Methods for the Production and Use of Nitrogen-13 In Studies of Denitrification

Methods were developed for use of the radioactive isotope of nitrogen, ¹³N, for short-term studies of denitrification. ¹³N was generated by irradiation of water with 12 to 15 MeV proton beams from a sector-focused cyclotron. Under typical operating conditions of 0.7 to 3 µA beam currents for 10 min, the ¹³N ionic species produced were NO₃⁻, 75–90%; NO₂⁻, 5–10% and NH₄⁺, 0.5–25%. Traces of [¹³N] N₂O and [¹³N] N₂ were also produced. The measured yield varied from 2 to 16 mCi/10 min irradiation depending on beam current. Vacuum evaporation at high pH was used to obtain ¹³NO₃⁻ + ¹³NO₂⁻ at > 99.8% purity, and high performance liquid chromatography (HPLC) was used to obtain ¹³NO₃⁻ or ¹³NO₂⁻ at > 99% purity. The HPLC system used a Partisil SAX anion exchange column eluted with phosphate buffer at pH 3.0 and was coupled to a coincidence NaI(T1) detector for counting ¹³N species in the effluent. Separation of NH₄⁺, NO₂⁻, and NO₃⁻ was achieved within 5 min. This system was used to monitor purity of ¹³N substrates and for studies of dissimilatory nitrate reduction to ammonia. A gas chromatograph-proportional counter detector system was developed to separate and measure [¹³N] N₂, [¹³N] N₂O and ¹³NO. Separation was by Poropak Q and Molecular Sieve 5A columns and was achieved in 5 min. Denitrification rates and products of soils and bacterial cultures incubated in sealed flasks were monitored with this system. Continuous rates of [¹³N] N₂ and [¹³N] N₂O production were monitored using a differential trapping, gas stripping system. Soil slurries amended with ¹³NO₃⁻ or ¹³NO₂⁻ were stripped of gases by continuously sparging with helium. N₂O was collected in a liquid nitrogen trap. Nitrogen gas passed through this trap but was retained in a Molecular Sieve trap immersed in liquid nitrogen. ¹³N gases collected in each trap were continuously counted by NaI (T1) detectors. Linear rates of gas production were typically observed from 15 min after addition of the ¹³N substrate to termination of the experiment after 1 to 1.5 hours. ¹³N has the advantage in denitrification studies of allowing direct measurement of N₂, very sensitive short-term rate measurements, and isotope exchange experiments at low substrate concentrations.