JP6542569B2 - Mass production method of cyanobacteria - Google Patents

Description

  The present invention relates to a method for mass-producing cyanobacteria such as Suizen nori, Ishikage, Ashitsuki and the like. More specifically, the present invention relates to a mass production method of cyanobacteria which is cultured in two steps of a stationary culture step in a closed small culture vessel and an aeration culture step in a closed substantially cylindrical culture vessel.

  The cyanobacteria are prokaryote algae, a group of eubacteria known as cyanobacteria (blue bacteria), and have photosynthetic ability. Some species that secrete an agar-like substrate outside the cell, such as Neisseria solanum, A. japonicus, etc., which form a colony with a small number of cells, such as the Schizosium sp. Including.

  Among these, Suizenjinori is a freshwater cyanobacterium endemic to Japan, which is native to a part of the Kyushu region and has been used as a food source since ancient times.

  In Suizenjinori, an oval single cell with a minor diameter of 3 to 4 μm and a major diameter of 6 to 7 μm repeats bipartite in asexual reproduction, secretes an agar-like substrate extracellularly, and intersperses cells in the substrate. , Forming an irregularly shaped colony. In the natural world, when a jellyfish-like flat colony is formed in a specific flooded land or the like, and the diameter of the largest growth group of the colony grows to about 20 to 50 mm, it breaks off and separates from the water bottom and floats while floating in the water.

  Suizenjinori is basically considered to grow only in the underflowing water that is rich in natural minerals from Kyushu and Aso mountains, which is originally the natural area, and it is also considered that proper water flow is necessary for growth.

  In recent years, natural change in the natural environment, deterioration of water quality, etc., the natural suizenjinori is in danger of extinction. In addition, in the Golden River in Asakura City, Fukuoka Prefecture, the pumped groundwater is flowed to a channel 25 m long, 4 m wide and 12 to 13 cm deep, and the water is bred from the bottom sand of the channel. The production volume is also decreasing year by year.

  Therefore, there is a need for a new method of cultivating and cultivating Suizenjinori in place of wild aquaculture, and several techniques for cultivating and cultivating Suizenjinori have been proposed.

  For example, Patent Document 1 has a protection mechanism and is about 25 m long, about 4 m wide, and about 0.5 m high, and is a culture tank covered with river gravel with a thickness of about 5 cm at the bottom. In the patent document 2, breeding water of about 1 m in height is stored with a length of about 1 m several dozens cm, a diameter of about 1 m, a capacity of about 1,000 L, etc. In addition to draining and rearing breeding water during the day in a cylindrical water tank, and washing the algal cells of Suizennori every 7 to 10 days, a drainage pipe with a large number of drainage holes in the water tank in the longitudinal direction The aquaculture method of the Suizenjinori is described, which is inserted, aeration is performed from the lower outer periphery of the drainage pipe, and air bubbles are brought into contact with the drainage hole.

  Further, in Patent Literatures 3 and 4, while circulating the use water, the algal cells of Suizenjinori are suspended in the water, air is supplied to the water, and the algal bodies are constantly vigorously exercised in the water to form the algal cells. Patent Document 5 discloses a culture method of Suizenjinori, which removes small fragments from the system out of the system, in Patent Document 5 by using a culture medium containing a metal element at a predetermined weight ratio and containing sulfate ion, nitrate ion, and phosphate ion at a predetermined concentration. The pure culture method of Suizenji nori to culture is described, respectively.

  In addition, the rock moth (scientific name "Nostoc commune", the same shall apply hereinafter) is a kind of land blue algae belonging to the genus Nenejumo, which grows on bare soils all over the world and forms agar-like colonies. It is excellent in environmental resistance, can survive even in a dry state, and is known to have antiviral activity, lipid metabolism improving activity and the like, and has also been used for food since ancient times. Ashitsuki (scientific name "Nostoc verrucosum", and so on) is a kind of blue-green alga belonging to the genus Nendjumo, and it grows in a clean stream place. Besides being known to have antibacterial activity etc., it was once used for food.

In recent years, it has been reported that the substrate portion of cyanobacteria contains a unique high molecular weight polysaccharide, and the present inventors have used its cyanobacteria-derived component based on its own research. Japanese Patent Application Laid-Open Publication No. H10-324118 discloses a method for treating coagulant water and the like.
JP, 2014-30398, A JP 2011-130704 A Japanese Patent Application Laid-Open No. 2-231019 JP-A-2-231020 Japanese Patent Application Laid-Open No. 2004-81022 Japanese Patent Application No. 2011-89492

  As described above, when industrially using cyanobacteria-derived components, it is necessary to culture cyanobacteria in large quantities and stably.

  However, for example, in the case of Suizen nori, it is believed that only a specific clear flow is required, and appropriate water flow is necessary for growth, so a large amount of Suizen nori can be cultured by conventional culture methods. Underground water or circulating water is required, and the labor required for securing / operating the facilities / sites for that purpose becomes excessive.

  In addition, since diatoms and other miscellaneous bacteria are easily attached to the substrate part of Suizenjinori, it is necessary to wash Suizenjinori frequently at the time of culture, and since contamination occurs frequently even after washing, a large amount of Suizenjinori is When culturing, it is substantially difficult to stably culture without causing contamination.

  In addition, there is little knowledge of culture technology itself about other cyanobacteria such as rock jellyfish and acidae.

  Therefore, the present invention has an object to provide a culture technique of cyanobacteria which can reduce the amount of water used for culture, and can be easily and stably mass-cultured.

  The present inventors have been able to cultivate Suizenjinori in a semi-closed culture vessel by performing aeration to such an extent that the algal cells are stirred, even without provision of (1) a configuration for circulating the culture solution. (2) A two-stage culture of stationary culture and aeration culture allows even if a high concentration of algal cells are added to a medium-sized culture vessel, it can culture and grow S. vulgaris and can also increase the growth efficiency, 3) The two-step culture makes it possible to significantly reduce the amount of liquid used for culture and to significantly increase the amount of algal growth per unit culture fluid volume, and (4) fragmented algal cells The risk of contamination occurring in the culture vessel can be reduced by (5) in the aeration culture, by performing stationary culture followed by feeding to aeration culture, and culturing in a closed or semi-closed culture vessel. As well as feeding high concentrations of algal By shortening the incubation period of the culture, can be harvested algal cells in high efficiency, contamination in the culture vessel can be also reduced risk of developing, found and new.

  Therefore, in the present invention, a stationary culture step of housing and culturing small pieces of fragmented cyanobacteria in a closed small-sized culture vessel in which the culture solution is stored, and a medium-sized semi-solid in which the culture solution is stored. A plurality of closed substantially cylindrical culture vessels are juxtaposed, and each semi-closed substantially cylindrical culture vessel is charged with medium-sized colonies of cyanobacteria grown in the static culture step, and aeration is carried out while culturing each while aeration And providing a method for mass-producing cyanobacteria including a culture step.

  The cyanobacteria mass production method according to the present invention includes a two-step process of a stationary culture process and an aeration culture process.

  In the stationary culture step, which is the first step, the small-piece clusters of finely divided cyanobacteria are accommodated and cultured in a closed compact culture vessel in which the culture solution is stored.

  By storing the culture solution in a closed small-sized culture container and circulating the culture in a static culture without circulating the culture solution, the inside and the outside of the container can be substantially blocked and contact between the inside and the outside of the container can be prevented as much as possible. It can prevent new contamination inside.

  In addition, in stationary culture, by separating algal cells into small pieces and storing them in the culture vessel, it is possible to reduce the contamination and growth of diatoms and the like attached to the substrate, so the risk of contamination occurring in the culture vessel is also reduced. In addition to the above, the risk of contamination occurring in the aeration culture step where subsequently grown algal cells are introduced can be reduced.

  In addition, since the algal cell washing step can be omitted or simplified, the labor can be greatly reduced, and mass production of cyanobacteria can be achieved easily and with low effort.

  Furthermore, since the culture is divided into small culture vessels, even if contamination occurs in one culture vessel, it is possible to prevent the spread of the contamination on the whole, and minimize the damage caused by the contamination.

  Subsequently, in the second step, the aeration culture step, a plurality of medium-sized semi-closed substantially cylindrical culture vessels storing the culture solution are juxtaposed, and stationary culture is performed in each semi-closed substantially cylindrical culture container. Medium-sized colonies of cyanobacteria grown in the process are introduced and cultured while being aerated respectively.

  The semi-closed, substantially cylindrical culture vessel used for aeration culture is a medium-sized culture vessel having a substantially cylindrical shape, and can aerate the culture solution in the vessel from the outside, and the volume of the volume supplied into the vessel by aeration is It has a configuration that can discharge air. In principle, the inside and outside of the container are substantially blocked except for the inflow and outflow of air due to aeration, and since the culture solution is not circulated, contact between the inside and the outside of the container can be prevented as much as possible. Pollution can be prevented.

  In the present invention, since aeration culture is performed in a medium-sized semi-closed, substantially cylindrical culture vessel, the installation and culture space can be easily secured, and large-scale facilities such as a large water tank and a water circulation pump are not necessary. Therefore, a plurality of culture vessels can be juxtaposed, and the culture vessels can be installed on each shelf of the multistage shelf. This enables mass production of cyanobacteria.

  In addition, since aeration culture is performed in a medium-sized semi-closed substantially cylindrical culture vessel, movement, removal, washing, maintenance, and management of the vessel are easy, and operating cost and labor can also be reduced. Therefore, the cyanobacteria can be mass-produced easily, with low labor and at low cost.

  In addition, even in the aeration culture step, culture can be divided into a plurality of culture vessels, so that even if contamination occurs in one culture vessel, it is possible to prevent the spread of the whole contamination, and the damage caused by the contamination is minimized. Can stop it.

  In this aeration culture step, medium-sized colonies of cyanobacteria grown in the static culture step are introduced. As a result, high concentration algal cells can be introduced, and cyanobacteria can be grown efficiently in a short period of time.

  In the aeration culture step, the culture is carried out with aeration in a medium-sized semi-closed culture vessel. Thus, the aeration can be performed with low energy and efficiently, and the algal cells and the culture solution can be effectively stirred to suppress the occurrence of unevenness and promote the growth of algal cells.

  In addition, by culturing while performing aeration in a medium-sized semi-closed culture vessel in aeration culture, algal bodies having a substantially spherical shape can be formed instead of the conventional flat shape. In order to be able to harvest such substantially spherical algal cells, (1) stirring efficiency in the culture vessel can be enhanced, and generation of stagnation and unevenness of the algal bodies and the culture solution can be suppressed (2) agitation in the culture vessel Since the efficiency can be increased, the growth efficiency of the algal cells does not decrease even if more algal cells are charged, that is, a large amount of algal cells can be introduced into the container, whereby per unit culture fluid volume of S. vulgaris (3) In addition to the above-mentioned ability to feed a large amount of algal cells, the volume per unit weight is also smaller compared to flat algal cells, so the production efficiency of Suizenji nori can be increased. (4) can be simplified culture procedures such as (4) Suizenjinori etc. (5) can facilitate recovery and processing of crops (6) compared to conventional flat algal cells The efficiency of capturing light is high. There is.

  As described above, in the present invention, algal bodies can be grown more efficiently by adopting the two-step culture of the stationary culture step and the aeration culture step. And, since a small culture vessel is used in the static culture process and a medium culture vessel is used in the aeration culture process, a plurality of culture vessels can be juxtaposed, and mass production of cyanobacteria can be achieved.

  Further, in the present invention, since a two-step culture is employed and culture is performed using a culture solution stored in a culture vessel, the amount of liquid used for culture can be significantly reduced, and algae per unit culture solution is used. Body gain can be significantly increased. This makes it possible to mass-produce cyanobacteria in a simple, low labor and low cost manner.

  In addition, in the present invention, (1) culture is performed in a closed or semi-closed culture vessel, (2) in the stationary culture step, algal cells are fragmented and then stored in the culture vessel, and (3) By introducing a high concentration of algal cells in the aeration culture step to shorten the culture period, it is possible to minimize the occurrence of contamination even in the case of mass culture. Therefore, since stable culture is possible according to the present invention, mass production of cyanobacteria is possible.

  The present invention enables simple and stable mass production of cyanobacteria with a small amount of culture solution.

<About the mass production method of cyanobacteria concerning the present invention>
In the present invention, a stationary culture step of housing and culturing small pieces of fragmented cyanobacteria in a closed small culture vessel in which the culture solution is stored, and a medium-sized semi-closed type in which the culture solution is stored Aeration culture process of arranging a plurality of substantially cylindrical culture vessels side by side, and introducing medium-sized assemblages of cyanobacteria grown in the stationary culture step into the respective semi-closed substantially cylindrical culture vessels, and culturing while respectively aeration And all of the cyanobacteria mass production methods, including and.

  Hereinafter, an example of the embodiment of the present invention will be described using FIG. The present invention is not narrowly limited to only the embodiment illustrated in FIG.

  FIG. 1 is a schematic view showing an example of the method for mass-producing cyanobacteria according to the present invention. In FIG. 1, the closed small-sized culture container 1 used in the stationary culture step A and the semi-closed substantially cylindrical culture container 2 used in the aeration culture step B are respectively attached to each shelf R1, R2, R3 of the multistage shelf R. Each is juxtaposed.

  The stationary culture step A, which is the first step, is a step of housing and culturing the fragmented small algae aggregate S1 in the closed compact culture vessel 1 in which the culture solution W is stored.

  The closed small-sized culture vessel 1 is basically a small-sized culture vessel in which the inside and the outside of the vessel are substantially blocked, and the culture fluid W is stored in the vessel, and the algal cells S1 are accommodated therein.

  A wide range of known closed culture vessels can be used. Although the size of the closed compact culture vessel 1 is not particularly limited, it is preferable that the size of the culture solution W is in the range of 10 to 80% of the volume. Since it is necessary to irradiate the algal cells S1 with light during culture, the closed compact culture vessel 1 is preferably formed of a transparent or translucent material. Moreover, in order to prevent the occurrence of contamination, the small-sized culture vessel 1 may have a structure that can be substantially sealed.

  The culture solution W is stored in the closed compact culture vessel 1. Although the liquid volume of the culture solution stored in the closed compact culture container 1 is not particularly limited, about 0.1 to 1.0 L, more preferably about 0.2 to 0.8 L is preferable. The composition of the culture solution W is not particularly limited, and known culture solutions for cyanobacterial culture can be widely used.

  In the stationary culture step A, the fragmented small algae clusters S1 of the cyanobacteria are stored and cultured in the closed compact culture vessel 1 in which the culture solution W is stored. Although the size of the algal cells to be subjected to fragmentation is not particularly limited, for example, algal cells having a maximum diameter of 1 to 10 mm may be used. Fragmentation of algal cells can be performed by a known method such as, for example, a blade, a mixer, and a homogenizer. Before being fragmented, the algal cells may be washed by a known method. Also, after being fragmented, it may be filtered through a mesh or the like and then subjected to stationary culture.

  The size of the algal cells S1 (pieces of fragmented cyanobacteria) contained in the closed small-sized culture vessel 1 is not particularly limited, but the maximum diameter is preferably 1 to 800 μm, more preferably 1 to 600 μm. It is preferable to be fragmented.

  The culture conditions for stationary culture may be any known conditions without particular limitation. For example, the light condition may be a light condition that repeats the light condition and the dark condition on a daily cycle, the light condition may be 6 to 12 hours, and the dark condition may be 18 to 12 hours. Under bright conditions, for example, light with an intensity of 5,000 to 20,000 lux, more preferably 8,000 to 15,000 lux, may be emitted from outside the container. The water temperature (the temperature of the culture solution W) may be adjusted, for example, in the range of 15 to 30 ° C., and the pH of the culture solution W may be adjusted in the range of 6 to 9, for example. Although the exchange of the whole culture solution does not need to be performed in principle in particular, depending on the condition of the algal cells, one or more additional culture of concentrated culture nutrient salts may be performed. In addition, since the growth rate of the algal cells S1 increases as the water temperature and / or the illuminance (light intensity) is increased, the growth rate of the algal cells S1 may be controlled by adjusting the water temperature and / or the illuminance (light intensity). it can.

  The culture period may be appropriately set according to the growth degree of the algal cells S1. For example, by carrying out stationary culture of the shredded algal cells S1 for 10 to 40 days, the algal bodies grow to medium size colonies, and the maximum diameter of the well grown colonies is about 3 to 5 mm ( Within the range of 1 to 10 mm).

  Subsequently, in the aeration culture step B, which is the second step, the medium-sized assemblage S2 of cyanobacteria grown in the stationary culture step A is charged into the semi-closed substantially cylindrical culture vessel 2 in which the culture solution W is stored. And culturing with aeration means 3 while aerating.

  In principle, the semi-closed substantially cylindrical culture vessel 2 is capable of performing aeration on the culture solution W in the vessel 2 from the outside, and having a configuration capable of discharging the amount of air supplied into the vessel by aeration. The other is a semi-closed culture vessel in which the inside and the outside of the vessel are substantially shut off, and the culture solution W is stored in the vessel, and the algal cells S2 are accommodated therein.

  Well-known things can be widely employ | adopted as the semi-closed substantially cylindrical culture container 2. FIG. The shape of the culture vessel 2 is preferably a substantially cylindrical shape such as a cylinder or a square tube in order to effectively stir the algal cells S2 and the culture solution W by aeration to suppress the occurrence of unevenness. The size of the semi-closed substantially cylindrical culture vessel 2 is not particularly limited, but it is preferable that the size of the culture fluid W is in the range of 10 to 80% of the volume. Since it is necessary to irradiate the algal cells S2 with light during culture, the semi-closed substantially cylindrical culture vessel 2 is preferably made of a transparent or translucent material. Moreover, in order to prevent the occurrence of contamination, the semi-closed substantially cylindrical culture vessel 2 may have a structure in which the portion other than the inflow and outflow portions of air due to aeration can be substantially sealed.

  The culture solution W is stored in the semi-closed substantially cylindrical culture container 2. The liquid volume of the culture solution stored in the semi-closed substantially cylindrical culture container 2 is not particularly limited, but in the aeration culture step B, the culture volume stored in the substantially cylindrical culture container 2 is 1 to 50 L each. In the aeration culture step B, the amount of culture fluid stored in the substantially cylindrical culture container 2 is more preferably 1 to 30 L each. In the aeration culture step B, the substantially cylindrical culture container 2 is preferably used. It is most preferable that the amount of culture solution to be stored in each is 1 to 20 L each. The composition of the culture solution W is not particularly limited, and known culture solutions for cyanobacterial culture can be widely used.

  In the aeration culture step B, the medium-sized colonies S2 of cyanobacteria grown in the stationary culture step A are charged into each semi-closed substantially cylindrical culture vessel 2 storing the culture solution W and cultured. The size of the algal cells to be introduced is not particularly limited. For example, it is preferable that the maximum diameter of the medium-sized assemblage S2 of cyanobacteria grown in the stationary culture step A is 1 to 10 mm, and 1 to 8 mm. One is more preferred, and 1 to 6 mm is most preferred. Also, the amount of algal cells to be added is not particularly limited, but for example, it is preferable that the amount of medium-sized clusters S2 input to the substantially cylindrical culture container 2 is 5 to 40 g in wet weight per 1 L of culture solution. And each 10 to 40 g is more preferable, and each 20 to 40 g is most preferable.

  In the aeration culture step B, the culture solution W in the semi-closed substantially cylindrical culture container 2 is aerated using the aeration means 3 during the culture period. The aeration means 3 is not particularly limited as long as it can supply air such as a compressor. For example, air may be guided into the culture vessel 2 by a hollow tube 31 or the like, and air may be supplied from the tip 32 into the culture solution W. In addition, as above-mentioned, the semi-closed substantially cylindrical culture container 2 is set as the structure which can discharge | emit the quantity of air supplied in the container by aeration. Here, since the inside of the semi-closed substantially cylindrical culture vessel 2 is always in a positive pressure atmosphere by continuously performing aeration, if the inside and the outside of the vessel are substantially shut off otherwise, in principle Thus, new contamination of the culture vessel 2 can be prevented.

  The aeration amount is not particularly limited as long as it is configured to supply air to such an extent that the algal cells S2 and the culture solution W are stirred, but for example, in the aeration culture step B, the aeration amount is 0.05 to 0.05 per liter of culture solution. If it is 0.25 L / min, more preferably about 0.10 to 0.20 L / min per 1 L of culture solution, the algal cells S2 and the culture solution W can be effectively stirred.

  The culture conditions of the aeration culture step B may be known conditions, and may be generally the same as the culture conditions in the static culture step A.

  The culture period may be appropriately set according to the growth degree of the algal cells S2. For example, by aeration culture of the algal cells S2 for 5 to 30 days, the maximum diameter of the well-grown colonies of the algal bodies becomes about 5 to 20 mm.

  In addition, as exemplified in FIG. 1, for example, a plurality of semi-closed substantially cylindrical culture vessels 2 are juxtaposed, and the cyanobacteria are cultured by culturing the algal cells S2 in each semi-closed substantially cylindrical culture vessel 2. It can be mass produced.

<About the Suizenji nori group according to the present invention>
The present invention includes all of the approximately spherical shape Suizenjinori clusters with a maximum diameter of 5 to 20 mm.

  The arid culture process is appropriately agitated during the culture period by the aeration in the above-mentioned aeration culture step, and the S. vulgaris group produced by the above-mentioned method for mass production of cyanobacteria is not a usual substantially flat shape but a substantially spherical shape. It has the form of

  As described above, in such a substantially spherical shape Suizenjinori cluster, as compared with the usual substantially flat shape, (1) the stirring efficiency in the culture vessel is enhanced, and retention and unevenness of algal cells and culture solution (2) Since the stirring efficiency in the culture vessel can be increased, the growth efficiency of the algal cells does not decrease even if more algal cells are introduced, that is, a large amount of algal bodies in the vessel In addition to being able to input a large amount of algal cells as described above, it is possible to increase the amount of production per unit culture fluid volume of Suizenjinori, as compared with flat algal cells, which makes it possible to Because the volume per unit weight is also small, the production efficiency of Suizenji nori can be enhanced, (4) culture procedures such as Suizenji nori can be simplified, (5) harvest and recovery can be facilitated, (6) conventional Culture compared to flat algal bodies Quantity incorporation efficiency in high, there is advantage of such of.

  In Example 1, culture of S. vulgaris was attempted in a semi-closed culture vessel, ie, a vessel not provided with a configuration for circulating the culture solution.

  2g / L (200g by wet weight) of washed algal cells are put into a semi-closed culture vessel storing 100 L of culture solution and cultured for 20 days while stirring algal bodies by aeration, then algal cells in the vessel Were harvested and their wet weight was measured. The semi-closed culture vessel is transparent, the culture solution is stored in the vessel, the culture solution in the vessel can be aerated from the outside, and the amount of air supplied into the vessel by aeration is It had the composition which can be discharged, and the thing in which the inside and outside of a container were substantially intercepted was used except it.

  The culture was conducted under a light condition of 10 hours of light conditions and a cycle of 14 hours of dark conditions. Under light conditions, light with an illuminance of 10,000 lux was applied from outside the container. The water temperature was adjusted to 24 ° C. The culture solution used had a known composition. Aeration was performed using a compressor during the culture period. As a result of appropriately adjusting the intensity of aeration to a level at which algal cells are stirred in the container, the air supply amount at the time of aeration was about 0.14 L / min with respect to 1 L of culture solution.

Table 1 shows the "daily gain rate" and "increment / liquid volume" when culturing is performed while aeration in a semi-closed culture vessel in which 100 L of culture solution is stored. Here, the “daily gain rate” is calculated by dividing the increment (amount obtained by subtracting the input amount (wet weight of algal cells input at the start of culture) from the harvested amount (wet weight of algal cells after culture) by the input amount. Then, it is a value obtained by dividing by the number of culture days (20 days) after calculating the multiplication rate by multiplying by 100. In Table 1, "incremental / fluid volume" represents the amount of algal growth per unit culture fluid volume, and is the value obtained by dividing the increment of wet algal biomass from the start of culture to the time after culture by the volume of culture fluid used. It is.

  As shown in Table 1, even if the medium was not provided with a configuration for circulating the culture solution, it was possible to culture a large amount of Suizenjinori in a semi-closed culture vessel by performing aeration to such an extent that the algal cells are stirred. In addition, by storing the culture solution in a container and culturing Suizenjinori, it is used for culture as compared with the conventional method (a culture method in which groundwater is flowed in a water channel or a culture method in which a culture solution is circulated). The liquid volume could be greatly reduced, and as shown in the item of "increment / liquid volume" in Table 1, the algal growth per unit culture volume could be increased. In addition, when culture | cultivation days approached 20 days, contamination generate | occur | produced in one part.

  In Example 2, culture of S. vulgaris was attempted in two stages: stationary culture in a closed small culture vessel and aeration culture in a semi-closed almost cylindrical culture vessel.

  First, static culture was performed. 100 closed small culture vessels of 1 L size and substantially clear and flat bottom were prepared, and 0.4 L of culture solution was put in each. The algal cells of 1 to 10 mm in size are cut into pieces of several to several hundreds of μm by a mixer, and 0.5 g / L (0.2 g wet weight) of the algal cells in each closed small culture vessel is stored for 30 days. After leaving still as it was, algal bodies were collected for each container, and the wet weight was measured.

  The light conditions for stationary culture were a cycle of light conditions for 10 hours and a dark condition for 14 hours. Under light conditions, light with an illuminance of 10,000 lux was irradiated from outside the container. During the culture, the temperature of the water was adjusted to 15 to 30 ° C., and the pH was adjusted to 6 to 9. The culture solution used had a known composition. The total amount of culture solution was not exchanged, and depending on the state of algal cells, fertilization of concentrated culture nutrient salts was performed once or several times.

  As a result, a total of 200 g of algal cells could be harvested per container in total of 200 g. The increment of wet algal body weight from the start of culture to the time after culture was 1.8 g per container on average, the weight gain rate was 900%, and the daily weight gain rate was 30.0% / day.

  Subsequently, aeration culture was performed. Two 6 L sized semi-closed, substantially cylindrical culture vessels were prepared, and 4 L of culture solution was placed in each. In the semi-closed substantially cylindrical culture vessel, the wall surface is transparent, the culture solution is stored in the vessel, and the culture solution in the vessel can be aerated from the outside, and is supplied into the vessel by aeration. The container had a configuration capable of discharging a large amount of air, and the others were used in which the inside and the outside of the container were substantially shut off.

  25g / L (100g by wet weight) of the algal cells of Suizenjinori grown to a size of 3 to 5 mm by static culture are charged into each semi-closed, substantially cylindrical culture vessel, and aeration is performed with a compressor during the culture period. After culturing the algal cells for 10 days while stirring, the algal cells in the container were harvested and their wet weights were measured.

  The conditions for culture were the same as for stationary culture. Aeration was performed while adjusting the air supply to be about 0.14 L / hour to 1 L of culture solution.

  Thus, in the present example, first, the shredded algal cells are grown in static culture, and then in a semi-closed, substantially cylindrical culture vessel, among the Suizen nori grown in the static culture. A large colony was introduced, and aeration culture was performed for 10 days on a 4 L scale.

Table 2 shows the "daily gain rate" and "incremental / liquid volume" when the culture is performed in two steps, a static culture process in a closed small culture vessel and an aeration culture process in a closed substantially cylindrical culture vessel. Indicates Each item in Table 2 is the same as Table 1 described above. In Table 2, brackets in static culture were the total amount when cultured in 100 closed small culture vessels, and brackets in aeration culture were cultured in two semi-closed almost cylindrical culture vessels The total amount of each case is shown.

  In Example 1 described above, as shown in Table 1, 200 g of algal cells were added to and cultured in a 100 L-scale culture solution, and the algal body concentration at the time of input was 2 g / L. On the other hand, in the aeration culture in this example, as shown in Table 2, 100 g of algal cells grown by static culture are added to a 4 L culture medium and cultured, and the algal body concentration at the time of input is It was 25 g / L. From this example, it was found that by adopting the above-described two-step culture, even when high-concentration algal cells are introduced into a medium-sized culture vessel, it is possible to culture and propagate Suizenjinori.

  Comparing Table 1 of Example 1 with Table 2 of this example, the aeration culture of this example has 9.4% of weight gain in comparison with the daily weight gain of 4.00% / day in Example 1. It was high with day. This indicates that the algal cells can be grown more efficiently by adopting the two-step culture in this example.

  Comparing Table 1 of Example 1 with Table 2 of this example, the “incremental / water amount” in Example 1 was 1.66 g / L, whereas it was 7.66 g / L in this example. . This indicates that by adopting the two-step culture in the present example, the amount of liquid used for culture can be significantly reduced, and the amount of algal growth per unit culture fluid volume can be significantly increased.

  In Example 1 described above, as a result of feeding the washed algal cells to a 100 L-scale culture solution, contamination occurred in part when the number of days of culture approached 20 days. On the other hand, in the present example, the minced algal cells were grown in static culture and then placed in aeration culture, and as a result, no contamination occurred in aeration culture for 10 days. This means that, in the present example, (1) employing finely divided algal cells in stationary culture, (2) employing a medium-sized semi-closed culture vessel for aeration culture, and ( 3) It is shown that the risk of occurrence of contamination in the culture vessel can be reduced by shortening the culture period by feeding high concentration algal cells in aeration culture.

  In addition, in the present embodiment, by adopting a medium-sized semi-closed culture vessel in aeration culture, aeration can be performed with low energy and efficiently, and the algae and culture solution are effectively agitated to make unevenness. Was able to suppress the occurrence of

  In the present embodiment, as shown in FIG. 2, by culturing while performing aeration in a medium-sized semi-closed culture vessel in aeration culture, a substantially spherical algal body is formed instead of the conventional flat shape. It was possible.

  In Example 3, a growth curve of Suizenjinori in the aeration culture step of the two-step culture according to the present invention was determined.

  In the same manner as in Example 2, 25 g / L (100 g by wet weight) of the algal cells of Suizenjinori grown to a size of 3 to 5 mm by static culture in a semi-closed substantially cylindrical culture vessel in which 4 L of culture fluid was stored A) During the culture period, aeration is performed with a compressor to culture the algal cells for 10 to 30 days while stirring, and the algal cells in the container are harvested 10, 20, and 30 days after the start of aeration culture. The wet weight was measured and a growth curve was determined.

  The results are shown in Figure 3. FIG. 3 is a graph showing the change in algal wet weight with respect to the number of days of culture in the aeration culture of the two-step culture according to the present invention. The horizontal axis in FIG. 3 represents the number of days of culture from the start of aeration culture, and the vertical axis represents the wet weight of algal cells.

  As shown in FIG. 3, in the aeration culture of the two-step culture in the present invention, when 25 g / L of algal cells are added to the 4 L-scale culture medium, the increase in algal wet weight is remarkable 10 days after the aeration culture starts. There was a high proliferation rate. On the other hand, almost no increase in algal wet weight was observed 20 and 30 days after the initiation of aeration culture. Further, as a result of visual observation of the culture state, at 20 and 30 days after the start of the aeration culture, although the change in algal mass was hardly observed, deterioration of the algal bodies was partially observed.

  From the present example, in the aeration culture of the two-step culture according to the present invention, a high concentration of algal cells (for example, 10 to 40 g / L) are added, and the culture period of the aeration culture is shortened to achieve high efficiency. It has been found that algal cells can be harvested and high quality algal cells can be recovered.

The schematic diagram which shows the example of the cyanobacteria mass production method which concerns on this invention. In Example 2, the photograph which shows the substantially spherical algal body obtained by aeration culture in a medium size semi-closed culture container. In Example 3, the graph showing the change of the algal wet weight with respect to each culture day in the aeration culture of the two-step culture according to the present invention.

1 Closed small culture vessel
2 Semi-closed almost cylindrical culture vessel
3 Aeration means
A stationary culture process
B aeration culture process
R multistage shelf
S1, S2 Alga
W culture solution

Claims (4)

A static culture step of housing and culturing small pieces of fragmented cyanobacteria in a closed compact culture vessel in which a culture solution is stored;
A plurality of medium-sized semi-closed substantially cylindrical culture vessels in which the culture solution is stored are juxtaposed, and medium-sized colonies of cyanobacteria grown in the static culture step are charged into the respective semi-closed substantially cylindrical culture vessels. and, only contains a ventilation culture step of culturing with aeration, respectively, the,
The cyanobacteria are at least one member selected from the group consisting of a sea bream, a rock snail and an aphid,
The cyanobacteria mass production method , wherein the aeration culture step is carried out by substantially blocking the inside and the outside of the semi-closed substantially cylindrical culture vessel except for the inflow and outflow of air due to aeration .   The method for mass producing cyanobacteria according to claim 1, wherein the amount of culture fluid stored in the substantially cylindrical culture vessel in the aeration culture step is 1 to 50 L each.   The maximum diameter of the medium-sized colony of cyanobacteria grown in the stationary culture step is 1 to 10 mm, and the input amount of the medium-sized colony to the substantially cylindrical culture container is each in wet weight per liter of culture solution The method for mass-producing cyanobacteria according to claim 1 or 2, which is 5 to 40 g.   The method according to any one of claims 1 to 3, wherein in the aeration culture step, the aeration amount is 0.05 to 0.25 L / min per liter of the culture solution.