Method for separating free fatty acids and method for culturing free fatty acid-producing microorganisms

Abstract

To provide a method for separating free fatty acids from a culture medium of a free fatty acid-producing microorganism, and to provide a method for culturing a free fatty acid-producing microorganism.SOLUTION: Provided are a method for separating free fatty acids, in which free fatty acids secreted extracellularly by free fatty acid-producing microorganisms are separated from the culture medium as water-insoluble or poorly water-soluble fatty acid salts; and a method for culturing free fatty acid-producing microorganisms, in which the free fatty acid-producing microorganisms are cultured in a culture medium having a concentration of metal ions, which form water-insoluble or poorly water-soluble fatty acid salts with the free fatty acids, of 0.3 mmol / L or more and 10 mmol / L or less.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for separating free fatty acids from a culture medium of a free fatty acid-producing microorganism, and a method for culturing a free fatty acid-producing microorganism. [Background technology]

[0002] With the aim of moving away from dependence on fossil fuels, efforts are underway worldwide to develop sustainable production technologies for energy materials. 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 cells, allowing FFA to be extracted without cell destruction, and it is expected that production volume can be dramatically increased. Therefore, 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 1-3).

[0003] However, there have been few studies on techniques for separating and recovering FFAs from culture solutions, and no efficient processes have 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; however, issues remain regarding 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). However, because the physicochemical properties of the organic solvent used are very similar to those of FFAs, it is impossible to extract FFAs from the organic solvent. Furthermore, increased FFA concentrations can adversely affect the microorganisms that produce free fatty acids (hereinafter referred to as free fatty acid-producing microorganisms) themselves, potentially resulting in their death (Non-Patent Document 5).

[0004] The present inventors have proposed a method of transferring free fatty acids in a culture medium out of the culture medium using a dialysis membrane (Patent Document 2), and a method of adsorbing free fatty acids in a culture medium onto an adsorbent and then desorbing the free fatty acids by washing the adsorbent with a solvent (Patent Documents 3 and 4).

Prior Technical Literature

Charter Documents

[0005] [Patent Document 1] Special Publication No. 2011-505838 [Patent Document 2] Japanese Patent Publication No. 2022-039612 [Patent Document 3] Japanese Patent Publication No. 2023-028686 [Patent Document 4] Japanese Patent Publication No. 2023-157283

Non-licensed literature

[0006]

Non-patent document 1

Non-patent document 2

Non-patent document 3

[0007] An object of the present invention is to provide a method for separating free fatty acids from a culture medium of a free fatty acid-producing microorganism, and a method for culturing a free fatty acid-producing microorganism. [Means for solving the problem]

[0008] The means for solving the problems of the present invention are as follows. 1. A method for separating free fatty acids, which comprises separating free fatty acids secreted extracellularly by free fatty acid-producing microorganisms from the culture medium as water-insoluble or poorly water-soluble fatty acid salts. 2. The separation method according to 1, wherein the fatty acid salt is a calcium salt. 3. A method for culturing free fatty acid-producing microorganisms, characterized by culturing free fatty acid-producing microorganisms in a culture medium having a metal ion concentration of 0.3 mmol / L or more and 10 mmol / L or less that forms water-insoluble or poorly water-soluble fatty acid salts with free fatty acids. [Effects of the Invention]

[0009] The separation method of the present invention separates free fatty acids in a culture medium from the culture medium as water-insoluble or poorly water-soluble fatty acid salts. The separation method of the present invention can separate free fatty acids in the culture medium from the culture medium, thereby keeping the free fatty acid concentration in the culture medium low and suppressing growth inhibition of free fatty acid-producing microorganisms. The culture method of the present invention can suppress growth inhibition of free fatty acid-producing microorganisms even when the fatty acid concentration in the medium becomes high. [Brief explanation of the drawings]

[0010] [Figure 1] Graph showing the change in bacterial cell turbidity over time during culture in an example. [Figure 2] 10 shows images showing the appearance of each culture medium after 14 days of culture in an example. DETAILED DESCRIPTION OF THE INVENTION

[0011] ·Separation method The separation method of the present invention is a method for separating free fatty acids secreted extracellularly by free fatty acid-producing microorganisms (hereinafter also referred to as microorganisms) from a culture medium as water-insoluble or poorly water-soluble fatty acid salts. The microorganisms used for producing free fatty acids are not particularly limited as long as they can produce free fatty acids, and any of genetically modified strains that have been genetically modified to produce free fatty acids, and non-genetically modified strains that have enhanced free fatty acid production, can be used from Escherichia coli, yeast, cyanobacteria, etc. Photosynthetic microorganisms are preferred as free fatty acid-producing microorganisms because they produce a high amount of free fatty acids relative to the amount of energy input. The method for culturing microorganisms is not particularly limited, and the composition of the medium, temperature, pH, whether or not light is irradiated, the wavelength of the irradiated light, oxygen concentration, carbon dioxide concentration, etc. may be adjusted depending on the type of microorganism to be cultured.

[0012] Free fatty acid-producing microorganisms secrete fatty acids outside the cells. The fatty acids produced by free fatty acid-producing microorganisms are a mixture, including 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). These fatty acids are inherently water-insoluble, but the fatty acids secreted by free fatty acid-producing microorganisms are present in water. The mechanism by which this occurs is still unknown, but one theory is that the fatty acids are transported out of the cells one molecule at a time and dispersed in water one molecule at a time, or that the fatty acids are secreted outside the cells in some form of interaction with surfactant, and then dispersed in water by the surfactant.

[0013] The separation method of the present invention separates the fatty acids secreted extracellularly and contained in the aqueous culture medium as water-insoluble or poorly water-soluble fatty acid salts. In this specification, "water-insoluble or poorly water-soluble" means a solubility of 1 g or less in 1 L of water at 25°C, and particularly "water-insoluble" means a solubility of 0.1 g or less in 1 L of water at 25°C.

[0014] The salt (metal) of the water-insoluble or poorly water-soluble fatty acid salt is not particularly limited, and examples thereof include calcium, barium, sodium, magnesium, zinc, cadmium, nickel, manganese, copper, aluminum, etc. Among these, calcium and barium are preferred, and calcium is more preferred, because free fatty acid-producing microorganisms can grow at relatively high metal concentrations. The water-insoluble or poorly water-soluble fatty acid salt separates into oil and water from the culture medium, forming an oil phase on the culture medium. The separated oil phase can be removed by a known method.

[0015] In the separation method of the present invention, the method for separating free fatty acids in the culture medium as water-insoluble or poorly water-soluble fatty acid salts from the culture medium is not particularly limited, and examples include a method in which free fatty acid-producing microorganisms are cultured in a culture medium containing a high concentration of salts such as calcium and the fatty acid salts are separated simultaneously with the culture, a method in which the culture medium of the free fatty acid-producing microorganisms is removed from the culture tank and salts are added to the removed culture medium to separate the fatty acid salts, etc. The method in which free fatty acid-producing microorganisms are cultured in a culture medium containing a high concentration of salts such as calcium and the fatty acid salts are separated simultaneously with the culture is preferred because it can prevent growth inhibition.

[0016] ·Culture method The culture method of the present invention involves culturing a free fatty acid-producing microorganism in a culture medium containing a metal ion concentration of 0.3 mmol / L to 10 mmol / L, which forms water-insoluble or poorly water-soluble fatty acid salts with free fatty acids. In the culture method of the present invention, fatty acids secreted extracellularly during culture are converted into water-insoluble or poorly water-soluble fatty acid salts and separated from the culture medium. However, a high metal ion concentration in the medium can prevent microbial growth inhibition even when the fatty acid concentration in the medium is high. While the mechanism is unclear, the inventors speculate that because microbial growth inhibition by fatty acids is due to high intracellular fatty acid concentrations, a high metal ion concentration in the medium efficiently excretes intracellular fatty acids. The metal ion concentration of the culture medium is more preferably 0.5 mmol / L or more, even more preferably 1 mmol / L or more, even more preferably 1.5 mmol / L or more, even more preferably 2 mmol / L or more, and more preferably 9 mmol / L or less, even more preferably 8 mmol / L or less, even more preferably 7 mmol / L or less, and even more preferably 6 mmol / L or less.

[0017] The metal ions to be used are not particularly limited as long as they can form water-insoluble or poorly water-soluble fatty acid salts, and examples thereof include calcium, barium, sodium, magnesium, zinc, cadmium, nickel, manganese, copper, aluminum, etc. Among these, calcium and barium are preferred, with calcium being more preferred, since free fatty acid-producing microorganisms can grow at relatively high metal ion concentrations. In the culture method of the present invention, the free fatty acid-producing microorganisms survive even after the free fatty acids in the culture medium are separated and recovered as water-insoluble or poorly water-soluble fatty acid salts. Therefore, by supplying a new medium containing a high concentration of salt (metal) after separating and recovering the fatty acid salts, the production of free fatty acids by culture can be continued. [Example]

[0018] CaCl2 was added to the basic medium (1xCa: Ca concentration 0.26 mmol / L) to make the Ca concentration 5 times (5xCa: Ca concentration 1.3 mmol / L), 10 times (10xCa: Ca concentration 2.6 mmol / L), and 20 times (20xCa: Ca concentration 5.2 mmol / L) to prepare media with different Ca concentrations. The free fatty acid producing microorganism, dAS1_g21r_KS strain, was grown at 25°C under a light intensity of 400 μE / m 2 ·s 1 The cells were cultured in each medium for 14 days under aeration of 2% CO2. 730The dAS1_g21r_KS strain was deposited internationally on December 22, 2023, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818)) under the accession number FERM BP-22489.

[0019] After 14 days of cultivation, the mixture was left to stand for two days, and fatty acid salts (calcium salts) were separated and collected as floating matter from the culture medium. Furthermore, the culture medium was centrifuged to separate the microorganisms, and the microorganism-free culture medium was collected. The supernatant and the collected culture medium were each dissolved in BCl3 methanol, and the fatty acids were methyl esterified. The mixture was then extracted with hexane and subjected to GCMS analysis to measure the FFA concentration. The amount of FFA contained in the supernatant and the collected culture medium, as well as the amount of extracellular FFA produced per culture medium, were calculated.

[0020] "result" OD 730 The time course of the turbidity (bacterial turbidity) is shown in Figure 1, and the appearance of each culture medium after 14 days of cultivation is shown in Figure 2. In the case of 1x Ca, the turbidity of the cells hardly increased, and the culture medium turned white after 14 days. In the case of 1x Ca, severe growth inhibition due to FFA in the culture medium was confirmed. The culture medium with high calcium concentrations (5xCa, 10xCa, and 20xCa) showed increased bacterial turbidity and turned green after 14 days. In particular, 10xCa showed the best growth, and the culture medium was also dark green.

[0021] Table 1 shows the amount of extracellular FFA produced per liter of culture medium under each condition. [Table 1]

[0022] The quantitative analysis of FFA components confirmed that the free fatty acids contained C14 to C18 fatty acids, with myristic acid (C14:0) and palmitic acid (C16:0) being the main components. In the 1x Ca medium, where growth was severely inhibited, almost no FFA was detected in the medium, and only a small amount of FFA was detected in the suspended matter. This is presumably because FFA accumulated in the algae, causing them to die or become dormant. The 5xCa, 10xCa, and 20xCa media showed good growth, with high FFA concentrations in both the suspension and the medium, and high FFA production per culture solution. Furthermore, over 40% of the extracellular FFAs were isolated as suspension in the 5xCa, 10xCa, and 20xCa media, and approximately 86% in the 5xCa media in particular, allowing for easy recovery of fatty acids. The FFA concentration in the medium in the 10xCa media was approximately 21 mg / L, which was higher than in the other media. Although algae die or become dormant when FFAs accumulate intracellularly, the algae remained active even in the 10xCa media, where the FFA concentration in the medium was high. This suggests that high calcium ions allow for efficient excretion of intracellular FFAs.

Claims

1. A method for separating free fatty acids, comprising separating free fatty acids secreted extracellularly by a free fatty acid-producing microorganism from a culture medium as water-insoluble or poorly water-soluble fatty acid salts.

2. 2. The method according to claim 1, wherein the fatty acid salt is a calcium salt.

3. A method for culturing free fatty acid-producing microorganisms, characterized in that the free fatty acid-producing microorganisms are cultured in a culture medium having a metal ion concentration of 0.3 mmol / L or more and 10 mmol / L or less, which metal ions form water-insoluble or poorly water-soluble fatty acid salts with the free fatty acids.

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