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New anti-biofouling carbon nanotubes-filled polydimethylsiloxane composites against colonization by pioneer eukaryotic microbes
International Biodeterioration & Biodegradation 110 (2016) 147e154
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New anti-biofouling carbon nanotubes-filled polydimethylsiloxane composites against colonization by pioneer eukaryotic microbes Yuan Sun a, b, Zhizhou Zhang a, b, c, * a
School of Chemical Engineering & Technology, 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 Shandong Province, Harbin Institute of Technology, Weihai 264209, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2016 Received in revised form 17 March 2016 Accepted 17 March 2016 Available online 26 March 2016
Microbial biofilm formation on composite surfaces has posed potential threats to the composite's structural integrity, durability and physical properties. Pioneer eukaryotes have been reported to be primarily responsible for the degradation of a wide range of composite materials. In this study, different carbon nanotubes (CNTs) were incorporated in the polydimethylsiloxane (PDMS) matrix respectively, in order to create CNTs-filled PDMS composites (PCs) with improved anti-biofouling properties. The antibiofouling properties of pure PDMS (P0) and PCs were examined through marine field assays. The diversity and richness of pioneer eukaryotic communities adhering to P0 and PCs surfaces were analyzed using the single-strand conformation polymorphism (SSCP) technique. PCs have exhibited differential and better anti-biofouling properties, when compared to the PDMS control. Two PCs with exceptional anti-biofouling properties were determined in the field. The Shannon diversity index and species richness of pioneer eukaryotic communities on most PCs surfaces were dramatically lower than those of the pure PDMS control (P < 0.05). This indicates a significant decrease in the diversity and richness of pioneer eukaryotic communities. The combined results suggest PCs can effectively reduce the colonization of pioneer eukaryotes, such as algae sporelings and invertebrate larvae. PCs with low cost and good compatibility in the marine environment give the potential for future anti-biofouling applications. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Biofouling Carbon nanotubes Diversity Pioneer eukaryotes Polydimethylsiloxane Single strand conformation polymorphism
1. Introduction Marine biofouling occurs when fouling organisms settle and colonize on surfaces of either natural or artificial substratum submerged in a marine environment (Fusetani, 2004). Biofouling can severely affect structures critical to the maritime industry, increase propulsive fuel consumption, and weaken ships' maneuverability, thereby resulting in considerable economic loss and environmental issues (Bixler and Bhushan, 2012). Broad-spectrum biocidal coatings containing tributyltin (TBT) or copper have been widely used to prevent biofouling (Banerjee et al., 2011). However, these biocidal coatings are toxic to a wide range of non-target marine organisms, and therefore are ecologically harmful to the marine aquatic environment (Domart-Coulon et al., 2000). As a result, extensive studies have been devoted to developing eco-friendly
* Corresponding author. School 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.ibiod.2016.03.019 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
antifouling (AF) coatings (Dafforn et al., 2011). Recently, the increasing usage of non-toxic silicone materials represents a promising approach for the development of ecofriendly polymers (Callow and Callow, 2011). Polydimethylsiloxane (PDMS) is a useful and attractive silicone elastomer with desirable chemical and physical properties, such as high hydrophobicity, low surface energy, contamination resistance and long-term endurance (Sokolova et al., 2012). However, because of the inherent disadvantages of PDMS, such as mechanical weakness and a propensity to sustain damage when immersed in the marine environment, the practical applications of PDMS against biofouling have been greatly limited. To date, one promising and effective method to overcome these drawbacks has been to introduce a variety of nanofillers into the polymeric matrix, in order to produce new composites with potentially improved anti-biofouling properties (Majumdar et al., 2008). Carbon nanotubes (CNTs) have been attracting considerable attention among the numerous nano-sized fillers due to its unique molecular structure, exceptional electrical, mechanical and thermal properties (Iijima, 1991). Beigbeder et al. have
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reported that the presence of a small amount of multi-walled carbon nanotubes (MWNTs) in the PDMS matrix can greatly improve its AF and fouling-release (FR) properties (Beigbeder et al., 2008a). CNTs seem to be the optimal fillers for preparing composites of various materials, including PDMS. Nevertheless, research on the preparation of novel PDMS composites (PCs) reinforced with different surface-modified CNTs, such as hydroxyl-modified MWNTs (hMWNTs) and carboxyl-modified MWNTs (cMWNTs) for anti-biofouling applications has not been systematically investigated. Furthermore, the anti-biofouling properties of most PCs failed to be examined via field assays under static conditions. Microbial biofilm formation on composite material surfaces under favorable conditions is usually considered as a prerequisite for biofouling and the subsequent degradation process, which can dramatically affect the composite's structural integrity, durability, chemical and physical properties (Fletcher, 1994; Gu, 2005). The growth of biofilm communities on composite surfaces has posed a major risk to composite materials. It has been reported that a wide range of composite materials are susceptible to microbial deterioration and degradation (Gu, 2003). It is well accepted that biodegradation of composite materials may be primarily attributed to a combined effect of both bacterial and fungi colonization in sequence, while fungi may have obvious advantages over bacteria during the early stage of surface colonization, particularly on relatively resistant composite surfaces (Gu et al., 1998; Gu, 2007). Various publications have reported that fungi are primarily responsible for the degradation of a variety of composite materials, such as fiber-reinforced polymeric composites (Gu et al., 1996a) and electronic packaging polyimides (Gu et al., 1996b), because some composite constituents can be readily degraded by fungi as carbon and energy sources. To date, only a few studies have attempted to describe biofilm communities on AF coating surfaces (Casse and Swain, 2006; Briand et al., 2012). Almost no detailed studies have focused exclusively on pioneer eukaryotic biofilm formation on silicone-based composite surfaces. Therefore, a better understanding of the effect of siliconebased composite materials on the dynamics of pioneer eukaryotic-community diversity and richness is essential for a comprehensive assessment of microbial damage, and also serves as the basis for future development of silicone-based AF strategies. In this study, culture-independent molecular biological techniques, based on a simple polymerase chain reaction (PCR) and a single-strand conformation polymorphism (SSCP), were employed to analyze pioneer eukaryotic-community diversity and richness variations on different silicone-based composite surfaces. The SSCP technique has been widely used to detect and measure the diversity of mixed microbial communities under various environmental conditions (Lee et al., 1996; Lin et al., 2007; Mnif et al., 2012). Nevertheless, the SSCP technique is rarely applied to study pioneer eukaryotic-community diversity adhering to different composites surfaces. The present study aims to improve the anti-biofouling properties of pure PDMS by incorporating different surface-modified CNT nanoparticles in the PDMS matrix. To the best of our knowledge, the use of a set of hMWNTs or cMWNTs as reinforcing agents to create PCs with enhanced anti-biofouling properties has yet to be reported. Anti-biofouling properties of the pure PDMS and PCs were examined through marine field assays. In addition, the results of pioneer eukaryotic-community diversity and richness variations on the PDMS and PCs surfaces based on Internal Transcribed Spacer 2 (ITS-2) sequence analysis are reported. Possible mechanisms for PCs against colonization of pioneer eukaryotic microbes are also discussed.
2. Materials and methods 2.1. Panel preparation Ship hull steel panels, measuring 100 mm 100 mm 3 mm, were carefully rubbed with sandpapers to obtain relatively uniform surfaces. All panels were thoroughly cleaned with sterile deionized water and 70% (v/v) ethyl alcohol, then rinsed again with sterile deionized water three times. Afterwards, these pre-treated panels were first coated with chlorinated rubber iron-red antirust paint (primer paint), in order to provide a bright background for the outer layer made of a transparent silicone elastomer. The primer paint was purchased from Jiamei Company (Weihai, China) which primarily consisted of chlorinated rubber resin, micaceous iron oxide, plasticizers, additives and a mixed solvent. It can be cured at room temperature in approximately 72 h. It is noteworthy that our previous study has confirmed that the primer paint demonstrated no anti-biofouling properties in the field, which was susceptible to microbial deterioration and degradation (Sun and Zhang, 2014). 2.2. Preparation of PDMS composites (PCs) The silicone-based material used in the study was a thermally curable PDMS-based material, namely the Sylgard 184 silicone elastomer kit (Dow Corning, USA), which was supplied as a twopart kit primarily consisting of a pre-polymer (base, component A) and a cross-linker (curing agent, component B). All CNT materials were purchased from Timesnano Company (Chengdu, China), including six MWNTs (F1 to F6), six hMWNTs (F7 to F12) and six cMWNTs (F13 to F18). Each CNT nanofiller was introduced into the PDMS matrix separately, in order to produce new PDMS composites (PCs). The PCs can be briefly grouped into three sets, namely the M set (MPs, M1eM6), H set (HPs, H1eH6) and C set (CPs, C1eC6). Detailed information and descriptions of the CNT fillers and PCs in this study are summarized as presented in Table 1. Pure PDMS (P0) was prepared by mixing component A and component B in the ratio 10:1 (weight) according to the manufacturer's instructions. Then the mixture was degassed at room temperature to remove the air bubbles. For the preparation of PCs, each of the CNT nanofillers was firstly well blended with component A for 10 min at room temperature. Then the mixture was mechanically stirred with component B for another 10 min, degassing until all air bubbles were completely removed. Each CNT nanofiller in the PDMS matrix adjusted to a final concentration of 0.1% (w/w) (Rosenhahn et al., 2008; Beigbeder et al., 2010). Afterwards, all silicone-based materials were painted on one side of the pre-treated steel panels using a bar-coater, in order to obtain a thin film as flat as possible. After 6 h of curing in a constant temperature oven at 105 C, the panels coated with either PCs or pure PDMS were well prepared for the subsequent field assays. The thickness of the silicone-based materials was approximately 300 mm. Similar preparation procedures have been described elsewhere by Beigbeder et al. in another study (Beigbeder et al., 2008a). Panels coated with pure PDMS served as the control. 2.3. Marine field assays The marine field assays were carried out at the XiaoShi Island harbor waters (37 3105100 N; 121580 1900 E), a natural preservation zone locating on the northwest of Weihai, China. A static, permanent wooden raft bridge allowed the immersion of numerous tested panels with the same dimension (measuring 100 mm 100 mm 3 mm) at different depths for long periods of time. Coated panels will face intense fouling pressure in natural marine environment under static conditions, because there existed
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Table 1 Detailed information on the CNT fillers and PDMS composites (PCs) in this study. CNT fillers
Hydroxyl content % (w/w)
Carboxyl content % (w/w)
Diameter (nm)
Length (mm)
SSA (m2/g)
Purity (%)
Composite codes
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F18 F17 F18
e e e e e e 5.58 3.70 3.06 1.76 1.06 0.71 e e e e e e
e e e e e e e e e e e e 3.86 2.56 2.00 1.23 0.73 0.64
10e20 8e15 10e20 20e30 30e50 >50 <8 8e15 10e20 20e30 30e50 >50 <8 8e15 10e20 20e30 30e50 >50
30e100 ~50 10e30 10e30 10e20 10e20 10e30 ~50 10e30 ~30 ~20 ~20 ~30 ~50 10e30 ~30 ~20 ~20
>165 >233 >200 >110 >60 >40 >500 >233 >200 >110 >60 >40 >500 >233 >200 >110 >60 >40
>95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95
M1 M2 M3 M4 M5 M6 H1 H2 H3 H4 H5 H6 C1 C2 C3 C4 C5 C6
Note: F1 to F6 represent different types of MWNTs; F7 to F12 represent different types of hydroxyl-modified MWNTs (hMWNTs); while F13 to F18 represent different types of carboxyl-modified MWNTs (cMWNTs). Here SSA is short for specific surface area.
relatively ample fouling organisms, including seaweeds, algae sporelings and juvenile invertebrates like barnacles, mussels and oysters at the immersion site during the testing period. Coating failures can be easily observed after four weeks of exposure to the marine environment, because of the presence of invertebrate larvae or algae sporelings using visual analysis. The tested panels were deployed to the stationary wooden raft bridge positioned in the inner Weihai harbor waters. For each silicone-based material, triplicate panels were used in field assays throughout. Tested panels were fixed to a frame using thin ropes so that they could be readily submerged at the depth of 0.8 m and 1.5 m. After a certain period of time, these panels were taken from the sea, gently washed with seawater, and then photographed. Afterwards, they were placed back into the sea as quickly as possible to continue the test. All tested panels were immersed in the sea from Oct.17 to Dec.17, 2013, in order to determine the longterm AF efficacy of the PCs. A set of treated panels coated with different PCs were examined. For comparison, the control panels coated with pure PDMS were also examined. 2.4. Marine in situ experiment and sampling Biofilm samples for SSCP analysis were collected from another short-term in situ experiment at the same site. Notably, our previous study has confirmed that pioneer eukaryotic biofilm communities began to colonize on the surface of cMWNTs-filled PDMS composite (namely C3 in this study) on day 2 and reached their mature state on day 14 (Sun et al., 2014). Therefore, a two-week in situ experiment (Nov. 11e25, 2013) was conducted to collect representative biofilm samples for SSCP analysis. For each siliconebased material, the panels for sampling (measuring 100 mm 100 mm 3 mm) were prepared in replicates of four throughout the two-week in situ experiment, in order to collect enough biological materials for SSCP analysis. Besides, sampling for each silicone-based material was carried out at five different points in time, namely on Nov. 13 (day 2), Nov. 16 (day 5), Nov. 19 (day 8), Nov. 22 (day 11), and Nov. 25 (day 14). The panels for sampling were brought back to the laboratory as quickly as possible using a cool-box. The biofilm sample was scraped out from each panel surface using sterile brushes. Each panel was gently washed with sterile deionized water before being scraped with the brushes. All replicate biofilm samples taken from the surfaces of the identical silicone-based material at the same
points in time were collected into one sterile Eppendorf tube, which was used as a representative of all replicate samples for further SSCP analysis. Afterwards, these mixed biofilm samples were suspended in 400 mL sterile deionized water, and quickly centrifuged at 4000 rpm for 5 min in order to obtain a cell pellet, and then they were preserved at 80 C for further SSCP analysis. 2.5. Polymerase chain reaction (PCR) Genomic DNA was extracted with hot phenol-chloroform method. Briefly, equal volumes of biofilm sample and phenol (pH 8.0)-chloroform were well mixed, then vigorously vortexed for 1 min, before being heated in a 65 C water bath for 15 min with frequent shaking. After incubation for 3 min at room temperature, the sample was centrifuged at 10,000 rpm for 10 min. The supernatant was extracted again, with an equal volume of phenol (pH 8.0)-chloroform. After a second centrifugation at the same speed and for the same length of time, the supernatant was collected and used directly as a PCR template. The amplification of the ITS-2 gene sequence was performed with specific primers ITS3 (50 -GCA TCG ATG AAG AAC GCA GC-30 ) and ITS4 (50 -TCC TCC GCT TAT TGA TAT GC-30 ) to identify pioneer eukaryotes as previously described (Hinrikson et al., 2005). Primers used in this study were synthesized from Sangon (Shanghai, China). PCR amplifications were carried out in a PCR Thermal Cycler Dice Gradient (TaKaRa) using a NPK02 kit (GREDBIO). The asymmetric PCR reaction was performed in 12 mL volumes, containing 0.2 mM primer, 200 nM of each dNTP, 2 U of Taq DNA polymerase (TaKaRa), and 0.4 ng of template. For each set of PCR reactions, a negative control was included. The PCR template was replaced with an equivalent volume of sterile deionized water. The PCR program included an initial denaturation at 94 C for 3 min, followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 56 C for 40 s, and extension at 72 C for 40 s, followed by a final extension at 72 C for 5 min. All PCR products were separated using 1.0% agarose gel electrophoresis and visualized using a WD-9413C gel imaging analysis system purchased from Liu Yi Company (Beijing, China). All the PCR products were stored at 40 C for further analysis. 2.6. SSCP gel electrophoresis All the PCR products were separated using the SSCP technique as Lee et al. previously described with modifications (Lee et al., 1996).
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A vertical gel electrophoresis apparatus (DYCZ-24DN) purchased from Liu Yi Company (Beijing, China) was used for SSCP analysis. The mixed biofilm samples collected from the surfaces of different silicone-based materials at the same points in time were analyzed using the same SSCP gel. SSCP electrophoresis was performed using an 8% polyacrylamide (29:1) gel submerged in 1 TBE buffer for 24 h at a constant voltage of 100 V. The gel temperature was maintained at 4 C using a circulating water bath. The PCR products were well mixed with equal volumes of a denaturing solution (95% formamide, 0.25% bromphenol blue, 0.25% xylene cyanol) to adjust to a final volume of 6 mL, then heated to 98 C for 10 min and snapfrozen on ice before loading. The gels were visualized using the silver-staining method and recorded using a digital camera (Samsung PL2000) for further analysis. 2.7. Data analysis The scanned gels were analyzed to construct SSCP community profiles, using Quantity One software (version4.6.2, Bio-Rad, USA). Both the lanes and bands were detected automatically, while bands with a relative intensity less than 0.3% were discarded. All such procedures mentioned above were performed three times. Peak intensity values were used to compare bands within SSCP profiles, in order to determine the diversity indices by considering each band to be analogous to a single species. Band position and intensity was determined in order to obtain data matrices. On the basis of these matrices, four diversity indices, including Shannon diversity index (H), species richness(S), total abundance and the Simpson index (l), were calculated using Biodap software (Thomas and Clay, 2000), in order to estimate the diversity and richness of pioneer eukaryotic communities (Hill, 1973). Multidimensional Scaling (MDS) analysis was performed using SPSS19.0 software (IBM, Armonk, NY, USA) by inputting diversity indices as variables. All data were presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism Software version 6.03 (GraphPad Software, CA, USA) to evaluate statistical differences. P-value < 0.05 was considered statistically significant.
panels immersed at the depth of 0.8 m and 1.5 m. To be specific, treated panels immersed at 0.8 m exhibited excellent antibiofouling properties in contrast to the PDMS surface, with extremely low attachment of early fouling organisms. In contrast, treated panels immersed at 1.5 m remained relatively clean for 4 weeks, but were prone to be heavily fouled at the end of the immersion with the exception of M1 and C3 (Fig. 1a and c), suggesting that the anti-biofouling properties of PCs may be closely related to the immersion depths. It is noticeable that only two reinforcing CNT fillers (i.e., F1 and F15) have been identified from a wide array of CNT candidates via marine exposure studies, suggesting that not all CNT fillers are well suited as reinforcing agents to enhance the anti-biofouling properties of PDMS. Similarly, not all PCs have demonstrated excellent anti-biofouling properties in the field. In this study, only two PCs (i.e., M1 and C3) with exceptional antibiofouling properties were determined in the field immersion testing, which can effectively prevent biofouling for another 12 weeks (Data not shown). Therefore, to identify more appropriate CNT fillers, establishing an effective selecting systems based on field assays is a central subject in our future work. To date, a majority of studies have been dedicated to the materials' AF efficacy against representative macrofoulers, such as sporelings of Ulva and adult barnacles (Balanus amphitrite). Nevertheless, the usage of the results obtained from laboratory assays may lead to catastrophic failure because of the paucity of comparisons between successful laboratory assays and field assays, although laboratory assays are less susceptible to seasonal, environmental and site-specific variations. It is possible that some antibiofouling potential coatings may perform exceptionally well against representative fouling species, while completely losing their AF efficacy in the field immersion assays. Therefore, this study highlights the significance of field assays for the assessment of antibiofouling properties of PCs. Furthermore, we have managed to identify the two PCs (M1 and C3) with excellent anti-biofouling properties from a wide arrays of PCs through field assays. Further studies to revalidate their long-term AF efficacy are required. 3.2. Clustering analysis based on MDS method
3. Results and discussion 3.1. Marine field assays In the present study, the anti-biofouling properties of PCs, including MPs (M1eM6), HPs (H1eH6) and CPs (C1eC6), were examined through field assays conducted in the marine environment under static conditions at different depths. A range of effects were observed and recorded as presented in Fig. 1. Fig. 1 showed that after four weeks of exposure in the sea, the PDMS surface was largely covered with some inorganics (mainly sea slime), algae sporelings and a few juvenile invertebrates. While at the end of the immersion, the PDMS surface was heavily fouled and showed extensive coverage of sea slime, algae spores, as well as some early colonizers like juvenile barnacles and ascidian, indicating that the PDMS surface does not exhibit excellent anti-biofouling properties when exposed to the marine environment, which might be susceptible to pioneer microbial colonization and deterioration. This result agrees well with the earlier findings by Sankar et al. (Sankar et al., 2015), which showed the pure PDMS surface was susceptible to microbial fouling triggered by microorganisms like bacteria and diatoms. The treated panels coated with different PCs exhibited better anti-biofouling properties in the field, as compared to PDMS control (Fig. 1), indicating that the anti-biofouling properties of PDMS can be fundamentally enhanced with the introduction of CNT fillers. Significant differences can be observed between treated
ITS-2 gene fragments were successfully amplified as expected, and separated using the SSCP technique. Fig. 2 shows SSCP fingerprints of pioneer eukaryotes adhering to different silicone-based material surfaces. Almost all silicone-based material surfaces were found to be readily colonized by a mixed culture of pioneer eukaryotes at five different points in time within the two-week in situ experiment, as observed from SSCP patterns. The results suggest that all silicone-based composite materials cannot fully prevent the colonization of pioneer eukaryotic microbes. Each siliconebased composite surface has presented specific eukaryotic SSCP patterns at different points in time. Based on the SSCP profiles, the four diversity indices were calculated using Quantity one 4.6.2 and BioDap software (Data not shown). Based on the four diversity indices, the MDS analysis was undertaken, as is shown in Fig. 3. In Fig. 3, pioneer eukaryotic microbial communities adhering to different PCs surfaces were clearly different from that of the PDMS surface, suggesting that different PCs surfaces may have differentially modulating effects on pioneer eukaryotic colonization. Pioneer eukaryotes attached to the surfaces of PCs belonging to the same set (M set, H set or P set) were prone to be closely grouped or distributed. In contrast, clear differences can be found among early eukaryotes adhering to the surfaces of PCs belonging to different sets. For example, pioneer eukaryotes attached to the surfaces of the M set (EM1eEM6), along with the C4 surface (EC4), were prone to cluster into one group; while most pioneer eukaryotes adhering to the surfaces of the H set
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Fig. 1. Images of the treated panels coated with (a) MPs(M1eM6), (b) HPs(H1eH6) and (c) CPs (C1eC6) after the immersion in seawater (OctobereDecember 2013, Weihai, China) as a function of time.
Fig. 2. The SSCP fingerprints of pioneer eukaryotic communities adhering to the surfaces of different silicone-based materials at different points in time.
(EH1eEH6) or the C set (except EC4) tended to cluster into another two groups. These combined results suggested that the types of CNT fillers may play a key role in deciding the clustering features of pioneer eukaryotic communities, which may contribute to accounting for the differentially modulating effects exerted by different PCs surfaces on the colonization of the pioneer eukaryotic microbes. 3.3. Effect of PCs surfaces on pioneer eukaryotic communities The analysis of pioneer eukaryotic-community diversity and richness was conducted based on the SSCP fingerprints. The comparison of the four diversity indices, namely the Shannon diversity index (H), species richness(S), total abundance and the Simpson index (l), is presented in Fig. 4. The Shannon diversity index (H), which takes account of both the number of species and the evenness of the species, was used as a measurement for the estimation of general biodiversity in habitant microbial communities. The Shannon diversity index provides a holistic view of a particular ecological niche, with H values usually ranging from 1.5 (low
species richness and evenness) to 3.5 (high species richness and evenness) (Bharathkumar et al., 2008). Fig. 4aec shows that the H value of the eukaryotic microbial community ranged between 1.49 ± 0.31 and 2.39 ± 0.17 for all the PCs surfaces, which was generally lower than that of the PDMS control (2.53 ± 0.23). Pioneer eukaryotes attached to CPs surfaces (C1eC6) showed the lowest level of diversity, with the H values ranging from 1.49 ± 0.31 to 1.97 ± 0.37, as compared to those of MPs surfaces (2.10 ± 0.27 to 2.26 ± 0.29) and HPs surfaces (1.98 ± 0.49 to 2.39 ± 0.17). This result indicates that CPs surfaces may have stronger modulating effect on pioneer eukaryotic colonization than that of the MPs and HPs surfaces. A significant decrease in pioneer eukaryotic-community diversity can be observed on the surfaces of MPs (except M2), H1, H4, and CPs (C1eC6) (P < 0.05), and particularly on the surfaces of H1, H4 and CPs (C1eC6) (P < 0.01). Species richness (S) simply describes the number of different species in a microbial community. Fig. 4d, e and f reveal that the S value of pioneer eukaryotic community (ranging from 6 ± 2 to 15 ± 3) was greatly down-regulated by most PCs surfaces, compared with that of the PDMS surface (19 ± 2), indicating a
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diversity and richness amongst different silicone-based material surfaces. Our study also confirmed that pioneer eukaryotic SSCP fingerprints can be highly helpful for the monitoring of the dynamics of pioneer eukaryotic community diversity and richness in a biologically relevant process. 3.4. Possible mechanisms for PCs against pioneer eukaryotic colonization
Fig. 3. Clustering analysis of pioneer eukaryotic communities adhering to the surfaces of different silicone-based materials using MDS method. EP0, EM, EH and EC represent pioneer eukaryotic microbial community attached to the surfaces of P0, MPs, HPs and CPs, respectively.
significant decrease in richness. In addition, total abundance is measured primarily by the total intensity of bands present in the SSCP profiles. Fig. 4gei show that the value of total abundance for pioneer eukaryotic communities (ranging from 49 ± 16 to 140 ± 68) was also greatly down-regulated by most PCs surfaces in contrast to PDMS control (153 ± 54). A significant decrease in total abundance can be found for pioneer eukaryotes adhering to the surfaces of M1, M2 and CPs (except C6) (P < 0.05), and particularly on the surfaces of C2 and C5 (P < 0.01). The significantly reduced eukaryoticcommunity diversity and richness suggest that most PCs surfaces may have strong modulating effects on pioneer eukaryotic communities, thereby dramatically reducing the colonization of pioneer eukaryotic microbes. Similarly, Camps et al. have reported that AF coatings could influence the abundance and community structure of colonizing biofilm communities in a recent study (Camps et al., 2014). In another study, Ling et al. have also found that microfabricated PDMS surfaces could regulate the development of microbial biofilm communities (Ling et al., 2014). The result obtained in this study agrees well with the findings reported by Camps et al. and Ling et al. Most PCs surfaces can effectively reduce pioneer eukaryotic-community diversity and richness by progressively weakening the invasion and colonization of pioneer eukaryotic microbes, thereby being less susceptible to pioneer eukaryotic microbial colonization and deterioration. The Simpson index (l) describes the number of dominant species in a particular microbial community. Fig. 4j, k and l reveal that the l value of pioneer eukaryotic communities (ranging from 0.117 ± 0.033 to 0.249 ± 0.082) was elevated on most PCs surfaces (except H4), significantly elevated on the surfaces of H4, C2, C3 and C5 (P < 0.05), and particularly on the surfaces of C2 and C5 (P < 0.01), when compared to that of the PDMS control (0.116 ± 0.045). This result indicates that the dominant eukaryotes in the biofilm communities on these surfaces may vary greatly in contrast to the PDMS control. The increase in the number of the dominant eukaryotes suggests that most PCs surfaces may have the potential to accelerate the succession progress of pioneer eukaryotic communities, and in turn change the succession patterns of pioneer eukaryotic communities. In addition, the four well-known diversity indices are found to be sensitive and complementary indicators to capture the variations of pioneer eukaryotic-community
In the current study, all the PCs surfaces exhibited better antibiofouling properties even under static conditions, when compared to the PDMS control, indicating that AF efficacy of pure PDMS can be further reinforced by the incorporation of 0.1% (w/w) CNT nanoparticles. Previously, Beigbeder et al. reported that the bulk mechanical properties of pure PDMS remained unchanged after the introduction of low quantities of CNT filler, indicating that the CNT filler incorporation into the PDMS matrix does not significantly change the surface properties of the PDMS matrix (Beigbeder et al., 2008a). Therefore, the general consistency of the enhanced anti-biofouling capacity may be primarily attributed to the CNTs themselves. Interestingly, differential anti-biofouling effects of PCs can also be observed from Fig. 1, implying that the CNT fillers may play key roles in determining anti-biofouling effects of PDMS, which agrees well with the findings reported by Kang et al. in an earlier study (Kang et al., 2008). In addition, most PCs performed well in marine exposure studies, although CNT fillers were not well dispersed in the PDMS matrix. The partial CNTs aggregation may also contribute to the reinforcement of composite' antibiofouling properties. Similarly, Kang et al. found SWNTs to be directly responsible for cell membrane damage because of SWNTs aggregation (Kang et al., 2007). It seems that the state of CNT filler dispersion in the PDMS matrix is not directly related to the composite' anti-biofouling properties, although Beigbeder et al. have previously emphasized that the significance of the dispersion quality of the CNT filler in the polymeric matrix (Beigbeder et al., 2010). Besides, Beigbeder et al. have also found that the reinforced AF efficacy to be mainly ascribed to the extremely favorable CH-pi electronic interactions (Beigbeder et al., 2008b). Yet we cannot exclude the possibility that the changes in surface micro/nanostructures of the PDMS may contribute to the improved anti-biofouling properties of PCs, although the hypothesis needs support from delicate AFM/SEM analysis in the future. It is probable that the incorporation of CNT fillers in PDMS matrix can produce novel PCs with different micro/nanostructures, which may be contributable to the weakening of the invasion and colonization of pioneer eukaryotic microbes. Several recent publications have also confirmed surface micro/nanostructures may have significant impacts on surface colonization (Hoipkemeier-Wilson et al., 2004; Cheng et al., 2006; Schumacher et al., 2007). Further research focusing exclusively on characterization of the surface micro/ nanostructures of different PCs using AFM/SEM is currently underway, in order to uncover the unrecognized anti-biofouling mechanisms of PCs surfaces. Combined with results obtained from MDS and SSCP analysis, it can be seen that pioneer eukaryotes adhering to the surfaces of the same PCs sets (M set, H set or C set) were closely distributed or grouped, suggesting that PDMS incorporated similar types of CNTs tending to have a similar modulating effect on the colonization of pioneer eukaryotic microbes. The differences among pioneer eukaryotes adhering to the surfaces of PCs belonging to different sets seemed larger than those attached to the surfaces of PCs belonging to the same set, indicating that the types of CNTs may be closely related to the composite' AF efficacy (Fig. 3). In addition, it seems that the differential anti-biofouling capacity of the PCs surfaces may not be directly associated with the growth process and
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Fig. 4. The comparison of the four diversity indices, (aec) Shannon diversity index, (def) Species richness, (gei) Total abundance, (jel) Simpson index of pioneer eukaryotic communities adhering to the surfaces of different silicone-based materials. Error bars represent the SD of the mean. One asterisk (*) represents significant difference (P < 0.05), whereas two asterisks (**) represent extremely significant difference (P < 0.01).
dynamics of pioneer eukaryotes in the biofilm. For example, CPs (C1eC6) in this study demonstrated stronger modulating effects on pioneer eukaryotic microbial communities than those of the other two PCs sets. Particularly, C2 and C5 surfaces can dramatically reduce pioneer eukaryotic-community diversity and richness (P < 0.01), thereby exerting the strongest perturbation effect on pioneer eukaryotic colonization. However, their anti-biofouling capacity was only intermediate or weak amongst the others (Fig. 1c). In contrast, M2 and C3 surfaces performed exceptionally well in the field, although the two surfaces failed to exert the greatest perturbation effect on the colonization of pioneer eukaryotic microbes.
biofouling applications. Interestingly, we found that most PCs surfaces were able to significantly reduce the diversity and richness, thereby being less susceptible to pioneer eukaryotic microbial colonization and deterioration. Our findings may facilitate the understanding of the dynamics of pioneer eukaryotic-community diversity and richness variations on different silicone-based material surfaces in the marine environment and also cast new light on the development of eco-friendly AF coatings. Further studies are in progress to identify more reinforcing CNT fillers and uncover the unrecognized anti-biofouling and degradation mechanisms of PCs.
Acknowledgments 4. Conclusion In the present study, we identified that the PDMS matrix incorporated with different surface-modified CNTs demonstrated excellent anti-biofouling properties as compared to the PDMS control. This simple and efficient method to produce functionalized PCs with improved AF efficacy gives potential for future anti-
The authors express their sincere gratitude and thanks to Tao Wang, Yunyang Cheng and Zihua Yang of Harbin Institute of Technology, school of Marine science and technology, for their constant assistance with marine field assays and sampling throughout the course of this investigation. This work was funded by National Natural Science Foundation of China (No. 31071170).
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
New anti-biofouling carbon nanotubes-filled polydimethylsiloxane composites against colonization by pioneer eukaryotic microbes Yuan Sun a, b, Zhizhou Zhang a, b, c, * a
School of Chemical Engineering & Technology, 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 Shandong Province, Harbin Institute of Technology, Weihai 264209, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2016 Received in revised form 17 March 2016 Accepted 17 March 2016 Available online 26 March 2016
Microbial biofilm formation on composite surfaces has posed potential threats to the composite's structural integrity, durability and physical properties. Pioneer eukaryotes have been reported to be primarily responsible for the degradation of a wide range of composite materials. In this study, different carbon nanotubes (CNTs) were incorporated in the polydimethylsiloxane (PDMS) matrix respectively, in order to create CNTs-filled PDMS composites (PCs) with improved anti-biofouling properties. The antibiofouling properties of pure PDMS (P0) and PCs were examined through marine field assays. The diversity and richness of pioneer eukaryotic communities adhering to P0 and PCs surfaces were analyzed using the single-strand conformation polymorphism (SSCP) technique. PCs have exhibited differential and better anti-biofouling properties, when compared to the PDMS control. Two PCs with exceptional anti-biofouling properties were determined in the field. The Shannon diversity index and species richness of pioneer eukaryotic communities on most PCs surfaces were dramatically lower than those of the pure PDMS control (P < 0.05). This indicates a significant decrease in the diversity and richness of pioneer eukaryotic communities. The combined results suggest PCs can effectively reduce the colonization of pioneer eukaryotes, such as algae sporelings and invertebrate larvae. PCs with low cost and good compatibility in the marine environment give the potential for future anti-biofouling applications. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Biofouling Carbon nanotubes Diversity Pioneer eukaryotes Polydimethylsiloxane Single strand conformation polymorphism
1. Introduction Marine biofouling occurs when fouling organisms settle and colonize on surfaces of either natural or artificial substratum submerged in a marine environment (Fusetani, 2004). Biofouling can severely affect structures critical to the maritime industry, increase propulsive fuel consumption, and weaken ships' maneuverability, thereby resulting in considerable economic loss and environmental issues (Bixler and Bhushan, 2012). Broad-spectrum biocidal coatings containing tributyltin (TBT) or copper have been widely used to prevent biofouling (Banerjee et al., 2011). However, these biocidal coatings are toxic to a wide range of non-target marine organisms, and therefore are ecologically harmful to the marine aquatic environment (Domart-Coulon et al., 2000). As a result, extensive studies have been devoted to developing eco-friendly
* Corresponding author. School 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.ibiod.2016.03.019 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
antifouling (AF) coatings (Dafforn et al., 2011). Recently, the increasing usage of non-toxic silicone materials represents a promising approach for the development of ecofriendly polymers (Callow and Callow, 2011). Polydimethylsiloxane (PDMS) is a useful and attractive silicone elastomer with desirable chemical and physical properties, such as high hydrophobicity, low surface energy, contamination resistance and long-term endurance (Sokolova et al., 2012). However, because of the inherent disadvantages of PDMS, such as mechanical weakness and a propensity to sustain damage when immersed in the marine environment, the practical applications of PDMS against biofouling have been greatly limited. To date, one promising and effective method to overcome these drawbacks has been to introduce a variety of nanofillers into the polymeric matrix, in order to produce new composites with potentially improved anti-biofouling properties (Majumdar et al., 2008). Carbon nanotubes (CNTs) have been attracting considerable attention among the numerous nano-sized fillers due to its unique molecular structure, exceptional electrical, mechanical and thermal properties (Iijima, 1991). Beigbeder et al. have
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reported that the presence of a small amount of multi-walled carbon nanotubes (MWNTs) in the PDMS matrix can greatly improve its AF and fouling-release (FR) properties (Beigbeder et al., 2008a). CNTs seem to be the optimal fillers for preparing composites of various materials, including PDMS. Nevertheless, research on the preparation of novel PDMS composites (PCs) reinforced with different surface-modified CNTs, such as hydroxyl-modified MWNTs (hMWNTs) and carboxyl-modified MWNTs (cMWNTs) for anti-biofouling applications has not been systematically investigated. Furthermore, the anti-biofouling properties of most PCs failed to be examined via field assays under static conditions. Microbial biofilm formation on composite material surfaces under favorable conditions is usually considered as a prerequisite for biofouling and the subsequent degradation process, which can dramatically affect the composite's structural integrity, durability, chemical and physical properties (Fletcher, 1994; Gu, 2005). The growth of biofilm communities on composite surfaces has posed a major risk to composite materials. It has been reported that a wide range of composite materials are susceptible to microbial deterioration and degradation (Gu, 2003). It is well accepted that biodegradation of composite materials may be primarily attributed to a combined effect of both bacterial and fungi colonization in sequence, while fungi may have obvious advantages over bacteria during the early stage of surface colonization, particularly on relatively resistant composite surfaces (Gu et al., 1998; Gu, 2007). Various publications have reported that fungi are primarily responsible for the degradation of a variety of composite materials, such as fiber-reinforced polymeric composites (Gu et al., 1996a) and electronic packaging polyimides (Gu et al., 1996b), because some composite constituents can be readily degraded by fungi as carbon and energy sources. To date, only a few studies have attempted to describe biofilm communities on AF coating surfaces (Casse and Swain, 2006; Briand et al., 2012). Almost no detailed studies have focused exclusively on pioneer eukaryotic biofilm formation on silicone-based composite surfaces. Therefore, a better understanding of the effect of siliconebased composite materials on the dynamics of pioneer eukaryotic-community diversity and richness is essential for a comprehensive assessment of microbial damage, and also serves as the basis for future development of silicone-based AF strategies. In this study, culture-independent molecular biological techniques, based on a simple polymerase chain reaction (PCR) and a single-strand conformation polymorphism (SSCP), were employed to analyze pioneer eukaryotic-community diversity and richness variations on different silicone-based composite surfaces. The SSCP technique has been widely used to detect and measure the diversity of mixed microbial communities under various environmental conditions (Lee et al., 1996; Lin et al., 2007; Mnif et al., 2012). Nevertheless, the SSCP technique is rarely applied to study pioneer eukaryotic-community diversity adhering to different composites surfaces. The present study aims to improve the anti-biofouling properties of pure PDMS by incorporating different surface-modified CNT nanoparticles in the PDMS matrix. To the best of our knowledge, the use of a set of hMWNTs or cMWNTs as reinforcing agents to create PCs with enhanced anti-biofouling properties has yet to be reported. Anti-biofouling properties of the pure PDMS and PCs were examined through marine field assays. In addition, the results of pioneer eukaryotic-community diversity and richness variations on the PDMS and PCs surfaces based on Internal Transcribed Spacer 2 (ITS-2) sequence analysis are reported. Possible mechanisms for PCs against colonization of pioneer eukaryotic microbes are also discussed.
2. Materials and methods 2.1. Panel preparation Ship hull steel panels, measuring 100 mm 100 mm 3 mm, were carefully rubbed with sandpapers to obtain relatively uniform surfaces. All panels were thoroughly cleaned with sterile deionized water and 70% (v/v) ethyl alcohol, then rinsed again with sterile deionized water three times. Afterwards, these pre-treated panels were first coated with chlorinated rubber iron-red antirust paint (primer paint), in order to provide a bright background for the outer layer made of a transparent silicone elastomer. The primer paint was purchased from Jiamei Company (Weihai, China) which primarily consisted of chlorinated rubber resin, micaceous iron oxide, plasticizers, additives and a mixed solvent. It can be cured at room temperature in approximately 72 h. It is noteworthy that our previous study has confirmed that the primer paint demonstrated no anti-biofouling properties in the field, which was susceptible to microbial deterioration and degradation (Sun and Zhang, 2014). 2.2. Preparation of PDMS composites (PCs) The silicone-based material used in the study was a thermally curable PDMS-based material, namely the Sylgard 184 silicone elastomer kit (Dow Corning, USA), which was supplied as a twopart kit primarily consisting of a pre-polymer (base, component A) and a cross-linker (curing agent, component B). All CNT materials were purchased from Timesnano Company (Chengdu, China), including six MWNTs (F1 to F6), six hMWNTs (F7 to F12) and six cMWNTs (F13 to F18). Each CNT nanofiller was introduced into the PDMS matrix separately, in order to produce new PDMS composites (PCs). The PCs can be briefly grouped into three sets, namely the M set (MPs, M1eM6), H set (HPs, H1eH6) and C set (CPs, C1eC6). Detailed information and descriptions of the CNT fillers and PCs in this study are summarized as presented in Table 1. Pure PDMS (P0) was prepared by mixing component A and component B in the ratio 10:1 (weight) according to the manufacturer's instructions. Then the mixture was degassed at room temperature to remove the air bubbles. For the preparation of PCs, each of the CNT nanofillers was firstly well blended with component A for 10 min at room temperature. Then the mixture was mechanically stirred with component B for another 10 min, degassing until all air bubbles were completely removed. Each CNT nanofiller in the PDMS matrix adjusted to a final concentration of 0.1% (w/w) (Rosenhahn et al., 2008; Beigbeder et al., 2010). Afterwards, all silicone-based materials were painted on one side of the pre-treated steel panels using a bar-coater, in order to obtain a thin film as flat as possible. After 6 h of curing in a constant temperature oven at 105 C, the panels coated with either PCs or pure PDMS were well prepared for the subsequent field assays. The thickness of the silicone-based materials was approximately 300 mm. Similar preparation procedures have been described elsewhere by Beigbeder et al. in another study (Beigbeder et al., 2008a). Panels coated with pure PDMS served as the control. 2.3. Marine field assays The marine field assays were carried out at the XiaoShi Island harbor waters (37 3105100 N; 121580 1900 E), a natural preservation zone locating on the northwest of Weihai, China. A static, permanent wooden raft bridge allowed the immersion of numerous tested panels with the same dimension (measuring 100 mm 100 mm 3 mm) at different depths for long periods of time. Coated panels will face intense fouling pressure in natural marine environment under static conditions, because there existed
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Table 1 Detailed information on the CNT fillers and PDMS composites (PCs) in this study. CNT fillers
Hydroxyl content % (w/w)
Carboxyl content % (w/w)
Diameter (nm)
Length (mm)
SSA (m2/g)
Purity (%)
Composite codes
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F18 F17 F18
e e e e e e 5.58 3.70 3.06 1.76 1.06 0.71 e e e e e e
e e e e e e e e e e e e 3.86 2.56 2.00 1.23 0.73 0.64
10e20 8e15 10e20 20e30 30e50 >50 <8 8e15 10e20 20e30 30e50 >50 <8 8e15 10e20 20e30 30e50 >50
30e100 ~50 10e30 10e30 10e20 10e20 10e30 ~50 10e30 ~30 ~20 ~20 ~30 ~50 10e30 ~30 ~20 ~20
>165 >233 >200 >110 >60 >40 >500 >233 >200 >110 >60 >40 >500 >233 >200 >110 >60 >40
>95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95
M1 M2 M3 M4 M5 M6 H1 H2 H3 H4 H5 H6 C1 C2 C3 C4 C5 C6
Note: F1 to F6 represent different types of MWNTs; F7 to F12 represent different types of hydroxyl-modified MWNTs (hMWNTs); while F13 to F18 represent different types of carboxyl-modified MWNTs (cMWNTs). Here SSA is short for specific surface area.
relatively ample fouling organisms, including seaweeds, algae sporelings and juvenile invertebrates like barnacles, mussels and oysters at the immersion site during the testing period. Coating failures can be easily observed after four weeks of exposure to the marine environment, because of the presence of invertebrate larvae or algae sporelings using visual analysis. The tested panels were deployed to the stationary wooden raft bridge positioned in the inner Weihai harbor waters. For each silicone-based material, triplicate panels were used in field assays throughout. Tested panels were fixed to a frame using thin ropes so that they could be readily submerged at the depth of 0.8 m and 1.5 m. After a certain period of time, these panels were taken from the sea, gently washed with seawater, and then photographed. Afterwards, they were placed back into the sea as quickly as possible to continue the test. All tested panels were immersed in the sea from Oct.17 to Dec.17, 2013, in order to determine the longterm AF efficacy of the PCs. A set of treated panels coated with different PCs were examined. For comparison, the control panels coated with pure PDMS were also examined. 2.4. Marine in situ experiment and sampling Biofilm samples for SSCP analysis were collected from another short-term in situ experiment at the same site. Notably, our previous study has confirmed that pioneer eukaryotic biofilm communities began to colonize on the surface of cMWNTs-filled PDMS composite (namely C3 in this study) on day 2 and reached their mature state on day 14 (Sun et al., 2014). Therefore, a two-week in situ experiment (Nov. 11e25, 2013) was conducted to collect representative biofilm samples for SSCP analysis. For each siliconebased material, the panels for sampling (measuring 100 mm 100 mm 3 mm) were prepared in replicates of four throughout the two-week in situ experiment, in order to collect enough biological materials for SSCP analysis. Besides, sampling for each silicone-based material was carried out at five different points in time, namely on Nov. 13 (day 2), Nov. 16 (day 5), Nov. 19 (day 8), Nov. 22 (day 11), and Nov. 25 (day 14). The panels for sampling were brought back to the laboratory as quickly as possible using a cool-box. The biofilm sample was scraped out from each panel surface using sterile brushes. Each panel was gently washed with sterile deionized water before being scraped with the brushes. All replicate biofilm samples taken from the surfaces of the identical silicone-based material at the same
points in time were collected into one sterile Eppendorf tube, which was used as a representative of all replicate samples for further SSCP analysis. Afterwards, these mixed biofilm samples were suspended in 400 mL sterile deionized water, and quickly centrifuged at 4000 rpm for 5 min in order to obtain a cell pellet, and then they were preserved at 80 C for further SSCP analysis. 2.5. Polymerase chain reaction (PCR) Genomic DNA was extracted with hot phenol-chloroform method. Briefly, equal volumes of biofilm sample and phenol (pH 8.0)-chloroform were well mixed, then vigorously vortexed for 1 min, before being heated in a 65 C water bath for 15 min with frequent shaking. After incubation for 3 min at room temperature, the sample was centrifuged at 10,000 rpm for 10 min. The supernatant was extracted again, with an equal volume of phenol (pH 8.0)-chloroform. After a second centrifugation at the same speed and for the same length of time, the supernatant was collected and used directly as a PCR template. The amplification of the ITS-2 gene sequence was performed with specific primers ITS3 (50 -GCA TCG ATG AAG AAC GCA GC-30 ) and ITS4 (50 -TCC TCC GCT TAT TGA TAT GC-30 ) to identify pioneer eukaryotes as previously described (Hinrikson et al., 2005). Primers used in this study were synthesized from Sangon (Shanghai, China). PCR amplifications were carried out in a PCR Thermal Cycler Dice Gradient (TaKaRa) using a NPK02 kit (GREDBIO). The asymmetric PCR reaction was performed in 12 mL volumes, containing 0.2 mM primer, 200 nM of each dNTP, 2 U of Taq DNA polymerase (TaKaRa), and 0.4 ng of template. For each set of PCR reactions, a negative control was included. The PCR template was replaced with an equivalent volume of sterile deionized water. The PCR program included an initial denaturation at 94 C for 3 min, followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 56 C for 40 s, and extension at 72 C for 40 s, followed by a final extension at 72 C for 5 min. All PCR products were separated using 1.0% agarose gel electrophoresis and visualized using a WD-9413C gel imaging analysis system purchased from Liu Yi Company (Beijing, China). All the PCR products were stored at 40 C for further analysis. 2.6. SSCP gel electrophoresis All the PCR products were separated using the SSCP technique as Lee et al. previously described with modifications (Lee et al., 1996).
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A vertical gel electrophoresis apparatus (DYCZ-24DN) purchased from Liu Yi Company (Beijing, China) was used for SSCP analysis. The mixed biofilm samples collected from the surfaces of different silicone-based materials at the same points in time were analyzed using the same SSCP gel. SSCP electrophoresis was performed using an 8% polyacrylamide (29:1) gel submerged in 1 TBE buffer for 24 h at a constant voltage of 100 V. The gel temperature was maintained at 4 C using a circulating water bath. The PCR products were well mixed with equal volumes of a denaturing solution (95% formamide, 0.25% bromphenol blue, 0.25% xylene cyanol) to adjust to a final volume of 6 mL, then heated to 98 C for 10 min and snapfrozen on ice before loading. The gels were visualized using the silver-staining method and recorded using a digital camera (Samsung PL2000) for further analysis. 2.7. Data analysis The scanned gels were analyzed to construct SSCP community profiles, using Quantity One software (version4.6.2, Bio-Rad, USA). Both the lanes and bands were detected automatically, while bands with a relative intensity less than 0.3% were discarded. All such procedures mentioned above were performed three times. Peak intensity values were used to compare bands within SSCP profiles, in order to determine the diversity indices by considering each band to be analogous to a single species. Band position and intensity was determined in order to obtain data matrices. On the basis of these matrices, four diversity indices, including Shannon diversity index (H), species richness(S), total abundance and the Simpson index (l), were calculated using Biodap software (Thomas and Clay, 2000), in order to estimate the diversity and richness of pioneer eukaryotic communities (Hill, 1973). Multidimensional Scaling (MDS) analysis was performed using SPSS19.0 software (IBM, Armonk, NY, USA) by inputting diversity indices as variables. All data were presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism Software version 6.03 (GraphPad Software, CA, USA) to evaluate statistical differences. P-value < 0.05 was considered statistically significant.
panels immersed at the depth of 0.8 m and 1.5 m. To be specific, treated panels immersed at 0.8 m exhibited excellent antibiofouling properties in contrast to the PDMS surface, with extremely low attachment of early fouling organisms. In contrast, treated panels immersed at 1.5 m remained relatively clean for 4 weeks, but were prone to be heavily fouled at the end of the immersion with the exception of M1 and C3 (Fig. 1a and c), suggesting that the anti-biofouling properties of PCs may be closely related to the immersion depths. It is noticeable that only two reinforcing CNT fillers (i.e., F1 and F15) have been identified from a wide array of CNT candidates via marine exposure studies, suggesting that not all CNT fillers are well suited as reinforcing agents to enhance the anti-biofouling properties of PDMS. Similarly, not all PCs have demonstrated excellent anti-biofouling properties in the field. In this study, only two PCs (i.e., M1 and C3) with exceptional antibiofouling properties were determined in the field immersion testing, which can effectively prevent biofouling for another 12 weeks (Data not shown). Therefore, to identify more appropriate CNT fillers, establishing an effective selecting systems based on field assays is a central subject in our future work. To date, a majority of studies have been dedicated to the materials' AF efficacy against representative macrofoulers, such as sporelings of Ulva and adult barnacles (Balanus amphitrite). Nevertheless, the usage of the results obtained from laboratory assays may lead to catastrophic failure because of the paucity of comparisons between successful laboratory assays and field assays, although laboratory assays are less susceptible to seasonal, environmental and site-specific variations. It is possible that some antibiofouling potential coatings may perform exceptionally well against representative fouling species, while completely losing their AF efficacy in the field immersion assays. Therefore, this study highlights the significance of field assays for the assessment of antibiofouling properties of PCs. Furthermore, we have managed to identify the two PCs (M1 and C3) with excellent anti-biofouling properties from a wide arrays of PCs through field assays. Further studies to revalidate their long-term AF efficacy are required. 3.2. Clustering analysis based on MDS method
3. Results and discussion 3.1. Marine field assays In the present study, the anti-biofouling properties of PCs, including MPs (M1eM6), HPs (H1eH6) and CPs (C1eC6), were examined through field assays conducted in the marine environment under static conditions at different depths. A range of effects were observed and recorded as presented in Fig. 1. Fig. 1 showed that after four weeks of exposure in the sea, the PDMS surface was largely covered with some inorganics (mainly sea slime), algae sporelings and a few juvenile invertebrates. While at the end of the immersion, the PDMS surface was heavily fouled and showed extensive coverage of sea slime, algae spores, as well as some early colonizers like juvenile barnacles and ascidian, indicating that the PDMS surface does not exhibit excellent anti-biofouling properties when exposed to the marine environment, which might be susceptible to pioneer microbial colonization and deterioration. This result agrees well with the earlier findings by Sankar et al. (Sankar et al., 2015), which showed the pure PDMS surface was susceptible to microbial fouling triggered by microorganisms like bacteria and diatoms. The treated panels coated with different PCs exhibited better anti-biofouling properties in the field, as compared to PDMS control (Fig. 1), indicating that the anti-biofouling properties of PDMS can be fundamentally enhanced with the introduction of CNT fillers. Significant differences can be observed between treated
ITS-2 gene fragments were successfully amplified as expected, and separated using the SSCP technique. Fig. 2 shows SSCP fingerprints of pioneer eukaryotes adhering to different silicone-based material surfaces. Almost all silicone-based material surfaces were found to be readily colonized by a mixed culture of pioneer eukaryotes at five different points in time within the two-week in situ experiment, as observed from SSCP patterns. The results suggest that all silicone-based composite materials cannot fully prevent the colonization of pioneer eukaryotic microbes. Each siliconebased composite surface has presented specific eukaryotic SSCP patterns at different points in time. Based on the SSCP profiles, the four diversity indices were calculated using Quantity one 4.6.2 and BioDap software (Data not shown). Based on the four diversity indices, the MDS analysis was undertaken, as is shown in Fig. 3. In Fig. 3, pioneer eukaryotic microbial communities adhering to different PCs surfaces were clearly different from that of the PDMS surface, suggesting that different PCs surfaces may have differentially modulating effects on pioneer eukaryotic colonization. Pioneer eukaryotes attached to the surfaces of PCs belonging to the same set (M set, H set or P set) were prone to be closely grouped or distributed. In contrast, clear differences can be found among early eukaryotes adhering to the surfaces of PCs belonging to different sets. For example, pioneer eukaryotes attached to the surfaces of the M set (EM1eEM6), along with the C4 surface (EC4), were prone to cluster into one group; while most pioneer eukaryotes adhering to the surfaces of the H set
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Fig. 1. Images of the treated panels coated with (a) MPs(M1eM6), (b) HPs(H1eH6) and (c) CPs (C1eC6) after the immersion in seawater (OctobereDecember 2013, Weihai, China) as a function of time.
Fig. 2. The SSCP fingerprints of pioneer eukaryotic communities adhering to the surfaces of different silicone-based materials at different points in time.
(EH1eEH6) or the C set (except EC4) tended to cluster into another two groups. These combined results suggested that the types of CNT fillers may play a key role in deciding the clustering features of pioneer eukaryotic communities, which may contribute to accounting for the differentially modulating effects exerted by different PCs surfaces on the colonization of the pioneer eukaryotic microbes. 3.3. Effect of PCs surfaces on pioneer eukaryotic communities The analysis of pioneer eukaryotic-community diversity and richness was conducted based on the SSCP fingerprints. The comparison of the four diversity indices, namely the Shannon diversity index (H), species richness(S), total abundance and the Simpson index (l), is presented in Fig. 4. The Shannon diversity index (H), which takes account of both the number of species and the evenness of the species, was used as a measurement for the estimation of general biodiversity in habitant microbial communities. The Shannon diversity index provides a holistic view of a particular ecological niche, with H values usually ranging from 1.5 (low
species richness and evenness) to 3.5 (high species richness and evenness) (Bharathkumar et al., 2008). Fig. 4aec shows that the H value of the eukaryotic microbial community ranged between 1.49 ± 0.31 and 2.39 ± 0.17 for all the PCs surfaces, which was generally lower than that of the PDMS control (2.53 ± 0.23). Pioneer eukaryotes attached to CPs surfaces (C1eC6) showed the lowest level of diversity, with the H values ranging from 1.49 ± 0.31 to 1.97 ± 0.37, as compared to those of MPs surfaces (2.10 ± 0.27 to 2.26 ± 0.29) and HPs surfaces (1.98 ± 0.49 to 2.39 ± 0.17). This result indicates that CPs surfaces may have stronger modulating effect on pioneer eukaryotic colonization than that of the MPs and HPs surfaces. A significant decrease in pioneer eukaryotic-community diversity can be observed on the surfaces of MPs (except M2), H1, H4, and CPs (C1eC6) (P < 0.05), and particularly on the surfaces of H1, H4 and CPs (C1eC6) (P < 0.01). Species richness (S) simply describes the number of different species in a microbial community. Fig. 4d, e and f reveal that the S value of pioneer eukaryotic community (ranging from 6 ± 2 to 15 ± 3) was greatly down-regulated by most PCs surfaces, compared with that of the PDMS surface (19 ± 2), indicating a
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diversity and richness amongst different silicone-based material surfaces. Our study also confirmed that pioneer eukaryotic SSCP fingerprints can be highly helpful for the monitoring of the dynamics of pioneer eukaryotic community diversity and richness in a biologically relevant process. 3.4. Possible mechanisms for PCs against pioneer eukaryotic colonization
Fig. 3. Clustering analysis of pioneer eukaryotic communities adhering to the surfaces of different silicone-based materials using MDS method. EP0, EM, EH and EC represent pioneer eukaryotic microbial community attached to the surfaces of P0, MPs, HPs and CPs, respectively.
significant decrease in richness. In addition, total abundance is measured primarily by the total intensity of bands present in the SSCP profiles. Fig. 4gei show that the value of total abundance for pioneer eukaryotic communities (ranging from 49 ± 16 to 140 ± 68) was also greatly down-regulated by most PCs surfaces in contrast to PDMS control (153 ± 54). A significant decrease in total abundance can be found for pioneer eukaryotes adhering to the surfaces of M1, M2 and CPs (except C6) (P < 0.05), and particularly on the surfaces of C2 and C5 (P < 0.01). The significantly reduced eukaryoticcommunity diversity and richness suggest that most PCs surfaces may have strong modulating effects on pioneer eukaryotic communities, thereby dramatically reducing the colonization of pioneer eukaryotic microbes. Similarly, Camps et al. have reported that AF coatings could influence the abundance and community structure of colonizing biofilm communities in a recent study (Camps et al., 2014). In another study, Ling et al. have also found that microfabricated PDMS surfaces could regulate the development of microbial biofilm communities (Ling et al., 2014). The result obtained in this study agrees well with the findings reported by Camps et al. and Ling et al. Most PCs surfaces can effectively reduce pioneer eukaryotic-community diversity and richness by progressively weakening the invasion and colonization of pioneer eukaryotic microbes, thereby being less susceptible to pioneer eukaryotic microbial colonization and deterioration. The Simpson index (l) describes the number of dominant species in a particular microbial community. Fig. 4j, k and l reveal that the l value of pioneer eukaryotic communities (ranging from 0.117 ± 0.033 to 0.249 ± 0.082) was elevated on most PCs surfaces (except H4), significantly elevated on the surfaces of H4, C2, C3 and C5 (P < 0.05), and particularly on the surfaces of C2 and C5 (P < 0.01), when compared to that of the PDMS control (0.116 ± 0.045). This result indicates that the dominant eukaryotes in the biofilm communities on these surfaces may vary greatly in contrast to the PDMS control. The increase in the number of the dominant eukaryotes suggests that most PCs surfaces may have the potential to accelerate the succession progress of pioneer eukaryotic communities, and in turn change the succession patterns of pioneer eukaryotic communities. In addition, the four well-known diversity indices are found to be sensitive and complementary indicators to capture the variations of pioneer eukaryotic-community
In the current study, all the PCs surfaces exhibited better antibiofouling properties even under static conditions, when compared to the PDMS control, indicating that AF efficacy of pure PDMS can be further reinforced by the incorporation of 0.1% (w/w) CNT nanoparticles. Previously, Beigbeder et al. reported that the bulk mechanical properties of pure PDMS remained unchanged after the introduction of low quantities of CNT filler, indicating that the CNT filler incorporation into the PDMS matrix does not significantly change the surface properties of the PDMS matrix (Beigbeder et al., 2008a). Therefore, the general consistency of the enhanced anti-biofouling capacity may be primarily attributed to the CNTs themselves. Interestingly, differential anti-biofouling effects of PCs can also be observed from Fig. 1, implying that the CNT fillers may play key roles in determining anti-biofouling effects of PDMS, which agrees well with the findings reported by Kang et al. in an earlier study (Kang et al., 2008). In addition, most PCs performed well in marine exposure studies, although CNT fillers were not well dispersed in the PDMS matrix. The partial CNTs aggregation may also contribute to the reinforcement of composite' antibiofouling properties. Similarly, Kang et al. found SWNTs to be directly responsible for cell membrane damage because of SWNTs aggregation (Kang et al., 2007). It seems that the state of CNT filler dispersion in the PDMS matrix is not directly related to the composite' anti-biofouling properties, although Beigbeder et al. have previously emphasized that the significance of the dispersion quality of the CNT filler in the polymeric matrix (Beigbeder et al., 2010). Besides, Beigbeder et al. have also found that the reinforced AF efficacy to be mainly ascribed to the extremely favorable CH-pi electronic interactions (Beigbeder et al., 2008b). Yet we cannot exclude the possibility that the changes in surface micro/nanostructures of the PDMS may contribute to the improved anti-biofouling properties of PCs, although the hypothesis needs support from delicate AFM/SEM analysis in the future. It is probable that the incorporation of CNT fillers in PDMS matrix can produce novel PCs with different micro/nanostructures, which may be contributable to the weakening of the invasion and colonization of pioneer eukaryotic microbes. Several recent publications have also confirmed surface micro/nanostructures may have significant impacts on surface colonization (Hoipkemeier-Wilson et al., 2004; Cheng et al., 2006; Schumacher et al., 2007). Further research focusing exclusively on characterization of the surface micro/ nanostructures of different PCs using AFM/SEM is currently underway, in order to uncover the unrecognized anti-biofouling mechanisms of PCs surfaces. Combined with results obtained from MDS and SSCP analysis, it can be seen that pioneer eukaryotes adhering to the surfaces of the same PCs sets (M set, H set or C set) were closely distributed or grouped, suggesting that PDMS incorporated similar types of CNTs tending to have a similar modulating effect on the colonization of pioneer eukaryotic microbes. The differences among pioneer eukaryotes adhering to the surfaces of PCs belonging to different sets seemed larger than those attached to the surfaces of PCs belonging to the same set, indicating that the types of CNTs may be closely related to the composite' AF efficacy (Fig. 3). In addition, it seems that the differential anti-biofouling capacity of the PCs surfaces may not be directly associated with the growth process and
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Fig. 4. The comparison of the four diversity indices, (aec) Shannon diversity index, (def) Species richness, (gei) Total abundance, (jel) Simpson index of pioneer eukaryotic communities adhering to the surfaces of different silicone-based materials. Error bars represent the SD of the mean. One asterisk (*) represents significant difference (P < 0.05), whereas two asterisks (**) represent extremely significant difference (P < 0.01).
dynamics of pioneer eukaryotes in the biofilm. For example, CPs (C1eC6) in this study demonstrated stronger modulating effects on pioneer eukaryotic microbial communities than those of the other two PCs sets. Particularly, C2 and C5 surfaces can dramatically reduce pioneer eukaryotic-community diversity and richness (P < 0.01), thereby exerting the strongest perturbation effect on pioneer eukaryotic colonization. However, their anti-biofouling capacity was only intermediate or weak amongst the others (Fig. 1c). In contrast, M2 and C3 surfaces performed exceptionally well in the field, although the two surfaces failed to exert the greatest perturbation effect on the colonization of pioneer eukaryotic microbes.
biofouling applications. Interestingly, we found that most PCs surfaces were able to significantly reduce the diversity and richness, thereby being less susceptible to pioneer eukaryotic microbial colonization and deterioration. Our findings may facilitate the understanding of the dynamics of pioneer eukaryotic-community diversity and richness variations on different silicone-based material surfaces in the marine environment and also cast new light on the development of eco-friendly AF coatings. Further studies are in progress to identify more reinforcing CNT fillers and uncover the unrecognized anti-biofouling and degradation mechanisms of PCs.
Acknowledgments 4. Conclusion In the present study, we identified that the PDMS matrix incorporated with different surface-modified CNTs demonstrated excellent anti-biofouling properties as compared to the PDMS control. This simple and efficient method to produce functionalized PCs with improved AF efficacy gives potential for future anti-
The authors express their sincere gratitude and thanks to Tao Wang, Yunyang Cheng and Zihua Yang of Harbin Institute of Technology, school of Marine science and technology, for their constant assistance with marine field assays and sampling throughout the course of this investigation. This work was funded by National Natural Science Foundation of China (No. 31071170).
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