Heavy metals tolerance of soil denitrifying communities: N2O dynamics

We investigated the effects of heavy metals (Cd, Cu, and Zn) on the enzyme activity of soil denitrifying community, and tolerance to the same heavy metals as indicated by denitrification rates. We focused on the rates of nitrate reduction to N2O and the N2O reductase activity, because the ratio between these two process rates is an indicator of the communityʹs intrinsic capacity to release N2O to the atmosphere. A sandy loam was given a single and double dose of a heavy metal mixture (single dose=0.32, 80, 120 mg kg−1 dry soil of Cd, Cu, and Zn, respectively). Ground straw was added together with the metals to enhance microbial growth, and the soil was incubated aerobically at 15 °C for 2 months. Kinetics of production and reduction of N2O by the denitrifying community of this soil was investigated by anaerobic incubation of soil slurries or extracted bacterial cells, using glutamate as a C source. Time courses of the N2O production and reduction curves (with and without acetylene) were used to estimate kinetic parameters to characterize the community. Heavy metal tolerance was tested by exposing extracted cells to heavy metals during such anaerobic incubations. The immediate effect (after 1 day) of heavy metals was a general reduction of the denitrification rate but also to decrease the N2O reduction more than N2O production rate. N2O production was partly recovered 8 days after heavy metal introduction, and completely restored (equal to the control soil) after 2 months. In contrast, the N2O reductase activity was still not completely restored after 2 months. Exposure of extracted cells to the different heavy metals showed that soil exposure of heavy metals had induced an increased Cd-, Cu-, and Zn-tolerance of N2O reductase activity. Simulation of the N2O production and reduction curves during the anaerobic incubation allowed an estimation of the apparent specific growth rate by fitting the simulated to the measured curves. Estimated growth rates were significantly lowered as the community heavy metal tolerance developed (heavy metal exposed soil after 2 months versus control soil), possibly reflecting a metabolic burden of the metal resistance mechanisms.