International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 1(8), 27-35, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 27 Growth, Total Lipid content and Fatty Acid Profile of a Native Strain of the Freshwater Oleaginous Microalgae Ankistrodesmus falcatus (Ralf) grown under Salt Stress Condition Talukdar Jayanta 1*, Kalita Mohan Chandra and Goswami Bhabesh Chandra1,2Department of Biotechnology, Gauhati University, Guwahati - 781014, Assam, INDIADepartment of Chemistry, Gauhati University, Guwahati - 781014, Assam, INDIAAvailable online at: www.isca.in Received 16th September 2012, revised 21st September 2012, accepted 26th September 2012Abstract Growth, fatty acid compositions and calorific value of a native freshwater oleaginous microalgae A.falcatus was studied in batch culture at light intensity 40 µ mol photons/m/s, temperature 25 ± 2 C and 16:8 h light and dark diurnal cycles. Improved growth and total lipid contents were determined with the culture grown under salinity up to 160 mM. The highest specific growth (µ=0.313 d-1) and least doubling time (T=2.21 days) with maximum increase in cell numbers (29.0 x 10 ml) were recorded in medium supplemented with 160 mM of NaCl compared to control medium (µ=0.209 d-1, T=3.32 days and 15.2 x 10 ml-1 respectively). Improved total lipid (55.3%), carbohydrate (14.5%) and protein (4.8%) contents were also determined in the culture under salinity compared to control medium (lipid 38.3%, carbohydrate 12.6% and protein 3.1% respectively). C16:0, C18:1 and C18:3 were found to be the major fatty acid components of the lipid content. Marginal increase in C18:1 (30.5%) was observed in culture grown under salinity stress. With maximum energy value of 27.9 ± 0.15 kJg-1, a close correlation (R = 0.949) between lipid content and calorific value was observed. With further augmentations of lipid content and improved fatty acids, the native microalga strain could be a potent candidate for biofuel production. Keywords:Ankistrodesmus falcatus, biofuel, fatty acid methyl ester, microalgae, salinity.IntroductionEnergy from renewable biomass sources has gained global attention with the potential to substitute for fossil fuels in all energy markets – in the production of heat, electricity, and fuels for transport. Concerted efforts are now underway globally to harness alternative energy from renewable biomass sources generated via photosynthesis to move towards more sustainable bio-based economies, where biomass-derived products substitute for petrochemical-derived products. Development of first and second generations of biofuels, which are based on different categories of biomass feedstock and their downstream processing and typically obtained from starch, vegetable oil, and cellulosic crops, require cultivated land and water supplies that can compete with land use for food production2-4. Extensive cropping of plants for biofuel production, which will certainly take place with the increasing fuel demand, raises a food vs fuel dilemma, and also natural resource demand problems5-8. With such limitations of first and second generation biofuels, microalgae based third and further generations of biofuel have gained great interest9-14. Microalgae are considered to represent the most promising among existing renewable biomass sources to generate biofuels. They are also likely to have much lower adverse effect on the environment and on the world’s food supply than conventional biofuel-producing crops. The unique diversity of microalgae and the spectrum of species available for amenability for biofuel production as compared to other advanced biomass feedstock have placed microalgae on the priority list. Various species may be selected to optimize the production of different biofuels. Microalgae biomass can be utilized to produce varieties of fuels, such as liquid fuels and gas, gas-or oil-based biofuels, bioethanol or methanol, biohydrogen and biodiesel2, 3, 5, 10, 11, 12, 15-24. Moreover, microalgae offer a diverse spectrum of valuable products and pollution solutions, such as food, nutritional compounds, omega-3 fatty acids, animal feed, organic fertilizers, biodegradable plastics, recombinant proteins, pigments, medicines, pharmaceuticals, and vaccines25. To enhance the economic feasibility of algal oil production, biomass productivity (production per unit volume per unit time), cellular lipid content, and overall lipid productivity are the three key parameters that need to be improved. These requirements are not always compatible, and in general, conditions favoring a high growth rate of cells result in a low lipid fraction in the cells and vice versa26. High lipid content with favorable fatty acid compositions27, faster growth rate, ease of harvest, and dominance in nature with the ability to adapt in the prevalent climatic conditions are some of the desirable criteria for choosing the right species for biofuel production23. Isolation of native strains from local water bodies can provide the best information regarding the selection of appropriate strain(s) in terms of biomass and lipid productivity, dominance in the native International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 27-35, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 28 algal flora, and hence the likelihood of being the most suitable selection as a biomass feedstock28-30. In addition to the species selection, nutritional requirement for culture optimization is another essential factor that significantly affects the growth rate and yield of products31. Some microalgae species can double its biomass in less than 24 h under optimum culture conditions10. Such high yield and high density biomass is ideal for intensive cultivation and can provide an excellent biomass source of biofuels. Oil levels 20 to 50% by dry weight biomass are quite common, and some can exceed 80%32. Significant increases in lipid contents have been reported in microalgae after being subjected to various stress conditions during cultivation such as nitrogen deprivation, high light intensity, high salinity, phosphate limitation or co-immobilized in alginate beads with the bacterium Azospirilium brasilense11,18,33-37 However, stress conditions also have negative influence on growth, resulting low overall biomass production. Therefore, it is more appropriate to apply stress conditions in the later stage of growth after initial biomass production38. Besides high lipid content, another important characteristic for any biodiesel feedstock is the suitability of the fatty acid profile for biodiesel production. Oils and fats are primarily composed of triacylglycerols (TAGs), which serve as a primary storage form of carbon and energy in microalgae; their fatty acid composition is also superior to that of other cellular lipids (phospholipids and glycolipids) for biodiesel production. Fatty acid compositions of a particular lipid influence the saponification number, iodine value of the lipids, and biodiesel quality, such as cetane number, heat of combustion, oxidative stability, cloud point, and lubricity39,40. Algal fatty acid composition is influenced by growth conditions such as temperature and nutrient availability41. The different carbon chain lengths and number and position of unsaturated bonds found in fatty acids influence the cetane number (CN), iodine value, oxidative stability, cold flow properties and viscosity of the fuel42. Production of liquid biofuels, such as biodiesel from microalgae involves to some extant or more extraction or conversion before the algae can be used as a biofuel. The direct uses of microalgae biomass do not involve such processing and thus minimize the cost. The use of dried powder of the microalgae Chlorella as fuel together with a mixture of 85% cellulose powder and 15% diesel to run a diesel engine was reported18. A high calorific value of the microalgae is essential to be act as a fuel, which chiefly depends on the lipid content besides carbohydrate and protein. An increasing calorific value is directly related to an increase in lipid content18. The present study was focused on influences of salinity on growth and lipid yield of a native Indian strain of the freshwater microalgae A.falcatus towards potential biomass utilization. The principal objectives were to investigate the influences of medium compositions and salinity on growth, lipid yield and calorific value of the biomass. Material and MethodsMicroalgae Strain Isolation: Freshwater microalgae A. falcatus was isolated from water samples collected from the wetland Deepor Beel (latitude 26 13´ N and longitude 91° 66´ E), Guwahati, Assam, India. Isolation of the microalgae and its mono-culture was raised in BG11 medium after several dilutions and streaking into agar plate according to following standard procedure43, 44. Purity of the culture was periodically checked using an optical microscope (LOBAMED, Model: ATC 2000) as well as streaking into agar plates. Subculture was done after every third week of inoculation. The isolated strain was studied under optical microscope for morphological identification. Microphotographs were taken with the help of a digital camera (SONY Model: DSC-W100; 8.1 MP). Growth medium and Culture conditions: Monocultures were raised and maintained aseptically in autoclaved BG11 medium under laboratory conditions. The various media compositions used in this investigation are depicted in table - 1. Culture pH was adjusted to 7.5 with either 1N HCl or 1N KOH prior to autoclaving. Growth conditions were: irradiance 40 mol photons m-2-1, temperature 25 ± 2 C and photoperiod of 16 h light: 8 h dark diurnal cycles. Table - 1 Culture medium composition Constituents (g L - 1 ) BG11 NaNO 3 1.5 CaCl 2 2H 2 O 0.036 MgSO 4 7H 2 O 0.075 K 2 HPO 4 0.4 EDTA 0.001 Citric acid 0.006 Na 2 CO 3 0.2 NaMoO 4 2.1 x 10 - 5 ZnSO 4 7H 2 O 2.2 x 10 - 4 MnCl 2 4H 2 O 1.8 x 10 - 3 CuSO 4 5H 2 O 0.8 x 10 - 5 H 3 BO 4 2.8 x 10 - 3 Influences of Salinity on Growth and Total Lipid Content: Salinity induced influences on growth responses of the microalgae strain were studied in BG11 medium supplemented with NaCl in increasing order (40, 80, 160 and 320 mM). BG11 medium without addition of NaCl was used as control. The study was conducted in batch mode using 100 ml of medium in 250 ml volume Erlenmeyer flasks. Exponentially growing culture (20% v/v) was used as inoculums. Studies were conducted in triplicate under similar growth conditions as stated earlier. Cells harvested after 10 days of culture durations were compared in terms of total lipid content in each of the treatments. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 27-35, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 29 Growth Evaluation and Biomass Determination: Growth characteristics were determined from the respective growth curves developed by plotting the observed cell numbers (cells/ml) against time of observations (days). Cell numbers were determined from direct cell counts using a Neubour Haemocytometer. The specific growth rate () and doubling time () were calculated using equations (1) and (2)45: = ln () / () (1) = 0.6931/µ (2) Where, and are the biomass at time () and time (), respectively. Biomass was determined as dry cell weight (DCW) and measured gravimetrically. A known volume of culture was filtered through pre-weighed and pre-combusted GF/C filter paper. The filtered cell mass was oven dried at 80°C for 6 hrs until constant weight, cooled down to room temperature in desiccators and measured the dry weight of the sample using an analytical balance with a precision of 0.1 mg. Biomass was expressed in grams per liter (g L-1). Determination of Total Cellular Contents: Total cellular protein and carbohydrate contents were determined from a known amount of freeze dried cell biomass harvested at the end of the logarithmic phase of growth following standard procedures46,47. Total lipid content was determined quantitatively. A known volume of culture was harvested during the late exponential phase of growth by centrifugation at 2000 for 2 minutes, rinsed with 1% NaCl solution, followed by rinsing with double-distilled water twice. The biomass was lyophilized using a freeze-dryer (ALPHA 1- 4, Germany) for 36-48 hrs and kept in desiccators until constant weight. Total lipid was extracted from 1 g of the lyophilized cell biomass using a solvent mixture of chloroform and methanol (2:1)48. The residue was re-extracted (2 to 3 times) with chloroform. The filtrate was taken in a separatory funnel, and lipids were obtained in the bottom chloroform layer formed upon addition of 0.9% NaCl and collected in a grease-free cleaned pre-weighted glass vial. The solvent was removed using a rotary vacuum evaporator (JSGW, India) at 50 şC to near dryness and dried in desiccators under reduced pressure over anhydrous silica gel (ACS, Sigma-Aldrich, USA). The dried lipid extract was measured gravimetrically and expressed as percent total lipid dry cell weight (% DCW). Analysis of Fatty Acid Methyl Ester (FAME): Approximately 20 mg of crude lipid extract was used to prepare the FAME. Transestrification of the lipid sample was performed using a reaction mixture of methanol: acetyl chloride (20:1 v/v) 49. Crude lipid (20 mg) was taken in Teflon lining glass vial and added 2 ml -hexane containing internal standard methyl C-13 (2 mg ml-1). Freshly prepared above stated methylation reaction mixture (5 ml) was then added to the mixture. The vial was filled with nitrogen gas and mixed well by vortexing at low speed followed by incubation at 75 C for 60 min. The mixture was allowed to cooled down to room temperature (30 ± 2 C), then added 5 ml of double-distilled water containing 2.5% CO and 2 ml of chloroform: -Hexane (1:4 v/v) followed by brief vortexing for about 30 to 40 seconds. The mixture was centrifuged at 1957 for 1 min and the upper hexane layer containing FAME was collected in a clean glass vial. The extracted FAME was dried by passing through anhydrous NaSO and analyzed using a gas chromatograph (DANI GC1000, Italy) equipped with a flame-ionized detector and DN WAX capillary column (30 m x 0.25 mm x 0.25 m). The column temperature was increased from 150 C to 230 C at a rate of 3.5 C min-1. Fatty acid components were identified by comparing the retention time against known standard. Determination of Calorific Value (CV): Biomass energy value in terms of calorific value (CV) was determined using an automatic adiabatic bomb calorimeter (Model: RBC 106/09) according to DIN 51900 T3 (testing of solid and liquid fuels, determination of gross calorific value by the bomb calorimeter and calculation of net calorific value; method with adiabatic jacket)18. A crucible containing moisture-free dry algal biomass (1 g) was ignited in presence of oxygen (99.99% purity) at 3000 kPa. Calorific value was calculated from the resulting increase of water temperature using equation (3). CV = (WE x WM) x (4.18400 x 10-3) kJ g-1 (3) Where, enthalpy of water equals to 2568.293 cal g, T differences in temperature, M sample weight Results and DiscussionMicroscopic observations revealed that the cells are fusiformis, solitary, or clustered of individual cells without colonial sheath; chloroplast parietal, pyrenoids absent, variable in size within the same plant mass; cells are 30 to 36 long and 2.5 to 4.5 in diameter (figure - 1). Based on the morphological data the newly isolated native strain of the freshwater microalga was initially identified as Ankistrodesmus falcatus (Corda) Ralfs, (Class: Chlorophyceae; Family: Selenastraceae). Figure - 1 A.falcatus cells under optical microscope (magnification 400x) International Research Journal of Biological Sciences ________________________________________________ Vol. 1(8), 27-35, December (2012) International Science Congress Association Influence of Salinity on Growth and Total Lipid Content: Influence of salinity (NaCl concentration) on growth and lipid content of the tested A.falcatus strain was observed with the growth beh avior, grown in BG11 medium supplemented with NaCl in increasing order. The strain was found to grow in all the five concentrations of NaCl (40 to 320 mM) (figure result was obtained in medium supplemented with 160 mM followed by 80 and 40 mM of NaCl. Growth rate was however retarded in the culture medium containing 320 mM of NaCl. Culture medium containing 160 mM of NaCl also revealed a net increase in cell numbers (29 x 10 ml-1 ) compared to that of control medium (15.2 x105 ml-1 ) at the termin the experiment (table 2). The highest specific growth rate (0.313 -1 ) and least doubling time (2.21 days) were observed in the medium supplemented with 160 mM of NaCl compared to control medium (0.209 d-1 and 3.32 days respectively). Ho wever, further increase in salt concentration beyond 320 mM retarded the growth of the studied microalgae strain. Similar finding on enhanced growth at low salinity level was also reported in Chlorella vulgaris and Chlorococcum humicola Significant increase in growth rate of C.vulgaris low salinity level of 50 to 100 mM. Further increase in salinity beyond 200 mM retarded the growth in both C.humicola . Similarly, increase in biomass production was reported in B.braunii at low salinity level of 17 to 34 mM However, the growth rate was significantly reduced at higher concentration of NaCl. Based on the salt tolerance extent algae were grouped as halophilic (salt is required for optimum growth) and halotolerant (having response mechanism to survive in saline condition). In both cases, the algae produce some secondary metabolites in order to cope up with salinity induced changes in growth conditions and also balance as per the surrounding osmotic51. Total lipid, carbohyd rate and protein contents were also found to be improved in medium containing NaCl up to 160 mM (table 2). Salinity induced enhanced lipid and other biochemical contents (such as keto carotenoid astaxanthin) were reported in a number of microalgae species34, 52-54 . The studied strain under a salinity stress revealed improved total lipid content. The highest lipid content (55.3% DCW) was recorded in the cultures containing 160 mM of NaCl, which was nearly 1.4 fold increase in total lipid content than the total lipid content (38.3% DCW) of cultures grown in control medium. However, slightly reduced amount of total lipid content (52.5% DCW) was recorded in culture grown at the highest salinity level (320 mM), which might be resulted due to the produ of less biomass in medium with highest salinity. Similarly, carbohydrate and protein content were also found to be increased with the increasing salinity. The highest carbohydrate (14.5% DCW) and protein (4.8% DCW) contents were recorded in the mediu m containing 160 mM of NaCl and the least (carbohydrate 12.6% and protein 3.1%) in control medium. Similar observation was also reported34 in the hydrocarbon rich B.braunii culture. International Research Journal of Biological Sciences ________________________________________________ International Science Congress Association Influence of Salinity on Growth and Total Lipid Content: Influence of salinity (NaCl concentration) on growth and lipid strain was observed with the avior, grown in BG11 medium supplemented with NaCl in increasing order. The strain was found to grow in all the five concentrations of NaCl (40 to 320 mM) (figure - 2). Best result was obtained in medium supplemented with 160 mM NaCl. Growth rate was however retarded in the culture medium containing 320 mM of NaCl. Culture medium containing 160 mM of NaCl also revealed a net ) compared to that of ) at the termin al day (day 10) of the experiment (table 2). The highest specific growth rate (0.313 ) and least doubling time (2.21 days) were observed in the medium supplemented with 160 mM of NaCl compared to and 3.32 days respectively). wever, further increase in salt concentration beyond 320 mM retarded the growth of the studied microalgae strain. Similar finding on enhanced growth at low salinity level was also Chlorococcum humicola 50. C.vulgaris was reported at low salinity level of 50 to 100 mM. Further increase in salinity beyond 200 mM retarded the growth in both C.vulgaris and . Similarly, increase in biomass production was at low salinity level of 17 to 34 mM 34. However, the growth rate was significantly reduced at higher concentration of NaCl. Based on the salt tolerance extent algae were grouped as halophilic (salt is required for optimum response mechanism to survive in saline condition). In both cases, the algae produce some secondary metabolites in order to cope up with salinity induced changes in growth conditions and also balance as per rate and protein contents were also found to be improved in medium containing NaCl up to 160 mM (table 2). Salinity induced enhanced lipid and other biochemical contents (such as keto carotenoid astaxanthin) were reported in . The studied A.falcatus strain under a salinity stress revealed improved total lipid content. The highest lipid content (55.3% DCW) was recorded in the cultures containing 160 mM of NaCl, which was nearly than the total lipid content (38.3% DCW) of cultures grown in control medium. However, slightly reduced amount of total lipid content (52.5% DCW) was recorded in culture grown at the highest salinity level (320 mM), which might be resulted due to the produ ction of less biomass in medium with highest salinity. Similarly, carbohydrate and protein content were also found to be increased with the increasing salinity. The highest carbohydrate (14.5% DCW) and protein (4.8% DCW) contents were recorded m containing 160 mM of NaCl and the least (carbohydrate 12.6% and protein 3.1%) in control medium. in the hydrocarbon rich Figure - 2 (A Influence of salinity on growth of characteristics under increasing order of salinity; (B) Increased in cell numbers and specific growth under various salt concentrations. Error bars represent standard error (n=3) Fatty Acid Methyl Ester (FAME) profile: compositions of A.falcatus biomass grown in control BG11 medium and under salinity- stressed (BG11 + 160 mM NaCl) conditions were evaluated (t able 3). FAME analysis revealed nearly similar fatty acid profile in the both culture conditions. Palmitic (C16:0), oleic (C18:1), linolenic (C18:2), and linolenic (C18:3) acids were found to be the major components of the oil derived from A.falcatus strain. The percentage of saturated fatty acids (SFA = 36.2%) was higher in culture grown under normal BG11 medium than the SFA (32.1%) monounsaturated fatty acid (MUFA = 31.1%) and polyunsaturated fatty acid (PUFA = 27.9%) were significantly higher in culture grown under NaCl stress condition. The percentage trienoic fatty acids (TFAs) were similar to that of C18:3 as other TFAs were not detected. Among the SFAs, C16:0 was the most dominant fatty acid of the studied A.falcatus strain in both normal and stress conditions (28.6% International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 30 2 (A -B) Influence of salinity on growth of A.falcatus. (A) Growth characteristics under increasing order of salinity; (B) Increased in cell numbers and specific growth under various salt concentrations. Error bars represent standard error Fatty Acid Methyl Ester (FAME) profile: FAME biomass grown in control BG11 stressed (BG11 + 160 mM NaCl) able 3). FAME analysis revealed nearly similar fatty acid profile in the both culture conditions. linolenic (C18:2), and linolenic (C18:3) acids were found to be the major components of the oil strain. The percentage of saturated fatty acids (SFA = 36.2%) was higher in culture grown under normal BG11 medium than the SFA (32.1%) of stress culture. Both monounsaturated fatty acid (MUFA = 31.1%) and polyunsaturated fatty acid (PUFA = 27.9%) were significantly higher in culture grown under NaCl stress condition. The percentage trienoic fatty acids (TFAs) were similar to that of as other TFAs were not detected. Among the SFAs, C16:0 was the most dominant fatty acid of the studied strain in both normal and stress conditions (28.6% International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 27-35, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 31 and 26.9% respectively). Oleic acid (30.5%) was the most dominant fatty acid of MUFAs, which increased significantly in culture grown under stress condition. Although the amount of C18:3 (15.7%) was slightly reduced in culture grown under salt stress condition than normal culture condition (16.7%), overall PUFA content was higher under stress condition. The degree of unsaturation (DU) calculated from individual fatty acid contents was also found higher in the studied A.falcatus strain grown under salinity stress (86.9%) than the DU of control medium (79.5%). The percentage of very long chain fatty acids (VLCFAs) was higher in the studied A.falcatus biomass grown in control medium (4.3%) compared to the VLCFAs of biomass grown under salinity stress (2.3%). Irrespective of the lipid types found in the different taxa of freshwater Chlorophyceae, fatty acids C16:0, C18:1, C18:2 and C18:3 were reported as the common fatty acid types55. In the present study, the FAME analysis of the native A.falcatus strain revealed C16:0, C18:1 and C18:3 were the major fatty acid components in the both control and salinity stress conditions of which, C16:0 and C18:1 contributed 28.6% and 26.8% relative to the control. The results were in accordance with the results from previous studies in Chlorella zofingiensis grown in presence of glucose56, 57. Significant increase in C18:1 (30.5%) was observed under salinity stress condition. Similar observation was also reported in the marine microalgae Dunaliella salina58. Among the PUFAs, slightly higher content (16.7%) of - linolenic (C18:3) acid was observed in the studied A.falcatus strain grown in control medium than in NaCl stress condition (15.7%). Otherwise, the content of C18:2 (12.2%) was significantly increased under salinity stress than under control medium (9.2%). Palmitic, linoleic, and - linolenic acids were reported as the major fatty acids in Ankistrodesmus sp, C. vulgarisand Chlamydomonas reinhardtii59, 60. Table - 2 Growth Characteristic, Total Lipid Content and Calorific Value of A.falcatus strain under Salt Stress Conditions Salinity (mM NaCl) Specific growth -1) Doubling time (T days) Cell numbers (x10 ml-1) Lipid (% DCW) Calorific value (kJgm-1) Control 0.209 3.32 15.0 38.3 ± 0.20 19.1 ± 0.05 40 0.263 2.64 19.3 38.6 ± 0.12 19.7 ± 0.11 80 0.275 2.52 22.8 43.0 ± 0.15 23.6 ± 0.05 160 0.313 2.21 29.0 55.3 ± 0.01 27.9 ± 0.15 320 0.179 3.87 9.7 52.4 ± 0.22 26.7 ± 0.05 values are mean of three independent values; values are mean of five independent values, ‘±’ represents standard error of means Table - 3 Fatty Acid Methyl Esters (FAME) Compositions (% wt) of A.falcatus strain under Salt Stress and Normal Conditions Fatty acid Salinity (mM) 0.0 mM 160 mM Palmitic acid (16:0) 28.6 26.9 Stearic acid (18:0) 4.2 3.1 Oleic acid (18:1) 26.8 30.5 Linoleic acid (18:2) 9.2 12.2 Linolenic acic (18:3) 16.7 15.7 Behenic acid (22:0) 0.5 0.8 Erucic acid (22:1) 0.9 0.6 Lignoceric acid (24:0) 2.9 1.3 Unidentified 10.2 8.9 SFA a 36.2 32.1 MUFA b 27.7 31.1 PUFA c 25.9 27.9 TFAs d 16.7 15.7 VLCFAs e 4.3 2.7 DU f 79.5 86.9 SFA (%wt) = Cn:0, b MUFA (%wt) = Cn:1, c PUFA (%wt) = Cn:2 + Cn:3, d TFA (%wt) = Cn:3, VLCFAs (%wt) = C20:n + C22:n + C24:n, DU ( %wt) = Cn:1 + 2 x ( Cn:2 + Cn:3) International Research Journal of Biological Sciences ________________________________________________ Vol. 1(8), 27-35, December (2012) International Science Congress Association Calorific Value (CV): The calorific value of the studied A.falcatus biomass was found to vary with total lipid content that influenced by increasing salinity (table value was improved with the increasing salinity up to 160 mM, which di rectly influenced the improved lipid content of the biomass (figure - 3A). The highest calorific value (27.9 kJ g was obtained with biomass produced from culture grown under 160 mM of NaCl. The microalgae A.falcatus BG11 medium containi ng a relatively less total lipid (38.3%) content revealed comparatively lesser CV (19.1 kJ g 2), which was in close agreement with earlier report principal contribution to the CV of cell biomass is from their total cellular carbohydrate, protein and lipid content Microalgae grown under normal conditions were reported to have CVs between 18 to 21 kJ g-1 , which is much lower than the CV of mineral diesel (42 kJ g-118 . However, an improved calorific value was reported by improvin g the total lipid content of the biomass18,61 , and also evident from the results of our present study (table - 2, figure - 3). The tested grown in BG11 medium containing elevated level of NaCl yielded an increased level of total lipid con increasing CVs (table - 2). Similar results were also reported by in C.vulgaris and C.emersonii18 . A linear trend of increasing CV with increasing total lipid content of the studied microalgae A.falcatus strain was observed with a value of - 3B). It is envisaged that microalgae biomass would be used as renewable biofuel for electricity generation using static diesel engines for which large microalgae biomass production with high productivity and high lipid content must need maintained18, 62. The native A.falcatus strain achieved a better growth and thereby higher biomass yield in BG11 medium supplemented with 160 mM NaCl compared to control medium (0.0 mM NaCl). The lipid content reached up to 55.3% with an increasing CV (27.9 kJ g-1 ), under the salt stressed condition. As seen from figure - 3B, a linear increase (R = 0.949) of CV with increasing lipid content was observed, which signify that the intercellular lipid content together with carbohydrate and protein largel y influence the resultant CV of the tested A.falcatus cell biomass. The results were in accordance with previous reports 18, 61-63. Conclusion Salinity induced improved growth rate, total lipid content, and energy value in terms of calorific value was rec studied native strain of oleaginous freshwater microalgae A.falcatus biomass. Salinity- induced improved growth and lipid content were evident from the salinity tests using BG11 with increasing concentration of NaCl. SFA (32.1%) was decreased under salinity stress, while MUFA (31.1%) was increased. Content of C18:1 (30.5%) was improved under salinity than that of control (26.8%). (15.7%) content was decreased slightly in the FAME contents of International Research Journal of Biological Sciences ________________________________________________ International Science Congress Association The calorific value of the studied biomass was found to vary with total lipid content that influenced by increasing salinity (table - 2). The calorific value was improved with the increasing salinity up to 160 mM, rectly influenced the improved lipid content of the 3A). The highest calorific value (27.9 kJ g -1) was obtained with biomass produced from culture grown under A.falcatus grown in control ng a relatively less total lipid (38.3%) content revealed comparatively lesser CV (19.1 kJ g -1) (table - 2), which was in close agreement with earlier report 18. The principal contribution to the CV of cell biomass is from their protein and lipid content 18, 61, 62. Microalgae grown under normal conditions were reported to , which is much lower than the . However, an improved g the total lipid content , and also evident from the results of our 3). The tested A.falcatus strain grown in BG11 medium containing elevated level of NaCl yielded an increased level of total lipid con tent revealed 2). Similar results were also reported by . A linear trend of increasing CV with increasing total lipid content of the studied microalgae strain was observed with a value of = 0.955 (figure It is envisaged that microalgae biomass would be used as renewable biofuel for electricity generation using static diesel engines for which large microalgae biomass production with high productivity and high lipid content must need to be strain achieved a better growth and thereby higher biomass yield in BG11 medium supplemented with 160 mM NaCl compared to control medium (0.0 mM NaCl). The lipid content reached up to 55.3% with an ), under the salt stressed condition. As = 0.949) of CV with increasing lipid content was observed, which signify that the intercellular lipid content together with carbohydrate and y influence the resultant CV of the tested cell biomass. The results were in accordance with Salinity induced improved growth rate, total lipid content, and energy value in terms of calorific value was rec orded in the studied native strain of oleaginous freshwater microalgae induced improved growth and lipid content were evident from the salinity tests using BG11 with was decreased under salinity stress, while  MUFA (31.1%) was increased. Content of C18:1 (30.5%) was improved under salinity than that of control (26.8%). TFA content was decreased slightly in the FAME contents of lipid fraction under salinity stress, however, content was marginally increased compared to that of control (25.9%). The VLCFA (2.7%) content was decreased considerably. The DU (86.9%) was increased considerably under salinity stress in comparison to control (79.5%). T of the studied A.falcatus strain biomass grown under normal BG11 medium revealed 19.1 kJ g-1 , which was much lower than that of petroleum diesel (42 kJ g-1 ). CV was improved up to 27.9 kJ g-1 in biomass produced under salinity stress. Further culture optimization for increased biomass yield with concomitant lipid content and improved of fatty acid profile, the native could be a potent biomass feedstock towards biofuel production. With the support from present research findings the native st of freshwater oleaginous microalgae potential renewable biomass source towards production biofuel, although further studies are needed for optimization of mass cultivation of the strain for utilization as renewable biomass feedstock of biofuels. Figure - 3 (A Calorific value and total lipid content of under salinity stress. (A) Increase in total lipid and calorific value with increasing salinity; (B) Linear increase in calorific value with increase in total lipid content of biomass (R2 = 0.949) was observed. Error bars represent standard error (n=3) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 32 stress, however, PUFA (27.9%) content was marginally increased compared to that of control VLCFA (2.7%) content was decreased The DU (86.9%) was increased considerably under salinity stress in comparison to control (79.5%). T he CV strain biomass grown under normal , which was much lower than ). CV was improved up to 27.9 in biomass produced under salinity stress. Further culture optimization for increased biomass yield with concomitant lipid content and improved of fatty acid profile, the native A.falcatus could be a potent biomass feedstock towards biofuel production. With the support from present research findings the native st rain of freshwater oleaginous microalgae A.falcatus was found to be potential renewable biomass source towards production biofuel, although further studies are needed for optimization of mass cultivation of the strain for utilization as renewable biomass 3 (A -B) Calorific value and total lipid content of A.falcatus biomass under salinity stress. (A) Increase in total lipid and calorific value with increasing salinity; (B) Linear increase in calorific value with increase in total lipid content of biomass = 0.949) was observed. Error bars represent standard (n=3) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 27-35, December (2012) Int. Res. J. Biological Sci. 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