Metagenomic insights into the symbiotic relationship in anammox consortia at reduced temperature

Anammox as a promising biological nitrogen removal technology has attracted much attention. However, cold temperature would limit its wide application and little is known about the microbial interactions between anammox bacteria (AnAOB) and heterotrophic bacteria at cold temperature. Here, we observed reduced temperature (25-15 °C) promoted the secretion of EPS and thus stimulated bigger size of granular sludge in a laboratory-scale anammox reactor. We further combined co-occurrence network analysis and genome-centered metagenomics to explore the potential interactions between AnAOB and heterotrophic bacteria. Network analysis suggested 22 out of 25 positively related species were reported as definite heterotrophic bacteria in subnetwork of AnAOB. Genome-centered metagenomics analysis yielded 23 metagenomic assembly genomes (MAGs), and we found that Acidobacteriota-affiliated bacteria could biosynthesize most polysaccharides (PS) precursors and contain the most glycosyltransferases and transporters to facilitate exopolysaccharides biosynthesis, together with partial PS precursors produced by AnAOB. AMX1 as the only anammox genome could synthesize most amino acids and cross feed with some heterotrophs to affect the extracellular protein function. Additionally, Bacteroidota, Planctomycetota, Chloroflexota, and Proteobacteria could contribute folate and molybdopterin cofactor for AMX1 to benefit their activity and growth. Superphylum Patescibacteria could survive by cross-feeding with AnAOB and heterotrophic organisms about organic compounds (Glyceraldehyde-3P and lactate). These cross-feedings maintained the stability of anammox reactor performance and emphasize the importance of heterotrophs in anammox system at reduced temperature.