Title of Invention

A NOVEL NON SALINE MEDIUM FOR THE PRODUCTION OF BETACAROTENE AND OTHER CAROTENOIDS FROM DUNALIELLA SALINA

Abstract

The present invention is a novel non saline medium for the production of Betacarotene and other carotenoids from cultivating Dunaliella salina. The novel medium developed using only Magnesium sulphate 3-10m preferably 8m with other ingredicnts ca²+ preferably 0.02mM, phosphate preferably 2uM, NO3- preferably O.O8mM, phosphate preferably O.4mM and carbonates as HCO3" preferably O.OO6M for the cultivation of Dunaliella salina and cultivating alga Dunaliella salina in the said medium in open ponds exposed to sunlight at pH between 7.5-8.5 and tomperature. about 32-45ºC. The cultivation of alga Dunaliella salina in the said medium produces about 6% dry weight basis of carotene. The extracted protein has higher protein content, usc as feed supplement for animal feed.

Full Text

The present invention relates to a non saline medium for the production of Betacarotene and other carotenoids from Dunaliella salina. The objective of this invention is the production of Betacarotene using Dunaliella salina in a culture medium devoid of sodium chloride (non saline medium) in open ponds. The other objective of the invention is the use of magnesium sulphate as an osmotic balance for mass cultivation of Dunaliella salina to produce carotenoids predominantly Betacarotene, its isomers and other carotenoids like lutein, zeaxanthin and alpha carotene. The following description in detail traces methods in respect of development of media for the cultivation of Dunaliella salina. STATE OF THE PRIOR ART The Dunaliella salina belongs to the family Chlorophyceae order volvocales, being a unicellular motile JP Patent 5,715,9484 describes the invention for the cultivation of Dunaliella salina in hypertonic solution of sodium chloride and high concentration (0.3-1 M) of Magnesium sulphate for the production of Betacarotene and its isomers, glycerol and single cell protein. JP Patent 5,605,1980 describes the invention to cultivate Dunaliella in extremely high productivity, by cultivating Dunaliella in a culture solution of Chlorella extract with hot water is added, having reduced NaCl, feeding CO2 gas to the culture solution. The culture solution is adjusted to have an NaCl concentration of 0.5-3wt%. The media used in the conventional process and novel process of cultivation is different. For example in the conventional process, sodium chloride is used as a medium whereas in this invention the media does not comprised of NaCl but only magnesium sulphate is used. Over the prior development the present invention is done for the cultivation of Dunaliella salina and production of carotenes using magnesium sulphate in the culture medium as the sole source of osmotic balance. The culture media for the cultivation of Dunaliella salina in the said invention is comprised of carbonates, sulphates, potassium, phosphates, and nitrates with trace elements. Prior to preparing the media to support growth of Dunaliella salina magnesium sulphate as hepta hydrate form is added at the concentration from 3M to 10 M preferably 8 M. A culture media comprises of Ca2+ 0.01- 20mM preferably 0.02mM, iron sources such as Fe EDTA 0.5 -4.5|iM preferably 2µM, NO+ 0.05 - 1 mM preferably 0.08 mM, phosphate 0.01 - 1 mM preferably 0.4 mM and carbonates as HCO3 0.001 - 0.05 M preferably 0,006 M. DETAIL DESCRIPTION OF THE INVENTION: In the said invention a culture media for the growth of Dunaliella salina and production of carotenoids is developed using only Magnesium sulphate. The algae Dunaliella salina is grown in this media to produce Betacarotene, its isomers and other carotenoids. The residue thus obtained after extraction of the said carotenes has high protein content and used as feed supplement for animal feed and for other similar purposes. It is the primary object of the invention to develop a medium for cultivation of Dunaliella salina for the production of Betacarotene, its isomers and other carotenoids. Further object of the invention will be clear from the ensuing description. The present invention provides a process for the cultivation of Dunaliella salina, which belongs to the Class of Chlorophyceae, Order of Volvocales. It is an embodiment of the invention that this invention leads to contrivance of medium for the cultivation of Dunaliella salina in open ponds especially exposed to sunlight. The osmolarity is contributed only by magnesium sulphate. It is another embodiment of the invention that this medium for the cultivation of Dunaliella salina in open ponds essentially exposed to sunlight wherein the optimum amount of magnesium sulphate is preferably in the range of 8M. It is another embodiment of the invention that this medium for the cultivation of Dunaliella salina in open ponds especially exposed essentially to sunlight wherein a medium for cultivation of Dunaliella salina the said medium comprising an artificial sea water having the following ingredients in proportionate concentrations are -MgS04 3-lOM preferably 8M, Ca2+ 0.01- 20mM preferably 0.02mM, iron sources such as Fe EDTA 0.5 - 4.5µM preferably 2µM, NO3 0.05 - 1 mM preferably 0.08 mM, phosphate 0.01 - 1 mM preferably 0.4 mM carbonates as HCO3 0.001 - 0.05 M preferably 0.006 M. It is another embodiment of the invention that this medium for the cultivation of Dunaliella salina in open ponds exposed to sunlight wherein this media initiates betacarotene synthesis in Dunaliella salina to generate mixed carotenoids in particular Betacarotene and its isomers having a concentration 6-8% on dry weight basis. The algae used in the process for the cultivation in the present invention are unicellular motile cells. The cells are broadly ovoid or ellipsoid in shape with a fine elastic periplast but with no rigid cell wall and has two flagella of 1.5 to 2 times the length of the cell emerge from one edge of the long cell axis, one large chloroplast occupies about half the cell volume and is often arranged in a cup-shape around the nucleus. A single median pyrenoid is embedded in the basal portion of the chloroplast and surrounded by starch granules. These cells reproduce vegetatively by longitudinal division of the motile cells. After extensive experiments, optimum conditions of cultivation have been defined. This results in the desired production of large quantities of carotene, providing as by-product a substance having a high protein content, which can be utilized for various purposes. When grown outdoors under suitable illumination, the cells are bright orange and have a high carotene content, which can reach values as high as 60 to 80 mg per gram of dry weight of algae. The alga is cultivated under an adequate high intensity of illumination and so it is the best to carry out cultivation outdoors in sunlight. A culture medium comprises the following nutrients. MgS04 3-lOM preferably 8M. The other ingredients comprise; Ca2+ 0.01-20mM preferably 0.02mM, iron sources such as Fe EDTA 0.5-4.5µM preferably 2µM, NO3-0.05 - 1 mM preferably 0.08 mM and phosphate 0.01 -ImM preferably 0,4 mM, carbonates as UCOz' 0.001 - 0.05 M preferably 0.006 M. A suitable source of carbon, such as HCO3- or CO2 in growth culture medium is provided. The optimum pH for cultivation is between?.5 and 8.5 and this is advantageously adjusted by adding required quantities of CO2 through a pH controlled solenoid valve. The optimum temperature of cultivation is in the range of about 32-45 ° C. In order to obtain a high content of carotenoids, it is necessary to provide adequate intensity of illumination. Thus cultivation is done in open environment under sunlight with less (lower than normal) concentration of NOs" for better productivity of carotenoids. Since the cultivation is done outdoors, the diurnal cycle is provided naturally. The media used in the conventional cultivation process and novel cultivation process is different. For example in the conventional process sodium chloride is used as a medium whereas in this media only magnesium sulphate is used. EXAMPLE: Cultivation of Dunaliella salina ARL 5 for Carotene Production. Dunaliella salina grown in a medium containing MgS04 8M, the other ingredients comprise; Ca2+ 0.02mM, iron sources such as Fe EDTA 2|xM, NO3+ (as potassium nitrate) O.OSmM and phosphate (as potassium hydrogen ortho phosphate) 0.4mM, carbonates as HCO3 (as sodium bi carbonate) 0.006M in open ponds either having wind to assist mixing or using a paddle wheel mixing assembly. Dunaliella salina at a rate of 0.5-1.0 division per day and was harvested when the cells attained a density in the media that is measured using a spectrophotometer. Cultures are harvested by flocculation using alum or ferric chloride followed by addition of a polyelectrolyte (food grade). Compressed air is sparged through the medium and the alga is allowed to stand for 30 min. The alga floats on top of the media and is harvested. The media is returned to the pond. The cells thus harvested are transferred to the kettle and extracted in vegetable oil directly. METHOD OF GROWING THE ALGAE The algal cell is grown in open ponds. The other nutrients comprise Nitrogen as Nitrates, phosphorous as phosphate, Magnesium as Mg2+, Sulphate as (S042 ), Iron as Ferric and calcium as Ca2+ This invention leading to production of carotenes has wide ramification in view of extensive application in various fields of technology and industry. The present invention produces Betacarotene; a precursor for Vitamin A, an antioxidant in pharmaceutical and nutraceutical industries, natural colouring agent for food and beverages, accumulates glycerol-a fuel substitute, used as feed for crusteaceans and feed additives. We claim, 1. A non saline culture medium for the production of Betacarotene, other carotenoids and its isomers from the alga Dunaliella Salina comprises MgS04 8M, Ca 0.02mM, iron source such as Fe EDTA 2|xM, NO3 (as potassium nitrate) 0-08mM, phosphate (as potassium hydrogen ortho phosphate) 0,4mM, HCO3' (as sodium hi carbonate) 0.006M; wherein cultivating the alga using the medium in open ponds under exposure of sunlight at pH between 7.5-8.5 and 32-45^C temperature. 2. A non saline culture medium for the production of Betacarotene, other carotenoids and its isomers from alga Dunaliella Salina as claimed in claim 1 wherein Dunaliella salina having identifying characteristics as: belongs to the family Chlorophyceae, order Volvocales, a unicellular motile green algae of the size ranging from 10-16 microns, the cells are broadly ovoid or ellipsoid in shape with a fine elastic periplast, with no rigid cell wall, two flagella 1.5 to 2 times the lengths of the cell emerge from one edge of the long cell axis, one large chloroplast occupies about half the nucleus, a single median pyrenoid is embedded in the basal portion of the chloroplast and surrounded by starch granules, the cells reproduce vegetatively by longitudinal division of the motile cells, the cells accumulate large quantities of carotenes. 3. The non saline culture medium for the production of Betacarotene, other carotenoids and its isomers from the alga Dunaliella Salina as claimed in claim 1 wherein the culture medium does not contain NaCl. 4. A non saline culture medium for the production of Betacarotene, other carotenoids and its isomers from the alga, Dunaliella Salina as claimed in claim 1 wherein production of Betacarotene, other carotenoids and its isomers are of atleast 6% on dry weight basis. 5. A non saline culture medium for the cultivation of Dunaliella salina as claimed in claim 1 wherein the carotenoids comprises such as lutein, zeaxanthin and alpha carotene. 6. A non saline culture medium for the production of Betacarotene, other carotenoids and its isomers from alga Dunaliella Salina as substantially herein described with foregoing description and examples.

Documents:

0957-mas-2002 abstract duplicate.pdf

0957-mas-2002 claims duplicate.pdf

0957-mas-2002 description (complete) duplicate.pdf

957-mas-2002-abstract.pdf

957-mas-2002-claims filed.pdf

957-mas-2002-claims granted.pdf

957-mas-2002-correspondnece-others.pdf

957-mas-2002-correspondnece-po.pdf

957-mas-2002-description(complete) filed.pdf

957-mas-2002-description(complete) granted.pdf

957-mas-2002-form 1.pdf

957-mas-2002-form 13.pdf

957-mas-2002-form 26.pdf

957-mas-2002-form 3.pdf

957-mas-2002-form 4.pdf

957-mas-2002-form 5.pdf