JP7539013B2 - Method for cultivating blue-green algae - Google Patents

Description

The present invention relates to a method for rapid, continuous, large-scale cultivation of cyanobacteria, and in particular to a method for rapid, continuous, large-scale cultivation of freshwater cyanobacteria, such as Aphanothece sacrum, Star Jellyfish, and Asatsuki, which form colonies in an agar matrix secreted by cells.

In recent years, there have been many attempts to search for active ingredients in various plants, animals, algae, fungi, etc. As a result, various useful substances have been discovered and are being used in a wide range of fields, including medicines, foods, and cosmetics. Recently, a polymeric polysaccharide with a molecular weight of over 10 million has been discovered in the cyanobacterium Aphanothece sacrum, and it is expected that its high moisturizing power will be useful for beauty effects and burn treatment, etc.

However, freshwater cyanobacteria such as A. saccharum 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. As a result, despite the high expected effectiveness, there has been an issue in that applications have not been fully developed. In response, attempts have been made to mass-produce cyanobacteria by combining a static culture process and an aerobic culture process (see Patent Document 1), but the process is complicated and efficient cultivation has not been possible.

Patent No. 6542569

The objective of the present invention is to enable highly efficient cultivation of cyanobacteria.

[1] A method for culturing cyanobacteria, comprising culturing the cyanobacteria in a liquid medium containing one or more salts selected from carbonates, bicarbonates, phosphates, and citrates, while maintaining the liquid medium at a pH of 10 or less.
[2] The culture method according to [1], characterized in that the cyanobacteria are freshwater cyanobacteria.
[3] The culture method according to either [1] or [2], characterized in that the pH during culture is maintained in the range of 7.5 to 10.
[4] The culture method according to any one of [1] to [3], wherein the liquid medium contains a carbonate or a bicarbonate.
[5] The culture method according to any one of [1] to [4], wherein the pH is adjusted by adding carbon dioxide.
[6] A cyanobacteria cultured by the culture method according to any one of [1] to [5] above.

Cyanobacteria such as Amorphophallus saccharum can only grow in water that is highly transparent and rich in minerals, so they prefer a nutrient-poor medium. If they are cultured in a nutrient-rich medium, as is usually the case when cultivating algae, they will not grow well and will lose out in the growth competition with the other algae that are mixed in.

The inventors confirmed that freshwater cyanobacteria such as Aphanothece saccharum efficiently utilize specific ions such as bicarbonate ions to carry out photosynthesis, and discovered that by adding a source of specific ions such as bicarbonate ions to the culture medium in advance and culturing the medium while maintaining a pH range in which these ions can be present in the medium, cyanobacteria can be cultured rapidly and continuously in large quantities using a reduced-salt concentration medium that can suppress the growth of other algae.

The present invention makes it possible to cultivate cyanobacteria with high efficiency.

The cyanobacteria cultivated by the cultivation method of the present invention are algae classified as prokaryotes, a group of photosynthetic eubacteria called cyanobacteria. Examples of the cyanobacteria include Aphanothece sacrum (scientific name: Aphanothece sacrum) of the Chroococcales order, and Nostoc commune and A. arbutifolia of the Nostoc order.

The cyanobacteria used in the present invention are not particularly limited as long as the effects of the present invention can be obtained, but it is preferable to use freshwater cyanobacteria that live in colonies in freshwater areas, such as Amorphophallus spp., Nostoc commune, and Aster gracilis, as these increase the cultivation efficiency, with Amorphophallus spp. and Aster gracilis being more preferable, and Amorphophallus spp. being the most preferable.

The aforementioned A. saccharum is a freshwater cyanobacteria that grows naturally in a specific area of Kyushu and is made up of multiple cells that form flat colonies. The outer surface of the colony is covered with a gel-like secretion made of polysaccharides and other substances, and the colony can grow to a diameter of about 50 mm.

The method for cultivating cyanobacteria according to the present invention is characterized in that the culture is carried out in a liquid medium containing one or more salts selected from carbonates, bicarbonates, phosphates, and citrates, and maintained at a pH of 10 or less.

The liquid medium used in the culture method of the present invention may be any liquid medium containing one or more salts selected from carbonates, bicarbonates, phosphates, and citrates, and may contain water, other minerals, etc., within the range in which the effects of the present invention can be obtained. Known media such as those described in patent literature (Japanese Patent No. 6590144) and non-patent literature (J. Gen Appl Microbiol., 65, 39-46, Mar. 2019) may be used. In the culture method of the present invention, the liquid medium may be appropriately replaced or components may be replenished.

The liquid medium of the present invention is characterized by containing one or more salts selected from carbonates, bicarbonates, phosphates, and citrates, as described above. These salts can improve the cultivation efficiency of cyanobacteria, and since they are particularly suitable as carbon sources in the photosynthesis of 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 and calcium bicarbonate.

The amount of one or more salts selected from carbonates, bicarbonates, phosphates, and citrates added in the liquid culture medium is preferably 1 ppm or more and 500 ppm or less, and in order to maintain better growth of cyanobacteria, it is particularly preferable that the amount be 20 ppm or more and 200 ppm or less.

The bicarbonate is consumed as a carbon source when the algae photosynthesize, but by adding carbon dioxide to the liquid culture medium by operations such as bubbling, it is regenerated as bicarbonate ions and can be used again as a carbon source for the algae, allowing for efficient cultivation.

The liquid medium used in the culture method of the present invention is preferably adjusted to maintain a pH of 10 or less during culture, more preferably in the range of 7.5 to 10, and most preferably in the range of 8 to 9, which significantly improves culture efficiency. The pH may be adjusted throughout the culture period, but it is preferable to adjust the pH during light irradiation, as this improves culture efficiency.

The method for adjusting the pH of the liquid medium is not particularly limited as long as the effects of the present invention can be obtained, but it is sufficient to observe the increase in pH accompanying the cultivation of cyanobacteria, and when the pH rises to around 10, add carbon dioxide to the liquid medium and repeat the process of lowering the pH. The addition of carbon dioxide is particularly preferable because it replenishes bicarbonate ions consumed by the proliferation of cyanobacteria and improves the photosynthetic efficiency of the cyanobacteria. The present invention may also involve an operation to maintain a constant pH so that the pH does not rise. The 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 the aeration process described below.

In the culture method of the present invention, the liquid medium may be filled into various containers for culture. These containers are not particularly limited, and either open containers or closed containers may be used, and containers having an aeration device for aeration treatment, stirring blades for stirring the culture liquid during culture, various light sources for irradiating light, a temperature adjustment device for adjusting the temperature during culture, etc. may be used. As mentioned above, these containers are not particularly limited, but it is preferable to use containers with high light transmittance such as glass, acrylic, polycarbonate, etc., because algae can efficiently carry out photosynthesis.

When culturing cyanobacteria using the culture method of the present invention, it is preferable to perform an aeration process by bubbling or the like during culture in order to efficiently photosynthesize the algae. The aeration process may include a process of aerating with air and a process of adding carbon dioxide and aerating, and the increase in pH can be confirmed by monitoring or the like and set appropriately. The amount of air added by the aeration process is preferably 0.3 L/min/Volume or more in terms of unit volumetric flow rate (VVM, L/min/Volume), and 0.5 L/min/Volume or more is preferable because the agitation of the cyanobacteria in the culture device is performed appropriately and the culture efficiency is improved. It is also preferable to adjust the amount of air added depending on the container size to efficiently agitate the algae.

The culture method according to the present invention provides a light period during which light from various light sources is irradiated onto the culture apparatus in order to improve the culture efficiency of cyanobacteria. Known light sources such as sunlight, incandescent bulbs, fluorescent lamps, arc lamps, and light-emitting diodes can be used as the light source for the irradiated light. These lights may be irradiated continuously or intermittently. The amount of light irradiated is not particularly limited as long as the effects of the present invention can be obtained, and may be appropriately adjusted after checking the progress of the culture. However, the photosynthetically active light quantum flux density is preferably 30 μmol/m ² /s or more, and more preferably 100 μmol/m ² /s or more in order to achieve a good culture rate.

In the culture method of the present invention, it is preferable to provide a dark period in which no light is irradiated, but culture may also be carried out in a manner in which light is irradiated constantly.

In addition, the temperature of the culture medium during cultivation can be set appropriately depending on the cyanobacteria to be cultivated, but when culturing Aphanothece sacrum, it is preferable to cultivate between 15 and 30°C, more preferably between 20 and 25°C, and most preferably between 23 and 25°C.

The cultivation method of the present invention is not particularly limited in terms of the cultivation time, which can be set by a person skilled in the art after confirming the amount of increase in cyanobacteria as appropriate.

The cyanobacteria used in the culture method of the present invention may be subjected to an acclimation treatment before the main culture treatment. These acclimation treatments are not particularly limited as long as they are treatments that allow the cyanobacteria obtained as wild strains to be cultured in a preliminary environment before the main culture so that the culture can be performed favorably during the main culture, and can be performed using known methods. In addition, the wild species may be directly subjected to culture. In addition, the cyanobacteria that have increased through culture may be cultured as seeds for culture in the next culture cycle.

[Example 1]
One liter of sodium bicarbonate-added medium listed in Table 1 was placed in a 1-liter glass container, and 20 g of cultured A. saccharifolia (FPU1 strain: Ohki, K et al. J Gen Appl Microbiol. 2019 Mar 8;65(1):39-46.) was added in wet weight. Culture was performed at 23°C, with an aeration rate of 0.5/min, photosynthetically active light quantum flux of 130 μmol/ ^m2 /s, and a 14-hour light/10-hour dark cycle. During the light period, when the pH rose to around pH 9, carbon dioxide gas was blown in to lower the pH to around 8. The medium was replaced one week after the start of culture, and after two weeks of culture, A. saccharifolia was collected and its wet weight was measured, and 214 g of A. saccharifolia was collected in wet weight.

[Comparative Example 1]
1 L of the sodium bicarbonate-added medium shown in Table 1 was placed in a 1 L glass container, and 20 g of cultured A. sacchariflorus (FPU1 strain) was added in wet weight. Cultivation was started in an atmosphere of 23°C, with an aeration rate of 0.5 L/min, a photosynthetically active light quantum flux of 130 μmol/ ^m2 /s, and a 14-hour light/10-hour dark cycle. Cultivation was carried out without pH control during the light period. The medium was replaced one week after the start of cultivation, and after two weeks of cultivation, A. sacchariflorus was collected and its wet weight was measured, and 114 g of A. sacchariflorus was collected in wet weight.

[Comparative Example 2]
A 1L glass container was filled with 1L of medium having the same composition as that described in Table 1 except that sodium bicarbonate was not added, and 20 g of cultured A. saccharifolia (FPU1 strain) was added in wet weight. Cultivation was started in an atmosphere of 23°C, with an aeration rate of 0.5 L/min, a photosynthetically active light quantum flux of 130 μmol/ ^m2 /s, and a 14-hour light/10-hour dark cycle. The medium composition was the same as that in Example 1 except that sodium was omitted. Cultivation was carried out without pH control during the light period. The medium was replaced one week after the start of cultivation, and after two weeks of cultivation, A. saccharifolia was collected and its wet weight was measured, and 82 g of A. saccharifolia was collected in wet weight.

[Comparative Example 3]
A 1L glass container was filled with 1L of medium with the same composition as that described in Table 1 except that sodium bicarbonate was not added, and 20g of cultured A. saccharum (FPU1 strain) was added in wet weight. Cultivation was started in an atmosphere of 23°C, with an aeration rate of 0.5L/min, a photosynthetically active light quantum flux of 130μmol/ ^m2 /s, and a 14-hour light/10-hour dark cycle. The pH control during the light period was not performed. The medium was replaced 1 week after the start of cultivation, and after 2 weeks of cultivation, A. saccharum was collected and its wet weight was measured, and 92g of A. saccharum was collected in wet weight.

[Comparative Example 4]
One liter of sodium bicarbonate-added medium shown in Table 1 was placed in a 2-liter glass container, and 20 g of cultured Aphanothece sacrum (FPU1 strain) was added in wet weight. Static culture was started in an atmosphere of 23°C, with a photosynthetically active light quantum flux of 130 μmol/ ^m2 /s and a 14-hour light/10-hour dark cycle (without aeration). One week after the start of culture, the color of Aphanothece sacrum was visually confirmed to have faded, and it was determined that the algae had died, so the experiment was discontinued.

As shown in Table 2 above, the examples in which sodium bicarbonate was added and pH control was performed showed a significant improvement in culture efficiency compared to the comparative examples in which these treatments were not performed.

Claims (4)

A method for culturing Aphanothece sacrum, comprising culturing a liquid medium containing bicarbonate at a pH level of 7.5 to 10 by adding carbon dioxide ,
The culture method is characterized in that the amount of one or more salts selected from carbonates, bicarbonates, phosphates and citrates added to the liquid medium is 20 to 200 ppm . 2. The method according to claim 1, wherein the culture is carried out while maintaining the pH in the range of 8 to 9. 3. The culture method according to claim 1, wherein the carbon dioxide is added during the light irradiation. A step of culturing Aphanothece sacrum by the culture method according to any one of claims 1 to 3;
and recovering the increased amount of A. cerevisiae after cultivation.