Rapid screening for bacterial phenotypes capable of biodegrading anionic surfactants: development and validation of a microtitre plate method.

Abstract

The Biolog microtitre plate assay, which is based on tetrazolium dye reduction as an indicator of sole-carbon-source utilization, has been evaluated as a rapid method to investigate the biodegradation of five classes of anionic surfactant by pure and mixed cultures of bacteria. The assay gave reproducible results over a fourfold range of inoculum optical density, and the surfactant concentration was selected to provide a compromise between the length of the lag period prior to colour production and the maximum colour produced. A kinetic model was developed and used to analyse the appearance of colour in the assay and was found to give rise to three biologically significant parameters describing the processes underlying the assay. No false-positives were obtained with environmental isolates. The small number of false-negatives obtained (< 8% of the total) could be explained by the methodology used to prepare the bacterial inoculum. All isolates which were positive in the Biology assay were shown to be both primary and ultimate degraders of the test surfactant. These results show that the method provides a useful means of studying the biodegradation of anionic surfactants by both pure and mixed cultures of bacteria and will find use in the rapid analysis of biodegradation kinetics and specificities of larger numbers of individual isolates than hitherto possible. In addition, an important benefit of the methodology is that it can be used for direct analysis of the biodegradation potential of whole bacterial communities without having to make an artificial selection during laboratory growth.