Soil organic matter plays a crucial role in maintaining soil fertility and stabilizing the global carbon cycle. The application of biochar (Bc) can be converted into soil organic matter and regulate the soil organic carbon (SOC) pool through the dual mechanisms of ”introducing new carbon inputs” and ”regulating old carbon turnover”. However, the temporal distribution patterns of biochar-derived new carbon across humus fractions and their underlying mechanisms remain unclear. To address this gap, this study used chemical extraction combined with δ 13C isotope techniques to quantify SOC content, extractable carbon (EC) and non-extractable carbon (nEC) contents, and their δ 13C values after Bc addition under different incubation periods, while further investigating the increment of new carbon and its distribution in soil carbon pools. The results showed that the Bc application rate significantly regulated the increment of new carbon, with the P3 treatment (21.2 g Bc kg -1) showing the most pronounced accumulation; moreover, new carbon exhibited selective allocation among humus fractions, as both the increment and proportion of new carbon in nEC were significantly higher than those in EC at all incubation times . Meanwhile, with prolonged incubation, the increment of new carbon in nEC decreased due to the decomposition of Bc residues, whereas the proportion of new carbon allocated to humic acid (HA) increased significantly under higher Bc application rates (e.g., in the P3 treatment after 540 days, HA-associated new carbon reached 4.32%); in addition, the increment of new carbon in EC also increased markedly driven by HA-associated new carbon accumulation, which reflects the transfer of Bc-derived new carbon from nEC to HA—a carrier of long-term stable carbon sequestration. This study provides a scientific basis for optimizing Bc-based carbon sequestration strategies and enhancing soil carbon sink potential, while also enriching the understanding of microscale processes governing humus-mediated soil carbon cycling.

Soil organic matter plays a crucial role in maintaining soil fertility and stabilizing the global carbon cycle. The application of biochar (Bc) can be converted into soil organic matter and regulate the soil organic carbon (SOC) pool through the dual mechanisms of ”introducing new carbon inputs” and ”regulating old carbon turnover”. However, the temporal distribution patterns of biochar-derived new carbon across humus fractions and their underlying mechanisms remain unclear. To address this gap, this study used chemical extraction combined with δ¹³C isotope techniques to quantify SOC content, extractable carbon (EC) and non-extractable carbon (nEC) contents, and their δ¹³C values after Bc addition under different incubation periods, while further investigating the increment of new carbon and its distribution in soil carbon pools. The results showed that the Bc application rate significantly regulated the increment of new carbon, with the P3 treatment (21.2 g Bc kg⁻¹) showing the most pronounced accumulation; moreover, new carbon exhibited selective allocation among humus fractions, as both the increment and proportion of new carbon in nEC were significantly higher than those in EC at all incubation times . Meanwhile, with prolonged incubation, the increment of new carbon in nEC decreased due to the decomposition of Bc residues, whereas the proportion of new carbon allocated to humic acid (HA) increased significantly under higher Bc application rates (e.g., in the P3 treatment after 540 days, HA-associated new carbon reached 4.32%); in addition, the increment of new carbon in EC also increased markedly driven by HA-associated new carbon accumulation, which reflects the transfer of Bc-derived new carbon from nEC to HA—a carrier of long-term stable carbon sequestration. This study provides a scientific basis for optimizing Bc-based carbon sequestration strategies and enhancing soil carbon sink potential, while also enriching the understanding of microscale processes governing humus-mediated soil carbon cycling.