Roughness coefficients are indicative of the degree of heterogeneity of the biofilms [58]. In fact, these values (Table 3), which are significantly different in function of the medium in which the biofilms were formed (Additional file 4: Table S3) agree with the visual evidence (Figure 3), and indicate see more a patchy, heterogeneous biofilm development in MB and SASW, and more uniform biofilm layers in MH2 and LMB. Table 3 Average values of different biofilm properties in the four selected media Medium Mean thickness (μm) Max. thickness (μm) Coverage (%) Roughness coefficient Young modulus (MPa) Adhesion (nN) MB 11.2 ± 0.8 25.3 ± 2.3 15.9 ± 1.7 1.92 ± 0.06
0.16 ± 0.10 1.33 ± 0.38 MH2 9.0 ± 1.2 13.5 ± 1.0 20.9 ± 2.4 0.97 ± 0.15 0.34 ± 0.16 0.73 ± 0.29 LMB 15.4 ± 2.2 20.5 ± 3.4 32.1 ± 4.6 0.65 ± 0.18 0.22 ± 0.13 0.85 ± 0.35 SASW 13.0 ± 0.8 29.5 ± 1.9 23.9 ± 3.9 1.40 ± 0.24 0.19 ± 0.09 1.11 ± 0.41 Biofilm thickness (n = 12), surface coverage (n = 12) and roughness
coefficients (n = 12) were determined from CLSM reconstructions. Young modulii and adhesion forces were quantified by AFM. In this case, at least 115 bacteria were individually analysed for each magnitude. Data represent the average ± SD. Figure 3 Effect of the medium on biofilm structure evidenced by CLSM. Projections (upper row) and sections (bottom row) of 24-h S. algae CECT 5071 biofilms (40x) developed in different media. Columns: (A) MB; enough (B) MH2; (C) LMB; (D) SASW. Thus, two trends were observed in biofilm development depending on the medium: a clear trend to a three-dimensional growth, with a variable MK-8669 manufacturer degree of homogeneity, in MB, LMB and SASW, and a relatively horizontal development in MH2, maximising cell-to-cell and cell-to-substrate interactions. According to this depiction, we will focus on the comparison between MB and MH2 since they have been considered representative enough of the two biofilm
growth behaviours. First of all, in order to show the topographic features exhibited by the studied cells at high resolution, the samples were imaged in air after being rinsed and dried. Thus, Figure 1A shows a representative picture of some S. algae cells attached to the treated polystyrene substrate. Since these images were obtained in air, some flagella belonging to neighbouring bacteria adsorbed on the surface could be observed as well. Bacterial cells were 2.2-3.5 μm in length and 0.4-0.7 μm in width. Some polishing lines resulting from the disc’s surface treatment are also visible. Additionally, in Figure 1B, some of these features can be observed in more detail, namely some flagella (white arrow), topographic details of the bacterial surface and submicrometer particles of EPS. Figures 4A-B correspond to AFM topographic images recorded in 0.22 μm filtered seawater (FSW) obtained in MB and MH2, respectively.
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