Bioenergetics of iron snow fueling life on Europa.

The main sources of redox gradients supporting high-productivity life in the Europan and other icy ocean world oceans were proposed to be photolytically derived oxidants, such as reactive oxygen species (ROS) from the icy shell, and reductants (Fe(II), S(-II), CH4, H2) from bottom waters reacting with a (ultra)mafic seafloor. Important roadblocks to maintaining life, however, are that the degree of ocean mixing to combine redox species is unknown, and ROS damage biomolecules. Here, we envisage a unique solution using an acid mine drainage (AMD)-filled pit lakes analog system for the Europan ocean, which previous models predicted to be acidic. We hypothesize that surface-generated ROS oxidize dissolved Fe(II) resulting in Fe(III) (hydr)oxide precipitates, that settle to the seafloor as "iron snow." The iron snow provides a respiratory substrate for anaerobic microorganisms ("breathing iron"), and limits harmful ROS exposure since they are now neutralized at the ice-water interface. Based on this scenario, we calculated Gibbs energies and maximal biomass productivities of various anaerobic metabolisms for a range of pH, temperatures, and H2 fluxes. Productivity by iron reducers was greater for most environmental conditions considered, whereas sulfate reducers and methanogens were more favored at high pH. Participation of Fe in the metabolic redox processes is largely neglected in most models of Europan biogeochemistry. Our model overcomes important conceptual roadblocks to life in icy ocean worlds and broadens the potential metabolic diversity, thus increasing total primary productivity, the diversity and volume of habitable environmental niches and, ultimately, the probability of biosignature detection.

Ohio Coronavirus Wastewater Monitoring Network: Implementation of Statewide Monitoring for Protecting Public Health.

CONTEXT: Prior to the COVID-19 pandemic, wastewater influent monitoring for tracking disease burden in sewered communities was not performed in Ohio, and this field was only on the periphery of the state academic research community. PROGRAM: Because of the urgency of the pandemic and extensive state-level support for this new technology to detect levels of community infection to aid in public health response, the Ohio Water Resources Center established relationships and support of various stakeholders. This enabled Ohio to develop a statewide wastewater SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) monitoring network in 2 months starting in July 2020. IMPLEMENTATION: The current Ohio Coronavirus Wastewater Monitoring Network (OCWMN) monitors more than 70 unique locations twice per week, and publicly available data are updated weekly on the public dashboard. EVALUATION: This article describes the process and decisions that were made during network initiation, the network progression, and data applications, which can inform ongoing and future pandemic response and wastewater monitoring. DISCUSSION: Overall, the OCWMN established wastewater monitoring infrastructure and provided a useful tool for public health professionals responding to the pandemic.

Uniform and Pitting Corrosion of Carbon Steel by Shewanella oneidensis MR-1 under Nitrate-Reducing Conditions.

Despite observations of steel corrosion in nitrate-reducing environments, processes of nitrate-dependent microbially influenced corrosion (MIC) remain poorly understood and difficult to identify. We evaluated carbon steel corrosion by Shewanella oneidensis MR-1 under nitrate-reducing conditions using a split-chamber/zero-resistance ammetry (ZRA) technique. This approach entails the deployment of two metal (carbon steel 1018 in this case) electrodes into separate chambers of an electrochemical split-chamber unit, where the microbiology or chemistry of the chambers can be manipulated. This approach mimics the conditions of heterogeneous metal coverage that can lead to uniform and pitting corrosion. The current between working electrode 1 (WE1) and WE2 can be used to determine rates, mechanisms, and, we now show, extents of corrosion. When S. oneidensis was incubated in the WE1 chamber with lactate under nitrate-reducing conditions, nitrite transiently accumulated, and electron transfer from WE2 to WE1 occurred as long as nitrite was present. Nitrite in the WE1 chamber (without S. oneidensis) induced electron transfer in the same direction, indicating that nitrite cathodically protected WE1 and accelerated the corrosion of WE2. When S. oneidensis was incubated in the WE1 chamber without an electron donor, nitrate reduction proceeded, and electron transfer from WE2 to WE1 also occurred, indicating that the microorganism could use the carbon steel electrode as an electron donor for nitrate reduction. Our results indicate that under nitrate-reducing conditions, uniform and pitting carbon steel corrosion can occur due to nitrite accumulation and the use of steel-Fe(0) as an electron donor, but conditions of sustained nitrite accumulation can lead to more-aggressive corrosive conditions.IMPORTANCE Microbially influenced corrosion (MIC) causes damage to metals and metal alloys that is estimated to cost over $100 million/year in the United States for prevention, mitigation, and repair. While MIC occurs in a variety of settings and by a variety of organisms, the mechanisms by which microorganisms cause this damage remain unclear. Steel pipe and equipment may be exposed to nitrate, especially in oil and gas production, where this compound is used for corrosion and "souring" control. In this paper, we show uniform and pitting MIC under nitrate-reducing conditions and that a major mechanism by which it occurs is via the heterogeneous cathodic protection of metal surfaces by nitrite as well as by the microbial oxidation of steel-Fe(0).

Response of soil-associated microbial communities to intrusion of coal mine-derived acid mine drainage.

A system has been identified in which coal mine-derived acid mine drainage (AMD) flows as a 0.5-cm-deep sheet over the terrestrial surface. This flow regime enhances the activities of Fe(II) oxidizing bacteria, which catalyze the oxidative precipitation of Fe from AMD. These activities give rise to Fe(III) (hydr)oxide-rich deposits (referred to as an iron mound) overlying formerly pristine soil. This iron mound has developed with no human intervention, indicating that microbiological activities associated with iron mounds may be exploited as an inexpensive and sustainable approach to remove Fe(II) from AMD. To evaluate the changes in microbial activities and communities that occur when AMD infiltrates initially pristine soil, we incubated AMD-unimpacted soil with site AMD. Continuous exposure of soil to AMD induced progressively greater rates of Fe(II) biooxidation. The development of Fe(II) oxidizing activities was enhanced by inoculation of soil with microorganisms associated with mature iron mound sediment. Evaluation of pyrosequencing-derived 16S rRNA gene sequences recovered from incubations revealed the development of microbial community characteristics that were similar to those of the mature iron mound sediment. Our results indicate that upon mixing of AMD with pristine soil, microbial communities develop that mediate rapid oxidative precipitation of Fe from AMD.

An assessment of microbial communities associated with surface mining-disturbed overburden.

To assess the microbiological changes that occur during the maturation of overburden that has been disturbed by surface mining of coal, a surface mining-disturbed overburden unit in southeastern Ohio, USA was characterized. Overburden from the same unit that had been disturbed for 37 and 16 years were compared to undisturbed soil from the same region. Overburden and soil samples were collected as shallow subsurface cores from each subregion of the mined area (i.e., land 16 years and 37 years post-mining, and unmined land). Chemical and mineralogical characteristics of overburden samples were determined, as were microbial respiration rates. The composition of microbial communities associated with overburden and soil were determined using culture-independent, nucleic acid-based approaches. Chemical and mineralogical evaluation of overburden suggested that weathering of disturbed overburden gave rise to a setting with lower pH and more oxidized chemical constituents. Overburden-associated microbial biomass and respiration rates increased with time after overburden disturbance. Evaluation of 16S rRNA gene libraries that were produced by "next-generation" sequencing technology revealed that recently disturbed overburden contained an abundance of phylotypes attributable to sulfur-oxidizing Limnobacter spp., but with increasing time post-disturbance, overburden-associated microbial communities developed a structure similar to that of undisturbed soil, but retained characteristics of more recently disturbed overburden. Our results indicate that over time, the biogeochemical weathering of disturbed overburden leads to the development of geochemical conditions and microbial communities that approximate those of undisturbed soil, but that this transition is incomplete after 37 years of overburden maturation.

Evaluation of intra- and inter-lab variability in quantifying SARS-CoV-2 in a state-wide wastewater monitoring network.

In December 2019, SARS-CoV-2, the virus that causes coronavirus disease 2019, was first reported and subsequently triggered a global pandemic. Wastewater monitoring, a strategy for quantifying viral gene concentrations from wastewater influents within a community, has served as an early warning and management tool for the spread of SARS-CoV-2 in a community. Ohio built a collaborative statewide wastewater monitoring network that is supported by eight labs (university, government, and commercial laboratories) with unique sample processing workflows. Consequently, we sought to characterize the variability in wastewater monitoring results for network labs. Across seven trials between October 2020 and November 2021, eight participating labs successfully quantified two SARS-CoV-2 RNA targets and human fecal indicator virus targets in wastewater sample aliquots with reproducible results, although recovery efficiencies of spiked surrogates ranged from 3 to 75%. When SARS-CoV-2 gene fragment concentrations were adjusted for recovery efficiency and flow, the proportion of variance between laboratories was minimized, serving as the best model to account for between-lab variance. Another adjustment factor (alone and in different combinations with the above factors) considered to account for sample and measurement variability includes fecal marker normalization. Genetic quantification variability can be attributed to many factors, including the methods, individual samples, and water quality parameters. In addition, statistically significant correlations were observed between SARS-CoV-2 RNA and COVID-19 case numbers, supporting the notion that wastewater surveillance continues to serve as an effective monitoring tool. This study serves as a real-time example of multi-laboratory collaboration for public health preparedness for infectious diseases.

Iron transformations induced by an acid-tolerant Desulfosporosinus species.

The mineralogical transformations of Fe phases induced by an acid-tolerant, Fe(III)- and sulfate-reducing bacterium, Desulfosporosinus sp. strain GBSRB4.2 were evaluated under geochemical conditions associated with acid mine drainage-impacted systems (i.e., low pH and high Fe concentrations). X-ray powder diffractometry coupled with magnetic analysis by first-order reversal curve diagrams were used to evaluate mineral phases produced by GBSRB4.2 in media containing different ratios of Fe(II) and Fe(III). In medium containing Fe predominately in the +II oxidation state, ferrimagnetic, single-domain greigite (Fe3S4) was formed, but the addition of Fe(III) inhibited greigite formation. In media that contained abundant Fe(III) [as schwertmannite; Fe8O8(OH)6SO4 · nH2O], the activities of strain GBSRB4.2 enhanced the transformation of schwertmannite to goethite (α-FeOOH), due to the increased pH and Fe(II) concentrations that resulted from the activities of GBSRB4.2.

Enhanced terrestrial Fe(II) mobilization identified through a novel mechanism of microbially driven cave formation in Fe(III)-rich rocks.

Most cave formation requires mass separation from a host rock in a process that operates outward from permeable pathways to create the cave void. Given the poor solubility of Fe(III) phases, such processes are insufficient to account for the significant iron formation caves (IFCs) seen in Brazilian banded iron formations (BIF) and associated rock. In this study we demonstrate that microbially-mediated reductive Fe(III) dissolution is solubilizing the poorly soluble Fe(III) phases to soluble Fe(II) in the anoxic zone behind cave walls. The resultant Fe(III)-depleted material (termed sub muros) is unable to maintain the structural integrity of the walls and repeated rounds of wall collapse lead to formation of the cave void in an active, measurable process. This mechanism may move significant quantities of Fe(II) into ground water and may help to explain the mechanism of BIF dissolution and REE enrichment in the generation of canga. The role of Fe(III) reducing microorganism and mass separation behind the walls (outward-in, rather than inward-out) is not only a novel mechanism of speleogenesis, but it also may identify a previously overlooked source of continental Fe that may have contributed to Archaean BIF formation.

Identification of Microbiological Activities in Wet Flue Gas Desulfurization Systems.

Thermoelectric power generation from coal requires large amounts of water, much of which is used for wet flue gas desulfurization (wFGD) systems that minimize sulfur emissions, and consequently, acid rain. The microbial communities in wFGDs and throughout thermoelectric power plants can influence system performance, waste processing, and the long term stewardship of residual wastes. Any microorganisms that survive in wFGD slurries must tolerate high total dissolved solids concentrations (TDS) and temperatures (50-60°C), but the inocula for wFGDs are typically from fresh surface waters (e.g., lakes or rivers) of low TDS and temperatures, and whose activity might be limited under the physicochemically extreme conditions of the wFGD. To determine the extents of microbiological activities in wFGDs, we examined the microbial activities and communities associated with three wFGDs. O2 consumption rates of three wFGD slurries were optimal at 55°C, and living cells could be detected microscopically, indicating that living and active communities of organisms were present in the wFGD and could metabolize at the high temperature of the wFGD. A 16S rRNA gene-based survey revealed that the wFGD-associated microbial communities included taxa attributable to both thermophilic and mesophilic lineages. Metatranscriptomic analysis of one of the wFGDs indicated an abundance of active Burholderiaceae and several Gammaproteobacteria, and production of transcripts associated with carbohydrate metabolism, osmotic stress response, as well as phage, prophages, and transposable elements. These results illustrate that microbial activities can be sustained in physicochemically extreme wFGDs, and these activities may influence the performance and environmental impacts of thermoelectric power plants.

Hydrologic Alteration and Enhanced Microbial Reductive Dissolution of Fe(III) (hydr)oxides Under Flow Conditions in Fe(III)-Rich Rocks: Contribution to Cave-Forming Processes.

Previous work demonstrated that microbial Fe(III)-reduction contributes to void formation, and potentially cave formation within Fe(III)-rich rocks, such as banded iron formation (BIF), iron ore and canga (a surficial duricrust), based on field observations and static batch cultures. Microbiological Fe(III) reduction is often limited when biogenic Fe(II) passivates further Fe(III) reduction, although subsurface groundwater flow and the export of biogenic Fe(II) could alleviate this passivation process, and thus accelerate cave formation. Given that static batch cultures are unlikely to reflect the dynamics of groundwater flow conditions in situ, we carried out comparative batch and column experiments to extend our understanding of the mass transport of iron and other solutes under flow conditions, and its effect on community structure dynamics and Fe(III)-reduction. A solution with chemistry approximating cave-associated porewater was amended with 5.0 mM lactate as a carbon source and added to columns packed with canga and inoculated with an assemblage of microorganisms associated with the interior of cave walls. Under anaerobic conditions, microbial Fe(III) reduction was enhanced in flow-through column incubations, compared to static batch incubations. During incubation, the microbial community profile in both batch culture and columns shifted from a Proteobacterial dominance to the Firmicutes, including Clostridiaceae, Peptococcaceae, and Veillonellaceae, the latter of which has not previously been shown to reduce Fe(III). The bacterial Fe(III) reduction altered the advective properties of canga-packed columns and enhanced permeability. Our results demonstrate that removing inhibitory Fe(II) via mimicking hydrologic flow of groundwater increases reduction rates and overall Fe-oxide dissolution, which in turn alters the hydrology of the Fe(III)-rich rocks. Our results also suggest that reductive weathering of Fe(III)-rich rocks such as canga, BIF, and iron ores may be more substantial than previously understood.

Impact of acid mine drainage chemistry and microbiology on the development of efficient Fe removal activities.

Coal mine derived acid mine drainage (AMD) is formed when oxygenated water infiltrates mine voids and oxidizes FeS phases, generating acidic fluid rich in heavy metals, polluting thousands of miles of streams. Existing remediation options are cost-prohibitive and difficult to sustain. In some cases, AMD flows over previously pristine soil in thin sheets over terrestrial surface, enhancing AMD aeration and Fe(II) oxidizing activities, leading to oxidative Fe(II) precipitation from AMD, without any human intervention. Since robust Fe(II) biooxidation occurs in the mixture of intruding AMD and pristine soil, understanding the effects of chemically variant AMD can be exploited for effective Fe(II) removal. We hypothesized that chemistry and microbiology of AMD intruding pristine soil on surface would influence the development of Fe(II) oxidizing capabilities. Therefore, to investigate the response of pristine soil to the addition of AMD varying in chemical and microbial characteristics, we mixed soil with a near-neutral and moderately acidic AMD, in separate incubations. Incubations with near-neutral AMD developed microbial Fe(II) oxidation activities after 10 days. However, Fe(II) oxidation in moderately acidic AMD incubations was mostly abiotic. 16S rRNA gene sequences and metabolic functional prediction (Tax4Fun) analysis of near-neutral AMD and soil mixture indicated development of taxonomically different communities capable of activities similar to microorganisms in a mine void. In conclusion, results indicate that AMD chemistry and microbiology affects development of Fe(II) biooxidation. Therefore, understanding of the effect of AMD chemistry on the development of FeOB activities in soil can be exploited to design site-specific and sustainable solutions.

An integrated microbiological and electrochemical approach to determine distributions of Fe metabolism in acid mine drainage-induced "iron mound" sediments.

Fe(III)-rich deposits referred to as "iron mounds" develop when Fe(II)-rich acid mine drainage (AMD) emerges at the terrestrial surface, and aeration of the fluids induces oxidation of Fe(II), with subsequent precipitation of Fe(III) phases. As Fe(III) phases accumulate in these systems, O2 gradients may develop in the sediments and influence the distributions and extents of aerobic and anaerobic microbiological Fe metabolism, and in turn the solubility of Fe. To determine how intrusion of O2 into iron mound sediments influences microbial community composition and Fe metabolism, we incubated samples of these sediments in a column format. O2 was only supplied through the top of the columns, and microbiological, geochemical, and electrochemical changes at discrete depths were determined with time. Despite the development of dramatic gradients in dissolved Fe(II) concentrations, indicating Fe(II) oxidation in shallower portions and Fe(III) reduction in the deeper portions, microbial communities varied little with depth, suggesting the metabolic versatility of organisms in the sediments with respect to Fe metabolism. Additionally, the availability of O2 in shallow portions of the sediments influenced Fe metabolism in deeper, O2-free sediments. Total potential (EH + self-potential) measurements at discrete depths in the columns indicated that Fe transformations and electron transfer processes were occurring through the sediments and could explain the impact of O2 on Fe metabolism past where it penetrates into the sediments. This work shows that O2 availability (or lack of it) minimally influences microbial communities, but influences microbial activities beyond its penetration depth in AMD-derived Fe(III) rich sediments. Our results indicate that O2 can modulate Fe redox state and solubility in larger volumes of iron mound sediments than only those directly exposed to O2.

Use of an Electrochemical Split Cell Technique to Evaluate the Influence of Shewanella oneidensis Activities on Corrosion of Carbon Steel.

Microbially induced corrosion (MIC) is a complex problem that affects various industries. Several techniques have been developed to monitor corrosion and elucidate corrosion mechanisms, including microbiological processes that induce metal deterioration. We used zero resistance ammetry (ZRA) in a split chamber configuration to evaluate the effects of the facultatively anaerobic Fe(III) reducing bacterium Shewanella oneidensis MR-1 on the corrosion of UNS G10180 carbon steel. We show that activities of S. oneidensis inhibit corrosion of steel with which that organism has direct contact. However, when a carbon steel coupon in contact with S. oneidensis was electrically connected to a second coupon that was free of biofilm (in separate chambers of the split chamber assembly), ZRA-based measurements indicated that current moved from the S. oneidensis-containing chamber to the cell-free chamber. This electron transfer enhanced the O2 reduction reaction on the coupon deployed in the cell free chamber, and consequently, enhanced oxidation and corrosion of that electrode. Our results illustrate a novel mechanism for MIC in cases where metal surfaces are heterogeneously covered by biofilms.

A procedure for quantitation of total oxidized uranium for bioremediation studies.

A procedure was developed for the quantitation of complexed U(VI) during studies on U(VI) bioremediation. These studies typically involve conversion of soluble or complexed U(VI) (oxidized) to U(IV) (the reduced form which is much less soluble). Since U(VI) freely exchanges between material adsorbed to the solid phase and the dissolved phase, uranium bioremediation experiments require a mass balance of U in both its soluble and adsorbed forms as well as in the reduced sediment bound phase. We set out to optimize a procedure for extraction and quantitation of sediment bound U(VI). Various extractant volumes to sediment ratios were tested and it was found that between 1:1 to 8:1 ratios (v/w) there was a steady increase in U(VI) recovered, but no change with further increases in v/w ratio. Various strengths of NaHCO(3), Na-EDTA, and Na-citrate were used to evaluate complexed U(VI) recovery, while the efficiency of a single versus repeated extraction steps was compared with synthesized uranyl-phosphate and uranyl-hydroxide. Total recovery with 1 M NaHCO(3) was 95.7% and 97.9% from uranyl-phosphate and uranyl-hydroxide, respectively, compared to 80.7% and 89.9% using 450 mM NaHCO(3). Performing the procedure once yielded an efficiency of 81.1% and 92.3% for uranyl-phosphate and uranyl-hydroxide, respectively, as compared to three times. All other extractants yielded 7.9-82.0% in both experiments. Biologically reduced U(IV) was treated either alone or mixed with uncontaminated sediment slurries to ensure that the procedure was not interfering with subsequent U(IV) quantitation. While U(VI) was recovered, it represented 0.07% of the total uranium alone or 7.8% when mixed with sediments. Total uranium recovered did not change. The procedure was then used to monitor changes in complexed U(VI) levels during uranium-reduction in pure culture and sediments. There was no appreciable complexed U(VI) concentration in pure culture. In sediments however, once soluble U(VI) levels and reduction rates decreased, complexed U(VI) levels began to decrease while U(IV) levels continued to increase. This indicated that once soluble U(VI) was nearly exhausted, sorbed U(VI) became bioavailable and was reduced microbiologically.Typically, uranium is quantified in two steps, soluble U(VI) and U(IV). However, the present study shows that after successive washings with water to remove soluble U(VI), a significant pool of oxidized uranium remains which may be mistakenly quantified as U(IV). This procedure can be used to quantified this pool, does not interfere with U(IV) quantitation, and has an overall efficiency of 95.8%.

Depth-dependent geochemical and microbiological gradients in Fe(III) deposits resulting from coal mine-derived acid mine drainage.

We evaluated the depth-dependent geochemistry and microbiology of sediments that have developed via the microbially-mediated oxidation of Fe(II) dissolved in acid mine drainage (AMD), giving rise to a 8-10 cm deep "iron mound" that is composed primarily of Fe(III) (hydr)oxide phases. Chemical analyses of iron mound sediments indicated a zone of maximal Fe(III) reducing bacterial activity at a depth of approximately 2.5 cm despite the availability of dissolved O2 at this depth. Subsequently, Fe(II) was depleted at depths within the iron mound sediments that did not contain abundant O2. Evaluations of microbial communities at 1 cm depth intervals within the iron mound sediments using "next generation" nucleic acid sequencing approaches revealed an abundance of phylotypes attributable to acidophilic Fe(II) oxidizing Betaproteobacteria and the chloroplasts of photosynthetic microeukaryotic organisms in the upper 4 cm of the iron mound sediments. While we observed a depth-dependent transition in microbial community structure within the iron mound sediments, phylotypes attributable to Gammaproteobacterial lineages capable of both Fe(II) oxidation and Fe(III) reduction were abundant in sequence libraries (comprising ≥20% of sequences) from all depths. Similarly, abundances of total cells and culturable Fe(II) oxidizing bacteria were uniform throughout the iron mound sediments. Our results indicate that O2 and Fe(III) reduction co-occur in AMD-induced iron mound sediments, but that Fe(II)-oxidizing activity may be sustained in regions of the sediments that are depleted in O2.

Microbial communities associated with wet flue gas desulfurization systems.

Flue gas desulfurization (FGD) systems are employed to remove SO(x) gasses that are produced by the combustion of coal for electric power generation, and consequently limit acid rain associated with these activities. Wet FGDs represent a physicochemically extreme environment due to the high operating temperatures and total dissolved solids (TDS) of fluids in the interior of the FGD units. Despite the potential importance of microbial activities in the performance and operation of FGD systems, the microbial communities associated with them have not been evaluated. Microbial communities associated with distinct process points of FGD systems at several coal-fired electricity generation facilities were evaluated using culture-dependent and -independent approaches. Due to the high solute concentrations and temperatures in the FGD absorber units, culturable halothermophilic/tolerant bacteria were more abundant in samples collected from within the absorber units than in samples collected from the makeup waters that are used to replenish fluids inside the absorber units. Evaluation of bacterial 16S rRNA genes recovered from scale deposits on the walls of absorber units revealed that the microbial communities associated with these deposits are primarily composed of thermophilic bacterial lineages. These findings suggest that unique microbial communities develop in FGD systems in response to physicochemical characteristics of the different process points within the systems. The activities of the thermophilic microbial communities that develop within scale deposits could play a role in the corrosion of steel structures in FGD systems.

Characterization of Fe(II) oxidizing bacterial activities and communities at two acidic Appalachian coalmine drainage-impacted sites.

We characterized the microbiologically mediated oxidative precipitation of Fe(II) from coalmine-derived acidic mine drainage (AMD) along flow-paths at two sites in northern Pennsylvania. At the Gum Boot site, dissolved Fe(II) was efficiently removed from AMD whereas minimal Fe(II) removal occurred at the Fridays-2 site. Neither site received human intervention to treat the AMD. Culturable Fe(II) oxidizing bacteria were most abundant at sampling locations along the AMD flow path corresponding to greatest Fe(II) removal and where overlying water contained abundant dissolved O(2). Rates of Fe(II) oxidation determined in laboratory-based sediment incubations were also greatest at these sampling locations. Ribosomal RNA intergenic spacer analysis and sequencing of partial 16S rRNA genes recovered from sediment bacterial communities revealed similarities among populations at points receiving regular inputs of Fe(II)-rich AMD and provided evidence for the presence of bacterial lineages capable of Fe(II) oxidation. A notable difference between bacterial communities at the two sites was the abundance of Chloroflexi-affiliated 16S rRNA gene sequences in clone libraries derived from the Gum Boot sediments. Our results suggest that inexpensive and reliable AMD treatment strategies can be implemented by mimicking the conditions present at the Gum Boot field site.

Effect of oxidation rate and Fe(II) state on microbial nitrate-dependent Fe(III) mineral formation.

A nitrate-dependent Fe(II)-oxidizing bacterium was isolated and used to evaluate whether Fe(II) chemical form or oxidation rate had an effect on the mineralogy of biogenic Fe(III) (hydr)oxides resulting from nitrate-dependent Fe(II) oxidation. The isolate (designated FW33AN) had 99% 16S rRNA sequence similarity to Klebsiella oxytoca. FW33AN produced Fe(III) (hydr)oxides by oxidation of soluble Fe(II) [Fe(II)sol] or FeS under nitrate-reducing conditions. Based on X-ray diffraction (XRD) analysis, Fe(III) (hydr)oxide produced by oxidation of FeS was shown to be amorphous, while oxidation of Fe(II)sol yielded goethite. The rate of Fe(II) oxidation was then manipulated by incubating various cell concentrations of FW33AN with Fe(II)sol and nitrate. Characterization of products revealed that as Fe(II) oxidation rates slowed, a stronger goethite signal was observed by XRD and a larger proportion of Fe(III) was in the crystalline fraction. Since the mineralogy of Fe(III) (hydr)oxides may control the extent of subsequent Fe(III) reduction, the variables we identify here may have an effect on the biogeochemical cycling of Fe in anoxic ecosystems.

Role for Fe(III) minerals in nitrate-dependent microbial U(IV) oxidation.

Microbiological reduction of soluble U(VI) to insoluble U(IV) is a means of preventing the migration of that element in groundwater, but the presence of nitrate in U(IV)-containing sediments leads to U(IV) oxidation and remobilizaton. Nitrite or iron(III) oxyhydroxides may oxidize U(IV) under nitrate-reducing conditions, and we determined the rate and extent of U(IV) oxidation by these compounds. Fe(III) oxidized U(IV) at a greater rate than nitrite (130 and 10 microM U(IV)/day, respectively). In aquifer sediments, Fe(III) may be produced during microbial nitrate reduction by oxidation of Fe(II) with nitrite, or by enzymatic Fe(II) oxidation coupled to nitrate reduction. To determine which of these mechanisms was dominant, we isolated a nitrate-dependent acetate- and Fe(ll)-oxidizing bacterium from a U(VI)- and nitrate-contaminated aquifer. This organism oxidized U(IV) at a greater rate and to a greater extent under acetate-oxidizing (where nitrite accumulated to 50 mM)than under Fe(II)-oxidizing conditions. We showthatthe observed differences in rate and extent of U(IV) oxidation are due to mineralogical differences between Fe(III) produced by reaction of Fe(II) with nitrite (amorphous) and Fe(III) produced enzymatically (goethite or lepidocrocite). Our results suggest the mineralogy and surface area of Fe(III) minerals produced under nitrate-reducing conditions affect the rate and extent of U(IV) oxidation. These results may be useful for predicting the stability of U(IV) in aquifers.

In-situ evidence for uranium immobilization and remobilization.

The in-situ microbial reduction and immobilization of uranium was assessed as a means of preventing the migration of this element in the terrestrial subsurface. Uranium immobilization (putatively identified as reduction) and microbial respiratory activities were evaluated in the presence of exogenous electron donors and acceptors with field push-pull tests using wells installed in an anoxic aquifer contaminated with landfill leachate. Uranium(VI) amended at 1.5 microM was reduced to less than 1 nM in groundwater in less than 8 d during all field experiments. Amendments of 0.5 mM sulfate or 5 mM nitrate slowed U(VI) immobilization and allowed for the recovery of 10% and 54% of the injected element, respectively, as compared to 4% in the unamended treatment. Laboratory incubations confirmed the field tests and showed that the majority of the U(VI) immobilized was due to microbial reduction. In these tests, nitrate treatment (7.5 mM) inhibited U(VI) reduction, and nitrite was transiently produced. Further push-pull tests were performed in which either 1 or 5 mM nitrate was added with 1.0 uM U(VI) to sediments that already contained immobilized uranium. After an initial loss of the amendments, the concentration of soluble U(VI) increased and eventually exceeded the injected concentration, indicating that previously immobilized uranium was remobilized as nitrate was reduced. Laboratory experiments using heat-inactivated sediment slurries suggested that the intermediates of dissimilatory nitrate reduction (denitrification or dissimilatory nitrate reduction to ammonia), nitrite, nitrous oxide, and nitric oxide were all capable of oxidizing and mobilizing U(IV). These findings indicate that in-situ subsurface U(VI) immobilization can be expected to take place under anaerobic conditions, but the permanence of the approach can be impaired by disimilatory nitrate reduction intermediates that can mobilize previously reduced uranium.