Rapid exposure of anoxic microbial communities to oxygen (O₂) can have unpredictable effects, including strong suppression of their enzymatic activity. Nonetheless, most medium- and long-term incubation studies on soil organic matter transformations fail to consider aeration effects during sample post-processing and/or assays. Moreover, it remains unclear whether anoxic enzymatic systems are adapted to quick switch to oxic conditions. We evaluated the effects of short-term (2-h oxic (+O₂) vs. anoxic (–O₂) assays) and medium-term aeration (after 10-day oxic vs. anoxic pre-incubation) on the kinetic parameters (V_max, K_m) of phosphomonoesterase, β-glucosidase, and leucine aminopeptidase in top bulk, rooted, and bottom bulk paddy soil of flooded rice mesocosms. We hypothesized contrasting short- and medium-term responses of hydrolytic enzyme activities to aeration (i) a negative short-term effect caused by reactive O₂ species toxicity and/or other mechanisms, and (ii) adaptation of anoxic microbial communities to medium-term aeration reducing the impact of ongoing O₂ exposure.

Overall, 2-h aeration suppressed V_max values by 7–43% and catalytic efficiency K_a (V_max/K_m) by 3–22%, and extended the substrate turnover time T_t (7–33%) of three tested enzymes in all soil compartments pre-incubated without O₂. In contrast, no short-term suppressive effect of O₂ was observed on three tested enzymes after oxic pre-incubation. Medium-term aeration increased V_max (by 12–253%) and K_a (by 3–78%) of the enzymes and shortened T_t (4–42%) as compared to the anoxic counterpart. These findings support our hypothesis about anoxic microbial community adaptation over the medium-term aeration. Accordingly, the sensitivity of anoxic hydrolytic enzymes to a short-term O₂ exposure and the O₂ adaptation mechanisms require strong consideration (i) for enzyme assays of anoxic soils and (ii) for understanding the soil organic matter dynamics in environments with O₂ fluctuations.