Page 158 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 7. Methane cycling in Arctic thermokarst lake sediments
production and consumption rates of CH4 were calculated by subtracting each measure point by previous time point and divide it by days and grams of dry weight of 50 ml slurry. The Q10 coefficient was calculated as described in Sepulveda-Jaurequi et al. (2018). The Q10-max was determined by the comparison of the rates occurring at the same time point. The final Q10 was determined by using the final measurement. Two-sided t-tests were performed in Microsoft Office Excel (2016). Nitrate concentrations in the cultures were estimated with Merckoquant test strips (0-80 mg L-1; Merck, Germany), nitrite was measured using the nitrite determination protocol by Griess (1879).
Molecular analysis
DNA isolation. Sediment samples were taken aseptically from the original sediments and stored at -18°C until DNA isolation. Samples from batch incubation cultures were pelleted by centrifugation for 10 min at 20,000 x g (Eppendorf, Nijmegen, the Netherlands). Samples for DNA analysis were obtained by pooling equal amounts of pelleted slurry sample per triplicate bottle. DNA was extracted in duplicate per sample using the PowerSoil DNA Isolation Kit (MO BIO, Qiagen, Venlo, the Netherlands) following the manufacturer’s instructions with the following modifications: the initial PowerBead Tube vortex step was carried out using a bead beater at 30 bps for 10 minutes. The primary centrifugation step was done for 1 min at 10,000 x g. DNA elution was performed with 2 x 25 μL sterile Milli-Q with 2 min incubation at room temperature prior to centrifugation. The second elution centrifugation step was carried out for 1 min at 10,000 x g. DNA quality was checked by agarose gel electrophoresis, spectrophotometrically using the NanoDrop 1000 (Invitrogen, Thermo Fisher, Carlsbad CA, USA) and fluorometrically using the Qubit dsDNA HS Assay Kit (Invitrogen, Thermo Fisher, Carlsbad CA, USA) according to the manufacturer’s instructions. Duplicate samples with highest yield and quality were selected for downstream applications.
Phylogenetic analysis. To assess the diversity of Bacteria and Archaea, 16S rRNA gene amplicon sequencing was performed on an Illuminia Miseq Next Generation Sequencing by Macrogen, Korea. The primers for bacterial amplification were Bac341F (5’- CCTACGGGNGGCWGCAG-3’) (Herlemann et al. 2011) and Bac806R (5’- GGACTACHVGGGTWTCTAAT-3’) (Caporaso et al. 2012). For archaeal amplification the
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