Automated Robotic Assay of Phosphomonoesterase Activity in Soils

Phosphorus cycling in most ecosystems is dependent on plant and microbiologically derived phosphatase enzymes, and available P limits both microbial and plant growth. Measurements of enzymes in soil systems are often time-consuming and labor intensive. In this study, we examined phosphomonoesterase activity in soils using a Zymark XP laboratory robotic system for soil handling, solvent addition and exchange, filtration, incubation, reagent addition, and final sample preparation. Phosphomonoesterase activity was measured using phenyl phosphate as substrate, and samples were analyzed using a 96-well microplate reader. Results indicated that our robotic system was capable of effectively measuring phosphomonoesterase activity in soils differing in physical and chemical characteristics. The results obtained using manual and robotic procedures were comparable in accuracy and precision. The robotic system decreased labor associated with this assay by about a factor of 4.5 relative to the manual system, with considerable savings on reagent costs and labor. MANY KEY BIOCHEMICAL REACTIONS in soils have been shown to be due to microbial enzymes that catalyze the transformation of both organic and inorganic compounds. These enzymes are of up-most importance for the biogeochemical cycling of elements and the mineralization and immobilization of biogenic materials in soils. Analyses of soil enzymes have received recent attention as a means to examine small-scale distribution of soil microbial processes in different microhabitats (e.g., soil–litter interface, rhizosphere, or particle-size fractions) (Tscherko et al., 2004; Marx et al., 2005; Sessitsch et al., 2004). The phosphatases represent a broad group of enzymes catalyzing the hydrolysis of esters and anhydrides of phosphoric acid, and include phosphoric acid monoesterases (E.C. 3.1.3.), phosphoric acid diester hydrolases (E.C. 3.1.4.), triphosphoric monoesterhydrolases (E.C. 3.1.5.), phosphoryl-containing anhydrides hydrolases (E.C. 3.6.1.), phosphatases hydrolyzing P–N bonds (E.C. 3.9.), and phosphoamidases (E.C. 3.9.1.1.) (Tabatabai, 1994; Criquet et al., 2004). The phosphomonoesterase enzymes, often called “phosphatases,” comprise a large and important group of biocatalysts involved in the hydrolysis of ester-linked organophosphorus compounds to orthophosphate, and are thought to be the major phosphatases in most soils and litter (Turner et al., 2002). The activities of both acid and alkaline phosphatases are influenced by various soil properties, soil organism interactions, vegetation cover, leachate inputs, the presence of inhibitors, activators, and heavy metals (Juma and Tabatabai, 1977; Kandeler et al., 1996; Hysek and Sarapatka, 1998; Belyaeva et al., 2005) and organic matter content (Dick et al., 1994; Jordan et al., 1995), and as such, have been used to provide estimates of changes in soil quality (Gil-Sotres et al., 2005). Determination of phosphatase activity in soils often relies on the use of colorimetric substrates, including phenylphosphate, p-nitrophenyl phosphate, and fluorescent substrates (4-methylumbelliferyl phosphate, fluorescein diphosphate) (Tabatabai 1994; Schinner et al., 1996; Marx et al., 2001). Reactions are usually quantitative, follow Michaelis-Menton Kinetics, and are relatively easy to perform. However, some assays require long incubation times, contain multiple steps, or use substrates whose products require immediate analysis, thereby limiting throughput. Robotic-based high-throughput technologies have revolutionized biology, research methods, and opened the door for large-scale genomic analyses of macroand microorganisms. Such technologies will undoubtedly have an impact on the examination of soils and soil processes. To analyze the large number of samples generated in large field and laboratory experiments with high precision and accuracy, it is often necessary to automate analytical procedures (Koskinen et al., 1991). This is especially true of soil monitoring and landscapelevel studies where large numbers of samples are often collected. While robotic analysis systems have been developed for routine analysis of soil characteristics (Quigley and Reid, 1998; Hill et al., 2002), pesticides in soil and water (Koskinen et al., 1991; Kraemer 1997), and microbial and enzymatic activity in dairy and food products (Richardson et al., 1988), there have been no reports on the use of robotic systems to analyze microbial enzyme activities in soils. In this study we developed and evaluated an automated procedure to quantify alkaline phosphomonoesterase activity in two Midwestern soils using phenylphosphate as substrate. Enzyme reactions with this substrate release phenol, which is subsequently quantified spectrophotometrically using the color reagent 2,6-dibromoquinone4-chloroimide. The procedure utilized a commercially available laboratory robotic system and results were compared to those obtained using manual procedures. MATERIALS AND METHODS