JP2015171327A - Method and device for culturing cyanobacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily and efficiently culturing a large amount of cyanobacteria, and to provide a device therefor.SOLUTION: There is provide a method for culturing cyanobacteria, in which the cyanobacteria are statically cultured in a liquid culture medium whose depth is 3 cm or less, whose liquid surface area is 400 cmor more, and whose liquid surface contacts with air. There is also provided a culturing device which comprises: a placing shelf comprising a plurality of placing parts arranged at different positions in the vertical direction; a plurality of containers placed on the placing parts; and light dimming sheets which cover the plurality of containers. Each container is shaped so as to house the liquid culture medium whose depth is 3 cm or less, whose liquid surface area is 400 cmor more, and whose liquid surface contacts with air.

Description

  The present invention relates to a method for easily and efficiently cultivating cyanobacteria and an apparatus therefor.

  Extracellularly-released polysaccharides produced by cyanobacteria are functional biopolymers having high water retention, excellent metal adsorption ability, and the like. In order to industrially use the polysaccharide, a simple and efficient method for culturing cyanobacteria is required.

  Conventional mass culture of cyanobacteria uses an expensive and complex culture system with aeration / circulation devices, etc., to suspend algal bodies in a large amount of medium and to provide sufficient irradiation light intensity and carbon dioxide supply. Is generally performed (for example, Patent Document 1).

International Publication WO2005 / 102031

K. Ohki et al., Journal Appl. Phycol., Vol.26, P.265-272 (2014) Morel, F.M.M.Rueter, J. AQUIL: A chemically defined phytoplankton culture medium for trace metal studies.J. Phycol. 15: 135-141 (1979)

  An object of this invention is to provide the method and apparatus for mass-cultivating cyanobacteria simply and efficiently.

The present inventor can easily and efficiently use cyanobacteria by static culture in a liquid medium having a depth of 3 cm or less and a liquid surface area of 400 cm ² or more and the liquid surface being in contact with air. It was found that it can be cultivated automatically.

The inventor also provides, as an apparatus for performing the culture method, a mounting shelf including a plurality of mounting units disposed at different positions in the vertical direction, and a plurality of removably mounted units on the mounting unit. And a light reducing sheet that covers the plurality of containers and reduces the photosynthesis effective photon flux density of light reaching each container from an external light source, and each container has a liquid medium for cyanobacteria culture, A device characterized by being accommodated such that the depth is 3 cm or less and the area of the liquid level is 400 cm ² or more, and the liquid level is accommodated so as to be in contact with air, is suitable. I found it.

  According to the present invention, it is possible to cultivate a large amount of cyanobacteria simply and efficiently.

FIG. 1 is a photograph showing the results of cyanobacterial culture tests in Experiments 1 and 2. FIG. 2 is a photograph showing an example of the culture apparatus of the present invention installed in a green house. FIG. 3 is a diagram showing the biomass production in each stage of the container when static culture of cyanobacteria is performed using the culture apparatus shown in FIG. FIG. 4 is a graph showing the relationship between the light and temperature conditions during cultivation and the amount of extracellular polysaccharide (EPS) produced in Reference Test 1. FIG. 5 is a view showing a container 50 for culture. FIG. 6 is a view showing a container 50 containing a liquid medium 602 containing cyanobacteria 601 at a depth of H. FIG. FIG. 7 is a diagram for explaining the maximum value and the minimum value of the outer dimension and the inner dimension of the container body 51. FIG. 8 is a diagram showing an example in which a plurality of containers 50 are arranged at different positions in the vertical direction using the mounting shelf 80. FIG. 9 is a diagram showing a culture apparatus 90 for cultivating cyanobacteria according to the present invention.

1. Cyanobacteria The cyanobacterium cultivated by the method of culturing cyanobacteria and the method using the apparatus for cultivating cyanobacteria according to the present invention is not particularly limited as long as it is a microorganism classified as cyanobacteria, but preferably extracellularly. It is a cyanobacteria that releases a polysaccharide and adheres to each other to form a cell mass.

Particularly suitable cyanobacteria include Rippka et al. (R. Rippka, J. Deruelles, JB Waterbury, M. Herdman, RYStanier. Genetic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111: 1- 61 (1979)), cyanobacteria belonging to section I of single cell species, section III of filamentous species, section IV, and section V are included. The cyanobacteria belonging to a single cell type section I, specifically, Shianose (Cyanothece) genus Gureose (Gloeothece) genus, or cyanobacteria and the like belonging to Gureokapusa (Gloeocapsa) genus. The cyanobacteria belonging to Section III of the filamentous species, specifically, Ringubia (Lyngbia) genus Phormidium (Phormidium) genus, or cyanobacteria and the like belonging to Oscillatoria (Oscillatoria) genus. Specific examples of cyanobacteria belonging to section IV of the filamentous species include cyanobacteria belonging to the genus Nostoc or the genus Calothrix . The cyanobacteria belonging to Section V of the filament type, specifically, chloro gray Opsys (Chlorogloeopsis) genus, or cyanobacteria and the like belonging to Fissherera (Fischerella) genus.

The present invention is particularly suitable for cyanobacteria belonging to the genus Cyanothe among the cyanobacteria classified into the specific genera listed above, and among the cyanobacteria belonging to the genus Cyanose, Are particularly suitable for cyanobacteria having at least one, preferably at least 3, and more preferably all of properties 1-7.

(Characteristic 1) A large amount of polysaccharide is released extracellularly. Specifically, when culturing in a liquid medium (for example, the liquid medium used in the experimental culture of Experiment 1 of the present specification), the amount of extracellular polysaccharide produced is 10-50 mg (polysaccharide) per liter of culture solution per day. Dry weight).
(Characteristic 2) Despite being a single cell, cells cultured due to the produced extracellular polysaccharide form a large cell mass.
(Characteristic 3) It can be grown by stationary culture in a liquid medium having a depth of 3 cm or less (more specifically, a depth of 2.2 below).
(Characteristic 4) It can grow in fresh water.
(Characteristic 5) It has nitrogen fixing ability.
(Characteristic 6) The photosynthesis effective photon flux density optimal for growth when cultured in a liquid medium at 25 to 45 ° C. is 5 to 100 μmole · m ⁻² · sec ⁻¹ , more preferably 10 to 60 μmole · m. ^-2 · sec ^-1 .
(Characteristic 7) It can grow even when exposed to a low temperature of 10 to 20 ° C. or a high temperature of 46 to 48 ° C. for 1 to 5 hours a day for several days (for example, 2 to 4 days).

Examples of cyanobacteria belonging to Shianose (Cyanothece) genus with the above characteristics 1-7 Cyanothece sp. Viet Nam 01 strain, Cyanothes sp. PCC7822 strain etc. are mentioned.

Cyanothes sp. Viet Nam 01 strain is a novel cyanobacteria isolated by the present inventors from a paddy field in Vietnam by the method described in Non-Patent Document 1. The applicant tried to deposit the stock at the Patent Biological Deposit Center of the National Institute of Technology and Evaluation, but the acceptance was refused (notification date of the certificate of acceptance rejection March 7, 2014) Notification number 2013-1367). The present applicant and the present inventor have confirmed that the Cyanotheth sp. Viet Nam 01 stock is stored on its own and can be sold. Cyanothes sp. The range of Viet Nam 01 is Cyanothes sp. A mutant strain of Viet Nam 01 strain that retains the ability to produce extracellular polysaccharides is also included.

Cyanothes sp. The reason why the acceptance of the Viet Nam 01 strain was rejected was because other bacteria were mixed in the sample of this strain. Bacteria are considered to be contaminated in the extracellular polysaccharide of this strain and cannot be removed. Generally, as a method of removing bacteria from cyanobacteria, cyanobacteria and bacteria are separated as much as possible by homogenization or sonication, etc., and then cyanobacteria cells are spread on agar plates and cultured. A method of aseptically collecting colonies containing only cyanobacteria is used. However, Cyanotheth sp. The Viet Nam 01 strain releases a large amount of polysaccharide to the outside of the cell, so that the outside of the cell is covered with a thick polysaccharide layer, and the contaminated bacteria are buried or attached to the polysaccharide layer. Since the polysaccharide layer is thick, contaminated bacteria cannot be separated by weak homogenization or ultrasonic treatment. Further, when the intensity of the treatment is increased to such an extent that contaminating bacteria can be separated, Cyanotheth sp. Viet Nam 01 strain itself is killed. Therefore, Cyanothes sp. From which bacteria have been separated and removed. Viet Nam 01 strain cannot be obtained. Furthermore, when a sample of this strain contaminated with bacteria is treated with an antibiotic that kills the bacteria, both the bacteria and this strain are prokaryotes, so this strain is also sensitive and killed. After all, separation is impossible.

Cyanothes sp. The PCC7822 strain is available from the Pasteur Institute algal strain collection. Cyanothes sp. The range of PCC7822 strain includes Cyanothes sp. A mutant strain of PCC7822 strain, which maintains the ability to produce extracellular polysaccharides, is also included.

2. Culture conditions
2.1. Liquid medium for culturing cyanobacteria used in the present invention is a liquid medium in which components necessary for culturing cyanobacteria are added to water, and a known liquid medium used for culturing cyanobacteria, A liquid medium obtained by appropriately modifying the liquid medium may be used. Known liquid media include those disclosed in Non-Patent Document 2, and the liquid media used in the examples described in the present specification are modified versions thereof.

In order to adjust the pH of the liquid medium, a buffering agent such as trisaminomethane hydrochloride is generally added. However, since the buffering agent is expensive, in a preferred embodiment, the liquid medium is adjusted for pH adjustment. Therefore, it contains bicarbonate and does not contain a buffer such as trisaminomethane hydrochloride. More preferably, the component for adjusting the pH contains only bicarbonate. For this purpose, the concentration of bicarbonate in the liquid medium is preferably 0.2 to 0.4 g · L ⁻¹ .

  When cultivating cyanobacteria having nitrogen-fixing ability, it is not necessary to add a nitrogen source such as nitrate such as sodium nitrate to the liquid medium. Since the nitrogen source is generally expensive, the culture cost of cyanobacteria can be reduced by using a liquid medium containing no nitrogen source.

2.2. Static culture in a shallow liquid medium One of the features of the present invention is that a cyanobacteria has a depth of 3 cm or less and a liquid surface area of 400 cm ² or more, and the liquid surface is in contact with air. In, static culture. In a liquid medium with a shallow liquid depth (3 cm or less) and a large surface area in contact with air (400 cm ² or more), sufficient air is supplied to cyanobacteria without agitation or aeration. Growth is possible. Therefore, it is possible to perform static culture without disturbing the liquid medium by aeration, stirring, shaking, or the like. While culture that perturbs the liquid medium is not suitable for culturing cyanobacteria that form cell masses, stationary culture is suitable for culturing cyanobacteria that form cell masses.

  The depth of the liquid medium is preferably 2.5 cm or less, more preferably 2.0 cm or less. The lower limit of the depth is not particularly limited, but the depth is usually 0.5 cm or more, preferably 1 cm or more, and more preferably 1.5 cm or more. In the present invention, the depth of the liquid medium refers to the depth of the liquid medium at the time when the culture is started after the liquid medium and cyanobacteria are mixed.

In Although the area of the surface of the liquid medium is not particularly limited as long as 400 cm ² or more, preferably 500 cm ² or more, more preferably 900 cm ² or more, more preferably 1,000 cm ² or more, most preferably 2090 cm @ ² or more is there. Within this range, the level of the liquid medium is sufficiently wide relative to the depth of the liquid medium, so that static culture of cyanobacteria is facilitated. The upper limit of the liquid surface area of the liquid medium is not particularly limited, but from the viewpoint of improving the handleability of the culture vessel and suppressing the volatilization of the liquid medium, the liquid surface area of the liquid medium is usually 15,000 cm ² or less, preferably 10,000 cm ² or less, more preferably 8,100Cm ² or less, more preferably 6,400Cm ² or less, still more preferably 4,900Cm ² or less.

  The shape of the liquid surface of the liquid medium is not particularly limited, but typically the shape when the liquid surface is viewed in plan is a triangle, a rectangle (rectangle, square, parallelogram, trapezoid, rhombus, etc.), a pentagon to an octagon May be a polygon, a circle, an ellipse, a flat circle, a flat ellipse, or the like. Polygons such as triangles, quadrilaterals, pentagons and octagons may have shapes with smooth corners. More preferably, in the figure when the liquid level is viewed in plan, the maximum value of the distance between a pair of points on the periphery of the figure that are opposed to each other with the center of gravity of the figure in between (for example, in the case of a rectangle or a square) Is the length of the diagonal, the length of the diameter in the case of a circle, the length of the major axis in the case of an ellipse), and the minimum distance (for example, the length of the short side for a rectangle, the length of a side for a square) A / B is preferably 1.0 to 5.0, more preferably 1.0 to 3 where B is the length of the diameter, the length of the circle in the case of a circle, and the length of the minor axis in the case of an ellipse. 0.5, more preferably 1.0 to 2.5, and still more preferably 1.0 to 2.0.

  The air in contact with the liquid surface of the liquid medium is preferably in a state where it can be ventilated with outside air. Even if the container containing the liquid culture medium has a lid, the lid and the outside air are not completely blocked by the lid, and the problem is particularly problematic if the air and the outside air in the container can be vented. Absent. It is preferable that the container for storing the liquid medium has a lid because volatilization of the liquid medium can be suppressed and invasion of pests from outside the container can be prevented.

FIG. 5 shows a container 50 which is an example of a container suitable for storing a liquid medium so that the depth is 3 cm or less and the liquid surface area is 400 cm ² or more and the liquid surface is in contact with air. , 6. The container 50 includes a container main body 51 and a lid 52. The lid 52 is not essential as described above, and may be a container composed only of the container body 51. The lid 52 can close the container body 51 in a state where air inside the container body 51 and outside air can be vented. In order to prevent a pest or the like from entering due to a large gap between the lid 52 and the container body 51, the lid 52 is preferably made of a plastic material such as a thin plastic sheet. The container body 51 includes a bottom portion 501 and a peripheral wall 502 that stands up from the periphery of the bottom portion 501 and opens upward. A liquid medium 602 containing cyanobacteria 601 is accommodated inside the container body 51. Here, when the container for storing the liquid medium includes a bottom portion and a peripheral wall rising from the peripheral edge of the bottom portion, the inner shape and the inner dimension (inner dimension) of the portion surrounded by the peripheral wall are the liquid level of the stored liquid medium. Determine the shape and area. When the maximum value of the inner dimension of the portion is A and the minimum value is B, it is preferable that A / B is in the above range. Here, the maximum value of the inner dimension of the part is a figure when the outline inside the peripheral wall in the part is viewed in plan, on the periphery of the figure that is opposed to each other with the center of gravity of the figure in between. The maximum value of the distance between a pair of points indicates the length of a diagonal line when the figure is a rectangle or a square, the length of a diameter when it is a circle, and the length of a major axis when it is an ellipse. In addition, the minimum value of the inner dimension of the portion is a pair of shapes on the periphery of the figure facing each other with the center of gravity of the figure in between when the outline inside the peripheral wall in the part is viewed in plan. If the figure is a rectangle, the length of the short side, if it is a square, the length of one side, if it is a circle, the length of the diameter, if it is an ellipse, Refers to the length. For example, in the container main body 51, since the figure when the outline inside the peripheral wall 502 in the portion 504 surrounded by the peripheral wall 502 is viewed in plan is a rectangle, the maximum value A of the inner dimension of the portion 504 is the length of the diagonal line. The minimum value B of the inner dimension is the length of the short side (FIG. 7).
The depth H of the liquid culture medium 602 accommodated in the container main body 51 satisfies the above-described condition regarding the depth.

  The outer shape and outer dimensions (outer dimensions) of the vessel for culture are not particularly limited. When the container includes a bottom portion and a peripheral wall standing up from the peripheral edge of the bottom portion, the shape of the outline of the outer periphery of the peripheral wall at the portion surrounded by the peripheral wall is a triangle, a rectangle (rectangle, square, parallel) A quadrilateral, a trapezoid, a rhombus, etc.), a pentagonal octagonal polygon, a circle, an ellipse, a flattened circle, a flattened ellipse, and the like. Polygons such as triangles, quadrilaterals, pentagons and octagons may have shapes with smooth corners. More preferably, C / D is preferably 1.0 to 5.0, more preferably 1.0 to 3 when the maximum value of the outer dimension of the portion surrounded by the peripheral wall is C and the minimum value is D. 0.5, more preferably 1.0 to 2.5, and still more preferably 1.0 to 2.0. Here, the maximum value of the outer dimension of the part surrounded by the peripheral wall is a figure when the outline of the outer side of the peripheral wall in the part surrounded by the peripheral wall is viewed in plan, with the center of gravity of the figure interposed therebetween. The maximum distance between a pair of points on the edge of the figure facing each other. When the figure is a rectangle or square, the length of the diagonal line, when it is a circle, the length of the diameter, and when it is an ellipse Indicates the length of the major axis. The minimum value of the outer dimension of the part surrounded by the peripheral wall is opposed to the figure when the outline of the outer side of the peripheral wall at the part surrounded by the peripheral wall is viewed in plan with the center of gravity of the figure in between , Refers to the minimum distance between a pair of points on the periphery of the figure, the length of the short side if the figure is a rectangle, the length of one side if it is a square, the length of the diameter if it is a circle In the case of an ellipse, it indicates the length of the minor axis. For example, in the container main body 51, since the figure when the outline of the outer side of the peripheral wall 502 at the portion 504 surrounded by the peripheral wall 502 is viewed in plan is a rectangle, the maximum value C of the outer dimension of the portion 504 is the diagonal of the rectangle. It is the length, and the minimum value D of the outer dimension is the length of the short side of the rectangle (FIG. 7). The maximum value C of the outer dimension of the portion surrounded by the peripheral wall of the vessel for culture is preferably 30 cm to 140 cm, more preferably 40 to 120 cm, and still more preferably 50 to 90 cm. If the maximum value of the outer dimension of the container is within this range, it can be easily transported manually or by machine and has excellent handleability.

  Although the material which comprises the container main body 51 and the lid | cover 52 is not specifically limited, It is preferable that it is the material which can permeate | transmit the light from an external light source.

2.3. Various conditions such as light conditions, temperature, culture time and the like in stationary culture of cyanobacteria such as light conditions, temperature, and time can be appropriately determined according to the type of cyanobacteria to be cultured, and are not particularly limited.

In a preferred example, when cultivating cyanobacteria belonging to the genus Cyanothece , static culture is carried out with a photosynthetic effective photon flux density of 5 to 100 μmole · m ⁻² · sec ⁻¹ , more preferably 10 to 60 μmole · m ⁻² · sec. It is performed under the condition including the light period which is ^-1 . The photosynthesis effective photon flux density is the number of photons per unit area of light in a wavelength region (400 to 700 nm) effective for photosynthesis, and is also simply referred to as “light intensity” in this specification. The photosynthetic effective photon flux density can be measured using the instrument described in the examples. It is preferable that the light period of the above conditions is 12 to 24 hours per 24 hours. The photosynthesis effective photon flux density is lower than the daylight natural light photosynthesis effective photon flux density. For this reason, when using natural light as a light source, it is preferable to reduce the photosynthesis effective photon flux density of the light which reaches | attains a culture system by the means mentioned later. Photosynthetic photon flux density of the range, Cyanothece genus cyanobacteria are suitable for producing extracellular polysaccharides.

When culturing cyanobacteria belonging to the genus Cyanothece , the culture temperature is 25 to 45 ° C, preferably 25 to 40 ° C, more preferably 25 to 35 ° C, and most preferably 27.5 to 32.5 ° C. is there. It is preferable to always carry out the culture at a temperature within the above range, but it is not limited to this, and it is exposed to a low temperature (15 ° C. or lower) or a high temperature (45 ° C. or higher) for a time of about 1 to 3 hours / day during the culture period. May be.

Is not time limited particularly in static culture, when cultured cyanobacterium belonging to Cyanothece genus, usually 10 to 40 days, preferably 14-25 days.

3. Three-dimensional culture method In a preferred embodiment of the culture method of the present invention, the liquid medium is accommodated in each of the plurality of containers so that the depth is 3 cm or less and the liquid surface area is 400 cm ² or more, and The liquid level is accommodated in contact with the air, and the containers are arranged at different positions in the vertical direction, and the cyanobacteria are statically cultured in the liquid medium accommodated in the containers.

  The characteristics of the individual containers used in this embodiment are as described in 2.2 above, and the container 50 shown in FIGS. 5 and 6 can be illustrated as a specific example.

In the present embodiment, a plurality of containers each containing a liquid medium are arranged at different positions in the vertical direction, preferably along the vertical direction. As a method of arranging in this way, two or more containers may be stacked in the vertical direction, or a mounting shelf including a plurality of mounting units arranged at different positions in the vertical direction is prepared. A container may be placed on the container. When using a liquid medium with a shallow liquid depth of 3 cm or less, it is usually difficult to improve the production of cyanobacteria per unit land area, but a plurality of containers are placed at different positions in the vertical direction and placed in stationary culture. This makes it possible to increase the production of cyanobacteria per unit land area. The number of containers arranged at different positions in the vertical direction is not particularly limited, but is preferably 2 to 10, more preferably 3 to 7. Cyanobacteria, particularly cyanobacteria belonging to the genus Cyanothece, have a small photosynthetic effective photon flux density, for example, 5 to 100 μmole · m ⁻² · sec ⁻¹ , preferably 10 to 60 μmole · m ⁻² · sec ⁻¹ . Since it can proliferate and produce extracellular polysaccharides, it is sufficient in each container even when a plurality of containers containing liquid media are arranged at different positions in the vertical direction as in this embodiment. It is possible to proliferate and produce extracellular polysaccharides.

In FIG. 8, the drainage receiving portion 804 and a plurality of placement portions arranged at different positions in the vertical direction (consisting of four placement plates 802 and a placement surface 803 that is a part of the drainage receiving portion 804). And a frame 801 for fixing the mounting plate 802, an embodiment in which the container 50 is detachably mounted on the mounting plate 802 and the mounting surface 803 and static culture is performed. . When the drainage receiving portion 804 is not provided, the lowermost placement portion may be the ground or the floor. In the example of FIG. 8, the two placement shelves 80 share one drainage receiving portion 804, but the two placement shelves 80 may each independently have a drainage receiving portion.
The conditions for static culture in the present embodiment are as described above.

4). A preferred embodiment of a culture apparatus for realizing the three-dimensional culture method for culturing apparatus described above, a shelf mounting comprises a plurality of mounting portion disposed vertically different positions, removable before mounting section And a dimming sheet that covers the plurality of containers and reduces the photosynthetic effective photon flux density of light reaching each container from an external light source, and each container is cultivated by cyanobacteria The apparatus is characterized in that the liquid medium for use is stored so that the depth is 3 cm or less and the liquid surface area is 400 cm ² or more, and the liquid surface is stored in contact with air. It is.

  The characteristics of each container are as described in 2.2 above, and the container 50 shown in FIGS. 5 and 6 can be illustrated as a specific example.

  A specific example of the apparatus (culture apparatus 90) is shown in FIG. The characteristics of the mounting shelf 80 are as described in the above section 3.

  The culture apparatus 90 includes a light reducing sheet 91 that covers the plurality of containers 50 and reduces the photosynthetic effective photon flux density of light reaching the containers 50 from an external light source. In the illustrated example, the dimming sheet 91 is configured to cover the containers 50 placed on the two placing shelves 80, but is not limited thereto, and one dimming sheet is provided. May be configured to cover only the container 50 in a state of being placed on one placement shelf 80. In the illustrated example, the dimming sheet 91 is configured to cover the plurality of containers 50 in a state of being placed on the placement shelf 80, but is not limited thereto, and the culture apparatus includes a plurality of culture apparatuses. A dimming sheet may be provided, and each dimming sheet may be configured to individually cover the individual container 50.

  The light reducing sheet 91 is configured to perform photosynthesis of light when the photosynthesis effective photon flux density of light reaching each container 50 from an external light source (for example, the sun) is stronger than the photosynthesis effective photon flux density suitable for cyanobacteria culture. It plays the role of reducing the effective photon flux density to the photosynthetic effective photon flux density suitable for the cultivation of cyanobacteria. The photosynthetic effective photon flux density suitable for cultivation of cyanobacteria is as shown in 2.2 above. The dimming sheet 91 can be opened and closed, and the degree of dimming can be appropriately adjusted according to the change in the light intensity of the external light source.

Experiment 1: Comparison between stationary culture and agitation culture
experimental method
Material Single cell cyanobacteria Cyanothes sp. Viet Nam 01 strain was used. Since this strain releases macromolecular polysaccharides outside the cells, the cells have the property of growing in clusters.

Culture (stationary culture)
Maintenance culture of the cells is performed using a constant temperature incubator at a temperature of 30 ° C., light intensity of 20 μmole · m ⁻² · sec ⁻¹ daylight fluorescent lamp light, continuous bright light, cylindrical glass culture bottle (diameter 7 cm, height 9 cm, figure 1a) containing approximately 100 ml of medium was used. Table 1 shows the composition of the medium used for the culture (the composition of the components added in water). Since this species has nitrogen fixing ability (ability to convert nitrogen gas in the air into ammonia), a nitrogen source (NaNO ₃ ) was added to the maintenance culture but not to the experimental culture. The feature of this medium was that the bicarbonate concentration was relatively high, which allowed the optimum pH of the cyanobacterial strain to be maintained without using a buffer.

In the experimental culture, 300 ml of fresh medium (10 ml as a culture solution) from the late logarithmic growth phase to the linear growth phase of the maintenance culture contained in a cylindrical glass culture bottle having the same shape as that used for the maintenance culture. (See Table 1) Planting was followed by culturing for 14 days. The experimental culture was carried out for 14 days under the same conditions as in the maintenance culture (temperature 30 ° C., light intensity 20 μmole · m ⁻² · sec ⁻¹ daylight fluorescent light / continuous light) in a constant temperature and temperature chamber installed indoors.

  In addition, all the light intensities in this experiment and other experiments described in the specification are obtained by using an LI-250A photonometer (both manufactured by LI-COR Inc. (Nebraska, USA)) equipped with an LI-1905A photon sensor. It is the measured light intensity. With this instrument, the light intensity in the wavelength range of 400 to 700 nm related to photosynthesis was measured.

  In the static culture test section, the experimental culture was performed under the above conditions with the culture bottle still, without stirring and shaking.

(Stirring culture)
In the agitation culture test group, the culture contained in the cylindrical glass culture bottle was cultured under the same conditions as the above stationary culture test group except that the culture was performed while stirring using a magnetic stirrer. .

Results and Discussion In the stationary culture test group, the proliferated cells formed a cell mass and spread in layers on the bottom of the culture vessel.

  In the agitation culture test group, the cell mass was broken and made finer, but gradually faded with the lapse of the culture time, and eventually became white and died.

Experiment 2: Relationship between culture depth and biomass production
experimental method
Similar to Material Experiment 1, Cyanotheth sp. Viet Nam 01 strain (Non-patent Document 1) was used.

The composition and culture conditions of the medium used for the culture maintenance culture and the composition of the medium used for the experimental culture are the same as in Experiment 1 above.

In the experimental culture, cells from the late logarithmic phase to the linear growth phase of the maintenance culture (300 ml as the culture solution) were placed in a translucent plastic container (external dimensions 36 cm × 54 cm, height 19 cm, inner shape approximately 35 cm × 45 cm, depth approximately). The culture was carried out for 14 days after planting in three different volumes of fresh medium housed in 18 cm, liquid medium surface area of about 1,575 cm ² , FIG. After the addition of the cells, the medium depths were 2 cm, 4 cm, and 8 cm, respectively. The experimental culture was performed in a constant temperature and temperature chamber installed indoors under the same conditions as in the maintenance culture (temperature 30 ° C., light intensity 20 μmole · m ⁻² · sec ⁻¹ daylight fluorescent light, continuous light).

Evaluation of biomass production Cells after culturing for 14 days under the above conditions were collected using a stainless steel basket (mesh width of about 3 mm) (Fig. 1c), freeze-dried and then measured for dry weight.

Results and Discussion As the medium depth decreased, the biomass production increased, and the difference was significant (n = 3, P <0.05) between the medium depths of 4 cm and 2 cm (Table 2). In the culture with a medium depth of 2 cm, the proliferated cells spread in layers on the bottom of the incubator and could be recovered as a jelly-like cell mass after removing the supernatant medium (FIGS. 1b and 1c). It was thought that by reducing the culture depth and increasing the relative surface area of the culture, gas exchange was efficiently performed, and it was possible to improve biomass production without stirring and aeration.

Experiment 3: Multistage culture vessel
experimental method
Similar to Material Experiment 1, Cyanotheth sp. Viet Nam 01 strain (Non-patent Document 1) was used.

The composition and culture conditions of the medium used for the culture maintenance culture and the composition of the medium used for the experimental culture are the same as in Experiment 1 above.

Translucent plastic container similar to that used in Experiment 2 (external dimensions: 36 cm × 54 cm, height: 19 cm, inner shape: about 35 cm × 45 cm, depth: about 18 cm, liquid medium liquid surface area: about 1,575 cm ² , FIG. 1b) 10 pieces were prepared, and five pieces were arranged in the vertical direction on each of two iron angle shelves (mounting shelves). The shelf was installed in an unheated green house (Prefectural University Bioresource Development Research Center), covered with a black mesh (dimming sheet) and shielded from natural light (Fig. 2). The black mesh used was capable of blocking natural light by about 60%. The light intensity in the shelf after being covered with the black mesh varies greatly depending on the time of day and the weather, but typically the light intensity in the daytime is about 30 to 50 μmole · m ⁻² · sec ⁻¹ .

  Fresh plastic medium for experimental culture was placed in each plastic container so that the liquid depth was 2 cm. Then, 300 ml of cells from the late logarithmic growth phase to the linear growth phase of the maintenance culture were added to each container as a culture solution and cultured for 14 days. After the cultivation, biomass production was measured by the same method as in Experiment 2.

  Experimental culture was performed 6 times from August to November 2013. Regarding the evaluation of multi-stage culture, since the biomass production amount fluctuated depending on the culture experiment period, the biomass production amount at the top stage was set to 100 for each experiment period, and the biomass production amount at each stage was evaluated using relative values.

Results and Discussion FIG. 3 shows values of biomass production at each stage when the biomass production at the top stage is 100. There was no significant difference in biomass production between each stage (n = 6, P> 0.05). This result shows that the difference in light intensity caused by arranging the culture vessels in three dimensions did not become a limiting factor for biomass production. Three-dimensional culture was effective.

Experiment 4: Culture of other cyanobacterial strains
experimental method
material
Cyanothes sp. PCC7822 strain was purchased from the Pasteur Institute algal strain collection. Cyanothes sp. The PCC7822 strain is Cyanothes sp. Similar to the Viet Nam 01 strain, it has the property of producing extracellular polysaccharides to form cell mass and proliferate.

The composition and culture conditions of the medium used for the culture conditions maintenance culture, the composition of the media used in the experiment the culture is the same as the experiment 1. The culture experiment was performed under the condition that the culture medium depth was 2 cm in a cylindrical glass culture bottle (diameter 7 cm, height 9 cm, FIG. 1 a), temperature 30 ° C., daylight fluorescent lamp (20 μmol · m ⁻² · sec ^−1). , 12 hours light: 12 hours dark cycle).

Results The average value and standard deviation of the increase in dry weight after 14 independent cultures for 14 days are shown in the table below as biomass production per day / liter of culture. Cyanothes sp. The result of culturing Viet Nam 01 strain under the same conditions is shown as a control experiment.

Reference experiment 1: Relationship between light intensity during culture and polysaccharide production
experimental method
Similar to Material Experiment 1, Cyanotheth sp. Viet Nam 01 strain (Non-patent Document 1) was used.

The composition and culture conditions of the medium used for the culture maintenance culture and the composition of the medium used for the experimental culture are the same as in Experiment 1 above.
As the culture vessel, a cylindrical glass culture bottle (diameter 7 cm, height 9 cm, FIG. 1 a) was used.

Under different culture temperatures (20, 25, 30, 35 and 40 ° C.) and light intensity (8, 40, 80, 160 μmole · m ⁻² · sec ⁻¹ ), 20 hours in a 12 hour dark / 12 hour light cycle The culture was performed for a day.

Measurement of production amount of extracellular polysaccharide (EPS) The cultured cells were collected on a membrane filter (pore diameter: 3 mm), placed in a plastic tube and freeze-dried. Dry cells were stored at room temperature until measurement.

  The freeze-dried cells were treated with alkali (0.1N NaOH, 80 ° C., 4 hours), and then the extracellular polysaccharides were extracted and washed with isopropanol, and dried. Extracellular polysaccharides were quantified using the sulfate phenol method (Radhakrishnamurty and Berenson, Effect of temperature and time of heating on the carbazole reaction of uronic acids and acid mucopolysaccharides Anal. Chem. Vol. 35, 1316-1318, 1963).

  The results are shown in FIG. Values are expressed as the average value of the amount of extracellular polysaccharide (EPS) per liter of culture synthesized for 20 days in two independent experiments.

Results and Discussion Optimum light and temperature conditions for the amount of extracellular polysaccharide synthesized were 8 μmole · m ⁻² · sec ⁻¹ and 30 ° C. It can be said that the amount of extracellular polysaccharide synthesized by this strain increases at relatively low illuminance and high temperature.

50... Container 501 for culture .. Bottom 502 .. Peripheral wall 504.. Part 602 surrounded by peripheral wall... Liquid culture medium 601. · Mounting shelf 90 · · · Incubator 91 · Dimming sheet C · · · Maximum value H of the outer dimensions of the part surrounded by the peripheral wall · · · Depth of liquid medium

Claims (6)

A method for cultivating cyanobacteria,
A method comprising stationary culture of cyanobacteria in a liquid medium having a depth of 3 cm or less and a liquid surface area of 400 cm ² or more, wherein the liquid surface is in contact with air. The method of claim 1, wherein the cyanobacteria are cyanobacteria belonging to the genus Cyanothece . In each of a plurality of containers, the liquid medium is accommodated so that the depth is 3 cm or less and the area of the liquid surface is 400 cm ² or more, and the liquid surface is accommodated in contact with air,
Each container is arranged at a different position in the vertical direction,
The method according to claim 1 or 2, wherein the cyanobacterium is statically cultured in the liquid medium contained in each container.   The method according to claim 3, wherein each container includes a bottom portion and a peripheral wall rising from a peripheral edge of the bottom portion, and a maximum outer dimension of a portion surrounded by the peripheral wall of each container is 30 cm to 140 cm. An apparatus for cultivating cyanobacteria,
A mounting shelf comprising a plurality of mounting portions arranged at different positions in the vertical direction;
A plurality of containers removably placed on the placement section, and
A light reducing sheet that covers the plurality of containers and reduces the photosynthesis effective photon flux density of light reaching each container from an external light source;
Each container has a shape in which a liquid medium for cultivating cyanobacteria is stored so that the depth is 3 cm or less and the liquid surface area is 400 cm ² or more, and the liquid surface is in contact with air. A device characterized by comprising.   The apparatus according to claim 5, wherein each container includes a bottom portion and a peripheral wall rising from a peripheral edge of the bottom portion, and a maximum value of an outer dimension of a portion surrounded by the peripheral wall of each container is 30 cm to 140 cm.

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