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Effect of anti-biofouling potential of multi-walled carbon nanotubes-filled polydimethylsiloxane composites on pioneer microbial colonization
Colloids and Surfaces B: Biointerfaces 145 (2016) 30–36
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effect of anti-biofouling potential of multi-walled carbon nanotubes-filled polydimethylsiloxane composites on pioneer microbial colonization Yuan Sun a,c , Yanhe Lang c , Qian Sun b , Shuang Liang b , Yongkang Liu b , Zhizhou Zhang a,b,c,∗ a
School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, 150006, China School of Marine Science and Technology, Harbin Institute of Technology, Weihai, 264209, China c Marine Antifouling Engineering Technology Center of Shangdong Province, Harbin Institute of Technology, Weihai, 264209 China b
a r t i c l e
i n f o
Article history: Received 19 January 2016 Received in revised form 14 April 2016 Accepted 18 April 2016 Available online 23 April 2016 Keywords: Anti-biofouling Biofouling Carbon nanotubes Diversity Pioneer microbial communities Polydimethylsiloxane Single-strand conformation polymorphism
a b s t r a c t In this paper, two carbon nanotube (CNT) nanofillers, namely the multi-walled carbon nanotubes (MWCNTs) and the carboxyl-modified MWCNTs (cMWCNTs), were introduced into the polydimethylsiloxane (PDMS) matrix respectively, in order to produce the PDMS composites with reinforced anti-biofouling properties. The anti-biofouling capacity of the silicone-based coatings, including the unfilled PDMS (P0 ), the MWCNTs-filled PDMS (PM ) and the cMWCNTs-filled PDMS (PC ), was examined via the field assays conducted in Weihai, China. The effect of different silicone-based coatings on the dynamic variations of the pioneer microbial-community diversity was analyzed using the single-strand conformation polymorphism (SSCP) technique. The PM and PC surfaces have exhibited excellent anti-biofouling properties in contrast to that of the PDMS surface, with extremely low attachment of the early colonizers, such as juvenile invertebrates, seaweeds and algae sporelings. The PM and PC surfaces can effectively prevent biofouling for more than 12 weeks. These combined results suggest that the incorporation of MWCNTs or cMWCNTs into the PDMS matrix can dramatically reinforce its anti-biofouling properties. The SSCP analysis reveals that compared with the PDMS surfaces, the PM and PC surfaces have strong modulating effect on the pioneer prokaryotic and eukaryotic communities, particularly on the colonization of pioneer eukaryotic microbes. The significantly reduced pioneer eukaryotic-community diversity may contribute to the weakening of the subsequent colonization of macrofoulers. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Biofouling is a ubiquitous problem for the application of materials in the marine environment [1]. The undesired accumulation of the fouling organisms adhering to the artificial structures and surfaces submerged in natural seawater have resulted in considerable economic losses and environmental problems to the shipping industry as well as other marine associated activities [2]. To date, the most effective antifouling (AF) strategy should be coating ship hulls with biocidal coatings containing tributyltin (TBT). However, considering the potential damaging effect of the TBT on the nontarget marine organisms and also the aquatic environment, the application of the TBT-based coatings has been totally banned by the International Maritime Organization (IMO) in 2008 [3]. Fur-
∗ Corresponding author at: Faculty of Marine Science and Technology, Harbin Institute of Technology, Weihai, 264209, China. E-mail address: [email protected] (Z. Zhang). http://dx.doi.org/10.1016/j.colsurfb.2016.04.033 0927-7765/© 2016 Elsevier B.V. All rights reserved.
thermore, most TBT-free systems, like copper-based or zinc-based coatings, are also ecologically harmful, because of the bioaccumulation of heavy metals in the internal organs of the marine organisms [4,5]. Therefore, recent efforts have been dedicated to the development of eco-friendly AF materials that are more durable, stronger and safer [6,7]. Polydimethylsiloxane (PDMS) has been widely shown to function as an excellent fouling-release (FR) material in the marine environment. Considering its distinct advantages, such as non-toxicity, easy fabrication, high hydrophobicity, long-term endurance and contamination resistance [8], the PDMS has become an attractive material for anti-biofouling applications [9]. Nevertheless, the PDMS is extremely susceptible to damage because of the inherent disadvantages, such as the weak mechanical performance. Therefore, the usage of the PDMS against marine biofouling has been greatly restricted. Various biocides and nanofillers have been introduced into the PDMS matrix, in order to create the PDMS composites with improved anti-biofouling capacity [10,11]. Car-
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bon nanotubes (CNTs) seem to be the appropriate candidates of choice for the improvement of a wide range of polymeric materials. Beigbeder et al. have found that the AF and FR properties of the PDMS matrix to be significantly reinforced with the presence of 0.05% (w/w) multi-walled carbon nanotubes (MWCNTs) [12]. Despite many potential applications, only a few publications have focused on the preparation of the CNTs-reinforced PDMS composites for anti-biofouling applications, as compared to other polymeric matrices. In addition, the AF efficacy of most PDMS composites was only determined via the laboratory assays, rather than the field assays. Almost no detailed investigations have been performed to identify the reinforcing CNT nanoparticles in order to improve the anti-biofouling capacity of the PDMS via the long-term field studies. Currently, the microbial biofilms that formed on the surfaces of various relatively resistant composites in natural seawater have gained extensive attention. The presence of the biofilms can either facilitate or discourage the colonization of invertebrate larvae and algae sporelings [13]. It is well believed that the degradation of the composite materials may be primarily ascribed to the colonization of the pioneer biofilm communities, like bacterial and fungi [14], because some composite constituents, such as the matrix resins, additives and plasticizers, can be readily degraded by bacterial and fungi as nutrients. Previously, most studies have primarily focused on the biofilm communities adhering to the unpainted artificial surfaces, like stainless steel, glass slides and polystyrene [15]. Only a few studies attempted to describe the biofilm communities grown on the AF coating surfaces [16–18]. Furthermore, the dynamics of the surface attachment and colonization in situ, particularly during the early stages of the establishment of the biofilms on the silicone-based composite surfaces, has not been investigated in depth. A better understanding of the diversity variations of pioneer microbial communities and related succession phenomena among various silicone-based coating surfaces may contribute to the future development of silicone-based AF strategy. In this study, a culture-independent molecular approach, namely the single-strand conformation polymorphism (SSCP) technique, was employed to analyze and monitor the dynamic variations of the pioneer microbial-community diversity among different siliconebased coating surfaces. The aim of the present study was to improve the anti-biofouling capacity of the PDMS matrix by 0.1% (w/w) CNT nanoparticle incorporation. The anti-biofouling capacity of the silicone-based coatings was examined via the field studies under actual marine conditions. The wettability of the silicone-based coatings was measured by the static water contact angle. Furthermore, the effect of different silicone-based coating surfaces on the dynamic variations of the pioneer microbial-community diversity was analyzed using the SSCP technique, based on the 16SrRNA gene and the Internal Transcribed Spacer 2 (ITS-2) analysis. The possible mechanisms of the CNTs-filled PDMS composites against biofouling were also discussed.
2. Materials and methods 2.1. Description of the immersion site The field assays were carried out at the Xiaoshi Island harbor waters (37◦ 31 51 N; 121◦ 58 19 E) in Weihai, China. A static, permanent wooden raft bridge allowed the immersion of numerous tested panels of the same dimension at different depths for long periods if necessary. There were rich fouling organisms at the immersion site during the field studies, such as invertebrate larvae (i.e., juvenile barnacles, oysters, mussels and ascidian), algae sporelings, seaweeds as well as sponges. All the tested panels will
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Table 1 Detailed information on the CNT fillers used in this study. Diameter (nm)Length (m)SSAa (m2 /g)Purity (%) CNT fillers Carboxyl content% (w/w) MWCNTs – cMWCNTsb 2.00 a b
10–20 10–20
30–100 10–30
>165 >200
>95 >95
SSA: Specific surface area. cMWCNTs: carboxyl-modified MWCNTs.
be faced with heavy fouling pressure in the marine environment. Furthermore, the results obtained from the field studies can be readily identified, because coating failures can be easily observed after four weeks of exposure to the natural seawater, according to the colonization of the early colonizers using visual analysis. 2.2. Test panel preparation Ship hull steel panels, with the same dimension of 10 cm × 10 cm × 0.3 cm, were thoroughly rubbed using sandpapers, in order to obtain relatively uniform surfaces. Then all the panels were carefully washed with the sterile deionized water. Afterwards, the panels were first coated with the primer coat (i.e., the chlorinated rubber iron-red antirust paint), in order to provide a bright background for the outer layer made of a transparent silicone-based coating. The primer coat was kindly supplied by Jiamei Company (Weihai, China), which can be cured in about 72 h at room temperature. 2.3. Coating fabrication The silicone elastomer matrix used in this study was essentially the Sylgard 184 kit (Dow Corning, USA), which was supplied as a two-part kit mainly consisting of a pre-polymer (base, part A) and a cross-linker (curing agent, part B). The CNT fillers were purchased from the Timesnano Company (Chengdu, China). The detailed information on the CNT fillers was presented in Table 1. To prepare the unfilled PDMS (P0 ), the PDMS pre-polymer (part A) and the curing agent (part B) were thoroughly mixed in a ratio of 10:1 (w/w), in order to obtain a cross-linked PDMS via hydrosilylation following the manufacturer’s recommendations. Both the pre-polymer and the curing agent were well mixed for 15 min, and then degassed to remove air bubbles from the mixture at room temperature. For the preparation of the CNTs-reinforced PDMS composites, each CNT filler has the priority to well blend with the PDMS pre-polymer (part A) for 15 min, and then the mixture was mechanically blended with the curing agent (part B) for another 15 min, degassing at room temperature until all air bubbles were completely removed from the mixture, in order to ensure the complete mixing between the two parts. The final concentration of the CNT fillers in the PDMS matrix was 0.1% (w/w) [19]. Afterwards, these silicone mixtures were painted on one side of surfaces of the pre-treated panels respectively, using a bar-coater. After 6 h of curing at 105 ◦ C, the silicone-based coatings with a thickness of 300 m were obtained for the subsequent field studies. Similar preparation procedures have also been described elsewhere in our recent study [20]. The detailed information of the CNTs-filled PDMS composites was summarized as presented in Table 2. 2.4. Contact angle measurements The static contact angle measurements were performed on the silicone-coated microscopic slides via the sessile drop technique using a JGW-360A apparatus (Chengzhou, China). The surfaces were previously cleaned with the sterile deionized water. The hydrophobic character of the silicone-based coatings was evalu-
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Table 2 Detailed information on the CNTs-filled PDMS composites and the measured water contact angles. Silicone-based composites
Code
Producers
CNT-fillers
Water contact angles w (◦ )
Unfilled PDMS MWCNTs-filled PDMS cMWCNTs-filled PDMS
P0 PM PC
Dow Corning – –
– MWCNTs cMWCNTs
114.57 ± 0.38 112.71 ± 0.68 112.90 ± 0.82
ated by measuring the static contact angle between the surface of the coating and the drops of the sterile deionized water (50 L). The results were the mean of a minimum of three determinations. 2.5. Field studies The tested panels were deployed at the static experimental wooden raft bridge located in XiaoShi Island, Weihai, China. For each of the silicone-based coatings, one panel was prepared in triplicate throughout the field studies. All tested panels were attached to a wooden frame using thin ropes in order that they could be submerged at different depths. All the panels were taken out from time to time, and then photographed using a digital camera (Samsung PL2000). Afterwards, they were placed back into the sea quickly in order to continue the test. All tested panels were immersed into seawater from the static wooden raft bridge on Oct.17, 2013. The treated panels coated with different silicone-based composites were examined. For comparison, the control panel coated with the unfilled PDMS was also examined. In addition, other field assays (Mar.14–Jun.28, 2014) conducted at the same immersion site under the same immersion conditions were performed, in order to revalidate the long-term AF efficacy of the CNTs-reinforced PDMS composites (i.e., PM and PC ).
at 94 ◦ C, followed by 35 cycles of 30 s denaturation at 94 ◦ C, 40 s primer annealing at 56 ◦ C and 40 s extension at 72 ◦ C, followed by a final extension step at 72 ◦ C for 5 min. The asymmetric PCR amplification was performed in 12 L volumes using the NPK02 kit (GREDBIO), including 200 nM of each dNTP, 0.2 M primer, 0.4 ng of template, and 2 U of Taq DNA polymerase (TaKaRa). The PCR products were detected using 1.0% agarose gel electrophoresis and visualized using a WD-9413C gel imaging analysis system (LiuYi, Beijing). Then they were preserved at −40 ◦ C for SSCP analysis. 2.8. SSCP The denaturation solution, including 95% formamide, 0.25% bromophenol blue and 0.25% xylene cyanol, was well mixed with each PCR product at a ratio of 1:1(v/v), in order to adjust to a final volume of 6 L. After incubation for 10 min at 98 ◦ C, all the PCR products were immediately cooled on ice for 15 min before loading. The SSCP electrophoresis was performed in an 8% polyacrylamide (29:1) gel submerged in 1 × TBE buffer for 24 h at 100 V, using a DYCZ-24DN electrophoresis system (LiuYi, Beijing). The gel temperature was maintained at 4 ◦ C throughout. The gel was visualized using silverstaining method and then photographed using a digital camera (Samsung PL2000) for further analysis.
2.6. Marine in situ experiment and sampling
2.9. Data analysis
The three-week in situ experiment (Mar.12–Apr.1, 2014) was performed in order to collect the continuous biofilm samples from the silicone-based coating surfaces at different points in time for the SSCP analysis. For each of the silicone-based coatings, triplicate panels for sampling were used throughout, aiming to collect sufficient biofilm samples for the SSCP analysis. The panels for sampling were immersed in the sea at the same time on Mar.12, 2014 at a depth of 1.5 m, and the continuous sampling lasted for three weeks. The panels for sampling were brought back to the laboratory as quickly as possible. Biofilm samples were collected by scraping using the sterile brushes. The steel panels were carefully washed with the sterile deionized water before scraping. All the replicate biofilm samples from the identical silicone-based coating surface at the same point in time were collected into a sterile 2.0 mL Eppendorf tube. Then all the mixed biofilm samples were resuspended in 400 L of the sterile deionized water, and centrifuged at 4000 rpm for 5 min in order to obtain a cell pellet. Afterwards, they were preserved at −80 ◦ C for further analysis.
The Quantity One software (version 4.6.2, Bio-Rad, USA) was applied to determine the band position and intensity in order to obtain the data matrices. Bands with a relative intensity less than 0.3% were discarded. These analysis procedures mentioned above were performed three times. Based on these matrices, three diversity indices, namely the Shannon diversity index, the Evenness index and the Berger-Parke index were calculated in the context of the pioneer prokaryotic and eukaryotic communities using the Biodap software [22], in order to estimate the pioneer microbialcommunity diversity. All data were presented as mean ± standard deviation (SD). The statistical analysis was performed using the SPSS19.0 software (IBM, Armonk, NY, USA) to evaluate statistical differences. The P-value < 0.05 was considered as statistically significant.
2.7. The 16SrRNA and ITS gene fragments amplification The genomic DNA extraction was conducted using the phenolchloroform method as previously reported [20,21]. The 16SrRNA gene and the ITS-2 gene fragments were used to identify the pioneer prokaryotic and eukaryotic microbes, respectively. The universal primers 337F (5 - GAC TCC TAC GGG AGG CWG CAG-3 ) and 1100R (5 - GGG TTG CGC TCG TTG -3 ) were used to amplify the 16SrRNA gene fragments, while the specific primers ITS3 (5 -GCA TCG ATG AAG AAC GCA GC-3 ) and ITS4 (5 -TCC TCC GCT TAT TGA TAT GC-3 ) were used to amplify the ITS-2 gene fragments. The PCR amplification was performed in a PCR Thermal Cycler Dice Gradient (TaKaRa). The PCR program contained 3 min denaturation of DNA
3. Results and discussion 3.1. The wettability of the silicone-based coatings In this study, the static water contact angle ( w ) was used to determine the wettability of the silicone-based coatings, and the results were summarized as presented in Table 2. The contact angle measurements showed widespread values ranging from 112.71 ± 0.68◦ to 114.57 ± 0.38◦ , which were generally higher than 90◦ , indicating that all the silicone-based coatings have exhibited highly hydrophobic behavior, and consequently considered as the hydrophobic surfaces. The values of w for the PM (112.71 ± 0.68◦ ) and PC (112.90 ± 0.82◦ ) surfaces were slightly lower than that of the PDMS surface (114.57 ± 0.38◦ ) (P > 0.05), suggesting a slight decrease of surface hydrophobicity. No significant differences in w values can be observed between the PDMS surface (P0 ) and the silicone-based composites (i.e., PM and PC ). This result suggests
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Fig. 1. The results of field studies. (a–c): Images of panels coated with the unfilled PDMS control (P0 ), the MWCNTs-filled PDMS composites (PM ) and the cMWCNTs-filled PDMS composites (PC ) after the immersion in natural seawater at different depths (Oct.17–Dec.17, 2013, Weihai, China). (d): Images of the ultimate result of panels coated with P0 , PM and PC after the immersion in natural seawater at different depths (Mar.14–Jun.28, 2014, Weihai, China).
that the presence of 0.1% (w/w) CNT fillers in the PDMS matrix cannot significantly affect its wettability, which is slightly different from the findings previously reported for the silicone-based coatings [23]. 3.2. Marine field assays In the current study, the anti-biofouling capacity of the siliconebased coatings was examined via the field assays under static conditions. The results obtained from the field studies were presented in Fig. 1. Some early colonizers, such as juvenile invertebrates, algae sporelings, together with the sea slime, can be observed adhering to the PDMS surface after four-week exposure to the marine environment at different depths. The PDMS control was found to be severely fouled at the end of the immersion (Fig. 1a), indicating that the PDMS surface was susceptible to microbial colonization and deterioration, which did not perform well in the field. In contrast, both the PM and PC surfaces remain relatively clean throughout the field studies, with extremely low attachment of the early colonizers. This result indicates that the anti-biofouling capacity of the PDMS matrix can be greatly reinforced by 0.1% (w/w) CNT nanoparticle incorporation (Fig. 1b and c). The PM and PC surfaces can maintain their deterrent effects against biofouling for the next 12 weeks (Data not shown). In addition, the results obtained from other field assays (March.14–June.28, 2014) revalidated that the PM and PC surfaces could effectively prevent biofouling in relatively warm marine environment for 12 weeks (Fig. 1d). Previously, the PDMS matrix incorporated with other four CNT nanoparticles (see Table S1 of the Supporting information), including the amino-modified MWCNTs, the aligned MWCNTs, the helical MWCNTs and the Ni-coated MWCNTs, were prepared and examined respectively through the field assays at the same site following the same immersion conditions. Unfortunately, none of these CNTsfilled PDMS composites performed well in the field, as presented in Fig. S1 in the online version at DOI: 10.1016/j.colsurfb.2016.04.033 of the Supporting information. This result suggests that not all CNT
nanoparticles have similar reinforcing effect on the PDMS matrix. Therefore, the identification of the optimal CNT nanoparticles for the reinforcement of anti-biofouling capacity of the PDMS matrix requires the long-term screening process via the field assays in the future. Beigbeder et al. have found that the bulk mechanical properties and some surface properties of the PDMS matrix to be slightly changed after the addition of low quantities of the MWCNT fillers [12]. Since the presence of the CNT filler does not significantly change the basic properties of the PDMS matrix, the differential and improved anti-biofouling capacity may be primarily attributed to the physicochemical properties and types of CNT fillers themselves. This hypothesis agrees well the findings obtained by Kang et al. [24]. Furthermore, the CNT-silicone interface interactions were also found to be contributable to the differential anti-biofouling behaviors [25], because different CNT nanoparticles may have differential interactions with the silicone matrix. Further study focusing exclusively on the characterization of the surface morphology features of the PM and PC surfaces using the AFM analysis is currently underway, in order to elucidate the possible reasons for the reinforced anti-biofouling effect, and these observations will be reported in a later paper. 3.3. Effect of the silicone-based composites on pioneer microbial communities 3.3.1. SSCP fingerprints In the present study, the SSCP technique was employed to analyze and monitor the pioneer microbial-community diversity via the comparison of diversity indices. Both the 16SrRNA gene and the ITS gene fragments were successfully amplified (data not shown) and separated using the SSCP technique. The SSCP profiles have displayed diverse banding patterns, as illustrated in Fig. 2. Almost all the silicone-based coating surfaces were found to be quickly occupied by the pioneer microbial communities, indicating that the silicone-based materials cannot completely resist to the colo-
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Fig. 2. The SSCP fingerprints of the pioneer microbial communities adhering to different silicone-based coating surfaces. The PP0 , PPM and PPC represent the SSCP profiles for the pioneer prokaryotic communities grown on the P0 , PM and PC surfaces; whereas the EP0 , EPM and EPC represent the SSCP profiles for the pioneer eukaryotic communities grown on the P0 , PM and PC surfaces.
nization of the pioneer prokaryotic and eukaryotic microbes. Each of the silicone-based coating surfaces has presented differential succession patterns in the context of the pioneer prokaryotic and eukaryotic communities as a function of time. Both the pioneer prokaryotic and eukaryotic communities adhering to the siliconebased coating surfaces were prone to gradually increase over time, tending to evolve similar and stable community structures. Based on the SSCP profiles, the three diversity indices were calculated in terms of the prokaryotic and eukaryotic communities (Data not shown) using the Biodap software. The comparison of the three diversity indices is presented in Figs. 3–5 .
3.3.2. Shannon diversity index The Shannon diversity index is used to estimate the general biodiversity of environmental microbial communities, which usually ranges from 1.5 to 3.5 [26]. In Fig. 3, the Shannon diversity index of the pioneer prokaryotic microbial communities was generally stable for all the silicone-based coatings, which does not show clear differences as compared to that of the PDMS surface
Fig. 3. The comparison of the Shannon diversity index of the pioneer prokaryotic and eukaryotic communities adhering to different silicone-based coating surfaces. Error bars represent the SD of the mean. One asterisk (*) represents significant difference existed (P < 0.05), whereas two asterisk (**) represent extremely significant difference existed (P < 0.01).
(P > 0.05). In contrast, the Shannon diversity index of the pioneer eukaryotic microbial communities adhering to the silicone-based coating surfaces was generally unstable. Significant differences can be observed between the pioneer eukaryotic microbial communities attached to the surfaces of silicone-based composites (i.e., PM
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Fig. 4. The comparison of the Evenness index of pioneer prokaryotic and eukaryotic communities adhering to different silicone-based coating surfaces. Error bars represent the SD of the mean. One asterisk (*) represents significant difference existed (P < 0.05), whereas two asterisk (**) represent extremely significant difference existed (P < 0.01).
Fig. 5. The comparison of the Berger-Parker index of the pioneer prokaryotic and eukaryotic communities adhering to different silicone-based coating surfaces. Error bars represent the SD of the mean. One asterisk (*) represents significant difference existed (P < 0.05), whereas two asterisk (**) represent extremely significant difference existed (P < 0.01).
and PC ) and the PDMS control (P < 0.05), indicating that the diversity of the pioneer eukaryotic communities grown on the PM and PC surfaces was significantly reduced, compared with that of the PDMS surface. It is interesting to see that the PM and PC surfaces have exerted differential perturbation effect on the adherent pioneer prokaryotic and eukaryotic communities. Compared with the PDMS surface, it seems that the pioneer eukaryotic microbial community was significantly perturbed by the PM and PC surfaces, particularly by the PC surface (P < 0.01), thereby being more prone to be subjected to the major perturbation with respect to the diversity. The globally significant differences with regard to the eukaryotic microbial diversity amongst different silicone-based coatings may largely depend on the presence\absent of the CNT fillers. By contrast, the pioneer prokaryotic microbial community seemed unaffected at all, which was only subjected to the minor perturbation with respect to the diversity. This result suggests that the PM and PC surfaces can only exert slight perturbation effect on the colonization of pioneer prokaryotic microbes. 3.3.3. Evenness index The Evenness index of a community describes the consistency of the distribution of various species with different abundances [27]. According to Fig. 4, the evenness index of the pioneer prokaryotic and eukaryotic communities grown on the PM and PC surfaces was significantly reduced, when compared to that of the PDMS surface (P < 0.01), indicating that the distribution and the structure of pioneer prokaryotic and eukaryotic communities were dramatically affected by the PM and PC surfaces. This result indicates that the PM and PC surfaces may have significant impact on the distribution and stability of the adherent pioneer prokaryotic and eukaryotic communities. 3.3.4. Berger-Parker index The Berger-Parker index describes the species abundance and distribution of the disturbed communities, which was widely used
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to discriminate the differences between the disturbed and undisturbed communities [28]. High Berger-Parker index value indicates that there might exist significant differences in the species composition (-diversity) and the dominance patterns amongst different environmental microbial communities. In Fig. 5, the Berger-Parker index of the pioneer prokaryotic and eukaryotic communities grown on the PM and PC surfaces were significantly elevated, compared with that of the PDMS surface (P < 0.01). Significant differences can be found in the species composition and dominance patterns between the pioneer microbial communities grown on the surfaces of the PDMS and the silicone-based composites (i.e., PM and PC ). In addition, the pioneer eukaryotic communities seem to have been subjected to the stronger modulating effect by the PM and PC surfaces, particularly by the PC surfaces. These combined results suggest that the PM and PC surfaces can significantly affect the species composition and the dominance patterns of the adherent pioneer microbial communities. 3.3.5. Differential responses of pioneer prokaryotic and eukaryotic communities to the silicone-based composites Previously, the pioneer prokaryotic communities were found to be successfully colonized on a wide range of dissimilar AF composites surfaces, even on the TBT-based coating surfaces [18], implying that most pioneer prokaryotic microbes may have evolved the special mechanisms to adapt and colonize on almost all kinds of AF coatings with various surface properties. It seems that the adherent pioneer prokaryotic communities own less susceptibility to be influenced by most AF surfaces, either during the process of initial settlement or growth on artificial surfaces. Even the PM and PC surfaces in this study failed to exert enough perturbation effect on the colonization of pioneer prokaryotic microbes, although the PM and PC surfaces can significantly affect the species composition, distribution and stability of the pioneer prokaryotic communities. In contrast, the PM and PC surfaces can exert significant perturbation effect on the pioneer eukaryotic communities, which can dramatically reduce the colonization of pioneer eukaryotic microbes [20]. These combined results suggest that the PM and PC surfaces have differential modulating effects on the adherent pioneer prokaryotic and eukaryotic communities. The results correlated well to the findings reported by Camps et al. in an earlier study [29]. In addition, it can be concluded that the pioneer prokaryotic microbes seemed more likely to be responsible for the deterioration and degradation of the CNTs-filled PDMS composites. Further research focusing on the deterioration and degradation mechanism of the silicone-based PDMS composites using the SEM analysis and Electrochemical Impedance Spectroscopy (EIS) will be required in our future work. 3.4. Possible mechanisms for CNTs-filled PDMS composites against biofouling The mechanisms for the improved anti-biofouling effects of the CNTs-filled PDMS composites against biofouling have been well studied by researchers from various fields. Beigbeder et al. have found that the AF and FR properties of the PDMS matrix to be significantly reinforced with the introduction of low qualities of MWCNTs, while the bulk properties remain almost unchanged [12]. The dramatically reinforced FR properties may contribute to accounting for the improved anti-biofouling capacity. Several publications have also highlighted that the surface micro/nanostructures may have significant impacts on surface colonization [30]. Beigbeder et al. have found that the surface topography of the PDMS composites to be significantly changed by less than 1% (w/w) MWCNT nanoparticle incorporation, and this morphological change was found to be time-dependent yielding nanostructured surfaces [19]. In addition, it has been reported by Beigbeder et al. in an earlier study that the
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improved anti-biofouling capacity may be directly related to the micro/nanostructures of the immersed PDMS surface induced by the nanoparticle incorporation [23]. Furthermore, in another study, Beigbeder et al. have also found that the additive CH- interactions between the methyl groups of the polymer and the CNT surface to be closely associated with the improved anti-biofouling effects [25]. Our previous study has confirmed that the partial CNT nanoparticle aggregation may also contribute to the reinforced anti-biofouling capacity [20], which suggests that the dispersion of CNT filler in the PDMS matrix may not be necessarily associated with the enhanced AF and FR properties. The current study has confirmed that the PM and PC surfaces may have strong modulating effect on the adherent pioneer prokaryotic and eukaryotic communities, particularly on the colonization of pioneer eukaryotic microbes. The significantly reduced pioneer eukaryotic-community diversity may contribute to the weakening of the subsequent colonization of macrofoulers. Therefore, the combined results obtained from Beigbeder’s research group and other publications suggest that the mechanisms for the improved anti-biofouling effects still remain obscure and need to be elucidated in the future. It seems that the improved anti-biofouling capacity may be primarily attributed to the combined effects of the significant changes in FR properties as well as the surface micro/nanostructures, although this hypothesis needs support from the AFM/SEM analysis in our future study. 4. Conclusions In this study, two CNTs-filled PDMS composites (i.e., PM and PC ) with excellent anti-biofouling properties were determined in the field, suggesting that the incorporation of 0.1% (w/w) CNT nanoparticle in the PDMS matrix can greatly reinforce its anti-biofouling capacity. The wettability of the CNTs-filled PDMS composites was only found to be slightly affected by MWCNT nanoparticle incorporation. The PM and PC surfaces can effectively prevent biofouling for more than 12 weeks. In addition, the SSCP analysis revealed the functionalized PM and PC surfaces were capable of exerting significant perturbation effect on the adherent pioneer eukaryotic communities (i.e., algae sporelings and invertebrate larvae), and dramatically reduced the colonization of pioneer eukaryotic microbes. In addition, our study also confirmed that the SSCP technique could be highly useful for the monitoring of the dynamic variations of pioneer microbial-community diversity in a biologically relevant process. Further research focusing on the characterization of the surface micro/nanostructures of the silicone-based PDMS composites using AFM is currently in progress, and the bulk mechanical properties, the surface properties as well as the FR properties will be also examined, in order to uncover the possible mechanisms of the reinforced anti-biofouling effect. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos.31071170) and the Science and Technology Development Projects of Weihai (2011DXGJ13, 2012DXGJ02). References [1] N. Fusetani, Biofouling and antifouling, Nat. Prod. Rep. 21 (2004) 94–104. [2] G.D. Bixler, B. Bhushan, Biofouling: lessons from nature, Philos. Transact. A Math. Phys. Eng. Sci. 370 (2012) 2381–2417. [3] B. Antizar-Ladislao, Environmental levels, toxicity and human exposure to tributyltin (TBT)-contaminated marine environment. A review, Environ. Int. 34 (2008) 292–308. [4] W.R. Arnold, R.C. Santore, S. Cotsifas, Predicting copper toxicity in estuarine and marine waters using the biotic ligand model, Mar. Pollut. Bull. 50 (2005) 1634–1640.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effect of anti-biofouling potential of multi-walled carbon nanotubes-filled polydimethylsiloxane composites on pioneer microbial colonization Yuan Sun a,c , Yanhe Lang c , Qian Sun b , Shuang Liang b , Yongkang Liu b , Zhizhou Zhang a,b,c,∗ a
School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, 150006, China School of Marine Science and Technology, Harbin Institute of Technology, Weihai, 264209, China c Marine Antifouling Engineering Technology Center of Shangdong Province, Harbin Institute of Technology, Weihai, 264209 China b
a r t i c l e
i n f o
Article history: Received 19 January 2016 Received in revised form 14 April 2016 Accepted 18 April 2016 Available online 23 April 2016 Keywords: Anti-biofouling Biofouling Carbon nanotubes Diversity Pioneer microbial communities Polydimethylsiloxane Single-strand conformation polymorphism
a b s t r a c t In this paper, two carbon nanotube (CNT) nanofillers, namely the multi-walled carbon nanotubes (MWCNTs) and the carboxyl-modified MWCNTs (cMWCNTs), were introduced into the polydimethylsiloxane (PDMS) matrix respectively, in order to produce the PDMS composites with reinforced anti-biofouling properties. The anti-biofouling capacity of the silicone-based coatings, including the unfilled PDMS (P0 ), the MWCNTs-filled PDMS (PM ) and the cMWCNTs-filled PDMS (PC ), was examined via the field assays conducted in Weihai, China. The effect of different silicone-based coatings on the dynamic variations of the pioneer microbial-community diversity was analyzed using the single-strand conformation polymorphism (SSCP) technique. The PM and PC surfaces have exhibited excellent anti-biofouling properties in contrast to that of the PDMS surface, with extremely low attachment of the early colonizers, such as juvenile invertebrates, seaweeds and algae sporelings. The PM and PC surfaces can effectively prevent biofouling for more than 12 weeks. These combined results suggest that the incorporation of MWCNTs or cMWCNTs into the PDMS matrix can dramatically reinforce its anti-biofouling properties. The SSCP analysis reveals that compared with the PDMS surfaces, the PM and PC surfaces have strong modulating effect on the pioneer prokaryotic and eukaryotic communities, particularly on the colonization of pioneer eukaryotic microbes. The significantly reduced pioneer eukaryotic-community diversity may contribute to the weakening of the subsequent colonization of macrofoulers. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Biofouling is a ubiquitous problem for the application of materials in the marine environment [1]. The undesired accumulation of the fouling organisms adhering to the artificial structures and surfaces submerged in natural seawater have resulted in considerable economic losses and environmental problems to the shipping industry as well as other marine associated activities [2]. To date, the most effective antifouling (AF) strategy should be coating ship hulls with biocidal coatings containing tributyltin (TBT). However, considering the potential damaging effect of the TBT on the nontarget marine organisms and also the aquatic environment, the application of the TBT-based coatings has been totally banned by the International Maritime Organization (IMO) in 2008 [3]. Fur-
∗ Corresponding author at: Faculty of Marine Science and Technology, Harbin Institute of Technology, Weihai, 264209, China. E-mail address: [email protected] (Z. Zhang). http://dx.doi.org/10.1016/j.colsurfb.2016.04.033 0927-7765/© 2016 Elsevier B.V. All rights reserved.
thermore, most TBT-free systems, like copper-based or zinc-based coatings, are also ecologically harmful, because of the bioaccumulation of heavy metals in the internal organs of the marine organisms [4,5]. Therefore, recent efforts have been dedicated to the development of eco-friendly AF materials that are more durable, stronger and safer [6,7]. Polydimethylsiloxane (PDMS) has been widely shown to function as an excellent fouling-release (FR) material in the marine environment. Considering its distinct advantages, such as non-toxicity, easy fabrication, high hydrophobicity, long-term endurance and contamination resistance [8], the PDMS has become an attractive material for anti-biofouling applications [9]. Nevertheless, the PDMS is extremely susceptible to damage because of the inherent disadvantages, such as the weak mechanical performance. Therefore, the usage of the PDMS against marine biofouling has been greatly restricted. Various biocides and nanofillers have been introduced into the PDMS matrix, in order to create the PDMS composites with improved anti-biofouling capacity [10,11]. Car-
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bon nanotubes (CNTs) seem to be the appropriate candidates of choice for the improvement of a wide range of polymeric materials. Beigbeder et al. have found that the AF and FR properties of the PDMS matrix to be significantly reinforced with the presence of 0.05% (w/w) multi-walled carbon nanotubes (MWCNTs) [12]. Despite many potential applications, only a few publications have focused on the preparation of the CNTs-reinforced PDMS composites for anti-biofouling applications, as compared to other polymeric matrices. In addition, the AF efficacy of most PDMS composites was only determined via the laboratory assays, rather than the field assays. Almost no detailed investigations have been performed to identify the reinforcing CNT nanoparticles in order to improve the anti-biofouling capacity of the PDMS via the long-term field studies. Currently, the microbial biofilms that formed on the surfaces of various relatively resistant composites in natural seawater have gained extensive attention. The presence of the biofilms can either facilitate or discourage the colonization of invertebrate larvae and algae sporelings [13]. It is well believed that the degradation of the composite materials may be primarily ascribed to the colonization of the pioneer biofilm communities, like bacterial and fungi [14], because some composite constituents, such as the matrix resins, additives and plasticizers, can be readily degraded by bacterial and fungi as nutrients. Previously, most studies have primarily focused on the biofilm communities adhering to the unpainted artificial surfaces, like stainless steel, glass slides and polystyrene [15]. Only a few studies attempted to describe the biofilm communities grown on the AF coating surfaces [16–18]. Furthermore, the dynamics of the surface attachment and colonization in situ, particularly during the early stages of the establishment of the biofilms on the silicone-based composite surfaces, has not been investigated in depth. A better understanding of the diversity variations of pioneer microbial communities and related succession phenomena among various silicone-based coating surfaces may contribute to the future development of silicone-based AF strategy. In this study, a culture-independent molecular approach, namely the single-strand conformation polymorphism (SSCP) technique, was employed to analyze and monitor the dynamic variations of the pioneer microbial-community diversity among different siliconebased coating surfaces. The aim of the present study was to improve the anti-biofouling capacity of the PDMS matrix by 0.1% (w/w) CNT nanoparticle incorporation. The anti-biofouling capacity of the silicone-based coatings was examined via the field studies under actual marine conditions. The wettability of the silicone-based coatings was measured by the static water contact angle. Furthermore, the effect of different silicone-based coating surfaces on the dynamic variations of the pioneer microbial-community diversity was analyzed using the SSCP technique, based on the 16SrRNA gene and the Internal Transcribed Spacer 2 (ITS-2) analysis. The possible mechanisms of the CNTs-filled PDMS composites against biofouling were also discussed.
2. Materials and methods 2.1. Description of the immersion site The field assays were carried out at the Xiaoshi Island harbor waters (37◦ 31 51 N; 121◦ 58 19 E) in Weihai, China. A static, permanent wooden raft bridge allowed the immersion of numerous tested panels of the same dimension at different depths for long periods if necessary. There were rich fouling organisms at the immersion site during the field studies, such as invertebrate larvae (i.e., juvenile barnacles, oysters, mussels and ascidian), algae sporelings, seaweeds as well as sponges. All the tested panels will
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Table 1 Detailed information on the CNT fillers used in this study. Diameter (nm)Length (m)SSAa (m2 /g)Purity (%) CNT fillers Carboxyl content% (w/w) MWCNTs – cMWCNTsb 2.00 a b
10–20 10–20
30–100 10–30
>165 >200
>95 >95
SSA: Specific surface area. cMWCNTs: carboxyl-modified MWCNTs.
be faced with heavy fouling pressure in the marine environment. Furthermore, the results obtained from the field studies can be readily identified, because coating failures can be easily observed after four weeks of exposure to the natural seawater, according to the colonization of the early colonizers using visual analysis. 2.2. Test panel preparation Ship hull steel panels, with the same dimension of 10 cm × 10 cm × 0.3 cm, were thoroughly rubbed using sandpapers, in order to obtain relatively uniform surfaces. Then all the panels were carefully washed with the sterile deionized water. Afterwards, the panels were first coated with the primer coat (i.e., the chlorinated rubber iron-red antirust paint), in order to provide a bright background for the outer layer made of a transparent silicone-based coating. The primer coat was kindly supplied by Jiamei Company (Weihai, China), which can be cured in about 72 h at room temperature. 2.3. Coating fabrication The silicone elastomer matrix used in this study was essentially the Sylgard 184 kit (Dow Corning, USA), which was supplied as a two-part kit mainly consisting of a pre-polymer (base, part A) and a cross-linker (curing agent, part B). The CNT fillers were purchased from the Timesnano Company (Chengdu, China). The detailed information on the CNT fillers was presented in Table 1. To prepare the unfilled PDMS (P0 ), the PDMS pre-polymer (part A) and the curing agent (part B) were thoroughly mixed in a ratio of 10:1 (w/w), in order to obtain a cross-linked PDMS via hydrosilylation following the manufacturer’s recommendations. Both the pre-polymer and the curing agent were well mixed for 15 min, and then degassed to remove air bubbles from the mixture at room temperature. For the preparation of the CNTs-reinforced PDMS composites, each CNT filler has the priority to well blend with the PDMS pre-polymer (part A) for 15 min, and then the mixture was mechanically blended with the curing agent (part B) for another 15 min, degassing at room temperature until all air bubbles were completely removed from the mixture, in order to ensure the complete mixing between the two parts. The final concentration of the CNT fillers in the PDMS matrix was 0.1% (w/w) [19]. Afterwards, these silicone mixtures were painted on one side of surfaces of the pre-treated panels respectively, using a bar-coater. After 6 h of curing at 105 ◦ C, the silicone-based coatings with a thickness of 300 m were obtained for the subsequent field studies. Similar preparation procedures have also been described elsewhere in our recent study [20]. The detailed information of the CNTs-filled PDMS composites was summarized as presented in Table 2. 2.4. Contact angle measurements The static contact angle measurements were performed on the silicone-coated microscopic slides via the sessile drop technique using a JGW-360A apparatus (Chengzhou, China). The surfaces were previously cleaned with the sterile deionized water. The hydrophobic character of the silicone-based coatings was evalu-
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Table 2 Detailed information on the CNTs-filled PDMS composites and the measured water contact angles. Silicone-based composites
Code
Producers
CNT-fillers
Water contact angles w (◦ )
Unfilled PDMS MWCNTs-filled PDMS cMWCNTs-filled PDMS
P0 PM PC
Dow Corning – –
– MWCNTs cMWCNTs
114.57 ± 0.38 112.71 ± 0.68 112.90 ± 0.82
ated by measuring the static contact angle between the surface of the coating and the drops of the sterile deionized water (50 L). The results were the mean of a minimum of three determinations. 2.5. Field studies The tested panels were deployed at the static experimental wooden raft bridge located in XiaoShi Island, Weihai, China. For each of the silicone-based coatings, one panel was prepared in triplicate throughout the field studies. All tested panels were attached to a wooden frame using thin ropes in order that they could be submerged at different depths. All the panels were taken out from time to time, and then photographed using a digital camera (Samsung PL2000). Afterwards, they were placed back into the sea quickly in order to continue the test. All tested panels were immersed into seawater from the static wooden raft bridge on Oct.17, 2013. The treated panels coated with different silicone-based composites were examined. For comparison, the control panel coated with the unfilled PDMS was also examined. In addition, other field assays (Mar.14–Jun.28, 2014) conducted at the same immersion site under the same immersion conditions were performed, in order to revalidate the long-term AF efficacy of the CNTs-reinforced PDMS composites (i.e., PM and PC ).
at 94 ◦ C, followed by 35 cycles of 30 s denaturation at 94 ◦ C, 40 s primer annealing at 56 ◦ C and 40 s extension at 72 ◦ C, followed by a final extension step at 72 ◦ C for 5 min. The asymmetric PCR amplification was performed in 12 L volumes using the NPK02 kit (GREDBIO), including 200 nM of each dNTP, 0.2 M primer, 0.4 ng of template, and 2 U of Taq DNA polymerase (TaKaRa). The PCR products were detected using 1.0% agarose gel electrophoresis and visualized using a WD-9413C gel imaging analysis system (LiuYi, Beijing). Then they were preserved at −40 ◦ C for SSCP analysis. 2.8. SSCP The denaturation solution, including 95% formamide, 0.25% bromophenol blue and 0.25% xylene cyanol, was well mixed with each PCR product at a ratio of 1:1(v/v), in order to adjust to a final volume of 6 L. After incubation for 10 min at 98 ◦ C, all the PCR products were immediately cooled on ice for 15 min before loading. The SSCP electrophoresis was performed in an 8% polyacrylamide (29:1) gel submerged in 1 × TBE buffer for 24 h at 100 V, using a DYCZ-24DN electrophoresis system (LiuYi, Beijing). The gel temperature was maintained at 4 ◦ C throughout. The gel was visualized using silverstaining method and then photographed using a digital camera (Samsung PL2000) for further analysis.
2.6. Marine in situ experiment and sampling
2.9. Data analysis
The three-week in situ experiment (Mar.12–Apr.1, 2014) was performed in order to collect the continuous biofilm samples from the silicone-based coating surfaces at different points in time for the SSCP analysis. For each of the silicone-based coatings, triplicate panels for sampling were used throughout, aiming to collect sufficient biofilm samples for the SSCP analysis. The panels for sampling were immersed in the sea at the same time on Mar.12, 2014 at a depth of 1.5 m, and the continuous sampling lasted for three weeks. The panels for sampling were brought back to the laboratory as quickly as possible. Biofilm samples were collected by scraping using the sterile brushes. The steel panels were carefully washed with the sterile deionized water before scraping. All the replicate biofilm samples from the identical silicone-based coating surface at the same point in time were collected into a sterile 2.0 mL Eppendorf tube. Then all the mixed biofilm samples were resuspended in 400 L of the sterile deionized water, and centrifuged at 4000 rpm for 5 min in order to obtain a cell pellet. Afterwards, they were preserved at −80 ◦ C for further analysis.
The Quantity One software (version 4.6.2, Bio-Rad, USA) was applied to determine the band position and intensity in order to obtain the data matrices. Bands with a relative intensity less than 0.3% were discarded. These analysis procedures mentioned above were performed three times. Based on these matrices, three diversity indices, namely the Shannon diversity index, the Evenness index and the Berger-Parke index were calculated in the context of the pioneer prokaryotic and eukaryotic communities using the Biodap software [22], in order to estimate the pioneer microbialcommunity diversity. All data were presented as mean ± standard deviation (SD). The statistical analysis was performed using the SPSS19.0 software (IBM, Armonk, NY, USA) to evaluate statistical differences. The P-value < 0.05 was considered as statistically significant.
2.7. The 16SrRNA and ITS gene fragments amplification The genomic DNA extraction was conducted using the phenolchloroform method as previously reported [20,21]. The 16SrRNA gene and the ITS-2 gene fragments were used to identify the pioneer prokaryotic and eukaryotic microbes, respectively. The universal primers 337F (5 - GAC TCC TAC GGG AGG CWG CAG-3 ) and 1100R (5 - GGG TTG CGC TCG TTG -3 ) were used to amplify the 16SrRNA gene fragments, while the specific primers ITS3 (5 -GCA TCG ATG AAG AAC GCA GC-3 ) and ITS4 (5 -TCC TCC GCT TAT TGA TAT GC-3 ) were used to amplify the ITS-2 gene fragments. The PCR amplification was performed in a PCR Thermal Cycler Dice Gradient (TaKaRa). The PCR program contained 3 min denaturation of DNA
3. Results and discussion 3.1. The wettability of the silicone-based coatings In this study, the static water contact angle ( w ) was used to determine the wettability of the silicone-based coatings, and the results were summarized as presented in Table 2. The contact angle measurements showed widespread values ranging from 112.71 ± 0.68◦ to 114.57 ± 0.38◦ , which were generally higher than 90◦ , indicating that all the silicone-based coatings have exhibited highly hydrophobic behavior, and consequently considered as the hydrophobic surfaces. The values of w for the PM (112.71 ± 0.68◦ ) and PC (112.90 ± 0.82◦ ) surfaces were slightly lower than that of the PDMS surface (114.57 ± 0.38◦ ) (P > 0.05), suggesting a slight decrease of surface hydrophobicity. No significant differences in w values can be observed between the PDMS surface (P0 ) and the silicone-based composites (i.e., PM and PC ). This result suggests
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Fig. 1. The results of field studies. (a–c): Images of panels coated with the unfilled PDMS control (P0 ), the MWCNTs-filled PDMS composites (PM ) and the cMWCNTs-filled PDMS composites (PC ) after the immersion in natural seawater at different depths (Oct.17–Dec.17, 2013, Weihai, China). (d): Images of the ultimate result of panels coated with P0 , PM and PC after the immersion in natural seawater at different depths (Mar.14–Jun.28, 2014, Weihai, China).
that the presence of 0.1% (w/w) CNT fillers in the PDMS matrix cannot significantly affect its wettability, which is slightly different from the findings previously reported for the silicone-based coatings [23]. 3.2. Marine field assays In the current study, the anti-biofouling capacity of the siliconebased coatings was examined via the field assays under static conditions. The results obtained from the field studies were presented in Fig. 1. Some early colonizers, such as juvenile invertebrates, algae sporelings, together with the sea slime, can be observed adhering to the PDMS surface after four-week exposure to the marine environment at different depths. The PDMS control was found to be severely fouled at the end of the immersion (Fig. 1a), indicating that the PDMS surface was susceptible to microbial colonization and deterioration, which did not perform well in the field. In contrast, both the PM and PC surfaces remain relatively clean throughout the field studies, with extremely low attachment of the early colonizers. This result indicates that the anti-biofouling capacity of the PDMS matrix can be greatly reinforced by 0.1% (w/w) CNT nanoparticle incorporation (Fig. 1b and c). The PM and PC surfaces can maintain their deterrent effects against biofouling for the next 12 weeks (Data not shown). In addition, the results obtained from other field assays (March.14–June.28, 2014) revalidated that the PM and PC surfaces could effectively prevent biofouling in relatively warm marine environment for 12 weeks (Fig. 1d). Previously, the PDMS matrix incorporated with other four CNT nanoparticles (see Table S1 of the Supporting information), including the amino-modified MWCNTs, the aligned MWCNTs, the helical MWCNTs and the Ni-coated MWCNTs, were prepared and examined respectively through the field assays at the same site following the same immersion conditions. Unfortunately, none of these CNTsfilled PDMS composites performed well in the field, as presented in Fig. S1 in the online version at DOI: 10.1016/j.colsurfb.2016.04.033 of the Supporting information. This result suggests that not all CNT
nanoparticles have similar reinforcing effect on the PDMS matrix. Therefore, the identification of the optimal CNT nanoparticles for the reinforcement of anti-biofouling capacity of the PDMS matrix requires the long-term screening process via the field assays in the future. Beigbeder et al. have found that the bulk mechanical properties and some surface properties of the PDMS matrix to be slightly changed after the addition of low quantities of the MWCNT fillers [12]. Since the presence of the CNT filler does not significantly change the basic properties of the PDMS matrix, the differential and improved anti-biofouling capacity may be primarily attributed to the physicochemical properties and types of CNT fillers themselves. This hypothesis agrees well the findings obtained by Kang et al. [24]. Furthermore, the CNT-silicone interface interactions were also found to be contributable to the differential anti-biofouling behaviors [25], because different CNT nanoparticles may have differential interactions with the silicone matrix. Further study focusing exclusively on the characterization of the surface morphology features of the PM and PC surfaces using the AFM analysis is currently underway, in order to elucidate the possible reasons for the reinforced anti-biofouling effect, and these observations will be reported in a later paper. 3.3. Effect of the silicone-based composites on pioneer microbial communities 3.3.1. SSCP fingerprints In the present study, the SSCP technique was employed to analyze and monitor the pioneer microbial-community diversity via the comparison of diversity indices. Both the 16SrRNA gene and the ITS gene fragments were successfully amplified (data not shown) and separated using the SSCP technique. The SSCP profiles have displayed diverse banding patterns, as illustrated in Fig. 2. Almost all the silicone-based coating surfaces were found to be quickly occupied by the pioneer microbial communities, indicating that the silicone-based materials cannot completely resist to the colo-
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Fig. 2. The SSCP fingerprints of the pioneer microbial communities adhering to different silicone-based coating surfaces. The PP0 , PPM and PPC represent the SSCP profiles for the pioneer prokaryotic communities grown on the P0 , PM and PC surfaces; whereas the EP0 , EPM and EPC represent the SSCP profiles for the pioneer eukaryotic communities grown on the P0 , PM and PC surfaces.
nization of the pioneer prokaryotic and eukaryotic microbes. Each of the silicone-based coating surfaces has presented differential succession patterns in the context of the pioneer prokaryotic and eukaryotic communities as a function of time. Both the pioneer prokaryotic and eukaryotic communities adhering to the siliconebased coating surfaces were prone to gradually increase over time, tending to evolve similar and stable community structures. Based on the SSCP profiles, the three diversity indices were calculated in terms of the prokaryotic and eukaryotic communities (Data not shown) using the Biodap software. The comparison of the three diversity indices is presented in Figs. 3–5 .
3.3.2. Shannon diversity index The Shannon diversity index is used to estimate the general biodiversity of environmental microbial communities, which usually ranges from 1.5 to 3.5 [26]. In Fig. 3, the Shannon diversity index of the pioneer prokaryotic microbial communities was generally stable for all the silicone-based coatings, which does not show clear differences as compared to that of the PDMS surface
Fig. 3. The comparison of the Shannon diversity index of the pioneer prokaryotic and eukaryotic communities adhering to different silicone-based coating surfaces. Error bars represent the SD of the mean. One asterisk (*) represents significant difference existed (P < 0.05), whereas two asterisk (**) represent extremely significant difference existed (P < 0.01).
(P > 0.05). In contrast, the Shannon diversity index of the pioneer eukaryotic microbial communities adhering to the silicone-based coating surfaces was generally unstable. Significant differences can be observed between the pioneer eukaryotic microbial communities attached to the surfaces of silicone-based composites (i.e., PM
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Fig. 4. The comparison of the Evenness index of pioneer prokaryotic and eukaryotic communities adhering to different silicone-based coating surfaces. Error bars represent the SD of the mean. One asterisk (*) represents significant difference existed (P < 0.05), whereas two asterisk (**) represent extremely significant difference existed (P < 0.01).
Fig. 5. The comparison of the Berger-Parker index of the pioneer prokaryotic and eukaryotic communities adhering to different silicone-based coating surfaces. Error bars represent the SD of the mean. One asterisk (*) represents significant difference existed (P < 0.05), whereas two asterisk (**) represent extremely significant difference existed (P < 0.01).
and PC ) and the PDMS control (P < 0.05), indicating that the diversity of the pioneer eukaryotic communities grown on the PM and PC surfaces was significantly reduced, compared with that of the PDMS surface. It is interesting to see that the PM and PC surfaces have exerted differential perturbation effect on the adherent pioneer prokaryotic and eukaryotic communities. Compared with the PDMS surface, it seems that the pioneer eukaryotic microbial community was significantly perturbed by the PM and PC surfaces, particularly by the PC surface (P < 0.01), thereby being more prone to be subjected to the major perturbation with respect to the diversity. The globally significant differences with regard to the eukaryotic microbial diversity amongst different silicone-based coatings may largely depend on the presence\absent of the CNT fillers. By contrast, the pioneer prokaryotic microbial community seemed unaffected at all, which was only subjected to the minor perturbation with respect to the diversity. This result suggests that the PM and PC surfaces can only exert slight perturbation effect on the colonization of pioneer prokaryotic microbes. 3.3.3. Evenness index The Evenness index of a community describes the consistency of the distribution of various species with different abundances [27]. According to Fig. 4, the evenness index of the pioneer prokaryotic and eukaryotic communities grown on the PM and PC surfaces was significantly reduced, when compared to that of the PDMS surface (P < 0.01), indicating that the distribution and the structure of pioneer prokaryotic and eukaryotic communities were dramatically affected by the PM and PC surfaces. This result indicates that the PM and PC surfaces may have significant impact on the distribution and stability of the adherent pioneer prokaryotic and eukaryotic communities. 3.3.4. Berger-Parker index The Berger-Parker index describes the species abundance and distribution of the disturbed communities, which was widely used
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to discriminate the differences between the disturbed and undisturbed communities [28]. High Berger-Parker index value indicates that there might exist significant differences in the species composition (-diversity) and the dominance patterns amongst different environmental microbial communities. In Fig. 5, the Berger-Parker index of the pioneer prokaryotic and eukaryotic communities grown on the PM and PC surfaces were significantly elevated, compared with that of the PDMS surface (P < 0.01). Significant differences can be found in the species composition and dominance patterns between the pioneer microbial communities grown on the surfaces of the PDMS and the silicone-based composites (i.e., PM and PC ). In addition, the pioneer eukaryotic communities seem to have been subjected to the stronger modulating effect by the PM and PC surfaces, particularly by the PC surfaces. These combined results suggest that the PM and PC surfaces can significantly affect the species composition and the dominance patterns of the adherent pioneer microbial communities. 3.3.5. Differential responses of pioneer prokaryotic and eukaryotic communities to the silicone-based composites Previously, the pioneer prokaryotic communities were found to be successfully colonized on a wide range of dissimilar AF composites surfaces, even on the TBT-based coating surfaces [18], implying that most pioneer prokaryotic microbes may have evolved the special mechanisms to adapt and colonize on almost all kinds of AF coatings with various surface properties. It seems that the adherent pioneer prokaryotic communities own less susceptibility to be influenced by most AF surfaces, either during the process of initial settlement or growth on artificial surfaces. Even the PM and PC surfaces in this study failed to exert enough perturbation effect on the colonization of pioneer prokaryotic microbes, although the PM and PC surfaces can significantly affect the species composition, distribution and stability of the pioneer prokaryotic communities. In contrast, the PM and PC surfaces can exert significant perturbation effect on the pioneer eukaryotic communities, which can dramatically reduce the colonization of pioneer eukaryotic microbes [20]. These combined results suggest that the PM and PC surfaces have differential modulating effects on the adherent pioneer prokaryotic and eukaryotic communities. The results correlated well to the findings reported by Camps et al. in an earlier study [29]. In addition, it can be concluded that the pioneer prokaryotic microbes seemed more likely to be responsible for the deterioration and degradation of the CNTs-filled PDMS composites. Further research focusing on the deterioration and degradation mechanism of the silicone-based PDMS composites using the SEM analysis and Electrochemical Impedance Spectroscopy (EIS) will be required in our future work. 3.4. Possible mechanisms for CNTs-filled PDMS composites against biofouling The mechanisms for the improved anti-biofouling effects of the CNTs-filled PDMS composites against biofouling have been well studied by researchers from various fields. Beigbeder et al. have found that the AF and FR properties of the PDMS matrix to be significantly reinforced with the introduction of low qualities of MWCNTs, while the bulk properties remain almost unchanged [12]. The dramatically reinforced FR properties may contribute to accounting for the improved anti-biofouling capacity. Several publications have also highlighted that the surface micro/nanostructures may have significant impacts on surface colonization [30]. Beigbeder et al. have found that the surface topography of the PDMS composites to be significantly changed by less than 1% (w/w) MWCNT nanoparticle incorporation, and this morphological change was found to be time-dependent yielding nanostructured surfaces [19]. In addition, it has been reported by Beigbeder et al. in an earlier study that the
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improved anti-biofouling capacity may be directly related to the micro/nanostructures of the immersed PDMS surface induced by the nanoparticle incorporation [23]. Furthermore, in another study, Beigbeder et al. have also found that the additive CH- interactions between the methyl groups of the polymer and the CNT surface to be closely associated with the improved anti-biofouling effects [25]. Our previous study has confirmed that the partial CNT nanoparticle aggregation may also contribute to the reinforced anti-biofouling capacity [20], which suggests that the dispersion of CNT filler in the PDMS matrix may not be necessarily associated with the enhanced AF and FR properties. The current study has confirmed that the PM and PC surfaces may have strong modulating effect on the adherent pioneer prokaryotic and eukaryotic communities, particularly on the colonization of pioneer eukaryotic microbes. The significantly reduced pioneer eukaryotic-community diversity may contribute to the weakening of the subsequent colonization of macrofoulers. Therefore, the combined results obtained from Beigbeder’s research group and other publications suggest that the mechanisms for the improved anti-biofouling effects still remain obscure and need to be elucidated in the future. It seems that the improved anti-biofouling capacity may be primarily attributed to the combined effects of the significant changes in FR properties as well as the surface micro/nanostructures, although this hypothesis needs support from the AFM/SEM analysis in our future study. 4. Conclusions In this study, two CNTs-filled PDMS composites (i.e., PM and PC ) with excellent anti-biofouling properties were determined in the field, suggesting that the incorporation of 0.1% (w/w) CNT nanoparticle in the PDMS matrix can greatly reinforce its anti-biofouling capacity. The wettability of the CNTs-filled PDMS composites was only found to be slightly affected by MWCNT nanoparticle incorporation. The PM and PC surfaces can effectively prevent biofouling for more than 12 weeks. In addition, the SSCP analysis revealed the functionalized PM and PC surfaces were capable of exerting significant perturbation effect on the adherent pioneer eukaryotic communities (i.e., algae sporelings and invertebrate larvae), and dramatically reduced the colonization of pioneer eukaryotic microbes. In addition, our study also confirmed that the SSCP technique could be highly useful for the monitoring of the dynamic variations of pioneer microbial-community diversity in a biologically relevant process. Further research focusing on the characterization of the surface micro/nanostructures of the silicone-based PDMS composites using AFM is currently in progress, and the bulk mechanical properties, the surface properties as well as the FR properties will be also examined, in order to uncover the possible mechanisms of the reinforced anti-biofouling effect. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant nos.31071170) and the Science and Technology Development Projects of Weihai (2011DXGJ13, 2012DXGJ02). References [1] N. Fusetani, Biofouling and antifouling, Nat. Prod. Rep. 21 (2004) 94–104. [2] G.D. Bixler, B. Bhushan, Biofouling: lessons from nature, Philos. Transact. A Math. Phys. Eng. Sci. 370 (2012) 2381–2417. [3] B. Antizar-Ladislao, Environmental levels, toxicity and human exposure to tributyltin (TBT)-contaminated marine environment. A review, Environ. Int. 34 (2008) 292–308. [4] W.R. Arnold, R.C. Santore, S. Cotsifas, Predicting copper toxicity in estuarine and marine waters using the biotic ligand model, Mar. Pollut. Bull. 50 (2005) 1634–1640.
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