Title of Invention	A PROCESS FOR OUTDOOR CULTIVATION OF MICROALGA BOTRYOCOCCUS SPECIES
Abstract	The present invention relates to a process for out door cultivation of rnicroalga Botryococcus braunii species for mass production. Botryococcus belongs to chlorophyceae (a green alga). It is a colonial, slow growing alga known to accumulate hydrocarbons of unsaturated and saturated hydrocarbons (20 to 50% w/w) in the range of carbon chain C20 to C37. The organism also produces extracellular polysaccharides and fatty acids. In view of the changing energy scenario for renewable energy resources, Botryococcus sps are identified as a potential resource for production of hydrocarbons. However there are no reports on its mass cultivation in any part of the world owing to its slow growth, low light and temperature requirements. Therefore the present invention focused on developing a process for out door cultivation of rnicroalga Botryococcus braunii species for mass production.

Title of Invention

A PROCESS FOR OUTDOOR CULTIVATION OF MICROALGA BOTRYOCOCCUS SPECIES

Abstract

The present invention relates to a process for out door cultivation of rnicroalga Botryococcus braunii species for mass production. Botryococcus belongs to chlorophyceae (a green alga). It is a colonial, slow growing alga known to accumulate hydrocarbons of unsaturated and saturated hydrocarbons (20 to 50% w/w) in the range of carbon chain C20 to C37. The organism also produces extracellular polysaccharides and fatty acids. In view of the changing energy scenario for renewable energy resources, Botryococcus sps are identified as a potential resource for production of hydrocarbons. However there are no reports on its mass cultivation in any part of the world owing to its slow growth, low light and temperature requirements. Therefore the present invention focused on developing a process for out door cultivation of rnicroalga Botryococcus braunii species for mass production.

Full Text	Field of Invention The present invention relates to a process for outdoor cultivation of microalga Botryococcus species useful for its mass cultivation. In general, the present invention relates to microalga such as Botryococcus which also produces hydrocarbons and unsaturated fatty acids and other bioactive metabolites. Background of Invention The potential of micro algae for production of natural pigments and high value metabolites and fuels have been realized throughout the world and efforts are being focused on their culturing and cultivation systems. In this regard, the present invention is focused on a process for mass production of Botryococcus under out door conditions which is known for its high content of hydrocarbons. Botryococcus is a chlorophycean, colonial, slow growing fresh water microalga, which is characterized by unusually high hydrocarbon contents (Maxwell et al 1968, Phytochem. 7:2157-2171; Brown et al 1969, Phytochem, 8:543-547). This microalga is grouped under three different races A, B and L, based on the type of hydrocarbons synthesized (Metzger et al 1985, Phytochem, 24: 2305-2312 and J. Phycol 1990: 26:258). Race A produces C23 to C33 odd numbered n-alkadienes, mono-, tri-, tetra- and pentaenes and are derived from fatty acids. These linear olefins can constitute upto 61% of the dry cell mass of the green active state colonies (Glepi et al 1970, Phytochem, 9:603-612). The L race produces a single hydrocarbon C40-C78, a tetraterpene known as lycopadiene. This can constitute upto 2-8% of the dry biomass (Metzger et al J. Phycol 1990: 26:258). The B race produces polyunsaturated and branched C3o-C37 terpenoid hydrocarbons referred as polymethylated botryococcenes and are promising as a renewable energy source as they are known to be accumulated in very high levels (30-40% of algal dry weight). Botryococcenes are extracted from the total lipids as the hexane-soluble component and can be converted into useful fuels such as gasoline by catalytic cracking (Hillen et al Biotechnol and Bioeng 1982, 24: 193-205), when hydrocracked, the distillate constituted of 67% gasoline, 15% aviation turbine fuel, 15% diesel fuel and 3% residual oil. In natural population, botryococcenes content varies from 27 to 86% of the dry cell mass (Brown et al 1969, Phytochem, 8:543-547). However, under controlled cultured conditions (in door) the hydrocarbon content varies with the species and physiological conditions from 15-80 %. In view of the changing energy scenario for renewable energy sources, Botryococcus sp. are being identified as an untapped resource for production of hydrocarbons. In light of this, the biotechnological exploitation of this potential microalga for hydrocarbons needs to be explored. Besides hydrocarbons the organism also produces extracellular polysaccharides with unique properties along with carotenoids. The organism is also reported to produce bioactive molecules such as plant growth regulators etc. Botryococcus is identified as the promising organism for production of hydrocarbons. However, till date its cultivation under out door conditions is not reported any where in the literature. Therefore, the present invention focuses on the development of a process for out door cultivation for mass production of microalga Botryococcus braunii species. Procedures currently reported for the production of microalgal biomass for value added compounds and hydrocarbons are as follows: Reference may be made to the method of US patent (US 5476787) 1995, wherein Botryococcus was cultivated in waste water for removing nitrogen impurities. The drawback in this method is that it was limited to the organism growth in free form for removing impurities. The growth rates are very slow and also the hydrocarbon details are not mentioned. Reference may be made to another US patent (US 4689301), wherein transparent polyurethane foam which was one side permeable was used as lining of the fermentor for support of the microbial growth. The drawback is that the polyurethane foam used is permeable only on one side and thus the nutrients will not be available to the microalgae. Therefore, the technique remains limited to fermentor and not applicable for the outdoor / open system of cultivation. Reference may be made to another US patent (US 4235043), wherein a method for cultivating algae using a covering material was disclosed. The drawback is that the covering material limits the light intensity useful for growth of microalgae and further its effect on hydrocarbon production is not reported. Reference may be made to US patent (US 6602703), wherein a photo-bioreactor was used for cultivation of microalgae. The drawback is that the photo-bio reactor system is useful for the production of high value metabolites but not feasible for production of hydrocarbons which will be used as substitute for fuel. Also the process becomes very expensive. Reference may be made to Lee et al 1999, Korean Journal of Applied Microbiology and Biotechnology, 27: 166-171 wherein mass cultivation of Botryococcus braunii for the advanced treatment of swine wastewater and lipid production in a photo-bioreactor was reported. The drawback is that the system used is a closed photo-bioreactor under out door conditions for growing algae in wastewater. Therefore, it is not feasible for production of hydrocarbons which will be used as substitute for fuel and the process becomes very expensive. Reference may also be made to Dayananda et al 2007. Biomass and Bioenergy, 31: 87-93, wherein autotrophic cultivation of B. braunii for production of hydrocarbons and exopolysaccharides in various media is reported. The drawback is that the report is limited to laboratory conditions, which are not applicable to mass cultivation under out door conditions. Therefore, keeping in view the lack of any available techniques in the hitherto known prior art for outdoor/ mass cultivation of Botryococcus braunii species the inventors of the present invention realized that there exists a dire need to develop a process for the outdoor cultivation of microalga Botryococcus braunii species useful for its mass cultivation. The said realization is coupled to the fact that Botryococcus braunii species is a potential source for the production of an alternative energy source in an era wherein we are facing severe depletion of the existing renewable sources of energy. The novelty of the invention is that the mass cultivation of Botryococcus braunii species which produces hydrocarbons is successfully achieved under out door culture conditions in an aqueous medium which is not reported till date in any part of the world. Objects of the Invention The main object of the present invention is thus to provide a process for out door cultivation of microalga Botryococcus species which is useful for its mass production. Another object of the present invention is to provide a process which does not favor the growth of contaminants such as protozoan, fungal and other microalgae. Summary of the Invention The present invention relates to a process for out door cultivation for mass production of microalga Botryococcus species. Botryococcus belongs to chlorophyceae (a green alga). It is a colonial, slow growing alga known to accumulate hydrocarbons of unsaturated and saturated hydrocarbons (20 to 50% w/w) in the range of carbon chain C20 to C37. In view of the changing energy scenario for renewable energy resources, Botryococcus sps are identified as a potential resource for production of hydrocarbons. However, there are no reports on its mass cultivation in any part of the world owing to its slow growth, low light and temperature requirements. Therefore, the present invention focused on developing a process for out door cultivation for mass production of microalga Botryococcus braunii species. The invention involved a systematic maintenance of the strains, adaptation of the strains to out door conditions and modification of media to enhance growth and successful scaling up of the culture in out door ponds which is not reported else where in any part of the world. Accordingly, the present invention provides a process for outdoor cultivation of microalga Botryococcus species useful for its mass cultivation, wherein the process steps comprise: a) preparing the inoculum of Botryococcus braunii (SAG 30.81) obtained from Sammlung von Kulturen, Pflanzen Physiologisches Institut, Universitat Gottingen, Gottingen, Germany or Botryococcus braunii (LB 572) obtained from UTEX culture collection centre, USA or Botryococcus braunii (N-836) obtained from NIES culture collection centre, Japan and other Botryococcus species in an aqueous medium of chul3 consisting (g/L) of potassium nitrate -0.20, di potassium phosphate - 0.04, magnesium sulphate - 0.10, calcium chloride - 0.08, ferric citrate - 0.01 and citric acid - 0.02 at pH 7.5 and light intensity of 2 to 4 klux at about 26±2 degree C for a period of about 10 to 15 days; b) inoculating 50 to 150 1 capacity circular or raceway type outdoor ponds containing aqueous medium with 5 to 25 % v/v of the inoculum as obtained in step (a), wherein the medium to pond volume ratio is in the range of 0.1 to 0.4, adding an assimilable nitrogen source to the said medium and bubbling of air mixed with 1 to 5 % [v/v] carbon dioxide at a rate of 1 to 2 liters/minute intermittently or through a float of volume 0.05 to 0.1:1 of culture volume or addition of carbonate/bicarbonate in the concentration range of 0.1 to l.Og/L under light intensity of 2 to 15 klux intensity for 7-10 hours a day for a period of 10 to 20days at ambient temperature ranging from 17 to 30 degree C; c) further inoculating 1000 to 3000 1 capacity circular or raceway type outdoor ponds containing aqueous medium with 5 to 25 % v/v of the inoculum as obtained in step (b), wherein the medium to pond volume ratio is in the range of 0.1 to 0.4, adding an assimilable nitrogen source to the said medium and bubbling of air mixed with 1 to 5% [v/v] carbon dioxide at a rate of 1 to 2 L/minute intermittently or through a float of volume 0.05 to 0.2:1 of culture volume or addition of carbonate/bicarbonate in the concentration range of 0.1 to l.Og/L; d) allowing the culture of step (c) to grow for a period of about 7 to 30 days under light intensity of 2 to 15 klux at a temperature of 17 to 30 degree C, while maintaining the culture pH in the rangeof 6.5 to 9.0 to obtain a biomass yield of 0.25 to 2.0-g/l; e) harvesting the biomass as obtained in step (d) by known methods; f) optionally drying the biomass obtained in step (e) by known methods viz, cross flow drying, oven drying, sun drying and freeze drying. In an embodiment of the present invention the inoculum for Botryococcus braunii strains is grown in containers which may be selected from acrylic, glass or cement of circular type with a diameter of 0.5 to 3 metre with a height of 0.2 to 1 metre and race way type cement tanks with length to width ratio of 1/5 to 1/10th with 0.4 to 0.6 metre height. In another embodiment of the present invention Botryococcus braunii is grown in aqueous medium containing assimilable nitrogen source which may be selected from nitrate salts of sodium, potassium, ammonium or commercial nitrogen sources such as suphala [commercially available nitrogenous fertilizer], DAP in the concentration range of 0.2 to 1.5 g/L. In yet another embodiment of the present invention the aqueous medium is bubbled with air mixed CO2 in the concentration range of 1 to 5 % v/v at a rate of 1-2 L/minute or through a float of volume 0.05 to 0.2:1 of culture volume or sodium carbonate/ bicarbonate in the concentration range of 0.1 to 1.0 g/L. In yet another embodiment of the present invention the Botryococcus braunii species are grown preferably in the pH range of 6.5 to 8.5. In yet another embodiment of the present invention the Botryococcus braunii species may be cultivated in out door raceway ponds of length to width ratio of 5:1 to 10:1.5 with height of 0.4 to 0.8 metre. In yet another embodiment of the present invention the Botryococcus braunii species may be grown in the temperature range of 17 to 30 degree C. In yet another embodiment of the present invention the Botryococcus braunii species may be grown in the light intensity of 2 to 15 klux for a period of 7 to 10 hours a day. In yet another embodiment of the present invention the Botryococcus braunii species may be allowed to grow for a period of 7 to 30 days. Detailed description of the invention The Botryococcus braunii strains were procured from different culture centres viz., • Botryococcus braunii (SAG 30.81) from Sammlung von Kulturen, Pflanzen Physiologisches Institut, Universitat Gottingen, Gottingen, Germany, • Botryococcus braunii (LB 572) from UTEX culture collection, USA and • Botryococcus braunii (N-836) from NIES culture collection, Japan. • Other Botryococcus species including the Indigenous strains collected from different water bodies and purified (which are under deposition) The said cultures were maintained in 2x concentration of Chul3 medium on slants and liquid medium under laboratory conditions such as light intensity of 1.5±0.2 klux light intensity and temperature of 25±1°C temperature. The constituents of chul3 medium are (g/1) potassium nitrate - 0.20, di potassium phosphate - 0.04, magnesium sulphate - 0.10, calcium chloride -0.08, ferric citrate - 0.01 and citric acid - 0.02 at pH 7.5. The cultures were evaluated for tolerance to different environmental conditions such as pH (6.0 to 9.5), temperature (20 to 35°C) and light dark cycle (8 to 16 hours) and light intensity (2 to 20klux) and later adopted to out door conditions in phases. Thereafter, the cultures were scaled up from slant to 150 ml Erlenmeyer flask to 500ml to 1L flask and to 5L carboy to 20L glass tank. Then the cultures were scaled up to 100L circular and race way ponds to 500L and 1000L and finally to 3000L in complete out door conditions with manual controls for temperature and light intensity under extreme conditions. Different sources of carbon and nitrogen sources were used to simplify the cost and enhance the growth rates of the organism. This invention is the first report of its kind to develop an outdoor cultivation process for mass production of Botryococcus species in the world. The scope of this invention does not limit to the above mentioned species only and encompasses various species of Botryococcus including the indigenous ones. The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention. EXAMPLE-1 Sixty four liters of aqueous medium containing potassium nitrate (0.2 g/L) as nitrogen source in circular pond (0.8X0.24 M) was inoculated with log phase cells of Botryococcus at 20% v/v with an initial optical density of 0.09 at 560nm and incubated under a light intensity of 2-11 klux during the day at 23 - 28°C temperature with bubbling air mixed CO2 (2.0%) 500ml /minute intermittently and pH maintained at 7.5 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.4 to 1.7 g/L was obtained. EXAMPLE-2 Ninety six liters of aqueous medium containing ammonium nitrate (0.15 g/L) as nitrogen source in circular pond (1.21X0.2 M) was inoculated with log phase cells of Botryococcus at 15% v/v with an initial optical density of 0.12 at 560nm and incubated under shade net at a light intensity of 2-10 klux during the day at 21 - 27°C temperature with CO2 float at 2.5% v/v and pH maintained at 8.0 for a period of 18days. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.6 to 1.95 g/L was obtained with 15% hydrocarbon content EXAMPLE-3 Fifty six liters of aqueous medium containing commercial nitrogen source (suphala) (0.25 g/L) as nitrogen source in raceway type pond (1.20 x 0.6x0.3 M) was inoculated with log phase cells of Botryococcus at 18% v/v with an initial optical density of 0.07 at 560nm and incubated at a light intensity of 4-15 klux during the day at 21 - 29°C temperature with 0.5 g/L bicarbonate and pH maintained at 8.5 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.2 to 1.5 g/L was obtained with hydrocarbon content of 11%. EXAMPLE-4 Eighty liters of autotrophic medium containing calcium nitrate (0.30g/L) as nitrogen source in a raceway type pond (1.13 x 0.6x 0.3M) was inoculated with log phase cells of Botryococcus at 20% v/v an initial optical density of 0.16 at 560nm and incubated under shade net at a light intensity of 2-14 klux during the day at 22 - 30°C temperature with CO2 float at 2.0% v/ and pH maintained at 7.5 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.8 to 2.0 g/L was obtained. EXAMPLE-5 500 liters of aqueous medium containing calcium nitrate (0.30g/L) as nitrogen source in a raceway type pond (4.8 x 1.4 x 0.4M) was inoculated with log phase cells of Botryococcus at 150% v/v to an initial optical density of 0.16 at 560nm and incubated at a light intensity of 2-10 klux during the day at 20 - 28°C temperature with 0.3 g/L bicarbonate and pH maintained at 8.2 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.7 to 2.0 g/L was obtained. EXAMPLE-6 1500 liters of aqueous medium containing calcium nitrate (0.30g/L) as nitrogen source in a raceway type pond (10 x 2 x 0.5M) was inoculated with log phase cells of Botryococcus at 20% v/v an initial optical density of 0.16 at 560nm and incubated under shade net at a light intensity of 2-10 klux during the day at 22 - 30°C temperature with occasional bubbling of air mixed CO2 at 2.0% v/v at 2L per minute flow rate and the pH was maintained at 8.0 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.5 to 1.8 g/L was obtained. The main advantages of the present invention are: 1. Botryococcus species are successfully cultivated in aqueous medium under outdoor conditions. 2. The temperature and light intensity were controlled effectively by using shade net to such an extent that facilitated Botryococcus growth. 3. This process does not favor the growth of contaminants such as protozoan, fungal and other microalgae. 4. High productivity of biomass nearly 2g/l was obtained under out door conditions. We claim: 1. A process for outdoor cultivation of microalga Botryococcus species useful for its mass cultivation, wherein the process steps comprise: a) preparing the inoculum of Botryococcus braunii (SAG 30.81) obtained from Sammlung von Kulturen, Pflanzen Physiologisches Institut, Universitat Gottingen, Gottingen, Germany or Botryococcus braunii (LB 572) obtained from UTEX culture collection centre, USA or Botryococcus braunii (N-836) obtained from NIES culture collection centre, Japan and other Botryococcus species in an aqueous medium of chulS consisting (g/L) of potassium nitrate - 0.20, di potassium phosphate - 0.04, magnesium sulphate - 0.10, calcium chloride - 0.08, ferric citrate - 0.01 and citric acid - 0.02 at pH 7.5 and light intensity of 2 to 4 klux at about 26±2 degree C for a period of about 10 to 15 days; b) inoculating 50 to 150 1 capacity circular or raceway type outdoor ponds containing aqueous medium with 5 to 25 % v/v of the inoculum as obtained in step (a), wherein the medium to pond volume ratio is in the range of 0.1 to 0.4, adding an assimilable nitrogen source to the said medium and bubbling of air mixed with 1 to 5 % [v/v] carbon dioxide at a rate of 1 to 2 liters/minute intermittently or through a float of volume 0.05 to 0.1:1 of culture volume or addition of carbonate/bicarbonate in the concentration range of 0.1 to l.Og/L under light intensity of 2 to 15 klux intensity for 7-10 hours a day for a period of 10 to 20days at ambient temperature ranging from 17 to 30 degree C; c) further inoculating 1000 to 3000 1 capacity circular or raceway type outdoor ponds containing aqueous medium with 5 to 25 % v/v of the inoculum as obtained in step (b), wherein the medium to pond volume ratio is in the range of 0.1 to 0.4, adding an assimilable nitrogen source to the said medium and bubbling of air mixed with 1 to 5% [v/v] carbon dioxide at a rate of 1 to 2 L/minute intermittently or through a float of volume 0.05 to 0.2:1 of culture volume or addition of carbonate/bicarbonate in the concentration range of 0.1 to l.Og/L; d) allowing the culture of step (c) to grow for a period of about 7 to 30 days under light intensity of 2 to 15 klux at a temperature of 17 to 30 degree C, while maintaining the culture pH in the range of 6.5 to 9.0 to obtain a biomass yield of 0.25 to 2.0 g/1; e) harvesting the biomass as obtained in step (d) by known methods; f) optionally drying the biomass obtained in step (e) by known methods viz, cross flow drying, oven drying, sun drying and freeze drying. 2. A process as claimed in claim 1, wherein the containers in which the inoculum of Botryococcus braunii is grown may be selected from acrylic, glass or cement of circular type with a diameter of 0.5 to 3 metre, height of 0.2 to 1 metre and race way type cement tanks with length to width ratio of 1/5 to I/ 10th with 0.4 to 0.6 metre height. 3. A process as claimed in claim 1, wherein the assimilable nitrogen source may be selected from nitrate salts of sodium, potassium, ammonium or commercial nitrogen sources such as suphala, DAP preferably in the concentration range of 0.2 to 1.5 g/L. 4. A process as claimed in claim 1, wherein the pH of the medium is preferably in the range of 7.0 to 8.5. 5. A process as claimed in claim 1, wherein the outdoor raceway ponds are preferably having length to width ratio of 5:1 to 10:1.5 with height of 0.4 to 0.8 metre. 6. A process as claimed in claim 1, wherein the light intensity of 2 to 15 klux is preferably provided to the culture for a period of 7-10 hours a day. 7. A process as claimed in claim 1, wherein the optimum growth of Botryococcus braunii species is achieved in 12 to 18 days. 8. A process as claimed in claim 1, wherein maximum yield of the biomass about 2.0 g/L having hydrocarbon content in the range of 10 to 20% on dry weight basis, is obtained at temperature of 20-28 degree C under 2 to 10 klux light intensity for 7-10 hours a day for a period of 2 to 3 weeks at pH of 7.5 to 8.5. 9. A process for outdoor cultivation of microalga Botryococcus braunii species substantially as herein described with references to the foregoing examples.

Full Text

Field of Invention
The present invention relates to a process for outdoor cultivation of microalga Botryococcus species useful for its mass cultivation. In general, the present invention relates to microalga such as Botryococcus which also produces hydrocarbons and unsaturated fatty acids and other bioactive metabolites.
Background of Invention
The potential of micro algae for production of natural pigments and high value metabolites and fuels have been realized throughout the world and efforts are being focused on their culturing and cultivation systems. In this regard, the present invention is focused on a process for mass production of Botryococcus under out door conditions which is known for its high content of hydrocarbons.
Botryococcus is a chlorophycean, colonial, slow growing fresh water microalga, which is characterized by unusually high hydrocarbon contents (Maxwell et al 1968, Phytochem. 7:2157-2171; Brown et al 1969, Phytochem, 8:543-547). This microalga is grouped under three different races A, B and L, based on the type of hydrocarbons synthesized (Metzger et al 1985, Phytochem, 24: 2305-2312 and J. Phycol 1990: 26:258).
Race A produces C23 to C33 odd numbered n-alkadienes, mono-, tri-, tetra- and pentaenes and are derived from fatty acids. These linear olefins can constitute upto 61% of the dry cell mass of the green active state colonies (Glepi et al 1970, Phytochem, 9:603-612). The L race produces a single hydrocarbon C40-C78, a tetraterpene known as lycopadiene. This can constitute upto 2-8% of the dry biomass (Metzger et al J. Phycol 1990: 26:258).
The B race produces polyunsaturated and branched C3o-C37 terpenoid hydrocarbons referred as polymethylated botryococcenes and are promising as a renewable energy source as they are known to be accumulated in very

high levels (30-40% of algal dry weight). Botryococcenes are extracted from the total lipids as the hexane-soluble component and can be converted into useful fuels such as gasoline by catalytic cracking (Hillen et al Biotechnol and Bioeng 1982, 24: 193-205), when hydrocracked, the distillate constituted of 67% gasoline, 15% aviation turbine fuel, 15% diesel fuel and 3% residual oil. In natural population, botryococcenes content varies from 27 to 86% of the dry cell mass (Brown et al 1969, Phytochem, 8:543-547). However, under controlled cultured conditions (in door) the hydrocarbon content varies with the species and physiological conditions from 15-80 %.
In view of the changing energy scenario for renewable energy sources, Botryococcus sp. are being identified as an untapped resource for production of hydrocarbons. In light of this, the biotechnological exploitation of this potential microalga for hydrocarbons needs to be explored. Besides hydrocarbons the organism also produces extracellular polysaccharides with unique properties along with carotenoids. The organism is also reported to produce bioactive molecules such as plant growth regulators etc.
Botryococcus is identified as the promising organism for production of hydrocarbons. However, till date its cultivation under out door conditions is not reported any where in the literature. Therefore, the present invention focuses on the development of a process for out door cultivation for mass production of microalga Botryococcus braunii species.
Procedures currently reported for the production of microalgal biomass for value added compounds and hydrocarbons are as follows:
Reference may be made to the method of US patent (US 5476787) 1995, wherein Botryococcus was cultivated in waste water for removing nitrogen impurities. The drawback in this method is that it was limited to the organism

growth in free form for removing impurities. The growth rates are very slow and also the hydrocarbon details are not mentioned.
Reference may be made to another US patent (US 4689301), wherein transparent polyurethane foam which was one side permeable was used as lining of the fermentor for support of the microbial growth. The drawback is that the polyurethane foam used is permeable only on one side and thus the nutrients will not be available to the microalgae. Therefore, the technique remains limited to fermentor and not applicable for the outdoor / open system of cultivation.
Reference may be made to another US patent (US 4235043), wherein a method for cultivating algae using a covering material was disclosed. The drawback is that the covering material limits the light intensity useful for growth of microalgae and further its effect on hydrocarbon production is not reported.
Reference may be made to US patent (US 6602703), wherein a photo-bioreactor was used for cultivation of microalgae. The drawback is that the photo-bio reactor system is useful for the production of high value metabolites but not feasible for production of hydrocarbons which will be used as substitute for fuel. Also the process becomes very expensive.
Reference may be made to Lee et al 1999, Korean Journal of Applied Microbiology and Biotechnology, 27: 166-171 wherein mass cultivation of Botryococcus braunii for the advanced treatment of swine wastewater and lipid production in a photo-bioreactor was reported. The drawback is that the system used is a closed photo-bioreactor under out door conditions for growing algae in wastewater. Therefore, it is not feasible for production of

hydrocarbons which will be used as substitute for fuel and the process becomes very expensive.
Reference may also be made to Dayananda et al 2007. Biomass and Bioenergy, 31: 87-93, wherein autotrophic cultivation of B. braunii for production of hydrocarbons and exopolysaccharides in various media is reported. The drawback is that the report is limited to laboratory conditions, which are not applicable to mass cultivation under out door conditions.
Therefore, keeping in view the lack of any available techniques in the hitherto known prior art for outdoor/ mass cultivation of Botryococcus braunii species the inventors of the present invention realized that there exists a dire need to develop a process for the outdoor cultivation of microalga Botryococcus braunii species useful for its mass cultivation. The said realization is coupled to the fact that Botryococcus braunii species is a potential source for the production of an alternative energy source in an era wherein we are facing severe depletion of the existing renewable sources of energy.
The novelty of the invention is that the mass cultivation of Botryococcus braunii species which produces hydrocarbons is successfully achieved under out door culture conditions in an aqueous medium which is not reported till date in any part of the world.
Objects of the Invention
The main object of the present invention is thus to provide a process for out
door cultivation of microalga Botryococcus species which is useful for its mass
production.
Another object of the present invention is to provide a process which does not
favor the growth of contaminants such as protozoan, fungal and other
microalgae.

Summary of the Invention
The present invention relates to a process for out door cultivation for mass production of microalga Botryococcus species. Botryococcus belongs to chlorophyceae (a green alga). It is a colonial, slow growing alga known to accumulate hydrocarbons of unsaturated and saturated hydrocarbons (20 to 50% w/w) in the range of carbon chain C20 to C37. In view of the changing energy scenario for renewable energy resources, Botryococcus sps are identified as a potential resource for production of hydrocarbons. However, there are no reports on its mass cultivation in any part of the world owing to its slow growth, low light and temperature requirements. Therefore, the present invention focused on developing a process for out door cultivation for mass production of microalga Botryococcus braunii species. The invention involved a systematic maintenance of the strains, adaptation of the strains to out door conditions and modification of media to enhance growth and successful scaling up of the culture in out door ponds which is not reported else where in any part of the world.
Accordingly, the present invention provides a process for outdoor cultivation of microalga Botryococcus species useful for its mass cultivation, wherein the process steps comprise:
a) preparing the inoculum of Botryococcus braunii (SAG 30.81) obtained from Sammlung von Kulturen, Pflanzen Physiologisches Institut, Universitat Gottingen, Gottingen, Germany or Botryococcus braunii (LB 572) obtained from UTEX culture collection centre, USA or Botryococcus braunii (N-836) obtained from NIES culture collection centre, Japan and other Botryococcus species in an aqueous medium of chul3 consisting (g/L) of potassium nitrate -0.20, di potassium phosphate - 0.04, magnesium sulphate - 0.10, calcium chloride - 0.08, ferric citrate - 0.01 and citric acid - 0.02 at

pH 7.5 and light intensity of 2 to 4 klux at about 26±2 degree C for a period of about 10 to 15 days;
b) inoculating 50 to 150 1 capacity circular or raceway type outdoor
ponds containing aqueous medium with 5 to 25 % v/v of the
inoculum as obtained in step (a), wherein the medium to pond
volume ratio is in the range of 0.1 to 0.4, adding an assimilable
nitrogen source to the said medium and bubbling of air mixed with 1
to 5 % [v/v] carbon dioxide at a rate of 1 to 2 liters/minute
intermittently or through a float of volume 0.05 to 0.1:1 of culture
volume or addition of carbonate/bicarbonate in the concentration
range of 0.1 to l.Og/L under light intensity of 2 to 15 klux intensity
for 7-10 hours a day for a period of 10 to 20days at ambient
temperature ranging from 17 to 30 degree C;
c) further inoculating 1000 to 3000 1 capacity circular or raceway type
outdoor ponds containing aqueous medium with 5 to 25 % v/v of the inoculum as obtained in step (b), wherein the medium to pond volume ratio is in the range of 0.1 to 0.4, adding an assimilable nitrogen source to the said medium and bubbling of air mixed with 1 to 5% [v/v] carbon dioxide at a rate of 1 to 2 L/minute intermittently or through a float of volume 0.05 to 0.2:1 of culture volume or addition of carbonate/bicarbonate in the concentration range of 0.1 to l.Og/L;
d) allowing the culture of step (c) to grow for a period of about 7 to 30
days under light intensity of 2 to 15 klux at a temperature of 17 to
30 degree C, while maintaining the culture pH in the rangeof 6.5 to
9.0 to obtain a biomass yield of 0.25 to 2.0-g/l;
e) harvesting the biomass as obtained in step (d) by known methods;
f) optionally drying the biomass obtained in step (e) by known methods
viz, cross flow drying, oven drying, sun drying and freeze drying.

In an embodiment of the present invention the inoculum for Botryococcus braunii strains is grown in containers which may be selected from acrylic, glass or cement of circular type with a diameter of 0.5 to 3 metre with a height of 0.2 to 1 metre and race way type cement tanks with length to width ratio of 1/5 to 1/10th with 0.4 to 0.6 metre height.
In another embodiment of the present invention Botryococcus braunii is grown in aqueous medium containing assimilable nitrogen source which may be selected from nitrate salts of sodium, potassium, ammonium or commercial nitrogen sources such as suphala [commercially available nitrogenous fertilizer], DAP in the concentration range of 0.2 to 1.5 g/L.
In yet another embodiment of the present invention the aqueous medium is bubbled with air mixed CO2 in the concentration range of 1 to 5 % v/v at a rate of 1-2 L/minute or through a float of volume 0.05 to 0.2:1 of culture volume or sodium carbonate/ bicarbonate in the concentration range of 0.1 to 1.0 g/L.
In yet another embodiment of the present invention the Botryococcus braunii species are grown preferably in the pH range of 6.5 to 8.5.
In yet another embodiment of the present invention the Botryococcus braunii species may be cultivated in out door raceway ponds of length to width ratio of 5:1 to 10:1.5 with height of 0.4 to 0.8 metre.
In yet another embodiment of the present invention the Botryococcus braunii species may be grown in the temperature range of 17 to 30 degree C.

In yet another embodiment of the present invention the Botryococcus braunii species may be grown in the light intensity of 2 to 15 klux for a period of 7 to 10 hours a day.
In yet another embodiment of the present invention the Botryococcus braunii species may be allowed to grow for a period of 7 to 30 days.
Detailed description of the invention
The Botryococcus braunii strains were procured from different culture centres viz.,
• Botryococcus braunii (SAG 30.81) from Sammlung von Kulturen,
Pflanzen Physiologisches Institut, Universitat Gottingen, Gottingen,
Germany,
• Botryococcus braunii (LB 572) from UTEX culture collection, USA and
• Botryococcus braunii (N-836) from NIES culture collection, Japan.
• Other Botryococcus species including the Indigenous strains collected
from different water bodies and purified (which are under deposition)
The said cultures were maintained in 2x concentration of Chul3 medium on slants and liquid medium under laboratory conditions such as light intensity of 1.5±0.2 klux light intensity and temperature of 25±1°C temperature.
The constituents of chul3 medium are (g/1) potassium nitrate - 0.20, di potassium phosphate - 0.04, magnesium sulphate - 0.10, calcium chloride -0.08, ferric citrate - 0.01 and citric acid - 0.02 at pH 7.5. The cultures were evaluated for tolerance to different environmental conditions such as pH (6.0 to 9.5), temperature (20 to 35°C) and light dark cycle (8 to 16 hours) and light intensity (2 to 20klux) and later adopted to out door conditions in phases.
Thereafter, the cultures were scaled up from slant to 150 ml Erlenmeyer flask to 500ml to 1L flask and to 5L carboy to 20L glass tank. Then the cultures

were scaled up to 100L circular and race way ponds to 500L and 1000L and finally to 3000L in complete out door conditions with manual controls for temperature and light intensity under extreme conditions. Different sources of carbon and nitrogen sources were used to simplify the cost and enhance the growth rates of the organism. This invention is the first report of its kind to develop an outdoor cultivation process for mass production of Botryococcus species in the world. The scope of this invention does not limit to the above mentioned species only and encompasses various species of Botryococcus including the indigenous ones.
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.
EXAMPLE-1
Sixty four liters of aqueous medium containing potassium nitrate (0.2 g/L) as nitrogen source in circular pond (0.8X0.24 M) was inoculated with log phase cells of Botryococcus at 20% v/v with an initial optical density of 0.09 at 560nm and incubated under a light intensity of 2-11 klux during the day at 23 - 28°C temperature with bubbling air mixed CO2 (2.0%) 500ml /minute intermittently and pH maintained at 7.5 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.4 to 1.7 g/L was obtained.
EXAMPLE-2
Ninety six liters of aqueous medium containing ammonium nitrate (0.15 g/L) as nitrogen source in circular pond (1.21X0.2 M) was inoculated with log phase cells of Botryococcus at 15% v/v with an initial optical density of 0.12 at 560nm and incubated under shade net at a light intensity of 2-10 klux

during the day at 21 - 27°C temperature with CO2 float at 2.5% v/v and pH maintained at 8.0 for a period of 18days. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.6 to 1.95 g/L was obtained with 15% hydrocarbon content
EXAMPLE-3
Fifty six liters of aqueous medium containing commercial nitrogen source (suphala) (0.25 g/L) as nitrogen source in raceway type pond (1.20 x 0.6x0.3 M) was inoculated with log phase cells of Botryococcus at 18% v/v with an initial optical density of 0.07 at 560nm and incubated at a light intensity of 4-15 klux during the day at 21 - 29°C temperature with 0.5 g/L bicarbonate and pH maintained at 8.5 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.2 to 1.5 g/L was obtained with hydrocarbon content of 11%.
EXAMPLE-4
Eighty liters of autotrophic medium containing calcium nitrate (0.30g/L) as nitrogen source in a raceway type pond (1.13 x 0.6x 0.3M) was inoculated with log phase cells of Botryococcus at 20% v/v an initial optical density of 0.16 at 560nm and incubated under shade net at a light intensity of 2-14 klux during the day at 22 - 30°C temperature with CO2 float at 2.0% v/ and pH maintained at 7.5 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.8 to 2.0 g/L was obtained.
EXAMPLE-5
500 liters of aqueous medium containing calcium nitrate (0.30g/L) as nitrogen source in a raceway type pond (4.8 x 1.4 x 0.4M) was inoculated with log phase cells of Botryococcus at 150% v/v to an initial optical density of 0.16

at 560nm and incubated at a light intensity of 2-10 klux during the day at 20 - 28°C temperature with 0.3 g/L bicarbonate and pH maintained at 8.2 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.7 to 2.0 g/L was obtained.
EXAMPLE-6
1500 liters of aqueous medium containing calcium nitrate (0.30g/L) as nitrogen source in a raceway type pond (10 x 2 x 0.5M) was inoculated with log phase cells of Botryococcus at 20% v/v an initial optical density of 0.16 at 560nm and incubated under shade net at a light intensity of 2-10 klux during the day at 22 - 30°C temperature with occasional bubbling of air mixed CO2 at 2.0% v/v at 2L per minute flow rate and the pH was maintained at 8.0 for a period of 3 weeks. Biomass was harvested and growth was estimated in terms of dry weight. A biomass yield of 1.5 to 1.8 g/L was obtained.
The main advantages of the present invention are:
1. Botryococcus species are successfully cultivated in aqueous medium under
outdoor conditions.
2. The temperature and light intensity were controlled effectively by using
shade net to such an extent that facilitated Botryococcus growth.
3. This process does not favor the growth of contaminants such as protozoan,
fungal and other microalgae.
4. High productivity of biomass nearly 2g/l was obtained under out door
conditions.

We claim:
1. A process for outdoor cultivation of microalga Botryococcus species
useful for its mass cultivation, wherein the process steps comprise:
a) preparing the inoculum of Botryococcus braunii (SAG 30.81)
obtained from Sammlung von Kulturen, Pflanzen Physiologisches
Institut, Universitat Gottingen, Gottingen, Germany or Botryococcus
braunii (LB 572) obtained from UTEX culture collection centre, USA
or Botryococcus braunii (N-836) obtained from NIES culture
collection centre, Japan and other Botryococcus species in an
aqueous medium of chulS consisting (g/L) of potassium nitrate -
0.20, di potassium phosphate - 0.04, magnesium sulphate - 0.10,
calcium chloride - 0.08, ferric citrate - 0.01 and citric acid - 0.02 at
pH 7.5 and light intensity of 2 to 4 klux at about 26±2 degree C for
a period of about 10 to 15 days;
b) inoculating 50 to 150 1 capacity circular or raceway type outdoor
ponds containing aqueous medium with 5 to 25 % v/v of the
inoculum as obtained in step (a), wherein the medium to pond
volume ratio is in the range of 0.1 to 0.4, adding an assimilable
nitrogen source to the said medium and bubbling of air mixed with 1
to 5 % [v/v] carbon dioxide at a rate of 1 to 2 liters/minute
intermittently or through a float of volume 0.05 to 0.1:1 of culture
volume or addition of carbonate/bicarbonate in the concentration

range of 0.1 to l.Og/L under light intensity of 2 to 15 klux intensity for 7-10 hours a day for a period of 10 to 20days at ambient temperature ranging from 17 to 30 degree C;
c) further inoculating 1000 to 3000 1 capacity circular or raceway type
outdoor ponds containing aqueous medium with 5 to 25 % v/v of the
inoculum as obtained in step (b), wherein the medium to pond
volume ratio is in the range of 0.1 to 0.4, adding an assimilable
nitrogen source to the said medium and bubbling of air mixed with 1
to 5% [v/v] carbon dioxide at a rate of 1 to 2 L/minute intermittently
or through a float of volume 0.05 to 0.2:1 of culture volume or
addition of carbonate/bicarbonate in the concentration range of 0.1
to l.Og/L;
d) allowing the culture of step (c) to grow for a period of about 7 to 30
days under light intensity of 2 to 15 klux at a temperature of 17 to
30 degree C, while maintaining the culture pH in the range of 6.5 to
9.0 to obtain a biomass yield of 0.25 to 2.0 g/1;
e) harvesting the biomass as obtained in step (d) by known methods;
f) optionally drying the biomass obtained in step (e) by known methods
viz, cross flow drying, oven drying, sun drying and freeze drying.
2. A process as claimed in claim 1, wherein the containers in which the inoculum of Botryococcus braunii is grown may be selected from acrylic, glass or cement of circular type with a diameter of 0.5 to 3 metre, height

of 0.2 to 1 metre and race way type cement tanks with length to width ratio of 1/5 to I/ 10th with 0.4 to 0.6 metre height.
3. A process as claimed in claim 1, wherein the assimilable nitrogen
source may be selected from nitrate salts of sodium, potassium,
ammonium or commercial nitrogen sources such as suphala, DAP
preferably in the concentration range of 0.2 to 1.5 g/L.
4. A process as claimed in claim 1, wherein the pH of the medium is
preferably in the range of 7.0 to 8.5.
5. A process as claimed in claim 1, wherein the outdoor raceway ponds are
preferably having length to width ratio of 5:1 to 10:1.5 with height of
0.4 to 0.8 metre.
6. A process as claimed in claim 1, wherein the light intensity of 2 to 15
klux is preferably provided to the culture for a period of 7-10 hours a
day.
7. A process as claimed in claim 1, wherein the optimum growth of
Botryococcus braunii species is achieved in 12 to 18 days.

8. A process as claimed in claim 1, wherein maximum yield of the biomass
about 2.0 g/L having hydrocarbon content in the range of 10 to 20% on
dry weight basis, is obtained at temperature of 20-28 degree C under 2
to 10 klux light intensity for 7-10 hours a day for a period of 2 to 3
weeks at pH of 7.5 to 8.5.
9. A process for outdoor cultivation of microalga Botryococcus braunii
species substantially as herein described with references to the
foregoing examples.

Documents:

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Patent Number

279117

Indian Patent Application Number

2377/DEL/2007

PG Journal Number

02/2017

Publication Date

13-Jan-2017

Grant Date

11-Jan-2017

Date of Filing

13-Nov-2007

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	GOKARE ASWATHANARAYANA RAVISHANKAR	CFTRI,MYSORE
2	RAVI SARADA	CFTRI, MYSORE
3	TUMKUR RAMACHANDRIAH SHAMALA	CFTRI, MYSORE

PCT International Classification Number

A01G13/02; A01G1/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA