Comparison of the acid–base reactivity of free-living Pseudomonas putida cells and biofilm

The proton reactivity of a soil bacterium, Pseudomonas putida ATCC12633 was investigated in two physiologically different states: (i) as free-living cells, and (ii) as a 5-day biofilm formed in a sandy column. Acid–base data analysis and modeling based on a three-site non-electrostatic model showed that biofilm has a proton exchange capacity 3.8 times higher than that of free-living cells (13.2 mmol/g protein corresponding to 5.9 ± 1.2 mmol/g dry weight for biofilm and 3.8 mmol g protein; 1.56 ± 0.32 mmol/g dry weight for free cells). The higher proton exchange capacity of the biofilm fragments mainly results from the high content of the ‘neutral’ pK 6.5 sites. This increase was explained through sorption on the biofilm of mineral phosphate residues circulating with the nutrient medium during the 5 days of column biofilm growth. SEM biofilm observations show a dense network of excreted organic materials linking the individual cells and organic residues. On the contrary, free cells are clearly individualized. The differences between the morphology and reactivity of biofilms and free cells strongly indicate that the two substrates have different compositions. In opposition to free cells, acidic conditions lead to non reversible biofilm proton exchange properties that could be explained by biofilm coagulation processes. These results show the importance of the growing conditions of biofilm and especially its development on a solid surface in the determination of biofilm composition and reactivity. ► We compared the proton exchange property of Pseudomonas putida biofilm and free cells. ► The biofilm was grown in a sandy column under a dynamic nutrient supply. ► A large number of phosphate sites are responsible for the increase of biofilm reactivity. ► Exopolymeric substances may be responsible for the high biofilm reactivity. ► The growth conditions of biofilm influence its proton exchange properties.