Objectives To investigate the characteristics of carbon-cycling functional microorganisms in black soil under long-term different isonitrogenous fertilization regimes and their correlations with active carbon pools, so as to provide a theoretical basis for soil carbon sequestration and efficient utilization in the black soil region.

Methods The research was based on a long-term field experiment established in Gongzhuling, Jilin Province in 1989. Soil samples were collected from the plow layer (0−20cm) under five fertilization treatments: no fertilization (CK), nitrogen and potassium (NK), nitrogen, phosphorus and potassium (NPK), NPK plus manure (NPKM), and NPK plus straw (NPKS). Metagenomic sequencing and soil carbon fraction measurements were combined to systematically analyze the microbial community structure, abundance of key functional genes involved in the carbon cycle, and their relationships with labile carbon fractions.

Results The dominant bacterial and archaeal phyla in black soil did not change significantly across fertilization treatments, whereas the abundance of the fungal phylum Mucoromycota increased significantly under NK (P<0.05). Alpha diversity analysis showed that NK increased bacterial diversity, while NPKS reduced archaeal diversity. Beta diversity analysis indicated that combined organic and inorganic fertilization treatments (NPKS, NPKM) increased within-group similarity and stability of microbial communities (P<0.05). Regarding carbon cycle functional genes, compared with CK, chemical fertilization alone (NK, NPK) significantly reduced the relative abundance of carbon fixation and methane metabolism genes, but increased the abundance of carbon degradation genes. NPKS maintained carbon fixation potential while significantly reducing carbon degradation gene abundance. The reductive citric acid cycle was the dominant carbon fixation pathway; NK and NPK decreased the abundance of key genes ppdK, korA, and oorB, but increased the abundance of tktB in the Calvin cycle and ppc in the dicarboxylate/4-hydroxybutyrate cycle. Carbon degradation was dominated by the cellulose degradation pathway; NK, NPK, and NPKM significantly increased the abundance of cellulose degradation genes bglX and bglB, hemicellulose degradation gene abfA, and starch degradation gene treS, while NPKS decreased the abundance of the glyoxylate cycle gene aceB. Additionally, both chemical and organic fertilization significantly reduced the abundance of the key methane metabolism gene acs (P<0.05). Correlation and random forest model analyses revealed that microbial biomass carbon (MBC) and microbial quotient (MBC/SOC) were key factors regulating carbon cycle functional genes. Carbon fixation gene abundance showed a nonlinear positive correlation with both, declining significantly when MBC > 290 mg/kg or MBC/SOC > 1.76%. Carbon degradation gene abundance was linearly negatively correlated with MBC and MBC/SOC but positively correlated with the proportion of dissolved organic carbon (DOC/SOC). Methane metabolism gene abundance was linearly positively correlated with MBC and MBC/SOC (P<0.05).

Conclusions Long-term fertilization significantly alters the microbial community structure of black soil. The application of chemical fertilizers in combination with straw or organic fertilizers is conducive to community stability, while the application of chemical fertilizers alone causes obvious fluctuations in the community. The application of chemical fertilizers alone shifts the carbon cycle function from fixation to degradation, while the combined application of chemical fertilizers and straw maintains the carbon sequestration potential while weakening carbon degradation. Soil microbial biomass carbon and its entropy value are the core factors regulating the carbon cycle function. The abundance of carbon sequestration genes significantly decreases when MBC > 290 mg/kg or MBC/SOC > 1.76%. In actual management, excessive input of organic materials should be avoided to optimize the carbon sequestration function of black soil.