Greg Wanger

Selecting Anode-Respiring Bacteria Based on Anode Potential: Phylogenetic, Electrochemical, and Microscopic Characterization

Anode-respiring bacteria (ARB) are able to transfer electrons contained in organic substrates to a solid electrode. The selection of ARB should depend on the anode potential, which determines the amount of energy available for bacterial growth and maintenance. In our study, we investigated how anode potential affected the microbial diversity of the biofilm community. We used a microbial electrolysis cell (MEC) containing four graphite electrodes, each at a different anode potential (E(anode) = -0.15, -0.09, +0.02, and +0.37 V vs SHE). We used wastewater-activated sludge as inoculum, acetate as substrate, and continuous-flow operation. The two electrodes at the lowest potentials showed a faster biofilm growth and produced the highest current densities, reaching up to 10.3 A/m(2) at the saturation of an amperometric curve; the electrode at the highest potential produced a maximum of 0.6 A/m(2). At low anode potentials, clone libraries showed a strong selection (92-99% of total clones) of an ARB that is 97% similar to G. sulfurreducens. At the highest anode potential, the ARB community was diverse. Cyclic voltammograms performed on each electrode suggest that the ARB grown at the lowest potentials carried out extracellular electron transport exclusively by conducting electrons through the extracellular biofilm matrix. This is supported by scanning electron micrographs showing putative bacterial nanowires and copious EPS at the lowest potentials. Non-ARB and ARB using electron shuttles in the diverse community for the highest anode potential may have insulated the ARB using a solid conductive matrix from the anode. Continuous-flow operation and the selective pressure due to low anode potentials selected for G. sulfurreducens, which are known to consume acetate efficiently and use a solid conductive matrix for electron transport.

Environmental genomics reveals a single-species ecosystem deep within earth

DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator, composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earths crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.

Desulfotomaculum and Methanobacterium spp. dominate a 4-to 5-kilometer-deep fault

ABSTRACT Alkaline, sulfidic, 54 to 60°C, 4 to 53 million-year-old meteoric water emanating from a borehole intersecting quartzite-hosted fractures >3.3 km beneath the surface supported a microbial community dominated by a bacterial species affiliated with Desulfotomaculum spp. and an archaeal species related to Methanobacterium spp. The geochemical homogeneity over the 650-m length of the borehole, the lack of dividing cells, and the absence of these microorganisms in mine service water support an indigenous origin for the microbial community. The coexistence of these two microorganisms is consistent with a limiting flux of inorganic carbon and SO42− in the presence of high pH, high concentrations of H2 and CH4, and minimal free energy for autotrophic methanogenesis. Sulfide isotopic compositions were highly enriched, consistent with microbial SO42− reduction under hydrologic isolation. An analogous microbial couple and similar abiogenic gas chemistry have been reported recently for hydrothermal carbonate vents of the Lost City near the Mid-Atlantic Ridge (D. S. Kelly et al., Science 307:1428-1434, 2005), suggesting that these features may be common to deep subsurface habitats (continental and marine) bearing this geochemical signature. The geochemical setting and microbial communities described here are notably different from microbial ecosystems reported for shallower continental subsurface environments.

Quantification of Electron Transfer Rates to a Solid Phase Electron Acceptor through the Stages of Biofilm Formation from Single Cells to Multicellular Communities

Microbial fuel cell (MFC) technology has enabled new insights into the mechanisms of electron transfer from dissimilatory metal reducing bacteria to a solid phase electron acceptor. Using solid electrodes as electron acceptors enables quantitative real-time measurements of electron transfer rates to these surfaces. We describe here an optically accessible, dual anode, continuous flow MFC that enables real-time microscopic imaging of anode populations as they develop from single attached cells to a mature biofilms. We used this system to characterize how differences in external resistance affect cellular electron transfer rates on a per cell basis and overall biofilm development in Shewanella oneidensis strain MR-1. When a low external resistance (100 Omega) was used, estimates of current per cell reached a maximum of 204 fA/cell (1.3 x 10(6) e(-) cell(-1) sec(-1)), while when a higher (1 MOmega) resistance was used, only 75 fA/cell (0.4 x 10(6) e(-) cell(-1) sec(-1)) was produced. The 1 MOmega anode biomass consistently developed into a mature thick biofilm with tower morphology (>50 microm thick), whereas only a thin biofilm (<5 microm thick) was observed on the 100 Omega anode. These data suggest a link between the ability of a surface to accept electrons and biofilm structure development.

Geobacter daltonii sp. nov., an Fe(III)- and uranium(VI)-reducing bacterium isolated from a shallow subsurface exposed to mixed heavy metal and hydrocarbon contamination.

An Fe(III)- and uranium(VI)-reducing bacterium, designated strain FRC-32(T), was isolated from a contaminated subsurface of the USA Department of Energy Oak Ridge Field Research Center (ORFRC) in Oak Ridge, Tennessee, where the sediments are exposed to mixed waste contamination of radionuclides and hydrocarbons. Analyses of both 16S rRNA gene and the Geobacteraceae-specific citrate synthase (gltA) mRNA gene sequences retrieved from ORFRC sediments indicated that this strain was abundant and active in ORFRC subsurface sediments undergoing uranium(VI) bioremediation. The organism belonged to the subsurface clade of the genus Geobacter and shared 92-98 % 16S rRNA gene and 75-81 % rpoB gene sequence similarities with other recognized species of the genus. In comparison to its closest relative, Geobacter uraniireducens Rf4(T), according to 16S rRNA gene sequence similarity, strain FRC-32(T) showed a DNA-DNA relatedness value of 21 %. Cells of strain FRC-32(T) were Gram-negative, non-spore-forming, curved rods, 1.0-1.5 microm long and 0.3-0.5 microm in diameter; the cells formed pink colonies in a semisolid cultivation medium, a characteristic feature of the genus Geobacter. The isolate was an obligate anaerobe, had temperature and pH optima for growth at 30 degrees C and pH 6.7-7.3, respectively, and could tolerate up to 0.7 % NaCl although growth was better in the absence of NaCl. Similar to other members of the Geobacter group, strain FRC-32(T) conserved energy for growth from the respiration of Fe(III)-oxyhydroxide coupled with the oxidation of acetate. Strain FRC-32(T) was metabolically versatile and, unlike its closest relative, G. uraniireducens, was capable of utilizing formate, butyrate and butanol as electron donors and soluble ferric iron (as ferric citrate) and elemental sulfur as electron acceptors. Growth on aromatic compounds including benzoate and toluene was predicted from preliminary genomic analyses and was confirmed through successive transfer with fumarate as the electron acceptor. Thus, based on genotypic, phylogenetic and phenotypic differences, strain FRC-32(T) is considered to represent a novel species of the genus Geobacter, for which the name Geobacter daltonii sp. nov. is proposed. The type strain is FRC-32(T) (=DSM 22248(T)=JCM 15807(T)).

Microbial population and functional dynamics associated with surface potential and carbon metabolism

Microbial extracellular electron transfer (EET) to solid surfaces is an important reaction for metal reduction occurring in various anoxic environments. However, it is challenging to accurately characterize EET-active microbial communities and each member’s contribution to EET reactions because of changes in composition and concentrations of electron donors and solid-phase acceptors. Here, we used bioelectrochemical systems to systematically evaluate the synergistic effects of carbon source and surface redox potential on EET-active microbial community development, metabolic networks and overall electron transfer rates. The results indicate that faster biocatalytic rates were observed under electropositive electrode surface potential conditions, and under fatty acid-fed conditions. Temporal 16S rRNA-based microbial community analyses showed that Geobacter phylotypes were highly diverse and apparently dependent on surface potentials. The well-known electrogenic microbes affiliated with the Geobacter metallireducens clade were associated with lower surface potentials and less current generation, whereas Geobacter subsurface clades 1 and 2 were associated with higher surface potentials and greater current generation. An association was also observed between specific fermentative phylotypes and Geobacter phylotypes at specific surface potentials. When sugars were present, Tolumonas and Aeromonas phylotypes were preferentially associated with lower surface potentials, whereas Lactococcus phylotypes were found to be closely associated with Geobacter subsurface clades 1 and 2 phylotypes under higher surface potential conditions. Collectively, these results suggest that surface potentials provide a strong selective pressure, at the species and strain level, for both solid surface respirators and fermentative microbes throughout the EET-active community development.

Shewanella oneidensis MR-1 Bacterial Nanowires Exhibit p-Type, Tunable Electronic Behavior

The study of electrical transport in biomolecular materials is critical to our fundamental understanding of physiology and to the development of practical bioelectronics applications. In this study, we investigated the electronic transport characteristics of Shewanella oneidensis MR-1 nanowires by conducting-probe atomic force microscopy (CP-AFM) and by constructing field-effect transistors (FETs) based on individual S. oneidensis nanowires. Here we show that S. oneidensis nanowires exhibit p-type, tunable electronic behavior with a field-effect mobility on the order of 10(-1) cm(2)/(V s), comparable to devices based on synthetic organic semiconductors. This study opens up opportunities to use such bacterial nanowires as a new semiconducting biomaterial for making bioelectronics and to enhance the power output of microbial fuel cells through engineering the interfaces between metallic electrodes and bacterial nanowires.

Microbial diversity in The Cedars, an ultrabasic, ultrareducing, and low salinity serpentinizing ecosystem

Significance The Cedars, in coastal northern California, is an active serpentinization site. The spring waters emerging from this system feature very high pH (≈11.5), low redox potential (≈−550 mV), and low ionic concentrations, making it an exceptionally challenging environment for life. The microbial communities are different in different springs, strongly correlated with the source of the serpentinizing groundwater feeding the springs (shallow or deep). The shallow groundwater community was similar to those described in other terrestrial serpentinizing sites, while the deep community was distinctly different from any other previously described terrestrial serpentinizing community. These communities have the potential to yield important insights into survival mechanisms in these challenging, early-earth analog environments. The Cedars, in coastal northern California, is an active site of peridotite serpentinization. The spring waters that emerge from this system feature very high pH, low redox potential, and low ionic concentrations, making it an exceptionally challenging environment for life. We report a multiyear, culture-independent geomicrobiological study of three springs at The Cedars that differ with respect to the nature of the groundwater feeding them. Within each spring, both geochemical properties and microbial diversity in all three domains of life remained stable over a 3-y period, with multiple samples each year. Between the three springs, however, the microbial communities showed considerable differences that were strongly correlated with the source of the serpentinizing groundwater. In the spring fed solely by deep groundwater, phylum Chloroflexi, class Clostridia, and candidate division OD1 were the major taxa with one phylotype in Euryarchaeota. Less-abundant phylotypes include several minor members from other candidate divisions and one phylotype that was an outlier of candidate division OP3. In the springs fed by the mixture of deep and shallow groundwater, organisms close to the Hydrogenophaga within Betaproteobacteria dominated and coexisted with the deep groundwater community members. The shallow groundwater community thus appears to be similar to those described in other terrestrial serpentinizing sites, whereas the deep community is distinctly different from any other previously described terrestrial serpentinizing community. These unique communities have the potential to yield important insights into the development and survival of life in these early-earth analog environments.

Electrically conductive bacterial nanowires in bisphosphonate- related osteonecrosis of the jaw biofilms

OBJECTIVE Bacterial biofilms play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this preliminary study was to test the hypothesis that the extracellular filaments observed in biofilms associated with BRONJ contain electrically conductive nanowires. STUDY DESIGN Bone samples of patients affected by BRONJ were evaluated for conductive nanowires by scanning electron microscopy (SEM) and conductive probe atomic force microscopy (CP-AFM). We created nanofabricated electrodes to measure electrical transport along putative nanowires. RESULTS SEM revealed large-scale multispecies biofilms containing numerous filamentous structures throughout necrotic bone. CP-AFM analysis revealed that these structures were electrically conductive nanowires with resistivities on the order of 20 Ω·cm. Nanofabricated electrodes spaced along the nanowires confirmed their ability to transfer electrons over micron-scale lengths. CONCLUSIONS Electrically conductive bacterial nanowires to date have been described only in environmental isolates. This study shows for the first time that these nanowires can also be found in clinically relevant biofilm-mediated diseases, such as BRONJ, and may represent an important target for therapy.

A study of the flavin response by Shewanella cultures in carbon-limited environments

Mediated electron transfer has been implicated as a primary mechanism of extracellular electron transfer to insoluble electron acceptors in anaerobic cultures of the facultative anaerobe Shewanella oneidensis. In this work, planktonic and biofilm cultures of S. oneidensis exposed to carbon-limited environments trigger an electrochemical response thought to be the signature of an electrochemically active metabolite. This metabolite was detected via cyclic voltammetry for S. oneidensis MR-1 biofilms. The observed electrochemical potentials correspond to redox potentials of flavin-containing molecules. Chromatographic techniques were then used to quantify concentrations of riboflavin by the carbon-limited environmental response of planktonic S. oneidensis. Further evidence of flavin redox chemistry was associated with biofilm formation on multi-walled carbon nanotube-modified Toray paper under carbon-starved environments. By encapsulating one such electrode in silica, the encapsulated biofilm exhibits riboflavin redox activity earlier than a non-encapsulated system after media replacement. This work explores the electrochemical nature of riboflavin interaction with an electrode after secretion from S. oneidensis and in comparison to abiotic systems.