The amount of NO3‐N exported in a second‐order mountain stream draining a clearcut and logged mixed hardwood forest was studied over a 4‐year period. Calculations based on measurements of stream chemistry and discharge rates indicated a within‐stream depletion of NO3 from the upper reaches of the stream to the watershed outlet. Within‐stream depletion the first year of treatment was 127% of total NO3‐N discharged from the watershed outlet and declined in succeeding years after treatment to 99, 42, and 5%. Assays of the quantities of denitrifying enzymes in stream sediment samples suggested 1.7 kg N year−1 were lost via this pathway, compared with 3.9 kg N year−1 calculated from within‐stream depletion for the same time period. This study suggests sediment denitrification is a major pathway by which NO3‐N is lost. Within‐stream gaseous transformations are important when accounting for changes in N dynamics associated with forest management practices, and the measurement of only hydrologic discharge of NO3 could result in underestimation of N losses.

Reduction of NO3‐ to NH4+ was studied in fresh and air‐dried soils which were amended with 15NO3‐ and glucose, acetate, or water and incubated anaerobically. Soils were either untreated or heat‐shocked at 68°C for 1 hour prior to amendment. Ammonium was produced rapidly after the onset of anaerobiosis, and thereafter was incorporated into organic matter. 15NH4+ plus 15N‐organic matter production was observed in glucose‐amended fresh soil in quantities up to 43% of added 15NO3‐ in untreated samples and 55% in heat‐shocked samples after 5 days incubation. In soils unamended with organic carbon, NO3‐ reduction to NH4+ was minor. Pretreatment of the soils either by air‐drying or heat‐shocking increased to a similar extent the amount of NO3‐ reduced to NH4+. The activity of clostridia during the NO3‐ reduction was indicated by the absence of any effect exerted by heat‐treatment, the production of H2 and CO2, and the presence of higher numbers of anaerobic sporeforming bacteria relative to denitrifying bacteria in the air‐dried soil. Also, the most common isolate capable of reducing NO3‐ to NH4+ was a Clostridium spp. The addition of washed spores of a NO3‐‐reducing Clostridium isolated from the soil increased the formation of 15NH4+‐N plus 15N‐organic N fourfold, an accumulation equivalent to 83% of added 15NO3‐‐N.The reduction of NO3‐ to NH4+ in soils was not inhibited by NH4+ or glutamine, indicating that the mechanism of reduction was dissimilatory. This conclusion is supported by studies with several Clostrdium spp. isolated from the soils. The isolates were capable of reducing NO3‐ to NH4+ and exhibited increased cell yields when NO3‐ was included in the growth medium. These studies suggest that the potential for significant dissimilatory NO3‐ reduction to NH4+ exists in most soils, principally in the sporeforming genera of Clostridium and Bacillus, but that this potential is likely expressed only when soils become anoxic and are rich in C.

No 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.

Cultures of Clostridium KDHS2 reduced 15NO3- to 15NH4+ with a concurrent increase in molar growth yield of 15.7% compared with fermentatively grown bacteria. The bacteria exhibited a Ks (NO3-) of 0.5 mM and reduced NO3- maximally at a rate of 0.1 mumol h(-1) mg dry wt)-1. A partially purified nitrate reductase was obtained which had a Km (NO3-) of 0.15 mM. The reduction of 13NO3- to 13NH4+ by resting bacteria was not inhibited by NH4+, glutamate, glutamine, methionine sulphoximine or azaserine. Glutamine synthetase affected neither the synthesis nor the activity of the NO3(-)-reducing enzymes. The results are consistent with the hypothesis that NO3- reduction to NH4+ in this Clostridium sp. is dissimilative. SO32-, but not SO42-, inhibited the reaction, apparently at the level of NO2- reduction.