International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 4(6), 69-74, June (2015) Int. Res. J. Biological Sci. International Science Congress Association 69 Fatty acid profiling of filamentous non-heterocystous cyanobacteria from Loktak Lake, the largest freshwater lake in North-Eastern region of IndiaOjit SK1*, Thadoi DA, Indrama Th, Avijeet SO, Gunapati OTiwari ON and Sharma GD1*Freshwater Cyanobacterial and Microalgal Repository, Microbial Resources Division, Institute of Bioresources and Sustainable Development, A National Institute of DBT, Govt. of India, Takyelpat, Imphal-795001, Manipur, INDIA Dept. of Life Science and Bio-informatics, Hargobind Khurana School of Life Sciences, Assam University, Silchar-788011, Assam, INDIA Available online at: www.isca.in, www.isca.me Received 12th May 2015, revised 25th May 2015, accepted 7th June 2015 Abstract In the present study, twenty-one (21) strains of cyanobacteria isolated from freshwater habitats of Loktak Lake were characterized for total lipid and fatty acid composition. Total lipid content ranged between 0.20% to 7.00%. Maximum lipid content was observed in Phormidium tenue BTA-1073 followed by Phormidium corium BTA-64 and Phormidium tenue BTA-63 while Phormidium corium BTA-1065 showed the minimum.Profiling offatty acid composition showed that capric acid (10:0) and lauric acid (C12:0) were only the predominant component. Plectonema sp. BTA-65 and Phormidium tenue BTA-1076 showed high capric acid content as 21.03% and 17.26%. Caprylic acid (C8:0) was maximum in Phormidium fragile BTA-1020 (10.08%). Lauric acid (C12:0) content was maximum in Plectonema sp. BTA-65 (16.23%) and minimum in Phormidium corium BTA-1065 (2.37%). Of the polyunsaturated fatty acid, -linolenic acid (C18:3n3) and arachidic acid (C20:0) were present only in Lyngbya putealis BTA-1013. Linoleic acid (C18:2n6) was observed only in Phormidium tenue BTA-63 (0.48%) and Plectonema sp. BTA-65 (0.65%). One of the pharmaceutically potential componenet, Eicosapentaenoic acid (C20:5n3) was occurred in Phormidium tenue BTA-63 (0.44%) only. Palmitoleic acid (C16:1) and oleic acid (C18:1n9) were the most abundant MUFAs in all the strains ranging between 0.05% to 6.85% for palmitoleic acid and 0.24% to 8.71% for oleic acid except Phormidium tenue BTA-1076 and Lyngbya birgei BTA-1080. The levels of erucic acid (C22:1n9) was also present in low quantity in most strains except in few cases. The goal of the present study was to profile the fatty acid components of cyanobacteria isolated from Loktak Lake where potent strains could be used for the production of commercially important desired products. Keywords: Cyanobacteria, Fatty acids, Gas chromatography, Lipids, Loktak Lake, North-East India.Introduction Cyanobacteria are the photosynthetic, prokaryotic organisms capable to store lipids as reserve cellular constituents. Microalgae, being the important organisms in the production of food products, fine chemicals and compounds with industrial applications, an increasing awareness has been focussed on microalgal biotechnology1,2. Cyanobacteria contain significant quantities of lipids and some of them are also quite high in linoleic and -linolenic acids content. There are some health benefits of polyunsaturated fatty acids (PUFA) for aquatic organisms which has spurred interest in their commercial production. They store reserve food materials which can be used as the source of pigments, lipids, vitamins, proteins and certain secondary metabolites4,5. Spirulinaplatensis accumulating large amount of -linolenic acid was also reported. Linoleic, -linolenic, eicosapentaenoic and arachidonic are also the suitable for nutritionally product for aquaculture. The surface activity properties of cyanobacterial lipids can be applied as biosurfactants in emulsification and flocculation process. Fatty acids from microalgae such as linoleic acid, linolenic acid, -linolenic acid, arachidonic acid and eicosapentaenoic acid are in large demand in the paharmaceutical market. -linolenic acid has occupy a globally potential applications in healthcare industries. This fatty acid has been used in the treatment of inammatory diseases, Parkinson’s disease, multiple sclerosis, pre-menstrual syndrome, heart disease and other health disorders9-12. Spirulina, a potential microalgae accumulates high amount of -linolenic acid has been utilized as a health food13-15. Under the availability of adequate nutrients and growth conditions, cyanobacteria especially filamentous forms can produced high volume of biomass and desired products. Earlier contributions have also made on the diversity of cyanobacteria from Loktak Lake16-18. This present research reports for the rst time, the fatty acid profiling of lamentous non-heterocystous cyanobacterial strains isolated from natural freshwater habitats of Loktak Lake. Material and Methods Cyanobacterial strains and growth conditions: The present Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(6), 69-74, June (2015) Int. Res. J. Biological Sci. International Science Congress Association 70 studied cyanobacterial strains were obtained from Freshwater Cyanobacterial and Microalgal Repository (National facility created by the Department of Biotechnology, Government of India with reference No. BT/PR 11323/PBD/26/171/2008 dated 31-03-2009), Institute of Bioresources and Sustainable Development (IBSD), Imphal, Manipur, India. These strains were previously isolated from Loktak Lake, the only largest freshwater lake in the North-Eastern region of India. Cyanobacterial strains were inoculated in Hoffmann’s flasks containing BG-11(+N) broth medium19. The flasks were kept in the culture room under light:dark cycles of 14:10h conditions maintained at 28±2ºC under illumination provided by cool white fluorescent tubes of 54-67 µmol photons m-2-1. The flasks were shaken manually for 5 min daily to prevent cell clumping. Lipid extraction and transesterification: The total lipids were extracted using the protocol20. Dried cyanobacterial biomass (250 mg) was taken into 100 ml round bottom flask and added 15 ml of methanolic-sulphuric acid. The flask along with the content was refluxed for 4 h at 60°C. Then the content was filtered and collected into a separating funnel. The filtrate was separated using ethyl acetate followed by washing with distilled water until pH of the filtrate shows neutral. Once pH becomes neutral, the organic phase was collected and anhydrous NaSO4 was added to remove the impurities. Solvents were removed by vacuum rota evaporation (Buchi Rotavapor R-215) at a pressure of 175 mbar with temperature of 60°C. Then to it, 400-500 µl of dichloromethane was added to dissolve the organic phase followed by injecting 1 µl of the sample using syringe (Hamilton 701N) in GC for fatty acid profiling.Gas chromatography of FAME: Fatty acid profiling of cyanobacterial strains was carried out using GC-FID having SGE forte capillary column (60 m × 0.32 mm I.D. × 0.25 m film thicknesses). During analysis, temperature of the injector and detector were kept at 240°C and 250°C. On the other hand, oven temperature was adjusted to 140°C at 5 min and raised to 240°C at 4°C min-1 and later it was kept at 240°C at 52 min. Nitrogen gas was used as carrier gas which was maintained at the flow rate of 1.0 ml min-1. Sample of 1 l was used for analysis with a split ratio of 100:1. SupelcoTM 37 component FAME mix (Sigma-Aldrich) was used as standard. Retention time was recorded for each sample from which each component of fatty acid can be known. Each concentration of different FAMEs was calculated by the percentage area method comparing the peak areas of their corresponding concentrations of standard using Chemito Chrom-card software version 2.6. Total lipid content of twenty-one (21) of the strains was also presented in table-1. Total lipid ranged between 0.20% to 7.00% (% of total lipid content). Maximum lipid content was observed in Phormidium tenue BTA-1073 followed by Phormidium corium BTA-64 and Phormidium tenue BTA-63 while Phormidium corium BTA-1065 showed the minimum.The fatty acid prole of the strains were summarized in table-2a and b. Results and Discussion Photomicrographs of non-heterocystous cyanobacteria which were characterized for fatty acid composition was shown in figure-1. Total lipid content of twenty-one (21) of the strains was also presented in table-1. Total lipid ranged between 0.20% to 7.00% (% of total lipid content). Maximum lipid content was observed in Phormidium tenue BTA-1073 followed by Phormidium corium BTA-64 and Phormidium tenue BTA-63 while Phormidium corium BTA-1065 showed the minimum.The fatty acid prole of the strains were summarized in table-2a and b. Table-1 Total lipid content of non-heterocystous cyanobacteria isolated from freshwater habitats of Loktak Lake, Manipur Name of strains % of total lipid content Phormidium sp. BTA-521.70 ± 0.12 Phormidium tenue BTA-632.20 ± 0.23 Phormidium corium BTA-642.50 ± 0.08 Plectonema sp.BTA-652.00 ± 0.21 Lyngbya aestuarii BTA-661.90 ± 0.11 Phormidium sp. BTA-71 1.20 ± 0.28 Phormidium sp. BTA-75 1.90 ± 0.09 Plectonema sp. BTA-79 1.90 ± 0.07 Limnothrix redekei BTA-987 1.70 ± 0.10 Lyngbya putealis BTA-1013 1.90 ± 0.06 Phormidium fragile BTA-1020 2.10 ± 0.18 Phormidium fragile BTA-1042 1.30 ± 0.16 Phormidium tenue BTA-1045 1.60 ± 0.06 Phormidium sp. BTA-1048 1.70 ± 0.06 Limnothrix vacuolifera BTA-1051 2.10 ± 0.14 Phormidium fragile BTA-1052 0.30 ± 0.21 Phormidium tenue BTA-1064 0.50 ± 0.08 Phormidium corium BTA-1065 0.20 ± 0.09 Phormidium tenue BTA-1073 7.00 ± 0.04 Phormidium tenue BTA-10760.30 ± 0.12 Lyngbya birgei BTA-10801.50 ± 0.15 All experiments were replicated three times and results are presented as mean ±SD. Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(6), 69-74, June (2015) Int. Res. J. Biological Sci. International Science Congress Association 71 Figure-1 Photomicrographs of non-heterocystous cyanobacteria isolated from freshwater habitats of Loktak Lake, Manipur showing high fatty acid composition (A) Lyngbya aestuarii (B) Phormidium corium (C) Phormidium sp. (D) Phormidium fragile (E) Phormidium tenue (F) Limnothrix vacuolifera (G) Phormidium fragile (H) Phormidium corium (I) Phormidium tenue (J) Limnothrix redekei (K) Plectonema sp. (L) Phormidium fragile B A C D E F G H I J K L Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(6), 69-74, June (2015) Int. Res. J. Biological Sci. International Science Congress Association 72 Table-2 Fatty acid composition (% of total fatty acid) of non-heterocystous cyanobacteria Strain Saturated fatty acid content (% of total fatty acid) C 8:0 C 10:0 C 12:0 C 14:0 C 16:0 C 18:0 C 20:0 1 0.45 8.82 6.04 0.77 0.91 1.09 nil 2 0.48 0.77 9.35 0.79 2.61 nil nil 3 1.80 10.56 7.60 nil 3.09 nil nil 4 7.48 21.03 16.23 1.31 6.58 0.42 nil 5 0.12 7.56 5.33 0.25 2.67 nil nil 6 1.14 15.26 10.84 0.62 0.38 nil nil 7 4.26 5.39 3.90 nil 1.13 nil nil 8 3.58 7.07 5.12 nil 3.66 nil nil 9 0.19 6.96 4.79 0.19 0.38 nil nil 10 0.56 7.83 6.21 0.58 0.56 nil 0.51 11 10.08 5.57 3.97 nil 2.07 nil nil 12 0.33 8.82 5.96 0.25 4.08 nil nil 13 0.94 15.77 12.94 0.94 0.94 nil nil 14 nil 9.48 7.08 nil 2.65 nil nil 15 0.13 4.97 3.50 0.25 2.04 nil nil 16 0.51 7.06 4.86 0.13 1.94 nil nil 17 2.77 3.61 2.38 0.08 1.06 nil nil 18 1.62 3.99 2.37 nil 3.02 nil nil 19 0.55 7.19 5.22 0.24 0.40 nil nil 20 1.02 17.26 6.60 nil nil nil nil 21 nil 12.15 7.64 nil nil nil nil StrainMonounsaturated and polyunsaturated fatty acid content (% of total fatty acid) C 16:1 C 18:1n9 C 22:1n9 C 18:2n6 C 18:3n3 C 18:3n6 C 20:5n3 1 3.18 1.18 1.59 nil nil nil nil 2 3.20 1.19 0.21 0.48 nil 0.59 0.44 3 2.44 0.90 0.90 nil nil nil nil 4 6.85 2.21 1.68 0.95 nil 0.73 nil 5 0.19 0.68 0.87 nil nil 0.12 nil 6 3.80 0.24 nil nil nil nil nil 7 0.96 3.74 2.35 nil nil nil nil 8 2.44 0.33 nil nil nil 0.16 nil 9 1.79 3.58 nil 0.38 nil nil nil 10 0.05 0.62 nil 0.47 0.08 0.60 nil 11 1.45 nil nil nil nil nil nil 12 1.72 0.49 0.49 nil nil 0.33 nil 13 5.80 6.37 0.12 0.17 nil 0.44 nil 14 2.02 1.26 nil 0.76 nil nil nil 15 1.34 3.57 nil nil nil nil nil 16 0.21 0.42 0.21 0.13 nil 0.42 nil 17 1.10 8.71 1.58 0.88 0.40 nil nil 18 1.72 1.19 nil nil nil nil nil 19 1.90 5.22 nil nil nil nil nil 20 nil nil nil nil nil nil nil 21 nil 2.43 nil nil nil nil nil 1-Phormidium sp. BTA-52; 2-Phormidium tenue BTA-63; 3-Phormidium corium BTA-64; 4-Plectonema sp.BTA-65; 5-Lyngbya aestuarii BTA-66; 6-Phormidium sp. BTA-71; 7- Phormidium sp. BTA-75; 8-Plectonema sp. BTA-79; 9-Limnothrix redekei BTA-987; 10-Lyngbya putealis BTA-1013; 11-Phormidium fragile BTA-1020; 12-Phormidium fragile BTA-1042; 13-Phormidium tenue BTA-1045; 14-Phormidium sp. BTA-1048; 15- Limnothrix vacuolifera BTA-1051; 16-Phormidium fragile BTA-1052; 17-Phormidium tenue BTA-1064; 18- Phormidium corium BTA-1065; 19-Phormidium tenue BTA-1073; 20-Phormidium tenue BTA-1076; 21-Lyngbya birgei BTA-1080 Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(6), 69-74, June (2015) Int. Res. J. Biological Sci. International Science Congress Association 73 In the present analysis, the concentrations of fatty acids were found to vary. The result showed that seven types of saturated fatty acid (C8:0, C10:0, C12:0, C14:0, C16:0, C18:0 and C20:0), four types of polyunsaturated fatty acid (PUFA) (C18:2n6, C18:3n3, C18:3n6, C20:5n3) and three monounsaturated fatty acid (MUFA) (C16:1, C18:1n9 and C22:1n9) were observed. In this study, most of the strains had C8:0, C10:0, C12:0, C14:0 and C16:0 as the dominating fatty acid. Small quantities of C18:2n6, C18:3n3, C18:3n6 and C20:5n3 were also present. The content of C16:1, C18:1n9 and C22:1n9 were moderate. Plectonema sp. BTA-65 and Phormidium tenue BTA-1076 showed high content of capric acid as 21.03% and 17.26%. Caprylic acid (C8:0) content was maximum in Phormidium fragile BTA-1020 (10.08%). Lauric acid (C12:0) content was maximum in Plectonema sp.BTA-65 (16.23%) but minimum in Phormidium corium BTA-1065 (2.37%). Of the polyunsaturated fatty acids, -linolenic acid (C18:3n3) and arachidic acid (C20:0) were present only in Lyngbya putealis BTA-1013 (0.08% and 0.51%). Linoleic acid (C18:2n6) was observed only in Phormidium tenue BTA-63 (0.48%) and Plectonema sp. BTA-65 (0.65%). Phormidium tenue BTA-63 was the only strain which showed Eicosapentaenoic acid (C20:5n3) with 0.44%. This fatty acid has a great value in pharmaceutical applications. The levels of SAFAs was also high in all the strains except myristic acid (C14:0) and palmitic acid (C16:0). Capric acid (10:0) and lauric acid (C12:0) were the most abundant fatty acids which were present in all the strains. Palmitoleic acid (C16:1) and oleic acid (C18:1n9) were the most abundant MUFAs in all the strains ranging between 0.05% to 6.85% for palmitoleic acid and 0.24% to 8.71% for oleic acid except Phormidium tenue BTA-1076 and Lyngbya birgei BTA-1080. The levels of erucic acid (C22:1n9) was also present in low amount in most strains except a few cases. Low levels of fatty acids which was longer than 18C-atoms were reported in cyanobacteria. Major fatty acids 16:0, 16:1, 18:2, and ALA, which are present in cyanobacteria are also differs even among same species22,23. The four strains of Spirulina were analyzed for their total lipid content24 and values obtained were within the range 2.7-6.8%. In the present study, the levels of saturated fatty acids were high in all the strains. On the contrary, monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) contents were generally low. Similar results were also obtained25. High cis-palmitoleic acid content (54.5 and 54.4% of total fatty acid) in two marine species of Phormidium and Oscillatoria were also reported26. The total lipid and fatty acid composition against certain external environmental factors like light, temperature and salinity some species of cyanobacterial species have also been reported by many workers27,28,29. PUFA play an important role in regulating cell membrane properties, precursors for production in animal hormones, maintaining high growth, survival and reproductive rates, aquaculture studies30. Cyanobacteria have been applied for the production of a range of value-added pharmacological and industrial desired products. However, understanding the lipid content is not sufficient for its applications in the industries. They should be analyzed at the molecular levels or genetically modified for providing better product yield. Conclusion From the above investigation, it was concluded that these strains which showed the presence of essential fatty acids of biotechnologically importance could be useful in mass production. Since Loktak Lake lies in one of the remote place in North-East India, more biotechnological research should be focussed to explore the potential organisms from this lake. These potential cyanobacterial strains would be useful in the production of high value-added biomolecules and would gain much importance in the biotech industries. Acknowledgements Research grant to carry out this work was funded by Indian Council of Agricultural Research (ICAR), Government of India. The IBSD-DBT, Imphal, Manipur, India is greatly acknowledged for providing laboratory facilities and required infrastructure. References 1.Apt K.E. and Behrens P.W., Commercial developments in microalgal biotechnology, . Phycol., 35, 215-226, (1999)2.Sanghvi A.M. and Lo Y.M., Present and potential industrial applications of macro- and microalgae, RecentPat. FoodNutr. Agric., , 187-194 (2010) 3.Anupama P.R., Value added food: single cell protein, Biotech. Adv., 18(6), 459-479 (2000) 4.Cardozo K.H.M., Guaratini T., Barros M.P., Falcao V.R., Tonon A.P., Lopes N.P., Campos S., Torres M.A., Souza A.O., Colepicolo P. and Pinto E., Metabolites from algae with economical impact, Comp. Biochem. Physiol., 146, 60-78 (2007) 5.Tan L.T., Bioactive natural products from marine cyanobacteria for drug discovery, Phytochemistry, 68, 954-979 (2007) 6.Mahajan G. and Kamat M., Gamma linolenic acid production from Spirulinaplatensis, Appl. Microbiol. Biotechnol., 43, 466-469 (1995) 7.Borowitzka M.A., Fats, oils and hydrocarbons, In: Micro-algal biotechnology (Borowitzka M.A. and Borowitzka L.J. Eds.), Cambridge University Press, Cambridge, 257-87 (1988) 8.Becker E.W., Microalgae: biotechnology and microbiology, In: Studies in biotechnology (Baddiley J., Carey N.H., Higgins I.J. and Potter W.G. Eds.), Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(6), 69-74, June (2015) Int. Res. J. Biological Sci. International Science Congress Association 74 Cambridge University Press, Cambridge, 293 (1994) 9.Callegari J.P., Feu vert pour les microalgues, Biofutur, 76, 25-40 (1989) 10.Henrikson R., New medical research with Spirulina, In: Earth Food Spirulina (Ronose Enterprises, Inc. Ed.), Laguna Beach, California, 61-73 (1989)11.Richmond A., Large-scale microalgal culture and applications, In: Progress in phycological research (Round M. and Chapman S. Eds.), Biopress Ltd, 269-330 (1990) 12.Rodriguez H. and Guerrero M.G., Products and uses of cyanobacteria (blue-green algae), In: Proles on biotechnology (Villa T.G. and Abalde J. Eds.), Universidad de Santiago de Compostela, Santiago, Chile, 247-260 (1992) 13.Cohen Z., Didi S. and Heimer Y.M., Overproduction of -linolenic and eicosapentaenoic acids by algae, PlantPhysiol., 98, 569-72 (1992)14.Cohen Z., Vonshak A. and Richmond A., Fatty acid and composition of Spirulina strains grown under various environmental conditions, Phytochemistry, 26, 2255-2258 (1987) 15.Richmond A., Spirulina, In: Micro-algal biotechnology (Borowitzka M.A. and Borowitzka L.J. Eds.), Cambridge University Press, Cambridge, 85-121 (1988) 16.Chingkheihunba A. and Arvind K.S., Molecular typing and distribution of filamentous heterocystous cyanobacteria isolated from two distinctly located regions in North-Eastern India, World. Microbiol. Biotechnol., 27, 2187-2194 (2011) 17.Singh K.O., Oinam G. and Tiwari O.N., New record of potential cyanobacteria from Indian region falling Indo-Burma biodiversity hotspots (North-East region of India) and partial characterization for value additions, Philipp. J. Sci., 141(1), 57-66 (2012)18.Tiwari O.N. and Singh H.T., Biodiversity of cyanobacteria in Loktak Lake and rice fields of Manipur, India having acidic properties, Proc. Natl. Acad. Sci. India, 75(B) III, 209-213 (2005) 19.Stanier R.Y., Kunisawa R., Mandel M. and Cohen-Bazire G., Purification and properties of unicellular blue-green algae (order Chroococcales), Bacteriol. Rev., 35(2), 171-205 (1971) 20.Bligh E.G. and Dyer W.J., A rapid method of lipid extraction and purification, Can. . Biochem. Physiol., 37(8), 911-917 (1959)21.Ahlgren G., Gustafsson I.B. and Boberg M., Fatty acid content and chemical composition of freshwater microalgae, . Phycol., 28, 37-50 (1992) 22.Kenyon C.N., Rippka R. and Stanier R.Y., Fatty acid composition and physiological properties of some lamentous blue-green algae, Arch. Mikrobiol., 83, 216-236 (1972) 23.Murata N. and Nishida I., Lipids of blue-green algae (Cyanobacteria), In: The biochemistry of plants (Stumpf P.K. and Conn E.E. Eds.), Academic Press, Orlando, Florida, 315-347 (1987) 24.Fatma T., Sarada R. and Venkataraman L.V., Evaluation of selected strains of Spirulina for their constituents, Phykos, 33, 89-97 (1994) 25.Vargas M.A., Rodriguez H., Moreno J., Olivares H., Del Campo J.A., Rivas J. and Guerrero M.G., Biochemical composition and fatty acid content of filamentous nitrogen fixing cyanobacteria, . Phycol., 34, 812-817 (1998) 26.Matsunaga T., Takeyama H., Miura Y., Yamazaki T., Furuya H. and Sode K., Screening of marine cyanobacteria for high palmitoleic acid production, FEMSMicrobiol. Lett., 133, 137-141 (1995) 27.Liu X.J., Jiang Y. and Chen F., Fatty acid profile of the edible filamentous cyanobacterium Nostoc flagelliformeat different temperatures and developmental stages in liquid suspension culture, ProcessBiochem., 40, 371-377 (2005) 28.Maslova I.P., Mouradyan E.A., Lapina S.S., KlyachkoGurvich G.L. and Los D.A., Lipid fatty acid composition and thermophilicity of cyanobacteria, Russian. PlantPhysiol., 51, 353-360 (2004) 29.Rezanka T., Viden I., Go J.V., Dor I. and Dembitsky V.M., Polar lipids and fatty acids of three wild cyanobacterial strains of the genus Chroococcidiopsis, FoliaMicrobiol., 48, 781-786 (2003) 30.Brett M.T. and Muller-Navarra D.C., The role of highly unsaturated fatty acids in aquatic food web processes, Freshwat. Biol., 38, 483-499 (1997)