Active assimilators of soluble microbial products produced by wastewater anammox bacteria and their roles revealed by DNA-SIP coupled to metagenomics

Heterotrophic bacteria grow on influent organics or soluble microbial products (SMP) in wastewater anammox processes, playing key roles in facilitating microbial aggregation and reducing excess nitrate. The overgrowth of heterotrophs represents one of the major causes of anammox process failure, while the metabolic functions of coexisting heterotrophs and their roles in anammox process remain vague. This study aimed at revealing metabolic interactions between AnAOB and active SMP assimilators by integrating 13 C DNA-stable isotope probing, metabolomic and metagenomic approaches. Glycine, aspartate, and glutamate with low biosynthetic energy cost were the major SMP components produced by AnAOB (net yield: 44.8, 10.4, 8.1 mg·g NH 4 + -N −1 ). Glycine was likely synthesized by AnAOB via the reductive glycine pathway which is oxygen-tolerant, supporting heterotrophic growth. Fermentative Chloroflexi bacterium OLB13, denitrifying Gemmatimonadaceae and Burkholderiaceae bacterium JOSHI-001 were active SMP assimilators, which were prevalent in globally distributed wastewater anammox reactors as core taxa. They likely formed a mutualistic relationship with auxotrophic Ca. Kuenenia by providing necessities such as methionine, folate, 4′-phosphopantetheine, and molybdopterin cofactor, and receiving vitamin B 12 for methionine synthesis. For the first time, the identify and metabolic features of SMP assimilators in wastewater anammox communities were revealed. Supplying necessities secreted by heterotrophs could be helpful to the endeavor of AnAOB enrichment. Practically, maintaining active but not overgrown SMP assimilators is critical to efficient and stable operation of wastewater anammox processes.