15N Tracer Methods
The best method for quantification of nitrification rates remains the direct 15N tracer approach in which a small amount of either 15NHJ or NO2 is added to a sample and incubated. The labeled product, 15NO2 or 15NO2, is then extracted and measured on a mass spectrometer. This approach has suffered in the past from the necessity to enrich the substrate pool by the addition of large concentrations of 15N-ammonia or -nitrite, thus artificially enhancing the observed rate. Improvements in assay techniques and mass spectrometer sensitivity with small N masses have minimized this problem.
The same analytical approaches can be applied in a tracer or isotope dilution format. In the isotope dilution format, label is added to the product pool and its dilution by addition of new product with natural abundance iso-topic signature during the incubation provides an estimate of production rate.
The advantages of the direct 15N approaches, compared to inhibitor methods, include shorter, thus less artifactual, incubations, minimal perturbations to in situ conditions (ambient light and nutrient conditions can be used), and much greater sensitivity. Inhibitor-based assays have the advantage, however, of requiring simpler less expensive instrumentation, as colorimetric, rather than mass spectrometric, analysis usually suffices.
Inhibitor methods depend on the ability of many compounds to interact specifically with the active site of the ammonia monooxygenase (AMO) or nitrite oxidoreductase (NOR) enzyme. A large number of potential inhibitors have been used for AMO, while chlorate is still the only specific inhibitor reported for nitrite oxidation. Replicate incubations are carried out in the dark with additions of either AMO or NOR inhibitors and the changes in inorganic N concentrations are used to infer nitrification rates. A more sensitive permutation of this approach involves measurement of 1 CO2 fixation in the presence and absence of inhibitor, where the decrease in the rate of1 CO2 assimilation in the presence of nitrifier inhibitor is attributed to nitrification. A conversion between CO2 fixation and N oxidation rates is then used to estimate nitrification.
One of the main attractions of the inhibitor approaches is their ease of use and analysis. Scintillation counters are much more common and easier to use than instruments required for stable isotope analysis and inorganic N
determinations involve analytical methods that are already standard to most laboratories. Although the inhibitor approaches are usually not appropriate for absolute rate measurements (because of uncertainty in conversion factors and perturbations during incubations) they can be very useful for spatial or temporal comparisons within studies.
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