Cooperative microorganisms in nitrification-denitrification sludge as the hidden helper for rapid anammox enrichment

Nitrification-denitrification sludge is widely used as the seed sludge for anammox bacteria enrichment, yet little is known about which microorganisms it harbors can metabolically support the growth of anammox bacteria. This study focused on exploring the above-mentioned issue during the enrichment of anammox bacteria from nitrification-denitrification sludge collected from a wastewater treatment plant. After 88 days of cultivation in an upflow biological filter reactor, the removal efficiencies of ammonium and nitrite reached 95%, and the relative abundances of Candidatus Brocadia on day 130 were 9.7% in the polypyrrole-modified carrier biofilm (M-PPy), 7.1% in the blank carrier biofilm (B-BC), and 8.8% in the flocculent sludge (B-FS). Unexpectedly, even though the removal efficiencies of ammonium and nitrite remained stably above 95% for 42 days, the most abundant genus across all microbial samples was the heterotrophic bacterium Candidatus Promineifilum, which was also the dominant microorganism in the seed sludge (relative abundance 1.9%). Candidatus Promineifilum exhibited the capacity for folate and molybdopterin cofactor synthesis, as well as nitrite reduction to NO, which were consistent with the characteristics of a cooperative microorganism. Given the auxotrophic nature of anammox bacteria for certain nutrients and the significant positive correlation observed between Candidatus Promineifilum and anammox bacteria, it is plausible that Candidatus Promineifilum is involved in a metabolic interdependency with anammox bacteria and seed sludge enriched with cooperative microorganisms, such as Candidatus Promineifilum, may favor anammox enrichment.