Potential denitrification activity assay in soil - With or without chloramphenicol?

A common way to characterize denitrification in soil is to determine the potential denitrifying activity (PDA). Our objectives were to compare different techniques of mathematically describing experimental data obtained in the PDA assay, both with and without use of chloramphenicol (CAP), and to quantify the effect of CAP on the process. The PDA assay was carried out in the presence of acetylene in slurries of three agricultural soils containing 1 mM glucose and 1 mM KNO3. When CAP was not used in the assay, growth related curves of N2O-formation were obtained for all three soils. These data were used to calculate the initial rate by: (1) assuming the initial phase to be linear and using the four first data points for linear regression; and by (2) using a growth-associated product formation equation. The good fit to the data that was obtained with the latter method suggests that PDA is a continuous process without distinct phases. Moreover, our results clearly show that denitrifying activity is inhibited by CAP even at the lowest concentration tested, 20 mg 1(-1). The inhibiting effect increased with increasing concentrations of CAP. The PDA was 17-42% lower at 1 g CAP 1(-1) compared with assays without CAP. This shows that not only synthesis of new enzymes is affected but also that the activity of already existing enzymes is decreased. Results from our study strongly suggest that single concentrations of CAP must not be used in PDA assays. An alternative strategy could be to use multiple CAP concentrations and then extrapolate to the rate at 0 g CAP 1(-1). However, we recommend assays without CAP and that data should be fitted to the growth-associated product formation equation. By using this method, values of the PDA and the growth rate of the denitrifying bacterial population are objectively obtained.