Nitrogen-fixing shrubs shift deep-soil MAOC formation from microbial necromass to an EPS-associated pathway in desert ecosystems

Shrub restoration is a widely applied strategy for combating desertification and enhancing soil carbon sequestration in arid regions. Microbial necromass carbon (MNC) is considered a major precursor of mineral-associated organic carbon (MAOC), whereas the contribution of extracellular polymeric substances (EPS) to long-term carbon stabilization remains poorly understood. Here, we examined depth-dependent associations of MNC and EPS with MAOC under contrasting shrub functional types in a restored desert ecosystem in northwestern China. We compared bare land, non-nitrogen-fixing shrubs (NNFS), and nitrogen-fixing shrubs (NFS) across surface (20-40 cm) and deep (80-100 cm) soils by integrating carbon fractionation, molecular biomarkers, microbial community analyses, and partial least squares path modeling. Relative to bare land, both shrub types increased soil organic carbon (SOC), particulate organic carbon (POC), and MAOC, whereas significant increases in MAOC/SOC and MAOC/POC were observed only under NFS, particularly in deep soil. In surface soil, NFS increased MNC-related proportional indices, and integrated modeling revealed a significant positive association between MNC and MAOC. In contrast, deep soil exhibited a distinct EPS-associated pattern: EPS and its proportional indices were significantly higher under NFS than under NNFS, whereas MNC-related indices showed no significant differences between shrub functional types. EPS was strongly associated with deep-soil MAOC (R² = 0.76, P = 0.001). Elevated inorganic nitrogen availability was associated with extracellular protein accumulation, while microbial community analyses identified candidate indicator taxa associated with EPS-related variables and MAOC. Under equal carbon input, EPS amendment produced a significantly greater net increase in operationally defined MAOC than MNC amendment during laboratory incubation. Collectively, these findings highlight a depth-dependent difference in microbial associations with MAOC and identify extracellular microbial products as potentially important contributors to deep-soil carbon stabilization under nitrogen-fixing shrub restoration.