There is a considerable interest, demand and search world wide for novel and sustainablesource of carotenoids in general and - carotene in particular. This is mainly due to itsfunctional attributes viz., precursor for vitamin-A, antioxidant potentials and also asnatural color. With current consumer awareness regarding the benefits of naturallyderived carotenes over synthetic ones, extensive research is going on worldwide inutilizing of biotechnology as a tool for such compounds. Micro algae repertoire ofvarious pigments, have received the attention of food technologist. The green micro algaDunaliella, which was discovered in early 1800, was initially identified and used as asource of glycerol and with advance in research it was found that it is also a potentialsource of carotenoids. It was found to accumulate upto 5-8 % weight of carotenoidsunder different stress conditions. Carotenoids are tetraterpenes containing carbon andhydrogen and few of them contain oxygen. Even though - carotene produced bysynthetic route is economical (US $ 200-300 kg-1), it is losing the customer attention as itis reported to be cancerous on long term usage instead of anti cancerous agent(Woutersen et al., 1999) and also less efficient as provitamin-A due to monoisomericform compared to naturally derived ones. Production of - carotene by otherbiotechnological routes will be too expensive, as they require large-scale fermenters withsophisticated devices. Micro algae can serve as economically viable and feasibletechnology, which can be easily commercialized. The present cost for natural - carotenebeing US $ 2000-2500 kg-1, employing some of the natural resources and simplertechniques cost can be made still economical as it will be highly helpful for meeting theneeds especially in developing countries.The aim of present investigation was to study the method for maintenance of cultures ofDunaliella, factors responsible for growth as well as carotenoids production. Cultureconditions, for improved algal cells growth and high carotenoids production with respectto media composition, nutrient requirements, illumination conditions, temperature andpH of the media were studied. Enhancement of carotenoids production was carried outadopting various stress conditions like, salt, nutrients, light etc., Further scaleup studieswere done to grow algae in outdoor conditions in raceway ponds upto 2000 L. Effortswere also made for downstream processing of Dunaliella, analysis of carotenoidsqualitatively and quantitatively and studies on biological efficacy of algal carotenoids incomparison with synthetic - carotene.Summary and Conclusion180Initially cultures were maintained in reported medium as stock cultures and used forfurther studies. AS-100 medium, was employed for maintenance of culture and to studythe effect of various culture conditions on growth of algal cells. Some of the parametersstudied were range of pH, concentrations of sodium chloride, carbon, nitrate, phosphate ,sulfate and vital micronutrient zinc. Results have shown the requirement of mediumconstituents necessary for optimum cell growth. Based on these results, composition wasmodified and a medium was developed denoted as ‘modified medium’. This was able toproduce cell density of >10.0 million cells mL-1, which is significant compared tointernational benchmark.Further the grown cells of Dunaliella were subjected for carotenoids accumulationthrough stress induction in terms of salt, nitrate and light. High light stress could inducethe carotenoids accumulation upto 2.5 % w/w in terms of dry weight. Upon standardizingthe conditions for growth and carotenoids accumulation in small scale, scaleup studieswere done to grow the culture in semi-open using carboys, followed by open rectangular

tanks. Further it was grown in circular cement tanks and raceway ponds, cell count of~12.0 x 106 cells mL-1 was achieved in carboys and ~ 10.0 0.5 x 106 cells mL-1 wasachieved in both circular and raceway ponds.Efforts were made to find out the suitable method for harvesting of cells by variousmethods viz., batch, online centrifuge and flocculation using various flocculants. Resultshave shown that flocculation using alum followed by centrifugation will be efficient andless energy consuming rather than direct centrifugation. Harvesting efficiency of morethan 95% cells could be achieved by this method. Harvested biomass were subjected fordrying using different methods. Significant loss of carotenoids was encountered in all themethods and IR drying has shown to enhance the cis to trans ratio of - carotene.Different food grade solvents were used to extract the carotenoids from cells and nhexane:Isopropyl alcohol (1:1) and ethyl acetate were found to be efficient solvents.Studies on utilization of oil matrix for enhancing the shelf life has shown that palm andolive oils have best shelf-life enhancing ability when stored with BHA (stabilizer) or inlow temperature.Analysis of the carotenoids of Dunaliella using various techniques has shown that -carotene, lutein, zeaxanthin, phytoene and neoxanthin are major carotenoids along with- carotene. MALDI analysis has shown that lutein is associated with C11, neoxanthinwith C12:0 and C20: 3 and zeaxanthin with C20:4 fatty acids. Dunaliella has shown theSummary and Conclusion181presence of 20 2.0 % protein in both the species studied. Apart from this, fat (7.5 0.4%) and carbohydrate (25.0 2.0 %) are the major constituents.Dunaliella salina whole cell biomass is shown to be safe on feeding to rats and haspotential antioxidant and hepatoprotective abilities. Carotenoids of Dunaliella have alsoshown the significant antioxidant and hepatoprotective ability in carbon tetrachloridechallenged albino rats. The whole research work is represented schematically in Figure-75 and Representation of steps of process developed shown in Figure-76.The results of the present investigation based on the objectives mentioned underintroduction section, reveals that the marine micro algae Dunaliella can be cultivated inboth marine and fresh water using suitable media composition with sodium chloridesupplimentation. The algae need to be cultivated in two stages: vegetative stage andcarotenogenesis stage under stress conditions. Results also revealed that the alga is richin - carotene constituting ~80% of total carotenoids present (3.0 0.5 % w/w on dryweight basis). It is also found that algae contain other carotenoids and xanthophylls,which are significant for biological activity. Present work also elucidates idea ofenhancing the carotenoids content under low light using biotic (fungal extracts) andabiotic (statin) elicitors. Safety studies revealed non-toxic effect of Dunaliella salinawhen fed at a dose upto 5.0 g kg-1 b.w. Studies on antioxidant and hepatoprotectiveactivity against CCl4 challenged animals reveals that both algal biomass as well ascarotenoids of algae are significant protectants of vital organs like liver compared to thesynthetic all trans - carotene against oxidative damage.Salient features of the thesis.Development of eco-friendly and less energy consuming technology forproduction of Dunaliella for carotenoids .Identification of various problems and finding solutions for industrial utility ofthe process.Detailed characterization and quantification of carotenoids present in micro algae

Dunaliella.Evaluation of radical scavenging and hepatoprotective ability of both wholeDunaliella salina whole cells and carotenoids as applicable for utilizing algae fornutritional, prophylactic and therapeutic applications.Summary and Conclusion182Figure 75. Schematic representation of the various facets of research work presented inthesis.Dunaliella biomass in circularand raceway pond cultureHARVESTINGFlocculation (alum) andCentrifugation (online)WASHING 3 timesMediaRecoveryAseptic Culture of D.salina &D.bardawil in AS-100 mediumExtraction of carotenoidsin Edible oil and permitted SolventsCAROTENOIDSAnalysisRecycling of Medium Development of modified mediumCarotenogenesis by light and nutrient stress

Stability studies

in oil instabilizers, lightand temperatureDifferent dryingmethodsHPLCTLCMALDIMSLC-MSNutritional studiesin albino ratsIn vivo antioxidantactivity in ratsIn vivohepatoprotectiveactivity in ratsStudies of in vitroantioxidant and lipidperoxidation activityof successive extractsBIOMASSReplenishmentand pH adjustSummary and Conclusion183