Page 157 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

control contained only the sediment slurry; the abiotic control contained CH4 and 500 μM chloroform to inhibit general microbial activity. Triplicate serum bottles were incubated in the dark at 4°C and 10°C under continuous shaking at 150 rpm. After near complete oxidation of CH4 or stagnation of methanotrophic activity, bottles were flushed with three times the headspace volume with 0.2 μM filter sterilized air before addition of CH4. Due to high activity, CH4 concentrations were increased to 7.8 mM after two weeks of incubations (1:1.5 CH4:O2). Cultures were harvested after 62 days, using the same method as described above.
Anaerobic methanotroph incubations. About 50 mL of slurry was amended with 0.25 mM nitrite (NO2-) or 2 mM nitrate (NO3-) together with 1 mM 13CH4 in 120 ml culture bottles. Control incubations were prepared either without electron acceptors or 13CH4. Bottles were capped and gassed/degassed as described in the aerobic methanotrophic incubations; a final overpressure of 0.5 bar Argon was applied. Thereafter, 13CH4 was added. The triplicate serum bottles were incubated in the dark at 4°C and 10°C under continuous shaking at 150 rpm. After 125 days, 14 mL of 13CH4 (20% of headspace) was added to stimulate activity. After 246 days, the pH (Hanna Instruments, Betuwehaven, the Netherlands) and concentration of NO2- and NO3- (Griess 1879) were measured by taking an aliquot of the culture, when depleted NO2- and NO3- were added. After, concentrations of NO2- and NO3- were monitored twice a month and were replenished when they dropped below 20 μM for NO2- and 0.8 mM for NO3-. Headspace 13CO2 was monitored for 450 days.
Analysis of substrates and products
Gas samples (50 μL) were withdrawn with a gas-tight glass syringe (Hamilton, Reno NE, USA) and injected into a HP 5890 gas chromatograph (Hewlett Packard, Palo Alto CA, USA) equipped with a Porapaq Q 100/120 mesh (Sigma Aldrich, Saint Louis MI, USA) and a flame ionization detector (FID) for CH4 detection and a thermal conductivity detector (TCD) for measuring H2, CH4 and CO2 simultaneously using N2 as carrier gas. An Agilent 6890 series gas chromatograph coupled to a mass spectrometer (Agilent, Santa Clara CA, USA) equipped with a Porapak Q column heated at 80°C with helium as the carrier gas was used for measurements of CO2 and O2. Concentration of gases in the incubations were calculated by the sum of the gas concentration in the headspace and liquid phase divided by the slurry dry weight. Maximum
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