Long-term nitrogen fertilization-induced enhancements of acid hydrolyzable nitrogen are mainly regulated by the most vital microbial taxa of keystone species and enzyme activities.

It is well known that nitrogen (N) fertilizer input is required to improve crop productivity, but we lack a comprehensive understanding of how elevated N input changes the formation of soil acid hydrolyzable nitrogen (AHN) by adjusting the most vital microbial taxa of keystone species of microbial communities and enzyme activities. A 15-year field experiment comprising four levels of inorganic N fertilization was conducted to identify the most important bacterial and fungal taxa of the keystone species derived from cooccurrence networks as well as the vital enzyme activities at the bell mouth and maturity stages. Long-term N fertilization significantly increased the levels of AHN along with its four fractions, including amino acid N (AAN), ammonium N (AN), amino sugar N (ASN), and hydrolysable unidentified N (HUN), by 30.1-118.6 %, regardless of growth stage. Some most vital microbial taxa of keystone species and enzyme activities, which changed in response to N fertilization, mainly regulated each ANH fraction, that is, AHN and AN were mainly controlled by the enrichment of Nocardioides and ß-1,4-N-acetyl-glucosaminidase (NAG), as well as by the reduction of Anaerolinea and urease (UR), AAN was determined by the enrichment of Hannaella and depletion of Penicillium, ASN was regulated by the enrichment of Hannaella and Arthrobacter, and HUN was influenced by the reduction of Penicillium and enrichment of Nitrosospira. These microbial genera have been found to be involved in dissimilatory nitrate reduction to ammonium (DNRA) and nitrification/denitrification processes and the two enzyme activities involved in organic N degradation and N-releasing processes, suggesting that the formation of AHN fractions was closely associated with specific functional microbial taxa and enzyme activities induced by N fertilization. Our results provide new insights into the associations among increased N input, altered formation of soil organic N, and shifts in microbial communities and enzyme activities.