The methanotrophs were grown on a nitrate mineral salts medium  in a 4-l fermenter (BioFlo IIc, New Brunswick Scientific) fed with a continuous flow of methane/air (1:2, v/v). Oxygen concentration was followed with a dissolved oxygen probe, and the agitation speed was regulated to maintain the dissolved oxygen level below 5%. M. szegediense OR2 was grown at 52°C with an agitation speed of 250–400 rpm and harvested at 1.7 OD540, and M. tepidum LK6 was grown at 43°C with an agitation speed of 100–150 rpm, and harvested at 1.6 OD540. Cells were harvested by centrifugation (15 000×g, 60 min) and lyophilised.
2.3Detection and isolation of hopanoids
Lyophilised cells were extracted under reflux with CHCl3/CH3OH (3×50 ml, 2:1, v/v). An aliquot of the crude extract (1/10) was treated with H5IO6/NaBH4, in order to cleave the side chain of the hopanoids . The relative amounts of the hopanoids were determined on the resulting derivatives from the integration of the GC peak areas. The remaining part of the crude extracts was acetylated. TLC separation (ethyl acetate/cyclohexane, 7:3, v/v, Rf=0.24) afforded a mixture of the acetates of the aminobacteriohopanepentols 1, 2 and 2a from M. szegediense (9 mg from 1.4 g lyophilised cells), and of the acetates of aminobacteriohopanetetrols 1 and 2 and aminobacteriohopanepentols 3 and 4 from M. tepidum (4 mg from 0.6 g lyophilised cells).
1H NMR of the mixture of the hexa-acetates of 1 (24%), 2 (68%) and 2a (8%) isolated from M. szegediense (400 MHz, CDCl3): δ(ppm)=*0.628 (s, 18α-CH3), *°0.643 (broad s, *4α- and °18α-CH3), *0.774 (s, 4β-CH3), °0.786 (s, 4β-CH3), *0.807 (d, J=6.7, 3β-CH3), °0.808 (s, 4α-CH3); °0.840 (s, 10β-CH3), *0.857 (s, 10β-CH3), 0.921 and 0.931 (2 broad s, °8β- and °14α-CH3; *8β- and *14α-CH3), 0.944 (d, J=6.5 Hz, 22-CH3), 1.948 (3H, s, CH3CONH-), 2.073 (3H, s, CH3COO-), 2.078 (3H, s, CH3COO-), 2.103 (3H, s, CH3COO-), 2.105 (3H, s, CH3COO-), 2.117 (3H, s, CH3COO-), 3.33 (1H, ddd, J35a,35b=14.7 Hz, J34,35a=7.2 Hz, J35a,NH=6.2 Hz, 35-Ha), 3.62 (1H, ddd, J35a,35b=14.7 Hz, J35b,NH=6.2Hz, J34,35b=3.8 Hz, 35-Hb), 5.10 (1H, dt, J34,35a=7.2 Hz, J33,34=4.2 Hz, J34,35b=3.8 Hz, 34-H), 5.24 (3H, m, 30-H, 31-H, 32-H), 5.31 (1H, dd, J33,34=4.2 Hz, J33,32=6.4 Hz, 33-H), +5.53 and +5.64 (2 dd, J11,12=10.8 Hz, J=3.2 Hz, J=2.4 Hz, 11-H and 12-H), 5.69 (1 H, t, J35a,NH=J35b,NH=6.0 Hz, NH). Spectroscopic data labelled with a degree symbol (°) correspond to the spectrum of the acetylated hopanoid (1) and those labelled with an asterisk (*) correspond to the spectrum of its 3β-methyl homologue (2). Those without superscript are common to the spectra of both compounds. Those labelled with a plus symbol (+) correspond to the signals of the vinylic protons of acetylated 2a. Integration was only given for the side chain signals, which was identical in the three investigated hopanoids 1, 2 and 2a.
Acetate of 3-methylhop-11-en-30-ol (12) from M. szegediense (GC/MS, electron impact, 70 eV): m/z=482 (M+, 13%), 467 (M+-CH3, 4%), 381 (M+-side chain, 2%), 276 (retro Diels-Alder reaction, 100%), 249 (ring C cleavage, 9%), 232 (retro Diels-Alder reaction, 94%), 205 (ring C cleavage, 49%), 189 (ring C cleavage-AcOH, 39%). Acetate of adiantol (6) from M. tepidum (GC/MS, electron impact, 70 eV): m/z=496 (M+-AcOH, 3%), 369 (M+-side chain, 1%), 235 (ring C cleavage, 2%), 191 (ring C cleavage, 100%), 175 (ring C cleavage-AcOH, 21%). Acetate of 3-methyladiantol (7) from M. tepidum (GC/MS, electron impact, 70 eV): m/z=410 (M+-AcOH, 8%), 383 (M+-side chain, 3%), 235 (ring C cleavage, 1%), 205 (ring C cleavage, 100%), 175 (ring C cleavage-AcOH, 57%).