Hydrogen isotope fractionation during H2/CO2 acetogenesis: hydrogen utilization efficiency and the origin of lipid-bound hydrogen

Hydrogen metabolism was studied in the anaerobic bacterium, Sporomusa sp. strain DMG 58, by measuring natural abundance levels of deuterium in H-2, H2O, and individual fatty acids during acetogenic growth on H-2/CO2. Four cultures were grown, each in medium with a distinct hydrogen-isotopic composition (delta D-H2O). The delta D value of H-2 was quantified in the residual gas exiting the growth chambers and found to decrease concurrently with net H-2 consumption, indicating rapid isotope exchange between H-2 and H2O. An isotopic mass balance was used to constrain the efficiency with which H-2 was activated by the cell and the reducing equivalents catabolized, which we term the H-2 utilization efficiency. Results indicate that H-2 utilization efficiency in these cultures is less than 20% during the growth phase, and less than 2% after the growth phase. The gross rate of cellular H-2 activation was similar in the growth phase and afterward. Biomass harvested at the end of each experiment was used to analyse the D/H of individual membrane lipids. Values of delta D were highly correlated between lipids and water (delta D-lipids = 0.59 x delta D-water-381 parts per thousand; R-2 = 0.995), indicating the source of lipid hydrogen is in isotopic equilibrium with water. Results are consistent with two possibilities: (i) water is the sole source of hydrogen to lipids, and the fractionation during biosynthesis is significantly larger than previously observed (alpha = 0.59), or (ii) hydrogen from H-2 is incorporated into lipids, but only after reaching isotopic equilibrium with H2O. Fatty acids were strongly depleted in deuterium relative to all other organisms studied thus far, and such large depletions may prove useful as biomarkers for studying H-2 cycling in anoxic environments as well as in the geological record.