Representative autocorrelation curves [
g(τ)] obtained for fluorescent nanospheres within PRTP
−, PRTP
+, and PRTP* biofilms are presented in Fig.
1. Adjustments of these experimental curves were performed using the Brownian diffusion model with two components (Table
1):
where α and 1 − α are the fractions of the molar concentrations of the two diffusive species and τ
1 and τ
2 are their respective translational diffusion times,
N is the number of fluorescent species inside the excitation volume, and ω
0/
z0 is the lateral/axial radius ratio of the laser beam (ω
0/
z0 = 0.30 ± 0.03, determined elsewhere [
1]).
An initial diffusion time (τ
1) of ∼8 ms was obtained in water and in all biofilms and hence could reasonably be assigned to the free diffusion of monomeric nanospheres. Nonetheless, the proportion of probes (α
1) freely diffusing is statistically different (
P < 0.05) depending on the surface physicochemical properties of the strains used to grow the biofilms: a lower fraction of fluorescent particles freely diffuses in PRTP
+ and PRTP* biofilms composed of hydrophobic cells than in PRTP
− biofilms composed of hydrophilic cells, as presented in Table
1. The remaining populations (α
2) correspond to probes diffusing with a longer diffusion time (τ
2) that could be assigned to species whose diffusion inside
L. lactis biofilms is hindered. Although the observed obstruction of diffusion could be interpreted as being caused by particle aggregation, the obtained τ
2 value measured in biofilms would unlikely correspond to the diffusion of aggregates larger than 1 μm in diameter (which has never been measured with these nanospheres in such proportions). This τ
2 diffusion time may more likely be ascribed to particles colliding with the bacterial envelope. Furthermore, α
2 and τ
2 values are higher in the case of PRTP
+ and PRTP* than in the case of PRTP
− (
P < 0.05), suggesting softer impacts of nanoparticles with hydrophobic cell walls than with hydrophilic ones. Moreover, similar results were obtained in both PRTP
+ and PRTP* biofilms (
P > 0.05), suggesting that the presence of the anchored proteinase rather than its enzymatic activity is responsive in hindering particle diffusion inside
L. lactis biofilms. It cannot be excluded that modifications to the cell surface may exert an indirect effect on EPS properties that could affect diffusion properties in the biofilm matrix.
To summarize, the presented data demonstrated that bacterial cell wall properties can affect diffusion inside the biofilm matrix. Indeed, in addition to the exopolymeric substances, interfacial bacterial components, such as peptidoglycan, capsules, proteins, S-layer, and pili, among others, could also condition molecule and particle mobility inside biofilms and hence participate in specific biofilm phenotypes.