Industrial Training Report on algal biotechnology

HARVESTING, MORPHOLOGICAL IDENTIFICATION, OPTIMIZATION, ISOLATION STANDARDIZATION AND MASS CULTIVATION OF HAEMATOCOCCUS PLUVIALIS Work carried out under the guidance of Branch Manager Mr. Jixy Mittal AT RESEARCH AND DEVELOPMENT DEPARTMENT OF ABCA BIOSOLUTIONS PVT LIMITED Mohali (140110) REPORT SUBMITTED TO SCHOOL OF SCIENCE AND TECHNOLOGY, LOVELY PROFESSIONAL UNIVERSITY, PUNJAB, INDI (IN PARTIAL FULFILMENT OF B.TECH-BIOTECHNOLOGY DEGREE) TRAINING REPORT SUBMITTED BY: SONAL B.TECH-BIOTECHNOLOGY REG ID. 11200154 DECLARATION This is to declare that I have personally worked during the training period from 1-JUNE-2015 to 15-JULY-2015 on the topic “Preparation of algal medium, Inoculation of medium, Cultivation, Morphological identification, Optimization of Haematococcus Pluvialis, Harvesting of Microalgae, Isolation of algal culture, Mass cultivation, Transesterification and Standardization from mass cultivated algae.” in the area of PLANT BIOTECHNOLOGY under the guidance of Branch Manager Mr. JIXY MITTAL. The data mentioned in this report were obtained during genuine work done by myself. None of the findings and observations pertaining to this work has been concealed. I therefore declare that the data and results are true to the best of my knowledge. Place: SONAL Date ACKNOWLEDGEMENT I would like to express my deepest appreciation to all those who provided me the possibility to complete this report. It is with deepest sense of gratitude and reverence that I express my indebtedness to branch Manager Mr. Jixy Mittal, who granted permission to do six weeks training in the highly equipped and esteemed company. I express my profound sense of gratitude, appreciation to Dr. Rajiv Aneja, Director of Abca Bio-Solutions Pvt. Ltd. for allowing me to carry out training at Research and Development department under his guidance and for his keen interest and excellent supervision. I am heartily obliged and deeply indebted to Dr. Nita Raj Head of School of Biotechnology and Bioscience for her splendid guidance, and constant encouragement which enabled me to complete this monograph. I take privilege to express my sincere thanks and gratitude to my incomparable guide Mr. Sanjeev Singh able guidance, valuable suggestion and his longstanding efforts which bought this report in its present form. I feel honored to have him as my guide who readily and cheerfully extended every help required from the beginning till the end of this work. The support of my family and friends are worth mentioning. I am thankful to my father, mother and sister without whose living involvement, support and blessings, this report would not have been possible. Most of all, I thank the Almighty for blessing me with the strength, light and wisdom to peruse the work. Place: SONAL Date: TABLE OF CONTENTS S.NO. Contents Page No. 1. Declaration 2 2. Acknowledgement 3 3. Preface 5 4. Introduction 6-10 5. Review Of Literature 11-18 6. Materials and Methods 19-31 7. Results And Observations 32-57 8. Instruments Used 58-63 9. Summary And Conclusion 64-65 10. References 66-68 PREFACE Microalgae are known to efficiently use the process of water- splitting photosynthesis for the conversion of solar energy into useful chemical energy. They are therefore considered as excellent candidates for the production of bio-fuels and biopolymers. The solar powered synthesis of sustainable products directly from CO2, water and sunlight with microalgae has the potential to provide renewable source of fuels and is therefore of economic and environmental priority for the public. However Biotechnology driven research with microalgae is still in its infancy. Improvements are required for the molecular processes of sun to product conversion as well as for the development of efficient and profitable biomass production system with positive energy balances. To reach these goals, research is going in algae biotechnology projects include a wide range of activities from fundamental approaches like screening for suitable algal species to more applied approaches like the development of crucial technologies for genetic and bioprocess engineering. Today algae are very important in research field due to their ability to replicate rapidly and produce hydrocarbons i.e. they are attracting the attention of more researchers and industrialists. PP CHAPTER ONE INTRODUCTION NJNJHDJHJHJFHJFH INTRODUCTION The carotenoid astaxanthin is one of the most important pigments utilized in various industries. In salmon pigments farming, which is a biggest market for astaxanthin, mostly synthesized form is used. In recent years, however, natural source of astaxanthin have successfully been used on the pigmentation of the skin and flesh of aquatic animals and poultry. In addition, astaxanthin extracted from microbial source was shown clinical trials to be a potent antioxidant. Though many organisms are able to produce astaxanthin, only a few of them have commercially been cultivated. In this respect, the fresh microalgae Haematococcus pluvialis, the most promising organism due to the higher cellular astaxanthin content compared to other i.e., more than 4% of dry weight. In general, astaxanthin production from Haematococcus is achieved though a two-stage culture. Vegetative cultivation of Haematococcus is most problematic part due to the slow growth rate, low cell concentration and susceptibility to contamination. With this respect, optimization of the culture medium is necessary to support higher cell productivities. There are several reports on the optimization of culture medium in Haematococcus, but most of them mainly focused on the optimum concentration of KNO3 and NaNO3. The others such as urea ammonium salt have not been studied enough. Besides, vitamins have been reported to another important component increase the productivity of vegetative Haematococcus culture. The drawbacks in vegetative culture of Haematococcus are mostly related to the low specific growth rate are the lack of a selective growth medium. The term "algae" encompasses a variety of organisms found throughout the world in or near bodies of water. Algae are simply plants that are distributed in sea, in freshwater and in moist situations on land. Algae have chlorophyll and can manufacture their own food. Because algae are naturally able to replicate rapidly and produce oils, proteins, alcohols, and biomass, they have attracted the attention of researchers and industrial producers seeking alternatives to oil. Algae thrive on organic carbon or CO2 and nutrients such as nitrogen and phosphorus. Growth conditions and the availability of sunlight, carbon and nutrients affect the metabolism of algae and whether they produce lipids or carbohydrates.  Researchers, for instance, have found that when algae naturally produce hydrocarbons – molecules that can most readily substitute for today's petroleum uses –growth and reproduction are limited. CLASSIFICATION OF ALGAE Most algae contain chloroplasts that are similar to the structure of cyanobacteria. Chloroplasts contain circular DNA like that in cyanobacteria. The table below describes the composition of three major groups of algae. S.No ENDOSYMBI-ONT MEMBERS CHARACTERISTICS 1. Cyanobacteria Chlorophyta Rhodophyta Glaucophyta These algae have primary chloroplast i.e. surrounded by two membranes and developed through single endosymbiotic event. 2. Green algae Chlorarachni- ophytes Euglinids These groups have green chloroplast containing chlorophylls a and b. Their chloroplasts are surrounded by four and three membrane respectively. 3. Red algae Heterokonts Haptophyta Cryptomonads Dinoflagellates These algae have chloroplast containing chlorophylls a, c and phycobilins. In the first three groups chloroplast has four membranes and in last it has three membranes. CHLOROPHYTA It is a division of green algae called chlorophytes. It refers to highly paraphletic group of all the green algae with green plants. Green algae contain chlorophyll a and b and store food as starch in their plastids. The division contains both unicellular and multicellular species. Most species live in freshwater habitats and marine habitats. HAEMATOCOCCUS PLUVIALIS It is a freshwater species of chlorophyta from the family Haematococcaceae. It is green microalgae contains astaxanthin i.e. strong antioxidant important in aquaculture and cosmetics. It is usually found in temperate regions around the world. More than 40 g of astaxanthin can be obtained from 1 kg of dry biomass (4% of dry Weight). There are two stages for the production of astaxanthin from Haematococcus Pluvialis. One is vegetative (green stage) and another is aplanospore (red stage). Different factors affecting the growth of Haematococcus Pluvialis are light, PH, Vitamins, Aeriation, Temperature. ASTAXANTHIN It is naturally occurring high-value ketocarotenoid pigment with excellent antioxidant effects. It differs from other antioxidants in its ability to penetrate the blood brain and retina barriers. It protects the brain and nervous system from neurodegenerative disease and aging. Haematocyst of H.Pluvialis Astaxanthin APPLICATIONS OF ASTAXANTHIN It has application in the nutraceutical industry, pharmaceutical industry, food coloration industry. It is used as an animal feed additive to impart coloration to salmonids and Tai. It has potential cosmeceutical applications in protection against skin aging due to its wrinkling and moisturizing effect. MARKET SCOPE The demand for natural astaxanthin is now emerging in the multi-billion dollar nutraceutical. It is principally consumed by salmon feed industry. Most commonly used algae strain for Astaxanthin production is Haematococcus Pluvialis. The annual worldwide aquaculture market of this pigment is estimated at US$ 200 million with an average price of US$ 2500/kg. The global astaxanthin market is estimated at about $257 million, most of which is used in fish coloration (2009 data; estimates by BCC Research for astaxanthin market size are however lower). The human uses market is growing and estimated at about $27-$40 million. Market Sectors Market Size (as of 2009) ( Million USD) Potential Market (2020) (Million USD) Animal feed coloring agents 300 800 Antioxidant neutraceuticals 30 300 Pharmaceuticals Emerging 500 Cosmetics Emerging 30 Table: Market sector and FUTURE MARKET Potential of Astaxanthin The main objectives of our investigation are below: OBJECTIVES: Preparation of medium (BG11) and inoculation of medium with respect to H. Pluvialis. Morphological identification and cultivation of Microalgae. Optimization of H. Pluvialis as growth affected by different sources (nitrogen, vitamins, light, PH). Isolation of algal cultures. Harvesting of microalgae with electrofloculation. CHAPTER TWO REVIEW OF LITERATURE Astaxanthin: It is a xanthophylls carotenoid which is found in various microorganisms and marine animals. It is a red fat soluble pigment which does not have pro-Vitamin A activity in human body. The use of astaxanthin as a nutritional supplement has been rapidly growing in foods, feeds, nutraceuticals and pharmaceuticals. The United States Food and Drug Administration (USFDA) have approved the use of astaxanthin as food colorant in animal and fish feed. Haematococcus pluvialis is a green microalga, which accumulates high astaxanthin content under stress conditions such as high salinity, nitrogen deficiency, high temperature and light. Astaxanthin produced from Haematococcus Pluvialis is a main source for human consumption. For dietry supplement in humans and animals, astaxanthin is obtained from seafood and extracted from H.Pluvialis Sources of Astaxanthin There are several natural sources of astaxanthin available right in your own grocery store. Adding certain types of proteins that are rich in this pigment will help you increase your intake of this beneficial antioxidant. The natural sources of astaxanthin are algae, yeast, salmon, trout, krill, shrimp and crayfish. The commercial astaxanthin is mainly from Phaffia yeast, Haematococcus and through chemical synthesis. Haematococcus pluvialis is one of the best sources of natural astaxanthin. Among the wild salmonids, the maximum astaxanthin content in wild species was reported in the range of 26–38 mg/kg flesh. Astaxanthin content in farmed Atlantic salmon was reported as 6–8 mg/kg flesh. Shrimp, crab and salmon can serve as dietary sources of astaxanthin. Astaxanthin levels (mg/kg flesh) of wild and farmed (*) salmonids Structure of astaxanthin Astaxanthin is a member of the xanthophylls, because it contains not only carbon and hydrogen but also oxygen atoms. Astaxanthin consists of two terminal rings joined by a polyene chain. This molecule has two asymmetric carbons located at the 3, 3′ positions of the β-ionone ring with hydroxyl group (-OH) on either end of the molecule. When one hydroxyl group reacts with fatty acid then it forms mono-ester, whereas when both hydroxyl group reacts with fatty acids then the result is termed a di-ester. Astaxanthin exists in stereoisomers, geometric isomers, free and esterified forms. All of these forms are found in natural sources. The primary stereoisomer of astaxanthin found in the Antarctic krill which contains mainly esterified form. Astaxanthin has the molecular formula C40H52O4. Its molar mass is 596.84 g/mol. Plannar Structure of Astaxanthin Astaxanthin and its esters from various sources Extraction and Analysis of Astaxanthin Astaxanthin is a lipophilic compound and can be dissolved in solvents and oils. Solvents, acids, edible oils, microwave assisted and enzymatic methods are used for astaxanthin extraction. Astaxanthin is accumulated in encysted cells of Haematococcus. Astaxanthin in Haematococcus was extracted with different acid treatments, Hydrochloric acid giving up to 80% recovery of pigment. High astaxanthin yield was observed with treatment of hydrochloric acid at various temperatures for 15 and 30 min using sonication. Vegetable oils (soyabean, corn, olive and grape seed) were used to extract astaxanthin from Haematococcus. The culture was mixed with oils, and the astaxanthin inside the cell was extracted into the oils, with the highest recovery of 93% with olive oil. Astaxanthin yield from Haematococcus was 80%–90% using supercritical fluid extraction with ethanol and sunflower oil as co-solvent. Biochemistry of Astaxanthin Astaxanthin contains conjugated double bonds, hydroxyl and keto groups. It has both lipophilic and hydrophilic properties. The red color is due to the conjugated double bonds at the center of the compound. This type of conjugated double bond acts as a strong antioxidant by donating the electrons and reacting with free radicals to convert them to be more stable product and terminate free radical chain reaction in a wide variety of living organisms. Astaxanthin showed better biological activity than other antioxidants, because it could link with cell membrane from inside to outside. Superior position of Astaxanthin in cell membrane Biological Activities of Astaxanthin and Its Health Benefits Antioxidant effects: An antioxidant is a molecule which can inhibit oxidation. Astaxanthin had higher antioxidant activity when compared to various carotenoids such as lutein, lycopene, α-carotene and β-carotene reported by Naguib et al. Astaxanthin in H. pluvialis offered the best protection from free radicals in rats followed by β-carotene and lutein. Astaxanthin contains a unique molecular structure in the presence of hydroxyl and keto moieties that is responsible for high anti-oxidant properties. Anti- Inflammation Astaxanthin is a potent antioxidant to terminate the induction of inflammation in biological systems. Algal cells extracts from Haematococcus significantly reduced bacterial load and gastric inflammation in H. pylori-infected mice. Astaxanthin showed protective effect on high glucose induced oxidative stress, inflammation and apoptosis in proximal tubular epithelial cells. Astaxanthin is a promising molecule for the treatment of ocular inflammation in eyes as reported by the Japanese researchers. Astaxanthin can prevent skin thickening and reduce collagen reduction against UV induced skin damage. Anticancer activity Astaxanthin showed significant antitumor activity when compared to other carotenoids like canthaxanthin and β-carotene. Astaxanthin contributes to aging and degenerative diseases such as cancer and atherosclerosis through oxidation of DNA, proteins and lipids. Antioxidant compounds decrease mutagenesis and carcinogenesis by inhibiting oxidative damage to cells. Cell–cell communication through gap junctions is lacking in human tumors and its restoration tends to decrease tumor cell proliferation. Gap junctional communication was improved in between the cells by natural carotenoids and retinoids. Astaxanthin inhibited the growth of fibrosarcoma, breast, and prostate cancer cells and embryonic fibroblasts. It also inhibited cell death, cell proliferation and mammary tumors in chemically induced male/female rats and mice. Anti-Diabetic Activity Oxidative stress levels are very high in diabetes mellitus patients. Astaxanthin could reduce the oxidative stress caused by hyperglycemia in pancreatic β-cells and also improve glucose and serum insulin levels. Astaxanthin can protect pancreatic β-cells against glucose toxicity. In another study, ameliorate oxidative stress in streptozotocin-diabetes rats were inhibited by the combination of astaxanthin with α-tocopherol. Some of the studies demonstrated that astaxanthin prevents diabetic nephropathy by reduction of the oxidative stress and renal cell damage. Effects on Circulation As people age, their red blood cells (RBCs) can be more susceptible to oxidative attack, resulting in peroxidative damage to the RBC membrane phospholipids, impairing its oxygen-carrying capacity. In a 2011 double-blind RCT healthy subjects, ages 50-69 years (n=30), were randomly allocated to receive astaxanthin at 6 mg/day or 12 mg/day or a placebo for 12 weeks. Astaxanthin significantly lowered RBC hydroperoxid levels. Astaxanthin also improved an experimental measure of “rheology” (blood flow capacity) in healthy men. Effect on vision and Eye Fatigue Astaxanthin has been extensively researched for its benefits for vision, especially in Japan. Yuan11 and Kajita (2009) discussed double-blind and other controlled trials. They concluded that astaxanthin taken at 6 mg/day consistently improved visual sharpness, even in healthy subjects. Astaxanthin also might relieve eye fatigue in persons using computer monitors. Many individuals, as they age, suffer decline in the eye’s ability to focus on near objects (presbyopia). Astaxanthin significantly improved papillary constriction, and more than 60 percent of the people experienced improvement in the categories of “difficulty to see near objects,” “eyestrain,” “blurred vision,” and “shoulder and low-back stiffness.” Effect on Mitochondrial activity The mitochondria have double membranes crammed with catalytic proteins that utilize oxygen to generate energy. In a series of experiments with various cultured cell lines astaxanthin improved cell survival under oxidative stress. By adding an oxidant-sensitive molecular probe into the mitochondria, the researchers found that astaxanthin reduced the mitochondria’s endogenous production of oxygen radicals and protected the mitochondria against a decline of membrane function that typically occurs over time in these cultures. It increased mitochondrial activity in these cells by increasing oxygen consumption without increasing generation of ROS (Reactive oxygen species). Safety and Dose of Astaxanthin Astaxanthin is safe, with no side effects when it is consumed with food. It is lipid soluble, accumulates in animal tissues after feeding of astaxanthin to rats and no toxic effects were found. Excessive astaxanthin consumption leads to yellow to reddish pigmentation of the skin in animals. A study reported that blood pressure (bp) was reduced in stroke prone rats and in hypertensive rats by feeding 50 mg/kg astaxanthin for five weeks and 14 days, respectively. Astaxanthin was also shown significant protection against naproxen induced gastric, antral ulcer and inhibited lipid peroxidation levels in gastric mucosa. Research has so far reported no significant side effects of astaxanthin consumption in animals and humans. These results support the safety of astaxanthin for future clinical studies. Haematococcus Pluvialis for improved production of Astaxanthin by mutagenesis The fresh water green microalgae Haematococcus Pluvialis is a potent producer of Astaxanthin. Because of its potential clinical applications as an antioxidant, astaxanthin has received much attention for medical purposes as well as for a pigmenting agent in aquaculture feeds. Mutagenesis has been applied for strain improvement. However there is limited information available on astaxanthin hyperproducing mutants of Haematococcus. In the present study an attempt was made to obtain mutants of H. Pluvialis by exposure to U.V., EMS (Ethyl Methane sulphonate) and subsequently screening using inhibitors of the carotenoid biosynthetic pathway. Mutagenesis by U.V. radiation Commercial applications of Astaxanthin Astaxanthin has great demand in food, feed, nutraceutical and pharmaceutical applications. According to the current literature Astaxanthin is used in various commercial applications in the market. Astaxanthin products are available in the form of capsule, soft gel, tablet, powder, biomass, cream, energy drink, oil and extract in the market Some of the astaxanthin products were made with combination of other carotenoids, multivitamins, herbal extracts and omega-3, 6 fatty acids. Patent applications are available on astaxanthin for preventing bacterial infection, inflammation, vascular failure, cancer, cardiovascular diseases, improving brain function and skin thickness. Benefits of Astaxanthin Natural Astaxanthin is a dietary supplement with extremely powerful antioxidant benefits for human applications. Astaxanthin trap more free radicals than any other antioxidant. It enhances the action of other antioxidants like Vitamin C and Vitamin E It protects nucleic acid components of DNA, avoiding mutations to genetic material due to oxidative stress. It also protects muscle cells from damaging effects of active oxygen produced upon swimming upstream. Astaxanthin Products CHAPTER THREE MATERIALS AND METHODS From the “Review of Literature” it is well realized that astaxanthin is one of the most important pigment utilized in various industries. Today Astaxanthin is commercially produced from microalga: Haematococcus Pluvialis. Here attempts have been made to produce Haematococcus Pluvialis for further research. In this chapter various procedures/ methodologies used along with BG11 media, nitrogen source, Vitamin (biotin) are described. MATERIALS Stock Solutions It is a concentrated solution that will be diluted to some lower concentrated for actual use. These are used to save preparation time, conserve materials, reduce storage space and improve the accuracy. Chemical Requirement for 1 litre media Preparation Sodium nitrate (NaNO3): Prepared by adding 15g of sodium nitrate in 1000ml of water. Di Potassium Hydrogen Phosphate (K2HPO4): Prepared by adding 0.4g of Di Potassium Hydrogen Phosphate in 100ml of water. Magnesium Sulphate (MgSO4.7H2O): Prepared by adding 0.75g of Magnesium sulphate in 100ml of water. Calcium Chloride (CaCl2.2H2O): Prepared by adding 0.36g of calcium chloride in 100ml of water. Ferric Ammonium Citrate: Prepared by adding 0.06g of Ferric ammonium citrate in 100ml of water. Citric Acid: Prepared by adding 0.06g of citric acid in 100ml of water. EDTA: Prepared by adding 0.05g of EDTA in 500ml of water. Sodium Carbonate: Prepared by adding 0.2g of Sodium carbonate in 100ml of water. TRACE METALS: Prepared by adding Boric Acid (H3BO3) : 2.86 g/ltr Magnese Chloride (MnCl2.4H2O) : 1.81g/ltr Zinc Sulphate (ZnSO4.7H2O) : 0.222 g/ltr Sodium Molybedate ( Na2MoO4.2H2O): 0.39 g/ltr Cupric sulphate(CuSO4.5H2O): 0.079 g/ltr Cobalt Nitrate (Co(NO3)2.6H2O) : 0.0494 g/ltr These are added in 1000ml of water. 10. Vitamin: Prepared by adding 0.1g in 1000ml of water. It was added after autoclaving. PREPARATION OF 1000ml MEDIA (BG11) sodium nitrate (100ml) K2HPO4 (10ml) MgSO4.7H2O (10ml) CaCl2.2H2O (10ml) Ferric ammonium citrate(1ml) Citric acid (1ml) EDTA (1ml) Sodium carbonate (10ml) Trace metals (1ml) Vitamins (1ml) Inoculation of Medium Procedure: BG11 media was prepared. Take three Flasks (control, Test1, Test 2) and divide 100ml media in each flask. Then 1ml algal culture was added to Test 1 and Test 2 with the help of micropipette .Flasks were incubated under outdoor in direct sunlight. O.D and PH were maintained and observed on regular basis. Inoculation and cultivation of Microalgae Procedure: 1000ml BG11 media was prepared. Take three Flasks (control, Test1, Test 2) and divide 100ml media in each flask. Then 1ml algal culture was added to Test 1 and Test 2 with the help of micropipette. Flasks were incubated double tube light. O.D and PH were maintained and observed on regular basis Standardization of daily harvested volume from mass cultivated algae Algal cells divide and double itself in 4 hours. 20 Days – 4 hr. 20 Days Similarly 4 hr There are four phases of growth (lag, log, stationary, death). Harvesting should be done in log phase when O.D. reaches from 0.8 to 1.2. 4 hr 20-30% culture can be harvested daily Procedure: 10L media (BG11) was prepared and put it in a container Then 10ml Haematococcus Pluvialis culture was added to it. Container was placed in direct sun light for harvest. Four phases of growth Optimization of the algae Heamatococcus pluvialis as Growth affected by Nitrogen Source. There are several reports on the optimization of culture medium in Haematococcus, but most of them mainly focused on the optimum concentration of KNO3 and NaNO3. The nitrogen source used was sodium nitrate (NaNO3). In addition the effect of three concentrations (0.5g, 1g, 1.5g) was examined. Maximum growth was observed in the flask containing 0.5g nitrogen source. Procedure 1000ml BG11 Media was prepared (BG11) and nitrogen was added to medium at different concentrations (0.5g, 1g, 1.5g). Haematococcus Pluvialis culture was also added to medium containing nitrogen source. The flasks were kept under indoor in direct sunlight light. O.D. and PH were observed and maintained on daily basis. Effects of Nitrogen on astaxanthin content of H. Pluvialis Optimization of the algae Haematococcus Pluvialis as growth affected by light The effect of light on cultures was examined by exposure to double tube light, single tube light, indoor in direct sunlight, outdoor in direct sunlight. Procedure: 1000ml BG11 media was prepared. 100ml of media was poured into five flasks (including control). Then 1ml Haematococcus Pluvialis culture was added to each flask (excluding control). Flasks were incubated under double tube light, single tube light, Outdoor in direct sunlight, indoor in direct sun light with one control flask incubated under double tube light. O.D. and PH were observed and maintained on daily basis. Optimization of the algae Haematococcus Pluvialis as growth affected by PH The pH range for most cultured algal species is between 7 and 9, with the optimum range being 8.2-8.7. Complete culture collapse due to the disruption of many cellular processes can result from a failure to maintain an acceptable PH. That is accomplished by mixing the culture which is necessary to prevent sedimentation of algae, to avoid thermal stratification (e.g. in outdoor cultures) and to improve gas exchange between culture medium and air. Co2 is added to reduce PH of cultures. Procedure: 1000ml BG11 media was prepared. 100ml media was poured into four flasks (including control). Then add 1ml Haematococcus Pluvialis to three flasks (excluding control). PH of the flasks was set at 7, 8, and 9 and was maintained on daily basis with the help of Co2. Flasks were incubated under double tube light. O.D and PH were maintained and observed on regular basis Optimization of the algae Haematococcus Pluvialis as growth affected by vitamins Vitamins have been reported to be another important component increasing the productivity of Haematococcus cultures. Different concentrations of vitamin (biotin) were examined. Procedure 1000ml of BG11 media was prepared. 100ml media was poured into four flasks (including control). Then vitamin (biotin) was added to three flasks except control flask at different concentrations (0.5ml, 1ml, 1.5ml). 1ml Haematococcus Pluvialis culture was added to flasks contained vitamin and media. Flasks were incubated under indoor in direct sunlight. O.D. and PH were maintained and observed on daily basis. Effects of biotin on astaxanthin content of H. Pluvialis. Harvesting of microalgae with electrofloculation Microalgae are large diverse group of autotrophic organism ranging from unicellular to multi-cellular forms. Many methods like centrifugation, precipitation, flocculation, etc., were used to harvest the algal biomass. Flocculation is one among such efficient methods to separate algal cells. Electrofloculation: In electrofloculation, voltage is increased and the separation of the cells increases marginally. Electrofloculation at 5V for 30 min. is very much effective for 90% algal recovery. Procedure: O.D. of algal culture was measured 300-400ml algal culture was taken into a beaker Aluminium Electrode was dipped in a beaker and connected with a 5V current. O.D. was checked after every 10 min. till O.D. reached 0. Transesterification It is the process of exchanging the organic group R″ of an ester with the organic group R′ of an alcohol. These reactions are often catalyzed by the addition of an acid or base catalyst. Transesterification: alcohol + ester → different alcohol + different ester Eg: Formation of Biodiesel Biodiesel represents methyl esters of fatty acids and is perspective substitute for hydrocarbon fuel. Biodiesel refers to a vegetable oil - or animal fat-based diesel fuel consisting of long-chain alkyl (methyl, ethyl or propyl) esters. Biodiesel is typically made by chemically reacting lipids (e.g., vegetable oil, soybean oil, animal fat (tallow)) with an alcohol producing fatty acid esters. CH2-O-COR NaOH CH2-OH | | CH-O-COR + 3R’OH 3RCOOR’ + CH-OH | | CH2-O-COR CH2-OH Oil Alcohol NaOH Bio Diesel Glycerin Requirements Methanol Sodium hydroxide Methoxide Sunflower Oil Procedure: 22ml Methanol was taken and placed in a suitable container with cap. Then 0.4 g sodium hydroxide was added to it. Jar was labeled with “Danger Methoxide”. 100ml oil was measured and placed it in a 500ml beaker. Then oil was heated to 55°C in hot air oven. Oil was transferred to a seperatory funnel. Quickly but carefully methoxide solution was added to seperatory funnel. Jar was shaked quickly and slowly. Cork was removed to release air pressure. Shaking procedure was repeated for an hour after every 10min interval. Seperatory funnel was set onto the stand and allowed for settling. Lower layer was glycerol and upper layer was biodiesel. Precautions: Do not drop the jar while shaking Slowly transfer glycerol to seperatory funnel Morphological identification of Microalgae with example of Haematococcus Pluvialis Procedure: Glass slide was taken. One drop of Microalgae was placed on a slide Cover slip was placed on a sample Slide was observed under microscope on 10X, 45X, 100X. Isolation of algal culture according to skinner’s technique Procedure: Algal culture was taken from wastewater samples collected from pond of industrial Area, Mohali. BG11 media was prepared Agar solution was prepared by adding 25g agar in 100ml water. Then solution was kept in hot air oven at 55 oC for few minutes then cooled to maintain liquid condition. Then agar solution was added to media. This mixture was quickly added to test tube Control (10ml) and 9ml to Test1, Test 2, Test3,Test4. After that 1ml culture was added to test1, shake it and then 1ml from test1 is added to Test2 making dilutions corresponding to 10-1. The dilutions were made quickly before media get solidified. The media was allowed to solidify after dilution which immobilized algal cells. Test tubes containing immobilized algae were incubated away from direct air where they get a few hour of direct sunlight. Green colonies appeared in solid media after an incubation of week Then tubes were broken and agar cylinders were carefully placed on sterile petri plates. Then agar cylinders were cut and green colonies were transferred to other test tubes containing 5ml of liquid BG11 medium. These were incubated at 28 oC and 3000-3500 lux light intensity provided by cool white fluorescent lamps. After growth in tubes algal culture were examined under Microscope Isolation of the algal culture according to serial plate method Procedure: 10 ml suspension of algal culture was centrifuged, washed twice with 0.85% saline water and suspended to 1 ml sterile waste from which 100 µl suspension was transferred to 900 µl sterile distilled water making a dilution corresponding to 10-1. Further dilutions were prepared in a similar way up to 10-6. 100 µl of appropriate dilutions was added to the petri plate containing solidified BG-11 media and spread with the help of a sterilized spreader. The plates were incubated at 28°C and 3000-3500 lux light intensity provided by cool white fluorescent lamps till green colored colonies appeared on the plates. Then these colonies were transferred into test tubes containing 10 ml liquid BG-11 media. Axenicity of pure Chlorella sp. (R1) culture was regularly checked by inoculating the culture in BG-11 medium with 0.1 % yeast extract and 0.1 % glucose. CHAPTER FOUR RESULTS AND OBSERVATIONS Results and Observations Astaxanthin extracted from microbial sources is a potent antioxidant. Astaxanthin production from Haematococcus is achieved through a two-stage culture: vegetative (green) and aplanospore (red) stages. Vegetative cultivation of Haematococcus is the most problematic part due to the slow growth rate. So optimization of the culture medium is necessary to support higher cell productivities. Factors governing the stages of growth of Haematococcus Pluvialis are Light, pH, Vitamins, Aeriation, Temprature. In this chapter the experimental data obtained for each set of experiment are presented. The results showed maximum H. Pluvialis growth were selected for electrofloculation (Seperation of algae) and rest were for harvesting. Inoculation of medium and development of growth curve Observation table: DAY DATE CULTURE CONDITIONS O.D. pH 0th 09-06-15 CONTROL 0.00 7.02 TEST-1 0.00 7.24 TEST-2 0.00 7.25 3rd 12-06-15 CONTROL 0.00 6.33 TEST-1 0.01 6.45 TEST-2 0.03 6.48 6th 15-06-15 CONTROL 0.00 6.50 TEST-1 0.03 6.60 TEST-2 0.05 6.63 9th 18-06-15 CONTROL 0.00 7.95 TEST-1 0.07 7.91 TEST-2 0.08 7.92 13th 22-06-15 CONTROL 0.00 7.95 TEST-1 0.10 7.95 TEST-2 0.12 7.96 15th 24-06-15 CONTROL 0.00 7.90 TEST-1 0.13 7.97 TEST-2 0.16 7.99 Graph between time and absorbance Time (No. of days) Flasks incubated under outdoor sunlight shade (0th day) Morphological identification of Microalgae with example of Haematococcus Pluvialis At 10X At 10X At 45X At 45X At 100X At 100X B strain of Astaxanthin (AHB) observed under microscope. At 45X At 10X At 100X At 100X Images at 100X Inoculation and cultivation of microalgae Observation table: DAY DATE CULTURE CONDITIONS O.D. pH 0th 12-06-15 CONTROL 0.00 7.40 TEST-1 0.00 7.56 TEST-2 0.00 7.58 3rd 15-06-15 CONTROL 0.00 6.76 TEST-1 0.00 6.55 TEST-2 0.00 6.29 6th 18-06-15 CONTROL 0.00 7.97 TEST-1 0.02 7.97 TEST-2 0.03 7.96 9th 21-06-15 CONTROL 0.00 7.97 TEST-1 0.05 7.98 TEST-2 0.04 7.99 12th 24-06-15 CONTROL 0.00 8.02 TEST-1 0.07 8.05 TEST-2 0.06 8.06 14th 26-06-15 CONTROL 0.00 8.06 TEST-1 0.09 8.12 TEST-2 0.08 8.34 Graph between Time (No. of days ) and absorbance Time (No. of Days) Incubated under double tube light (0th day) Standardization of Daily harvested volume from Mass Cultivated algae in 1L media DAY DATE CULTURE CONDITIONS O.D. pH 0th 19-06-15 CONTAINER 0.00 7.19 3rd 22-06-15 CONTAINER 0.01 7.72 6th 25-06-15 CONTAINER 0.03 8.09 10th 29-06-15 CONTAINER 0.09 8.47 13th 02-07-15 CONTAINER 0.11 8.87 17th 06-07-15 CONTAINER 0.13 9.09 20th 09-07-15 CONTAINER 0.17 9.45 Graph between Time (No. of days) and absorbance Time (No. of days) 0th day After 19 days (incubated under outdoor sunlight shade) Optimization of the algae Haematococcus Pluvialis as Growth affected by Nitrogen Source Nitrogen source was added at different concentrations (0.5g, 1g, 1.5g) Observation table: DAY DATE CULTURE CONDITIONS O.D. pH 0th 15-06-15 CONTROL 0.00 7.58 TEST-1 (0.5g) 0.00 7.57 TEST-2(0.5g) 0.00 7.53 TEST-1(1g) 0.00 7.54 TEST-2(1g) 0.00 7.55 TEST-1(1.5g) 0.00 7.54 TEST2(1.5g) 0.00 7.54 3rd 18-06-15 CONTROL 0.00 7.95 TEST-1 (0.5g) 0.01 8.22 TEST-2(0.5g) 0.01 8.18 TEST-1(1g) 0.00 8.12 TEST-2(1g) 0.01 8.24 TEST-1(1.5g) 0.01 8.06 TEST2(1.5g) 0.01 8.13 7th 22-06-15 CONTROL 0.00 8.08 TEST-1 (0.5g) 0.05 8.63 TEST-2(0.5g) 0.02 8.50 TEST-1(1g) 0.02 8.26 TEST-2(1g) 0.03 8.41 TEST-1(1.5g) 0.02 8.38 TEST2(1.5g) 0.01 8.24 9th 24-06-15 CONTROL 0.00 8.17 TEST-1 (0.5g) 0.08 9.05 TEST-2(0.5g) 0.05 9.00 TEST-1(1g) 0.04 8.67 TEST-2(1g) 0.05 8.77 TEST-1(1.5g) 0.03 8.33 TEST2(1.5g) 0.02 8.64 11th 26-06-15 CONTROL 0.00 8.26 TEST-1 (0.5g) 0.11 9.04 TEST-2(0.5g) 0.08 9.04 TEST-1(1g) 0.06 8.89 TEST-2(1g) 0.06 8.84 TEST-1(1.5g) 0.05 8.49 TEST2(1.5g) 0.04 8.77 15th 29-06-15 CONTROL 0.00 6.47 TEST-1 (0.5g) 0.13 6.37 TEST-2(0.5g) 0.10 6.03 TEST-1(1g) 0.07 6.31 TEST-2(1g) 0.08 6.02 TEST-1(1.5g) 0.07 6.21 TEST2(1.5g) 0.06 6.27 Time (No. of days) Flasks (control, test 1(0.5g/100ml), test 2(0.5g/100ml)) Flasks (Test1 (1g/100ml), Test 2(1g/100ml), Test1 (1.5g), Test 2 (1.5g)) These were incubated under indoor in direct sunlight (0th day) After 15 days Control Flask Flasks ((test 1(0.5g), test 2(0.5g), test 1(1g), test 2(1g)) Flasks ((Test 1(1.5g), Test 2(1.5g)) After performing this experiment it was found that the best growth from the nitrogen source was achieved in NaNO3 (0.5g/100ml) when incubated under indoor in direct sunlight. Optimization of the algae Haematococcus Pluvialis as Growth affected by light Flasks were incubated under Double tube light(D.T.L) Single tube light(S.T.L) Indoor in direct sunlight(I) Outdoor in direct sunlight(O) Observation table: DAY DATE CULTURE CONDITIONS O.D. pH 0th 16-06-15 CONTROL 0.00 6.06 FLASK-1(D.T.L) 0.00 6.07 FLASK-2(S.T.L) 0.00 6.12 FLASK-3(I) 0.00 6.01 FLASK-4(O) 0.00 6.09 3rd 19-06-15 CONTROL 0.00 8.01 FLASK-1(D.T.L) 0.01 8.00 FLASK-2(S.T.L) 0.00 8.13 FLASK-3(I) 0.01 8.70 FLASK-4(O) 0.01 8.03 6th 22-06-15 CONTROL 0.00 8.00 FLASK-1(D.T.L) 0.03 8.28 FLASK-2(S.T.L) 0.01 8.23 FLASK-3(I) 0.04 8.48 FLASK-4(O) 0.03 8.56 9th 25-06-15 CONTROL 0.00 8.03 FLASK-1(D.T.L) 0.07 8.98 FLASK-2(S.T.L) 0.03 8.43 FLASK-3(I) 0.07 8.93 FLASK-4(O) 0.05 9.12 13th 29-06-15 CONTROL 0.00 6.64 FLASK-1(D.T.L) 0.10 6.43 FLASK-2(S.T.L) 0.04 6.16 FLASK-3(I) 0.12 6.14 FLASK-4(O) 0.09 6.07 15th 1-07-15 CONTROL 0.00 6.17 FLASK-1(D.T.L) 0.13 6.07 FLASK-2(S.T.L) 0.06 6.06 FLASK-3(I) 0.17 6.20 FLASK-4(O) 0.14 6.11 20th 06-07-15 CONTROL 0.00 6.23 FLASK-1(D.T.L) 0.17 6.38 FLASK-2(S.T.L) 0.10 6.26 FLASK-3(I) 0.21 6.19 FLASK-4(O) 0.19 6.41 Graph between Time (No. of days ) and absorbance Time ( No. of days) Flasks incubated under double tube light and single tube light (0th day) Flask incubated under indoor in direct sunlight. Outdoor in direct sunlight After 20 days Control Flask Flasks incubated under double tube light and single tube light. Flasks incubated under indoor and outdoor in direct sunlight. Result shows that H.Pluvialis growth was maximum under indoor and outdoor in direct sunlight rather than double tube light and single tube light. Optimization of the algae Haematococcus Pluvialis as Growth affected by PH In this three flasks were taken and their pH was set at 7, 8, 9. Observation Table: (Incubated under double tube light.) DAY DATE CULTURE CONDITIONS O.D. pH 0th 22-06-15 CONTROL 0.00 7.28 FLASK-1(pH 7) 0.00 7.34 FLASK-2(pH 8) 0.00 7.32 FLASK-3(pH 9) 0.00 7.34 3rd 25-06-15 CONTROL 0.00 7.90 FLASK-1(pH 7) 0.01 7.99 FLASK-2(pH 8) 0.01 8.02 FLASK-3(pH 9) 0.00 8.00 7th 29-06-15 CONTROL 0.00 7.93 FLASK-1(pH 7) 0.04 7.23 FLASK-2(pH 8) 0.03 8.24 FLASK-3(pH 9) 0.02 8.12 9th 01-07-15 CONTROL 0.00 7.88 FLASK-1(pH 7) 0.07 7.95 FLASK-2(pH 8) 0.05 8.58 FLASK-3(pH 9) 0.04 8.23 11th 3-07-15 CONTROL 0.00 7.88 FLASK-1(pH 7) 0.10 7.98 FLASK-2(pH 8) 0.09 8.63 FLASK-3(pH 9) 0.07 8.45 14th 06-07-15 CONTROL 0.00 7.89 FLASK-1(pH 7) 0.14 7.90 FLASK-2(pH 8) 0.16 8.88 FLASK-3(pH 9) 0.10 8.57 17th 09-07-15 CONTROL 0.00 7.88 FLASK-1(pH 7) 0.16 7.92 FLASK-2(pH 8) 0.18 8.86 FLASK-3(pH 9) 0.12 8.65 21st 13-07-15 CONTROL 0.00 7.91 FLASK-1(pH 7) 0.19 7.95 FLASK-2(pH 8) 0.22 8.87 FLASK-3(pH 9) 0.14 8.89 Graph between Time (No. of days) and absorbance Time (No. of days) After 21 days Optimization of the algae Haematococcus Pluvialis as growth affected by Vitamins Vitamins are added at different concentrations (0.5ml, 1ml, 1.5ml). After adding Co2, pH reduces (that is written in brackets). Observation table: DAY DATE CULTURE CONDITIONS O.D. pH 1st 19-06-15 CONTROL 0.00 8.02 FLASK-1(0.5ml) 0.00 8.06 FLASK-2 (1ml) 0.00 8.03 FLASK-3(1.5ml) 0.00 8.05 4th 22-06-15 CONTROL 0.00 7.92 FLASK-1(0.5ml) 0.01 8.34 FLASK-2 (1ml) 0.02 8.08 FLASK-3(1.5ml) 0.00 8.11 7th 25-06-15 CONTROL 0.00 7.97 FLASK-1(0.5ml) 0.04 8.54 FLASK-2 (1ml) 0.05 8.78 FLASK-3(1.5ml) 0.02 8.34 11th 29-06-15 CONTROL 0.00 8.11(6.11) FLASK-1(0.5ml) 0.07 8.79 (6.16) FLASK-2 (1ml) 0.07 8.80(6.20) FLASK-3(1.5ml) 0.05 8.17(6.22) 14th 2-07-15 CONTROL 0.00 8.12(6.12) FLASK-1(0.5ml) 0.10 9.43(6.12) FLASK-2 (1ml) 0.13 9.52(6.16) FLASK-3(1.5ml) 0.08 8.10(6.06) 18th 6-07-15 CONTROL 0.00 8.04 FLASK-1(0.5ml) 0.15 9.55 FLASK-2 (1ml) 0.19 9.56 FLASK-3(1.5ml) 0.10 8.56 21st 09-07-15 CONTROL 0.00 8.34 FLASK-1(0.5ml) 0.18 9.67 FLASK-2 (1ml) 0.23 9.60 FLASK-3(1.5ml) 0.11 8.88 25th 13-07-15 CONTROL 0.00 8.50 FLASK-1(0.5ml) 0.20 9.70 FLASK-2 (1ml) 0.27 9.67 FLASK-3(1.5ml) 0.13 9.00 Graph between time (No. of days) and absorbance Time (No. of days) Incubated under indoor in direct sunlight (1st day) AFTER 25 days Control Flask Flasks Contained 0.5ml, 1ml, 1.5ml vitamins In vitamins maximum growth was achieved in (1ml/100ml) concentration of biotin when incubated under indoor in direct sunlight. Transesterification Biodiesel was formed Glycerol has been separated from biodiesel Separated biodiesel Separated Glycerol Separated biodiesel and glycerol Harvesting of Nitrogen culture Half of the Nitrogen culture was taken for harvesting and other half for electrofloculation (Separation of algae). For harvesting flask was exposed to direct sunlight after green stage has been reached. This harvested culture was submitted to high light for 15 days in order to stimulate the transition to aplanospore (red) stage and the accumulation of astaxanthin. Haematococcus Pluvialis is usually found in temperate regions around the world. Their resting cysts are often responsible for the blood-red colour seen in the bottom of dried out rock pools and bird baths. This color is caused by astaxanthin which is believed to protect the resting cysts from the detrimental effect of UV-radiation, when exposed to direct sunlight. Electrofloculation Before passing current Formation of algae CHAPTER FIVE INSTRUMENTS USED INSTRUMENTS USED RW1- 170P- PHOTOBIOREACTOR: It is the combination of easy and mass algal growth. It represents the latest algal growth technology. It is a modern pilot scale growth module with an operating capacity of 170 liters. It is designed for the cultivation of algae and cyanobacteria. RW1 - 170P-PHOTOBIOREACTOR provides a flexible system for culture volume 170L. A standard light source of fluorescent tube light offer a reproducible, high quality light source with the possibility of simulates a daylight curve. With layer thickness of 2cm transparent acrylic, the raceway design vessel with impeller mediated mixing bioreactor offers uniform irradiation strength for entire culture without showing effects. This is also used to measure PH of the culture flasks and Co2 is added to reduce the PH. Artificially Photobioreactor allow much higher growth rate and purity level than natural habitat, as species condition are carefully controlled. AUTOCLAVE: It is a device used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C (249°F) for around 15–20 minutes depending on the size of the load and the contents. Sterilization autoclaves are widely used in microbiology, medicine, veterinary science, etc; they vary in size and function depending on media to be sterilized. It is very important to ensure that all of the trapped air is removed from the autoclave before activation, as trapped air is a very poor medium for achieving sterility. Principle: It was recognized that the boiling point of water goes up when exposed to increased pressures. In a pressure cooker where the pressure is 15 pounds/sq inch (1 atmosphere) above standard pressure (760 mm Hg), water boils at 121C. This temperature kills all life forms in 15 minutes or less. Heat labile components cannot be sterilized in autoclave. They are sterilized using filteration method. COLORIMETER: It is a device used in scientific laboratories to measure absorbance of particular wavelength of light by a specific solution. It is most commonly used to determine the concentration of a known solute in a given solution by the application of Beer- Lambert law, which states that concentration of a solute is proportional to the absorbance. Parts of Colorimeter A light source An adjustable aperture A set of colored filters A cuvette to hold the working solution A detector to measure transmitted light A meter to display output from the detector. In addition there may be A voltage regulator A second light path, cuvette, detector WEIGHING BALANCE: It is a class of balance which is used to measure small mass in the gram range. The capacity of weighing balance is 0.01g. This balance (electronic compact scale )also come with transparent enclosure with doors so that dust doesn’t collect and air current in the room do not affect the balance’s operations that is called draft shield. This instrument is also used in Kitchens to measure weight of fruits and vegetables. HOT AIR OVEN: It is a device which uses dry heat to sterilize. It was originally developed by Pasteur. Hot air ovens can be operated from 50 to 300 °C, using a thermostat to control the temperature. Their double walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor and outer layer being metallic. The capacities of these ovens vary. Power supply needs vary from country to country, depending on the voltage and frequency (hertz) used. Principle: Sterilizing by dry heat is accomplished by conduction. The heat is absorbed by outside surface of the item, then passes towards the centre of the item layer by layer. The entire item will eventually reach the temperature required for sterilization to take place. Dry heat does most of the damage by oxidizing molecules.  The essential cell constituents are destroyed and the organism dies and the temperature is maintained for almost an hour. MICROSCOPE: It is an optical instrument used for viewing very small objects such as mineral samples or animal or plant cells. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope. Fundamentally there are two types of microscope- Light and electron. In light both living and dead specimens can be viewed and in real color but in electron only dead once can be viewed and never in real color. Light microscope is of three type’s simple, compound, stereoscopic. Compound microscopes have great application and mostly used in laboratories. COMPOUND MCROSCOPE CHAPTER SIX SUMMARY & CONCLUSION .  SUMMARY AND CONCLUSION Haematococcus Pluvialis (species of Chlorophyta) culture was used for different experiments. It was inoculated into BG11 medium to observe growth under double tube light and outdoor in direct sunlight Optimization of Haematococcus Pluvialis was done by adding nitrogen source (NaNO3) or vitamins (Biotin) along with medium and culture and best growth was achieved when incubated under indoor in direct sunlight. Various factors governing the stages of growth of Haematococcus Pluvialis are light, pH, Vitamins, Temperature, and Aeration. Algal cultures were also kept under Double tube light, single tube light, indoor in direct sunlight and outdoor in direct sunlight to achieve maximum growth. Half of the culture was used for harvesting after green stage has been reached and kept under direct sunlight to stimulate transition to red stage and accumulation of astaxanthin. Another half of the culture was used to separate algae from media by passing a current using electrode (electrofloculation). Algae can manufacture their own food and able to replicate i.e. it is attracting various producers to produce biofuels e.g oil. Each algal cell contains 3 to 80% oil. Haematococcus Pluvialis is a microalga that contains Astaxanthin that is most powerful carotenoid and has highest antioxidant activities than other carotenoids. It is very useful in pharmaceutical and nutraceutical industries. It has anti-cancer, anti-inflammation, anti-diabetic activities. CHAPTER SEVEN REFERENCES REFERENCES T. Goksan, I.AK, Cenker Kihc, 2011. Growth Characteristics of the Alga Haematococcus pluvialis Flotow as affected by Nitrogen Source, Vitamin, Light and Aeration. Turkish Journal of Fisheries and Aquatic Sciences 11: 377-383 (2011) R. R. Ambati, S. M. Phang, S. Ravi and R. G. Aswathanarayana, 2014. Astaxanthin: Sources, Extraction, Stability, Biological Activities and Its Commercial Applications. Mar. Drugs 2014, 12, 128-152; doi: 10.3390/md12010128 U. Tripathi, G. Venkateshwaran, R. Sarada,  G.A. Ravishankar, 2001. Studies on Haematococcus pluvialis for improved production of astaxanthin by mutagenesis. World Journal of Microbiology and Biotechnology March 2001, Volume 17, Issue 2, pp 143-14 Li-xin Li, Zhi-wei Song, You Zhan, Shun-shan Duan, Qian-shen Zhao and Yan Liu, 2013. Effect of Vitamin-B12 and Vitamin-H on the Growth and Astaxanthin Content of Haematococcus pluvialis CH-1. Advance Journal of Food Science and Technology 5(9): 1139-1142, 2013 G. Kavitha, C. Kurinjimalar, R. Thevanathan and R. Rengasamy, 2015. Impact of UV-B Radiation on Haematococcus pluvialis Flotow Isolated from Himachal Pradesh under Laboratory Conditions. Journal of Academia and Industrial Research (JAIR) Volume 3, Issue 11 April 2015 Nurul Asmidar Hanan, Najeeb Kaid Al-Shorgani, Hafiza Shukor, Norliza Abd. Rahman, Mohd Sahaid Kalil. Pre-Optimization Conditions for Haematococcus pluvialis Growth. International Journal on advance science Engineering Information Technology Vol.3 (2013) No. 2ISSN: 2088-5334 Parris Kidd. Astaxanthin, Cell Membrane Nutrient with Diverse Clinical Benefits and Anti- Aging Potential. 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