Glacial meltwater and seasonality influence community composition of diazotrophs in Arctic coastal and open waters.

The Arctic Ocean is particularly affected by climate change with unknown consequences for primary productivity. Diazotrophs-prokaryotes capable of converting atmospheric nitrogen to ammonia-have been detected in the often nitrogen-limited Arctic Ocean but distribution and community composition dynamics are largely unknown. We performed amplicon sequencing of the diazotroph marker gene nifH from glacial rivers, coastal, and open ocean regions and identified regionally distinct Arctic communities. Proteobacterial diazotrophs dominated all seasons, epi- to mesopelagic depths and rivers to open waters and, surprisingly, Cyanobacteria were only sporadically identified in coastal and freshwaters. The upstream environment of glacial rivers influenced diazotroph diversity, and in marine samples putative anaerobic sulphate-reducers showed seasonal succession with highest prevalence in summer to polar night. Betaproteobacteria (Burkholderiales, Nitrosomonadales, and Rhodocyclales) were typically found in rivers and freshwater-influenced waters, and Delta- (Desulfuromonadales, Desulfobacterales, and Desulfovibrionales) and Gammaproteobacteria in marine waters. The identified community composition dynamics, likely driven by runoff, inorganic nutrients, particulate organic carbon, and seasonality, imply diazotrophy a phenotype of ecological relevance with expected responsiveness to ongoing climate change. Our study largely expands baseline knowledge of Arctic diazotrophs-a prerequisite to understand underpinning of nitrogen fixation-and supports nitrogen fixation as a contributor of new nitrogen in the rapidly changing Arctic Ocean.

Long-term patterns of hydrocarbon biodegradation and bacterial community composition in epipelagic and mesopelagic zones of an Arctic fjord.

Oil spill attenuation in Arctic marine environments depends on oil-degrading bacteria. However, the seasonally harsh conditions in the Arctic such as nutrient limitations and sub-zero temperatures limit the activity even for bacteria capable of hydrocarbon metabolism at low temperatures. Here, we investigated whether the variance between epipelagic (seasonal temperature and inorganic nutrient variations) and mesopelagic zone (stable environmental conditions) could limit the growth of oil-degrading bacteria and lead to lower oil biodegradation rates in the epipelagic than in the mesopelagic zone. Therefore, we deployed absorbents coated with three oil types in a SW-Greenland fjord system at 10-20 m (epipelagic) and 615-650 m (mesopelagic) water depth for one year. During this period we monitored the development and succession of the bacterial biofilms colonizing the oil films by 16S rRNA gene amplicon quantification and sequencing, and the progression of oil biodegradation by gas chromatography - mass spectrometry oil fingerprinting analysis. The removal of hydrocarbons was significantly different, with several polycyclic aromatic hydrocarbons showing longer half-life times in the epipelagic than in the mesopelagic zone. Bacterial community composition and density (16S rRNA genes/ cm2) significantly differed between the two zones, with total bacteria reaching to log-fold higher densities (16S rRNA genes/cm2) in the mesopelagic than epipelagic oil-coated absorbents. Consequently, the environmental conditions in the epipelagic zone limited oil biodegradation performance by limiting bacterial growth.

Biodegradation of water-accommodated aromatic oil compounds in Arctic seawater at 0 °C.

Oil spills in Arctic marine environments are expected to increase concurrently with the expansion of shipping routes and petroleum exploitation into previously inaccessible ice-dominated regions. Most research on oil biodegradation focusses on the bulk oil, but the fate of the water-accommodated fraction (WAF), mainly composed of toxic aromatic compounds, is largely underexplored. To evaluate the bacterial degradation capacity of such dissolved aromatics in Greenlandic seawater, microcosms consisting of 0 °C seawater polluted with WAF were investigated over a 3-month period. With a half-life (t1/2) of 26 days, m-xylene was the fastest degraded compound, as measured by gas chromatography - mass spectrometry. Substantial slower degradation was observed for ethylbenzene, naphthalenes, phenanthrene, acenaphthylene, acenaphthene and fluorenes with t1/2 of 40-105 days. Colwellia, identified by 16S rRNA gene sequencing, was the main potential degrader of m-xylene. This genus occupied up to 47 % of the bacterial community until day 10 in the microcosms. Cycloclasticus and Zhongshania aliphaticivorans, potentially utilizing one-to three-ringed aromatics, replaced Colwellia between day 10 and 96 and occupied up to 6 % and 23 % of the community, respectively. Although most of the WAF can ultimately be eliminated in microcosms, our results suggest that the restoration of an oil-impacted Arctic environment may be slow as most analysed compounds had t1/2 of over 2-3 months and the detrimental effects of a spill towards the marine ecosystem likely persist during this time.

Distinct methane-dependent biogeochemical states in Arctic seafloor gas hydrate mounds.

Archaea mediating anaerobic methane oxidation are key in preventing methane produced in marine sediments from reaching the hydrosphere; however, a complete understanding of how microbial communities in natural settings respond to changes in the flux of methane remains largely uncharacterized. We investigate microbial communities in gas hydrate-bearing seafloor mounds at Storfjordrenna, offshore Svalbard in the high Arctic, where we identify distinct methane concentration profiles that include steady-state, recently-increasing subsurface diffusive flux, and active gas seepage. Populations of anaerobic methanotrophs and sulfate-reducing bacteria were highest at the seep site, while decreased community diversity was associated with a recent increase in methane influx. Despite high methane fluxes and methanotroph doubling times estimated at 5-9 months, microbial community responses were largely synchronous with the advancement of methane into shallower sediment horizons. Together, these provide a framework for interpreting subseafloor microbial responses to methane escape in a warming Arctic Ocean.

The Impact of Methane on Microbial Communities at Marine Arctic Gas Hydrate Bearing Sediment.

Cold seeps are characterized by high biomass, which is supported by the microbial oxidation of the available methane by capable microorganisms. The carbon is subsequently transferred to higher trophic levels. South of Svalbard, five geological mounds shaped by the formation of methane gas hydrates, have been recently located. Methane gas seeping activity has been observed on four of them, and flares were primarily concentrated at their summits. At three of these mounds, and along a distance gradient from their summit to their outskirt, we investigated the eukaryotic and prokaryotic biodiversity linked to 16S and 18S rDNA. Here we show that local methane seepage and other environmental conditions did affect the microbial community structure and composition. We could not demonstrate a community gradient from the summit to the edge of the mounds. Instead, a similar community structure in any methane-rich sediments could be retrieved at any location on these mounds. The oxidation of methane was largely driven by anaerobic methanotrophic Archaea-1 (ANME-1) and the communities also hosted high relative abundances of sulfate reducing bacterial groups although none demonstrated a clear co-occurrence with the predominance of ANME-1. Additional common taxa were observed and their abundances were likely benefiting from the end products of methane oxidation. Among these were sulfide-oxidizing Campilobacterota, organic matter degraders, such as Bathyarchaeota, Woesearchaeota, or thermoplasmatales marine benthic group D, and heterotrophic ciliates and Cercozoa.

Methane-fuelled biofilms predominantly composed of methanotrophic ANME-1 in Arctic gas hydrate-related sediments.

Sedimentary biofilms comprising microbial communities mediating the anaerobic oxidation of methane are rare. Here, we describe two biofilm communities discovered in sediment cores recovered from Arctic cold seep sites (gas hydrate pingos) in the north-western Barents Sea, characterized by steady methane fluxes. We found macroscopically visible biofilms in pockets in the sediment matrix at the depth of the sulphate-methane-transition zone. 16S rRNA gene surveys revealed that the microbial community in one of the two biofilms comprised exclusively of putative anaerobic methanotrophic archaea of which ANME-1 was the sole archaeal taxon. The bacterial community consisted of relatives of sulphate-reducing bacteria (SRB) belonging to uncultured Desulfobacteraceae clustering into SEEP-SRB1 (i.e. the typical SRB associated to ANME-1), and members of the atribacterial JS1 clade. Confocal laser scanning microscopy demonstrates that this biofilm is composed of multicellular strands and patches of ANME-1 that are loosely associated with SRB cells, but not tightly connected in aggregates. Our discovery of methanotrophic biofilms in sediment pockets closely associated with methane seeps constitutes a hitherto overlooked and potentially widespread sink for methane and sulphate in marine sediments.

Microbial methane formation in deep aquifers of a coal-bearing sedimentary basin, Germany.

Coal-bearing sediments are major reservoirs of organic matter potentially available for methanogenic subsurface microbial communities. In this study the specific microbial community inside lignite-bearing sedimentary basin in Germany and its contribution to methanogenic hydrocarbon degradation processes was investigated. The stable isotope signature of methane measured in groundwater and coal-rich sediment samples indicated methanogenic activity. Analysis of 16S rRNA gene sequences showed the presence of methanogenic Archaea, predominantly belonging to the orders Methanosarcinales and Methanomicrobiales, capable of acetoclastic or hydrogenotrophic methanogenesis. Furthermore, we identified fermenting, sulfate-, nitrate-, and metal-reducing, or acetogenic Bacteria clustering within the phyla Proteobacteria, complemented by members of the classes Actinobacteria, and Clostridia. The indigenous microbial communities found in the groundwater as well as in the coal-rich sediments are able to degrade coal-derived organic components and to produce methane as the final product. Lignite-bearing sediments may be an important nutrient and energy source influencing larger compartments via groundwater transport.

Enhanced gene detection assays for fumarate-adding enzymes allow uncovering of anaerobic hydrocarbon degraders in terrestrial and marine systems.

The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. Fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) have already been established as specific functional marker genes for anaerobic hydrocarbon degraders. Several recent studies based on pure cultures and laboratory enrichments have shown the existence of new and deeply branching FAE gene lineages, such as clostridial benzylsuccinate synthases and homologues, as well as naphthylmethylsuccinate synthases. However, established FAE gene detection assays were not designed to target these novel lineages, and consequently, their detectability in different environments remains obscure. Here, we present a new suite of parallel primer sets for detecting the comprehensive range of FAE markers known to date, including clostridial benzylsuccinate, naphthylmethylsuccinate, and alkylsuccinate synthases. It was not possible to develop one single assay spanning the complete diversity of FAE genes alone. The enhanced assays were tested with a range of hydrocarbon-degrading pure cultures, enrichments, and environmental samples of marine and terrestrial origin. They revealed the presence of several, partially unexpected FAE gene lineages not detected in these environments before: distinct deltaproteobacterial and also clostridial bssA homologues as well as environmental nmsA homologues. These findings were backed up by dual-digest terminal restriction fragment length polymorphism diagnostics to identify FAE gene populations independently of sequencing. This allows rapid insights into intrinsic degrader populations and degradation potentials established in aromatic and aliphatic hydrocarbon-impacted environmental systems.

Accelerated methanogenesis from aliphatic and aromatic hydrocarbons under iron- and sulfate-reducing conditions.

The impact of four electron acceptors on hydrocarbon-induced methanogenesis was studied. Methanogenesis from residual hydrocarbons may enhance the exploitation of oil reservoirs and may improve bioremediation. The conditions to drive the rate-limiting first hydrocarbon-oxidizing steps for the conversion of hydrocarbons into methanogenic substrates are crucial. Thus, the electron acceptors ferrihydrite, manganese dioxide, nitrate or sulfate were added to sediment microcosms acquired from two brackish water locations. Hexadecane, ethylbenzene or 1-(13)C-naphthalene were used as model hydrocarbons. Methane was released most rapidly from incubations amended with ferrihydrite and hexadecane. Ferrihydrite enhanced only hexadecane-dependent methanogenesis. The rates of methanogenesis were negatively affected by sulfate and nitrate at concentrations of more than 5 and 1 mM, respectively. Metal-reducing Geobacteraceae and potential sulfate reducers as well as Methanosarcina were present in situ and in vitro. Ferrihydrite addition triggered the growth of Methanosarcina-related methanogens. Additionally, methane was removed concomitantly by anaerobic methanotrophy. ANME-1 and -2 methyl coenzyme M reductase genes were detected, indicating anaerobic methanotrophy as an accompanying process [Correction added 16 December after online publication: 'methyl coenzyme A' changed to 'methyl coenzyme M' in this sentence]. The experiments presented here demonstrate the feasibility of enhancing methanogenic alkane degradation by ferrihydrite or sulfate addition in different geological settings.