Long-term chemical fertilizer application enhances ammonia oxidizers-mediated soil carbon neutrality

Abstract

Ammonia-oxidizing microorganisms (AOMs) primarily use chemoautotrophic CO₂ fixation for growth, while their decay substantially contributes to soil organic carbon (SOC) that may be respired as CO₂, leaving their net impact on soil carbon neutrality unclear. This study employed two-phase microcosm incubation to examine how AOM cell proliferation and death affect SOC accumulation in long-term unfertilized CK (continuous maize) and chemically fertilized CC (continuous maize), CS (continuous soybean), and RCS (rotation maize-soybean) treatments. During the 28-day incubation with ¹³CO₂ and urea (Phase I), net production of soil organic ¹³C (¹³C-SOC) showed no significant differences (p > 0.05) among treatments: CK (23.6 μg g^-1), CC (20.9 μg g^-1), CS (22.8 μg g^-1), and RCS (25.0 μg g^-1). This ¹³C-SOC originated entirely from active ammonia-oxidizing bacteria (AOB) and archaea (AOA), with fertilized treatments showing significantly higher AOB: AOA protein-C ratios (CC: 4.48; CS: 5.88; RCS: 12.5) than CK (1.56). The mortality of active cells was further assessed (Phase II) by measuring AOM-related ¹³C-CO₂ mineralization, which was approximately twice as high in the CK compared with the fertilized treatments (p < 0.05) within 30 days. This derived mortality rate followed the same trend, which confirmed that the respired portion of the newly generated microbial carbon was lower under chemical fertilizer application. We conclude that long-term chemical fertilizer application increases the AOB: AOA protein-C ratio and promotes the ammonia oxidizer-derived SOC accumulation through their life cycles, ultimately supporting carbon neutrality.