Biofilm proliferation in engineered water systems is an ongoing concern for both municipal water infrastructure and medical equipment in healthcare environments. Research into those systems under flow conditions is especially important due to their prevalence and known effects of flow on biofilm mechanical properties and bacterial proliferation. Pseudomonas aeruginosa is an opportunistic pathogen primarily known for its harmful role in the lungs of cystic fibrosis (CF) patients, but it also inhabits other environments, including human-made water installations. The P. aeruginosa PPF-1 strain was previously isolated from a dental unit waterline (DUWL) and is representative of other strains found in the same environment. Interestingly, similar to P. aeruginosa strains isolated from chronic CF lung infections, the gacS gene of PPF-1 is inactivated. This gene encodes a sensor kinase that is a component of a signalling network that regulates the expression of various virulence factors. PPF-1 also contains a lasR gene alteration, as observed in other P. aeruginosa known to proliferate in engineered and natural water systems such as swimming pools, rivers and hydrocarbon-contaminated soils, hospital sink drains, and meat/fish market environments. The PPF-1 strain is an important producer of biofilms, and consequently, studying biofilm formation by PPF-1 as a model strain is of interest. Biofilms are surface-attached structures made of cells embedded in a self-produced extracellular polymeric matrix. This helps to create a niche for cell growth and proliferation in otherwise inhospitable environments. As such, biofilm formation is likely the main ARTICLE ARTICLE Journal Name strategy for efficient colonization of DUWLs and other engineered water systems. PPF-1 can form biofilms in static conditions and in pseudodynamic environments, such as within lightly shaken microplate cultures where bacteria exist under uncontrolled shear stress. The imposition of shear stresses produced by flow systems such as DUWLs may enhance biofilm downstream colonization through biofilm removal (sloughing) and control of oxygen availability. Flow systems also provide a supply of substrate and other molecules and avoid build-up of metabolic byproducts. To investigate flow-assisted biofilm sloughing, welldefined flow conditions should be applied while passive measurements are acquired. Amongst the numerous laboratory approaches available to study biofilms, the main tools (microtiter dish biofilm and Calgary Biofilm Devices ) provide a static growth environment and are therefore not useful for this study. Common flow reactors (drip flow reactors and the Center for Disease Control biofilm reactor ) do not provide a high degree of control over shear stresses, and other flow reactor types are not well adapted for long duration studies due to the large volumes of liquids required. To address these challenges, the biofilm research community is increasingly turning to microfluidic tools. In addition to reduced solution consumption, even for fast flow and long-duration experiments, the excellent optical properties of microfluidic fabrication materials enable straightforward transmission microscopy as a tool for high-quality time-lapse imaging. Due to their small dimensions, parallel flow microchannels, e.g., containing a sample and reference strain, can be monitored in the same field of view. Previously, we exploited these benefits to create threedimensional biofilm re-constructions and then used them to study the dynamic properties of Pseudomonas sp. biofilms in microchannels. This, combined with a complete lack of turbulence, results in highly predictable applied shear stress against wall-adhered biofilms, opening the way for computational fluidic dynamic simulations with realistic boundary conditions. In a previous study, we demonstrated that in a shaken culture, divalent magnesium ions (Mg 2+ ) from added MgCl2 (20 mM) to the LB medium resulted in an increase in adhered biofilms. It should be noted that LB medium typically contains only 0.05 to 0.3 mM of Mg 2+ . The increased adherence was unique among the P. aeruginosa strains included in this study (PPF-1, PAO1, LESB58, and Urg-7). The effect of di-and trivalent ions on biofilm mechanical properties and their ability to resist sloughing from applied shear stresses are contradictory in the literature. Studies using microfluidics stand to clarify such discrepancies, especially, for example, contemporary questions related to the role of Mg 2+ in biofilm accumulation and mechanical properties. In this work, we investigate the role of Mg 2+ in stabilizing PPF-1 biofilm adhesion using new advances on previous microfluidic experiments and simulations studying biofilm mechanical properties. The results are compared with those from PPF-1 biofilms under control conditions (no additional Mg 2+ ). Comparisons to biofilms from the P. aeruginosa clinical strain PAO1 isolated from a human wound were used as a reference because they are well represented in a range of earlier microfluidic studies, albeit differently focused than the present research into the consequences of applied shear stress on biofilms. Specifically, we present quantitative results on critical shear stress for biofilm sloughing and cumulative biofilm release profiles over time. We also investigated the effect of Mg 2+ ions, which caused an increased in mechanical stability and a propensity in PPF-1 for infrequent but catastrophic biofilm sloughing events, similar to the PAO1 reference strain. In contrast, PPF-1 that is not exposed to an increased Mg 2+ ion concentration may more easily lead to downstream contamination.