ASSAY FOR FLUORESCEIN DIACETATE HYDROLYTIC ACTIVITY: OPTIMIZATION FOR SOIL SAMPLES

Authors: GREEN, V - PURDUE UNIVERSITY; Stott, Diane; DIACK, M - SENEGAL

Submitted to: Journal of Soil Biology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/2005
Publication Date: 7/1/2006
Citation: Green, V.S., Stott, D.E., Diack, M. 2006. Assay for fluorescein diacetate hydrolytic activity: optimization for soil samples. Journal of Soil Biology and Biochemistry. 38:693-701.

Interpretive Summary: In recent years there has been an increased interest in soil ecosystem health and soil quality. Soil enzymatic activities are among the measurements of soil quality that are the most frequently and consistently able to distinguish between management systems. The soil enzymatic activity is a product of extracellular enzymes. This group of enzymes is a combination of exoenzymes that are released from living cells, endoenzymes released from disintegrating cell, and enzymes still bound to cell fragments. These extracellular enzymes become immobilized in the soil matrix of organic and mineral components. The immobilized enzymes are protected from degradation in the soil environment, but are often less active than their cell-bound counterparts. The protection allows these enzymes to remain active in the soil environment for weeks or even months. As a result, diurnal shifts in activity seen in microbial respiration measurements, are avoided in the enzymatic measurements, yet they are still sensitive to seasonal shifts and changes in soil management. The enzymatic hydrolysis of fluorescein diacetate (FDA) has been suggested as a possible enzymatic method for measuring overall soil microbial activity. Enzymes such as lipase, protease, and esterase are capable of catalyzing the hydrolysis of FDA. These enzymes are ubiquitous in the soil environment. When FDA is hydrolyzed, fluorescein is released and can be measured with a simple spectrophotometer. The original FDA hydrolysis assay was developed for measuring the enzyme activity in pure cultures of microbes. The first attempts to use this assay with soil samples were unsuccessful in that it had poor precision, and unreproducible results. The objective of this study was to optimize the FDA hydrolysis assay for soil samples in order to overcome these problems and determine the kinetic parameters involved in this reaction. The optimized method involves extraction and measurement of the fluorescein released when 1.0 g of soil is incubated with 50 mL of 60 mM Na-phosphate solution (buffered at pH 7.6) at 37°C for 3 hours. Results showed that FDA hydrolysis was optimum at buffer pH 7.6 and the soil enzymes were denatured at temperatures above 60°C. Three soils were used in the optimization of this method: a clay from Texas, a silt loam from Indiana, and a sandy loam from Georgia. This procedure is simple, precise, and reproducible. The impact is that we now have a method for estimating changes in microbial activity with time and from alterations in soil managagement. It is simple enough to be used in commercial soil testing laboratories for determination of general microbial activity and as a soil quality indicator.

Technical Abstract: With the increased interest in integrated soil bioecosystem studies, there has been a need to have a method of measuring overall microbial activity potential. Hydrolysis of fluorescein diacetate (3',6'-diacetylfluorescein [FDA]) has been suggested as a possible method because the ubiquitous lipase, protease, and esterase enzymes are involved in the hydrolysis of FDA. Following hydrolysis of FDA, fluorescein is released and can be measured spectrophotometrically. The objective of this study was to optimize the FDA hydrolysis assay for soil samples and determine the kinetic parameters involved in this reaction. The optimized method involves extraction and measurement of the fluorescein released when 1.0 g of soil is incubated with 50 mL of 60 mM Na-phosphate solution (buffered at pH 7.6) at 37°C for 3 hours. Results showed that FDA hydrolysis was optimum at buffer pH 7.6 and the soil enzymes were denatured at temperatures above 60°C. Three soils were used in the optimization of this method: Heiden clay (fine, smectitic, thermic Udic Haplustert), Raub silt loam (fine-silty, mixed, superactive, mesic Aquic Argiudoll), and Cecil sandy loam (clayey, kaolinitic, thermic Typic Kanhapludult). Further, results from eight soils show that the assay gives repeatable results. This procedure is simple, precise, and can be used in commercial soil testing laboratories for determination of general microbial activity and as a soil quality indicator.