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

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.

Nitrogen‐13 as a Biochemical Tracer

“…These authors found that in some autoclaved soils, some 13N20 (but not [13N]N2) was produced in an abiotic reaction from "NO3-(113). However, Tiedje et al (67) did not observe such a reaction with their soil samples.…”

“…The predominant metabolic fate of ammonia taken up by rabbit heart tissue was the same as that found for rat brain: incorporation into the amide of glutamine. Thus, in two separate groups of experiments carried out by Krivokapich et al,67 ? 19 % (SD, n = 3) and 78 ? 11 % (SD, n = 4) of the 13N in the 6-min septum specimens were in glutamine amide.…”

“…These authors used the gas stripping and acetyleneinhibition techniques to assess denitrification in soil slurries, lake sediments and microbial cultures (67,127,(131)(132)(133)(134)(135)(136). Measured rates for various soils were 0.1-2 pg of Nlgihr, but these may have been artificially high because the assay method provided conditions for enhanced denitrification.…”

“…The Michigan group has relied on rapid TLC and electrophoretic separation of 13N-amino acids which are quantitated by a scanning technique (67,95,100,110). However, ~-['~N]aspartate is poorly separated from ~-['~N]glutamate and 13NH4+ has to be quantitated following steam distillation.…”

Denitrification Process as an Indicator of Soil Health

“…Methods based on the use of 13N have also been described (Smith et al, 1978;Tiedje et al, 1979), but these cannot be applied in the field (Tiedje et al, 1989).…”