Title of Invention	A NOVEL MEDIUM FOR THE PRODUCTION OF BETACAROTENE AND OTHER CAROTENOIDS FROM DUNALIELLA SALINA (ARL 5)AND STRAIN OF DUNALIELLA SALINA FOR PRODUCTION OF CAROTENES USING THE NOVEL MEDIA
Abstract	The invention relates to the development of a novel artificial sea water medium comprising conventional salts such as NaC! and MgSO4 with the addition of KCI in the proportion defined in the specification to generate biomass of higher carotenogenesis in particular betacarotene and its isomers using Dunaliella salina (ARL 5). The strain has an unique property of having a wide range of pH and temperature tolerance which is beneficial in production terms as it is grown in external conditions(i. e., Outdoors).

Title of Invention

A NOVEL MEDIUM FOR THE PRODUCTION OF BETACAROTENE AND OTHER CAROTENOIDS FROM DUNALIELLA SALINA (ARL 5)AND STRAIN OF DUNALIELLA SALINA FOR PRODUCTION OF CAROTENES USING THE NOVEL MEDIA

Abstract

The invention relates to the development of a novel artificial sea water medium comprising conventional salts such as NaC! and MgSO4 with the addition of KCI in the proportion defined in the specification to generate biomass of higher carotenogenesis in particular betacarotene and its isomers using Dunaliella salina (ARL 5). The strain has an unique property of having a wide range of pH and temperature tolerance which is beneficial in production terms as it is grown in external conditions(i. e., Outdoors).

Full Text	A PROCESS FOR THE PRODUCTION OF BETACAROTENE USING DUNALIELLA SALINA. FIELD OF INVENTION This invention in general relates the production of higher concentrations of betacarotene than conventionally known strains of Dunaliella salina (ARL 5) in a novel medium comprising concentrations of potassium chloride, sodium chloride and magnesium sulphate which constitutes the artificial sea water and is the one responsible for the productivity of higher concentrations of betacarotene. The said alga is a strain of alga which is isolated from the coast of chennai tamilnadu which has a high tolerance rage of pH and temperature. The invention also relates to the use of KC1 in the artificial sea water medium which is responsible for the alga to produce higher concentration of beta carotene (upto 12 % dry weight) and also the use of KC1 in a specified combination with NaCl and MgS04 is responsible simultaneously achieving growth and higher yields of betacarotene. for the higher yields of betacarotene. All exixting strains of Dunaliella suiina are capauxe of producing not more than 8 - 10 % betacarotene on dry weight basis. The products obtained of value are in particular (1) Carotenes — are of value as coloring materials which are safe, as they are natural products, and can be used as substances having vitamin A activity. (2) Glycerol (3) Biomass (single cell producing beta carotens and its bio-mass) The patent application is related to a culture media developed for a specific alga known as Dunaliella salina. Prior application No. 800, 521, field on May 25, 1977 now U.S. Pat.No. 4, 115, 949. STATE OF THE PRIOR ART •The US patent application is related to a cuuuic mcuia ueveiopea ior a specific algae known as Dunaliella salina. Prior application No. 800,521, filed on May 25, 1977 now U.S. Pat.No. 4,115,949. Cultivating this algae in mass scale has been previously reported in U.S.Patent No. 4,115,949 using 1.5 M sodium chloride. In this patent growth and carotenogenesis are achieved in two stages wherein maximisation is achieve in the first stage in 1.5M NaCl and productivity is achieved in 3M NaCl. Another patent was filed in continuation for culturing this algae in artificial sea water containing 1 - 5 M concentration of sodium chloride (U.S.Patent No. 4,199,895). A patent was filed in Japan (Japanese Patent No. JP 57159484) for the cultivation of Dunaliella salina was also performed in hypertonic solution of sodium chloride and high concentrations of Magnesium sulphate (0.3 - 1M).. In all these patents, the culturing media has been clearly defined for the production of beta carotene and its isomers, glycerol and single cell protein. It is also known that some of the genus oi Dunaliella algae have a content of carotenes and carotene like substances and that these impart to such algae a reddish-brown color. The content of carotenes is normally quite low and generally does not exceed about 1% by weight. It is clear that on perusal of the said patent application the US patent describes the development of Dunaliella salina algae using sodium chloride (1.5M-3M) concentration. Likewise Japanese patent describes the development of Dunaliella salina algae using a combination of sodium chloride and high concentrations of magensium sulphate (0.3 - 1M). Cultivation of Dunaliella salina and production of carotenes using potassium chloride is not known in the art nor mentioned in any of these patents. From the cited American as well as Japanese patent specifications so far an attempt is made to prepare a artificial sea water medium such as NaCl and or a combination of NaCl and MgS04. The very object of the invention is to evaluate exactly whether KC1 can be used in the preparation of the artificial sea water medium and its effect on carotenogenesis. ■ Substituting NaCl with KC1 as an artificial sea water medium did not support growth but in combination with sodium chloride and magnesium sulphate as the artificial sea water medium has been very successful in the cultivation of D.salina (ARL 5) for the production of biomass, betacarotene and its isomers , other carotenoids and glycerol. The addition of KC1 in this proportion h^s benefited by generating more betacarotene i.e. 12% on dry weight basis as compared to 6 to 8% under conventional methods. The study also confirms the Dunaliella salina (ARL 5). of this algae has better properties as far as growth condition are concerned, which has not been reported till date. The characteristics are that has a wide pH range from 5.0 - 10.0 pH and a temperature range of 18°C to 55°C. The existing strains till date has a 6.0 -7.5 pH range and a temperature range from 20°C to 45°C. The study also confirms that the strain also grows on salinity contributed by either sodium chloride or magnesium sulphate. The advantages of this novel strain are that it can withstand a wide pH range and temperature shifts and also continue to grow (biomass) and while generating betacarotene, other carotenoids and glycerol. The strain grows comfortably in artificial sea water media prepared using NaCl alone, NaCl and MgS04 combination and also in NaCl + MgSC>4 + KC1 combination. The artificial medium comprises of KC1 - 0.2 - 1M preferably 0.35M; NaCl - 1 -5M preferably - 2M; Mg 0.41M - 1.5 M preferably 0.6 M; Ca2+ 0.01 - 20mM preferably 0.4mM iron sources such as Fe EDTA 0.5 - 4.5uM preferably 2uM; NO3" 0.5 - 1 mM preferably 0.5 mM and phosphate 0.01 - ImM preferably 0.5 mM The findings of the research is given in the following specification. It has been a break through in the findings that use of potassium chloride as a single ingredient is toxic to the development of algae {Dunaliella salina) but use of potassium chloride in combination with other ingredients (contributing to salinity viz sodium chloride and magnesium sulphate)' as invented by virtue of extensive research results in an unexpectedly high yield of carotenes with respect to the biomass on a dry weight basis. Further this invention is directed and focussed to the following area namely 1. Synthesis of a novel Medium for the production of betacarotene. 2. A novel algae (Dunaliella salina) cultured using the novel medium for beta carotene production. 3. A process for the production of algal strain and extensive application of the invention. SUMMARY OF THE INVENTION It is generally stated that a new culturing media has been developed using potassium chloride. The culture media also contains high concentration of sodium chloride and magnesium sulphate. The algae Dunaliella salina is grown in this media to produce betacarotene and its isomers and glycerol. The residue thus obtained after extraction of the said carotenes has a high protein content and can be used as feed supplement for animal feed and for other similar purposes. Taking the various aspects of the invention together various objectives of the invention are stated herein under. It is the primary object of the invention to construct/develop a novel medium for cultivation of Dunaliella salina for the production of betacarotene and its isomers and other carotenoids. It is another object of the invention to develop a novel strain of Dunaliella salina using the said medium. It is another object of the invention to develop a process for the cultivation of the novel strain of Dunaliella salina. It is another object of the invention to use the novel strain of Dunaliella salina to produce carotenoids. It is another object of the invention to use the novel strain of Dunaliella salina to produce betacarotene and its isomers having provitamin A activity. It is another object of the invention to use the novel strain of Dunaliella salina for the production of glycerol. It is another object of the invention to use the novel strain of Dunaliella salina whole cells and or cell constituents as feed for crusteaceans and animals. Further object of the invention will be clear from the ensuing description. It is an embodiment of the invention that this invention leads to contrivance of novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL5) in open raceway ponds especially exposed to sunlight. The salinity is contributed by sodium chloride, magnesium sulphate with the addition of a new chemical component i.e., potassium chloride in a particular proportion defined in the specification. A novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL5) in open raceway ponds is essentially exposed to sunlight and wherein the novel media being in the proportion of KC1 0.2 - 1M, NaCl 1 - 5M and MgS04 0.41 - 1.5M. It is another embodiment of the invention that the production of betacarotene and artificial sea water medium for the cultivation of Dunaliella salina (ARL 5) in open raceway ponds essentially exposed to sunlight wherein the optimum amount of potassium chloride is preferably in the range of 0.35M. It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds essentially exposed to sunlight wherein the optimum amount of magnesium sulphate is preferably in the range of 0.6M. It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds essentially exposed to sunlight wherein the optimum amount of sodium chloride is preferably in the range of 2M. It is another embodiment of the invention that this novel artificial sea water media for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) which comprises of conventional nutrient such as Nitrates, Phosphates, Carbon (such as C02, HC03) Iron as Chelate, Magnesium, Calcium and Potassium and in a salinity contributed of a proportion of KC1 0.2 - 1 M preferably0.35M; NaCl 1- 5 M preferably 2.0M; MgS04 0.41M -1.5 M preferably 0.6M. It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds especially exposed essentially to sunlight wherein a novel medium for the production of betacarotene and cultivation of Dunaliella salina the said medium comprising an artificial sea water medium having the following ingredients (1) KC1 (2) NaCl (3) MgS04 and its proportionate concentrations are - (1) KC1 0.2 - 1M preferably 0.35M; (2) NaCl - 1 - 5M preferably 2M; (3) MgS04 0.41M - 1.5M preferably 0.6M. The other ingredients comprise; Ca 2+, 0.01-20mM preferably 0.02-0.04mM; Iron source such as FeEDTA 0.5-4.5uM, preferably l-3uM; N03- 0.5-1.0M preferably 0.5mM; phosphate 0.01-1M preferably 0.5mM and CO2 as the carbon source. It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds exposed to sunlight and wherein a novel medium for cultivation of Dunaliella salina wherein this novel media induces the algae Dunaliella salina (ARL 5) to generate a higher concentration of carotenes in particular betacarotene and its isomers having a concentration of 10 -12% on dry weight basis, as compared to the use of conventional method (use of sodium chloride) yield of 6-8% on dry weight basis. It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds exposed to sunlight wherein a novel medium for the production of betacarotene and the cultivation of Dunaliella salina and wherein the Dunaliella salina(ARL5) is an unique strain isolated from the coastline of Chennai, Tamil Nadu, India which has a wide pH, temperature and potassium chloride tolerance. It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds exposed to sunlight the said novel medium for the production of betacarotene and the cultivation of Dunaliella salina is used to develop a new strain. The Dunaliella salina belongs to the family Chlorophycae order volvocales, being 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 but 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 (predominantly betacarotene and its isomers), providing as by-product a substance having a high protein content (biomass) and which can be utilized for various purposes such as a precursor for Vitamin A. It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds essentially exposed to sunlight and wherein a novel medium being used for generation of a new strain of Dunaliella salina has a pH range of 5.0 - 10.0 and a temperature range of 18°C - 55°C and high potassium chloride tolerence from 0.2- 1.0M. It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL5) in open raceway ponds essentially exposed to sunlight and wherein a novel medium being used for the generation of anew strain of Dunaliella salina, the Dunaliella salina (ARL 5) has a preferable range of 7.5 - 8.5 pH and a preferable temperature range of 32°C - 45°C. DETAILED DESCRIPTION OF PREFERRED EMBODIMENT OF INVENTION . The nature of the invention and the manner in which it is to be performed is clearly described in the specification. The invention has various components and they are clearly described in the following pages of complete specification. Now the invention will be described in detail and specifically with reference to figure I contained in this complete specification. The present invention provides a process for the controlled cultivation of an algae designated by us as Dunaliella salina ARL 5 (a local isolate of Chennai coast, India), which belongs to the Class of Chlorophyceae, Order of Volvocales. The algae used in the process of 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 100 to 120 mg per gram of dry weight of algae. The algae must be cultivated under an adequate high intensity of illumination and this is best carried out outdoors in sunlight. An artificial medium ought to contain the following nutrients. KC1 0.2 - 1M preferably 0.35M : NaCl : 1.0 - 5.0 and preferably 2.0 M ; MgS04 0.41M - 1.5M preferably 0.6M ; Ca 2++, 0.01-20mM preferably 0.02-0.04mM; Iron source such as FeEDTA 0.5-4.5uM, preferably 1-3 \xM; NO32' 0.5-1.0M preferably 0.5mM; phosphate 0.01-1M preferably 0.5mM. A suitable source of carbon, such as HCO3" or C02 in growth medium is provided. The optimum pH for cultivation is between7.5 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. Various contaminant microorganisms such as fungi, zooplankton, protozoans etc. constitute a problem under conditions of large scale cultivation and these can be overcome to a very large extent by carrying out the cultivation at higher concentrations of potassium chloride (0.35M), sodium chloride (2.0M) and Magnesium sulphate (0.41M). In order to obtain a high content of carotenes, it is necessary to provide adequate intensity of illumination. To effect better productivity of carotenes concentrations of nitrogen has to be lower than normal. Since the cultivation is done outdoors the diurnal cycle is provided naturally. Continuous illumination severely inhibits growth. At lower concentrations of salinity contributed by KC1, MgSC>4 and NaCl proportions the culture doubles very fast though the concentrations of beta carotene drops to 5-8 %. Optimisation of the culture media (salinity and nutrient proportion) has been done to balance growth (biomass) and production of betacarotene and its isomers. The carotenes are recovered from the algae and there remains a residue having a high protein content. The novel process of cultivation under controlled conditions on a predetermined culture medium provides the possiblility to obtain simultaneously three valuable products viz., (1) carotenes (betacarotene and its isomers and other carotenoids), (2) algae meal and (3)glycerol. In the ensuing figure the conventional route for the cultivation of Dunaliella. salina ARL 5 and producing various carotenes and the route according to the invention is illustrated. Various steps involving the production of carotenes in both the routes are clearly illustrated On perusal of the figure I the following differences are very clear 'The media used in the conventional process and novel process are different. For example in the conventional process sodium chloride is used as a medium whereas in this novel process a combination of potassium chloride, magnesium sulphate and sodium chloride are used as medium. In the conventional process betacarotene is achevied in two stages. In the first stage maximisation of growth and in the second stages carotenogenesis is achieved. In the novel process growth and carotenogenesis is achieved in the same stage. Both algae and medium constitute the subject matter of our invention. A Comparison is made between the conventional process and patented process in terms of parameters namely pH, temperature and the novel media tolerance. pH range tolerance : Existing strains of Dunaliella salina Novel Dunaliella salina (ARL5) Temperature Tolerance : Existing strains Dunaliella salina Novel Dunaliella salina (ARL 5) Novel Media Tolerance Existing strains of Dunaliella salina Novel Dunaliella salina (ARL 5) 6.5-8.0 5.0-10.0 20°C - 45°C 18°C- 55°C Causes death in the presence of potassium chloride High tolerance to Potassium chloride (upto 1.0M) EXAMPLE Cultivation of Dunalliella salina ARL 5 for Carotene Production. Dunaliella salina grown in a seawater medium containing KC1 0.35M; NaCl 2.0M; MgSC>4 0.6M, the other ingredients comprise Ca ++, 0.04mM; Iron source such as FeEDTA 3.0 \\M; NO32' (as potassium nitrate) 0.5mM; phosphate (as potassium hydrogen ortho phosphate) 0.5mM. CO2 as carbon source was added by demand via a pH controlled solenoid valve. The pH is maintained automatically at pH 7.5 - 8.2 by adding CO2 on demand. The algae was grown outdoors in a 15 cm deep open raceway pond under natural conditions of solar irradiation and temperature. The culture was mixed by a paddle wheel agitator at about 20 rpm. Dunaliella salina ARL5 grew 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 were 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 algae is allowed to stand for 30 min. The algae 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 conventional raceway ponds with a continuous agitation for a period of 8 hrs. during the day. CO2 is sparged through the system through a solenoid valve controlled electronically by means of pH probe. The set point ensures constant control of pH and also ensures carbon addition for growth. The other nutrients comprise Nitrogen as Nitrates, phosphorous as phosphate, Magnesium as Mg2+, Sulphate as (SO42" ), Iron as Ferric and calcium as Ca2+ It is to be noted that the invention introduces a new paradigm concept in the scientific community. This invention leading to production of carotenes has wide ramification in view of extensive application in various fields of technology and industry nevertheless specific use of invention is listed for perusal. USES 1. To produce betacarotene precursor for Vitamin A 2. Used as an antioxidant in pharmaceutical and neutraceutical industries 3. Used as a natural colouring agent for food and beverages. 4. Also accumulates glycerol - a fuel substitute 5. Used as feed for crusteaceans and feed additives. An algae, Dunaliella salina belonging to chlorophyceae possess the characteristics herein described. 1. Unicellular motile algae having two flagella. 2. Having a cup shaped chloroplast. 3. Has a wide pH range tolerance from 5 to 10.0 pH 4. Has a wide temperature range tolerance of 18°C to 55°C 5. Accumulates betacarotene under stress condition Viz., nitrogen and phosphorous depletion. It is to be noted that the very objective of the description of the invention is to indicate salient features of the invention. It is to be further noted that this description is in no way to abridge the scope of the invention. Further it is clear that within the scope of the invention various modifications are permissible. This scope of the invention is appended in the ensuing description. We claim, 1. A method of manufacturing betacarotene from the green alga Dunaliella salina (ARL 5) a unique strain which has a high pH (from 5.0 - 10.0) and temperature (from 18 ° C - 55 ° C) tolerance, using a novel artificial sea water medium starting from fresh water containing a ratio of potassium chloride, sodium chloride and magnesium sulphate with ratio of 0.2 - 1.0 M of KCI, 1 - 5M of NaCI and 0.41 - 1.5 M of MgS04, the said process conforming to the above specified combination of the artificial seawater medium wherein the other nutrients are added in the proportion of; Calcium, 0.01-20mM preferably 0.02-0.04mM; Iron source such as FeEDTA 0.5-4.5M, preferably 1-3 \|iM; Nitrate 0.5-1.0M preferably 0.5mM; phosphate 0.01-1M preferably 0.5mM which enables to generate algal growth and carotenogenesis in a single stage whereby betacarotene is synthesised not less than 12 % dry on weight basis by Dunaliella salina (ARL 5) and extraction of betacarotene from the Dunaliella salina (ARL 5) are by known methods. 2. A method of manufacturing beta carotene as claimed in claim 1 wherein the optimum concentration of KCI is 0.35 M. 3. A method of manufacturing beta carotene as claimed in claim 1 wherein the optimum concentration of MgS04 is 0.6 M. 4. A method of manufacturing beta carotene as claimed in claim 1 wherein the optimum concentration of NaCI is 2.0 M.

Full Text

A PROCESS FOR THE PRODUCTION OF BETACAROTENE USING
DUNALIELLA SALINA.
FIELD OF INVENTION
This invention in general relates the production of higher concentrations of betacarotene than conventionally known strains of Dunaliella salina (ARL 5) in a novel medium comprising concentrations of potassium chloride, sodium chloride and magnesium sulphate which constitutes the artificial sea water and is the one responsible for the productivity of higher concentrations of betacarotene. The said alga is a strain of alga which is isolated from the coast of chennai tamilnadu which has a high tolerance rage of pH and temperature.
The invention also relates to the use of KC1 in the artificial sea water medium which is responsible for the alga to produce higher concentration of beta carotene (upto 12 % dry weight) and also the use of KC1 in a specified combination with NaCl and MgS04 is responsible simultaneously achieving growth and higher yields of betacarotene. for the higher yields of betacarotene. All exixting strains of Dunaliella suiina are capauxe of producing not more than 8 - 10 % betacarotene on dry weight basis.
The products obtained of value are in particular (1) Carotenes — are of value as coloring materials which are safe, as they are natural products, and can be used as substances having vitamin A activity. (2) Glycerol (3) Biomass (single cell producing beta carotens and its bio-mass)
The patent application is related to a culture media developed for a specific alga known as Dunaliella salina. Prior application No. 800, 521, field on May 25, 1977 now U.S. Pat.No. 4, 115, 949.

STATE OF THE PRIOR ART
•The US patent application is related to a cuuuic mcuia ueveiopea ior a specific algae known as Dunaliella salina. Prior application No. 800,521, filed on May 25, 1977 now U.S. Pat.No. 4,115,949.
Cultivating this algae in mass scale has been previously reported in U.S.Patent No. 4,115,949 using 1.5 M sodium chloride. In this patent growth and carotenogenesis are achieved in two stages wherein maximisation is achieve in the first stage in 1.5M NaCl and productivity is achieved in 3M NaCl. Another patent was filed in continuation for culturing this algae in artificial sea water containing 1 - 5 M concentration of sodium chloride (U.S.Patent No. 4,199,895). A patent was filed in Japan (Japanese Patent No. JP 57159484) for the cultivation of Dunaliella salina was also performed in hypertonic solution of sodium chloride and high concentrations of Magnesium sulphate (0.3 - 1M).. In all these patents, the culturing media has been clearly defined for the production of beta carotene and its isomers, glycerol and single cell protein.
It is also known that some of the genus oi Dunaliella algae have a content of carotenes and carotene like substances and that these impart to such algae a reddish-brown color. The content of carotenes is normally quite low and generally does not exceed about 1% by weight.
It is clear that on perusal of the said patent application the US patent describes the development of Dunaliella salina algae using sodium chloride (1.5M-3M) concentration. Likewise Japanese patent describes the development of Dunaliella salina algae using a combination of sodium chloride and high concentrations of magensium sulphate (0.3 - 1M). Cultivation of Dunaliella salina and production of carotenes using potassium chloride is not known in the art nor mentioned in any of these patents.
From the cited American as well as Japanese patent specifications so far an attempt is made to prepare a artificial sea water medium such as NaCl and or a combination of NaCl and MgS04. The very object of the invention is to

evaluate exactly whether KC1 can be used in the preparation of the artificial sea water medium and its effect on carotenogenesis.
■
Substituting NaCl with KC1 as an artificial sea water medium did not support growth but in combination with sodium chloride and magnesium sulphate as the artificial sea water medium has been very successful in the cultivation of D.salina (ARL 5) for the production of biomass, betacarotene and its isomers , other carotenoids and glycerol. The addition of KC1 in this proportion h^s benefited by generating more betacarotene i.e. 12% on dry weight basis as compared to 6 to 8% under conventional methods. The study also confirms the Dunaliella salina (ARL 5). of this algae has better properties as far as growth condition are concerned, which has not been reported till date. The characteristics are that has a wide pH range from 5.0 - 10.0 pH and a temperature range of 18°C to 55°C. The existing strains till date has a 6.0 -7.5 pH range and a temperature range from 20°C to 45°C. The study also confirms that the strain also grows on salinity contributed by either sodium chloride or magnesium sulphate.
The advantages of this novel strain are that it can withstand a wide pH range and temperature shifts and also continue to grow (biomass) and while generating betacarotene, other carotenoids and glycerol. The strain grows comfortably in artificial sea water media prepared using NaCl alone, NaCl and MgS04 combination and also in NaCl + MgSC>4 + KC1 combination. The artificial medium comprises of KC1 - 0.2 - 1M preferably 0.35M; NaCl - 1 -5M preferably - 2M; Mg 0.41M - 1.5 M preferably 0.6 M; Ca2+ 0.01 - 20mM preferably 0.4mM iron sources such as Fe EDTA 0.5 - 4.5uM preferably 2uM; NO3" 0.5 - 1 mM preferably 0.5 mM and phosphate 0.01 - ImM preferably 0.5 mM
The findings of the research is given in the following specification. It has been a break through in the findings that use of potassium chloride as a single ingredient is toxic to the development of algae {Dunaliella salina) but use of potassium chloride in combination with other ingredients (contributing

to salinity viz sodium chloride and magnesium sulphate)' as invented by virtue of extensive research results in an unexpectedly high yield of carotenes with respect to the biomass on a dry weight basis. Further this invention is directed and focussed to the following area namely 1. Synthesis of a novel Medium for the production of betacarotene. 2. A novel algae (Dunaliella salina) cultured using the novel medium for beta carotene production. 3. A process for the production of algal strain and extensive application of the invention.
SUMMARY OF THE INVENTION
It is generally stated that a new culturing media has been developed using potassium chloride. The culture media also contains high concentration of sodium chloride and magnesium sulphate. The algae Dunaliella salina is grown in this media to produce betacarotene and its isomers and glycerol. The residue thus obtained after extraction of the said carotenes has a high protein content and can be used as feed supplement for animal feed and for other similar purposes. Taking the various aspects of the invention together various objectives of the invention are stated herein under. It is the primary object of the invention to construct/develop a novel medium for cultivation of Dunaliella salina for the production of betacarotene and its isomers and other carotenoids.
It is another object of the invention to develop a novel strain of Dunaliella salina using the said medium.
It is another object of the invention to develop a process for the cultivation of the novel strain of Dunaliella salina.
It is another object of the invention to use the novel strain of Dunaliella salina to produce carotenoids.

It is another object of the invention to use the novel strain of Dunaliella salina to produce betacarotene and its isomers having provitamin A activity.
It is another object of the invention to use the novel strain of Dunaliella salina for the production of glycerol.
It is another object of the invention to use the novel strain of Dunaliella salina whole cells and or cell constituents as feed for crusteaceans and animals.
Further object of the invention will be clear from the ensuing description.
It is an embodiment of the invention that this invention leads to contrivance of novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL5) in open raceway ponds especially exposed to sunlight. The salinity is contributed by sodium chloride, magnesium sulphate with the addition of a new chemical component i.e., potassium chloride in a particular proportion defined in the specification.
A novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL5) in open raceway ponds is essentially exposed to sunlight and wherein the novel media being in the proportion of KC1 0.2 - 1M, NaCl 1 - 5M and MgS04 0.41 - 1.5M.
It is another embodiment of the invention that the production of betacarotene and artificial sea water medium for the cultivation of Dunaliella salina (ARL 5) in open raceway ponds essentially exposed to sunlight wherein the optimum amount of potassium chloride is preferably in the range of 0.35M.
It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds essentially exposed to sunlight

wherein the optimum amount of magnesium sulphate is preferably in the range of 0.6M.
It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds essentially exposed to sunlight wherein the optimum amount of sodium chloride is preferably in the range of
2M.
It is another embodiment of the invention that this novel artificial sea water media for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) which comprises of conventional nutrient such as Nitrates, Phosphates, Carbon (such as C02, HC03) Iron as Chelate, Magnesium, Calcium and Potassium and in a salinity contributed of a proportion of KC1 0.2 - 1 M preferably0.35M; NaCl 1- 5 M preferably 2.0M; MgS04 0.41M -1.5 M preferably 0.6M.
It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds especially exposed essentially to sunlight wherein a novel medium for the production of betacarotene and cultivation of Dunaliella salina the said medium comprising an artificial sea water medium having the following ingredients (1) KC1 (2) NaCl (3) MgS04 and its proportionate concentrations are - (1) KC1 0.2 - 1M preferably 0.35M; (2) NaCl - 1 - 5M preferably 2M; (3) MgS04 0.41M - 1.5M preferably 0.6M. The other ingredients comprise; Ca 2+, 0.01-20mM preferably 0.02-0.04mM; Iron source such as FeEDTA 0.5-4.5uM, preferably l-3uM; N03- 0.5-1.0M preferably 0.5mM; phosphate 0.01-1M preferably 0.5mM and CO2 as the carbon source.
It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds exposed to sunlight and wherein a

novel medium for cultivation of Dunaliella salina wherein this novel media induces the algae Dunaliella salina (ARL 5) to generate a higher concentration of carotenes in particular betacarotene and its isomers having a concentration of 10 -12% on dry weight basis, as compared to the use of conventional method (use of sodium chloride) yield of 6-8% on dry weight basis.
It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds exposed to sunlight wherein a novel medium for the production of betacarotene and the cultivation of Dunaliella salina and wherein the Dunaliella salina(ARL5) is an unique strain isolated from the coastline of Chennai, Tamil Nadu, India which has a wide pH, temperature and potassium chloride tolerance.
It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds exposed to sunlight the said novel medium for the production of betacarotene and the cultivation of Dunaliella salina is used to develop a new strain. The Dunaliella salina belongs to the family Chlorophycae order volvocales, being 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 but 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 (predominantly betacarotene and its isomers), providing as by-product a substance having a high protein content (biomass) and which can be utilized for various purposes such as a precursor for Vitamin A.

It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL 5) in open raceway ponds essentially exposed to sunlight and wherein a novel medium being used for generation of a new strain of Dunaliella salina has a pH range of 5.0 - 10.0 and a temperature range of 18°C - 55°C and high potassium chloride tolerence from 0.2- 1.0M.
It is another embodiment of the invention that this novel artificial sea water medium for the production of betacarotene and the cultivation of Dunaliella salina (ARL5) in open raceway ponds essentially exposed to sunlight and wherein a novel medium being used for the generation of anew strain of Dunaliella salina, the Dunaliella salina (ARL 5) has a preferable range of 7.5 - 8.5 pH and a preferable temperature range of 32°C - 45°C.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENT OF
INVENTION .
The nature of the invention and the manner in which it is to be performed is clearly described in the specification. The invention has various components and they are clearly described in the following pages of complete specification.
Now the invention will be described in detail and specifically with reference to figure I contained in this complete specification.
The present invention provides a process for the controlled cultivation of an algae designated by us as Dunaliella salina ARL 5 (a local isolate of Chennai coast, India), which belongs to the Class of Chlorophyceae, Order of Volvocales.
The algae used in the process of 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 100 to 120 mg per gram of dry weight of algae. The algae must be cultivated under an adequate high intensity of illumination and this is best carried out outdoors in sunlight.
An artificial medium ought to contain the following nutrients.
KC1 0.2 - 1M preferably 0.35M : NaCl : 1.0 - 5.0 and preferably 2.0 M ; MgS04 0.41M - 1.5M preferably 0.6M ; Ca 2++, 0.01-20mM preferably 0.02-0.04mM; Iron source such as FeEDTA 0.5-4.5uM, preferably 1-3 \xM; NO32' 0.5-1.0M preferably 0.5mM; phosphate 0.01-1M preferably 0.5mM.
A suitable source of carbon, such as HCO3" or C02 in growth medium is provided. The optimum pH for cultivation is between7.5 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.
Various contaminant microorganisms such as fungi, zooplankton, protozoans etc. constitute a problem under conditions of large scale cultivation and these can be overcome to a very large extent by carrying out the cultivation at

higher concentrations of potassium chloride (0.35M), sodium chloride (2.0M) and Magnesium sulphate (0.41M).
In order to obtain a high content of carotenes, it is necessary to provide adequate intensity of illumination. To effect better productivity of carotenes concentrations of nitrogen has to be lower than normal. Since the cultivation is done outdoors the diurnal cycle is provided naturally. Continuous illumination severely inhibits growth.
At lower concentrations of salinity contributed by KC1, MgSC>4 and NaCl proportions the culture doubles very fast though the concentrations of beta carotene drops to 5-8 %. Optimisation of the culture media (salinity and nutrient proportion) has been done to balance growth (biomass) and production of betacarotene and its isomers. The carotenes are recovered from the algae and there remains a residue having a high protein content.
The novel process of cultivation under controlled conditions on a predetermined culture medium provides the possiblility to obtain simultaneously three valuable products viz., (1) carotenes (betacarotene and its isomers and other carotenoids), (2) algae meal and (3)glycerol. In the ensuing figure the conventional route for the cultivation of Dunaliella. salina ARL 5 and producing various carotenes and the route according to the invention is illustrated. Various steps involving the production of carotenes in both the routes are clearly illustrated

On perusal of the figure I the following differences are very clear
'The media used in the conventional process and novel process are different. For example in the conventional process sodium chloride is used as a medium whereas in this novel process a combination of potassium chloride, magnesium sulphate and sodium chloride are used as medium.
In the conventional process betacarotene is achevied in two stages. In the first stage maximisation of growth and in the second stages carotenogenesis is achieved. In the novel process growth and carotenogenesis is achieved in the same stage.
Both algae and medium constitute the subject matter of our invention. A Comparison is made between the conventional process and patented process in terms of parameters namely pH, temperature and the novel media tolerance.

pH range tolerance :
Existing strains of Dunaliella salina Novel Dunaliella salina (ARL5)
Temperature Tolerance :
Existing strains Dunaliella salina Novel Dunaliella salina (ARL 5)
Novel Media Tolerance
Existing strains of Dunaliella salina
Novel Dunaliella salina (ARL 5)

6.5-8.0
5.0-10.0
20°C - 45°C
18°C- 55°C
Causes death in the presence of potassium chloride
High tolerance to Potassium chloride (upto 1.0M)

EXAMPLE
Cultivation of Dunalliella salina ARL 5 for Carotene Production.
Dunaliella salina grown in a seawater medium containing KC1 0.35M; NaCl 2.0M; MgSC>4 0.6M, the other ingredients comprise Ca ++, 0.04mM; Iron source such as FeEDTA 3.0 \\M; NO32' (as potassium nitrate) 0.5mM; phosphate (as potassium hydrogen ortho phosphate) 0.5mM. CO2 as carbon source was added by demand via a pH controlled solenoid valve. The pH is maintained automatically at pH 7.5 - 8.2 by adding CO2 on demand. The algae was grown outdoors in a 15 cm deep open raceway pond under natural conditions of solar irradiation and temperature. The culture was mixed by a paddle wheel agitator at about 20 rpm.
Dunaliella salina ARL5 grew 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 were 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 algae is allowed to stand for 30 min. The algae 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 conventional raceway ponds with a continuous agitation for a period of 8 hrs. during the day. CO2 is sparged through the system through a solenoid valve controlled electronically by means of pH probe. The set point ensures constant control of pH and also ensures carbon addition for growth. The other nutrients comprise Nitrogen as Nitrates,

phosphorous as phosphate, Magnesium as Mg2+, Sulphate as (SO42" ), Iron as Ferric and calcium as Ca2+
It is to be noted that the invention introduces a new paradigm concept in the scientific community. This invention leading to production of carotenes has wide ramification in view of extensive application in various fields of technology and industry nevertheless specific use of invention is listed for perusal.

USES
1. To produce betacarotene precursor for Vitamin A
2. Used as an antioxidant in pharmaceutical and neutraceutical industries
3. Used as a natural colouring agent for food and beverages.
4. Also accumulates glycerol - a fuel substitute
5. Used as feed for crusteaceans and feed additives.
An algae, Dunaliella salina belonging to chlorophyceae possess the characteristics herein described.
1. Unicellular motile algae having two flagella.
2. Having a cup shaped chloroplast.
3. Has a wide pH range tolerance from 5 to 10.0 pH
4. Has a wide temperature range tolerance of 18°C to 55°C
5. Accumulates betacarotene under stress condition Viz., nitrogen and phosphorous depletion.
It is to be noted that the very objective of the description of the invention is to indicate salient features of the invention. It is to be further noted that this description is in no way to abridge the scope of the invention. Further it is clear that within the scope of the invention various modifications are permissible. This scope of the invention is appended in the ensuing description.

We claim,
1. A method of manufacturing betacarotene from the green alga Dunaliella salina (ARL 5) a unique strain which has a high pH (from 5.0 - 10.0) and temperature (from 18 ° C - 55 ° C) tolerance, using a novel artificial sea water medium starting from fresh water containing a ratio of potassium chloride, sodium chloride and magnesium sulphate with ratio of 0.2 - 1.0 M of KCI, 1 - 5M of NaCI and 0.41 - 1.5 M of MgS04, the said process conforming to the above specified combination of the artificial seawater medium wherein the other nutrients are added in the proportion of; Calcium, 0.01-20mM preferably 0.02-0.04mM; Iron source such as FeEDTA 0.5-4.5M, preferably 1-3 |iM; Nitrate 0.5-1.0M preferably 0.5mM; phosphate 0.01-1M preferably 0.5mM which enables to generate algal growth and carotenogenesis in a single stage whereby betacarotene is synthesised not less than 12 % dry on weight basis by Dunaliella salina (ARL 5) and extraction of betacarotene from the Dunaliella salina (ARL 5) are by known methods.
2. A method of manufacturing beta carotene as claimed in claim 1 wherein the optimum concentration of KCI is 0.35 M.
3. A method of manufacturing beta carotene as claimed in claim 1
wherein the optimum concentration of MgS04 is 0.6 M.
4. A method of manufacturing beta carotene as claimed in claim 1
wherein the optimum concentration of NaCI is 2.0 M.

Documents:

1088-mas-1999 abstract.pdf

1088-mas-1999 claims.pdf

1088-mas-1999 correspondence others.pdf

1088-mas-1999 correspondence po.pdf

1088-mas-1999 description (complete).pdf

1088-mas-1999 drawing.pdf

1088-mas-1999 form-1.pdf

1088-mas-1999 form-26.pdf

1088-mas-1999 form-4.pdf

1088-mas-1999 form-6.pdf

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Patent Number

189919

Indian Patent Application Number

1088/MAS/1999

PG Journal Number

30/2009

Publication Date

24-Jul-2009

Grant Date

12-Feb-2004

Date of Filing

11-Nov-1999

Name of Patentee

M/S. PROALGEN BIOTECH LIMITED

Applicant Address

NO.6(old no.58/1), Josier street, Nungambakkam, Chennai-600 034

Inventors:

#	Inventor's Name	Inventor's Address
1	NIDAMANGALA SRINIVASA,VENKATESH	NO.6,RAGHAVIAH ROAD,THYAGARAYA NAGAR, CHENNAI-600 017
2	RAJGOPAL,BALAJI	L NO.6B,10th CROSS STREET,DHANDESSWARAM NAGAR,VELACHERRY,CHENNAI-600 042
3	PARTHASARATHY,SATHYA MURTHY	#6(old no.58/1),JOISER STREET,NUNGAMBAKKM,CHENNAI-600 034

PCT International Classification Number

C12N1/12

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA