Abstract:
The present invention provides a cost effective biotechnological process for production of bio-fuels from isolated and characterized microalgae. The algal strains used in the present invention having higher biomass, higher lipid productivity, higher pH and temperature tolerance are selected from the group consisting of  Chlorella vulgaris  iOC-1,  Chlorella vulgaris  iOC-2,  Chlorella kessleri, Botrococcus bruni, Dunaliella salina  and  Nannochloris oculat  or a combination thereof having 95-100% similarity with 18s ribosomal nucleic acids nucleotide sequences (rDNA) given for Seq. ID I, Seq. ID 2, Seq. ID 3, Seq. ID 4, Seq. ID 5 and Seq. ID 6. The present process of bio-fuel production comprises the steps of producing lipid from green algae in bioreactors by various novel steps and extracting oil from dried algal cells and ultimately producing biodiesel by transesterification of the said extracted oil.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention is related to the field of biofuel. More particularly, the present invention is directed to a process for the production of lipids suitable for biofuel production from microalgae by heterotrophic cultivation. 
       BACKGROUND OF THE INVENTION 
       [0002]    The need of energy is increasing continuously, because of increase in industrialization and population. The basic sources of this energy are petroleum, natural gas, coal, hydro and nuclear. The major disadvantage of using petroleum based fuels is atmospheric pollution created by the use of petroleum diesel. Petroleum diesel combustion is a major source of greenhouse gas (GHG). Apart from these emissions, petroleum diesel is also major source of other air contaminants including NOx, SOx, CO, particulate matter and Volatile Organic Compounds (VOC). 
         [0003]    Biomass is one of the better sources of energy. Large-scale introduction of biomass energy could contribute to sustainable development on several fronts, environmentally, socially and economic. Bio-diesel (monoalkyl esters) is one of such alternative fuel, which is obtained by the transesterification of triglyceride oil with monohydric alcohols. Biodiesel fuel can be prepared from waste cooking oil, such as palm, soybean, canola, rice bran, sunflower, coconut, corn oil, fish oil, chicken fat and algae which would partly decrease the dependency on petroleum-based fuel. 
         [0004]    Macroalgae has also been used in the production of biodiesel. Microalgae with higher oil content than plants are known and they are faster and easier to grow. Microalgae can provide several different types of renewable biofuels. These include methane produced by anaerobic digestion of the algal biomass, biodiesel derived from microalgal oil and photobiologically produced biohydrogen. The idea of using microalgae as a source of fuel is not new but it is now being taken seriously because of the extinguishing petroleum resources and, more significantly, the emerging concern about global warming that is associated with burning fossil fuels. 
         [0005]    Microalgae comprise a vast group of unicellular photosynthetic, heterotrophic organisms which have an extraordinary potential for cultivation as energy crops. Microalgae are the great source of many highly valuable products such as polyunsaturated fatty acids, astaxanthin and bioactive compounds. 
         [0006]    Microalgae can be grown in two different modes: Photoautotrophic and Heterotrophic mode of growth. Large-scale production of these products, however, has hindered by an ability to obtain high cell densities and productivities in photoautotrophic systems because of light penetration issues and uncontrolled growth conditions. High cell density processes suitable for heterotrophic cultures of microalgae may provide an alternative means for large-scale production of algal products of high value. The heterotrophic growth of algae holds many practical applications in industrial scale especially in a controlled manner to obtain highest biomass as well as lipid productivity. 
         [0007]    In heterotrophic conditions algae can be grown on organic carbon sources, such as sugars and organic acids. This mode of culture eliminates the requirement for light and therefore, offers the possibility of greatly increased cell density and productivity. Some microalgae show rapid heterotrophic growth. Heterotrophic algal cultivation has been reported to provide not only a high algal biomass productivity, but high cellular oil content as well. Additionally the culture suffers from the contamination by undesired microbes. However, to date, the very few reports of such processes for microalgal cultivation have mostly been on lab-scale work/plant scale. 
         [0008]    Xu et al 2006 (Han Xu, Xiaoling Miao and Qingyu Wu High quality biodiesel production from a microalga  Chlorella protothecoides  by heterotrophic growth in fermenters. Journal of Biotechnology Volume 126, Issue 4, 1 Dec. 2006, Pages 499-507) discussed high quality biodiesel production from a microalga  Chlorella protothecoids  through the technology of transesterification. The technique of metabolic controlling through heterotrophic growth of  C. protothecoides  was applied, and the heterotrophic  C. protothecoides  contained the crude lipid content of 55.2%. To increase the biomass and reduce the cost of alga, corn powder hydrolysate instead of glucose was used as organic carbon source in heterotrophic culture medium in fermenters. The result showed that cell density significantly increased under the heterotrophic condition, and the highest cell concentration reached 15.5 g L-1. Large amount of microalgal oil was efficiently extracted from the heterotrophic cells by using n-hexane, and then transmuted into biodiesel by acidic transesterification. The biodiesel was characterized by a high heating value of 41 MJ kg-1, a density of 0.864 kg L-1, and a viscosity of 5.2×10−4 Pa s (at 40° C.). The method has great potential in the industrial production of liquid fuel from microalga. 
         [0009]    Li et al. 2007 reported (Li X F, Xu H, Wu Q Y (2007) Large-scale biodiesel production from microalga  Chlorella protothecoides  through heterotrophic cultivation in bioreactors. Biotechnol Bioeng 98:764-771) an integrated approach of biodiesel production from heterotrophic  Chlorella protothecoides  focused in bioreactors. Through substrate feeding and fermentation process controls, the cell density of  C. protothecoides  achieved 15.5 g L −1  in 5 L, 12.8 g L −1  in 750 L, and 14.2 g L −1  in 11,000 L bioreactors, respectively. Resulted from heterotrophic metabolism, the lipid content reached 46.1%, 48.7%, and 44.3% of cell dry weight in samples from 5 L, 750 L, and 11,000 L bioreactors, respectively. 
         [0010]    Liang et al (2009) reported (Liang Y, Sarkany N, Cui Y 2009 Biomass and lipid productivities of  Chlorella vulgaris  under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnology Letters July; 31(7):1043-9) biomass and lipid productivities of  Chlorella vulgaris  under different growth conditions. While autotrophic growth did provide higher cellular lipid content (38%), the lipid productivity was much lower compared with those from heterotrophic growth with acetate, glucose, or glycerol. Optimal cell growth (2 g l(-1)) and lipid productivity (54 mg l(-1) day(-1)) were attained using glucose at 1% (w/v) whereas higher concentrations were inhibitory. Growth of  C. vulgaris  on glycerol had a similar dose effects as those from glucose. Overall,  C. vulgaris  is mixotrophic. 
         [0011]    US patent application 2009/0211150A1 discloses a method to produce biodiesel from algae using a strain of microalga  chlorella protothecoids,  by screening a specific strain with characteristics of high yield of biomass and high oil content, cultivating the screened strain for high-cell-density growth for up to 108 grams of dry cell weight per liter of the suspension in a bioreactor using solutions containing carbohydrates as feed, harvesting and drying the high density cultivated algal cells to extract oil from the dried algal cells, and producing the biodiesel by catalyzed transesterification using the extracted oil as feedstock. 
         [0012]    In the prior art few microalgal strains have been cultured to produce lipids for biodiesel. However, lipids produced from these microalgal strains are mostly rich in unsaturated fatty acids which makes them unsuitable for biodiesel production. Therefore, there exists a need to have increased saturated fatty acids content in lipids from microalgal strain with higher biomass oil content using various cheap carbon sources for cultivation which ultimately makes the process more cost effective and applicable in terms of its industrial success. 
       OBJECT OF THE INVENTION 
       [0013]    The principal object of the present invention is to provide a process to produce suitable lipids from heterotrophic cultivation of microalgae for biodiesel. 
         [0014]    Another object is to provide a process where lipids are produced from microalgal strain with higher biomass and oil content. 
         [0015]    Yet another object of the invention is to provide a process where lipids is produced from the microalgae which can withstand high temperature and high pH. 
         [0016]    Still another object of the invention is to provide a process for production of biofuel where the microalgae is fed cheap source of carbohydrates like wheat flour, hydrolysate of lignocellulosic biomass, organic waste water streams like sewage treatment plant, water from distillery, fruit processing industry, dairy industry etc. 
       SUMMARY OF THE INVENTION 
       [0017]    Accordingly, the present invention provides a novel and cost effective method to produce oil feedstock for the production of biofuel by heterotrophic growth of green microalgae in a bioreactor. The process of the present invention comprises the steps of isolation of high oil producing green algae with characteristics of high yield of biomass and high oil content; screening of the same for heterotrophic growth, inoculating the strain in a bioreactor for algal-seed-cells cultivation; transferring the cultivated algal-seed-cells into a second bioreactor for high-cell-density cultivation; feeding a second solution containing carbohydrates into the second bioreactor; harvesting the high density cultivated algal cells from the second bioreactor; drying the high density cultivated algal cells; extracting oil from dried algal cells; and producing the biodiesel by reaction of transesterification using the extracted oil as feedstock or using the biomass for gasification, fermentative biohydrogen, bioethanol, and biomethane production. The high-cell-density cultivation of the present invention further comprises heterotrophic cultivation of  Chlorella vulgaris, C. kessleri, Botrococcus bruni, Dunaliella salina  and  Nannochloris oculata  high oil content, preferably up to 58% dry cell weight. The carbohydrates solutions include but not limit to glucose or other monosaccharides, and/or disaccharides, or polysaccharides preferably with the concentration of glucose or other monosaccharides, disaccharides, polysaccharides, or hydrolysates of corn starch, wheat flour, hydrolysate of lignocellulosic biomass e.g. cheaper lignocellulosic biomass belonging to Lemna, baggase, sugar cane top, pine needle, wheat straw, rice straw etc, organic waste water streams like sewage treatment plant, water from distillery and fruit processing industry, dairy industry containing organic sugars, molasses preferably with concentration of the carbohydrates solutions being controlled between 0.01 and 100 gL.sup.-1. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0018]    The present invention comprises the steps of isolation of high oil producing green alga with characteristics of high yield of biomass and high oil content; screening of the same for heterotrophic growth, inoculating the strain in a bioreactor for algal-seed-cells cultivating; transferring the cultivated algal-seed-cells into a second bioreactor for high-cell-density cultivation; feeding a second solution containing carbohydrates into the second bioreactor; harvesting the high density cultivated algal cells from the second bioreactor; drying the high density cultivated algal cells; extracting oil from dried algal cells; and producing the biodiesel by reaction of transesterification using the extracted oil as feedstock or using the biomass for gasification, fermentative biohydrogen, bioethanol, and biomethane production. 
         [0019]    The algal strains used for the process are  Chlorella vulgaris  iOC-1,  Chlorella vulgaris  iOC-2,  Chlorella kessleri, Botrococcus bruni, Dunaliella salina  and  Nannochloris oculat  or combination thereof. The said algae can be grown separately as well as in different combination providing better yield in terms of biomass and lipid content/composition. These algal strains have been well characterized by their 18s ribosomal nucleic acids. The partial genomic DNA sequences shows 95% to 100% sequence identities to the nucleic acid sequences selected from the group consisting of Seq. ID 1, Seq. ID 2, Seq. ID 3, Seq. ID 4, Seq. ID 5 and Seq. ID 6. and the algal strains shows higher temperature tolerance up to 52° C. 
         [0020]    The source of carbohydrates fed in the bioreactor selected from the group consisting of pure sugar(s), hydrolysates of corn starch, wheat flour, hydrolysate of lignocellulosic biomass, organic waste water streams like sewage treatment plant, water from distillery and fruit processing industry, dairy industry containing organic sugars, molasses etc. The process of the present invention optionally comprises feeding of nitrogen into the bioreactor. The nitrogen is organic nitrogen which may be selected from the group consisting of glycine, yeast powder, yeast extract, peptone, ammonium chloride, urea, KNO 3 , Ammonium nitrate, ammonia, or corn syrup. 
         [0021]    feeding of phosphorus into the bioreactor. The phosphorus used in the bioreactor may be selected from the group consisting of di-ammonium phosphate, K 2 HPO 4  or KH 2 PO 4 . 
       Isolation of Specific Algae 
       [0022]    Soil and water samples were collected from different locations e.g. Effluent treatment plant (ETP), Yamuna River, Agra, agricultural soil. The water and soil sample were inoculated in 0.8% agar medium was prepared using media containing KH.sub.2PO.sub.4: 0.7 g/L, K.sub.2HPO.sub.4: 0.3 g/L, MgSO.sub.4.7H.sub.2O: 0.3 g/L, FeSO.sub.4.7H.sub.2O: 3 mg/L, Glycine: 0.1 g/L, vitamin B.sub.1: 0.01 mg/L, A5 trace mineral solution 1.0 ml/L, wherein the A5 trace mineral solution comprises H.sub.3BO.sub.3, Na.sub.2MoO.sub.4.2H.sub.2O, ZnSO.sub.4.7H.sub.2O, MnCl.sub.2.4H.sub.2O, and CuSO.sub.4.5H.sub.2O. A preferred A5 trace mineral solution comprises: H.sub.3BO.sub.3: 2.86 g/L, Na.sub.2MoO.sub.4.2H.sub.2O: 0.039 g/L, ZnSO.sub.4.7H.sub.2O: 0.222 g/L, MnCl.sub.2.4H.sub.2O: 1.81 g/L, CuSO.sub.4.5H.sub.2O: 0.074 g/L. 1 g L-1 peptone, 2 g L-1 yeast extract, 4 g L-1 glucose, and antibiotics including ampicillin (sodium form), streptomycin sulfate, and kanamycin sulfate (100 mg L-1 each). After inoculation, the plates were wrapped and stored at 26° C. for 2-5 days. Single green colonies were picked and carefully transferred to a new plate. The purified colonies are selectively picked up and inoculated into flasks containing growth medium including but not limiting to components of basal medium under light conditions, for further culture. 
       Screening for Heterotrophic Growth 
       [0023]    The micro-algal strains were inoculated into a 500-mL Erlenmeyer flasks containing 100-mL medium at 28° C. under continuous shaking at 180 rpm. Glucose with a concentration of 30 g/L and yeast extract with a concentration of 4 g/L are added into the basal medium. The heterotrophic media was incubated in the dark. The cell growth rates and cellular oil contents in different culture are then compared with each other to determine a specific strain with characteristics of the highest oil content and a high cell growth rate. The selected strain having ability to utilize sugar as carbon source and grow in heterotrophic conditions were selected for evaluation of their pH tolerance, temperature tolerance, ability to grow in wastewater, ability to grow in presence of different contaminants like hydrocarbon, heavy metals etc and strains having ability to grow in stringent condition with high cell density and higher lipid accumulation were selected for further study. 
       Identification and Characterization of Algal Strains 
       [0024]    The selected algal strains were identified by their physiological, morphological characteristics. The 18S rRNA gene sequences as well as some specific morphological characteristics have been extensively studied by the present inventors. The resulting 18S rRNA gene sequences were aligned insilico and compared to the nucleotide sequences of some known microalge in GenBank database of the National Center for Biotechnology Information by using Basic Local Alignment Search Tool (BLAST®). 
         [0025]    The partial genomic DNA sequences shows 95% to 100% sequence identities to the nucleic acid sequences selected from the group consisting of Seq. ID 1, Seq. ID 2, Seq. ID 3, Seq. ID 4, Seq. ID 5 and Seq. ID 6. 
       Algal-Seed-Cells Cultivation 
       [0026]    The autotrophically grown cells of selected strain inoculated in the bioreactor aseptically containing culture medium for algal-seed-cells cultivation. The components of basal culture medium are: (mg/l) Glucose—9000, KNO 3 —1011.1, NaH 2 PO 4 .H 2 O—621, Na 2 HPO 4 .2H 2 O—89, MgSO 4 .7H 2 O—246.5, EDTA—9.3, H 3 BO 3 —0.061, CaCl 2 .2H 2 O—14.7, FeSO 4 .7H 2 O—6.95, ZnSO 4 .7H 2 O—0.287, MnSO 4 .H 2 O—0.169, (NH 4 ) 6 Mo 7 O 24 .4H 2 O—0.01235, CuSO 4 .5H 2 O—0.00249, 1 g L-1 peptone, 2 g L-1 yeast extract, 4 g L-1 glucose, and antibiotics including ampicillin (sodium form), streptomycin sulfate, and kanamycin sulfate (100 mg L-1 each). The bioreactor was operated 28° C. in dark at 480 rpm. The pH maintained for pH range 6-7 and sampling was done every day to estimate the dry cell weight, chlorophyll content and lipid content regularly. Algal cell yield can be determined using various methods, including but not limiting to light intensity measurement of the cell suspension, such as OD540 nm of cell suspension. Preferable conditions such as glucose concentration, different nitrogen sources in the basal medium, temperatures, and shaking rate during algal-seed-cells cultivation in shaking flasks are determined by real-time light intensity measurement of the cell suspension. A preferable glucose concentration in a range of 2 to 40 g/L glucose is added in the basal medium. A preferable yeast extract in a range of 05 to 15 g/L yeast extract is added in the basal medium. A temperature in incubator is set between 10-50° C., preferably at 30° C. The shaking rate is controlled between 50 to 700 rpm, preferably at 300 rpm. The cells are harvested till the culture of algal seed cell enters into late-exponential-phase. The cell harvesting time before reaching the late exponential-phase is approximately at 120 hours. 
       High-Cell-Density Cultivation 
       [0027]    The late-exponential-phase algal-seed-cells in the small bioreactor are transferred to a second bioreactor of 200 L containing 150 L media, for high-cell-density cultivation by process control and optimization. Glucose and yeast extract solutions are added into the basal culture medium initiate in the second bioreactor, preferably with 2 to 80 g/L glucose and 0.5 to 15 g/L yeast extract, further preferably with 45 g/L glucose and 6 g/L yeast extract. Parameters, such as the amount of inoculum, substrate (organic carbon and nitrogen) feeding, oxygen supply, stirring rate, temperature, pH, and time of cell harvest, and adjusted to optimize cell growths in the second bioreactor. Among the parameters, dissolved oxygen (DO) in the fermentation suspension for high-cell-density cultivation of heterotrophic algal cells in the bioreactor can be used to monitor the growth conditions, such as organic carbon sources in the reactor, biomass and accumulation of lips. On-line monitoring of the DO parameter is preferably used to monitor the growth conditions, and to adjust agitation speed and aerating rate. The carbohydrates solutions include but not limit to glucose or other monosaccharides, and/or disaccharides, or polysaccharides, preferably with the concentration of glucose or other monosaccharides, disaccharides, polysaccharides, or hydrolysates of corn starch, wheat flour, hydrolysate of lignocellulosic biomass, organic waste water streams like sewage treatment plant, water from distillery and fruit processing industry, dairy industry containing organic sugars, molasses preferably with concentration of the carbohydrates solutions being controlled between 0.01 and 100 gL.sup.-1. 
         [0028]    The conditions for high-cell-density heterotrophic cultivation of the different strains were automatically monitored and set as follows:
       Inoculum amount of seed algal cells (V/V): 10-30%, preferably of 20%;   Temperature at 15-52° C., preferably at 30±.0.5° C.; Aeration 100-200 L/h (1:1 vvm), preferably at 180 L/h;   pH 6.0 to 9.0, preferably at 6.3.±.0.1;   Concentration of glucose in medium: 20 g/L;
 
DO over 20% controlled by increasing agitation and airflow, gradually increasing agitation speed from 100 to 600 rpm after a period of cultivation for about 88 hours, to maintain the dissolved oxygen at above 20% of saturation;
       
 
         [0033]    When the cell density and/or the oil content reach desired values, preferably with the dry cell density reaching 24 g/L and the oil content reaching 58% of dry cell weight, the growth of the cells in the second reactor is terminated. The growth duration in said second bioreactor lasts about 120 hours. 
         [0034]    The extreme conditions of pH and high temperature provide an advantage of inhibiting the growth of undesired microbes and obtaining less unsaturated fatty acids. 
       Harvesting the High Density Microalgal Cells from Bioreactor 
       [0035]    After determining a sample of the high-cell-density heterotrophic cultivation to reach a desired dry biomass concentration, preferable between 12 to 24 g/L, dry biomass of the algal cell suspension from bioreactor is separated using a separation process, including but not limited to flocculation, filtration or centrifuge. The separated dry biomass may be in a form of powder or other solid forms. 
       Extracting the Oil from Dried Algal Cells 
       [0036]    Lipids (oil) in heterotrophic cell powder are subsequently extracted by any well known solvent extraction methodology, e.g. the Soxhlet method, wherein N-hexane is used as the standard Soxhlet solvent for extracting oil from cells. Extraction is achieved by washing the cells repeatedly with pure solvent until no lipid is left in cells. Then the solvent in the extract is removed under reduced pressure. In an embodiment the selected microalgae was cultivated at temperature as high as 40-50° C. The lipid extracted from the algae on analysis for fatty acid composition were found to have more than 70% fatty acids saturated as compared to only 40% at 30° C. for the same algae. The selected algae have the ability to grow at extreme pH i.e., pH 5-9 and at temperature as high as upto 52 degree C. 
       Producing the Biodiesel from Microalgal Oil 
       [0037]    These extracted microalgal oil can then be converted into biodiesel by known methods of transesterification, e.g. the enzymatic transesterification and/or acids and/or base catalyst. 
       EXAMPLE 
     Isolation and Selection of Heterotrophic Algal Strains 
       [0038]    In the present invention, the soil and water samples were collected from Effluent Treatment Plant (ETP) of hydrocarbon processing industry. The collected water and soil samples were inoculated in 0.8% agar medium. The preparation was made using media containing KH2.sub.2PO.sub.4: 0.7 g/L, K.sub.2HPO.sub.4: 0.3 g/L, MgSO.sub.4.7H.sub.2O: 0.3 g/L, FeSO.sub.4.7H.sub.2O: 3 mg/L, Glycine: 0.1 g/L, vitamin B.sub.1: 0.01 mg/L, A5 trace mineral solution 1.0 ml/L, wherein the A5 trace mineral solution comprises H.sub.3BO.sub.3, Na.sub.2MoO.sub.4.2H.sub.2O, ZnSO.sub.4.7H.sub.2O, MnCl.sub.2.4H.sub.2O, and CuSO.sub.4.5H.sub.2O. A preferred A5 trace mineral solution comprises: H.sub.3BO.sub.3: 2.86 g/L, Na.sub.2MoO.sub.4.2H.sub.2O: 0.039 g/L, ZnSO.sub.4.7H.sub.2O: 0.222 g/L, MnCl.sub.2.4H.sub.2O: 1.81 g/L, CuSO.sub.4.5H.sub.2O: 0.074 g/L. 1 g L-1 peptone, 2 g L-1 yeast extract, 4 g L-1 glucose, and antibiotics including ampicillin (sodium form), streptomycin sulfate, and kanamycin sulfate (100 mg L-1 each). After inoculation, the plates were wrapped and stored at 26° C. for 2-5 days. Single green colonies were picked and carefully transferred to a new plate. The purified colonies are selectively picked up and inoculated into flasks containing growth medium, including but not limited to components of basal medium, for further culture. 
         [0039]    The selected algal strains were characterized according to their 18S rRNA gene sequences, as well as some morphological characteristics. Six algae having six different sequences for 18S rRNA gene were obtained. These sequences were named as Seq ID1, Seq ID2, Seq ID3, Seq ID4, Seq ID5, Seq ID6. Seq. ID 1 represents DNA sequence of  Chlorella vulgaris  IOC-1 18S ribosomal RNA gene; Seq. ID 2 represents DNA sequence of  Chlorella vulgaris  IOC-2 18S ribosomal RNA gene; Seq. ID 3 represents DNA sequence of  Chlorella kessleri  18S ribosomal RNA gene; Seq. ID 4 represents DNA sequence of  Botryococcus braunii  18S ribosomal RNA gene; Seq. ID 5 represents  Dunaliella salina  18S ribosomal RNA gene and Seq. ID 6 represents  Nannochloris oculata  18S small subunit ribosomal RNA gene. 
         [0040]    The resulting 18S rRNA gene sequences were aligned and compared to the nucleotide sequences of some known microalge in GenBank database of the National Center for Biotechnology Information by using Basic Local Alignment Search Tool (BLAST®). Five potential culture having ability to grow in heterotrophic conditions and accumulate high lipid content and higher biomass was identified as  Chlorella vulgaris, Chlorella kessleri, Botrococcus brunii, Dunaliella salina  and  Nannochloris oculata.    
       Heterotrophic Growth in Bioreactor 
       [0041]    The selected strain inoculated in the bioreactor aseptically containing culture medium for algal-seed-cells cultivation. The components of basal culture medium are: (Mg/l) Glucose—9000, KNO 3 —1011.1, NaH 2 PO 4 .H 2 O—621, Na 2 HPO 4 .2H 2 O—89, MgSO 4 .7H 2 O—246.5, EDTA—9.3, H 3 BO 3 —0.061, CaCl 2 .2H 2 O—14.7, FeSO 4 .7H 2 O—6.95, ZnSO 4 .7H 2 O—0.287, MnSO 4 .H 2 O—0.169, (NH 4 ) 6 Mo 7 O 24 .4H 2 O—0.01235, CuSO 4 .5H 2 O—0.00249, 1 g L-1 peptone, 2 g L-1 yeast extract, 4 g L-1 glucose, and antibiotics including ampicillin (sodium form), streptomycin sulfate, and kanamycin sulfate (100 mg L-1 each). The bioreactor was operated 28° C. in dark at 480 rpm. The pH maintained for pH range 6-7 and sampling was done every day to estimate the dry cell weight, chlorophyll content and lipid content regularly. Algal cell yield can be determined using various methods, including but not limiting to light intensity measurement of the cell suspension, such as OD540 nm of cell suspension. Preferable conditions such as glucose concentration, different nitrogen sources in the basal medium, temperatures, and shaking rate during algal-seed-cells cultivation in shaking flasks are determined by real-time light intensity measurement of the cell suspension. A preferable glucose concentration in a range of 2 to 40 g/L glucose is added in the basal medium. A preferable yeast extract in a range of 05 to 15 g/L yeast extract is added in the basal medium. A temperature in incubator is set between 10-50° C., preferably at 30° C. The shaking rate is controlled between 50 to 700 rpm, preferably at 300 rpm. The cells are harvested till the culture of algal seed cell enters into late-exponential-phase. The cell harvesting time before reaching the late exponential-phase is approximately at 168 hours. 
         [0042]    The late-exponential-phase algal-seed-cells in the small bioreactor are transferred to a second bioreactor of 200 L containing 150 L media, for high-cell-density cultivation by process control and optimization. Glucose and yeast extract solutions are added into the basal culture medium initiate in the second bioreactor, preferably with 2 to 80 g/L glucose and 0.5 to 15 g/L yeast extract, further preferably with 45 g/L glucose and 6 g/L yeast extract. Parameters, such as the amount of inoculum, substrate (organic carbon and nitrogen) feeding, oxygen supply, stirring rate, temperature, pH, and time of cell harvest, and adjusted to optimize cell growths in the second bioreactor. Among the parameters, dissolved oxygen (DO) in the fermentation suspension for high-cell-density cultivation of heterotrophic algal cells in the bioreactor can be used to monitor the growth conditions, such as organic carbon sources in the reactor, biomass and accumulation of lips. On-line monitoring of the DO parameter is preferably used to monitor the growth conditions, and to adjust agitation speed and aerating rate. The carbohydrates solutions include but not limit to glucose or other monosaccharides, and/or disaccharides, or polysaccharides, preferably with the concentration of glucose or other monosaccharides, disaccharides, polysaccharides, or hydrolysates of corn starch, wheat flour, hydrolysate of lignocellulosic biomass, organic waste water streams like sewage treatment plant, water from distillery and fruit processing industry, dairy industry containing organic sugars, molasses preferably with concentration of the carbohydrates solutions being controlled between 0.01 and 100 gL.sup.-1. 
         [0043]    The conditions for high-cell-density heterotrophic cultivation of the different strains were automatically monitored and set as follows:
       Inoculum amount of seed algal cells (V/V): 20%;   Temperature at 30.+−.0.5.° C.   Aeration 180 L/h;   pH 7.3.±.0.1;   Concentration of glucose in medium: 20% (w/v)
 
DO over 20% controlled by increasing agitation and airflow, gradually increasing agitation speed from 100 to 600 rpm after a period of cultivation for about 88 hours, to maintain the dissolved oxygen at above 20% of saturation;
       
 
         [0049]    When the cell density and/or the oil content reach desired values, preferably with the dry cell density reaching24 g/L and the oil content reaching 41% of dry cell weight, the growth of the cells in the second reactor is terminated. The growth duration in said second bioreactor lasts about 120 hours. Cell growth is measured by the absorbance of the suspension at 540 nm and dry cell weight. 1.5 ml of algal culture was taken in pre-weighed Eppendorf tubes, centrifuged at 8000 rpm for 5 minutes. The supernatant media was removal using micropipette and the algae pellet at the bottom was dried at 105° C. until the constant weight was achieved. The dry weight of algae biomass was determined gravimetrically and growth was expressed in terms of dry weight. Lipid measurements were made by using a mixture of methanol, chloroform, and water. A culture sample is collected at three points during the experiments for lipid analysis. The culture sample is centrifuged at 3,500 rpm for 10 minutes in a large (200 ml) plastic centrifuge tube; the pelleted cells along with 35 ml of supernatant are then transferred to a plastic centrifuge tube (45 ml) to be re-centrifuged again at 5000 rpm for 10 minutes. The supernatant is removed by pipette. The pellet is then resuspended with 4 ml of DI H 2 O, then 10 ml of methanol and 5 ml of chloroform is added, resulting in a 10:5:4 ratio of methanol:chloroform:water. At this ratio, all solvents are miscible and form one layer. After overnight extraction on a shaker table, 5 ml of water and 5 ml of chloroform are added which results in a 10:10:9 ratio of methanol:chloroform:water. Tubes are centrifuged for 10 minutes at 5000 rpm. At this solvent ratio, two layers are formed, a water methanol upper layer and chloroform lower layer. The chloroform lower layer which contains the extracted lipids is then removed by Pasteur pipette and placed into a pre-weighed vial. After the first extraction, 10 ml of additional chloroform is added to conduct a second extraction. The additional 10 ml of chloroform again results is a 10:10:9 methanol:chloroform:water ratio and two layers are formed. The tube is centrifuged at 3,500 rpm for 10 minutes, and the lower chloroform layer is removed by Pasteur pipette and placed into another pre-weighed vial. The chloroform is evaporated by heating in a 55° C. water bath under a constant stream of nitrogen gas. After 1 hour in a 105° C. oven, vials are weighed again. The weight difference represents weight of lipids extracted from the culture sample. The extracted lipid was analysed by gas chromatography as per method described in prior art. The lipid showed fatty acid suitable for biodiesel production. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 The Bio-mass and oil content of micro-algal species 
               
               
                 obtained under heterotrophic conditions 
               
             
          
           
               
                   
                   
                 Biomass 
                 Oil Content 
               
               
                   
                 Strain 
                 (g (DCW)/l) 
                 (% w/w (DCW) 
               
               
                   
                   
               
             
          
           
               
                   
                   Chlorella vulgaris  IOC-1 
                 16.82 
                 29.8 
               
               
                   
                   C. vulgaris  IOC-2 
                 12.01 
                 27.4 
               
               
                   
                 
                   C. kessleri 
                 
                 11.08 
                 29.4 
               
               
                   
                   Botrococcus bruni , 
                 12.8 
                 41.5 
               
               
                   
                 
                   Dunaliella salina 
                 
                 24.5 
                 32.5 
               
               
                   
                 
                   Nannochloris oculata 
                 
                 15.4 
                 29.7 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 SEQ ID 1 to 6: 
                   
               
               
                 &gt; Chlorella vulgaris  for 18S ribosomal RNA strain: IOC-1 
               
               
                 SEQ 1 
                   
               
               
                 TTTCATTCAAATTTCTGCCCTATCAACTTTTGATGGTAGGATAGAGGCCTACCATGGTGGTAAC 
                   
               
               
                 GGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAG 
               
               
                 GAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACAGGGAGGTAGTGACAATAAATAACAAT 
               
               
                 ACTGGGCCCGATCAGGTCTGGTAATTGGAATGAGTACAATCTAAACCCCTTAACGAGGATCAAT 
               
               
                 TGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTG 
               
               
                 CTGCAGTTAAAAAGCTCGTAGTTGGATTTCGGGTGGGACCTGCCGGTCCGCCGTTTCGGTGTGC 
               
               
                 ACTGGCAGGGCTCACCTTGTTGCCGGGGACGGGCTCCTGGGCTTCACTGACCGGGACTCGGAGT 
               
               
                 CGGCGCTGTTACTTTGAGTAAATTAGAGTGTTCAAAGCAGGCCTACGCTCTGAATACATTAGCA 
               
               
                 TGGAATAACACGATAGGACTCTGGCCTATCCTGTTGGTCTGTAGGACCGGAGTAATGATTAAGA 
               
               
                 GGGACAGTCGGGGGCATTCGTATTTCATTGTCAGAGGTGAAATTCTTGGATTTATGAAAGACGA 
               
               
                 ACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGTCCGCGAGTTGGGGGCTCGAA 
               
               
                 GACGATTAGATACCGTCCTAGTCTCAACCATAAACGATGCCGACTAGGGATCGGCGGATGTTTC 
               
               
                 TTCGATGACTCCGCCGGCACCTTATGAGAAATCAAAGTTTTTGGGTTCCGGGGGGAGTATGGTC 
               
               
                 GCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGATTCTGGCTTAAT 
               
               
                 TTGACTCAACACGGGAAAACTTACCAGGTCCAGACATAGTGAGGACTGACAGATTGAGACTCTT 
               
               
                 TCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGGTTGCCTTGTCAGGTTGAT 
               
               
                 TCCGGTAACGAACGAGACCTCAGCCTGCTAAATAGTCACGGTTGGTTCGCCAGCCGGCGGACTT 
               
               
                 CTTAGAGGGACTATTGGCGACTAGCCAATGGAAGCATGAGGCTATAACAGGTCTGTGATGCCCT 
               
               
                 TAGATGTTCTGGGCCGCACGCGCGCTACACTGATGCATTCAACGAGATTAGCCTTGGCCGAGAG 
               
               
                 GCCCGGGTAATCTTCGAAACTGCATCGTGATG 
               
               
                   
               
               
                 &gt; Chlorella vulgaris  for 18S ribosomal RNA strain: IOC-2 
               
               
                 SEQ 2 
                   
               
               
                 AAAAGGCCGACCGGGCTTCTGCCCGACTCGCGGTGAATCATGATAACTTCACGAATCGCATGGC 
                   
               
               
                 CTTGTGCCGGCGATGTTTCTTTCAAATTTCTGCCCTATCAACTTTTGATGGTAGGATAGAGGCC 
               
               
                 TACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACG 
               
               
                 GCTACCACATCCAAGGAAGGCAGCAGGCACGCAAATTACCCAATCCTGACACAGGGAGGTAGTG 
               
               
                 ACAATAAATAACAATACTGGGCCTTGTCAGGTCTGGTAATTGGAATGAGTACAATCTAAACCCC 
               
               
                 TTAACGAGGATCAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAG 
               
               
                 CGTATATTTAAGTTGCTGCAGTTAAAAAGCTCGTAGTTGGATTTCGGGTGGGACCTGCCGGTCC 
               
               
                 GCCGTTTCGGTGTGCACTGGCAGGGCTCACCTTGTTGCCGGGGACGGGCTCCTGGGCTTCACTG 
               
               
                 TCCGGGACTCGGAGTCGGCGCTGTTACTTTGAGTAAATTAGAGTGTTCAAAGCAGGCCTACGCT 
               
               
                 CTGAATACATTAGCATCGAATAACACGATAGGACTCTGGCATATCCTGTTGGTCTGTAGGACCG 
               
               
                 GAGTAATGATTAAGAGGGACAGTCTGGGGCATTCGTATTTCATTGTCAGAGGTGAAATTCTTGG 
               
               
                 ATTTATGAAAGACGAACTACTGCCCTAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAA 
               
               
                 AGTTGGGGGCTCGAAGACGATTAGATACCGTCCTAGTCTCAAGCATAAACGATGCCGACTAGGG 
               
               
                 ATCGGCGGATGTTTCTTCGATGACTCCGCCGGCACCTTATGAGATATCAAAGTTTTTAGCTTCC 
               
               
                 GGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGG 
               
               
                 AGCCTGCGGCTTAAGGAGACTCAACACGGGAAAACTTACGAGGTCCAGACATAGTGAGGATTGA 
               
               
                 CAGATTGAGAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGGTT 
               
               
                 GCCTTGTCAGGTTGATTCCGGTAACGAACGAGACCTCAGCCTGCTAAATAGTCACGGTTGGTTC 
               
               
                 GCCAGCCGGCGGACTTCTTAGAGGGAAATGCCTAGTAAGCGC 
               
               
                   
               
               
                 &gt; Chlorella kessleri  18S ribosomal RNA 
               
               
                 SEQ 3 
                   
               
               
                 CGTAAATCCCGACTTCTGGAAGGGACGTATTTATTAGATTTAAGGCCGACCCGGCTCTGCCGGT 
                   
               
               
                 CTCGCGGTGAATCATGATAACTTCACGAATCGCATGGCCTTGCGCCGGCGATGTTTCATTCTTT 
               
               
                 TTTCTGCCCTATCAACTTTCGATGGTAGGATAGAGGCCTACCATGGTGGTAACGGGTGACGGAG 
               
               
                 GATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGCCAGCAG 
               
               
                 GCGCGCAAATTACCCAATCCTGACACAGGGAGGTAGTGACAATAAATTTCAATACCGGGCCTTT 
               
               
                 TCAGGTCTGGTAATTGGAATGAGTACAATCTAAACCCCTTAACGAGGATCAATTGGAGGGCAAG 
               
               
                 TCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGCTGCAGTTAAA 
               
               
                 AAGCTCGTAGTTGGATTTCGGGCGGGGCCTGCCGGTCCGCCGTTTGGGTGTGCACAGGCAGGGC 
               
               
                 CCGCCTTGTTGCCGGGGACGGGCTCCTGGGCTTCACTGTCCGGGACTCGGAGTTGGCGCTGTTA 
               
               
                 CTTTGAGTAAATTAGAGTGTTCAAAGCAGGCCTACGCTCTGAATGCATTAGCATGGAATAACAC 
               
               
                 GATAGGACTCTGGCCTATCCTGTTGGTCTGTAGGACCGGAGTAATGATTAAGAGGGACAGTCGG 
               
               
                 GGGCATTCGTATTTCGATGTCAGAGGTGAAATTCTTGGATTTTCGAAAGACGAACTACTGCGAA 
               
               
                 AGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGATGGGGGCTCGAAGACGATTAGAT 
               
               
                 ACCGTCCTAGTCTCAACCATAAACGATGCCGACTAGCGATCGGCGGATGTTTCTTCGATGACTC 
               
               
                 CGCCGGCACCTTATGAGAAATCAAAGTTTTTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAA 
               
               
                 ACTTGGGGGAATTGACGGAAGGGCACCACCATGCGTGGAGCCTGCGGCTTAATTTGACTCAACA 
               
               
                 CGGGAAAACTTACCAGGTCCAGACATAGTGCGGATTGACAGATTGAGAGCTCTTTCTTGATTCT 
               
               
                 ATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGGTTGCCTTGTCAGGTTGATTCCGGTAACG 
               
               
                 AACGAGACCTCAGCCTGCTAAATCGTCACGGCCTCCTCGGGGGCCGGCAGACTTCTTAGAGGGA 
               
               
                 CTATTGGCGACTAGCCAATGGAATCATGAGGCAATAACAGGTCTGTGATGCCCTTAGATGCCCT 
               
               
                 GGGCCGCACGCGCGCTACTCTGATGCAATCAACGAGCCTAGCCTTGG 
               
               
                   
               
               
                 &gt;  Botryococcus braunii  18S ribosomal RNA gene 
               
               
                 SEQ 4 
                   
               
               
                 TATTTATTAGATAAAAGGCTGACCGGGCTCGCCCGACTCTTGCTGACTCATGATAACTCGACGG 
                   
               
               
                 ATCGCACGGGCTTGTCCCGGCGACGTTTCATTCGCTTTTCTGCCCTATCAACTGTCGATGGTAC 
               
               
                 GGTAGTGGCCTACCATGGTGTTCACGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGCGC 
               
               
                 CTGAGAGACGGCGACCACATCCAAGGCCGGCAGCAGGCGCGCAAATTACCCAATCCTGACACAG 
               
               
                 GGAGGTAGTGACAATAAATAACAATATCGGGGTTTCCAAACTCTGATAATTGGAATGAGTACAA 
               
               
                 TCTAAAATCCTTAACGAGGATCAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCA 
               
               
                 GCTCCAATAGCGTATACCCAAGTTGTTGCAGTTAAGCTGCTCGTAGTCGGACTTCGGGTGGGGG 
               
               
                 CCGGCGGTCCGCCGACTGGTGTGCCATGCCGGGCCCCGCCTTGCTGCCGGAGATGGGATCCTGG 
               
               
                 GCTTCGCTGTCCGGGACCCGGACTCGGCGTGGTTACTTTGAGTAAATTAGAGTGTTCAAAGCAG 
               
               
                 GCCTACGCTCTGAATATGTTAGCATGGAATAACGCGATAGGACTCTGGCCTATCTTGTTGGTCT 
               
               
                 GTGGGACCGGAGTAATGATTAAGAGGGACAGTCGGGGGCATTCGTATTTCATTGTCAGGGGTGA 
               
               
                 AATTCTTGGATTTATGAAAGACGGACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTGATC 
               
               
                 AAGAACGAAAGTTGGGGGCTCGAAGACGATTAGATACCGTCCTAGTCTCAACCATAAACGATGC 
               
               
                 CGACTAGGGATTGGTGGGTGTTCTTTTGACGACCCCTCCAGCACCTTATGAGAAATCAAAGTTT 
               
               
                 TTGGGTTCCGGGGCGAGTATGGTCGTAAGGCTGGAACTTAAAGGAATTGACGGAAGGGCACCAC 
               
               
                 CAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAGACATAGT 
               
               
                 GAGGATTGACAGATTGAGAGCTCTTCCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGT 
               
               
                 TGGTGGGTTGGCTTGTCAGGTTGATTCCGGTAACGAACGAGACCTCAGCCTGCTAAATAGTCCG 
               
               
                 ACCAGGTTCGCCCAGGCCGCCGACTTCTTAGAGGGACTCTCGGCGACTAGCCGGAGGAAGTGTG 
               
               
                 AGGCGATAACAGGACTGTG 
               
               
                   
               
               
                 SEQ 5 
                   
               
               
                 &gt;  Dunaliella salina  18S ribosomal RNA gene 
                   
               
               
                 ATTAGATGGTACCTTTACTCGGATAACCGTAGTAATTCTAGAGCTAATACGTGCGTAAATCCCG 
               
               
                 ACTTCTGGAAGGGACGTATTTATTAGATAAAAGGCCAGCCGGGCTTGCCCGACTCTTGGCGAAT 
               
               
                 CATGATAACTTAACGAATCGCACGGCTTTATGCCGGCGATGTTTCATTCAAATTTCTGCCCTAT 
               
               
                 CAACTTTCGATGGTAGGATAGAGGCCTACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCG 
               
               
                 ACCCCGGAGAGGGAGCCTGAGATTCGGCTACCAAATCCCAGGAAGGCAGCAGGCGCGCAAATTA 
               
               
                 CCCAATCCCAACACGGGGATGTAGTGACAATAAATAACAATACCGGGCATTTTTGTCTGGTAAT 
               
               
                 TGGAATGAGTACAATCTAAATCCCTTAACGAGTATCCATTGGAGGGCAAGTCTGGTGAAAGCAG 
               
               
                 CCGCGGTAATTCCAGCTCCAATAGCGTATATGTAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGG 
               
               
                 ATTTCGGGTGGGTTGTAGCGGTCAGCCTTTGGTTAGTACTGCTACGGCCTACCTTTCTGCCGGG 
               
               
                 GACGAGCTCCTGGGCTTAACTGTCCGGGACTCGGAATCGGCGAGGTTACTCTGAGTAAATTAGA 
               
               
                 GTGTTCAAAGCAAGCCTACGCTCTGAATACATTAGCATGGAATAACACGATCGGACTCTGGCTT 
               
               
                 ATCTTGTTGGTCTGTAAGACCGGAGTAATGATTAAGAGGGACAGTCGGGGCCATTCGTATTTCA 
               
               
                 CTGTCAGAGGTGAAATTCTTGGATTTTGAAAGACGAACTTCCTGCGAAAGCATTTGCCAAGGAT 
               
               
                 GTTTTCATTAATCCAAGAACGAAAGTTGGGGGCTCGAAGACGATTAGATACCAGTCGTAGTCTC 
               
               
                 AACCATAAACGATGCCGACTTAGGGATTGGCAGGTGTTTCGTTGATGACCCTGCCAGCACCTTT 
               
               
                 ATGAGAAATCACAGTTTTTTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAA 
               
               
                 TTGACGGAAGGGCACCACCAGGCGTGGAGCATGCGGCTTAATTAGACTCAACACGGGAAAACTT 
               
               
                 ACCAGGTCCAGACACGGGGAGGATTGACAGATTGAGAGCTCTTTCTTGATTCTGTGGGTGGTGG 
               
               
                 TGCATGGCCGTTCTTAGTTGGTGGGTTGCCTTGTCAGGTTGATTCCGG 
               
               
                   
               
               
                 SEQ 6 
                   
               
               
                 &gt;  Nannochloris oculata  18S small subunit ribosomal RNA gene 
                   
               
               
                 TATAAACTGCTTTATACTATGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTATTTGATG 
               
               
                 GTACCTACTTACTCGGATAACCGTAGTAATTCTAGACGTAATACGTGCGCACATCCCGACTTCT 
               
               
                 GGAAGGGACGTATTTATTAGATAAAAGGCCGACCGGATTTTTCCGACTCGCGGTGACTCATGAT 
               
               
                 AACTTCACGAATCGCATGGCCTCGTGCCGGCGATGTTTCATTCAAATTTCTGCCCTATCGGCTT 
               
               
                 TTGATGGTAGGATAGAGGCCTACCATGGTGGTAACGGGTGACGGAGAATTAGGGTTCGATTCCG 
               
               
                 GAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAAT 
               
               
                 CCTGACACAGGGAGGTAGTGACAATAAATAACAATACCGGGCCTTTGGTCTGGTAATTGGAATG 
               
               
                 AGTACAACCTAAACACCTTAACGAGGATCAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGT 
               
               
                 AATTCCAGCTCCAATAGCGTATATTTAAGTTGCTGCAGTTAAAAAGCTCGTAGTTGGATTACGG 
               
               
                 GTGGGGCCTGCCGGTCCGCCGTTTCGGTGTGCACTGGCCGGGCCCACCTTGTTGCCGGGGACGG 
               
               
                 GCTCCTGGGCTTCGCTGTCCGGGACCCGGAGTCGGCGAGGTTACTTTGAGTAAAATAGAGTGTT 
               
               
                 CAAAGCAGGCCTACGCTCTGAATAATTAGCATGGAATAACACGATAGGACTCAGGCCTATCCTG 
               
               
                 TTGGTCTGTAGGACCGGAGTAATGATTAAGAGGGACAGTCGGGGGCATTCGTATTTCATTGTCA 
               
               
                 GAGGTGAAATTCTTGGATTTATGAAAGACGAACTACTGCGAAAGCATTTGCCAAGGATGTTTTC 
               
               
                 ATTAATCAAGAACGAAAGTTGGGGGCTCGAAGACGATTAGATACCGTCCTAGTCTCAACCATAA 
               
               
                 ACGATGCCGACTAGGGATCGGCGGGTGTTTTTTTGATGACCCCGCCCCCACCTTATGAGAAATC 
               
               
                 AAAGTTTTTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGG 
               
               
                 GCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAG 
               
               
                 ACATAGTGAGGATTGACAGATTGAGAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCGT 
               
               
                 TCTTAGTTGGTGGGTTGCCTTGTCAGGTTGATTCCGGTGACGAACGAGACCTCAGCCTGCTAAC 
               
               
                 TAGTCACGCGTGCTCCGGCACGCGGCGGACTTCTTAGAGGGACTATTGGCGACTAGCCAATGGA 
               
               
                 TGCATGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTG 
               
               
                 ATGCATTCAACGAGCCTATCCTTGGCCGAGAGG