Strain of chlorella vulgaris microalgae for obtaining lipids as raw material for production of motor fuel

FIELD: biotechnologies.

SUBSTANCE: invention propose a strain of Chlorella vulgaris IPPAS C-616 microalgae for obtaining lipids as raw material for production of motor fuel.

EFFECT: strain of microalgae has an ability to produce lipids with high content of saturated and mono non-saturated fatty acids.

1 dwg, 1 tbl, 3 ex

The invention relates to strains of microalgae and can be used to obtain lipids as raw material for the production of motor fuel.

The last decade has been marked by growing interest towards the development of technologies based on the use of alternative raw materials for industry, including the use of plant biomass. However, with the rapid increase in the production of bioenergy carriers emerged is a common global problem that is associated with the occupation of arable land for growing energy crops, competitive renewable energy sector food products for agricultural raw materials, primarily for corn, soybeans, wheat. In connection with this important research to find the best source of plant biomass, which is characterized by high productivity and availability, and low resource and energy during cultivation and processing. As such a source of raw materials considering the biomass of algae, which in its main indicators as energy raw material is superior to other raw biological resources. The biomass of algae is a source of lipids and hydrocarbons used in the production of biofuels.

The productivity of microalgae biomass and lipids on p is the row exceeds the productivity of terrestrial plants. For example, some species of algae under optimal culture conditions, the lipids exceeds those in oilseed plants: Botryococcus braunii 25-75% of the dry mass, Chlorella sp.28-32%, Crypthecodinium cohnii 20%, Cylindrotheca sp.16-37%, Dunaliella primolecta 23%, Isochrysis sp.25-33%o, Nannochloris sp.20-35%, Nannochloropsis sp.31-68%, Neochloris okoabundans 35-54%, Nitzschia sp.45-47%, Phaeodactylum tricornutum 20-30%. The space required for their cultivation, is 50-100 times less than, for example, rapeseed [Chisti Y. Biodizel from Microalgae// Biotechnology Advances. 2007. - Vol.25. - P. 294-306].

Most strains of microalgae have a high content of unsaturated fatty acids that can not be obtained without additional processing on the basis of their lipid fuel with low cetane number and good resistance to oxidation. Standard fuel is characterized by a cetane number of 40 to 45, and the highest quality fuel has a cetane number of 45 to 50. When the values of cetane number in excess of 60 decreases the combustion efficiency, increases the opacity of the exhaust gases increases fuel consumption. Too high cetane number increase the viscosity of the fuel at low temperatures. Therefore, the mixture of fatty acids C18:1, C18:0 and C16:0 included in the composition of the lipids of microalgae oil is the most suitable for the production of biofuels [Knothe G. Improving biodiesel fuel properties by modifying fatty ester composition //Energy Environ. Sci. 2009. V.2. P. 759-766].

The known method the product of the AI polyunsaturated fatty acids by strains of microalgae Cryptheconidium conii with decreasing chloride concentration in the culture fluid [U.S. Patent No. 7163811, IPC SR 7/64; A23D 9/00]. The disadvantage of this method is the high content of unsaturated fatty acids (up to 53.9% of the weight of the oil) in the biomass of microalgae, which greatly complicates obtaining high-quality biofuels because of their possible oxidation of unsaturated bonds.

Known strains Pseudochoricystis ellipsoidea MBIC11204 and MBIC11220, producing different amounts of hydrocarbons n-heptadecane, n-heptadecane, n-nonadecane and n-nonadecane. The disadvantage of this strain is that under cultivation, the use of vitamins (In₁In₁₂and Biotin), which significantly increases the cost of production of biomass and lipids these microalgae. It also uses the method of cultivation, including the changing environment rich in nitrogenous compounds depleted, which complicates the production technology.

Known cuts Pseudochoricystis ellipsoidea, producing hydrocarbons, which can be used as a substitute for diesel fuel is light oil [European patent No. 1873233]. Also presented is a method of obtaining hydrocarbons, involving the cultivation of this microalga capable of producing hydrocarbons.

The closest analogue of the proposed strain of Chlorella vulgaris E is a strain of Chlorella vulgaris ESP-31 [Yeh K.L., Chang J.S. Effects of cultivation conditions and media composition on cell growth and lipid productivity ofindigenous microalga Chlorella vulgaris ESP-31 // Bioresource Technology. 2012. V. 105. P. 120-127], which allows to obtain lipids, including 60-68% saturated and monounsaturated fatty acids in the composition, including palmitic (16:0), stearic (18:0) and oleic acid (C18:1).

However, this strain under cultivation is not possible to obtain lipids with a high content of saturated acids: the content of palmitic acid is 16.44-26.46%, stearic 7.07-14.92% of total fatty acids in lipids, depending on used for culturing environment.

The invention solves the problem of obtaining strains of microalgae have the ability to produce lipids with a high content of saturated and monounsaturated fatty acids suitable for use as raw material for production of motor fuels of high quality.

The problem is solved in that a strain of microalgae Chlorella vulgaris A with a high content of saturated and monounsaturated fatty acids in the lipid composition, including palmitic, as raw material for the production of motor fuels.

The strain of the microalgae Chlorella vulgaris A isolated from a hot spring at the foot of Mutnovsky volcano (Kamchatka), identified in the Institute of Cytology and genetics, and deposited in the Collection of microalgae IGF RAS under registration number IPPAS With-616.

The strain of Chlorella vulgaris A has the following morpho is ideologicheskuyu characteristic. The cells are round, spherical, thin-walled, without mucus, the size of 3-5 microns with a single pyrenaicum size of 1 μm, reproduction occurs 4-8 autospare. On agar medium IIM forms colonies of green with a smooth edge. Strain auto - and mixotrophic. The cultivation temperature from 20 to 35°C. the Strain grows on mineral environment of the IIM, does not require to increase the supply in the atmosphere of carbon dioxide. Sufficient lighting period for growth - up to 16-18 hours a day. When staining with a fluorescent dye Nile Red in the cytoplasm of cells visualized drops of lipids. The strain is not prone to precipitation in the culture, and the walls of cultivation install, saves axenically during storage. Strain develops regardless of the season.

The resulting strain in comparison with the previously described strains has a higher content of lipids with saturated and monounsaturated fatty acids. The total content of fatty acids determined by gas chromatography-spectrometry, was 75%, as shown in the Table, including palmitic - 38%, which is higher than the strain of Chlorella vulgaris ESP-31 and allows to obtain raw materials for biofuel production with a high content of saturated fatty acids with improved properties after processing biomass ultrasound and solvent extraction. The content of lepidostoma Chlorella vulgaris A when mixotrophic cultivation amounted to 32% of the dry weight, in addition, the cultivation of the strain does not require adding in an environment of carbon dioxide and vitamins.

The invention is illustrated by the following examples.

Example 1. Visualization of the accumulation of lipids in the cells of the microalgae Chlorella vulgaris Al 123 when the color of the fluorescent dye Nile Red.

Cultivation of the strain is carried out in laboratory conditions at 25°C in the environment of the IIM pH 6.8, and the following composition per 1 litre:

NaNC₃- 0,75,

CaCl₂·2H₂O - 0,025 g

MgSO₄·7H₂O - 0.075 g

K₂HPO₄- 0.075 g

KH₂PO₄- 0,175 g

NaCl - 0.025 g

Na₂EDTA·2H₂O 10 mg

FeSO₄·7H₂O - to 4.98 mg

MnCl₂·4H₂O - 1,81 mg

ZnSO₄- 0,222 mg

Co(NO₃)₂·6H₂O - 0,049 mg

NaMoO₄·5H₂O - 0,39 mg

CuSO₄·5H₂O - 0,079 mg

H₃BO₃·2H₂0 - 19,28 mg

Cultivation of a strain of microalgae in limited nitrogen conditions hold for 7 days in medium volume of 200 ml in flasks 500 ml, with continuous bubbling suspension with sterile air at 200 ml/min, light intensity of 120 W/m²with a photoperiod of 16 h of the cell Suspension is mixed with 1 mm solution of Nile Red in water, followed by fluorescent microscopy at a wavelength of excitation 450-490 nm and the wavelength of detection of 515-565 nm. The results are shown in ounce, showing cells of the microalgae Chlorella vulgaris A: A. - microscopy in transmitted light, b - fluorescence microscopy staining dye Nile Red. The scale 10 µm. Arrows indicate lipid droplets in the cytoplasm of cells.

Example 2. Mixotrophic cultivation of a strain of microalgae Chlorella vulgaris Al 123 in the medium with glucose.

Cultivation of the strain is carried out in laboratory conditions on the environment of the IIM at pH 6.8 and 25°C as in example 1, but with the addition of glucose to 10 g/l, the cells are precipitated by centrifugation at 3000 rpm for 5 minutes Extraction of lipids from biomass carried out according to the method of Folch (Folch J., Lees, M. and Sloane Stanley GH A simple method for the isolation and purification of total lipids from animal tissues // Journal of biological chemistry. - 1957. V.226. - P. 497-509.) with the following modification: the cells are homogenized using an ultrasonic generator for 2 min, at 30 kHz and power of 130 watts in a mixture of chloroform:methanol (2:1). To the resulting mixture add a quarter of the volume of 0.9% solution of potassium chloride and intensively stirred. After separation of the liquid organic phase is distilled in a stream of nitrogen, the resulting residue is weighed to determine the content of lipids on a dry weight basis. Lipid content in the biomass of the microalgae Chlorella vulgaris Al 123 on the medium with glucose at mixotrophic cultivation is 32% (dry weight).

Example 3. Analysis of fatty acid composition is of Obidov microalgae Chlorella vulgaris A.

For analysis of fatty acid composition comprising the composition of the lipids of microalgae Chlorella vulgaris Al 123, use the lipids obtained as described in example 2, for which conduct their pereeterifikacii order to obtain the methyl esters of fatty acids as in [W.W. Christie Gas chromatography and lipids.Publisher: The Oily Press. - 1989. - P. 37-38]. The addition of lipids (5 mg) dissolved in toluene (1 ml), after add a solution of 1%sulfuric acid in methanol (2 ml), intensively stirred. The mixture is boiled for two hours, after which add 5 ml of 5% NaCl solution. The resulting mixture was twice extracted with 5 ml of hexane. The hexane layer is washed with a solution of 2% NaHCO₃and dried over anhydrous Na₂SO₄the mixture is filtered and removed solvent in a stream of nitrogen.

The composition of the methyl esters of fatty acids of the lipids analyzed by gas chromatography spectrometry instrument Agilent 7000 with ionization by electron impact (70 eV), column DB-5 (30 m × 0.25 mm), with a temperature gradient from 80°C to 290°C at a rate of 4°C/min, the Temperature of injector and detector was set at 250 and 230°C respectively. The flow of carrier gas is 1.2 ml/min Identification of compounds produced by comparing the obtained data with a library of spectra of known compounds. The results of the analysis shown in the Table, show that cuts Chlorella vularis Al 123 has a high content of saturated fatty acids C16:0, C18:0 (57%)and monounsaturated C18:1, the total number of which is 75%.

The strain of microalgaeChlorella vulgarisIPPAS With-616 to obtain lipids as raw material for the production of motor top is Yves.