Determination of phenol-degrader distribution in biofilms using gene probes

This research presents a methodology for coupling microsectioning of biofilms, the subsequent extraction of DNA from the biofilm sections, and the analysis of the DNA by gene probes to determine the distribution of phenol-degrading organisms in the biofilm. A mixed species biofilm was grown in an aqueous feed that simulates a municipal wastewater. Frozen biofilm samples were sliced parallel to the substratum, using a cryostat. From representative layers of the biofilm sectioned parallel to the biofilm substratum, ethidium bromide dot tests on extracted DNA and radioactively labelled DNA probes [toluene-3-monooxygenase (tbu) or toluene dioxygenase (tod)] were used to examine the distribution of total bacterial organisms and phenol-degrading organisms, respectively, in the biofilm. The concentration of total bacterial DNA increased significantly as depth increased, for biofilms over 300 μm thick (depth from substratum to biofilm-water interface). Under conditions of stress (decreasing organic feed), however, the relationship began to fail. As with total bacterial DNA, DNA from phenol degraders showed a direct relationship with depth. The results of this study confirm that biofilm sectioning followed by DNA extraction provides suitable samples for genetic probing. Results of genetic probing reflect the heterogeneity of the biofilms grown in this study.