Page 203 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

each time point) to follow the general trend of microbial response to long-term warming exposure of the three distinct zones of a thaw-affected permafrost soil. Here we report the results of the first time point (5 years) after incubation.
Pore water and bulk soil analysis
All initial samples were analyzed for pore water chemical composition and bulk element content. Major cations (NH4+, Ca2+, K+, Mg2+, Na+) and anions (Cl-, NO3-, NO2-, SO42-) in pore water of the initial samples were analyzed by using non-suppressed and suppressed ion chromatography (IC) (Sykam, Fürstenfeldbruck, Germany). Ferric [Fe(III)] and ferrous [Fe(II)] iron were measured with spectrophotometry (DR3900, Hach, Germany) according to the ferrozine method (Viollier et al. 2000). The measurements were conducted in an anaerobic glove box to inhibit oxidation of Fe(II). In addition, bulk soil samples were dried at 70°C and milled for measuring the total carbon and nitrogen content (VarioMAX cube Elementar, Germany). Soil water contents were calculated as the weight difference between wet and dried (105°C) samples. pH values were measured in a suspension of 5 g thawed soil in 12.5 ml distilled water (CG820, Schott AG, Germany).
Anoxic incubations
Samples from AL, TL and PF were incubated in the dark at 4°C under anoxic conditions for
over five years. Here, the selected incubation temperature was the MST of 4.1°C at a depth of 9 20 cm in a warm August (Boike et al. 2013) under oxygen depleted conditions, which are
common in this area (Liebner et al. 2011). No nutrient amendment mimicked the boundary
response and minimized additional biases. During the incubation, GHG production was
followed for 1163 days (Walz et al. 2017; Knoblauch et al. 2018). As the CH4 production rates
remained fairly constant and low on the long term, production rate measurements were stopped
after 1163 days but the incubations were kept as mentioned above. The anoxic incubations were
prepared under a N2 atmosphere in a glove box. Briefly, samples were thawed and equilibrated
in a refrigerator at about 2°C for 1 day. Approximately 20 g of thawed soil was weighed into
glass bottles and sealed with butyl-rubber stoppers. Each bottle was amended with 10 ml of N2-
flushed, O2-free demineralized water and the headspace was thoroughly exchanged with N2 for
30 minutes. Subsequently, CO2 and CH4 concentrations were measured repeatedly, with a
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