Characterization and in vitro anticancer potential of exopolysaccharide extracted from a freshwater diatom Nitzschia palea (Kütz.) W.Sm. 1856

Diatoms are photoautotrophic microalgae classified under class Bacillariophyceae, engulfed by hard silicate frustules, which give mechanical support and protection from bacterial infections. They exude polysaccharides extracellularly that help them with their gliding motion (locomotion). However, the bioactivity of such compounds was least explored from freshwater diatoms. In the present study, a single species of pennate diatom identified as Nitzschia palea was isolated and molecularly characterized by 18S rRNA smaller subunit gene (partial) sequencing and submitted to GenBank NCBI and accession number retrieved as ON360983. Based on logarithmic growth curve analysis, the exponential phase was obtained from 3rd to 4th day of diatom culture. The exopolysaccharide was extracted by the hot-water extraction method, and characterized by FT-IR. The total yield of exopolysaccharide from Nitzschia palea was estimated as 1.56 mg in 100 mL of culture after 7 days of incubation. The estimated carbohydrate content was 51.35 µg/100 µL. The monosaccharide constituents were determined by acid hydrolysis of exopolysaccharide, silylation (derivatization), followed by GC–MS analysis and tabulated. The extracted exopolysaccharide was evaluated for its anti-cancer potential against the Human Adenocarcinoma lung cancer cell line (A549) and the estimated IC50 value was 62.64 µg/mL. Acridine orange staining assay and DNA fragmentation assay also confirmed the apoptotic activity of exopolysaccharide derived from the diatom Nitzschia palea.

Isolation of diatom. The collected freshwater sample was subjected to filtration with a nylon mesh to remove debris from the water. About 20 mL of water sample was centrifuged at 3000 rpm for 5 min. in a cooling centrifuge (Remi C-24 plus). The obtained pellet was diluted in 2 mL of distilled water and vortexed. Then, 50 µL of the sample was observed under the light microscope (Lawrence and Mayo equipped with ScopeImage 9.0), and microphotographs were recorded. For the isolation of diatom, about 1 mL of the concentrated water sample was used as an inoculum in an autoclaved 100 mL of PM culture broth medium 22 (with pH 6.0) in 250 mL of the conical flask. After inoculation, the flask was kept under incubation for two weeks at 12 h of light and dark respectively in an Algal culture room from 18 to 20 °C. Then, about 100 µL of broth culture grown in a 250 mL Conical flask was taken as inoculum for the spread plate technique in a Petri dish with 20 mL of PM medium solidified with 2.5% Agar. Again the plates were kept for incubation under 12 h of light and dark conditions for two weeks in the Algal culture room at 18-20 °C.
A distinct diatom colony growing on the spread plate was further transferred (loop full pull of inoculum) to a fresh Petri dish with PM solidified culture medium using quadrant streak plate technique for obtaining a pure culture of diatom and incubated at the same conditions discussed above for two weeks. Then the pure diatom culture was introduced into a PM broth culture medium, sub-cultured, and maintained in the Algal culture room.
Morphological identification of diatom. The morphological identification of diatom was based on the morphological structure and features of frustules. The isolated pure diatom culture was identified based on www.nature.com/scientificreports/ unique morphological features including the presence of raphae, the number of raphae, length, and the number of striae, and fibulae found on the frustules of the diatom.
Acid digestion of organic matter. For morphological identification of the diatom, the organic content present in the diatom should be removed to obtain clear frustules. About 10 mL of the diatom broth culture was subjected to centrifugation at 1000 rpm for 3 min. to yield a pellet of diatom without breaking the frustules (high rpm may brittle frustule). The pellet was then treated with acetic acid and the sulphuric acid solution mixed at a ratio of 9:1 for a duration of 30 min in a boiling water bath (90 °C). After successful acid digestion of organic matter, the content was again centrifuged at 3000 rpm for 3 min. Then other debris found in the pellet was washed with 2 mL of ethanol and again centrifuged at 1000 rpm for 2 min. Then the obtained pellet containing frustules devoid of organic matter was diluted in 1 mL of distilled water, observed under a light microscope, and subjected to Scanning Electron Microscopic (SEM) analysis.
Scanning electron microscope (SEM) imaging. The acid-digested frustules were fixed on a glass slide covered with aluminum foil and air-dried at room temperature. Then coated with chromium in an ion sputter and observed under SEM (Theromofischer Apreo S Scanning electron microscope) and imaged at 6500X to 25000X magnification. However, initial denaturation was extended up to 2 min. before starting denaturation initially, whereas, elongation was also extended after the completion of PCR cycles for 10 min. Then the amplified PCR products were qualified using 1% agarose gel with EtBr as a dye and observed under UV transillumination and photographed. Sangers method of dideoxy sequencing was employed in this study to obtain 18S rRNA sequence amplified from the DNA template of Nitzschia palea using a 3500 Genetic analyzer (Applied Biosystems). From the obtained forward and reverse sequences, a contig sequence (18S rRNA partial gene) was generated by using DNA Baser Assembler v5.15.0.

Molecular characterization of
Phylogenetic analysis. The 18S rRNA partial gene of Nitzschia palea sequenced was subjected to Nucleotide BLAST in NCBI, and similar sequences aligned with high hits were retrieved from NCBI. Then the retrieved sequences were further aligned pair-wise, and multiple-sequence by ClustalW sequence alignment with the 18S rRNA gene of Nitzschia palea using the MEGA X package. Simultaneously, the 18S rRNA gene of Nitzschia palea was submitted to GenBank, NCBI (National Centre for Biotechnology Information), and the accession num- www.nature.com/scientificreports/ ber was retrieved. The phylogenetic tree was constructed using the MEGA X package (Molecular Evolutionary Genetics Analysis X). The neighbor-joining statistical method was employed to construct the phylogenetic tree with 500 numbers of bootstrap replications, gamma distributed, and pairwise deletion with a sum of branch length (SBL) value of 0.63.

Logarithmic growth curve.
To study the logarithmic growth curve, 100 mL of PM culture broth was prepared in 250 mL of a glass conical flask and inoculated with 1% inoculum. The logarithmic growth rate of Nitzschia palea was determined by enumerating the number of cells in each quadrant of the Haemocytometer using a light microscope every 24 h of intervals for 14 days. About 50 µL of culture was placed on a haemocytometer and observed under the light microscope under 4X magnification. The number of cells in each quadrant was enumerated for four quadrants, and the average number of cells was noted every day for a period of 14 days. Then a logarithmic growth curve was plotted in a graph and resulted.
Extraction of exopolysaccharide. The exopolysaccharide was extracted from the diatom Nitzschia palea cultured in 400 mL of PM broth inoculated and incubated for six days under 12:12 h of light and dark conditions at room temperature in an algal culture room. The exopolysaccharide synthesized extracellularly by the diatom was extracted by the hot-water extraction method. The 400 mL of culture broth was filtered through Whatman No.1 filter paper to obtain cell-free aqueous filtrate, and it was kept in a water bath for 70 °C for 40 min. Simultaneously, twice the volume of absolute ethanol was added to the heat-treated aqueous filtrate and kept for incubation at −20 °C for overnight to enhance the precipitation. Then the precipitated exopolysaccharide was obtained in pellet by the centrifugation at 8000 rpm for 5 min. The pellet was dissolved in 1 mL of Milli-Q water and kept in a hot mantle at 10 °C to obtain a dry yield.

Characterization of Exopolysaccharide.
Estimation of Carbohydrate. The total carbohydrate content was determined from the extracted exopolysaccharide of Diatom Nitzschia palea by Dubois et al. 23 Phenol-Sulphuric acid method with dextrose as standard.
Fourier Transform Infra-Red FT-IR analysis of Exopolysaccharide. About 1 mg of the dried exopolysaccharide sample obtained from Nitzschia palea was blended with potassium bromide (KBr) and compressed into a small tablet. Then the sample was analyzed for FT-IR (Broker α-E (ATR, Lab India Instruments Pvt. Ltd., Mumbai)) frequency range between 500 and 4000 cm −1 .

Acid hydrolysis of exopolysaccharide and Thin Layer Chromatography Analysis (TLC)
. About 10 mg of the exopolysaccharide isolated from Nitzschia palea was diluted in 1 mL of Milli-Q water in a microcentrifuge tube. Then 500 µL of 10% HCl was added and boiled at 80 °C for 1 h. Once acid hydrolysis was carried out, TLC was performed using a silica gel plate as the stationary phase, and the mobile phase was a mixture of chloroform, acetic acid, and water in a ratio of 6:7:1. The acid hydrolyzed sample was spotted on the gel plate using a capillary tube and TLC was carried out. After performing the TLC, the plate was removed from the coupling jar and sprayed with a 5% sulphuric acid solution (dissolved in ethanol). Once the spray was dried, the gel plate was heated at 50 °C to obtain a clear spot.

Gas Chromatography-Mass Spectrometry (GC−MS) analysis of Monosaccharide composition of an exopolysaccharide isolated from Nitzschia palea.
About 500 µL of acid-hydrolyzed exopolysaccharide sample was subjected to derivatized with 500 µL of a mixture of pyridine, hexamethyldisilazane, and trimethylchlorosilane in a ratio of 9:3:1 in a watch glass. Then the mixture was subjected to GC-MS analysis using a Gas chromatograph Agilent 7890B, with HP-5MS (5% Phenyl methyl siloxane-) column of size 30 m × 250 µm × 0.25 µm connected to a 5977A mass-spectrometry detector. A 1 µL of the sample was injected at a temperature of 250 °C and the detector was kept at 280 °C. The initial temperature of the column was set at 60 °C for 1 min and was gradually increased to 325 °C and was maintained for 10 min. Helium was used as the carrier gas at a flow rate of 1 mL/minute. The split ratio was kept as 1:1. The compounds detected by the detector were identified by comparing them to the data present in the National Institute of Standards and Technology MS2011 library.
Anticancer potential of Exopolysaccharide isolated from Nitzschia palea. MTT

Results
Collection of freshwater sample. The freshwater sample was collected from the inland Chembarambakkam Lake of Kanchipuram District, Tamil Nadu, India which is geographically located at a latitude of The pH of the water sample was 6.1, which was 13.7 mV, whereas, the oxidation-reduction potential was 58.3 ORP (Table 2).
Microscopic observation of water sample. The microscopic observation of water samples collected from Chembarambakkam inland lake showed microalgal diversity of diatom (Bacillariophyceae) Nitzschia sp., cyanobacterium (Cyanophyceae) Merismopedia sp., and Green alga (Chlorophyceae) Chlorella sp., and Scenedesmus sp. (Fig. 2A). Pure culture of diatom Nitzschia sp. was isolated from the Chembarambakkam Lake and sub-cultured in the Algal Culture Laboratory (Fig. 2B-D).
Morphological identification of diatom. For morphological identification of diatoms, frustule was isolated by acid-digestion of organic matter. The frustule of the diatom Nitzschia sp. was morphologically identified based on the characteristic features such as the isolated diatom structure pennate shape, and raphid with a length of 60 µm. The presence of 30 striae and 10 fibulae were evident, each of 10 µm in length (Fig. 3). Hence, based on the unique morphological features, the isolated diatom was morphologically identified as Nitzschia palea.

Molecular characterization of Nitzschia palea.
The genomic DNA isolated from the pure culture of Nitzschia palea showed pure, single, and visible genomic DNA bands in agarose gel (Fig. 4) and the estimated concentration of genomic DNA was 61 µg/mL. The 18S rRNA smaller subunit partial gene of Nitzschia palea was amplified and the PCR product was qualitatively analyzed by agarose gel electrophoresis (Fig. 5). Based on the neighbour-joining phylogenetic tree, the 18S rRNA smaller subunit partial gene of the isolated diatom Nitzschia palea was closely related to the 18S rRNA partial gene of Nitzschia palea accession Number: KU561133 (Fig. 6). Hence, based on the molecular characterization, the isolated diatom was identified as Nitzschia palea. The 18S rRNA smaller subunit partial gene of the isolated diatom Nitzschia palea constitutes 285 bp and was submitted to GenBank and the Accession number retrieved as ON360983 (https:// www. ncbi. nlm. nih. gov/ nucco re/ ON360 983.1/).

Logarithmic growth curve. Binary cell division is the only mode of asexual reproduction in this diatom
Nitzschia palea within 24 h. Based on the haemocytometer cell count study, the diatom starts initiating its exponential phase from 72 h (3rd day) of inoculation until 120 h (5th day) in the PM culture broth medium. However, the growth gradually declines after 144 h (6th day) of culturing (Fig. 7). Therefore, the 4th and 5th day of diatom culture at its exponential phase is preferably good for the sub-culturing of this diatom.  Thin layer chromatography analysis (TLC). After acid-hydrolysis of the exopolysaccharide isolated from the diatom Nitzschia palea showing hydrolyzed exopolysaccharide as separate oligosaccharide units in the TLC sheet (Fig. 9). Two different black spots were visible with Rf values of 0.53, and 0.44.   (Table 4). Among them, the top 10 monosaccharide units based on their abundance were xylose, lyxose, galactose, altrose, ribose, glucose, mannose, fucose, talose, and gulose (Fig. 11).

Extraction of exopolysaccharide.
Anticancer potential of exopolysaccharide isolated from Nitzschia palea. The cell viability of human lung adenocarcinoma cells was decreased while increasing the concentration of the test sample (Exopolysaccharide isolated from the diatom Nitzschia palea), hence, the concentration of the test sample was inversely proportional to the cell viability of the cancer cells (Fig. 12). Microscopic evaluation also confirms the above results, in which, the number of cells was found decreased while increasing the concentration of the test sample (Fig. 13). Moreover, the estimated IC 50 value of the test sample (exopolysaccharide from Nitzschia palea) was 62.64 µg/mL. The control cells (untreated human lung adenocarcinoma cells), and treated cells (test sample treated human lung adenocarcinoma cells) were stained with acridine orange stain, in which, the control cells were stained with green fluorescence. The treated cells were stained with yellow to bright orange colored fluorescence resulting that the treated cells undergoing apoptosis (Fig. 14). DNA fragmentation assay reveals that unlike control cells (Untreated cells), extracted DNA of the treated cells was found fragmented and dragged as a ladder in agarose gel (Fig. 15) and hence the treated cells underwent apoptosis.

Discussion
In the present study, the freshwater sample was collected from the inland Chembarambakkam Lake (freshwater) of Kanchipuram District, Tamil Nadu, India. This is a man-made lake built by Emperor Rajendra Chola I (son of Emperor Rajaraja Chola) during the Cholas Era. This lake is one of the rainwater storage reservoirs supplying water to Chennai City other than Puzhal Lake. The physicochemical parameters tested for the collected water sample had shown that the water has optimal conductivity, resistivity, less TDS, and salinity, with high dissolved oxygen content with a pH of 6.1. Therefore, the water is safe for other aquatic flora and fauna and hence, safe to use as drinking water after further treatment. In this current investigation, for isolation of diatom, a freshwater sample was collected from the Chembarambarambakkam Lake, a PM culture medium was used and the favorable pH was set between 6 and 6.5 as the pH of the water was 6.1. The cosmopolitan dispersion of diatoms in various environments like marine ecosystems, www.nature.com/scientificreports/ brackish water, and freshwater ecosystems makes them an important primary producer in the food chain, and nutrient cycling in the ecosystem, including several industrial applications 24 . However, the identification of diatoms plays a crucial role in biotechnological applications. Morphological features such as the length, and width of diatoms, including frustule characteristics like striae, fibulae, and raphe are fruitful in the identification of diatoms at species level 25 . Diatom cultures were treated before microscopic studies with acid to obtain clear and transparent frustules as described by Hendey 26 . Therefore, in our study, based on morphological identification, the isolated diatom was identified as Nitzschia palea.
Henceforth, due to the wide variety of diatom species, morphological identification is quite challenging and remains problematic to survey the biodiversity of diatoms. To solve this issue, studies on molecular phylogeny were performed for the identification and classification of diatoms. Molecular characterization is a feasible,  www.nature.com/scientificreports/ rapid, accurate, and convenient method for the morphological identification of diatoms. The 18S rRNA gene is the most widely used common gene marker for the molecular identification of microbes 27 . However, the ribulose-1,5-bisphosphate carboxylase large subunit (rbcL) gene from chloroplast was also reported to analyze the phylogeny of diatoms 28,29 . Hence, in the present study, the 18S rRNA smaller subunit gene was selected for molecular characterization of the diatom isolated from the Chembarambakkam Lake, the 285 bp length of 18S RNA smaller subunit partial gene was submitted to NCBI GenBank and the accession number was also retrieved The total carbohydrate content was enhanced under nutrient supply, whereas, hampered when the condition was nutrient scarce and cell density was also found lowered in diatoms 30 . However, nitrogen and phosphate deprivation affect the growth rate of diatom isolated from the Adriatic Sea 31 . Contrastingly, under nutrient deprivation, condition exopolysaccharide synthesis like mannose, glucose, galactose, and uronic acids was found enhanced 32 . In the case of Nitzschia palea, (current study) the exponential phase of the diatom was 72-120 h of incubation (3rd-5th day), and the growth rate declined after 144 h (6th day) of incubation. Extraction of polysaccharides can be done with several techniques, in which the most appropriate and effective way of extracting the polysaccharide was done with the procedure of hot water extraction method described by Li and Shah 33 in which the temperature was changed to 70 °C for only 45 min to avoid interference of any other compounds present in the sample solution. After 12 h of incubation, the exopolysaccharide was precipitated. The total yield of exopolysaccharide content was 1.56 mg of dry weight from 100 mL of PM culture medium inoculated with Nitzschia palea on the 7th day of incubation. The estimated carbohydrate content was 51.35 µg/100 µL.
According to Xu et al. 34 the polysaccharide sample showed a distinguished functional group with higher peaks at C = O 24 , which was moreover similar to the FT-IR analysis of the diatom exopolysaccharide showed peaks for C = O and C-O groups denoting the presence of carboxyl groups in our study. The peak near 873 cm −1 corresponds to the existence of α-configuration of polysaccharides 34 . Bacterial exopolysaccharide was incubated for 30 min. in HCl and was efficient for complete hydrolysis of exopolysaccharide 35 . But for the eukaryotic exopolysaccharide 60 min. incubation was required for complete hydrolysis. However, in our study, the TLC chromatogram had shown distinct spots of oligosaccharides derived from the exopolysaccharide of Nitzschia palea. The optimized mobile phase for TLC was chloroform, acetic acid, and water in the ratio of 6:7:1 reported according to Reiffova 36 .
In another study, exopolysaccharide isolated from a bacterium Bifidobacterium breve composed of monosaccharide units like rhamnose, arabinose, glucose, galactose, and mannose had shown anticancer activity on head and neck squamous cell carcinoma by promoting apoptosis 39 . Similarly, arabinose-rich exopolysaccharide   www.nature.com/scientificreports/ containing glucuronic acid, and galacturonic acids isolated from Bacillus sp. had anticancer potential on HepG2 liver cancer cells with an IC 50 value of 218 µg/mL 40 . Simultaneously, glucan and mannose-rich exopolysaccharides extracted from Leuconostoc mesenteroides were reported to possess anticancer activity on hepatocellular carcinoma 41 . However, the bioactive potential of exopolysaccharides from freshwater diatoms is least studied. However, Lakshmegowda et al. 42 reported that the hexane extract (volatile compounds) of the freshwater diatom Nitzschia palea had shown to possess potent anti-inflammatory activity with an IC 50 value of 93.5 µg/ mL by suppressing proinflammatory cytokines including nitric oxide, IL-6, TNF-α, and PGE2. In this present investigation, the exopolysaccharide extracted from a pennate diatom Nitzschia palea was observed to show significant results when treated against human adenocarcinoma lung cancer cell line A549 with an IC 50 value of 62.64 µg/ml. The mode of its activity was found due to its apoptosis activity was also observed when the cells were viewed under a fluorescence microscope when stained with acridine orange, and DNA fragmentation assay. Therefore, the exopolysaccharide extracted from the freshwater diatom Nitzschia palea isolated from the Chembarambakkam Lake, Tamil Nadu, India found to exhibit potential anticancer activity on Human Adenocarcinoma Lung cancer. This is a hitherto report on the anticancer potential of exopolysaccharide from the diatom Nitzschia palea.

Conclusion
A pure isolate of Nitzschia palea belongs to the pennate diatom was isolated from the freshwater lake Chembarambakkam, Tamil Nadu, India. Based on the morphological and molecular characterization, it was identified as Nitzschia palea. Intriguingly, the exopolysaccharide extracted from the diatom Nitzschia palea has potential anticancer activity on the Human Adenocarcinoma lung cancer cell line (A549). Therefore, this diatom exopolysaccharide is a novel bioactive compound with potential anticancer activity.

Data availability
The 18S rRNA partial gene of Nitzschia palea sequenced was submitted to National Centre for Biotechnology Information (NCBI), and the Accession number retrieved as ON360983 (https:// www. ncbi. nlm. nih. gov/ nucco re/ ON360 983.1/).