Potential for natural attenuation of crude oil hydrocarbons in benthic microbiomes near coastal communities in Kivalliq, Nunavut, Canada.

Oil spilled in marine environments can settle to the seafloor through aggregation and sedimentation processes. This has been predicted to be especially relevant in the Arctic due to plankton blooms initiated by melting sea ice. These conditions exist in the Kivalliq region in Nunavut, Canada, where elevated shipping traffic has increased the risk of accidental spills. Experimental microcosms combining surface sediment and crude oil were incubated at 4 °C over 21 weeks to evaluate the biodegradation potential of seabed microbiomes. Sediments sampled near the communities of Arviat and Chesterfield Inlet were assessed for biodegradation capabilities by combining hydrocarbon geochemistry with 16S rRNA gene and metagenomic sequencing, revealing decreased microbial diversity but enrichment of oil-degrading taxa. Alkane and aromatic hydrocarbon losses corresponded to detection of genes and genomes that encode enzymes for aerobic biodegradation of these compounds, pointing to the utility of marine microbiome surveys for predicting the fate of oil released into Arctic marine environments.

Hyperthermophilic endospores germinate and metabolize organic carbon in sediments heated to 80°C.

Cold surface sediments host a seedbank of functionally diverse thermophilic bacteria. These thermophiles are present as endospores, which are widely dispersed in aquatic environments. Here, we investigated the functional potential of endospore populations in cold surface sediments heated to 80°C. Microbial production of acetate was observed at 80°C and could be enhanced by supplying additional organic carbon substrates. Comparison of 16S rRNA gene amplicon libraries from 80°C enrichments to sediments heated to lower temperatures (50-70°C) showed that temperature selects for distinct populations of endospore-forming bacteria. Whereas sulfate-reducing thermophiles were enriched in 50-70°C incubations, 80°C exceeds their thermal tolerance and selects for hyperthermophilic organotrophic bacteria that are similarly detected in amplicon libraries from sediments heated to 90°C. Genome-resolved metagenomics revealed novel carbon cycling members of Symbiobacteriales, Thermosediminibacteraceae, Thermanaeromonas and Calditerricola with the genomic potential for the degradation of carbohydrates, sugars, amino acids and nucleotides. Endospores of thermophilic bacteria are deposited on seabed sediments worldwide where they remain dormant as they are buried in the accumulating sediments. Our results suggest that endospore populations could be activated by temperature increases encountered during burial and show the potential for organotrophic metabolic activity contributing to acetate generation in deep hot sediments.

Metabolic responses of thermophilic endospores to sudden heat-induced perturbation in marine sediment samples.

Microbially mediated processes in a given habitat tend to be catalyzed by abundant populations that are ecologically adapted to exploit specific environmental characteristics. Typically, metabolic activities of rare populations are limited but may be stimulated in response to acute environmental stressors. Community responses to sudden changes in temperature and pressure can include suppression and activation of different populations, but these dynamics remain poorly understood. The permanently cold ocean floor hosts countless low-abundance microbes including endospores of thermophilic bacteria. Incubating sediments at high temperature resuscitates viable spores, causing the proliferation of bacterial populations. This presents a tractable system for investigating changes in a microbiome's community structure in response to dramatic environmental perturbations. Incubating permanently cold Arctic fjord sediments at 50°C for 216 h with and without volatile fatty acid amendment provoked major changes in community structure. Germination of thermophilic spores from the sediment rare biosphere was tracked using mass spectrometry-based metabolomics, radiotracer-based sulfate reduction rate measurements, and high-throughput 16S rRNA gene sequencing. Comparing community similarity at different intervals of the incubations showed distinct temporal shifts in microbial populations, depending on organic substrate amendment. Metabolite patterns indicated that amino acids and other sediment-derived organics were decomposed by fermentative Clostridia within the first 12-48 h. This fueled early and late phases of exponential increases in sulfate reduction, highlighting the cross-feeding of volatile fatty acids as electron donors for different sulfate-reducing Desulfotomaculia populations. The succession of germinated endospores triggered by sudden exposure to high temperature and controlled by nutrient availability offers a model for understanding the ecological response of dormant microbial communities following major environmental perturbations.

Endospores associated with deep seabed geofluid features in the eastern Gulf of Mexico.

Recent studies have reported up to 1.9 × 1029 bacterial endospores in the upper kilometre of deep subseafloor marine sediments, however, little is understood about their origin and dispersal. In cold ocean environments, the presence of thermospores (endospores produced by thermophilic bacteria) suggests that distribution is governed by passive migration from warm anoxic sources possibly facilitated by geofluid flow, such as advective hydrocarbon seepage sourced from petroleum deposits deeper in the subsurface. This study assesses this hypothesis by measuring endospore abundance and distribution across 60 sites in Eastern Gulf of Mexico (EGM) sediments using a combination of the endospore biomarker 2,6-pyridine dicarboxylic acid or 'dipicolinic acid' (DPA), sequencing 16S rRNA genes of thermospores germinated in 50°C sediment incubations, petroleum geochemistry in the sediments and acoustic seabed data from sub-bottom profiling. High endospore abundance is associated with geologically active conduit features (mud volcanoes, pockmarks, escarpments and fault systems), consistent with subsurface fluid flow dispersing endospores from deep warm sources up into the cold ocean. Thermospores identified at conduit sites were most closely related to bacteria associated with the deep biosphere habitats including hydrocarbon systems. The high endospore abundance at geological seep features demonstrated here suggests that recalcitrant endospores and their chemical components (such as DPA) can be used in concert with geochemical and geophysical analyses to locate discharging seafloor features. This multiproxy approach can be used to better understand patterns of advective fluid flow in regions with complex geology like the EGM basin.

Geological processes mediate a microbial dispersal loop in the deep biosphere.

The deep biosphere is the largest microbial habitat on Earth and features abundant bacterial endospores. Whereas dormancy and survival at theoretical energy minima are hallmarks of microbial physiology in the subsurface, ecological processes such as dispersal and selection in the deep biosphere remain poorly understood. We investigated the biogeography of dispersing bacteria in the deep sea where upward hydrocarbon seepage was confirmed by acoustic imagery and geochemistry. Thermophilic endospores in the permanently cold seabed correlated with underlying seep conduits reveal geofluid-facilitated cell migration pathways originating in deep petroleum-bearing sediments. Endospore genomes highlight adaptations to life in anoxic petroleum systems and bear close resemblance to oil reservoir microbiomes globally. Upon transport out of the subsurface, viable thermophilic endospores reenter the geosphere by sediment burial, enabling germination and environmental selection at depth where new petroleum systems establish. This microbial dispersal loop circulates living biomass in and out of the deep biosphere.

Sensitive quantification of dipicolinic acid from bacterial endospores in soils and sediments.

Endospore-forming bacteria make up an important and numerically significant component of microbial communities in a range of settings including soils, industry, hospitals and marine sediments extending into the deep subsurface. Bacterial endospores are non-reproductive structures that protect DNA and improve cell survival during periods unfavourable for bacterial growth. An important determinant of endospores withstanding extreme environmental conditions is 2,6-pyridine dicarboxylic acid (i.e. dipicolinic acid, or DPA), which contributes heat resistance. This study presents an improved HPLC-fluorescence method for DPA quantification using a single 10-min run with pre-column Tb3+ chelation. Relative to existing DPA quantification methods, specific improvements pertain to sensitivity, detection limit and range, as well as the development of new free DPA and spore-specific DPA proxies. The method distinguishes DPA from intact and recently germinated spores, enabling responses to germinants in natural samples or experiments to be assessed in a new way. DPA-based endospore quantification depends on accurate spore-specific DPA contents, in particular, thermophilic spores are shown to have a higher DPA content, meaning that marine sediments with plentiful thermophilic spores may require spore number estimates to be revisited. This method has a wide range of potential applications for more accurately quantifying bacterial endospores in diverse environmental samples.

Thermogenic hydrocarbon biodegradation by diverse depth-stratified microbial populations at a Scotian Basin cold seep.

At marine cold seeps, gaseous and liquid hydrocarbons migrate from deep subsurface origins to the sediment-water interface. Cold seep sediments are known to host taxonomically diverse microorganisms, but little is known about their metabolic potential and depth distribution in relation to hydrocarbon and electron acceptor availability. Here we combined geophysical, geochemical, metagenomic and metabolomic measurements to profile microbial activities at a newly discovered cold seep in the deep sea. Metagenomic profiling revealed compositional and functional differentiation between near-surface sediments and deeper subsurface layers. In both sulfate-rich and sulfate-depleted depths, various archaeal and bacterial community members are actively oxidizing thermogenic hydrocarbons anaerobically. Depth distributions of hydrocarbon-oxidizing archaea revealed that they are not necessarily associated with sulfate reduction, which is especially surprising for anaerobic ethane and butane oxidizers. Overall, these findings link subseafloor microbiomes to various biochemical mechanisms for the anaerobic degradation of deeply-sourced thermogenic hydrocarbons.

Taxonomic and functional insights into rock varnish microbiome using shotgun metagenomics.

Rock varnish is a microbial habitat, characterised by thin (5-500 µm) and shiny coatings of iron (Fe) and manganese (Mn) oxides associated with clay minerals. This structure is well studied by geologists, and recently there have been reports about the taxonomical composition of its microbiome. In this study, we investigated the rock varnish microbiome using shotgun metagenomics together with analyses of elemental composition, lipid and small molecule biomarkers, and rock surface analyses to explore the biogeography of microbial communities and their functional features. We report taxa and encoded functions represented in metagenomes retrieved from varnish or non-varnish samples, additionally, eight nearly complete genomes have been reconstructed spanning four phyla (Acidobacteria, Actinobacteria, Chloroflexi and TM7). The functional and taxonomic analyses presented in this study provide new insights into the ecosystem dynamics and survival strategies of microbial communities inhabiting varnish and non-varnish rock surfaces.

Metabolic potential of uncultured bacteria and archaea associated with petroleum seepage in deep-sea sediments.

The lack of microbial genomes and isolates from the deep seabed means that very little is known about the ecology of this vast habitat. Here, we investigate energy and carbon acquisition strategies of microbial communities from three deep seabed petroleum seeps (3 km water depth) in the Eastern Gulf of Mexico. Shotgun metagenomic analysis reveals that each sediment harbors diverse communities of chemoheterotrophs and chemolithotrophs. We recovered 82 metagenome-assembled genomes affiliated with 21 different archaeal and bacterial phyla. Multiple genomes encode enzymes for anaerobic oxidation of aliphatic and aromatic compounds, including those of candidate phyla Aerophobetes, Aminicenantes, TA06 and Bathyarchaeota. Microbial interactions are predicted to be driven by acetate and molecular hydrogen. These findings are supported by sediment geochemistry, metabolomics, and thermodynamic modelling. Overall, we infer that deep-sea sediments experiencing thermogenic hydrocarbon inputs harbor phylogenetically and functionally diverse communities potentially sustained through anaerobic hydrocarbon, acetate and hydrogen metabolism.

Modes of carbon fixation in an arsenic and CO2-rich shallow hydrothermal ecosystem.

The seafloor sediments of Spathi Bay, Milos Island, Greece, are part of the largest arsenic-CO2-rich shallow submarine hydrothermal ecosystem on Earth. Here, white and brown deposits cap chemically distinct sediments with varying hydrothermal influence. All sediments contain abundant genes for autotrophic carbon fixation used in the Calvin-Benson-Bassham (CBB) and reverse tricaboxylic acid (rTCA) cycles. Both forms of RuBisCO, together with ATP citrate lyase genes in the rTCA cycle, increase with distance from the active hydrothermal centres and decrease with sediment depth. Clustering of RuBisCO Form II with a highly prevalent Zetaproteobacteria 16S rRNA gene density infers that iron-oxidizing bacteria contribute significantly to the sediment CBB cycle gene content. Three clusters form from different microbial guilds, each one encompassing one gene involved in CO2 fixation, aside from sulfate reduction. Our study suggests that the microbially mediated CBB cycle drives carbon fixation in the Spathi Bay sediments that are characterized by diffuse hydrothermal activity, high CO2, As emissions and chemically reduced fluids. This study highlights the breadth of conditions influencing the biogeochemistry in shallow CO2-rich hydrothermal systems and the importance of coupling highly specific process indicators to elucidate the complexity of carbon cycling in these ecosystems.

Pyrococcus kukulkanii sp. nov., a hyperthermophilic, piezophilic archaeon isolated from a deep-sea hydrothermal vent.

A novel hyperthermophilic, piezophilic, anaerobic archaeon, designated NCB100T, was isolated from a hydrothermal vent flange fragment collected in the Guaymas basin at the hydrothermal vent site named 'Rebecca's Roost' at a depth of 1997 m. Enrichment and isolation were performed at 100 °C under atmospheric pressure. Cells of strain NCB100T were highly motile, irregular cocci with a diameter of ~1 µm. Growth was recorded at temperatures between 70 and 112 °C (optimum 105 °C) and hydrostatic pressures of 0.1-80 MPa (optimum 40-50 MPa). Growth was observed at pH 3.5-8.5 (optimum pH 7) and with 1.5-7 % NaCl (optimum at 2.5-3 %). Strain NCB100T was a strictly anaerobic chemo-organoheterotroph and grew on complex proteinaceous substrates such as yeast extract, peptone and tryptone, as well as on glycogen and starch. Elemental sulfur was required for growth and was reduced to hydrogen sulfide. The fermentation products from complex proteinaceous substrates were CO2 and H2. The G+C content of the genomic DNA was 41.3 %. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain NCB100T belongs to the genus Pyrococcus, showing 99 % similarity with the other described species of the genus Pyrococcus. On the basis of physiological characteristics, DNA G+C content, similarity level between ribosomal proteins and an average nucleotide identity value of 79 %, strain NCB100T represents a novel species for which the name Pyrococcus kukulkanii sp. nov. is proposed. The type strain is NCB100T (=DSM 101590T=Souchothèque de Bretagne BG1337T).

Impact of temperature on ladderane lipid distribution in anammox bacteria.

Anaerobic ammonium-oxidizing (anammox) bacteria have the unique ability to synthesize fatty acids containing linearly concatenated cyclobutane rings, termed "ladderane lipids." In this study we investigated the effect of temperature on the ladderane lipid composition and distribution in anammox enrichment cultures, marine particulate organic matter, and surface sediments. Under controlled laboratory conditions we observed an increase in the amount of C(20) [5]-ladderane fatty acids compared with the amount of C(18) [5]-ladderane fatty acids with increasing temperature and also an increase in the amount of C(18) [5]-ladderane fatty acids compared with the amount of C(20) [5]-ladderane fatty acids with decreasing temperature. Combining these data with results from the natural environment showed a significant (R(2) = 0.85, P = <0.0001, n = 121) positive sigmoidal relationship between the amounts of C(18) and C(20) [5]-ladderane fatty acids and the in situ temperature; i.e., there is an increase in the relative abundance of C(18) [5]-ladderane fatty acids at lower temperatures and vice versa, particularly at temperatures between 12 degrees C and 20 degrees C. Novel shorter (C(16)) and longer (C(22) to C(24)) ladderane fatty acids were also identified, but their relative amounts were small and did not change with temperature. The adaptation of ladderane fatty acid chain length to temperature changes is similar to the regulation of common fatty acid composition in other bacteria and may be the result of maintaining constant membrane fluidity under different temperature regimens (homeoviscous adaptation). Our results can potentially be used to discriminate between the origins of ladderane lipids in marine sediments, i.e., to determine if ladderanes are produced in situ in relatively cold surface sediments or if they are fossil remnants originating from the warmer upper water column.

A comparative genomics study of genetic products potentially encoding ladderane lipid biosynthesis.

BACKGROUND: The fatty acids of anaerobic ammonium oxidizing (anammox) bacteria contain linearly concatenated cyclobutane moieties, so far unique to biology. These moieties are under high ring strain and are synthesised by a presently unknown biosynthetic pathway. RESULTS: Gene clusters encoding enzymes of fatty acid biosynthesis in the anammox bacterium Kuenenia stuttgartiensis and 137 other organisms were analysed and compared in silico to gain further insight into the pathway of (ladderane) fatty acid biosynthesis. In K. stuttgartiensis four large gene clusters encode fatty acid biosynthesis. Next to the regular enzyme complex needed for fatty acid biosynthesis (FASII), the presence of four putative S-adenosyl-methionine (SAM) radical enzymes, two enzymes similar to phytoene desaturases and many divergent paralogues of beta-ketoacyl-ACP synthase (fabF) were unusual. Surprisingly, extensive synteny was observed with FASII gene clusters in the deltaproteobacterium Desulfotalea psychrophila. No ladderane lipids were detected in lipid extracts of this organism but we did find unusual polyunsaturated hydrocarbons (PUHC), not detected in K. stuttgartiensis. CONCLUSION: We suggest that the unusual gene clusters of K. stuttgartiensis and D. psychrophila encode a novel pathway for anaerobic PUFA biosynthesis and that K. stuttgartiensis further processes PUFA into ladderane lipids, in similar fashion to the previously proposed route of ladderane lipid biosynthesis. However, the presence of divergent paralogues of fabF with radically different active site topologies may suggest an alternative pathway where ladderane moieties are synthesised externally and are recruited into the pathway of fatty acid biosynthesis.

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway.

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-(13)C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field (13)C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C. Brocadia fulgida synthesizes C(16:0) and isoC(16:0) fatty acids according to the known pathway of type II fatty acid biosynthesis. The (13)C-labelling pattern of the C(8) alkyl chain of the C(20) [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.

Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production.

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems.

Ladderane lipid distribution in four genera of anammox bacteria.

Intact ladderane phospholipids and core lipids were studied in four species of anaerobic ammonium oxidizing (anammox) bacteria, each representing one of the four known genera. Each species of anammox bacteria contained C18 and C20 ladderane fatty acids with either 3 or 5 linearly condensed cyclobutane rings and a ladderane monoether containing a C20 alkyl moiety with 3 cyclobutane rings. The presence of ladderane lipids in all four anammox species is consistent with their putative physiological role to provide a dense membrane around the anammoxosome, the postulated site of anammox catabolism. In contrast to the core lipids, large variations were observed in the distribution of ladderane phospholipids, i.e. different combinations of hydrophobic tail (ladderane, straight chain and methyl branched fatty acid) types attached to the glycerol backbone sn-1 position, in combination with different types of polar headgroup (phosphocholine, phosphoethanolamine or phosphoglycerol) attached to the sn-3 position. Intact ladderane lipids made up a high percentage of the lipid content in the cells of "Candidatus Kuenenia stuttgartiensis", suggesting that ladderane lipids are also present in membranes other than the anammoxosome. Finally, all four investigated species contained a C27 hopanoid ketone and bacteriohopanetetrol, which, indicates that hopanoids are anaerobically synthesised by anammox bacteria.

Candidatus 'Brocadia fulgida': an autofluorescent anaerobic ammonium oxidizing bacterium.

Anaerobic ammonium oxidizing (anammox) bacteria are detected in many natural ecosystems and wastewater treatment plants worldwide. This study describes the enrichment of anammox bacteria in the presence of acetate. The results obtained extend the concept that the anammox bacteria can be enriched to high densities in the presence of substrates for heterotrophic growth. Batch experiments showed that among the tested biomass, the biomass from the Candidatus 'Brocadia fulgida' enrichment culture oxidizes acetate at the highest rate. Continuous cultivation experiments showed that in the presence of acetate, ammonium, nitrite and nitrate, Candidatus 'Brocadia fulgida' out-competed other anammox bacteria. The results indicated that Candidatus 'Brocadia fulgida' did not incorporate acetate directly into their biomass. Candidatus 'Brocadia fulgida' exhibited the common characteristics of anammox bacteria: the presence of an anammoxosome and ladderane lipids and the production of hydrazine in the presence of hydroxylamine. Interestingly, the biofilm aggregates of this species showed strong autofluorescence. It is the only known anammox species exhibiting this feature. The autofluorescent extracellular polymeric substance had two excitation (352 and 442 nm) and two emission (464 and 521 nm) maxima.

Candidatus "Anammoxoglobus propionicus" a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria.

The bacteria that mediate the anaerobic oxidation of ammonium (anammox) are detected worldwide in natural and man-made ecosystems, and contribute up to 50% to the loss of inorganic nitrogen in the oceans. Two different anammox species rarely live in a single habitat, suggesting that each species has a defined but yet unknown niche. Here we describe a new anaerobic ammonium oxidizing bacterium with a defined niche: the co-oxidation of propionate and ammonium. The new anammox species was enriched in a laboratory scale bioreactor in the presence of ammonium and propionate. Interestingly, this particular anammox species could out-compete other anammox bacteria and heterotrophic denitrifiers for the oxidation of propionate in the presence of ammonium, nitrite and nitrate. We provisionally named the new species Candidatus "Anammoxoglobus propionicus".

Improved analysis of ladderane lipids in biomass and sediments using high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry.

Ladderane lipids, containing three or five linearly concatenated cyclobutane moieties, are considered to be unique biomarkers for the process of anaerobic ammonium oxidation, an important link in the oceanic nitrogen cycle. Due to the thermal lability of the strained cyclobutane moieties, the ladderane lipids are difficult to analyze by gas chromatography. A method combining high-performance liquid chromatography coupled to positive ion atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS) was developed for the analysis of the most abundant ladderane lipids, occurring as fatty acids and ether-bound to glycerol. Detection was achieved by selective reaction monitoring of four specific fragmentations per ladderane lipid. Detection limits of 30-35 pg injected on-column and a linear response (r(2) > 0.99) over nearly 3 orders of magnitude were achieved for all compounds. Using this method, these unique ladderane lipids were for the first time identified in a surface sediment from the Gullmarsfjorden, in concentrations ranging from 1.1-5.5 ng/g for the ladderane fatty acids and of 0.7 ng/g for the monoether. It is foreseen that this method will allow the investigation of the occurrence of anaerobic ammonium oxidation in natural settings in much greater detail than before.