Oil and Gas Wastewater Components Alter Streambed Microbial Community Structure and Function.

The widespread application of directional drilling and hydraulic fracturing technologies expanded oil and gas (OG) development to previously inaccessible resources. A single OG well can generate millions of liters of wastewater, which is a mixture of brine produced from the fractured formations and injected hydraulic fracturing fluids (HFFs). With thousands of wells completed each year, safe management of OG wastewaters has become a major challenge to the industry and regulators. OG wastewaters are commonly disposed of by underground injection, and previous research showed that surface activities at an Underground Injection Control (UIC) facility in West Virginia affected stream biogeochemistry and sediment microbial communities immediately downstream from the facility. Because microbially driven processes can control the fate and transport of organic and inorganic components of OG wastewater, we designed a series of aerobic microcosm experiments to assess the influence of high total dissolved solids (TDS) and two common HFF additives-the biocide 2,2-dibromo-3-nitrilopropionamide (DBNPA) and ethylene glycol (an anti-scaling additive)-on microbial community structure and function. Microcosms were constructed with sediment collected upstream (background) or downstream (impacted) from the UIC facility in West Virginia. Exposure to elevated TDS resulted in a significant decrease in aerobic respiration, and microbial community analysis following incubation indicated that elevated TDS could be linked to the majority of change in community structure. Over the course of the incubation, the sediment layer in the microcosms became anoxic, and addition of DBNPA was observed to inhibit iron reduction. In general, disruptions to microbial community structure and function were more pronounced in upstream and background sediment microcosms than in impacted sediment microcosms. These results suggest that the microbial community in impacted sediments had adapted following exposure to OG wastewater releases from the site. Our findings demonstrate the potential for releases from an OG wastewater disposal facility to alter microbial communities and biogeochemical processes. We anticipate that these studies will aid in the development of useful models for the potential impact of UIC disposal facilities on adjoining surface water and shallow groundwater.

Microbial community composition of a hydrocarbon reservoir 40 years after a CO2 enhanced oil recovery flood.

Injecting CO2 into depleted oil reservoirs to extract additional crude oil is a common enhanced oil recovery (CO2-EOR) technique. However, little is known about how in situ microbial communities may be impacted by CO2 flooding, or if any permanent microbiological changes occur after flooding has ceased. Formation water was collected from an oil field that was flooded for CO2-EOR in the 1980s, including samples from areas affected by or outside of the flood region, to determine the impacts of CO2-EOR on reservoir microbial communities. Archaea, specifically methanogens, were more abundant than bacteria in all samples, while identified bacteria exhibited much greater diversity than the archaea. Microbial communities in CO2-impacted and non-impacted samples did not significantly differ (ANOSIM: Statistic R = -0.2597, significance = 0.769). However, several low abundance bacteria were found to be significantly associated with the CO2-affected group; very few of these species are known to metabolize CO2 or are associated with CO2-rich habitats. Although this study had limitations, on a broad scale, either the CO2 flood did not impact the microbial community composition of the target formation, or microbial communities in affected wells may have reverted back to pre-injection conditions over the ca. 40 years since the CO2-EOR.

Common Hydraulic Fracturing Fluid Additives Alter the Structure and Function of Anaerobic Microbial Communities.

The development of unconventional oil and gas (UOG) resources results in the production of large volumes of wastewater containing a complex mixture of hydraulic fracturing chemical additives and components from the formation. The release of these wastewaters into the environment poses potential risks that are poorly understood. Microbial communities in stream sediments form the base of the food chain and may serve as sentinels for changes in stream health. Iron-reducing organisms have been shown to play a role in the biodegradation of a wide range of organic compounds, and so to evaluate their response to UOG wastewater, we enriched anaerobic microbial communities from sediments collected upstream (background) and downstream (impacted) of an UOG wastewater injection disposal facility in the presence of hydraulic fracturing fluid (HFF) additives: guar gum, ethylene glycol, and two biocides, 2,2-dibromo-3-nitrilopropionamide (DBNPA) and bronopol (C3H6BrNO4). Iron reduction was significantly inhibited early in the incubations with the addition of biocides, whereas amendment with guar gum and ethylene glycol stimulated iron reduction relative to levels in the unamended controls. Changes in the microbial community structure were observed across all treatments, indicating the potential for even small amounts of UOG wastewater components to influence natural microbial processes. The microbial community structure differed between enrichments with background and impacted sediments, suggesting that impacted sediments may have been preconditioned by exposure to wastewater. These experiments demonstrated the potential for biocides to significantly decrease iron reduction rates immediately following a spill and demonstrated how microbial communities previously exposed to UOG wastewater may be more resilient to additional spills.IMPORTANCE Organic components of UOG wastewater can alter microbial communities and biogeochemical processes, which could alter the rates of essential natural attenuation processes. These findings provide new insights into microbial responses following a release of UOG wastewaters and are critical for identifying strategies for the remediation and natural attenuation of impacted environments.

Identification of Mn(II)-oxidizing bacteria from a low-pH contaminated former uranium mine.

Biological Mn oxidation is responsible for producing highly reactive and abundant Mn oxide phases in the environment that can mitigate metal contamination. However, little is known about Mn oxidation in low-pH environments, where metal contamination often is a problem as the result of mining activities. We isolated two Mn(II)-oxidizing bacteria (MOB) at pH 5.5 (Duganella isolate AB_14 and Albidiferax isolate TB-2) and nine strains at pH 7 from a former uranium mining site. Isolate TB-2 may contribute to Mn oxidation in the acidic Mn-rich subsoil, as a closely related clone represented 16% of the total community. All isolates oxidized Mn over a small pH range, and isolates from low-pH samples only oxidized Mn below pH 6. Two strains with different pH optima differed in their Fe requirements for Mn oxidation, suggesting that Mn oxidation by the strain found at neutral pH was linked to Fe oxidation. Isolates tolerated Ni, Cu, and Cd and produced Mn oxides with similarities to todorokite and birnessite, with the latter being present in subsurface layers where metal enrichment was associated with Mn oxides. This demonstrates that MOB can be involved in the formation of biogenic Mn oxides in both moderately acidic and neutral pH environments.

Linking specific heterotrophic bacterial populations to bioreduction of uranium and nitrate in contaminated subsurface sediments by using stable isotope probing.

Shifts in terminal electron-accepting processes during biostimulation of uranium-contaminated sediments were linked to the composition of stimulated microbial populations using DNA-based stable isotope probing. Nitrate reduction preceded U(VI) and Fe(III) reduction in [¹³C]ethanol-amended microcosms. The predominant, active denitrifying microbial groups were identified as members of the Betaproteobacteria, whereas Actinobacteria dominated under metal-reducing conditions.

Microbial community changes in response to ethanol or methanol amendments for U(VI) reduction.

Microbial community responses to ethanol, methanol, and methanol plus humics amendments in relationship to U(VI) bioreduction were studied in laboratory microcosm experiments using sediments and ground water from a uranium-contaminated site in Oak Ridge, TN. The type of carbon source added, the duration of incubation, and the sampling site influenced the bacterial community structure upon incubation. Analysis of 16S rRNA gene clone libraries indicated that (i) bacterial communities found in ethanol- and methanol-amended samples with U(VI) reduction were similar due to the presence of Deltaproteobacteria and Betaproteobacteria (members of the families Burkholderiaceae, Comamonadaceae, Oxalobacteraceae, and Rhodocyclaceae); (ii) methanol-amended samples without U(VI) reduction exhibited the lowest diversity and the bacterial community contained 69.2 to 92.8% of the family Methylophilaceae; and (iii) the addition of humics resulted in an increase of phylogenetic diversity of Betaproteobacteria (Rodoferax, Polaromonas, Janthinobacterium, Methylophilales, and unclassified) and Firmicutes (Desulfosporosinus and Clostridium).

Microbial links between sulfate reduction and metal retention in uranium- and heavy metal-contaminated soil.

Sulfate-reducing bacteria (SRB) can affect metal mobility either directly by reductive transformation of metal ions, e.g., uranium, into their insoluble forms or indirectly by formation of metal sulfides. This study evaluated in situ and biostimulated activity of SRB in groundwater-influenced soils from a creek bank contaminated with heavy metals and radionuclides within the former uranium mining district of Ronneburg, Germany. In situ activity of SRB, measured by the (35)SO(4)(2-) radiotracer method, was restricted to reduced soil horizons with rates of < or =142 +/- 20 nmol cm(-3) day(-1). Concentrations of heavy metals were enriched in the solid phase of the reduced horizons, whereas pore water concentrations were low. X-ray absorption near-edge structure (XANES) measurements demonstrated that approximately 80% of uranium was present as reduced uranium but appeared to occur as a sorbed complex. Soil-based dsrAB clone libraries were dominated by sequences affiliated with members of the Desulfobacterales but also the Desulfovibrionales, Syntrophobacteraceae, and Clostridiales. [(13)C]acetate- and [(13)C]lactate-biostimulated soil microcosms were dominated by sulfate and Fe(III) reduction. These processes were associated with enrichment of SRB and Geobacteraceae; enriched SRB were closely related to organisms detected in soils by using the dsrAB marker. Concentrations of soluble nickel, cobalt, and occasionally zinc declined < or =100% during anoxic soil incubations. In contrast to results in other studies, soluble uranium increased in carbon-amended treatments, reaching < or =1,407 nM in solution. Our results suggest that (i) ongoing sulfate reduction in contaminated soil resulted in in situ metal attenuation and (ii) the fate of uranium mobility is not predictable and may lead to downstream contamination of adjacent ecosystems.

Geobacter daltonii sp. nov., an Fe(III)- and uranium(VI)-reducing bacterium isolated from a shallow subsurface exposed to mixed heavy metal and hydrocarbon contamination.

An Fe(III)- and uranium(VI)-reducing bacterium, designated strain FRC-32(T), was isolated from a contaminated subsurface of the USA Department of Energy Oak Ridge Field Research Center (ORFRC) in Oak Ridge, Tennessee, where the sediments are exposed to mixed waste contamination of radionuclides and hydrocarbons. Analyses of both 16S rRNA gene and the Geobacteraceae-specific citrate synthase (gltA) mRNA gene sequences retrieved from ORFRC sediments indicated that this strain was abundant and active in ORFRC subsurface sediments undergoing uranium(VI) bioremediation. The organism belonged to the subsurface clade of the genus Geobacter and shared 92-98 % 16S rRNA gene and 75-81 % rpoB gene sequence similarities with other recognized species of the genus. In comparison to its closest relative, Geobacter uraniireducens Rf4(T), according to 16S rRNA gene sequence similarity, strain FRC-32(T) showed a DNA-DNA relatedness value of 21 %. Cells of strain FRC-32(T) were Gram-negative, non-spore-forming, curved rods, 1.0-1.5 microm long and 0.3-0.5 microm in diameter; the cells formed pink colonies in a semisolid cultivation medium, a characteristic feature of the genus Geobacter. The isolate was an obligate anaerobe, had temperature and pH optima for growth at 30 degrees C and pH 6.7-7.3, respectively, and could tolerate up to 0.7 % NaCl although growth was better in the absence of NaCl. Similar to other members of the Geobacter group, strain FRC-32(T) conserved energy for growth from the respiration of Fe(III)-oxyhydroxide coupled with the oxidation of acetate. Strain FRC-32(T) was metabolically versatile and, unlike its closest relative, G. uraniireducens, was capable of utilizing formate, butyrate and butanol as electron donors and soluble ferric iron (as ferric citrate) and elemental sulfur as electron acceptors. Growth on aromatic compounds including benzoate and toluene was predicted from preliminary genomic analyses and was confirmed through successive transfer with fumarate as the electron acceptor. Thus, based on genotypic, phylogenetic and phenotypic differences, strain FRC-32(T) is considered to represent a novel species of the genus Geobacter, for which the name Geobacter daltonii sp. nov. is proposed. The type strain is FRC-32(T) (=DSM 22248(T)=JCM 15807(T)).

Impact of biostimulated redox processes on metal dynamics in an iron-rich creek soil of a former uranium mining area.

Understanding the dynamics of metals and radionuclides in soil environments is necessary for evaluating risks to pristine sites. An iron-rich creek soil of a former uranium-mining district (Ronneburg, Germany) showed high porewater concentrations of heavy metals and radionuclides. Thus, this study aims to (i) evaluate metal dynamics during terminal electron accepting processes (TEAPs) and (ii) characterize active microbial populations in biostimulated soil microcosms using a stable isotope probing (SIP) approach. In biostimulated soil slurries, concentrations of soluble Co, Ni, Zn, As, and unexpectedly U increased during Fe(III)-reduction. This suggests that there was a release of sorbed metals and As during reductive dissolution of Fe(III)-oxides. Subsequent sulfate-reduction was concurrent with a decrease of U, Co, Ni, and Zn concentrations. The relative contribution of U(IV) in the solid phase changed from 18.5 to 88.7% after incubation. The active Fe(III)-reducing population was dominated by delta-Proteobacteria (Geobacter) in (13)C-ethanol amended microcosms. A more diverse community was present in (13)C-lactate amended microcosms including taxa related to Acidobacteria, Firmicutes, delta-Proteobacteria, and beta-Proteobacteria. Our results suggested that biostimulated Fe(III)-reducing communities facilitated the release of metals including U to groundwater which is in contrast to other studies.

Functional diversity and electron donor dependence of microbial populations capable of U(VI) reduction in radionuclide-contaminated subsurface sediments.

In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.

Metabolically active microbial communities in uranium-contaminated subsurface sediments.

In order to develop effective bioremediation strategies for radionuclide contaminants, the composition and metabolic potential of microbial communities need to be better understood, especially in highly contaminated subsurface sediments for which little cultivation-independent information is available. In this study, we characterized metabolically active and total microbial communities associated with uranium-contaminated subsurface sediments along geochemical gradients. DNA and RNA were extracted and amplified from four sediment-depth intervals representing moderately acidic (pH 3.7) to near-neutral (pH 6.7) conditions. Phylotypes related to Proteobacteria (Alpha-, Beta-, Delta- and Gammaproteobacteria), Bacteroidetes, Actinobacteria, Firmicutes and Planctomycetes were detected in DNA- and RNA-derived clone libraries. Diversity and numerical dominance of phylotypes were observed to correspond to changes in sediment geochemistry and rates of microbial activity, suggesting that geochemical conditions have selected for well-adapted taxa. Sequences closely related to nitrate-reducing bacteria represented 28% and 43% of clones from the total and metabolically active fractions of the microbial community, respectively. This study provides the first detailed analysis of total and metabolically active microbial communities in radionuclide-contaminated subsurface sediments. Our microbial community analysis, in conjunction with rates of microbial activity, points to several groups of nitrate-reducers that appear to be well adapted to environmental conditions common to radionuclide-contaminated sites.