Method and system for recovering free fatty acids

Abstract

To provide a method for readily recovering free fatty acid produced by free fatty acid-producing microorganisms.SOLUTION: A method for recovering free fatty acid includes an adsorption step in which free fatty acid produced by free fatty acid-producing microorganisms is adsorbed onto an adsorbent containing oxide or hydroxide of at least one of magnesium, aluminum and silicon; and a desorption step in which the adsorbent having adsorbed free fatty acid is washed with a solvent having an octanol-water partition coefficient of-0.85-0.8 to desorb the free fatty acid.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and a system for recovering free fatty acids produced by a free fatty acid-producing microorganism. [Background technology]

[0002] 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 amount 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).

[0003] However, there are few research examples on techniques for recovering FFAs from culture solutions, and no efficient process has been established. For example, Patent Document 1 proposes a method for removing FFAs by adding a microparticle adsorbent to the culture solution or by filling a fixed-bed column with the culture solution and passing it through the column, but there are still problems with the method for recovering the microparticle adsorbent and the method for transporting the culture solution. As an alternative 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 FFAs, it is impossible to extract FFAs from the organic solvent. Furthermore, when the FFA concentration increases, it can 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 FFAs from culture medium. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2011-505838

Non-licensed literature

[0005] [Non-licensed document 1] 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)

Non-licensed Document 2

Non-licensed Document 4

[0006] An objective of the present invention is to provide a simple method for recovering free fatty acids produced by free fatty acid-producing microorganisms. [Means for solving the problem]

[0007] 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, or 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 described in 1., characterized in that the adsorbent is one or more 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, characterized in that the free fatty acids 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, or 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. Effect of the Invention

[0008] 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. [Brief description of the drawings]

[0009] [Figure 1] 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. [Diagram 2] Measurement results of free fatty acid concentration in Experiment 1. [Diagram 3] Measurement results of the recovery rate of free fatty acids using activated clay in experiment 2. [Figure 4] Measurement results of the recovery rate of free fatty acids using a silica-magnesia adsorbent in Experiment 2. [Diagram 5] Measurement results of free fatty acid concentration in experiment 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 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., a genetically modified strain 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. Furthermore, the method for culturing 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 depending on the type of microorganism to be cultured.

[0011] Collection method The method for recovering free fatty acids of the present invention includes an adsorption step of adsorbing free fatty acids secreted by microorganisms 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; has.

[0012] "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 in terms of adsorptivity and desorption properties.

[0013] 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, more preferably 20 μm or more, and the upper limit of the median diameter is preferably 5 mm or less, more preferably 3 mm or less.

[0014] The adsorbent adsorbs free fatty acids simply by contacting it with an aqueous layer containing free fatty acids. The adsorbent can be directly contacted with a culture solution of microorganisms, but the adsorption performance may be reduced due to the attachment of microorganisms to the surface of the adsorbent. In addition, when the adsorbent with microorganisms attached to its surface is collected and washed, the amount of the microorganisms in the culture system is reduced. Therefore, 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 the microorganisms, and then contact and adsorb the free fatty acids with the adsorbent. 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 effect of the free fatty acids on the microbial culture can be suppressed.

[0015] 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, it is preferable that the molecular weight cutoff is 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, more preferably 200 kDa or less. When a filtration membrane is used, it is preferable that the pore size is 0.1 μm or more, more preferably 0.3 μm or more. In addition, the filtration membrane is preferably one with a pore size of 2 μm or less, more preferably 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 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 from the culture solution.

[0016] "Detachment process" By simply washing the adsorbent that has adsorbed the free fatty acids with a solvent having an octanol-water distribution 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, and is preferably 15 ml or less, more preferably 10 ml or less, per gram of the adsorbent slurry. 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. Furthermore, the adsorbent after washing can be reused in the adsorption step.

[0017] Collection system Fig. 1 shows a configuration diagram of one embodiment of the free fatty acid recovery system of the present invention. The recovery system shown in Fig. 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. Note that the recovery system shown in Figure 1 is just one example. For example, two or more columns can be set up in parallel, and the adsorption line and washing line can be branched to connect to each column, and the FFA desorption process can be performed simultaneously in at least one column and the adsorption process can be performed in at least one column. A device for adding nutrients, etc. can also be provided downstream of the column in the adsorption line. EXAMPLES

[0018] "Experiment 1" The FFA dissolution solution was prepared using the amphoteric surfactant CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate) and 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.

[0019] 2 g of activated clay (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 50 mL of the FFA solution and incubated overnight with shaking at room temperature. 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 all FFAs were adsorbed almost 100% onto the activated clay. The adsorption rate of saturated fatty acids tended to be faster than that of unsaturated fatty acids.

[0020] "Experiment 2" Palmitic acid (16:0) was adsorbed onto activated clay or a silica-magnesia adsorbent (Mizusawa Industrial Chemicals, Mizuka Life) using 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 added to this Eppendorf tube, which was then stirred for 30 minutes to wash, after which the activated clay was precipitated by centrifugation (14,000 rpm, 1 min) and the supernatant 1 was separated. 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 prepare the 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 cleaning and the FFA concentration in the measurement solution after cleaning. The results for activated clay and silica-magnesia adsorbents are shown in Figures 3 and 4, respectively.

[0021] By washing with a solvent with an octanol-water partition coefficient in the range of -0.85 to 0.8, more than 90% of the free fatty acids could be recovered from the activated clay and silica-magnesia adsorbents. In contrast, the recovery rates of free fatty acids were low when using water, chloroform (1.97), and hexane (3.9).

[0022] "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 Corporation, activated carbon (powder, neutral)) and water, methanol (-0.83), and hexane (3.9) were used as the extraction solvent. No FFAs were detected in water, methanol (-0.83), or hexane (3.9), and the recovery rate was 0%. FFAs could not be desorbed from activated carbon.

[0023] "Experiment 4" The FFA solution prepared in the same manner as in Experiment 1 was filtered through a membrane filter (Advantec, hydrophilic PTFE membrane filter, 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 Figure 5. All the FFAs were able to permeate the filtration membrane. This suggests that by using a filtration membrane as a separation membrane, free fatty acids can be migrated outside the culture solution, and 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. [Explanation of symbols]

[0024] Culture tank 1 Separation membrane 2 Column 3 Suction line 4 Washing Line 5

Claims

1. 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. 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. 3. The method for recovering free fatty acids according to claim 2, wherein the separation membrane is in the form of a hollow fiber.

4. 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-based 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.

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