Rates and signatures of methane turnover in sediments of continental margins

In this thesis, a variety of different cold seep systems (mud volcanoes and a gas seep) were investigated using a multidisciplinary approach to gain a more systematic understanding of these, methane-driven biogeosystems. The main goals were the detection and quantification of hot spots of methane oxidation as well as an assessment of environmental factors determining the activity and the distribution of methanotrophic communities. Furthermore, key microbial players were identified and the impact of Anaerobic Oxidation Of Methane (AOM) and Aerobic Oxidation Of Methane (MOx) on the surrounding, marine environment was studied. The investigations revealed the following:1. Submarine mud volcanoes are colonized by specialized microbial communities utilizing the fluxes of reduced substrates such as methane and sulphide as energy source. At the actively methane-seeping Haakon Mosby mud volcano (HMMV, Barents Sea), a distinct spatial zonation of several novel clades of free-living and symbiotic aerobic and anaerobic methanotrophs was found. The main selection mechanism determining vertical and horizontal distribution and dominance of the methanotrophic communities were fluid flow rates controlling access to electron acceptors for methane oxidation. 2. The analysis of archaeal and bacterial specific lipid concentrations and their associated delta 13C-values from three seepage areas at HMMV (thermal centre, grey mats and Beggiatoa site) showed a distinct distribution of methanotrophic biomass. At the centre, MOx mediated by a type I methanotroph was the primary biomass-generating process in surface sediments. In patches of reduced sediment, covered by greyish, thiotrophic, microbial mats at the boundary of the centre, a four-fold increase in 13C-depleted lipids specific for anaerobic methanotrophs, gave evidence of active microbial communities, which mediate AOM in the upper 20 cm of sediment. Further away from the centre, in the zone covered by Beggiatoa mats, sharp, vertical gradients of 13C-depleted archaeal and bacterial lipids indicate that AOM communities were restricted to a narrow surface horizon of no more than 4 cm. A combination of molecular techniques (DAPI, FISH, gene libraries) and biomarker fingerprints provided evidence that the AOM community was dominated by a novel strain of archaea (termed ANME-3) and SRB of the Desulfobulbus cluster.3. Biogeochemical investigations at HMMV revealed a high upward flow of sulphate-free subsurface fluids in the centre, strongly limiting the penetration of sulphate and oxygen from seawater. Here, MOx was restricted to the top sediment layer with rates of 0.9 mol m-2 yr-1 and AOM was absent. In the patches of reduced sediments covered with grey mats, a deeper penetration of sulphate was observed, fueling AOM activity down to >12 cm with rates of 12.4 mol m-2 yr-1. Adjacent to the centre at the Beggiatoa site, decreased upward fluid flow allowed for an AOM zone of ca 4 cm at the sediment surface with rates of 4.5 mol m-2 yr-1. At the outer rim of the HMMV, bioventilation of the pogonophoran worms irrigated a much deeper zone with oxygen- and sulphate-rich seawater. Just beneath the roots of the worms, aOM activity was high with 7.1 mol m-2 yr-1. With respect to the area size of the different habitats at HMMV, microbial consumption reduces the methane efflux of HMMV by ca 7* 10-5 Tg yr-1, i.e. 22 to 55 %. 4. The mud volcanoes of the Gulf of Cadiz are currently much less active than the HMMV. Here, thermogenic methane was completely consumed anaerobically in subsurface sediments. AOM and SR rates showed maxima in distinct subsurface sediment horizons between 20 to 200 cm below sea floor. In comparison to other methane dominated environments of the world oceans, AOM activity and diffusive methane fluxes (<0.4 mol m-2 yr-1, respectively) were low to mid range. AOM was generally exceeded by SRR, most likely because methane related, higher hydrocarbons were oxidised anaerobically by SR microbes. Lipid biomarker and 16S rDNA clone library analyses gave evidence that AOM was mediated by a mixed community of ANME-2 and ANME-1 archaea and associated SRB (Seep-SRB1 group). 5. The Tommeliten gas seep is located in the central North Sea. Here, cracks in a buried marl horizon allow methane to migrate into overlying clay-silt sediments. Hydroacoustic sediment echosounding showed several gas flares coinciding with the apex of the marl domes where methane is released into the water column and potentially to the atmosphere. Carbonates in the vicinity of the gas seep contained 13C-depleted, archaeal lipids indicating long-term AOM activity. In the sediment, the zone of active methane consumption was restricted to a distinct horizon of no more than 20 cm. Diagnostic, 13C-depleted archaeal and bacterial lipids as well as 16S rDNA clone libraries provided evidence that AOM was mediated by ANME-1b archaea and SRB most likely belonging to the Seep-SRB1 cluster

Field-scale labelling and activity quantification of methane-oxidizing bacteria in a landfill-cover soil

Aerobic methane-oxidizing bacteria (MOB) play an important role in soils, mitigating emissions of the greenhouse gas methane (CH4) to the atmosphere. Here, we combined stable isotope probing on MOB-specific phospholipid fatty acids (PLFA-SIP) with field-based gas push-pull tests (GPPTs). This novel approach (SIP-GPPT) was tested in a landfill-cover soil at four locations with different MOB activity. Potential oxidation rates derived from regular- and SIP-GPPTs agreed well and ranged from 0.2 to 52.8 mmol CH4 (L soil air)−1 day−1. PLFA profiles of soil extracts mainly contained C14 to C18 fatty acids (FAs), with a dominance of C16 FAs. Uptake of 13C into MOB biomass during SIP-GPPTs was clearly indicated by increased δ13C values (up to c. 1500‰) of MOB-characteristic FAs. In addition, 13C incorporation increased with CH4 oxidation rates. In general, FAs C14:0, C16:1ω8, C16:1ω7 and C16:1ω6 (type I MOB) showed highest 13C incorporation, while substantial 13C incorporation into FAs C18:1ω8 and C18:1ω7 (type II MOB) was only observed at high-activity locations. Our findings demonstrate the applicability of the SIP-GPPT approach for in situ quantification of potential CH4 oxidation rates and simultaneous labelling of active MOB, suggesting a dominance of type I MOB over type II MOB in the CH4-oxidizing community in this landfill-cover soi

Methanotrophs: Discoveries, Environmental Relevance, and a Perspective on Current and Future Applications

Methane is the final product of the anaerobic decomposition of organic matter. The conversion of organic matter to methane (methanogenesis) as a mechanism for energy conservation is exclusively attributed to the archaeal domain. Methane is oxidized by methanotrophic microorganisms using oxygen or alternative terminal electron acceptors. Aerobic methanotrophic bacteria belong to the phyla Proteobacteria and Verrucomicrobia, while anaerobic methane oxidation is also mediated by more recently discovered anaerobic methanotrophs with representatives in both the bacteria and the archaea domains. The anaerobic oxidation of methane is coupled to the reduction of nitrate, nitrite, iron, manganese, sulfate, and organic electron acceptors (e.g., humic substances) as terminal electron acceptors. This review highlights the relevance of methanotrophy in natural and anthropogenically influenced ecosystems, emphasizing the environmental conditions, distribution, function, co-existence, interactions, and the availability of electron acceptors that likely play a key role in regulating their function. A systematic overview of key aspects of ecology, physiology, metabolism, and genomics is crucial to understand the contribution of methanotrophs in the mitigation of methane efflux to the atmosphere. We give significance to the processes under microaerophilic and anaerobic conditions for both aerobic and anaerobic methane oxidizers. In the context of anthropogenically influenced ecosystems, we emphasize the current and potential future applications of methanotrophs from two different angles, namely methane mitigation in wastewater treatment through the application of anaerobic methanotrophs, and the biotechnological applications of aerobic methanotrophs in resource recovery from methane waste streams. Finally, we identify knowledge gaps that may lead to opportunities to harness further the biotechnological benefits of methanotrophs in methane mitigation and for the production of valuable bioproducts enabling a bio-based and circular economy

Microbial communities on plastic particles in surface waters differ from subsurface waters of the North Pacific Subtropical Gyre

The long-term fate of plastics in the ocean and their interactions with marine microorganisms remain poorly understood. In particular, the role of sinking plastic particles as a transport vector for surface microbes towards the deep sea has not been investigated. Here, we present the first data on the composition of microbial communities on floating and suspended plastic particles recovered from the surface to the bathypelagic water column (0-2000 m water depth) of the North Pacific Subtropical Gyre. Microbial community composition of suspended plastic particles differed from that of plastic particles afloat at the sea surface. However, in both compartments, a diversity of hydrocarbon-degrading bacteria was identified. These findings indicate that microbial community members initially present on floating plastics are quickly replaced by microorganisms acquired from deeper water layers, thus suggesting a limited efficiency of sinking plastic particles to vertically transport microorganisms in the North Pacific Subtropical Gyre.HN, AV were financed through the European Research Council (ERC-CoG Grant Nr 772923, project VORTEX). PDM was supported by NWO (VI.Veni.212.040)

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

Anaerobic oxidation of methane in hypersaline cold seep sediments

Life in hypersaline environments is typically limited by bioenergetic constraints. Microbial activity at the thermodynamic edge, such as the anaerobic oxidation of methane (AOM) coupled to sulphate reduction (SR), is thus unlikely to thrive in these environments. In this study, carbon and sulphur cycling was investigated in the extremely hypersaline cold seep sediments of Mercator mud volcano. AOM activity was partially inhibited but still present at salinity levels of 292 g L−1 (c. eightfold sea water concentration) with rates of 2.3 nmol cm−3 day−1 and was even detectable under saturated conditions. Methane and evaporite-derived sulphate comigrated in the ascending geofluids, which, in combination with a partial activity inhibition, resulted in AOM activity being spread over unusually wide depth intervals. Up to 79% of total cells in the AOM zone were identified by fluorescence in situ hybridization (FISH) as anaerobic methanotrophs of the ANME-1. Most ANME-1 cells formed monospecific chains without any attached partner. At all sites, AOM activity co-occurred with SR activity and sometimes significantly exceeded it. Possible causes of these unexpected results are discussed. This study demonstrates that in spite of a very low energy yield of AOM, microorganisms carrying this reaction can thrive in salinity up to halite saturatio

Differentiation of bacterial communities on five common plastics after six days of exposure to Caribbean coastal waters

Plastic pollution in coastal areas, particularly in subtropical and tropical regions, remains a pervasive environmental issue. Marine plastic debris provides an artificial surface that rapidly accumulates a dynamic microbial biofilm upon entering the marine ecosystem. Especially the early stages of colonization are critical in shaping the microbial community. This study investigates the early microbial colonization, in less than a week, on five different plastic polymers in Caribbean coastal waters through 16S rRNA gene amplicon sequencing. We discovered shared bacterial taxa among the various plastic polymers and sampling timepoints, with dominant orders being Flavobacteriales, Rhodobacterales, Rhizobiales, and Pseudomonadales. Statistical analysis confirmed significant differences in community composition between the two sampling points, with polystyrene exhibiting a distinct microbial community on day 6 compared to polyethylene, polypropylene, and nylon. We found the same for polyethylene compared to nylon and polyethylene-terephthalate. Further examination identified 47 genera responsible for these differences, primarily belonging to the phyla Proteobacteria and Bacteroidota. Our data indicate an influence of both environmentally related stochastic processes and plastic-related specific factors during early colonization. Interestingly, we noticed an increase in the relative abundance of hydrocarbon and potentially plastic-degrading bacteria (PDB) from 12.4 to 34.5 % between the first and sixth day, suggesting their vital role in shaping the epiplastic community. Notably, some identified PDB have been reported to degrade the specific polymers studied, thus the monitored increase in relative abundance supports their role in plastic degradation. However, more research is required to fully understand their functioning and potential role in the epiplastic community. Our study provides insights into the prokaryotic colonization of marine plastics in the Caribbean basin, where to date studies have been limited despite high pollution rates

Diel and seasonal methane dynamics in the shallow and turbulent Wadden Sea

The Wadden Sea is a coastal system along the fringe of the land–sea borders of Denmark, Germany and the Netherlands. The Wadden Sea is extremely productive and influenced by strong variations in physical and biological forcing factors that act on timescales of hours to seasons. Productive coastal seas are known to dominate the ocean's methane emission to the atmosphere, but knowledge of controls and temporal variations in methane dynamics in these vastly dynamic systems is scarce. Here we address this knowledge gap by measuring methane inventories and methanotrophic activity at a temporal resolution of 1 h over a period of 2 d, repeatedly during four successive seasons in the central Dutch Wadden Sea. We found that methane dynamics varied between colder and warmer seasons, with generally higher water column methane concentrations and methanotrophic activity in the warmer seasons. The efflux of methane to the atmosphere was, on the other hand, lower in the warmer seasons because of lower wind speeds. On a diel scale, tides controlled methanotrophic activity, which increased ∼40 % at low tide compared to high tide. We estimate that methane oxidizing bacteria reduce the methane budget of the Dutch Wadden Sea by only 2 %, while escapes to the atmosphere and are flushed out into the open North Sea at ebb tide. Our findings indicate that tides play a key role in controlling methane dynamics and methanotrophic activity and highlight the importance of high-resolution and repeated sampling strategies to resolve methane dynamics in fast-changing coastal systems