Title of Invention

"A PROCESS FOR PRODUCTION OF CAROTENOID FROM MICROALGAE"

Abstract

The present invention relates to a process for production of carotenoid from microalgae. The potential of microalgae for production of high value compounds and natural pigments have realized throughout the world and efforts are being focused on their culturing and cultivation systems. In this regard, the present invention focused on a process for growth and carotenoid accumulation in microalgae with reference to Haematococcus.

Full Text

The present invention relates to a process for production of carotenoid from microalgae. The potential of microalgae for production of high value compounds and natural pigments have realized throughout the world and efforts are being focused on their culturing and cultivation systems. In this regard, the present invention focused on a process for growth and carotenoid accumulation in microalgae with reference to Haematococcus. Haematococcus is a unicellular, biflagellate, motile organism, which can grow both autotrophically and heterotrophically. The organism loses its motility and forms a cyst under unfavorable growth conditions. This encystment is accompanied by the synthesis and accumulation of carotenoids especially astaxanthin. During the encystment process, the relative quantities of individual carotenoids change considerably from 75-80% lutein and 10-20% p-carotene to over 80-90% astaxanthin in encysted cells. The astaxanthin content in Haematococcus accounts to more than 2% on dry weight basis. The conditions which are favourable for growth of Haematococcus does not facilitate astaxanthin formation and it takes several weeks for encystment and astaxanthin formation. Astaxanthin is a ketocarotenoid (3,3'-dihydroxy p ,p carotene- 4, 4' dione) used extensively in aquaculture and poultry as pigmentation source. It is a high value product with applications in pharmaceuticals, nutraceuticals, agriculture and animal nutrition (Miki 1991; Torrissen & Christiansen 1995; Nishino 1998; Wu et al 1998). Carotenoids also have important metabolic functions in animals and man including conversion to vitamin A, enhancement of the immune response and protection against diseases such as cancer by scavenging of oxygen radicals (Johnson and An 1991, Astaxanthin from microbial sources, Critical Reviews in Biotechnology, 11: 249-326). Since animals can not synthesize carotenoids, the pigments must be supplemented in the feeds of farmed species. The growing trend towards using natural sources of feed nutrients, the health benefits of carotenoids placed greater demand for natural sources of carotenoids. H. pluvialis has been identified as the prime natural source of astaxanthin pigment for commercial exploitation (Hohnson & An, 1991;Margalith, 1999). Therefore the present invention focussed on development of autotrophic bioreaction vessel for the growth process of Haematococcus. Procedures currently reported for the production of microalgal biomass for high value compounds are the following Reference may be made to US patent (US 5882849) wherein a method for the control of Haematococcus sp. growth process and astaxanthin synthesis, in a reaction chamber is reported. The degree of turbulence in the growth medium, scale of apparatus, light exposure, nutrient supply, sedimentation rate, bulk density, gas exchange rate and cell integrity, pH, temperature, NO3 concentration etc. were discussed. The drawback is that the patent focussed on growth process to maintain viable cells in a closed reaction chamber using an heterotrophic medium, making it prohibitively expensive. Reference may be made to Indian patent 1284/Del/1999 wherein a method for the preparation of carotenoids from encysted Haematococcus cells is reported. The drawback is that the patent is limited to extraction of carotenoids but not the growth and enhancement of carotenoids in the microalgae. Reference may be made to Indian patent 267/Del/2000 wherein two step process is reported. A modified medium is reported for enhanced carotenoid in the stationary phase of cells. The drawback is that the method is limited to only pigmentation phase but is it not suitable for growth phase and the medium used is heterotrophic which makes the process expensive. Reference may be made to US patent (US 5541056) wherein a method for the control of aqueous microoganisms growth process in reaction chamber transparent to visible light in an aqueous medium is disclosed. The drawback is that the patent focussed on growth process to maintain viable cells in a closed reaction chamber using heterotrophic medium making it prohibitively expensive. Reference may be made to US patent (US 3951742) wherein a process for the preparation of zeaxanthin from Flavobacterium cultured in a medium was disclosed. The drawback is that the culture conditions are suitable for bacterial culture while conditions for algae would be different. Reference may be made to the method of Miguel Olaizola 2000 ( Journal of Applied Phycology 12:499-506 Commercial production of astaxanthin from Haematococcus pluvialis using 25,000 liter photobioreactors) wherein cultivation of Haematococcus was carried out in a closed tubular module where temperature is maintained by flooding with cold seawater . The drawback in this method is that it is limited to growth phase only and the astaxanthin accumulation phase was carried out in outdoor ponds. Main object of the present invention is to provide a process for production of carotenoid from microalgae, as detailed above. Accordingly, the present invention re;ates to a process for production of carotenoid from microalgae, comprising: a) inoculation of a photosynthetic microalgae Haematococcus pluvialis (SAG 19-a), in an aqueous medium in bioreactor vessel with medium to vessel volume ratio in the range of 0.1 to 0.4; b) maintaing nitrogen sources in the range of 0.1-1.0 g/L, carbon dioxide 1-5% v/v through a float volume of 0.05 to 0.2 : 1 of culture volume or bubbling air along with CO2 1-5% v/v at a rate of 1-2 l/min.; c) exposing the bioreactor vessel to visible light to provide 0.5 to 2.5 klux light intensity for a period ranging 12 to 18 hrs a day for a period ranging 8 to 15 days; d) maintaing the culture medium pH in the range 6.8- 7.5 during the growth period; e) maintaing the culture medium temperature in the range of 20 -27 °C during the culture growth; f) exposing the culture to a fluorescent light intensity in the range of 3-10 klux or to the sunlight 10-26klux for a period about 3-8 days; g) harvesting the biomass obtained from step (f) using known methods; h) drying the biomass obtained from step (g) in a mechanical dryer or freeze drier or spray drier by known processes; In an embodiment the bioreactor vessel for growth of microalgae may be selected from transparent materials such as acrylic, glass or polycarbonate; In an another embodiment the assimilable nitrogen source may be selected from nitrate salts of sodium, potassium or ammonium; In yet an another embodiment the CO2 in the range of 1 -5% v/v through a float volume of 0.05 to 0.2 : 1 of the culture volume or bubbling air along with CO2 1 -5% v/v at a rate of 1 - 2 l/min. In yet an another embodiment the incubation temperature of the culture is in the range of 20-27°C under 0.5 to 2.5 klux light intensity for a period ranging 5-12 days. In yet an another embodiment the the microalgal cells grown for 5-12 days and is further exposed to a fluorescent light intensity in the range of 3-10 klux or to the sunlight 10-26 klux for about 3-8 days in the presence of sodium chloride in the range of 0.05 to 0.5% and sodium acetate in the range of 2 -10mM or CO2 in the range of 1-5% v/v and harvesting the cells with a carotenoid content in the range of 0.5 to 2.0% on dry weight basis. The novelty of the invention is that culturing of microalgal cells in a bioreaction vessel, which is transparent to visible light and hence facilitated faster growth. Another novelty is that by use of CO2 supply through float or bubbling enhanced the growth leading to high biomass yields, which maintained the pH of the media in a range which is favorable to growth in autotrophic media which is very economical compared to heterotrophic media. Yet another novelty in the process is exposing the cells to higher light after growth period especially to sunlight facilitated both early and enhanced accumulation of carotenoid content. EXAMPLE-1 Four litres of aqueous medium containing sodium nitrate (0.5g/L) as nitrogen source, in a glass bioreaction vessel, was inoculated with Haematococcus cells (log phase) to a cell count (2 x 105 cells/ml) and incubated under 1 klux light intensity at 25 ±1°C for a period of 10 days with a CO2 2.5% v/v float and pH maintained at 7.0. Growth in terms of cell count was monitored using Haemocytometer. The cells were further exposed to sunlight of an intensity in the range of 5 to 25klux during the day for a period of 5 days with sodium chloride 0.3%. The encysted cells were harvested. Chlorophyll and total carotenoid contents were determined according to the method of Lichtenthaler 1987 (Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology, 148: 350-382) by reading the absorbance at 470, 645 and 661.5nm using Shimadzu Spectrophotometer. The astaxanthin content was estimated by Davies method 1976, (Carotenoids, In: Chemistry and biochemistry of plant pigments, Goodwin T.W editor. Academic press, London). A cell count of 4.5x1 O5cells/ml was achieved. The cells contained 1.6% astaxanthin (w/w). EXAMPLE-2 Four litres of aqueous medium containing ammonium carbonate (0.2g/L) as nitrogen source, in a glass bioreaction vessel, was inoculated with Haematococcus cells (log phase) to a cell count (2x105 cells/ml) and incubated under 1.5 klux light intensity at 25 +1°C bubbling air mixed with CO2 (2.0%) 500ml /minute and pH maintained at 7.2 for a period of 8 days. Growth in terms of cell count was monitored using Haemocytometer. The cells were further exposed to sunlight of an intensity in the range of 5 to 25klux during the day for a period of 7 days with sodium chloride 0.22%. The encysted cells were harvested, Chlorophyll and total carotenoid contents were determined according to the method of Lichtenthaler 1987 (Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology, 148: 350-382) by reading the absorbance at 470, 645 and 661.5nm using Shimadzu Spectrophotometer. The astaxanthin content was estimated by Davies method 1976, Carotenoids, In: Chemistry and biochemistry of plant pigments, Goodwin T.W editor. Academic press, London). A cell count of 5x105cells/ml was achieved. The cells contained 1.8% astaxanthin (w/w). EXAMPLE-3 Three litres of aqueous medium containing commercial nitrogen source (0.25g/L) as nitrogen source, in a glass bioreaction vessel, was inoculated with Haematococcus cells (log phase) to a cell count (2 x 105 cells/ml) and incubated under 1 klux light intensity at 25 ±1°C for a period of 12 days with a CO2 3.0% v/v float and pH maintained at 7.1. Growth in terms of cell count was monitored using Haemocytometer. The cells were further exposed to sunlight of an intensity in the range of 10 to 25klux during the day for a period of 6 days with sodium chloride 0.27% and sodium acetate 4 mM. The encysted cells were harvested. Chlorophyll and total carotenoid contents were determined according to the method of Lichtenthaler 1987 (Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology, 148: 350-382) by reading the absorbance at 470, 645 and 661.5nm using Shimadzu Spectrophotometer. The astaxanthin content was estimated by Davies method 1976, (Carotenoids, In: Chemistry and biochemistry of plant pigments, Goodwin T.W editor. Academic press, London). A cell count of 4.5x1 O5cells/ml was achieved. The cells contained 1.9% astaxanthin (w/w). The main advantages of the present invention are 1. Culturing of microalgal cells in a bioreaction vessel which is transparent to visible light facilitated faster growth. 2. The cells will continue to be in growth phase for a longer period unlike in heterotrophic medium where the cells enter stationary phase in five days. 3. Control of physical parameters such as light, temperature and pH. 4. The problem of contamination in autotrophic medium was minimal due to absence of organic carbon source and yeast extract as in the heterotrophic medium. 5. The process is simple to scale up. 6. High carotenoid content was obtained in a shorter duration than the conventional method which takes several weeks for carotenoid production especially under autotrophic conditions. 7. Autotrophic media employed is more economical as compared to heterotrophic media. We Claim: 1. A process for production of carotenoid from microalgae, comprising: i) inoculation of a photosynthetic microalgae Haematococcus pluvialis (SAG 19-a), in an aqueous medium in bioreactor vessel with medium to vessel volume ratio in the range of 0.1 to 0.4; j) maintaing nitrogen sources in the range of 0.1-1.0 g/L, carbon dioxide 1-5% v/v through a float volume of 0.05 to 0.2 : 1 of culture volume or bubbling air along with CO2 1-5% v/v at a rate of 1-2 l/min. ; k) exposing the bioreactor vessel to visible light to provide 0.5 to 2.5 klux light intensity for a period ranging 12 to 18 hrs a day for a period ranging 8 to 15 days; I) maintaing the culture medium pH in the range 6.8- 7.5 during the growth period; m) maintaing the culture medium temperature in the range of 20 -27 °C during the culture growth; n) exposing the culture to a fluorescent light intensity in the range of 3-10 klux or to the sunlight 10-26klux for a period about 3-8 days; o) harvesting the biomass obtained from step (f) using known methods; p) drying the biomass obtained from step (g) in a mechanical dryer or freeze drier or spray drier by known processes; 2. A process for production of carotenoid from microalgae as claimed in claim 1, wherein the bioreactor vessel for growth of microalgae is selected from transparent materials such as acrylic, glass or polycarbonate. 3. A process for production of carotenoid from microalgae as claimed in claims 1, wherein the assimilable nitrogen source is selected from nitrate salts of sodium, potassium or ammonium. 4. A process for production of carotenoid from microalgae substantially as herein described with reference to the examples accompanying this specification.

Documents:

490-DEL-2004-Abstract-(01-07-2010).pdf

490-del-2004-abstract.pdf

490-DEL-2004-Claims-(01-07-2010).pdf

490-del-2004-claims.pdf

490-DEL-2004-Correspondence-Others-(01-07-2010).pdf

490-del-2004-correspondence-others.pdf

490-del-2004-correspondence-po.pdf

490-DEL-2004-Description (Complete)-(01-07-2010).pdf

490-del-2004-description (complete).pdf

490-del-2004-form-1.pdf

490-del-2004-form-18.pdf

490-DEL-2004-Form-2-(01-07-2010).pdf

490-del-2004-form-2.pdf

490-DEL-2004-Form-3-(01-07-2010).pdf

490-del-2004-form-3.pdf

490-del-2004-form-5.pdf