Page 156 - 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
pyridoxine HCl 100 mg L-1, thiamine HCl·2H2O 5 mg L-1, riboflavin 5 mg L-1, nicotinic acid 5 mg L-1, Ca-P-pantothenate 5 mg L-1, cobalamin 0.10 mg L-1, p-aminobenzoic acid 5 mg L-1 and lipoic acid 5 mg L-1. The final pH of the slurries was 6.3.
Methanogenic incubations. 50 mL aliquots of the slurry were distributed over 120 mL glass serum bottles. Triplicate incubations for 4°C and 10°C were prepared for acetoclastic, hydrogenotrophic, methylotrophic and hydrogen-dependent methylotrophic methanogens and biotic controls without any substrates. Final concentrations of 2 mM acetate, TMA or MeOH/H2 were added to the respective incubations. Culture bottles were sealed with airtight red butyl rubber stoppers and secured with open-top aluminum crimp caps. Anaerobic conditions were created by three 15-min cycles of vacuuming and subsequent gassing for three minutes with 1 bar Argon overpressure; in the end the overpressure was removed. For the incubations with hydrogen, H2 (8 mM) and CO2 (2 mM) were added to the serum bottles. To remove trace oxygen, 0.1 mL sterile 150 g L-1 L-cysteine and 150g L-1 Na2S were added. The serum bottles were incubated in the dark at 4°C and 10 °C under continuous shaking at 150 rpm. During incubation, substrates were replenished when needed. For acetate amended cultures, in total 14 mM of acetate was added (pulses of 4 mM at day 64 and 2 mM at day 141, 190, 211 and 234) and 12 mM of TMA (pulses of 2 mM at day 141, 190, 211, 234, 269) during the incubation period of 279 days. For H2/CO2 incubations a total of 56 mM H2 and 22 mM CO2 was added (at day 28, 36, 44, 48, 50, 51, 52, 55, 57, 62) and for H2/methanol incubations this was 16 mM H2 and 16 mM methanol (in pulses of 2 mM on day 10, 21, 28, 36, 44, 141, 255). All concentrations were calculated based on 50 ml liquid volume, assuming all of the substrate dissolves over time. Due to high substrate consumption in the H2/CO2 incubations, biomass was harvested after 64 days. All other enrichment cultures were terminated after 279 days. pH was measured at the end of the incubation period (Metrohm, Herisau, Switzerland; Hanna Instruments, Betuwehaven, the Netherlands). Contents from triplicate individual bottles were pooled and centrifuged for 10 min at 2,800 x rcf (Eppendorf, Nijmegen, the Netherlands). Pellets were stored at -18oC until further analysis.
Aerobic methanotrophic incubations. 50 mL slurry was transferred to 120 mL culture bottles and 2 mM of CH4 (final concentration for 50 mL) was added to the headspace. The biotic
154