Influence of Inorganic Particle Incorporation on Biofilm Mechanical Stability

Laboratory-grown biofilms are widely used as controlled models to investigate biofouling processes and mechanics. However, they typically exclude inorganic particulate matter, despite the widespread occurrence of suspended particles in feedwaters of engineered water systems and their accumulation within biofouling layers, resulting in simplified structures and underestimation of biofouling layer mechanical strength. This study investigated how particle embedding influences the structural, chemical, biological, and mechanical development of biofouling layers. A mature, particle-rich biofouling layer recovered from a spent full-scale seawater reverse osmosis (SWRO) membrane was first characterized as a reference system. Synthetic biofilms were then cultivated with and without embedded kaolin and diatomaceous earth (DE) particles to isolate particle-driven effects. The SWRO fouling layer exhibited a clay-mineral-rich matrix, low microbial activity, and high mechanical strength (yield point = 25262 Pa), indicating that although inorganic constituents were not dominant by mass, they disproportionately contributed to mechanical reinforcement through the formation of dense, particle-reinforced networks. Particle-free biofilms formed soft EPS-dominated matrices (yield point = 1048 Pa), whereas embedding kaolin and diatomaceous earth (DE) increased inorganic content, reduced microbial activity, and enhanced viscoelastic strength of biofilms (kaolin: yield point = 1603 Pa; DE: yield point = 4741 Pa). Mechanistically, particle embedding promoted EPS-particle interactions (adsorption, bridging, particle-induced confinement), strengthening the biofilm matrix by increasing network connectivity and resistance to deformation. Consequently, matrix stiffness and yield strength increased with inorganic fraction, demonstrating that particle embedding promotes the formation of mechanically reinforced fouling layers and that particle-free biofilm models systematically underestimate mechanical resilience.