FATTY ACID PROFILING AND EFFECTS OF NUTRIENT DEFICIENCY ON LIPID ACCUMULATION IN THE CYANOPHYTE Nostoc muscorumLalrinkimi and S.K. MehtaDepartment of BotanyMizoram University.Email: [email protected], Phone: 9774633697.Introduction There has been a tremendous exploitation of cyanobacterial resources in the past few decades which has led to increase in mass production of blue green algae (cyanobacteria), from laboratory-scale to industrial-scale using modern biotechnologies (Milledge, 2011). The Chinese were recorded to have used the cyanobacterium Nostoc to survive famine thousands of years ago (Milledge, 2011).

Presently, many cyanobacteria and microalgae species are used in foods and health foods, aquaculture feeds, and for production of biofuels, pigments, PUFAs (polyunsaturated fatty acids) and other fine chemicals (Spolaore et al., 2006). Several species have high contents of PUFAs of >18C atoms which are essential fatty acids required in healthy diets (Harwood and Guschina, 2009). Essential fatty acids are precursors of prostaglandins and so have importance in the pharmaceutical industry (Becker, 1994). Many species accumulate high levels of lipids in the form of glycerolipids, such as triacylglycerols (TAGs), phospholipids (PLs) and glycolipids (GLs).

TAGs are sources of edible oils and biodiesel, and PLs are useful as aquaculture feed. Reports have also indicated that lipid contents and compositions in microalgae and cyanobacteria can change significantly with nutrient deprivation (Gardner et al., 2010, Liu et al., 2016). Cyanobacteria are a group of diverse gram-negative phototrophic prokaryotes having oxygenic photosynthesis similar to the higher plants. Nostoc is a heterocystous, filamentous blue green alga of the family Nostocaceae. It is commonly seen in moist soils and on rocks, as well as in and around stagnant and running freshwater and marine water-bodies. In this study, Nostoc muscorum was investigated for its lipid content, fatty acid profiles and growth in response to nutrient availability, namely phosphorus and nitrogen. The results presented here show the potential of N. muscorum for the production of lipid to be used for general or specific applications. MATERIALS AND METHODSCollection, isolation and identification of cyanobacteriumThe present strain of Nostoc was collected from damp soil along Tuirial River, Mizoram, India. The river is situated between 24°21І5ІІ N Latitude and 92°53І2ІІ E Longitude. It serves as a place for several anthropogenic activities resulting in mildly polluted water at certain areas. The filaments were scratched and collected in sample tubes. Isolation was done in the laboratory using standard microbial streaking method in Chu-10 agar medium (Gerloff et al., 1950) under aseptic conditions. Identification was done according to Deshikachary (1959). Culture media and culture conditionsPure unialgal filaments from distinct colonies in the agar plates were transferred to 250 ml Erlenmeyer’s flasks with 150 ml of Chu-10 medium containing KNO3 (0.04 g l-1), K2HPO4 (0.01 g l-1), CaCl2 (0.04 g l-1), MgSO4.7H2O (0.025 g l-1), Na2SiO3 (0.02 g l-1), Ferric Citrate (0.003 g l-1), Citric acid (0.003 g l-1), MnCl2.4H2O (0.5 mg l-1), Na2MoO4.2H2O (0.01 mg l-1), H3BO3 (0.5 mg l-1), CuSO4.5H2O (0.02 mg l-1), CoCl2 (0.04 mg l-1), and ZnSO4.7H2O (0.05 mg l-1). Cultures were maintained at a temperature of 25°C and under 10:14 h light:dark period using cool white fluorescent tubes. The initial medium pH was adjusted to 7.5 with 0.1N HCl / NaOH. Culture flasks were hand shaken twice in a day for adequate nutrient mixing and to allow uniform light penetration. Growth measurements Growth was estimated by taking optical density (OD) reading of culture and chlorophyll a contents for a period of 20 days at regular intervals. The culture flasks were thoroughly shaken for homogenization and 5 ml of culture was aseptically withdrawn from each flask and OD at 440 nm was recorded using UV-Vis Spectrophotometer 117 (Systronics). A reference blank of basal culture medium was used. Specific growth rate (ј day-1) was calculated using the formula ј day-1 = ln (n2 ” n1) / ”t, where n2 and n1 are absorbance readings at which exponential growth was seen, and ”t = time interval of n1 and n2 in days (Guillard, 1973).Extraction and determination of chlorophyll a content5 ml of culture was taken and centrifuged at 12,000 rpm for 15 minutes. To the pellet, 5 ml of 80% acetone was added and left for 12 hours at 4°C. This suspension was centrifuged and absorbance of the supernatant was taken at 665 nm. Chlorophyll a pigment in terms of mg l-1 was calculated using the equation: C = D / d± X 1000, where ± = absorption coefficient (82.04), D = optical density at 665 nm, d = inside path length of cuvette in cm, C = concentration of pigment in mg l-1 (Mackinney, 1941). Extraction of Lipid The total lipid content was determined by using a modified Bligh and Dyer’s method (Bligh and Dyer, 1959). 1.0 g of sun dried biomass was mixed with 30 ml of chloroform: methanol (1:2) in a conical flask and sonicated using an ultrasonicator (Labsonic, Sartorius) for 30 sec at full power. The mixture was vortexed for 20 min and allowed to settle for the next 30 min. 15 ml of the supernatant was then removed in another tube and 10 ml of chloroform was again added to the mixture. This was vortexed for 10 min and 10 ml of water was added to it. The mixture was again vortexed for 1 min and then centrifuged at 3000 rpm for 10 min. The middle layer containing cell debris was discarded along with the upper methanol/water phase. The bottom chloroform/oil phase was collected and filtered into a test tube. The chloroform was completely evaporated at 40°C and the residual lipid content was stored at -20°C until analysis. Determination of lipid contentLipid content of the species was determined gravimetrically. Lipid extract was placed in a pre-weighed glass tube. Percentage of lipid content was determined by the following equation:% Total lipid content= (wt.of lipid in g)/(wt.of cyanobacterial sample in g) X 100Effect of nitrogen and phosphorus depletion The effect of nutrient depletion on growth, lipid accumulation and lipid profile of N. muscorum was investigated by modifying the composition of Chu-10 medium as follows: (i) P-depletion (-P): complete elimination of the phosphorus source K2HPO4 (ii) N-depletion (-N): complete elimination of the nitrogen source KNO3 (iii) Both N- and P-depletion (-N-P): complete elimination of N and P from medium (iv) Control (+N+P): full strength Chu-10 mediumDetermination of growth under nutrient depletion The growth pattern and specific growth rate (µ day-1) of N. muscorum under N- and P-depletion was determined for a period of 20 days. Three replicates each were maintained for different treatments. Lipid accumulation under nutrient depletion N. muscorum was inoculated to the modified growth media as described above, and incubated under the various depletion conditions for 7 consecutive days keeping other culture conditions unchanged. After 7 days, total lipid content was determined.Effect of N concentrations on lipid content of N. muscorum The effect of different nitrogen concentrations (0.02 g l-1, 0.04 g l-1, 0.06 g l-1, 0.08 g l-1 KNO3) in Chu-10 medium on the lipid accumulation in N. muscorum was also tested under culture conditions similar to the conditions mentioned above. The treatment period was 7 days. Quantification of lipid classes under nutrient depletion of culture N. muscorum was grown in nutrient deplete and replete medium as described above. Total lipids from known biomass were extracted. The lipid extracts were re-suspended in 1 ml of chloroform. TLC was performed and TAG, DAG, MAG and PL were quantified. Lipid profiling using thin layer chromatography TLC was performed for separation of the lipid classes – neutral lipids (TAG), diacylglycerides (DAG), monoacylglycerides (MAG) and polar lipids (PL). The solvent system used to elute neutral lipids was hexane: diethyl ether: acetic acid (85:15:1) mixture. Lipid extracts from known biomass were suspended in 1 ml of chloroform. 40 µl each of the lipid extracts were loaded on pre-coated TLC Silica gel plates (Merck 60 F254, 20×20 cm, layer thickness 0.2 mm, Germany) and simultaneously run with triolein in chloroform (0.04 µg ml-1 and 0.08 µg ml-1) as standard for TAG. Bands were visualized using iodine vapors inside a glass chamber. The TLC plates with bands were scanned with HP scanner. Lipid class contents were quantified using the open source software GelAnalyzer2010.Fatty acid analysis using gas chromatography An analysis of the fatty acid profiles of N. muscorum was performed using gas chromatography. 100 mg of dried biomass was taken and 1.0 ml methylating reagent containing methanol : acetyl chloride : benzene (20:1:4) was added. The tubes were placed in a water bath at 60°C for 1 h. After cooling, 1 ml n-hexane was added and centrifuged at 1500 rpm where the FAME separated out in the upper n-hexane layer. The hexane layer containing FAMEs was analyzed using Agilent 6890 GC equipped with an FID detector. A fused silica capillary column DB-225 (30 m x 25 mm id x 0.25 јm, film thickness) was used for the analysis of FAMEs. The oven temperature was held at 160°C for 2 min and increased at 5°C min-1 to 230°C and maintained for 15 min. The injector and FID temperature were kept at 220°C and 255°C, respectively. The carrier gas was N2 and the flow rate was kept at 1.5 ml min-1. The area percentage was recorded with a standard HP chemstation data system.Statistical analysis Statistical analyses of the results were performed by Student’s t-test and treatments were compared with the control. A significance level of P

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