In vitro study of microbial carbon cycling in subseafloor sediments

Over geological time, the majority of organic matter that escapes the internal cycling in the biosphere is deposited and preserved in marine sediments. Although the biodegradation of organic matter during early diagenesis beneath the seafloor has long been supported by geochemical evidence, it is only recently that the central parts of microbial carbon cycling, i.e. the microorganisms and their metabolic intermediates, have been intensively examined in subseafloor sediments. Recent studies based on intact polar lipids (IPLs) contributed significantly to the proposition of a 'marine deep biosphere' dominated by live, heterotrophic archaea. In this study, an IPL-stable carbon isotope probing experiment was performed to evaluate the connection between sedimentary archaeal IPLs and benthic archaea. An analytical protocol was also developed to determine the isotopic composition of both the head groups and hydrocarbon chains of the archaeal glycolipids. Among the four 13C-labeled substrates tested (bicarbonate, methane, acetate, and Spirulina platensis cells), only S. platensis cells resulted in significant labeling signals. The glycosidic headgroups exhibited stronger signals of 13C incorporation than the hydrocarbon chains. These results suggest that marine benthic archaea are heterotrophic, and may generate IPLs via an anabolic shortcut that bypasses the energy-costly tetraether biosynthesis. Hydrogen (H2) is a metabolic intermediate that is poorly understood, although there has been a persistent interest in hydrogenotrophic processes in subseafloor sediments. In the present study, the first step to elucidate the H2-fueled carbon cycling was to determine sedimentary H2 concentrations with both the classical 'headspace equilibration technique' and a newly-developed extraction-based procedure. The H2 concentrations obtained by both methods were orders of magnitude higher than the level predicted by thermodynamic calculations, and would be high enough to fuel some hydrogenotrophic trace volatile formation proposed in earlier studies. In the subsequent laboratory experiments, the supplementation of H2 induced the formation of trace volatiles, mainly methylated sulfides. In the lake sediment, the formation of dimethyl sulfide by CO2 reduction was found to be a biological process, whereas in marine sediments, the formation of thiols was an abiotic reaction. The carbon of the thiols was not from CO2 but from another uncharacterized source.