Method for culturing blue-green algae and blue-green algae

Abstract

To provide a method for culturing blue-green algae in high efficiency.SOLUTION: The method for culturing blue-green algae includes a step (1) of culturing blue-green algae in a liquid in a culture tank, a step (2) of selecting precipitated cells by removing blue-green algae cells floating in the liquid from the culture tank or transferring blue-green algae cells precipitated in the liquid to a new culture tank, and a step (3) of culturing the precipitated cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for culturing cyanobacteria with high efficiency and to cyanobacteria cultivated by said method. [Background technology]

[0002] In recent years, attempts to search for active ingredients from various plants, animals, algae, fungi, etc. have been widely undertaken. As a result, various useful substances have been discovered and are being used in a wide range of fields, including pharmaceuticals, foods, and cosmetics. Recently, a sulfated polysaccharide with a molecular weight exceeding 10 million has been discovered from the cyanobacterium Aphanothece sacrum, and its high moisturizing power is expected to be useful for beauty treatments, burn treatments, and other purposes (see Patent Document 1).

[0003] However, freshwater cyanobacteria such as A. sacchariflora can only grow in highly transparent, mineral-rich spring water, and are easily affected by weather conditions, making it difficult to ensure a stable supply even when cultivating or cultivating on exposed surfaces. Therefore, despite the high efficacy and industrial value expected from sulfated polysaccharides extracted from A. sacchariflora, there has been a problem in that applications for sulfated polysaccharides have not been fully developed. In response to this problem, attempts have been made to mass-produce cyanobacteria by combining static culture and aerobic culture processes (see Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4066443 [Patent Document 2] Patent No. 6542569 Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, industrial mass production of cyanobacteria is desirable from the viewpoint of utilizing cyanobacteria and components derived from cyanobacteria. However, a technology for sustainably and efficiently cultivating cyanobacteria has not yet been established, and there is room for improvement in the method described in Patent Document 2. In particular, when cyanobacteria are cultivated over a long period of time, there is a problem that the growth rate of the cyanobacteria gradually decreases, resulting in a deterioration in cultivation efficiency. The present invention has been made in view of the above problems, and has as its object to highly efficiently culture cyanobacteria. [Means for solving the problem]

[0006] As a result of intensive research to solve the above problems, the inventors discovered that as the culture is continued, there are cell groups that float in the liquid used for culture and cell groups that settle, and that there is a difference in proliferation ability between these cell groups, thereby completing the present invention. That is, the present invention includes the following aspects. (1) a step of culturing cyanobacteria in a liquid in a culture tank (1); (2) removing the cyanobacterial cells floating in the liquid from the culture tank or transferring the cyanobacterial cells settling in the liquid to a new culture tank to select precipitated cells; and Step (3) of culturing the precipitated cells A method for culturing cyanobacteria, comprising: (2) The culture method according to the above aspect (1), wherein the cyanobacteria is Aphanothece sacrum. (3) The step (1) is carried out by generating a water flow in the liquid, and The culture method according to aspect (1) or (2), wherein the liquid is allowed to stand before the precipitation cell selection in step (2). (4) Cyanobacteria cultured by the culture method according to any one of the above aspects (1) to (3). [Effects of the Invention]

[0007] According to the present invention, it is possible to culture cyanobacteria with high efficiency. DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention will be described in further detail below, but the present invention is not limited to the following embodiments. In this specification, unless otherwise specified, "parts", "%" and "ppm" mean "parts by mass", "% by mass" and "ppm by mass". In this specification, unless otherwise specified, "XX or more and YY or less" or "XX to YY" means a range of values ​​including the lower and upper limit values, which are the endpoints. When a range of values ​​is described in stages, the upper and lower limit values ​​of each range can be combined in any way.

[0009] <Cyanobacteria cultivation method> In the present invention, cyanobacteria are prokaryotic algae capable of photosynthesis, and are also known as cyanobacteria (blue-green bacteria). Examples of cyanobacteria include the genera Aphanothece, Spirulina, Arthrospira, Aphanizomenon, Fisherella, Anabaena, Nostoc, Synechocystis, Synechococcus, Tolypothrix, Mastigoclaus, and Pleurocapsa. The cyanobacteria targeted by the present invention are preferably Aphanothece sacrum (Suizenji moss), Nostoc commune, and Nostoc verrucosum, with Aphanothece sacrum being particularly preferred. Aphanothece sacrum is a freshwater cyanobacterium that grows naturally in a specific region of Kyushu and forms flat colonies of multiple cells. The outer surface of the colonies is covered with a gel-like secretion formed from polysaccharides and the like, and the colonies can grow to a diameter of approximately 50 mm. The cyanobacteria to be cultured in the present invention may be wild strains collected from nature, or may be bred or genetically modified strains. Furthermore, the cyanobacteria may be acclimatized before the main culture treatment.

[0010] (Process (1)) Step (1) is a step of culturing cyanobacteria in a liquid in a culture tank. The culture tank is not particularly limited as long as it is capable of storing a liquid and capable of culturing cyanobacteria inside. Specifically, it may be a culture vessel widely used for relatively small-scale algae culture, such as a tube, flask, or bottle, or a culture vessel incorporated into a culture system such as a (photo)bioreactor, such as a flat panel or tube, or a culture tank used for large-scale algae culture, such as a tank or outdoor culture pond. When a culture vessel is used as a culture tank, the material is not particularly limited, but from the viewpoint of transmitting natural light and artificial light, a material that combines transparency and strength, such as glass, acrylic, or polycarbonate, is preferable. The culture tank may be an open or closed system, but an open system is preferred from the viewpoint of ease of management and connection to peripheral devices and facilities. The culture tank may be equipped with an aeration device for aeration treatment, stirring blades (stirring impellers) for stirring the culture solution during culture, various light sources for irradiating light, a temperature control device for adjusting the temperature during culture, etc.

[0011] The liquid is not particularly limited as long as it can maintain and / or grow cyanobacteria. Specifically, it may be a natural liquid present in nature, such as freshwater or seawater, or an artificial liquid medium (liquid composition) containing nutrients necessary for maintenance and / or growth.

[0012] Known artificial media can be used. Among these, preferred are artificial media containing one or more salts selected from carbonate, bicarbonate, phosphate, and citrate. In addition to these salts, water and other minerals may be added to the artificial media. Known media such as those described in patent literature (Japanese Patent No. 6590144) and non-patent literature (J. Gen Appl Microbiol., 65, 39-46, 2019 Mar.) to which the above salts have been added may also be used. Furthermore, when natural liquids such as freshwater are used for cultivation, the above salts may be added to the natural liquid. These salts can further improve the cultivation efficiency of cyanobacteria, and since they are particularly suitable as carbon sources for photosynthesis by cyanobacteria, it is particularly preferable to add one or more salts selected from carbonates and bicarbonates, and specifically, it is most preferable to use sodium bicarbonate or calcium bicarbonate. For example, when the cyanobacteria are freshwater cyanobacteria such as Aphanothece sacrum, Aphanothece sacrum and the like efficiently utilize specific ions such as bicarbonate ions for photosynthesis, making it possible to rapidly and continuously cultivate large quantities of the target cyanobacteria while suppressing the growth of other algae.

[0013] The amount of one or more salts selected from carbonates, bicarbonates, phosphates, and citrates added to the liquid medium is preferably 1 mass ppm or more and 500 mass ppm or less, and particularly preferably 20 mass ppm or more and 200 mass ppm or less to maintain better growth of the cyanobacteria. Bicarbonates are consumed as a carbon source when the cyanobacteria photosynthesize, but when carbon dioxide is added to the liquid medium by a procedure such as bubbling, they are regenerated as bicarbonate ions, which can be used again as a carbon source for the cyanobacteria, allowing for efficient cultivation.

[0014] The pH of the liquid is not particularly limited, and in the case of natural liquids, pH adjustment may or may not be performed. Furthermore, when using an artificial medium, it is preferable to maintain and adjust the pH during cultivation within a range of pH 10 or less, more preferably within a range of pH 7.5 to 10, and most preferably within a range of pH 8 to 9, as this significantly improves cultivation efficiency. Although pH adjustment may be performed throughout the cultivation period, it is preferable to adjust the pH during light irradiation, as this particularly improves cultivation efficiency. The method for adjusting the pH of the liquid medium is not particularly limited, but examples include a step of observing the increase in pH associated with the cultivation of cyanobacteria, and adding carbon dioxide to the liquid medium when the pH rises to around 10 to lower the pH. Details regarding the addition of carbon dioxide will be described later.

[0015] The culture can be initiated by inoculating a liquid with an appropriate amount of cyanobacteria. The algae to be inoculated into the medium may be those that have been passaged in a solid medium, those that have been precultured in a liquid medium, or those that have been collected from nature. The culture in step (1) may be performed as a preculture or a main culture. The amount of algal cells to be inoculated at the start of cultivation is not particularly limited. For example, the amount can be set to about 10 to 200 g, preferably about 20 to 100 g, and more preferably about 40 to 80 g, in wet weight per liter.

[0016] The culture conditions are not particularly limited as long as they allow the growth of the cyanobacteria to be cultured. The light conditions are not particularly limited, but the light intensity for cyanobacteria that grow autotrophically through photosynthesis is 10 to 2000 μE m -2 sec -1 30 to 1500 μE m -2 sec -1 is preferred, and 50 to 1200 μE m -2 sec -1 More preferably, 50 to 500 μE m -2 sec -1is more preferable. The cells may be cultured in a bright place using natural light or artificial light; they may be cultured in a dark place and constantly exposed to artificial light; or they may be cultured in a dark place and a light-dark cycle (L / D) may be established using artificial light. Examples of the light-dark cycle include 10L:14D to 14L:10D. For artificial light irradiation, known light sources such as incandescent lamps, fluorescent lamps, arc lamps, and light-emitting diodes can be used.

[0017] The culture temperature is not particularly limited as long as it is a temperature at which the cyanobacteria to be cultured can grow. Examples of the culture temperature include 15 to 50°C. When culturing Aphanothece sacrum, the culture temperature is preferably between 15 and 30°C, more preferably between 20 and 25°C, and even more preferably between 23 and 25°C.

[0018] Although the culture may be performed by static culture, it is preferable to perform the culture by generating a water current in the liquid in order to prevent a shortage of carbon dioxide during photosynthesis and a shortage of oxygen during non-photosynthesis or in heterotrophic cyanobacteria. Here, "generating a water current" refers to bringing the liquid in the culture tank into a motile state by applying some external factor (addition of gas or liquid, or imparting energy) to the liquid. Examples of culture methods that generate a water current include aeration culture (aeration culture), shaking culture, and culture with stirring. The conditions for aeration (aeration), shaking (swirl), or stirring may be such that the dissolved carbon dioxide and dissolved oxygen in the liquid are not depleted and that the cyanobacteria to be cultured can grow.

[0019] In the case of cyanobacteria that grow autotrophically through photosynthesis, the concentration of dissolved carbon dioxide in the liquid may decrease due to the consumption of carbon dioxide under light irradiation conditions, resulting in an increase in pH. In such cases, aeration or aeration is preferably performed during light irradiation to adjust the pH and replenish carbon dioxide. The use of a liquid medium containing a salt such as bicarbonate as described above is particularly preferred, as the addition of carbon dioxide replenishes the consumed bicarbonate ions, improving the photosynthetic efficiency of the cyanobacteria. In addition, an operation may be carried out to maintain a constant pH so as not to increase the pH. Carbon dioxide added for the purpose of adjusting the pH may be ordinary air to which carbon dioxide has been added, or air to which carbon dioxide has been further added, or carbon dioxide alone may be added by aeration treatment as described below. Furthermore, in the case of cyanobacteria that grow heterotrophically by utilizing organic carbon sources or that grow mixotrophically (a combination of autotrophic and heterotrophic growth), the amount of oxygen consumed increases as the number of cyanobacteria increases, which can lead to a decrease in the dissolved oxygen concentration in the liquid. In such cases, it is preferable to perform aeration or ventilation to replenish oxygen.

[0020] The aeration rate is preferably as high as possible, and although there is no particular upper limit for the aeration rate, it can be set to 1 vvm or less in practice. Examples of the lower limit for the aeration rate include 0.05 vvm or more, or 0.1 vvm or more. vvm (volume per volume per minute) is a unit that represents the amount of aeration per unit volume of culture solution per minute. Shaking and stirring can be carried out using a known shaking device, stirring device, or stirring blade. The rotation speed for shaking and stirring is, for example, 50 to 500 rpm, preferably 100 to 300 rpm, and more preferably 50 to 200 rpm.

[0021] The number of rotations for shaking or stirring and / or the amount of aeration may be changed depending on the concentrations of bicarbonate ions, dissolved carbon dioxide and dissolved oxygen in the liquid during the culture period.

[0022] (Process (2)) Step (2) is a step of selecting precipitated cells. In the present invention, "floating" refers to floating near the liquid surface, and includes cases where the cyanobacterial cells float near the liquid surface only after the liquid containing the cyanobacterial cells has been allowed to stand for a certain period of time. Here, "near the liquid surface" refers to a liquid height from the bottom of the culture tank, where the bottom of the culture tank is 0 and the liquid surface is 100, in the range of more than 50 and 100, preferably 75 to 100, and more preferably 80 to 100. In the present invention, "settled" refers to the cells sinking near the bottom of the culture tank, and includes the case where the cells sink near the bottom only after the liquid containing the cyanobacterial cells has been allowed to stand for a certain period of time. Here, "near the liquid surface" refers to the height of the liquid from the bottom of the culture tank, where the bottom of the culture tank is 0 and the liquid surface is 100, and is in the range of 0 to 50, preferably 0 to 25, and more preferably 0 to 20.

[0023] The cyanobacterial cells may float as single cells or may form colonies and float. For example, in the case of Aphanothece sacrum, the cyanobacterial cells form colonies and float or sink as colonies.

[0024] If the culture was carried out in step (1) by generating a water current in the liquid, it is preferable to stop the means for generating the water current (e.g., a device used for aeration, ventilation, shaking, swirling, or stirring) and allow the liquid to stand, and then select the precipitated cells. The time for leaving the solution to stand is not particularly limited, and should be long enough for the cells to separate into an upper layer (near the liquid surface) and a lower layer (near the bottom of the culture tank) to the extent that sedimented cells can be selected, and long enough for the cyanobacterial cells not to proliferate. Generally, the time is 1 to 60 minutes after leaving the solution to stand, with 3 to 45 minutes being preferred, and 5 to 30 minutes being more preferred.

[0025] The selection of precipitated cells can be carried out by either removing the cyanobacterial cells floating in the liquid (hereinafter referred to as "floating cells") from the culture tank, leaving only precipitated cells in the culture tank, or by transferring the cyanobacterial cells that have settled in the liquid (hereinafter referred to as "settled cells") to a new culture tank, and using the new culture tank in the subsequent step (3) and thereafter. If, as a result of leaving the mixture to stand, the cyanobacterial cells are separated into at least three groups: floating cells, settling cells, and middle layer-forming cells, the middle layer-forming cells may be used together with the settling cells in step (3) or later, or the middle layer-forming cells may be removed together with the floating cells. The handling of the middle layer-forming cells can be determined appropriately depending on the appearance and state of the middle layer-forming cells, the amount of cells forming each cell group, etc. The amount of the liquid containing the selected precipitated cells is not particularly limited, but examples include 5 to 60% by volume, 5 to 50% by volume, 10 to 40% by volume, or 10 to 30% by volume of the total liquid in step (1).

[0026] The timing of selecting precipitated cells in step (2) is not particularly limited, but from the viewpoint of improving the culture efficiency, it is preferable to collect the cells at a time when the algal cell concentration is close to the maximum algal cell concentration. For example, the culture medium may be collected when the algal cell concentration reaches 150 g / L or more. By collecting the culture medium at such a time, a culture medium with a high algal cell concentration can be obtained. The culture medium may be collected when the algal cell concentration reaches 50 g / L or more.

[0027] Cultivation after recovery of the culture medium can be carried out under the same culture conditions as above. If the amount of culture liquid decreases during selection and this interferes with the culture in the subsequent step (3), a liquid equivalent to the amount of the recovered culture liquid can be added. The liquid added may be the same as that in step (1), or it may be different. When an artificial medium is added, the concentration of each component may be adjusted so that the concentration of each component after the addition of the medium is approximately the same as that at the initial stage.

[0028] The floating cells that were not selected in step (2) may be cultured again after some measures have been taken, or may be recovered and used industrially as raw materials for foods, cosmetics, pharmaceuticals, pesticides, textile treatment agents, etc.

[0029] (Step (3)) Step (3) is a step of culturing the sedimented cells selected in step (2). The culturing method in step (3) can be performed in the same manner as in step (1). Step (2) can be added after step (3), and steps (1) to (3) can be repeated multiple times. Furthermore, the cyanobacteria increased by steps (1) to (3) of the present invention can be used as seeds for the next culture cycle.

[0030] According to the culture method of the present invention, the following advantages can be obtained by removing floating cells and continuing culture with precipitated cells. 1) In cyanobacteria such as Aphanothece saccharin, floating cells tend to have lower proliferation ability than sedimentary cells. By removing floating cells with reduced proliferation ability and maintaining (passaging) the sedimentary cells, cyanobacteria can be grown more efficiently. 2) By removing the floating cells, sunlight reaching outdoor ponds and artificial light irradiating the culture vessel from the top surface are no longer obstructed, which improves the growth of autotrophic blue-green algae. [Example]

[0031] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0032] [Example 1] One liter of sodium bicarbonate-supplemented medium described in Table 1 was placed in a 10-liter glass container, and 500 g of cultured Aphanothece sacrum (FPU1 strain: Ohki, K et al. J Gen Appl Microbiol. 2019 Mar 8;65(1):39-46.) was added in wet weight. The culture was maintained at 23°C in an atmosphere with an aeration rate of 0.5 L / min and a photosynthetic photon flux of 130 μmol / m 2 The culture was conducted under a 14-hour light / 10-hour dark cycle during the light period. During the light period, when the pH rose to around pH 9, carbon dioxide gas was injected to lower the pH to around pH 8. After one week of cultivation, the aeration was stopped for 10 minutes, the container was left to stand, and 50 g of Aphanothece sacrum cell colonies that had sunk to the bottom were collected. The collected Aphanothece sacrum cell colonies were black, mulberry-like in color, and approximately 0.06 to 2 cm in diameter. The collected A. saccharinum was then added to a 1-liter glass container in a wet weight of 50 g, and the culture was resumed under the same culture conditions as above. One week after the resumption of culture, the entire A. saccharinum was collected and its wet weight was measured. The collected amount was 113 g in wet weight. The wet weight is the weight of the Amorphophallus saccharinus collected from the culture medium and measured after leaving it to stand on a sieve for 30 minutes to separate the solid and liquid.

[0033] [Table 1]

[0034] [Comparative Example 1] In Example 1, 50 g of the Aphanothece sacrum cell colonies floating in the container were collected in wet weight, and the culture was resumed using these. Except for the above, the same procedure was followed as in Example 1, and the entire amount of Aphanothece sacrum in Comparative Example 1 was finally collected. When the wet weight after collection was measured, the collected amount was 91 g in wet weight.

[0035] Comparative Example 2 In Example 1 above, 25 g (wet weight) of the A. saccharinii cell colonies floating in the container and 25 g (wet weight) of the A. saccharinii cell colonies settling in the container were collected together, and the culture was resumed using these. Except for the above, the same procedure was followed as in Example 1, and finally, the entire amount of A. saccharinii of Comparative Example 2 was collected. When the wet weight was measured after collection, the collected amount was 106 g wet weight.

[0036] Comparative Example 3 In Example 1 above, 30 g (wet weight) of the A. saccharinii cell colonies floating in the container and 20 g (wet weight) of the A. saccharinii cell colonies settling in the container were collected together, and the culture was resumed using these. Except for the above, the same procedure was followed as in Example 1, and finally, the entire amount of A. saccharinii of Comparative Example 3 was collected. When the wet weight was measured after collection, the collected amount was 95 g wet weight.

[0037] [Table 2]

[0038] From the above results, it was confirmed that selective cultivation of precipitated cyanobacteria can improve the recovery amount and growth rate of cyanobacteria, thereby making cultivation more efficient.

Claims

1. (1) a step of culturing cyanobacteria in a liquid in a culture tank; (2) removing the cyanobacterial cells floating in the liquid from the culture tank or transferring the cyanobacterial cells settling in the liquid to a new culture tank to select precipitated cells; and Step (3) of culturing the precipitated cells A method for culturing cyanobacteria, comprising:

2. The culture method according to claim 1, wherein the cyanobacterium is Aphanothece sacrum.

3. The step (1) is carried out by generating a water flow in the liquid, and The culture method according to claim 1 , wherein the liquid is allowed to stand before the sedimentation cell selection in step (2).

4. A cyanobacteria cultured by the culture method according to any one of claims 1 to 3.

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