Azo dye method for mapping relative sediment enzyme activity in situ at precise spatial locations

Existing methodology for measuring microbial enzyme activity in aquatic sediments involves horizontal sectioning of sediment cores into centimeter slices, followed by determination of the enzyme activity of each homogenized sediment slice. At best, this approach provides only one-dimensional information on the distribution of microbial activity. This paper describes the development of a novel technique to map sediment enzyme activity in situ at millimeter spatial resolution. Naphthol AS enzyme substrates were loaded onto filter membranes by evaporation from an organic solvent. The membranes were attached to plastic cards to form rigid probes, which were deployed vertically in sediments for a fixed time period. The exposed membranes were developed in a diazonium salt solution, resulting in the formation of a colored precipitate where substrate hydrolysis had occurred. The chromogenic reaction was calibrated and quantified by immersing substrate-loaded membranes in a series of solutions of known enzyme activity. A flatbed scanner and image analysis software were used to produce digitized images and to generate two-dimensional maps of enzyme activity. The technique was used to map the spatial features of esterase activity in aquatic sediment samples from wetland areas and enabled the precise locations of microbial activity "hotspots" to be identified.