Isolation of effective 3-chlorobenzoate-degraders in soil using community analyses by PCR-DGGE

Abstract

The screening of pollutant degraders by relying solely on cultivation techniques such as liquid enrichment often fails to isolate the actual degraders in the environment. Community analyses by PCR-denaturing gradient gel electrophoresis (DGGE) were performed to isolate bacteria that can degrade 3-chlorobenzoate (3CB) effectively in soil. A forest soil sample was repeatedly dosed with 3CB (500 mg kg-1) to enrich it with indigenous 3CB-degraders, and changes in the bacterial community were monitored by PCR-DGGE of the 16S rRNA gene and benzoate 1,2-dioxygenase alpha subunit gene (benA). Initially, it required about 3 weeks to degrade 3CB in the soil, whereas it took only 3 days after the third dose. With this accelerated degradation, several intensified bands appeared in the DGGE profiles of both 16S rRNA gene and benA. We succeeded in isolating five 3CB-degrading Burkholderia strains corresponding to these bands by direct plating, while most of them were eliminated by liquid enrichment. Inoculation of the strains into the soil demonstrated that the five strains could degrade 3CB effectively in the soil. This study clearly shows significant bias during the liquid enrichment process and the advantage of using PCR-DGGE in screening effective degraders under environmental conditions.