Patent Application
20240263128
The present invention relates to a method of producing a biological material.
Most algae increase an amount of accumulated fat and oil and carbohydrate such as starch in an environment with lacking nutrients, e.g. nitrogen (Non-Patent Literature 1). The fat and oil produced by algae can be utilized as a raw material for biofuel, for example. In addition, carbohydrates produced by algae can be used as a raw material for, for example, fuel additives, pharmaceuticals, cosmetics and plastic products.
Accumulation of fat and oil and carbohydrate by algae can be promoted without causing nitrogen to be lacking in the environment. For example, accumulation of fat and oil and carbohydrates by algae can be promoted by using a target of rapamycin (TOR) inhibitor that specifically inhibits the activity of TOR kinase possessed by microalgae that are eukaryotic algae (Non-Patent Literatures 2 and 3).
An object of the present invention is to provide a technique capable of achieving production of a biological material using photosynthetic microorganisms at low cost.
According to a first aspect of the present invention, there is provided a method of producing a biological material, including culturing a photosynthetic microorganism in a culture medium containing dimethyl 2-oxoglutarate and then collecting a substance produced by or accumulated in the photosynthetic microorganism.
According to a second aspect of the present invention, there is provided a method of producing a biological material, including culturing a photosynthetic microorganism in a culture medium, then adding dimethyl 2-oxoglutarate to the culture medium to continue the culture of the photosynthetic microorganism in the culture medium, and then collecting a substance produced by or accumulated in the photosynthetic microorganism.
According to a third aspect of the present invention, there is provided a promoter for use in promoting accumulation of a substance by a photosynthetic microorganism, containing dimethyl 2-oxoglutarate.
According to the present invention, there is provided a technique capable of achieving production of a biological material using photosynthetic microorganisms at low cost.
Hereinafter, embodiments of the present invention will be described. The embodiments described below are more specific implementations of any of the above-mentioned aspects. The items described below can be incorporated into each of the above-mentioned aspects alone or in combination.
In a method of producing a biological material according to an embodiment of the present invention, first, photosynthetic microorganisms are cultured in a culture medium. In this culture, the photosynthetic microorganisms are irradiated with light, for example, in the presence of carbon dioxide. For example, the photosynthetic microorganisms are irradiated with sunlight under the air atmosphere. This causes photosynthesis in the photosynthetic microorganisms and causes the photosynthetic microorganisms to proliferate.
The photosynthetic microorganisms are, for example, microorganisms that carry out oxygenic photosynthesis. The photosynthetic microorganisms are, for example, algae such as eukaryotic algae. The algae are preferably microalgae. Here, “microalgae” are, for example, photosynthetic eukaryotes, and is a unicellular organism or a colony thereof. The microalgae are, for example, unicellular green algae such as Chlamydomonas reinhardtii and Botryococcus, unicellular red algae such as Cyanidioschyzon merolae, or a colony thereof. The photosynthetic microorganisms may not be a eukaryote. The photosynthetic microorganisms may be a prokaryotic organism, for example, bacteria such as cyanobacterium. The prokaryotic organisms may be archaea.
As the culture medium, a culture medium containing all nutrients necessary for the proliferation and photosynthesis of photosynthetic microorganisms in a sufficient concentration is used. The culture medium is, for example, a liquid culture medium.
The above-mentioned cultivation is, for example, suspended cultivation. The above-mentioned cultivation may be another type of cultivation, e.g., attached cultivation. In attached cultivation, a biofilm of photosynthetic microorganisms is formed on a support, and the cultivation is carried out in a state where the biofilm and the culture medium are brought into contact with each other.
Next, a promoter to promote accumulation of a substance by photosynthetic microorganisms is added to the above-mentioned culture medium. Then, the cultivation of the photosynthetic microorganisms is continued in the culture medium. In other words, the photosynthetic microorganisms are irradiated with light, for example, in the presence of carbon dioxide. For example, the photosynthetic microorganisms are irradiated with sunlight under the air atmosphere.
The promoter contains dimethyl 2-oxoglutarate represented by the following chemical formula (1). Dimethyl 2-oxoglutarate slows cell proliferation of photosynthetic microorganisms, thereby promoting accumulation of substances produced by the photosynthetic microorganisms.
The promoter may further contain a solvent. As the solvent, for example, a liquid having the same or almost the same composition as that of the culture medium used for culturing the photosynthetic microorganisms can be used. When dimethyl 2-oxoglutarate is diluted with a solvent, for example, dimethyl 2-oxoglutarate can be supplied uniformly to photosynthetic microorganisms.
The substances produced and accumulated by the photosynthetic microorganisms are various, such as fat and oil, carbohydrates, hydrocarbons, amino acids, and the like. The substances produced by and accumulated in the photosynthetic microorganisms are different according to the kind of the photosynthetic microorganisms. The fat and oil are, for example, neutral lipids such as triacylglycerols. The hydrocarbons are, for example, botryococcene. The carbohydrates are, for example, starch or a combination of starch and one or more other carbohydrate components.
A timing at which the promoter is added is determined based on any of, for example, a light transmittance and turbidity of a dispersion containing the photosynthetic microorganisms and the culture medium, the number of cells per volume, a specific growth rate of the photosynthetic microorganisms and a rate of change thereof. According to one example, a timing at which the promoter is added is determined by comparing any of a light transmittance and turbidity of a dispersion, the number of cells per volume, a specific growth rate of the photosynthetic microorganisms and a rate of change thereof with a predetermined threshold.
The addition of the promoter to the culture medium is preferably performed so that the number of cells of the photosynthetic microorganisms per volume immediately before the addition of the promoter is within the range of 100,000 to 10,000,000,000 cells/mL, and more preferably performed so that the number of cells of the photosynthetic microorganisms per volume is within the range of 10,000,000 to 100,000,000 cells/mL. Here, the number of cells of the photosynthetic microorganisms per volume is the ratio of the number of cells of the photosynthetic microorganisms to the total volume of the photosynthetic microorganisms and the culture medium.
In order to efficiently produce the biological material, it is desirable that the number of cells of the photosynthetic microorganisms be large to some extent. However, when the photosynthetic microorganisms proliferate excessively, hardly any light reaches a part deep inside thereof.
The promoter is preferably added so that the dimethyl 2-oxoglutarate concentration of a mixed liquid containing the culture medium, the photosynthetic microorganisms, and the promoter is in the range of 0.001 to 1,000 mmol/L, and more preferably added so that the concentration is in the range of 0.5 to 5 mmol/L. When the concentration is lowered, the effect of promoting the accumulation of the substances in the photosynthetic microorganisms is reduced. When the concentration is increased, the cost becomes higher.
The cultivation period after the addition of the promoter is different according to the kind of the photosynthetic microorganisms. In general, the cultivation period is preferably in the range of 3 hours to 14 days, and more preferably in the range of 12 hours to 72 hours.
Thereafter, the substances produced by or accumulated in the photosynthetic microorganisms are collected. When the substances accumulated by the photosynthetic microorganisms in the cell are collected, for example, hydrophobic substances are extracted from the photosynthetic microorganisms to obtain the extract containing the hydrophobic substances and the residue containing hydrophilic substances. Alternatively, the hydrophilic substances are extracted from the photosynthetic microorganisms to obtain an extract containing the hydrophilic substances and a residue containing the hydrophobic substances. When substances produced by the photosynthetic microorganisms and released to the outside of the cells are collected, the substances are collected from, for example, a culture medium. Here, as an example, a method for acquiring fat and oil or carbohydrates from the photosynthetic microorganisms will be described.
For example, the photosynthetic microorganisms are first separated from the culture medium. In the case of suspended cultivation, at least a part of the culture medium is removed from the mixed solution of the photosynthetic microorganisms and the culture medium, for example, by means of centrifugal separation or compression. Thereby, a concentrate containing photosynthetic microorganisms in a higher concentration than the previous mixed liquid is obtained. Then, the concentrate is dried to obtain a dried product composed of the photosynthetic microorganisms.
Next, fat and oil are extracted from the dried product composed of the photosynthetic microorganisms. An organic solvent is used as an extraction medium for extracting the fat and oil. Thus, the extract containing the fat and oil is obtained and the residue containing carbohydrates is obtained.
In the case of producing fat and oil, the extract is then purified if necessary. The purified product may be modified. In this way, fat and oil are obtained from photosynthetic microorganisms. The fat and oil thus obtained can be used as a biofuel or a raw material thereof, for example.
In the case of producing a carbohydrate, for example, a carbohydrate is extracted from the above residue. In the case of extracting polysaccharides such as starch, for example, water is used as the extraction medium. Then, if necessary, the extract is purified. The purified product may be modified. In this way, carbohydrates are obtained from photosynthetic microorganisms. The carbohydrates can be used as a raw material for, for example, fuel additives, pharmaceuticals, cosmetics, and plastic products.
For example, fat and oil, carbohydrates, or a purified product or a modified product thereof can be obtained in the above-described method. In addition, using a method similar thereto, other substances such as hydrocarbons and amino acids accumulated in the photosynthetic microorganisms, or a purified product or modified products thereof can be obtained.
The substances produced by or accumulated in the photosynthetic microorganisms and acquired as described above, substances obtained by performing post-treatment such as purification and modification to the above substances, or substances obtained by using the above substances as raw materials are biological materials. The biological material may be a product, for example, a biofuel, a fuel additive, a pharmaceutical, a supplement, a physiologically active substance, a food, a cosmetic, or a plastic product. Alternatively, the biological material is one or more components or raw materials of the above-mentioned products.
As described above, the accumulation of substances in the photosynthetic microorganisms can be promoted by reducing nitrogen in the environment. However, in order to change an environment from one in which all nutrients are sufficiently present to one in which a specific nutrient is deficient, for example, a culture medium in which all nutrients are sufficiently present needs to be replaced with one in which a specific nutrient is deficient. In order to replace a culture medium used for culturing photosynthetic microorganisms, it is necessary to collect the photosynthetic microorganisms by using a centrifugal separator or the like. Therefore, in order to produce a biological material using photosynthetic microorganisms on a large scale, a large amount of energy, time and effort are required.
If a TOR inhibitor is used, exchange of a culture medium for changing the environment from one in which all nutrients are sufficiently present to one in which a specific nutrient is deficient is unnecessary. However, TOR inhibitors are expensive.
In the method described above, the cell proliferation of the photosynthetic microorganism slows by adding a promoter to the culture medium, thereby promoting the accumulation of substances in the photosynthetic microorganisms. That is, in this method, there is no need to exchange the culture medium for changing the environment from one in which all nutrients are sufficiently present to one in which a particular nutrient is deficient.
The promoter used in this method contains dimethyl 2-oxoglutarate as an active ingredient. Dimethyl 2-oxoglutarate is much cheaper than a TOR inhibitor.
For this reason, the method can realize production of a biological material using photosynthetic microorganisms at low cost.
The TOR inhibitor is not effective for all photosynthetic microorganisms. On the other hand, 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 above-described effects of the addition of the promoter containing dimethyl 2-oxoglutarate can be exhibited in all photosynthetic microorganisms.
The tests conducted by the inventors will be described below.
Unicellular red algae were cultured in suspension under a light condition and a promoter was added thereto. An MA2 culture medium was used as a culture medium. Cyanidiosyzon merolae were used as unicellular red algae. Dimethyl 2-oxoglutarate was used as a promoter. The promoter was added so that the final concentration of dimethyl 2-oxoglutarate would be 2 mmol/L.
After the addition of the promoter, the cultivation was continued for 24 hours more. Thereafter, a bright field image was obtained for the cultured unicellular red algae using a microscope. In addition, the fat and oil of the unicellular red algae were stained with commercially available BODIPY (registered trademark) from the Cosmo Bio, Inc. Then, a fluorescence observation image of the unicellular red algae with stained fat and oil was acquired by using a fluorescence microscope.
In addition, cultivation was similarly conducted as described above except that no promoter was added. Then, acquisition of a bright field image, staining of fat and oil, and acquisition of a fluorescence observation image were carried out also for the cultured unicellular red algae.
As shown in
The same cultivation as in test 1 was performed, and the amount of starch contained in the unicellular red algae after the cultivation was determined. The results are shown in
As shown in
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/022698 | 6/15/2021 | WO |
