Exploring denitrifying communities in the environment

Denitrifiers are aerobically respiring bacteria that under anoxic conditions have the ability to switch to anaerobic respiration, so that nitrate and nitrite are stepwise reduced to nitric oxide, nitrous oxide and dinitrogen. Denitrifiers are a very diverse functional group with members from almost all phylogenetic bacterial groups. One aim of this thesis was to re-evaluate published primers targeting the functional genes, nirS, nirK and nosZ, encoding different enzymes in the denitrification pathway. New primer combinations for nirS and nosZ were designed, whereas the existing primers for nirK still seemed satisfactory. It was possible to PCR-amplify nirS genotypes from soil samples using the new nirS primer pair. In addition, denaturing gradient gel electrophoresis was introduced as a fingerprinting method for nirK- and nosZ-denitrifiers. The methods developed in this initial project were then used to study denitrifying communities in two environments where denitrification is especially important from a human perspective. In the first, methanol and ethanol were added to a wastewater treatment plant with an activated sludge process to increase the denitrification rate. As a result of the additions of external carbon sources the denitrifying communities altered their metabolic function, and specific nirS- and nirK-denitrifiers developed. In the second project, the nirK-denitrifiers were used as a model community to investigate the toxicity of the heavy metal silver to soil denitrifiers. The use of silver is increasing because of its well-known antimicrobial effects, and this may lead to increased environmental contamination. The addition of silver reduced activity and number of denitrifiers, whereas their diversity increased. Because of this demonstrated sensitivity, it is proposed that denitrifiers are suitable indicator organisms for environmental pollution. In conclusion, this thesis shows that molecular methods show great potential for investigating diversity of denitrifiers in various environments, much of which is yet to be discovered. However, in order to fully understand the ecology of denitrifiers, methods targeting the active organisms must be developed, and more denitrifiers must be isolated and characterised