Carbon and hydrogen isotope fractionation during uncultured aerobic oxidation of short-chain alkanes that discharged from a natural hydrothermal system

Carbon isotope fractionation factors (εC) of methane (C1), ethane (C2), and propane (C3), and hydrogen isotope fractionation factor (εH) of C1 were determined from uncultured closed system experiment of gases discharged from an intertidal Lutao hydrothermal system. Although we did not introduce any strains or substrates, the Lutao short-chain alkanes experienced significant aerobic oxidation by methanotrophs (dominated by Methylomicrobium and Methylophaga) via the ribulose monophosphate pathway (RuMP). The relative oxidation rates rank in an order of C1 > C2 > C3 (vent A samples) or C2 > C3 > C1 (vent B samples). The estimated turnover time for methane against oxidation was 2.2 years. Fractionation factors were calculated using first-order equations (Rayleigh model) by comparing the stable isotope values with their residual concentrations. The εC values decreased with increasing carbon number, as -37.1 ± 1.9‰ for C1, -12.8 ± 1.9‰ for C2, and -5.2 ± 1.9‰ for C3. The εC × n increases linearly with carbon number (n), showing a slope of 10.75 and a correlation coefficient of 0.9984. The εH of C1 was determined as -263 ± 60‰. Both the εC and εH in this study are larger than previously reported values, possibly ascribed to the limited O2 supply and low cell density for our gas samples. Furthermore, after certain degrees of aerobic oxidation with oxidation rates and εC of this study, thermogenic or microbial alkanes would exhibit reversed carbon isotope patterns that comparable to abiotic hydrocarbons. This study demonstrates that aerobic oxidation could produce significant isotope fractionation in alkanes discharged from natural settings similar to this study (closed systems with moderate temperatures, and limited supply of substrates and O2), e.g., cold seeps, mud volcanoes, gas hydrate reservoirs, and hydrothermal aquifers.