METHANE- AND WATER-DERIVED H IN LIPIDS PRODUCED BY METHANOTROPHS

Hydrogen isotopic compositions of individual lipids vary significantly, both between similar lipids in a single organism, and between similar organisms. This variability provides opportunities to study both biochemical and organic geochemical processes, but understanding the factors that control such variability is often difficult because multiple hydrogen sources and biochemical pathways exist. To demonstrate a method for studying biological fractionation of hydrogen isotopes where multiple hydrogen sources are present, we grew the aerobic, obligate methanotroph Methylococcus capsulatus on isotopically distinct CH₄ and H₂O. Analysis of the resulting changes in dD values of seven different lipids allows us to estimate that 31 ± 2% of hydrogen in every lipid we isolated, including fatty acids, sterols, and hopanols, is derived from methane. Examination of the relevant biochemical pathways indicates that no hydrogen is transferred directly (with C-H bonds intact) from methane to lipids, so other indirect pathways must exist. Possibilities include NADPH, and H₂O resulting from the oxidation of CH₄. Isotope fractionation associated with the utilization of methane (i.e. a_{lipid/methane}) averages 0.986 for fatty acids and 0.789 for isoprenoid lipids. For water, fractionation averages 0.938 for fatty acids and 0.831 for isoprenoid lipids. Given typical dD values for seawater (0‰) and methane (–200‰), fatty acids from M. capsulatus should have dD values near –100‰, and isoprenoids should have dD values near –230‰. Thus lipids produced by methanotrophs may be slightly D-enriched relative to those from photoautotrophs. Significant differences in dD values also exist between similar lipids from the same culture. 3-methylhopan-30-ol is consistently enriched in D by ~32‰ relative to hopan-30-ol, and 16:0 fatty acid is enriched in D by ~43‰ relative to 16:1 fatty acid. These isotopic differences cannot be explained by the small differences in their structures, and seem to indicate some fundamental difference in their biosynthesis, such as different mediating enzymes, or different pools of substrates or cofactors.