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Phylogenetic analysis of bacteria and archaea on the seafloor surface of the Nankai Trough was conducted. DNA was extracted from a total of 14 samples (2 samples from 1 methane seep area and 12 samples from 12 general seafloor areas) and analyzed, targeting the 16S rRNA gene. As a result of the phylogenetic analysis of bacteria, 1 clone was found to have 96% homology with the 16S rRNA gene sequence of Methylomicrobium alcaliphilum, a halophilic methane-oxidizing bacterium, in a sample taken from the methane seep area. However, overall, no characteristic pattern was observed in the bacterial community structure between the methane seep area and the general seafloor. In contrast, in archaea, the genus Methanosarcina was predominantly detected in the samples from the methane seep area. Among them, many sequences that were closely related to anaerobic methane-oxidizing archaea, which perform anaerobic methane oxidation, were detected.

Background Bioelectrochemical methane oxidation catalysed by anaerobic methanotrophic archaea (ANME) is constrained by limited methane bioavailability as well as by slow kinetics of extracellular electron transfer (EET) of ANME. In this study, we tested a combination of two strategies to improve the performance of methane-driven bioelectrochemical systems that includes (1) the use of hollow fibre membranes (HFMs) for efficient methane delivery to the ANME organisms and (2) the amendment of ferricyanide, an effective soluble redox mediator, to the liquid medium to enable electrochemical bridging between the ANME organisms and the anode, as well as to promote EET kinetics of ANME. Results The combined use of HFMs and the soluble mediator increased the performance of ANME-based bioelectrochemical methane oxidation, enabling the delivery of up to 196 mA m−2, thereby outperforming the control system by 244 times when HFMs were pressurized at 1.6 bar. Conclusions Improving methane delivery and EET are critical to enhance the performance of bioelectrochemical methane oxidation. This work demonstrates that by process engineering optimization, energy recovery from methane through its direct oxidation at relevant rates is feasible.

Methane is a potent greenhouse gas and one of the major contributors to global warming. Aquatic systems, both marine and freshwater, contribute to methane emissions, as methane is produced in anoxic sediments. Methane consumption, which can decrease methane emissions, can take place in both oxic and anoxic environments, and is performed by methane oxidizing archaea or bacteria, called methanotrophs. As methane consumption proceeds via a redox reaction – methane oxidation coupled to the reduction of another compound – the availability of suitable electron acceptors has a large impact on the methane removal rates. When considering relevant electron acceptors for methane oxidation, it is, however, important to know which methanotrophs are present in the system, and which electron acceptors they are capable of using. In this thesis, methane oxidizing bacteria are studied in the eutrophic, temperate zone Lacamas Lake (WA, US). Incubation experiments showed enhanced methane oxidation rates after nitrate or sulfate were added to anoxic water column samples. Genomic analysis showed, however, that no complete denitrification pathway was present in the dominant methanotroph, a Methylobacter species. Enrichment culturing and laboratory incubation experiments were performed to further unravel the interaction between methane oxidation and nitrate. One non-methanotrophic species, belonging to the genus Methylotenera, was discovered to be highly abundant. Further analysis showed that the genome of this species contained all genes required for denitrification. Stable isotope labeling studies showed that methane-derived carbon was incorporated in both Methylobacter and Methylotenera cells, suggesting that the methanotroph Methylobacter produced methane-derived carbon compounds that were used by the non-methanotrophic Methylotenera. This thesis also contains a study on anaerobic methane oxidizing archaea (ANME) of the Black Sea water column. Marine anaerobic methane oxidation (AOM) is generally assumed to be coupled to sulfate reduction, via a consortium of ANME and sulfate reducing bacteria (SRB). ANME-1 are, however, often found as single cells, or only loosely aggregated with SRB, suggesting they perform a form of AOM independent of sulfate reduction. Oxidized metals and humic substances have been suggested as potential electron acceptors for ANME, but up to now, AOM linked to reduction of these compounds has only been shown for the ANME-2 and ANME-3 clades. The Black Sea methanotrophic archaea belong to the ANME-1b clade. Our incubation studies suggest that humic compounds increase methane oxidation rates. Large shifts in the microbial community were observed after the addition of humic substances, opening up the opportunity that partner species may be involved in humic substance stimulated methane oxidation.

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 degrees C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed "Mat Mound") were characterized by porewater geochemistry of methane, C-2-C-6 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 Glade 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.

Cytochromes c (Cytc) are widespread electron transfer proteins and important enzymes in the global nitrogen and sulfur cycles. The distribution of Cytc in more than 300 archaeal proteomes deduced from sequence was analyzed with computational methods including pattern and similarity searches, secondary and tertiary structure prediction. Two hundred and fifty-eight predicted Cytc (with single, double, or multiple heme c attachment sites) were found in some but not all species of the Desulfurococcales, Thermoproteales, Archaeoglobales, Methanosarcinales, Halobacteriales, and in two single-cell genome sequences of the Thermoplasmatales, all of them Cren- or Euryarchaeota. Other archaeal phyla including the Thaumarchaeota are so far free of these proteins. The archaeal Cytc sequences were bundled into 54 clusters of mutual similarity, some of which were specific for Archaea while others had homologs in the Bacteria. The cytochrome c maturation system I (CCM) was the only one found. The highest number and variability of Cytc were present in those species with known or predicted metal oxidation and/or reduction capabilities. Paradoxical findings were made in the haloarchaea: several Cytc had been purified biochemically but corresponding proteins were not found in the proteomes. The results are discussed with emphasis on cell morphologies and envelopes and especially for double-membraned Archaea-like Ignicoccus hospitalis. A comparison is made with compartmentalized bacteria such as the Planctomycetes of the Anammox group with a focus on the putative localization and roles of the Cytc and other electron transport proteins.

To obtain knowledge on how regional variations in methane seepage rates influence the stratification, abundance, and diversity of anaerobic methanotrophs (ANME), we analyzed the vertical microbial stratification in a gravity core from a methane micro-seeping area at Nyegga by using 454-pyrosequencing of 16S rRNA gene tagged amplicons and quantitative PCR. These data were compared with previously obtained data from the more active G11 pockmark, characterized by higher methane flux. A down core stratification and high relative abundance of ANME were observed in both cores, with transition from an ANME-2a/b dominated community in low-sulfide and low methane horizons to ANME-1 dominance in horizons near the sulfate-methane transition zone. The stratification was over a wider spatial region and at greater depth in the core with lower methane flux, and the total 16S rRNA copy numbers were two orders of magnitude lower than in the sediments at G11 pockmark. A fine-scale view into the ANME communities at each location was achieved through operational taxonomical units (OTU) clustering of ANME-affiliated sequences. The majority of ANME-1 sequences from both sampling sites clustered within one OTU, while ANME-2a/b sequences were represented in unique OTUs. We suggest that free-living ANME-1 is the most abundant taxon in Nyegga cold seeps, and also the main consumer of methane. The observation of specific ANME-2a/b OTUs at each location could reflect that organisms within this clade are adapted to different geochemical settings, perhaps due to differences in methane affinity. Given that the ANME-2a/b population could be sustained in less active seepage areas, this subgroup could be potential seed populations in newly developed methane-enriched environments.