury contaminated environments, there is a relative lack of information on the total bacterial community in highly contaminated mercury sediments. Streams draining DOE facilities in Oak Ridge, TN, have been exposed to discharges of mercury and we are examining the response of streambed microbial communities to this exposure across a Hg contamination gradient using a functional gene array (FGA) and by phylogenetic characterization (a 16s rDNA approach). The version of the FGA used for this study contains 23,864 probes covering 14,000 known microbial functional genes. We hypothesized that there would be a greater diversity of genes related to pollutants at the contaminated sites. In repeated sampling at 2-6 sites, there was a notable response in the FGA results that appears to be related to seasonal changes. We observed low numbers of genes in all categories at all sites during the winter months. Results from warmer months indicate greater differences among sites. In general, during the warmer months the contaminated sites (e.g., mercury at 33.3 ug/g and numerous other contaminants) exhibited elevated gene frequencies in all general categories compared to the control site (e.g., mercury at 0.065 ug/g). In addition to the genes that could be associated with a response to contaminants (e.g., metal resistance and contaminant degradation), genes involved in metabolism (sulfate reduction, denitrification, carbon utilization) were also elevated at the contaminated sites. We also observed an elevation in the number of different rubisco genes present with a much higher number at the most highly contaminated site compared to the control site. The only two currently completed 16s clone libraries are from these sites and interestingly the proportion of cyanobacteria is much higher in the clone library from the contaminated site. Also, the 16s diversity evident in the contaminated site is lower than that at the control site. In November 2007 (when differences among sites were reduced), a synoptic snapshot of 6 sites where total mercury in sediments ranged from 0.071ug/g to 39.1 ug/g and where there were wide ranges in concentration of SRP, nitrate, sulfate, uranium, and total organic carbon (TOC) shows that there was a poor correlation between mercury in stream sediments and mercury in the water (r = 0.71). This observation is consistent with the complex relationship between stream sediment and stream water concentration that is likely influenced by geochemical factors and mercury speciation. During this sampling, concentrations of uranium and nitrate were correlated (r = 0.71 to 0.90) with gene frequency for nitrogen cycling, sulfate cycling, metal resistance, and organic contaminant degradation. Aside from these relationships, the highest correlations (r= 0.65 and 0.66) were between TOC and gene frequency for organic contaminant degradation and sulfate and gene frequency for organic contaminant degradation. Thus, for the samples analyzed to date, the FGA appears to be able to detect differences in the diversity of genes for specific functions that can be related to site geochemistry.
B33C-0441
Comparison of Two Mercury Contaminated Surface Water Bodies
Due to the methyl mercury (MeHg) burden in fish tissues, the Virginia Department of Health and the Tennessee Department of Environment and Conservation have posted fish advisories for the South River, VA and the East Fork Poplar Creek, TN (EFPC), respectively. This presentation will compare and contrast the hydrogeochemical characteristics of these two industrially contaminated water bodies. Both streams share broad similarities in terms of their general chemistry and underlying geology. Nevertheless, patterns of waterborne mercury (Hg) and, importantly, MeHg concentration are different. For example, in the South River both Hg and MeHg concentrations increase with increasing distance downstream from the industrial site of mercury origin whereas in EFPC Hg decreases while MeHg increases with increasing distance downstream. Although both sites are the focus of concerted research efforts to identify effective remediation, the underlying mechanisms that drive the patterns within each system and therefore account for the differences between them are poorly understood. We intend for this presentation to provide a context within which attendees can frame their discussion of the challenges inherent to studying the biogeochemical cycling of Hg in general and at contaminated sites in particular where effective remedies can be elusive.
B33C-0442
Evidence for the Presence of Colloidal Metacinnabar in Mercury-DOM-Sulfide Systems as Determined by a Chromatographic-EXAFS Method
Mercury speciation and bioavailability is frequently thought to be controlled by the presence of dissolved organic matter (DOM) and sulfide. However, the speciation of mercury in these systems is poorly understood due to the complex interactions of mercury, DOM, and sulfide. We have developed a combined chromatographic-extended x-ray absorption fine structure (EXAFS) spectroscopy approach to determine the speciation of the hydrophobic fraction of mercury species in both sulfide-free and sulfide-rich (100 μM) experimental systems that also contain dissolved organic matter isolated from several locations, including the Florida Everglades. Chromatographic experiments were carried out with and without sulfide at varied mercury concentrations ranging from 0.1 nM to 1 μM in the presence of 10 mg L-1 DOM. The method consists of equilibrating the mercury-DOM with or without sulfide for 20 h (pH 6.5, I 0.1M) followed by chromatographic fractionation and concentration on a small column of C18 resin. Greater than 80% of the mercury in all solutions was found to be hydrophobic with respect to the resin when the mercury was interacting with the strong-binding DOM sites. The chromatographic behavior of solutions with and without sulfide was distinctly different. Sulfide-free mercury-DOM systems exhibited typical chromatographic behavior exemplified by resin saturation and subsequent breakthrough of mercury species. The sulfide-rich system exhibited very high resin affinity for almost all mercury species in solution and no apparent breakthrough, regardless of the ratio of mercury to DOM. Similar chromatographic experiments were carried out with and without sulfide at mercury concentrations as low as 250 nM and a DOM concentration of 50 mg L-1. EXAFS spectroscopy at the mercury LIII edge clearly showed spectra consistent with metacinnabar (HgS) as the dominant form of mercury adsorbed to the resin under sulfidic conditions despite the fact that no bulk precipitation was observed. EXAFS spectra from resins loaded with solutions of only mercury and DOM showed a distinctly different mercury binding environment that was not consistent with metacinnabar, although the local mercury binding environment for these sulfide-free solutions also contained sulfur as the nearest mercury neighbor.
B33C-0443
Product Dissociation Pathways in MerB Catalysis: A Molecular Dynamics Study
The bacterial organomercurial lyase, MerB, catalyzes the protonolysis of organomercurial compounds. MerB cleaves Hg-C bonds of various substrates ranging from the methylmercury cation (MeHg) to merbromin. Upon Hg-C bond cleavage, Hg2+ and an organic molecule are produced. For example, methane is the product resulting from the protonolysis of MeHg. The release pathway and mechanism of the organic product are unclear. Here, we have applied molecular dynamics and free energy simulations to study the dissociation of a series of organic molecules. The x-ray crystallographic structure of MerB with a bound Hg2+ cation was used as the starting model, and the organic products were manually placed in the active site. The umbrella sampling method was used to obtain free energy profiles for the dissociation pathways. Several hydrophobic sidechains of MerB were found to interact with the organic molecules and may have important roles in the dissociation processes. The relatively low free energy barriers of dissociation suggest that organic product dissociation is not rate limiting.
B33C-0444
Reduction of Cr(VI) and survival in Cr-contaminated sites by Caulobacter crescentus
The Caulobacter spp. is known to be able to live in low-nutrient environments, a characteristic of most heavy metal-contaminated sites. Recent studies have shown that Caulobacter crescentus can grow in chemically defined medium containing up to 1 mM uranium. Whole-genome transcriptional analysis and electron microscopic imaging of heavy metal stresses in Caulobacter crescentus also provided insight and evidence that the bacterium used an array of defensive mechanisms to deal with heavy metal stresses. In addition to up-regulated enzymes protecting against oxidative stress, DNA repair and down-regulated potential chromium transport, one of the major gene groups respond to chromium stress is "electron transport process and cytochrome oxidases", including cytochrome c oxidases, raising the possibility that the cells can employ the cytochromes to reduce chromium. Analysis of the microbial community at the chromium contaminated DOE site at Hanford, WA revealed the presence of Caulobacter spp. As an oligotroph, Caulobacter can play a significant role in chromium reduction in the environment where the nutrients are limited. This result was confirmed by both 16S rDNA based microarray (Phylochip) as well as by MDA-based clone library data. Based on these results we further investigated the capability of this organism to reduce Cr(VI) using the well known model strain Caulobacter crescentus CB15N. Preliminary cell suspension experiments were set up with glucose as the electron donor and Cr(VI) as the electron acceptor in phosphate based M2 salts buffer. After 22 hours almost 27% of Cr(VI) was reduced in the incubations containing active cells relative to the controls containing heat killed cells. Also, in another set of controls with no electron acceptor added, cells showed no increase in cell density during that time demonstrating that the reduction of Cr(VI) by cells of Caulobacter was due to biological activity. Future experiments will investigate the components responsible and the mechanism of Cr(VI) reduction by Caulobacetr crescentus.
B33C-0445
Proteogenomic Analysis of Geobacter Populations in a low Nutrient Contaminated Aquifer Under Stimulated Conditions.
Proteogenomic samples were obtained from a U(VI)-contaminated aquifer undergoing acetate-stimulated bioreduction at the U.S. Department of Energy Integrated Field Challenge (IFC) site in Western Colorado. Analysis of these samples using ICP-MS/MS indicated that they were dominated by Geobacter species, with over 2,500 proteins identified per sample. The detected proteins revealed a wealth of information about how Geobacter species are able to dominate subsurface environments under nutrient-poor conditions such as those at Rifle. The presence of nitrogenase proteins indicates that the Geobacter populations are fixing nitrogen, although the absence of other proteins indicative of nitrogen stress, such as the uridylylated version of the P-II regulatory protein and NtrB, suggests that low-level N2 fixation occurs without the community undergoing extreme nitrogen stress. The detection of a large number of proteins involved in two- component sensor and chemotaxis systems, along with flagella subunits, indicates that Geobacter species are able to rapidly detect and respond to chemical gradients in the environment. Pathways for the efficient utilization of the elevated acetate concentrations in the subsurface have also been elucidated, with an important role suggested for acetyl-CoA transferase in controlling flux between succinyl-CoA and succinate. Other proteins detected that are clearly important for growth in the subsurface include those involved in phosphate acquisition and heavy-metal efflux.
B33C-0446
Bacteriophage Infection of Model Metal Reducing Bacteria
Microbially-mediated metal reduction plays a significant role controlling contaminant mobility in aqueous, soil, and sedimentary environments. From among environmentally relevant microorganisms mediating metal reduction, Geobacter spp. have been identified as predominant metal-reducing bacteria under acetate- oxidizing conditions. Due to the significance of these bacteria in environmental systems, it is necessary to understand factors influencing their metabolic physiology. Examination of the annotated finished genome sequence of G. sulfurreducens PCA, G. uraniumreducens Rf4, G. metallireduceans GS-15 as well as a draft genome sequence of Geobacter sp. FRC-32 have identified gene sequences of putative bacteriophage origin. Presence of these sequences indicates that these bacteria are susceptible to phage infection. Polymerase chain reaction (PCR) primer sets designed tested for the presence of 12 of 25 annotated phage-like sequences in G. sulfurreducens PCA and 9 of 17 phage-like sequences in FRC- 32. The following genes were successfully amplified in G. sulfurreducens PCA: prophage type transcription regulator, phage-induced endonuclease, phage tail sheath, 2 phage tail proteins, phage protein D, phage base plate protein, phage-related DNA polymerase, integrase, phage transcriptional regulator, and Cro-like transcription regulator. Nine of the following sequences were present in FRC-32: 4 separate phage- related proteins, phage-related tail component, viron core protein, phage Mu protein, phage base plate, and phage tail sheath. In addition to the bioinformatics evidence, incubation of G. sulfurreducens PCA with 1 μg mL-1 mytomycin C (mutagen stimulating prophage induction) during mid-log phase resulted in significant cell lysis relative to cultures that remained unamended. Cell lysis was concurrent with an increase in viral like particles enumerated using epifluorescent microscopy. In addition, samples collected following this lytic event (~44hours) were filtered through a 0.22 μ m sterile nylon filter, stained with phosphotungstic acid (PTA), and examined using transmission electron microscopy (TEM). TEM revealed the presence of viral like particles in the culture exposed to mytomycin C. Together these results suggest an active infection with a lysogenic bacteriophage in the model metal reducing bacteria, Geobacter spp., which could affect metabolic physiology and subsequently metal reduction in environmental systems.
B33C-0447
Robustness of flux distribution in Shewanella oneidensis MR-1 under environmental stress and genetic perturbation
The environmental important bacterium, Shewanella oneidensis MR-1, has the significantly different growth rates under normal growth (doubling time=3hrs) in the minimal lactate medium, salt stress (doubling time > 6 hrs), and enhanced growth with amino acids supplementation (doubling time <1.5hrs). 13C based metabolic flux analysis of Shewanella oneidensis MR-1 indicates that the relative central metabolism maintains stable under all growth conditions. Furthermore, screen of MR-1 transposon mutants using high throughput 13C isotopomer analysis method also indicates the robustness of central metabolism under genetic perturbations. These observations reveal a rigid physiology in the level of flux distribution in MR-1, in contrast to the significant change of transcriptomes and metabolite profiles under various growth conditions. This study provides evidence that microbial metabolism maintains metabolic stability under various environmental conditions, rather than being geared towards growth rate maximization.
B33C-0448
Electron microscopic characterization of the sulfate reducer Desulfovibrio vulgaris: biofilms and clumps
Numerous studies have helped characterize the stress response of the anaerobic sulfate reducer Desulfovibrio vulgaris Hildenborough (DvH). Yet all of these techniques represent bulk analyses of cells grown mostly under liquid culture conditions in large reactors. Such results represent an average over a large variety of individual cellular responses, hence assuming a homogeneous distribution of physiological traits. Moreover, only recently are those techniques applied to the environmentally more relevant condition of microbial communities (biofilms). What is missing is a detailed ultrastructural analysis of such biofilms in order to determine biofilm organization and its extracellular metal deposition distribution. Using sophisticated sample cryo-preparation approaches such as high-pressure freezing, freeze-substitution or microwave- assisted processing, followed serial section TEM imaging, we have found a large heterogeneity with respect to metal precipitation with some cells being surrounded by metal precipitates whereas neighboring cells, being genetically identical and seeing virtually the exact same microenvironment, completely lack extracellular metal deposits. Interestingly, apart from metal deposits near cell surfaces, we also found string- and sheet- like metal deposits in between neighboring cells that in mature biofilms can extend for hundreds of micrometers. In mature DvH biofilms such deposits were predominantly associated with areas of intact cells in biofilms, with areas devoid of such metal deposits displayed predominantly cell debris, suggesting a role of such deposits for cell survival, which may be of high significance to biofilms at DOE sites. Upon tomographic imaging we found that extracellular metal deposits were often associated with thin filaments and vesicle-like features. To complement our serial section 2D analysis of resin-embedded samples and the resulting limitation of sampling 3D biofilm as thin sections of arbitrary orientation, we have developed an on-grid culturing and whole-mount imaging approach, which under electron-acceptor limiting conditions resulted in the presence of filaments and vesicles making this system an interesting surrogate assay for DvH-related metal reduction under a number of environmentally relevant conditions, including stress conditions. Moreover efforts, as part of the GTL-PCAP project, are underway to correlate intracellular protein expression and localization patterns, as obtained by SNAP-tag labeling and photoconversion, with extracellular metal deposition in order to determine the respective role of the various proteins in physiology and metal reduction. We have further started to characterize by SEM and TEM the clumping behavior of DvH both wildtype and megaplasmid minus under batch liquid culture conditions, and found differences in the extracellular abundance of filaments as well as differences metal deposition patterns that occur at the onset of clumping and which may promote or indeed by responsible for clumping behavior. Clumping may be a first step of biofilm formation. For a complete understanding such morphological studies need to be accompanied by studies of protein expression through microarray analysis and possibly protein localization patterns.
B33C-0449
Biochemical Mechanisms and Energy Strategies of Geobacter sulfurreducens for Long- Term Survival
Numerous species of bacteria have been observed to exhibit a growth advantage in stationary phase (GASP) phenotype, indicating that microorganisms starved of an energy source may adapt to allow for long-term survival. Understanding how Geobacter sulfurreducens persists using various metal forms as energy sources and whether a GASP phenotype develops during long-term growth are important for efficient application of this bacterium to sites requiring engineered bioremediation of soluble metals. Thus, we investigated the growth kinetics and survival of G. sulfurreducens. The growth rate of G. sulfurreducens was highest when cultured with soluble iron and generally higher on iron oxide than manganese oxide, suggesting that soluble metal forms are more readily utilized as energy sources by G. sulfurreducens. By monitoring the abundance of G. sulfurreducens in batch cultures for >6 months, distinct growth, stationary, and prolonged starvation phases were observed and a cell density of 105- 106 cells/mL persisted under long-term starvation conditions. The outgrowth of an aged G. sulfurreducens strain co-cultured with a young strain was monitored as a measure of the existence of the GASP phenotype. As the strains aged, the rpoS gene was cloned and sequenced at different stages of growth to identify mutations corresponding to a growth advantage. The results of these studies provide insight into the use of various metal forms for growth by G. sulfurreducens and its ability to persist when starved of energy sources.
B33C-0450
Biodegradation of the french reference nuclear glass SON 68 by Acidithiobacillus thiooxidans : protective effect of the biofilm,U and REE retention
Although underground nuclear waste repositories are not expected to be favourable places for microbial activity, one should not exclude localized action of extremophilic bacteria on some materials involved in the storage concept. Among endogenous or accidentally introduced acidophiles, some are susceptible to lead to a locally drastic decreased in pH with potential consequences on materials corrosion. Experiments were performed with Acidithiobacillus thiooxidans on 100-125 μm french reference nuclear glass SON68 grains in a mineral medium under static conditions during 60 days at 25°C. Growth medium was periodically renewed and analyzed by ICP-AES and ICP-MS spectrometry for both major, traces and ultra-traces elements. Biofilm formation was evidenced by confocal laser microscopy, staining DNA with ethidium bromide and exopolysaccharides with calcofluor white. Biofilm thickness around material grains exceeded 20 μm under the chosen experimental conditions. It can be noticed that while numerous studies on biofilm formation upon interaction between Acidithiobacillus ferrooxidans and materials can be found in the literature, evidence for biofilm formation is still scarce for the case of the acidophilic bacterium A. thiooxidans. Presence of biofilm is a key parameter for material alteration at the solid/solution interface in biotic systems. Indeed, various constitutive elements of materials trapped in the polyanionic polymer of biofilm may also influence the alteration process. In particular, biofilm may reduce the alteration rate of materials by forming a protective barrier at their surface (Aouad et al., 2008). In this study, glass alteration rates, determined using strontium, molybdenum and caesium as tracers, showed that the biofilm has a protective effect against glass alteration. U and REE are efficiently trapped in the biogenic compartment of the system (exopolysaccharides (EPS) + bacterial cells). Biofilm analysis are in progress to determine whether these elements are in bacterial cells or in the EPS. . Aouad G., Crovisier J.-L., Damidot D., Stille P., Hutchens E., Mutterer J., Meyer J.-M., and Geoffroy V. A. (2008) Interactions between municipal solid waste incinerator bottom ash and bacteria (Pseudomonas aeruginosa). Science of The Total Environment 393(2-3), 385-393.
B33C-0451
Uptake of Mg, Sr and Na in valves of the ostracod "Heterocypris salina" determined from culture experiments
Experiments with the ostracod "Heterocypris salina" controlled-cultures were carried out in five kinds of water with conductivities from 1 to 17 mS/cm and different hydrochemistries. The ranges of molar ratios of the waters are Mg/Ca: 0.6 to 13, Sr/Ca: 0.004 to 0.023, and Na/Ca: 0.9 to 130. Individual A-1 moult stage ostracods were introduced and weekly monitored in individual microaquaria within those waters under different constant temperature regimes of 18º, 20º, 25 º and 28°C. The juvenile individuals were grown in these cultures to obtain adults moulted in controlled water and temperature conditions. Well-calcified adult valves grown in the controlled conditions were analysed for Ca, Mg, Na and Sr contents. For the studied waters, in the temperature range of the experiments, the valve Sr/Ca and Na/Ca molar ratios are proportional to the Sr/Ca and Na/Ca molar ratios of the waters where the ostracods moulted. The Mg/Ca of the valves seems to be proportional to the [Mg], instead of to the Mg/Ca molar ratio of the water. Regarding to the water temperature, there seems to be a direct relationship between the Mg/Ca and the Sr/Ca of the valves and the temperature of the water where ostracods moulted. The Na/Ca of the valves seems to not be dependent on the temperature of the water. KdMg and KdNa follow potential functions in relation with the Mg/Ca and Na/Ca in water. KdSr has a constant value of 0.23, being independent of the Sr/Ca in water. This experiment has been funded by the Spanish Ministry of Science and Innovation CGL-2005-01467 grant, and the BES-2006-13920 pre-doctoral grant (I. Sayyad).