Metagenomics and metatranscriptomics uncover the regulatory mechanism of nitrogen metabolism response to aggregation status of anammox bacteria

Abstract

Auto-aggregation of anammox bacteria is known to enhance their activities and growth, yet the regulatory mechanism remains elusive. We address this issue using two anammox reactors: Nspd (with the aggregation inhibitor-norspermidine) and Aggre (control). After the 155 days of incubation, the mean diameter of the Nspd sludge (232.09 μm) was significantly lower than the Aggre sludge (510.56 μm), and the Nspd sludge exhibited lower extracellular protein content (1.10 mg/g-VSS) compared to Aggre (1.33 mg/g-VSS) but higher neutral exopolysaccharide content (23.97 vs. 17.37 mg/g-VSS). It means that norspermidine leads to disaggregation of anammox bacteria by breaking and altering EPS structure which further decreased ammonia removal efficiency (80.0 % and 98.3 % in Aggre and Nspd). The negative impact was also verified by physical grinding separation method of anammox granules. Metagenomic and metatranscriptomic analysis indicated that a Candidatus Brocadia strain, Bro-MAG_N75, dominated in both reactors and disaggregation induced a decrease of cAMP and quorum sensing (QS) signals levels as well as the increase of the c-di-GMP level. Then, this modulation of signal levels up-regulated the expression levels of exopolysaccharides and extracellular proteins (two fold), however down-regulated those of the principal nitrogen metabolic enzymes of Bro-MAG_N75 by approximately 50 %, namely hydrazine synthase and hydrazine oxidoreductase. Concurrently, there was an up-regulation of non-mainstream nitrogen metabolic pathways, like DNRA and potential denitrification, which enhance metabolic diversity. These findings reveal the mechanism of aggregation through QS signals and second messengers regulating nitrogen metabolic activity and finally enhancing the activities and growth rate of anammox bacteria