Type × methanotrophs use primarily the ribulose monophosphate pathway, but possess the enzymes needed for the serine pathway as well [20]. Stable isotope probing and sequencing of 16S rDNA and pmoA, as well as lipid biomarker analysis, have APO866 solubility dmso detected type-I aerobic methanotrophs in sediments and biofilms at the COP Shane and Brian seeps [21, 22]. Recently, measurements of average δ13C of carbonates and lipid biomarkers associated with ANME and SRB also indicated occurrence of AOM at the Brian seep [23]. Another survey at the Brian seep detected ANME-2 at 6-9 cm bsf (below sea floor) by FISH (Fluorescent in situ hybridization) [24]. In
the present study, we have used metagenomics to characterize the taxonomic and metabolic potential for both aerobic and anaerobic methane oxidation in two sediment samples from different depths at the Tonya seep (COP). By avoiding PCR amplification and primer target specificity, the metagenomics approach offered further insight into the taxonomy and metabolic potential of the prokaryotic communities of the methane seep sediments. Results Gas measurements and methane oxidation rate The average methane oxidation rate based
on 11 measurements in the top 15 cm of the seep sediments was 156 ± 64 nmol cm-3 day-1. Still, the gas emitted from the Tonya seep sediments into the water phase contained a large fraction of methane. Even after travelling 25 DAPT mouse m through the water column, where dissolved O2 and N2 entered the bubbles, the two gas samples contained 80.4% (gas PRIMA-1MET sample I) and 68.1% (gas sample II) methane. When O2 and N2 were excluded, and the hydrocarbon and CO2 content were normalized, methane accounted for 93.6% in both gas samples.
The remainder consisted of CO2 and short chain hydrocarbons (C2, C3, i-C4 and n-C4). Metagenome creation through filtering of reads 454 sequencing resulted in 395540 reads for the 0-4 cm sample and 282964 reads for the 10-15 cm sample. Replicate filtering of the metagenomes removed 33.03% of the reads from the 0-4 cm sample and 31.31% of the reads in the 10-15 cm sample. The resulting metagenomes consisted of 264902 reads (average length 413 ± 138 bases, range 29-1907 bases) for the 0-4 cm sample and 194360 reads (average length of 419 ± 134 bases, range 29-1458 bases) for the Thalidomide 10-15 cm sample. All further analyses were performed on these metagenomes (Figure 1). Unless other ways specified, all percentages throughout the text are given as percent of total reads for each filtered metagenome. Figure 1 Flowchart showing the workflow for taxonomic binning, marker gene annotation and pathway mapping. Abbreviations used in the figure: ncbiP-nr (NCBIs non-redundant Protein Database), mcrA (methyl-coenzyme M reductase), pmoA (particulate methane monooxygenase), dsrAB (dissimilatory sulphite reductase), KAAS (KEGG Automatic Annotation Server) and KEGG (Kyoto Encyclopedia of Genes and Genomes). Estimated effective genome sizes (EGS) were 4.8 Mbp and 4.