The goal of this study was to develop an enzyme assay protocol for Everglades

soils to be utilized in further investigations of enzyme activities and their relation to

nutrient regimes. The objectives of this study were to address the effects of (1) varying

substrate concentrations; (2) incubation times; and (3) the use of multiple time point

measurements in determining optimum enzyme assay conditions.

 Materials and Methods

 Soil cores were collected in Water Conservation Area 2A, a 447 km2 impounded

wetland in the northern Everglades that has received agricultural drainage for over 30

years (Davis, 1991). Three soil cores were collected at nutrient enriched (Fl) and

reference (U3) transect sites along a distinct P gradient. Soil sampling was accomplished

using a 5 cm diameter piston type corer in the spring of 2001. The benthic layer or

flocculent detrital layer was separated from the remaining soil to be utilized in this

experiment. This layer, composed of active algal remains and plant components in

various stages of decay, was chosen due to greater microbial activity and variability

between sites. The samples were stored on ice until transfer to the laboratory. Large

roots and rocks were removed. The samples were homogenized for 5 min using a

handheld Biospec BiohomogenizerTM in a 500 mL beaker. Approximately 10 g wet

weight was transferred to a second 250 mL beaker and 100 mL deionized H20 was

added. The suspension was homogenized for an additional 5 min and 1 mL was

transferred to another 250 mL beaker to which 99 mL of DI H20 was added. The

suspension was mixed and 50 mL of the suspension was transferred to a 50 mL

Eppendorf centrifuge tube. This suspension was used for the assays and refrigerated until

use. Previous studies have shown that freezing generally caused increased enzyme

activities due to the disruption of enzyme complexes and cell lysis as compared to