Lithogenic hydrogen supports microbial primary production in subglacial and proglacial environments.

Life in environments devoid of photosynthesis, such as on early Earth or in contemporary dark subsurface ecosystems, is supported by chemical energy. How, when, and where chemical nutrients released from the geosphere fuel chemosynthetic biospheres is fundamental to understanding the distribution and diversity of life, both today and in the geologic past. Hydrogen (H2) is a potent reductant that can be generated when water interacts with reactive components of mineral surfaces such as silicate radicals and ferrous iron. Such reactive mineral surfaces are continually generated by physical comminution of bedrock by glaciers. Here, we show that dissolved H2 concentrations in meltwaters from an iron and silicate mineral-rich basaltic glacial catchment were an order of magnitude higher than those from a carbonate-dominated catchment. Consistent with higher H2 abundance, sediment microbial communities from the basaltic catchment exhibited significantly shorter lag times and faster rates of net H2 oxidation and dark carbon dioxide (CO2) fixation than those from the carbonate catchment, indicating adaptation to use H2 as a reductant in basaltic catchments. An enrichment culture of basaltic sediments provided with H2, CO2, and ferric iron produced a chemolithoautotrophic population related to Rhodoferax ferrireducens with a metabolism previously thought to be restricted to (hyper)thermophiles and acidophiles. These findings point to the importance of physical and chemical weathering processes in generating nutrients that support chemosynthetic primary production. Furthermore, they show that differences in bedrock mineral composition can influence the supplies of nutrients like H2 and, in turn, the diversity, abundance, and activity of microbial inhabitants.

Environmental predictors of electroactive bacterioplankton in small boreal lakes.

Extracellular electron transfer (EET) by electroactive bacteria in anoxic soils and sediments is an intensively researched subject, but EET's function in planktonic ecology has been less considered. Following the discovery of an unexpectedly high prevalence of EET genes in a bog lake's bacterioplankton, we hypothesized that the redox capacities of dissolved organic matter (DOM) enrich for electroactive bacteria by mediating redox chemistry. We developed the bioinformatics pipeline FEET (Find EET) to identify and summarize predicted EET protein-encoding genes from metagenomics data. We then applied FEET to 36 bog and thermokarst lakes and correlated gene occurrence with environmental data to test our predictions. Our results provide indirect evidence that DOM may participate in bacterioplankton EET. We found a similarly high prevalence of genes encoding putative EET proteins in most of these lakes, where oxidative EET strongly correlated with DOM. Numerous novel clusters of multiheme cytochromes that may enable EET were identified. Taxa previously not considered EET-capable were found to carry EET genes. We propose that EET and DOM interactions are of ecologically important to bacterioplankton in small boreal lakes, and that EET, particularly by methylotrophs and anoxygenic phototrophs, should be further studied and incorporated into methane emission models of melting permafrost.

In Situ Measurements of Dynamic Bacteria Transport and Attachment in Heterogeneous Sand-Packed Columns.

Prevention, mitigation, and regulation of bacterial contaminants in groundwater require a fundamental understanding of the mechanisms of transport and attachment in complex geological materials. Discrepancies in bacterial transport behaviors observed between field studies and laboratory experiments indicate an incomplete understanding of dynamic bacterial transport and immobilization processes in realistic heterogeneous geologic systems. Here, we develop a new experimental approach for in situ quantification of dynamic bacterial transport and attachment distribution in geologic media that relies on radiolabelingEscherichia coliwith positron-emitting radioisotopes and quantifying transport with three-dimensional (3D) positron emission tomography (PET) imaging. Our results indicate that the highest bacterial attachment occurred at the interfaces between sand layers oriented orthogonal to the direction of flow. The predicted bacterial attachment from a 3D numerical model matched the experimental PET results, highlighting that the experimentally observed bacterial transport behavior can be accurately captured with a distribution of a first-order irreversible attachment model. This is the first demonstration of the direct measurement of attachment coefficient distributions from bacterial transport experiments in geologic media and provides a transformational approach to better understand bacterial transport mechanisms, improve model parametrization, and accurately predict how local geologic conditions can influence bacterial fate and transport in groundwater.

Vertical Hydrologic Exchange Flows Control Methane Emissions from Riverbed Sediments.

CH4 emissions from inland waters are highly uncertain in the current global CH4 budget, especially for streams, rivers, and other lotic systems. Previous studies have attributed the strong spatiotemporal heterogeneity of riverine CH4 to environmental factors such as sediment type, water level, temperature, or particulate organic carbon abundance through correlation analysis. However, a mechanistic understanding of the basis for such heterogeneity is lacking. Here, we combine sediment CH4 data from the Hanford reach of the Columbia River with a biogeochemical-transport model to show that vertical hydrologic exchange flows (VHEFs), driven by the difference between river stage and groundwater level, determine CH4 flux at the sediment-water interface. CH4 fluxes show a nonlinear relationship with the magnitude of VHEFs, where high VHEFs introduce O2 into riverbed sediments, which inhibit CH4 production and induce CH4 oxidation, and low VHEFs cause transient reduction in CH4 flux (relative to production) due to reduced advective CH4 transport. In addition, VHEFs lead to the hysteresis of temperature rise and CH4 emissions because high river discharge caused by snowmelt in spring leads to strong downwelling flow that offsets increasing CH4 production with temperature rise. Our findings reveal how the interplay between in-stream hydrologic flux besides fluvial-wetland connectivity and microbial metabolic pathways that compete with methanogenic pathways can produce complex patterns in CH4 production and emission in riverbed alluvial sediments.

Microbial chemolithotrophy mediates oxidative weathering of granitic bedrock.

The flux of solutes from the chemical weathering of the continental crust supplies a steady supply of essential nutrients necessary for the maintenance of Earth's biosphere. Promotion of weathering by microorganisms is a well-documented phenomenon and is most often attributed to heterotrophic microbial metabolism for the purposes of nutrient acquisition. Here, we demonstrate the role of chemolithotrophic ferrous iron [Fe(II)]-oxidizing bacteria in biogeochemical weathering of subsurface Fe(II)-silicate minerals at the Luquillo Critical Zone Observatory in Puerto Rico. Under chemolithotrophic growth conditions, mineral-derived Fe(II) in the Rio Blanco Quartz Diorite served as the primary energy source for microbial growth. An enrichment in homologs to gene clusters involved in extracellular electron transfer was associated with dramatically accelerated rates of mineral oxidation and adenosine triphosphate generation relative to sterile diorite suspensions. Transmission electron microscopy and energy-dispersive spectroscopy revealed the accumulation of nanoparticulate Fe-oxyhydroxides on mineral surfaces only under biotic conditions. Microbially oxidized quartz diorite showed greater susceptibility to proton-promoted dissolution, which has important implications for weathering reactions in situ. Collectively, our results suggest that chemolithotrophic Fe(II)-oxidizing bacteria are likely contributors in the transformation of rock to regolith.

Electron acceptor availability alters carbon and energy metabolism in a thermoacidophile.

The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe3+ ) or oxygen (O2 ) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H2 ) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe3+ or O2 grown cells. During growth on S° or Fe3+ with H2 as an electron donor, the amount of CO2 assimilated into biomass decreased when cultures were provided with acetate. The addition of CO2 to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H2 /Fe3+ grown cells but had no effect on H2 /S° grown cells. In acetate/Fe3+ grown cells, the presence of H2 decreased the amount of acetate mineralized as CO2 in cultures compared to those without H2 . These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H2 to cultures overcomes this limitation and alters heterotrophic metabolism.

Stable Isotope Probing for Microbial Iron Reduction in Chocolate Pots Hot Spring, Yellowstone National Park.

Chocolate Pots hot springs (CP) is a circumneutral-pH Fe-rich geothermal feature located in Yellowstone National Park. Previous Fe(III)-reducing enrichment culture studies with CP sediments identified close relatives of known dissimilatory Fe(III)-reducing bacterial (FeRB) taxa, including Geobacter and Melioribacter However, the abundances and activities of such organisms in the native microbial community are unknown. Here, we used stable isotope probing experiments combined with 16S rRNA gene amplicon and shotgun metagenomic sequencing to gain an understanding of the in situ Fe(III)-reducing microbial community at CP. Fe-Si oxide precipitates collected near the hot spring vent were incubated with unlabeled and 13C-labeled acetate to target active FeRB. We searched reconstructed genomes for homologs of genes involved in known extracellular electron transfer (EET) systems to identify the taxa involved in Fe redox transformations. Known FeRB taxa containing putative EET systems (Geobacter, Ignavibacteria) increased in abundance under acetate-amended conditions, whereas genomes related to Ignavibacterium and Thermodesulfovibrio that contained putative EET systems were recovered from incubations without electron donor. Our results suggest that FeRB play an active role in Fe redox cycling within Fe-Si oxide-rich deposits located at the hot spring vent.IMPORTANCE The identification of past near-surface hydrothermal environments on Mars emphasizes the importance of using modern Earth environments, such as CP, to gain insight into potential Fe-based microbial life on other rocky worlds, as well as ancient Fe-rich Earth ecosystems. By combining stable carbon isotope probing techniques and DNA sequencing technology, we gained insight into the pathways of microbial Fe redox cycling at CP. The results suggest that microbial Fe(III) oxide reduction is prominent in situ, with important implications for the generation of geochemical and stable Fe isotopic signatures of microbial Fe redox metabolism within Fe-rich circumneutral-pH thermal spring environments on Earth and Mars.

Van Krevelen diagram visualization of high resolution-mass spectrometry metabolomics data with OpenVanKrevelen.

INTRODUCTION: Van Krevelen (VK) diagrams provide a promising but uncommon solution to a number of challenges associated with the visualization of metabolomics data. VK diagrams are created by plotting H:C ratios against O:C ratios of the compounds in a chemical mixture. OBJECTIVES: The aim of this manuscript is to present an open-source software tool and reference map that we have developed to make VK diagrams for visualization of metabolomics data. METHODS: Software was created with a prompt-driven command line user interface and was written using Python 2.7. We empirically derived an accompanying map by plotting where compounds from seven biomolecule types fall within the VK plot space. RESULTS: We've created an easy to use, open source software tool named OpenVanKrevelen for making a range of VK diagrams that is available on GitHub: https://github.com/HegemanLab/VanKrevelenLocal . The empirical mapping approach has produced several improvements from previously published maps. CONCLUSIONS: OpenVanKrevelen provides the metabolomics community with access to a new tool for visualization of complex metabolomics datasets.

Dual Role of Humic Substances As Electron Donor and Shuttle for Dissimilatory Iron Reduction.

Dissimilatory iron-reducing bacteria (DIRB) are known to use humic substances (HS) as electron shuttles for dissimilatory iron reduction (DIR) by transferring electrons to HS-quinone moieties, which in turn rapidly reduce Fe(III) oxides. However, the potential for HS to serve as a source of organic carbon (OC) that can donate electrons for DIR is unknown. We studied whether humic acids (HA) and humins (HM) recovered from peat soil by sodium pyrophosphate extraction could serve as both electron shuttles and electron donors for DIR by freshwater sediment microorganisms. Both HA and HM served as electron shuttles in cultures amended with glucose. However, only HA served as an electron donor for DIR. Metagenomes from HA-containing cultures had an overrepresentation of genes involved in polysaccharide and to a lesser extent aromatic compound degradation, suggesting complex OC metabolism. Genomic searches for the porin-cytochrome complex involved in DIR resulted in matches to Ignavibacterium/Melioribacter, DIRB capable of polymeric OC metabolism. These results indicate that such taxa may have played a role in both DIR and decomposition of complex OC. Our results suggest that decomposition of HS coupled to DIR and other anaerobic pathways could play an important role in soil and sediment OC metabolism.

Stability of Ferrihydrite-Humic Acid Coprecipitates under Iron-Reducing Conditions.

Recent studies have suggested the potential for release of iron (hydr)oxide-bound organic carbon (OC) during dissimilatory iron oxide reduction (DIR). However, the stability of iron (hydr)oxide-bound OC in the presence of a natural microbial consortium capable of driving both OC metabolism and DIR has not been resolved. Pure ferrihydrite (Fhy) and Fhy-humic acid coprecipitates (Fhy-HA) were inoculated with a small quantity of freshwater sediment and incubated under anoxic conditions in the presence and absence of H2 or glucose as electron donors for DIR. H2 promoted DIR led to release of ca. 1 mM dissolved organic carbon (DOC). However, comparable amounts of DOC were released from both pure Fhy and Fhy-HA, similar to DOC levels in mineral-free, inoculum-only controls. These results suggest that the observed DOC release during H2-promoted DIR originated from OC contained in the inoculum as opposed to the much larger pool (ca. 38 mM) of OC in the Fhy-HA. Thus, DIR preferentially released sorbed OC with low aromaticity (inoculum OC) versus highly aromatic OC (HA) coprecipitated with iron oxide. Our findings provide new insight into the extent and mechanisms by which DIR is likely to influence aqueous/solid-phase OC partitioning in anoxic soils and sediments.

Colonization Habitat Controls Biomass, Composition, and Metabolic Activity of Attached Microbial Communities in the Columbia River Hyporheic Corridor.

Hydrologic exchange plays a critical role in biogeochemical cycling within the hyporheic zone (the interface between river water and groundwater) of riverine ecosystems. Such exchange may set limits on the rates of microbial metabolism and impose deterministic selection on microbial communities that adapt to dynamically changing dissolved organic carbon (DOC) sources. This study examined the response of attached microbial communities (in situ colonized sand packs) from groundwater, hyporheic, and riverbed habitats within the Columbia River hyporheic corridor to "cross-feeding" with either groundwater, river water, or DOC-free artificial fluids. Our working hypothesis was that deterministic selection during in situ colonization would dictate the response to cross-feeding, with communities displaying maximal biomass and respiration when supplied with their native fluid source. In contrast to expectations, the major observation was that the riverbed colonized sand had much higher biomass and respiratory activity, as well as a distinct community structure, compared with those of the hyporheic and groundwater colonized sands. 16S rRNA gene amplicon sequencing revealed a much higher proportion of certain heterotrophic taxa as well as significant numbers of eukaryotic algal chloroplasts in the riverbed colonized sand. Significant quantities of DOC were released from riverbed sediment and colonized sand, and separate experiments showed that the released DOC stimulated respiration in the groundwater and piezometer colonized sand. These results suggest that the accumulation and degradation of labile particulate organic carbon (POC) within the riverbed are likely to release DOC, which may enter the hyporheic corridor during hydrologic exchange, thereby stimulating microbial activity and imposing deterministic selective pressure on the microbial community composition.IMPORTANCE The influence of river water-groundwater mixing on hyporheic zone microbial community structure and function is an important but poorly understood component of riverine biogeochemistry. This study employed an experimental approach to gain insight into how such mixing might be expected to influence the biomass, respiration, and composition of hyporheic zone microbial communities. Colonized sands from three different habitats (groundwater, river water, and hyporheic) were "cross-fed" with either groundwater, river water, or DOC-free artificial fluids. We expected that the colonization history would dictate the response to cross-feeding, with communities displaying maximal biomass and respiration when supplied with their native fluid source. By contrast, the major observation was that the riverbed communities had much higher biomass and respiration, as well as a distinct community structure compared with those of the hyporheic and groundwater colonized sands. These results highlight the importance of riverbed microbial metabolism in organic carbon processing in hyporheic corridors.

Metagenomic Analyses of the Autotrophic Fe(II)-Oxidizing, Nitrate-Reducing Enrichment Culture KS.

Nitrate-dependent ferrous iron [Fe(II)] oxidation (NDFO) is a well-recognized chemolithotrophic pathway in anoxic sediments. The neutrophilic chemolithoautotrophic enrichment culture KS originally obtained from a freshwater sediment (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl Environ Microbiol 62:1458-1460, 1996) has been used as a model system to study NDFO. However, the primary Fe(II) oxidizer in this culture has not been isolated, despite extensive efforts to do so. Here, we present a metagenomic analysis of this enrichment culture in order to gain insight into electron transfer pathways and the roles of different bacteria in the culture. We obtained a near-complete genome of the primary Fe(II) oxidizer, a species in the family Gallionellaceae, and draft genomes from its flanking community members. A search of the putative extracellular electron transfer pathways in these genomes led to the identification of a homolog of the MtoAB complex [a porin-multiheme cytochromec system identified in neutrophilic microaerobic Fe(II)-oxidizing Sideroxydans lithotrophicus ES-1] in a Gallionellaceae sp., and findings of other putative genes involving cytochromecand multicopper oxidases, such as Cyc2 and OmpB. Genome-enabled metabolic reconstruction revealed that this Gallionellaceae sp. lacks nitric oxide and nitrous oxide reductase genes and may partner with flanking populations capable of complete denitrification to avoid toxic metabolite accumulation, which may explain its resistance to growth in pure culture. This and other revealed interspecies interactions and metabolic interdependencies in nitrogen and carbon metabolisms may allow these organisms to cooperate effectively to achieve robust chemolithoautotrophic NDFO. Overall, the results significantly expand our knowledge of NDFO and suggest a range of genetic targets for further exploration.

Influence of Oxygen and Nitrate on Fe (Hydr)oxide Mineral Transformation and Soil Microbial Communities during Redox Cycling.

Oscillations between reducing and oxidizing conditions are observed at the interface of anaerobic/oxic and anaerobic/anoxic environments, and are often stimulated by an alternating flux of electron donors (e.g., organic carbon) and electron acceptors (e.g., O2 and NO3(-)). In iron (Fe) rich soils and sediments, these oscillations may stimulate the growth of both Fe-reducing bacteria (FeRB) and Fe-oxidizing bacteria (FeOB), and their metabolism may induce cycling between Fe(II) and Fe(III), promoting the transformation of Fe (hydr)oxide minerals. Here, we examine the mineralogical evolution of lepidocrocite and ferrihydrite, and the adaptation of a natural microbial community to alternating Fe-reducing (anaerobic with addition of glucose) and Fe-oxidizing (with addition of nitrate or air) conditions. The growth of FeRB (e.g., Geobacter) is stimulated under anaerobic conditions in the presence of glucose. However, the abundance of these organisms depends on the availability of Fe(III) (hydr)oxides. Redox cycling with nitrate results in decreased Fe(II) oxidation thereby decreasing the availability of Fe(III) for FeRB. Additionally, magnetite is detected as the main product of both lepidocrocite and ferrihydrite reduction. In contrast, introduction of air results in increased Fe(II) oxidation, increasing the availability of Fe(III) and the abundance of Geobacter. In the lepidocrocite reactors, Fe(II) oxidation by dissolved O2 promotes the formation of ferrihydrite and lepidocrocite, whereas in the ferrihydrite reactors we observe a decrease in magnetite stoichiometry (e.g., oxidation). Understanding Fe (hydr)oxide transformation under environmentally relevant redox cycling conditions provides insight into nutrient availability and transport, contaminant mobility, and microbial metabolism in soils and sediments.

Biochar-Facilitated Microbial Reduction of Hematite.

As an important component of soil organic matter (SOM), the transformation of pyrogenic carbon plays a critical role in the biogeochemical cycles of carbon and other redox-active elements such as iron (Fe). Herein, we studied the influences of wheat straw-derived biochars on the microbial reduction of 100 mM of hematite by the dissimilatory metal reducing bacteria Shewanella oneidensis MR-1 under anoxic conditions. The long-term microbial reduction extent and initial reduction rate of hematite were accelerated by more than 2-fold in the presence of 10 mg L(-1) biochar. Soluble leachate from 10 mg L(-1) biochar enhanced Fe(III) reduction to a similar degree. Microbially prereduced biochar leachate abiotically reduced hematite, consistent with the apparent electron shuttling capacity of biochar leachate. Electron paramagnetic resonance (EPR) analysis suggested that biochar leachate-associated semiquinone functional groups were likely involved in the redox reactions. In addition to electron shuttling effects, biochar particles sorbed 0.5-1.5 mM biogenic Fe(II) and thereby increased the long-term extent of hematite reduction by 1.4-1.7 fold. Our results suggest that Fe redox cycling may be strongly impacted by pyrogenic carbon in soils with relatively high content of indigenous pyrogenic carbon or substantial application of biochar.

Iron Isotope Fractionations Reveal a Finite Bioavailable Fe Pool for Structural Fe(III) Reduction in Nontronite.

We report on stable Fe isotope fractionation during microbial and chemical reduction of structural Fe(III) in nontronite NAu-1. (56)Fe/(54)Fe fractionation factors between aqueous Fe(II) and structural Fe(III) ranged from -1.2 to +0.8‰. Microbial (Shewanella oneidensis and Geobacter sulfurreducens) and chemical (dithionite) reduction experiments revealed a two-stage process. Stage 1 was characterized by rapid reduction of a finite Fe(III) pool along the edges of the clay particles, accompanied by a limited release to solution of Fe(II), which partially adsorbed onto basal planes. Stable Fe isotope compositions revealed that electron transfer and atom exchange (ETAE) occurred between edge-bound Fe(II) and octahedral (structural) Fe(III) within the clay lattice, as well as between aqueous Fe(II) and structural Fe(III) via a transient sorbed phase. The isotopic fractionation factors decreased with increasing extent of reduction as a result of the depletion of the finite bioavailable Fe(III) pool. During stage 2, microbial reduction was inhibited while chemical reduction continued. However, further ETAE between aqueous Fe(II) and structural Fe(III) was not observed. Our results imply that the pool of bioavailable Fe(III) is restricted to structural Fe sites located near the edges of the clay particles. Blockage of ETAE distinguishes Fe(III) reduction of layered clay minerals from that of Fe oxyhydroxides, where accumulation of structural Fe(II) is much more limited.

Aerobic and Anaerobic Thiosulfate Oxidation by a Cold-Adapted, Subglacial Chemoautotroph.

Geochemical data indicate that protons released during pyrite (FeS2) oxidation are important drivers of mineral weathering in oxic and anoxic zones of many aquatic environments, including those beneath glaciers. Oxidation of FeS2 under oxic, circumneutral conditions proceeds through the metastable intermediate thiosulfate (S2O3 (2-)), which represents an electron donor capable of supporting microbial metabolism. Subglacial meltwaters sampled from Robertson Glacier (RG), Canada, over a seasonal melt cycle revealed concentrations of S2O3 (2-) that were typically below the limit of detection, despite the presence of available pyrite and concentrations of the FeS2 oxidation product sulfate (SO4 (2-)) several orders of magnitude higher than those of S2O3 (2-). Here we report on the physiological and genomic characterization of the chemolithoautotrophic facultative anaerobe Thiobacillus sp. strain RG5 isolated from the subglacial environment at RG. The RG5 genome encodes genes involved with pathways for the complete oxidation of S2O3 (2-), CO2 fixation, and aerobic and anaerobic respiration with nitrite or nitrate. Growth experiments indicated that the energy required to synthesize a cell under oxygen- or nitrate-reducing conditions with S2O3 (2-) as the electron donor was lower at 5.1°C than 14.4°C, indicating that this organism is cold adapted. RG sediment-associated transcripts of soxB, which encodes a component of the S2O3 (2-)-oxidizing complex, were closely affiliated with soxB from RG5. Collectively, these results suggest an active sulfur cycle in the subglacial environment at RG mediated in part by populations closely affiliated with RG5. The consumption of S2O3 (2-) by RG5-like populations may accelerate abiotic FeS2 oxidation, thereby enhancing mineral weathering in the subglacial environment.

Geochemical and microbiological responses to oxidant introduction into reduced subsurface sediment from the Hanford 300 Area, Washington.

Pliocene-aged reduced lacustrine sediment from below a subsurface redox transition zone at the 300 Area of the Hanford site (southeastern Washington) was used in a study of the geochemical response to introduction of oxygen or nitrate in the presence or absence of microbial activity. The sediments contained large quantities of reduced Fe in the form of Fe(II)-bearing phyllosilicates, together with smaller quantities of siderite and pyrite. A loss of ca. 50% of 0.5 M HCl-extractable Fe(II) [5-10 mmol Fe(II) L(-1)] and detectable generation of sulfate (ca. 0.2 mM, equivalent to 10% of the reduced inorganic sulfur pool) occurred in sterile aerobic reactors. In contrast, no systematic loss of Fe(II) or production of sulfate was observed in any of the other oxidant-amended sediment suspensions. Detectable Fe(II) accumulation and sulfate consumption occurred in non-sterile oxidant-free reactors. Together, these results indicate the potential for heterotrophic carbon metabolism in the reduced sediments, consistent with the proliferation of known heterotrophic taxa (e.g., Pseudomonadaceae, Burkholderiaceae, and Clostridiaceae) inferred from 16S rRNA gene pyrosequencing. Microbial carbon oxidation by heterotrophic communities is likely to play an important role in maintaining the redox boundary in situ, i.e., by modulating the impact of downward oxidant transport on Fe/S redox speciation. Diffusion-reaction simulations of oxygen and nitrate consumption coupled to solid-phase organic carbon oxidation indicate that heterotrophic consumption of oxidants could maintain the redox boundary at its current position over millennial time scales.

U(VI) reduction in sulfate-reducing subsurface sediments amended with ethanol or acetate.

An experiment was conducted with subsurface sediments from Oak Ridge National Laboratory to determine the potential for reduction of U(VI) under sulfate-reducing conditions with either ethanol or acetate as the electron donor. The results showed extensive U(VI) reduction in sediments supplied with either electron donor, where geochemical and microbiological analyses demonstrated active sulfate reduction.

Microbial iron-redox cycling in subsurface environments.

In addition to its central role in mediating electron-transfer reactions within all living cells, iron undergoes extracellular redox transformations linked to microbial energy generation through utilization of Fe(II) as a source of chemical energy or Fe(III) as an electron acceptor for anaerobic respiration. These processes permit microbial populations and communities to engage in cyclic coupled iron oxidation and reduction within redox transition zones in subsurface environments. In the present paper, I review and synthesize a few case studies of iron-redox cycling in subsurface environments, highlighting key biochemical aspects of the extracellular iron-redox metabolisms involved. Of specific interest are the coupling of iron oxidation and reduction in field and experimental systems that model redox gradients and fluctuations in the subsurface, and novel pathways and organisms involved in the redox cycling of insoluble iron-bearing minerals. These findings set the stage for rapid expansion in our knowledge of the range of extracellular electron-transfer mechanisms utilized by subsurface micro-organisms. The observation that closely coupled oxidation and reduction of iron can take place under conditions common to the subsurface motivates this expansion in pursuit of molecular tools for studying iron-redox cycling communities in situ.

Microbial lithotrophic oxidation of structural Fe(II) in biotite.

Microorganisms are known to participate in the weathering of primary phyllosilicate minerals through the production of organic ligands and acids and through the uptake of products of weathering. Here we show that the lithotrophic Fe(II)-oxidizing, nitrate-reducing enrichment culture described by Straub et al. (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl. Environ. Microbiol. 62:1458-1460, 1996) can grow via oxidation of structural Fe(II) in biotite, a Fe(II)-rich trioctahedral mica found in granitic rocks. Oxidation of silt/clay-sized biotite particles was detected by a decrease in extractable Fe(II) content and simultaneous nitrate reduction. Mössbauer spectroscopy confirmed structural Fe(II) oxidation. Approximately 1.5 × 10(7) cells were produced per µmol of Fe(II) oxidized, in agreement with previous estimates of the growth yield of lithoautotrophic circumneutral-pH Fe(II)-oxidizing bacteria. Microbial oxidation of structural Fe(II) resulted in biotite alterations similar to those found in nature, including a decrease in the unit cell b dimension toward dioctahedral levels and Fe and K release. Structural Fe(II) oxidation may involve either direct enzymatic oxidation, followed by solid-state mineral transformation, or indirect oxidation as a result of the formation of aqueous Fe, followed by electron transfer from Fe(II) in the mineral to Fe(III) in solution. Although it is not possible to distinguish between these two mechanisms with available data, the complete absence of aqueous Fe in oxidation experiments favors the former alternative. The demonstration of microbial oxidation of structural Fe(II) suggests that microorganisms are directly responsible for the initial step in the weathering of biotite in granitic aquifers and the plant rhizosphere.