Activities and metabolic versatility of distinct anammox bacteria in a full-scale wastewater treatment system

Anaerobic ammonium oxidation (anammox) is a key N₂-producing process in the global nitrogen cycle. Major progress in understanding the core mechanism of anammox bacteria has been made, but our knowledge of the survival strategies of anammox bacteria in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Here, by combining metagenomics with in situ metatranscriptomics, complex anammox-driven nitrogen cycles in an anoxic tank and a granular activated carbon (GAC) biofilm module of a full-scale WWTP treating landfill leachate were constructed. Four distinct anammox metagenome-assembled genomes (MAGs), representing a new genus named Ca. Loosdrechtii, a new species in Ca. Kuenenia, a new species in Ca. Brocadia, and a new strain in "Ca. Kuenenia stuttgartiensis", were simultaneously retrieved from the GAC biofilm. Metabolic reconstruction revealed that all anammox organisms highly expressed the core metabolic enzymes and showed a high metabolic versatility. Pathways for dissimilatory nitrate reduction to ammonium (DNRA) coupled to volatile fatty acids (VFAs) oxidation likely assist anammox bacteria to survive unfavorable conditions and facilitate switches between lifestyles in oxygen fluctuating environments. The new Ca. Kuenenia species dominated the anammox community of the GAC biofilm, specifically may be enhanced by the uniquely encoded flexible ammonium and iron acquisition strategies. The new Ca. Brocadia species likely has an extensive niche distribution that is simultaneously established in the anoxic tank and the GAC biofilm, the two distinct niches. The highly diverse and impressive metabolic versatility of anammox bacteria revealed in this study advance our understanding of the survival and application of anammox bacteria in the full-scale wastewater treatment system.