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The activity of individual microorganisms can be measured within environmental samples by detecting uptake of isotope-labelled substrates using nano-scale secondary ion mass spectrometry (nanoSIMS). Recent studies have demonstrated that sample preparation can decrease 13 C and 15 N enrichment in bacterial cells, resulting in underestimates of activity. Here, we explore this effect with a variety of preparation types, microbial lineages and isotope labels to determine its consistency and therefore potential for correction. Specifically, we investigated the impact of different protocols for fixation, nucleic acid staining and catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) on >14 500 archaeal and bacterial cells (Methanosarcina acetivorans, Sulfolobus acidocaldarius and Pseudomonas putida) enriched in 13 C, 15 N, 18 O, 2 H and/or 34 S. We found these methods decrease isotope enrichments by up to 80% - much more than previously reported - and that the effect varies by taxa, growth phase, isotope label and applied protocol. We make recommendations for how to account for this effect experimentally and analytically. We also re-evaluate published nanoSIMS datasets and revise estimated microbial turnover times in the marine subsurface and nitrogen fixation rates in pelagic unicellular cyanobacteria. When sample preparation is accounted for, cell-specific rates increase and are more consistent with modelled and bulk rates.

Marine cold seeps are natural sites of methane emission and harbor distinct microbial communities capable of oxidizing methane. The majority of known cold seeps are on tectonically active continental margins, but recent discoveries have revealed abundant seeps on passive margins as well, including on the U.S. Atlantic Margin (USAM). We sampled in and around four USAM seeps and combined pore water geochemistry measurements with amplicon sequencing of 16S rRNA and mcrA (DNA and RNA) to investigate the microbial communities present, their assembly processes, and how they compare to communities at previously studied sites. We found that the USAM seeps contained communities consistent with the canonical seep microbiome at the class and order levels but differed markedly at the sequence variant level, especially within the anaerobic methanotrophic (ANME) archaea. The ANME populations were highly uneven, with just a few dominant mcrA sequence variants at each seep. Interestingly, the USAM seeps did not form a distinct phylogenetic cluster when compared with other previously described seeps around the world. Consistent with this, we found only a very weak (though statistically significant) distance-decay trend in seep community similarity across a global data set. Ecological assembly indices suggest that the USAM seep communities were assembled primarily deterministically, in contrast to the surrounding nonseep sediments, where stochastic processes dominated. Together, our results suggest that the primary driver of seep microbial community composition is local geochemistry—specifically methane, sulfide, nitrate, acetate, and ammonium concentrations—rather than the geologic context, the composition of nearby seeps, or random events of dispersal. IMPORTANCE Cold seeps are now known to be widespread features of passive continental margins, including the northern U.S. Atlantic Margin (USAM). Methane seepage is expected to intensify at these relatively shallow seeps as bottom waters warm and underlying methane hydrates dissociate. While methanotrophic microbial communities might reduce or prevent methane release, microbial communities on passive margins have rarely been characterized. In this study, we investigated the Bacteria and Archaea at four cold seeps on the northern USAM and found that despite being colocated on the same continental slope, the communities significantly differ by site at the sequence variant level, particularly methane-cycling community members. Differentiation by site was not observed in similarly spaced background sediments, raising interesting questions about the dispersal pathways of cold seep microorganisms. Understanding the genetic makeup of these discrete seafloor ecosystems and how their microbial communities develop will be increasingly important as the climate changes.

Marine microbial communities are a vital component of global carbon cycling, and numerous studies have shown that populations of petroleum-degrading bacteria are ubiquitous in the oceans. Few studies have attempted to distinguish all of the taxa that might contribute to petroleum biodegradation (including, e.g., heterotrophic and nondesignated microbes that respond positively to petroleum and microbes that grow on petroleum as the sole carbon source). This study quantifies the subpopulations of microorganisms that are expected to be involved in petroleum hydrocarbon biodegradation, which is important information during the decision-making process in the event of a petroleum spill accident., The environmental surveys following the 2010 Deepwater Horizon (DWH) spill identified a variety of hydrocarbon-degrading microorganisms, and laboratory studies with field-collected water samples then demonstrated faster-than-expected hydrocarbon biodegradation rates at 5°C. Knowledge about microbial community composition, diversity, and functional metabolic capabilities aids in understanding and predicting petroleum biodegradation by microbial communities in situ and is therefore an important component of the petroleum spill response decision-making process. This study investigates the taxonomic composition of microbial communities in six different global basins where petroleum and gas activities occur. Shallow-water communities were strikingly similar across basins, while deep-water communities tended to show subclusters by basin, with communities from the epipelagic, mesopelagic, and bathypelagic zones sometimes appearing within the same cluster. Microbial taxa that were enriched in the water column in the Gulf of Mexico following the DWH spill were found across marine basins. Several hydrocarbon-degrading genera (e.g., Actinobacteria, Pseudomonas, and Rhodobacteriacea) were common across all basins. Other genera such as Pseudoalteromonas and Oleibacter were highly enriched in specific basins.

Bisphenol A (BPA) belongs to a group of chemicals used in the production of polycarbonate, polysulfone, and polyethersulfone which are used, among other applications, in the manufacture of dialyzers. While exposure to BPA is widespread in the general population, dialysis patients represent a population with potentially chronic parenteral BPA exposures. To assess the potential risk of BPA exposure to dialysis patients through dialyzer use, exposure estimates were calculated based on BPA levels measured by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry following extractions from dialyzers manufactured by Fresenius Medical Care. Extraction conditions included both simulated-use leaching and exaggerated extractions to evaluate possible leachable and extractable BPA, respectively, from the devices. The mean BPA concentrations were 3.6 and 108.9 ppb from simulated-use and exaggerated extractions, respectively, from polycarbonate-containing dialyzers. No BPA was detected from polypropylene-containing dialyzers. Margins of Safety (MOS) were calculated to evaluate the level of risk to patients from estimated BPA exposure from the dialyzers, and the resulting MOS were 229 and 45 for simulated-use and exaggerated extractions, respectively. The findings suggest that there is an acceptable level of toxicological risk to dialysis patients exposed to BPA from use of the dialyzers tested in the current study.

Nitrogen fixation, the biological conversion of N_2 to NH_3, is critical to alleviating nitrogen limitation in many marine ecosystems. To date, few measurements exist of N_2 fixation in deep‐sea sediments. Here, we conducted > 400 bottle incubations with sediments from methane seeps, whale falls and background sites off the western coast of the United States from 600 to 2893 m water depth to investigate the potential rates, spatial distribution and biological mediators of benthic N_2 fixation. We found that N2 fixation was widespread, yet heterogeneously distributed with sediment depth at all sites. In some locations, rates exceeded previous measurements by > 10×, and provided up to 30% of the community anabolic growth requirement for nitrogen. Diazotrophic activity appeared to be inhibited by pore water ammonium: N_2 fixation was only observed if incubation ammonium concentrations were ≤ 25 μM, and experimental additions of ammonium reduced diazotrophy. In seep sediments, N_2 fixation was dependent on CH_4 and coincident with sulphate reduction, consistent with previous work showing diazotrophy by microorganisms mediating sulphate‐coupled methane oxidation. However, the pattern of diazotrophy was different in whale‐fall and associated reference sediments, where it was largely unaffected by CH_4, suggesting catabolically different diazotrophs at these sites.

The nitrogen cycle in the marine environment is strongly affected by ammonia-oxidizing Thaumarchaeota. In some marine settings, Thaumarchaeotes can comprise a large percentage of the prokaryotic population. To better understand the biogeographic patterns of Thaumarchaeotes, we sought to investigate differences in their abundance and phylogenetic diversity between geographically distinct basins. Samples were collected from four marine basins (The Caspian Sea, the Great Australian Bight, and the Central and Eastern Mediterranean). The concentration of bacterial and archaeal 16S rRNA genes and archaeal amoA genes were assessed using qPCR. Minimum entropy decomposition was used to elucidate the fine-scale diversity of Thaumarchaeotes. We demonstrated that there were significant differences in the abundance and diversity of Thaumarchaeotes between these four basins. The diversity of Thaumarchaeotal oligotypes differed between basins with many oligotypes only present in one of the four basins, which suggests that their distribution showed biogeographic patterning. There were also significant differences in Thaumarchaeotal community structure between these basins. This would suggest that geographically distant, yet geochemically similar basins may house distinct Thaumarchaeaotal populations. These findings suggest that Thaumarchaeota are very diverse and that biogeography in part contributes in determining the diversity and distribution of Thaumarchaeotes., Archaea are dominant in the water column in most deep marine basins; however, geographically distant, yet geochemically similar basins may house distinct ammonium-oxidizing Thaumarchaeaotal populations.

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., This study describes microbial biomass, community composition and diversity in Caspian Sea sediments using lipid and genomic techniques.

Humid tropical forest soils are characterized by low and fluctuating redox, conditions which are thought to inhibit organic matter degradation by microbes. However, evidence suggests that soil microbial communities are adapted to the redox conditions in these ecosystems. In this study we tested the hypothesis that soil oxygen (O2) availability as an index of redox conditions structures patterns in litter decomposition and associated microbial community dynamics over space and time in humid tropical forests. We conducted a two year decomposition experiment on a common litter substrate in four sites along an elevational gradient with well described climate and redox dynamics. Microbial community sequencing, potential enzyme activities, and litter chemistry measurements were made on litter and soil to determine the relationship between soil and litter communities and biogeochemistry. Decomposition was slowest in the upper elevation site, which was the wettest and had the lowest average soil O2 availability. However, soil hydrolytic and litter phenol oxidase activities were greatest at this site. Small subunit ribosomal RNA genes were sequenced with universal primers for bacteria, archaea and eukaryotes, yielding 40,850 unique taxa after quality filtering and clustering. Across all sites, microbial succession was observed as increasing litter richness, converging bacterial community profiles, and diverging fungal community profiles. Initial decomposers (1–4 weeks) included many r-selected bacteria, including Alpha-, Beta- and Gammaproteobacteria, Clostridia, Bacteroidetes. We also found evidence of anaerobic fungi such as Cryptococcus, as well as the plant-associated Phialocephala and Phyllachora species, suggesting that anaerobic and plant-associated fungi are prevalent later in decomposition in soils with low and fluctuating redox conditions. Because of the striking similarities between sites in functional potential despite differences in wet tropical soil decomposing communities and litter chemistry, we suggest that future climate-driven disruptions to redox fluctuations could significantly alter the terrestrial carbon (C) cycle in tropical forests.

Biological soil crusts (BSCs) cover extensive portions of the earth’s deserts. In order to survive desiccation cycles and utilize short periods of activity during infrequent precipitation, crust microorganisms must rely on the unique capabilities of vegetative cells to enter a dormant state and be poised for rapid resuscitation upon wetting. To elucidate the key events involved in the exit from dormancy, we performed a wetting experiment of a BSC and followed the response of the dominant cyanobacterium, Microcoleus vaginatus, in situ using a whole-genome transcriptional time course that included two diel cycles. Immediate, but transient, induction of DNA repair and regulatory genes signaled the hydration event. Recovery of photosynthesis occurred within 1 h, accompanied by upregulation of anabolic pathways. Onset of desiccation was characterized by the induction of genes for oxidative and photo-oxidative stress responses, osmotic stress response and the synthesis of C and N storage polymers. Early expression of genes for the production of exopolysaccharides, additional storage molecules and genes for membrane unsaturation occurred before drying and hints at preparedness for desiccation. We also observed signatures of preparation for future precipitation, notably the expression of genes for anaplerotic reactions in drying crusts, and the stable maintenance of mRNA through dormancy. These data shed light on possible synchronization between this cyanobacterium and its environment, and provides key mechanistic insights into its metabolism in situ that may be used to predict its response to climate, and or, land-use driven perturbations.

The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

Deep-sea bacteria enriched by oil and dispersant from the Deepwater Horizon spill Jacob Baelum 1,2 , Sharon Borglin 1 , Romy Chakraborty 1 , Julian L. Fortney 1 , Regina Lamendella 1 , Olivia U. Mason 1 , Manfred Auer 1 , Marcin Zemla 1 , Markus Bill 1 , Mark E. Conrad 1 , Stephanie A. Malfatti 3 , Susannah G. Tringe 3 , Hoi-Ying Holman 1 , Terry C. Hazen 1 , and Janet K. Jansson 1,3* MS 70A-3317, 1 Cyclotron Rd., Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA The Geological Survey of Denmark and Greenland, Oster Voldgade 10, 1350 Copenhagen, Denmark Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA Corresponding author phone: 1-510-486-7487; e-mail: jrjansson@lbl.gov; fax: 1-510- Running title: Enrichment of oil degraders from Gulf of Mexico

Environmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.

Diving into Deep Water The Deepwater Horizon oil spill in the Gulf of Mexico was one of the largest oil spills on record. Its setting at the bottom of the sea floor posed an unanticipated risk as substantial amounts of hydrocarbons leaked into the deepwater column. Three separate cruises identified and sampled deep underwater hydrocarbon plumes that existed in May and June, 2010—before the well head was ultimately sealed. Camilli et al. (p. 201 ; published online 19 August) used an automated underwater vehicle to assess the dimensions of a stabilized, diffuse underwater plume of oil that was 22 miles long and estimated the daily quantity of oil released from the well, based on the concentration and dimensions of the plume. Hazen et al. (p. 204 ; published online 26 August) also observed an underwater plume at the same depth and found that hydrocarbon-degrading bacteria were enriched in the plume and were breaking down some parts of the oil. Finally, Valentine et al. (p. 208 ; published online 16 September) found that natural gas, including propane and ethane, were also present in hydrocarbon plumes. These gases were broken down quickly by bacteria, but primed the system for biodegradation of larger hydrocarbons, including those comprising the leaking crude oil. Differences were observed in dissolved oxygen levels in the plumes (a proxy for bacterial respiration), which may reflect differences in the location of sampling or the aging of the plumes.

Producing cellulosic biofuels from plant material has recently emerged as a key US Department of Energy goal. For this technology to be commercially viable on a large scale, it is critical to make production cost efficient by streamlining both the deconstruction of lignocellulosic biomass and fuel production. Many natural ecosystems efficiently degrade lignocellulosic biomass and harbor enzymes that, when identified, could be used to increase the efficiency of commercial biomass deconstruction. However, ecosystems most likely to yield relevant enzymes, such as tropical rain forest soil in Puerto Rico, are often too complex for enzyme discovery using current metagenomic sequencing technologies. One potential strategy to overcome this problem is to selectively cultivate the microbial communities from these complex ecosystems on biomass under defined conditions, generating less complex biomass-degrading microbial populations. To test this premise, we cultivated microbes from Puerto Rican soil or green waste compost under precisely defined conditions in the presence dried ground switchgrass (Panicum virgatum L.) or lignin, respectively, as the sole carbon source. Phylogenetic profiling of the two feedstock-adapted communities using SSU rRNA gene amplicon pyrosequencing or phylogenetic microarray analysis revealed that the adapted communities were significantly simplified compared to the natural communities from which they were derived. Several members of the lignin-adapted and switchgrass-adapted consortia are related to organisms previously characterized as biomass degraders, while others were from less well-characterized phyla. The decrease in complexity of these communities make them good candidates for metagenomic sequencing and will likely enable the reconstruction of a greater number of full-length genes, leading to the discovery of novel lignocellulose-degrading enzymes adapted to feedstocks and conditions of interest.

© 2016, Public Library of Science. All rights reserved. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Many microbial ecology experiments use sequencing data to measure a community's response to an experimental treatment. In a common experimental design, two units, one control and one experimental, are sampled before and after the treatment is applied to the experimental unit. The four resulting samples contain information about the dynamics of organisms that respond to the treatment, but there are no analytical methods designed to extract exactly this type of information from this configuration of samples. Here we present an analytical method specifically designed to visualize and generate hypotheses about microbial community dynamics in experiments that have paired samples and few or no replicates. The method is based on the Poisson lognormal distribution, long studied in macroecology, which we found accurately models the abundance distribution of taxa counts from 16S rRNA surveys. To demonstrate the method's validity and potential, we analyzed an experiment that measured the effect of crude oil on ocean microbial communities in microcosm. Our method identified known oil degraders as well as two clades, Maricurvus and Rhodobacteraceae, that responded to amendment with oil but do not include known oil degraders. Our approach is sensitive to organisms that increased in abundance only in the experimental unit but less sensitive to organisms that increased in both control and experimental units, thus mitigating the role of "bottle effects".

Tolumonas lignolytica BRL6-1T sp. nov. is the type strain of T. lignolytica sp. nov., a proposed novel species of the Tolumonas genus. This strain was isolated from tropical rainforest soils based on its ability to utilize lignin as a sole carbon source. Cells of Tolumonas lignolytica BRL6-1T are mesophilic, non-spore forming, Gram-negative rods that are oxidase and catalase negative. The genome for this isolate was sequenced and returned in seven unique contigs totaling 3.6Mbp, enabling the characterization of several putative pathways for lignin breakdown. Particularly, we found an extracellular peroxidase involved in lignin depolymerization, as well as several enzymes involved in β-aryl ether bond cleavage, which is the most abundant linkage between lignin monomers. We also found genes for enzymes involved in ferulic acid metabolism, which is a common product of lignin breakdown. By characterizing pathways and enzymes employed in the bacterial breakdown of lignin in anaerobic environments, this work should assist in the efficient engineering of biofuel production from lignocellulosic material. Electronic supplementary material The online version of this article (doi:10.1186/s40793-015-0100-3) contains supplementary material, which is available to authorized users.

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.

The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of ∼4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using 14 C-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, both of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of 14 C-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)'s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem. © 2014 International Society for Microbial Ecology All rights reserved.

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated Klebsiella sp. strain BRL6-2 on minimal media with alkali lignin as the sole carbon source. This organism was isolated anaerobically from tropical forest soils collected from the Bisley watershed at the Ridge site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are characterized by cycles of iron oxidation and reduction. Genome sequencing was targeted because of its ability to grow on lignin anaerobically and lignocellulolytic activity via in vitro enzyme assays. The genome of Klebsiella sp. strain BRL6-2 is 5.80 Mbp with no detected plasmids, and includes a relatively small arsenal of genes encoding lignocellulolytic carbohydrate active enzymes. The genome revealed four putative peroxidases including glutathione and DyP-type peroxidases, and a complete protocatechuate pathway encoded in a single gene cluster. Physiological studies revealed Klebsiella sp. strain BRL6-2 to be relatively stress tolerant to high ionic strength conditions. It grows in increasing concentrations of ionic liquid (1-ethyl-3-methyl-imidazolium acetate) up to 73.44 mM and NaCl up to 1.5 M.

The Deepwater Horizon oil spill resulted in a massive influx of hydrocarbons into the Gulf of Mexico (the Gulf). To better understand the fate of the oil, we enriched and isolated indigenous hydrocarbon-degrading bacteria from deep, uncontaminated waters from the Gulf with oil (Macondo MC252) and dispersant used during the spill (COREXIT 9500). During 20 days of incubation at 5°C, CO(2) evolution, hydrocarbon concentrations and the microbial community composition were determined. Approximately 60% to 25% of the dissolved oil with or without COREXIT, respectively, was degraded, in addition to some hydrocarbons in the COREXIT. FeCl(2) addition initially increased respiration rates, but not the total amount of hydrocarbons degraded. 16S rRNA gene sequencing revealed a succession in the microbial community over time, with an increase in abundance of Colwellia and Oceanospirillales during the incubations. Flocs formed during incubations with oil and/or COREXIT in the absence of FeCl(2) . Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy revealed that the flocs were comprised of oil, carbohydrates and biomass. Colwellia were the dominant bacteria in the flocs. Colwellia sp. strain RC25 was isolated from one of the enrichments and confirmed to rapidly degrade high amounts (approximately 75%) of the MC252 oil at 5°C. Together these data highlight several features that provide Colwellia with the capacity to degrade oil in cold, deep marine habitats, including aggregation together with oil droplets into flocs and hydrocarbon degradation ability.

Tropical forest soils decompose litter rapidly with frequent episodes of anoxic conditions, making it likely that bacteria using alternate terminal electron acceptors (TEAs) play a large role in decomposition. This makes these soils useful templates for improving biofuel production. To investigate how TEAs affect decomposition, we cultivated feedstock-adapted consortia (FACs) derived from two tropical forest soils collected from the ends of a rainfall gradient: organic matter-rich tropical cloud forest (CF) soils, which experience sustained low redox, and iron-rich tropical rain forest (RF) soils, which experience rapidly fluctuating redox. Communities were anaerobically passed through three transfers of 10 weeks each with switchgrass as a sole carbon (C) source; FACs were then amended with nitrate, sulfate, or iron oxide. C mineralization and cellulase activities were higher in CF-FACs than in RF-FACs. Pyrosequencing of the small-subunit rRNA revealed members of the Firmicutes, Bacteroidetes, and Alphaproteobacteria as dominant. RF- and CF-FAC communities were not different in microbial diversity or biomass. The RF-FACs, derived from fluctuating redox soils, were the most responsive to the addition of TEAs, while the CF-FACs were overall more efficient and productive, both on a per-gram switchgrass and a per-cell biomass basis. These results suggest that decomposing microbial communities in fluctuating redox environments are adapted to the presence of a diversity of TEAs and ready to take advantage of them. More importantly, these data highlight the role of local environmental conditions in shaping microbial community function that may be separate from phylogenetic structure., IMPORTANCE After multiple transfers, we established microbial consortia derived from two tropical forest soils with different native redox conditions. Communities derived from the rapidly fluctuating redox environment maintained a capacity to use added terminal electron acceptors (TEAs) after multiple transfers, though they were not present during the enrichment. Communities derived from lower-redox soils were not responsive to TEA addition but were much more efficient at switchgrass decomposition. Though the communities were different, diversity was not, and both were dominated by many of the same species of clostridia. This reflects the inadequacy of rRNA for determining the function of microbial communities, in this case the retained ability to utilize TEAs that were not part of the selective growth conditions. More importantly, this suggests that microbial community function is shaped by life history, where environmental factors produce heritable traits through natural selection over time, creating variation in the community, a phenomenon not well documented for microbes.

The Deepwater Horizon oil spill in the Gulf of Mexico is the deepest and largest offshore spill in the United State history and its impacts on marine ecosystems are largely unknown. Here, we showed that the microbial community functional composition and structure were dramatically altered in a deep-sea oil plume resulting from the spill. A variety of metabolic genes involved in both aerobic and anaerobic hydrocarbon degradation were highly enriched in the plume compared with outside the plume, indicating a great potential for intrinsic bioremediation or natural attenuation in the deep sea. Various other microbial functional genes that are relevant to carbon, nitrogen, phosphorus, sulfur and iron cycling, metal resistance and bacteriophage replication were also enriched in the plume. Together, these results suggest that the indigenous marine microbial communities could have a significant role in biodegradation of oil spills in deep-sea environments.

Lignin is often the most difficult portion of plant biomass to degrade, with fungi generally thought to dominate during late stage decomposition. Lignin in feedstock plant material represents a barrier to more efficient plant biomass conversion and can also hinder enzymatic access to cellulose, which is critical for biofuels production. Tropical rain forest soils in Puerto Rico are characterized by frequent anoxic conditions and fluctuating redox, suggesting the presence of lignin-degrading organisms and mechanisms that are different from known fungal decomposers and oxygen-dependent enzyme activities. We explored microbial lignin-degraders by burying bio-traps containing lignin-amended and unamended biosep beads in the soil for 1, 4, 13 and 30 weeks. At each time point, phenol oxidase and peroxidase enzyme activity was found to be elevated in the lignin-amended versus the unamended beads, while cellulolytic enzyme activities were significantly depressed in lignin-amended beads. Quantitative PCR of bacterial communities showed more bacterial colonization in the lignin-amended compared to the unamended beads after one and four weeks, suggesting that the lignin supported increased bacterial abundance. The microbial community was analyzed by small subunit 16S ribosomal RNA genes using microarray (PhyloChip) and by high-throughput amplicon pyrosequencing based on universal primers targeting bacterial, archaeal, and eukaryotic communities. Community trends were significantly affected by time and the presence of lignin on the beads. Lignin-amended beads have higher relative abundances of representatives from the phyla Actinobacteria, Firmicutes, Acidobacteria and Proteobacteria compared to unamended beads. This study suggests that in low and fluctuating redox soils, bacteria could play a role in anaerobic lignin decomposition.

The waters of the Eastern Mediterranean are characterized by unique physical and chemical properties within separate water masses occupying different depths. Distinct water masses are present throughout the oceans, which drive thermohaline circulation. These water masses may contain specific microbial assemblages. The goal of this study was to examine the effect of physical and geological phenomena on the microbial community of the Eastern Mediterranean water column. Chemical measurements were combined with phospholipid fatty acid (PLFA) analysis and high-throughput 16S rRNA sequencing to characterize the microbial community in the water column at five sites. We demonstrate that the chemistry and microbial community of the water column were stratified into three distinct water masses. The salinity and nutrient concentrations vary between these water masses. Nutrient concentrations increased with depth, and salinity was highest in the intermediate water mass. Our PLFA analysis indicated different lipid classes were abundant in each water mass, suggesting that distinct groups of microbes inhabit these water masses. 16S rRNA gene sequencing confirmed the presence of distinct microbial communities in each water mass. Taxa involved in autotrophic nitrogen cycling were enriched in the intermediate water mass suggesting that microbes in this water mass may be important to the nitrogen cycle of the Eastern Mediterranean. The Eastern Mediterranean also contains numerous active hydrocarbon seeps. We sampled above the North Alex Mud Volcano, in order to test the effect of these geological features on the microbial community in the adjacent water column. The community in the waters overlaying the mud volcano was distinct from other communities collected at similar depths and was enriched in known hydrocarbon degrading taxa. Our results demonstrate that physical phenomena such stratification as well as geological phenomena such as mud volcanoes strongly affect microbial community structure in the Eastern Mediterranean water column.

© 2016 The Authors The detection and quantification of naturally occurring microbial cellular densities is an essential component of environmental systems monitoring. While there are a number of commonly utilized approaches for monitoring microbial abundance, capacitance-based biosensors represent a promising approach because of their low-cost and label-free detection of microbial cells, but are not as well characterized as more traditional methods. Here, we investigate the applicability of enhanced alternating current electrokinetics (ACEK) capacitive sensing as a new application for rapidly detecting and quantifying microbial cellular densities in cultured and environmentally sourced aquatic samples. ACEK capacitive sensor performance was evaluated using two distinct and dynamic systems – the Great Australian Bight and groundwater from the Oak Ridge Reservation in Oak Ridge, TN. Results demonstrate that ACEK capacitance-based sensing can accurately determine microbial cell counts throughout cellular concentrations typically encountered in naturally occurring microbial communities (103−106 cells/mL). A linear relationship was observed between cellular density and capacitance change correlations, allowing a simple linear curve fitting equation to be used for determining microbial abundances in unknown samples. This work provides a foundation for understanding the limits of capacitance-based sensing in natural environmental samples and supports future efforts focusing on evaluating the robustness ACEK capacitance-based within aquatic environments.