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The candidate phylum Omnitrophica-recently termed Omnitrophota, and originally known as OP3-is an understudied bacterial clade that has primarily been found in aquatic ecosystems. To characterize the diversity and ecology of this phylum, we reconstructed 55 Omnitrophota metagenome-assembled genomes (MAGs) from a well-characterized groundwater system within central Germany and placed them within the context of publicly available genomes. Seven clades were identified, four of which contained novel genomes obtained from our groundwater system. All clades exhibited the capacity for type IV pili, type II secretion systems, glycogen storage, and carbohydrate degradation. Only the characterized Cand. Omnitrophus magneticus genome exhibited functions associated with magnetosome construction. Clades were characterized by sets of traits rather than unique pathways, which were then used to infer ecological strategies. These lifestyles consisted of mixotrophs, obligate fermenters, and versatile respiratory heterotrophs. Patterns in 16S rRNA gene amplicons from a 6 years, monthly sampled groundwater time-series dataset reflected the persistent and widespread occurrence of Clade 7 Wood-Ljungdahl utilizing mixotrophs and highlight this group as a core member of the groundwater community. Overall, this study uncovered, characterized, and contextualized the metabolic and phylogenetic diversity within phylum Omnitrophota, and predicts that environmental populations may mediate both nitrogen and sulfur cycling, along with organic matter production and degradation within aquatic ecosystems.

To better understand the influence of habitat on the genetic content of Candidate Phyla Radiation (CPR) bacteria, we studied the effects of transitioning from soil to groundwater on genomic divergences of these organisms. Bacterial metagenome-assembled genomes (318 total, 35 of CPR bacteria) were generated from seepage waters and compared directly to groundwater counterparts. Seepage water CPR bacteria exhibited 1.24-fold greater mean genome size, while their inferred mean replication rate was 21.1% lower than groundwater lineages. While exploring gene loss and adaptive gains in closely related lineages in groundwater, we identified a zinc transporter, a surface protein, and a lipogylcopeptide resistance gene unique to a seepage Parcubacterium. A nitrite reductase gene unique to the groundwater Parcubacterium, likely acquired from pelagic microbes via horizontal gene transfer, was also identified. Groundwater Parcubacteria harbored nearly double the fraction (9.4%) of pseudogenes than their seepage kin (4.9%), suggesting further genome streamlining.

Current understanding of organic carbon inputs into ecosystems lacking photosynthetic primary production is predicated on data and inferences derived almost entirely from metagenomic analyses. The elevated abundances of putative chemolithoautotrophs in groundwaters suggest that dark CO2 fixation is an integral component of subsurface trophic webs. To understand the impact of autotrophically fixed carbon, the flux of CO2-derived carbon through various populations of subsurface microbiota must first be resolved, both quantitatively and temporally. Here we implement novel Stable Isotope Cluster Analysis to render a time-resolved and quantitative evaluation of 13CO2-derived carbon flow through a groundwater community in microcosms stimulated with reduced sulfur compounds. We demonstrate that mixotrophs, not strict autotrophs, were the most abundant active organisms in groundwater microcosms. Species of Hydrogenophaga, Polaromonas, Dechloromonas, and other metabolically versatile mixotrophs drove the production and remineralization of organic carbon. Their activity facilitated the replacement of 43% and 80% of total microbial carbon stores in the groundwater microcosms with 13C in just 21 and 70 days, respectively. The mixotrophs employed different strategies for satisfying their carbon requirements by balancing CO2 fixation and uptake of available organic compounds. These different strategies might provide fitness under nutrient-limited conditions, explaining the great abundances of mixotrophs in other oligotrophic habitats, such as the upper ocean and boreal lakes.

Large amounts of methane (CH4) could potentially be formed as a result of the gradual or abrupt thawing of Arctic permafrost due to global warming. Upon its release, this potent greenhouse gas can be emitted into the atmosphere, or transported laterally into aquatic ecosystems via hydrologic connectivity at surface or groundwaters. While high northern latitudes contribute up to 5 % of total global CH4 emissions, the specific contribution of Arctic rivers and streams is largely unknown. In this study, we measured high-resolution continuous CH4 concentrations in a ~120 km section of the Kolyma River in Northeast Siberia navigated twice between 15–17 June 2019 (late freshet). The average partial pressure of CH4 (pCH4) in tributaries (66.8–206.8 µatm) was 2–7 times higher than in the main river channel (28.3 µatm). In the main channel, CH4 was up to 1600 % supersaturated with respect to atmospheric equilibrium. At key sites located near the riverbank and tributary confluences, pCH4 (41±7 µatm) and emissions (0.03±0.004 mmol m–2 d–1) were higher compared to other sites within the main channel. Warm waters (T>14.5 °C) and low specific conductivities (κ–1) defined these key sites. The distribution of methane in the river could also be linked statistically to T and κ of the water, as well as to the distance to the shore z, as indicators used to predict CH4 concentrations in unsampled river areas. Similarly, the abundance of methane consuming bacteria and methane producing archaea strongly correlated mainly to T and κ, and less to the pCH4, and were similar to those previously detected in nearby soils, suggesting the source of CH4 to be associated with sites close to land. The average total CH4 flux densities in the investigated Kolyma River section were 0.02±0.006 mml m–2 d–1, equivalent to a total CH4 flux of 12.4 mmol m–2. Key sites with highest CH4 concentrations contributed from 13 to 20 % to the total flux. Our study highlights the importance of high-resolution continuous CH4 measurements in Arctic Rivers for identifying spatial and temporal variabilities, and offers a glimpse to the magnitude of riverine methane emissions in the Arctic and their potential relevance to regional methane budgets.

The biogeochemical cycling of carbon in Arctic rivers is perturbed as more terrestrial organic carbon and nutrients are released upon active layer and permafrost thaw. The majority of the carbon dioxide (CO2) in rivers is emitted into the atmosphere, but it can also be utilized during photosynthesis, especially with more availability of nutrients, influencing the carbon flow and aquatic ecosystem metabolism. However, the timing and amount of photosynthetic primary production in Arctic rivers are unknown.Water samples from the Kolyma River in Northeast Siberia were collected in June (late freshet) and August (summer) 2019. For the first time in an Arctic river, we measured biological oxygen supersaturations using the relative oxygen-to-argon ratio above equilibrium, Δ(O2/Ar), which is an indicator of the presence of biologically produced oxygen. This ratio is influenced in approximately equal parts by physical processes, while biological processes unilaterally influence the oxygen content.In addition, we measured the partial pressure of CO2, p(CO2), dissolved oxygen and inorganic nutrients concentrations. Mass spectrometry was employed to chemically characterize the composition of dissolved organic matter (DOM) and better understand its origin. Microbial communities were elucidated using 16S and metagenomic based sequencing approaches.In June, the oxygen saturation in turbid and warm waters (average: 14 °C) in the main river channel was on average 10% above atmospheric equilibrium. The p(CO2) values were well above equilibrium (2000 µatm). Unlike oxygen saturation, Δ(O2/Ar) was negative (undersaturation); thus, physical processes contributed most to the total oxygen supersaturation (up to 20%), apparently due to contributions of freshet cold gas-rich meltwater, while the net biological oxygen concentration was between –10 and –15%.In August, the water was colder (3 °C drop), and the total oxygen was mostly undersaturated (up to –10%). However, lower p(CO2) and a decrease in the biological oxygen deficit (between 0 and –5%) indicated net biological oxygen input. At the confluence of the main river channel and some tributaries, an algal bloom was observed resulting in up to 6.4% supersaturation in Δ(O2/Ar) and p(CO2) near atmospheric equilibrium.Concentrations of nitrate and silica were higher in August than in June. Dissolved phosphate concentrations were low at both sampling times, but apparently did not limit primary productivity. The microbial community composition varied greatly between sampling times, with differential shifts across the transect. Compared to June, the DOM pattern in August was less diverse in the river due to more stable stream conditions and defined hydrologic connectivity between land and river, promoting also nutrient supply for biological productivity.Unlike anticipated, the O2/Ar ratios suggested that net biological oxygen production in the river did not profit during the late freshet, despite unlimited light and CO2 availability and warm temperatures. Contrastingly, the summer low-flow allowed for photosynthetically-driven oxygen production and CO2 uptake in some sites. We conclude that the O2/Ar ratios were essential for quantifying the contribution of biological production, and understand better the fate of CO2 in an Arctic river influenced by thawing permafrost, as well as the land-aquatic-continuum in the context of climate change.

The terrestrial subsurface contains nearly all of Earth’s freshwater reserves1 and harbors upwards of 60% of our planet’s total prokaryotic biomass2,3. While genetic surveys suggest these organisms rely on in situ carbon fixation, rather than the translocation of photosynthetically derived organic carbon4–6, corroborating measurements of carbon fixation in the subsurface are absent. Using a novel ultra-low level 14C-labeling technique, we show that in situ carbon fixation rates in a carbonate aquifer reached 10% of the median rates measured in oligotrophic marine surface waters, and were up to six-fold greater than those observed in lower euphotic zone waters where deep chlorophyll levels peak. Empirical carbon fixation rates were substantiated by both nitrification and anammox rate data. Metagenomic analyses revealed a remarkable abundance of putative chemolithoautotrophic members of an uncharacterized order of Nitrospiria – the first representatives of this class expected to fix carbon via the Wood-Ljungdahl pathway. Based on these fixation rates, we extrapolate global primary production in carbonate groundwaters to be 0.11 Pg of carbon per year.

The highly diverseCand. Patescibacteria are predicted to have minimal biosynthetic and metabolic pathways, which hinders understanding of how their populations differentiate to environmental drivers or host organisms. Their metabolic traits to cope with oxidative stress are largely unknown. Here, we utilized genome-resolved metagenomics to investigate the adaptive genome repertoire of Patescibacteria in oxic and anoxic groundwaters, and to infer putative host ranges.Within six groundwater wells,Cand. Patescibacteria was the most dominant (up to 79%) super-phylum across 32 metagenomes obtained from sequential 0.2 and 0.1 µm filtration. Of the reconstructed 1275 metagenome-assembled genomes (MAGs), 291 high-quality MAGs were classified asCand. Patescibacteria.Cand. Paceibacteria andCand. Microgenomates were enriched exclusively in the 0.1 µm fractions, whereas candidate division ABY1 andCand. Gracilibacteria were enriched in the 0.2 µm fractions. Patescibacteria enriched in the smaller 0.1 µm filter fractions had 22% smaller genomes, 13.4% lower replication measures, higher fraction of rod-shape determining proteins, and genomic features suggesting type IV pili mediated cell-cell attachments. Near-surface wells harbored Patescibacteria with higher replication rates than anoxic downstream wells characterized by longer water residence time. Except prevalence of superoxide dismutase genes in Patescibacteria MAGs enriched in oxic groundwaters (83%), no major metabolic or phylogenetic differences were observed based on oxygen concentrations. The most abundant Patescibacteria MAG in oxic groundwater encoded a nitrate transporter, nitrite reductase, and F-type ATPase, suggesting an alternative energy conservation mechanism. Patescibacteria consistently co-occurred with one another or with members of phyla Nanoarchaeota, Bacteroidota, Nitrospirota, and Omnitrophota. However, only 8% of MAGs showed highly significant one-to-one association, mostly with Omnitrophota. Genes coding for motility and transport functions in certain Patescibacteria were highly similar to genes from other phyla (Omnitrophota, Proteobacteria and Nanoarchaeota).Other than genes to cope with oxidative stress, we found little genomic evidence for niche adaptation of Patescibacteria to oxic or anoxic groundwaters. Given that we could detect specific host preference only for a few MAGs, we propose that the majority of Patescibacteria can attach to multiple hosts just long enough to loot or exchange supplies with an economic lifestyle of little preference for geochemical conditions.

The microbial ecology of oligotrophic deep ocean sediments is understudied relative to their shallow counterparts, and this lack of understanding hampers our ability to predict responses to current and future perturbations. The Gulf of Mexico has experienced two of the largest accidental marine oil spills, the 1979 Ixtoc-1 blowout and the 2010 Deepwater Horizon (DWH) discharge. Here, microbial communities were characterized for 29 sites across multiple years in > 700 samples. The composition of the seafloor microbiome was broadly consistent across the region and was well approximated by the overlying water depth and depth within the sediment column, while geographic distance played a limited role. Biogeographical distributions were employed to generate predictive models for over 4000 OTU that leverage easy-to-obtain geospatial variables which are linked to measured sedimentary oxygen profiles. Depth stratification and putative niche diversification are evidenced by the distribution of taxa that mediate the microbial nitrogen cycle. Furthermore, these results demonstrate that sediments impacted by the DWH spill had returned to near baseline conditions after 2 years. The distributions of benthic microorganisms in the Gulf can be constrained, and moreover, deviations from these predictions may pinpoint impacted sites and aid in future response efforts or long-term stability studies.

The current understanding of organic carbon inputs into ecosystems lacking photosynthetic primary production is predicated on data and inferences derived almost entirely from metagenomic analyses. The elevated abundances of putative chemolithoautotrophs in groundwaters suggest that dark CO2 fixation is an integral component of subsurface trophic webs. To understand the impact of autotrophically-fixed carbon, the flux of CO2-derived carbon through various subpopulations of subsurface microbiota must first be resolved, both quantitatively and temporally. Here, we implement novel stable isotope cluster analysis to render a time-resolved and quantitative evaluation of 13CO-derived carbon flow through a groundwater microbiome stimulated with reduced sulfur compounds. We demonstrate that mixotrophs, not obligate chemolithoautotrophs, were the most abundant active organisms in groundwater microcosms. Species of Hydrogenophaga, Polaromonas, Dechloromonas, and other metabolically versatile mixotrophs drove the recycling of organic carbon and, when chance afforded, supplemented their carbon requirements via chemolithoautotrophy and uptake of available organic compounds. Mixotrophic activity facilitated the replacement of 43 and 80% of total microbial carbon stores with 13C in just 21 and 70 days, respectively. This opportunistic “utilize whatever pathways net the greatest advantage in fitness” strategy may explain the great abundances of mixotrophs in other oligotrophic habitats, like the upper ocean and boreal lakes.

Crude oil buried in intertidal sands may be exposed to alternating oxic and anoxic conditions but the effect of this tidally induced biogeochemical oscillation remains poorly understood, limiting the effectiveness of remediation and managing efforts after oil spills. Here, we used a combination of metatranscriptomics and genome-resolved metagenomics to study microbial activities in oil-contaminated sediments during oxic-anoxic cycles in laboratory chambers that closely emulated in situ conditions. Approximately 5-fold higher reductions in the total petroleum hydrocarbons were observed in the oxic as compared to the anoxic phases with a relatively constant ratio between aerobic and anaerobic oil decomposition rates even after prolonged anoxic conditions. Metatranscriptomics analysis indicated that the oxic phases promoted oil biodegradation in subsequent anoxic phases by microbially mediated reoxidation of alternative electron acceptors like sulfide and by providing degradation-limiting nitrogen through biological nitrogen fixation. Most population genomes reconstructed from the mesocosm samples represented uncultured taxa and were present typically as members of the rare biosphere in metagenomic data from uncontaminated field samples, implying that the intertidal communities are adapted to changes in redox conditions. Collectively, these results have important implications for enhancing oil spill remediation efforts in beach sands and coastal sediments and underscore the role of uncultured taxa in such efforts.

Assembling microbial and viral genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. To compare the recovery of genomes from microorganisms and their viruses from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore Technologies (ONT) sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from an Illumina-only assembly, ONT-only assembly, and a hybrid assembly approach. The hybrid approach recovered 2× more mid to high-quality MAGs compared to the Illumina-only approach and 4× more than the ONT-only approach. A similar number of viral genomes were reconstructed using the hybrid and ONT methods, and both recovered nearly fourfold more viral genomes than the Illumina-only approach. While yielding fewer MAGs, the ONT-only approach generated MAGs with a high probability of containing rRNA genes, 3× higher than either of the other methods. Of the shared MAGs recovered from each method, the ONT-only approach generated the longest and least fragmented MAGs, while the hybrid approach yielded the most complete. This work provides quantitative data to inform a cost-benefit analysis of the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.

Assembling microbial and phage genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. In order to compare the recovery of genomes from microorganisms and their phages from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from Illumina-only assemblies and a hybrid assembly approach. Strikingly, the hybrid approach more than doubled the number of mid to high-quality MAGs (> 50% completion, < 10% redundancy), generated nearly four-fold more phage genomes, and improved all associated genome metrics relative to the Illumina only method. The hybrid assemblies yielded MAGs that were on average 7.8% more complete, with 133 fewer contigs and a 14 kbp greater N50. Furthermore, the longer contigs from the hybrid approach generated microbial MAGs that had a higher proportion of rRNA genes. We demonstrate this usefulness by linking microbial MAGs containing 16S rRNA genes with extensive amplicon dataset. This work provides quantitative data to inform a cost-benefit analysis on the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.

Benthic ecosystems often act as a repository for oil contamination that washes ashore or is deposited onto sediments following a major oil spill. Sedimentary microorganisms mediate central ecosystem services on the coast, such as carbon and nutrient cycling, and these services may be adversely impacted by oil perturbation. Thus, during the response to the Deepwater Horizon (DWH) oil discharge in the Gulf of Mexico, considerable effort went into characterizing the response of benthic microbial communities to oil deposition on shorelines of the Northern Gulf where oil came ashore. Oil perturbation elicited a pronounced microbial response in coastal ecosystems, altering the abundance, diversity, and community composition of sedimentary microorganisms. Next-generation gene sequencing and metagenomic approaches, which were not available during previous large oil spills, have revolutionized the field of microbiology, providing new insights into the microbial response after the DWH discharge. This review centers on a case study of the fate of oil contamination in Pensacola Beach sands, which sheds light on the mechanisms of microbially mediated hydrocarbon degradation and the impacts of oiling to ecosystem functions. Analysis of field and laboratory results is discussed along with the technological advances that made these observations possible. Metagenomics enabled the application of ecological theory, thereby building a stronger foundation for the effective prediction of baseline microbial community structure/function and response to oiling. Oil perturbation was shown to resemble a press ecosystem disturbance according to the disturbance-specialization hypothesis. Benthic microbial communities were shown to be resilient, maintained ecosystem functions, and recovered quickly after oil disturbance.

The Deepwater Horizon (DWH) discharge is unique in that it represents the first large spill that occurred in the deep sea, and unparalleled volumes of chemical dispersant were applied during emergency response efforts. Thus, the DWH incident raised new challenges with regard to predictions of petroleum hydrocarbon (PHC) biodegradation and the fate of discharged hydrocarbons in the deep sea, which is permanently cold (~4 °C) and exposed to high hydrostatic pressure (1 MPa per 100 m). Although extensive information is available on the rates and controls of PHC biodegradation in marine environments, relatively few studies have been conducted under conditions resembling the deep sea. In particular, hydrostatic pressure is a key environmental parameter that has been largely overlooked in biodegradation studies, due to methodological challenges and the difficulty to obtain samples. Considering the rapid expansion of oil and gas drilling into deeper waters, there is an urgent need to improve understanding of the influence of low temperature and high pressure on biodegradation in order to better constrain the fate of hydrocarbons in the deep sea. This chapter addresses the current understanding of deep sea PHC biodegradation, highlighting discoveries made during the scientific response to the DWH disaster.

To advance understanding of the fate of hydrocarbons released from the Deepwater Horizon oil spill and deposited in marine sediments, this study characterized the microbial populations capable of anaerobic hydrocarbon degradation coupled with sulfate reduction in non-seep sediments of the northern Gulf of Mexico. Anaerobic, sediment-free enrichment cultures were obtained with either hexadecane or phenanthrene as sole carbon source and sulfate as a terminal electron acceptor. Phylogenetic analysis revealed that enriched microbial populations differed by hydrocarbon substrate, with abundant SSU rRNA gene amplicon sequences from hexadecane cultures showing high sequence identity (up to 98%) to Desulfatibacillum alkenivorans (family Desulfobacteraceae), while phenanthrene-enriched populations were most closely related to Desulfatiglans spp. (up to 95% sequence identity; family Desulfarculaceae). Assuming complete oxidation to CO2, observed stoichiometric ratios closely resembled the theoretical ratios of 12.25:1 for hexadecane and 8.25:1 for phenanthrene degradation coupled to sulfate reduction. Phenanthrene carboxylic acid was detected in the phenanthrene-degrading enrichment cultures, providing evidence to indicate carboxylation as an activation mechanism for phenanthrene degradation. Metagenome-assembled genomes (MAGs) revealed that phenanthrene degradation is likely mediated by novel genera or families of sulfate-reducing bacteria along with their fermentative syntrophic partners, and candidate genes linked to the degradation of aromatic hydrocarbons were detected for future study.

Modeling crude-oil biodegradation in sediments remains a challenge due in part to the lack of appropriate model organisms. Here we report the metagenome-guided isolation of a novel organism that represents a phylogenetically narrow (>97% 16S rRNA gene identity) group of previously uncharacterized, crude-oil degraders. Analysis of available sequence data showed that these organisms are highly abundant in oiled sediments of coastal marine ecosystems across the world, often comprising ~30% of the total community, and virtually absent in pristine sediments or seawater. The isolate genome encodes functional nitrogen fixation and hydrocarbon degradation genes together with putative genes for biosurfactant production that apparently facilitate growth in the typically nitrogen-limited, oiled environment. Comparisons to available genomes revealed that this isolate represents a novel genus within the Gammaproteobacteria, for which we propose the provisional name “Candidatus Macondimonas diazotrophica” gen. nov., sp. nov. “Ca. M. diazotrophica” appears to play a key ecological role in the response to oil spills around the globe and could be a promising model organism for studying ecophysiological responses to oil spills., This research was made possible by a grant from The Gulf of Mexico Research Initiative (Grant No 321611-00; RFP V). Data are publicly available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC) at https://data.gulfresearchinitiative.org (UDI: R5.x278.000:0014, UDI: R5.x278.000:0002). RR-M acknowledges the financial support for his sabbatical stay at Georgia Tech from the Spanish Ministry of Sciences, Innovation and Universities (Grant No. PRX18/00048).

The microbial ecology of oligotrophic deep ocean sediments is understudied relative to their shallow counterparts, and this lack of understanding hampers our ability to predict responses to current and future perturbations. The Gulf of Mexico has experienced two of the largest accidental marine oil spills, i.e., the 1979 Ixtoc-1 blowout in the Southern Gulf of Mexico (GoM) and the 2010 Deepwater Horizon (DWH) discharge in the Northern GoM. Microbial communities were characterized via next generation sequencing of SSU rRNA gene sequences for 29 sites across multiple years in the Gulf of Mexico, represented by >700 samples. The distribution of seafloor microbial communities was elucidated and found to be surprisingly consistent across the entire region in terms of the OTUs detected and their relative abundances. The composition of the seafloor microbiome was well approximated by the overlying water depth and depth within the sediment column, which together explained 38% of the observed variation. In contrast, geographic distance had a limited role and explained only 6%. Biogeographical distributions were used to generate a depth-stratified machine-learning based predictive model for over 4000 dominant OTUs that relies on easy-to-obtain geospatial variables. Microbial community structure is linked to oxygen penetration depth and sediment geochemical regime, which are likely controlled through carbon delivery. Our results further demonstrate that sediments impacted by the DWH spill had returned to near baseline conditions after two years. The distributions of key microbial populations can now be calculated and constrained across the region while deviations from these predictions may be evaluated to pinpoint impacted sites, and more importantly, in future response efforts or long-term stability studies.

Although petroleum hydrocarbons discharged from the Deepwater Horizon (DWH) blowout were shown to have a pronounced impact on indigenous microbial communities in the Gulf of Mexico, effects on nearshore or coastal ecosystems remain understudied. This study investigated the successional patterns of functional and taxonomic diversity for over 1 year after the DWH oil was deposited on Pensacola Beach sands (FL, USA), using metagenomic and 16S rRNA gene amplicon techniques. Gamma- and Alphaproteobacteria were enriched in oiled sediments, in corroboration of previous studies. In contrast to previous studies, we observed an increase in the functional diversity of the community in response to oil contamination and a functional transition from generalist populations within 4 months after oil came ashore to specialists a year later, when oil was undetectable. At the latter time point, a typical beach community had reestablished that showed little to no evidence of oil hydrocarbon degradation potential, was enriched in archaeal taxa known to be sensitive to xenobiotics, but differed significantly from the community before the oil spill. Further, a clear succession pattern was observed, where early responders to oil contamination, likely degrading aliphatic hydrocarbons, were replaced after 3 months by populations capable of aromatic hydrocarbon decomposition. Collectively, our results advance the understanding of how natural benthic microbial communities respond to crude oil perturbation, supporting the specialization-disturbance hypothesis; that is, the expectation that disturbance favors generalists, while providing (microbial) indicator species and genes for the chemical evolution of oil hydrocarbons during degradation and weathering.

Summary The temperature dependency of denitrification and anaerobic ammonium oxidation (anammox) rates from Arctic fjord sediments was investigated in a temperature gradient block incubator for temperatures ranging from −1 to 40°C. Community structure in intact sediments and slurry incubations was determined using Illumina SSU rRNA gene sequencing. The optimal temperature (Topt) for denitrification was 25–27°C, whereas anammox rates were optimal at 12–17°C. Both denitrification and anammox exhibited temperature responses consistent with a psychrophilic community, but anammox bacteria may be more specialized for psychrophilic activity. Long-term (1–2 months) warming experiments indicated that temperature increases of 5–10°C above in situ had little effect on the microbial community structure or the temperature response of denitrification and anammox. Increases of 25°C shifted denitrification temperature responses to mesophilic with concurrent community shifts, and anammox activity was eliminated above 25°C. Additions of low molecular weight organic substrates (acetate and lactate) caused increases in denitrification rates, corroborating the hypothesis that the supply of organic substrates is a more dominant control of respiration rates than low temperature. These results suggest that climate-related changes in sinking particulate flux will likely alter rates of N removal more rapidly than warming.

After Deepwater Horizon oil reached the Florida coast, oil was buried in Pensacola Beach (PB) sands to ~70cm depth, resulting in Total Petroleum Hydrocarbon (TPH) concentrations up to ~2kg per meter of beach. This study followed the decomposition of the buried oil and the factors influencing its degradation. The abundance of bacteria in oiled sand increased by 2 orders of magnitude within one week after oil burial, while diversity decreased by ~50%. Half-lives of aliphatic and aromatic hydrocarbons reached 25 and 22days, respectively. Aerobic microbial oil decomposition, promoted by tidal pumping, and human cleaning activities effectively removed oil from the beach. After one year, concentrations of GC-amenable hydrocarbons at PB were similar to those in the uncontaminated reference beach at St. George Island/FL, and microbial populations that disappeared after the oil contamination had reestablished. Yet, oxihydrocarbons can be found at PB to the present day.

Microorganisms are very sensitive to environmental change and can be used to gauge anthropogenic impacts and even predict restoration success of degraded environments. Here, we report assessment of bauxite mining activities on soil biogeochemistry and microbial community structure using un-mined and three post-mined sites in Jamaica. The post-mined soils represent a chronosequence, undergoing restoration since 1987, 1997, and 2007. Soils were collected during dry and wet seasons and analyzed for pH, organic matter (OM), total carbon (TC), nitrogen (TN), and phosphorus. The microbial community structure was assessed through quantitative PCR and massively parallel bacterial ribosomal RNA (rRNA) gene sequencing. Edaphic factors and microbial community composition were analyzed using multivariate statistical approaches and revealed a significant, negative impact of mining on soil that persisted even after greater than 20 years of restoration. Seasonal fluctuations contributed to variation in measured soil properties and community composition, but they were minor in comparison to long-term effects of mining. In both seasons, post-mined soils were higher in pH but OM, TC, and TN decreased. Bacterial rRNA gene analyses demonstrated a general decrease in diversity in post-mined soils and up to a 3-log decrease in rRNA gene abundance. Community composition analyses demonstrated that bacteria from the Proteobacteria (α, β, γ, δ), Acidobacteria, and Firmicutes were abundant in all soils. The abundance of Firmicutes was elevated in newer post-mined soils relative to the un-mined soil, and this contrasted a decrease, relative to un-mined soils, in proteobacterial and acidobacterial rRNA gene abundances. Our study indicates long-lasting impacts of mining activities to soil biogeochemical and microbial properties with impending loss in soil productivity.

A significant portion of oil from the recent Deepwater Horizon (DH) oil spill in the Gulf of Mexico was transported to the shoreline, where it may have severe ecological and economic consequences. The objectives of this study were (i) to identify and characterize predominant oil-degrading taxa that may be used as model hydrocarbon degraders or as microbial indicators of contamination and (ii) to characterize the in situ response of indigenous bacterial communities to oil contamination in beach ecosystems. This study was conducted at municipal Pensacola Beach, FL, where chemical analysis revealed weathered oil petroleum hydrocarbon (C 8 to C 40 ) concentrations ranging from 3.1 to 4,500 mg kg −1 in beach sands. A total of 24 bacterial strains from 14 genera were isolated from oiled beach sands and confirmed as oil-degrading microorganisms. Isolated bacterial strains were primarily Gammaproteobacteria , including representatives of genera with known oil degraders ( Alcanivora x, Marinobacter , Pseudomonas , and Acinetobacter ). Sequence libraries generated from oiled sands revealed phylotypes that showed high sequence identity (up to 99%) to rRNA gene sequences from the oil-degrading bacterial isolates. The abundance of bacterial SSU rRNA gene sequences was ∼10-fold higher in oiled (0.44 × 10 7 to 10.2 × 10 7 copies g −1 ) versus clean (0.024 × 10 7 to 1.4 × 10 7 copies g −1 ) sand. Community analysis revealed a distinct response to oil contamination, and SSU rRNA gene abundance derived from the genus Alcanivorax showed the largest increase in relative abundance in contaminated samples. We conclude that oil contamination from the DH spill had a profound impact on the abundance and community composition of indigenous bacteria in Gulf beach sands, and our evidence points to members of the Gammaproteobacteria ( Alcanivorax , Marinobacter ) and Alphaproteobacteria ( Rhodobacteraceae ) as key players in oil degradation there.

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

The fields of microbial ecology and environmental microbiology have been revolutionized by the development of next-generation sequencing technologies. In this chapter we specifically address the use of PCR amplification coupled with high-throughput sequencing for the analysis of microbial community composition and structure, and for subsequent visualization and statistical analyses of this community data.

The Deepwater Horizon blowout in April 2010 represented the largest accidental marine oil spill and the largest release of chemical dispersants into the environment to date. While dispersant application may provide numerous benefits to oil spill response efforts, the impacts of dispersants and potential synergistic effects with crude oil on individual hydrocarbon-degrading bacteria are poorly understood. In this study, two environmentally relevant species of hydrocarbon-degrading bacteria were utilized to quantify the response to Macondo crude oil and Corexit 9500A-dispersed oil in terms of bacterial growth and oil degradation potential. In addition, specific hydrocarbon compounds were quantified in the dissolved phase of the medium and linked to ecotoxicity using a U.S. Environmental Protection Agency (EPA)-approved rotifer assay. Bacterial treatment significantly and drastically reduced the toxicity associated with dispersed oil (increasing the 50% lethal concentration [LC 50 ] by 215%). The growth and crude oil degradation potential of Acinetobacter were inhibited by Corexit by 34% and 40%, respectively; conversely, Corexit significantly enhanced the growth of Alcanivorax by 10% relative to that in undispersed oil. Furthermore, both bacterial strains were shown to grow with Corexit as the sole carbon and energy source. Hydrocarbon-degrading bacterial species demonstrate a unique response to dispersed oil compared to their response to crude oil, with potentially opposing effects on toxicity. While some species have the potential to enhance the toxicity of crude oil by producing biosurfactants, the same bacteria may reduce the toxicity associated with dispersed oil through degradation or sequestration.

Bacteria associated with sap-feeding insect herbivores include not only symbionts that may increase their hosts' fitness but also harmful plant pathogens. Calophya spp. gall-inducing psyllids (Hemiptera: Calophyidae) are being investigated for their potential as biological control agents of the noxious weed, Brazilian peppertree (Schinus terebinthifolia), in Florida. Although there are no examples of plant pathogen transmission by members of the family Calophyidae, several insects in the superfamily Psylloidea are known to transmit pathogenic bacteria in the genera Candidatus Liberibacter and Candidatus Phytoplasma. To determine whether Calophya spp. harbor potentially harmful plant pathogenic bacteria, we sequenced small subunit (SSU) ribosomal RNA (rRNA) gene amplicons generated from individuals from four Calophya spp. populations: All microbial SSU gene sequences fell into the bacterial domain, with 98-99% belonging to the Proteobacteria. The Calophya microbiomes contained a relatively simple community, with 49-79 operational taxonomic units (OTUs; 97%) detected, and only 5-8 OTUs with greater than 1% abundance. Candidatus Carsonella showed the highest relative abundance, with OTUs from this candidate genus representing between 51-65% of all recovered sequences. The next most abundant clade observed was an unclassified Enterobacteriacae group closely related to bacteria from the genera Buchnera and Blochmannia that ranged from 20-31% in relative abundance. Wolbachia populations were the third most abundant group and represented 7-27% of the diversity in microbial OTUs. No SSU rRNA gene sequences from putative pathogenic bacteria from the genera Ca. Liberibacter or Ca. Phytoplasma were detected in the microbiomes of the four Calophya populations. The probability that infected psyllids were present in our colonies, but were not sampled, was extremley low (1.39 x 10(-10)). As far as we are aware, our study is the first to characterize the microbiome of a candidate biological control agent, and coupled with previous work demonstrating a high degree of host specificity and absence of plant viruses, suggests that releasing Calophya spp. in United States poses minimal risk to non-target plants.

The objective of this study was to characterize fungal communities in a subsurface environment cocontaminated with uranium and nitrate at the watershed scale and to determine the potential contribution of fungi to contaminant transformation (nitrate attenuation). The abundance, distribution, and diversity of fungi in subsurface groundwater samples were determined using quantitative and semiquantitative molecular techniques, including quantitative PCR of eukaryotic small-subunit rRNA genes and pyrosequencing of fungal internal transcribed spacer (ITS) regions. Potential bacterial and fungal denitrification was assessed in sediment-groundwater slurries amended with antimicrobial compounds and in fungal pure cultures isolated from the subsurface. Our results demonstrate that subsurface fungal communities are dominated by members of the phylum Ascomycota , and a pronounced shift in fungal community composition occurs across the groundwater pH gradient at the field site, with lower diversity observed under acidic (pH Coniochaeta , which were detected in abundance in pyrosequence libraries of site groundwater samples, were shown to reduce nitrate to nitrous oxide. Denitrifying fungal isolates recovered from the site were classified and found to be distributed broadly within the phylum Ascomycota and within a single genus of the Basidiomycota . Potential denitrification rate assays with sediment-groundwater slurries showed the potential for subsurface fungi to reduce nitrate to nitrous oxide under in situ acidic pH conditions.

Volume 82, no. 2, p. 518–527, 2016. Page 525: The second paragraph of the Funding Information should read, “This work was made possible in part by a grant from BP/The Gulf of Mexico Research Initiative to the C-IMAGE, C-IMAGE-2, and Deep-C consortia and in part by a National Science Foundation

The effect of long-term mixed-waste contamination, particularly uranium and nitrate, on the microbial community in the terrestrial subsurface was investigated at the field scale at the Oak Ridge Integrated Field Research Challenge (ORIFRC) site in Oak Ridge, TN. The abundance, community composition, and distribution of groundwater microorganisms were examined across the site during two seasonal sampling events. At representative locations, subsurface sediment was also examined from two boreholes, one sampled from the most heavily contaminated area of the site and another from an area with low contamination. A suite of DNA- and RNA-based molecular tools were employed for community characterization, including quantitative PCR of rRNA and nitrite reductase genes, community composition fingerprinting analysis, and high-throughput pyrotag sequencing of rRNA genes. The results demonstrate that pH is a major driver of the subsurface microbial community structure and that denitrifying bacteria from the genus Rhodanobacter (class Gammaproteobacteria ) dominate at low pH. The relative abundance of bacteria from this genus was positively correlated with lower-pH conditions, and these bacteria were abundant and active in the most highly contaminated areas. Other factors, such as the concentration of nitrogen species, oxygen level, and sampling season, did not appear to strongly influence the distribution of Rhodanobacter bacteria. The results indicate that these organisms are acid-tolerant denitrifiers, well suited to the acidic, nitrate-rich subsurface conditions, and pH is confirmed as a dominant driver of bacterial community structure in this contaminated subsurface environment.

Bacterial strains 2APBS1T and 116-2 were isolated from the subsurface of a nuclear legacy waste site where the sediments are co-contaminated with large amounts of acids, nitrate, metal radionuclides and other heavy metals. A combination of physiological and genetic assays indicated that these strains represent the first member of the genus Rhodanobacter shown to be capable of complete denitrification. Cells of strain 2APBS1T and 116-2 were Gram-negative, non-spore-forming rods, 3–5 µm long and 0.25–0.5 µm in diameter. The isolates were facultative anaerobes, and had temperature and pH optima for growth of 30 °C and pH 6.5; they were able to tolerate up to 2.0 % NaCl, although growth improved in its absence. Strains 2APBS1T and 116-2 contained fatty acid and quinone (ubiquinone-8; 100 %) profiles that are characteristic features of the genus Rhodanobacter . Although strains 2APBS1T and 116-2 shared high 16S rRNA gene sequence similarity with Rhodanobacter thiooxydans LCS2T (>99 %), levels of DNA–DNA relatedness between these strains were substantially below the 70 % threshold used to designate novel species. Thus, based on genotypic, phylogenetic, chemotaxonomic and physiological differences, strains 2APBS1T and 116-2 are considered to represent a single novel species of the genus Rhodanobacter , for which the name Rhodanobacter denitrificans sp. nov. is proposed. The type strain is 2APBS1T ( = DSM 23569T = JCM 17641T).

The terrestrial subsurface contains nearly all of Earth's freshwater reserves and harbours the majority of our planet's total prokaryotic biomass. Although genetic surveys suggest these organisms rely on in situ carbon fixation, rather than the photosynthetically derived organic carbon transported from surface environments, direct measurements of carbon fixation in the subsurface are absent. Using an ultra-low level C-14-labelling technique, we estimate in situ carbon fixation rates in a carbonate aquifer. We find these rates are similar to those measured in oligotrophic marine surface waters and up to six-fold greater than those observed in the lower euphotic zone. Our empirical carbon fixation rates agree with nitrification rate data. Metagenomic analyses reveal abundant putative chemolithoautotrophic members of an uncharacterized order of Nitrospiria that may be behind the carbon fixation. On the basis of our determined carbon fixation rates, we conservatively extrapolate global primary production in carbonate groundwaters (10% of global reserves) to be 0.11 Pg carbon per year. These rates fall within the range found for oligotrophic marine surface waters, indicating a substantial contribution of in situ primary production to subsurface ecosystem processes. We further suggest that, just as phototrophs are for marine biogeochemical cycling, such subsurface carbon fixation is potentially foundational to subsurface trophic webs. Direct measurements of carbon fixation rates in groundwater suggest a substantial contribution of in situ primary production to subsurface ecosystem processes.