A three-step method for analysing bacterial biofilm formation under continuous medium flow
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For the investigation and comparison of microbial biofilms, a variety of analytical methods have been established, all focusing on different growth stages and application areas of biofilms. In this study, a novel quantitative assay for analysing biofilm maturation under the influence of continuous flow conditions was developed using the interesting biocatalyst Pseudomonas taiwanensis VLB120. In contrast to other tubular-based assay systems, this novel assay format delivers three readouts using a single setup in a total assay time of 40 h. It combines morphotype analysis of biofilm colonies with the direct quantification of biofilm biomass and pellicle formation on an air/liquid interphase. Applying the Tube-Assay, the impact of the second messenger cyclic diguanylate on biofilm formation of P. taiwanensis VLB120 was investigated. To this end, 41 deletions of genes encoding for protein homologues to diguanylate cyclase and phosphodiesterase were generated in the genome of P. taiwanensis VLB120. Subsequently, the biofilm formation of the resulting mutants was analysed using the Tube-Assay. In more than 60 % of the mutants, a significantly altered biofilm formation as compared to the parent strain was detected. Furthermore, the potential of the proposed Tube-Assay was validated by investigating the biofilms of several other bacterial species.
KeywordsPseudomonas taiwanensis VLB120 Tubular biofilm Biofilm dry weight Pellicle Single species biofilm Cyclic diguanylate
We are grateful to Dr. Nick Wierckx of RWTH Aachen University, Institute of Applied Microbiology (iAMB), Aachen and Prof. Victor de Lorenzo of CNB, CSIC, Madrid for providing required plasmids. We thank Prof. Katja Ickstadt of TU Dortmund University, Faculty of Statistics, for her support during statistical data analysis.
Compliance with ethical standards
This study was funded by the Ministry of Innovation, Science and Research of North Rhine-Westphalia in the frame of CLIB-Graduate Cluster Industrial Biotechnology (contract no: 314–108 001 08).
Conflict of interest
The authors declare that they have no conflict of interest.
Human and animal rights and informed consent
This article does not contain any studies with human or animal subjects.
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