Metagenomic Insights into <i>Candidatus</i> Scalindua in a Long-term Cultivated Marine Anammox Consortium: The Important Role of Tetrahydrofolate-mediated Carbon Fixation

Marine anammox bacteria have been an exciting research area in recent years due to their high effectiveness in treating ammonia-containing saline wastewater. However, their direct implementation in the wastewater industry faces challenges due to slow growth, difficulty obtaining pure cultures, and their tendency to exist as part of an anammox consortium, interacting symbiotically with other bacteria. In the present study, 91 draft genome metagenome-assembled genomes (MAGs) from a long-term-operated reactor were recovered to clarify detailed symbiotic interactions within an anammox consortium. One marine anammox bacterial MAG, identified as Candidatus Scalindua, was successfully recovered and was abundant within the sampled microbial community. A comprehensive metabolic pathway ana-lysis revealed that Ca. Scalindua exhibited the complete anammox pathway and the Wood-Ljungdahl pathway for carbon fixation. The folate biosynthesis pathway in Ca. Scalindua was incomplete, lacking dihydrofolate reductase, a key enzyme for tetrahydrofolate (THF) production. The folate biopterin transporter, essential for transporting folate-related metabolites among coexisting bacteria, was identified exclusively in Ca. Scalindua. In addition, the impact of exogenously supplied THF on microbial activity and carbon uptake rates was investigated in batch experiments using ¹⁴C-labeled bicarbonate. The results obtained revealed that 2‍ ‍mg L^-1 of exogenous THF resulted in a 43% increase in the carbon uptake rate, while anammox activity remained unaffected. The present results suggest that THF is a key intermediate for carbon fixation in Ca. Scalindua and may be essential for their growth.