6533b82dfe1ef96bd12912f9

RESEARCH PRODUCT

Molecular tools to assess the diversity and density of denitrifying bacteria in their habitats

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Publisher Summary This chapter describes the molecular tools to assess the diversity and density of denitrifying bacteria in their habitats. Genome sequencing and metagenomic projects might even provide new denitrification gene sequences, which could aid in designing more broad range primers. Most information is obtained by cloning and sequencing the polymerase chain reaction (PCR) amplicons, but a more rapid analysis is achieved using fingerprinting techniques. As all PCR-based analyses, the fingerprinting techniques are subjected to well-known biases introduced by, e.g., DNA extraction procedures, primer selection, and PCR conditions. For denitrifiers, the PCR-RFLP (restriction fragment length polymorphism) fingerprint technique has mainly been applied to study the diversity of the narG gene encoding the membrane-bound nitrate reductase. Several studies used Terminal restriction fragment length polymorphism based on nir and nosZ genes as functional marker genes to explore denitrifier communities in different habitats. The use of denaturing gradient gel electrophoresis to fingerprint denitrifier communities in the environment is rather new, although the technique has been exploited since around 1990. Different approaches based on either PCR or hybridization have been developed to quantify denitrifiers without any cultivation step. Each soil has a nearly unique denitrifier community, because of inherent soil properties and specific environmental conditions at the site. The estimations of the diversity of denitrification genes from environmental clone libraries using statistical approaches clearly show that there is awareness of a very small fraction of this diversity.