3.1. Fouling and Surface Evaluation
In this study, we directly examined the AF capacity of the aforementioned PDMS-based coatings in natural seawater and established a fouling evaluation system based on the MDS method in order to further quantify the fouling conditions among different PC surfaces, in the context of the adhesive number of five representative major macrofoulers, including barnacles, mussels, ascidians, Ulva
and seaweeds (see Figure 2
and Figure 3
From Figure 2
, it is clear that the AF properties of the plain PDMS were greatly improved after the incorporation of a low amount of nanosized CNTs (0.1 wt %). Each PC displayed a differential but reinforced AF efficacy against the representative macrofoulers compared with the PDMS standard. The M1, H1 and C3 coatings performed exceptionally well in the field exposure assays, while the M3, C2 and C5 coatings were found to be heavily fouled. Furthermore, it seems that most PCs of the identical set (e.g., coatings in the M Set, except M3) tended to exhibit similar AF properties, although there were some exceptions (e.g., C2 and C5 coatings in the C Set). These differential AF behaviors may be largely owing to the differences within different CNT fillers.
In addition, in Figure 3
, most PCs, including M1, M2 and M4-M6 coatings from the M set, H1–H6 coatings from the H set, and C1, C3, C4 and C6 coatings from the C set, were liable to cluster into the same group, suggesting that these PCs may have possessed similar AF performances. Nevertheless, it is noticeable that M3, C2 and C5 coatings were liable to cluster separately, and their AF properties were clearly different from those of the PDMS standards (P0) and the aforementioned PCs. This result further revealed that the physicochemical properties of the CNT filler may have differential reinforcing impacts on the AF properties of the PDMS matrix. Recently, CNTs have been applied as additives to improve the membrane properties of various polymeric matrixes worldwide, and a host of highly promising functionalized nanocomposites with excellent properties have been obtained for marine AF applications [23
]. However, most fouling evaluation systems are still confined to laboratory assays, only involving the measurement of the adhesive number of representative hard foulings (e.g., B. Amphitrite
and Mytilus edulis
] or soft foulings (e.g., Ulva
]. It is obvious that laboratory biological assays still remain insufficient and limited, although laboratory assays are insusceptible to environmental disturbances, unlike field exposure assays [29
]. Here, we provided a feasible and effective way to solve this problem and established a novel fouling evaluation system targeting the measurement of the adhesive number of multiple natural fouling organisms in natural seawater using the MDS method, based on the data obtained from rigorous marine field assays. The advantage of this approach is obvious, since the adhesive behaviors of multiple adherent marcofoulers on different coating surfaces can be dynamically observed and recorded directly in the natural seawater, which can give a more comprehensive and objective assessment on their actual AF performance. Besides, the variations within different AF coatings can also be easily observed and captured simply using visual inspection.
3.2. SSCP Patterns of the Bacterial Biofilm Communities
shows the SSCP profiles of the pioneer bacterial communities in the natural biofilms developed on the PDMS-based material surfaces at different exposure times. Each band within the SSCP profiles is approximately identical to a single microbial species. As observed from SSCP patterns, eighteen kinds of PCs were generally colonized by a mixture of the early-adherent bacterial communities without exception during the two-week in situ experiment, and no significant differences were screened compared with the PDMS standards (P0) via the visual inspection. This indicated that no PCs completely resisted or deterred the colonization of pioneer prokaryotic microbes.
Early bacterial communities formed on the PDMS-based coatings belonging to the same PC set were liable to evolve similar SSCP patterns at different exposure times, while differential SSCP patterns were screened within different PC sets. For example, in the 5-day biofilm, clear differences were observed in the SSCP patterns of the pioneer biofilm communities developed on different PC sets, owing to the differences within various coating types. It is estimated that the physicochemical properties of the CNT types may be closely related to the differential and improved AF properties of the PCs. In addition, as the natural biofilm grew older (e.g., the 14-day biofilm), the early adhered bacterial communities on the PDMS-based coatings were found to be clearly increased. This result suggested that the deterrence effects of PCs against the colonization of the early bacterial communities may become increasingly weakened over time. These combined results indicated that the PCs were susceptible to microfouling when immersed in the marine environment during the short-term in situ experiment.
3.4. Analysis of Pioneer Bacterial Communities in the Natural Biofilms
Three diversity indices, including the Shannon diversity index (H
), species richness(S
), and the Simpson index (λ
), were calculated and compared, as presented in Figure 6
The Shannon diversity index (H
) describes the general biodiversity in environmental microbial communities, and was used to estimate the early bacterial community diversity in the natural biofilms developed on different PCs [30
]. Figure 6
a–c show that the H
value of the bacterial communities ranged between 2.53 ± 0.27 and 2.73 ± 0.23 for all the PC surfaces, compared with the PDMS control (2.56 ± 0.26), indicating that different PC surfaces may have differential modulating effects on the colonization of pioneer bacterial communities. The highest level of early bacterial community diversity was screened on the M1 surfaces (H
= 2.73 ± 0.23) among all of the PC surfaces, while the lowest level of diversity was found on the C6 surfaces (H
= 2.53 ± 0.27). The pioneer prokaryotic microbial communities attached to the PCs surfaces belonging to the M set and H
set (H1–H6) shared a relatively high level of diversity, with H
values ranging from 2.53 ± 0.27 to 2.73 ± 0.23 and 2.54 ± 0.19 to 2.64 ± 0.23 (Figure 6
a,b), respectively. However, the diversity of the early bacterial communities on the CP surfaces (C1–C6) was lower than that of the PDMS control (2.56 ± 0.26), with H
values ranging from 2.09 ± 0.44 to 2.45 ± 0.22 (Figure 6
c), particularly on the C6 surfaces (p
< 0.05). No significant differences were found in the diversity level between the PCs (except C6) and the PDMS standards (p
> 0.05). This indicated that the PC surfaces may only have weak modulating effects on colonized pioneer prokaryotic microbes, and most PC surfaces were still susceptible to the colonization and deterioration of the pioneer prokaryotic microbes.
Furthermore, species richness (S) describes the number of different species in an environmental microbial community, which was applied to give descriptions about the number of species of the early bacterial communities in the natural biofilms developed on different PCs [31
]. Figure 6
d–f revealed that the S value of the pioneer bacterial community (ranging from 9 ± 2 to 17 ± 5) was slightly downregulated by most PC surfaces (except C6), compared with the PDMS surface (15 ± 4). Specifically, the pioneer bacterial biofilm communities adhering to the PC surfaces belonging to the MPs surfaces and the HPs surfaces shared a relatively high level of richness, while the early bacterial communities adhering to the PCs belonging to the C set shared a relatively low level of richness, significantly on the C6 surfaces (p
< 0.01), compared to the PDMS control, which correlated well with the diversity level.
The Simpson index (λ) describes the number of dominant species in a particular microbial community and was used to measure the number of dominant populations in pioneer bacterial communities in the natural biofilms formed on different PCs [32
]. Figure 6
g–i reveal that the λ
value of pioneer bacterial communities was slightly decreased on the MP surfaces (0.066 ± 0.013 to 0.083 ± 0.023), and remained almost unchanged on the HP surfaces, while it slightly increased on the CP surfaces (0.093 ± 0.021 to 0.160 ± 0.088), particularly on the C6 surfaces (p
< 0.05), compared to the PDMS control (0.088 ± 0.027). This result indicates that the dominant bacterial population in the biofilm developed on most PCs surfaces (except C6) varied slightly, in contrast to the PDMS control. The slightly changed dominant bacterial communities in the biofilm suggest that most PC surfaces may only have a weak capacity to exert enough perturbations on the biological colonization and successional patterns of early adherent bacterial communities in natural biofilms.
Previously, a host of publications reported that AF coatings could influence and regulate the development of early-colonized bacterial communities [33
]. However, a few publications have focused on the modulating effects of different PDMS-based nanocomposites on colonized pioneer bacterial communities in natural biofilms [37
]. In the current study, most PCs demonstrated differential modulating effects on the colonization of pioneer prokaryotic microbes. The pioneer bacterial communities were only found to be subjected to the minor perturbations exerted by most PCs (except for coating C6). This slightly modulating effect suggested that the PCs may not exert sufficient perturbations on the biological succession patterns of the pioneer bacterial communities in the natural biofilms, which may contribute to the mechanisms causing the plain PDMS surfaces, along with most PCs surfaces (except coating C6), to be extremely susceptible heavy fouling after long-term exposure to the marine environment, since the bacterial communities in the biofilms have been found to play key roles in the biodeterioration and biodegradation of synthetic polymeric materials [39
]. Data on this hypothesis still requires further study in our future work. Coating C6 seems to be promising for future marine anti-biofouling applications, owing to its strong perturbation effects on the colonization of early bacterial communities.