Introduction

Cyanobacteria are photosynthetic organism found in a variety of ecosystems, including fresh, marine, brackish water and terrestrial environments. Cyanobacteria were found to produce different types of secondary metabolites which inhibit the growth of other microorganisms in their surrounding.^1,2 Cyanobacteria possess natural bioactive substances that are difficult to produce by chemical synthesis³. These compounds has a wide range of biological activities such as antimicrobial, antioxidant, anticancer etc^4,5-7. The search for antibiotics and other pharmacologically active substances in cyanobacteria has received more attention at present time⁸. A number of literatures are available on antimicrobial substances produced from microalgae and cyanobacteria⁹. However, the compounds responsible for antimicrobial properties in cyanobacteria are yet to be identified for its maximum efficacy^10,11. In the marine environment cyanobacteria were found to synthesize a large array of chemical compounds due to biotic and abiotic stress¹². Antimicrobial substances produced by cyanobacteria are of interest for research not only protective for pathogens, but also as pharmacological important bioactive secondary metabolites¹³. Moreover, the differential antimicrobial activity of cyanobacteria is correlated to the selected species and the solvents used in experiments^14-17. Despite the fact that several work has been carried out on antimicrobial property of cyanobacteria of different habitats but a limited reports were available on the species found across Odisha coast. Thus the current study intends to find out the antimicrobial properties of two selected predominant species (Oscillatoria boryana and Oscillatoria pseudogeminata), obtained from Odisha coast.

Materials and Methods

Collection, culture condition and isolation of cyanobacteria

The test organism (Cyanobacteria) were collected from different sites across Odisha coast (Fig 1A & B) by using a plankton net (2μ) and the samples attached to different substratum using forceps. The samples were isolated to unialgal culture by repeated subculture and serial dilution, observed under trinocular research microscope (Make: Dewinter Model: “SELECT” DGI-1000) and identified by standard procedure^18-20. The isolated cyanobacterial species were grown in 1 litre flasks with aeration to obtain large biomass using ASN III medium with illumination of 30 μmol photon m^-2 s^-1alongwith light/dark cycle of 16 h/ 8h  at 25±1°C temperature.

Test Cyanobacteria

Morpho-taxonomic identification

Oscillatoria boryana

Class- Cyanophyceae; Order- Nostocales; Family- Oscillatoriaceae; Genus- Oscillatoria; Species-       boryana

Trichome coiled like a screw at the apices or totally, sometimes straight compressed near cross walls, 6-8µ broad, sometimes slight granulation observed adjacent to cross walls, cellsat the apices 4-6µ elongated or rounded or more or less pointed, not capitates, calyptra absent, the breadth of the trichome varies very much between 3.3-5.0µ, 4-6.6µ, 5-7µ and 6-8µ(Fig.1B-i).

Oscillatoria pseudogeminata

Class- Cyanophyceae; Order- Nostocales; Family- Oscillatoriaceae; Genus- Oscillatoria; Species- pseudogeminata

Thallus blue-green in colour, trichomes coiled, ends are not attenuated, cells are 1.3-2.2µ broad, about 2.6µ long, the cross walls are not squeezed but thickened, non granulated and cells are spherical, calyptras absent (Fig.1B-ii).

Figure 1: (A). Sample collection sites across Odisha Coast Click here to view figure

Figure 1: (B). Microscopic Photograph of two test cyanobacteria (i) Oscillatoria boryana (ii) Oscillatoria pseudogeminata Click here to view figure

Cyanobacterial extract preparation

The cyanobacterial cultures (10-14days old) were harvested by centrifugation (3500 rpm for 15min) and dried at 45 °C in a hot air oven. The dried samples were grounded using a sterile mortar and pestle. Then, 0.5 g of grounded sample was mixed with 10 ml of solvents (acetone, methanol, benzene and aqueous) used and kept overnight at room temperature in a rotary shaker for complete extraction. At 4000rpm for 15minutes the extracts were centrifuged and concentrated at 50°C under reduced pressure. The dried residue was dissolved in DMSO to obtain final concentrations of 1mg/ml, and used for antimicrobial activity²¹.

Pathogenic strains

Four pathogenic bacteria i.e two Gram +ve (Staphylococcus aureus MTCC-96, Bacillus subtilis MTCC-441) and two Gram -ve (Vibrio Cholerae MTCC-3906, Eschercichia coli MTCC-443) and four pathogenic fungi (Candida albicans MTCC 183, Aspergillus niger MTCC-1344, Penicillium verrucosun MTCC-1758, Fusarium oxysporum MTCC-284) were used for the experimental purpose. These microorganisms were procured from Institute of Microbial Technology, Chandigarh and kept in the Department of Biotechnology, Maharaja Sriram Chandra Bhanja Deo University, Takatpur, Mayurbhanj, Odisha. 

Antibacterial assay

The agar cup method²²is used for the antibacterial study, using Mueller-Hinton agar (MHA) media for bacteria. Petriplates with 20ml of sterilised solid media was inoculated with 20μl of test pathogens (10⁶ CFU/ml) were uniformly swabbed. 6 mm diameter wells were formed and 50 μl of cyanobacterial extracts (DMSO, 1 mg/ml) were put into the wells and placed for incubation at 37 ± 1 ºC for 24 hours. The inhibition zone formed around the wells was measured after incubation period. The average value of diameter of three plates was recorded. Ampicillin at a concentration of 100µg/ml are used as positive control.

Antifungal assay

The antifungal activity study was carried out on potato dextrose agar medium (PDA). ²² Sterilized potato dextrose agar (PDA) was poured into sterile petriplates (20ml), solidified and inoculated with 20μl of fungal test pathogens (10⁶ CFU/ml) and swabbed uniformly. 6 mm diameter wells were formed and 50 μl of cyanobacterial extracts (DMSO, 1 mg/ml) were put into the wells and placed for incubation at 25 ± 2 ºC for 72 hours. The inhibition zone formed around the wells was measured after incubation period. The average value of diameter of three plates was recorded. Clotrimazole at a concentration of 100µg/ml are used as positive control.

Study of Minimum inhibitory concentration (MIC)

MIC experiment was done using 96 well micro titer plates containing broth medium. The first series of well was given 50 µl of Mueller-Hinton broth (MHB) and 50 µl bacteria culture as negative control. Starting from second series of wells  until eighth row were embedded with various concentration of extract(15.62, 31.25,62.5,125, 250,500 and 1000µg/ml) by two fold dilution in MHB media with pathogenic microorganisms as control and kept at 37º C for 24 h and subsequently were analysed with microplate reader (make-TECAN, model-M200PRO)and interpreted.^23-25

Gas Chromatography/Mass Spectrometry (GC-MS) analysis

The GC-MS evaluation was determined at Analytical Chemistry, Triyat Scientific Co., Nagpur, Maharashtra, India. The GC-MS analysis was done using a Agilent 7890A Gas Chromatography system equipped with a HP-5 column (30ml x 250µm ID, 0.25µm thick film) and Agilent 5975C inert Mass Spectrometer Detector with Helium as carrier gas. The sample extract was injected into the injector port and the Helium gas expel the sample down the column where different chemical constituents get separated. The injection port was maintained at 80 ºC for 4 minutes. The run time was continued for 40 minutes. The flow rate of 1.50ml/min; injector and column oven temperature of 280ºC and 80ºC; injector split ratio of 20:1 was set. GC-MS mass spectrum interpretation was conducted and compared with the reference data base from NIST. 

Statistical analysis

Statistical analysis of results is performed. The values are expressed as mean with standard deviations (±SD) and were subjected to one way ANOVA, and the results were the average of three replicates (p ≤ 0.05).

Results

Antibacterial Activity

The results obtained in the evaluation of the antibacterial activity of different solvent extracts of two marine cyanobacteria Oscillatoria boryana and Oscillatoria pseudogeminata against four pathogenic bacteria (S. aureus, B. Subtilis, V. cholerae and E. coli) strains using the agar well diffusion method was shown in Table 1(A&B). Differential antimicrobial activity was observed in separate solvent extracts against particular pathogen. The maximum zone of inhibition of 20mm was detected against V. cholerae using methanol extracts of O. boryana, followed by 18 mm and 17 mm with B. Subtilis and S. aureus respectively (Table 1A). A Smaller zone of inhibition of 9mm was found in aqueous extract of O. Pseudogeminata against B. Subtilis and E. coli (Table 1B). Further, larger inhibition zone was observed in methanol extract than by acetone and benzene whereas minimum zone of inhibition was found in aqueous extract in both the test species of marine cyanobacteria. The study result reveals that the methanolic extract of O. boryana found to have significant antibacterial activity in V. cholerae and in contrary neglible activity was marked in aqueous solvent in agar well diffusion assay.

Antifungal Activity

The results exhibiting the in-vitro antifungal activity of different solvent extracts of two marine cyanobacteria O. boryana and O. pseudogeminata against four fungal pathogens (A. niger, C. albicans, P. verrucosun and F. oxysporum) strains using the agar cup method was shown in Table 1A&B. Maximum zone of inhibition of 17mm against C. albicans and 14mm against A. niger in O. boryana and O. pseudogeminata respectively in methanol extract and the minimum zone of inhibition of 9mm was found in aqueous extract of O. boryana against A. niger and 8mm in O. pseudogeminata against F. oxysporum (Table 1A& B).

Table 1: (A). Antibacterial and antifungal activity of different solvent extract of Oscillatoria boryana with bacterial and fungal pathogens.

Table 1: (B). Antibacterial and antifungal activity of different solvent extracts of Oscillatoria pseudogeminata with bacterial fungal pathogens.

Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration was estimated at a concentrations range of 1000µg/ml to 15.62µg/ml and optimum dilution showing inhibition was observed as MIC value (Table 2).The MICs of the extracts were measured using serial dilutions up to 1000µg/ml of extracts in DMSO. The MIC detected for O. boryana was 31.2 µg/ml and was 125µg/ml for O. pseudogeminata. The MIC result thus reveals that O. boryana has significant antimicrobial property as compared to O. Pseudogeminata.

Table 2: Minimum inhibitory concentration (MIC) of O. boryana and O. pseudogeminata against bacterial and fungal pathogens.

GC-MS Analysis of methanolic extract of O. boryana 

The chemical constituents of methanol extract of O. boryana detected by GC-MS was presented in Fig. 2, Table 3. Major chemical components present in the methanol extract of O. boryana indicated the occurence of many novel bioactive compounds. Thirteen compounds are detected in methanolic extract of O. boryana with the highest value of n-hexadecanoic acid (15.42 %) followed by 5-Methyl-2-phenyl indolizine (13.3%). The other prevailing compounds were Acetic acid (12.47%), 5-Nitro-3-cyano-2(1H)-pyridone(10.37%), 9-Oxo-9,10-dihydro acridine-4-yl (6.8%), Hexadecanoic acid and Silicic acid  5.02% each. The analysis also revealed the presence of few biologically active compounds in low concentration such as Benzenemethanol (1.9%), Undecanoic acid (1.73 %), Phytol(1.58%), Propane,1,1,3-triethoxy (0.92 %), Erucic acid (0.87%) and Benzoic acid (0.78 %). The study results infers that possibly these chemical compounds are important in terms of their antimicrobial activity ^27,28.

Figure 2: GC/MS analysis of methanolic extract of O. boryana Click here to view figure

Table 3: Compounds identified in methanol extract of O. boryana as revealed by GC-MS analysis.

Discussion

Unregulated use of antibiotics gives rise antibiotic resistance in bacteria, fungs and thus raises an issue of health hazards. The alarming increase in microbial contamination has boosted the search for new, novel and secure candidate to fight against infection. Thus more specific research on identification of bioactive compounds derived from various microalgae and cyanobacteria is of interest at present. Cyanobacteria are potential organism for producing a variety of bioactive secondary metabolites. These compounds derived from cyanobacteria were found to possess antimicrobial activity. Based on this fact; two species of marine cyanobacteria Oscillatoria boryana and Oscillatoria pseudogeminata were studied for antimicrobial activity. The findings of present work explained that among the two test cyanobacteria, methanol extract of O. boryana exhibited significant antimicrobial activity than O. pseudogeminata. In the similar pattern, ²⁹also reported that Oscillatoria brevis has antibacterial activity against S. aureus(ATCC 13565);B. cereus(EMCC 1080); S. typhi (ATCC25566); P. aeruginosa (NRRL B-272) and K. Pneumonia (LMD 7726). Earlier study by Sethubathi and Prabu³⁰ which is in agreement to our result revealed that the crude extracts of Oscillatoria sp.; Phormidium sp. and Lyngbya majuscule were found to possess antimicrobial activity against pathogens such as S. mutants; S. aureus; P. aeruginosa; B. Subtilis and K. pneumonia. The MIC value as recorded in the present piece of work was promising and the cyanobacterial extracts showed significant antimicrobial potency against V. cholerae; B. subtilis and C. albicans which match to the work carried out by Wijesekara and Manage ³¹ with Oscillatoria boryana extract showing maximum antimicrobial activity against S. aureus as compared to S. typhi. Moreover, the findings of the present investigation on GC-MS analysis showed the presence of n-hexadecanoic acid, Acetic acid, 5-Nitro-3-cyano-2(1H)-pyridoneetcas predominant compounds which might be responsible for antimicrobial activity in Oscillatoria boryana, which matches to the work carried out by Mundt et al.³² and Shanab^33,34. Similarly, El-Syad et al.³⁵ detected presence of hexadecanoic acid and bis(2-ethylhexyl) phthalate in the phenolic extract of Spirulina and in addition, phytol was previously found in a marine cyanobacteria, N. muscorum and in the S. platensis ³⁶showing antimicrobial property. Recently, Hobby et al.³⁷ stated that modification of phospholipid composition by fatty acids suppress the antimicrobial compounds. However, there is no any specific reason established between chemical classes and their role in bioactive properties such as antimicrobial activity³⁸. The saturated fatty acids might be binding the cell membrane and alter the cellular construct thereby enhancing in antimicrobial activity. Thus, deeper study is required to focus on the bioactivity of metabolites synthesise in cyanobacteria for the production of novel antimicrobial compounds that maybe useful for commercial purpose.

Conclusion

This study demonstrates that both the cyanobacteria have the potential to produce potent antimicrobial compounds that act against human pathogens. Among different solvents used, the methanolic extract of both the species of Oscillatoria showed better activity, inhibiting the growth of test pathogens. The present research has shown that the antimicrobial activity of different species of cyanobacteria is dependent on the solvents used to make the extracts and the effect of those solvents. It is thought that the findings obtained from this investigation can be used for future research and for the production of antibacterial drugs of cyanobacterial origin. Moreover, this study has paved the way towards demonstrating the presence of potential high-value pharmaceutical antimicrobials having ample scope for further investigation towards drug development in pharmaceutical and health care industry.

Acknowledgment

The authors are thankful to Head, Department of Biotechnology, Maharaja Sriram Chandra Bhanja Deo University for providing facilities for conducting the experiments.

Conflict of Interest

The authors declares no conflict of Interest. 

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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