FIELD OF INVENTION This invention in general relates to a novel artificial sea water medium for the production of Betacarotene using Dunaliella salina (ARL 5) in open raceway ponds exposed to sunlight.

This invention further relates to development of novel Dunaliella salina (ARL 5) using the said novel medium as herein described. Further the invention relates to a novel process for developing the said strain.

This invention relates to the use of Potassium chloride in combination with sodium chloride and magnesium sulphate in a proportion in the culture medium for mass cultivation of Dunaliella salina. A process for cultivation of a novel strain of algae Dunaliella salina by addition of Potassium chloride of a particular concentration for the production of biomass (single cell protein), betacarotene and its isomers. The conditions of cultivation and working up of the media are defined herein. The products obtained of value are in particular (1) Carotenoids-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 protein) This invention assumes significance in view of varied applications. This invention with respect to application of Potasium Choloride and its

addition in combination with sodium chloride and magnesium sulphate) introduces a new concept which has wide ramifications.

The following discription in detail traces the various prior art technologies, methods or practices in respect of creation/development of media for the cultivation of Dunaliella salina. The description traces various patent specifications granted in respect of this field of technology and summarises the information contained therein. The company has a rich and varied expertise was committed to reassess the scientific knowledge and information known in this relevant field and the findings of the research are described in the following specification.

STATE OF THE PRIOR ART The US patent application is related to a culture media developed for 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. 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 of 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 has 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 + MgS04 + KCl combination. The artificial medium comprises of KCl-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 0.5-4.5pM preferably 2pM ; N03-0. 5-1 mM preferably 0.5 mM and phosphate 0.01-1mM 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 algae. 2. A novel algae (Dunaliella salina) cultured using the novel medium. 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 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 cultivation of Dunaliella salina (ARL5) in open raceway ponds is essentially exposed to sunlight and wherein the novel media being in the proportion of KCl 0.2-1M, NaCl 1-5M and MgSO4 0. 41-1.5M.

It is another embodiment of the invention that this novel 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 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 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 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 cultivation of Dunaliella salina (ARL 5) in open raceway ponds especially exposed essentially to sunlight wherein a novel medium for cultivation of Dunaliella salina the said medium comprising an artificial sea water medium having the following ingredients (1) KC1 (2) NaCl (3) MgSO4 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. ol- 20mM preferably 0.02-0.04mM; Iron source such as FeEDTA 0.5-4.5, uM, preferably l-3uM ; N03-0. 5-1. OM preferably 0. 5mM ; phosphate 0.01-1M preferably 0. 5mM and C02 as the carbon source.

It is another embodiment of the invention that this novel artificial sea water medium for 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 cultivation of Dunaliella salina (ARL 5) in open raceway ponds exposed to sunlight wherein a novel medium for 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 cultivation of Dunaliella salina (ARL 5) in open raceway ponds exposed to sunlight the said novel medium for 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 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 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.

KCl 0.2-1M preferably 0.35M: NaCl : 1. 0-5.0 and preferably 2.0 ; MgS04 0.41M-1.5M preferably 0.6M ; Ca 2++, 0. 01-20mM preferably 0.02-0.04mM; Iron source such as FeEDTA 0.5-4.5pM, preferably 1-3 u. M ; No32-0. 5-1. OM preferably 0. 5mM ; phosphate 0.01-1M preferably 0. 5mM.

A suitable source of carbon, such as HC03-or CO2 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. OM) 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, MgS04 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 CULTIVATION OF DUNALIELLA SALINAI PRODUCTION OF CAROTENES PROCESS FLOW CHART PRIOR TECHNOLOGY INVENTION TOLERANCE. ALGAE TOLERANCE (Dunaliella salinaARL 5) pH: 6.0-7.5pH PH: 5.0-10. OpH Temperature: 18°C-45°C Temperature-18°C-55°C Ideal Conditions-Potassium chloride tolerance: 1M pH: 6.8-7.0 Ideal : Temperature: 28°C-35°C pH: 7.5-8.5 Temnersalre 3voS4ioC X b Salinity-contributed by Salinity-INOCULUM ADDITION combination of KCl, MgS04 Contributed by NaCl and NaCl in the proportion. 0. 35M KCL+2. 0M NaCl+ 0. 6M MgS04 PRODUCTION RACEWAY PONDS Alga reaches a maximum of 12 % of betacarotene Alga reaches a maximum on dry weight basis foo/i- - on dry weight basis of 8% betacarotene on dry weight Basis4, < HARVEST FIGURE I

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.

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-6. 5-8.0 Novel Dunaliella salina (ARL 5)-5. 0-10.0 Temperature Tolerance : Existing strains Dunaliella salina-20°C-45°C Novel Dunaliella salina (ARL 5)-18°C-55°C Novel Media Tolerance Existing strains of Dunaliella salina-Causes death in the presence of potassium chloride Novel Dunaliella salina (ARL 5)-High tolerance to Potassium chloride (upto 1. OM)

EXAMPLE Cultivation of Dunalliella salina ARL 5 for Carotene Production.

Dunaliella salina grown in a seawater medium containing KC1 0.35M; NaCl 2.0M; MgS04 0.6M, the other ingredients comprise Ca ++, 0.04mM; Iron source such as FeEDTA 3.0 u. M ; Nô32- (as potassium nitrate) 0. 5mM ; phosphate (as potassium hydrogen ortho phosphate) 0. 5mM. C02 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 C02 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. C02 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.