Abstract: The effect of calcium chloride on the biofilm forming-ability of Pseudomonas fluorescens was analyzed in this study. The influences of Ca2+ on biofilm formation, planktonic bacterial growth, polysaccharide secretion, biofilm structure and the expression levels of related genes in P. fluorescens were measured by crystal violet method, bacterial counting, phenol-sulfuric acid method, confocal laser scanning microscopy (CLSM) and qPCR, respectively. The results showed that Ca2+ at 0.1 mmol/L promoted biofilm formation of P. fluorescens. Biofilm biomass gradually increased with increasing concentration of Ca2+ up to 1 mmol/L, and then decreased at Ca2+ concentrations higher than 10 mmol/L. Planktonic bacterial growth was not significantly affected by various concentrations of calcium. Similarly, the production of extracellular polysaccharides, the pellicle and swimming motility were enhanced with the addition of Ca2+ at 0.1 and 1 mmol/L while an inhibitory effect was observed at higher concentrations. CLSM observations revealed that the thickness of matured biofilm in the control, 1 mmol/L Ca2+ and 20 mmol/L Ca2+ treated groups was 20.0, 40.0 and 25.0 μm, respectively and that 1 mmol/L Ca2+ resulted in significantly higher biofilm thickness and more adhered cells/extracellular secretions together with a more compact biofilm structure. qPCR revealed that the expression levels of lapA, alg and flgA genes were up-regulated about 3 to 4 folds by 1 mmol/L Ca2+. Furthermore, Ca2+ at low or high concentration positively stimulated the expression level of luxI, an acyl homoserine lactone (AHL) synthetase, suggesting that effect of Ca2+ on the biofilm may be closely related with quorum sensing. Thus, these results indicated that calcium chloride induced changes in the characteristics and structure of biofilm formation in P. fluorescens by affecting the adherence, extracellular secretions and the expression of related genes. This study provides a basis for exploring the biofilm formation and adherence characteristics of spoilage bacteria in complex food matrices.

Key words: Pseudomonas fluorescens, biofilm, extracellular secretions, calcium chloride