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 via 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 Actinobacteriota (11%) and exopolysaccharide-producing Brevifollis, 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.