Selection of Indigenous Algal Species for Potential Biodiesel Production

Currently, India utilizes an enormous amount of fossil fuels and a major quantity of fossil fuels are imported from other countries. It’s a giant load on the Indian Economy. The burning of fossil fuels causes global warming. Carbon neutral, renewable fuels are essential for environmental protection and it’s economically sustainable for India. Biofuels attention day by day due to a rise in energy demands and environmental concerns. Biodiesel produced from algal oil a possible renewable and carbonneutral substitute to fossil fuels. The feasibility of the algal-based biodiesel industry depends on the selection of adequate species regarding commercial oil yields and oil quality. Present research work to bioprospecting and screening of 19 algal and blue-green algal species, the oil percentage and the fatty acid profiles, used for analyzing the biodiesel fuel properties. Oil from Tolypothrix phyllophila algal strain and compared it with another eighteen algal and blue-green algal strains from different literature. Tolypothrix phyllophila algal strain contains approximately 12.6% lipid on a dry weight basis. We also compared the FAME profile of 19 algal and blue-green algal strains and calculated and compared the fuel properties such as cetane number, Iodine Value, etc. of the biodiesel derived from these algal and blue-green algal oils based on chain length and saturation. We also investigated the 19 algal and blue-green algal fatty acid profiles and its suitability for biodiesel production and strains selection through PROMETHEE (Preference Ranking Organization Method for Enrichment Evaluations) and GAIA (geometrical analysis for interactive aid) analysis.

of potential feedstocks (algae and blue-green algae) if the fatty acid composition is well-known.
Suitability of any material as fuel, including biodiesel, is depend on the skeleton of the fatty acids as well as ester derived from the base alcohol 16  In the current study, we investigated the lipid content in Tolypothrix phyllophila and FAME examinations of lipid extracted by this strain by GC-MS. We also compared the FAME profile of 19 algal and blue-green algal species. On the basis of FAME profile of fatty acids of these 19 algal and blue-green algal species, we calculated the fuel properties such as Cetane Number, Iodine Value, Saponification Value, Degree of Unsaturation, Long Chain Saturation Factor, and Cold Filter Plugging Point. Based on these fuel properties, we compared the biodiesel produced by these 19 algal and blue-green algal species by PROMETHEE and GAIA software.
The current research describes the importance of the fatty acid profile on these fuel properties. Not all of these fuel properties have been incorporated as specifications in biodiesel standards, although all of are necessary for the optimal performance of the fuel.

MATERIAlS And METHOdS Algal Culture
Tolypothrix phyllophila (NAIMCC-C-00047), a microalgae strain was collected from ICAR-National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, Uttar Pradesh, India. A slant culture was procured and subcultured in BG-11 media 17 for further studies at the Harcourt Butler Technical University, Kanpur. We also took the 18 algal and blue-green algal species ( Table no.

1) from different literature for comparative analysis. lipid content and FAME Analysis
Total lipid content of Tolypothrix phyllophila was estimated using the Soxhlet method 18 , as a percentage of the total biomass (in % dry weight). The algal strain was grown aseptically. Algae pellet was dried at 80°C for 4 h in hot air oven (Navyug Udyog, Haryana, India). The

Journal of Pure and Applied Microbiology
Soxhlet extraction unit (Borosil, India) was filled with the solvent mixture containing Chloroform: Methanol (2:1) 19 in the heating mantle (Perfit, India) of the unit, followed by heating the solvent mixture to extract the lipid out of the dried algal sample from a thimble, which is subsequently collected in the bottom flask. The solvent was then separated from the lipid content by the distillation process. The mixture was heated to the boiling point of the solvent that selectively evaporate the solvent, leaving behind the lipid content. The lipid content was measured based on the weight of lipid extract compare to the dry weight of algal biomass.
The resultant methyl esters was evaluated by Gas Chromatograph-Mass Spectrometry (GC-MS, model 7890/5977B, Agilent Technologies, USA) 25 connected to autosampler, split-splitless injector, flame ionization detector (FID), and a 60 m BPX70 capillary column (SGE, Ringwood, Australia) having 0.25 mm internal diameter and 0.25 µm film thickness. Samples of 1 µL were injected at 60°C, held for 3 min, before raising by 40°C/min to 150°C and then sequentially by 1.5°C/min to 230°C. The constant flow mode of helium carrier gas with an estimated average velocity of 30 cm/s was used in the analysis. As per manufacturer instructions, injector and detector temperatures were set at 250°C and 300°C, correspondingly. The fatty acid profile was recorded using Mass Hunter workstation software version B.04.07 by Agilent Technologies USA.
The analysis showed that in the above 19 algal and blue-green algal strains, Microcystis aeruginosa has the highest percentage of oil (28.15%) in comparison to other algal and bluegreen algal strains, while Phormidium purpurescens species have 26.45% of oil. The Phormidium ambiguum has a minimum oil percentage (10.48%) in comparison to other strains.

Calculation of Biodiesel Properties Based on FAME Profiles
Based on oil percentage we could not decide that this is the most excellent algal strain for biodiesel production, because the fuel properties of microalgal biodiesel were dependent on fatty acid profile 29 . Continuous research has been undertaken to improve the quality of algal biodiesel. Biodiesel production and its commercialization were done since the 1980s. Commercialization requires the standardization of algal biodiesel. US, Europe, the Republic of South Africa, Brazil, Australia and India now have their biodiesel guidelines 30,31,32 .
Fatty acid methyl ester (FAME) of algal biodiesel may change according to carbon chain sizes and the position and/or number of double bonds 16,33 . The main properties of biodiesel such as Iodine value (IV), Cetane Number, oxidation stability, and Cold Filter Plugging Point (CFPP) was directly influenced by these molecular structures 16,34,35,36 . Biodiesel properties such as Saponification Value (SV), Cetane Number (CN), Iodine Value (IV), Degree of Unsaturation (DU), Cold Filter Plugging Point (CFPP), and Long Chain Saturated Factor (LCSF) were determined by the empirical equations.

Cetane number
The cetane number (CN) is the determination of the ignition properties of the fuel. It is connected to the ignition delay time 30; 31,37,38 . Generally, the higher the CN, the better the ignition quality of the diesel and vice versa. The CN of fuel depends upon the structure of the compound comprising the mixture. The CN increases with increasing saturation and increasing chain length. Aromatic and branched compounds have a low cetane number 38,39 .
For the No. 2D diesel ASTM standard was determined to be 53.2 min in the case of the cetane number 40 . According to Indian Standards (IS 15607:2005), the minimum cetane number for Biodiesel (B 100) is 51 41 .
The CN of the FAME mixture was calculated by the empirical equation given by Krisnangkura 42 . The CN can be calculated with the following equation no. 1 43,44 .

Saponification value
Saponification Value (SV) means milligrams of potassium hydroxide required to saponify 1 g of oil, it is inversely connected to the esters molecular weight. It can be calculated by The Degree of unsaturation was calculated by the following equations no. 4 43; 44 ... (4) Where MUFA means the weight percentage of Monounsaturated fatty acids and PUFA means the weight percentage of Polyunsaturated fatty acids.

Cold-flow characteristics
More saturated and longer chain FAMEs have higher melting points in compare to shorter chains and more unsaturated FAMEs. Fuels with a huge concentration of SFAs can have poor coldflow properties and unsuitable viscosities. At low temperatures, saturated components of the fuel crystallize and precipitate causing troubles, such as clogged pumps, fuel lines, filters, and injectors 16,31,33,45 . To avoid problems with cold flow properties, it is essential for biodiesel have relatively high levels of unsaturated FAMEs and so relatively low concentrations of saturated FAMEs 45 .
The Long Chain Saturation Factor (LCSF) was measured by the following equations no. 5.
It should be well-known that the properties of Fatty Acids/Fatty Acid Mehtyl Esters that impart a favorable Cetane Number on a fuel (long-chain and low degree of unsaturation) impart minimum cold-flow properties and viscosities 39,47 . It is, so, essential to achieve a balance between Cetane Number and cold-flow properties. Fatty Acid Methyl Esters that attain this balance are the monounsaturated methyl hexadecenoate (C16:1) and methyl octadecenoate (C18:1) 48,49 . Biofuels rich in these Fatty Acid Methyl Esters will have good enough Cetane Numbers, viscosities, and cold-flow parameters. The ideal biodiesel fuel feedstock would be composed totally of C18:1 and C16:1 Mono Unsaturated Fatty Acids 48 , thus in practice, a biodiesel feedstock should contain hudge concentrations of C18:1 and C16:1, that are as hudge as feasible and so maintain the concentrations of all other Fatty Acids as minimum as feasible. However, feedstocks will contain mixed Fatty Acid compositions and will have non-ideal Fatty Acids. It is, so, essential to quantify the maximum acceptable levels of other Fatty Acids (not Mono Unsaturated Fatty Acids) in biodiesel fuel and to make this model that predicts the fuel properties such as CN, CFPP, and viscosity from FA composition have to be used.

Selection of Suitable Algal Species for Biodiesel
The selection of best algal strains done by using PROMETHEE and GAIA analysis 29,50 . with the selected criteria Yield, CN, SV, IV, CFPP. Here we use Visual PROMETHEE Software 1.4.0.0 Edition for comparative analysis of algal and blue-green algal strains. We draw a graph by PROMETHEE-GAIA software with multiple criteria by giving equal weightage to all criteria. This graph a comparative analysis and gives the phi value to all 19 strains. Based on phi value the best strain for biodiesel production.
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Statistical Analysis
A variety of multi-criteria decision analyses (MCDA) are available. Here we utilized Preference Ranking Organisation Method for Enrichment Evaluation (PROMETHEE) and Graphical Analysis for Interactive Assistance (GAIA) for selection of best algal strains. These softwares are utilized in various research paper for suitability analysis of algal strain for biodiesel production.

lipid content and FAME Analysis
Based on the Soxhlet method, Tolypothrix phyllophila culture was estimated to contain approximately 12.6% lipid on a dry weight basis. The lipid content obtained by the above method was converted into esters of fatty acids (biodiesel) by standard transesterification method, and the resultant product was analyzed by GC-MS. FAME analysis led to the identification of constituent of the trans-esterified product or biofuel, principally containing, Palmitic acid (C 16:0 ) (31.6 %) and oleic acids (C 18:1 ) (23.3 %) in excess amount ( Table 2). We also observed that Tolypothrix phyllophila contains 46.7 % total saturated fatty acids, 28.7 % MUFA, and 20.5 % PUFA.

Calculation of Biodiesel Fuel Properties Based on Fatty Acid Methyl Esters Profiles
Based on FAME profiles of algal and blue-green algal strains, we estimated the total saturated fatty acids, MUFA, and PUFA present in different algal and blue-green algal species as shown in Table 3.
On the basis of the equations no. 1 to 6, we calculated the CN, SV, IV, DU, LCSF, and CFPP as shown in table 4.

Selection of best Algal Strain for Biodiesel production
To be a perfect source of sustainable biodiesel, the selected algal strain should have the sufficient proportion of lipids with suitable fatty acids. A multi-criteria decision method (MCDM) software PORMETHEE-GAIA was used to construct objective selections for hudge amount production.
The selection of suitable species was done based on above calculated biodiesel fuel properties (CN, IV, SV, DU, LCSF, and CFPP) with the help of PROMETHEE-GAIA software.
The    51 . The decision vector shows the most appropriate strain, i.e., those that align with the direction of this vector, and the outermost criteria in the direction of the decision vector are the most preferable 52 .
In contrast, Db > 4, SAFs, and C18:3 were extremely variable criteria (Table 4) and they had a strong effect on the decision vector. According to Fig. 2 and the calculated outranking flows, the most appropriate strains are Chlorella sp. and Phormidium ambiguum with the highest phi value in all 19 species (Fig. 2, Table no. 5). Calothrix fusca and Lyngbya dendrobia remained their low ranking with low phi value and are the least appropriate strain for biodiesel production.

DisCussiON
Biofuels are the current need of the world, given the depleting carbon resources and the footprint it generates. Microalgae are one of the potential sources of bio-fuels.
Oil content (% in dwt) and growth rate have been the two criteria for the success of large scale cultivation of algae for biofuel production 53 . The qualitative lipid composition and the lipid volumetric productivity should be considered as the most suitable parameters to facilitate decision making on strain selection for biodiesel production 54 .
The present study, pertains to the analysis of lipid content in Tolypothrix phyllophila. The algal strain is native to the Baharaich region (Geographical coordinate 27.7525° N, 81.4279° E) of Uttar Pradesh, India.
In this present study Tolypothrix phyllophila culture was estimated to contain approximately 12.6 % lipid on a dry weight basis. The present study showed that among 19 algal and blue-green algal strains the lipid % on dry weight varies between 10.48 to 26.45 %, in these strains more than 25 % lipid on a dry weight basis found in Chlorella sp. 26 % 27 , Oscillatoria calcuttenis 25.7 %, Phormidium purpurescens 26.45 % lipid on a dry weight basis 28 .  FAME of Tolypothrix phyllophila, principally containing, C 16:0 (up to 31.6%). It is also rich in oleic acids (C 18:1). In the present study, we also compare the FAME of Tolypothrix phyllophila and 18 algal and blue-green algal strains found in India, with different literature. FAME analysis shows that highest palmitic acid (C 16:0) was found in Scytonema bohnerii and highest total saturated fatty acid was also found in Scytonema bohnerii, highest Oleic acid (C 18:1) and total monounsaturated fatty acid were found in Lyngbya limnetica, highest linoleic acid (C 18:2) was found in Scytonema bohnerii, and highest linolenic acid (C 18:3) was found in Chlorella sp., and total polyunsaturated fatty acids were found in Chlorococcum sp. and total highest total unsaturated fatty acids were also found in Chlorococcum sp. Microalgae rich in MUFAs (particularly, oleic acid (C 18:1) and palmitoleic acid (C 16:1) and SFAs are suitable for biodiesel production 55 .

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Various types of lipids, such as glycolipids, phospholipids, monoglyceride, diglyceride, and triglycerides, among others are produced by algae  42 . This parameter, on the other hand, is highly changeable Journal of Pure and Applied Microbiology because it is also directly linked by the technology used for biodiesel production.
The Iodine Value is a parameter represents the Degree of Unsaturation, concerning with the weighted sum of the masses of Mono Unsaturated Fatty Acid and Poly Unsaturated Fatty Acid. It is significant for biodiesel oxidative stability. High unsaturation levels may result in the polymerization of glycerides and the synthesis of deposits 43 . In comparison to biodiesel from vegetable oils 42 most of the estimated Iodine Value for the biodiesels from the algal and blue-green algal species (Table 4) (Table 4) were below than, or similar to palm oil (48)(49)(50)(51)(52)(53)(54)(55)(56), indicative of lesser susceptibility to oxidative attack. Saturated fatty acids have higher melting points in compare to unsaturated fatty acids. When most saturated fatty acid esters molecules are present in biodiesel, crystallization may take place at temperatures within the usual engine operation range 43 . Biodiesel rich in stearic acid methyl ester and palmitic acid methyl esters tend to present a poor Cold Filter Plugging Point (similar to a higher temperature of plugging point) because when liquid biodiesel is cooled, these fatty acid methyl esters are the first to precipitate [56]. In the current research work, the percentage of Stearic acid (C 18:0) (  An equal weighted to parameters, PROMETHEE analyses recognized that the Chlorella sp. and Phormidium ambiguum outranked while Calothrix fusca and Lyngbya dendrobia are the least suitable species in above mentioned nineteen species for biodiesel production.

COnCluSIOn And FuTuRE PROSPECTS
The microalgae species isolated from other countries/continents may not perform better in India (Asia region) due to the remarkable variations in the regional climatic conditions. Therefore, domestic microalgal species must be isolated to serve the purpose. Indigenously isolated species may have adapted to the particular climatic conditions over their evolutionary period 57 .
In this study, 19 algal and blue-green algal strains were evaluated according to their lipid profiles and productivities. The total lipids percentage in dry biomass varied between 10.48 % (Phormidium ambiguum) to 26.45 % (Phormidium purpurescens).
As the physio-chemical properties of biodiesel are analysed by the molecular structures of the constituents fatty acid methyl esters, present research work proposes that the fattyacids composition of algae and blue-green algal oil must be main criteria for species selection, to make feasible the algal-based biodiesel industry.
According to their fatty acid profiles, various algae and blue-green algae exhibit immense potential to produce biodiesel within most of the biodiesel standards. Interestingly, most of the investigated species would hardly produce a lipid profile capable of fulfilling all the standard requirements for biodiesel production. But most of them harbour one or more main characteristics mandated for standard quality. Hence, it is worthwhile to generate designer biodiesel by calculated mixture of the distinct oil extracts, obtained from different species.
Realizing the importance of fatty acid methyl esters and the scarce information on the qualitative composition of algal oil, this research provides an important contribution for further bioprospection related to algae and blue-green algae for biodiesel production.