Quantifying in situ beta-glucosidase and phosphatase activity in groundwater

Enzymes play an important role in the environment, they breakdown natural-occurring and anthropogenic molecules so that they can be transported into cells and utilized. Enzyme assays are routinely used in soil science and oceanography to measure the activities of specific processes and to serve as general indicators of microbial activity. Conventional enzyme assays are conducted as batch incubation of sediment and water samples. During these assays the concentration of product is measured and enzyme activity is then determined as the rate of product formation. Few studies have measured enzyme activities of groundwater. This work investigates the use of beta-glucosidase and phosphatase assays for quantifying in situ enzyme activities in groundwater. Improvements to conventional enzyme assays using p-nitrophenyl substituted compounds were made by developing a high performance liquid chromatography method to improve quantitation limits of the product and to quantify concentrations of both the substrate and the product. An in situ single-well push pull test was then conducted to measure beta-glucosidase activity in situ and to estimate the Michaelis constant (Km) and the maximum reaction velocity (Vmax) in petroleum-contaminated groundwater at a field site near Newberg, Oregon. An important feature of the single-well push pull test is the nonlinear drop in pore water velocity that the test solution experiences as it moves out from the injection point. The nonlinear drop in pore water velocity is of particular interest because enzyme-mediated reactions are very fast and changes in the hydraulic properties during the test may give rise to mass-transport limitations. Fast reactions lead to the simultaneous depletion of substrate and accumulation of product at the site of the reaction so substrate and product concentrations near the enzyme can be different then the concentrations in bulk solution. And the rates obtained from a single-well push pull tests may be a combination of the rates at which substrate diffuses to the microorganism and at which the reaction occurs. Laboratory experiments with sediment-packed columns were conducted with a range of pore water velocities typically achieved in the subsurface during as push-pull test as a means for examining the potential effects of inhibition and diffusion on phosphatase enzyme kinetics. In this set of column experiments rates of phosphatase-mediated reactions were investigated instead of beta-glucosidase, which is an inducible enzyme. (Abstract shortened by UMI.)...