Matthias Y. Kellermann

Thermophilic anaerobic oxidation of methane by marine microbial consortia

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. AOM is performed by microbial consortia of archaea (ANME) associated with partners related to sulfate-reducing bacteria. In vitro enrichments of AOM were so far only successful at temperatures ⩽25 °C; however, energy gain for growth by AOM with sulfate is in principle also possible at higher temperatures. Sequences of 16S rRNA genes and core lipids characteristic for ANME as well as hints of in situ AOM activity were indeed reported for geothermally heated marine environments, yet no direct evidence for thermophilic growth of marine ANME consortia was obtained to date. To study possible thermophilic AOM, we investigated hydrothermally influenced sediment from the Guaymas Basin. In vitro incubations showed activity of sulfate-dependent methane oxidation between 5 and 70 °C with an apparent optimum between 45 and 60 °C. AOM was absent at temperatures ⩾75 °C. Long-term enrichment of AOM was fastest at 50 °C, yielding a 13-fold increase of methane-dependent sulfate reduction within 250 days, equivalent to an apparent doubling time of 68 days. The enrichments were dominated by novel ANME-1 consortia, mostly associated with bacterial partners of the deltaproteobacterial HotSeep-1 cluster, a deeply branching phylogenetic group previously found in a butane-amended 60 °C-enrichment culture of Guaymas sediments. The closest relatives (Desulfurella spp.; Hippea maritima) are moderately thermophilic sulfur reducers. Results indicate that AOM and ANME archaea could be of biogeochemical relevance not only in cold to moderate but also in hot marine habitats.

Microbial Communities in Methane- and Short Chain Alkane-Rich Hydrothermal Sediments of Guaymas Basin

The hydrothermal sediments of Guaymas Basin, an active spreading center in the Gulf of California (Mexico), are rich in porewater methane, short-chain alkanes, sulfate and sulfide, and provide a model system to explore habitat preferences of microorganisms, including sulfate-dependent, methane- and short chain alkane-oxidizing microbial communities. In this study, hot sediments (above 60°C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed “Mat Mound”) were characterized by porewater geochemistry of methane, C2–C6 short-chain alkanes, sulfate, sulfide, sulfate reduction rate measurements, in situ temperature gradients, bacterial and archaeal 16S rRNA gene clone libraries and V6 tag pyrosequencing. The most abundantly detected groups in the Mat mound sediments include anaerobic methane-oxidizing archaea of the ANME-1 lineage and its sister clade ANME-1Guaymas, the uncultured bacterial groups SEEP-SRB2 within the Deltaproteobacteria and the separately branching HotSeep-1 Group; these uncultured bacteria are candidates for sulfate-reducing alkane oxidation and for sulfate-reducing syntrophy with ANME archaea. The archaeal dataset indicates distinct habitat preferences for ANME-1, ANME-1-Guaymas, and ANME-2 archaea in Guaymas Basin hydrothermal sediments. The bacterial groups SEEP-SRB2 and HotSeep-1 co-occur with ANME-1 and ANME-1Guaymas in hydrothermally active sediments underneath microbial mats in Guaymas Basin. We propose the working hypothesis that this mixed bacterial and archaeal community catalyzes the oxidation of both methane and short-chain alkanes, and constitutes a microbial community signature that is characteristic for hydrothermal and/or cold seep sediments containing both substrates.

Autotrophy as a predominant mode of carbon fixation in anaerobic methane-oxidizing microbial communities.

The methane-rich, hydrothermally heated sediments of the Guaymas Basin are inhabited by thermophilic microorganisms, including anaerobic methane-oxidizing archaea (mainly ANME-1) and sulfate-reducing bacteria (e.g., HotSeep-1 cluster). We studied the microbial carbon flow in ANME-1/ HotSeep-1 enrichments in stable-isotope–probing experiments with and without methane. The relative incorporation of 13C from either dissolved inorganic carbon or methane into lipids revealed that methane-oxidizing archaea assimilated primarily inorganic carbon. This assimilation is strongly accelerated in the presence of methane. Experiments with simultaneous amendments of both 13C-labeled dissolved inorganic carbon and deuterated water provided further insights into production rates of individual lipids derived from members of the methane-oxidizing community as well as their carbon sources used for lipid biosynthesis. In the presence of methane, all prominent lipids carried a dual isotopic signal indicative of their origin from primarily autotrophic microbes. In the absence of methane, archaeal lipid production ceased and bacterial lipid production dropped by 90%; the lipids produced by the residual fraction of the metabolically active bacterial community predominantly carried a heterotrophic signal. Collectively our results strongly suggest that the studied ANME-1 archaea oxidize methane but assimilate inorganic carbon and should thus be classified as methane-oxidizing chemoorganoautotrophs.

Assessing sub-seafloor microbial activity by combined stable isotope probing with deuterated water and 13C-bicarbonate

Sub-seafloor sediments are populated by large numbers of microbial cells but not much is known about their metabolic activities, growth rates and carbon assimilation pathways. Here we introduce a new method enabling the sensitive detection of microbial lipid production and the distinction of auto- and heterotrophic carbon assimilation. Application of this approach to anoxic sediments from a Swedish fjord allowed to compare the activity of different functional groups, the growth and turnover times of the bacterial and archaeal communities. The assay involves dual stable isotope probing (SIP) with deuterated water (D(2) O) and (13) C(DIC) (dissolved inorganic carbon). Culture experiments confirmed that the D content in newly synthesized lipids is in equilibrium with the D content in labelled water, independent of whether the culture grew hetero- or autotrophically. The ratio of (13) C(DIC) to D(2) O incorporation enables distinction between these two carbon pathways in studies of microbial cultures and in environmental communities. Furthermore, D(2) O-SIP is sufficiently sensitive to detect the formation of few hundred cells per day in a gram of sediment. In anoxic sediments from a Swedish fjord, we found that > 99% of newly formed lipids were attributed to predominantly heterotrophic bacteria. The production rate of bacterial lipids was highest in the top 5 cm and decreased 60-fold below this depth while the production rate of archaeal lipids was rather low throughout the top meter of seabed. The contrasting patterns in the rates of archaeal and bacterial lipid formation indicate that the factors controlling the presence of these two lipid groups must differ fundamentally.

Systematic fragmentation patterns of archaeal intact polar lipids by high‐performance liquid chromatography/electrospray ionization ion‐trap mass spectrometry

Archaea are ubiquitous and abundant microorganisms on Earth that mediate key global biogeochemical cycles. The headgroup attached to the sn-1 position of the glycerol backbone and the ether-linked isoprenoid lipids are among the diagnostic traits that distinguish Archaea from Bacteria and Eukarya. Over the last 30 years, numerous archaeal lipids have been purified and described in pure cultures. Coupled high-performance liquid chromatography (HPLC) ion-trap mass spectrometry (ITMS) now enables the detection and rapid identification of intact polar lipids in relatively small and complex samples, revealing a wide range of archaeal lipids in natural environments. Although major structural groups have been identified, the lack of a systematic evaluation of MS/MS fragmentation patterns has hindered the characterization of several atypical components that are therefore considered as unknowns. Here, we examined mass spectra resulting from lipid analysis of natural microbial communities using HPLC/electrospray ionization (ESI)-ITMS(n), and depicted the systematics in MS(2) fragmentation of intact archaeal lipids. This report will be particularly useful for environmental scientists interested in a rapid and straightforward characterization of intact archaeal membrane lipids.

Metabolic Capabilities of Microorganisms Involved in and Associated with the Anaerobic Oxidation of Methane

In marine sediments the anaerobic oxidation of methane with sulfate as electron acceptor (AOM) is responsible for the removal of a major part of the greenhouse gas methane. AOM is performed by consortia of anaerobic methane-oxidizing archaea (ANME) and their specific partner bacteria. The physiology of these organisms is poorly understood, which is due to their slow growth with doubling times in the order of months and the phylogenetic diversity in natural and in vitro AOM enrichments. Here we study sediment-free long-term AOM enrichments that were cultivated from seep sediments sampled off the Italian Island Elba (20°C; hereon called E20) and from hot vents of the Guaymas Basin, Gulf of California, cultivated at 37°C (G37) or at 50°C (G50). These enrichments were dominated by consortia of ANME-2 archaea and Seep-SRB2 partner bacteria (E20) or by ANME-1, forming consortia with Seep-SRB2 bacteria (G37) or with bacteria of the HotSeep-1 cluster (G50). We investigate lipid membrane compositions as possible factors for the different temperature affinities of the different ANME clades and show autotrophy as characteristic feature for both ANME clades and their partner bacteria. Although in the absence of additional substrates methane formation was not observed, methanogenesis from methylated substrates (methanol and methylamine) could be quickly stimulated in the E20 and the G37 enrichment. Responsible for methanogenesis are archaea from the genus Methanohalophilus and Methanococcoides, which are minor community members during AOM (1–7‰ of archaeal 16S rRNA gene amplicons). In the same two cultures also sulfur disproportionation could be quickly stimulated by addition of zero-valent colloidal sulfur. The isolated partner bacteria are likewise minor community members (1–9‰ of bacterial 16S rRNA gene amplicons), whereas the dominant partner bacteria (Seep-SRB1a, Seep-SRB2, or HotSeep-1) did not grow on elemental sulfur. Our results support a functioning of AOM as syntrophic interaction of obligate methanotrophic archaea that transfer non-molecular reducing equivalents (i.e., via direct interspecies electron transfer) to obligate sulfate-reducing partner bacteria. Additional katabolic processes in these enrichments but also in sulfate methane interfaces are likely performed by minor community members.

Recurrent Oil Sheens at the Deepwater Horizon Disaster Site Fingerprinted with Synthetic Hydrocarbon Drilling Fluids

We used alkenes commonly found in synthetic drilling-fluids to identify sources of oil sheens that were first observed in September 2012 close to the Deepwater Horizon (DWH) disaster site, more than two years after the Macondo well (MW) was sealed. While explorations of the sea floor by BP confirmed that the well was sound, they identified the likely source as leakage from an 80-ton cofferdam, abandoned during the operation to control the MW in May 2010. We acquired sheen samples and cofferdam oil and analyzed them using comprehensive two-dimensional gas chromatography. This allowed for the identification of drilling-fluid C16- to C18-alkenes in sheen samples that were absent in cofferdam oil. Furthermore, the spatial pattern of evaporative losses of sheen oil alkanes indicated that oil surfaced closer to the DWH wreckage than the cofferdam site. Last, ratios of alkenes and oil hydrocarbons pointed to a common source of oil found in sheen samples and recovered from oil-covered DWH debris collected shortly after the explosion. These lines of evidence suggest that the observed sheens do not originate from the MW, cofferdam, or from natural seeps. Rather, the likely source is oil in tanks and pits on the DWH wreckage, representing a finite oil volume for leakage.

Respiratory quinones in Archaea: phylogenetic distribution and application as biomarkers in the marine environment.

The distribution of respiratory quinone electron carriers among cultivated organisms provides clues on both the taxonomy of their producers and the redox processes these are mediating. Our study of the quinone inventories of 25 archaeal species belonging to the phyla Eury-, Cren- and Thaumarchaeota facilitates their use as chemotaxonomic markers for ecologically important archaeal clades. Saturated and monounsaturated menaquinones with six isoprenoid units forming the alkyl chain may serve as chemotaxonomic markers for Thaumarchaeota. Other diagnostic biomarkers are thiophene-bearing quinones for Sulfolobales and methanophenazines as functional quinone analogues of the Methanosarcinales. The ubiquity of saturated menaquinones in the Archaea in comparison to Bacteria suggests that these compounds may represent an ancestral and diagnostic feature of the Archaea. Overlap between quinone compositions of distinct thermophilic and halophilic archaea and bacteria may indicate lateral gene transfer. The biomarker potential of thaumarchaeal quinones was exemplarily demonstrated on a water column profile of the Black Sea. Both, thaumarchaeal quinones and membrane lipids showed similar distributions with maxima at the chemocline. Quinone distributions indicate that Thaumarchaeota dominate respiratory activity at a narrow interval in the chemocline, while they contribute only 9% to the microbial biomass at this depth, as determined by membrane lipid analysis.

Starvation and recovery in the deep‐sea methanotroph Methyloprofundus sedimenti

In the deep ocean, the conversion of methane into derived carbon and energy drives the establishment of diverse faunal communities. Yet specific biological mechanisms underlying the introduction of methane‐derived carbon into the food web remain poorly described, due to a lack of cultured representative deep‐sea methanotrophic prokaryotes. Here, the response of the deep‐sea aerobic methanotroph Methyloprofundus sedimenti to methane starvation and recovery was characterized. By combining lipid analysis, RNA analysis, and electron cryotomography, it was shown that M. sedimenti undergoes discrete cellular shifts in response to methane starvation, including changes in headgroup‐specific fatty acid saturation levels, and reductions in cytoplasmic storage granules. Methane starvation is associated with a significant increase in the abundance of gene transcripts pertinent to methane oxidation. Methane reintroduction to starved cells stimulates a rapid, transient extracellular accumulation of methanol, revealing a way in which methane‐derived carbon may be routed to community members. This study provides new understanding of methanotrophic responses to methane starvation and recovery, and lays the initial groundwork to develop Methyloprofundus as a model chemosynthesizing bacterium from the deep sea.

Determining the flux of methane into Hudson Canyon at the edge of methane clathrate hydrate stability

Methane seeps were investigated in Hudson Canyon, the largest shelf-break canyon on the northern US Atlantic Margin. The seeps investigated are located at or updip of the nominal limit of methane clathrate hydrate stability. The acoustic identification of bubble streams was used to guide water column sampling in a 32 km2 region within the canyons thalweg. By incorporating measurements of dissolved methane concentration with methane oxidation rates and current velocity into a steady-state box model, the total emission of methane to the water column in this region was estimated to be 12 kmol methane per day (range: 6 – 24 kmol methane per day). These analyses suggest this methane is largely retained inside the canyon walls below 300 m water depth, and that it is aerobically oxidized to near completion within the larger extent of Hudson Canyon. Based on estimated methane emissions and measured oxidation rates, the oxidation of this methane to dissolved CO2 is expected to have minimal influences on seawater pH. This article is protected by copyright. All rights reserved.