Community and diversity surveys of bacteria have for nearly 10 years focused on the phylogenetic diversity of bacteria in the environment. However, species composition based on 16S rRNA analysis only provides marginal information on communities and certain metabolic groups. An exciting direction in molecular ecology is the analysis of genes encoding important functions for the ecosystems. The analysis of functional diversity and its physiological dynamics is essential for improved understanding of the microbial ecology and biogeochemistry of different environments. Generally, functional genes have more sequence variation than the relatively conserved 16S rRNA genes. They can therefore be exploited as biomarkers to discriminate between closely related but ecologically different populations . Moreover, some important functions are not associated with a specific taxonomic group. One example is denitrification, which is a functional trait found within more than 50 genera .
Denitrification is the stepwise reduction of nitrate (NO3−) to dinitrogen (N2), associated with oxidative phosphorylation and release of nitric oxide (NO) and nitrous oxide (N2O) gases. This process is of global concern since it leads to nitrogen losses from agricultural soils and because nitrous oxide contributes to ozone depletion in the stratosphere and is a potent greenhouse gas . Denitrifying bacteria can also be used to remove excess nitrogen in wastewater treatment plants and to degrade organic pollutants. Most bacteria with this functional trait belong to a wide range of various subclasses of Proteobacteria. However, the ability to denitrify has also been found in some Archaea, in the halophilic and hyperthermophilic branches, and in mitochondria of certain fungi . Lateral gene transfer is the most likely explanation for this widespread ability to denitrify .
Functional genes that encode for the enzymes involved in the denitrification pathway, such as nitrite, nitric oxide and nitrous oxide reductases, can be exploited by targeting conserved regions. The reduction of nitrite (NO2−) to nitric oxide distinguishes denitrifiers from other nitrate-respiring bacteria . This reaction is central to denitrification and is catalysed by two different types of nitrite reductases (Nir), either a cytochrome cd1 enzyme encoded by nirS or a Cu-containing enzyme encoded by nirK. The reduction of nitrous oxide is the last step in the denitrification pathway and is catalysed by nitrous oxide reductase encoded by the nosZ gene. However, some denitrifiers lack this enzyme. The nosZ gene can be used as a target for the different populations of the denitrifying bacteria capable of nitrous oxide reduction. Most work on molecular ecology of denitrifying bacteria has been based on nirK and nirS, as well as nosZ, e.g. [5,6]. Recently, the gene, norB, encoding nitric oxide reductase was used as a marker for denitrifying bacteria in freshwater and marine sediments .
Surveys of bacterial community composition in environmental samples are often based on the polymerase chain reaction (PCR). Reliable PCR primers are a prerequisite for microbial community surveys since they ultimately determine what is detected in the environmental sample. Ward  made the first attempt to design PCR primers targeting the nirS gene and they were based on only three sequences from two different species. A better attempt to amplify nirK and nirS was published in 1998 , and alternatives or modifications followed [10–15]. Primers for detection of the nosZ gene were first published in 1998  and these were later made more degenerate . Kloos et al.  developed more pertinent broad range primers for nosZ. The different nir and nosZ primers have mainly been used to study community composition of denitrifying bacteria in marine sediments [5,6,16,19–22] but also in estuarine sediments , cyanobacterial bloom , soil [13,24–29], wastewater treatment reactors [30,31] and groundwater . The oldest and most frequently used primers to detect denitrifying bacteria target nirS and nirK and were designed based on a limited number of sequences, mainly from laboratory strains. Since then the number of partial nir and nosZ sequences deposited in the GenBank have increased almost a 100-fold and it has become apparent that the primer sites are more variable than previously shown.
The aim of our study was to re-evaluate primers for amplification of nirS, nirK and nosZ gene fragments in silico and in vitro, and to introduce denaturing gradient gel electrophoresis (DGGE)  as a tool to survey the denitrifying community composition in environmental samples. A limiting factor when designing primers for DGGE is that the fragments should not be much longer than 500 bp for successful analysis . Nevertheless, the most commonly used primers for the denitrifying genes are not suitable for DGGE since they amplify fragments that are approximately 600–1100 bp. DGGE of partial 16S rDNA has been successfully employed for analysis of community DNA even in such complex environments as soil (e.g. ). However, the use of DGGE with functional genes is still in its beginnings.