An improved high-throughput method for p-nitrophenol based enzyme assays to aid soil ecology studies

Abstract:

The activities of soil enzymes, influence critical ecological processes including decomposition, and transformation of organic matter, biogeochemical cycling and applications for industry, medicine, and energy. The p-nitrophenol (pNP)-based methods are widely used in agronomic and ecological studies but an adequate high-throughput, microplate method has yet to be adopted. The overall goal of this study is to further refine current pNP microplate methods to reduce the high variability and low sensitivity previously reported. Specific objectives were to: 1) determine the correct ratio of soil:water to ensure similar quantities of soil are compared regardless of texture; 2) reduce variability between replicates by removing soil particles from wells using filters embedded within microplate wells; 3) identify the influence of homogenization timing on enzyme activity; and 3) compare results to the traditional bench scale method. b-glucosidase activity was determined in sandy loam and clay loam soils with four homogenization times (2, 3, 4, and 5 minutes) with filtered microplates. Soil textural class significantly affected the actual quantity of soil pipetted into microplate wells, which supports the necessity to quantify actual soil used and adjust soil:water ratios. Results from the microplate method were significantly correlated with those of the bench scale results with an average r² of 0.98 and an average slope of 1.37. The filtered plates removed the variability otherwise imparted by soil particles in the non-filtered technique with an average coefficient of variation of 7.3% (range was 2-24%). Slope increased with increasing homogenization with no significant difference between the 2 and 3 minute tests and the 4 and 5 minute tests. Results from additional tests will be discussed that evaluate a larger variety of soils under different management and environmental conditions.