Sub-Parts-Per-Billion Nitrate Method: Use of an N2O-Producing Denitrifier to Convert NO3− or 15NO3− to N2O †

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A more sensitive analytical method for NO3− was developed based on the conversion of NO3− to N2O by a denitrifier that could not reduce N2O further. The improved detectability resulted from the high sensitivity of the 63Ni electron capture gas chromatographic detector for N2O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO3− to N2O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO3− N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO3− concentrations of <2 ppb of NO3− N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of 15N in NO3−. This method avoids the incomplete reduction and contamination of the NO3− -N by the NH4+ and N2 pools which can occur by the conventional method of 15NO3− analysis. N2O-producing denitrifier strains were also used to measure the apparent Km values for NO3− use by these organisms. Analysis of N2O production by use of a progress curve yielded Km values of 1.7 and 1.8 μM NO3− for the two denitrifier strains studied.

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