Matrix matters: How extracellular substances shape biofilm structure and mechanical properties

Biofilms possess unique mechanical properties that are vital to their stability and function. Biofilms are made of extracellular polymeric substances (EPS) secreted by microorganisms and comprise polysaccharides, proteins, extracellular DNA (eDNA), and lipids. EPS is the primary contributor and driver of the biofilm structure and mechanical properties such as stiffness, cohesion, and adhesion. EPS enhances the elasticity and viscosity of biofilms, allowing them to withstand mechanical stresses, shear forces, and deformation. Therefore, biofilms are notoriously difficult to remove and can result in billions of dollars in losses for various industries due to their adverse effects, such as contamination, pressure loss, and corrosion. As a result, it is essential to comprehend the mechanical properties of biofilms to control or remove them in various scenarios. We undertook a fundamental study to determine the relationship between individual EPS components and biofilm mechanical properties. In this study, a CDC biofilm reactor was used to grow pure culture biofilms (Staphylococcus epidermidis) which were treated with six EPS modifier agents (Ca²⁺, Mg²⁺, periodic acid, protease K, lipase, and DNAase I) to modify or cleave specific EPS components. The mechanical properties (Young's Modulus) of treated biofilms were subsequently tested using atomic force microscopy (AFM), the biofilm EPS functional groups were measured via the Fourier transform infrared (FTIR) spectroscopy, and biofilm structural characteristics using confocal imaging. The FTIR results showed that EPS modifier agents successfully reduced their target EPS components. Similarly, the confocal microscopic analysis results showed that most of these modifier agents (except lipase) significantly reduced (P-value <0.05) the biovolume and thickness of treated biofilms. Conversely, most of these modifier agents (except protease K) significantly increased (P-value <0.05) the roughness coefficient of the biofilms. Finally, data from AFM showed that biofilm mechanical properties (Young’s modulus) significantly (P-value <0.05) changed with their EPS composition. These results have significant ramifications for biofilm management and control in myriad scenarios.