Assimilation overwhelms nitrification in saline wastewater nitrogen removal

Abstract

Heterotrophic nitrification and aerobic denitrification (HN-AD) represents an innovative biological nitrogen removal strategy for saline wastewater treatment. However, how HN-AD microbes could be applied to environmental microbiomes and conduct nitrogen metabolic performance remains ambiguous. Here we established synthetic heterotrophic microbiomes using halophilic HN-AD strains - biofilm-forming Pseudomonas kunmingens 8-C and Acinetobacter johnsonii 2–1-H, and characterized nitrogen metabolism in pure-cultured strains and microbiomes. The pure-cultured HN-AD strains removed ammonium primarily via ammonium assimilation (> 46 % contribution) and heterotrophic nitrification, comprehensively validated by nitrogen balance, ¹⁵N stable isotopic labeling tests, enzyme activity assays and functional gene identification. Four synthetic halophilic microbiomes constructed by biofilm-forming and HN-AD strains achieved ammonium and total nitrogen removal efficiencies of 76-92 % and 72–86 %. Biofilm-forming strains facilitated heterotrophic microbiome assembly by shaping microbial communities through the deterministic assembly process. Notably, initial functional strains selectively recruited environmental microbes with efficient ammonium-assimilating capacity, manifested as a stable and relatively high abundance of glnA gene in microbiomes. But the invasion of microbes consequently led to the overwhelming dominance of ammonium assimilation over nitrification in microbiomes. Our results provided a framework for constructing environmental microbiomes using functional microbes and highlighted the distinct nitrogen metabolism shifting from HN-AD pure-cultured bacteria to microbial consortia.