| Biofilms, microbial communities growing on surfaces, are extremely important in a wide variety of environmental, engineered, and biomedical systems. Cells in biofilms interact with each other synergistically and competitively, and they are over 500 times more resistant to antimicrobials than planktonic cells. Coupling between physical-chemical-biological processes is known to regulate the development of biofilms. However, current experimental systems do not provide sufficient control of environmental conditions to enable detailed investigation of these complex interactions. Here, a novel planar flow cell is developed to support biofilm growth under complex two-dimensional flow conditions. This device provides precise control of flow conditions and can be used to create well-defined physical and chemical gradients that significantly affect biofilm heterogeneity. Under velocity gradients, a positive relationship was found between patterns of fluid velocity and biomass in Pseudomonas aeruginosa biofilms, but this relationship eventually reversed because high hydrodynamic shear lead to the detachment of cells from the surface. In addition, P. aeruginosa and Flavobacterium CDC-65 were chosen to form a model biofilm because they are opportunistic pathogens and are commonly found coexisting in natural/engineered aquatic systems. Mono-/multi- species biofilms were cultured under highly controlled flow gradients. Both organisms behave differently in co-cultures than in pure cultures, and their interactions and spatial distributions are significantly affected by external flow conditions. P. aeruginosa was the dominant organism in co-cultures under stagnant conditions, while Flavobacterium CDC-65 was the dominant one under flow-through conditions. Finally, the efficacy of tobramycin in killing P. aeruginosa biofilms was evaluated, revealing that spatial patterns of killing efficiency have different relationships with biofilm morphology and flow conditions at different scales. Multiple analyses indicated that cell clusters protect cells from antimicrobial. These results provide deeper understanding of biofilm heterogeneity under flow gradients and the response of biofilms to antimicrobial treatment. This study reveals the critical role of flow gradients in the development and treatment of biofilm communities, and serves as the basis to improve the design of bioreactors and other biofilm-based technologies. The findings contribute to an improved knowledge base between microscopic and environmental system scales, which connect basic microbiology to applied and environmental microbiology. |
