Chemical Conversion of PON to Nitrous Oxide for Nitrogen Isotopic Analysis : High- Sensitivity Tracer Assay for Nitrogen Fixation

We present a method for high-sensitivity nitrogen isotopic analysis of particulate organic nitrogen (PON) in seawater and freshwater, for the aim of measuring the rate of nitrogen fixation through 15N2 tracer technique for samples that contain low numeric abundance of organisms. The method composed of the traditional oxidation/reduction methods: such as oxidation of PON to nitrate using persulfate, the reduction of nitrate to nitrite using spongy cadmium, and further reduction of nitrite to nitrous oxide using sodium azide in an acetic acid buffer. Then, Nitrous oxide is purged from the water and trapped cryogenically with subsequent release into a gas chromatography column to analyze stable nitrogen isotopic compositions using a continuous-flow isotope ratio mass spectrometer. A standard deviation less than 0.3 ‰ for nitrogen was found for PON samples containing more than 80 nmolN, with a blank nitrogen of 20 nmolN. Nitrogen isotopic fractionation were consistent within each batch of analysis. Besides, we found that we can also determine nitrogen isotopic compositions of total dissolved nitrogen (TDN, sum of NO3-, NO2-, ammonium, and DON), by applying the method for filtrate samples. Our method may prove to obtain isotopic information for lower quantities of PON and TDN than other methods, so that we can reduce quantities of water samples needed for incubation to determine the nitrogen fixation rate in both seawater and freshwater. Besides, the method is also useful to determine the rate of nitrogen fixation by the small size fraction in ocean. We will also present the results obtained at western subtropical North Pacific to support our conclusions.