JP7646964B2 - Method and system for recovering free fatty acids - Google Patents

Description

The present invention relates to a method and a recovery system for free fatty acids produced by free fatty acid-producing microorganisms.

Aiming to break away from dependence on fossil fuels, the development of sustainable production technologies for energy materials is underway around the world. Among these, extracellular production systems for free fatty acids (FFA) using genetically modified microorganisms have attracted attention. In extracellular production systems, the produced FFA is released outside the bacterial body, and FFA can be extracted without destroying the cells, so it is expected that the production volume can be dramatically increased. For this reason, genetically modified strains that release FFA outside the cells have been created using various microorganisms such as Escherichia coli, yeast, and cyanobacteria (Non-Patent Documents 1 to 3).

However, there are few research examples on the technology of recovering FFA from culture liquid, and an efficient process has not been established. For example, Patent Document 1 proposes a method of removing FFA by adding a microparticle adsorbent to the culture liquid, or by filling a fixed bed column and passing the culture liquid through it, but there are still problems with the method of recovering the microparticle adsorbent and the method of transferring the culture liquid. As another method, a two-phase culture method has been reported in which a non-cytotoxic organic solvent is layered on the medium (Non-Patent Document 4), but since the physicochemical properties of the organic solvent used are very close to those of FFA, it is impossible to extract FFA from the organic solvent. Furthermore, if the FFA concentration increases, it may have a negative effect on the microorganisms themselves that produce free fatty acids, causing them to die (Non-Patent Document 5).
Therefore, it is necessary to develop a more practical method for recovering FFA from culture medium.

Special Publication No. 2011-505838

Rebecca M. Lennen., Drew J. Braden., Ryan M. West., James A. Dumesic., Brian F. Pfleger. (2010) A Process for Microbial Hydrocarbon Synthesis: Overproduction of Fatty Acids in Escherichia coli and Catalytic Conversion to Alkanes. Biotechnol Bioeng. June 1; 106(2) Yongjin J. Zhou1., Nicolaas A. Buijs1., Zhiwei Zhu., Jiufu Qin., Verena Siewers., Jens Nielsen. (2016) Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories. Nat. Commun. 7:11709 Liu, X., Sheng, J. and Curtiss, R. (2011) Fatty acid production in genetically modified cyanobacteria.Proc. Natl. Acad. Sci. USA. 108: 6899-6904. Kato, A., Takatani, N., Ikeda, K., Maeda, S., Omata, T. (2017) Removal of the product from the culture medium strongly enhances free fatty acid production by genetically engineered Synechococcus elongatus. Biotechonology for Biofuels Vol. 10, 141 Kato, A., Use, K., Takatani, N., Ikeda, K., Matsuura, M., Kojima, K., Aichi, M., Maeda, S., Omata, T. (2016) Modulation of the balance of fatty acid production and secretion is crucial for enhancement of growth and productivity of the engineered mutant of the cyanobacterium Synechococcus elongatus. Biotechonology for Biofuels Vol. 9, 91

The objective of the present invention is to provide a simple method for recovering free fatty acids produced by free fatty acid-producing microorganisms.

The means for solving the problems of the present invention are as follows.
1. An adsorption step of adsorbing free fatty acids secreted by a free fatty acid producing microorganism onto an adsorbent containing at least one oxide or hydroxide of magnesium, aluminum, and silicon;
a desorption step of washing the adsorbent having adsorbed the free fatty acids with a solvent having an octanol-water partition coefficient in the range of −0.85 to 0.8 to desorb the free fatty acids;
A method for recovering free fatty acids, comprising the steps of:
2. The method for recovering free fatty acids according to 1., characterized in that the adsorbent is at least one of activated clay, diatomaceous earth, zeolite, silica gel, bentonite, perlite, Mg-Al hydrotalcite-based adsorbent, and silica-magnesia-based adsorbent.
3. The method for recovering free fatty acids according to 1. or 2., wherein the free fatty acids are those that have migrated from a culture solution of a free fatty acid-producing microorganism to the outside of the culture solution through a separation membrane.
4. The method for recovering free fatty acids according to 3., wherein the separation membrane is in the form of a hollow fiber.
5. A culture tank for culturing free fatty acid producing microorganisms;
A separation membrane that transfers the free fatty acids secreted by the free fatty acid producing microorganism out of the culture solution;
A column packed with an adsorbent containing at least one oxide or hydroxide of magnesium, aluminum, and silicon;
an adsorption line for passing the free fatty acids that have passed through the separation membrane through the column;
a washing line for passing a solvent having an octanol-water partition coefficient in the range of −0.85 to 0.8 through the column;
A free fatty acid recovery system having the above structure.

The recovery method of the present invention can recover free fatty acids by a very simple method of adsorbing free fatty acids onto a specific adsorbent and then washing with a specific solvent. The recovery method of the present invention can recover free fatty acids efficiently because the free fatty acids are adsorbed onto the adsorbent and concentrated.
By transferring the free fatty acids secreted by the free fatty acid producing microorganisms from the culture medium to outside the culture medium through the separation membrane, it is possible to prevent adverse effects on the cultivation of the free fatty acid producing microorganisms caused by an increase in the concentration of free fatty acids in the culture medium and adverse effects caused by the free fatty acid producing microorganisms adhering to the adsorbent.
The recovery system of the present invention can recover free fatty acids by a very simple method of adsorbing the free fatty acids onto a specific adsorbent and then washing the adsorbed fatty acids with a specific solvent.

FIG. 1 is a block diagram of an embodiment of a recovery system for carrying out the method for recovering free fatty acids of the present invention. Measurement results of free fatty acid concentration in Experiment 1. Measurement results of the recovery rate of free fatty acids using activated clay in Experiment 2. Measurement results of the recovery rate of free fatty acids using a silica-magnesia based adsorbent in Experiment 2. Measurement results of free fatty acid concentration in Experiment 4.

The present invention relates to a method and a system for recovering free fatty acids secreted by free fatty acid-producing microorganisms.
The free fatty acid producing microorganism (hereinafter also referred to as microorganism) to be cultured is not particularly limited as long as it can produce free fatty acids, and among Escherichia coli, yeast, cyanobacteria, etc., genetically modified strains capable of producing free fatty acids can be used. Among these, photosynthetic microorganisms are preferred because they can directly synthesize free fatty acids from carbon dioxide. The free fatty acids produced by the microorganisms are also not particularly limited, and examples thereof include C12 to C18 saturated and unsaturated fatty acids.
The method for culturing the microorganisms is not particularly limited, and the composition of the medium, temperature, pH, the presence or absence of light irradiation, the wavelength of the irradiated light, oxygen concentration, carbon dioxide concentration, etc. may be adjusted according to the type of the microorganism to be cultured.

The recovery method of the present invention includes an adsorption step of adsorbing the free fatty acids secreted by the microorganisms to an adsorbent containing at least one oxide or hydroxide of magnesium, aluminum, and silicon;
a desorption step of washing the adsorbent having adsorbed the free fatty acids with a solvent having an octanol-water partition coefficient in the range of −0.85 to 0.8 to desorb the free fatty acids;
has.

"Adsorption process"
The adsorbent may contain at least one oxide or hydroxide of magnesium, aluminum, or silicon, and may also contain zinc, titanium, zirconium, etc. As the adsorbent, for example, one or more of activated clay, diatomaceous earth, zeolite, silica gel, bentonite, perlite, Mg-Al hydrotalcite-based adsorbent, silica-magnesia-based adsorbent, acid clay, activated alumina, aluminum silicate, etc. can be used, and among these, activated clay, diatomaceous earth, zeolite, silica gel, bentonite, perlite, Mg-Al hydrotalcite-based adsorbent, and silica-magnesia-based adsorbent are preferred from the viewpoints of adsorptivity and desorption property.

The smaller the particle size of the adsorbent, the larger its specific surface area and the higher its adsorptivity. On the other hand, if the particle size is too small, the adsorbent becomes difficult to recover, clean, and reuse. Therefore, the lower limit of the median diameter of the adsorbent is preferably 10 μm or more, and more preferably 20 μm or more, and the upper limit of the median diameter is preferably 5 mm or less, and more preferably 3 mm or less.

The adsorbent adsorbs free fatty acids simply by contacting it with an aqueous layer containing free fatty acids. The adsorbent can also be brought into direct contact with the culture solution of microorganisms, but microorganisms may adhere to the surface of the adsorbent, reducing the adsorption performance. In addition, when an adsorbent with microorganisms adhering to its surface is collected and washed, the amount of microbial cells in the culture system decreases. For this reason, it is preferable to transfer the free fatty acids from the culture solution to the outside of the culture solution through a separation membrane to separate them from the microorganisms, and then to bring the free fatty acids into contact with the adsorbent and adsorb them. By transferring the free fatty acids to the outside of the culture solution, the concentration of free fatty acids in the culture solution can be kept low, and the adverse effects of free fatty acids on microbial culture can be suppressed.

As the separation membrane, any membrane that does not allow microorganisms to pass and allows free fatty acids to pass can be used without any particular limitations, and a dialysis membrane, a filtration membrane, etc. can be used. When a dialysis membrane is used, the molecular weight cutoff is preferably 5 kDa or more, more preferably 10 kDa or more, and even more preferably 25 kDa or more. In addition, the dialysis membrane is preferably one with a molecular weight cutoff of 300 kDa or less, and more preferably one with a molecular weight cutoff of 200 kDa or less. When a filtration membrane is used, the pore size is preferably 0.1 μm or more, and more preferably 0.3 μm or more. In addition, the filtration membrane is preferably one with a pore size of 2 μm or less, and more preferably one with a pore size of 1 μm or less. The larger the size that can pass through the separation membrane, the more efficiently the free fatty acids can be transferred, but the transfer of compounds other than the free fatty acids also increases, so the effort required to purify the recovered free fatty acids may be increased.
As the separation membrane, a strong filtration membrane is preferable, and a filtration membrane made of a fluororesin is more preferable because microorganisms are less likely to adhere to the filtration membrane. Moreover, the filtration membrane is more preferably a hollow fiber type because it can migrate and extract free fatty acids outside the culture solution.

"Desorption process"
By simply washing the adsorbent that has adsorbed the free fatty acids with a solvent having an octanol-water partition coefficient in the range of -0.85 to 0.8, the free fatty acids can be desorbed from the adsorbent.
Examples of solvents with an octanol-water partition coefficient in the range of -0.85 to 0.8 include methanol (-0.82, the number in parentheses is the octanol-water partition coefficient), acetonitrile (-0.34), ethanol (-0.31), acetone (-0.24), isopropanol (0.05), ethyl acetate (0.73), etc. Also, mixed solvents can be used as long as the octanol-water partition coefficient is in the range of -0.85 to 0.8.
The washing time is preferably 1 minute or more, more preferably 5 minutes or more, and even more preferably 10 minutes or more, and is preferably 180 minutes or less, more preferably 120 minutes or less, and even more preferably 60 minutes or less.
The amount of the solvent used for washing is preferably 3 ml or more, more preferably 5 ml or more, per gram of the adsorbent slurry, and is preferably 15 ml or less, more preferably 10 ml or less.
The free fatty acids desorbed from the adsorbent and transferred to the solvent can be recovered by a known method such as vacuum concentration, etc. The adsorbent after washing can be reused in the adsorption step.

- Recovery system Figure 1 shows a configuration diagram of one embodiment of the free fatty acid recovery system of the present invention. The recovery system shown in Figure 1 has a culture tank 1 for culturing free fatty acid producing microorganisms, a hollow fiber separation membrane 2 for transferring free fatty acids secreted by the microorganisms to the outside of the culture solution, a column 3 filled with an adsorbent, an adsorption line 4 for passing the free fatty acids that have passed through the separation membrane through the column, and a washing line 5 for passing a solvent having an octanol-water distribution coefficient in the range of -0.85 to 0.8 through the column. This recovery system allows the culture of free fatty acid producing microorganisms and the recovery of free fatty acids to be performed simultaneously.
The recovery system shown in FIG. 1 is just one example. For example, two or more columns can be provided in parallel, and an adsorption line and a washing line can be branched to connect to each column, with the FFA desorption process in at least one column and the adsorption process in at least one column being carried out simultaneously, and a device for adding nutrients, etc. can be provided downstream of the column in the adsorption line.

"Experiment 1"
The FFA dissolution solution was prepared using CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate), an amphoteric surfactant, and an FFA reagent.
The FFAs used were lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3).
1 g of each FFA was added to 200 mL of 1% (w/w) CHAPS solution and stirred overnight at room temperature. Undissolved FFA was removed by filtration through a glass filter, and the filtrate was collected as an FFA solution.

2 g of activated clay (FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to 50 mL of the FFA solution and cultured overnight with shaking at room temperature, and the FFA concentration in the solution before and after shaking was measured using a Free Fatty Acid Quantification Kit (Biovision). The results are shown in Figure 2.
It was confirmed that almost 100% of all FFAs were adsorbed onto the activated clay. The adsorption rate of saturated fatty acids tended to be faster than that of unsaturated fatty acids.

"Experiment 2"
Palmitic acid (16:0) was adsorbed onto activated clay or a silica-magnesia adsorbent (Mizuka Life, Mizusawa Industrial Chemicals, Ltd.) by the method described above in Experiment 1. The FFA concentration after adsorption was measured using a Free Fatty Acid Quantification Kit (Biovision), and the amount of adsorption was calculated.
The liquid after adsorption was transferred to a 50 ml Falcon tube and centrifuged (5,000 rpm, 10 min) to precipitate the activated clay. The supernatant was discarded, and about 0.2 g of the activated clay was dispensed into a 2 ml Eppendorf tube, and the weight of the dispensed activated clay was precisely weighed.
1 ml of the extraction solvent was placed in this Eppendorf tube, which was then stirred for 30 minutes to wash, and the activated clay was precipitated by centrifugation (14,000 rpm, 1 min) to separate the supernatant 1. 1 ml of the extraction solvent was added again to the precipitate, which was then stirred for 30 minutes and centrifuged (14,000 rpm, 1 min), and the supernatant was combined with the previous supernatant 1 to obtain a measurement solution.
The FFA concentration in the measurement solution was measured using a Free Fatty Acid Quantification Kit (Biovision). The recovery rate of FFA was calculated from the amount of FFA adsorbed on the adsorbent before washing and the FFA concentration in the measurement solution after washing. The results for activated clay and silica-magnesia adsorbents are shown in Figures 3 and 4, respectively.

By washing with a solvent with an octanol-water partition coefficient in the range of -0.85 to 0.8, it was possible to recover more than 90% of the free fatty acids from the activated clay and silica-magnesia adsorbent. In contrast, the recovery rate of free fatty acids was low when using water, chloroform (1.97), and hexane (3.9).

"Experiment 3"
The recovery rate of FFA was calculated in the same manner as in Experiment 2, except that activated carbon (Fujifilm Wako Pure Chemical Industries, Ltd., activated carbon (powder, neutral)) and water, methanol (-0.83), and hexane (3.9) were used as the extraction solvent.
No FFA was detected in water, methanol (-0.83), or hexane (3.9), resulting in a recovery rate of 0%. FFA could not be desorbed from activated carbon.

"Experiment 4"
The FFA solution prepared in the same manner as in Experiment 1 was filtered through a membrane filter (Advantec, membrane filter, hydrophilic PTFE type, pore size 0.2 μm or 0.5 μm), and the FFA concentration in the solution before and after filtration was measured using a Free Fatty Acid Quantification Kit (Biovision). The results are shown in FIG.
All of the FFAs were able to permeate the filtration membrane.
This suggests that by using a filtration membrane as a separation membrane, it is possible to migrate free fatty acids out of the culture solution, and that the migrated free fatty acids can be recovered by using an adsorbent containing at least one oxide or hydroxide of magnesium, aluminum, or silicon, and a solvent with an octanol-water partition coefficient in the range of -0.85 to 0.8.

Culture tank 1
Separation membrane 2
Column 3
Suction line 4
Washing Line 5

Claims (4)

an adsorption step of adsorbing the C12 to C18 free fatty acids secreted by the free fatty acid producing microorganism onto an adsorbent selected from one or more of activated clay and silica-magnesia based adsorbents ;
a desorption step of washing the adsorbent having adsorbed the free fatty acids with a solvent having an octanol-water partition coefficient in the range of −0.85 to 0.8 to desorb the free fatty acids;
A method for recovering free fatty acids, comprising the steps of: 2. The method for recovering free fatty acids according to claim 1 , wherein the free fatty acids are those that have migrated from a culture solution of a free fatty acid-producing microorganism to the outside of the culture solution through a separation membrane. 3. The method for recovering free fatty acids according to claim 2 , wherein the separation membrane is in the form of a hollow fiber. A culture tank for culturing free fatty acid producing microorganisms;
A separation membrane that transfers the C12 to C18 free fatty acids secreted by the free fatty acid producing microorganism out of the culture solution;
A column packed with an adsorbent selected from the group consisting of activated clay and silica-magnesia adsorbents ;
an adsorption line for passing the free fatty acids that have passed through the separation membrane through the column;
a washing line for passing a solvent having an octanol-water partition coefficient in the range of −0.85 to 0.8 through the column;
A free fatty acid recovery system having the above structure.