Effect of Temperature on the Gaseous Nitrogen Products of Denitrification in a Silt Loam Soil

The amount and relative proportion of N₂ and N₂O evolved from a nitrate-amended surface soil under an atmosphere of He in the range of 7 to 75°C was examined. The objective was to evaluate the effect of temperature on N₂O evolution and investigate the optimal temperature for denitrification. The soil had 2.43% organic C and pH of 6.8. Incubations were performed in all-glass systems which were sealed throughout the incubation period. Denitrification products (N₂O, NO, and N₂) were determined periodically by gas chromatography using an ultrasonic detector. At the end of the incubation period, inorganic N remaining was determined. Above 15°C, the rate of denitrification increased with temperature, with an apparent optimum of 60 to 67°C. The percentage as N₂O of the total (N₂O + N₂)-N declined with time, but was still 44 to 50% of the gas produced at 16 days with the 7 and 15°C experiments. Nitrous oxide comprised up to 88% of the gas produced at 40°C after 2 days. At 50°C, most of the gas was N₂O at one day, but at 60 to 67°C, N₂O had disappeared by 1 day. Denitrification was still occurring at 70°C, but at a reduced rate. More than 85% of the N evolved at 70°C was as N₂O. No denitrification occurred at 75°C. At 50 to 67°C, much more N was recovered as gaseous N than was initially present in the system as NO₃-N, and no (NO₂⁻+NO₃⁻)-N remained at 4 days. These results are explained by the hypothesis that thermophilic Bacillus sp. (and perhaps others) rapidly reduces NO₃⁻ to NO₂⁻, with the NO₂⁻ reacting with oxidized N functional groups to form nitrogenous gases. This hypothesis was partially confirmed by experiments involving treatment of steam-sterilized soil with NO₂⁻ at 40 and 60°C. Our results suggest that the commonly-stated optimal temperature for denitrification may be too high for true biological denitrification due to the dominance of temperature-dependent chemical reactions at >50°C.