B33A-0840
Magnetite Formation in Magnetospirillum Gryphiswaldense Studied With FORC Diagrams
First-order reversal curve (FORC) diagrams, obtained from partial hysteresis curves, were measured on a set of cultured Magnetospirillum gryphiswaldense in order to study the formation of magnetite in magnetotactic bacteria. We used a cell-suspension assay for the growth-independent study of magnetite formation under highly controlled conditions. FORC diagrams showed a clear evolution from size distribution with a majority of superparamagnetic (SP) grains, to a distribution dominated by stable, single-domain grains, but still containing some SP particles. FORC diagrams also suggest that magnetostatic interactions increase during the formation and growth of the magnetite grains. TEM observations confirm this evolution. According to the saturation isothermal remanent magnetization (SIRM) cooling and warming curves, the Verwey transition can only be seen in the most mature samples, and well below 120 K. This suggests that the samples may have suffered from some partial oxidation
B33A-0841
Measurement of Microbially Induced Transformation of Magnetic Iron Minerals in Soils Allows Localization of Hydrocarbon Contamination
Soil contamination by crude oil and other hydrocarbons represents a severe environmental problem, but often the location and extent of contamination is not known. Hydrocarbons, or their degradation products, can stimulate iron-metabolizing microorganisms, leading to the formation or dissolution of (magnetic) iron minerals and an associated change of soil magnetic properties. Therefore, the screening of soil magnetic properties has the potential to serve as an efficient and inexpensive tool to localize such contaminations. In order to identify the influence of different biogeochemical factors on the microbially influenced changes of magnetic iron minerals after hydrocarbon contamination, oil spills were simulated in laboratory batch experiments. The parameters tested in these experiments included soils with different bedrocks, type and amount of added hydrocarbon, and microbiological parameters (sterile and autochthonous microorganisms). In order to follow the changes of the soil magnetic properties, the magnetic susceptibility of the samples was measured weekly. First results show that changes in the magnetic mineralogy are caused by microbial activity, as sterile samples showed no changes. In the microbially active set-ups, the magnetic susceptibility increased or decreased up to 10% in comparison to the initial magnetic susceptibility within a few weeks. In one iron-rich soil even a decrease of the magnetic susceptibility of ~40% was observed. Although the amount and type of hydrocarbons did not effect the changes in magnetic susceptibility, DGGE fingerprints revealed that they influenced microbial communities. These results show that the magnetic susceptibility changes in the presence of hydrocarbons and that this change is microbially induced. This suggests that the screening of soil magnetic properties can be applied to localize and assess hydrocarbon contamination. In order to understand the biogeochemical processes better, the change of the iron mineralogy will be followed by Moessbauer spectroscopy in future batch experiments. Furthermore, iron-metabolizing microorganisms are currently isolated and identified.
B33A-0842
Solid-state Au/Hg Microelectrode for the Investigation of Fe and Mn Cycling in a Freshwater Wetland: implications for methane production
The solid-state voltammetric gold-amalgam microelectrode was used to measure multiple redox species (O, S, Fe and Mn) in situ at (sub)millimeter vertical resolution to elucidate rhizosphere processes in Jug Bay wetlands. In vegetated soil, a classic diagenetic redox sequence without any dissolved sulfide was observed in summer. However, the rhizosphere can be quite variable which is due to the introduction of O2 to the anoxic sediments by plants. In non-vegetated soil, the vertical concentration-depth profiles were relatively constant. The presence of Fe(II), Mn(II) and soluble Fe(III) in deeper sediments indicates the oxidation of Fe(II) as well as the non-reductive dissolution of Fe(III) and the reductive dissolution of Fe(III) and Mn(III, IV) solids. Mn(III, IV) and Fe(III) redox chemistry is important in organic matter mineralization mediated by bacteria and in suppressing methane formation. In addition, Mn(III, IV) also can oxidize Fe(II) to supply Fe(III) for bacterial Fe(III) reduction. Studying Fe and Mn cycling via voltammetric methods can give insights to methane production and loss as there is no methane sensor for sediment work at present.
B33A-0843
Seasonal Changes in the Biogeochemistry of Bacteriogenic Iron Oxides in a Groundwater- Supplied Wetland
Bacteriogenic iron oxides (BIOS), composed of a mixture of poorly ordered hydrous ferric oxides and intact and partially decomposed bacterial cells, are thought to play an important role in regulating aqueous trace element concentrations in groundwater-supplied wetland environments. We have initiated a study examining the seasonal variation in the biogeochemistry of BIOS and its role in contaminant sequestration in a wetland site at Chalk River, Ontario, Canada, an area that hosts an exceptional abundance of BIOS. Surficial BIOS and subsurface cores were collected in spring (April) and summer (July) for microbial and bulk geochemical characterization. In addition, dialysis `peepers` samplers were deployed to examine the changes in selected dissolved elements with depth. SEM inspection of representative BIOS samples found a predominance of sheaths similar to Leptothrix spp., Fe(II)-oxidizing microbes known to thrive in the circumnetural pH, high dissolved Fe(II) (up to 5 mg L-1) and low dissolved oxygen (<5 mg L-1) environment found at the groundwater springs of our study site. Helical stalks reminiscent of iron-oxidizing Gallionella spp. were also occasionally observed. The BIOS mineralogy was dominated by 2-line ferrihydrite, although interestingly samples collected further downstream from the groundwater discharge zone appear more closely related to 6-line ferrihydrite. Iron reduction was noted at depth in both the sedimentary and porewater profiles, with the dissolved Fe(II) peak in the latter occurring at shallower depths in summer compared to the spring. Strontium, a contaminant of concern at this site, showed little seasonal variation, but its concentration was broadly correlated with that of dissolved iron and manganese. In addition, porewater strontium concentrations were 2-3 fold higher at a nearby control site where BIOS was absent, indicating the likely importance of BIOS in attenuating migration of this contaminant.
B33A-0844
Iron oxidation and biomineralization by Mariprofundus ferrooxydans, a deep-sea microaerophilic lithoautotroph
The ocean crust contains a large reservoir of reduced iron, available for microbial energy generation. Some of this ferrous iron is mobilized by fluids in hydrothermal fields at seamounts and mid-ocean ridges. A microaerophilic iron oxidizer, Mariprofundus ferrooxydans has been identified (by molecular methods and microscopy) at various sites, and appears to be a key iron-oxidizing bacterium (FeOB) in the deep sea. Originally isolated from microbial mats near vents at the Loihi Seamount in Hawaii, Mariprofundus is distinctive because it forms an extracellular iron-mineralized stalk-like structure. We aim to understand its metabolism and mineral formation using a multidisciplinary approach, including electron microscopy, x-ray spectroscopy, time-lapse light microscopic imaging of live cells, and genomic and biochemical analyses. Microscopy and spectroscopy work shows that as the cells grow, they excretes iron and organic-rich fibrils that make up the stalk, at a rate of ~2 microns/hr. Stalk growth appears to be parallel to the direction of Fe and oxygen gradients. The Mariprofundus genome contains several terminal oxidases/peroxidases, including two cbb3-type cytochrome oxidases with a high affinity for oxygen, consistent with the microaerophilic lifestyle of these organisms. However, we have not identified genes for metabolisms other than aerobic iron oxidation, nor have we found any genes similar to known or suspected iron oxidases, though the genome (2.87 Mb) is rich in cytochromes (32 of 2922 genes). Thus, we are performing experiments to extract and analyze proteins from both cultured and environmental samples in order to find ones that will oxidize iron. UV-Vis spectra of extracts suggest that c-type cytochromes are particularly abundant, so these are candidates for further investigation. In combination with the microscopy and spectroscopy studies, these are the first steps towards understanding the complete pathway of iron from uptake through mineral formation and growth.
B33A-0845
In Situ Chemical Profiling of an Extremely low Temperature Hydrothermal System at Loihi Seamount, Hawaii
Loihi Seamount is a submarine, active volcano located on the southeast flanks of the Big Island of Hawaii. It is considered to be the youngest volcano in the chain, sharing the hot spot magma chamber with Mauna Loa and Kilauea. Sites of both vigorous and diffuse hydrothermal venting can be found surrounding the pit crater summit (1000m) and on the flanks of the seamount, down to its base (5000m). Vent fluids at Loihi are chemically distinct from other well-studied marine hydrothermal systems and have been shown to be enriched in carbon dioxide, iron(II), and manganese(II), and deplete in sulfur species. The Loihi summit is located within a zone of low oxygen, further enabling elevated iron(II) concentrations and support for a dominant community of iron-oxidizing bacteria. We deployed a sensor wand consisting of up to four voltammetric working electrodes and the ROV Jason temperature probe, and/or a submersible micromanipulator with voltammetric electrodes to provide real time in situ redox characterizations of hydrothermal fluids and geochemical gradients associated with iron-oxidizing microbial mats and flocs. In addition to surveying known areas of warm temperature (10-60 degrees C) venting at the Loihi summit, we performed widespread profiling of a previously undescribed site at 5000m, that exhibits temperature anomalies of just 0.2 degrees C. Extensive iron-oxidizing microbial mats were shown to occur up to 2m in thickness over several hundred square meters. Bottom water oxygen concentrations were near-saturation, and we observed steep gradients at the mat-interface, with little oxygen penetration and iron(II) concentrations of up to 150 micromolar. Our in situ electrochemical analyses provided an efficient and valuable means for directed discrete sampling of hydrothermal fluids and microbial flocs, as well as previously unattainable high spatial resolution geochemical profiles through the mats.
B33A-0846
Zeta-Proteobacteria dominate the formation of microbial mats in low-temperature hydrothermal vents at Loihi Seamount
Loihi Seamount is Hawaii's youngest volcano and one of the earth's most active. Loihi is located 30 km SE of the big island of Hawaii and rises over 3000m above the sea floor and summits at 1100m below sea level. An eruption in 1996 of Loihi led to the formation of Pele's Pit, a 300 meter deep caldera. The current observations have revealed diffuse hydrothermal venting causing low to intermediate temperatures (10 to 65°C). The elevated temperatures, coupled with high concentrations of Fe(II) (ranging from 50 to 750 μM) support conditions allowing for extensive microbial mat formation. The focus of this study was to identify the colonizing populations of bacteria generated by the microbial mats at Loihi Seamount. Twenty-six microbial growth chambers were deployed and recovered after placement in the flow of hydrothermal vents for 3 to 8 days from within Loihi's caldera. Genomic DNA was extracted from samples and analyzed by Terminal-Restriction Fragment Length Polymorphism (T-RFLP) using eight restriction enzyme treatments to generate fingerprints from bacterial amplicons of small subunit rRNA genes (SSU rDNAs). Pearson product-moment coupled with UPGMA cluster analysis of these T-RFLP fingerprints showed that these communities bifurcated into two primary clusters. The first (Group 1) had an average vent effluent temperature of 44°C, and the second (Group 2) had an average vent effluent temperature of 64°C. Representative samples from within the two clusters (or groups) were chosen for further clone library and sequencing analysis. These libraries revealing a dominance of the recently discovered zeta- Proteobacteria in the lower temperature group (Group 1) indicating that they were the dominant colonizers of the microbial mats. These microaerophilic, obligately lithotrophic, Fe-oxidizing bacteria are most closely related to Mariprofundus ferrooxydans. The higher temperature group (Group 2) was dominated by epsilon- Proteobacteria primarily of the genus Sulfurimonas, which are sulfur- and thiosulfate-oxidizing bacteria. http://fire.biol.wwu.edu/cmoyer/research.html
B33A-0847
Bacterial Diversity and Spatial Variability Found in a Mn-Fe Oxide Encrusted Microbial Mat From the 5000 Meter-Deep Hydrothermal Vent 'Ula Nui, Hawaii
'Ula Nui Vent Field was discovered on the southern flank of Loihi Seamount during the 2006 FeMO Microbial Observatory expedition at over 5000 meters depth. The vent field exhibited abundant low temperature (<2°C above ambient) seeps which were covered with extensive mineral-encrusted microbial mat material. The microbial mats consisted of a 0.5-3 cm thick upper mat which was comprised of laminated layers of manganese oxides and iron oxides which overlayed a flocculent iron oxide mat that could attain depths of over 1 m deep. Bacterial communities from the top and bottom mats were analyzed using SSU rRNA terminal-restriction fragment polymorphisms (T-RFLP) coupled with traditional clone library analysis. T-RFLP chromatograms indicate dominance of the ζ- Proteobacteria in both the top and lower mat. Cluster analysis of the T- RFLP fingerprints show a strong correlation between the bottom mat and iron oxide-encrusted microbial mats found in the hydrothermally active Pele's Pit near the summit of Loihi Seamount. The top mat clusters with iron and manganese oxide encrusted microbial mats found at various sites on Loihi Seamount not associated with measurable active hydrothermal venting. Clone library analysis show that the top mat was dominated by phylotypes related to the δ- γ- and the recently described ζ- Proteobacteria, along with members of the Planctomycete Division. The dominance of ζ- Proteobacteria and Planctomycete phylotypes implies that neutrophilic iron oxidation and anaerobic ammonia oxidation are active metabolisms in the top mat bacterial community. Anaerobic ammonia oxidation coupled with nitrite reduction may also be an integral metabolism in this community since some Planctomycete phylotypes from the top mat cluster within the anammox clade.
B33A-0848
Dinitrogen Gas Production From Epi- and Endolithic Basalt Communities at Loihi Seamount: A New Challenge for the Marine Nitrogen Budget
Current knowledge of the nitrogen cycle in the oceans indicates a net loss of biologically available nitrogen from the oceans. One marine habitat that is conspicuously absent from current nitrogen budget estimations is seafloor-exposed lavas, such as occur at young ridge flanks around mid-ocean ridge systems and seamounts. Basalt from two locations on Loihi Seamount, an actively venting volcano southeast of the island of Hawai'i, were incubated shipboard with 15N labelled nitrate and ammonium additions to determine the rate of nitrogen loss mediated by epi- and endolithic microbial communities. In all incubations a net production of dinitrogen gas occurred with concurrent consumption of oxidized nitrogen species (nitrate plus nitrite) and ammonium. The availability of reduced iron and manganese from the basalts could serve as potential electron donors for microbial metabolisms, including denitrification and anaerobic ammonia oxidizing (anammox) bacteria. The presence of 16S rRNA gene sequences that align with the anammox clade of Planctomycetes and ongoing anammox enrichment cultures inoculated with Loihi basalt suggest a role for anammox bacteria as well. This initial study indicates an active and significant role for basalt associated microbial communities in the nitrogen budget of the oceans.
B33A-0849
Deciphering Life in Young Ocean Crust: Development and Testing of Methods for Subsurface Microbial Observatories and In-Situ Incubations
Biogeochemical processes within the ocean crust, a hydrologically active global habitat, are hypothesized to balance key elemental budgets in the ocean (i.e. Si, Fe), yet the importance of microbial activity in mediating these processes remains unexplored. Reduced iron makes up roughly 10% of the basalt that comprises ocean crust, making iron cycling a likely source of energy for microbial activity in these systems. Available data indicates that young sub-seafloor ocean crust may be the most redox active: weathering features are established early (within the first 10-20 Ma) and change little thereafter. Laboratory studies, field examinations, and in-situ colonization and weathering experiments have shown that chemoautotrophic Fe- and S-oxidizing microorganisms are abundant and active in weathering rocks exposed at the seafloor at low temperatures. A current challenge is adaptation of available techniques for use in subsurface hard rock environments to evaluate the extent and activity of the deep biosphere. Because of the importance to study microbial communities under their relevant environmental conditions, significant resources must be dedicated to investigate this realm in situ. To address these challenges, sub-seafloor microbial observatories designed to yield high-quality, undisturbed samples that are amenable to genomic, taxonomic, spatial and geochemical profiling are in development and testing. Deployment of these observatories within drilled boreholes will allow the scientific community unprecedented opportunities to investigate microbial processes experimentally in sediments as well as subsurface hard rock environments. Initial experiments during ocean drilling expeditions at both the young and relatively warm Juan de Fuca flank as well as the young and cold ridge flank of North Pond along the Mid-Atlantic Ridge are crucial for evaluating whether the sub-seafloor ridge flank habitat supports a unique microbial community, how the microbial community compares in diversity and activity to those of deep sediments and seawater, and the global significance of microbes in biologically-mediated oxidative weathering of basalts.
B33A-0850
Optimization of DNA Extraction from Deep-sea Basalt
Studies on the microorganisms that inhabit deep-sea basalt can provide information on this dark ecosystem, which will contribution to our understanding of mass transformation and energy flow in the deep ocean. However, molecular methods for use with metal- and clay-rich rock materials such as basalt have not been suitably developed at present, yet are critically required in order to be able to fully evaluate the basalt biotope. For example, inefficient DNA extraction might lead to loss of information about important components of this community, and misinterpretation about the total community diversity and function. In order to investigate the effects of sample pretreated method, particle size, different DNA extraction methods and cell density on extracted DNA yields, two basalt samples were collected from the East Pacific Rise 9° N during research cruise AT11- 20 in Nov 2004. Basalt samples were crushed to different particle size, washed with ddH2O and 100% ethanol respectively, and autoclaved. Marinobacter aquaeolei cultures with different cell densities were inoculated into differently treated basalt samples. Pure culture and basalt samples without inoculation were used as positive and negative control to evaluate the extracting efficiency. FastDNA„¥ spin for soil kit, GeneClean„¥ for ancient DNA kit and UltraCleanTM soil DNA Kit are used for DNA extraction. Results showed that DNA yields increased with culture density. FastDNA„¥ spin for soil kit gave the highest DNA yields, which is almost 10 times more than that of UltraCleanTM soil DNA Kit. Ethanol washing and ddH2O washing did not make big difference to DNA yields. Mineral composition and surface areas might also affect DNA yields.
B33A-0851
The impact of hydrothermally-sourced Fe on ocean biogeochemistry: new investigations from the East Pacific Rise, 9-10°N.
We have initiated a new study as part of NSF's Ridge 2000 program to investigate the fate of dissolved Fe(II) released from high-temperature hydrothermal venting on the East Pacific Rise and its potential impact on local deep-ocean biogeochemical budgets. Our principal focus is on the biogeochemical cycling of Fe and the role of microbial activity in that cycle: Fe is released in abundance from high-temperature vents, it plays a major role in modifying the gross flux of other elements from hydrothermal vents to the oceans and hydrothermal sources have the potential to dominate the global deep ocean budget of dissolved Fe: an essential micronutrient. To achieve our goals we have collected samples from close to vent-sites at 9-10°N on the East Pacific Rise over a period extending from late 2004 to late 2007 that brackets an episode of recent volcanism (Winter 2005-2006). Thus, we are in a position to investigate both the "steady state" input from a deep-ocean vent-system and also how that system has been perturbed following volcanic activity out to a period of 1-2 years post-eruption. Sampling has been conducted using time-series sediment traps deployed on long-term moorings that have sampled particles settling from buoyant and non-buoyant plumes at 9°50`N and at a contrasting (sulfide- free) vent-site at 9°30`N. Our project is nested within a larger study (LADDER) that spans across disciplines from segment-scale ocean circulation in the 9-10°N area to larval transport between adjacent vent sites. Our specific on-going work includes: i) analyses for major and trace element flux variations; ii) investigation for Fe isotope fractionation within our hydrothermal plume samples; and iii) a study of the role of Fe- oxidising bacteria in catalysing Fe oxidation and/or Fe isotope fractionation within these hydrothermal plumes. For example, the mineralogical structure and character of Fe oxyhydroxide minerals may, themselves, result from the microbial catalysis of Fe oxidation.
B33A-0852
Aging of the Glass to Altered-Glass Boundary in Seamount Basalts
In oceanic basalts, fresh volcanic glass reacts with seawater, resulting in the loss of cations from the silica network and the formation of altered glass that retains insoluble cations and potassium. Microorganisms are associated with the glass to altered-glass (GAG) boundary, although their role is not known. It has been hypothesized that microorganisms promote the alteration of volcanic glass to take advantage of the reducing power of iron and manganese in the silicate glass, or alternatively, that microorganisms colonize the GAG boundary to use iron and manganese that are released during alteration. We examined the chemical composition of the GAG boundary of seamount basalts from the Gulf of Alaska that span the age range of <0.1 Ma to 49 Ma. All samples in the study were collected by manned or unmanned submersibles from outcrops on the sea floor, and were presumably exposed to bottom seawater from the time of eruption to the time of collection. The underlying assumptions of this study are that (1) the glass would become less reactive as it aged due to the closing off of fluid pathways as fractures became filled and as the surface of the basalt became coated with manganese crust; (2) the diminished fluid movement would result in the extinction of the original community of microorganisms, or in a change in the microbial community at the GAG boundary; and (3) the changing fluid flow and biological conditions in the rocks would result in changes in the composition of the altered glass at the GAG boundary. The younger basalts (<7 Ma) have the granular texture at the GAG boundary that has been associated with microorganisms. The older samples in this study (30 Ma and 49 Ma) have no, or only minor, granular texture. The altered glass of the older samples have higher Ti and lower Al, Mg, and Mn than the altered glass of the younger samples. Fe is enriched in the altered glass relative to the glass in samples of all ages. Phosphorus, an element that could be elevated by the presence of microorganisms, is only enriched in one area of altered glass in the 4 Ma sample. The aging of the GAG boundary does result in changes in composition of the altered glass, however, the influence, if any, of microorganisms on this change is not known.
B33A-0853
Mineralization of Fe/Mn-precipitates From Hot Springs in the Sanbe Volcanic Area, Japan
Mt. Sanbe is an active volcano located in western Japan. At southwest slope of Mt.Sanbe, hot springs are discharged with flow rate of 2000 liter per minute, which are accomapnied by iron and manganese precipitates. We studied chemical composition of hot spring waters by IC, ICP-AES and AA analysis, and of precipitates by XRF analysis. Water chemistry of the main hot spring (t= 30-39 °C) is Na-Cl type and contains significant iron (= 5.6-7.5 mg/l) and manganese (= 1.1-1.6 mg/l). Around the main spring discharge, both ferrihydrites (contains 67-76% of Fe) and manganese oxides (contains 71-79% of Mn) are precipitated. About 30 to 100m east from the main hot spring, black precipitates mainly consists of manganese oxides (contains 5-83% of Mn) are distributed in the area of 90 m × 45 m. Cold spring waters (t= 12-20 °C) in this area also contains Mn (0.1-0.6 mg/l), but their Fe concentration was under detection limit. Relative chemical comoposition of the cold spring waters are similar to that of the main hot spring, which implies dilution with groundwater. Total bacterial count measured by DAPI staining was 16,000-37,000 cells/ml in these waters. In order to estimate microbial activity, we conducted manganese oxidizing experiments. The oxidation rate was estiamted as 4-5 nM per hour for the experiment in which 5 g of fresh manganese wad was added to 200ml of the 0.20 μm filtered hot spring water. This rapid oxidation rate would suggest that microbial activity plays important role in manganese precipitation from the Sanbe hot/cold spring waters.
B33A-0854
Diversity of Thermophilic Microorganisms within Hawaiian Fumaroles
Fumaroles provide heat and moisture characteristic of an environment suitable for thermophilic microorganisms. On the Island of Hawaii, fumaroles are scattered across the southeastern portion of the island as a result of the volcanic activity from Kilauea Crater and Pu'u' O'o vent. We used metagenomics to detect 16S rDNA from archaeal and bacterial thermophilic microorganisms indicating their presence in Hawaiian fumaroles. The fumaroles sampled exist along elevation and precipitation gradients; varying from sea level to 4,012ft and annual rainfall from less than 20in to greater than 80in. To determine the effects of environmental gradients (including temperature, pH, elevation, and precipitation) on microbial diversity within and among fumaroles, we obtained 22 samples from 7 fumaroles over a three-day period in February of 2007. Temperature variations within individual fumaroles vary from 2.3oC to 35oC and the pH variances that range from 0.4 to 2.0. Temperatures of the different fumaroles range from 29.9oC to greater than 105oC, with pH values that vary from 2.55 to 6.93. Further data on the microbial diversity within fumaroles and among fumaroles will be determined once the sequencing of the microbial 16S rDNA regions is completed. We are currently assembling and sequencing clone libraries of bacterial and archaeal 16S rDNA fragments from fumaroles.
B33A-0855
Evolution of the Microbial Community Structure and Iron Reduction Rate in a Column Biostimulation Experiment During the Transition From Iron to Sulfate Reduction
During the biostimulation of iron reducers for the purpose of concurrent biological reduction of U(VI), it has been postulated that iron reduction proceeds while bioavailable iron is present, after which the system switches to sulfate reduction if sulfate is present. Field experiments from the Rifle Integrated Field Challenge (IFC) site in Colorado showing that the onset of sulfate reduction has been associated with decreased removal of U(VI) from groundwater support this hypothesis. However, column experiments using sediments from the Rifle site and synthetic groundwater with comparable (7 mM) sulfate levels as in the field, showed that the onset of sulfate reduction occurred within a month with no negative effect on U(VI) reduction. Separate column experiments using low (9 uM) sulfate concentrations showed that iron reduction can be maintained for over 200 days with no indication of iron limitations. To address the discrepancy between field and column experiments, an experiment is being conducted to determine the activity of iron reducers before and after the onset of sulfate reduction. Since Fe(II) buildup is difficult to quantify in the presence of sulfate reduction, the sediments were augmented with Fe- 57 goethite. Minute changes in the Fe-57 goethite can be detected via Mössbauer spectroscopy. Ten columns (2.5 cm internal diameter and 15 cm in length), loaded with sediment from the Rifle site, have been set up and are being operated at 17 °C. Groundwater from the Rifle site, amended with 3mM acetate and 20 μM U(VI), is pumped through the columns at a rate of 0.035 ml/min. Column effluent concentrations are being monitored for acetate, Fe(II), U(VI), and sulfate. Columns are sacrificed at 10 day intervals and the sediment samples are analyzed for Fe(II), U(IV), and acid volatile sulfides using standard analytical procedures. Changes in Fe-57 goethite measured using Mössbauer spectroscopy during biostimulation of the native microorganisms at 10-day intervals are showing measurable decreases in the goethite over these time intervals. Initial characterization of the microbial community in the sediment via DGGE of 16S rDNA shows the presence of Geobacter, Rhodoferax, Flexibacter, Flavobacterium, and Desulfuromonaceae. Very similar microbial populations were observed in column effluents using TRFLP analysis. Changes in individual bacterial cell numbers are being quantified via qPCR, while changes in bacterial activity are being quantified via proteomics.
B33A-0856
Kinetics of Microbially Mediated Iron Reduction: The Role of Biomass, Ferrous Inhibition, and Electron Shuttles
Microbially mediated reduction of poorly crystalline iron oxides in the subsurface controls the fate and transport of many organic and inorganic contaminants and plays a role in biogeochemical nutrient cycling. Despite recent advances in understanding this important process, much remains undiscovered. In particular, the influence of biomass on iron reducing kinetics is not well understood for surface bound microbes undergoing direct electron transfer to ferric iron oxides. Nor do we understand how Fe(II) sorption to ferric iron influences this process under direct electron transfer versus transfer via redox shuttles. Accordingly, this study investigates the iron reducing kinetics and mineralogy of geobacter sulfurreducens reducing poorly crystalline iron coated sand using acetate as electron donor, with and without AQDS, an electron shuttle and humic analog. This research demonstrates that the rate of iron reduction depends logarithmically on initial biomass for surface bound geobacter, suggesting surface area limitations with increasing biomass. However, electron shuttles remove these limitations and shift the kinetics such that the rate depends linearly on biomass over the range of cell concentrations explored. This study also finds that ferrous iron inhibits the rate of reduction with or without AQDS, but has a weaker effect in the presence of AQDS, and adding an Fe(II) specific chelator removes the negative feedback mechanism altogether.
B33A-0857
Comparative Effects of Electron Transfer Mediators on the Bioreduction of Fe(III) Oxide
The transfer of electrons from microbes to sparingly-soluble, extracellular electron acceptors such as Fe(III) oxides can occur via direct contact with the mineral surface, by dissolution of the mineral facilitated by exogenous or endogenous ligands and subsequent reduction of the dissolved Fe(III) ligand complex, and by facilitated electron transfer involving endogenous or exogenous electron transfer mediators (ETMs, also commonly referred to as electron shuttles) that are reduced by the microbes and then subsequently diffuse away from the cell and transfer electrons to the Fe(III) mineral surface, regenerating the oxidized form of the ETM. This study examines the effects of a series of compounds representing major classes of natural and synthetic organic ETMs (including low molecular-mass quinones, humic substances, phenazines, phenoxazines, phenothiazines, and indigo derivatives) on the bioreduction of lepidocrocite (γ-FeOOH) by the dissimilatory Fe(III)-reducing bacterium Shewanella putrefaciens CN32. S. putrefaciens CN32 was able to reduce lepidocrocite in the absence of exogenous ETMs; however, relative to the control, all of the synthetic ETMs examined in this study enhanced the bioreduction of lepidocrocite. The extent of the enhanced bioreduction increased with decreasing reduction potential of the given ETM redox couple. However, the addition of Suwannee River fulvic acid, humic acid, or unfractionated NOM (10 mg organic C L-1) resulted in, at best, a minimal enhancement of lepidocrocite bioreduction relative to the control that did not contain any added exdogenous ETM. These results suggest that the relative contribution of humic substances to microbially mediated Fe(III) reduction may be minimal in low-carbon environments such as oligotrophic lakes and typical groundwaters.
B33A-0858
Immobilization of Arsenic by Vivianite Biomineralization
Microbial metal reduction and biomineralization has the potential for immobilizing metals and radionuclides in subsurface environments. The objective of this study was to examine Fe reduction and biomineralization in the presence of arsenite or arsenate using metal-reducing bacteria, Shewanella sp. (Haejae-1), enriched from an intertidal flat sediment, South Korea. The bacteria was able to use glucose as an electron donor and Fe(III)-citrate as an electron acceptor in the presence of arsenite or arsenate using phosphate buffered saline medium. The reduction of Fe(III)-citrate in the presence of arsenite or arsenate resulted in the precipitation of white µm-sized crystalline minerals. XRD analysis of the white precipitate after 14-day incubation identified the mineral phase as vivianite. SEM with EDX analysis of the vivianite precipitated by the metal reducing bacteria confirmed the presence of Fe, As, O, and P. This study indicates that formation of sparingly soluble vivianite precipitates, mediated by the metal -reducing bacteria, may sequester iron, phosphate, and arsenic into more stable and less toxic forms. The formation of phosphate minerals has frequently been observed in sedimentary environments under high biological productivity, where organic matter serves as a source of phosphate to sediment pore water through bacterial degradation. Therefore, formation of sparingly soluble iron precipitates, mediated by the metal- reducing bacteria, may sequester iron, phosphate, and other metals into more stable and less toxic forms in subsurface environments.
B33A-0859
Localisation and Functional Analysis of a Manganese Oxidase Protein From the Marine Alpha-Proteobacterium Aurantimonas manganoxydans (sp. SI85-9A1)
The marine α-proteobacterium SI85-9A1 was isolated in 1985 from the oxic-anoxic transition zone in Sannich Inlet, Vancouver Island, Canada. This organism was originally isolated due to its ability to oxidise manganese from Mn(II) to Mn(IV) oxides. It later proved to have a functional Type I RuBisCO suggesting it can potentially grow autotrophically with Mn(II) acting as the electron donor. Until now the mechanism of Mn(II) oxidation has remained enigmatic although clues from the genome and preliminary experiments suggested SI85-9A1 possessed a manganese oxidase similar to the Mox oxidase produced by Pedomicrobium sp. ACM 3067. Whole cell fractionation techniques were used to localise this protein to the outer membrane where it is loosely bound and can be washed off. Manganese oxidation activity in non-pure crude extracts was analysed using an LBB colorimetric assay and monitoring Mn(III)-pyrophosphate complex formation at 258 nm in a Mn(III) capture assay. Both these assays not only confirmed function but suggested that this protein is extremely active with a manganese oxidation rate approaching 8.9 μM min-1 mg-1. Partial FPLC purification confirmed that this protein was approximately 50 KD in size and may be part of a larger holoenzyme.
B33A-0860
Characterization of Microbial Communities in Coal Mine Drainage Treatment Systems With Elevated Manganese
Sediment samples were collected from two coal mine drainage treatment sites in western Pennsylvania. Both of the sites use constructed limestone beds to passively treat acidic coal mine drainage containing elevated manganese (Mn). Site #1 has influent manganese of 150 mg/L and effluent manganese between 40-100 mg/L. Site #2 has influent manganese of 20 mg/L and effluent manganese of less than 0.5 mg/L. Large quantities of black crusts were deposited throughout the beds at both sites. X-ray diffraction showed these crusts constituted of buserite, which is a layered structure manganese oxide mineral. Both culture-dependent and nucleic acid- based techniques were used to characterize the bacterial and fungal communities in these beds. 16S rRNA gene analysis showed that bacterial communities were very diverse and included Cyanobacter, Proteobacteria, Bacteroidete, Planctomyceta, Acidobacter, Actinobacter and Gemmatimonade taxa. The archaeal diversity was lower and most sequences were related to uncultivated species. Two Mn-oxidizing fungi strains were isolated from one of the sites. One of the fungi is capable of oxidizing Mn(II) at both low and netural pH (3-7) while the other fungi can only oxidze Mn(II) at circumneutral pH. 18S rRNA gene analysis showed the low pH Mn-oxidizing fungus was closely related to Menispora tortuosa, Chaetosphaeria curvispora and Kionochaeta spissa, and the circumneutral Mn-oxidizing fungus was closely related to Myrothecium verrucaria, Didymostilbe echinofibrosa and Myrothecium roridum.
B33A-0861
Biodiversity of Rock Varnish at Yungay, Atacama Desert, Chile
Rock varnish is a very slow-growing nanostratigraphic coating consisting of approximately 70% clay and 30% iron and manganese oxides of fine-grained clay minerals rich in manganese and iron oxides, which forms on the surfaces of rocks in most semi-arid to hyper-arid climates. Rock varnish has even been postulated to exist on Mars based on surface imagery from several landed missions, and is considered a potential biomarker. However, the mechanism of varnish nucleation and growth remains unknown. Whether or not microbes are involved in the nucleation and growth of rock varnish, the detection of microbes using cultivation or cultivation- independent techniques has demonstrated that varnish provides a microhabitat for microbes. We hypothesized that rock varnish in the Mars-like Yungay region of the Atacama Desert may provide such a microhabitat for microbial life where none has been found to date in the surface soil (< 1 cm). The presence of microbes was investigated using adenosine triphosphate (ATP) assay techniques and culture-independent biomolecular methods. High levels of both total and intracellular ATP were associated with the rock varnish while negligible ATP was found in the surrounding surface soil, suggesting that viable organisms were present. Total DNA was extracted from ground varnish and surrounding surface soil and subjected to trifurcate polymerase chain reactions (PCR). No DNA was recovered from the soil. Amplicons were used to generate ribosomal DNA (rDNA) clone libraries, which suggest the presence of numerous phylogenetically distinct microorganisms in eight Eubacterial clades, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Cytophaga-Flavobacterium- Bacteroides (CFB), Chloroflexi (green non-sulfur bacteria (GNS)), Gemmatimonadetes, Actinobacteria and Cyanobacteria. The diversity of bacteria found and presence of cyanobacteria suggests that rock varnish provides a niche environment for a cryptoendolithic microbial community where life is unable to colonize the surface soil in a Mars-like environment.
B33A-0862
Nutrient Dynamics and Carbon Microbial Mineralization Processes in the Pichavaram Intertidal Mangrove Sediments from India
Various nutrient parameters of the mangrove water were analyzed for one year (July 2003-June2004) to delineate the driving forces behind the biogeochemical nutrient dynamics in the Pichavaram mangrove waters of south east coast of India. With few exceptions, the over all trend was higher concentrations of nutrients in monsoon and post-monsoon periods. Regression analyses between salinity and various nutrients explain that salinity had firm control over the nutrient distribution in the mangrove water. Stoichiometric ratios deviated greatly from the Redfield ratio, reflecting nutrient behavior that was non-conservative because of long residence time and high primary productivity associated with organic matter decomposition. Microbial nutrient mineralization rates were estimated at two different locations (i.e. impacted and un-impacted). Sulfate reduction is the major microbial mineralization pathway followed by metal (Fe-Mn) and aerobic decomposition. Total carbon oxidation (TCOX) were calculated and found that TCOX rates were higher in the pristine A. marina zone than in the two impacted stands (43 and 79 mol C m−2 y−1); rates of total carbon oxidation in the R. apiculata forest averaged 75 mol C m−2 y−1. The sulfate reduction was peaked at the surface to a depth of 1 m, leading to little carbon burial (~5% of total C input). The microbial activities are actively dampened by the anthropogenic activities and it alters the plant-soil relationship which ultimately influences the biogeochemical cycling of nutrients in the coastal environment.
B33A-0863
Arsenite-Dependent Anoxygenic Photosynthesis by Bacteria from Mono Lake, California
Chemolithoautotrophic growth of bacteria capable of using inorganic arsenic compounds is well documented. Energy conservation and carbon fixation have been demonstrated in both arsenite oxidizers and arsenate reducers from many bacterial lineages under both aerobic and anaerobic conditions. However, phototrophic growth using reduced arsenic as an electron donor has not been described. Here we report the light-dependent oxidation of arsenite to arsenate, coupled with autotrophic growth, by an enrichment culture of bacteria from Mono Lake, California. The culture is dominated by a phototrophic member of the Ectothiorhodospiraceae that is >99% similar to Ectothiorhodospira sp. (strain Borgoria Red). It also contains an uncultured member of the Bacteriodetes and another member of the Ectothiorhodospiraceae that is 96% similar to Alkalilimnicola erhlichii. The bacteria grow more slowly and clump when grown on arsenite versus sulfide or thiosulfate. Scanning electron micrographs reveal that they are closely associated and enmeshed in an organic matrix.
B33A-0864
Aerobic and Anaerobic Oxidation of Organic Acids in Yellowstone Hot Spring Ecosystems
Thermodynamic analysis of energy supply based on samples collected from continental hot spring ecosystems at Yellowstone show that aerobic reactions yield the greatest energy. In terms of energy per mole of electrons transferred, aerobic oxidation of organic acids rivals or exceeds the energy supply from aerobic oxidation of hydrogen, CO, hydrogen sulfide, pyrite, sulfur or ammonia. This analysis is derived from samples collected where hot spring fluid are in contact with the atmosphere. It is likely that oxygen will be present at lower concentrations deeper in the system, which will place hard constraints on aerobic lifestyles. If so, which metabolisms could be supported deeper in the system? How will other oxidants be used to release energy? What characterizes the transition from aerobic to anaerobic oxidation? To answer these questions, pH, temperature, and alkalinity were measured in the field while measurements of dissolved oxygen and other redox-sensitive species (nitrate, ammonia, ferrous iron, and sulfide) were made with field-portable spectrophotometers and samples were taken for analysis of organic and inorganic ions by ion chromatography. Conditions in the subsurface can be predicted by starting from measured oxygen concentrations and calculating the effect of decreasing the concentration on the overall energetics of the system. Depending on hot spring composition, the amount of energy from aerobic oxidation of organic acid anions like succinate matches that from anaerobic oxidation (by nitrate or sulfate) once the log of the activity of dissolved oxygen drops to -6 to -8. These activities are 1 to 4 orders of magnitude lower that values determined for surface water in the hot springs. At lower oxygen activities aerobic oxidation gives way to anaerobic oxidation, and organic oxidation is more likely to involve nitrate and sulfate. Preliminary estimates indicate that these changes may occur at shallow depths in hot spring sediments (perhaps within the first centimeter), which suggests great differences between conditions inferred from fluids and those inferred from genomic data based on sediments or isolates from the same hot spring system.
B33A-0865
Clay-Bacteria Systems and Biofilm Production
Soil clots and the aerosol transport of bacteria and spores are promoted by the formation of biofilms (bacteria cells in an extracellular polymeric matrix). Biofilms protect microorganisms by promoting adhesion to both organic and inorganic surfaces. Time series experiments on bacteria-clay suspensions demonstrate that biofilm growth is catalyzed by the presence of hectorite in minimal growth media for the studied species: Gram negatives (Pseudomonas syringae and Escherichia coli,) and Gram positives (Staphylococcus aureus and Bacillus subtilis). Soil organisms (P. syringae, B. subtilis) and organisms found in the human population (E. coli, S. aureus) are both used to demonstrate the general applicability of clay involvement. Fluorescent images of the biofilms are acquired by staining with propidium iodide, a component of the BacLightTM Live/Dead bacterial viability staining kit (Molecular Probes, Eugene, OR). The evolving polysaccharide-rich biofilm reacts with the clay interlayer site causing a complex substitution of the two-water hectorite interlayer with polysaccharide. The result is often a three-peak composite of the (001) x-ray diffraction maxima resulting from polysaccharide-expanded clays and an organic-driven contraction of a subset of the clays in the reaction medium. X-ray diffractograms reveal that the expanded set creates a broad maximum with clay subsets at 1.84 nm and 1.41 nm interlayer spacings as approximated by a least squares double Lorentzian fit, and a smaller shoulder at larger 2q, deriving from a contraction of the interlayer spacing. Washing with chlorox removes organic material from the contracted clay and creates a 1-water hectorite single peak in place of the double peak. The clay response can be used as an indirect indicator of biofilm in an environmental system.
B33A-0866
Heavy Metal Contaminated Soils in Riverside Park, Milwaukee, WI: Character, Bioavailability, and Distribution
Prior to being breached in 1990, the North Avenue Dam on the Milwaukee River had created a 2.5-mile impoundment for over 150 years. Upstream urban runoff and industrial pollution resulted in the deposition of heavy metal rich sediments in the slow moving waters of the impoundment. After the dam removal, the river returned to a more natural flowpath and as the river narrowed, newly exposed riverbed was annexed as part of Riverside Park, enabling ecological recovery efforts on the river and riparian zones. However, these newly exposed soils are enriched with heavy metal contaminants, most notably, Pb, Zn, Cd, Cu, and Ni, concentrated by the impoundment. The current study has analyzed the location and concentrations of these trace metals, as well as their mobility and availability. This study is being conducted in conjunction with the Urban Ecology Center, a nonprofit environmental organization located in Riverside Park that is dedicated to serving the local community and urban youth while restoring and protecting the natural areas along the Milwaukee River. Analyses have included determination of general soil parameters such as particle size, organic content, and point of zero charge analyses. Beyond bulk chemical analysis, we have conducted selective sequential extractions to estimate the chemical speciation of these elements, which showed that approximately 30 percent of contaminants are highly available. Additionally, the soils have been analyzed with an Electron Microprobe to directly observe phase relationships of metals in the soils. Microprobe and other analyses have shown that heavy metals are associated with a variety of phases, including Mn and Fe oxy-hydroxides, and vary in concentration and phase relationships with depth and distance from the river. Finally, a field-portable x-ray fluorescence spectrometer (pXRF), coupled with GPS data, is being used to create a geochemical map of heavy metal distributions throughout the park.