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Rheometry is an experimental technique widely used to determine the mechanical properties of biofilms. However, it characterizes the bulk mechanical behavior of the whole biofilm. The effects of biofilm mechanical heterogeneity on rheometry measurements are not known. We used laboratory experiments and computer modeling to explore the effects of biofilm mechanical heterogeneity on the results obtained by rheometry. A synthetic biofilm with layered mechanical properties was studied, and a viscoelastic biofilm theory was employed using the Kelvin–Voigt model. Agar gels with different concentrations were used to prepare the layered, heterogeneous biofilm, which was characterized for mechanical properties in shear mode with a rheometer. Both experiments and simulations indicated that the biofilm properties from rheometry were strongly biased by the weakest portion of the biofilm. The simulation results using linearly stratified mechanical properties from a previous study also showed that the weaker portions of the biofilm dominated the mechanical properties in creep tests. We note that the model can be used as a predictive tool to explore the mechanical behavior of complex biofilm structures beyond those accessible to experiments. Since most biofilms display some degree of mechanical heterogeneity, our results suggest caution should be used in the interpretation of rheometry data. It does not necessarily provide the “average” mechanical properties of the entire biofilm if the mechanical properties are stratified.

Biofilms commonly develop in flowing aqueous environments, where the flow causes the biofilm to deform. Because biofilm deformation affects the flow regime, and because biofilms behave as complex heterogeneous viscoelastic materials, few models are able to predict biofilm deformation. In this study, a phase‐field (PF) continuum model coupled with the Oldroyd‐B constitutive equation was developed and used to simulate biofilm deformation. The accuracy of the model was evaluated using two types of biofilms: a synthetic biofilm, made from alginate mixed with bacterial cells, and aPseudomonas aeruginosabiofilm. Shear rheometry was used to experimentally determine the mechanical parameters for each biofilm, used as inputs for the model. Biofilm deformation under fluid flow was monitored experimentally using optical coherence tomography. The comparison between the experimental and modeling geometries, for selected horizontal cross sections, after fluid‐driven deformation was good. The relative errors ranged from 3.2 to 21.1% for the synthetic biofilm and from 9.1 to 11.1% for theP. aeruginosabiofilm. This is the first demonstration of the effectiveness of a viscoelastic PF biofilm model. This model provides an important tool for predicting biofilm viscoelastic deformation. It also can benefit the design and control of biofilms in engineering systems.

The mechanical properties of biofilms can be used to predict biofilm deformation under external forces, for example, under fluid flow. We used magnetic tweezers to spatially map the compliance ofPseudomonas aeruginosabiofilms at the microscale, then applied modeling to assess its effects on biofilm deformation. Biofilms were grown in capillary flow cells with Reynolds numbers (Re) ranging from 0.28 to 13.9, bulk dissolved oxygen (DO) concentrations from 1 mg/L to 8 mg/L, and bulk calcium ion (Ca²⁺) concentrations of 0 and 100 mg CaCl₂/L. Higher Re numbers resulted in more uniform biofilm morphologies. The biofilm was stiffer at the center of the flow cell than near the walls. Lower bulk DO led to more stratified biofilms. Higher Ca²⁺concentrations led to increased stiffness and more uniform mechanical properties. Using the experimental mechanical properties, fluid–structure interaction models predicted up to 64% greater deformation for heterogeneous biofilms, compared with a homogeneous biofilms with the same average properties. However, the deviation depended on the biofilm morphology and flow regime. Our results show significant spatial mechanical variability exists at the microscale, and that this variability can potentially affect biofilm deformation. The average biofilm mechanical properties, provided in many studies, should be used with caution when predicting biofilm deformation.