The bacterium isolated from the alga U. lactuca was identified as Kocuria flava based on the phylogenetic analysis. This strain was designated as Kocuria flava KAU-MB and the 16S rRNA gene sequence was submitted to NCBI GenBank (accession number: MN381105). The genus Kocuria is coming under the class Actinobacteria, which is one of the important bacterial groups as nearly 50% of the bioactive metabolites from the microbial origin are reported from this group (Bérdy, 2005). Further, Kocuria is considered as a source of novel thiazolyl peptide antibiotic kocurin (Palomo et al., 2013). While many studies highlighted the bioactivity of Kocuria strains isolated from different sources (Shiyamala et al., 2014; Bibi et al., 2018; Elbendary et al., 2018), the biotechnological potentials of the extracellular polymeric substances are not studied in detail. In a previous study, Mallick et al. (2018) reported that K. flava could produce a large amount of EPS under salt stress. Results obtained in this study further widened the bioactivity of the EPS produced by the bacteria K. flava.
The EPS isolated from the K. flava was completely soluble in seawater under laboratory temperature conditions (28–30°C). Biochemical composition of the EPS isolated from K. flava revealed that carbohydrates and proteins were the major components. The total protein and carbohydrate content of the EPS are 56 µg mg–1 and 120 µg mg–1 respectively. The FT-IR spectrum of the EPS is presented in Fig. 1. The FT-IR spectrum of the EPS revealed the presence of a prominent absorption peak (broad absorption peak) at 3409 cm–1 which indicated the presence of O-H stretching vibration peak (Wang et al., 2018). Peaks were also observed around 2927 cm–1, 1629 cm–1, 1149 cm–1 and 615 cm–1. The peak at 2927 cm–1 indicated the presence of the CH2 group (Wang et al., 2018). The FT-IR peak observed at 1629 cm–1 indicated the presence of alkene group (C=C stretch). The peaks observed around 1149 cm–1 revealed the presence of monosaccharides in the EPS (Badireddy et al., 2008; Wang et al., 2018). Specifically, peaks around 1100 cm–1 in FT-IR spectrum of the EPS may indicate the presence of monosaccharides in pyran form (Wang et al., 2018) The peak around 615 cm–1 could be assigned to the presence of glycosidic linkage groups between the glycosyl groups (Rani et al., 2017).
The proton NMR spectrum of exopolysaccharides in DMSO is shown in Fig. 2. The 1H NMR spectral peaks of EPS were observed at δ 01.21, 2.06, 2.48, 2.49, 2.94, 3.20, 3.27, 3.40 – 3.64, 4.47 ppm. The peaks around δ 2.48 – 2.49 ppm were attributed to alkyl region. The proton shift at δ 3.40 – 3.64 revealed the presence of sugar ring resonances. The peak at δ 4.47 ppm was ascribed to the anomeric proton region. Additionally, the peaks below 3.2 ppm indicate the presence of proteins, acetyl and succinyl groups. The chemical shift at 3.4 ppm was attributed to polysaccharides, proteins, acetyl and succinyl groups (Gonzalez-Gil et al., 2015). The spectral shift at δ 2.06 and 4.47 ppm showed the presence of N-acetyl glucosamine and N-acetyl lactosamine (Vliegenthart et al., 1981; Liu et al., 2011).
The XRD spectrum (Fig. 3) of the EPS with their respective inter-planar spacings (d-spacings) indicate the crystalline nature of exopolymers. The XRD spectral patterns of EPS were attributed to the amorphous characteristics, with a crystalline phase. The crystalline index (CIXRD) of extracted EPS was calculated for the determination of crystalline and amorphous phase and the values were found as 11 and 89% respectively. XRD analysis indicated that the EPS produced by the K. flava was amorphous as it showed less crystalline index. Previous studies also reported the amorphous structure of bacterial exopolymers (Kavita et al., 2011; Solmaz et al., 2018).
The SEM images of EPS clearly described the compact nature of exopolymers (Fig. 4). The exopolymer obtained from K. flava strain KAU-MB consisted of aggregated and irregular sphere-shaped particles. Upon higher magnification (5.0 kx magnification), the coarse surface with irregular lumps of different size was visible (Fig. 4). Previous studies on the SEM analysis of EPS isolated from the Lactobacillus indicated highly compact porous web-like structure (Yadav et al., 2011; Wang et al., 2015). The irregular coarse shaped structure of the EPS of the bacterium Streptococcus thermophilus isolated from the milk was also previously reported by Kanamarlapudi and Muddada (2017).
Results revealed that the EPS isolated from the strain did not affect the growth of the biofilm-forming bacteria significantly (Fig. 5). The growth was considerably reduced on the cultures treated with a higher concentration of EPS (higher than 100 µg ml–1) particularly against V. harveyi and Planomicrobium sp. The EPS of the epibiotic bacterium revealed the biofilm inhibition activity in the microtitre plate assay. Concentration dependent biofilm growth inhibition was observed against all the three biofilm-forming bacteria strains used in this study (Fig. 6). One-way ANOVA indicated a significant variation in the biofilm reduction with different concentrations of EPS against P. shioyasakiensis (F=59.27, df=7, 16, P<0.05), V. harveyi (F=38.17, df=7, 16, P<0.05) and Planomicrobium sp. (F=7.31, df=7, 16, P<0.05). Post-hoc Tukey test indicated that P. shioyasakiensis culture treated with 10, 25, 50, 75 and 100 µg ml–1 of EPS differed significantly with the control (Table 1). However, higher concentrations (125 and 150 µg ml–1) of EPS treated P. shioyasakiensis did not show significant difference from the control. For V. harveyi, significant variation was observed between control and groups treated with 25, 50, 125 and 150 µg ml–1 of EPS. Further, significant reduction in biofilm formation was observed only between the Planomicrobium sp. treated with 25 µg ml–1 and control (Table 1).
Figure 5. Bacterial growth inhibiting activity of the extracellular polymeric substances. The EPS was tested against three biofilm-forming bacteria isolated from the hard substrates. a). activity against Pseudoalteromonas shioyasakiensis b). activity against Vibrio harveyi c). activity against Planomicrobium sp.
Figure 6. Antibiofilm activity of the extracellular polymeric substances isolated from the epibiotic bacterial strain K. flava. a). activity against Pseudoalteromonas shioyasakiensis b). activity against Vibrio harveyi c). activity against Planomicrobium sp.
Treatment group 1 Treatment group 2 EPS concentration (µg ml–1) P. shioyasakiensis V. harveyi Planomicrobium sp. Control 10 0.000 1 0.022 4 0.114 5 Control 25 0.000 1 0.000 1 0.017 1 Control 50 0.000 1 0.203 1 0.092 9 Control 75 0.000 1 1 0.129 5 Control 100 0.000 2 0.324 2 0.999 4 Control 125 0.448 7 0.009 2 0.999 9 Control 150 0.780 8 0.000 8 0.986 6
Table 1. Post-hoc Tukey test between control and different treatment groups of antibiofilm assay. P<0.05 is considered as significant.
The survival of barnacle nauplii was affected by the higher concentration of the EPS (750 and 1000 µg L–1). The control and other two treatment groups (250 and 500 µg L–1) showed more than 90% of larval survival after 96 h (Fig. 7). The 96 h LC50 value for the EPS was calculated as 1817.46 µg L–1. Moreover, one-way ANOVA indicated a significant difference in the survival of nauplii treated with different concentrations of EPS (F=13.49; df=4, 29; P<0.05). The settlement of cyprid larva of barnacle A. amphitrite was considerably reduced after treated with different concentration of EPS. In the control groups (without EPS treatment), 91% of cyprids settled on the Petri dishes after 96 h. The cyprids treated with EPS showed a reduction in the settlement with 80, 55, 50 and 43% respectively for 250, 500, 750 and 1000 µg L–1 treatment (Fig. 7). Results also indicated that the EPS inhibited the barnacle larval settlement at a moderate EC50 value of 748.54 µg L–1. One-way ANOVA revealed a significant variation in the settlement of larvae between the different concentration of EPS (F=25.21; df=4, 29; P<0.05). Further, the settlement of cyprids treated with EPS showed significant difference with control groups except those treated with 250 µg L–1 (Post-hoc Tukey test, Table 2).
Figure 7. Antifouling activity of the extracellular polymeric substances against barnacle larvae. a). Toxicity of EPS against stage III nauplii. b). Anti-settlement activity of EPS against cyprids.
Treatment group 2 EPS
concentration (µg L–1)
P value Control 250 0.297 7 Control 500 0.000 1 Control 750 0.000 1 Control 1 000 0.000 1
Table 2. Results of Post-hoc Tukey test carried out on the barnacle cyprid settlement between control and different treatment groups. P<0.05 is considered as significant.
Extracellular polymeric substances from microbial sources are gaining importance over other biopolymers due to the applications in different fields (Angelina and Vijayendra, 2015). Considering the significance of biofilm formation in later stages of biofouling (Satheesh et al., 2016), it is important to search compounds which could prevent or control the biofilm formation on the surfaces. The compounds which interfere with the attachment of biofilm-forming bacteria may serve as a potential antifoulant (Camacho-Chab et al., 2016). In this study, the EPS isolated from the strain K. flava reduced the biofilm formation by three predominant biofilm-forming bacteria. While the actual mechanism of biofilm reduction needs to be studied, the EPS affected the attachment of bacteria on surfaces. The reduction may be due to the presence of polysaccharides or proteins. For instance, Hwang et al. (2012) reported that the presence of protein in the EPS might have responsible for the inhibition of bacterial adhesion on surfaces. The antibiofilm and antifouling activities of the EPS observed in this study was in parallel with the results of the previous studies (Sayem et al., 2011; Pradeepa et al., 2016; Wu et al., 2016). For instance, the EPS isolated from the biofilm-forming bacterium Pseudoaltermonas ulvae showed antibiofilm activity against marine bacteria (Brian-Jaisson et al., 2016). Also, the EPS isolated from the marine bacteria Vibrio sp. showed antibiofilm activity without antibacterial activity against different bacterial strains belonging to both Gram-negative and Gram-positive bacteria (Jiang et al., 2011). Another study by Kavita et al., (2014) reported the antibiofilm activity of the EPS isolated from the bacteria Oceanobacillus iheyensis against the pathogenic strain Staphylococcus aureus.
The barnacle nauplii survival test of this study indicated that the EPS did not affect the barnacle larval survival at low concentration. The survival rate was reduced at higher EPS concentration. However, inhibition barnacle larval settlement was observed even at low EPS concentrations. The EC50 value observed in this study confirmed that the EPS isolated from the bacterium K. flava could inhibit the settlement of bacteria and barnacle larvae at low concentrations without causing much toxicity to the fouling organisms. Ideally, a good antifoulant should prevent biofouling growth at low concentration to avoid environmental concerns or toxicity to non-target organisms (Salama et al., 2018). While many natural antifouling compounds were reported from the marine organisms including microbial sources (Qian et al., 2013; Satheesh et al., 2016), most of these compounds are tested under laboratory conditions. A previous field study using the EPS isolated from the cyanobacteria as an auxiliary biocide along with copper oxide based paint found promising results which indicate that these biopolymers could be used as potential biocides for the control of biofouling (De Siqueira Melo et al., 2016).
The major advantage of the EPS for antifouling coatings is that they do not have toxic metals or other harmful compounds to the environment (Camacho-Chab et al., 2016). Natural product antifoulants from microorganisms will also have some advantages than other marine organisms (Satheesh et al., 2016). Many bacteria may produce good amount EPS under optimum laboratory conditions (Delbarre-Ladrat et al., 2014) and that could negate any product supply constraints for industrial applications. As like other natural products, the use of EPS as an antifouling biocide is not without challenges. Most of the bacterial EPS are soluble in water (Lembre et al., 2012) and the rate of solubility could affect the stability of the compound for long-term applications. A suitable coating which is biodegradable and controls the release of the biocides for a reasonable period may solve this problem for some extend but more needs to be investigated under field conditions.
Characterization and antifouling activity analysis of extracellular polymeric substances produced by an epibiotic bacterial strain Kocuria flava associated with the green macroalga Ulva lactuca
- Available Online: 2021-04-01
Abstract: Extracellular polymeric substances (EPS) are present externally to the microorganisms and play an important role in attachment and biofilm formation. These polymers possess antibacterial and antifouling activities. In this study, the antifouling activity of EPS produced by an epibiotic bacterium associated with macroalga Ulva lactuca was assessed against fouling bacteria and barnacle larvae. Results indicate that the EPS isolated from the epibiotic bacterium inhibits the biofilm formation of the bacteria without much antibacterial activity. Also, the EPS reduced the settlement of barnacle larvae on the hard substrate under laboratory conditions. The epibiotic bacterium was identified as Kocuria flava based on 16S rRNA gene sequencing. The EPS was further analysed using FT-IR, NMR and XRD to understand the biochemical composition. NMR analysis revealed the presence of polysaccharides, proteins, acetyl amine and succinyl groups. Scanning electron microscope analysis indicated that the EPS consisted of aggregated and irregular sphere-shaped particles.
|Citation:||Mohammad Abdulaziz Ba-akdah, Sathianeson Satheesh. Characterization and antifouling activity analysis of extracellular polymeric substances produced by an epibiotic bacterial strain Kocuria flava associated with the green macroalga Ulva lactuca[J]. Acta Oceanologica Sinica.|