ReseaRch PaPeR Journal of Agricultural and Marine Sciences Vol. 23 : 92– 98 DOI: 10.24200/jams.vol23iss1pp92-98 Reveived 01 Mar 2018 Accepted 27 Sep 2018 Antifouling properties of chitosan coatings on plastic substrates 1,2Laila Al-Naamani, 3Thirumahal Muthukrishnan, 4Joydeep Dutta, 5Sergey Dobretsov 1,2Laila Al-Naamani ( ) lnaamani@hotmail.com 1Department of Marine Science and Fisheries’ Sultan Qaboos University, 123 Al-Khoud, Oman.2Ministry of Municipalities and Water Resources, Muscat, Sul- tanate of Oman.3Department of Biology, Sultan Qaboos University, 123 Al-Khoud, Oman.4Functional Materials Division, Materials and Nano Physics Department, ICT School, KTH Royal Institute of Technology, SE-164 40, Kista, Stockholm, Sweden. 5Center of Excellence in Marine Biotechnology, Sultan Qaboos University, 123 Al-Khoud, Oman. Introduction Natural and biodegradable biopolymers are cur-rently receiving great interest to be used as al-ternative to petroleum polymers in terms of raw material supply and waste product reduction (Leceta et al. 2013). Chitosan is a biocompatible, biodegradable and bioactive biopolymer, which can be used in diverse industrial applications (Chatelet et al. 2001). It exhibits numerous interesting physicochemical and biological properties with various applications in water treatment, agriculture, biomedicine, food industry, and marine an- tifouling management (Cestari et al 2007; El-Sawy et al. 2010; Ramya et al. 2012; Luo & Wang 2013; Cai et al. 2016; Al-Naamani et al. 2017). Chitosan possesses antimicrobial properties against a number of bacteria, fungi and algae (Benhabiles et al. 2012; Lam & Diep 2015; Park et al. 2016). Antimicrobial properties of chitosan are due to the positively charged NH2+ ions which interact with the negatively charged molecules in the microbial cell membranes leading to disruption of the cells (Coma et al. 2003; Alisashi & Aïder 2012). The efficiency of the bactericidal effect of chitosan is dependent on various factors, such as micro- organism strain or species, environmental conditions (pH, temperature), molecular weight and concentration of chitosan as well as its physical state (solution or film) (Kong et al. 2010; Leceta et al. 2013). According to the field of application, chitosan can be modified to form powder, flakes, gel beads, fibres or membranes and several other forms (Wang & Shen 2000; Pitakpoolsil & Hunsom 2014; Zhang et al. 2015). Chitosan films or membranes have found their ways in various industrial applications such as water desalina- tion, as coating for food applications, wound dressing, and tissue engineering (Goy 2009; Sudha et al. 2015). Films can be easily prepared by dissolving chitosan pow- der or flakes in diluted acid solutions like acetic acid, and then casting the resulting solution on a flat surface or خصائص طالء الشيتوزان املضاد للحشف على ألواح البالستيك ليلى النعماين1,2 وثريومهال موثوكريشنان3 وجويديب دوتّا4 وسرجي دوبريتسوف5 Abstract. In the current study, chitosan coatings were fabricated on plastic substrata and investigated for their anti- fouling activities. Scanning Electron Microscopy (SEM) and water contact angle measurement (WCA) of the fabricated chitosan films showed smooth and hydrophilic surface with WCA below 60°C. In the first experiment, chitosan coating on plastic substrate showed 88% reduction in settlement of bryozoan Bugula neritina larvae compared to the control after 3 hours incubation at dark conditions with no larval mortality. In the second experiment, the antimicrobial activ- ity of chitosan was evaluated by coating plastic panels with the prepared chitosan solution and immersing the coated samples in seawater at controlled environmental conditions for two weeks. Biofilms scraped from immersed chitosan coated panels showed no bacteria after 1 week of immersion. After the second week of immersion, less than 1500 bac- teria/mm2 were observed on the chitosan-coated panels compared to more than 105 bacteria/mm2 on uncoated ones. Thus, this study proved the efficiency of chitosan coatings against micro- and macro-fouling. Keywords: Chitosan; anti-larval activity; antifouling activity; bryozoan; biofilms. املســتخلص:يف الدراســة احلاليــة، مت طــالء مــادة الكيتــوزان علــى ألــواح بالســتيكية وحبــث إمكانيــة اســتخدامها كمــادة مضــادة للرتســبات احليويــة البحريــة. أظهــرت حتاليــل املســح باجملهــر اإللكــرتوين وقيــاس زاويــة االتصــال بامليــاه علــى أن أغشــية الكيتــوزان تتكــون مــن ســطح أملــس وحمــب للمــاء بزاويــة اتصــال مــع املــاء بدرجــة اأقــل مــن ٦٠. طــالء الكيتــوزان علــى األســطح البالســتيكية أظهــر معــدل اخنفــاض يف جتمــع يرقــات Bugula neritina بنســبة ٨٨٪ مقارنــًة باألســطح غــري املطليــة بعــد ٣ ســاعات مــن احلضانــة يف ظــروف مظلمــة. كمــا مل يتــم مالحظــة أي وفيــات يف الريقــات خــالل التجربــة. مت تقييــم النشــاط املضــاد للميكروبــات ملــادة الكيتــوزان عــن طريــق غمــر األلــواح البالســتيكية املطليــة هبــذه املــادة يف كميــة مــن ميــاه البحــر مــع وجــود ظــروف بيئيــة متحكــم هبــا. حتليــل األغشــية احليويــة املرتســبة يف األلــواح املطليــة بالكيتــوزان واملغمــورة يف املــاء أظهــر عــدم وجــود أي بكرتيــا بعــد أســبوع واحــد مــن التجربــة. بعــد األســبوع الثــاين ، تالحــظ وجــود أقــل مــن ١٥٠٠ بكرتيا/ملــم علــى األلــواح املطليــة بالكيتــوزان مقارنــة بأكثــر مــن ١٠٥ بكرتيا/ملــم علــى األلــواح الغــري مطليــة. بالتــايل، فــإن هــذه الدراســة تثبــت كفــاءة طــالء الكيتــوزان ضــد الرتســبات احليويــة البحريــة. الكلمات املفتاحية: الرتاكم احليوي على األسطح املغمورة، 93Research Article Naamani dipping or coating of any substrate, and finally allowing them to dry (Krajewska 2005; Goy 2009). Biofouling is the undesirable attachment and growth of micro- (bacteria and diatoms) and macro-fouling (bryozoans, barnacles, mussels, etc.) organisms on man- made installations (Wahl 1989). Maritime industries spent billions of US dollars to prevent and control bio- fouling. Current ways of controlling biofouling include the use of toxic antifouling coatings that kill organisms and pollute the environment (Hellio & Yebra 2009). Thus, non-toxic antifouling solutions are urgently needed. Due to chitosan biodegradability, low toxicity to eukary- otes and environmental safety, chitosan films have been proposed as a green approach to prevent biofouling and as an alternative to toxic biocides (Pelletier et al. 2009). Chitosan was proven as a successful antifouling coating for membranes (Zhao et al. 2003; Zhou et al. 2010). A study by Yang et al. (2011) found that stainless steel func- tionalised with chitosan and hydroxyethylmethacrylate (HEMA) polymer reduced protein adsorption, bacterial adhesion, and exhibited antibacterial activity against E. coli. Chitosan films in laboratory experiments inhibited growth of fouling microorganisms, such as Pseudomo- nas and Bacillus (Machul et al. 2015; Zhou et al. 2013). A two months field study in northern Canadian waters was conducted with chitosan-based coatings (Pelletier et al. 2009). While the results of the study demonstrat- ed promising antibacterial activity, the coating did not have any activity against algae. In our previous study, chitosan-zinc oxide nanocomposite coatings prevented growth of fouling diatoms and marine bacteria in lab- oratory and mesocosm experiments (Al Naamani et al. 2017). At the same time, the antifouling effect of chi- tosan films on larval settlement of major fouling species, like the bryozoan Bugula neritina (Dahms et al. 2004), has not been investigated. The aims of this study were to: 1) fabricate chitosan coatings and characterise their physical and chemical properties, and 2) determine antifouling activity of chi- tosan coatings against micro- and macro-fouling organ- isms in laboratory experiments. Materials and Methods Preparation of chitosan solution Two and a half grams of commercial chitosan powder of medium molecular weight with 110 cps viscosity and 95.6% deacetylation (Tru-Nutra Nutraceuticals LLC, India) were mixed with a volume of 100mL of 2% ace- tic acid (Sigma Aldrich, USA) to prepare 2.5% chitosan solution. The solution was kept under constant stirring for 24h at 25°C. The viscous solution was coated on plastic substrates and allowed to dry for 24h at 26°C. The resulting coatings were characterized using FTIR spectrophotometery, scanning electron microscopy (SEM) and water contact angle. The chitosan coatings were analysed directly us- ing FTIR spectroscopic equipment (PerkinElmer, USA, FrontierTM), in a spectral range from 4000 to 400 cm-1 at a resolution of 4 cm-1. Surface morphologies of dry coatings were characterized using JEOL JSM-7200 (Ja- pan) field emission scanning electron microscope (FE- SEM) working at 20 kV. The water contact angle (WCA) of coated substrates was measured using a Theta Lite Attension tensiometer (Biolin Scientific, Sweden) using a sessile drop technique to determine the films hydro- phobicity (Al-Fori et al. 2014). A drop of 5µL water was placed in five different positions on each coating’s sur- face. The right and left contact angles of each drop were measured and a mean water contact angle (WCA) was calculated from the resulting two values. Activity assessments of chitosan coatings on surfaces Anti-larval activity assessment To determine the anti-larval activity of chitosan, the prepared chitosan solution in acetic acid was coated on plastic panels (low density polyethylene, size 1cm2) and allowed to dry overnight. Three replicates of chitosan coated plastic panels were placed into a 24-well plate (Corning Costar, USA). Uncoated clean plastic pan- els were used as a control. Each well was filled with 1 mL seawater containing the bryozoan Bugula neritina larvae. Adult broodstocks of B. neritina were collected from pilings and floating rafts at Marina Bandar Row- dah. Larvae were obtained according to the method de- scribed by Bryan et al. (1997) and only newly (i.e., within 10min) released larvae were included in the bioassays. Experiments were conducted under dark conditions by covering the 24-well plate with aluminium foil. In each well, the number of dead larvae, attached larvae and the total number of larvae were counted under a dissecting microscope (Zeiss, Germany, magnification 10X) after 1h, 2h and 3h. The percentages of larval mor- tality and larval settlement were calculated as follows (equations 1 and 2). Larval mortality (%)=(NDL)/(TL)×100 Eq.(1) Larval settlement (%)=(NSL)/(TL)×100 Eq.(2) Where NDL is the number of dead larvae, TL is the to- tal number of larvae and NSL is the number of settled larvae. Anti-microfouling activity To determine the antifouling activity of the chitosan in a small scale microcosm experiment, three different coat- ings were prepared: (a) 2.5% chitosan solution, (b) com- mercial two component non-toxic paint (Hempadur 45182, Hempel, Denmark) mixed with 2.5% chitosan solution at a ratio of 1:1 (v:v), (c) commercial two com- ponent non-toxic paint (Hempadur 45182, Hempel, Denmark). Plastic panels (Acrylic, 7.5 cm × 2.5 cm) were cleaned and both sides of the panels then painted with 94 SQU Journal of Agricultural and Marine Sciences, 2018, Volume 23, Issue 1 Antifouling properties of chitosan coatings on plastic substrates a brush with each of the coatings. The coatings were allowed to dry for 24 hours at room temperature. Un- coated clean plastic panels were used as a control. Each panel was immersed vertically in a separate beaker con- taining seawater. Beakers were incubated at 26°C for 2 weeks. Each treatment and the control were replicated three times. After 1 week and 2 weeks, panels were re- moved. Biofilms from the whole area of the panel were scraped from the surface into Eppendorf tubes using a sterile scalpel. The remaining traces of the biofilm were rinsed with distilled water into the tube. Ten µL of the resulting biofilm suspension from each tube were add- ed on a microscope glass slide and mixed with 10µL of SYBR Green 1 stain (SIGMA, Aldrich, USA) and incu- bated for 10 min. Finally, slides were analysed by an epi- fluorescence microscope. The number of bacteria in 20 randomly selected fields of view (Muthukrishnan et al. 2017) was counted and the total abundance of bacteria within 1 mm2 was calculated. Results and Discussion Chitosan film characterization The FTIR spectra of chitosan coatings (Figure 1a) showed a characteristic peak at 3362 cm-1 for N–H and O–H stretching. Peaks corresponding to amide I and amide II were observed at 1662 cm-1 and 1598 cm-1, respec- tively. The characteristic peak at 1046 is attributed to C-O stretching. The main absorption peaks of chitosan films have been reported to be at 1650 cm−1, attribut- ed to C=O stretching (amide I), 1558 cm-1 attributed to N-H bending (amide II) and 1382 cm-1 attributed to C-N stretching (amide III). The broad bands above 3000 cm-1 assigned to O-H and N-H bonds while absorption peaks at 1050 cm-1 were attributed to C-O stretching (Fernan- dez-Saiz et al. 2007; Leceta et al. 2013). The SEM images and WCA results showed a smooth and hydrophilic sur- face of chitosan coatings as shown in Figures 1b and 1c. Anti-larval activity of chitosan coating Bugula neritina is a common fouling marine bryozoan with a short pelagic larval stage which can be found in warm temperate and subtropical waters worldwide (Ry- land et al. 2011; Linneman et al. 2014). Bugula larvae have barrel-shaped bodies with their surfaces mostly covered with cilia that are referred to as a ciliated co- rona. Those cilia assist the larvae in swimming (Price et al. 2017). The effect of chitosan coatings on the mortality and settlement of the bryozoan Bugula neritina larvae after 3 hours incubation in dark conditions is shown in Table Table 1. Effect of chitosan coating on Bugula neritina larval mortality and settlement after 1, 2, and 3 hours of incubation at dark conditions. Control is seawater. % Larval mortality % Larval settlement 1h 2h 3h 1h 2h 3h Control 0 0 0 100±0 100±0 100±0 Chitosan 0 0 0 12±6.3 12±6.3 12±6.3 Figure 1. (A) FTIR spectra of chitosan coating on plastic panels using 2.5% chitosan dissolved in 2% acetic acid, (B) SEM image of the chitosan coating, (C) Water control angle of the chitosan coating. 95Research Article Naamani 1. Results showed no larval mortality during the whole experiment. A 10-fold significant decrease in settlement of the larvae on chitosan films in comparison with the control was observed (Table 1). All larvae settled in the control and no changes in the settlement rate were ob- served during the incubation period. Overall, there was no difference in the larval settlement between 1h, 2h and 3h on chitosan film and the control. This can be ex- plained by the fact that B. neritina has fast settling larvae that attach to the substratum within one hour (Bryan et al. 1997). To our knowledge, this is the first study stating the effect of chitosan on the mortality and settlement of B. neritina larvae. Previously, Rasmussen et al. (2002) in- vestigated the settlement of cyprids of Balanus amphi- trite on chitosan gel crosslinked with glutaraldehyde. The authors observed a reduction in larval settlement to 35% when chitosan concentration was 2%. The set- tlement of B. neritina larvae on the surface of 8×10 cm plastic panels coated with low density polyethylene (LDPE), polypropylene (PP), polyvinyl chloride (PVC) and high density polyethylene (HDPE) was studied by Li et al. (2016). The authors reported higher larval settle- ment on the surface of panels coated with LDPE, PP, and PVC, compared to HDPE and glass panels after immer- sion in water for 4 days. The influences of organic films, such as chitosan, PP, PVC, etc., on larval settlement is not yet clear. It was suggested that physical, chemical and biological factors of the substratum, such as rough- ness, chemical properties of the substratum, presence of biofilms and other species, could affect larval settlement in the field (Clare et al. 1992; Faimali et al. 2004; Do- bretsov et al. 2006; Qian et al. 2007; Li et al. 2016). Antimicrobial experiment The experiment showed that the lowest bacterial abun- dances were found on chitosan coatings; no bacteria af- ter 1 week and <1500 bacteria/mm2 after 2 weeks of im- mersion were observed (Fig. 2). The highest densities of bacteria were found on the control substrata after 1 and 2 weeks of the experiment. The non-toxic antifouling paint was more effective than the mixture of non-toxic paint and chitosan (Fig. 2). Only a few algal cells were observed in the control samples. Comparatively, higher numbers of algal cells were observed on samples coat- ed with non-toxic paint. However, no algal cells were observed on both the panels coated with a mixture of non-toxic paint and chitosan solution and that of only chitosan solution. In the marine environment, the antifouling activi- ty of chitosan was directly evaluated by Pelletier et al. (2009). They demonstrated antibacterial activity of 20% chitosan coating after 14 days of immersion in the sea, while 5% chitosan coating did not have any antifouling activity. Antibacterial activity of chitosan with concen- tration less that 2% chitosan was observed against Ba- 0 5 10 Week 1 Week 2 B ac te ria l c ou nt (1 04 m m −2 ) Coating Control Non−toxic paint Chitosan + Non−toxic paint Chitosan Figure 2. Bacterial abundances on plastic panels coated with commercial non-toxic paint, commercial non-toxic paint mixed with 2.5% chitosan coating in ratio (1:1) and 2.5% chitosan coating. The control represents an uncoated panel. Figure 3. Bacterial cells as observed by epifluorescence microscopy at 1000X magnification in (a) biofilms scraped from the uncoated control plastic panels, and (b) biofilm scraped from panels coated with a mixture of chitosan and non-toxic paint. Cells were stained with CYBR Green I dye. 96 SQU Journal of Agricultural and Marine Sciences, 2018, Volume 23, Issue 1 Antifouling properties of chitosan coatings on plastic substrates cillus sp., Vibrio and Pseudomonas sp., which are known to be involved in the biofouling process (Sekiguchi et al. 1994; Jumaa et al. 2002; No et al. 2002; Rasmussen & Østgaard 2003). It is well known that the cationic amine group in the chitosan molecule has a major role in its antimicrobial activity, as it forms electrostatic interac- tions with anionic group on the cell membrane of bac- terial cells, which eventually lead to cell death (Rabea et al. 2003; Alisashi & Aïder 2012). However, in our exper- iment chitosan charge has minor effect because of high pH of seawater (6.9-7.2) which neutralize most of the positive charges in chitosan’s amino groups. Rasmussen & Østgaard (2003) suggested that surface energy was the crucial factor to prevent bacterial adhesion to the hy- drophilic surface provided by chitosan at conditions of high pH. The anti-algal effect of chitosan was previously reported by Ravi Kumar (2000). However, no anti-algal activity of chitosan was observed by other researchers (Pelletier et al. 2009). Conclusion In this experiment, chitosan solution was used to fab- ricate coatings characterised by FTIR, SEM and WCA. This chitosan solution was applied as a coating on acryl- ic plastic and antifouling effect against macro- and mi- cro-fouling organisms was studied. The results of this study proved the effectiveness of chitosan coatings on the settlement inhibition of Bugula neritina compared to the control. 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