Use of a “least square” optimization procedure to estimate enzyme characteristics and substrate affinities in the denitrification reactions in soil
Predicting temporal variation in the production of gaseous forms of N generated in the denitrification process over a short time scale requires a quantification of the characteristics and kinetics of the denitrification enzymes. The treatments used soil aerobically incubated for 5 days, amended with or without C2H2 and a solution containing 3.4 mM KNO3 with or without 3.7 mM chloramphenicol. Samples were then anaerobically incubated for 48 h at 25°C. Gas fluxes and mineral N pools were monitored periodically from 1 to 48 h. Experiments were set up to determine the initial concentrations of the reduction enzymes involved in denitrification and their rates of de novo synthesis when anaerobiosis was induced for a soil from a permanent pasture. The dynamics of the system were explored with the DETRAN model using an iterative curve-fitting procedure based on the “least square” criterion. The NO3− reduction to CO2 production ratio was ca. 0.6 but increased to 0.8 when glucose was added. The link between the C mineralization and denitrification in the model was adjusted accordingly: “readily” decomposable organic material had a ratio of NO3− reduced to CO2 produced of 1.17 while soil organic matter had a ratio of 0.6. Chloramphenicol did not inhibit the reduction of nitrate so de novo synthesis of nitrate reductase did not occur. The concentration of nitrate reductase could therefore be set at 1. Best fits (lowest residual sum of squares) for the simulation of the NO2− concentrations were obtained when the initial concentration of nitrite reductase, relative to the nitrate reductase concentration, was ca. 0.3, its affinity for NO2− was between 300 and 450, and its de novo synthesis occurred between ca. 4 and 8 h after the onset of anaerobiosis. Best fits for the simulation of the N2O and N2 concentrations were obtained when the relative initial concentration of nitrous oxide reductase was ca. 0.1, its affinity for N2O was between 3 and 4, and its de novo synthesis occurred between 27 and 42 h after the onset of anaerobiosis. Chloramphenicol reduced the activity of the nitrite and nitrous oxide reductases; pointing at a possible inhibitory effect of the antibiotic. It was concluded that the iterative curve-fitting procedure enabled the estimation of the characteristics of the reduction enzymes involved in the denitrification process, i.e. initial concentrations and the rates of de novo synthesis, which could be used to simulate the kinetics of the denitrification process. The curve-fitting procedure can be used to test the validity of the concepts used to simulate the denitrification process and it can reveal anomalies, e.g. the inhibitory effect of chloramphenicol on the nitrite and nitrous oxide reductase.