Long-term manure shifts microbial growth control from chemical constraints to substrate-quality regulation

Microbial growth governs the accumulation of microbial-derived carbon and the persistence of soil organic matter, yet the dominant constraints limiting microbial growth under long-term fertilization remain poorly resolved. Using a 34-year field experiment in a subtropical acidic soil in China, we quantified microbial biomass-specific potential growth rate (Gmass) across bulk soil and aggregate microhabitats and disentangled its environmental controls through nested cross-validation modeling and structural equation analysis. Manure significantly increased potential Gmass, whereas mineral fertilization exerted comparatively weaker effects. Under mineral fertilization, potential Gmass was primarily constrained by soil chemical conditions, particularly acidity and nutrient imbalance and remained tightly coupled to extracellular enzyme investment, indicating acquisition- and stress-limited growth. In contrast, manure alleviated chemical constraints, weakened enzyme-growth coupling, and shifted growth regulation toward substrate quality and metabolic return, with labile carboxylic compounds emerging as key drivers. Although soil aggregate structure remained physically stable, aggregates modulated these regimes differently by structuring spatial heterogeneity under mineral inputs and regulating effective substrate accessibility under manure through mineral-organic interactions. These findings demonstrate that manure does not simply enhance microbial growth but restructures the dominant limitation regime from chemical constraint to substrate-quality-mediated control. Incorporating management-induced shifts in microbial growth limitation into soil carbon models is critical for improving predictions of carbon dynamics in agroecosystems.