Synergistic effects of sodium acetate and calcium on structure and function in multispecies biofilms

This investigation scrutinizes the manner in which sodium acetate (SA) and calcium cations (Ca²⁺) independently and collaboratively affect biofilm development. Confocal microscopy revealed that SA (1 mM) increased biofilm biovolume (5.5-fold) and thickness by enhancing microbial growth, while Ca²⁺ (1.5 mM) stabilized the matrix <i>via</i> EPS crosslinking. Combined, SA and Ca²⁺ synergistically boosted biovolume (1.5-fold) and thickness (21.3 µm) compared to SA alone. 16S rRNA sequencing showed SA-enriched <i>Actinobacteriota</i> (11%) and exopolysaccharide-producing <i>Brevifollis</i>, whereas Ca²⁺ improved surface coverage (22.3%). Functional predictions linked SA to purine degradation and Ca²⁺ to fatty acid oxidation, aligning with EPS modifications. These findings highlight how carbon sources and divalent cations collaboratively shape biofilm resilience, offering insights for biofilm management in environmental, industrial, and medical settings where SA and Ca²⁺ gradients exist.