Microfluidic investigation of the impacts of flow fluctuations on the development of Pseudomonas putida biofilms

Biofilms play critical roles in wastewater treatment, bioremediation, and medical-device-related infections. Understanding the dynamics of biofilm formation and growth is essential for controlling and exploiting their properties. However, the majority of current studies have focused on the impact of steady flows on biofilm growth, while flow fluctuations are common in natural and engineered systems such as water pipes and blood vessels. Here, we reveal the effects of flow fluctuations on the development of Pseudomonas putida biofilms through systematic microfluidic experiments and the development of a theoretical model. Our experimental results showed that biofilm growth under fluctuating flow conditions followed three phases: lag, exponential, and fluctuation phases. In contrast, biofilm growth under steady-flow conditions followed four phases: lag, exponential, stationary, and decline phases. Furthermore, we demonstrated that low-frequency flow fluctuations promoted biofilm growth, while high-frequency fluctuations inhibited its development. We attributed the contradictory impacts of flow fluctuations on biofilm growth to the adjustment time ( T 0 ) needed for biofilm to grow after the shear stress changed from high to low. Furthermore, we developed a theoretical model that explains the observed biofilm growth under fluctuating flow conditions. Our insights into the mechanisms underlying biofilm development under fluctuating flows can inform the design of strategies to control biofilm formation in diverse natural and engineered systems.