Effect of Water Chemistry and Hydrodynamics on Nitrogen Transformation Activity and Microbial Community Functional Potential in Hyporheic Zone Sediment Columns.

Hyporheic zones (HZ) are active biogeochemical regions where groundwater and surface water mix. N transformations in HZ sediments were investigated in columns with a focus on understanding how the dynamic changes in groundwater and surface water mixing affect microbial community and its biogeochemical function with respect to N transformations. The results indicated that denitrification, DNRA, and nitrification rates and products changed quickly in response to changes in water and sediment chemistry, fluid residence time, and groundwater-surface water exchange. These changes were accompanied by the zonation of denitrification functional genes along a 30 cm advective flow path after a total of 6 days' elution of synthetic groundwater with fluid residence time >9.8 h. The shift of microbial functional potential toward denitrification was correlated with rapid NO3- reduction collectively affected by NO3- concentration and fluid residence time, and was resistant to short-term groundwater-surface water exchange on a daily basis. The results implied that variations in microbial functional potential and associated biogeochemical reactions in the HZ may occur at space scales where steep concentration gradients present along the flow path and the variations would respond to dynamic HZ water exchange over different time periods common to natural and managed riverine systems.

Internal Domains of Natural Porous Media Revealed: Critical Locations for Transport, Storage, and Chemical Reaction.

Internal pore domains exist within rocks, lithic fragments, subsurface sediments, and soil aggregates. These domains, termed internal domains in porous media (IDPM), represent a subset of a material's porosity, contain a significant fraction of their porosity as nanopores, dominate the reactive surface area of diverse media types, and are important locations for chemical reactivity and fluid storage. IDPM are key features controlling hydrocarbon release from shales in hydraulic fracture systems, organic matter decomposition in soil, weathering and soil formation, and contaminant behavior in the vadose zone and groundwater. Traditionally difficult to interrogate, advances in instrumentation and imaging methods are providing new insights on the physical structures and chemical attributes of IDPM, and their contributions to system behaviors. Here we discuss analytical methods to characterize IDPM, evaluate information on their size distributions, connectivity, and extended structures; determine whether they exhibit unique chemical reactivity; and assess the potential for their inclusion in reactive transport models. Ongoing developments in measurement technologies and sensitivity, and computer-assisted interpretation will improve understanding of these critical features in the future. Impactful research opportunities exist to advance understanding of IDPM, and to incorporate their effects in reactive transport models for improved environmental simulation and prediction.

Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals.

The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decaheme cytochromes, MtrC and MtrA, brought together inside a transmembrane porin, MtrB, to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system containing a pool of internalized electron carriers was used to investigate how the topology of the MtrCAB complex relates to its ability to transport electrons across a lipid bilayer to externally located Fe(III) oxides. With MtrA facing the interior and MtrC exposed on the outer surface of the phospholipid bilayer, the established in vivo orientation, electron transfer from the interior electron carrier pool through MtrCAB to solid-phase Fe(III) oxides was demonstrated. The rates were 10(3) times higher than those reported for reduction of goethite, hematite, and lepidocrocite by S. oneidensis, and the order of the reaction rates was consistent with those observed in S. oneidensis cultures. In contrast, established rates for single turnover reactions between purified MtrC and Fe(III) oxides were 10(3) times lower. By providing a continuous flow of electrons, the proteoliposome experiments demonstrate that conduction through MtrCAB directly to Fe(III) oxides is sufficient to support in vivo, anaerobic, solid-phase iron respiration.

(99)Tc(VII) Retardation, Reduction, and Redox Rate Scaling in Naturally Reduced Sediments.

An experimental and modeling study was conducted to investigate pertechnetate (Tc(VII)O4(-)) retardation, reduction, and rate scaling in three sediments from Ringold formation at U.S. Department of Energy's Hanford site, where (99)Tc is a major contaminant in groundwater. Tc(VII) was reduced in all the sediments in both batch reactors and diffusion columns, with a faster rate in a sediment containing a higher concentration of HCl-extractable Fe(II). Tc(VII) migration in the diffusion columns was reductively retarded with retardation degrees correlated with Tc(VII) reduction rates. The reduction rates were faster in the diffusion columns than those in the batch reactors, apparently influenced by the spatial distribution of redox-reactive minerals along transport paths that supplied Tc(VII). X-ray computed tomography and autoradiography were performed to identify the spatial locations of Tc(VII) reduction and transport paths in the sediments, and results generally confirmed the newly found behavior of reaction rate changes from batch to column. The results from this study implied that Tc(VII) migration can be reductively retarded at Hanford site with a retardation degree dependent on reactive Fe(II) content and its distribution in sediments. This study also demonstrated that an effective reaction rate may be faster in transport systems than that in well-mixed reactors.

Effect of subgrid heterogeneity on scaling geochemical and biogeochemical reactions: a case of U(VI) desorption.

The effect of subgrid heterogeneity in sediment properties on the rate of uranyl[U(VI)] desorption was investigated using a sediment collected from the U.S. Department of Energy Hanford site. The sediment was sieved into 7 grain size fractions that each exhibited different U(VI) desorption properties. Six columns were assembled using the sediment with its grain size fractions arranged in different spatial configurations to mimic subgrid heterogeneity in reactive transport properties. The apparent rate of U(VI) desorption varied significantly in the columns. Those columns with sediment structures leading to preferential transport had much lower rates of U(VI) desorption than those with relatively homogeneous transport. Modeling analysis indicated that the U(VI) desorption model and parameters characterized from well-mixed reactors significantly overpredicted the measured U(VI) desorption in the columns with preferential transport. A dual domain model, which operationally separates reactive transport properties into two subgrid domains, improved the predictions significantly. A similar effect of subgrid heterogeneity, albeit to a lesser degree, was observed for denitrification, which also occurred in the columns. The results imply that subgrid heterogeneity is an important consideration in extrapolating reaction rates from the laboratory to field.

Investigation of U(VI) adsorption in quartz-chlorite mineral mixtures.

A batch and cryogenic laser-induced time-resolved luminescence spectroscopy investigation of U(VI) adsorbed on quartz-chlorite mixtures with variable mass ratios have been performed under field-relevant uranium concentrations (5×10(-7) M and 5×10(-6) M) in pH 8.1 synthetic groundwater. The U(VI) adsorption Kd values steadily increased as the mass fraction of chlorite increased, indicating preferential sorption to chlorite. For all mineral mixtures, U(VI) adsorption Kd values were lower than that calculated from the assumption of component additivity possibly caused by surface modifications stemming from chlorite dissolution; The largest deviation occurred when the mass fractions of the two minerals were equal. U(VI) adsorbed on quartz and chlorite displayed characteristic individual luminescence spectra that were not affected by mineral mixing. The spectra of U(VI) adsorbed within the mixtures could be simulated by one surface U(VI) species on quartz and two on chlorite. The luminescence intensity decreased in a nonlinear manner as the adsorbed U(VI) concentration increased with increasing chlorite mass fraction-likely due to ill-defined luminescence quenching by both structural Fe/Cr in chlorite, and trace amounts of solubilized and reprecipitated Fe/Cr in the aqueous phase. However, the fractional spectral intensities of U(VI) adsorbed on quartz and chlorite followed the same trend of fractional adsorbed U(VI) concentration in each mineral phase with approximate linear correlations, offering a method to estimate of U(VI) concentration distribution between the mineral components with luminescence spectroscopy.

Structure of a bacterial cell surface decaheme electron conduit.

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split ß-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

The 'porin-cytochrome' model for microbe-to-mineral electron transfer.

Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species electrons cross the outer membrane to extracellular substrates via 'porin-cytochrome' electron transport modules. The molecular structure of an outer-membrane extracellular-facing deca-haem terminus for such a module has recently been resolved. It is debated how, once outside the cells, electrons are transferred from outer-membrane cytochromes to insoluble electron sinks. This may occur directly or by assemblies of cytochromes, perhaps functioning as 'nanowires', or via electron shuttles. Here we review recent work in this field and explore whether it allows for unification of the electron transport mechanisms supporting extracellular mineral respiration in Shewanella that may extend into other genera of Gram-negative bacteria.

A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella.

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H(2)O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately -240 mV) and low-spin (approximately -110, -190 and -265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (E(m) = -80 mV) in the presence of NADH (E(m) = -320 mV) and an NADH-menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.

Electron transport at the microbe-mineral interface: a synthesis of current research challenges.

Many bacterial and archaeal species can couple growth to the respiratory reduction or oxidation of insoluble mineral oxides of transition metals. These solid substrates are abundant electron sinks and sources for life on Earth, but, since they are insoluble in water, they cannot enter the bacterial cells. So, to exploit these electron sinks and sources, specific respiratory electron-transfer mechanisms must overcome the physical limitations associated with electron transfer between a microbe and extracellular metal oxides. Recent microbiological, geochemical, biochemical, spectroscopic and structural work is beginning to shed light on the molecular mechanism and impacts of electron transfer at the microbe-mineral interface from a nanometre to kilometre scale. The research field is attracting attention in applied quarters from those with interests in nanowires, microbial fuel cells, bioremediation and microbial cell factories.

Mtr extracellular electron-transfer pathways in Fe(III)-reducing or Fe(II)-oxidizing bacteria: a genomic perspective.

Originally discovered in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), key components of the Mtr (i.e. metal-reducing) pathway exist in all strains of metal-reducing Shewanella characterized. The protein components identified to date for the Mtr pathway of MR-1 include four multihaem c-Cyts (c-type cytochromes), CymA, MtrA, MtrC and OmcA, and a porin-like outer membrane protein MtrB. They are strategically positioned along the width of the MR-1 cell envelope to mediate electron transfer from the quinone/quinol pool in the inner membrane to Fe(III)-containing minerals external to the bacterial cells. A survey of microbial genomes has identified homologues of the Mtr pathway in other dissimilatory Fe(III)-reducing bacteria, including Aeromonas hydrophila, Ferrimonas balearica and Rhodoferax ferrireducens, and in the Fe(II)-oxidizing bacteria Dechloromonas aromatica RCB, Gallionella capsiferriformans ES-2 and Sideroxydans lithotrophicus ES-1. The apparent widespread distribution of Mtr pathways in both Fe(III)-reducing and Fe(II)-oxidizing bacteria suggests a bidirectional electron transfer role, and emphasizes the importance of this type of extracellular electron-transfer pathway in microbial redox transformation of iron. The organizational and electron-transfer characteristics of the Mtr pathways may be shared by other pathways used by micro-organisms for exchanging electrons with their extracellular environments.

Analysis of structural MtrC models based on homology with the crystal structure of MtrF.

The outer-membrane decahaem cytochrome MtrC is part of the transmembrane MtrCAB complex required for mineral respiration by Shewanella oneidensis. MtrC has significant sequence similarity to the paralogous decahaem cytochrome MtrF, which has been structurally solved through X-ray crystallography. This now allows for homology-based models of MtrC to be generated. The structure of these MtrC homology models contain ten bis-histidine-co-ordinated c-type haems arranged in a staggered cross through a four-domain structure. This model is consistent with current spectroscopic data and shows that the areas around haem 5 and haem 10, at the termini of an octahaem chain, are likely to have functions similar to those of the corresponding haems in MtrF. The electrostatic surfaces around haem 7, close to the ß-barrels, are different in MtrF and MtrC, indicating that these haems may have different potentials and interact with substrates differently.

Molecular structure and free energy landscape for electron transport in the decahaem cytochrome MtrF.

The free energy profile for electron flow through the bacterial decahaem cytochrome MtrF has been computed using thermodynamic integration and classical molecular dynamics. The extensive calculations on two versions of the structure help to validate the method and results, because differences in the profiles can be related to differences in the charged amino acids local to specific haem groups. First estimates of reorganization free energies λ yield a range consistent with expectations for partially solvent-exposed cofactors, and reveal an activation energy range surmountable for electron flow. Future work will aim at increasing the accuracy of λ with polarizable forcefield dynamics and quantum chemical energy gap calculations, as well as quantum chemical computation of electronic coupling matrix elements.

The roles of CymA in support of the respiratory flexibility of Shewanella oneidensis MR-1.

Shewanella species are isolated from the oxic/anoxic regions of seawater and aquatic sediments where redox conditions fluctuate in time and space. Colonization of these environments is by virtue of flexible respiratory chains, many of which are notable for the ability to reduce extracellular substrates including the Fe(III) and Mn(IV) contained in oxide and phyllosilicate minerals. Shewanella oneidensis MR-1 serves as a model organism to consider the biochemical basis of this flexibility. In the present paper, we summarize the various systems that serve to branch the respiratory chain of S. oneidensis MR-1 in order that electrons from quinol oxidation can be delivered the various terminal electron acceptors able to support aerobic and anaerobic growth. This serves to highlight several unanswered questions relating to the regulation of respiratory electron transport in Shewanella and the central role(s) of the tetrahaem-containing quinol dehydrogenase CymA in that process.

Development of a proteoliposome model to probe transmembrane electron-transfer reactions.

The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decahaem cytochromes brought together inside a transmembrane porin to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system has been developed that contains Methyl Viologen as an internalized electron carrier and valinomycin as a membrane-associated cation exchanger. These proteoliposomes can be used as a model system to investigate MtrCAB function.

Exploring the biochemistry at the extracellular redox frontier of bacterial mineral Fe(III) respiration.

Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources. In the present paper, we consider the common structural features of two of these outer-membrane decahaem cytochromes, MtrC and MtrF, and bring this together with biochemical, spectroscopic and voltammetric data to identify common and distinct properties of these prototypical members of different clades of the outer-membrane decahaem cytochrome superfamily.

Quantitative 3-D elemental mapping by LA-ICP-MS of a basaltic clast from the Hanford 300 Area, Washington, USA.

Laser ablation with inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used to measure elemental concentrations at the 100-µm scale in a 3-dimensional manner within a basaltic clast sample collected from the Hanford 300 Area in south-central Washington State, United States. A calibration method was developed to quantify the LA-ICP-MS signal response using a constant-sum mass fraction of eight major elements; the method produced reasonable concentration measurements for both major and trace elements when compared to a standard basalt sample with known concentrations. 3-Dimensional maps (stacked 2-D contour layers, each representing 2100 µm × 2100 µm) show relatively uniform concentration with depth for intrinsic elements such as Si, Na, and Sr. However, U and Cu accumulation were observed near the sample surface, consistent with the site's release history of these contaminants. U and Cu show substantial heterogeneity in their concentration distributions within horizontal slices, while the intrinsic elements are essentially uniformly distributed. From these measured U concentrations and published grain size distributions, gravel and cobbles were estimated to contain about 1% of the contaminant U, implicating the coarse fraction as a long-term release source.

Redox reactions of reduced flavin mononucleotide (FMN), riboflavin (RBF), and anthraquinone-2,6-disulfonate (AQDS) with ferrihydrite and lepidocrocite.

Flavins are secreted by the dissimilatory iron-reducing bacterium Shewanella and can function as endogenous electron transfer mediators. To assess the potential importance of flavins in Fe(III) bioreduction, we investigated the redox reaction kinetics of reduced flavin mononucleotide (i.e., FMNH(2)) and reduced riboflavin (i.e., RBFH(2)) with ferrihydrite and lepidocrocite. The organic reductants rapidly reduced and dissolved ferrihydrite and lepidocrocite in the pH range 4-8. The rate constant k for 2-line ferrihydrite reductive dissolution by FMNH(2) was 87.5 ± 3.5 M(-1)·s(-1) at pH 7.0 in batch reactors, and k was similar for RBFH(2). For lepidocrocite, k was 500 ± 61 M(-1)·s(-1) for FMNH(2) and 236 ± 22 M(-1)·s(-1) for RBFH(2). The surface area normalized initial reaction rates (r(a)) were between 0.08 and 77 µmol·m(-2)·s(-1) for various conditions in stopped-flow experiments. Initial rates (r(o)) were first-order with respect to iron(III) oxide concentration, and r(a) increased with decreasing pH. Poorly crystalline 2-line ferrihydrite yielded the highest r(a), followed by more crystalline 6-line ferrihydrite and crystalline lepidocrocite. Compared to a previous whole-cell study with Shewanella oneidensis strain MR-1, our findings suggest that the reduction of electron transfer mediators by the Mtr (i.e., metal-reducing) pathway coupled to lactate oxidation is rate limiting, rather than heterogeneous electron transfer to the iron(III) oxide.

Pore-scale characterization of biogeochemical controls on iron and uranium speciation under flow conditions.

Etched silicon microfluidic pore network models (micromodels) with controlled chemical and redox gradients, mineralogy, and microbiology under continuous flow conditions are used for the incremental development of complex microenvironments that simulate subsurface conditions. We demonstrate the colonization of micromodel pore spaces by an anaerobic Fe(III)-reducing bacterial species (Geobacter sulfurreducens) and the enzymatic reduction of a bioavailable Fe(III) phase within this environment. Using both X-ray microprobe and X-ray absorption spectroscopy, we investigate the combined effects of the precipitated Fe(III) phases and the microbial population on uranium biogeochemistry under flow conditions. Precipitated Fe(III) phases within the micromodel were most effectively reduced in the presence of an electron shuttle (AQDS), and Fe(II) ions adsorbed onto the precipitated mineral surface without inducing any structural change. In the absence of Fe(III), U(VI) was effectively reduced by the microbial population to insoluble U(IV), which was precipitated in discrete regions associated with biomass. In the presence of Fe(III) phases, however, both U(IV) and U(VI) could be detected associated with biomass, suggesting reoxidation of U(IV) by localized Fe(III) phases. These results demonstrate the importance of the spatial localization of biomass and redox active metals, and illustrate the key effects of pore-scale processes on contaminant fate and reactive transport.

Characterization of an electron conduit between bacteria and the extracellular environment.

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning beta-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.