The responses of microbial extracellular enzyme activities to soil sample storage conditions across different soil types

Storage at −20 °C optimally preserves hydrolytic enzyme activities in all soils. Oxidative enzyme responses to storage conditions are highly soil-specific. Storage dictates hydrolytic activity, while climate drives oxidative enzymes. Tailoring storage protocols to soil and enzyme types ensures cross-study comparability. Microbial extracellular enzymes are key indicators of soil biogeochemical functioning, yet their sensitivity to storage conditions remains unclear and limits cross-study comparability. We evaluated how three storage conditions (−20 °C, air-drying, and freeze-drying) affect hydrolytic and oxidative enzyme activities across forest soils spanning an acidic-to-alkaline pH gradient in China (Acrisols, Luvisols, and Calcisols). Storage at −20 °C consistently preserved higher hydrolytic activities, whereas air-drying and freeze-drying caused significant reductions across all soils. Conversely, oxidative enzymes showed soil-specific responses: -20 °C was optimal for Acrisols; both -20 °C and freeze-drying outperformed air-drying in Luvisols; while storage conditions little affected Calcisols. Multivariate analysis revealed that, although both enzyme types were influenced by key soil properties (e.g., pH, carbon, nutrients), their primary drivers differed. Hydrolytic activities were predominantly governed by storage conditions, whereas oxidative activities were mainly regulated by climatic factors (temperature and precipitation). These findings underscore the necessity of tailoring storage protocols to specific soil and enzyme types. Standardizing and explicitly reporting these conditions will improve the reproducibility of enzyme assays, providing a robust methodological foundation for future ecological research.