Adaptive Synthesis of a Rough Lipopolysaccharide in Geobacter sulfurreducens for Metal Reduction and Detoxification.

The ability of some metal-reducing bacteria to produce a rough (no O-antigen) lipopolysaccharide (LPS) could facilitate surface interactions with minerals and metal reduction. Consistent with this, the laboratory model metal reducer Geobacter sulfurreducens PCA produced two rough LPS isoforms (with or without a terminal methyl-quinovosamine sugar) when growing with the soluble electron acceptor fumarate but expressed only the shorter and more hydrophilic variant when reducing iron oxides. We reconstructed from genomic data conserved pathways for the synthesis of the rough LPS and generated heptosyltransferase mutants with partial (ΔrfaQ) or complete (ΔrfaC) truncations in the core oligosaccharide. The stepwise removal of the LPS core sugars reduced the hydrophilicity of the cell and increased outer membrane vesiculation. These changes in surface charge and remodeling did not substantially impact planktonic growth but disrupted the developmental stages and structure of electroactive biofilms. Furthermore, the mutants assembled conductive pili for extracellular mineralization of the toxic uranyl cation but were unable to prevent permeation and mineralization of the radionuclide in the cell envelope. Hence, not only does the rough LPS promote cell-cell and cell-mineral interactions critical to biofilm formation and metal respiration but it also functions as a permeability barrier to toxic metal cations. In doing so, the rough LPS maximizes the extracellular reduction of soluble and insoluble metals and preserves cell envelope functions critical to the environmental survival of Geobacter bacteria in metal-rich environments and their performance in bioremediation and bioenergy applications. IMPORTANCE Some metal-reducing bacteria produce an LPS without the repeating sugars (O-antigen) that decorate the surface of most Gram-negative bacteria, but the biological significance of this adaptive feature was not previously investigated. Using the model representative Geobacter sulfurreducens strain PCA and mutants carrying stepwise truncations in the LPS core sugars, we demonstrate the importance of the rough LPS in the control of cell surface chemistry during the respiration of iron minerals and the formation of electroactive biofilms. Importantly, we describe hitherto overlooked roles for the rough LPS in metal sequestration and outer membrane vesiculation that are critical for the extracellular reduction and detoxification of toxic metals and radionuclides. These results are of interest for the optimization of bioremediation schemes and electricity-harvesting platforms using these bacteria.

Maghemite (γ-Fe2O3) nanoparticles enhance dissimilatory ferrihydrite reduction by Geobacter sulfurreducens: Impacts on iron mineralogical change and bacterial interactions.

Microbially mediated bioreduction of iron oxyhydroxide plays an important role in the biogeochemical cycle of iron. Geobacter sulfurreducens is a representative dissimilatory iron-reducing bacterium that assembles electrically conductive pili and cytochromes. The impact of supplementation with γ-Fe2O3 nanoparticles (NPs) (0.2 and 0.6 g) on the G. sulfurreducens-mediated reduction of ferrihydrite was investigated. In the overall performance of microbial ferrihydrite reduction mediated by γ-Fe2O3 NPs, stronger reduction was observed in the presence of direct contact with γ-Fe2O3 NPs than with indirect contact. Compared to the production of Fe(II) derived from biotic modification with ferrihydrite alone, increases greater than 1.6- and 1.4-fold in the production of Fe(II) were detected in the biotic modifications in which direct contact with 0.2 g and 0.6 g γ-Fe2O3 NPs, respectively, occurred. X-ray diffraction analysis indicated that magnetite was a unique representative iron mineral in ferrihydrite when active G. sulfurreducens cells were in direct contact with γ-Fe2O3 NPs. Because of the sorption of biogenic Fe(II) onto γ-Fe2O3 NPs instead of ferrihydrite, the addition of γ-Fe2O3 NPs could also contribute to increased duration of ferrihydrite reduction by preventing ferrihydrite surface passivation. Additionally, electron microscopy analysis confirmed that the direct addition of γ-Fe2O3 NPs stimulated the electrically conductive pili and cytochromes to stretch, facilitating long-range electron transfer between the cells and ferrihydrite. The obtained findings provide a more comprehensive understanding of the effects of iron oxide NPs on soil biogeochemistry.

Bread-derived 3D macroporous carbon foams as high performance free-standing anode in microbial fuel cells.

Microbial fuel cells (MFCs) are a promising clean energy source to directly convert waste chemicals to available electric power. However, the practical application of MFCs needs the increased power density, enhanced energy conversion efficiency and reduced electrode material cost. In this study, three-dimensional (3D) macroporous N, P and S co-doped carbon foams (NPS-CFs) were prepared by direct pyrolysis of the commercial bread and employed as free-standing anodes in MFCs. As-obtained NPS-CFs have a large specific surface area (295.07 m2 g-1), high N, P and S doping level, and excellent electrical conductivity. A maximum areal power density of 3134 mW m-2 and current density of 7.56 A m-2 are generated by the MFCs equipped with as-obtained NPS-CF anodes, which is 2.57- and 2.63-fold that of the plain carbon cloth anodes (areal power density of 1218 mW m-2 and current density of 2.87 A m-2), respectively. Such improvement is explored to mainly originate from two respects: the good biocompatibility of NPS-CFs favors the bacterial adhesion and enrichment of electroactive Geobacter species on the electrode surface, while the high conductivity and improved bacteria-electrode interaction efficiently promote the extracellular electron transfer (EET) between the bacteria and the anode. This study provides a low-cost and sustainable way to fabricate high power MFCs for practical applications.

Evaluation of microbial fuel cells for electricity generation from oil-contaminated wastewater.

Large quantities of oils and fats are discharged into wastewater from food industries. We evaluated the possibility of using microbial fuel cells (MFCs) for the generation of electricity from food-industry wastewater containing vegetable oils. Single-chamber MFCs were supplied with artificial wastewater containing soybean oil, and oil removal and electric output were examined under several different conditions. We found that MFC performance could be improved by supplementing wastewater with an emulsifier, inoculating MFCs with oil-contaminated soil, and coating the graphite-felt anodes with carbon nanotubes, resulting in a power output of more than 2 W m-2 (based on the projected area of the anode). Sequencing of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments detected abundant amount of Burkholderiales bacteria (known to include oil degraders) in the oil-contaminated soil and anode biofilm, whereas those affiliated with the genus Geobacter were only detected in the anode biofilm. These results suggest that MFCs can be used for energy recovery from food industry wastewater containing vegetable oils.

Thermally activated charge transport in microbial protein nanowires.

The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors.

Enhanced uranium immobilization and reduction by Geobacter sulfurreducens biofilms.

Biofilms formed by dissimilatory metal reducers are of interest to develop permeable biobarriers for the immobilization of soluble contaminants such as uranium. Here we show that biofilms of the model uranium-reducing bacterium Geobacter sulfurreducens immobilized substantially more U(VI) than planktonic cells and did so for longer periods of time, reductively precipitating it to a mononuclear U(IV) phase involving carbon ligands. The biofilms also tolerated high and otherwise toxic concentrations (up to 5 mM) of uranium, consistent with a respiratory strategy that also protected the cells from uranium toxicity. The enhanced ability of the biofilms to immobilize uranium correlated only partially with the biofilm biomass and thickness and depended greatly on the area of the biofilm exposed to the soluble contaminant. In contrast, uranium reduction depended on the expression of Geobacter conductive pili and, to a lesser extent, on the presence of the c cytochrome OmcZ in the biofilm matrix. The results support a model in which the electroactive biofilm matrix immobilizes and reduces the uranium in the top stratum. This mechanism prevents the permeation and mineralization of uranium in the cell envelope, thereby preserving essential cellular functions and enhancing the catalytic capacity of Geobacter cells to reduce uranium. Hence, the biofilms provide cells with a physically and chemically protected environment for the sustained immobilization and reduction of uranium that is of interest for the development of improved strategies for the in situ bioremediation of environments impacted by uranium contamination.

Fluctuations in species-level protein expression occur during element and nutrient cycling in the subsurface.

While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.

Enrichment of specific protozoan populations during in situ bioremediation of uranium-contaminated groundwater.

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uranium-contaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100% of the sequences recovered during this phase of the bioremediation. These results suggest a prey-predator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.

Model-based analysis of the role of biological, hydrological and geochemical factors affecting uranium bioremediation.

Uranium contamination is a serious concern at several sites motivating the development of novel treatment strategies such as the Geobacter-mediated reductive immobilization of uranium. However, this bioremediation strategy has not yet been optimized for the sustained uranium removal. While several reactive-transport models have been developed to represent Geobacter-mediated bioremediation of uranium, these models often lack the detailed quantitative description of the microbial process (e.g., biomass build-up in both groundwater and sediments, electron transport system, etc.) and the interaction between biogeochemical and hydrological process. In this study, a novel multi-scale model was developed by integrating our recent model on electron capacitance of Geobacter (Zhao et al., 2010) with a comprehensive simulator of coupled fluid flow, hydrologic transport, heat transfer, and biogeochemical reactions. This mechanistic reactive-transport model accurately reproduces the experimental data for the bioremediation of uranium with acetate amendment. We subsequently performed global sensitivity analysis with the reactive-transport model in order to identify the main sources of prediction uncertainty caused by synergistic effects of biological, geochemical, and hydrological processes. The proposed approach successfully captured significant contributing factors across time and space, thereby improving the structure and parameterization of the comprehensive reactive-transport model. The global sensitivity analysis also provides a potentially useful tool to evaluate uranium bioremediation strategy. The simulations suggest that under difficult environments (e.g., highly contaminated with U(VI) at a high migration rate of solutes), the efficiency of uranium removal can be improved by adding Geobacter species to the contaminated site (bioaugmentation) in conjunction with the addition of electron donor (biostimulation). The simulations also highlight the interactive effect of initial cell concentration and flow rate on U(VI) reduction.

Proteogenomic monitoring of Geobacter physiology during stimulated uranium bioremediation.

Implementation of uranium bioremediation requires methods for monitoring the membership and activities of the subsurface microbial communities that are responsible for reduction of soluble U(VI) to insoluble U(IV). Here, we report a proteomics-based approach for simultaneously documenting the strain membership and microbial physiology of the dominant Geobacter community members during in situ acetate amendment of the U-contaminated Rifle, CO, aquifer. Three planktonic Geobacter-dominated samples were obtained from two wells down-gradient of acetate addition. Over 2,500 proteins from each of these samples were identified by matching liquid chromatography-tandem mass spectrometry spectra to peptides predicted from seven isolate Geobacter genomes. Genome-specific peptides indicate early proliferation of multiple M21 and Geobacter bemidjiensis-like strains and later possible emergence of M21 and G. bemidjiensis-like strains more closely related to Geobacter lovleyi. Throughout biostimulation, the proteome is dominated by enzymes that convert acetate to acetyl-coenzyme A and pyruvate for central metabolism, while abundant peptides matching tricarboxylic acid cycle proteins and ATP synthase subunits were also detected, indicating the importance of energy generation during the period of rapid growth following the start of biostimulation. Evolving Geobacter strain composition may be linked to changes in protein abundance over the course of biostimulation and may reflect changes in metabolic functioning. Thus, metagenomics-independent community proteogenomics can be used to diagnose the status of the subsurface consortia upon which remediation biotechnology relies.

Detection and quantification of Geobacter lovleyi strain SZ: implications for bioremediation at tetrachloroethene- and uranium-impacted sites.

Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 x 10(6) and 1 x 10(4) 16S rRNA gene copies per mul of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per mul of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 x 10(7) +/- 0.1 x 10(7) per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.