Page 232 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 10. Co-culturing methanogens and methanotrophs in an MBR
Technologies, Santa Clara CA, USA) equipped with a Porapaq Q column heated at 80°C with helium as carrier gas. Data were analyzed with the MSD ChemStation F01.01.2317 (Agilent Technologies, Santa Clara, CA, USA). All gas measurements were performed in duplicate.
Acetate. Acetate concentrations were determined according to the protocol described by Kage et al. with the following modifications: An internal standard (IS) was prepared by dissolving 0.1 mM methylstearate (MS) in n-hexane (Kage et al. 2004). For each reaction, 40 μl supernatant sample, 40 μl 0.5 M phosphate-buffered saline (PBS 65 mM NaCl, 5 mM phosphate buffer pH 7.4 [80% Na2HPO4 and 20% NaH2PO4]), and 200 μl pentafluorobenzyl bromide (PFBBr) in acetone at a concentration of 100 mM were mixed and incubated for 1 h at 60°C. Acetate standard solutions were prepared according to Kage et al. (2004) using sodium acetate in Milli-Q water with a concentration range from 0 to 10 mM. Four hundred microliters of the 0.1 mM MS solution in n-hexane was added, and samples were vortexed for 1 min at RT and centrifuged for 2 min at maximum speed. One hundred microliter aliquots were divided into 12 x 32 mm clear Cronus crimp vials capped with rubber/PTFE snap caps (SMI-LabHut Ltd, Maisemore, UK); 0.1 ml 15 mm tip clear glass inserts were used to reduce sample volume (VWR International BV, Amsterdam, the Netherlands). Pure n-hexane was added to the vials to avoid excess sample evaporation. All samples were injected five times on a JEOL AccuTOF- GCv JMS-100GCv (JEOL Ltd., Akishima, Tokyo, Japan).
Ammonium and nitrite. Ammonium concentrations were measured using 50 μl supernatant sample and 750 μl OPA Reagent (0.54% (w/v) ortho-phthaldialdehyde, 0.05% (v/v) β- mercaptanol and 10% (v/v) ethanol in 400 mM potassium phosphate buffer [pH 7.3]). Samples were vortexed shortly and incubated at room temperature (RT) for 20 min. Absorbance was measured at 420 nm and values compared to a calibration curve with ammonium chloride in Milli-Q. Nitrite concentrations were measured using 100 μl sample, 100 μl nitrite reagent (1% (w/v) sulfanilic acid in 1M HCl and 100 μl 0.1% (w/v) naphtylethylene diaminedihydrochloride in water). Samples were mixed by pipetting and incubated for 20 min at RT. Standard curves were prepared by using a dilution series of sodium nitrite in Milli-Q. Absorbance was measured at 540 nm. Both assays were performed in triplicate in a 96-well plate set-up as described for the protein assay.
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