Large D/H variations in bacterial lipids reflect central metabolic pathways

Abstract

Large hydrogen-isotopic (D/H) fractionations between lipids and growth water have been observed in most organisms studied to date. These fractionations are generally attributed to isotope effects in the biosynthesis of lipids, and are frequently assumed to be approximately constant for the purpose of reconstructing climatic variables. Here, we report D/H fractionations between lipids and water in 4 cultured members of the phylum Proteobacteria, and show that they can vary by up to 500 per thousand in a single organism. The variation cannot be attributed to lipid biosynthesis as there is no significant change in these pathways between cultures, nor can it be attributed to changing substrate D/H ratios. More importantly, lipid/water D/H fractionations vary systematically with metabolism: chemoautotrophic growth (approximately -200 to -400 per thousand), photoautotrophic growth (-150 to -250 per thousand), heterotrophic growth on sugars (0 to -150 per thousand), and heterotrophic growth on TCA-cycle precursors and intermediates (-50 to +200 per thousand) all yield different fractionations. We hypothesize that the D/H ratios of lipids are controlled largely by those of NADPH used for biosynthesis, rather than by isotope effects within the lipid biosynthetic pathway itself. Our results suggest that different central metabolic pathways yield NADPH--and indirectly lipids--with characteristic isotopic compositions. If so, lipid deltaD values could become an important biogeochemical tool for linking lipids to energy metabolism, and would yield information that is highly complementary to that provided by (13)C about pathways of carbon fixation.

Fig. 1.

Summary of D/H fractionations between fatty acids and water observed in culture experiments, native specimens, and marine organic matter fractions. Plotted fractionations are based on the average δD value of palmitic acid, or the fatty acid/alkane of nearest chain length, in the culture with water δD closest to 0‰. Bacterial cultures from this study (*) are C. oxalaticus (Co), C. necator (Cn), E. coli (Ec), and R. palustris (Rp). Organisms from other studies (, , –21, 61) are cultured bacteria D. autotrophicum (Da), Sporumusa sp. (Sp), and M. capsulatus (Mc), cultured phytoplankton B. braunii (Bb), Alexandrium fundyense (Af), Isochrysis galbana (Ig), and natural specimens of brown alga Undaria pinnatifida (Up), red alga Binghamia californica (Bc), and seagrass Zostera marina (Zm). The gray box covers the range of fractionations observed in marine POM and sediments (17, 18). Growth substrates are oxalate (ox), formate (fo), fructose (fr), glucose (gc), gluconate (gl), pyruvate (py), acetate (ac), succinate (su, sut), LB (lb), H₂ + CO₂ (hc), H₂ + SO₄²⁻ (hs), methane (me), acetate+light (ph), CO₂ + light (pa), S₂O₃²⁻ + CO₂ + light (apa). Error bars for culture Co*-ox are the standard deviation (±1σ) for 4 sets of biological replicates; other cultures were not replicated. Typical analytical uncertainties are ± 3.5‰ for all cultures.

Fig. 2.

Regressions of δD values for palmitic acid versus water for C. oxalaticus grown on oxalate, formate, acetate, and succinate. Error bars represent 1σ uncertainty for biological replicates. Data for other lipids are in Fig. S2 and Table S3. Each regression provides constraints on fractionations that can be described most succinctly as a single fractionation curve (see Fig. 3).

Fig. 3.

Fractionation factor curves for palmitic acid in C. oxalaticus grown on formate, acetate, and succinate, and in E. coli grown on glucose and LB. Each curve represents the set of all possible combinations of α_l/s and α_l/w satisfying the constraints imposed by parallel cultures with differing R_w (i.e., 1 linear regression in Fig. 2). Filled circles indicate values corresponding to X_w = 0.5. Gray shaded area defines up to 600‰ variation between α_l/s and α_l/w. Increases in the true value of α_l/s, α_l/w, and X_w shift curves up, to the right, or down a diagonal as detailed in Fig. S3.