JP6590144B2 - Indoor closed aquaculture system by static culture method using clonal monoalgae strain of Suizinori - Google Patents

Description

  The present invention relates to a method for stably culturing a large amount of a sardine squirrel, more simply and efficiently.

  Suizenjinori (Aphanothace sacrum) is a freshwater unicellular cyanobacterium that has been cultivated in the Kyushu region by a traditional method for over 300 years since the Edo period and has been used for food (Non-patent Documents 1-2). Although native species are designated as natural monuments, they are threatened with extinction due to environmental pollution.

  The extracellularly-released polysaccharide produced by this species is known as “Sakuran (registered trademark)” and has a high water retention capacity (Non-patent Document 3). The use for is also considered.

  The traditional aquaculture method of suizenjinori has been practiced outdoors using low-temperature, oligotrophic river water, but in recent years, along with the decrease in river water flow, outdoor aquaculture has been carried out using a method in which groundwater is pumped and poured. Has been done.

  However, in such outdoor aquaculture, the contamination of microalgae and red squirrels other than swallowtail butterflies due to eutrophication of groundwater has increased dramatically, and there are many zooplankton, insects and insect larvae that feed on the contaminants. Appear. This not only causes a drastic decrease in the production amount of suizenjinori, but also lowers the quality of Sakuran (registered trademark) because such suizenjinori is used as a raw material.

  In order to solve these problems, attempts have been made to cultivate swallowtail in an indoor environment where contamination with other organisms is avoided, but most of them have been cultivated in the field where microalgae have already been mixed. The algae of the plant is used as it is, and groundwater is poured or aeration and agitation are used. As in the case of outdoor aquaculture, there is no drastic improvement against contamination with other organisms. Since the cost of aeration and agitation is large, it has not been put into practical use (Non-patent Document 4). In addition, according to a culture report using a sterilized strain (Patent Document 1), only two weeks of growth was confirmed in a wet weight mg level culture system. Furthermore, although a method has been reported in which suizendinori purified by physically crushing or removing contaminating organisms using crushing / washing is left to stand in an indoor environment (Patent Document 2), it survived. In order to remove the contaminated organisms, it is necessary to wash the swallowtail and change the medium every two weeks, and the growth rate is not sufficient.

  Therefore, in this field, it is possible to continue culturing for a long time on a large scale without washing or changing the medium. A new technique is eagerly desired.

JP 2004-81022 A JP2015-122991A

Yamagata stag wolf Kumamoto natural monument Suizenji moss self-dough survey report (Kumamoto Prefecture historic site natural product survey report Vol. 5) Kumamoto (1931) Toshio Hara Suizenjinori (Ask the taste) Chuko Bunko pp145-150 (1971) Okajima, MK. Et al., Pure. Appl. Chem. 79: 2039-2046 (2007). Seida Hamada et al., Culture of Aphanothes sacrun (Sur.) Okada and Search for Functional Monosaccharides and Functionality Bulletin of Kyushu Tokai University 24: 37-43 (2005)

  It is an object of the present invention to provide a method for mass-culturing suizenjinori stably, more simply and efficiently.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have been able to cultivate suizendinori simply and efficiently by culturing the clonal monoalgae strain of suizendinori in a liquid medium. It has been found that by including a salt at a predetermined concentration in the medium, it is possible to cultivate suizendinori simply and efficiently by static culture, and the present invention has been completed. That is, the present invention includes the following inventions.

[1] A method for cultivating suizenjinori,
The method as described above, comprising stationary culture of a Suizendinori clone monoalgae strain in a liquid medium whose liquid surface is in contact with air.
[2] The method of [1], wherein the total salt concentration in the liquid medium is in the range of 0.003% to 1.3%.
[3] The method of [2], wherein the phosphate concentration in the liquid medium is 0.5 to 1 ppm in terms of phosphorus ion concentration.
[4] The method of [2] or [3], wherein the nitrate concentration in the liquid medium is 1 to 14 ppm in terms of nitrogen ion concentration.
[5] The method according to any one of [1] to [4], comprising stationary culture under conditions where the depth of the liquid medium is 3 cm or less.
[6] 20 ℃ ~30 ℃ temperature, and to static culture under conditions of light intensity of ^{20μmol · m -2 · sec -1 ~120μmol} · m -2 · sec -1, [1] Any method of [5].
[7] The liquid medium is accommodated in each of the plurality of containers,
Each container is arranged at a different position in the vertical direction,
The method according to any one of [1] to [6], wherein the Suizendinori clone monoalgae strain is statically cultured in the liquid medium contained in each container.

  ADVANTAGE OF THE INVENTION According to this invention, the method of cultivating a sardine sardine stably, more simply and efficiently can be provided.

FIG. 1 is a view showing a container 50 for culture. FIG. 2 is a view showing a container 50 containing a liquid culture medium 602 containing a suizendinori clone monoalgae strain 601. FIG. 3 is a schematic diagram illustrating an example in which a plurality of containers 50 are stacked in the vertical direction. FIG. 4 is a schematic 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. 5 is a graph showing the relationship between the light intensity and temperature conditions during culturing and the average growth rate (white squares) and biomass growth rate (black squares) of the Suizendinori clone monoalgae strain. (A) High phycoerythrin (PE) strain, (B) Low PE strain.

1. Suizendinori clone monoalgae strain The Suizendinori clone monoalgae strain used in the present invention can be obtained by a technique generally used in isolating cyanobacteria. That is, after pulverizing the swallowtail collected from rivers and farms by homogenization, ultrasonic treatment, etc., the cells forming the clumps are removed through a net having the same size (approximately 10 μm) as the cell diameter of swallowtail, A cell suspension is obtained in which the cells are not adhered to each other. The cell suspension can be confirmed that the cells adhere to each other using an optical microscope and no cell mass is formed.

  Next, the collected scorpion ginseng is spread and cultured on a plate of an agar medium to which components necessary for the growth of suizendinori are added, and the emergence of the suizenjinori colony is aseptically recovered to obtain a suizendinori clone monoalgae strain. be able to.

  In the present invention, the term “suizendinori clone monoalgae strain” means a genetically uniform cell population that does not contain other microalgae (diatoms, green algae, etc.) and is grown from a single cell. .

  As the “Suizendinori clone monoalgae strain” that can be used in the present invention, a known Suizendinori clone monoalgae strain (established strain) may be used, or a mutant strain in which the ability to produce extracellular polysaccharides is maintained. May be used.

  It is preferable that the Suizendinori clone monoalgae strain is used by dividing it into maintenance culture for the purpose of stock preservation and culture for sugar production for use in the production of Suizendinori. By performing the maintenance culture separately from the suizendinori production, it is possible to prevent the strain used for the suizenjinori production from disappearing.

2. Culture conditions 2.1. Medium The medium for cultivating suizendinori used in the present invention may be a liquid medium in which components necessary for culturing suizendinori are added to water, and a known medium (for example, AST medium (for example, AST medium ( Fujishiro et al., (2004) Appl. Environ. Microbiol. 70: 3338.), AQUIL medium (More et al., (1979) J. Physol. 15: 135; Ohki et al., (2014) J. Appl. Physol. 26: 265-272), MDM medium (Watanabe, AJ (1960) Gen. Microbiol. 6: 283-292), KMC medium (Kratz, WA & Myers, J. (1955). Am.J.Bot.42: 2 2), BG11 medium J.Gen.Microbiol.111 (Rippka et al, (1979.): 1) Although the like are not limited to) or can utilize their modified medium.

  In the present invention, the total salt concentration in the liquid medium is preferably 0.03 ‰ to 13 ‰ (0.003% to 1.3%), preferably 0.05 ‰ to 3 ‰ (0.005% to 0.3%). %).

The liquid medium in the present invention is preferably
Liquid medium does not contain ammonium salt (ammonium salt has a strong inhibitory effect on the growth of scorpionate);
The liquid medium does not contain silicon (diatoms have a shell composed mainly of silicon and require silicon for growth. For this reason, the liquid medium does not contain silicon, so that it does not contain silicon in the air during culture. In the unlikely event that diatoms scattered in the environment are mixed, it is possible to suppress the growth of mixed diatoms by making the environment in which silicon cannot be used); and the liquid medium is copper, manganese, zinc, boron, Including one or more (two, three, four, five or six) trace metals selected from cobalt and molybdenum,
It can be characterized by one or more (two or three) selected.

  Further, the liquid medium in the present invention may or may not contain nickel or cesium. Conventionally, nickel and cesium were considered to be necessary for the culture medium for suizendinori. However, as shown in the following examples, these components are not essential for culturing suizendinori.

  For example, in the medium that can be used in the present invention, Mg is 0.3 to 19 ppm, preferably 2 to 10 ppm, more preferably 9.7 ppm; K is 1 to 14 ppm, preferably 2 to 7 ppm, more preferably 6. 1 ppm; Na 9-200 ppm, preferably 18-40 ppm, more preferably 39 ppm; Ca 1.5-50 ppm, preferably 6-40 ppm, more preferably 27 ppm; Fe 0.02-0.4 ppm, preferably 0.06 to 0.25 ppm, more preferably 0.13 ppm; N is 1 to 20 ppm, preferably 1 to 14 ppm, preferably 1.4 ppm; P is 0.1 to 2 ppm, preferably 0.5 to 1 ppm, and more Preferably 0.9 ppm; S 2-100 ppm, preferably 4-50 ppm, more preferably 32.5 p m; a Cl 20-200 ppm, preferably 40～130Ppm, more preferably may be included in the ion concentration of 108 ppm,. In addition, since N, S, Cl, and Na contained in trace metals are trace amounts, they are not included in the above numerical range.

  The culture medium that can be used in the present invention is further 0.000002 to 0.00007 ppm of Cu, preferably 0.000006 to 0.000025 ppm, more preferably 0.000013 ppm; Mn is 0.00003 to 0.0006 ppm, preferably 0. 0.0008-0.0003 ppm, more preferably 0.00016 ppm; Zn is 0.00001-0.0002 ppm, preferably 0.00002-0.0001 ppm, more preferably 0.00005 ppm; B is 0.2-3 ppm, preferably 0.5 to 2 ppm, more preferably 1.08 ppm; Co 0.000006 to 0.00008 ppm, preferably 0.00001 to 0.00005 ppm, more preferably 0.00002 ppm; Mo 0.000006 to 0.000 8 ppm, preferably 0.00001～0.00005Ppm, more preferably may be included in the ion concentration 0.00002Ppm.

  The pH of the medium can be 7.2 to 7.8, preferably 7.4 to 7.6. Here, the pH value refers to a value measured when the temperature of the medium is 25 ° C. Although adjustment of the pH of a culture medium is not specifically limited, It can carry out using hydroxide (NaOH, KOH, etc.), hydrochloric acid, etc.

2.2. Static culture in a shallow liquid medium In the present invention, a Suizendinori clone monoalgae strain is statically cultured in a liquid medium whose liquid surface is in contact with air.

The liquid medium preferably has a shallow liquid depth. The depth of the liquid medium can be preferably 3 cm or less (for example, 3 cm, 2.5 cm, 2 cm, 1.5 cm). 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 the Suizendinori clone monoalgae strain are mixed. The liquid medium preferably has a large liquid surface area in contact with air. The area of the liquid surface of the liquid medium is not particularly limited, and can be appropriately selected according to the culture vessel to be used. For example, 15 cm ² or more, 50 cm ² or more, 150 cm ² or more, 300 cm ² or more, 500 cm ² or more, 1,000 cm ² or more, 1,500Cm ² or more, it may be 2,000 cm ² or more and the like. 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, It is preferably 10,000 cm ² or less, more preferably 5,000 cm ² or less.

  The liquid medium has a shallow liquid depth, and the surface area of the liquid surface is increased with respect to the depth, so that sufficient air is supplied to the Suizendinori Clone monoalgae strain without agitation or aeration. It is possible to grow a Suizendinori clone monoalgae strain without aeration or to perform static culture well.

  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, 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.

  1 and 2 show a container 50 as an example of a container suitable for containing a liquid medium so that the liquid surface is in contact with air. 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 only needs to be able to close the container main body 51 in a state in which the air inside the container main body 51 and the outside air can be vented, and can include a vent 53. 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, More preferably, a packing made of a plastic material is provided between the lid 52 and the container body 51. The vent 53 need only be in a state in which the air inside the container main body 51 and the outside air can be vented through the vent, and the shape, size, number, etc. thereof are not particularly limited. The vent 53 can be provided with a stopper for preventing the entry of other organisms such as pests, as long as it is in a ventable state. The stopper is not particularly limited, and a culture stopper having air permeability such as a cotton stopper, a paper stopper, a urethane stopper, or a silicone stopper can be used.

The depth H of the liquid culture medium 602 accommodated in the container main body 51 satisfies the above-described condition regarding the depth.
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, for example, transparent plastics (acrylic resin, vinyl chloride resin, etc.) Can be used. Preferably, the container body 51 is made of a material that does not contain glass (more specifically, silicon). Diatoms can utilize the silicon dissolved from the container material for growth. By making the container body 51 into a material that does not contain glass (more specifically, silicon), it is possible to make an environment in which silicon cannot be used, and even if diatoms are mixed in the Suizinori clone clones, Proliferation can be suppressed.

2.3. Light conditions, temperature, time, etc. Various conditions such as light conditions, temperature, culture time, etc. in stationary culture of the Suizendinori clone monoalgae strain can be appropriately determined and are not particularly limited.

In a preferred example, the static culture is performed under conditions including a light period in which the photosynthetic effective photon flux density is 10 μmol · m ⁻² · sec ⁻¹ or more, preferably 20 μmol · m ⁻² · sec ⁻¹ or more. 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 upper limit of the photosynthetic effective photon flux density in the stationary culture is not particularly limited, but can be, for example, 150 μmol · m ⁻² · sec ⁻¹ or less, preferably 120 μmol · m ⁻² · sec ⁻¹ or less. The photosynthetic effective photon flux density can be measured using the instrument described in the examples. The light period of the above conditions is 12 to 16 hours, preferably 12 to 14 hours per 24 hours. As the light source, a fluorescent lamp or a white LED can be used. When using the mounting shelf described below, a light source may be provided for each mounting unit. Although natural light can be used, the above-mentioned 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 using light reduction means, such as a light reduction sheet.

  The temperature at the time of culture can be a temperature selected from 15 ° C to 30 ° C, preferably from 20 ° C to 25 ° C. Although it is preferable to always perform the culture at a temperature within the above range, the present invention is not limited to this, and a predetermined time during the culture period may be set as another temperature condition (for example, different in light period and dark period in one day). It may be a temperature condition).

  The time for stationary culture is not particularly limited, but it can usually be performed for about 1 week to 8 weeks. By sterilizing the container, equipment, and medium used for stationary culture, growth of undesired microorganisms can be suppressed, and culture for the above period can be performed without medium replacement. Preferably, static culture is carried out using a pre-cultured (pre-cultured) Suizendinori clone monoalgae strain. By performing pre-culture, the Suizendinori clone monoalgae strain grown to a certain amount can be used for static culture, and the stationary culture time can be shortened and the Suizendinori clone monoalgae strain can be efficiently propagated. it can. Pre-culture can be performed on a small scale as compared with the scale of stationary culture, and can be performed in a culture container smaller than the culture container used for static culture and / or in a small liquid medium. The pre-culture time is not particularly limited, but it can usually be performed for about 1 week to 4 weeks. The pre-culture light conditions and temperature can be the same as those for stationary culture. If necessary, one or more pre-cultures may be performed before the pre-culture.

3. Three-dimensional culture method In a preferred embodiment of the culture method of the present invention, a liquid medium is accommodated in each of a plurality of containers so that the liquid surface is in contact with air, and each container is at a different position in the vertical direction. The Suizendinori clone monoalgae strain is statically cultured in the liquid medium that is placed and accommodated in each container.

  The characteristics of the individual containers used in this embodiment are as described in 2.2 above, and the container 50 shown in FIGS.

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 manner, as shown in FIG. 3, a mounting shelf 80 including a plurality of mounting portions 802 arranged and fixed to a frame 801 is prepared, and two or more containers are placed on each mounting portion. You may pile up and down. Alternatively, as illustrated in FIG. 4, a mounting shelf 80 including a plurality of mounting portions 802 arranged and fixed to the frame 801 at different positions in the vertical direction is prepared, and a container is placed on each mounting portion. May be. When using a liquid medium with a shallow liquid depth, it is usually difficult to improve the yield of swallowtail butter per unit land area, but by placing multiple containers at different positions in the vertical direction, It is possible to increase the production of suizenjinori per 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. Since suizenjinori can proliferate and produce extracellular polysaccharides under conditions of a small photosynthetic effective photon flux density, for example, 20 μmol · m ⁻² · sec ⁻¹ or less, as in this embodiment, a liquid medium is used. Even in the case where a plurality of containers in which are stored are arranged at different positions in the vertical direction, it is possible to sufficiently proliferate in each container and produce an extracellular polysaccharide.
The conditions for static culture in the present embodiment are as described above.

  The growth rate of the swallowtail is the fastest in the container closest to the light source (for example, the uppermost container if the light source is in the upper part), and the container farther away from the light source (if the light source is in the upper part, the lower stage) The growth rate can be reduced because the amount of light received is reduced. Therefore, harvesting of swallowtail can be done from the container closest to the light source. For example, if the light source is at the top, after removing the uppermost container, move the container at the lower stage to the upper stage one by one, and at the bottom, the liquid level that is newly inoculated with the suizeninori clone monoalgae is air. You may add and arrange the container accommodated so that it may touch. By repeating this process, it is possible to always harvest the swallowtail from the uppermost container with the highest production volume, and it is possible to stably produce the swallowtail in a stable manner.

Example 1: Establishment of Suizenjinori clone monoalgae strains The Suizenjinori used was provided by Suizenji Nori Honpo Tanseido (Kumamoto City) and Fukuoka Prefectural Fisheries and Marine Technology Center.

  The establishment of a euglena clonal monoalgae strain was performed according to a general method for establishing a cyanobacterium clonal monoalgae strain (Ohki, K. et al., Microbes. Environ. 27: 171-178 (2012)). . That is, it was not crushed by crushing suizendinori in a sterilized medium using a Teflon homogenizer, followed by sonication, further crushing, and filtering with a sterilized nylon mesh (NYTAL: opening 10 μm). Cell mass was removed. The obtained filtrate (hereinafter referred to as “cell suspension”) was subjected to microscopic observation, and it was confirmed that the cells were separated without forming a cell mass.

About 10 ml of a culture solution containing 0.8% (w / v) agar (Nacalai Tesque) previously autoclaved in a plastic petri dish (commercially sterilized polystyrene petri dish, diameter 90 mm) was added and solidified at room temperature (hereinafter referred to as “ Agar medium ”). The collected cell suspension is mixed in a culture solution containing the same amount of 1.5% (w / v) agarose (low melting point agarose, Sigma-Aldrich Inc), which has been autoclaved in advance and cooled to about 35 ° C. The mixture was shaken with care to avoid the occurrence of This was spread over the agar medium and solidified at room temperature, and then turned upside down, 20-25 ° C., ˜30 μmol · m ⁻² · sec ⁻¹ (daylight fluorescent lamp), light 14 hours: dark 10 The culture was continued under the conditions of time until colonies appeared.

  After culturing, a scorpionate clone monoalgae strain was established by picking up a scorpionate colony that appeared on the agar medium. It was confirmed by identifying the base sequence of the 16S rDNA gene isolated from the obtained clonal monoalgae strain that the obtained clonal monoalgae strain was Suizendinori.

  Six clone monoalgae strains were obtained from Suizenori from Suizenji Nori Honpo Tanseido, and 20 clone monoalgae strains were obtained from Suizenjiori from the Inland Water Research Institute, Fukuoka Prefectural Fisheries and Marine Technology Center.

Example 2: Examination of medium (1) Examination of modified AQUIL medium (i) Culture method The examination of the medium was carried out using a plurality of the clonal monoalgae strains obtained above.
The culture conditions were a temperature of 20 to 25 ° C., a fluorescent lamp (daylight color light), light intensity of 40 μmol · m ² · sec ⁻¹ , 14 hours of light, and 10 hours of darkness.

A clonal monoalgae strain was planted in each medium, subcultured at intervals of 3 to 5 weeks, and the influence of the medium on the growth of the clonal monoalgae strain was evaluated according to the following criteria:
・ It will die within a few days after planting ・ It will not die within a few days after planting, but its growth will be gradually inhibited and it cannot be subcultured twice or more ・ Subcultured 10 times or more Can do.

(Ii) Production of modified AQUIL medium Based on “AQUIL medium” (More et al., Supra; Ohki et al., Supra), which is a marine cyanobacteria culture medium, the following salt composition, nutrient composition, and trace amount Various modifications of the metal composition and various additions of vitamins were combined to prepare a modified AQUIL medium.

(Iii) Results Table 5 shows the growth results of the clonal monoalgae strains in various modified AQUIL media. In the table, “x” is killed within a few days after planting, “△” is not killed within a few days after planting, but growth is gradually inhibited and subcultured more than once Cannot, “◯” indicates that subculture can be performed 10 times or more, and “nd” indicates no data.

From this result, the following points became clear.
-The salt composition showed good growth in the range of 3 to 0.05 ‰ (0.3 to 0.005%) (a composition including modifications of the salt compositions 197, 98, and 39 in Table 5).
-Growth was inhibited by addition of high-concentration calcium salt (about 60 ppm in Ca) (including modifications of the salt compositions 197-H-Ca, 98-H-Ca, and 39-H-Ca in Table 5).
-Strong growth inhibition was observed at a phosphate concentration of 4 to 5 ppm (including alteration of HP with the nutrient composition shown in Table 5).
・ Growth inhibition was observed at a nitrate concentration of 20 ppm or more, but not as strong as phosphate (including alteration of HN in the nutrient composition shown in Table 5).
Ammonia salt is very strong inhibitory even low concentrations (0.07 ppm) was observed (at nutrient composition in Table 5 including the modification of the _NH 3 -N).
-Growth was very slow in the medium containing no nitrogen source (including -N modification in the nutrient composition of Table 5). Nitrogen fixing ability is suggested to be low even if expressed.
・ Use trace concentrations of 1/5 (including LM modification in the nutrient composition shown in Table 5) instead of the original concentration of AQUIL medium (including HM modification in the nutrient composition shown in Table 5). Good growth was seen.
-The addition effect of vitamin was not recognized (including the modification of Vit in the nutrient composition of Table 5).

(2) Examination of Modified MDM Medium (i) Culture Method The culture method was carried out in the same manner as the above “(1) Examination of modified AQUIL medium”.

(Ii) Production of Modified MDM Medium Based on the results of the modified AQUIL medium, “MDM medium” (Watanabe, AJ, supra), which is a marine cyanobacteria medium, is shown below. A modified medium was prepared by combining various modifications of the metal composition. Note that the MDM medium is easier to prepare and autoclave sterilized than the AQUIL medium.

(Iii) Results Table 9 shows the growth results of the clonal monoalgae strains in various modified MDM media. In the table, “x”, “Δ”, and “◯” indicate the same evaluation as in “(1) Examination of modified AQUIL medium”.

  From this result, as in the modified AQUIL medium, the phosphate concentration is 0.9 ppm for P, the nitrate concentration is about 1.4 ppm for N, and trace metals are contained at a low concentration in the modified MDM medium. It was revealed that a good growth of can be obtained.

(3) Examination of modified KMC medium (i) Culture method The culture method was carried out in the same manner as in the above "(1) Examination of modified AQUIL medium".

(Ii) Preparation of Modified KMC Medium Based on the results of the modified AQUIL medium, “KMC medium” (Kratz, WA & Myers, J., supra), which is a medium for marine cyanobacteria, A modified medium was prepared by combining various modifications of the nutrient salt composition and the trace metal composition.

  Various modifications of the nutrient composition are the same as those of HP / HN, HP / LN, LP / HN, and LP / LN (Table 7) of the nutrient composition in “(2) Examination of modified MDM medium”. The trace metal composition was the same as LM (Table 8) of the trace metal composition in “(2) Examination of modified MDM medium”.

(Iii) Results Table 11 shows the growth results of the clonal monoalgae strains in various modified KMC media. In the table, “x”, “Δ”, and “◯” indicate the same evaluation as in “(1) Examination of modified AQUIL medium”.

  Similar to the modified MDM medium, the phosphate concentration is 0.9 ppm for P, the nitrate concentration is about 1.4 ppm for N, and the trace metal is contained at a low concentration, so that the modified KMC medium has good swift dinosaur. It became clear that proliferation was obtained.

(4) New Suizen Ginori Medium Composition From the above results, the following was found as a medium composition for obtaining good growth of Suizen Ginori.

  In the following experiments, a medium having the following composition was used.

Example 3 Examination of Growth and Biological Production A Suizendinori clone monoalgae strain (2 strains) derived from Suizenji Nori Honpo Tanseido was used in this example. In addition, each eucalyptus monoalgae strain used has different phycoerythrin (PE) content, and those with relatively high PE content are described as “high PE strains” and those with relatively low PE content are described as “low PE strains”. To do.

Place the above medium in a transparent container so that the depth of the medium is about 3 cm, and plant each pre-cultured clonal monoalgae strain in logarithmic phase to ensure transparency while maintaining air permeability. And covered for 5 weeks. The culture was performed at a temperature of 20 ° C., 25 ° C., or 30 ° C., and a light intensity of 20, 40, 80, or 120 μmol · m ⁻² · sec ⁻¹ . As the light source, a daylight color fluorescent lamp was used, and the light cycle was 14 hours light / 10 hours dark. The container, equipment, and medium used for the culture were used after sterilization.

The “average growth rate” was calculated based on the change in the chlorophyll concentration obtained by collecting the cells in the container every week and extracting with methanol. In addition, the cells in the container were collected on day 0 and week 5 of the culture, the weight obtained by lyophilization was measured, and the “biomass increase rate” was calculated from the ratio. In the experiment, three sets of different cultures were performed for each condition, and an average value and a standard deviation were obtained.
The results are shown in FIG.

In the above-mentioned medium, under the conditions of a temperature of 20 ° C. to 30 ° C. and a light intensity of 20 to 120 μmol · m ⁻² · sec ⁻¹ , the Suizendinori clonal monoalgae strain is cultured and propagated satisfactorily without changing the medium. In some cases, the rate of increase in biomass reached about 15 times. The rate of increase in biomass obtained in this example is significantly higher than the rate of increase in biomass in the conventionally reported suizendinori culture method. It was confirmed to be effective.

50... Container 501 for culture .. Bottom 502 .. Peripheral wall 504... Surrounded by peripheral wall 51... Container body 52... Lid 53. Suizen Ginori clone monoalgae strain 80 ... Placement shelf 801 ... Frame 802 ... Placement part H ... Depth of liquid medium

Claims (6)

A method of cultivating suizenjinori,
Look including to stationary culture in a liquid medium in which the liquid surface Sui Zen Ginori clonal unialgal strain is in contact with air,
The total salt concentration in the liquid medium is in the range of 0.003% to 1.3%;
The liquid medium is Mg 2-10 ppm; K 2-7 ppm; Na 18-40 ppm; Ca 6-40 ppm; Fe 0.06-0.25 ppm; N 1-14 ppm; P 0.5 ~ 1 ppm; S 4-50 ppm; Cl 40-130 ppm; Cu 0.000006-0.000025 ppm; Mn 0.00008-0.0003 ppm; Zn 0.00002-0.0001 ppm; B 0.5 ˜2 ppm; Co containing 0.00001 to 0.00005 ppm; Mo containing 0.00001 to 0.00005 ppm in ionic concentration and no ammonia salt . The method according to claim 1 , wherein the phosphate concentration in the liquid medium is 0.5 to 1 ppm in terms of phosphorus ion concentration. The method according to claim 1 or 2 , wherein the nitrate concentration in the liquid medium is 1 to 14 ppm in terms of nitrogen ion concentration. The method according to any one of claims 1 to 3 , comprising stationary culture under conditions where the depth of the liquid medium is 3 cm or less. 20 ° C. to 30 ° C. of temperature, and that the static culture under conditions of light intensity of ^{20μmol · m -2 · sec -1 ~120μmol} · m -2 · sec -1, according to claim 1-4 The method according to any one of the above. Each of the plurality of containers contains the liquid medium,
Each container is arranged at a different position in the vertical direction,
The method according to any one of claims 1 to 5 , wherein the Suizendinori clone monoalgae strain is statically cultured in the liquid medium accommodated in each container.

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