Spontaneous enhancement of nitrate assimilation facilitates the adaptation of anaerobic microbiome to hyperosmotic stress by promoting osmoprotectants synthesis

• About 1.5-2 % salinity is the threshold for freshwater anaerobic microbiome collapse. • Denitrification dominates below 2% salinity while ANR prevails beyond this threshold. • N assimilation genes ( nasE, glnA, gdhA ) were enriched under hyperosmotic stress. • High salinity selectively enriches species harboring N assimilation genes. • N assimilation facilitates osmoprotectant synthesis for hyperosmotic adaptation. The biotransformation of nitrate (NO 3 − -N), one of the main reactive nitrogen (N) types produced by human activities, is profoundly changing global climate and biodiversity. In human N-managed systems, NO 3 − -N transformation is often disturbed by salinity fluctuations in wastewater, ranging from 1% to 3.5%. As an important component of central nutrient metabolism, NO 3 − -N metabolism may facilitate the adaptation of freshwater microorganisms to withstand hypertonic stress, yet the regulatory mechanism remains unclear. Here, we demonstrate that increasing salinity redistributed NO 3 − -N transformation pathways and fluxes of freshwater anaerobic sludge, causing over 70% declines in both NO 3 − -N and total nitrogen removal efficiency, despite sufficient organic carbon and prolonged domestication time are provided. Denitrification dominated NO 3 − -N transformation at a salinity <2%, whereas assimilatory nitrate reduction outperformed denitrification and contributed an average of 53% to NO 3 − -N removal within the salinity range of 2–3%. Salinity levels ≥2% also triggered steep microbial biomass and diversity losses. Metagenomics revealed that the abundance of genes linked to denitrification and dissimilatory nitrate reduction to ammonium declined with rising salinity, while genes coding for nitrite assimilation and the synthesis and transport of osmoprotectants were up-regulated compared to low salinity conditions. Additionally, species harboring nitrogen assimilation genes nasE, glnA , and gdhA were enriched as salinity increased from 0% to 3%. These results highlighted that spontaneous enhancement of N assimilation facilitates amino acid metabolism and osmoprotectants synthesis, thereby reinforcing the metabolic adaptation of freshwater anaerobic communities to hyperosmotic stress. These findings offer new perspectives on the coupling of microbial nitrogen metabolism and osmoregulatory mechanisms.