Nova Biotechnol Chim (2018) 17(2): 115-124 DOI: 10.2478/nbec-2018-0012  Corresponding author: vijuuu333@gmail.com Nova Biotechnologica et Chimica Antifouling activities of extracellular polymeric substances produced by marine bacteria associated with the gastropod (Babylonia sp.) Nadarajan Viju1,, Nagarajan Ezhilraj1, Chellamnadar Vaikundavasagom Sunjaiy Shankar1, Stanislaus Mary Josephine Punitha1 and Sathianeson Satheesh2 1Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari, Tamilnadu, India 2Department of Marine Biology, Faculty of Marine Sciences, King Abdulaziz University, Jeddah, Saudi Arabia Article info Article history: Received: 3rd April 2018 Accepted: 2nd November 2018 Keywords: Antifouling coating Biofilm Biofouling Extracellular polymeric substances Surface-associated bacteria Abstract Bacteria associated with surfaces have been frequently cited as a potential source for the isolation of bioactive metabolites. In this study, bacteria associated with marine gastropod, Babylonia sp. were isolated and screened for antibacterial activity against biofilm-forming bacteria. The antibiofilm and antifouling effect of the selected surface- associated bacterial strains were examined under in vitro and in vivo conditions. Results showed that the extracellular polymeric substances (EPS) of the bacterial strain CML associated with gastropod species considerably reduced the adhesion of biofilm-forming bacteria on glass coupons. Besides, the antifouling coat prepared by incorporating of this EPS into polyurethane varnish prevented the settlement of biofoulers on test substratum submerged in marine waters. The functional groups present in the EPS were analyzed using FT-IR. The bacterium responsible for the production of the bioactive EPS was identified as Bacillus subtilis subsp. by 16S rRNA gene sequencing. More detailed characterization of the identified bioactive EPS could lead to the isolation of a novel natural antifouling product.  University of SS. Cyril and Methodius in Trnava Introduction Biofouling continues to be one of the important problems in the maritime sector, which causes severe problems on the surfaces of the marine living organisms and submerged structures (Almedia et al. 2017). Although, highly effective biocides are used as coatings for avoiding the settlement of organisms, TBT (Tributyltin) like biocides has created environmental problems to non-target organisms (Thomas and Brooks 2010). There is a growing need for the effective and eco-friendly antifoulants for marine applications particularly after the ban of TBT based paints; therefore research interests on natural antifoulants has been increased (Satheesh et al. 2016). The marine environment is a rich source of biological and chemical diversity, and also an exceptional reservoir for unique bioactive compounds (Fenical 1993). Therefore, the marine organisms have been placed into focus discovering novel bioactive compounds (Clare 1996). A number of bioactive metabolites have been isolated from marine sources (Kijjoa and Sawangwong 2004; Xu et al. 2010). Notably, marine bacteria have been the source for the isolation of many such compounds in medicine, Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 11:16 UTC Nova Biotechnol Chim (2018) 17(2): 115-124 116 industry and agriculture (Bowman 2007). The bacteria associated with marine algae and other organisms are reported to produce a higher percentage of bioactive compounds comparing to free floating bacteria (Zheng et al. 2005). Hence, a consequent attention has been paid on marine bacteria associated with the surface of invertebrates for the isolation of novel antifouling compounds (Satheesh et al. 2012; Viju et al. 2017). The production of compounds such as Giffinisterone B, Lobocompacto and Dicitrinin A etc. by the symbiotic bacteria has been reported (Cho 2012; Cho and Kim 2012). Therefore, the aim of the present study was to identify novel antifouling compound from the bacteria living at the surface of marine molluscs. For the screening, fouling bacteria isolated from the acrylic panel submerged in the Kudankulam coastal water (Satheesh et al. 2012) were used as the target. It has been reported that the biofilm formed by such bacteria could act as a cue for the settlement of following macrofouling organisms (Lau et al. 2003). Therefore preventing formation of this initial biofilm (microfouling) would also prevent the subsequent macrofouling (Armstrong et al. 2000). With this reasoning the selected fouling bacteria have been used for the preliminary screening of extracellular polymeric substances (EPS) for antifouling property. Such study might contribute to knowledge about biofouling and antifouling activities expressed by surface associated bacteria. Experimental Collection and isolation of surface associated bacteria The marine gastropod Babylonia sp. collected from Colachel coast (West coast of India) were brought to the laboratory in a sterile plastic container with seawater. The bacteria associated with the gastropod were isolated according to the method described by Viju et al. (2016). In brief, the loosely attached organisms on the surface of gastropod were removed by washing with sterile seawater. Following this, the bacteria associated with the surface of gastropod were scraped out using a nylon brush and suspended in 1 mL of filter- sterilized seawater. This suspension was serially diluted and spread on agar plate prepared with Zobell marine agar (ZMA). These plates were then incubated for 24 h at 37 °C for the growth of bacterial colonies. The colonies grown were isolated based on the morphology and maintained on slants at 4 oC for further studies. Isolation of extracellular polymeric substance The bacterial colonies were inoculated into a beaker containing a 500 mL of Zobell marine broth (ZMB) and placed in a shaker at 37 °C for 72 h of incubation. After that, the culture broth was centrifuged (at 5,000 rpm for 15 min 4 oC) and the supernatant was collected. The collected supernatant was added with adequate amount of ethanol (1 : 1 ratio) and kept in a safe place (overnight) for the precipitation of EPS. The collected precipitate (EPS) was filtered using a membrane filter and diluted in double distilled water. This suspension was then dialyzed using a tubular dialysis membrane (8 kDa) and the obtained fraction was used for further studies (Rajasree et al. 2012). Antimicrobial Assay Agar disc diffusion method described by Bauer et al. (1966) was used to test the antimicrobial activity of the EPS. For this, the sterile disc (6 mm, Himedia, India) was loaded with 50 µL of bacterial EPS and placed on Mueller-Hinton agar plates swabbed with target (biofilm-forming) bacteria such as Gallionella sp., Alteromonas sp. and Pseudomonas sp. These bacteria were isolated from the artificial surfaces immersed in the seawater (Satheesh et al. 2012). Control discs loaded with distilled water were also maintained. All the plates were incubated at 37 °C for 48 h. Following incubation, the inhibition zone appeared around the discs were measured. The presented data represent the average of two measured values. Preparation of bacterial cell suspension for laboratory assays The biofilm-forming bacteria were grown at 37 °C for overnight in Zobell marine broth. This broth Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 11:16 UTC Nova Biotechnol Chim (2018) 17(2): 115-124 117 was then centrifuged at 5,000 rpm for 15 min and the cell pellets obtained were washed with phosphate-buffered saline (10 mL PBS). Following this, the washed pellet was re-suspended in the same buffer in order to obtain OD540 = 0.2 and used for the further assay (Jain et al. 2007). Adhesion assay The bacterial adhesion assay was performed according to the procedure outlined by Rajasree et al. (2014). Briefly, the microscopic slides (7.5×2.5 cm) were treated with EPS by placing them in beaker (500 mL) containing seawater (300 mL) and 0.5 mL of EPS. After 24 hours, the slides treated with EPS were taken out and placed into another beaker which contained 300 mL of sterile seawater, 3 mL of ZMB and 3 mL of biofilm-forming bacterial suspension. Following 5 h of incubation, the microscopic slides were retrieved and the bacteria adhered on the slides were stained with crystal violet. The stained bacterial cells were then enumerated under a microscope. The variation between the bacterial cells adhered on the control and EPS treated slides were statistically analysed using Student’s t-test (Microsoft Excel program). Influence of surface-associated bacterial extract (EPS) on the EPS production of biofilm-forming bacteria Five hundred microliter of EPS was taken in a test tube containing 3 mL of biofilm forming bacterial suspension and 3 mL of Zobell Marine broth was added in order to provide the nutrients for bacterial growth. Control tubes were prepared without the addition of bacterial EPS and all the tubes were incubated at 37 °C for 24 h. After that, the culture broth was centrifuged at 5,000 rpm for 5 min at 4 °C and the supernatant was collected and filtered through Whatman No.1 filter paper (0.47 µm). The filtered supernatant was added with equal volume of cold absolute ethanol and allowed for the precipitation of EPS by keeping at room temperature for overnight. The precipitated EPS was isolated and the carbohydrate (Dubois et al. 1956) and protein (Lowry et al. 1951) content of the EPS were estimated (Viju et al. 2013). The obtained data were subjected to Student’s t-test (Microsoft Excel program) in order to analysis the significant influence of gastropod associated bacterial EPS on the production of carbohydrate and protein by biofilm-forming bacteria. Preparation of EPS mixed coating for biofilm control (Field assay) An insoluble matrix antifouling coating was prepared in order to study the antifouling activity of bacterial EPS in the coastal waters. In brief, the commercially available binder polyurethane varnish (Berger) was mixed with the EPS (biocide) at 1 : 1 ratio. This mixture (coating) was coated on fiber glass plates (10 cm×6 cm) using a nylon brush. The varnish coated fiber glass plate was used as a control for the comparison of results. All these plates were fitted on to an iron frame and immersed in to the coastal water for a period of 15 days. After immersion, the plates were taken out and the settlement of biofoulers on the plates was analysed (Viju et al. 2014). Partial purification of the bioactive EPS using Thin-Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) A drop of bacterial EPS was placed on a silica gel- G pre-coated glass plate and placed in a TLC chamber containing the mobile phase prepared by mixing benzene, acetic acid and methanol in 1 : 1 : 3 ratio. The movement of the mobile phase on the TLC plate was watched carefully and the plate was taken out once the mobile phase reached the other end of the TLC plate. Following this, the plate was air dried and placed in a glass chamber containing iodine crystal for the visualization of EPS. The spot developed on the TLC plate was scraped off and mixed with adequate amount of distilled water. This mixture was then centrifuged at 5,000 rpm for 15 min and the supernatant was collected. This supernatant was then concentrated and subjected to HPLC analysis. In HPLC, acetonitrile and water (1 : 1) were used as the solvent system and the flow rate was fixed as 1 mL.min-1 (Satheesh et al. 2012). Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 11:16 UTC Nova Biotechnol Chim (2018) 17(2): 115-124 118 Fig. 1. Antibacterial activity of Extracellular Polymeric Substances from different bacterial strains colonizing the surface of Babylonia sp. Activities were tested against biofilm-forming Pseudomonas sp. (A) Gallionella sp. (B) and Alteromonas sp. (C). EPS (50 µL) were loaded on sterile discs on the agar plate swabbed with target bacteria. The inhibitory activity was observed after 24 h of incubation at room temperature. Analysis of the functional groups present in the EPS using Fourier Transform Infrared (FT-IR) spectroscopy The FT-IR spectrometer was used to identify the functional groups present in the EPS. A small quantity of TLC purified EPS was placed on the face of a highly polished KBr salt plate and another KBr plate was positioned on the top to spread the compound as a thin layer. The frequencies were measured as wave numbers over the range of 400 to 4,000 cm-1 (Viju et al. 2017). Identification of bacteria The bacterial identification was performed according to the procedure described by Satheesh et al. (2012). Briefly, the 16S rRNA of the 24 h old bacterial culture was extracted using phenol chloroform method. The 16S rRNA was amplified by polymerase chain reaction (PCR) using universal primers 16S (F-5`- AGAGTTTGATCCTGGCTCAG-3`) and 16S (R- 5`- GGTTACCTTGTTACGACTT-3`). The PCR reaction steps such as denaturation, annealing and extension were maintained at 95 °C, 52 °C and 60 °C for the time duration of 30 sec, 30 sec and 4 min, respectively. The whole procedure was repeated in order to get adequate number of nucleotides (bps). Following this, the obtained PCR product was purified and sequenced using the same PCR primers and other internal primers (F-5´ACAAACAACGTGAAACGTCAA 3´ and R- 5´-AAACGAAACACGGAAACTT 3´) to confirm the sequence. The obtained sequence of the bacterial isolate was analyzed using Basic Local Alignment Search Tool (BLAST) and the phylogenetic tree was constructed by comparing the sequences available in NCBI database. Results Antimicrobial activity of EPS Bacteria were isolated from the surface of Babylonia sp. and single colonies were picked and cultivated individually. The EPS was isolated from a total of 21 bacterial strains and tested for antibacterial activity against three biofilm- forming bacterial strains Alteromonas sp., Gallionella sp. and Pseudomonas sp., respectively. The assay revealed the growth inhibitory activity of EPS isolated from 10 bacterial isolates against biofilm-forming bacteria (Fig. 1). Notably, the EPS produced by the strains CMS and CML showed maximum zone of inhibition against all the tested bacteria reproducibly in 2 independent tests (Table 1). The former strain CMS was selected and used for further analyses. Bacterial adhesion assay The antifouling effect of EPS was first tested in laboratory conditions using glass plates. The EPS of CMS strain considerably reduced the Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 11:16 UTC Nova Biotechnol Chim (2018) 17(2): 115-124 119 Table 1. Antibacterial activity of the Extracellular Polymeric Substances (EPS) isolated from the bacterial clones associated with the surface of Babylonia sp. Biofilm-forming bacteria Zone of growth inhibition [mm] CMS CML CBO 26 27 32 51 52 53 54 Alteromonas sp. 12 11 10 10 8 11 9 7 11 10 Gallionella sp. 13 13 10 9 9 8 8 8 9 11 Pseudomonas sp. 12 12 - 9 - 7 - - 7 - biofilm bacterial density on the microscopic slides; the number of Alteromonas sp. cells adhered to glass surface was significantly reduced upon treatment with EPS (Student’s t-test, t stat = 2.29, P < 0.041). Similar restriction of bacterial adhesion was achieved for Gallionella sp. (Student’s t-test, t stat = 1.66, P < 0.085) and also for the Pseudomonas sp. cell density (Student’s t-test, t stat = 3.59, P < 0.011) (Fig. 2). Influence of surface associated bacteria on EPS production in biofilm-forming bacteria The antifouling agents can have direct effect on biofilm-forming bacteria by means of their growth rate (Fig. 2) or metabolic performance. Therefore, we analysed some metabolic products of previous experiment (Fig. 2). The gastropod associated bacterium CMS considerably reduced the carbohydrate and protein concentration of EPS Fig. 2. Influence of surface – associated bacterial EPS on the adhesion of biofilm-forming bacteria. EPS treated glass slides were placed in a beaker containing biofilm bacteria and the slides were retrieved and stained with crystal violet. The number of cells adhered was counted under a microscope. Data presented are average ± standard deviation of three replicates. * indicates significance at P < 0.05. produced by biofilm-forming bacteria. The carbohydrate concentration of EPS produced by Alteromonas sp. culture was significantly reduced (Student’s t-test, t stat = 3.18, P < 0.016) after treated with the EPS of the strain CMS. Similarly, there was a significant reduction in the concentration of carbohydrate produced by Gallionella sp. culture treated with the EPS of the strain CMS (Student’s t-test, t stat = 14.56, P < 6.46E-05). The carbohydrate concentration of EPS produced by the Pseudomonas sp. significantly reduced when treated with the EPS of the strain CMS (Student’s t-test, t stat = 2.42, P < 0.036). In the same way, the EPS of gastropod associated bacterium CMS substantially reduced the protein content of EPS produced by biofilm- forming bacteria Alteromonas sp., Gallionella sp. and Pseudomonas sp.; the obtained results were statistically significant (Student’s t-test, t stat = 3.10, P < 0.018; t stat = 2.5, P < 0.033 and t stat = 4.87, P < 0.004, respectively) (Fig. 3). Fig. 3: Carbohydrate and protein concentrations in the EPS produced by biofilm-forming bacteria after treated with the EPS from bacteria associated with the gastropod Babylonia sp. Data presented are average ± standard deviation of three replicates. Symbol * above the bars indicate the significant differences at P < 0.05.……………………………………... Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 11:16 UTC Nova Biotechnol Chim (2018) 17(2): 115-124 120 Fig. 4. Antifouling effect of EPS: Control plate was coated with polyurethane varnish and experimental plate was coated with polyurethane varnish and EPS. Both plates were submerged in to the coastal water for a period of 15 days. The growth of biofouling in the plates was observed and it was found that the (experiment) plate coated with EPS considerably reduced the settlement of biofoulers. The control plate coated with polyurethane varnish was completely covered by the biofouling growth. Preparation of EPS mixed coating for biofilm control (Field assay) Since laboratory results not necessarily reflect the possible scenarios in a natural (and often much more complex) environment, we tested the antifouling efficiency of antifouling coating prepared with EPS in the coastal water against marine biofoulers using fiber plate as substratum. The fiber plate coated with the EPS of strain CMS considerably inhibited the settlement of marine biofoulers for a short period of 15 days (Fig. 4). Partial purification of the bioactive EPS using TLC Thin-layer chromatography was used to separate and partly purify the studied EPS substance from the CMS bacteria. The resulting chromatogram showed a single spot with the Rf value of 0.80 cm (Fig. 5). After elution of the compounds into water Fig. 6. HPLC spectrum of the EPS fraction resolved through thin-layer chromatography analysis. HPLC analysis was performed using acetonitrile and water (1 : 1) as the solvent system. and subsequent analysis by HPLC, the corresponding spectrum showed two major peaks at the retention time of 3.490 and 4.390 min respectively. The heights of the peaks were 0.504 and 1.266 mv, respectively (Fig. 6) Fourier transform infrared (FT-IR) analysis The EPS may contain the functional groups such as alcohol, carboxylic acid and esters, therefore FT-IR analysis of eluted EPS was performed. The obtained spectrum showed an O-H stretch at 3600 ‒ 3200 and a C-O stretch between 300 ˗ 1000 cm-1 indicating the presence of alcohol. Table 2: Details of the peaks obtained from the HPLC spectrum of EPS isolated from the bacterial strain CMS (Bacillus subtilis subsp.). Peaks R. Time [min] Area [mV] Height [mV] Area [%] Height [%] WOS [min] 1 3.490 193.330 0.504 87.0 28.5 3.76 2 4.390 28.999 1.266 13.0 77.5 0.26 Total 222.330 1.770 100.0 100.0 Fig. 5. Thin layer chromato- graphy analysis of the EPS isolated from the strain CMS. The sample was placed on silica gel coated plates and the solvent system used was benzene, acetic acid and methanol (1 : 1 : 3). Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 11:16 UTC Nova Biotechnol Chim (2018) 17(2): 115-124 121 Likewise, a C=O between 1,730 ‒ 1,700, an O-H stretch between 3,400 ‒ 2,400 and a C–O stretch at 1,320 ‒ 1,210 cm-1 indicate the presence of carboxylic acid. Besides, a C=O stretch at between 1,740 ‒ 1,715 and a C–O stretch at 1,300 ‒ 100 cm-1 indicates the presence of esters. (Fig. 7). It has been reported that the broad peak appeared between 3,600 ‒ 3,200 could indicate the presence of saccharides (Iyer et al. 2005) while the peaks appearing between 1,000 – 1,300 could be due to the presence of polysaccharides (Biswas et al. 2015). Identification and phylogenetic analysis of the strain CMS The gram positive bacterium (CMS) associated with the surface of gastropod was identified by sequencing the 16S rRNA gene. The sequence data of the bacterium CMS was related to the database of Bacillus subitilis subsp. available in NCBI with 99 % homology. Discussion This study showed that bacteria associated with gastropods possess antagonistic activities against the biofilm-forming bacteria. Ten out of 21 isolates inhibited the growth of biofilm-forming bacteria on the agar plates. It has been reported that bacteria associated with the surface of marine organisms are more pronounced reservoir of bioactive compounds than the bacteria obtained from other sources (Burgess et al. 1999). Zheng et al. (2005) also confirmed that at least 20 % of bacteria associated with marine invertebrates were likely to produce antibacterial compounds. There are plenty of studies which demonstrate the bioactivity of bacteria associated with marine invertebrates (Shankar et al. 2015). For instance, Punitha et al. (2013) reported the bioactivity of bacteria associated with the surface of marine gastropod. Microorganisms naturally have the tendency to attach to surfaces which are considered as one of the initial steps leading to biofilm formation (Costerton et al. 1987; Simoes et al. 2010). Hence, preventing initial adhesion process of bacteria would disrupt subsequent biofilm formation. Our adhesion assay results showed that the adhesion of biofilm-bacteria on the glass surface was considerably reduced by the EPS isolated from the bacterium CMS (Bacillus subtilis subsp.). Generally, bacterial metabolites are reported to possess anti-adhesion or anti-attachment activity against the biofilm-forming bacterial strains. For example, the inhibitory activity of polysaccharide produced by a bacterial species was previously reported by Valle et al. (2006). Similarly, biofilm inhibitory activities of EPS produced by surface- associated marine bacteria have been demonstrated by Rajasree et al. (2012, 2014) and Viju et al. (2016). Following adhesion, bacteria produce extracellular polymeric substances which are essential for cell- to-cell adhesion (Azedero and Oliveira 2000) cell aggregation (Burdman et al. 2000) and biofilm formation (Decho 2000). The EPS is mainly composed of polysaccharides (Fletcher and Floodgate 1973; Costerton et al. 1985) and proteins Fig. 7. FT-IR analysis of the EPS isolated from the strain CMS (Bacillus subtilis ssp.). Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 11:16 UTC Nova Biotechnol Chim (2018) 17(2): 115-124 122 (Danielsson et al. 1977). Hence, to prevent or reduce the biofilm formation on surfaces, it is necessary to screen the natural products targeting the EPS production of bacteria involving in biofilm formation. In this study, EPS isolated from the bacteria associated with the gastropod affected the carbohydrate and protein concentration of the EPS produced by the biofilm-forming bacteria. This observation showed that the metabolite present in the surface-associated bacterial EPS has the potential to reduce the bacterial adhesion on the surface. This observation correlated with a previous study by Satheesh et al. (2012) in which the authors reported that the sponge-associated bacterial extract significantly reduced the carbohydrate and protein content of the EPS produced by the biofilm- forming bacteria. Incorporation of bacterial inhibitory compounds into a polymer matrix is a model system representing a method to discover eco-friendly antifouling coating (Hellio and Yebra 2009). The active compounds could be incorporated into the matrix at a concentration higher than those in the natural environment without altering the physical characteristics of the settlement surface (Henrikson and Pawlik 1995). Previously, Armstrong et al. (2000) incorporated the sponge extract into a paint matrix and it was active against barnacle larvae. Similarly, in our study the EPS of strain CMS (Bacillus subtilis subsp.) was incorporated into synthetic wood polish (Varnish) and tested for antifouling activity in the coastal waters. Many previous studies have also reported the antifouling performance of coatings developed with the crude bacterial extracts (Satheesh et al. 2012; Viju et al. 2014, 2017). However, these studies including the present study were conducted for a short period of field trials. Further long-term field studies using the coatings developed with natural products may provide sufficient information on the performance of these compounds in marine waters Generally, chromatography methods are used for the separation and purification of the organic compounds (Iyyeparaj et al. 2014). In this study, TLC and HPLC methods were used for the purification of the EPS. The HPLC chromatogram revealed the presence of minor and major peaks at retention time of 3.490 and 4.390, respectively. These peaks likely correspond to the polysaccharide units (sugars) such as arabinose (3.63), maltose or galactose (4.50 – 5.00). The presence of these sugars (polysaccharide) has been reported in the HPLC spectrum of the exopolysaccharide produced by Micromonospora sp. along with respective standards (Ledezma et al. 2016). Moreover, the FT-IR spectrum showed the presence of polysaccharides in the purified EPS with the full stretch appeared between 1,000 – 1,300 (Biswas et al. 2015). It has been reported that polysaccharides are the major components of EPS (Vu et al. 2009) which include homo- polysaccharides (α-D-glucan, β-D-glucans, fructans and polygalactan) and heteropolysaccharides (D-glucose, D-galactose, L-rhamnose, N-acetylglucosamine and glucuronic acid) (Ruas- Madiedo et al. 2002). The production of polysaccharides by wide variety of bacteria have been reported (Guo et al. 2010), which is based on the physiochemical factors such as pH, temperature, and medium composition (Heumann et al. 1994). Saccharides are considered as potential antifouling agents (Almedia et al. 2017) since their hydrophilic groups have the ability to form hydrogel when they react with water (Piehler et al. 1999). The antifouling activity of hydrogels has been studied extensively and reported ideal antifouling agents (Rasmussen et al. 2002). For instance, in a study Katsuyama et al. (2002) reported the antifouling activity of polyelectrolyte hydrogels against the spores of seaweed. It is believed that the softness of the hydrogels could be the reason for their antifouling properties, as surface roughness plays major role in biofouling (Petronis et al. 2000). However, the exact antifouling mechanism of hydrogels is unclear. The bacterium responsible for the production of EPS which showed promising results was identified as Bacillus subtilis subsp. with 99 % homology (with the sequences available in NCBI GenBank). The genus Bacillus is widely distributed in the marine environment and produces a variety of useful products (Arias et al. 2003). The species Bacillus subtilis is known to produce compounds with biotechnological and biopharmaceutical applications (Stain 2005). For instance, Satheesh et al. (2012) reported the antifouling activity of Bacillus sp. isolated from the surface of sponge Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 11:16 UTC Nova Biotechnol Chim (2018) 17(2): 115-124 123 (Sigmadocia sp.). Similarly, Rajasree et al. (2012) studied the antifouling activity of Bacillus sp. isolated from the surface of seaweed Sargassum wightii. Conclusions The present study demonstrated the antifouling activity of EPS produced by the marine bacterium Bacillus subtilis subsp. in laboratory as well field conditions. Further characterization and confirming the lack of toxicity of the responsible compound in the EPS would promote the isolation of novel eco- friendly antifouling agent from the marine bacterium Bacillus subtilis subsp. Moreover, selecting a suitable binder (matrix) for its incorporation to the antifouling coat, as well as long-term field trials of the applied coating would provide further required knowledge for the development of an effective eco-friendly antifouling coating based on marine bacterial metabolites. Acknowledgements The authors would like to thank UGC for providing financial support through the major project scheme (Ref. F. No: 39- 294/2010 SR). References Almedia JR, Da-Silva MC, Sousa E, Antunes J, Pinto M, Vasconcelos V, Cunha I (2017) Antifouling potential of Nature inspired sulfated compounds. 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