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 while being irradiated with light in a medium including dimethyl 2-oxoglutaric acid thereby proliferating the photosynthetic micro-organism, 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] The oil produced by algae can be used, for example, as a raw material for biofuels. Also, the carbohydrates produced by algae can be used, for example, as raw materials for fuel additives, pharmaceuticals, cosmetics, and plastic products.

[0003] When producing oil and starch on a large scale using microalgae, an open pond may be used for culturing the algae. As the growth of the algae progresses, the density of algal cells in the dispersion containing the algae and the liquid medium increases. When such a situation occurs, for example, in an open pond, much of the incident light is absorbed by the algal cells near the liquid surface.

[0004] When algal cells are irradiated with light having an intensity exceeding the light saturation point, among the energy of the absorbed photons, the surplus not used for photosynthesis is dissipated as heat to protect their photosynthetic function. When the intensity of the light irradiated to the algal cells exceeds the light saturation point near the liquid surface, a part of the energy absorbed by the algal cells near the liquid surface is dissipated as heat as surplus and is not used for the photosynthesis of the algal cells in the deep part.

[0005] Also, in a situation where much of the incident light is absorbed by the algal cells near the liquid surface, the light does not reach the algal cells in the deep part with sufficient intensity. That is, when the growth of the algae progresses and the intensity of the light reaching the algal cells in the deep part decreases, the photosynthesis rate in the algal cells in the deep part decreases. Therefore, as the growth of the algae progresses, the growth rate of the algae decreases.

[0006] Non-Patent Document 1 describes a transformant of Chlamydomonas reinhardtti that has a lower amount of chlorophyll per cell and a higher ratio a / b of chlorophyll a to chlorophyll b compared to the wild type. Chlorophyll b serves as an antenna for collecting light, i.e., as a light-harvesting pigment. On the other hand, chlorophyll a serves as a reaction center directly involved in the electron transfer reaction of photosynthesis and as a light-harvesting pigment.

[0007] The above-mentioned transformant has a lower amount of chlorophyll per cell. Therefore, the decrease in the intensity of light reaching the deep cells associated with its growth is small. Also, since the above-mentioned transformant has a high ratio a / b, there is less surplus energy in its culture as described above.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

[0009] An object of the present invention is to provide a technique that can easily alleviate the decrease in the growth rate of photosynthetic microorganisms caused by an increase in cell density.

[0010] According to a first aspect of the present invention, there is provided a method for producing a bio-derived material, including culturing photosynthetic microorganisms while irradiating light in a medium containing dimethyl 2-oxoglutaric acid to grow the photosynthetic microorganisms, and then recovering a substance produced or accumulated by the photosynthetic microorganisms.

[0011] According to a second aspect of the present invention, there is provided a method for producing a bio-derived material, including culturing photosynthetic microorganisms while irradiating light in a medium to grow the photosynthetic microorganisms, then adding dimethyl 2-oxoglutaric acid to the medium, further culturing the photosynthetic microorganisms while irradiating light in the medium to which dimethyl 2-oxoglutaric acid has been added to further grow the photosynthetic microorganisms, and then recovering a substance produced or accumulated by the photosynthetic microorganisms.

[0012] According to a third aspect of the present invention, there is provided an adjusting agent containing dimethyl 2-oxoglutaric acid and used for reducing the amount of light-harvesting pigments in photosynthetic microorganisms.

[0013] According to the present invention, there is provided a technique that enables easy mitigation of a decrease in the growth rate of photosynthetic microorganisms caused by an increase in cell density.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

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Modes for Carrying Out the Invention

[0015] Embodiments of the present invention will be described below. The embodiments described below are more specific forms of any of the above aspects. The matters described below can be incorporated into each of the above aspects alone or in combination.

[0016] In a method for producing a bio-derived 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 occurs in the photosynthetic microorganisms and the photosynthetic microorganisms are grown.

[0017] 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.

[0018] 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 a liquid medium here. Also, the above culture is, for example, a suspension culture. The suspension culture can be carried out, for example, in an open-type culture tank called an open pond. The suspension culture may be carried out in a closed system called a closed-type photobioreactor.

[0019] When photosynthetic microorganisms are grown, a dispersion containing the photosynthetic microorganisms and the medium is obtained, and here the cell density in the dispersion increases. Along with this, the intensity of light reaching the cells in the deep part away from the light incident surface decreases, and the photosynthesis rate in the deep cells decreases. As a result, the growth rate of the photosynthetic microorganisms decreases.

[0020] Therefore, at any point in time when the growth rate of the photosynthetic microorganisms has decreased, an adjusting agent containing dimethyl 2-oxoglutarate represented by the following chemical formula (1) is added to the above medium.

[0021]

Chemical formula

[0022] This adjusting agent contains dimethyl 2-oxoglutarate. Dimethyl 2-oxoglutarate reduces the amount of light-harvesting pigments in photosynthetic microorganisms, as will be described below.

[0023] 2-Oxoglutarate represented by the following chemical formula (2) is considered as one of the signal substances for nitrogen deficiency in microorganisms. In photosynthetic microorganisms, generally, under nitrogen-deficient conditions, light-harvesting pigments are decomposed and decreased. Therefore, by adding 2-oxoglutarate to the above medium, a pseudo-nitrogen-deficient state can be created in the photosynthetic microorganism cells, and it is considered that this can induce pigment decomposition.

[0024]

Chemical formula

[0025] However, even if 2-oxoglutarate is added to the above medium, pigment decomposition in the photosynthetic microorganism cells is not induced, and the growth rate does not increase either. This is because 2-oxoglutarate is not taken up into the photosynthetic microorganism cells.

[0026] Unlike 2-oxoglutaric acid, dimethyl 2-oxoglutarate is cell membrane permeable. In addition, dimethyl 2-oxoglutarate exhibits the same effects in photosynthetic microbial cells as those of 2-oxoglutaric acid in photosynthetic microbial cells. Therefore, when an adjusting agent containing dimethyl 2-oxoglutarate is added to the above medium, the amount of light-harvesting pigments in photosynthetic microorganisms can be reduced.

[0027] The adjusting agent 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. Diluting dimethyl 2-oxoglutarate with a solvent makes it easy to supply dimethyl 2-oxoglutarate uniformly, for example, to the liquid surface of the above dispersion.

[0028] The timing of adding the adjusting agent is determined based on, for example, any of the light transmittance and turbidity of the dispersion, the number of cells per volume, the specific growth rate of photosynthetic microorganisms, and the rate of change thereof. According to one example, the timing of adding the adjusting agent is determined by comparing any of the light transmittance and turbidity of the dispersion, the number of cells per volume, the specific growth rate of photosynthetic microorganisms, and the rate of change thereof with a predetermined threshold value.

[0029] The adjusting agent is preferably added such that the concentration of dimethyl 2-oxoglutarate in the mixture containing the medium, photosynthetic microorganisms, and the adjusting agent is 5 mmol / L or more, and more preferably 20 mmol / L or more. When this concentration is lowered, the effect of reducing the amount of light-harvesting pigments in photosynthetic microorganisms becomes smaller. This concentration is preferably 100 mmol / L or less. When this concentration is increased, in addition to the high cost, as described later, the growth of photosynthetic microorganisms tends to stagnate.

[0030] The regulator is supplied to the culture medium so that, for example, the dimethyl-2-oxoglutaric acid concentration becomes substantially uniform throughout the culture medium. The regulator may be supplied so that the dimethyl-2-oxoglutaric acid concentration is higher near the surface (light incident surface) of the dispersion containing the culture medium and the photosynthetic microorganism than in the deeper part of this dispersion. Dimethyl-2-oxoglutaric acid can reduce the amount of light-harvesting pigments in photosynthetic microorganisms while arresting the growth of photosynthetic microorganisms. For example, when performing suspension culture of photosynthetic microorganisms in an open culture tank, if the dimethyl-2-oxoglutaric acid concentration near the liquid surface is made higher than the dimethyl-2-oxoglutaric acid concentration in the deeper part, it is possible to reduce the amount of light-harvesting pigments contained in the photosynthetic microorganisms near the liquid surface while preventing the growth arrest by dimethyl-2-oxoglutaric acid from occurring in the deeper part. Incidentally, the above concentration gradient can be realized, for example, by spraying the regulator onto the liquid surface.

[0031] After adding the regulator to the culture medium, the same culture as above is continued to further grow the photosynthetic microorganisms. By adding the regulator to the culture medium, for example, the amount of light-harvesting pigments contained in the photosynthetic microorganisms near the liquid surface decreases. As a result, light reaches the photosynthetic microorganism cells in the deeper part with sufficient intensity, and the growth rate increases.

[0032] The time required from adding the regulator until the amount of light-harvesting pigments contained in the photosynthetic microorganisms sufficiently decreases varies depending on the type of photosynthetic microorganism. Generally, this time is within the range of 3 hours to 48 hours. Therefore, in order to further grow the photosynthetic microorganisms after adding the regulator, the culture period after adding the regulator is preferably 3 hours or more, and more preferably 12 hours or more. Although there is no upper limit to this culture period, considering productivity, it is preferably 14 days or less.

[0033] Next, if necessary, the cycle consisting of adding the regulator to the culture medium and subsequent culturing of the photosynthetic microorganisms is repeated one or more times.

[0034] Subsequently, the substances produced or accumulated by the photosynthetic microorganisms are recovered. When recovering the substances accumulated intracellularly by the photosynthetic microorganisms, for example, hydrophobic substances are extracted from the photosynthetic microorganisms to obtain an extract containing the hydrophobic substances and a residue containing hydrophilic substances. Alternatively, hydrophilic substances are extracted from the photosynthetic microorganisms to obtain an extract containing the hydrophilic substances and a residue containing hydrophobic substances. When recovering the substances released extracellularly among the substances produced by the photosynthetic microorganisms, this substance is recovered from, for example, the culture medium. Here, as an example, a method for obtaining oils or sugars from photosynthetic microorganisms will be described.

[0035] For example, first, the 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 the photosynthetic microorganisms and the culture medium by centrifugation or pressing. Thereby, a concentrate containing the photosynthetic microorganisms at a higher concentration than the previous mixture is obtained. Next, the concentrate is dried to obtain a dried product composed of the photosynthetic microorganisms.

[0036] Next, oils are extracted from the dried product composed of the photosynthetic microorganisms. An organic solvent is used as the extraction medium for the extraction of the oils. Thereby, an extract containing the oils and a residue containing sugars are obtained.

[0037] When producing oils, subsequently, purification is performed on the extract as necessary. This purified product may be modified. In the above manner, oils are obtained from the photosynthetic microorganisms. The oils thus obtained can be used, for example, as biofuels or raw materials therefor.

[0038] When producing sugars, for example, sugars are extracted from the above-mentioned residue. When extracting polysaccharides such as starch, water is used, for example, as the extraction medium. Next, purification is performed on this extract as necessary. This purified product may be modified. In the above manner, sugars are obtained from the photosynthetic microorganisms. These sugars can be used, for example, as raw materials for fuel additives, pharmaceuticals, cosmetics, and plastic products.

[0039] Incidentally, the substances produced and accumulated by photosynthetic microorganisms are diverse, 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 microorganism. 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.

[0040] For example, by such a method, oils and fats, 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.

[0041] Incidentally, the substances produced or accumulated by photosynthetic microorganisms obtained in this way, the substances obtained by performing post-treatments such as purification and modification on this substance, 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.

[0042] In the method described above, an adjusting agent is added to the medium. Thereby, it is possible to mitigate the decrease in the growth rate of photosynthetic microorganisms caused by the increase in cell density. This will be described with reference to FIGS. 1 and 2.

[0043] FIG. 1 is a diagram schematically showing a method for producing a bio-derived material according to an embodiment of the present invention. FIG. 2 is a diagram schematically showing a method for producing a bio-derived material according to a comparative example. FIGS. 1 and 2 depict a state in which sunlight L is irradiated onto a dispersion liquid containing a liquid medium 2 and photosynthetic microorganisms 3 and housed in an open-type culture tank 1.

[0044] Even though the growth of photosynthetic microorganism 3 has progressed and the cell density has increased, when the regulator is not added to the liquid medium 2, as shown in FIG. 2, much of the sunlight L is absorbed by the photosynthetic microorganism 3 near the liquid surface, and sunlight L of sufficient intensity does not reach the photosynthetic microorganism 3 in the deep part. Therefore, in the deep part, the photosynthesis rate in the photosynthetic microorganism 3 is low, and the growth rate is also low.

[0045] On the other hand, for example, when the regulator is sprayed into the liquid medium 2 after the growth of the photosynthetic microorganism 3 has progressed and the cell density has increased, the amount of light-harvesting pigment contained in the photosynthetic microorganism can be reduced at least near the liquid surface. Therefore, as shown in FIG. 1, sunlight L of sufficient intensity can reach the photosynthetic microorganism 3 in the deep part.

[0046] As described above, a transformant of Paramecium bursaria with a smaller amount of chlorophyll per cell and a larger ratio a / b of chlorophyll a to chlorophyll b compared to the wild type is known. This transformant has a smaller amount of chlorophyll per cell. Therefore, the decrease in the intensity of light reaching the cells in the deep part associated with growing this is small. Also, since this transformant has a large ratio a / b, there is little excess energy dissipated as heat in its culture.

[0047] However, to obtain a transformant having properties similar to the above transformant, it is necessary to introduce a foreign gene into the photosynthetic microorganism cell or cause genetic modification by ultraviolet irradiation, and isolate a strain having desirable properties from those obtained in this way, for example, a strain similar to the wild type except that the amount of light-harvesting pigment per cell is small. Photosynthetic microorganisms into which genes can be introduced into cells are limited. Also, especially when using genetic modification by ultraviolet irradiation, trial and error is required to obtain a transformant having desirable properties, which requires a long time and a great deal of labor. Therefore, reports on transformants of photosynthetic microorganisms having the above properties are limited to only a few examples.

[0048] As described with reference to FIG. 1, in the above method, by adding an adjusting agent to the medium, it is possible to reduce the amount of light-harvesting pigments contained in photosynthetic microorganisms, at least in the vicinity of the liquid surface. Adding an adjusting agent to the medium is much easier compared to obtaining a recombinant.

[0049] Therefore, according to this method, it is possible to easily alleviate the decrease in the growth rate of photosynthetic microorganisms caused by an increase in cell density.

[0050] Also, the above transformant is a recombinant. Therefore, even if a transformant having the above properties is obtained, the facilities for culturing it are limited.

[0051] In contrast, in the method described above, it is possible to alleviate the decrease in the growth rate of photosynthetic microorganisms caused by an increase in cell density without using a recombinant as the photosynthetic microorganism. Therefore, when culturing wild-type photosynthetic microorganisms, it is not necessary to use a closed system. And dimethyl 2-oxoglutarate is relatively inexpensive. Therefore, according to this method, it is possible to realize the production of bio-derived materials using photosynthetic microorganisms at low cost.

[0052] Furthermore, dimethyl 2-oxoglutarate is a derivative of 2-oxoglutarate, which is a metabolite common to all organisms. Therefore, the effects described above regarding the addition of an adjusting agent containing dimethyl 2-oxoglutarate can be achieved in any photosynthetic microorganism.

[0053] [Test] The tests conducted by the present inventors are described below. (Test 1) Single-celled red algae were suspension-cultured under light conditions, and an adjusting agent was added thereto. As the medium, MA2 medium was used. As the single-celled red algae, Cyanidioschyzon merolae was used. As the adjusting agent, dimethyl 2-oxoglutarate was used. And after adding the adjusting agent, the culture was continued for another 18 hours.

[0054] The above culturing was carried out for each case where the final concentration of dimethyl 2-oxoglutarate was 10, 20, 40, and 80 mmol / L (= mM). Also, similar culturing was carried out except that dimethyl 2-oxoglutarate was not added. Thereafter, each of the dispersions containing the unicellular red alga and the medium was imaged.

[0055] Also, culturing similar to the above was carried out except that 2-oxoglutaric acid was used instead of dimethyl 2-oxoglutaric acid. Thereafter, each of these dispersions containing the unicellular red alga and the medium was imaged.

[0056] Figure 3 is an image obtained by imaging the dispersion after culturing. In Figure 3, "2-OG" and "dimethyl 2-OG" represent 2-oxoglutaric acid and dimethyl 2-oxoglutaric acid, respectively.

[0057] As shown in Figure 3, when 2-oxoglutaric acid was added to the medium, even when its final concentration was increased, the color of the dispersion was the same as that in the case of no addition. That is, the addition of 2-oxoglutaric acid to the medium contributed little to the decrease in the light-harvesting pigment.

[0058] In contrast, when dimethyl 2-oxoglutaric acid was added to the medium, even when its final concentration was low, the color of the dispersion was lighter compared to the case of no addition. In particular, when the final concentration of dimethyl 2-oxoglutaric acid was 20 mmol / L or more, the dispersion became almost colorless. That is, the addition of dimethyl 2-oxoglutaric acid to the medium contributed greatly to the decrease in the light-harvesting pigment.

[0059] (Test 2) Under light conditions, unicellular red algae were cultured in suspension, and an adjusting agent was added thereto. As the medium, MA2 medium was used. As the unicellular red algae, Cyanidioschyzon merolae was used. As the adjusting agent, dimethyl 2-oxoglutaric acid was used. The adjusting agent was added such that the final concentration of dimethyl 2-oxoglutaric acid was 2 mmol / L. After the addition of the adjusting agent, the culture was continued. Then, immediately before the addition of the adjusting agent, 6 hours after the addition of the adjusting agent, and 24 hours after the addition of the adjusting agent, the amount of chlorophyll a contained in the dispersion was measured.

[0060] In parallel with this culture, a culture was performed in the same manner as above except that the adjusting agent was not added. Also here, in the same manner as above, the amount of chlorophyll a contained in the dispersion was measured.

[0061] Figure 4 is a graph showing an example of the influence of the addition of the adjusting agent on the amount of chlorophyll a. Figure 4 shows the average value of the results of three independent tests, and the standard deviation is shown as error bars. Also, in Figure 4, "Control" shows the results obtained for unicellular red algae cultured without adding the adjusting agent to the medium. And "dimethyl 2-OG" shows the results obtained for unicellular red algae cultured with dimethyl 2-oxoglutaric acid added as the adjusting agent to the medium.

[0062] As shown in Figure 4, in the unicellular red algae cultured without adding the adjusting agent, the amount of chlorophyll a increased according to the culture time. In contrast, in the unicellular red algae cultured with the adjusting agent added, the amount of chlorophyll a decreased according to the culture time.

[0063] (Test 3) The same culture as in Test 2 was performed, and the amount of phycocyanin contained in the dispersion was measured for each culture time.

[0064] Figure 5 is a graph showing an example of the effect of the addition of the regulator on the amount of phycocyanin. Figure 5 shows the average value of the results of three independent tests, and the standard deviation is shown as error bars. Also, in Figure 5, "Control" shows the results obtained for unicellular red algae cultured without adding the regulator to the medium. And "Dimethyl 2-OG" shows the results obtained for unicellular red algae cultured with dimethyl 2-oxoglutaric acid added as the regulator to the medium.

[0065] As shown in Figure 5, in the unicellular red algae cultured without adding the regulator, the amount of phycocyanin increased according to the culture time. In contrast, in the unicellular red algae cultured with the regulator added, the amount of phycocyanin decreased according to the culture time.

Explanation of symbols

[0066] 1…Open culture tank 2…Liquid medium 3…Photosynthetic microorganism L…Sunlight

Claims

1. Culturing photosynthetic microorganisms while irradiating them with light in a medium containing dimethyl 2-oxoglutarate to grow the photosynthetic microorganisms; Then, recovering the substances produced or accumulated by the photosynthetic microorganisms; A method for producing a biological material containing a photosynthetic microorganism including unicellular red algae, comprising the above steps.

2. Culturing photosynthetic microorganisms while irradiating them with light in a medium to grow the photosynthetic microorganisms; Then, adding dimethyl 2-oxoglutarate to the medium; Further culturing the photosynthetic microorganisms while irradiating them with light in the medium to which dimethyl 2-oxoglutarate has been added to further grow the photosynthetic microorganisms; Then, recovering the substances produced or accumulated by the photosynthetic microorganisms; A method for producing a biological material containing a photosynthetic microorganism including unicellular red algae, comprising the above steps.

3. The method for producing a biological material according to claim 1 or 2, wherein the photosynthetic microorganism is a wild type.

4. The method for producing a biological material according to any one of claims 1 to 3, wherein the photosynthetic microorganism is cultured in an open culture tank.

5. The method for producing a biological material according to any one of claims 1 to 4, wherein the concentration of dimethyl 2-oxoglutarate is made higher near the surface of the dispersion containing the medium and the photosynthetic microorganisms compared to the depth of the dispersion.

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

7. An agent used to reduce the amount of light-harvesting pigments in photosynthetic microorganisms, which contains dimethyl 2-oxoglutarate, and the photosynthetic microorganisms include unicellular red algae.

Images