Halophilic Anaerobic Cultures Enriched with CO2:H2 from Different Saline Environments Reveal Unknown Autotrophic Bacterial Diversity and Modular Carbon Fixation Pathways

The subsurface sediments of saline-aquatic systems host diverse microbes, with unclear ecological roles and challenging lab cultivability. Chemolithotrophic anaerobes involved in CO 2 -fixation are one of the poorly studied groups. This study focused on understanding these bacteria from subsurface sediments of four representative saline environments, two marine (i.e., Coastal Arabian and Bay of Bengal seas) and two lake (Sambhar and Lonar) systems through enrichment and metagenomics. Enrichment cultures with bicarbonate/CO 2 and hydrogen as the carbon and energy sources, respectively, showed CO 2 fixation, producing acetic and formic acids as the major organic products. Enriched culture with Sambhar Lake sediment produced more formic acid (391 ± 8 mg/L) than acetic acid (92 ± 20 mg/L); however, other enriched cultures produced considerably higher acetic acid (up to 966 ± 24 mg/L) than formic acid (up to 367 ± 30 mg/L). The organics production was accompanied by unique thread-like (up to 500 μm long) aggregates, harbouring chains of rod and oval-shaped microbes in all cultures. Metagenome sequencing revealed dominance of Vibrio spp. (relative sequence abundance of 91% to 97%) across all cultures, while canonical CO 2 -fixing taxa were nearly absent (< 0.01%). KEGG analysis revealed partial genes for various CO 2 fixation pathways, including Wood-Ljungdahl, reverse-TCA, dicarboxylate-hydroxybutyrate, hydroxypropionate bicycle, hydroxypropionate-hydroxybutyrate, and the reductive-glycine pathway. The presence of a near-complete serine variant of the reductive glycine pathway, which has been demonstrated in engineered systems, suggests that this pathway may play an operational role in natural systems. The consistent production of organic acids and incomplete pathway representation suggests modular CO 2 fixation within the Vibrio -dominated enriched mixed cultures.