Diel mercury concentration variations in a mercury-impacted stream.

Filtered and particulate mercury (Hg) and methylmercury (MMHg), and associated water chemistry parameters, were evaluated bi-hourly for several 30 h periods during the summer and winter seasons at several distinct locations (downstream forested, midstream urban/suburban, upstream industrial) along a creek contaminated with high levels of inorganic Hg to determine if biogeochemical Hg and MMHg cycles respond to the daily photocycle. In summer particulate Hg and MMHg concentrations doubled overnight (excluding the upstream industrial site) concurrent with increases in turbidity and total suspended sediment; no such pattern was evident in winter. Seasonal and diel changes in the activity of macrobiota affecting the suspension of contaminated sediments are likely responsible for these patterns as other potential explanatory variables (e.g., instrument drift, pH, discharge) could not account for the range and timing of our observations. Diel patterns in filtered Hg (HgD) were significant only at locations and times of the year when channel shading was not present and daytime concentrations increased 22-89% above nighttime minima likely caused by direct and indirect photochemical reactions. Relationships between HgD and dissolved organic carbon (DOC) concentration or character were inconsistent between sites. Unlike HgD, there were significant diel patterns in filtered MMHg (MMHgD) at all sites and times of year, with summer concentrations peaking in mid to late afternoon while the timing differed in winter, with concentrations peaking after sunset. Daily variability in MMHgD concentration ranged between 25 and 75%. The results imply key controls on net methylation occur within the stream or on the stream bed and include factors such as small-scale temperature changes in the water column and photosynthetic activity of stream biofilm. With respect to stream monitoring, results from this study indicate (1) consistent timing in stream Hg and MMHg sampling is required for accurate assessment of long-term trends, (2) in situ measurements of turbidity can be used to quantify diel dynamics of both particulate Hg and MMHg concentrations, and (3) in situ fluorescing dissolved organic matter (FDOM), a potential proxy for DOC, was not capable of resolving diel dynamics of filtered Hg or MMHg.

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.

Dissolved organic matter reduces the effectiveness of sorbents for mercury removal.

Mercury (Hg) contamination of soils and sediments impacts numerous environments worldwide and constitutes a challenging remediation problem. In this study, we evaluate the impact of dissolved organic matter (DOM) on the effectiveness of eight sorbent materials considered for Hg remediation in soils and sediments. The materials include both engineered and unmodified materials based on carbon, clays, mesoporous silica and a copper alloy. Initially, we investigated the kinetics of Hg(II) complexation with DOM for a series of Hg:DOM ratios. Steady-state Hg-DOM complexation occurred within 48 to 120 h, taking longer time at higher Hg:DOC (dissolved organic carbon) molar ratios. In subsequent equilibrium experiments, Hg(II) was equilibrated with DOM at a defined Hg:DOC molar ratio (2.4 ·â€¯10-6) for 170 h and used in batch experiments to determine the effect of DOM on Hg partition coefficients and sorption isotherms by comparing Hg(II) and Hg-DOM. Hg sorption capacities of all sorbents were severely limited in the presence of DOM as a competing ligand. Thiol-SAMMS®, SediMite™ and pine biochar were most effective in reducing Hg concentrations. While pine biochar and lignin-derived carbon processed at high temperatures released negligible amounts of anions into solution, leaching of sulfate and chloride was observed for most engineered sorbent materials. Sulfate may stimulate microbial communities harboring sulfate reducing bacteria, which are considered one of the primary drivers of microbial mercury methylation in the environment. The results highlight potential challenges arising from the application of sorbents for Hg remediation in the field.

Replicates, Read Numbers, and Other Important Experimental Design Considerations for Microbial RNA-seq Identified Using Bacillus thuringiensis Datasets.

RNA-seq is being used increasingly for gene expression studies and it is revolutionizing the fields of genomics and transcriptomics. However, the field of RNA-seq analysis is still evolving. Therefore, we specifically designed this study to contain large numbers of reads and four biological replicates per condition so we could alter these parameters and assess their impact on differential expression results. Bacillus thuringiensis strains ATCC10792 and CT43 were grown in two Luria broth medium lots on four dates and transcriptomics data were generated using one lane of sequence output from an Illumina HiSeq2000 instrument for each of the 32 samples, which were then analyzed using DESeq2. Genome coverages across samples ranged from 87 to 465X with medium lots and culture dates identified as major variation sources. Significantly differentially expressed genes (5% FDR, two-fold change) were detected for cultures grown using different medium lots and between different dates. The highly differentially expressed iron acquisition and metabolism genes, were a likely consequence of differing amounts of iron in the two media lots. Indeed, in this study RNA-seq was a tool for predictive biology since we hypothesized and confirmed the two LB medium lots had different iron contents (~two-fold difference). This study shows that the noise in data can be controlled and minimized with appropriate experimental design and by having the appropriate number of replicates and reads for the system being studied. We outline parameters for an efficient and cost effective microbial transcriptomics study.

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.

A limited microbial consortium is responsible for extended bioreduction of uranium in a contaminated aquifer.

Subsurface amendments of slow-release substrates (e.g., emulsified vegetable oil [EVO]) are thought to be a pragmatic alternative to using short-lived, labile substrates for sustained uranium bioimmobilization within contaminated groundwater systems. Spatial and temporal dynamics of subsurface microbial communities during EVO amendment are unknown and likely differ significantly from those of populations stimulated by soluble substrates, such as ethanol and acetate. In this study, a one-time EVO injection resulted in decreased groundwater U concentrations that remained below initial levels for approximately 4 months. Pyrosequencing and quantitative PCR of 16S rRNA from monitoring well samples revealed a rapid decline in groundwater bacterial community richness and diversity after EVO injection, concurrent with increased 16S rRNA copy levels, indicating the selection of a narrow group of taxa rather than a broad community stimulation. Members of the Firmicutes family Veillonellaceae dominated after injection and most likely catalyzed the initial oil decomposition. Sulfate-reducing bacteria from the genus Desulforegula, known for long-chain fatty acid oxidation to acetate, also dominated after EVO amendment. Acetate and H(2) production during EVO degradation appeared to stimulate NO(3)(-), Fe(III), U(VI), and SO(4)(2-) reduction by members of the Comamonadaceae, Geobacteriaceae, and Desulfobacterales. Methanogenic archaea flourished late to comprise over 25% of the total microbial community. Bacterial diversity rebounded after 9 months, although community compositions remained distinct from the preamendment conditions. These results demonstrated that a one-time EVO amendment served as an effective electron donor source for in situ U(VI) bioreduction and that subsurface EVO degradation and metal reduction were likely mediated by successive identifiable guilds of organisms.

Decreasing lead bioaccessibility in industrial and firing range soils with phosphate-based amendments.

In-situ stabilization using phosphate (P) amendments, such as P-based fertilizers and rock, are a potentially cost-effective and minimally disruptive alternative for stabilizing Pb in soils. We examined the effect of time (0-365 d), in vitro extraction pH (1.5 vs. 2.3), and dosage of three P-based amendments on the bioaccessibility (as a surrogate for oral bioavailability) of Pb in 10 soils from U.S. Department of Defense facilities. Initial untreated soil bioaccessibility consistently exceeded the U.S. Environmental Protection Agency default value of 60% relative bioavailability, with higher bioaccessibility consistently observed at an in vitro extraction pH of 1.5 vs. 2.3. Although P-based amendments statistically (P < 0.05) reduced bioaccessibility in many instances, with reductions dependent on the amendment and dosage, large amendment dosages (approximately 20-25% by mass to yield 5% P by mass) were required to reduce average bioaccessibility by approximately 25%. For most amendment combinations, reductions continued to occur for periods up to 1 yr, indicating that the observed reductions were not merely experimental artifacts of the in vitro extraction procedure. Although our results indicated that reductions in Pb bioaccessibility with P amendments are technically feasible, relatively large amendment masses were required to achieve relatively modest reductions in bioaccessibility. The cost and potential environmental implications of adding such large amounts of P may limit the practicality of in situ immobilization for some Pb-contaminated soils, industrial and firing range soils in particular.

A nested-cell approach for in situ remediation.

We characterize the hydraulics of an extraction-injection well pair in arbitrarily oriented regional flow by the recirculation ratio, area, and average residence time in the recirculation zone. Erratic regional flow conditions may compromise the performance of the reactor between a single well pair. We propose an alternative four-well system: two downgradient extraction and two upgradient injection wells creating an inner cell nested within an outer cell. The outer cell protects the inner cell from the influence of regional flow. Compared to a two-well system, the proposed four-well system has several advantages: (1) the recirculation ratio within the nested inner cell is less sensitive to the regional flow direction; (2) a transitional recirculation zone between the inner and outer cells can capture flow leakage from the inner cell, minimizing the release of untreated contaminants; and (3) the size of the recirculation zone and residence times can be better controlled within the inner cell by changing the pumping rates. The system is applied at the Field Research Center in Oak Ridge, Tennessee, where experiments on microbial in situ reduction of uranium (VI) are under way.

A parametric transfer function methodology for analyzing reactive transport in nonuniform flow.

We analyze reactive transport during in-situ bioremediation in a nonuniform flow field, involving multiple extraction and injection wells, by the method of transfer functions. Gamma distributions are used as parametric models of the transfer functions. Apparent parameters of classical transport models may be estimated from those of the gamma distributions by matching temporal moments. We demonstrate the method by application to measured data taken at a field experiment on bioremediation conducted in a multiple-well system in Oak Ridge, TN. Breakthrough curves (BTCs) of a conservative tracer (bromide) and a reactive compound (ethanol) are measured at multi-level sampling (MLS) wells and in extraction wells. The BTCs of both compounds are jointly analyzed to estimate the first-order degradation rate of ethanol. To quantify the tracer loss, we compare the approaches of using a scaling factor and a first-order decay term. Results show that by including a scaling factor both gamma distributions and inverse-Gaussian distributions (transfer functions according to the advection-dispersion equation) are suitable to approximate the transfer functions and estimate the reactive rate coefficients for both MLS and extraction wells. However, using a first-order decay term for tracer loss fails to describe the BTCs at the extraction well, which is affected by the nonuniform distribution of travel paths.

Mass-transfer limitations for nitrate removal in a uranium-contaminated aquifer.

A field test on in situ subsurface bioremediation of uranium(VI) is underway at the Y-12 National Security Complex in the Oak Ridge Reservation, Oak Ridge, TN. Nitrate has a high concentration at the site, which prevents U(VI) reduction, and thus must be removed. An acidic-flush strategy for nitrate removal was proposed to create a treatment zone with low levels of accessible nitrate. The subsurface at the site contains highly interconnected fractures surrounded by matrix blocks of low permeability and high porosity and is therefore subject to preferential flow and matrix diffusion. To identify the heterogeneous mass transfer properties, we performed a novel forced-gradient tracer test, which involved the addition of bromide, the displacement of nitrate, and the rebound of nitrate after completion of pumping. The simplest conceptualization consistent with the data is that the pore-space consists of a single mobile domain, as well as a fast and a slowly reacting immobile domain. The slowly reacting immobile domain (shale matrix) constitutes over 80% of the pore volume and acts as a long-term reservoir of nitrate. According to simulations, the nitrate stored in the slowly interacting immobile domain in the fast flow layer, at depths of about 12.2-13.7 m, will be reduced by an order of magnitude over a period of about a year. By contrast, the mobile domain rapidly responds to flushing, and a low average nitrate concentration can be maintained if the nitrate is removed as soon as it enters the mobile domain. A field-scale experiment in which the aquifer was flushed with acidic solution confirmed our understanding of the system. For the ongoing experiments on microbial U(VI) reduction, nitrate concentrations must be low in the mobile domain to ensure U(VI) reducing conditions. We therefore conclude that the nitrate leaching out of the immobile pore space must continuously be removed by in situ denitrification to maintain favorable conditions.