Determination of Carbon-Fixing Potential of Bathyarchaeota in Marine Sediment by DNA Stable Isotope Probing Analysis

Bathyarchaeota is believed to play a crucial role in the global carbon cycle due to its vast biomass,broad distribution,and diverse habitat.However,its physiological and metabolic features are hard to determine without pure culture.While metagenomic analyses have shown that Bathyarchaeota has a complete inorganic carbon fixation (Wood-Ljungdahl,WL)pathway,no direct functional confirmation has been reported.To explore the inorganic carbon fixation ability of Bathyarchaeota,we used lignin and sodium bicarbonate- 13 C (NaH 13 CO 3 ) in the long-term incubation of marine sediment samples.We found that Bathyarchaeota grew continuously in the cultivation system with lignin,and its abundance increased up to 15.3 times after10 months,increasing its fraction of all archaea from 30%to 80%.We monitored the δ 13 C of total organic carbon to identify microbial carbon fixation in the cultivation systems,finding that it increased in the first month while NaH 13 CO 3 was present but only increased continuously afterward when lignin was also present.Furthermore,ultracentrifugation was performed on DNA extracted from samples at different cultivation stages to separate DNA of different buoyant densities,and bathyarchaeotal and bacterial 16S ribosomal RNA (r RNA) gene abundance were quantified using qPCR.Compared to bacteria,bathyarchaeotal 16S rRNA tended to be concentrated in heavy layers after 4 months of incubation with lignin and NaH 13 CO 3 ,indicating that Bathyarchaeota DNA contained 13 C through proliferation based on lignin utilization and NaH 13 CO 3 assimilation,proving the carbon fixation capacity of Bathyarchaeota.