JP7551990B2 - Methods for separating and recovering free fatty acids - Google Patents

Description

The present invention relates to a method for efficiently separating and recovering free fatty acids from the culture medium of a free fatty acid-producing microorganism.

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 technique of separating and recovering FFA from the culture solution, 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 solution or by filling a fixed bed column and passing the culture solution through it, but there are still problems with the method of recovering the microparticle adsorbent and the method of transferring the culture solution. 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 separating and 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 separating free fatty acids from the culture medium of a free fatty acid-producing microorganism, and a simple method for recovering the separated free fatty acids.

The means for solving the problems of the present invention are as follows.
1. A method for separating free fatty acids, comprising transferring free fatty acids present in a culture solution of a microorganism to the outside of the culture solution through a dialysis membrane.
2. The method for separating free fatty acids according to 1., wherein the molecular weight cutoff of the dialysis membrane is 5 kDa or more.
3. A method for recovering free fatty acids, comprising adsorbing the free fatty acids transferred outside the culture solution by the separation method according to 1. or 2. to an adsorbent.
4. The method for recovering free fatty acids according to 3., characterized in that the adsorbent is at least one of activated carbon, activated clay, diatomaceous earth, zeolite, silica gel, bentonite, perlite, and Mg-Al hydrotalcite compounds.

The separation method of the present invention can separate free fatty acids from the culture medium of a free fatty acid-producing microorganism by a very simple method using a dialysis membrane.
In the recovery method of the present invention, the free fatty acids are adsorbed to the adsorbent and concentrated, so that the free fatty acids can be recovered efficiently. In addition, since the adsorbent and the free fatty acid-producing microorganisms do not come into contact with each other, the free fatty acid-producing microorganisms do not adhere to the adsorbent.

Measurement results of the concentration of free fatty acids that permeated the dialysis membrane in Experiment 1. Measurement results of free fatty acid concentration in Experiment 2.

The present invention relates to a method for efficiently separating and recovering free fatty acids from a culture medium of a free fatty acid-producing microorganism (hereinafter also referred to as a microorganism).
The microorganism to be cultured is not particularly limited as long as it can produce free fatty acids, and genetically modified strains capable of producing free fatty acids can be used from Escherichia coli, yeast, cyanobacteria, etc. Among these, photosynthetic microorganisms are preferred because they produce a superior amount of free fatty acids relative to the amount of energy input. 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.

-Separation method The separation method of the present invention is characterized in that free fatty acids present in the culture solution secreted by the microorganisms to the outside of the culture solution are transferred to the outside of the culture solution by a dialysis membrane. The free fatty acids are transferred to the outside of the culture solution by passing through the dialysis membrane due to osmotic pressure. 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. The dialysis membrane used may be one through which the free fatty acids can pass, and for example, one with a molecular weight cutoff of 5 kDa or more is preferable, one with a molecular weight cutoff of 10 kDa or more is more preferable, and one with a molecular weight cutoff of 25 kDa or more is even more preferable. If the molecular weight cutoff is small, some types of free fatty acids may not pass through the dialysis membrane. 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. The larger the molecular weight cutoff, the more efficiently the free fatty acids can be transferred, but the transfer of compounds other than the free fatty acids also increases, so that the effort required to purify the free fatty acids later may be increased.

The recovery method of the present invention is characterized in that the free fatty acids transferred to the outside of the culture solution by the above-mentioned separation method are adsorbed by an adsorbent. The adsorbent is not particularly limited as long as it can adsorb the free fatty acids in the aqueous layer, and examples of the adsorbent that can be used include activated carbon, activated clay, diatomaceous earth, zeolite, silica gel, bentonite, perlite, and Mg-Al hydrotalcite compounds.
The recovery method of the present invention is preferably carried out simultaneously with the above separation method, that is, the culture tank is divided by a dialysis membrane, one side is a culture compartment and the other is a recovery compartment, free fatty acids are produced by free fatty acid producing bacteria in the culture compartment, and free fatty acids are recovered in the recovery compartment. In the above separation method, when the molar concentrations of the solutions on both sides of the dialysis membrane become equal, free fatty acids cannot be separated any more, but by carrying out recovery and separation simultaneously, the free fatty acids that have migrated to the recovery compartment (outside the culture solution) can be adsorbed by an adsorbent to reduce the free fatty acid concentration in the recovery compartment, so that many free fatty acids can be transferred from the culture compartment to the recovery compartment for separation and recovery.

"Experiment 1" Separation of free fatty acids using a dialysis membrane An FFA dissolution solution was prepared using CHAPS (3-[(3-cholamidopropyl)dimethylamino]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.

15 mL of the FFA solution was dispensed into test tubes with an outer diameter of 18 mm. Three types of dialysis membranes with different molecular weight cutoffs (REPLIGEN, Biotech RC, molecular weight cutoffs: 10 kD, 100 kD, 1000 kD) were cut to a length of 15 cm, one end was tightly tied, 3 mL of distilled water was added to the membrane, and the other end was tightly tied. Then, each dialysis membrane was immersed in the dispensed FFA solution and left to stand overnight at room temperature, and the water in the dialysis membrane was sampled to measure the FFA concentration. A Free Fatty Acid Quantification Kit (Biovision) was used to measure the FFA concentration. The results are shown in Figure 1.

In the solution of lauric acid, which has 12 carbon atoms, and myristic acid, which has 14 carbon atoms, FFA migrated into the inside of the dialysis membrane regardless of the molecular weight cutoff of the dialysis membrane. In the solution of palmitic acid, which has 16 carbon atoms, FFA migrated into all dialysis membranes, but there was a tendency for the amount of FFA to increase as the molecular weight cutoff of the dialysis membrane increased.
Stearic acid and oleic acid, which have 18 carbon atoms, did not transfer at all through a dialysis membrane with a molecular weight cutoff of 10 kD, but transferred through dialysis membranes with molecular weight cutoffs of 100 kD and 1000 kD. Furthermore, linoleic acid and linolenic acid, which also have 18 carbon atoms, transferred FFAs even through a dialysis membrane with a molecular weight cutoff of 10 kD, but transferred significantly through dialysis membranes with molecular weight cutoffs of 100 kD or more.

"Experiment 2" Adsorption of free fatty acids (FFA) present in culture solution 1 g of activated carbon (FUJIFILM Wako Pure Chemical Industries, activated carbon (powder, neutral)) was added to 25 mL of FFA solution and cultured overnight at room temperature with shaking, and the FFA concentration in the solution before and after shaking was measured using a Free Fatty Acid Quantification Kit (Biovision).
The measurements were performed in the same manner, except that stearic acid (18:0) and activated clay (Fujifilm Wako Pure Chemical Industries, Ltd., activated clay) were used and the culture was incubated with shaking for 6 hours.
For each condition, a negative control was also measured, which was the same except that no adsorbent was added.
The results are shown in Figure 2.

In all of the FFA solutions, the FFA concentration in the solution after shaking with activated charcoal and activated clay was below the detection limit, indicating that activated charcoal and activated clay adsorb FFA. In addition, in the negative control, the measurement results showed that the concentration of some FFA increased, but based on the principle of the measurement kit used, this is presumed to be due to unsaturated bonds in the fatty acids.
From the above results, it was confirmed that free fatty acids in the culture medium can be separated using a dialysis membrane, and the separated free fatty acids can be recovered by adsorbing them onto an adsorbent.

Claims (4)

A method for separating free fatty acids, characterized in that free fatty acids present in a culture solution of a microorganism are transferred out of the culture solution through a dialysis membrane (excluding a method in which the culture solution is contacted with a draw solution having an osmotic pressure higher than that of the culture solution through a dialysis membrane) . The method for separating free fatty acids according to claim 1, characterized in that the molecular weight cutoff of the dialysis membrane is 5 kDa or more. A method for recovering free fatty acids, comprising adsorbing the free fatty acids transferred outside the culture solution by the separation method according to claim 1 or 2 onto an adsorbent. The method for recovering free fatty acids according to claim 3, characterized in that the adsorbent is one or more of the following: activated carbon, activated clay, diatomaceous earth, zeolite, silica gel, bentonite, perlite, and Mg-Al hydrotalcite compounds.