Detection of Nitrate-Reducing/Denitrifying Bacteria from Contaminated and Uncontaminated Tallgrass Prairie Soil: Limitations of PCR Primers

Contamination of soil by spills of crude oil and oilfield brine is known to affect the species composition and functioning of soil microbial communities. However, the effect of such contamination on nitrogen cycling, an important biogeochemical cycle in tallgrass prairie soil, is less well known. Detecting nitrate-reducing (NR) and denitrifying (DN) bacteria via PCR amplification of the genes essential for these processes depends on how well PCR primers match the sequences of these bacteria. In this study, we enriched for NR and DN bacteria from oil/brine tallgrass prairie soil contaminated 5-10 years previously versus those cultured from uncontaminated soil, confirmed the capacity of 75 strains isolated from the enrichments to reduce nitrate and/nitrite, then screened the strains with primers specific to seven nitrogen cycle functional genes. The strains comprised a phylogenetically diverse group of NR and DN bacteria, with proportionately more γ-Proteobacteria in oil-contaminated sites and more Bacilli in brine-contaminated sites, suggesting some residual effect of the contaminants on the NR and DN species distribution. Around 82% of the strains shown to reduce nitrate/nitrite would not be identified as NR and DN bacteria by the battery of NR and DN primers used. Our results indicate an urgent need to expand the NR/DN functional gene primer database by first identifying novel NR/DN strains through their capacity to reduce nitrate/nitrite.