FR3038619A1 - PROCESS FOR OBTAINING FATTY ACIDS FROM MICROORGANISMS - Google Patents

Abstract

The invention relates to a method for obtaining fatty acids from microorganisms comprising a microorganism maceration step in which the microorganisms are mixed with an aqueous solution of a quaternary amine hydroxide in order to extract the lipids. contained in the microorganisms and transform them into fatty acids.

Description

The invention relates to a process for extracting, transforming and separating fatty acids in one step and without the use of organic solvent from microorganisms.

For the production of biofuel (in particular the biojetfuel), the fatty acids are converted either into alkanes (bio-kerosene) or into methyl esters (biofuel). The fatty acids are obtained from the hydrolysis of lipids previously extracted from microorganisms (yeasts in particular). These processes require several steps of drying the biomass, extracting lipids from the dried biomass with an organic solvent, usually hexane, and then acid or basic hydrolysis of lipids extracted into fatty acids for further processing into biokerosene.

Microorganisms (yeast, micro-algae and bacteria) can accumulate up to 80% of lipids (monoglyceride, diglyceride, triglyceride, phospholipids and sterols) in their organisms. They are composed of a cell wall, consisting mainly of lipids, polysaccharides and chitin. These compounds make the wall rigid and strong; which requires mechanical, chemical or physico-chemical pretreatment to facilitate accessibility to the lipids located inside the cell.

Currently, the extraction of lipids from wet microorganisms takes place as follows: 1 - drying and pretreatment of biomass 2 - extraction with organic solvents (hexane) 3 - centrifugation or filtration (solvent recycling) 4- Evaporation of the solvent The drying step is necessary to overcome the water / solvent interactions; which makes water a limiting parameter in lipid extraction. The drying stage is considered the most energy consuming and the most expensive.

In addition, this process uses organic solvents that are likely in the medium term to be banned for their toxicity to humans and the environment.

There is therefore a need for a process for obtaining fatty acids from microorganisms which consumes less energy and is more respectful of the environment.

The inventors have the merit of having developed such a method.

The present invention describes a process for obtaining fatty acids from microorganisms comprising a microorganism maceration step in which the microorganisms are mixed with an aqueous solution of a quaternary amine hydroxide in order to extract the lipids contained in microorganisms and turn them into fatty acids.

Typically, the quaternary amine hydroxides which can be used for this maceration step are, for example, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, or choline hydroxide.

According to a preferred embodiment, the amine hydroxide used is choline hydroxide.

Typically, the microorganisms that can be used for the invention are lipid-rich microorganisms such as yeasts, microalgae, bacteria or filamentous fungi.

Typically, there may be mentioned among the microorganisms particularly suitable for the invention, Rhodotorula glutinis, Yarrowia lipolytica, Lipomyces starkeyi among yeasts, Phaeodactylum tricornutum Chlorella vulgaris, Nannochloropsis oculata among microalgae, Rhodococcus opacus, Streptomyces sp, Escherichia coli among bacteria, and Rhizopus arrhizus, Mucor circinelloides, penicillium lilacinum among filamentous fungi.

According to a preferred embodiment, the microorganisms used are yeasts and in particular a yeast of the species Yarrowia lipolytica or Rhodotorula glutinis.

Typically, the biomass consisting of microorganisms is in the wet phase. It comes directly from the bioreactor that made it possible to produce it or it has been centrifuged at the outlet of the bioreactor.

The biomass is mixed with an aqueous solution of an amine hydroxide, which allows the destructuration of the cell walls, to extract and transform the lipids (tri-glycerides, di-glycerides, mono-glycerides, phospholipids) into acids free fatty acids (FFA).

Typically, the maceration step is carried out with an aqueous solution having a concentration C of amine hydroxide, with Cmin <C <Cmax, the concentrations Cmin and Cmax being chosen independently of one another, Cmin being chosen from the values 5%, 10%, 20% and 30%, and C max being selected from 35%, 40%, 45%, 50% and 60%, the percentages being percentages of amine hydroxide / volume of solution. aqueous, preferably Cmin is equal to 30% and Cmax to 50%. According to a particular embodiment, the concentration C is approximately 45%.

The higher the concentration C, the higher the kinetics of the reaction.

Typically, the ratio of quaternary amine hydroxide mass to mass of microorganisms is between 20 and 70 or 30 and 60, preferably between 35 and 50. According to a particular embodiment, the ratio is about 40.

Typically, the maceration step is carried out for a time d, with dmin <d <dmax the durations dmin and dmax being chosen independently of one another, dmin being chosen from values of 5 min, 30 min, 60 min. , 2 h, 4 h and dmax being chosen from the values 4 h, 8 h, 12 h, 16 h, 24 h, 1 day, 2 days and 7 days, preferably dmin is equal to 1 h and dmax to 2 days. According to a particular embodiment, the duration d is approximately 2 h.

Typically, the maceration step is carried out at a temperature T with Tmin <T <Tmax, the temperatures Tmin and Tmax being chosen independently of one another, Tmin being chosen from values 20, 25, 30, 35, 40, 45, 50, 55, and 60 ° C and Tmax being selected from the values 60, 65, 70, 75, 80, and 90 ° C, preferably Tmin is equal to 25 ° C and Tmax to 80 ° C. According to a particular embodiment, the temperature T is approximately 40 ° C.

The higher the temperature T, the higher the kinetics of the reaction.

Following this maceration step, two phases are obtained: a solid phase and a liquid phase. The solid phase contains cellular debris, while the liquid phase contains fatty acids in the form of salt (water-soluble quaternary amine / carboxylate). Typically these two phases can be separated by filtration or centrifugation.

After separation, the liquid phase is acidified to precipitate the fatty acids. Typically, a weak acid is used to acidify the liquid phase, for example, acetic acid or citric acid may be used.

Then the fatty acids are separated from the acidified liquid phase, for example by filtration.

As an alternative to the acidification of the liquid phase, the fatty acids can be obtained by adding to the liquid phase an excess of sodium, potassium or calcium salt, such as

NaCl, KC1, or CaCl2, so that the fatty acids precipitate as sodium salt, potassium or calcium. The precipitate thus obtained is separated from the liquid phase, the fatty acids are then obtained by acid attack.

Advantageously, the quaternary amine contained in the liquid phase is regenerated to quaternary amine hydroxide, and is recycled.

Typically, the fatty acids thus obtained can be subsequently converted into alkanes to obtain bio-kerosene, or methyl ester to obtain biofuel. EXAMPLES Example 1 0.2 g of Yarrowia lipolytica dry matter was mixed with 10 ml of 45% choline in water for 2 h at 40 ° C. The mixture was subsequently centrifuged to separate cell debris from the salt-containing liquid phase (FFA-Choline).

This solution was washed twice with 15 mL of hexane and then analyzed by gas chromatography and by high performance thin layer chromatography. 70% of the lipids have been converted into FFA-choline salts. Unconverted lipids are in the form of triglycerides (TAGs) and free fatty acids (FFAs).

Example 2 Example 1 was reproduced except that the maceration time is 48 hours. 76.77% of the lipids were converted into FFA-Choline salts. Unconverted lipids are in the form of free fatty acids (FFA).

Example 3 Example 1 was repeated except that the maceration temperature was 80 ° C. 74% of the lipids were converted into FFA-Choline salts. Unconverted lipids are in the form of free fatty acids (FFA).

Example 4 Example 1 was repeated except that the choline concentration was 5%. 65% of the lipids were converted into FFA-Choline salts. Unconverted lipids are in the form of triglycerides (TAGs) and free fatty acids (FFAs).

Claims (14)

A process for obtaining fatty acids from microorganisms comprising a step of macerating the microorganisms in which the microorganisms are mixed with an aqueous solution of a quaternary amine hydroxide in order to extract the lipids contained in the microorganisms and to turn them into fatty acids. The process of claim 1, wherein the quaternary amine hydroxide is tetraethylammonium hydroxide, tetrapropylammonium hydroxide, or choline hydroxide. The process of claim 1, wherein the quaternary amine hydroxide is choline hydroxide. 4. A process according to any one of claims 1 to 3, wherein the microorganisms are yeasts, microalgae, bacteria or filamentous fungi. 5. The method of claim 4, wherein the microorganisms are yeasts. 6. The method of claim 5, wherein the microorganisms are yeasts of the species Yarrowia lipolytica or Rhodotorula glutinis. 7. Process according to any one of claims 1 to 6, in which the maceration step is carried out with an aqueous solution having a concentration C of amine hydroxide, with 30% <C <50%, the percentages being mass / volume percentages. 8. Process according to any one of claims 1 to 7, wherein the mass ratio of quaternary amine hydroxide mass of microorganisms is between 30 and 60. 9. A process according to any one of claims 1 to 8, wherein the maceration step is carried out at a temperature T with 25 ° C <T <80 ° C. 10. Process according to any one of claims 1 to 9, wherein the liquid phase obtained at the end of the maceration step is separated from the solid phase. 11. The method of claim 10, comprising a step of precipitation of fatty acids contained in the liquid phase. 12. The method of claim 11, wherein the precipitation is obtained by acidification of the liquid phase. 13. The method of claim 12, wherein the acidification is carried out with acetic acid or citric acid. 14. The method of claim 11, wherein the precipitation is obtained by adding to the liquid phase an excess of sodium salt, potassium or calcium.