Effects of incubation temperatures on soil extracellular enzyme activity across different soil pH conditions

Abstract Soil extracellular enzymes are essential regulators of biogeochemical cycles, yet the conditions under which their activities are assayed, particularly incubation temperature, remain poorly standardized. This variation among studies can introduce substantial bias and complicate cross-study comparisons. Here, we examined hydrolase and oxidase activities across different soil pH conditions, with samples separately incubated at 10, 20, and 30 °C in acidic, neutral, and alkaline forest soils. Our results showed that hydrolase activities increased with increasing incubation temperature, though the pattern differed among soil pH conditions. In acidic soil, hydrolase activities were lower at 10 °C compared to 20 and 30 °C, with little difference between 20 and 30 °C. In contrast, neutral and alkaline soils showed a continuous increase in hydrolase activities from 10 to 30 °C. When data from all soils were combined, oxidase activities displayed no overall temperature effect. However, distinct pH-specific trends emerged: in acidic and neutral soils, oxidase activities increased with incubation temperature, whereas in alkaline soils it declined at higher temperatures. Across all incubation temperatures, soil pH was the primary environmental driver of both hydrolase and oxidase activities, whereas incubation temperature itself exerted no significant influence. This strong soil pH dependence masked the temperature effect, highlighting that pre-existing soil conditions fundamentally influenced the enzymatic thermal response. We propose a decision-making framework that selects incubation temperatures based on soil pH and enzyme class to improve the accuracy and cross-study comparability of measurements of soil extracellular enzyme activities.