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This article in SSSAJ

  1. Vol. 43 No. 4, p. 709-716
    Received: Dec 3, 1978
    Accepted: Apr 3, 1979

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

  1. J. M. Tiedje,
  2. R. B. Firestone,
  3. M. K. Firestone,
  4. M. R. Betlach,
  5. M. S. Smith and
  6. W. H. Caskey2



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.

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