Substitution of organic fertilizer did not change the dominant role of biochar on bacterial community stability but the soil carbon fractions

Biochar and organic fertilizers are used to improve soil carbon retention. However, the interactive roles of biochar and organic fertilizer in stabilizing soil organic carbon (SOC) at different horizons remain unclear. A field experiment with biochar application (0 %, 1 %, and 2 % of dry soil; Control, LB, and HB, respectively) and organic fertilizer substitution (0 %, 20 %, 40 %, and 60 % of inorganic N; T0, T2, T4, and T6, respectively) (fermented sheep manure replacing urea-N)) was conducted to reveal the mechanisms. The SOC, particulate organic carbon (POC), and mineral-associated organic carbon (MAOC) contents increased with the biochar addition and organic fertilizer substitution. In the topsoil (0–15 cm), values ranged from 17 to 24 g kg⁻¹ (SOC), 2.03–6.6 g kg⁻¹ (POC), and 15–19 g kg⁻¹ (MAOC). In the subsoil (16–30 cm), the ranges were 16–20 g kg⁻¹ (SOC), 1.2–3.4 g kg⁻¹ (POC), and 14–18 g kg⁻¹ (MAOC). HB combined with organic fertilizer substitution promoted SOC, POC, and MAOC more effectively than biochar alone. The β-1,4-glucosidase and cellobiohydrolase (CBH) activities in the topsoil were 1.2–1.6 times higher than those in the subsoil. Increases in biochar reduced the POC/MAOC ratio by limiting CBH activity (P < 0.05). Substituting organic fertilizer increased the POC/MAOC ratio by alleviating the limitation of biochar on bacterial biomass in the soil horizons. Bacterial community composition varied significantly with biochar addition and between soil horizons (P < 0.05). Biochar enhanced the dominant role of heterogeneous selection and increased bacterial network complexity in both the topsoil and subsoil. Based on structural equation modeling, an increase in bacterial negative/positive cohesion significantly improved the proportion of MAOC in the topsoil (P < 0.05). This study provides evidence that biochar (but not organic fertilizer substitution) helped improve C storage by decreasing the CBH activity and bacterial biomass, and enhancing network complexity. Thus, it is essential to consider soil horizons when evaluating the dynamic effects of agricultural practices on SOC pools.