Radioactivity was quantified by scintillation counting (Beckman LSC 6500). The ex-situ CH4 oxidation rates (MOR) were calculated by the following selleck screening library equation: (1) where 14CO2 is the activity of the microbially-produced

CO2, CH4 is the amount of CH4 in the sample, 14CH4 is the activity of the injected CH4, www.selleckchem.com/products/OSI-906.html v is the volume of the sediment and t is the incubation time. DNA extraction For metagenomic analysis, cores I and II were pushed out from the liners and the 0-4 cm bsf and the 10-15 cm bsf horizons were removed for DNA extraction. Multiple parallel 0.5 g subsamples of the cores at each horizon were used for DNA extraction. Total genomic DNA was extracted with a FastDNA®SPIN for Soil Kit (MP Biomedicals) and cleaned using eFT508 purchase Wizard DNA Clean-Up (Promega) according to the manufacturer’s instructions. The DNA quality was assessed by agarose gel electrophoresis and by optical density using a NanoDrop instrument (NanoDrop Products, Thermo Scientific). To get enough high quality DNA for the subsequent 454 sequencing DNA, subsamples from the same horizon were pooled. Of the total DNA isolated from the 0-4 cm horizon, 35% originated from core I and 65% from core

II. For the 10-15 cm horizon, 38% was isolated from core I and 62% from core II. 454 sequencing For creation of the metagenomic libraries, 9.8 μg DNA of the 0-4 cm sample and 6.8 μg of the 10-15 cm sample were used. Sample preparation and sequencing of the extracted DNA were performed at the Norwegian High-Throughput Sequencing Centre (NSC) at CEES [55], University of Oslo according to standard GS FLX Titanium

protocols, except that after the initial dsDNA immobilization, ssDNA was brought into solution by adding 50 μl 1 × TE to the beads, followed by Depsipeptide manufacturer 2 min at 90°C and rapid cooling on ice. The samples were tagged (fusion primers with tag sequences were used to mark sample origin), mixed and sequenced on a 70 × 75 format PicoTiterPlate™ on a GS FLX titanium instrument. The metagenomic reads have been submitted to the Genbank Sequence Read archive [GeneBank: SRP005641]. The average of the mean quality score per sequence was 33.1 (standard deviation: 3.6) and 32.9 (standard deviation: 3.5) for the 0-4 cm metagenome and 10-15 cm metagenome respectively. Replicate removal Replicate reads were removed from the two metagenomes using the 454 Replicate filter [56, 57]. Standard settings of a sequence identity cut off of 0.9, a length difference requirement of 0 and a number of beginning base pairs to check of 3, were used. After removal of replicates, the 0-4 cm metagenome contained 525 reads with more than 2 ambiguous bases and 1222 reads with long homopolymers (> 10 nt), making a total of 1733 (0.65%) low quality reads. The 10-15 cm metagenome contained 395 reads with more than 2 ambiguous bases and 143 reads with long homopolymers (> 10 nt), making a total of 535 (0.