An improved method for extracellular enzyme assays in paddy soil: a comparative study under aerobic and anaerobic conditions

Soil enzymes produced by microorganisms and plants are very sensitive to the variations in microclimate, e.g. aeration, and respond quickly to the induced changes. The majority of the enzyme assays are conducted under normal (temperature and air) conditions irrespectively of the origin of the environmental samples. However, it remains unclear how conditions of assays may affect results in anaerobic systems. In the present study, we have clarified this key gap in current methods by measuring the kinetics of phosphatase, β-glucosidase, and leucine aminopeptidase in paddy soil under aerobic and anaerobic conditions by means of a glovebox. Specifically, we quantified V_max and K_m in soil from three compartments in a rhizobox (top bulk (2-5 cm), rhizosphere, and bottom bulk (15-18 cm)) during rice growth. We demonstrate that the activities of three tested enzymes were significantly lower under aerobic conditions compared to anaerobic conditions at three consecutive dates of rice growth. Lower V_max values for phosphatase in top bulk soil and rhizosphere soil and β-glucosidase in top bulk soil, rhizosphere soil, and bottom bulk soil confirmed that aerobic conditions limited enzyme activities. For leucine aminopeptidase, although the difference in V_max values between anaerobic and aerobic conditions was not significant, the values always increased under anaerobic conditions compared to aerobic conditions. Compared with anaerobic conditions, the K_m values for phosphatase under aerobic conditions decreased by 10.11-22.78%. The maximum difference in the K_m values for β-glucosidase and leucine aminopeptidase between aerobic and anaerobic conditions was 30.93% and 40.53%, respectively. We conclude that enzyme activities of samples taken from the anaerobic or low-redox environment have to be assayed under anoxic conditions to avoid 10-40% underestimation (for V_max) due to suppression by oxygen.