Anaerobic oxidation of ethane by archaea from a marine hydrocarbon seep.

Ethane is the second most abundant component of natural gas in addition to methane, and-similar to methane-is chemically unreactive. The biological consumption of ethane under anoxic conditions was suggested by geochemical profiles at marine hydrocarbon seeps1-3, and through ethane-dependent sulfate reduction in slurries4-7. Nevertheless, the microorganisms and reactions that catalyse this process have to date remained unknown8. Here we describe ethane-oxidizing archaea that were obtained by specific enrichment over ten years, and analyse these archaea using phylogeny-based fluorescence analyses, proteogenomics and metabolite studies. The co-culture, which oxidized ethane completely while reducing sulfate to sulfide, was dominated by an archaeon that we name 'Candidatus Argoarchaeum ethanivorans'; other members were sulfate-reducing Deltaproteobacteria. The genome of Ca. Argoarchaeum contains all of the genes that are necessary for a functional methyl-coenzyme M reductase, and all subunits were detected in protein extracts. Accordingly, ethyl-coenzyme M (ethyl-CoM) was identified as an intermediate by liquid chromatography-tandem mass spectrometry. This indicated that Ca. Argoarchaeum initiates ethane oxidation by ethyl-CoM formation, analogous to the recently described butane activation by 'Candidatus Syntrophoarchaeum'9. Proteogenomics further suggests that oxidation of intermediary acetyl-CoA to CO2 occurs through the oxidative Wood-Ljungdahl pathway. The identification of an archaeon that uses ethane (C2H6) fills a gap in our knowledge of microorganisms that specifically oxidize members of the homologous alkane series (CnH2n+2) without oxygen. Detection of phylogenetic and functional gene markers related to those of Ca. Argoarchaeum at deep-sea gas seeps10-12 suggests that archaea that are able to oxidize ethane through ethyl-CoM are widespread members of the local communities fostered by venting gaseous alkanes around these seeps.

Aerobic methane synthesis and dynamics in a river water environment.

Reports of aerobic biogenic methane (CH4) have generated new views about CH4 sources in nature. We examine this phenomenon in the free-flowing Yellowstone river wherein CH4 concentrations were tracked as a function of environmental conditions, phototrophic microorganisms (using chlorophyll a, Chl a, as proxy), as well as targeted methylated amines known to be associated with this process. CH4 was positively correlated with temperature and Chl a, although diurnal measurements showed CH4 concentrations were greatest during the night and lowest during maximal solar irradiation. CH4 efflux from the river surface was greater in quiescent edge waters (71-94 µmol m-2 d) than from open flowing current (~ 57 µmol m-2 d). Attempts to increase flux by disturbing the benthic environment in the quiescent water directly below (~ 1.0 m deep) or at varying distances (0-5 m) upstream of the flux chamber failed to increase surface flux. Glycine betaine (GB), dimethylamine and methylamine (MMA) were observed throughout the summer-long study, increasing during a period coinciding with a marked decline in Chl a, suggesting a lytic event led to their release; however, this did not correspond to increased CH4 concentrations. Spiking river water with GB or MMA yielded significantly greater CH4 than nonspiked controls, illustrating the metabolic potential of the river microbiome. In summary, this study provides evidence that: (1) phototrophic microorganisms are involved in CH4 synthesis in a river environment; (2) the river microbiome possesses the metabolic potential to convert methylated amines to CH4; and (3) river CH4 concentrations are dynamic diurnally as well as during the summer active months.

Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea.

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate-methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors.

Cultivation and characterization of snowbound microorganisms from the South Pole.

Little is known about microbial ecosystems of interior Antarctica, if indeed such ecosystems exist. Although considerable research has assessed microorganisms indigenous to coastal regions of Antarctica, particularly their lakes, ponds, and soils, to our knowledge only one characterized bacterium, a strain of Pseudomonas, has been isolated from South Pole ice or snow. Metagenomic community analyses described in this work and elsewhere reveal that a diversity of bacteria exists in inland polar snows, yet attempts to culture and characterize these microbes from this extreme environment have been few to date. In this molecular and culture-dependent investigation of the microbiology of inland Antarctica, we enriched and isolated two new strains of bacteria and one strain of yeast (Fungi) from South Pole snow samples. The bacteria were of the genera Methylobacterium and Sphingomonas, and the yeast grouped with species of Naganishia (class Tremellocytes). In addition to phylogenetic analyses, characterization of these isolates included determinations of cell morphology, growth as a function of temperature, salinity tolerance, and carbon and energy source versatility. All organisms were found to be cold-adapted, and the yeast strain additionally showed considerable halotolerance. These descriptions expand our understanding of the diversity and metabolic activities of snowbound microorganisms of interior Antarctica.

Microbial metabolism of methanol and methylamine in the Gulf of Mexico: insight into marine carbon and nitrogen cycling.

One carbon (C1) metabolism plays an important role in marine carbon cycling but the dynamics and modes of C1 transformations are not fully understood. We made contemporaneous measurements of methylamine and methanol metabolism to elucidate the role of C1 compounds as sources of carbon, energy and nitrogen. Methanol and methylamine were predominantly used as an energy source in offshore waters (oxidation rate constant: kmethanol : 0.02-0.10 day-1 ; kmethylamine : 0.01-0.18 day-1 ), but were also important sources of biomass carbon in coastal waters (assimilation rate constant: kmethanol : 0.04-0.10 day-1 ; kmethylamine : 0.01-0.05 day-1 ). The relative extent of assimilation versus oxidation for these substrates correlated positively with chlorophyll, nutrients and heterotrophic bacterial production. Methanol oxidation and assimilation were stimulated significantly by nutrient addition. In contrast, methylamine metabolism was inhibited by ammonium or nitrate, suggesting that methylamine served as a nitrogen source. A preliminary metagenomic survey revealed a diverse population of putative C1-utilizing microorganisms. These results show that the remineralization of methylamine could provide both C and N sources for microbes. Both methanol and methylamine contribute to microbial energetic and carbon substrate demands with a distinctly different signature in nearshore versus offshore environments.

Chemical dispersants can suppress the activity of natural oil-degrading microorganisms.

During the Deepwater Horizon oil well blowout in the Gulf of Mexico, the application of 7 million liters of chemical dispersants aimed to stimulate microbial crude oil degradation by increasing the bioavailability of oil compounds. However, the effects of dispersants on oil biodegradation rates are debated. In laboratory experiments, we simulated environmental conditions comparable to the hydrocarbon-rich, 1,100 m deep plume that formed during the Deepwater Horizon discharge. The presence of dispersant significantly altered the microbial community composition through selection for potential dispersant-degrading Colwellia, which also bloomed in situ in Gulf deep waters during the discharge. In contrast, oil addition to deepwater samples in the absence of dispersant stimulated growth of natural hydrocarbon-degrading Marinobacter. In these deepwater microcosm experiments, dispersants did not enhance heterotrophic microbial activity or hydrocarbon oxidation rates. An experiment with surface seawater from an anthropogenically derived oil slick corroborated the deepwater microcosm results as inhibition of hydrocarbon turnover was observed in the presence of dispersants, suggesting that the microcosm findings are broadly applicable across marine habitats. Extrapolating this comprehensive dataset to real world scenarios questions whether dispersants stimulate microbial oil degradation in deep ocean waters and instead highlights that dispersants can exert a negative effect on microbial hydrocarbon degradation rates.

Characterization of a cold-active bacterium isolated from the South Pole "Ice Tunnel".

Extremely cold microbial habitats on Earth (those below -30 °C) are rare and have not been surveyed for microbes as extensively as environments in the 0 to -20 °C range. Using cryoprotected growth media incubated at -5 °C, we enriched a cold-active Pseudomonas species from -50 °C ice collected from a utility tunnel for wastewater pipes under Amundsen-Scott South Pole Station, Antarctica. The isolate, strain UC-1, is related to other cold-active Pseudomonas species, most notably P. psychrophila, and grew at -5 °C to +34-37 °C; growth of UC-1 at +3 °C was significantly faster than at +34 °C. Strain UC-1 synthesized a surface exopolymer and high levels of unsaturated fatty acids under cold growth conditions. A 16S rRNA gene diversity screen of the ice sample that yielded strain UC-1 revealed over 1200 operational taxonomic units (OTUs) distributed across eight major classes of Bacteria. Many of the OTUs were Clostridia and Bacteriodia and some of these were probably of wastewater origin. However, a significant fraction of the OTUs were Proteobacteria and Actinobacteria of likely environmental origin. Our results shed light on the lower temperature limits to life and the possible existence of functional microbial communities in ultra-cold environments.

Selective quantification of DOSS in marine sediment and sediment-trap solids by LC-QTOF-MS.

At the onset of the 2010 Gulf oil spill, analytical methods for the quantification of the surfactants in Corexit did not exist in the peer-reviewed literature. To date, only a single study reports the presence of bis-(2-ethylhexyl) sodium sulfosuccinate (DOSS) in deep-sea Gulf sediment collected in 2010 from a single location. There are no data on the occurrence of DOSS in association with settling solids (i.e., sediment-trap solids). To address this data gap, DOSS was initially quantified by liquid chromatography tandem quadrupole mass spectrometry (LC-MS/MS) in sediment and sediment-trap solids collected from multiple sites in the Gulf between 2010 and 2013. However, interferences confounded analyses using only a quadrupole (MS/MS) system; therefore, a LC-high mass accuracy quadruple time of flight mass spectrometry (LC-QTOF-MS) method was developed. The LC-QTOF method was validated and applied to eight representative samples of sediment and of sediment-trap solids. The presented method quantifies DOSS in solids of marine origin at concentrations above the limit of quantification of 0.23 µg kg-1 with recoveries of 97 ± 20 % (mean ± 95 CI). Gulf sediment and sediment-trap solids gave DOSS concentrations of

A halophilic bacterium inhabiting the warm, CaCl2-rich brine of the perennially ice-covered Lake Vanda, McMurdo Dry Valleys, Antarctica.

Lake Vanda is a perennially ice-covered and stratified lake in the McMurdo Dry Valleys, Antarctica. The lake develops a distinct chemocline at about a 50-m depth, where the waters transition from cool, oxic, and fresh to warm, sulfidic, and hypersaline. The bottom water brine is unique, as the highly chaotropic salts CaCl2 and MgCl2 predominate, and CaCl2 levels are the highest of those in any known microbial habitat. Enrichment techniques were used to isolate 15 strains of heterotrophic bacteria from the Lake Vanda brine. Despite direct supplementation of the brine samples with different organic substrates in primary enrichments, the same organism, a relative of the halophilic bacterium Halomonas (Gammaproteobacteria), was isolated from all depths sampled. The Lake Vanda (VAN) strains were obligate aerobes and showed broad pH, salinity, and temperature ranges for growth, consistent with the physicochemical properties of the brine. VAN strains were halophilic and quite CaCl2 tolerant but did not require CaCl2 for growth. The fact that only VAN strain-like organisms appeared in our enrichments hints that the highly chaotropic nature of the Lake Vanda brine may place unusual physiological constraints on the bacterial community that inhabits it.

Groundwater controls ecological zonation of salt marsh macrophytes.

Ecological zonation of salt marsh macrophytes is strongly influenced by hydrologic factors, but these factors are poorly understood. We examined groundwater flow patterns through surficial sediments in two saltmarshes in the southeastern United States to quantify hydrologic differences between distinct ecological zones. Both sites included tall- or medium-form Spartina alterniflora near the creek bank; short-form Spartina alterniflora in the mid-marsh; salt flats and Salicornia virginica in the high marsh; and Juncus roemarianus in brackish-to-fresh areas adjacent to uplands. Both sites had relatively small, sandy uplands and similar stratigraphy consisting of marsh muds overlying a deeper sand layer. We found significant hydrologic differences between the four ecological zones. In the zones colonized by S. alterniflora, the vertical flow direction oscillated with semi-diurnal tides. Net flow (14-day average) through the tall S. alterniflora zones was downward, whereas the short S. alterniflora zones included significant periods of net upward groundwater flow. An examination of tidal efficiency at these sites suggested that the net flow patterns rather than tidal damping controlled the width of the tall S. alterniflora zone. In contrast to the S. alterniflora zones, hypersaline zones populated by S. virginica were characterized by sustained periods (days) of continuous upward flow of saline water during neap tides. The fresher zone populated by J. roemarianus showed physical flow patterns that were similar to the hypersaline zones, but the upwelling porewaters were fresh rather than saline. These flow patterns were influenced by the hydrogeologic framework of the marshes, particularly differences in hydraulic head between the upland water table and the tidal creeks. We observed increases in hydraulic head of approximately 40 cm from the creek to the upland in the sand layers below both marshes, which is consistent with previous observations that sandy aquifers below fine-grained marsh soils act as conduits for flow from uplands to tidal creeks. This hydrologic framework supports relatively good drainage near the creek, increased waterlogging in the mid-marsh, and the development of hypersalinity adjacent to the freshwater upland. These hydrologic differences in turn support distinct ecological zones.

Spatial distribution of nitrogen fixation in methane seep sediment and the role of the ANME archaea.

Nitrogen (N2) fixation was investigated at Mound 12, Costa Rica, to determine its spatial distribution and biogeochemical controls in deep-sea methane seep sediment. Using (15)N2 tracer experiments and isotope ratio mass spectrometry analysis, we observed that seep N2 fixation is methane-dependent, and that N2 fixation rates peak in a narrow sediment depth horizon corresponding to increased abundance of aggregates of anaerobic methanotrophic archaea (ANME-2) and sulfate-reducing bacteria (SRB). Using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS), we directly measured (15)N2 uptake by ANME-2/SRB aggregates (n = 26) and observed maximum (15)N incorporation within ANME-2-dominated areas of the aggregates, consistent with previous analyses. NanoSIMS analysis of single cells (n = 34) from the same microcosm experiment revealed no (15)N2 uptake. Together, these observations suggest that ANME-2, and possibly physically associated SRB, mediate the majority of new nitrogen production within the seep ecosystem. ANME-2 diazotrophy was observed while in association with members of two distinct orders of SRB: Desulfobacteraceae and Desulfobulbaceae. The rate of N2 fixation per unit volume biomass was independent of the identity of the associated SRB, aggregate size and morphology. Our results show that the distribution of seep N2 fixation is heterogeneous, laterally and with depth in the sediment, and is likely influenced by chemical gradients affecting the abundance and activity of ANME-2/SRB aggregates.

Dispersant addition, but not nutrients, stimulated blooms of multiple hydrocarbonoclastic genera in nutrient-replete coastal marine surface waters.

The range of impacts of chemical dispersants on indigenous marine microbial communities and their activity remains poorly constrained. We tested the response of nearshore surface waters chronically exposed to oil leakage from a downed platform and supplied with nutrients by the Mississippi River to Corexit dispersant and nutrient additions. As assessed using 14C-labeled tracers, hexadecane mineralization potential was orders of magnitude higher in all unamended samples than in previously assessed bathypelagic communities. Nutrient additions stimulated microbial mortality but did not affect community composition and had no generalizable effect on hydrocarbon mineralization potential. By contrast, Corexit amendments caused a rapid shift in community composition and a drawdown of inorganic nitrogen and orthophosphate though no generalizable effect on hydrocarbon mineralization potential. The hydrocarbonoclastic community's response to dispersants is largely driven by the relative availability of organic substrates and nutrients, underscoring the role of environmental conditions and multiple interacting stressors on hydrocarbon degradation potential.

Evidence of giant sulphur bacteria in Neoproterozoic phosphorites.

In situ phosphatization and reductive cell division have recently been discovered within the vacuolate sulphur-oxidizing bacteria. Here we show that certain Neoproterozoic Doushantuo Formation (about 600 million years bp) microfossils, including structures previously interpreted as the oldest known metazoan eggs and embryos, can be interpreted as giant vacuolate sulphur bacteria. Sulphur bacteria of the genus Thiomargarita have sizes and morphologies similar to those of many Doushantuo microfossils, including symmetrical cell clusters that result from multiple stages of reductive division in three planes. We also propose that Doushantuo phosphorite precipitation was mediated by these bacteria, as shown in modern Thiomargarita-associated phosphogenic sites, thus providing the taphonomic conditions that preserved other fossils known from the Doushantuo Formation.

Anaerobic oxidation of short-chain hydrocarbons by marine sulphate-reducing bacteria.

The short-chain hydrocarbons ethane, propane and butane are constituents of natural gas. They are usually assumed to be of thermochemical origin, but biological formation of ethane and propane has been also observed. Microbial utilization of short-chain hydrocarbons has been shown in some aerobic species but not in anaerobic species of bacteria. On the other hand, anaerobic utilization of short-chain hydrocarbons would in principle be expected because various anaerobic bacteria grow with higher homologues (> or =C(6)). Indeed, chemical analyses of hydrocarbon-rich habitats with limited or no access of oxygen indicated in situ biodegradation of short-chain hydrocarbons. Here we report the enrichment of sulphate-reducing bacteria (SRB) with such capacity from marine hydrocarbon seep areas. Propane or n-butane as the sole growth substrate led to sediment-free sulphate-reducing enrichment cultures growing at 12, 28 or 60 degrees C. With ethane, a slower enrichment with residual sediment was obtained at 12 degrees C. Isolation experiments resulted in a mesophilic pure culture (strain BuS5) that used only propane and n-butane (methane, isobutane, alcohols or carboxylic acids did not support growth). Complete hydrocarbon oxidation to CO2 and the preferential oxidation of 12C-enriched alkanes were observed with strain BuS5 and other cultures. Metabolites of propane included iso- and n-propylsuccinate, indicating a subterminal as well as an unprecedented terminal alkane activation with involvement of fumarate. According to 16S ribosomal RNA analyses, strain BuS5 affiliates with Desulfosarcina/Desulfococcus, a cluster of widespread marine SRB. An enrichment culture with propane growing at 60 degrees C was dominated by Desulfotomaculum-like SRB. Our results suggest that diverse SRB are able to thrive in seep areas and gas reservoirs on propane and butane, thus altering the gas composition and contributing to sulphide production.

Species-specific responses of marine bacteria to environmental perturbation.

Environmental perturbations shape the structure and function of microbial communities. Oil spills are a major perturbation and resolving spills often requires active measures like dispersant application that can exacerbate the initial disturbance. Species-specific responses of microorganisms to oil and dispersant exposure during such perturbations remain largely unknown. We merged metatranscriptomic libraries with pangenomes to generate Core-Accessory Metatranscriptomes (CA-Metatranscriptomes) for two microbial hydrocarbon degraders that played important roles in the aftermath of the Deepwater Horizon oil spill. The Colwellia CA-Metatranscriptome illustrated pronounced dispersant-driven acceleration of core (~41%) and accessory gene (~59%) transcription, suggesting an opportunistic strategy. Marinobacter responded to oil exposure by expressing mainly accessory genes (~93%), suggesting an effective hydrocarbon-degrading lifestyle. The CA-Metatranscriptome approach offers a robust way to identify the underlying mechanisms of key microbial functions and highlights differences of specialist-vs-opportunistic responses to environmental disturbance.

Metagenomics and metatranscriptomics reveal broadly distributed, active, novel methanotrophs in the Gulf of Mexico hypoxic zone and in the marine water column.

The northern Gulf of Mexico (nGOM) hypoxic zone is a shallow water environment where methane, a potent greenhouse gas, fluxes from sediments to bottom water and remains trapped due to summertime stratification. When the water column is destratified, an active planktonic methanotrophic community could mitigate the efflux of methane, which accumulates to high concentrations, to the atmosphere. To investigate the possibility of such a biofilter in the nGOM hypoxic zone we performed metagenome assembly, and metagenomic and metatranscriptomic read mapping. Methane monooxygenase (pmoA) was an abundant transcript, yet few canonical methanotrophs have been reported in this environment, suggesting a role for non-canonical methanotrophs. To determine the identity of these methanotrophs, we reconstructed six novel metagenome-assembled genomes (MAGs) in the Planctomycetota, Verrucomicrobiota and one putative Latescibacterota, each with at least one pmoA gene copy. Based on ribosomal protein phylogeny, closely related microbes (mostly from Tara Oceans) and isolate genomes were selected and co-analyzed with the nGOM MAGs. Gene annotation and read mapping suggested that there is a large, diverse and unrecognized community of active aerobic methanotrophs in the nGOM hypoxic zone and in the global ocean that could mitigate methane flux to the atmosphere.

Expanding our view of the cold-water coral niche and accounting of the ecosystem services of the reef habitat.

Coral reefs are iconic ecosystems that support diverse, productive communities in both shallow and deep waters. However, our incomplete knowledge of cold-water coral (CWC) niche space limits our understanding of their distribution and precludes a complete accounting of the ecosystem services they provide. Here, we present the results of recent surveys of the CWC mound province on the Blake Plateau off the U.S. east coast, an area of intense human activity including fisheries and naval operations, and potentially energy and mineral extraction. At one site, CWC mounds are arranged in lines that total over 150 km in length, making this one of the largest reef complexes discovered in the deep ocean. This site experiences rapid and extreme shifts in temperature between 4.3 and 10.7 °C, and currents approaching 1 m s-1. Carbon is transported to depth by mesopelagic micronekton and nutrient cycling on the reef results in some of the highest nitrate concentrations recorded in the region. Predictive models reveal expanded areas of highly suitable habitat that currently remain unexplored. Multidisciplinary exploration of this new site has expanded understanding of the cold-water coral niche, improved our accounting of the ecosystem services of the reef habitat, and emphasizes the importance of properly managing these systems.

Anaerobic methane oxidation in metalliferous hydrothermal sediments: influence on carbon flux and decoupling from sulfate reduction.

The anaerobic oxidation of methane (AOM) is a globally significant sink that regulates methane flux from sediments into the oceans and atmosphere. Here we examine mesophilic to thermophilic AOM in hydrothermal sediments recovered from the Middle Valley vent field, on the Juan de Fuca Ridge. Using continuous-flow sediment bioreactors and batch incubations, we characterized (i) the degree to which AOM contributes to net dissolved inorganic carbon flux, (ii) AOM and sulfate reduction (SR) rates as a function of temperature and (iii) the distribution and density of known anaerobic methanotrophs (ANMEs). In sediment bioreactors, inorganic carbon stable isotope mass balances results indicated that AOM accounted for between 16% and 86% of the inorganic carbon produced, underscoring the role of AOM in governing inorganic carbon flux from these sediments. At 90°C, AOM occurred in the absence of SR, demonstrating a striking decoupling of AOM from SR. An abundance of Fe(III)-bearing minerals resembling mixed valent Fe oxides, such as green rust, suggests the potential for a coupling of AOM to Fe(III) reduction in these metalliferous sediments. While SR bacteria were only observed in cooler temperature sediments, ANMEs allied to ANME-1 ribotypes, including a putative ANME-1c group, were found across all temperature regimes and represented a substantial proportion of the archaeal community. In concert, these results extend and reshape our understanding of the nature of high temperature methane biogeochemistry, providing insight into the physiology and ecology of thermophilic anaerobic methanotrophy and suggesting that AOM may play a central role in regulating biological dissolved inorganic carbon fluxes to the deep ocean from the organic-poor, metalliferous sediments of the global mid-ocean ridge hydrothermal vent system.