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Methods for the Production and Use of Nitrogen-13 In Studies of Denitrification¹

ABSTRACT

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.

¹ Contribution from the Dep. of Crop & Soil Sciences, Dep. of Microbiology & Public Health, and the Heavy Ion Laboratory, Michigan State Univ., E. Lansing, MI 48824. Published as Journal Article no. 8928 of the Michigan Agric. Exp. Stn. This work was supported by NSF Grants DEB-77-19273 and PHY-78-01684 and USDA Regional Research Project NE-39.

² Professor of Soil Microbiology, Assistant Professor of Chemistry and Heavy Ion Laboratory, and Graduate Students, respectively. Current addresses are: M. K. F., Dept. of Soils and Plant Nutrition, Univ. of Calif., Berkeley; M. S. S., Dept. of Agronomy, Univ. of Kentucky; and W. H. C., Inst. of Ecology, Univ. of Georgia.

Received for publication December 3, 1978. Accepted for publication April 3, 1979.

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