Page 110 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 5. Early Holocene carbon storage and microbial activity in North Sea peats
Activity studies
Incubation experiments. Sediment cores for the activity study were drilled on 27 and 28 June 2018 and stored for 10 months at 4°C until further processing. Material was taken aseptically from the carbon-rich (dark black/brown) peat layer and stored in sterile 50-ml falcon tubes kept on ice at 4°C during transport. A total sediment slurry volume of 1.5 l was obtained by mixing 750 g (0.5 l volume) of peat with artificial sea water (0.546 M Cl-, 0.469 M Na+, 0.0528 M Mg2+, 0.0282 SO42-, 0.0103 M Ca2+, 0.0102 M K+, 0.0012 M CO32-, 0.000844 M Br-, 0.000091 M Sr2+, 0.000416 M B-, 0.00935 M NH3+, 0.00367 M PO43-) (Dickson, A. G. & Goyet 1994) amended with 1 ml/l 1000x trace element solution SL-10 with 24 mg l−1 CeCl3 · 7H2O, 30 mg l−1 Na2SeO3 · 5H2O and 40 mg l−1 Na2WO4 · 2H2O (DSMZ) and adjusted to pH 7.0. Under continuous mixing, 50-ml sludge aliquots were transferred to 120-ml sterile glass serum bottles. The bottles were sealed with airtight butyl rubber stoppers and capped with open-top aluminium crimp caps. All incubations were carried out in triplicate per condition.
Methanogenic incubations were carried out anoxically with acetate (20 mM), H2/CO2 (20 mM H2 with 20% CO2 in headspace), H2/methanol (10 mM H2, 10 mM MeOH), and trimethylamine (20 mM). For methoxydotrophic methanogenesis, incubations were started with methoxyphenol (3 mM) and trimethylbenzoate (3 mM). For sulfate-dependent methanotrophy, samples were incubated with 28.2 mM sulfate, the concentration present in the artificial seawater, and 5% (~2 mM) 13C-CH4. The anoxic control incubations were unamended. Anoxic conditions were created by three 15-min cycles of vacuuming and subsequent gassing for 3 min with 1 bar overpressure. The overpressure was removed before starting the incubations. The gas mixture contained 80% N2 and 20% CO2 except for the incubations for hydrogen-dependent methylotrophic methanogenesis, which were gassed with 100% N2. To remove trace oxygen, 0.5 ml of 150 g/l L-cysteine-HCl and 0.5 ml of 150 g/l Na2S were added. To inhibit excessive growth of sulfate-reducing bacteria, a sterile sodium molybdate solution was added at a final concentration of 1.5 mM to all incubations with H2 (Banat Nedwell and Balba, 1983). A new dose of 10 mM H2 was added to the H2/CO2 incubations at 30 and 49 days of incubation and to the H2/methanol incubations at 35 and 49 days of incubation. A second dose of 10 mM MeOH was added to the H2/methanol incubations at 63 days of incubation.
For aerobic methanotrophic incubations, air was used as the headspace and amended with 10 mM CH4. Oxic control incubations contained only air. All substrate concentrations were
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