Freshwater anammox bacteria leverage mixotrophy to regulate osmoadaptation under saline stress

The application of freshwater anammox bacteria in saline wastewater treatment is often limited by osmotic stress, despite these bacteria exhibiting faster growth kinetics than marine lineages. Although saline effluents such as anaerobic digestate often contain residual organics, the mechanisms enabling freshwater lineages to inhabit these environments remain unclear. The present study integrated comparative genomics with physiological characterization to elucidate the bioenergetic role of acetate in the osmoadaptation of anammox bacteria. Genome-centric analysis of 29 anammox genomes revealed that the marine genus Ca. Scalindua encoded diverse ion pumps, whereas Ca. Brocadia developed distinct organotrophic versatility by encoding multiple acetate metabolic pathways and crucial genes related to dissimilatory nitrate reduction to ammonium. Short-term batch assays and long-term bioreactor operation combined with transcriptomic profiling revealed that Ca. Brocadia sapporoensis shifts its bioenergetic strategy in response to salinity (7 g L^-1 NaCl). Although glycogen degradation was triggered for survival, exogenous acetate supplementation significantly upregulated the expression of genes encoding AMP-forming acetyl-CoA synthetase, isocitrate dehydrogenase, and glutamate dehydrogenase (t test, p < 0.05). These results suggest a metabolic rewiring in which acetate is strategically diverted to synthesize glutamate (a key compatible solute), thereby bypassing the energetic cost of autotrophic osmoadaptation. Collectively, our findings indicate mixotrophic osmoadaptation in Ca. Brocadia sapporoensis, which suggests that residual organics can act as a bioenergetic subsidy rather than a liability. Accordingly, maintaining a low C/N ratio may be an effective strategy for supporting the resilience and performance of freshwater anammox systems in saline wastewater treatment.