The effects of incubation temperature, incubation duration, and sample storage condition on the activity of β-1,4-glucosidase across different soil types

Soil β-1,4-glucosidase is a key enzyme involved in microbial cellulose degradation and the terrestrial carbon cycle. However, reliable determination of its potential activity is often hampered by methodological uncertainties arising from a lack of explicit knowledge regarding the standardized requirements for incubation and soil sample storage conditions across different studies. To evaluate these sources of variation, we quantified soil potential β-1,4-glucosidase activity using a fluorometric microplate assay across three contrasting soil types (Acrisols, Luvisols, and Calcisols), three incubation temperatures (10, 20, and 30 °C), three incubation durations (1, 4, and 12 h), and two soil sample storage conditions (–20 °C and air-drying). Among the experimental treatments, incubation temperature emerged as the primary factor regulating soil potential β-1,4-glucosidase activity, with more than a 60% increase as temperature rose from 10 to 30 °C. This temperature effect did not vary with incubation durations, storage conditions, or soil types. Incubation duration interacted strongly with both storage condition and soil type; likewise, the effect of storage condition was modulated by soil type. These findings challenge the universality of common methodological assumptions by demonstrating that the influence of incubation duration and storage condition is modulated by their interactive effects and respective interactions with soil type. Standardization of soil potential β-1,4-glucosidase activity assays requires strict control of incubation temperature while adjusting incubation duration and storage condition in accordance to soil type for multi-site comparison studies. Our study provides a methodological framework that improves the reliability and comparability of soil potential β-1,4-glucosidase activity assays, thereby enhancing their application in studies of soil carbon cycling. • Incubation temperature is the dominant factor controlling the measured potential activity of soil β-1,4-glucosidase, with activity increasing by over 60% as temperature rises from 10 to 30 °C. • Strong interactions exist between incubation duration and storage condition, showing that their effects are not independent but can reverse the measured enzyme activity depending on the specific combination. • The influence of storage condition and incubation duration is modulated by soil type, challenging the assumption of universally optimal protocols across different soils. • The effect of incubation temperature is consistent and not significantly altered by variations in incubation duration, storage condition, or soil type. • Assay standardization requires strict temperature control and soil type-specific adjustments to incubation duration and storage condition for reliable cross-site comparisons.