In natural or man-made environments, microorganisms exist predominantly as biofilms forming surface-associated bacterial communities embedded in extracellular polymeric substances (EPSs). Often, biofilm reactors used for endpoint and disruptive analyses of biofilm are not suitable for periodic observation of biofilm formation and development. In this study, a microfluidic device designed with multiple channels and a gradient generator was used for high-throughput analysis and real-time monitoring of dual-species biofilm formation and development. We compared the structural parameters of monospecies and dual-species biofilms containing Pseudomonas aeruginosa (expressing mCherry) and Escherichia coli (expressing green fluorescent protein [GFP]) to understand the interactions in the biofilm. The rate of biovolume increase of each species in monospecies biofilm (2.7 × 105 μm3) was higher than those in a dual-species biofilm (9.68 × 104 μm3); however, synergism was still observed in the dual-species biofilm due to overall increases in biovolume for both species. Synergism was also observed in a dual-species biofilm, where P. aeruginosa forms a "blanket" over E. coli, providing a physical barrier against shear stress in the environment. The microfluidic chip was useful for monitoring the dual-species biofilm in the microenvironment, indicating that different species in a multispecies biofilm exhibit different niches for the survival of the biofilm community. Finally, we demonstrated that the nucleic acids can be extracted from the dual-species biofilm in situ after biofilm imaging analysis. In addition, gene expression supported that the activation and suppression of different quorum sensing genes resulted in the different phenotype seen in the biofilm. This study showed that the integration of microfluidic device with microscopy analysis and molecular techniques could be a promising tool for studying biofilm structure and gene quantification and expression simultaneously. IMPORTANCE In natural or man-made environments, microorganisms exist predominantly as biofilms forming surface-associated bacterial communities embedded in extracellular polymeric substances (EPSs). Often, biofilm reactors used for endpoint and disruptive analyses of biofilm are not suitable for periodic observation of biofilm formation and development. Here, we demonstrate that a microfluidic device with multiple channels and a gradient generator can be useful for high-throughput analysis and real-time monitoring of dual-species biofilm formation and development. Our study revealed synergism in the dual-species biofilm, where P. aeruginosa forms a "blanket" over E. coli, providing a physical barrier against shear stress in the environment. Furthermore, different species in a multispecies biofilm exhibit different niches for the survival of the biofilm community. This study showed that the integration of microfluidic device with microscopy analysis and molecular techniques could be a promising tool for studying biofilm structure and gene quantification and expression simultaneously.
Keywords: dual-species biofilm; gene expression; gene quantification; imaging technique; microfluidics.