Complex impacts of hydraulic fracturing return fluids on soil microbial community respiration, structure and functional potentials.

The consequences of soils exposed to hydraulic fracturing (HF) return fluid, often collectively termed flowback and produced water (FPW), are poorly understood, even though soils are a common receptor of FPW spills. Here, we investigate the impacts on soil microbiota exposed to FPW collected from the Montney Formation of western Canada. We measured soil respiration, microbial community structure and functional potentials under FPW exposure across a range of concentrations, exposure time and soil types (luvisol and chernozem). We find that soil type governs microbial community response upon FPW exposure. Within each soil, FPW exposure led to reduced biotic soil respiration, and shifted microbial community structure and functional potentials. We detect substantially higher species richness and more unique functional genes in FPW-exposed soils than in FPW-unexposed soils, with metagenome-assembled genomes (e.g. Marinobacter persicus) from luvisol soil exposed to concentrated FPW being most similar to genomes from HF/FPW sites. Our data demonstrate the complex impacts of microbial communities following FPW exposure and highlight the site-specific effects in evaluation of spills and agricultural reuse of FPW on the normal soil functions.

Comparison of the Hydraulic Fracturing Water Cycle in China and North America: A Critical Review.

There is considerable debate about the sustainability of the hydraulic fracturing (HF) water cycle in North America. Recently, this debate has expanded to China, where HF activities continue to grow. Here, we provide a critical review of the HF water cycle in China, including water withdrawal practices and flowback and produced water (FPW) management and their environmental impacts, with a comprehensive comparison to the U.S. and Canada (North America). Water stress in arid regions, as well as water management challenges, FPW contamination of aquatic and soil systems, and induced seismicity are all impacts of the HF water cycle in China, the U.S., and Canada. In light of experience gained in North America, standardized practices for analyzing and reporting FPW chemistry and microbiology in China are needed to inform its efficient and safe treatment, discharge and reuse, and identification of potential contaminants. Additionally, conducting ecotoxicological studies is an essential next step to fully reveal the impacts of accidental FPW releases into aquatic and soil ecosystems in China. From a policy perspective, the development of China's unconventional resources lags behind North America's in terms of overall regulation, especially with regard to water withdrawal, FPW management, and routine monitoring. Our study suggests that common environmental risks exist within the world's two largest HF regions, and practices used in North America may help prevent or mitigate adverse effects in China.

Bacterial community succession in a high-altitude subarctic glacier foreland is a three-stage process.

Alpine glaciers are retreating rapidly, exposing foreland minerals, which develop into soils. Bacterial communities in glacier forelands exhibit high rates of turnover and undergo dramatic shifts in composition within the first 50 years after deglaciation, followed by relative stabilization and convergence. This period of microbial development occurs simultaneously with plant colonization in most systems; thus, it remains unclear whether the changes in the bacterial communities occur primarily as the result of edaphic, climatic or biotic factors. We examined bacterial community structure along two replicate chronosequences within the glacial foreland of Duke River Glacier, Yukon, Canada. This foreland is estimated to include >200 years of bare soils before an appreciable grassline, likely due to the high latitude and altitude of the glacier. This enabled us to examine bacterial community development prior to plant colonization over a longer period than previous studies. We observed three successional groups in the chronosequence: (i) an 'early' group in soils of less than approximately 50 years since deglaciation; (ii) an 'intermediate' group within bare soils, after the early period but before the grassline, containing communities with a relatively high degree of variability in composition; and (iii) a 'grassline' group in soils collected after plant colonization with higher diversity but lower age-group variability in community composition. These findings suggest rapid replacement and addition of species better adapted to glacier foreland conditions followed by slower community shifts over the next 150 years and, finally, indications of a possible response to plant colonization.

Bacterial and archaeal diversity in sediments of west Lake Bonney, McMurdo Dry Valleys, Antarctica.

Bacterial and archaeal diversity was examined in a sediment core from Lake Bonney, Antarctica. Members of the Archaea showed both low abundance and diversity, whereas bacterial diversity was moderately high and some phyla were fairly abundant, even in geologically old samples. Microbial diversity correlated with sample texture and differed in silty and coarse samples.

Desulfohalophilus alkaliarsenatis gen. nov., sp. nov., an extremely halophilic sulfate- and arsenate-respiring bacterium from Searles Lake, California.

A haloalkaliphilic sulfate-respiring bacterium, strain SLSR-1, was isolated from a lactate-fed stable enrichment culture originally obtained from the extreme environment of Searles Lake, California. The isolate proved capable of growth via sulfate-reduction over a broad range of salinities (125-330 g/L), although growth was slowest at salt-saturation. Strain SLSR-1 was also capable of growth via dissimilatory arsenate-reduction and displayed an even broader range of salinity tolerance (50-330 g/L) when grown under these conditions. Strain SLSR-1 could also grow via dissimilatory nitrate reduction to ammonia. Growth experiments in the presence of high borate concentrations indicated a greater sensitivity of sulfate-reduction than arsenate-respiration to this naturally abundant anion in Searles Lake. Strain SLSR-1 contained genes involved in both sulfate-reduction (dsrAB) and arsenate respiration (arrA). Amplicons of 16S rRNA gene sequences obtained from DNA extracted from Searles Lake sediment revealed the presence of close relatives of strain SLSR-1 as part of the flora of this ecosystem despite the fact that sulfate-reduction activity could not be detected in situ. We conclude that strain SLSR-1 can only achieve growth via arsenate-reduction under the current chemical conditions prevalent at Searles Lake. Strain SLSR-1 is a deltaproteobacterium in the family Desulfohalobiacea of anaerobic, haloalkaliphilic bacteria, for which we propose the name Desulfohalophilus alkaliarsenatis gen. nov., sp. nov.

Coupled arsenotrophy in a hot spring photosynthetic biofilm at Mono Lake, California.

Red-pigmented biofilms grow on rock and cobble surfaces present in anoxic hot springs located on Paoha Island in Mono Lake. The bacterial community was dominated ( approximately 85% of 16S rRNA gene clones) by sequences from the photosynthetic Ectothiorhodospira genus. Scraped biofilm materials incubated under anoxic conditions rapidly oxidized As(III) to As(V) in the light via anoxygenic photosynthesis but could also readily reduce As(V) to As(III) in the dark at comparable rates. Back-labeling experiments with (73)As(V) demonstrated that reduction to (73)As(III) also occurred in the light, thereby illustrating the cooccurrence of these two anaerobic processes as an example of closely coupled arsenotrophy. Oxic biofilms also oxidized As(III) to As(V). Biofilms incubated with [(14)C]acetate oxidized the radiolabel to (14)CO(2) in the light but not the dark, indicating a capacity for photoheterotrophy but not chemoheterotrophy. Anoxic, dark-incubated samples demonstrated As(V) reduction linked to additions of hydrogen or sulfide but not acetate. Chemoautotrophy linked to As(V) as measured by dark fixation of [(14)C]bicarbonate into cell material was stimulated by either H(2) or HS(-). Functional genes for the arsenate respiratory reductase (arrA) and arsenic resistance (arsB) were detected in sequenced amplicons of extracted DNA, with about half of the arrA sequences closely related ( approximately 98% translated amino acid identity) to those from the family Ectothiorhodospiraceae. Surprisingly, no authentic PCR products for arsenite oxidase (aoxB) were obtained, despite observing aerobic arsenite oxidation activity. Collectively, these results demonstrate close linkages of these arsenic redox processes occurring within these biofilms.

Optimization of subsampling, decontamination, and DNA extraction of difficult peat and silt permafrost samples.

This study aims to act as a methodological guide for contamination monitoring, decontamination, and DNA extraction for peaty and silty permafrost samples with low biomass or difficult to extract DNA. We applied a biological tracer, either only in the field or both in the field and in the lab, via either spraying or painting. Spraying in the field followed by painting in the lab resulted in a uniform layer of the tracer on the core sections. A combination of bleaching, washing, and scraping resulted in complete removal of the tracer leaving sufficient material for DNA extraction, while other widely used decontamination methods did not remove all detectable tracer. In addition, of four widely used commercially available DNA extraction kits, only a modified ZymoBIOMICS DNA Microprep kit was able to acquire PCR amplifiable DNA. Permafrost chemical parameters, age, and soil texture did not have an effect on decontamination efficacy; however, the permafrost type did influence DNA extraction. Based on these findings, we developed recommendations for permafrost researchers to acquire contaminant-free DNA from permafrost with low biomass.

Response of aquatic microbial communities and bioindicator modelling of hydraulic fracturing flowback and produced water.

The response of microbial communities to releases of hydraulic fracturing flowback and produced water (PW) may influence ecosystem functions. However, knowledge of the effects of PW spills on freshwater microbiota is limited. Here, we conducted two separate experiments: 16S rRNA gene sequencing combined with random forests modelling was used to assess freshwater community changes in simulated PW spills by volume from 0.05% to 50%. In a separate experiment, live/dead cell viability in a freshwater community was tested during exposure to 10% PW by volume. Three distinct patterns of microbial community shifts were identified: (i) indigenous freshwater genera remained dominant in <2.5% PW, (ii) from 2.5% to 5% PW, potential PW organic degraders such as Pseudomonas, Rheinheimera and Brevundimonas became dominant, and (iii) no significant change in the relative abundance of taxa was observed in >5% PW. Microbial taxa including less abundant genera such as Cellvibrio were potential bioindicators for the degree of contamination with PW. Additionally, live cells were quickly damaged by adding 10% PW, but cell counts recovered in the following days. Our study shows that the responses of freshwater microbiota vary by spill size, and these responses show promise as effective fingerprints for PW spills in aquatic environments.

Bacteria beneath the West Antarctic ice sheet.

Subglacial environments, particularly those that lie beneath polar ice sheets, are beginning to be recognized as an important part of Earth's biosphere. However, except for indirect indications of microbial assemblages in subglacial Lake Vostok, Antarctica, no sub-ice sheet environments have been shown to support microbial ecosystems. Here we report 16S rRNA gene and isolate diversity in sediments collected from beneath the Kamb Ice Stream, West Antarctic Ice Sheet and stored for 15 months at 4 degrees C. This is the first report of microbes in samples from the sediment environment beneath the Antarctic Ice Sheet. The cells were abundant ( approximately 10(7) cells g(-1)) but displayed low diversity (only five phylotypes), likely as a result of enrichment during storage. Isolates were cold tolerant and the 16S rRNA gene diversity was a simplified version of that found in subglacial alpine and Arctic sediments and water. Although in situ cell abundance and the extent of wet sediments beneath the Antarctic ice sheet can only be roughly extrapolated on the basis of this sample, it is clear that the subglacial ecosystem contains a significant and previously unrecognized pool of microbial cells and associated organic carbon that could potentially have significant implications for global geochemical processes.

Ecophysiology of "Halarsenatibacter silvermanii" strain SLAS-1T, gen. nov., sp. nov., a facultative chemoautotrophic arsenate respirer from salt-saturated Searles Lake, California.

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1(T) was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced arrABD gene cluster, cell envelope lipids, and its phylogenetic alignment within the order Halanaerobacteriales, assigning it the name "Halarsenatibacter silvermanii" strain SLAS-1(T). We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order Desulfovibriales, the archaeon Halorhabdus utahensis, as well as a close homolog of strain SLAS-1(T).

Molecular ecological analysis of planktonic bacterial communities in constructed wetlands invaded by Culex (Diptera: Culicidae) mosquitoes.

The succession of the planktonic bacterial community during the colonization by Culex (Diptera: Culicidae) mosquitoes of 0.1-ha treatment wetlands was studied using denaturing gradient gel electrophoresis (DGGE) methodology. Relationships between apparent bacterial diversity and ecological factors (water quality, total bacterial counts, and immature mosquito abundance) were determined during a 1-mo flooding period. Analysis of DGGE banding patterns indicated that days postflooding and temporal changes in water quality were the primary and secondary determinants, respectively, of diversity in bacterial communities. Lower levels of diversity were associated with later postflood stages and increases in ammoniacal nitrogen concentration and total bacterial counts. Diversity was therefore most similar for bacteria present on the same sampling date at wetland locations with similar flooding regimes and water quality, suggesting that wastewater input was the driving force shaping bacterial communities. Comparatively small changes in bacterial diversity were connected to natural processes as water flowed through the wetlands. Greater immature mosquito abundance coincided with less diverse communities composed of greater total numbers of bacteria. Five individual DGGE bands were directly associated with fluctuations in mosquito production, and an additional 16 bands were associated with hydrological aspects of the environment during the rise and fall of mosquito populations. A marked decline in mosquito numbers 21 d after inundation may have masked associations of bacterial communities and mosquito recruitment into the sparsely vegetated wetlands. DGGE was an effective tool for the characterization of bacteria in mosquito habitat in our study, and its potential application in mosquito ecology is discussed.

Bacterial communities from Arctic seasonal sea ice are more compositionally variable than those from multi-year sea ice.

Arctic sea ice can be classified into two types: seasonal ice (first-year ice, FYI) and multi-year ice (MYI). Despite striking differences in the physical and chemical characteristics of FYI and MYI, and the key role sea ice bacteria play in biogeochemical cycles of the Arctic Ocean, there are a limited number of studies comparing the bacterial communities from these two ice types. Here, we compare the membership and composition of bacterial communities from FYI and MYI sampled north of Ellesmere Island, Canada. Our results show that communities from both ice types were dominated by similar class-level phylogenetic groups. However, at the operational taxonomic unit (OTU) level, communities from MYI and FYI differed in both membership and composition. Communities from MYI sites had consistent structure, with similar membership (presence/absence) and composition (OTU abundance) independent of location and year of sample. By contrast, communities from FYI were more variable. Although FYI bacterial communities from different locations and different years shared similar membership, they varied significantly in composition. Should these findings apply to sea ice across the Arctic, we predict increased compositional variability in sea ice bacterial communities resulting from the ongoing transition from predominantly MYI to FYI, which may impact nutrient dynamics in the Arctic Ocean.

A viable microbial community in a subglacial volcanic crater lake, Iceland.

We describe a viable microbial community in a subglacial lake within the Grímsvötn volcanic caldera, Iceland. We used a hot water drill to penetrate the 300-m ice shelf and retrieved lake water and volcanic tephra sediments. We also acquired samples of borehole water before and after penetration to the lake, overlying glacial ice and snow, and water from a nearby subaerial geothermal lake for comparative analyses. Lake water is at the freezing point and fresh (total dissolved solids = 260 mg L(-1)). Detectable numbers of cells were found in samples of the lake water column and tephra sediments: 2 x 10(4) ml(-1) and 4 x 10(7) g(-1), respectively. Plate counts document abundant cold-adapted cultivable organisms in the lake water, but not in the borehole (before penetration) or glacial ice. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from genomic DNA extracted from Grímsvötn samples indicates that the lake community is distinct from the assemblages of organisms in borehole water (before penetration) and the overlying ice and snow. Sequencing of selected DGGE bands revealed that many sequences are highly similar to known psychrophilic organisms or cloned DNA from other cold environments. Significant uptake of 14C-labeled bicarbonate occurred in dark, low-temperature incubations of lake water samples, indicating the presence of autotrophs. Acetylene reduction assays under similar incubation conditions showed no significant nitrogen fixation potential by lake water samples. This may be a consequence of the inhibition of diazotrophy by nitrogen in the lake.

Distinct bacterial assemblages reside at different depths in Arctic multiyear sea ice.

Bacterial communities in Arctic sea ice play an important role in the regulation of nutrient and energy dynamics in the Arctic Ocean. Sea ice has vertical gradients in temperature, brine salinity and volume, and light and UV levels. Multiyear ice (MYI) has at least two distinct ice layers: old fresh ice with limited permeability, and new saline ice, and may also include a surface melt pond layer. Here, we determine whether bacterial communities (1) differ with ice depth due to strong physical and chemical gradients, (2) are relatively homogenous within a layer, but differ between layers, or (3) do not vary with ice depth. Cores of MYI off northern Ellesmere Island, NU, Canada, were subsectioned in 30-cm intervals, and the bacterial assemblage structure was characterized using 16S rRNA gene pyrotag sequencing. Assemblages clustered into three distinct groups: top (0-30 cm); middle (30-150 cm); and bottom (150-236 cm). These layers correspond to the occurrence of refrozen melt pond ice, at least 2-year-old ice, and newly grown first-year ice at the bottom of the ice sheet, respectively. Thus, MYI houses multiple distinct bacterial assemblages, and in situ conditions appear to play a less important role in structuring microbial assemblages than the age or conditions of the ice at the time of formation.

Composition, diversity, and stability of microbial assemblages in seasonal lake ice, miquelon lake, central alberta.

The most familiar icy environments, seasonal lake and stream ice, have received little microbiological study. Bacteria and Eukarya dominated the microbial assemblage within the seasonal ice of Miquelon Lake, a shallow saline lake in Alberta, Canada. The bacterial assemblages were moderately diverse and did not vary with either ice depth or time. The closest relatives of the bacterial sequences from the ice included Actinobacteria, Bacteroidetes, Proteobacteria, Verrucomicrobia, and Cyanobacteria. The eukaryotic assemblages were less conserved and had very low diversity. Green algae relatives dominated the eukaryotic gene sequences; however, a copepod and cercozoan were also identified, possibly indicating the presence of complete microbial loop. The persistence of a chlorophyll a peak at 25-30 cm below the ice surface, despite ice migration and brine flushing, indicated possible biological activity within the ice. This is the first study of the composition, diversity, and stability of seasonal lake ice.

An oligarchic microbial assemblage in the anoxic bottom waters of a volcanic subglacial lake.

In 2006, we sampled the anoxic bottom waters of a volcanic lake beneath the Vatnajökull ice cap (Iceland). The sample contained 5 x 10(5) cells per ml, and whole-cell fluorescent in situ hybridization (FISH) and PCR with domain-specific probes showed these to be essentially all bacteria, with no detectable archaea. Pyrosequencing of the V6 hypervariable region of the 16S ribosomal RNA gene, Sanger sequencing of a clone library and FISH-based enumeration of four major phylotypes revealed that the assemblage was dominated by a few groups of putative chemotrophic bacteria whose closest cultivated relatives use sulfide, sulfur or hydrogen as electron donors, and oxygen, sulfate or CO(2) as electron acceptors. Hundreds of other phylotypes are present at lower abundance in our V6 tag libraries and a rarefaction analysis indicates that sampling did not reach saturation, but FISH data limit the remaining biome to <10-20% of all cells. The composition of this oligarchy can be understood in the context of the chemical disequilibrium created by the mixing of sulfidic lake water and oxygenated glacial meltwater.

Maribius salinus gen. nov., sp. nov., isolated from a solar saltern and Maribius pelagius sp. nov., cultured from the Sargasso Sea, belonging to the Roseobacter clade.

Two strictly aerobic, Gram-negative bacteria, designated strains CL-SP27T and B5-6T, were isolated from the hypersaline water of a solar saltern in Korea and from the surface water of the Sargasso Sea, respectively. The two strains were rod-shaped, non-motile and grew on marine agar 2216 as beige colonies. Phylogenetic analyses of 16S rRNA gene sequences revealed a clear affiliation of the novel strains to the family Rhodobacteraceae. However, the novel strains were only distantly related to members of the Roseobacter clade, forming a distinct lineage. Although the 16S rRNA gene sequence similarity between strains CL-SP27T and B5-6T was very high (99.6 %), DNA-DNA relatedness between the strains was 48.4 %, suggesting that the strains be categorized as two genospecies. Additionally, the two novel strains could be differentiated by DNA G+C contents, fatty acid profiles, carbon source utilization patterns, antibiotic susceptibilities and biochemical characteristics. Based on taxonomic data obtained in this study, strains CL-SP27T and B5-6T represent separate species within a novel genus of the family Rhodobacteraceae, for which the names Maribius salinus gen. nov., sp. nov. (type species) and Maribius pelagius sp. nov. are proposed. The type strains of Maribius salinus and Maribius pelagius are CL-SP27T (=KCCM 42113T=JCM 13037T) and B5-6T (=KCCM 42336T=JCM 14009T), respectively.

Comparison of microbial community compositions of two subglacial environments reveals a possible role for microbes in chemical weathering processes.

Viable microbes have been detected beneath several geographically distant glaciers underlain by different lithologies, but comparisons of their microbial communities have not previously been made. This study compared the microbial community compositions of samples from two glaciers overlying differing bedrock. Bulk meltwater chemistry indicates that sulfide oxidation and carbonate dissolution account for 90% of the solute flux from Bench Glacier, Alaska, whereas gypsum/anhydrite and carbonate dissolution accounts for the majority of the flux from John Evans Glacier, Ellesmere Island, Nunavut, Canada. The microbial communities were examined using two techniques: clone libraries and dot blot hybridization of 16S rRNA genes. Two hundred twenty-seven clones containing amplified 16S rRNA genes were prepared from subglacial samples, and the gene sequences were analyzed phylogenetically. Although some phylogenetic groups, including the Betaproteobacteria, were abundant in clone libraries from both glaciers, other well-represented groups were found at only one glacier. Group-specific oligonucleotide probes were developed for two phylogenetic clusters that were of particular interest because of their abundance or inferred biochemical capabilities. These probes were used in quantitative dot blot hybridization assays with a range of samples from the two glaciers. In addition to shared phyla at both glaciers, each glacier also harbored a subglacial microbial population that correlated with the observed aqueous geochemistry. These results are consistent with the hypothesis that microbial activity is an important contributor to the solute flux from glaciers.