15 CHAPTER 3 BLUE GREEN ALGAE 3.1 GENERAL INTRODUCTION OF BLUE GREEN ALGAE Generally, blue green algae are microorganisms and show their natural presence in lakes and running water. BGA can be seen abundantly in areas with more sunlight and rich nutrient water sources. Cyanobacteria are oxygenic prototroph and constitute the diverse group of photosynthetic prokaryotes and grouped under gram negative bacteria. Cyanobacteria are photosynthetic in nature and aquatic microorganism. They are unicellular and are usually seen in colonies and clusters. They also act as important agent in fixation of atmospheric nitrogen and thereby help in cultivation with good supply of nitrogenous compounds. BGA represents a wide variety of photosynthetic species which may be autotrophic or heterotrophic in nature. The autotrophic BGA s uses sunlight to fix the carbon from CO 2 which is present in the atmosphere, while the heterotrophic BGA s consumes organic molecule present in the environment and convert them into building blocks of their own mainly lipids and proteins (Demirbas et al.2011). These autotrophic and heterotrophic algae mainly make use of sunlight which is abundantly and freely available and capable of synthesizing proteins, lipids, carbohydrates, astaxanthin, omega 3 fatty acids and many more.
16 The major requirements for the growth of BGA are land, sunlight, water, carbon-di-oxide, macro and micro nutrients (Chaumont 2005). They can be even grown on non agricultural land and in coastal areas as they will not create competition for food products. Water is necessary for the algal growth with the presence of nutrients and minerals. Even waste water from industries and residential areas can be treated and used for algal growth so the environmental impact and sewage disposal problems can be minimized to a great extent. Recently, oil rich microalgae which are non edible gains more importance as a better source for biodiesel. It is estimated that, there are more than 3,00,000 species of unicellular and multicellular algae. Stain Selection Growth media Lipid Triggers Growth of algae Carbon-di-oxide Micro & Macro Nutrients Power Generation Energy Ether & Hexane Harvesting & Oil Extraction Biomas Anaerobic Digestion NaOH Methanol Transesterific ation Glycerol Biodiesel Figure 3.1 Algal biofuel life cycle BGA s contain 30% to 75% of lipids (Cholesterol) by its dry weight and can be also called as oilgae (oleaginous algae). Algae are theoretically and practically a very best source of biodiesel. It takes in CO 2 as
17 photosynthetic reaction and gives out O 2 to the atmosphere as shown in Figure 3.1. Microscopic algae produce oil and lipids at higher rate when compared with Jatropha, Neem and Pongamia seed through photosynthetic reactions (Guan et al.2010). Advantages of BGA s The microbial biomass gets doubled in a short period of time. The doubling rate for microalgae is 2 to 6 hrs. The space required for mass culture is relatively low when compared with conventional biodiesel. Suitable nutrient along with sunlight and carbon-di-oxide can be a cheap raw material for the growth of BGA. Climatic conditions have minimal or negligible effect on the continuous culture and can be located anywhere. The algal biomass is rich in easily digestible proteins, lipids, carbohydrates and vitamins. Secondary metabolism in blue green alga produces alkaloids, cyclic peptides, terpens and lipopolysaccharides Amino acid composition can also be manipulated to increase the biomass and thereby increase the oil content. 3.2 TYPES OF OIL PRODUCING ALGAE There are various species of oil producing algae. The Table 3.1 shows the chemical composition of various algae, based on their protein, carbohydrates, lipids and nucleic acid content.
18 Table 3.1 Comparison of chemical composition of various oil yielding algae S.No Species Protein Lipids Carbohydrates Nucleic % % % acid % 1 Scenedesmus obliquus 50-60 12-14 10-17 3-6 2 Scenedesmus quadricauda 47-50 19-20 - - 3 Scenedesmus dimorphus 8-18 16-20 21-52 - 4 Chlamydomonas rheinhardii 48-50 21-22 17-18 - 5 Chlorella vulgaris 51-58 14-22 12-17 4-5 6 Chlorella pyrenoidosa 57-58 20-22 26-28 - 7 Spirogyra sp. 6-20 11-21 33-64 - 8 Dunaliella bioculata 49 8 4-9 Dunaliella salina 57 3 15-10 Euglena parvum 28-45 22-38 25-33 1-2 11 Porphyridium cruentum 28-29 9-14 40-57 - 12 Spirulina platensis 46-63 14-19 8-14 2-5 13 Spirulina maxima 60-71 10-20 13-16 3-5 14 Anabena cylindrical 43-56 4-7 25-30 - 15 Synechoccus Spp. 63 11 15 5 Table 3.2 Comparison of oil content of various algae used for biodiesel production S.No Species Oil content (% of dry mass) 1 Botryococcus braunii 25 75 2 Chlorella spp. 28 32 3 Cryothecodinium cohnii 20 4 Cylindrotheca spp. 16 37 5 Dunaliella primolecla 23 6 Isochrysis spp. 25-33 7 Manollanthus salina > 20 8 Nannochlorospsis spp. 20 35 9 Nannochloropsis spp 31 68 10 Neochloris oleabundans 35 54 11 Nitzschia spp. 45 47 12 Schizochytrium spp. 50 77 13 Spirulina spp. 20 47 14 Tetraselmis sueica 15 23 15 Phaeodactylum tricornutum 20 30
19 The production of algal oil requires a huge quantity of algal biomass. The algae containing lipids / oil content between 20% to 40% are extremely suitable for oil extraction as biodiesel. The open pond cultivation technique is one of the most successful, economical and viable method for algal (Spirulina Spp.) cultivation in India. Photobioreactor, a closed culture technique can also be employed which is expensive. The yield of algae can be increased by proper supply of CO 2 (aeration) with micro and macro nutrients ( Hossain et al.2008). Among the different species of algae listed in Table 3.1 and 3.2, species namely Chlorella and Spirulina Spp. were selected for cultivation based on protein, carbohydrate and lipid content in which Spirulina Spp. was successful. 3.3 CHARACTERISTICS OF Chlorella Spp. AND Spirulina Spp. Characteristics of Chlorella Spp. Chlorella sp. is unicellular green algae which belong to the phylum of chlorephyta. It is spherical in shape with a diameter of 4 to 10 µm. It contains chlorophyll A and chlorophyll B in its chloroplast as a photosynthetic pigment. It replicates itself in the presence of sunlight along with CO 2, micro and macro nutrients. Chlorella pyrenoidosa is obtained from NCIM, Pune in agar broth. It contains more than 20% of lipid content and fatty acids like C18: 1, C16: 0 and C18: 3. The BG11 medium was identified to be the best suited medium for growth. The species was allowed to grow in a aseptic condition. Due to high illumination and contamination, negative growth with loss in biomass was observed. The major constituents of Chlorella Spp. are given in Table 3.3.
20 Table 3.3 Composition of Chlorella Spp. type of oil producing algae S.No Constituents Composition 1 Crude protein 60.5 % 2 Crude lipid 18 % - 22 % 3 Carbohydrates 20.1 % 4 Ash content 4.6 % 5 Fibers 0.2 % 6 Calcium 203 mg 7 Phosphorous 989 mg 8 Vitamin A 55000 IU 9 Vitamin B 134 mg 10 Vitamin C 15.5 mg 11 Vitamin E 1.01 IU 12 Niacine 23.8 mg 13 Folic acid 26.9 mg 14 Biotin 191.6 mg Characteristics of Arthrospira maxima (Spirulina maxima) Spirulina Spp. are planktonic Cyanobacteria which are found as mass population in tropical and sub-tropical regions. They are primarily characterized by high carbonates and bicarbonates with high ph level. The classification of Spirulina Spp.is given in Table 3.4. Table 3.4 Classification of Arthrospira maxima Empire Kingdom Subkingdom Phylum Class Sub class Order Family Sub family Genus Prokaryota Bacteria Negibacteria Cyanobacteria Cyanophyceae Synecholocco phycideae Pseudanabaenales Pseudanabaenaceae Spirulinoideae Spirulina
21 The Spirulina Spp. is widely accepted as Arthrospira platensis and Arthrospira maxima. Spirulina maxima are filamentous Cyanobacteria with the arrangement of unicellular cylindrical trichomes in an open left hand helix along the entire length under. In the microscopic study, a non heterocystous filament is seen which is blue green in color, composed of cells under binary fission in a signal plane with cross walls. The filaments have motility and are freely floating. The trichomes are covered by a thin sheet with thin diameter and are non attenuated at the ends. Spirulina are symbiotic, filamentous algae with symbiotic bacteria that fix nitrogen from atmosphere. Generally, Spirulina maxima has a rod shaped structure with a blue-green photosynthetic pigment called phyocyanin. It also contains Chlorophyll A, Chlorophyll B and Cartenoids. 3.4 MORPHOLOGICAL STUDY OF Spirulina Spp. The trichome, a helical shaped filament is the characteristic feature of the genus and is seen only in the culture media. The presence of gas filled vacuoles in the cell along with the filament makes it to float as mats. The filament has a length of 50 to 500µm and width of 3 to 4µm. It is easily digestible by simple enzymatic systems as shown from Figure 3.2 to 3.5. Generally Spirulina is found in soil, marshes, fresh water, brackish water and sea water. Alkaline and saline water (salinity less than 30 g/l and ph of 8.5 to 11.0) yields good biomass of Spirulina. It reduces carbon-di-oxide releasing oxygen and assimilates mainly nitrates. It shows optimum growth between 35 o C to 45 o C under laboratory condition which eliminates microbial mesophilic contaminants.
22 Figure 3.2 Microscopic view of Arthrospira maxima Figure 3.3 Electron microscopic view at 0.5 m of Arthrospira maxima in longitudinal section along the trichome showing sheath (SH) junctional pores (JP) around the circumference of the trichome
23 Figure 3.4 Electron microscopic view at 0.5 m of Arthrospira maxima in longitudinal section showing the divisions of trichome by cross walls 3.5 COMPOSITION AND PROPERTIES OF Spirulina Spp. Composition of Spirulina Spp. The biochemical composition of Spirulina Spp.. is given in Table 3.5 and can be summarized as follows as given by Hariram et al (2011). Proteins Spirulina contains 50% to 70% of proteins of its dry mass. It depends on the environment in which it is cultivated. Fatty acid It contains high quantity of poly unsaturated fatty acids (PUF s) like Linolenic acid, Linolenic acid, Stearidonic acid, Eicosapentaenoic acid, Docosahexaenoic acid and Arachidonic acid.
24 Vitamins It contains Thiamine, Riboflavin, Nicotinamide, Pyridoxine, Folic acid, Cyanocobalamine, Vitamin C, D and E. Minerals Spirulina Spp. is a rich source of Potassium, Calcium, Chromium, Copper, Iron, Magnesium, Manganese, Phosphorous, Selenium, Sodium and Zinc. Pigments Since Spirulina is an abligate photoautotroph, it contains photosynthetic pigments which include Chlorophyll A, Xanthophylls, Betacarotene, Echinenone, Myxoxanthophyll, Zeaxanthin, Canthaxanthin, Diatoxanthin, 3-hydroxyechinenone, Beta-cryptoxanthin, Oscillaxanthin, Phycobiliproteins, C-phycocyanin and Allophycocyanin. Amino acids Various amino acids are present in Spirulina depending upon the culture media used. They are Lysine, Phenylalanine, Tyrosine, Leucine, Methionine, Glutamic acid, Aspartic acid, Tryptophan, Cystine, Serine, Arginine, Histidine, Threonine, Proline, Valine, Isoleuline, Alanine and Glycine. Table 3.5 Composition of Arthrospira maxima S.No Contents % by dry mass 1 Crude protein 50 to 70 2 Carbohydrates 15 to 20 3 Lipids 10 to 20 4 Fibers 3 to 7 5 Ash 3 to 11 6 Moisture 4 to 9
25 3.6 ESTIMATION OF PROTEIN IN Arthrospira maxima. The protein analysis of Arthrospira maxima was carried out using Lowry s method which comprises of the following steps. 1. Homogenization 2. Centrifugation 3. Alkaline solution preparation 4. Analysis using UV Spectrometer The chemicals used in Lowry s method were Folin ciocalteau reagent, Bovine serum albumin, Sodium hydroxide, Sodium carbonate, Sodium phosphate and Copper sulphate. ways, Three different types of solutions were prepared by the following 1. Bovine serum albumin solution 0.1 gram of Bovine serum albumin was dissolved in 100 ml of distilled water. 2. Folius phenol solution 5 ml of Folin ciocalteau reagent was dissolved in 5 ml of distilled water. 3. The alkaline solution was prepared by mixing 2 ml of Solution B with 100 ml of Solution A. (Solution A 0.4 gram of Sodium hydroxide was dissolved in 100 ml of water and 2 grams of Sodium carbonate was added. Solution B 0.1 gram of Sodium phosphate was dissolved in 10 ml of water and 0.5 gram of Copper sulphate was added.)
26 One ml of Centrifuged Spirulina biomass was added to all the three samples and mixed thoroughly. The solutions were kept in the UV spectrometer. The protein content was analyzed based on the absorbing capacity of the ultra violet rays. The Protein content was estimated to by 68% using this method.