Hydrogen isotope fractionation in lipids of the methane-oxidizing bacterium Methylococcus capsulatus

Hydrogen isotopic compositions of individual lipids from Methylococcus capsulatus, an aerobic, methane-oxidizing bacterium, were analyzed by hydrogen isotope-ratio-monitoring gas chromatography-mass spectrometry (GC-MS). The purposes of the study were to measure isotopic fractionation factors between methane, water, and lipids and to examine the biochemical processes that determine the hydrogen isotopic composition of lipids. M. capsulatus was grown in six replicate cultures in which the deltaD values of methane and water were varied independently. Measurement of concomitant changes in deltaD values of lipids allowed estimation of the proportion of hydrogen derived from each source and the isotopic fractionation associated with the utilization of each source. All lipids examined, including fatty acids, sterols, and hopanols, derived 31.4 +/- 1.7% of their hydrogen from methane. This was apparently true whether the cultures were harvested during exponential or stationary phase. Examination of the relevant biochemical pathways indicates that no hydrogen is transferred directly (with C-H bonds intact) from methane to lipids. Accordingly, we hypothesize that all methane H is oxidized to H2O, which then serves as the H source for all biosynthesis, and that a balance between diffusion of oxygen and water across cell membranes controls the concentration of methane-derived H2O at 31%. Values for alpha(l/w), the isotopic fractionation between lipids and water, were 0.95 for fatty acids and 0.85 for isoprenoid lipids. These fractionations are significantly smaller than those measured in higher plants and algae. Values for alpha(l/m), the isotopic fractionation between lipids and methane, were 0.94 for fatty acids and 0.79 for isoprenoid lipids. Based on these results, we predict that methanotrophs living in seawater and consuming methane with typical deltaD values will produce fatty acids with deltaD between -50 and -170parts per thousand, and sterols and hopanols with deltaD between - 150 and -270parts per thousand, Copyright (C) 2002 Elsevier Science Ltd.