A review of methods for field measurements of denitrification

Abstract Field methods for measuring denitrification require special attention for avoidance of the threat of large and unpredictable error arising from even the slightest contamination with 14N2, which comprises 79% of the Earth's atmosphere. Lack of a radioactive tracer isotope of nitrogen longer lived than 13N (approximately 10 min) or a stable tracer more easily assayed for than 15N places further strains on the acceptability of assays for N2, the final product of denitrification. Mass spectrometry remains an option; but, even with quadrupole detectors, the process remains cumbersome and its usefulness is limited for field studies. An older method recently reassessed involves gas chromatographic determination of rates of reduction of nitrous oxide in the field, in effect drawing inference about the entire process by assaying for the terminal step alone. In selected situations, this technique may provide useful information, but only when compared with findings from use of other procedures. Emission spectroscopy offers some simplification in the use of 15N-labeled materials to permit measurement of only the 28N2/29N2 ratio in denitrification products arising from soil. The method has shown early promise in experiments performed close enough to the laboratory for rapid sample testing to be feasible. Assays for accumulated intermediates appear to be effective more often than actual measurements of the end-product, N2. Now that it is accepted with certainty as an intermediate product, new assay procedures for measurement of nitric oxide as an indicator of denitrification offer promise for broad applicability. These methods require sparging to drive test gases from test soils and into the argon flow stream of a specific ozone-NO chemiluminescence detector or into a trap containing hemoglobin. In solution, the hemoglobin-NO complex exhibits a reliable light-absorption spectrum that can serve as the basis for colorimetric determination. The simplicity of this method has much to recommend it. Even without sparging, amperometric probes, specific for NO and N2O, also appear promising for use in soils in which moisture is continuous throughout the system. Specially constructed microprobes may be required; but, for many experiments, the ordinary Clark-type O2 electrode will serve, providing that the polarizing voltage against the reference Ag/AgCl electrode is −0.95 V for detection of N2O and − 0.7 V for measurement of NO. Until these procedures are tested in the field, current practices are likely to continue to be used. Our present standard method, the acetylene blockage technique, involves gas chromatographic detection of nitrous oxide, the penultimate product of denitrification, arising from field samples in which the reduction of N2O is selectively blocked by acetylene. A 63Ni-electron capture detector provides the most sensitive assay system, but thermal conductivity is often used. Porous underground tubing for even distribution of acetylene throughout the soil in experimental plots, and covered, stop cock-vented boxes placed over the test soils have been found to be effective collection devices. Because microbial degradation of acetylene sets in after 4–7 days of exposure in soil, use of acetylene blockage is restricted to relatively short-term experiments. Acetylene also inhibits nitrification, a factor that must be taken into account in design of experiments. Microsite and temporal variability in gas output is often encountered, and stochastic modeling of results may be required. Not all the nitrogen oxides released come from true denitrification. Whether produced by plants, algae, fungi, cyanobacteria or eubacteria, nitrate reductases act anomalously upon nitrite to liberate small amounts of NO and N2O, which may, in aggregate, contribute significantly to global atmospheric stocks of nitrogen oxides. Bacteria that produce a copper protein nitrite reductase, and appear sparsely distributed in nature in relation to true denitrifiers, also release nitrogen oxides during detoxification of nitrite. Classical nitrifying bacteria utilize such as enzyme to function in this manner and reduce nitrite when oxygen is limiting.