B24D-01
Manganese Dependent Anaerobic Oxidation of Methane
Understanding the anaerobic oxidation is not only important for understanding hydrocarbon degradation but it also important for understanding the global carbon cycle. The anaerobic oxidation of methane (AOM) is a large sink for methane consuming 5-20% of today's methane flux (Valentine and Reeburgh, 2000), yet the requirements for this process are not well understood. It has been suggested that no other electron acceptors other than sulfate can be used in the AOM (Nauhaus, 2005). However, our new data suggests that manganese, in the form of birnessite, can be used as an electron acceptor instead of sulfate (Beal et al., in prep). Methane seep sediment from the Eel River Basin, CA was incubated with methane, 13C-labeled methane, and carbon dioxide. Because the net result of the AOM is the production of carbon dioxide from methane, the rate of the AOM in each of the incubations can be determined by measuring the incorporation of 13C in the carbon dioxide. Using this method, it was found that cultures incubated with nitrate showed inhibition of the AOM, while cultures incubated with iron gave inconclusive results. The only positive results that were found for alternate electron acceptors are the incubations that were given manganese and no sulfate, which showed methane oxidation. Further, when more manganese was injected into these incubations, the rate of AOM increased. Preliminary analysis of the microbial population using terminal restriction fragment length polymorphism (TRFLP) targeting the mcr gene showed an unidentified organism in these cultures. Future work with TRFLP, as well as clone libraries, will help to identify the organisms responsible for this process. Nauhaus, K., 2005, Environmental regulation of the anaerobic oxidation of methane: a comparison of ANME-I and ANME-II communities: Environmental microbiology, v. 7, p. 98. Valentine, D.L., and Reeburgh, W.S., 2000, New perspectives on anaerobic methane oxidation: Environmental Microbiology, v. 2, p. 477-484.
B24D-02
Carbon and Manganese Cycling in the Columbia River's Estuarine Turbidity Maxima in the South Channel
The Columbia River represents the largest input (60-90%) of fresh water to the California Current System, and provides a major source of dissolved manganese and nutrients to the coastal waters. Researchers have identified upper Estuarine Turbidity Maxima (ETM(s)) as hot spots for microbial activity, and it is here that extensive manganese cycling is thought to occur. Most probable number counts of microorganisms within the ETM have revealed that the cultivable numbers of manganese-oxidizing bacteria are not statistically significantly different than that of other heterotrophs when grown on defined media with simple carbon sources or low concentrations (0.05%) of casamino acids and were in the range of 103 - 104 cells per mL. Similar numbers of heterotrophs (9.3 X 103 cells/mL) were found using a nutrient-rich complex medium; however, the numbers of manganese-oxidizers were significantly lower (~13 cells/mL). Approximately 100 different manganese-oxidizing bacteria were isolated from different media and are being phylogenetically characterized. Measurements of dissolved, ascorbate-reducible and total Mn by inductively coupled plasma- optical emission spectroscopy revealed that concentrations of Mn are positively correlated with turbidity and thus are higher during an ETM event. In addition, dissolved, total, and ascorbate-reducible Mn were all negatively correlated with salinity, supporting the idea that the manganese originates in the river and is diluted by the seawater originating off the coast. Uptake of 14C-labeled bicarbonate in response to various electron donors (nitrite, ammonium, thiosulfate, or Mn(II)) was stimulated during an ETM event but not before or after, indicating that these electron donors may serve as potential energy sources for carbon fixation. Taken together, our results further demonstrate that ETMs are zones with high microbial activity and that the ETM microbial communities harbor the potential for carbon fixation even in the relatively nutrient-rich environment of the upper Columbia River estuary.
B24D-03
Diversity and As-adsorption properties of Mn(II)-oxidizing bacteria within tropical wetlands of the Mekong Delta
Manganese (Mn) and iron (Fe) oxides are ubiquitous minerals that occur under similar redox conditions in terrestrial systems and have high sorptive capacities for many trace metals, including arsenic (As). In most natural environments, Fe oxidation is dominated by abiotic processes, while Mn oxides are primarily formed via bacterial Mn(II) oxidation, and both processes can profoundly impact the mobility of metal(loid) contaminants. Deciphering the mechanisms involved in arsenic transport within soils and sediments is essential for aiding many Southeast Asian countries, including Cambodia, where naturally occurring As is in groundwater at concentrations well above the WHO recommended limit. Although numerous past studies have characterized the effects of As adsorption onto Fe and Mn oxides individually, it is unknown whether, in the presence of both oxides, there is preferential adsorption of As onto one oxide over the other. In the present study, we examine the competitive retention of As(III) and As(V) on goethite and biogenic Mn oxides using Donnan membranes--where each oxides is isolated by a semi-permeable membrane through which arsenic can migrate. Mn(II)-oxidizing bacteria, isolated from several Mn-rich sites along the Mekong River and wetland areas within the Mekong delta, were dominated by a diverse array of Bacillus strains that rapidly oxidize Mn(II) within three to five days in liquid culture. The results of this study not only expand our knowledge of the diversity and biogeochemical importance of terrestrial Mn(II)-oxidizing bacteria, but also contribute to our understanding the relative impact of Fe and Mn oxides on arsenic retention within natural wetlands.
B24D-04
The Integrated Field-Scale Subsurface Research Challenge Site (IFC) at Rifle, Colorado: Preliminary Results on Microbiological, Geochemical and Hydrologic Processes Controlling Iron Reduction and Uranium Mobility
The IFC at Rifle, Colorado was recently funded by the U.S. Department of Energy to address knowledge gaps in 1) geochemical and microbial controls on stimulated U(VI) bioreduction by iron-reducers, 2) U(VI) sorption under Fe-reducing conditions, 3) post-biostimulation U(VI) stability and removal, and 4) rates of natural bioreduction of U(VI). The over-arching goal of the project is to develop a mechanistic understanding of bioreductive and abiotic processes that control uranium mobility targeting new knowledge that can be translated into scientifically defensible flow and reactive transport process models. The Rifle IFC will conduct a focused set of field and lab experiments that use recently developed sciences of proteogenomics and stable isotope probing to track microbial metabolic status during acetate amendment. This information will be linked to changes in Fe redox status and sulfide minerals, with field-scale changes detected by non-invasive hydrogeophysics, including 3-D resistivity tomography. A key goal of the project is to combine abiotic sorption processes under reducing conditions with biotic processes controlling U(VI) reduction. The initial field-scale experiment for the Rifle IFC was conducted during the summer of 2007 with the objectives of collecting simultaneous metagenomic and proteomic samples during acetate amendment and to assess the impact of intentionally decreasing electron donor concentration on the metabolic processes of iron reducers. The 2007 experiment replicated previous field experiments, producing dominance of Geobacter sp. in groundwater within 10 days after the start of acetate amendment. The experiment also confirmed the importance of heterogeneities in controlling the flux of electron donor and the impact of naturally reduced zones on the duration of Fe reduction.
B24D-05
Sulfidogenesis Controls on Ferrihydrite Transformation and Repartitioning of Sorbed Arsenic
Iron (hydr)oxides are ubiquitous sorbents of arsenic (As) that undergo reductive dissolution and transformation upon reaction with dissolved sulfide. Here, we examine diverging pathways of solid phase iron (Fe) transformation during sulfate reduction in the presence of varying As loadings. Columns initially containing As(V)- ferrihydrite coated sand, inoculated with the sulfate reducing bacteria Desulfovibrio vulgaris (Hildenborough), were eluted with artificial groundwater containing sulfate and lactate. Additionally, abiotic batch reaction experiments were conducted to examine Fe secondary products rapidly formed during sulfidization of As-loaded ferrihydrite. Rapid and consistent sulfate reduction coupled with lactate oxidation is observed within column solids possessing low As(V) surface coverage (10% of the adsorption maximum). Column experiments illustrated that at high As(V) surface coverage (50% of the adsorption maximum), sulfate reduction and lactate oxidation are initially slow but gradually increase over time, and all As(V) is reduced to As(III) by the end of experimentation. The dominant Fe solid-phase transformation products at low As coverage include amorphous FeS within the zone of sulfate reduction (near the inlet of the column) and magnetite downstream where Fe(II)aq concentrations exceed 1 mM. Arsenic(V) is reduced to As(III) and displaced from the zone of sulfidogenesis and Fe(III)s depletion. At higher As coverage, green rust carbonate, as opposed to magnetite, is a dominant Fe solid phase product. Independent of loading, As is strongly associated with magnetite and residual ferrihydrite, while being excluded from green rust and iron sulfide. Abiotic batch reactor experiments illustrate that As is readily released from ferrihydrite during sulfidization, and that low As loadings yield initial Fe secondary products of lepidocrocite and FeS, while high loadings inhibit rapid secondary Fe mineral formation. Our observations illustrate that sulfidogenesis occurring in proximity with Fe (hydr)oxides may govern pathways of Fe solid phase transformation and As partitioning; formation of As sulfide minerals, in particular, is inhibited by reactive Fe either through sulfide oxidation or complexation.
B24D-06
Assessing the Geochemical Reactivity of Fe-DOM Complexes in Lacustrine Sediments Using Nitroaromatic Probe Compounds
The complexation environment of Fe in anoxic sediment porefluids is important for understanding its role in biogeochemical reactions in these systems. These include the microbial dissolution of iron-bearing sediments and the degradation of hydrophobic pesticides. We studied the effect of naturally occurring Fe-DOM complexes on the reduction of two nitroaromatic compounds (NACs) as a surrogate for probing electron-transfer kinetics, and monitored the geochemical parameters affecting reactivity. We found that Fe(II) was necessary for rapid NAC reduction (< 24 hr), and observed faster reduction with increased pH. NAC reduction in `pH-adjusted' porefluids (acidified to pH 2.5 after porefluid extraction and raised to the native pH (between 6.5 to 7.8) prior to reaction) was similar to that observed in model systems containing Fe(II) and aquatic fulvic acids. Conversely, NAC reduction in fresh, unaltered porefluids was slower than that observed in `pH-adjusted' porefluids, indicating that the natural metal redox speciation differs between fresh and `pH-adjusted' samples. Electrochemical scans demonstrate a vertical gradient in concentrations of Fe(III), Fe(II), and Mn(II) from 0 to 30 cm depth. Furthermore, the electrochemical data indicate that ferric iron in these systems is predominantly complexed with organic ligands, with a half-cell potential of -0.45 V. The magnitude of this ferric-organic peak decreases when porefluids are pH- adjusted for short durations (acidified to pH 2.5 and re-raised to circumneutral levels) and disappears completely when the acidified porefluid is allowed to equilibrate overnight. These data show that pH-adjustment of porefluids presumably alters both their complexation chemistry and reactivity towards NACs, and shows how small changes in Fe complexation chemistry potentially affects electron transfer reactions in anoxic environments.
B24D-07
Electrochemical Characterization Shewanella oneidensis () Mr-1 MtrABC
Dissimilatory iron-reducing bacteria have the ability to use a wide range of terminal electron acceptors including solid state iron (oxihydr)oxides. It is generally accepted that electrons are transferred by electron transfer proteins to a series of multiheme c-type cytochromes which enable the electron transport from the periplasm to the extracellular side of the outer cell membrane and across the bacteria-mineral interface to the terminal electron acceptor. In the last decade, the facultative anaerobe organism Shewanella oneidensis Mr-1 has been used as a model organism to identify, purify, and sequence single proteins involved in Fe(III) and Mn(IV) reduction, but these studies have provided little biochemical information on the actual electron transfer process within the bacterial cell. In order to extend the knowledge on electron transfer, Ross et al.(in press) have recently purified a complex from Shewanella oneidensis Mr-1 which includes the membrane proteins MtrA, MtrB, and MtrC and spans the space from the periplasm to the extracellular side of the outer membrane. In our study we applied the relatively new technique of protein film electrochemistry to the MtrABC complex to gain more biochemical information on electron transport in the membrane of Shewanella oneidensis Mr-1. A wealth of information on the reaction of redox-active sites in proteins like MtrABC can be acquired by voltammetric studies in which the protein sample is immobilized as a layer onto an electrode surface. By carrying out cyclic voltammetry over a wide range of scan rates, the data can be analyzed in terms of peak potentials versus scan rate. A simple reversible electron transfer process gives rise to a trumpet-shaped plot because the oxidation and the reduction peaks increasingly separate at high scan rate. In this contribution we show a detailed electrochemical picture of the MtrABC complex, which gives insight into the electron transfer from the periplasm to the extracellular side of the outer membrane of Shewanella oneidensis Mr-1. Such electrochemical analysis will help to understand how electrons are transferred to solid state electron acceptors such as ferrihydrite or goethite with different mineralogical and thermodynamic properties. Ross, D. E., Ruebush, S. S., Brantley, S. L., Hartshorne, R. S., Clarke, T. A., Richardson, D. J., and Tien, M., in press. Characterization of Protein/Protein Interactions Involved in Iron Reduction by Shewanella oneidensis MR-1. Applied and Environmental Microbiology.
B24D-08
Secondary Iron Mineral Formation by Shewanellae Using Different Carbon Sources
It has been known for some time that microbes play an important role in the redox cycling of iron. A considerable amount of work has been done investigating the various factors that play a role in solid phase iron reduction such as the type of iron oxide available for reduction, medium composition, surface area and ferrous iron concentration. However, the rate at which a single cell reduces iron oxides and the impact of dissimilatory iron reduction kinetics on mineral speciation are not well understood. In order to determine the relationship between iron oxide reduction rates and secondary mineral formation, we have tested 4 different strains of Shewanella with different carbon sources. Strains MR-1, MR-4, CN32 and W3-18-1 were incubated with HFO (hydrous ferric oxide) as electron acceptor and either lactate, pyruvate, isoleucine or uridine as the organic carbon source. Mineral products were analyzed using X-ray diffraction, electron microscopy and Mossbauer spectroscopy. Initial results suggest that reduction rates are similar among strains when using the same carbon source. However, qualitative assessment of mineral products suggests that while reduction rates for the tested strains may be similar, the secondary mineral products can be quite different.