Influence of electron donor/acceptor concentrations on hydrous ferric oxide (HFO) bioreduction

Dissimilatory metal-reducing bacteria (DMRB) facilitate the reduction of Feand Mn oxides in anoxic soils and sediments and play an important role inthe cycling of these metals and other elements such as carbon in aqueousenvironments. Previous studies investigating the reduction of Fe(III) oxidesby DMRB focused on reactions under constant initial electron donor (lactate)and electron acceptor (Fe oxide) concentrations. Because the concentrationsof these reactants can vary greatly in the environment and would be expectedto influence the rate and extent of oxide reduction, the influence of variableelectron acceptor and donor concentrations on hydrous ferric oxide (HFO)bioreduction was investigated. Batch experiments were conducted in pH 7 HCO₃– buffered media using Shewanella putrefaciens strain CN32. In general, the rate of Fe(III) reduction decreased with increasing HFO:lactateratios, resulting in a relatively greater proportion of crystalline Fe(III) oxidesof relatively low availability for DMRB. HFO was transformed to a variety ofcrystalline minerals including goethite, lepidocrocite, and siderite but was almostcompletely dissolved at high lactate to HFO ratios. These results indicate thatelectron donor and acceptor concentrations can greatly impact the bioreductionof HFO and the suite of Fe minerals formed as a result of reduction. The respirationdriven rate of Fe(II) formation from HFO is believed to be a primary factor governingthe array of ferrous and ferric iron phases formed during reduction.     

Adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to crystalline Fe(III) oxides

Shewanella alga BrY adhesion to hydrous ferric oxide, goethite, and hematite was examined. Adhesion to each oxide followed the Langmuir adsorption model. No correlation between adhesion and Fe(III) oxide surface area or crystallinity was observed. Zeta potential measurements suggested that electrostatic interactions do not influence S. alga BrY adhesion to these minerals. Cell adhesion does not appear to explain the recalcitrance of crystalline Fe(III) oxides to bacterial reduction. Received: 12 May 2000 / Accepted: 19 June 2000     

Adhesion of Dissimilatory Fe(III)-Reducing Bacteria to Fe(III) Minerals

The metabolism of dissimilatory iron-reducing bacteria (DIRB) may provide a means of remediating contaminated subsurface soils. The factors controlling the rate and extent of bacterial F(III) mineral reduction are poorly understood. Recent research suggests that molecular-scale interactions between DIRB cells and Fe(III) mineral particles play an important role in this process. One of these interactions, cell adhesion to Fe(III) mineral particles, appears to be a complex process that is, at least in part, mediated by a variety of surface proteins. This study examined the hypothesis that the flagellum serves as an adhesin to different Fe(III) minerals that range in their surface area and degree of crystallinity. Deflagellated cells of the DIRB Shewanella algae BrY showed a reduced ability to adhere to hydrous ferric oxide (HFO) relative to flagellated cells. Flagellated cells were also more hydrophobic than deflagellated cells. This was significant because hydrophobic interactions have been previously shown to dominate S. algae cell adhesion to Fe(III) minerals. Pre-incubating HFO, goethite, or hematite with purified flagella inhibited the adhesion of S. algae BrY cells to these minerals. Transposon mutagenesis was used to generate a flagellum-deficient mutant designated S. algae strain NF. There was a significant difference in the rate and extent of S. algae NF adhesion to HFO, goethite, and hematite relative to that of S. algae BrY. Amiloride, a specific inhibitor of Na + -driven flagellar motors, inhibited S. algae BrY motility but did not affect the adhesion of S. algae BrY to HFO. S.algae NF reduced HFO at the same rate as S. algae BrY. Collectively, the results of this study support the hypothesis that the flagellum of S. algae functions as a specific Fe(III) mineral adhesin. However, these results suggest that flagellum-mediated adhesion is not requisite for Fe(III) mineral reduction.     

Protein-mediated adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to hydrous ferric oxide.

The rate and extent of bacterial Fe(III) mineral reduction are governed by molecular-scale interactions between the bacterial cell surface and the mineral surface. These interactions are poorly understood. This study examined the role of surface proteins in the adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO). Enzymatic degradation of cell surface polysaccharides had no effect on cell adhesion to HFO. The proteolytic enzymes Streptomyces griseus protease and chymotrypsin inhibited the adhesion of S. alga BrY cells to HFO through catalytic degradation of surface proteins. Trypsin inhibited S. alga BrY adhesion solely through surface-coating effects. Protease and chymotrypsin also mediated desorption of adhered S. alga BrY cells from HFO while trypsin did not mediate cell desorption. Protease removed a single peptide band that represented a protein with an apparent molecular mass of 50 kDa. Chymotrypsin removed two peptide bands that represented proteins with apparent molecular masses of 60 and 31 kDa. These proteins represent putative HFO adhesion molecules. S. alga BrY adhesion was inhibited by up to 46% when cells were cultured at sub-MICs of chloramphenicol, suggesting that protein synthesis is necessary for adhesion. Proteins extracted from the surface of S. alga BrY cells inhibited adhesion to HFO by up to 41%. A number of these proteins bound specifically to HFO, suggesting that a complex system of surface proteins mediates S. alga BrY adhesion to HFO.     

Protein-Mediated Adhesion of the Dissimilatory Fe(III)-Reducing Bacterium Shewanella alga BrY to Hydrous Ferric Oxide

The rate and extent of bacterial Fe(III) mineral reduction are governed by molecular-scale interactions between the bacterial cell surface and the mineral surface. These interactions are poorly understood. This study examined the role of surface proteins in the adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO). Enzymatic degradation of cell surface polysaccharides had no effect on cell adhesion to HFO. The proteolytic enzymes Streptomyces griseus protease and chymotrypsin inhibited the adhesion of S. alga BrY cells to HFO through catalytic degradation of surface proteins. Trypsin inhibited S. alga BrY adhesion solely through surface-coating effects. Protease and chymotrypsin also mediated desorption of adhered S. alga BrY cells from HFO while trypsin did not mediate cell desorption. Protease removed a single peptide band that represented a protein with an apparent molecular mass of 50 kDa. Chymotrypsin removed two peptide bands that represented proteins with apparent molecular masses of 60 and 31 kDa. These proteins represent putative HFO adhesion molecules. S. alga BrY adhesion was inhibited by up to 46% when cells were cultured at sub-MICs of chloramphenicol, suggesting that protein synthesis is necessary for adhesion. Proteins extracted from the surface of S. alga BrY cells inhibited adhesion to HFO by up to 41%. A number of these proteins bound specifically to HFO, suggesting that a complex system of surface proteins mediates S. alga BrY adhesion to HFO.     

Shewanella oneidensis MR‐1 mutants selected for their inability to produce soluble organic‐Fe(III) complexes are unable to respire Fe(III) as anaerobic electron acceptor

Summary Recent voltammetric analyses indicate that Shewanella putrefaciens strain 200 produces soluble organic‐Fe(III) complexes during anaerobic respiration of sparingly soluble Fe(III) oxides. Results of the present study expand the range of Shewanella species capable of producing soluble organic‐Fe(III) complexes to include Shewanella oneidensis MR‐1. Soluble organic‐Fe(III) was produced by S. oneidensis cultures incubated anaerobically with Fe(III) oxides, or with Fe(III) oxides and the alternate electron acceptor fumarate, but not in the presence of O₂, nitrate or trimethylamine‐N‐oxide. Chemical mutagenesis procedures were combined with a novel MicroElectrode Screening Array (MESA) to identify four (designated Sol) mutants with impaired ability to produce soluble organic‐Fe(III) during anaerobic respiration of Fe(III) oxides. Two of the Sol mutants were deficient in anaerobic growth on both soluble Fe(III)‐citrate and Fe(III) oxide, yet retained the ability to grow on a suite of seven alternate electron acceptors. The rates of soluble organic‐Fe(III) production were proportional to the rates of iron reduction by the S. oneidensis wild‐type and Sol mutant strains, and all four Sol mutants retained wild‐type siderophore production capability. Results of this study indicate that the production of soluble organic‐Fe(III) may be an important intermediate step in the anaerobic respiration of both soluble and sparingly soluble forms of Fe(III) by S. oneidensis.     

Influence of pH and ionic strength on adhesion of a wild strain of Pseudomonas sp. to titanium

The adherence of an environmental strain of Pseudomonas sp. to titanium was evaluated modifying the pH (2 to 8) and ionic strength (0.1 and 0.6 M NaCl) of the electrolyte solution. Results were analyzed considering the participation of the different interfacial forces under the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory. At 0.1 M, maximal bacterial adhesion was at pH 6, in agreement with the point of zero charge of the titanium surface. Similar adhesion values were observed at both sides of this point despite the opposite electrostatic condition of the surface oxide. At 0.6 M an absence of bacterial adhesion was observed throughout the pH range tested. The changes in bacterial adhesion are in agreement with the changes in the number of reinforced H-bond-forming sites on the titanium surface calculated using a simple model for the ionization of OH group adsorbed to the surface. Journal of Industrial Microbiology & Biotechnology (2001) 26, 303–308. Received 25 May 2000/ Accepted in revised form 14 February 2001     

Influence of Biogenic Fe(II) on Bacterial Crystalline Fe(III) Oxide Reduction

Bacterial crystalline Fe(III) oxide reduction has the potential to significantly influence the biogeochemistry of anaerobic sedimentary environments where crystalline Fe(III) oxides are abundant relative to poorly crystalline (amorphous) phases. A review of published data on solid-phase Fe(III) abundance and speciation indicates that crystalline Fe(III) oxides are frequently 2- to S 10-fold more abundant than amorphous Fe(III) oxides in shallow subsurface sediments not yet subjected to microbial Fe(III) oxide reduction activity. Incubation experiments with coastal plain aquifer sediments demonstrated that crystalline Fe(III) oxide reduction can contribute substantially to Fe(II) production in the presence of added electron donors and nutrients. Controls on crystalline Fe(III) oxide reduction are therefore an important consideration in relation to the biogeochemical impacts of bacterial Fe(III) oxide reduction in subsurface environments. In this paper, the influence of biogenic Fe(II) on bacterial reduction of crystalline Fe(III) oxides is reviewed and analyzed in light of new experiments conducted with the acetate-oxidizing, Fe(III)-reducing bacterium (FeRB) Geobacter metallireducens . Previous experiments with Shewanella algae strain BrY indicated that adsorption and/or surface precipitation of Fe(II) on Fe(III) oxide and FeRB cell surfaces is primarily responsible for cessation of goethite ( f -FeOOH) reduction activity after only a relatively small fraction (generally < 10%) of the oxide is reduced. Similar conclusions are drawn from analogous studies with G. metallireducens . Although accumulation of aqueous Fe(II) has the potential to impose thermodynamic constraints on the extent of crystalline Fe(III) oxide reduction, our data on bacterial goethite reduction suggest that this phenomenon cannot universally explain the low microbial reducibility of this mineral. Experiments examining the influence of exogenous Fe(II) (20 mM FeCl 2 ) on soluble Fe(III)-citrate reduction by G. metallireducens and S. algae showed that high concentrations of Fe(II) did not inhibit Fe(III)-citrate reduction by freshly grown cells, which indicates that surface-bound Fe(II) does not inhibit Fe(III) reduction through a classical end-product enzyme inhibition mechanism. However, prolonged exposure of G. metallireducens and S. algae cells to high concentrations of soluble Fe(II) did cause inhibition of soluble Fe(III) reduction. These findings, together with recent documentation of the formation of Fe(II) surface precipitates on FeRB in Fe(III)-citrate medium, provide further evidence for the impact of Fe(II) sorption by FeRB on enzymatic Fe(III) reduction. Two different, but not mutually exclusive, mechanisms whereby accumulation of Fe(II) coatings on Fe(III) oxide and FeRB surfaces may lead to inhibition of enzymatic Fe(III) oxide reduction activity (in the absence of soluble electron shuttles and/or Fe(III) chelators) are identified and discussed in relation to recent experimental work and theoretical considerations.     

Seasonal cycling of Fe in saltmarsh sediments

This study combines an analysis of porewater chemistry with new, solid phase wet chemical extractions to examine the seasonal cycling of Fe in vegetated and unvegetated (cyanobacterial mat) saltmarsh sediments. Saltmarsh sediments are shown to contain more solid phase reactive Fe than other marine sediments studied so far. From the partitioning and speciation of solid Fe, and solid/soluble reduced S analysis in 10 sediment cores, we have observed that a majority of solid Fe in these sediments is cycled rapidly and completely between oxidized reactive Fe and reduced Fe as pyrite. Vegetated porewaters showed a lower pH and much higher Fe(II) concentrations on average than unvegetated porewaters in the top 10 cm, whereas sulfate, alkalinity, and sulfide concentrations were similar in the two environments. The amorphous Fe(III) oxide fraction showed a high negative correlation to solid and soluble reduced S (r ² = –0.86 and –0.71, respectively) in surface vegetated sediments whereas the crystalline Fe(III) oxide fraction showed a high negative correlation (r ² = –0.96) to sulfide only at depth. Though reactive Fe was observed in unvegetated sediments, no seasonal trend was apparent and the speciation of solid Fe revealed that most of it was reduced. Solid phase and porewater chemistry support the dominant role of the biota (Spartina alterniflora and bacteria) in controlling the reactivity of Fe and suggest that the current definition of solid phase, reactive Fe should be expanded to include crystalline Fe(III) minerals which are available for pyrite formation in saltmarsh sediments. In support of previous saltmarsh studies, we present evidence that the redox cycle of solid Fe is controlled by sulfate reduction and sediment oxidation which respond to both annual cycles (light, temperature) and to short-term, episodic effects such as weather and tides.     

A Comparison of the Rates of Fe(III) Reduction in Synthetic and Bacteriogenic Iron Oxides by Shewanella putrefaciens CN32

The susceptibility of various bacteriogenic iron oxides (BIOS) to bacterial Fe(III) reduction was examined. Reduction resulted in complete dissolution of the iron mineral from the surfaces of the Fe-oxidizing consortium. Reduction rates were compared to that of synthetic ferrihydrite (HFO). The reduction rate of HFO (0.162 day^{? 1}) was significantly lower than that of Äspö (Gallionella dominated) BIOS (0.269 day^{? 1}). Two Canadian (Leptothrix dominated) BIOS samples showed statistically equivalent rates of reduction (0.541 day^?1 and 0.467 day^{? 1}), which were higher than both Äspö BIOS and HFO. BIOS produced by different iron-oxidizing genera have different susceptibilities to microbial reduction.     

Interactions Between Fe(III)-oxides and Fe(III)-phyllosilicates During Microbial Reduction 2: Natural Subsurface Sediments

Dissimilatory microbial reduction of solid-phase Fe(III)-oxides and Fe(III)-bearing phyllosilicates (Fe(III)-phyllosilicates) is an important process in anoxic soils, sediments and subsurface materials. Although various studies have documented the relative extent of microbial reduction of single-phase Fe(III)-oxides and Fe(III)-phyllosilicates, detailed information is not available on interaction between these two processes in situations where both phases are available for microbial reduction. The goal of this research was to use the model dissimilatory iron-reducing bacterium (DIRB) Geobacter sulfurreducens to study Fe(III)-oxide vs. Fe(III)-phyllosilicate reduction in a range of subsurface materials and Fe(III)-oxide stripped versions of the materials. Low-temperature (12 K) Mossbauer spectroscopy was used to infer changes in the relative abundances of Fe(III)-oxide, Fe(III)-phyllosilicate, and phyllosilicate-associated Fe(II) (Fe(II) phyllosilicate). A Fe partitioning model was employed to analyze the fate of Fe(II) and assess the potential for abiotic Fe(II)-catalyzed reduction of Fe(III)-phyllosilicates. The results showed that in most cases Fe(III)-oxide utilization dominated (70–100%) bulk Fe(III) reduction activity, and that electron transfer from oxide-derived Fe(II) played only a minor role (ca. 10–20%) in Fe partitioning. In addition, the extent of Fe(III)-oxide reduction was positively correlated to surface area-normalized cation exchange capacity and the Fe(III)-phyllosilicate/total Fe(III) ratio. This finding suggests that the phyllosilicates in the natural sediments promoted Fe(III)-oxide reduction by binding of oxide-derived Fe(II), thereby enhancing Fe(III)-oxide reduction by reducing or delaying the inhibitory effect that Fe(II) accumulation on oxide and DIRB cell surfaces has on Fe(III)-oxide reduction. In general our results suggest that although Fe(III)-oxide reduction is likely to dominate bulk Fe(III) reduction in most subsurface sediments, Fe(II) binding by phyllosilicates is likely to play a key role in controlling the long-term kinetics of Fe(III) oxide reduction     

Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor

The mechanisms for Fe(III) oxide reduction in Geobacter species are of interest because Fe(III) oxides are the most abundant form of Fe(III) in many soils and sediments and Geobacter species are prevalent Fe(III)-reducing microorganisms in many of these environments. Protein abundance in G. sulfurreducens grown on poorly crystalline Fe(III) oxide or on soluble Fe(III) citrate was compared with a global accurate mass and time tag proteomic approach in order to identify proteins that might be specifically associated with Fe(III) oxide reduction. A total of 2991 proteins were detected in G. sulfurreducens grown with acetate as the electron donor and either Fe(III) oxide or soluble Fe(III) citrate as the electron acceptor, resulting in 86% recovery of the genes predicted to encode proteins. Of the total expressed proteins 76% were less abundant in Fe(III) oxide cultures than in Fe(III) citrate cultures, which is consistent with the overall slower rate of metabolism during growth with an insoluble electron acceptor. A total of 269 proteins were more abundant in Fe(III) oxide-grown cells than in cells grown on Fe(III) citrate. Most of these proteins were in the energy metabolism category: primarily electron transport proteins, including 13 c-type cytochromes and PilA, the structural protein for electrically conductive pili. Several of the cytochromes that were more abundant in Fe(III) oxide-grown cells were previously shown with genetic approaches to be essential for optimal Fe(III) oxide reduction. Other proteins that were more abundant during growth on Fe(III) oxide included transport and binding proteins, proteins involved in regulation and signal transduction, cell envelope proteins, and enzymes for amino acid and protein biosynthesis, among others. There were also a substantial number of proteins of unknown function that were more abundant during growth on Fe(III) oxide. These results indicate that electron transport to Fe(III) oxide requires additional and/or different proteins than electron transfer to soluble, chelated Fe(III) and suggest proteins whose functions should be further investigated in order to better understand the mechanisms of electron transfer to Fe(III) oxide in G. sulfurreducens.     

Ferric iron reduction by Desulfovibrio vulgaris Hildenborough wild type and energy metabolism mutants

Desulfovibrio vulgaris Hildenborough wild type and its hyn1, hyd and hmc mutants, lacking genes for periplasmic [NiFe] hydrogenase-1, periplasmic [FeFe] hydrogenase or the transmembrane high molecular weight cytochrome (Hmc) complex, respectively, were able to reduce Fe(III) chelated with nitrilotriacetic acid (NTA), but not insoluble ferric oxide, with lactate as the electron donor. The rate and extent of Fe(III)-NTA reduction followed the order hyn = WT > hmc >> hyd, suggesting that reduction of soluble Fe(III) is a periplasmic process that requires the presence of periplasmic [FeFe] hydrogenase. Reduction of Fe(III)-NTA was not coupled to cell growth. In fact cell concentrations declined when D. vulgaris was incubated with Fe(III)-NTA as the only electron acceptor. Wild type and mutant cells reducing a limiting concentration of sulfate (2 mM), reduced Fe(III)-NTA with similar rates. However, these were similarly incapable of catalyzing subsequent lactate-dependent reduction of Fe(III)-NTA to completion. Periplasmic reduction of Fe(III)-NTA appeared to inhibit the productive, sulfate-reducing metabolism of D. vulgaris, possibly because it prevents the cycling of reducing equivalents needed to achieve a net bioenergetic benefit.     

Meeting reports

This investigation documents the formation of Green Rust (GR) and immobilization of Ni 2+ in response to bacterial reduction of hydrous ferric oxide (HFO). In the absence of Ni 2+ , 79% of the total Fe(III) present as HFO was reduced; at 10 -3 and 10 -4 M Ni 2+ , 36% of the total Fe(III) was reduced, whereas 45 to 50% of the total Fe(III) was reduced at 10 -5 M Ni 2+ . The inhibitory effect of 10 -3 and 10 -4 M Ni 2+ on Fe(III)-reduction corresponded to a 50% decrease in number of viable cells relative to the Ni 2+ -free condition, and a 25% decrease at 10 -5 M Ni 2+ . A prominent GR peak at d = 10.9 nm was evident in X-ray diffraction patterns of postreduction residual solids from the cultures. Minor peaks arising for vivianite and magnetite were also present. In samples prepared for scanning electron microscopy, thin hexagonal plates of GR were easily distinguished as a solid phase transformation product of HFO. Small hexagonal sheets and fragments of larger GR plates were also observed in transmission electron microscopy whole mounts together with bacteria that were mineralized by surface precipitates of microcrystalline magnetite. Energy dispersive spectroscopy (EDS) confirmed that GR contained Fe and P, as well as Ni in those samples taken from the Ni 2+ -amended experiments. EDS detected neither P nor Ni in the magnetite precipitates associated with the bacterial cells. Dissolved Ni2 + concentrations decreased in an exponential fashion with respect to time in all experimental systems, corresponding to an overall first-order rate constant k of -0.030 day -1 . At the same time, a strong linear relationship (r 2 = 0.99) between the dissolved and solid phase Ni 2+ /Fe 2+ ratios over the entire period of the Fe(III)reduction experiments provided evidence that the solid-phase partitioning of Ni 2+ in GR extended from equilibrium solid-solution behavior.     

Fe(III) reduction during pyruvate fermentation by Desulfotomaculum reducens strain MI‐1

Desulfotomaculum reducens MI‐1 is a Gram‐positive, sulfate‐reducing bacterium also capable of reducing several metals, among which is Fe(III). Very limited knowledge is available on the potential mechanism(s) of metal reduction among Gram‐positive bacteria, despite their preponderance in the microbial communities that inhabit some inhospitable environments (e.g., thermal or hyperthermal ecosystems, extreme pH or salinity environments, heavy metal or radionuclide contaminated sediments). Here, we show that in the presence of pyruvate, this micro‐organism is capable of reducing both soluble Fe(III)‐citrate and solid‐phase hydrous ferric oxide, although growth is sustained by pyruvate fermentation rather than Fe(III) respiration. Despite the fact that Fe(III) reduction does not support direct energy conservation, D. reducens uses it as a complementary means of discarding excess reducing equivalent after H₂ accumulation in the culture headspace renders proton reduction unfavorable. Thus, Fe(III) reduction permits the oxidation of greater amounts of pyruvate than fermentation alone. Fe(III) reduction by D. reducens is mediated by a soluble electron carrier, most likely riboflavin. Additionally, an intracellular electron storage molecule acts as a capacitor and accumulates electrons during pyruvate oxidation for slow release to Fe(III). The reductase responsible for the transfer of electrons from the capacitor to the soluble carrier has not been identified, but data presented here argue against the involvement of c‐type cytochromes.     

Iron Redox Transformation by the Thermo-Acidophilic Archaea from the Genus Sulfolobus

Iron redox transformations by five representative Sulfolobus strains (S. metallicus Kra23, S. tokodaii 7, S. acidocaldarius 98-3, S. solfataricus P1, S. shibatae B12) were studied to clarify the general trend of Fe metabolism across different species of the genus Sulfolobus. Negligible to major Fe(II) oxidation was detected in cell suspensions of the strains. Fe(III)-reducing ability was differently expressed in each strain with dependence on the oxygen level and growth status; growth-uncoupled cell suspensions of all strains reduced Fe(III) under either anaerobic or microaerobic conditions, or under both conditions. A linear correlation between cell growth and Fe(III) reduction was also found in S. tokodaii 7, S. solfataricus P1, and S. shibatae B12. In addition to Fe redox behaviors, the strains were also tested for reduction of highly toxic Cr(VI) to less toxic and soluble Cr(III), as an application possibility; the trend in degree of Cr(V) reduction did not necessarily correspond to that of Fe(III) reduction, suggesting the involvement of different reduction mechanisms.     

Linking geochemical processes with microbial community analysis: successional dynamics in an arsenic-rich, acid-sulphate-chloride geothermal spring

The source waters of acid‐sulphate‐chloride (ASC) geothermal springs located in Norris Geyser Basin, Yellowstone National Park contain several reduced chemical species, including H₂, H₂S, As(III), and Fe(II), which may serve as electron donors driving chemolithotrophic metabolism. Microorganisms thriving in these environments must also cope with high temperatures, low pH (～3), and high concentrations of sulphide, As(III), and boron. The goal of the current study was to correlate the temporal and spatial distribution of bacterial and archaeal populations with changes in temperature and geochemical energy gradients occurring throughout a newly formed (redirected) outflow channel of an ASC spring. A suite of complimentary analyses including aqueous geochemistry, microscopy, solid phase identification, and 16S rDNA sequence distribution were used to correlate the appearance of specific microbial populations with biogeochemical processes mediating S, Fe, and As cycling and subsequent biomineralization of As(V)‐rich hydrous ferric oxide (HFO) mats. Rapid As(III) oxidation (maximum first order rate constants ranged from 4 to 5 min^?1, t_1/2 = 0.17 ? 0.14 min) was correlated with the appearance of Hydrogenobaculum and Thiomonas–like populations, whereas the biogenesis of As(V)‐rich HFO microbial mats (mole ratios of As:Fe ～0.7) was correlated with the appearance of Metallosphaera, Acidimicrobium, and Thiomonas–like populations. Several 16S sequences detected near the source were closely related to sequences of chemolithotrophic hyperthermophilic populations including Stygiolobus and Hydrogenobaculum organisms that are known H₂ oxidizers. The use of H₂, reduced S(–II,0), Fe(II) and perhaps As(III) by different organisms represented throughout the outflow channel was supported by thermodynamic calculations, confirming highly exergonic redox couples with these electron donors. Results from this work demonstrated that chemical energy gradients play an important role in establishing distinct community structure as a function of distance from geothermal spring discharge.     

Enrichment of Geobacter Species in Response to Stimulation of Fe(III) Reduction in Sandy Aquifer Sediments

Abstract Engineered stimulation of Fe(III) has been proposed as a strategy to enhance the immobilization of radioactive and toxic metals in metal-contaminated subsurface environments. Therefore, laboratory and field studies were conducted to determine which microbial populations would respond to stimulation of Fe(III) reduction in the sediments of sandy aquifers. In laboratory studies, the addition of either various organic electron donors or electron shuttle compounds stimulated Fe(III) reduction and resulted in Geobacter sequences becoming important constituents of the Bacterial 16S rDNA sequences that could be detected with PCR amplification and denaturing gradient gel electrophoresis (DGGE). Quantification of Geobacteraceae sequences with a PCR most-probable-number technique indicated that the extent to which numbers of Geobacter increased was related to the degree of stimulation of Fe(III) reduction. Geothrix species were also enriched in some instances, but were orders of magnitude less numerous than Geobacter species. Shewanella species were not detected, even when organic compounds known to be electron donors for Shewanella species were used to stimulate Fe(III) reduction in the sediments. Geobacter species were also enriched in two field experiments in which Fe(III) reduction was stimulated with the addition of benzoate or aromatic hydrocarbons. The apparent growth of Geobacter species concurrent with increased Fe(III) reduction suggests that Geobacter species were responsible for much of the Fe(III) reduction in all of the stimulation approaches evaluated in three geographically distinct aquifers. Therefore, strategies for subsurface remediation that involve enhancing the activity of indigenous Fe(III)-reducing populations in aquifers should consider the physiological properties of Geobacter species in their treatment design. Received: 19 October 1999; Accepted: 28 December 1999; Online Publication: 25 April 2000     

Dependence of In Situ Bacterial Fe(II)-Oxidation and Fe(III)-Precipitation on Sequential Reactive Transport

A study was conducted to determine in situ rates of Fe(II) oxidation and Fe(III) precipitation along a 5.0 m reach of a ferruginous groundwater discharge zone under two distinct conditions; (i) the natural state featuring abundant flocculent mats of bacteriogenic iron oxides (BIOS) produced by Fe(II)-oxidizing bacteria, and (ii) after a manual washout of the streambed to remove the microbial mat. Examination of mat samples by differential interference contrast light microscopy revealed tangled meshworks of filamentous Leptothrix sheaths and helical Gallionella stalks intermixed with fine-grained hydrous ferric oxide (HFO) precipitates. The greatest accumulation of BIOS mat was 1.0 m downstream of the groundwater spring. Redox potential (Eh) increased sharply from 200 mV to over 300 mV over the last 2.0 m of the reach. Similarly, dissolved oxygen increased from < 10% saturation to almost 100% saturation over the last 2.0 m of the reach, whereas pH increased from 6.4 to 7.3. Pseudo-first-order rate constants determined on the basis of analytical solutions to sequential partial differential advection-dispersion-reaction equations for the linear Fe(II)→Fe(III)→HFO reaction network yielded in situ Fe(II) oxidation rate constants (k_ox) of 1.70 ± 0.20 min^?1 in natural conditions and 0.48 ± 0.14 min^?1 after washout. Corresponding Fe(III)-precipitation rates (k_p) before and after washout were 3.45 ± 0.10 min^?1 and 0.90 ± 0.01 min^?1, respectively. These values for k_ox and k_p are higher than estimates obtained from closed batch microcosm and laboratory experiments, underscoring the crucial dependence of in situ Fe(II) oxidation and Fe(III) precipitation rates on advective and dispersive mass transport. The results also highlight the influence that BIOS microbial mats exert on the reaction kinetics of the multiple heterogeneous reactions contributing not only to Fe(II)/Fe(III) redox transformations in groundwater discharge zones, but also the precipitation of HFO.