3, with OD440 nm of 0.1 corresponding to 24 mg dry biomass (L)−1 was used. Protein was quantified according to Bradford (1976) using bovine serum albumen standards. Methylococcus capsulatus (Bath) was grown in 500 mL

volumes of NMS medium in 2-L Erlenmeyer flasks under air supplemented with 19% (v/v) methane and 1% (v/v) carbon dioxide. Flasks were sealed with red rubber ‘Suba Seal’ vaccine stoppers and were incubated at 45 °C in SB431542 cost an orbital incubator (Gallenkamp, Loughborough, UK) at 120 r.p.m. Cells were harvested at late exponential phase by centrifugation at 13 000 g for 30 min at 4 °C with one wash in NMS and resuspension in 50 mM PIPES-HCl at pH 7.2. QuickFit Erlenmeyer flasks of capacity 250-mL were modified by the Glass Workshop at the University of Warwick to attach QuickFit test tubes of 8-mL volume with a short length of glass tubing (Fig. 1), in the style of BioMeter flasks. Hereafter, the Erlenmeyer (A) is termed ‘flask’ and the test tube (B),

‘trap’. Both openings were sealed using ‘Suba Seal’ vaccine stoppers (C) with three coats of polytetrafluoroethylene dry lubricant spray (RS see more Components) to minimize methane adsorption. Vaccine stoppers were pierced with 19G, blunt-ended, hollow surgical steel needles (Beckton, Dickinson & Co., Rutherford, NJ or Studley Surgical Needle Co., Studley, UK), sufficiently long to reach the bottom of the flask and the trap (D), with integral Luer-Slip™ female tapers. Luer-Lok™ Nintedanib (BIBF 1120) polycarbonate taps (E; Cole-Parmer Instrument Company Ltd, Hanwell, UK) were attached to the needles. Cell suspensions in

NMS (50 mL containing 12 mg dry biomass) were placed in flasks with 5 mL 21.6 M KOH solution in the trap, ensuring that it could not enter the flask. Killed controls were prepared by incubating flasks containing cells suspended in 5.2 M formaldehyde in NMS on ice for 10 min. Experimental flasks were also preincubated in this way to avoid any variance. Cell-free controls were performed with or without the addition of formaldehyde, and no significant difference in carbon partitioning between trap and flask was observed (data not shown). Radiorespirometry experiments were conducted at 45 °C in a gyrotory water bath shaker (New Brunswick, Edison, NJ) with moderate agitation. Nine millilitres of methane and 1 mL of carbon dioxide were injected, and flasks were allowed to preincubate for 10 min. 2.3μCi [14C]-methane (43 nmol) was injected along with, in some flasks, 0.5 mL of 1 M HgCl2 solution to give a final concentration of 10 mM. At 5 min intervals, 2 mL volumes of cell suspension were removed with 0.5 mL volumes from the trap for scintillation counting. Cells were harvested onto nitrocellulose filters of 0.2-μm pore size (Whatman LTD, Maidstone, UK) supported on 0.45-μm glass fibre filters (Whatman LTD) using a vacuum and were washed with 25 mL 0.1 M HCl to remove [14C]-carbonates followed by 25 mL of NMS.