Optimizing pH for Soil Enzyme Assays Reveals Important Biochemical Functions in Low pH Soil

Abstract Soil enzyme assays are often used as indicators of potential biological functions. The objective of this study was to understand enzyme activity across a range of soil pH. Soils (0–15 cm) were collected from a heathland restoration project (established 1999) on the Isle of Purbeck, UK with treatments of elemental sulphur or ferrous sulphate compared to a control, acid grassland and heathland. Enzyme assays were conducted using fluorescent substrates for β-1,4-glucosidase, β-N-acetylglucosaminidase (NAG) and phosphatase with a range of buffer pH from 3.0 to 12.0. Differences in soil pH were still evident with the control (pH 5.3) and ferrous sulphate (pH 5.2) significantly higher than elemental sulphur (pH 4.5), acid grassland (pH 4.3) and heathland (pH 4.0). The optimum buffer pH for enzyme assays varied from pH 3-4.5 for β-glucosidase, pH 4–5 for NAG and pH 4–6 for phosphatase. Comparisons using a standard MUB pH resulted in different conclusions compared to optimum pH. For example, β-glucosidase activity at pH 5 for the control was significantly higher than elemental sulphur, acid grassland, and heathland. However, there were no differences when the pH optimums were considered. Comparisons of phosphatase activity at MUB pH 6.5 resulted in higher activity in the control plots compared to the heathland, despite the heathland soils showing the highest activity at optimum buffer pH. By examining the relationships between soil pH, enzyme activity, and assay conditions, this study highlights the importance of optimizing pH in enzyme assays when comparing diverse soil types.