Extracellular polymeric substances (EPS) are metabolic products that accumulate on the cell surface and are composed of organic substances such as polysaccharides, proteins, lipids and nucleic acids (Frolund et al. 1996). Most of the microbial cells produce EPS for the purpose of protection (Sutherland 1997; Wozniak et al. 2003). However, production of extracellular polymeric substances by bacteria is important (Ferris, Beveridge 1985) and due to their unique physical and chemical properties they are widely used in the food, pharmaceutical, biomedical, bioremediation, waste water treatment and bioleaching fields. Previous investigations showed the antimicrobial activity (Gauthier, Flatau 1976; Shankar et al. 2010), antibiofilm activity (Fletcher, Floodgate 1973; Sayem et al. 2011) and antifouling activity (Rajasree et al. 2012) of the EPS produced by marine bacteria.
Settlement of organisms on the surface of man-made structures submerged in the marine environment commonly known as biofouling, has severe economic impact through increasing the use of manpower for the periodic cleaning and maintenance of the structures (Clare 1998; Armstrong et al. 2000). There are various antifouling methods available to solve this problem, among them antifouling coatings containing organotin and copper compounds were used as biocides to prevent fouling (Evans 2001; Yebra et al. 2004). These chemical antifouling agents were not only toxic to fouling organisms but also toxic to non-target organisms (Fingerman 1998). Due to the negative effect of the toxic biocides, a global ban was imposed by the International Maritime Organization (IMO) on the toxic organotin biocides used for biofouling control (Champ 2000), which necessitated the research focused on environment friendly biocides (LimnaMol et al. 2009).
Since most of the natural products are biodegradable and nontoxic or less toxic than synthetic compounds toward other organisms, compounds of natural origin are suggested as the best alternative of toxic biocides (Omae 2003; Fusetani 2004). Marine environment is considered as an underexplored source of novel bioactive natural products (Gulder, Moore 2009; Imhoff 2011). Many bioactive compounds have been extracted from various marine animals like ascidians, sponges, soft corals, sea hares, nudibranchs, bryozoans, sea slugs and marine microorganisms (Donia, Hamann 2003; Haefner 2003). Especially, marine bacteria isolated from the surface of marine algae and invertebrates produce a higher percentage of antimicrobial metabolites (Lemos et al. 1986; Burgess et al. 1999; Zhang et al. 2005).Since the previous investigations were mainly focused on the microbes associated with sponges and ascidians, an attempt has been made in this study to investigate the bioactivity of EPS isolated from bacteria associated with a gastropod species. Results obtained in this study will further advance our knowledge on the bacteria associated with marine invertebrates and their application as a potential source for natural antifoulants.
The gastropod (
A loop full of bacterial culture was inoculated into the flask containing 100 ml Zobell marine broth and incubated for 72 h in a shaker. Following the incubation, the culture broth was centrifuged at 10000 ×
The overnight grown culture broth of biofilm-forming bacteria (
The bacterial EPS (50 μl) was collected in a test tube containing 5 ml of Zobell marine broth (ZMA) along with 500 μl of biofilm-forming bacterial (
The bacterial EPS (20 μl) was transferred into a well of a polystyrene microtiter plate (Cat No. 911296) containing 200 μl of the bacterial cell suspension and the reference was prepared by adding 20 μl of saline in place of bacterial EPS. The plate was incubated at 37°C for 24 h. Following the incubation, the content of each well was gently removed by tapping the plates and washed with 0.2 ml of phosphate buffer saline (PBS pH 7.4) to remove bacteria. The biofilm formed by adherent bacteria was fixed with sodium acetate (2%) and stained with crystal violet (0.1% wv−1). The excess stain was rinsed off with deionized water and the plates were kept for drying. Finally, the optical density (OD) of stained bacterial cells attached to the plates was determined with a micro ELISA auto reader at a wavelength of 570 nm.
To prepare antifouling coating, the epoxy primer (3%) was added with epoxy resin (1%), hardener (1%) and bacterial EPS (1%). The control coat was prepared by adding saline (1%) in place of EPS. After proper mixing, the antifouling and reference coatings were coated on the fiber plates (18 × 12 cm) and kept in a sterile chamber to dry for a week. The dried plates were firmly fitted on a frame (iron) and submerged into the sea (Colachel coast, the west coast of India) about 2 meters from the mean sea level for a period of 50 days (3/8/2014 to 21/9/2014). After submersion, the plates were retrieved at 10 day intervals and brought to the laboratory. The fouling community recruited on the plates was scraped off and dried overnight in an oven at 60°C. The biomass of fouling on the reference and plates coated with EPS was calculated by using the following formula:
where, IWFC–the initial weight of the fouling community; FWFC–the final weight of the fouling community; N–the number of measurements taken.
The analysis of functional groups present in the bioactive EPS was performed by FT-IR (2000, SHIMADZU) analysis. A small quantity of bioactive EPS was placed on the face of a highly polished K Br salt plate, and another KBr plate was positioned on the top to spread the compound in a thin layer.
The bacterial strain was cultured in marine broth at 37°C, and the total genomic DNA of the strain was extracted using the phenol chloroform method. The 16S rRNA was amplified by polymerase chain reaction (PCR) using primers 16S (5`-AGAGTRTGATCMTYGCTWAC-3`) and 16S (5`-CGYTAMCTTWTTACGRCT-3`). The PCR product was sequenced using the same PCR primers and other internal primers to confirm the sequence. The obtained 16S rRNA sequence of the bacterial strain was analyzed using the Basic Local Alignment Search Tool (BLAST) and the phylogenetic tree was constructed using the 16SrRNA sequence of others obtained from the NCBI Gen Bank.
Student’s
The EPS isolated from the marine bacteria associated with
Antibacterial activity of EPS isolated from the marine bacteria associated with
S. No | Bioflim bacteria | EPS - EPS of surface associated marine bacteria and their zone of inhibition against test bacteria |
||||
---|---|---|---|---|---|---|
K1 | K2 | K7 | K8 | K9 | ||
1 | Alteromonas sp | 12 | 9 | 9 | 12 | 10 |
2 | Pseudomonas sp | 13 | 10 | 9 | 10 | 7 |
The growth rate of biofilm-forming bacteria on the culture medium treated with bioactive EPS was much smaller than the control medium. The growth rate of
The biofilm formation of the bacterium
The test panel coated with antifouling coating significantly (Student’s
The FT-IR spectrum of EPS showed the presence of alcohol, alkanes, alkynes, amines, carboxylic acid and ethers. The broad O-H stretch between 3400-3300 and the C-O-H bending between 1550-1220 and the C-O stretch between 1260-1000 cm−1 indicate the presence of alcohol. Similarly, the C-H stretch between 3000-2840, the CH2 bending mode near 1450 and the CH3 bending absorption near 1375 cm−1 indicate the presence of alkanes. Likewise, the C-H stretching frequency near 3300 and the C-C stretch near 2150 cm−1 indicate the presence of alkynes. Furthermore, the N-H stretch between 3500-3300, the N-H bending between 1640-1560 and the C-N stretch 1350-1000 cm−1 indicate the presence of an amine. Moreover, the O-H broad peak between 3400-2400, the C=O stretch at 1730-1700 and the C-O stretch between 1320-1200 cm−1 indicates the carboxylic acid group. The C=O stretch at 1300-1000 and the C-O-C asymmetric stretch near 1250 and the symmetric stretch near 1040 cm−1 revealed the presence of ethers (Fig. 4).
A total of 1,158 consensus nucleotides representing the 16S rRNA genes were obtained from the amplified DNA fragments of the good active strain KT1, and the phylogenetic tree constructed showed 99% similarity with
Production of bioactive metabolites by marine bacteria is a unique biological property (Fenical 1993). It has also been reported that bacteria associated with the surface of marine organisms are the higher antimicrobial and antifouling compound producers (Isnansetyo, Kamei 2003; Uzairet al. 2006; Satheeshet al. 2012). Moreover, bacteria can produce bioactive compounds including EPSs which have fascinating industrial applications (Querellou 2003). Some previous studies reported the antibacterial and antifouling activities of EPS isolated from marine bacteria (Balamurugan, Prakash 2012; Rajasreeet al. 2014). In this study, a total of 13 strains were isolated from the surface of
At the beginning of adhesion, bacteria colonize the surface and build up a biofilm (Kirchman et al. 1982) which leads to biofouling. Biofilm formation is a multistep process, including bacterial adhesion as the first step. Since bacterial adhesion is the beginning of biofilm/biofouling formation, there are many methods for screening of antibiofilm/antifouling compounds (Deighton et al. 2001; Arciola et al. 2002; Harraghy et al. 2006). In this study, the antibiofilm activity of EPS was assessed by the microtiter plate (directed) method modified after Ghaima et al. (2013). The results revealed that the EPS significantly reduced the biofilm formation of
The development of antifouling coatings by incorporating natural product as biocides into the binder has been done by many researchers (Armstrong et al. 2000; Peppiatt et al. 2000). For example, Satheesh et al. (2012) incorporated the sponge associated bacterial extract into paint and studied the activity against microalgal settlement. In this study, we prepared in the same way the antifouling coat by incorporating the bacterial EPS into epoxy primer along with epoxy resin and amine hardener. The results revealed that the fiber plates coated with the antifouling coating considerably reduced the biofouling for a period of 50 days. Similarly, Bazes et al. (2009) incorporated the dichloromethane extract of the seaweed
EPS is a complex mixture consisting of polysaccharides, proteins, nucleic acids, lipids and humic substances. Hence, it’s necessary to isolate and characterize the bioactive molecule present in the crude EPS. But in the present study, we have only analyzed the functional groups present in the EPS using FT-IR. Based on the obtained FT-IR spectrum, the bioactive EPS may contain six functional groups such as alcohol, alkanes, alkynes, amines, carboxylic acid and ethers. Likewise, Viju et al. (2014) reported the presence of alcohol, alkenes, carboxylic acid, esters and amines in the bioactive EPS produced by the surface associated bacterium
In conclusion, the present study presents the antifouling potential of marine bacteria associated with the surface of gastropod