RU2610675C1 - Method for extraction of lipids from biomass of microalgae chlorella and yeast yarrowia lipolytica - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention refers to biotechnology. A method is claimed for extraction of lipids from microalgae of Chlorella genus and yeast Yarrowia lipolytica for producing biodiesel fuel. The method includes supplementation of the culturing stage of microalgae Chlorella with culturing stage of yeast Yarrowia lipolytica W23. Lipid fraction is extracted from the microalgae and yeast biomass using organic solvents chloroform - methanol in ratio 2:1-1:2, the residual microalgae biomass and residual yeast biomass being hydrolyzed at a temperature of 80-140°C for 20-90 minutes, and this biomass in the form of aqueous suspension is used as a nutrient medium for culturing the yeast Yarrowia lipolytica W23.

EFFECT: invention provides for high lipid output: up to 37% based on dry weight.

6 cl, 1 dwg, 1 tbl, 6 ex

Description

The invention relates to the field of biotechnology, in particular to methods for producing biodiesel from biomass of microalgae, previously grown in photobioreactors of various designs (open, closed, cylindrical, flat and tubular), as well as yeast biomass. The collected biomass of microalgae is intended for further processing in order to isolate lipids and obtain biodiesel from them, the residual biomass is used to build up yeast biomass, from which lipids are also isolated to produce biodiesel.

The use of microalgae as lipid producers is a new promising approach to obtaining energy from renewable sources (Singh A. et. Al., 2011; Lee D.H. 2011).

The microalgae productivity in biomass and lipids is an order of magnitude higher than the productivity of terrestrial plants. So, for example, in some types of algae under optimal cultivation conditions, the lipid content of the dry mass exceeds that in oil plants: Botryococcus braunii 25-75%, Chlorella sp. 28-32%, Crypthecodinium cohnii 20%, Cylindrotheca sp. 16-37%, Dunaliella primolecta 23%, Isochrysis sp. 25-33%, Monallanthus salina> 20, Nannochloris sp. 20-35%, Nannochloropsis sp. 31-68%, Neochloris oleoabundans 35-54%, Nitzschia sp. 45-47%, Phaeodactylum tricornutum 20-30%, Tetraselmis sueica 15-23. Moreover, the area required for their cultivation is 50-100 times and 350-750 times less than, for example, for rapeseed and corn, respectively (Chisti Y., 2007).

Based on published data, it can be concluded that a sufficiently large number of microalgae species have the ability to increase lipid production. However, for the full realization of the biosynthetic potential of a given strain, it is necessary to select the most suitable conditions for both cultivation and lipid accumulation.

Various methods are known for cultivating microalgae biomass to produce lipids.

According to the method (RF Patent 2507251) inoculate microalgae Desmodesmus sp. strain 2C166E is introduced into the mineral medium BG-11 to a final concentration of chlorophyll in a mixture of 4-6 μg / ml. Cultivate with constant lighting and bubbling the medium with atmospheric air for 12-16 days at a temperature of 25-27 ° C, followed by separation of the microalgae biomass from the nutrient medium to obtain microalgae biomass containing 33-35% fatty acids from the dry weight of the cells.

According to the method (RF Patent 2497944), the cultivation of microalgae for biofuel purposes involves two stages of algolization. At the first stage, the primary inoculum is algolized with a culture, mainly Chlorella vulgaris BIN, obtained in a photobioreactor, synchronously or with a time shift of multifunctional indoor pools with translucent fences. The total volume of these pools is from 1/30 to 1/15 of the total volume of open water bodies. A secondary inoculum is grown with a bulk density of 10 ⁹ -10 ¹¹ cells / l, while cultivation begins in the spring with an average daily temperature of water in the pools in the range of 12-18 ° C. The second stage of the cultivation of microalgae begins by selection of the secondary inoculum from the pools at a water temperature of 12–18 ° C in open water and its supply to open water is continued until the microalgae has a bulk density of 2 × 10 ⁸ -10 ⁹ . A secondary inoculum is partially taken from the pool as a finished product in the spring and autumn at a water temperature in the range of 8-12 ° C, while an equal amount of water with dissolved biogens is added to the multifunctional pools. The invention provides an increase in the productivity of the method of cultivating microalgae. The resulting microalgae contain 33.9% fiber, 51.0% protein, 7.3% fat.

A known method (RF Patent 2508398), in which the cultivation of the microalgae strain Chlorella vulgaris Al 123 is carried out in laboratory conditions at a temperature of 25 ° C on BBM medium pH 6.8 under nitrogen-limited conditions for 7 days, in a volume of medium 200 ml in flasks on 500 ml, with continuous suspension of the suspension with sterile air at a speed of 200 ml / min, at an illumination of 120 W / m ² with a photoperiod of 16 hours. The lipid content in the biomass of the microalga Chlorella vulgaris Al 123 on a medium with glucose during mixotrophic cultivation is 32% (dry weight).

Based on this, it is proposed to use microalgae of the genus Chlorella for processing.

The closest technical solution, selected as a prototype, is a method for extracting biologically active substances from the biomass of microalgae of the genus Chlorella [Patent RU 2044770 C1, C12N 1/12, C12P 21/00, A23J 3/20, A23K 1/00, 1992] - a two-stage method for extracting biologically active substances from the biomass of microalgae of the genus Chlorella, which allows you to get products of different nature. At the first stage, to obtain a lipid-pigment complex with maximum yield, dry microalgae biomass is poured with 6 L of extractant (ethanol, gasoline or ethanol-gasoline mixture (1: 2)) and stirred for 6 hours at room temperature. The extract was separated by filtration, and the remaining biomass was poured with a fresh portion of the solvent in a volume of 4 L, stirred for 3 hours and filtered. The extracts are combined and concentrated in a vacuum evaporator at a temperature not exceeding 45 ° C. The final products are the lipid-pigment complex with a yield of 11-15% (mass) and non-fat biomass. At the second stage of the process, fat-free biomass is subjected to hydrolysis with cellulolytic and proteolytic enzymes, heat treatment is performed to inactivate the enzymes, and the protein hydrolyzate and meal are separated.

In this way, lipids are isolated only in the first stage with a yield of 11-15% (mass).

The objective of the invention is to provide a method that allows you to process microalgae of the genus Chlorella in a closed waste-free cycle to obtain lipids in high yield (up to 37% by dry weight), which are then used to produce biodiesel.

To achieve this result, a method for extracting lipids from the biomass of a microalgae of the genus Chlorella is proposed, which consists in separating the microalgae biomass from the culture medium, treating it with organic solvents to extract the lipid fraction, and the residual microalgae biomass is sent for further processing, while the residual microalgae biomass is subjected to hydrolysis and in the form of an aqueous suspension is used as a nutrient medium for the cultivation of yeast Yarrowia lipolytica W23, after which the biomass dr the yeast is separated from the culture medium and the lipid fraction is extracted from it, and the residual biomass of the yeast is hydrolyzed and sent to the stage of their growth.

Besides,

- as an organic solvent, chloroform-methanol is used in a ratio of 2: 1 - 1: 2;

- the biomass of the microalgae Chlorella is hydrolyzed at a temperature of 80-140 ° C for 20-90 minutes;

- the residual biomass of the yeast Yarrowia lipolytica W23 is subjected to hydrolysis at a temperature of 80-140 ° C for 20-90 minutes;

- use a 5% - 20% aqueous suspension of residual microalgae biomass;

- the amount of residual biomass of yeast in the nutrient medium for their cultivation is 5-10%.

A schematic diagram of the implementation of the method is shown in the drawing.

Chlorella vulgaris GKV-1 was used as microalgae.

The choice of yeast Yarrowia lipolytica W23 is due to their ability to accumulate a large amount of lipids, as well as the possibility of gene modification by the developed methods in order to increase the yield of lipids.

Stage of the method:

1. Cultivate the microalgae Chlorella vulgaris GKV-1;

2. The biomass of the microalgae Chlorella vulgaris GKV-1 is separated from the culture medium and dried;

3. Grind the microalgae biomass and extract the lipid fraction with an organic solvent;

4. Separate the lipid fraction from the solvents;

5. Get biodiesel from the lipid fraction;

6. The residual biomass of microalgae is subjected to hydrolysis; An aqueous suspension is prepared from the hydrolyzed residual biomass of the microalgae and sent to grow Yarrowia lipolytica yeast.

7. Yarrowia lipolytica W23 yeast biomass is grown on the residual biomass of microalgae, yeast biomass is separated and dried;

8. The yeast biomass is ground and the lipid fraction is extracted with an organic solvent .;

9. Separate the lipid fraction and direct it to obtain biodiesel;

10. The residual biomass is hydrolyzed and used to grow Yarrowia lipolytica W23 yeast.

The method parameters were selected based on the following considerations.

The biomass of the microalgae Chlorella vulgaris and the yeast Yarrowia lipolytica is hydrolyzed at a temperature of from 80 ° C to 140 ° C for 20-90 minutes; this temperature and processing time range is most suitable for hydrolysis of microalgae and yeast biomass for use as a component of nutrient media for the production of lipids: at temperatures below 80 ° C, the required destruction of biomass is not achieved, above 140 ° C, additional destruction of biomass does not occur.

As an organic solvent, chloroform-methanol is used at a ratio of 2: 1-1: 2; at these ratios of organic solvents, the largest amount of lipids recovered from the biomass of microalgae and yeast is observed.

The concentration of the aqueous suspension from the hydrolyzed biomass of the microalgae Chlorella vulgaris, which is sent for the cultivation of Yarrowia lipolytica yeast, is 5% -20%, at such concentrations the highest lipid yield during yeast processing is observed.

Hydrolyzed biomass of Yarrowia lipolytica yeast is introduced as a component into the growth medium of Yarrowia lipolytica yeast. The sowing dose of yeast is 5-15% of the volume of the medium; at such concentrations, the highest yield of lipids during yeast processing is observed.

Method implementation examples

Example 1

The microalgae Chlorella vulgaris GKV-1 is grown phototrophically for 7 days, temperature 24 ± 2 ° C, on standard Basal medium ((Yeh and Chang, 2012), pH 7.0, in a volume of 250 ml in 500 ml flasks at continuous bubbling of the suspension with sterile air at a speed of 250 ml / min, at an illumination of 3000 lux with a photoperiod of 24 hours

Microalgae cells are separated by centrifugation at 8500g for 7 minutes. The resulting microalgae biomass is dried. Next, the dry biomass of microalgae is ground in a homogenizer. The extraction of lipids is carried out by shaking on a rocking chair with a rotation speed of 200 rpm with organic solvents - chloroform-methanol (2: 1) at room temperature. Then the supernatant (lipid fraction) is separated by centrifugation. The lipid fraction is concentrated in vacuo on a rotary evaporator and then used to produce biodiesel. After extraction of the lipids, the residual biomass of the microalgae is hydrolyzed in an autoclave at 121 ° C for 30 minutes, a 10% aqueous suspension is prepared from it, sterilized in an autoclave at 121 ° C for 20 minutes and used as a nutrient medium for growing Yarrowia lipolytica W23 yeast. The sowing dose of yeast is 5% of the volume of the medium. Yeast is grown in a medium volume of 250 ml in 500 ml flasks for 48 hours at 25 ± 2 ° C on a shaker with a rotation speed of 150 rpm. Yeast cells are separated by centrifugation at 8500g for 7 minutes. The resulting biomass of the yeast is dried. Next, dry biomass of yeast is ground in a homogenizer. The extraction of lipids is carried out by shaking with a rotation speed of 200 rpm on a rocking chair with organic solvents - chloroform-methanol (2: 1) at room temperature. Then the supernatant (lipid fraction) is separated by centrifugation. The lipid fraction is concentrated in vacuo on a rotary evaporator and then used to produce biodiesel. The content of total lipids was determined gravimetrically.

The output of lipids from the microalgae biomass was 15% (by dry weight), from the yeast biomass 21.5% (by dry weight). In total, the lipid yield is 36.5% (by dry weight).

Example 2

The same as in example 1, only the residual biomass of microalgae is hydrolyzed in an autoclave at 121 ° C for 60 minutes, a 15% aqueous suspension is prepared from it, which is used for growing yeast.

The yield of lipids from the microalgae biomass was 15% (by dry weight), from the yeast biomass 21.7% (by dry weight). In total, the lipid yield is 36.7% (dry weight).

Example 3

The same as in example 1, only the residual biomass of microalgae is hydrolyzed in an autoclave at 121 ° C for 30 minutes, a 10% aqueous suspension is prepared from it, 5% residual yeast biomass hydrolyzed at 121 ° C is added to it for 30 minutes. This culture medium is used to grow yeast.

The output of lipids from the microalgae biomass was 15% (by dry weight), from the yeast biomass 21.8% (by dry weight). In total, the lipid yield is 36.8% (by dry weight).

Example 4

The same as in example 1, only the residual biomass of microalgae is hydrolyzed in an autoclave at 121 ° C for 60 minutes, a 10% aqueous suspension is prepared from it, 10% residual yeast biomass hydrolyzed at 121 ° C of 60 minutes is added to it. This culture medium is used to grow yeast.

The output of lipids from the microalgae biomass was 15% (by dry weight), from yeast biomass 22% (by dry weight). In total, the lipid yield is 37% (dry weight).

Example 5

The same as in example 1, only the extraction of lipids from the biomass of microalgae was carried out with organic solvents - chloroform-methanol in a ratio of 1: 1.

The output of lipids from the biomass of microalgae was 14.9% (by dry weight), from the biomass of yeast 21.3% (by dry weight). In total, the lipid yield is 36.2% (by dry weight).

Example 6

The same as in example 1, only the extraction of lipids from the biomass of microalgae was carried out with organic solvents - chloroform-methanol in a ratio of 1: 2.

The output of lipids from the biomass of microalgae was 14.8% (by dry weight), from the biomass of yeast 21.2% (by dry weight). In total, the lipid yield is 36% (dry weight).

The table shows a comparison of the proposed method for producing lipids from the biomass of microalgae and yeast with the method presented in Patent RU 2044770.

The use of residual biomass of microalgae and residual biomass of yeast can increase the total lipid yield in a closed waste-free cycle to 37.0% (by dry weight)

Claims (6)

1. The method of extracting lipids from the biomass of microalgae of the genus Chlorella and the yeast Yarrowia lipolytica, which consists in the fact that the biomass of the microalgae is separated from the culture medium, treated with organic solvents to extract the lipid fraction, and the residual biomass of the microalgae is sent for further processing, and the residual microalgae biomass is subjected to and in the form of an aqueous suspension is used as a nutrient medium for the cultivation of yeast Yarrowia lipolytica W23, after which the yeast biomass is separated from the culture medium and the lipid fraction is extracted from it, and the residual biomass of the yeast is hydrolyzed and sent to the stage of their growth. 2. The method according to p. 1, characterized in that the organic solvent used is chloroform-methanol in a ratio of 2: 1-1: 2. 3. The method according to p. 1, characterized in that the biomass of the microalgae Chlorella is hydrolyzed at 80-140 ° C for 20-90 minutes. 4. The method according to p. 1, characterized in that the residual biomass of the yeast Yarrowia lipolytica W23 is subjected to hydrolysis at 80-140 ° C for 20-90 minutes. 5. The method according to p. 1, characterized in that use a 5% -20% aqueous suspension of residual biomass of microalgae. 6. The method according to p. 1, characterized in that the amount of residual biomass of yeast in the nutrient medium for their cultivation is 5-10%.

Images