Temporal Dynamics of In-Field Bioreactor Populations Reflect the Groundwater System and Respond Predictably to Perturbation.

Temporal variability complicates testing the influences of environmental variability on microbial community structure and thus function. An in-field bioreactor system was developed to assess oxic versus anoxic manipulations on in situ groundwater communities. Each sample was sequenced (16S SSU rRNA genes, average 10,000 reads), and biogeochemical parameters are monitored by quantifying 53 metals, 12 organic acids, 14 anions, and 3 sugars. Changes in dissolved oxygen (DO), pH, and other variables were similar across bioreactors. Sequencing revealed a complex community that fluctuated in-step with the groundwater community and responded to DO. This also directly influenced the pH, and so the biotic impacts of DO and pH shifts are correlated. A null model demonstrated that bioreactor communities were driven in part not only by experimental conditions but also by stochastic variability and did not accurately capture alterations in diversity during perturbations. We identified two groups of abundant OTUs important to this system; one was abundant in high DO and pH and contained heterotrophs and oxidizers of iron, nitrite, and ammonium, whereas the other was abundant in low DO with the capability to reduce nitrate. In-field bioreactors are a powerful tool for capturing natural microbial community responses to alterations in geochemical factors beyond the bulk phase.

Reproducible responses of geochemical and microbial successional patterns in the subsurface to carbon source amendment.

Carbon amendments designed to remediate environmental contamination lead to substantial perturbations when injected into the subsurface. For the remediation of uranium contamination, carbon amendments promote reducing conditions to allow microorganisms to reduce uranium to an insoluble, less mobile state. However, the reproducibility of these amendments and underlying microbial community assembly mechanisms have rarely been investigated in the field. In this study, two injections of emulsified vegetable oil were performed in 2009 and 2017 to immobilize uranium in the groundwater at Oak Ridge, TN, USA. Our objectives were to determine whether and how the injections resulted in similar abiotic and biotic responses and their underlying community assembly mechanisms. Both injections caused similar geochemical and microbial succession. Uranium, nitrate, and sulfate concentrations in the groundwater dropped following the injection, and specific microbial taxa responded at roughly the same time points in both injections, including Geobacter, Desulfovibrio, and members of the phylum Comamonadaceae, all of which are well established in uranium, nitrate, and sulfate reduction. Both injections induced a transition from relatively stochastic to more deterministic assembly of microbial taxonomic and phylogenetic community structures based on 16S rRNA gene analysis. We conclude that geochemical and microbial successions after biostimulation are reproducible, likely owing to the selection of similar phylogenetic groups in response to EVO injection.

Characterization of soils from an industrial complex contaminated with elemental mercury.

Historical use of liquid elemental mercury (Hg(0)l) at the Y-12 National Security Complex in Oak Ridge, TN, USA, resulted in large deposits of Hg(0)l in the soils. The fate and distribution of the spilled Hg(0) are not well characterized. In this study we evaluated analytical tools for characterizing the speciation of Hg in the contaminated soils and then used the analytical techniques to examine the speciation of Hg in two soil cores collected at the site. These include x-ray fluorescence (XRF), soil Hg(0) headspace analysis, and total Hg determination by acid digestion coupled with cold vapor atomic absorption (HgT). XRF was not found to be suitable for evaluating Hg concentrations in heterogeneous soils containing low concentration of Hg or Hg(0) because Hg concentrations determined using this method were lower than those determined by HgT analysis and the XRF detection limit is 20 mg/kg. Hg(0)g headspace analysis coupled with HgT measurements yielded good results for examining the presence of Hg(0)l in soils and the speciation of Hg. The two soil cores are highly heterogeneous in both the depth and extent of Hg contamination, with Hg concentrations ranging from 0.05 to 8400mg/kg. In the first core, Hg(0)l was distributed throughout the 3.2m depth, whereas the second core, from a location 12m away, contained Hg(0)l in a 0.3m zone only. Sequential extractions showed organically associated Hg dominant at depths with low Hg concentration. Soil from the zone of groundwater saturation showed reducing conditions and the Hg is likely present as Hg-sulfide species. At this depth, lateral Hg transport in the groundwater may be a source of Hg detected in the soil at the deeper soil depths. Overall, characterization of soils containing Hg(0)l is difficult because of the heterogeneous distribution of Hg within the soils. This is exacerbated in industrial facilities where fill materials make up much of the soils and historical and continued reworking of the subsurface has remobilized the Hg.

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.

Evaluation of engineered sorbents for the sorption of mercury from contaminated bank soils: a column study.

As a global environmental pollutant, mercury (Hg) threatens our water resources and presents a substantial risk to human health. The rate and extent of immobilization of Hg2+ (hereafter, Hg) on engineered sorbents (Thiol-SAMMS®, pine biochar, SediMite™, Organoclay™ PM-199, and quartz sand as a control) was evaluated using flow-through column experiments. The effectiveness of the sorbents was based on (1) the percentage of Hg removed in relation to the total amount of Hg passing the sorbent column, and (2) the rate of Hg uptake compared to the nonreactive tracer bromide (Br-). All sorbents removed Hg to a certain extent, but none of the sorbents removed all the Hg introduced to the columns. Thiol-SAMMS showed the highest mean percentage of Hg removed (87% ± 2.9%), followed by Organoclay PM-199 (71% ± 0.4%), pine biochar (57% ± 22.3%), SediMite (61% ± 0.8%), and the control quartz sand (11% ± 5.6%). Thiol-SAMMS was the only sorbent to exhibit retardation of Hg in comparison to the conservative tracer Br-. For the remaining sorbents, Br- along with low concentrations of Hg were eluted within the first 3 pore volumes, indicating limited retardation of Hg. Overall, removal of Hg by sorbents was substantial, suggesting that sorbents might be suitable for deployment in contaminated environments. High concentrations of DOM leaching from the soil columns likely influenced the speciation of Hg and inhibited sorption to the sorbents. Incomplete removal of Hg by any sorbent suggests that additional optimization is needed to increase efficiency.

In-field bioreactors demonstrate dynamic shifts in microbial communities in response to geochemical perturbations.

Subsurface microbial communities mediate the transformation and fate of redox sensitive materials including organic matter, metals and radionuclides. Few studies have explored how changing geochemical conditions influence the composition of groundwater microbial communities over time. We temporally monitored alterations in abiotic forces on microbial community structure using 1L in-field bioreactors receiving background and contaminated groundwater at the Oak Ridge Reservation, TN. Planktonic and biofilm microbial communities were initialized with background water for 4 days to establish communities in triplicate control reactors and triplicate test reactors and then fed filtered water for 14 days. On day 18, three reactors were switched to receive filtered groundwater from a contaminated well, enriched in total dissolved solids relative to the background site, particularly chloride, nitrate, uranium, and sulfate. Biological and geochemical data were collected throughout the experiment, including planktonic and biofilm DNA for 16S rRNA amplicon sequencing, cell counts, total protein, anions, cations, trace metals, organic acids, bicarbonate, pH, Eh, DO, and conductivity. We observed significant shifts in both planktonic and biofilm microbial communities receiving contaminated water. This included a loss of rare taxa, especially amongst members of the Bacteroidetes, Acidobacteria, Chloroflexi, and Betaproteobacteria, but enrichment in the Fe- and nitrate- reducing Ferribacterium and parasitic Bdellovibrio. These shifted communities were more similar to the contaminated well community, suggesting that geochemical forces substantially influence microbial community diversity and structure. These influences can only be captured through such comprehensive temporal studies, which also enable more robust and accurate predictive models to be developed.

Small and mighty: adaptation of superphylum Patescibacteria to groundwater environment drives their genome simplicity.

BACKGROUND: The newly defined superphylum Patescibacteria such as Parcubacteria (OD1) and Microgenomates (OP11) has been found to be prevalent in groundwater, sediment, lake, and other aquifer environments. Recently increasing attention has been paid to this diverse superphylum including > 20 candidate phyla (a large part of the candidate phylum radiation, CPR) because it refreshed our view of the tree of life. However, adaptive traits contributing to its prevalence are still not well known. RESULTS: Here, we investigated the genomic features and metabolic pathways of Patescibacteria in groundwater through genome-resolved metagenomics analysis of > 600 Gbp sequence data. We observed that, while the members of Patescibacteria have reduced genomes (~ 1 Mbp) exclusively, functions essential to growth and reproduction such as genetic information processing were retained. Surprisingly, they have sharply reduced redundant and nonessential functions, including specific metabolic activities and stress response systems. The Patescibacteria have ultra-small cells and simplified membrane structures, including flagellar assembly, transporters, and two-component systems. Despite the lack of CRISPR viral defense, the bacteria may evade predation through deletion of common membrane phage receptors and other alternative strategies, which may explain the low representation of prophage proteins in their genomes and lack of CRISPR. By establishing the linkages between bacterial features and the groundwater environmental conditions, our results provide important insights into the functions and evolution of this CPR group. CONCLUSIONS: We found that Patescibacteria has streamlined many functions while acquiring advantages such as avoiding phage invasion, to adapt to the groundwater environment. The unique features of small genome size, ultra-small cell size, and lacking CRISPR of this large lineage are bringing new understandings on life of Bacteria. Our results provide important insights into the mechanisms for adaptation of the superphylum in the groundwater environments, and demonstrate a case where less is more, and small is mighty.

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.

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.