Method for producing a bio-derived material

Abstract

Provided is a technology that enables a biological material utilizing a photosynthetic micro-organism to be produced at low cost. A production method for a biological material according to the present invention includes cultivating a photosynthetic micro-organism in a medium including dimethyl 2-oxoglutaric acid, and then recovering a material in which the photosynthetic micro-organism has been produced or accumulated.

Description

Technical Field

[0001] The present invention relates to a method for producing a bio-derived material.

Background Art

[0002] Many algae increase the accumulation amounts of lipids and carbohydrates such as starch in an environment lacking nutrients, for example, nitrogen (Non-Patent Document 1). The lipids produced by algae can be used, for example, as raw materials for biofuels. In addition, the carbohydrates produced by algae can be used, for example, as raw materials for fuel additives, pharmaceuticals, cosmetics, and plastic products.

[0003] The accumulation of lipids and carbohydrates by algae can be promoted without depleting nitrogen in the environment. For example, by using a TOR inhibitor that specifically inhibits the activity of TOR (target of rapamycin) kinase possessed by microalgae, which are eukaryotic algae, the accumulation of lipids and carbohydrates by algae can be promoted (Non-Patent Documents 2 and 3).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0005] An object of the present invention is to provide a technique capable of realizing the production of bio-derived materials using photosynthetic microorganisms at low cost.

[0006] According to a first aspect of the present invention, there is provided a method for producing a bio-derived material, including culturing a photosynthetic microorganism in a medium containing dimethyl 2-oxoglutarate, and then recovering the substance produced or accumulated by the photosynthetic microorganism.

[0007] According to a second aspect of the present invention, there is provided a method for producing a bio-derived material, including culturing photosynthetic microorganisms in a medium, then adding dimethyl 2-oxoglutarate to the medium and continuing the culturing of the photosynthetic microorganisms in this medium, and then recovering the substances produced or accumulated by the photosynthetic microorganisms.

[0008] According to a third aspect of the present invention, there is provided an accelerator containing dimethyl 2-oxoglutarate and used for promoting the accumulation of substances by photosynthetic microorganisms.

[0009] According to the present invention, there is provided a technology that enables the production of bio-derived materials using photosynthetic microorganisms to be realized at low cost.

Brief Description of the Drawings

[0010]

Figure 1

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Embodiments for Carrying Out the Invention

[0011] Hereinafter, embodiments of the present invention will be described. The embodiments described below are more specific embodiments of any of the above aspects. The matters described below can be incorporated into each of the above aspects alone or in combination.

[0012] In the method for producing a biological material according to an embodiment of the present invention, first, photosynthetic microorganisms are cultured in a medium. In this culture, for example, the photosynthetic microorganisms are irradiated with light in the presence of carbon dioxide. For example, the photosynthetic microorganisms are irradiated with sunlight under an air atmosphere. Thereby, photosynthesis is caused in the photosynthetic microorganisms and the photosynthetic microorganisms are grown.

[0013] The photosynthetic microorganisms are, for example, microorganisms that perform oxygen-generating photosynthesis. The photosynthetic microorganisms are, for example, algae such as eukaryotic algae. The algae are preferably microalgae. Here, the "microalgae" are, for example, photosynthetic eukaryotes that are single-celled organisms or colonies thereof. The microalgae are, for example, single-celled green algae such as Chlamydomonas reinhardtti and Botryococcus, single-celled red algae such as Cyanidioschyzon merolae, or colonies thereof. The photosynthetic microorganisms do not have to be eukaryotes. The photosynthetic microorganisms may be prokaryotes, for example, bacteria such as cyanobacteria. The prokaryotes may be archaea.

[0014] The medium used is one that contains all the nutrients necessary for the growth and photosynthesis of the photosynthetic microorganisms at a sufficient concentration. The medium is, for example, a liquid medium.

[0015] The above culture is, for example, a suspension culture. The above culture may be other cultures, for example, a carrier culture. In the carrier culture, a biofilm of photosynthetic microorganisms is formed on the carrier, and the culture is performed in a state where this biofilm is in contact with the medium.

[0016] Next, an accelerator for promoting the accumulation of substances by the photosynthetic microorganisms is added to the above medium. Then, the culture of the photosynthetic microorganisms is continued in this medium. That is, for example, the photosynthetic microorganisms are irradiated with light in the presence of carbon dioxide. For example, the photosynthetic microorganisms are irradiated with sunlight under an air atmosphere.

[0017] The above accelerator contains dimethyl 2-oxoglutarate represented by the following chemical formula (1). Dimethyl 2-oxoglutarate arrests the cell growth of photosynthetic microorganisms, thereby promoting the accumulation of substances produced by the photosynthetic microorganisms.

[0018]

Chemical formula

[0019] The accelerator can further contain a solvent. As this solvent, for example, a liquid having the same or almost the same composition as the medium used for culturing photosynthetic microorganisms can be used. When dimethyl 2-oxoglutarate is diluted with a solvent, for example, it becomes easy to uniformly supply dimethyl 2-oxoglutarate to photosynthetic microorganisms.

[0020] Note that the substances produced and accumulated by photosynthetic microorganisms are various, such as oils and fats, carbohydrates, hydrocarbons, and amino acids. The substances produced and accumulated by photosynthetic microorganisms vary depending on the type of photosynthetic microorganisms. Oils and fats are, for example, neutral lipids such as triacylglycerol. Hydrocarbons are, for example, botryococcene. Carbohydrates are, for example, starch, or a combination of starch and one or more other carbohydrate components.

[0021] The timing of adding the accelerator is determined based on, for example, any one of the light transmittance and turbidity of the dispersion containing photosynthetic microorganisms and the medium, the number of cells per volume, the specific growth rate of photosynthetic microorganisms, and the change rate thereof. According to one example, the timing of adding the accelerator is determined by comparing any one of the light transmittance and turbidity of the dispersion, the number of cells per volume, the specific growth rate of photosynthetic microorganisms, and the change rate thereof with a predetermined threshold value.

[0022] The addition of the promoter to the medium is preferably carried out so that the number of photosynthetic microorganism cells per volume is in the range of 100,000 to 1,000,000,000 cells / mL immediately before adding the promoter, and more preferably in the range of 10,000,000 to 100,000,000 cells / mL. Here, the number of photosynthetic microorganism cells per volume is the ratio of the number of photosynthetic microorganism cells to the total volume of the photosynthetic microorganism and the medium.

[0023] In order to efficiently produce bio-derived materials, it is desirable that the number of photosynthetic microorganism cells is somewhat large. However, if the photosynthetic microorganisms grow excessively, it becomes difficult for light to reach deep parts.

[0024] The promoter is preferably added so that the dimethyl-2-oxoglutaric acid concentration in the mixed solution containing the medium, the photosynthetic microorganism, and the promoter is in the range of 0.001 to 1000 mmol / L, and more preferably in the range of 0.5 to 5 mmol / L. If this concentration is lowered, the effect of promoting the accumulation of substances in the photosynthetic microorganism becomes small. If this concentration is increased, the cost becomes high.

[0025] The culture period after adding the promoter varies depending on the type of photosynthetic microorganism. Generally, this culture period is preferably in the range of 3 hours to 14 days, and more preferably in the range of 12 hours to 72 hours.

[0026] Thereafter, the substance produced or accumulated by the photosynthetic microorganism is recovered. When recovering the substance accumulated inside the photosynthetic microorganism cells, for example, a hydrophobic substance is extracted from the photosynthetic microorganism to obtain an extract containing the hydrophobic substance and a residue containing the hydrophilic substance. Alternatively, a hydrophilic substance is extracted from the photosynthetic microorganism to obtain an extract containing the hydrophilic substance and a residue containing the hydrophobic substance. When recovering the substance released extracellularly among the substances produced by the photosynthetic microorganism, this substance is recovered from the medium, for example. Here, as an example, a method for obtaining oil or sugar from photosynthetic microorganisms will be described.

[0027] For example, first, photosynthetic microorganisms are separated from the culture medium. When performing suspension culture, for example, at least a part of the culture medium is removed from the mixture of photosynthetic microorganisms and the culture medium by centrifugation or pressing. Thereby, a concentrate containing photosynthetic microorganisms at a higher concentration than the previous mixture is obtained. Next, the concentrate is dried to obtain a dried product composed of photosynthetic microorganisms.

[0028] Next, oil and fat are extracted from the dried product composed of photosynthetic microorganisms. An organic solvent is used as the extraction medium for the extraction of oil and fat. Thereby, an extract containing oil and fat is obtained, and a residue containing carbohydrates is obtained.

[0029] When producing oil and fat, then, if necessary, the extract is purified. This purified product may be modified. In the above manner, oil and fat are obtained from photosynthetic microorganisms. The oil and fat obtained in this way can be used, for example, as biofuel or its raw material.

[0030] When producing carbohydrates, for example, carbohydrates are extracted from the above-mentioned residue. When extracting polysaccharides such as starch, for example, water is used as the extraction medium. Next, if necessary, the extract is purified. This purified product may be modified. In the above manner, carbohydrates are obtained from photosynthetic microorganisms. These carbohydrates can be used, for example, as raw materials for fuel additives, pharmaceuticals, cosmetics, and plastic products.

[0031] For example, by such a method, oil and fat, carbohydrates, or their purified or modified products can be obtained. Also, by a similar method, other substances such as hydrocarbons and amino acids accumulated in photosynthetic microorganisms, or their purified or modified products can be obtained.

[0032] In addition, the substances produced or accumulated by photosynthetic microorganisms obtained in this manner, the substances obtained by performing post-treatments such as purification and modification on these substances, or the substances obtained using them as raw materials are bio-derived materials. Bio-derived materials are, for example, articles such as biofuels, fuel additives, pharmaceuticals, supplements, physiologically active substances, foods, cosmetics, and plastic products. Alternatively, bio-derived materials are one or more components or raw materials of the above articles.

[0033] As described above, the accumulation of substances by photosynthetic microorganisms can be promoted by depleting nitrogen in the environment. However, in order to change from an environment where all nutrients are sufficiently present to an environment where a specific nutrient is lacking, for example, it is necessary to replace the medium used for culturing photosynthetic microorganisms from one where all nutrients are sufficiently present to one where a specific nutrient is lacking. To replace the medium used for culturing photosynthetic microorganisms, it is necessary to recover the photosynthetic microorganisms using a centrifugal separator or the like. Therefore, a large amount of energy, time, and labor are required to produce bio-derived materials using photosynthetic microorganisms on a large scale.

[0034] When using a TOR inhibitor, medium replacement for changing from an environment where all nutrients are sufficiently present to an environment where a specific nutrient is lacking is unnecessary. However, TOR inhibitors are expensive.

[0035] In the method described above, by adding a promoter to the medium, the cell growth of photosynthetic microorganisms is arrested, thereby promoting the accumulation of substances in the photosynthetic microorganisms. That is, in this method, medium replacement for changing from an environment where all nutrients are sufficiently present to an environment where a specific nutrient is lacking is unnecessary.

[0036] In addition, the promoter used in this method contains dimethyl 2-oxoglutarate as an active ingredient. Dimethyl 2-oxoglutarate is much cheaper compared to TOR inhibitors.

[0037] Therefore, according to this method, the production of bio-derived materials using photosynthetic microorganisms can be realized at low cost.

[0038] In addition, TOR inhibitors do not have an effect on all photosynthetic microorganisms. In contrast, dimethyl 2-oxoglutarate is a derivative of 2-oxoglutarate represented by the following chemical formula (2). 2-oxoglutarate is a metabolite common to all organisms. Therefore, the effects described above regarding the addition of the promoter containing dimethyl 2-oxoglutarate can be achieved in all photosynthetic microorganisms.

[0039] [Chemical formula]

[0040] [Test] The tests conducted by the present inventors are described below. (Test 1) Single-celled red algae were suspension-cultured under light conditions, and a promoter was added thereto. As the medium, MA2 medium was used. As the single-celled red algae, Cyanidioschyzon merolae was used. As the promoter, dimethyl 2-oxoglutarate was used. The promoter was added so that the final concentration of dimethyl 2-oxoglutarate was 2 mmol / L.

[0041] After the addition of the promoter, the culture was continued for another 24 hours. Thereafter, for the single-celled red algae after this culture, a bright-field image was acquired using a microscope. Also, the oil of this single-celled red algae was stained with BODIPY (registered trademark) commercially available from Cosmo Bio Co., Ltd. Then, for the single-celled red algae stained with oil, a fluorescence observation image was acquired using a fluorescence microscope.

[0042] In addition, the same culture as above was performed except that the promoter was not added. Then, for the single-celled red algae after this culture, acquisition of a bright-field image and staining of the oil and acquisition of a fluorescence observation image were also performed.

[0043] Figure 1 is a bright-field image obtained using a microscope for single-celled red algae cultured with a promoter added to the medium. Figure 2 is a fluorescence observation image obtained using a microscope for single-celled red algae cultured with a promoter added to the medium and stained with oil. Figure 3 is a bright-field image obtained using a microscope for single-celled red algae cultured without adding a promoter to the medium. Figure 4 is a fluorescence observation image obtained using a microscope for single-celled red algae cultured without adding a promoter to the medium and stained with oil.

[0044] For single-celled red algae cultured without adding a promoter, as shown in Figure 4, almost no fluorescence due to staining was confirmed. In contrast, for single-celled red algae cultured with a promoter added, as shown in Figure 2, a region strongly fluorescing due to staining (the region indicated by the arrow) was confirmed. That is, it was confirmed that the addition of the promoter significantly promoted the accumulation of oil.

[0045] (Test 2) The same culture as in Test 1 was performed, and the amount of starch contained in the cultured single-celled red algae was measured. The results are shown in Figure 5.

[0046] Figure 5 is a graph showing an example of the effect of the addition of the promoter on the starch accumulation amount. Figure 5 shows the average value of the results of three independent tests and the standard deviation. Also, in Figure 5, "Control" shows the results obtained for single-celled red algae cultured without adding a promoter to the medium. And "Dimethyl 2-OG" shows the results obtained for single-celled red algae cultured with a promoter added to the medium.

[0047] As shown in Figure 5, for single-celled red algae cultured with a promoter added, the starch accumulation amount was significantly higher compared to single-celled red algae cultured without adding a promoter. From this result, it was confirmed that the addition of the promoter significantly promoted the accumulation of starch.

Claims

1. Culturing photosynthetic microorganisms in a medium containing dimethyl 2-oxoglutarate, and then recovering the substances produced or accumulated by the photosynthetic microorganisms, A method for producing a biological material, comprising the above steps, wherein the photosynthetic microorganisms include unicellular red algae, and the substances include at least one of oil and starch.

2. Culturing photosynthetic microorganisms in a medium, then adding dimethyl 2-oxoglutarate to the medium and continuing the culturing of the photosynthetic microorganisms in this medium, and then recovering the substances produced or accumulated by the photosynthetic microorganisms, A method for producing a biological material, comprising the above steps, wherein the photosynthetic microorganisms include unicellular red algae, and the substances include at least one of oil and starch.

3. The method for producing a biological material according to claim 2, wherein the number of cells of the photosynthetic microorganisms per volume is in the range of 100,000 to 1,000,000,000,000 cells / mL immediately before adding dimethyl 2-oxoglutarate to the medium.

4. The method for producing a biological material according to any one of claims 1 to 3, wherein the concentration of dimethyl 2-oxoglutarate is in the range of 0.001 to 1000 mmol / L.

5. The method for producing a biological material according to any one of claims 1 to 4, wherein recovering the substances includes extracting one of the hydrophobic and hydrophilic substances from the photosynthetic microorganisms to obtain an extract and a residue.

6. An accelerator used to promote the accumulation of substances by photosynthetic microorganisms, containing dimethyl 2-oxoglutarate, wherein the photosynthetic microorganisms include unicellular red algae, and the substances include oil and starch.

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