Ammonia-oxidizing bacteria dominate active ammonia oxidation during nitrogen fertilizer-induced acidification of a maize-cultivated black soil

Nitrogen fertilization accelerates soil acidification by stimulating ammonia oxidation, yet the dominant active ammonia oxidizers and their acid-adaptation strategies remain unresolved. We combined DNA stable isotope probing (DNA-SIP) with enrichment cultivation, metagenomics, and metatranscriptomics to identify active ammonia oxidizers and characterize acid-tolerance responses in a maize-cultivated black soil from Northeast China. Repeated urea and NH4Cl additions stimulated nitrification and acidified the soil, with pH decreasing from 7.35 to 4.73 under NH4Cl addition. DNA-SIP showed that ammonia-oxidizing bacteria (AOB), rather than ammonia-oxidizing archaea (AOA), dominated active ammonia oxidation during acidification. Phylogenetic analysis of amoA sequences from isotope-labelled higher-density DNA fractions identified Nitrosospira-related D11 and D12 clusters among active AOB. An acid-tolerant enrichment culture retained nitrification activity at pH 5.0 and yielded a D11-affiliated Nitrosospira MAG, designated Nitrosospira sp. NT. Its amoA sequence showed 98.2% nucleotide identity to a SIP-derived D11 amoA sequence, linking the enrichment-derived population to an active soil D11 cluster. Under acidic conditions, the enrichment culture showed pronounced aggregation with increased fungal occurrence, suggesting that fungal occurrence may be associated with the aggregated phenotype. Genomic and transcriptomic analyses indicated coordinated responses involving substrate acquisition and acid buffering, respiratory energy metabolism, intracellular pH homeostasis, envelope stress responses, and candidate systems potentially related to surface-associated growth and stress responses. These findings link community-level SIP evidence with genomic and transcriptional insights from an enrichment-derived representative of an active D11 cluster and show that acid-tolerant AOB can dominate active ammonia oxidation and sustain nitrification during nitrogen fertilizer-induced acidification in the black soil examined.