Screening of Lipopeptide-Producing Strains of Bacillus sp. Using a New Automated and Sensitive Fluorescence Detection Method

Abstract

Lipopeptides, such as surfactins are important biosurfactants produced by Bacillus sp. that find applications in many areas (environment, medicine, and food industries). Giving their importance, the use of simple detection methods will facilitate screening and quantification. In the present work, the authors describe a completely automated workflow for the screening of lipopeptide-producing strains, including quantification. First, isolated colonies from environmental samples are automatically picked and inoculated in 96 wells growth plate. After overnight incubation, surfactin produced in the broth is quantified, using a new sensitive fluorescent method. The method uses fluorescein (FL), which is an anionic dye at neutral to alkaline pH and forms a stable complex with the cationic surfactant cetylpiridinium chloride (CPC), quenching fluorescence. Upon addition of surfactin or other lipopeptides, fluorescein is released from the CPC-FL complex and quantified. The robustness of this method is assessed by comparing the quantification results to those conventionally measured by RP-UPLC and the results of strain screening are confirmed by MALDI-ToF analysis. The authors report for the first time the successful application of this analytical method for high-throughput screening of novel lipopeptide-producing strains.

Keywords: Bacillus; fluorescence; high-throughput screening; lipopeptides; surfactin.