A PCR test to identify Bacillus subtilis and closely related species and its application to the monitoring of wastewater biotreatment

A PCR test based on the 16S rRNA gene was set up that could identify any of the five species of the 'Bacillus subtilis group' (B. subtilis, B. pumilus , B. atrophaeus, B. licheniformis and B. amyloliquefaciens). The test was d irectly applicable to single colonies and showed excellent specificity. In the mixed population context of wastewater analysis, direct detection of th e target Bacillus species by PCR on either crude or purified DNA extracts h ad poor sensitivity. When assayed on cell suspensions derived from enriched wastewater samples, sensitivity was increased. Using a simple calibration method, it was possible to estimate the proportion of the target organisms. This method was found suitable for easy monitoring of a wastewater bioaugm entation experiment carried out with a mixture of sporulated Bacillus strai ns.