Page 231 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

50 ml reactor fluid from the upper layer in 20 min. Triplicate samples were taken every 3-4 days. Cells and supernatant were taken aseptically from the MBR and separated by centrifugation for 10 min at maximum speed (20,348 x g) and used for protein determination, FISH microscopy, and activity tests.
Monitoring of growth and substrate metabolism
OD600 and protein assay. After sterilization, the bioreactor was inoculated with M. sporium. Batch culture and reactor optical density were measured on a Bio-Rad SmartSpecTM 3000 spectrophotometer with the pre-set OD600 method (Bio-Rad Laboratories, Veenendaal, the Netherlands) using 1.6 ml semi-micro polystyrene cuvettes (Sarstedt AG & Co. KG, Nümbrecht, Germany). For analysis of total protein contents, duplicate 1.5 ml reactor liquid pellets were resuspended in 0.3 ml 3M NaOH and boiled for 10 min at 95°C. Samples were cooled down to RT, neutralized with 0.3 ml 3 M HCl, and centrifuged for 1 min at maximum speed. Ten microliters supernatant was loaded onto a polystyrene flat-based 96-well microtest plate (Sarstedt AG & Co. KG, Nümbrecht, Germany). To each well, 200 μl working solution (50 parts of Pierce BCA Protein Assay Kit, reagent A [article no 23223] and 1 part of 4% cupric sulfate in Milli-Q water was added and mixed by pipetting. Samples were incubated for 30 min at 60°C. After cooling down to room temperature, the absorbance was measured at 562 nm on a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale CA, USA), and data were analyzed using SoftMax Pro 6.4 (Molecular Devices, Sunnyvale CA, USA). Values were compared to a standard curve with bovine serum albumin (BSA) in Milli-Q. OD600 values and total protein analysis achieved similar results (Supplementary Information Fig. S1).
Methane and oxygen. For all gas determinations, 50μl gas samples were withdrawn with a gas- tight glass syringe (Hamilton, Reno NE, USA). Methane in- and outflow concentrations were measured daily using a HP5890a gas chromatograph (Hewlett Packard 5890a, Agilent Technologies, Santa Clara CA, US) equipped with a Porapaq Q 100/120 mesh and a thermal conductivity detector (TCD) using N2 as carrier gas (Sigma-Aldrich, Saint Louis MI, USA). Data were analyzed using GC ChemStation Rev. A.10.02 (Agilent Technologies, Santa Clara CA, USA). Oxygen concentrations were determined with the same gas sampling and volume on an Agilent 6890 series gas chromatograph coupled to a mass spectrometer (Agilent
10
229