Page 107 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

dry mass was measured using an air pycnometer. The bulk density of the soil material was calculated by dividing the volume of the dried sediment by the dry mass. The total pore space was calculated by subtracting the measured volumes of water from the original fixed sample volume.
Total carbon storage was estimated using the following formula (Erkens, Van Der Meulen and Middelkoop 2016):
Total C (kg) = OMd * V * Cf
where OMd is organic matter density (kg m-3), V is peat volume (m-3), and Cf is an OM-to-
carbon conversion factor for peat deposits.
Pore water analysis
Samples for pore water analysis were extracted using 5-cm MOM Rhizon samplers (produced by Rhizosphere Research Products, Wageningen) at 10-cm resolution and stored at 4°C until further analysis. The samples were acidified with 1% HNO3 and analyzed by inductive coupled plasma-optical emission spectrometry (ICP-OES) for Al, Ca, Fe, K, Mg, Mn, Na, P, S, Si, and Zn (iCap 6300, Thermo Scientific, Waltham, MA) and continuous flow analysis (CFA) for NO3-, NH4+, PO43-, Na+, K+ and Cl- (Bran+Luebbe Auto Analyzer, SPX Flow, Norderstedt, Germany; Seal Analytical AutoAnalyzer 3, Seal Analytical, Southampton, UK; Table S4.).
Molecular analyses
Four cores in the southern North Sea were selected for 16S rRNA amplicon sequencing, and four cores from the Doggerbank area were selected for microbial activity studies.
DNA isolation. Samples for DNA isolation were immediately extracted aseptically upon sampling. Samples were stored at -20°C until further analysis. DNA was extracted in duplicate per sample using the Qiagen DNeasy Power Soil Kit (Qiagen, Venlo, the Netherlands) following the manufacturer’s instructions with the following modifications: the initial PowerBead Tube vortex step was carried out using a TissueLyser LT (Qiagen, Venlo, the Netherlands) at 50 Hz for 10 min, and the primary centrifugation step was increased to 1 min at 10,000 xg. DNA was eluted with 2×30 μl of sterile Milli-Q incubated for 2 min at room temperature prior to centrifugation. The second elution centrifugation step was carried out for
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