Iron- and aluminium-induced depletion of molybdenum in acidic environments impedes the nitrogen cycle.

Anthropogenic nitrate contamination is a serious problem in many natural environments. Nitrate removal by microbial action is dependent on the metal molybdenum (Mo), which is required by nitrate reductase for denitrification and dissimilatory nitrate reduction to ammonium. The soluble form of Mo, molybdate (MoO4 2- ), is incorporated into and adsorbed by iron (Fe) and aluminium (Al) (oxy) hydroxide minerals. Herein we used Oak Ridge Reservation (ORR) as a model nitrate-contaminated acidic environment to investigate whether the formation of Fe- and Al-precipitates could impede microbial nitrate removal by depleting Mo. We demonstrate that Fe and Al mineral formation that occurs as the pH of acidic synthetic groundwater is increased, decreases soluble Mo to low picomolar concentrations, a process proposed to mimic environmental diffusion of acidic contaminated groundwater. Analysis of ORR sediments revealed recalcitrant Mo in the contaminated core that co-occurred with Fe and Al, consistent with Mo scavenging by Fe/Al precipitates. Nitrate removal by ORR isolate Pseudomonas fluorescens N2A2 is virtually abolished by Fe/Al precipitate-induced Mo depletion. The depletion of naturally occurring Mo in nitrate- and Fe/Al-contaminated acidic environments like ORR or acid mine drainage sites has the potential to impede microbial-based nitrate reduction thereby extending the duration of nitrate in the environment.

Contaminant mobilization from the vadose zone to groundwater during experimental river flooding events.

Natural river flooding events can mobilize contaminants from the vadose zone and lead to increased concentrations in groundwater. Characterizing the mass and transport mechanisms of contaminants released from the vadose zone to groundwater during these recharge events is particularly challenging. Therefore, conducting highly-controlled in-situ experiments that simulate natural flooding events can help increase the knowledge of where contaminants can be stored and how they can move between hydrological compartments. This study specifically targets uranium pollution, which is accompanied by high sulfate levels in the vadose zone and groundwater. Two novel experimental river flooding events were conducted that utilized added non-reactive halides (bromide and iodide) and 2,6-difluorobenzoate tracers. In both experiments, about 8 m3 of traced water from a nearby contaminant-poor river was flooded in a 3-m diameter basin and infiltrated through the vadose zone and into a contaminant-rich unconfined aquifer for an average of 10 days. The aquifer contained 13 temporary wells that were monitored for solute concentration for up to 40 days. The groundwater analysis was conducted for changes in contaminant mass using the Theissen polygon method and for transport mechanisms using temporal moments. The results indicated an increase in uranium (21 and 24%), and sulfate (24 and 25%) contaminant mass transport to groundwater from the vadose zone during both experiments. These findings confirmed that the vadose zone can store and release substantial amounts of contaminants to groundwater during flooding events. Additionally, contaminants were detected earlier than the added tracers, along with higher concentrations. These results suggested that contaminant-rich pore water in the vadose zone was transported ahead of the traced flood waters and into groundwater. During the first flooding event, elevated concentrations of contaminants were sustained, and that chloride behaved similarly. The findings implied that contaminant- and chloride-rich evaporites in the vadose zone were dissolved during the first flooding event. For the second flooding event, the data suggested that the contaminant-rich evaporites continued to dissolve whereas chloride-rich evaporites were previously flushed. Overall, these findings indicated that contaminant-rich pore water and evaporites in the vadose zone can play a significant role in contaminant transport during flooding events.

Halophilic bacteria in a Lake Michigan drainage basin as potential biological indicators of chloride-impacted freshwaters.

There are few biological indicators for freshwater systems subjected to high chloride levels. Freshwater systems receive many forms of chloride such as road salts (e.g., NaCl, CaCl2, MgCl2), fertilizers (e.g., KCl), and year-round water softener pollution. The goal our study was to investigate Halomonadaceae populations as prospective biological indicators of chloride-impacted freshwaters. The bacterial family Halomonadaceae are halophiles that generally require the presence of salt to survive, which make them an attractive candidate in determining chloride impaired areas. Field sediment surveys assessed how salt tolerant and halophilic bacteria abundance corresponded to chloride and conductivity measurements. Colony forming unit (CFU) counts on modified M9 6% NaCl plates (w/v) at urbanized sites compared to the rural sites had highest counts during winter and spring when chloride concentrations were also highest. Select isolates identified as Halomonadaceae through 16S rRNA sequencing were kept as active cultures to determine the NaCl concentration and temperature preference that resulted in the isolates optimal growth. Isolates tested under 5 °C (cold) grew optimally in 2 % NaCl (w/v), whereas under 18 °C (warm), isolates showed optimal growth at 6 % NaCl. The majority of isolates had maximum growth in the warmer temperature, however, select isolates grew better in the cold temperature. Culture-independent methods were used and identified Halomonadaceae were widespread and permeant members of the microbial community in a Lake Michigan drainage basin. Quantitative polymerase chain reaction (qPCR) targeting Halomonadaceae genera demonstrated that abundance varied by site, but overall were present throughout the year. However, community sequencing revealed there were a large relative proportion of specific Halomonadaceae populations present in winter versus summer. Methods targeting salt tolerant bacteria and specific members of Halomonadaceae appears to be a promising approach to assess chloride-impacted areas to better understand the long-term ecological impacts as we continue to salinize freshwater resources.

Single-Well Injection-Drift Test to Estimate Groundwater Velocity.

A simple algebraic equation is presented here to estimate the magnitude of groundwater velocity based on data from a single-well injection-drift test thereby eliminating the time-consuming and costly extraction phase. A volume of tracer-amended water was injected by forced-gradient into a single well followed by monitoring of the conservative solute tracers under natural-gradient conditions as their upgradient portions drifted back through the well. The breakthrough curve data from the single well during the drift phase was analyzed to determine the mean travel times of the tracers. The estimated mean upgradient travel distance back through the single well and the mean travel times of the tracers were used in a simple algebraic equation to estimate groundwater velocity. The groundwater velocity based on the single-well injection-drift test was estimated to be approximately 0.64 ft per day. Two transects of observation wells were used to monitor the natural-gradient tracer transport downgradient of the injection well. The one-dimensional, or dual-well, transport of the tracer from the injection well to the nearest downgradient observation well indicated that the groundwater velocity was 0.55 ft per day. The two-dimensional, or multi-well, transport of the center of mass of the tracers indicated that the groundwater velocity was 0.60 ft per day; the dual- and multi-well results were in excellent agreement with those from the single-well and validated the simple algebraic equation. The new single-well method presented here is relatively simple, rapid, and does not require an extraction phase.

Elucidating mobilization mechanisms of uranium during recharge of river water to contaminated groundwater.

The recharge of stream water below the baseflow water table can mobilize groundwater contaminants, particularly redox-sensitive and sorptive metals such as uranium. However, in-situ tracer experiments that simulate the recharge of stream water to uranium-contaminated groundwater are lacking, thus limiting the understanding of the potential mechanisms that control the mobility of uranium at the field scale. In this study, a field tracer test was conducted by injecting 100 gal (379 l) of oxic river water into a nearby suboxic and uranium-contaminated aquifer. The traced river water was monitored for 18 days in the single injection well and in the twelve surrounding observation wells. Mobilization of uranium from the solid to the aqueous phase was not observed during the tracer test despite its pre-test presence being confirmed on the aquifer sediments from lab-based acid leaching. However, strong evidence of oxidative immobilization of iron and manganese was observed during the tracer test and suggested that immobile uranium was likely in its oxidized state as U(VI) on the aquifer sediments; these observations ruled out oxidation of U(IV) to U(VI) as a potential mobilization mechanism. Therefore, desorption of U(VI) appeared to be the predominant potential mobilization mechanism, yet it was clearly not solely dependent on concentration as evident when considering that uranium-poor river water (<0.015 mg/L) was recharged to uranium-rich groundwater (≈1 mg/L). It was possible that uranium desorption was limited by the relatively higher pH and lower alkalinity of the river water as compared to the groundwater; both factors favor immobilization. However, it was likely that the immobile uranium was associated with a mineral phase, as opposed to a sorbed phase, thus desorption may not have been possible. The results of this field tracer study successfully ruled out two common mobilization mechanisms of uranium: (1) oxidative dissolution and (2) concentration-dependent desorption and ruled in the importance of advection, dispersion, and the mineral phase of uranium.

Sustained Ability of a Natural Microbial Community to Remove Nitrate from Groundwater.

Microbial-mediated nitrate removal from groundwater is widely recognized as the predominant mechanism for nitrate attenuation in contaminated aquifers and is largely dependent on the presence of a carbon-bearing electron donor. The repeated exposure of a natural microbial community to an electron donor can result in the sustained ability of the community to remove nitrate; this phenomenon has been clearly demonstrated at the laboratory scale. However, in situ demonstrations of this ability are lacking. For this study, ethanol (electron donor) was repeatedly injected into a groundwater well (treatment) for six consecutive weeks to establish the sustained ability of a microbial community to remove nitrate. A second well (control) located upgradient was not injected with ethanol during this time. The treatment well demonstrated strong evidence of sustained ability as evident by ethanol, nitrate, and subsequent sulfate removal up to 21, 64, and 68%, respectively, as compared to the conservative tracer (bromide) upon consecutive exposures. Both wells were then monitored for six additional weeks under natural (no injection) conditions. During the final week, ethanol was injected into both treatment and control wells. The treatment well demonstrated sustained ability as evident by ethanol and nitrate removal up to 20 and 21%, respectively, as compared to bromide, whereas the control did not show strong evidence of nitrate removal (5% removal). Surprisingly, the treatment well did not indicate a sustained and selective enrichment of a microbial community. These results suggested that the predominant mechanism(s) of sustained ability likely exist at the enzymatic- and/or genetic-levels. The results of this study demonstrated the in situ ability of a microbial community to remove nitrate can be sustained in the prolonged absence of an electron donor.

Field experiments of surface water to groundwater recharge to characterize the mobility of uranium and vanadium at a former mill tailing site.

Characterizing the mobility of uranium and vanadium in groundwater with a hydraulic connection to surface water is important to inform the best management practices of former mill tailing sites. In this study, the recharge of river water to the unsaturated and saturated zones of a uranium-contaminated alluvial aquifer was simulated in a series of forced-gradient single- and multi-well injection-extraction tests. The injection fluid (river water) was traced with natural and artificial tracers that included halides, fluorobenzoates, lithium, and naphthalene sulfonate to characterize the potential mass transport mechanisms of uranium and vanadium. The extraction fluid (river water/groundwater mixture) was analyzed for the tracers, uranium, and vanadium. The results from the tracers indicated that matrix diffusion was likely negligible over the spatiotemporal scales of the tests as evident by nearly identical breakthrough curves of the halides and fluorobenzoates. In contrast, the breakthrough curves of lithium and naphthalene sulfonate indicated that sorption by cation exchange and sorption to organic matter, respectively, were potential mass transport mechanisms of uranium and vanadium. Uranium was mobilized in the saturated zone containing gypsum (gypsum-rich zone), the vadose zone (vadose-rich zone), and the saturated zone containing organic carbon (organic-rich zone) whereas vanadium was mobilized only in the saturated gypsum-rich zone. The mechanisms responsible for the mobilization of uranium and vanadium were likely dissolution of uranium- and vanadium-bearing minerals and/or desorption from the gypsum-rich zone, flushing of uranium from the vadose-rich zone, and desorption of uranium from the organic-rich zone due to the natural contrast in the geochemistry between the river water and groundwater. The experimental design of this study was unique in that it employed the use of multiple natural and artificial tracers coupled with a direct injection of native river water to groundwater. These results demonstrated that natural recharge and flooding events at former mill tailing sites can mobilize uranium, and possibly vanadium, and contribute to persistent levels of groundwater contamination.

Characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer.

The processing of sediment to accurately characterize the spatially-resolved depth profiles of geophysical and geochemical properties along with signatures of microbial density and activity remains a challenge especially in complex contaminated areas. This study processed cores from two sediment boreholes from background and contaminated core sediments and surrounding groundwater. Fresh core sediments were compared by depth to capture the changes in sediment structure, sediment minerals, biomass, and pore water geochemistry in terms of major and trace elements including pollutants, cations, anions, and organic acids. Soil porewater samples were matched to groundwater level, flow rate, and preferential flows and compared to homogenized groundwater-only samples from neighboring monitoring wells. Groundwater analysis of nearby wells only revealed high sulfate and nitrate concentrations while the same analysis using sediment pore water samples with depth was able to suggest areas high in sulfate- and nitrate-reducing bacteria based on their decreased concentration and production of reduced by-products that could not be seen in the groundwater samples. Positive correlations among porewater content, total organic carbon, trace metals and clay minerals revealed a more complicated relationship among contaminant, sediment texture, groundwater table, and biomass. The fluctuating capillary interface had high concentrations of Fe and Mn-oxides combined with trace elements including U, Th, Sr, Ba, Cu, and Co. This suggests the mobility of potentially hazardous elements, sediment structure, and biogeochemical factors are all linked together to impact microbial communities, emphasizing that solid interfaces play an important role in determining the abundance of bacteria in the sediments.

Improved Method for Estimating Reaction Rates During Push-Pull Tests.

The breakthrough curve obtained from a single-well push-pull test can be adjusted to account for dilution of the injection fluid in the aquifer fluid. The dilution-adjusted breakthrough curve can be analyzed to estimate the reaction rate of a solute. The conventional dilution-adjusted method assumes that the ratios of the concentrations of the nonreactive and reactive solutes in the injection fluid vs. the aquifer fluid are equal. If this assumption is invalid, the conventional method will generate inaccurate breakthrough curves and may lead to erroneous conclusions regarding the reactivity of a solute. In this study, a new method that generates a dilution-adjusted breakthrough curve was theoretically developed to account for any possible combination of nonreactive and reactive solute concentrations in the injection and aquifer fluids. The newly developed method was applied to a field-based data set and was shown to generate more accurate dilution-adjusted breakthrough curves. The improved dilution-adjusted method presented here is simple, makes no assumptions regarding the concentrations of the nonreactive and reactive solutes in the injection and aquifer fluids, and easily allows for estimating reaction rates during push-pull tests.

In situ decay of polyfluorinated benzoic acids under anaerobic conditions.

Polyfluorinated benzoic acids (PBAs) can be used as non-reactive tracers to characterize reactive mass transport mechanisms in groundwater. The use of PBAs as non-reactive tracers assumes that their reactivities are negligible. If this assumption is not valid, PBAs may not be appropriate to use as non-reactive tracers. In this study, the reactivity of two PBAs, 2,6-difluorobenzoic acid (2,6-DFBA) and pentafluorobenzoic acid (PFBA), was tested in situ. A series of two single-well push-pull tests were conducted in two hydrogeologically similar, yet spatially distinct, groundwater monitoring wells. Bromide, 2,6-DFBA, and PFBA were added to the injection fluid and periodically measured in the extraction fluid along with chloride, nitrate, sulfate, and fluoride. Linear regression of the dilution-adjusted breakthrough curves of both PBAs indicated zero-order decay accompanied by nitrate and subsequent sulfate removal. The dilution-adjusted breakthrough curves of chloride, a non-reactive halide similar to bromide, showed no evidence of reactivity. These results strongly suggested that biodegradation of both PBAs occurred under anaerobic conditions. The results of this study implied that PBAs may not be appropriate to use as non-reactive tracers in certain hydrogeologic settings, presumably those where they can serve as carbon and/or electron donors to stimulate microbial activity. Future studies would benefit from using ring-14C-labeled PBAs to determine the fate of carbon combined with microbial analyses to characterize the PBA-degrading members of the microbial community.

In situ mobility of uranium in the presence of nitrate following sulfate-reducing conditions.

Reoxidation and mobilization of previously reduced and immobilized uranium by dissolved-phase oxidants poses a significant challenge for remediating uranium-contaminated groundwater. Preferential oxidation of reduced sulfur-bearing species, as opposed to reduced uranium-bearing species, has been demonstrated to limit the mobility of uranium at the laboratory scale yet field-scale investigations are lacking. In this study, the mobility of uranium in the presence of nitrate oxidant was investigated in a shallow groundwater system after establishing conditions conducive to uranium reduction and the formation of reduced sulfur-bearing species. A series of three injections of groundwater (200 L) containing U(VI) (5 µM) and amended with ethanol (40 mM) and sulfate (20 mM) were conducted in ten test wells in order to stimulate microbial-mediated reduction of uranium and the formation of reduced sulfur-bearing species. Simultaneous push-pull tests were then conducted in triplicate well clusters to investigate the mobility of U(VI) under three conditions: 1) high nitrate (120 mM), 2) high nitrate (120 mM) with ethanol (30 mM), and 3) low nitrate (2 mM) with ethanol (30 mM). Dilution-adjusted breakthrough curves of ethanol, nitrate, nitrite, sulfate, and U(VI) suggested that nitrate reduction was predominantly coupled to the oxidation of reduced-sulfur bearing species, as opposed to the reoxidation of U(IV), under all three conditions for the duration of the 36-day tests. The amount of sulfate, but not U(VI), recovered during the push-pull tests was substantially more than injected, relative to bromide tracer, under all three conditions and further suggested that reduced sulfur-bearing species were preferentially oxidized under nitrate-reducing conditions. However, some reoxidation of U(IV) was observed under nitrate-reducing conditions and in the absence of detectable nitrate and/or nitrite. This suggested that reduced sulfur-bearing species may not be fully effective at limiting the mobility of uranium in the presence of dissolved and/or solid-phase oxidants. The results of this field study confirmed those of previous laboratory studies which suggested that reoxidation of uranium under nitrate-reducing conditions can be substantially limited by preferential oxidation of reduced sulfur-bearing species.

Microbial community composition and diversity in Caspian Sea sediments.

The Caspian Sea is heavily polluted due to industrial and agricultural effluents as well as extraction of oil and gas reserves. Microbial communities can influence the fate of contaminants and nutrients. However, insight into the microbial ecology of the Caspian Sea significantly lags behind other marine systems. Here we describe microbial biomass, diversity and composition in sediments collected from three sampling stations in the Caspian Sea. Illumina sequencing of 16S rRNA genes revealed the presence of a number of known bacterial and archaeal heterotrophs suggesting that organic carbon is a primary factor shaping microbial communities. Surface sediments collected from bottom waters with low oxygen levels were dominated by Gammaproteobacteria while surface sediments collected from bottom waters under hypoxic conditions were dominated by Deltaproteobacteria, specifically sulfate-reducing bacteria. Thaumarchaeota was dominant across all surface sediments indicating that nitrogen cycling in this system is strongly influenced by ammonia-oxidizing archaea. This study provides a baseline assessment that may serve as a point of reference as this system changes or as the efficacy of new remediation efforts are implemented.

Natural bacterial communities serve as quantitative geochemical biosensors.

UNLABELLED: Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. IMPORTANCE: Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.