Genome repository of oil systems: An interactive and searchable database that expands the catalogued diversity of crude oil-associated microbes.

Microbial communities ultimately control the fate of petroleum hydrocarbons (PHCs) that enter the natural environment, but the interactions of microbes with PHCs and the environment are highly complex and poorly understood. Genome-resolved metagenomics can help unravel these complex interactions. However, the lack of a comprehensive database that integrates existing genomic/metagenomic data from oil environments with physicochemical parameters known to regulate the fate of PHCs currently limits data analysis and interpretations. Here, we curated a comprehensive, searchable database that documents microbial populations in natural oil ecosystems and oil spills, along with available underlying physicochemical data, geocoded via geographic information system to reveal their geographic distribution patterns. Analysis of the ~2000 metagenome-assembled genomes (MAGs) available in the database revealed strong ecological niche specialization within habitats. Over 95% of the recovered MAGs represented novel taxa underscoring the limited representation of cultured organisms from oil-contaminated and oil reservoir ecosystems. The majority of MAGs linked to oil-contaminated ecosystems were detectable in non-oiled samples from the Gulf of Mexico but not in comparable samples from elsewhere, indicating that the Gulf is primed for oil biodegradation. The repository should facilitate future work toward a predictive understanding of the microbial taxa and their activities that control the fate of oil spills.

Integrated Omics Elucidate the Mechanisms Driving the Rapid Biodegradation of Deepwater Horizon Oil in Intertidal Sediments Undergoing Oxic-Anoxic Cycles.

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.

Temperature response of denitrification and anaerobic ammonium oxidation rates and microbial community structure in Arctic fjord sediments.

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.

Aquatic Eddy Covariance: The Method and Its Contributions to Defining Oxygen and Carbon Fluxes in Marine Environments.

Aquatic eddy covariance (AEC) is increasingly being used to study benthic oxygen (O2) flux dynamics, organic carbon cycling, and ecosystem health in marine and freshwater environments. Because it is a noninvasive technique, has a high temporal resolution (∼15 min), and integrates over a large area of the seafloor (typically 10-100 m2), it has provided new insights on the functioning of aquatic ecosystems under naturally varying in situ conditions and has given us more accurate assessments of their metabolism. In this review, we summarize biogeochemical, ecological, and biological insightsgained from AEC studies of marine ecosystems. A general finding for all substrates is that benthic O2 exchange is far more dynamic than earlier recognized, and thus accurate mean values can only be obtained from measurements that integrate over all timescales that affect the local O2 exchange. Finally, we highlight new developments of the technique, including measurements of air-water gas exchange and long-term deployments.

Fleshy red algae mats act as temporary reservoirs for sessile invertebrate biodiversity.

Many coastal ecosystems, such as coral reefs and seagrass meadows, currently experience overgrowth by fleshy algae due to the interplay of local and global stressors. This is usually accompanied by strong decreases in habitat complexity and biodiversity. Recently, persistent, mat-forming fleshy red algae, previously described for the Black Sea and several Atlantic locations, have also been observed in the Mediterranean. These several centimetre high mats may displace seagrass meadows and invertebrate communities, potentially causing a substantial loss of associated biodiversity. We show that the sessile invertebrate biodiversity in these red algae mats is high and exceeds that of neighbouring seagrass meadows. Comparative biodiversity indices were similar to or higher than those recently described for calcifying green algae habitats and biodiversity hotspots like coral reefs or mangrove forests. Our findings suggest that fleshy red algae mats can act as alternative habitats and temporary sessile invertebrate biodiversity reservoirs in times of environmental change.

Hydrocarbon-degrading bacteria and the bacterial community response in gulf of Mexico beach sands impacted by the deepwater horizon oil spill.

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 (C8 to C40) 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 (Alcanivorax, 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 × 107 to 10.2 × 107 copies g⁻¹) versus clean (0.024 × 107 to 1.4 × 107 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.

A novel, divergent alkane monooxygenase (alkB) clade involved in crude oil biodegradation.

Alkanes are ubiquitous in marine ecosystems and originate from diverse sources ranging from natural oil seeps to anthropogenic inputs and biogenic production by cyanobacteria. Enzymes that degrade cyanobacterial alkanes (typically C15-C17 compounds) such as the alkane monooxygenase (AlkB) are widespread, but it remains unclear whether or not AlkB variants exist that specialize in degradation of crude oil from natural or accidental spills, a much more complex mixture of long-chain hydrocarbons. In the present study, large-scale analysis of available metagenomic and genomic data from the Gulf of Mexico (GoM) oil spill revealed a novel, divergent AlkB clade recovered from genomes with no cultured representatives that was dramatically increased in abundance in crude-oil impacted ecosystems. In contrast, the AlkB clades associated with biotransformation of cyanobacterial alkanes belonged to 'canonical' or hydrocarbonoclastic clades, and based on metatranscriptomics data and compared to the novel clade, were much more weakly expressed during crude oil biodegradation in laboratory mesocosms. The absence of this divergent AlkB clade in metagenomes of uncontaminated samples from the global ocean survey but not from the GoM as well as its frequent horizontal gene transfer indicated a priming effect of the Gulf for crude oil biodegradation likely driven by natural oil seeps.

Beach sand oil spills select for generalist microbial populations.

The specialization-disturbance hypothesis predicts that, in the event of a disturbance, generalists are favored, while specialists are selected against. This hypothesis has not been rigorously tested in microbial systems and it remains unclear to what extent it could explain microbial community succession patterns following perturbations. Previous field observations of Pensacola Beach sands that were impacted by the Deepwater Horizon (DWH) oil spill provided evidence in support of the specialization-disturbance hypothesis. However, ecological drift as well as uncounted environmental fluctuations (e.g., storms) could not be ruled out as confounding factors driving these field results. In this study, the specialization-disturbance hypothesis was tested on beach sands, disturbed by DWH crude oil, ex situ in closed laboratory advective-flow chambers that mimic in situ conditions in saturated beach sediments. The chambers were inoculated with weathered DWH oil and unamended chambers served as controls. The time series of shotgun metagenomic and 16S rRNA gene amplicon sequence data from a two-month long incubation showed that functional diversity significantly increased while taxonomic diversity significantly declined, indicating a decrease in specialist taxa. Thus, results from this laboratory study corroborate field observations, providing verification that the specialization-disturbance hypothesis can explain microbial succession patterns in crude oil impacted beach sands.

Coral mucus functions as an energy carrier and particle trap in the reef ecosystem.

Zooxanthellae, endosymbiotic algae of reef-building corals, substantially contribute to the high gross primary production of coral reefs, but corals exude up to half of the carbon assimilated by their zooxanthellae as mucus. Here we show that released coral mucus efficiently traps organic matter from the water column and rapidly carries energy and nutrients to the reef lagoon sediment, which acts as a biocatalytic mineralizing filter. In the Great Barrier Reef, the dominant genus of hard corals, Acropora, exudes up to 4.8 litres of mucus per square metre of reef area per day. Between 56% and 80% of this mucus dissolves in the reef water, which is filtered through the lagoon sands. Here, coral mucus is degraded at a turnover rate of at least 7% per hour. Detached undissolved mucus traps suspended particles, increasing its initial organic carbon and nitrogen content by three orders of magnitude within 2 h. Tidal currents concentrate these mucus aggregates into the lagoon, where they rapidly settle. Coral mucus provides light energy harvested by the zooxanthellae and trapped particles to the heterotrophic reef community, thereby establishing a recycling loop that supports benthic life, while reducing loss of energy and nutrients from the reef ecosystem.

Succession of microbial populations and nitrogen-fixation associated with the biodegradation of sediment-oil-agglomerates buried in a Florida sandy beach.

The Deepwater Horizon (DWH) oil spill contaminated coastlines from Louisiana to Florida, burying oil up to 70 cm depth in sandy beaches, posing a potential threat to environmental and human health. The dry and nutrient-poor beach sand presents a taxing environment for microbial growth, raising the question how the biodegradation of the buried oil would proceed. Here we report the results of an in-situ experiment that (i) characterized the dominant microbial communities contained in sediment oil agglomerates (SOAs) of DWH oil buried in a North Florida sandy beach, (ii) elucidated the long-term succession of the microbial populations that developed in the SOAs, and (iii) revealed the coupling of SOA degradation to nitrogen fixation. Orders of magnitude higher bacterial abundances in SOAs compared to surrounding sands distinguished SOAs as hotspots of microbial growth. Blooms of bacterial taxa with a demonstrated potential for hydrocarbon degradation (Gammaproteobacteria, Alphaproteobacteria, Actinobacteria) developed in the SOAs, initiating a succession of microbial populations that mirrored the evolution of the petroleum hydrocarbons. Growth of nitrogen-fixing prokaryotes or diazotrophs (Rhizobiales and Frankiales), reflected in increased abundances of nitrogenase genes (nifH), catalyzed biodegradation of the nitrogen-poor petroleum hydrocarbons, emphasizing nitrogen fixation as a central mechanism facilitating the recovery of sandy beaches after oil contamination.

Decomposition of sediment-oil-agglomerates in a Gulf of Mexico sandy beach.

Sediment-oil-agglomerates (SOA) are one of the most common forms of contamination impacting shores after a major oil spill; and following the Deepwater Horizon (DWH) accident, large numbers of SOAs were buried in the sandy beaches of the northeastern Gulf of Mexico. SOAs provide a source of toxic oil compounds, and although SOAs can persist for many years, their long-term fate was unknown. Here we report the results of a 3-year in-situ experiment that quantified the degradation of standardized SOAs buried in the upper 50 cm of a North Florida sandy beach. Time series of hydrocarbon mass, carbon content, n-alkanes, PAHs, and fluorescence indicate that the decomposition of golf-ball-size DWH-SOAs embedded in beach sand takes at least 32 years, while SOA degradation without sediment contact would require more than 100 years. SOA alkane and PAH decay rates within the sediment were similar to those at the beach surface. The porous structure of the SOAs kept their cores oxygen-replete. The results reveal that SOAs buried deep in beach sands can be decomposed through relatively rapid aerobic microbial oil degradation in the tidally ventilated permeable beach sand, emphasizing the role of the sandy beach as an aerobic biocatalytical reactor at the land-ocean interface.

"Candidatus Macondimonas diazotrophica", a novel gammaproteobacterial genus dominating crude-oil-contaminated coastal sediments.

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.

Characterization of nitrifying, denitrifying, and overall bacterial communities in permeable marine sediments of the northeastern Gulf of Mexico.

Sandy or permeable sediment deposits cover the majority of the shallow ocean seafloor, and yet the associated bacterial communities remain poorly described. The objective of this study was to expand the characterization of bacterial community diversity in permeable sediment impacted by advective pore water exchange and to assess effects of spatial, temporal, hydrodynamic, and geochemical gradients. Terminal restriction fragment length polymorphism (TRFLP) was used to analyze nearly 100 sediment samples collected from two northeastern Gulf of Mexico subtidal sites that primarily differed in their hydrodynamic conditions. Communities were described across multiple taxonomic levels using universal bacterial small subunit (SSU) rRNA targets (RNA- and DNA-based) and functional markers for nitrification (amoA) and denitrification (nosZ). Clonal analysis of SSU rRNA targets identified several taxa not previously detected in sandy sediments (i.e., Acidobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, and Firmicutes). Sequence diversity was high among the overall bacterial and denitrifying communities, with members of the Alphaproteobacteria predominant in both. Diversity of bacterial nitrifiers (amoA) remained comparatively low and did not covary with the other gene targets. TRFLP fingerprinting revealed changes in sequence diversity from the family to species level across sediment depth and study site. The high diversity of facultative denitrifiers was consistent with the high permeability, deeper oxygen penetration, and high rates of aerobic respiration determined in these sediments. The high relative abundance of Gammaproteobacteria in RNA clone libraries suggests that this group may be poised to respond to short-term periodic pulses of growth substrates, and this observation warrants further investigation.

Degradation of Deepwater Horizon oil buried in a Florida beach influenced by tidal pumping.

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.

Microbial community successional patterns in beach sands impacted by the Deepwater Horizon oil spill.

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.

Benthic exchange and biogeochemical cycling in permeable sediments.

The sandy sediments that blanket the inner shelf are situated in a zone where nutrient input from land and strong mixing produce maximum primary production and tight coupling between water column and sedimentary processes. The high permeability of the shelf sands renders them susceptible to pressure gradients generated by hydrodynamic and biological forces that modulate spatial and temporal patterns of water circulation through these sediments. The resulting dynamic three-dimensional patterns of particle and solute distribution generate a broad spectrum of biogeochemical reaction zones that facilitate effective decomposition of the pelagic and benthic primary production products. The intricate coupling between the water column and sediment makes it challenging to quantify the production and decomposition processes and the resultant fluxes in permeable shelf sands. Recent technical developments have led to insights into the high biogeochemical and biological activity of these permeable sediments and their role in the global cycles of matter.

Dispersants as used in response to the MC252-spill lead to higher mobility of polycyclic aromatic hydrocarbons in oil-contaminated Gulf of Mexico sand.

After the explosion of the Deepwater Horizon oil rig, large volumes of crude oil were washed onto and embedded in the sandy beaches and sublittoral sands of the Northern Gulf of Mexico. Some of this oil was mechanically or chemically dispersed before reaching the shore. With a set of laboratory-column experiments we show that the addition of chemical dispersants (Corexit 9500A) increases the mobility of polycyclic aromatic hydrocarbons (PAHs) in saturated permeable sediments by up to two orders of magnitude. Distribution and concentrations of PAHs, measured in the solid phase and effluent water of the columns using GC/MS, revealed that the mobility of the PAHs depended on their hydrophobicity and was species specific also in the presence of dispersant. Deepest penetration was observed for acenaphthylene and phenanthrene. Flushing of the columns with seawater after percolation of the oiled water resulted in enhanced movement by remobilization of retained PAHs. An in-situ benthic chamber experiment demonstrated that aromatic hydrocarbons are transported into permeable sublittoral sediment, emphasizing the relevance of our laboratory column experiments in natural settings. We conclude that the addition of dispersants permits crude oil components to penetrate faster and deeper into permeable saturated sands, where anaerobic conditions may slow degradation of these compounds, thus extending the persistence of potentially harmful PAHs in the marine environment. Application of dispersants in nearshore oil spills should take into account enhanced penetration depths into saturated sands as this may entail potential threats to the groundwater.

Sulfide assimilation by ectosymbionts of the sessile ciliate, Zoothamnium niveum.

We investigated the constraints on sulfide uptake by bacterial ectosymbionts on the marine peritrich ciliate Zoothamnium niveum by a combination of experimental and numerical methods. Protists with symbionts were collected on large blocks of mangrove-peat. The blocks were placed in a flow cell with flow adjusted to in situ velocity. The water motion around the colonies was then characterized by particle tracking velocimetry. This shows that the feather-shaped colony of Z. niveum generates a unidirectional flow of seawater through the colony with no recirculation. The source of the feeding current was the free-flowing water although the size of the colonies suggests that they live partly submerged in the diffusive boundary layer. We showed that the filtered volume allows Z. niveum to assimilate sufficient sulfide to sustain the symbiosis at a few micromoles per liter in ambient concentration. Numerical modeling shows that sulfide oxidizing bacteria on the surfaces of Z. niveum can sustain 100-times higher sulfide uptake than bacteria on flat surfaces, such as microbial mats. The study demonstrates that the filter feeding zooids of Z. niveum are preadapted to be prime habitats for sulfide oxidizing bacteria due to Z. niveum's habitat preference and due to the feeding current. Z. niveum is capable of exploiting low concentrations of sulfide in near norm-oxic seawater. This links its otherwise dissimilar habitats and makes it functionally similar to invertebrates with thiotrophic symbionts in filtering organs.

Activity and distribution of bacterial populations in Middle Atlantic Bight shelf sands.

Abstract Spatiotemporal variation and metabolic activity of the microbial community were studied in coarse-grained Middle Atlantic Bight shelf sediments in relation to pools of dissolved and particulate carbon. Algal cells were present 8->70 mum) fraction of the sediment held the major share (61-98%) of benthic bacteria. Bacterial and algal cell abundances, exoenzymatic activity, and [DOC] generally showed higher values in May/July 2001 than in August/December 2000. Carbohydrates and proteins were hydrolyzed at potential rates of 1-12 nmol cm(-3) h(-1) (beta-glucosidase) and 3-70 nmol cm(-3) h(-1) (aminopeptidase), respectively. Fluorescence in situ hybridization analyses of the benthic microbes assigned 45-56% of DAPI-stained cells to Eubacteria and less than 2% to Eukarya. The prokaryotic community was dominated by planctomycetes and members of the Cytophaga/Flavobacterium cluster. Near the sediment surface, iodonitrotetrazolium violet reducing cells, that are considered actively respiring, amounted to 15-29% of total bacteria. Despite a low organic content (particulate organic carbon <0.03%) and relatively low bacterial abundances (<10(9) cm(-3)), the Middle Atlantic Bight shelf sediments showed organic matter turnover rates that are comparable to those found in organic-rich finer-grained deposits. Our findings suggest a high biocatalytic filtration activity in these coarse permeable sediments.