High-throughput spectrophotometric assay of potential soil nitrate reductase activity

Soil denitrification can either be measured as denitrification rates by quantifying N2O production with the acetylene inhibition method or by assessing the activity of nitrate reductase - NRA (EC 1.7.99.4). We updated the traditional single-tube assay of potential NRA based on soil anaerobic incubation by developing an high-throughput, sensitive and low-cost spectrophotometric assay. We measured NRA across a range of soil types, soil depths and contrasting cropping systems to determine the relative sensitivity of this alternative method as an indicator of biological NO3 removal. The method involves the determination of NO2-N production after adding KNO3 as a substrate and 2,4-dinitrophenol (DNP) as an inhibitor of nitrite reductase but not of nitrate reductase. We combined the use of 2-mL 96-well deep-wells and 96-well microplates for incubation, extraction, centrifugation and determination of NRA. Our results show that the method has a low detection limit (<2 μg NO2-N gsoil-1 day-1), high precision (CV 0.3-2.1%) and enables the detection of significant differences on NRA among: 1) different perennial energy crops cultivated as buffer strips, 2) contrasting soil management practices (including cover crops and tillage systems), 3) different crop growing seasons. NRA was significantly related to NO3 content, DOC and fine root biomass. With this method one hundred samples in two eight-hour working days can easily be processed by a well-trained operator in a well-equipped laboratory, and even more if the weighting and liquid handling are automatized. Our results demonstrate the usefulness of our high-throughput NRA assay in quickly and inexpensively assessing changes in the soil biological NO3 removal as affected by cropping systems.