Characterization and assessment of barnacle larval settlement-inducing activity of extracellular polymeric substances isolated from marine biofilm bacteria

Extracellular polymeric substances (EPSs) are the hydrated gelatinous matrix produced by microorganisms for attachment in a biofilm environment. In this study, the compositional variation between EPSs of three marine biofilm bacteria (Pseudoalteromonas shioyasakiensis, Vibrio harveyi and Planomicrobium sp.) were analysed by GC-MS, 1H NMR, FT-IR and XRD and SEM. The ecological significance of exopolymers was assessed in vivo using marine model organism barnacle larvae for their settlement-inducing activity. Chemical analysis revealed the presence of glycan fucosylated oligosaccharides, tetraose, trisaccharides, iso-B-Pentasaccharides, sialyllactose, oligomannose, galacto-N-biose, difucosyl-para-lacto-N-neohexaose, 3′-sialyl N-acetyllactosamine and isoglobotriaose-β-N(Acetyl)-Propargyl in all extracted EPSs. Bioassay results indicated that treatment of the barnacle larvae with EPSs from three bacterial strains enhanced settlement on substrates. In conclusion, this study highlighted the role of water-soluble EPSs in the invertebrate larval settlement on artificial materials.


Results
Larval toxicity and settlement assay. Exopolymers obtained from P. shioyasakiensis, V. harveyi and Planomicrobium sp. showed no toxicity towards barnacle nauplii after 24 and 48 h. Mortality was not observed during the treatment. Further, EPSs enhanced the settlement of barnacle larvae. The larval settlement of A. amphitrite increased considerably with increasing concentrations of EPSs (Fig. 1a-c). In the control group, 11.66% of cyprid settlement was observed after 24 h. After 48 h, 66.66% of cyprid settlement was noticed in the control group. Moreover, the maximum percentage of settlement (96.66%) was observed in cyprids treated with 750 µg l −1 concentrations of EPS-Pla. The larval groups treated with EPS-Psh and EPS-Vha showed a maximum cyprid settlement of 83.33% and 90% (750 µg l −1 ) respectively. The EC 50 values of EPS-Psh, EPS-Vha and EPS-Pla were 498.29, 494.36 and 500.09 µg l −1 respectively. Two-way analysis of variance (ANOVA) revealed a significant variation in the settlement of larvae treated with different concentrations of EPS-Vha and EPS-Pla (Table 1). The settlement of larvae treated with different concentrations of EPS-Psh did not show significant difference between   www.nature.com/scientificreports www.nature.com/scientificreports/ treatments. However, the interaction between concentration and observation time revealed significant variation (Table 1).
Fluorescence microscopy imaging of biofilm. Microscopy images confirmed the biofilm-forming ability of the three bacterial strains under laboratory conditions. The direct fluorescence microscopy imaging of 24-h-grown biofilms is shown in Fig. 2. The biofilm images were captured using different filters: red filter (Fig. 2a-c), green filter ( Fig. 2e-g), colourless (visible) filter ( Fig. 2i-k) and DAPI filter (Fig. 2d,h,l). The biofilm mats formed by bacterial cells were visible at the scale of 1000 µm (4x objective). An enlarged view of biofilm bacterial cells captured using 40x objective (100 µm scale) is presented in Supplementary Fig. 2. DAPI stain clearly showed the presence of bacterial cells in biofilm matrix.
FT-IR analysis. The FT-IR spectra of exopolymers EPS-Psh, EPS-Vha, and EPS-Pla are shown in Fig. 6. The IR spectra of all the three exopolymers were similar and displayed a broad stretching peak at 3407-3414 cm −1 possibly due to the presence of a hydroxyl group. Peaks were also observed around 2926-2929 cm −1 , 1637-1646 cm −1 , 1421-1424 cm −1 , 1127-1147 cm −1 and 500-650 cm −1 . A small peak was observed around 2356 cm −1 in EPS-Psh, but not in EPS-Vha and EPS-Pla. X-ray diffraction analysis of EPS. The XRD spectra (Fig. 7a-c) of three EPSs with their respective inter-planar spacings (d-spacings) indicate the crystalline nature of exopolymers (Fig. 7). The XRD spectral patterns of EPS are attributed to amorphous characteristics, with a crystalline phase. The crystalline index (CI XRD ) of extracted EPSs was calculated for determining the crystalline and amorphous phases and the values are shown in Table 6.
SEM analysis of EPS. The SEM images of three EPSs clearly described the compact nature of exopolymers ( Fig. 8). There was no visible difference in the morphology of all the three EPSs. The exopolymers obtained from P. shioyasakiensis, V. harveyi and Planomicrobium sp. consisted of aggregated and irregular sphere-shaped particles under 5.0 kx magnification.  Table 3. GC-MS analysis of glycosyl composition of exopolymers isolated from Pseudoalteromonas shioyasakiensis (EPS-Psh).

Discussion
The settlement assay results of the present study indicated the importance of water-soluble cues as settlement inducers for barnacle larvae. The EPSs isolated from P. shioyasakiensis, V. harveyi and Planomicrobium sp. showed better settlement induction rate compared with the control. However, the percentage of larval settlement induced by EPS-Pla was high when compared with EPS-Psh and EPS-Vha ( Fig. 1). This indicates that the larval settlement-inducing activity of the exopolymers may differ with the source microbial species 31 . The present results are consistent with previous work that barnacle cyprids depend on water-soluble polymeric cues for larval response due to the absence of substratum-coated cues 27,31 . The active substrate selection and settlement of marine invertebrates are induced by cues believed to be chemical in nature 28 . The chemical cues responsible for settlement has been recognized by the chemoreceptors of marine invertebrate larvae 32,33 . Earlier, the role of chemical cues for settlement was reported only for few marine invertebrate larvae, including mud snail Ilyanassa obsoleta 34 , sand dollar Dendraster excentricus 35 , coral-eating nudibranch Phestilla 36 and the oyster Crassostrea gigas 37 . Induction of larval settlement by bacteria-derived metabolites has been well documented in the literature 23 . For instance, water-soluble exopolymers and bacterial metabolites induced settlement of the tube worm H. elegans 25 .
1 H NMR analysis is a powerful tool for chemical fingerprinting and in this study, polymeric samples from different sources were analysed for the presence of different types of chemicals 38 . Results revealed chemical shifts indicating the presence of sugar ring protons, proteins and acetyl groups (Fig. 3). An anomeric proton region in the NMR spectra was revealed through the presence of proton signals between 4.1 and 5.4 ppm 39 . The peaks around 2.2 ppm spectra have been assigned to acetyl amines of hexose or pentose 40 . The chemical shift at 3.4 ppm was attributed to exopolymers, proteins, acetyl and succinyl groups 41 . The proton signals at 4.1 ppm were attributed to fucosylated compounds in the EPS samples 42 . The spectral shift at 2.0, 4.4 and 4.6 ppm showed the presence of N-acetyl and N-acetyllactosamine 43 . The chemical shift at 5.3 ppm was ascribed to tetraose compounds 44 . The proton signals between 3.5 and 4.0 ppm were common in all the three exopolymeric samples. The chemical shift in the particular region was ascribed to N-glycans and linked to mannose compounds 45 .
The biochemical characterization of exopolymers showed that the polysaccharide was the major component and the protein content was lower than the total carbohydrates (Table 2). GC-MS analysis of the glycosyl composition of the exopolymers produced by EPS-Psh, EPS-Vha and EPS-Pla were composed of N-glycans, fucosylated compounds and oligomannose glycans, N-acetyllactosamine (Tables 3-5). The glycans has a complementary function in governing the strength and viscosity of a biofilm matrix produced by bacteria 46 . Previous research has shown that N-glycans, fucosylated oligomannose and oligomannose glycan moiety induce the settlement of A. amphitrite cyprids 47,48 . N-acetyllactosamine identified in our study has been reported earlier for its settlement-inducing activity of coral larvae Acropora spp. and Agaricia humilis 23,24 . Earlier it has been identified www.nature.com/scientificreports www.nature.com/scientificreports/ that exogenous polysaccharides induced larval settlement and metamorphosis through mannose-binding receptors in spirorbids, ascidians and corals [49][50][51] . Furthermore, transcriptomics and proteomics studies revealed the presence of Malectin-A and mannose-binding receptors in A. amphitrite cyprid stage 52,53 . Therefore, the results of this study suggest that N-glycans and oligomannose-containing exogenous polysaccharides could bind to the mannose-binding receptors of larvae and induce larval attachment.
XRD analysis revealed the crystalline nature of polymers. EPSs were mostly amorphous in nature, which was reported previously for Vibrio parahaemolyticus, Bacillus licheniformis B22 and Scenedesmus sp. SB1 40,60,61 . Since very few pieces of evidence are available for the X-ray diffraction analysis of exopolymers, results obtained in this study will further enhance our knowledge of the properties of exopolymers isolated from marine biofilms. The SEM analysis is commonly used to study the surface morphology of exopolymers. The surface features of EPS isolated in the present study exhibit similar morphological characteristics of irregular and aggregated particles as described earlier 62 .
In conclusion, the results of this study indicated the ecological role of N-acetyl, N-glycans and oligomannose present in exopolymers of marine bacteria. Further, the compounds identified from the marine bacterial exopolymers were similar to compounds reported from the settlement-inducing protein complex (SIPC) of A. amphitrite and coralline algal cell wall polysaccharides. Hence, the present study suggests that the cues for larval settlement may possess some kind of similarity in chemical composition irrespective of origin. Further research focusing on the structural characterization of biofilm exopolymers and larval binding receptors will provide more insights into the larval settlement process on hard substrates in marine ecosystems. www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Barnacle larval rearing for settlement assay. Adult barnacles (A. amphitrite) attached to rocky substrates were collected from the Obhur Creek near the King Abdulaziz University marine station. Adult organisms along with the substrate were maintained in seawater at an ambient temperature (28 °C). The barnacles were exposed to air for less than an hour to induce nauplii release. The nauplii released were collected using a hand net (80 µm) and maintained in a 2 L polypropylene aquarium containing 0.7 µm filtered seawater (FSW). The larvae were fed Chaetoceros calcitrans at a density of 2 × 10 5 cells ml −1 . The photoperiod of 12:12 h light and the dark cycle were maintained during cyprid culture. Water was changed daily and renewed with fresh microalgal diet. Nauplii stages were checked under a stereomicroscope (Leica S6E) until the cyprid stage was noticed (5-6 days). Cyprids were collected from the aquarium using a Pasteur pipette and used for settlement studies.
Extraction and purification of exopolymers. Biofilm-forming bacteria were isolated from natural biofilms that developed on aquaculture cage nets submerged in the Obhur Creek, Central Red Sea. P. shioyasakiensis (NCBI GenBank accession number: KY224086), V. harveyi (NCBI GenBank accession number: KY266820) and Planomicrobium sp. (NCBI GenBank accession number: KY224087) were the dominant biofilm bacteria isolated from the cage nets 63 . EPSs were extracted from the marine bacteria by following the method of Zhang et www.nature.com/scientificreports www.nature.com/scientificreports/ al. 64 with few modifications. The bacteria were cultured in modified seawater nutrient broth (peptone 10 g, beef extract 10 g, sodium chloride 5 g, 0.2 µm FSW 1 L) at an ambient temperature of 25 °C for 2 days. The bacterial cultures were centrifuged at 10000 × g at 4 °C for 20 min. The bacterial pellets were removed and the supernatants were filtered through nitrocellulose membrane filter (0.45 µm). The filtered supernatants were used for EPS extraction. EPSs were precipitated by adding equal volume of ice-cold absolute ethanol and refrigerated at 4 °C for 48 h. The precipitated crude EPSs were collected from the medium and washed with double distilled water and freeze-dried. The freeze-dried crude EPSs were purified using dialysis membrane (12-14 kDa cut-off). The crude EPSs were dialysed against double distilled water overnight. The purified EPSs were freeze-dried and used for characterization and bioassays. The isolated EPSs were then named as EPS-Psh (P. shioyasakiensis), EPS-Vha (V. harveyi) and EPS-Pla (Planomicrobium sp.). The total carbohydrate content of the EPSs was determined by the phenol-sulphuric acid assay using d-glucose as a standard solution 65 . The total protein content was measured by using the protein quantification assay (Roti-Quant, Carl Roth GmbH) according to the methods described by Bradford 66 (1976). Bovine serum albumin (99.8%) crystals were used as a protein standard and the assay was carried out in 96-microwell culture plates 67 .
Fluorescence microscopy imaging of biofilm. In order to confirm the biofilm-forming ability of the bacteria strains, the biofilms of P. shioyasakiensis, V. harveyi and Planomicrobium sp. were grown in gamma-sterilized crystal grade polystyrene 24-well cell culture plates (SPL Life Sciences) for 24 h under static conditions at 28 °C. After 24 h, the broth was removed and the wells were slightly rinsed with 0.2 µm FSW to remove planktonic bacterial cells. For nucleus staining, the biofilm bacterial cells were stained with 50 µl of 0.01% DAPI solution (4′,6-diamidino-2-phenylindole) and incubated for 5 min in the dark. After incubation, the wells were washed with 1x PBS and dried before imaging. Images were collected using fluorescence microscope EVOS  www.nature.com/scientificreports www.nature.com/scientificreports/ Larval settlement assay. Barnacle larval settlement assay was performed by adding 10 cyprids to six-well plates (Falcon) containing 10 ml of different EPS concentrations (0, 250, 500, 750 µg L −1 ) in 0.2 µm FSW. The experiment was carried out (in replicates, n = 6) for 48 h in a climate-controlled walk-in environmental chamber at an ambient temperature of 28 °C. After 24 and 48 h, the number of settled and metamorphosed cyprids in the multi-well plates was checked under a stereomicroscope (Leica S6E) (Supplementary Figure S1). Data obtained from larval settlement assay were subjected to two-way ANOVA to understand the variations between treatments. Concentration of EPS and larval settlement observation time (24 and 48 h) were used as factors. For statistical tests, P < 0.05 was considered as significant. Also, the EC 50 values (the concentration in which 50% settlement induction was observed) were calculated based on regression analysis between the EPS concentration and percentage of settlement observed.

H NMR analysis.
For proton NMR analysis, 30 mg of lyophilized EPS samples was dissolved in 500 µl of deuterated DMSO and kept in a 5 mm NMR tube. The NMR spectra were recorded at 300 K using a Bruker AVANCE III 600 MHz equipped with a CryoProbe. The chemical shifts were expressed in parts per million (ppm) relative to internal chemical shift reference, 4-dimethyl-4-silapentane-1-sulfonic acid. The NMR peaks obtained were integrated and picked by Bruker Topspin 3.2 software.
GC-MS analysis. Glycosyl composition of the exopolymers from three marine biofilm bacteria was carried out as described by Hammi et al. 68 with little modifications. The purified EPSs (4 mg) added with 50 μL of myo-inositol (2 mg mL −1 , internal standard) were hydrolysed by 2 M trifluoroacetic acid at 90 °C for 2 h in a sealed glass tube. Then, the hydrolysed lysates were freeze-dried. Trimethylsilyl derivatives were prepared by adding 150 μL of pyridine and 150 μL of N, O-bis trimethylsilyl trifluoroacetamide to the dried samples. The samples were heated to 80 °C for 30 min and then evaporated to dryness. The residues were dissolved in 750 μL of dichloromethane and trimethylsilyl derivatives were analysed by a 7000 C triple quadrupole gas chromatography-mass www.nature.com/scientificreports www.nature.com/scientificreports/ spectrometry system (Agilent Technologies). The instrument was equipped with a high-sensitivity extractor ion source and a triple-axis HED-EM detector which can identify trace components even in complex mixtures at the femtogram level. For statistical tests, the samples were analysed by GC-MS in triplicate. The triplicate samples of EPS-Psh were labelled as Psh1, Psh2 and Psh3. Similarly, the EPS-Vha and EPS-Pla samples were named as Vha1, Vha2, Vha3 and Pla1, Pla2, Pla3 respectively.
The Agilent MassHunter, Openchrom (Dalton, Lablicate and Scientific community), Automated Mass Spectral Deconvolution and Identification System (AMDIS v2.73), NIST MS search, NIST glycan MS library (National Institute of Standards and Technology, USA), MetaboAnalyst 4.0 were used for mass spectral acquisition, peak processing, spectral deconvolution, compound identification, data visualization and statistical analysis. GC-MS spectra for the exopolymers samples were acquired using an Agilent MassHunter software and the data were exported (.mzdata.xml file) to OpenChrom mass spectral analysis software for peak integration and active peak identification. The MS peaks were deconvoluted using AMDIS software; the retention indices were searched against NIST MS library and NIST glycan MS library search for compound identification. The compounds from the top hit list of the library search were reported. The statistical analysis for the MS peak list and intensity data were performed using MetaboAnalyst 4.0 software. The MS peak list data were uploaded as a CSV (.csv) file; a further quality check of data and normalization by autoscaling were done. The normalized data were used for PCA and correlation heatmap analysis.
FT-IR analysis. All the three EPSs were subjected to functional group analysis using Fourier-transform infrared (FT-IR) spectroscopy. Three milligrams of lyophilized EPS samples was pressed in KBr pellets and the spectral wavelength was recorded in the range of 4000-400 cm −1 with a resolution of 4 cm −1 using an FT-IR spectrometer www.nature.com/scientificreports www.nature.com/scientificreports/ (Perkin Elmer) 69 . The FT-IR were analysed and the functional groups were interpreted using KnowItAll(R) Informatics System from Bio-Rad Laboratories. The FT-IR spectra of all the EPSs were stacked together to elucidate similarities in spectral regions.

X-ray diffraction analysis of EPSs.
Powder XRD analysis is extensively used for the characterization of polymers and is useful for studying the crystalline nature of exopolymers derived from different origins 68 . EPSs (EPS-Psh, EPS-Vha and EPS-Pla) were analysed by XRD using a multipurpose X-ray diffraction system (Rigaku Ultima IV XRD) with 2Θ ranging from 2 to 80° at 25 °C. The irradiated length and specimen length were 10 mm, with a receiving slit size of 0.2 mm at a 285 mm goniometer radius. The distance from the focus to divergence slit was 100 mm. Lyophilized EPS samples were mounted on quartz substrata, and the intensity peaks of diffracted X-rays were continuously recorded with a scan step time of 1 s; d-spacings appropriate to the diffracted X-rays at each Θ value were calculated from Bragg's law (Eq. 1).
where d is the interplanar distance, λ is the incident wavelength and Θ is the scattering angle measured from the incident beam. The crystallinity index (CI xrd ) was calculated as the ratio of the crystalline phase peak areas to the sum of the crystalline peak areas and the amorphous profile (Eq. 2) 70 .
xrd c rystal crystal a morphous SEM analysis of EPS. The surface morphology of EPSs was identified using scanning electron microscopy (VEGA3 TESCAN) with an accelerating voltage of 20 kV and 10 mm working distance. Lyophilized EPS samples were mounted on the metal and gold sputtered prior to imaging. SEM micrographs were captured at 500x, 2.0 kx and 5.0 kx magnification 71 .

Data availability
All generated data used for the analysis are available upon request from the corresponding author.