Title of Invention

"A MEDIA COMPOSITION USEFUL FOR THE PRODUCTION OF DUNALIELLA SP."

Abstract The present invention relates to a media composition useful for the production of Dunaliella, which is of significance biologically, nutritionally and economically. Dunaliella are green algae belong to Phylum: Chlorophyta Class: Chlorophycae, Order: Volvocales Family: Chlorophycea or Dunalillacae Genus: Dunaliella bardawil. Different strains of Dunaliella exist notably the spices bardawil and other species salina, parva and tertiolecta. Dunaliella is halotolerant algae, with a large economical and biological value. Molecular identification of p-Carotene hyper producing strains of Dunaliella from saline environment using species specific Oligonucleotids .
Full Text The present invention relates to an improved medium for the production of Dunaliella, which is of significance biologically, nutritionally and economically.
Dunaliella are green algae belong to Phylum: Chlorophyta Class: Chlorophycae, Order: Volvocales Family: Chlorophycea or Dunalillacae Genus: Dunaliella bardawil. Different strains of Dunaliella exist notably the spices bardawil and other species salina, pan/a and tertiolecta. Dunaliella is halotolerant algae, with a large economical and biological value (Soto JO, Michel JP, Contreras R & Trujillo T. 2002 Molecular identification of p-Carotene hyper producing strains of Dunaliella from saline environment using species specific Oligonucleotids Biotech. Letters 24: 365-369). Owing to its very high content of p-Carotene. The later is one of high demand molecules due to its benefits as nutritional, prophylactic, and therapeutic agent. It is a precursor of Vitamin A.
D. bardawil is also rich in other nutrient such as protein (17-22%), Lipid (7-12w/w), glycerol (5-8%) p carotene (2-5%), Carbohydrate(20-24%) and ash (22-30%). therefore the whole cell can be used as a nutritional supplement. It was found safe when fed to rats up to three generations, without any symptoms of toxicity (Mokdy ef a/., 1989). It is rich in calcium and contain nucleic acid and heavy metals below the maximum permitted levels. ( Mokady S, Abramovier A and Cogan U1989. "The safety evaluation of Dunaliella bardawil as a potential food supplement". J. Chem. Toxic 27.14:221-226). The Dunaliella has been used as a protein supplement in bread (Finney K F, Pomeranz Y and B.C Bruinsmsa 1984. Use of algae Dunaliella as a protein supplement in bread. Cereal Chemistry, 61: 5: 402-406)
P-Carotene acts as a potential antioxidant agent by quenching singlet oxygen, this is due to number of conjugated double bonds in the molecule. It is also known to reduce risk of cancer by inducing the synthesis of one of the connexon proteins which involved in gap junction, which are very important for cell to cell communication (Burton GW and Ingold KV 1984 p- carotene: An unusual type of lipid antioxidant. Science 11; 569-573.) Other biological benefits include, cardiac disorders, lung cancer (Albanes. D. 1999. p- carotene & lung cancer: A case

study. Am. J. Clin. Nutr. 69: 1345S-1350S). p- carotene acts as hepatoprotective by xenobiotic metabolizing enzyme enhancement in liver (Astrong P., Gradelet S., Leclerc. J, Canivenc MC and Siess MH. 1994 "Effect of p carotene & Astaxanthin on liver xenobiotic- Metabolizing enzyme in the rats. Food .Chem. toxic. 32: 8. 735-742).
Bioavailability of p- carotene in natural form is more than synthetic form. This is mainly due to changes in their solubility pattern and also they are associated with one or more oxygenated carotenoids. (Yeum K J. & Russel RM 2002. Carotenoids bioavailability & bioconversion. Ann. Re//. Nutrition 22:483-504). Other biological benefits of p Carotene includes anticancer, nephroprotection often proving beneficial in malabsorption problem, pancreas disorders, cystic fibrosis, and such activities are attributed to antioxidant or vitamin A activity.
Dunaliella is cultivated generally either in AS-100 medium (Vonshok A. 1986, Laboratory techniques for cultivation of microalgal biomass culture CRC press Florida, USA pp 155 -161) and Ben - Amortz (Ben amortz A. Avron M. 1983 'On the factors which determines massive accumulation in halotolerant alga Dunaliella bardawil' Plant Physiology 72: 593-597.) medium. The composition of these are shown in Table-1. Which contain Magnesium sulfate, Sodium nitrate, Ammonium Chloride as major nutrient and Sodium chloride 1.5M. This media uses TRIS (Tris (Hydroxy Methyl) amino methane C4H11NO3 as source of carbon
The advantages over AS-100 and Ben amortz media is that the modified medium does not require additional external carbon dioxide, as reported in some of the work this does not require two stage cultivation. Here the modified medium after growth for 14 days by diluting with 20 %w/v of sodium chloride can serve as carotenogenesis media. The yield of biomass is 2 folds more than reported media and the carotene content is also high.
Accordingly, the present invention provides an improved medium for the production of Dunaliella comprising,

Chemicals Quantity
Magensium sulphate 1.0-5.0 g/L
Sodium nitrate 0.5 - 3.0 g /L
Sodium bicarbonate 2.0 -10.0g/L
Potassium chloride 0.2 -1.5g/L
Potassium dihydrogen phosphate 0.01 -0.5 g/L
Sodium chloride 30.0-100 g/L
Trace elements mix 5.0-20.0 mL/L
Chelated Iron 1.0-10.0 mL/L
In an embodiment of the improved medium the growth period for the production of Dunaliella is in the range of 12-14 hours per day for a total cultivation period of 6-7 days.
In an another embodiment of the improved medium the biomass yield of Dunaliella using the improved medium is;

Days Yield (Cell count x 104 Cells/ mL Culture)
Initial 34 ±1.2
3 125 + 0.59
6 195 ±0.29
9 309 ±1.36
12 432 ± 0.29
15 696 ±1.11
Biomass dry weight on 15th day 3.94 g/L
p- carotene content in % w/w 2.88
In yet an another embodiment of the improved medium the nutritional composition of Dunaliella grown in improved medium has the following composition.

Composition %w/w
Protein 21.51 ±0.75
Fat 7.8 ±0.38
Carbohydrate 26.7 ±1.11
Carotenoids 2.8 ±0.3
Ash 22.8 ±1.80

Table"!. Chemical composition of different media used. Chemicals in units/ L. (% units in w/v)

Chemicals AS-100 Ben Amortz Modified
MgSO4 0.244 g 0.123 g 0.224 g
MgCl3 - - -
CaCl2 0.03 g 0.044 g -
KH2PO4 0.005 g 0.027 g 0.005 g
KC1 0.06 g - 0.06 g
KNO3 - 0.05 g -
NaNO3 0.1 g - 0.1 g
NaHCO3 - 0.42 g 0.4 g
NaCl 5.0 g 8.78 g 5.0 g
Tris buffer 0.1 g - -
NH4CI 0.00267 g - -
EDTA 0.012 mg(1.2x 10"4) -
FeCl3 -
0.08 mg (8.3 x
Trace metal solution 10"5)
{H3BO3 3.426g/L
CoC12.6H2O 1.215 g/L
MnCl2.4H2O 0.432 g/L 10.0 ml/Lt. 10.0 ml/
ZnCl2 31.5 mg/L (1.0% v/v) Lt.
Conc.H2S04 l.OmL 10.0 ml/Lt. (1.0%
(NH4)6.MO7O2.4H2O 31.19 (1.0% v/v) v/v)
g/L} stock
Chelated Iron solution. 3.0 mL/Lt.
{Na2EDTA (0.3% v/v) -
FeCl3.6H2O} Stock - 3.0
mL/Lt
(0.3%
v/v)
Preparation of Chelated Iron stock solution:
To 500mL of hotglass distilled water was added to 10g of Na2EDTA and stirred. Dissolved 0.81g of FeCI3.6H2O in 500mL of 0.1 N HCI and slowly added this to the hot EDTA solution, while stirring. This solution was cooled before addition to the media.
The following examples are given by way of illustration of the present invention therefore, should not be considered to limit the scope of the present invention.

Example 1.
Among the different species of Dunaliella, the Species bardawil was selected as a best performing strain under various light, temperature and nutrient conditions and preserved for future use. The nutrient medium used for maintaining the stock culture, namely the AS-100 medium, was prepared using the following chemicals:
Table 3. Composition of medium for maintenance of Dunaliella on agar slants

Chemicals (% units in w/v)
MgSO4 0.244 g
CaCI2 0.03 g
KH2PO4 0.005 g
KCI 0.06 g
NaNO3 0.1 g
NaCI 5.0 g
Tris buffer 0.1 g
NH4CI 0.00267 g
Trace metal solution
{H3BO3 3.426g/L
COCI2.6H2O 1.215 mg/L
MnCI2.4H2O 0.432 mg/L
ZnCI2 31.5 mg/L 10.0 ml/ Lt.
Conc.H2SO4 1 .OmL (1.0% v/v)
(NH4)6.MO7O2.4H2O 31.19 mg/L}
Stock
Chelated Iron solution. 3.0 mULt.
(Na2EDTA (0.3% v/v)
FeCI3.6H2O) Stock
The chemicals are dissolved in distilled water and the final volume is made up to one litre, the pH is adjusted to about 7.5, after which 20 grams of agar is added, and agar slants preparation and Dunaliella culture inoculations are done as per standard microbiological methods. Cultures are maintained at a temperature ranging from 20 to 22°C and an illumination cycle of about 16 h of white light of 1000 - 2000 lux and 8 h in dark is maintained. The cells are allowed to grow for a period of 25-40 days.

Example 2
For indoor liquid inoculum development, the Dunaliella cells cultured in agar slants grown as described in example 1 are taken by aseptically scraping the surface of the medium with a spatula and the cells are suspend in media developed containing Magensium sulphate 1-5g/L Sodium nitrate 0.5-3.0/L, Sodium bicarbonate 2 -1Og/L, Potassium chloride 0.2-1.5/L, Potassium dihydrogen phosphate 0.01-0.5 g/L, Sodium chloride 30 -100g/L and trace elements mix 5-20 mL/L and cheated Iron 1-10 mL/L solution, Composition as in table-1. Where the compounds (grams) are dissolved in domestic water to make up a final volume of 1 litre, and an appropriate volume of medium is thus prepared. The suspended cells are incubated at ambient condition with daily 3 to 4 times by manual agitation. The initial optical density of 0.1 at 560nm and cell count of 25-50 X104 cells/mL is ensured and further cell growth is allowed at 25-35°C under 20-30 Klux illumination for a photoperiod of 12-14h per day for a total cultivation period of about one week or till the culture reaches more than 1.0 optical density at 560 nm or cell count of 2-2.5 X106cells/mL (OD 1.0, which corresponds to 200-250 mg dry algae/liter). For further increase in the inoculum volume, the culture broth of optical density 1.0 is diluted 10 times using modified medium and the required level of inoculum is thus developed.
Example 3.
The cell culture broth developed as described in example 2 is subsequently used to produce large quantity of cell biomass in open conventional raceway ponds. The basic design we suggest for mass cultivation of Dunaliella consists of oblong, shallow raceway type pond / tank stirred with or without paddle wheel. Commercial pond area varies between 5 to 5000 m2. The Dunaliella cultures from several carboys is transferred to 5 m2 tank with the addition of 0.90 m3 water fortified with the nutrients of medium the composition of which as above:

The following factors are important for large-scale cultivation of Dunaliella.
Optimum condition
a. Light (Kilolux) 30-35
b. Temperature 25-35°C
c. Inoculum size of OD 0.1 (25-50 X104 cells/mL)
d. Nutrient Modified medium
e. Culture depth (cms) 18-20
Table 5. Growth of Dunaliella in modified medium and biomass yield.

Days Cell count x 10* Cells/ mL Culture
As-100 medium Ben- Amortz Modified
Initial 35 ± 2.5 38 ± 0.55 34 ± 1.2
3 75 ±3.0 98 ± 0.65 125 ±0.59
6 116±2.5 124 ±1.1 195 ±0.29
9 188 ±0.96 213 ±2.0 309 ±1.36
12 296 ±1.23 268 ± 0.56 432 ± 0.29
15 350 ±1.1 295 ± 2.05 696 ±1.11
Biomass dry weight on 15th day 1.96 g/L 1.85 g/L 3.94 g/L
p- carotene content in % w/w 2.22 2.05 2.88
Table 6. Nutritional composition of Dunaliella grown in Modified Medium

Composition %w/w
Protein 21.51 ±0.75
Fat 7.8 ±0.38
Carbohydrate 26.7 ±1.11
Carotenoids 2.8 ±0.3
Ash 22.8 ±1.80
Further the 14-day grown cultures were transferred to larger tanks by diluting the culture to cell count to 25-50x104 cells/mL concentration and transferring to

bigger tank for suitable height under 30-35 Klux illumination for carotenoid accumulation.
Harvesting of the Dunaliella cells was done by centrifugation or any other available conventional method. Harvested biomass was washed with distilled water in order to remove surface coated Sodium chloride and bound minerals. Moreover, the salt concentration of the rinsing solution, which is similar to one of the culture medium, permits to avoid breaking of the cellular membranes due to osmotic pressure.
The harvested Dunaliella biomass can be lyophilized or sprayed, preferably immediately, in order to avoid a secondary contamination. Dunaliella may also be used directly for extraction of carotene using oil or other permitted solvents. It can also be dried by any other standard dying method such as spray-drying, vacuum-drying, cross-flow drying, sun-drying etc.
Advantages
1. Requires less chemicals compared to other reported media and hence
cost effective.
2. Growth is better and yield is good compared to other media and
nutritional composition remains unaffected.
3. Does not require second stage culture media i.e. single media can be
used for carotenogenesis by suitable dilution.










We claim:
1. A media composition useful for the production of Dunaliella sp. comprising of:
A. Magnesium sulphate 1.0-5.0 g/L
B. Sodium nitrate 0.5 - 3.0 g /L
C. Sodium bicarbonate 2.0-10.0g/L
D. Potassium chloride 0.2 -1.5g/L
E. Potassium dihydrogen phosphate 0.01-0.5 g/L
F. Sodium chloride 30.0-100 g/L
G. Trace elements mix 5.0-20.0 ml/L
H. Chelated Iron 1.0-10.0 ml/L
2. A media composition as claimed in claim 1 wherein the trace element mix comprises of:
A. H3BO3 3.426g/L
B. CoCl2.6H2O 1.215 g/L
C. MnCl2.4H2O 0.432 g/L
D. ZnCl2 31.5 mg/L
E. Conc.H2SO4 1.0ml
F. (NH4)6.MO7O2.4H2O 31.19g/L ,wherein the final concentration of the
mix is nearly 1% v/v.
3. A media composition as claimed in claim 1 wherein the chelated iron comprises of:
A. Na2EDTA
B. FeCl3.6H2O ,wherein the final concentration of the solution is 0.3% v/v.
4. A media composition useful for the production of Dunaliella sp. as herein described with
reference to the examples accompanying this specification.


Documents:

587-del-2004-Abstract-(08-11-2010).pdf

587-del-2004-abstract.pdf

587-del-2004-Claims-(08-11-2010).pdf

587-del-2004-claims.pdf

587-del-2004-Correspondence-Others-(08-11-2010).pdf

587-del-2004-correspondence.pdf

587-del-2004-description.pdf

587-del-2004-Form-1-(08-11-2010).pdf

587-del-2004-Form-2-(08-11-2010).pdf

587-del-2004-form1.pdf

587-del-2004-form2.pdf

587-del-2004-form3.pdf

587-del-2004-form5.pdf


Patent Number 244659
Indian Patent Application Number 587/DEL/2004
PG Journal Number 51/2010
Publication Date 17-Dec-2010
Grant Date 14-Dec-2010
Date of Filing 24-Mar-2004
Name of Patentee COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110 001, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 GOKARE ASWATHANARAYANA RAVISHANKAR CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE INDIA
2 KOTAMBALLI NAGENDRA MURTHY CHIDAMBARA MURTHY CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE,INDIA
3 MALLAPPA MAHADEVASWAMY CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE INDIA
4 RAVI SARADA CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE INDIA
PCT International Classification Number C08C
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA