WO2016175302A1

WO2016175302A1 - Method for culturing and collection of microalgae culture

Info

Publication number: WO2016175302A1
Application number: PCT/JP2016/063410
Authority: WO
Inventors: 若田　裕一; 金原　秀行; 岩戸　薫
Original assignee: 富士フイルム株式会社
Priority date: 2015-04-30
Filing date: 2016-04-28
Publication date: 2016-11-03
Also published as: JPWO2016175302A1; JP2016208972A

Abstract

Provided is a method for culturing and collecting a microalgae culture by using a culturing device provided with a storage member that stores a culture fluid, wherein the method includes the following: a main culturing step for culturing microalgae in a culture fluid while keeping an air layer present above the culture fluid; a liquid amount reduction step for reducing the amount of liquid in the culture fluid; and a collection step for collecting the microalgae culture after the liquid amount reduction step.

Description

Method of culturing and recovering cultures of microalgae

The present invention relates to a method for culturing and recovering cultures of microalgae.

In recent years, with the development of industrial activities, global warming is progressing due to the soaring fuel price by using a large amount of fossil fuel and the greenhouse effect by carbon dioxide released into the atmosphere by using fossil fuel Is a problem.

As a means to solve such problems, there is increasing hope for the use of microalga which has the ability to fix carbon dioxide with light energy and convert it to biofuels such as hydrocarbon compounds and biodiesel (triglyceride). Already, various studies have been conducted to fix carbon dioxide using light energy by culturing microalgae and to produce biofuels such as biodiesel and hydrocarbon compounds.

However, in order to commercialize biofuel production using microalgae and carry it out on a commercial scale, efficient microalgal culture methods, microalgae recovery methods, and extraction methods of biofuels such as oil are also available. The problem is that it has not been developed and its cost is high. One of the biggest causes is the lack of efficient culture, drying and recovery methods for microalgae. Specifically, since microalgae usually grow suspended in liquid, in order to use microalgae as biomass, it is necessary to recover very thin concentrations of microalgae from a large amount of liquid . In addition, since light energy is required for the growth of the microalgae, the concentration of the microalgae present in the liquid can not be excessively high in order to secure sufficient light irradiation. As a result, in order to recover the microalgae suspended in the liquid, it was necessary to filter a large amount of water. In addition, the size of microalgae was generally small and filtration was not easy. As a study of recovery methods for solving such problems, a method using a precipitant, a method using a centrifuge, a method of recovering large organisms after using microalga as feed for larger organisms, etc. Although no attempt was made, none of the methods led to a fundamental solution.

On the other hand, in Patent Document 1, as a simple and inexpensive method, a suspension or dispersion of Botryococcus sudeticus AVFF 007 strain is prepared, and a culture vessel (storage member) is allowed to stand. A method of culturing microalga comprising forming a biofilm on a liquid surface by settling microalgae on the bottom and then culturing for a while, and forming the formed biofilm as a substrate A method of recovering microalgae is disclosed, which comprises transferring or depositing on the surface for recovery. In addition, Patent Document 2 discloses, as a simpler and lower-cost method, by culturing Botryococcus sudeticus AVFF 007 strain in a culture solution in a first incubator (storage member), The first step of culture in which a biofilm consisting of microalgae is formed on the liquid surface of the culture solution, and the microalgae by using at least a part of the biofilm formed on the liquid surface of the culture solution as a seed algae A culture method of microalgae comprising culturing the second step on the liquid surface, and transferring or depositing the formed biofilm on the surface of the substrate for recovery. Methods of recovery of microalgae are described. In any of the method described in Patent Document 1 and the method described in Patent Document 2, in the stage of collecting microalgae, a biofilm composed of an aggregate of microalgae floats on the liquid surface, and Since the biofilm is to be recovered, it is not necessary to recover microalgae from a large amount of culture solution as in the prior art, and the recovery cost of microalgae can be significantly reduced.

International Publication No. 2013/161832 International Publication No. 2014/088010

The liquid surface floating culture methods described in Patent Document 1 and Patent Document 2 can form a biofilm on the liquid surface, so that stirring is not necessary and the water depth can be made shallow compared to conventional culture methods. It is characterized in that the recovery of the culture is easy, it is strong against the invasion of contamination microorganisms, and the moisture content of the recovered product is low.
However, according to the study by the present inventors, culture of microalgae by liquid surface suspension culture method is carried out on a practical scale, and in order to produce algal biomass and algal biofuel, it is more preferable to recover the culture. It was found that there is room for efficiency.

Then, this invention makes it a subject to provide the culture | cultivation and collection | recovery method of the culture thing of a micro algae which can collect | recover the cultured micro algae efficiently, after culture | cultivating a micro algae on a practical scale. .

The present inventors diligently studied to solve the above-mentioned problems, and it is a method for culturing and recovering a culture of microalgae using a culture apparatus provided with a storage member for holding a culture solution, After the main culture step of cultivating the microalga with the culture solution while maintaining the air layer above, the liquid amount reduction step of reducing the liquid amount of the culture solution, and the liquid amount reduction step, the culture of the microalga is According to the culture and recovery method of the microalgal culture including the recovery step, the microalga culture can be efficiently recovered after the microalgae is cultured on a practical scale. The present invention has been completed.
That is, the present invention provides the following (1) to (17).

(1) A method for cultivating and collecting a microalga culture using a culture apparatus provided with a storage member for holding a culture solution,
A main culture step of culturing the microalga in the culture solution while maintaining an air layer above the culture solution;
A volume reduction step of reducing the volume of the culture solution,
And a recovery step of recovering the microalgal culture after the liquid volume reduction step.
(2) After the volume reduction step and before the recovery step,
The culture | cultivation and collection | recovery method of the culture of micro algae as described in (1) including the post-culture process of culture | cultivating micro algae in presence of a culture solution.
(3) In the post-culture step, the mass ratio of the microalga algal cells to the culture solution is: mass of algal cells / mass of culture solution = 20/80 to 0.1 / 99.9, described in (2) And a method of culturing and collecting microalga cultures.
(4) The culture apparatus is further disposed above the storage member so as to cover at least a part of the liquid surface of the culture solution when viewed in the vertical direction, and can transmit light of at least a part of the wavelengths A culture and recovery of the culture of the microalga according to any one of (1) to (3), further comprising a shielding member, wherein an air layer is formed between the liquid surface of the culture solution and the shielding member. Method.
(5) The culture apparatus further includes at least one of an air temperature control unit that controls the temperature of the air layer and a liquid temperature control unit that controls the temperature of the culture solution,
In the main culture step, the culture of the microalga according to any one of (1) to (4), wherein at least one of the temperature of the air layer and the temperature of the culture solution is controlled to a temperature range suitable for culture of the microalga. Method of culture and recovery of
(6) The method for cultivating and recovering a microalgal culture according to any one of (1) to (5), wherein the culture apparatus further comprises an aeration unit in communication with the air layer.
(7) The method for cultivating and recovering a culture of microalga according to (6), wherein the culture apparatus further comprises an aeration control unit for controlling passage of gas in the aeration unit.
(8) The storage member has a flexible first film and a support for supporting the first film,
The microalgal culture according to any one of (1) to (7), wherein the support supports the first film to form a pool-like recess, and the culture solution is retained in the recess. Culture and recovery method.
(9) The culture apparatus has a first film moving member for moving the first film,
In the volume reduction step, the culture volume of the culture solution is decreased to bring the culture of the cultured microalga into contact with the first film,
The method for cultivating and collecting a microalgal culture according to (8), wherein the first film is moved from the support in the collection step, and the microalgal culture is collected.
(10) The culture apparatus has a first winding machine for winding the first film in a roll,
In the recovery step, the first winder recovers the microalgal culture by rolling up the first film with the microalgal culture in a roll, according to (8) or (9). Method for culturing and recovering cultures of microalgae.
(11) The culture apparatus has a first winding machine for winding the first film in a roll, and a first recovery member for peeling the culture of the microalga from the first film,
In the recovery step, while the first winder winds the first film into a roll, the first recovery member exfoliates the culture of microalgae from the first film to recover the culture of microalgae The culture | cultivation and collection | recovery method of the culture of the micro algae as described in (8) or (9).
(12) The culture of the microalga according to any one of (8) to (11), wherein the culture apparatus comprises one or more third films between the first film and the support. Culture and recovery method.
(13) The microalgae according to any one of (1) to (12), wherein the storage member comprises a second film in which a plurality of through holes are formed on the outermost surface in contact with the culture solution. Method for culturing and recovering a culture of
(14) The culture apparatus has a second film moving member for moving the second film,
The method for cultivating and collecting a microalgal culture according to (13), wherein the second film is moved in the collection step, and the microalgal culture is collected.
(15) The culture apparatus has a second winding machine that winds the second film in a roll,
In the recovery step, the second winding machine recovers the microalgal culture by rolling up the second film together with the microalgal culture in a roll, according to (13) or (14). Method for culturing and recovering cultures of microalgae.
(16) The culture apparatus has a second winding machine for winding the second film in a roll, and a second recovery member for peeling the culture of microalgae from the second film,
In the recovery step, while the second winder winds up the second film in a roll, the second recovery member exfoliates the culture of microalgae from the second film to recover the culture of microalgae The culture | cultivation and collection | recovery method of the culture of the micro algae as described in (13) or (14).
(17) The method for cultivating and recovering a culture of microalga according to any one of (1) to (16), wherein the microalga is a microalga capable of forming a biofilm on the liquid surface of a culture solution.

According to the present invention, it is possible to provide a method for cultivating and collecting a microalgal culture, which comprises efficiently collecting the cultured microalga after culturing the microalga on a practical scale. .

According to the culture and recovery method of the culture according to the present invention, recovery of the culture of microalgae becomes extremely easy as compared with the conventional suspension culture, and the culture of microalgae can be recovered with high recovery rate . That is, in the present invention, it is not necessary to recover a culture of microalgae from a large amount of culture solution as in the prior art at the stage of recovering a culture of microalgae, and the recovery cost of cultured microalgae can be significantly reduced. . Moreover, there is no need to handle a large amount of liquid culture solution, and it is not necessary to use a large amount of water.

FIG. 1 is a schematic perspective view showing an example of a culture apparatus. FIG. 2 is a cross-sectional view taken along line II-II of FIG. FIG. 3 is a cross-sectional view taken along line II-II of FIG. However, the mode of existence of microalgae differs from FIG. FIG. 4 is a cross-sectional view taken along line II-II of FIG. However, the mode of existence of microalgae differs from FIG. FIG. 5 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 6 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 7 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 8 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 9 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 10 is a schematic cross-sectional view for explaining the installation method of the culture apparatus. FIG. 11 is a schematic cross-sectional view for explaining the installation method of the culture apparatus. FIG. 12 is a schematic cross-sectional view for explaining the installation method of the culture apparatus. FIG. 13 is a schematic cross-sectional view for explaining the installation method of the culture apparatus. FIG. 14 is a schematic cross-sectional view showing an example of a suitable installation surface. FIG. 15 is a schematic cross-sectional view for explaining the installation method of the culture apparatus. FIG. 16 is a schematic cross-sectional view for explaining the installation method of the culture apparatus. FIG. 17 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 18 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 19 is a schematic perspective view showing another example of the culture apparatus. FIG. 20 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 21 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 22 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 23 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 24 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 25 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 26 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 27 is a schematic cross-sectional view showing another example of the culture apparatus. FIG. 28 is a schematic cross-sectional view for explaining the liquid amount reduction step and the recovery step. FIG. 29 is a schematic cross-sectional view for explaining the liquid amount reduction step and the recovery step. FIG. 30 is a schematic cross-sectional view for explaining the liquid amount reduction step and the recovery step. FIG. 31 is a schematic cross-sectional view for explaining the liquid amount reduction step and the recovery step. FIG. 32 is a schematic cross-sectional view for explaining the liquid amount reduction step and the recovery step. FIG. 33 is a schematic cross-sectional view for explaining the recovery step. FIG. 34 is a schematic cross-sectional view for explaining the recovery step. FIG. 35 is a schematic cross-sectional view for explaining the recovery step. FIG. 36 is a schematic cross-sectional view for explaining the recovery step. FIG. 37 is a schematic cross-sectional view for explaining the recovery step. FIG. 38 is a schematic plan view showing another example of the culture apparatus. FIG. 39 is a photograph showing the state of the first film after drying a microalgal culture in Example 1. FIG. 40 is a photograph showing the results of testing, in Example 31, the ease of desorption of the microalga dry matter, on the three types of materials. FIG. 41 is a view schematically showing a microscopic image obtained by observing a biofilm of microalgae forming a biofilm on a liquid surface with an optical microscope. FIG. 42 is a view schematically showing a microscopic image obtained by observing a biofilm of microalgae forming a biofilm on a liquid surface with an optical microscope. FIG. 43 is a view schematically showing a microscopic image obtained by observing a microalga floating in a culture solution with an optical microscope. FIG. 44 is a diagram schematically showing a state in which a biofilm formed on a liquid surface in Example 32 is observed from the horizontal direction. A single foam and overlapping foams and cages are shown. FIG. 45 is a view schematically showing a state in which a biofilm formed on a liquid surface in Example 32 is observed from above in the vertical direction. The biofilm on the liquid surface shows a foamy structure and a scaly structure. FIG. 46 is a view showing a process of transferring a biofilm (algal cells) of microalgae formed on the liquid surface to the surface of the substrate using the first substrate. FIG. 47 is a view showing a process of depositing a biofilm (algal cells) of microalgae formed on the liquid surface on the surface of the substrate using the second substrate.

Hereinafter, preferred embodiments of the present invention will be described in detail.
In the present specification, a numerical range represented using “to” means a range including numerical values described before and after “to” as the lower limit value and the upper limit value.

[Method for culturing and recovering cultures of microalgae]
The method for culturing and recovering a culture of microalgae according to the present invention (hereinafter, sometimes referred to simply as the “culture and recovery method of the present invention”) is a method using a culture apparatus comprising a storage member for holding a culture solution. A method for cultivating and collecting algal culture, comprising a main culture step of culturing microalga in a culture solution while reducing an amount of the culture solution while maintaining an air layer above the culture solution It is a culture | cultivation and collection | recovery method of the culture of micro algae including a process and the collection process of collect | recovering culture of micro algae after a liquid volume reduction process.

The characteristic point of the method for cultivating and recovering microalga of the present invention in comparison with the conventional method for cultivating and recovering microalgae is to cultivate the microalga after culturing the microalga to reduce the liquid volume of the culture solution It is in the point of collecting things.
In the conventional culture and recovery method of microalgae, since the microalgae cultured is recovered by centrifuging or filtering out the microalgae from a large amount of culture solution, the microalgae are recovered from the culture solution It was necessary to handle a large amount of culture fluid when
However, according to the culture and recovery method of the present invention, since it is not necessary to culture microalgae on a practical scale and handle a large amount of culture solution when recovering the culture, it is not necessary to recover the cultured microalga. It can be reduced compared to the prior art.

Moreover, in the culture and recovery method of the present invention, the recovery cost can be reduced by further including a post-culture step of culturing the microalga in the presence of the culture solution after the fluid volume reduction step and before the recovery step. In addition, algal productivity and / or oil productivity can be improved, and the production cost can be further reduced.

In the following description, the cultured microalgae culture may be simply referred to as "microalgae" or "culture".

First, the culture apparatus used in the production method of the present invention will be described using FIGS. 1 to 27, and then the production method of the present invention will be described.

<Culture device>
FIG. 1 is a schematic perspective view schematically showing an example of a culture apparatus used in the culture and recovery method of the present invention, and FIGS. 2, 3 and 4 are cross-sectional views taken along line II-II of FIG. In addition, in FIG. 1, illustration of the shielding member 14a is partially abbreviate | omitted for description.
The culture apparatus 10a shown in FIG. 1, FIG. 2, FIG. 3 and FIG. 4 is a storage member 12a for holding the culture solution M, and a storage member so as to cover the liquid surface of the culture solution M when viewed from the vertical direction. A shielding member 14a, which can transmit light of at least a part of wavelengths, is disposed above the 12a.
In addition, when culturing the microalgae P, the culture apparatus 10a forms an air layer N between the liquid surface of the culture fluid M and the shielding member 14a, as shown in FIG. 2, FIG. 3 and FIG. Do. Even in culturing microalgae P _2, as shown in FIGS. 3 and 4, it may form a Kiso N between the shielding member 14a and the liquid surface of the culture solution M, microalgae P ₃ Also when culturing C, as shown in FIG. 4, an air layer N may be formed between the liquid surface of the culture solution M and the shielding member 14a.
2, microalgae P 3 and 4 forms a biofilm on the liquid surface of the culture medium M, microalgae P ₂ in FIGS. 3 and 4 is adhered to the surface in contact with the culture medium M of the container 16a and has, microalgae P ₃ in FIG. 4 are suspended in culture M.
Each component will be described in detail below with reference to FIGS. 1 to 27.

<< Reserved member >>
The storage member is a culture solution tank capable of holding the culture solution M in which the microalgae P can be cultured for a certain period of time. The storage member is not particularly limited in its configuration, shape, size, material, and the like, as long as the culture solution M can be held during the culture period of the microalgae P.
For example, the storage member 12a of the culture apparatus 10a shown in FIG. 1, FIG. 2, FIG. 3 and FIG. 4 is a substantially rectangular parallelepiped box-shaped container 16a whose one surface is open. It is a holding part.

As such a container 16a, what was shape | molded in the container-like form using plastic, soil, concrete, wood, metal, clay, pottery, glass, or the combination of 2 or more types of these is mentioned.
Among them, as the material of the container 16a, it is preferable to use a material composed of an organic polymer compound, an inorganic compound, a metal, or a complex thereof. It is also possible to use mixtures thereof.
By using a material having a high thermal conductivity as the material of the container, for example, it is possible to suppress a temperature rise when culturing is performed under conditions of high temperature such as summer daytime. As a material with high thermal conductivity, for example, clay, a metal plate and the like can be mentioned, and clay is particularly preferable because it is inexpensive and has high thermal conductivity.
Alternatively, for example, the liquid holding portion may be formed of a resin, and a layer made of a material having high thermal conductivity such as clay may be bonded to the periphery of the liquid holding portion.
By using a material having a low thermal conductivity as the material of the container, for example, it is possible to suppress a temperature drop when culturing is performed under conditions of low temperature such as nighttime in winter. Examples of the material having a low thermal conductivity include, for example, plastics, expanded polystyrene, wood, pottery, gases and the like, and plastics are particularly preferable because they are inexpensive and have a low thermal conductivity.
Also, for example, the liquid holding portion may be formed of glass, and a layer made of a material having a low thermal conductivity such as glass may be bonded to the periphery of the liquid holding portion.

In addition, as described in detail later, the culture and recovery method of the present invention is a microalgal algae having a property of floating on the liquid surface by being left standing, bubbling treatment with air, etc., floating by oil accumulation, addition of a standing step. The present invention can be widely applied to microalgae in which microalgae is floated to the liquid surface by floating or the like, and microalgae capable of stationary culture. It is particularly preferable when using a microalga capable of forming a biofilm on the liquid surface. When using a microalga that forms a biofilm on the liquid surface, the depth of the culture solution can be reduced, so a so-called bat-shaped shallow container (container with a small ratio of height to bottom area) is used. can do. The use of a shallow container facilitates the recovery operation of the biofilm obtained by the culture, and is also advantageous in terms of the cost of the container.
In addition, according to the production method of the present invention, a deep container (a container having a large ratio of height to bottom area as compared to a shallow container) can be used, as long as the culture solution can be held depending on the topography and the place to be cultured You may use.

The size (area) and depth of the liquid holding portion of the culture solution M of the storage member 12a (container 16a) are not particularly limited, and may be appropriately selected according to the type of algae to be cultured, the culture method, and the like.
The bottom surface of the liquid holding portion of the storage member 12a (the container 16a) is preferably flat in order to keep the amount of the culture solution small and in a substantially uniform state.

Moreover, in the example shown in FIG.2, FIG3 and FIG.4, although the container 16a was made into the box shape of a substantially rectangular parallelepiped, limitation is not carried out like this, like the container 16b of the culture apparatus 10b shown in FIG. The shape may be inclined toward the opening side in the direction in which the side surface is open, or may be inclined in the direction in which the side surface is closed. Also, the open surface is not limited to a rectangular shape, and may be a triangular shape, a polygonal shape, a circular shape, an elliptical shape, a raceway shape, or the like.

Further, in the case of reducing the liquid amount by draining the culture solution M in the storage member in the liquid amount reduction step described in detail later, as shown in FIG. 2, FIG. 3 and FIG. The drainage pipe 18 and the valve 17 are connected to the side surface of the container 16a).
Here, in the case of using the microalga which forms a biofilm on the liquid surface to which the present invention is suitably applied, most of the microalgae are present on the liquid surface side of the culture solution, so the drainage pipe 18 is Preferably, it is connected to the side near the bottom of the member 12a. Alternatively, the drain pipe 18 may be connected to the bottom surface of the storage member 12a.

Moreover, although the storage member 12a was comprised by the one container 16a in the example shown in FIG.2, FIG3 and FIG.4, limitation is not carried out to this, The storage member holding the culture solution M is comprised with several members May be

For example, the storage member has a flexible first film and a support for supporting the first film, and the support supports the first film to form a pool-like recess. Alternatively, the culture solution may be held in a recess. Here, the first film may be a film of one sheet or may be a film of two or more sheets, but when it is formed of two or more films, the culture and recovery steps Preferred to repeat. The storage member 12f of the culture apparatus 10f shown in FIG. 6 has a flexible first film 22f and a support 20f for supporting the first film 22f. The support 20f is a first film. 22f is supported to form a pool-like recess. Therefore, the storage member 12 f holds the culture solution M, using the recess as a storage portion. The first film 22f has two surfaces, the front side (the side in contact with the culture solution M or the side closer to the culture solution M) and the rear side (the side farther from the culture solution M).

As described above, when the storage member 12f is configured of the first film 22f and the support 20f supporting the first film 22f, the liquid amount of the culture solution M is reduced in the liquid amount reduction step described later. After bringing the cultured microalgae P into contact with the first film 22f, the microalgae P can be easily recovered by moving (detaching) the first film 22f from the support 20f in the recovery step. Can.
Moreover, after collection of the microalgae P, the first film 22f can be repeatedly used by setting the first film 22f on the support 20f again.
This point will be described in detail later.

The first film 22f is not particularly limited as long as the culture solution M can be maintained for a certain period, that is, a period for culturing the microalgae P, and a resin film, a metal film, a woven fabric, a non-woven fabric, a combination thereof, etc. It is preferable to use a resin film. In addition, as a fiber which comprises a woven fabric and a nonwoven fabric, plastics, metals, wood material, grasses, glass fiber, a carbon fiber etc. are mentioned, for example.

When the obtained microalgae P is separated and recovered from the first film 22f in the recovery step to be described later, a material having a surface property having a suitable affinity with the microalgae P should be used. Thus, the recovery rate of microalgae P can be improved.
Here, as a material having a surface property having appropriate affinity with the microalgae P, for example, glass (silicate glass), silicone (silicone rubber, silicone resin, etc.), fluorocarbon resin (PTFE, PCTFE, CTFE, etc.) , PVDF, PVF, PFA, FE, ETFE, ECTFE, etc.), polyvinyl chloride, polyolefins (polyethylene, polypropylene, etc.) and the like.

Further, in the recovery step, when the obtained microalgae P is collected as it is without peeling from the first film 22f and used as a film-like fuel, the surface having high affinity with the microalgae P It is preferred to use a material having properties.
When the first film 22f is also used as a fuel, a film (polyethylene, polyolefin such as polypropylene, polyethylene terephthalate, etc. which does not generate toxic substances such as chlorine even if it is burned together with the dried algal cells as the first film 22f) And polyesters, polyurethanes, polyamides, silicone resins, fluorine resins, composite films combining these, etc. are preferable, and biomass-derived films (bioPE, biopolyester, polylactic acid, and the like) from the viewpoint of environmental load (preventing global warming). Acetyl cellulose etc. is preferred.

A recovery step may be performed after performing a plurality of main culture steps and a liquid volume reduction step. By performing the recovery step after performing the main culture step and the liquid volume reduction step a plurality of times, the number of times of implementation of the recovery step can be reduced, and the overall cost can be reduced. In this case, it is preferable to dry the algal cells after the liquid amount reduction step. By drying the algal cells, it is possible to prevent the reduction of accumulated oil and to prevent the reduction of algal biomass.

The support 20 f is a substantially rectangular box-shaped container whose one surface is open. As shown in FIG. 6, the open surface of the support 20f is formed smaller than the first film 22f, and while the end of the first film 22f is locked to the edge of the open surface, the inside of the container is formed. By arranging the first film 22f, the first film 22f is concavely supported.
As the support 20f, the same container as the container 16a shown in FIG. 2 can be used.

Further, the shape of the support is not limited to the container shown in FIG. 6, and a frame having no bottom may be used as the support 20h as in the culture apparatus 10h shown in FIG. The support 20h of the culture device 10h shown in FIG. 7 holds the end of the first film 22f in the frame and supports the first film 22f so as to form a recess in the frame.
Similar to the above-mentioned support 20f, such a frame-shaped support 20h may be a frame-like form using plastic, soil, concrete, wood, metal, clay, pottery, or a combination of two or more of them. What was formed into is mentioned.

Alternatively, a plurality of pillars may be used as a support. For example, the four columns are arranged so that the line connecting the columns forms a rectangular shape, and the end of the first film 22f is engaged with the tip of each column to form a recess. Alternatively, the first film 22f may be supported.

When the storage member is constituted by the first film and the support, the drainage pipe 18 may be connected to the support, and the first film may be formed with a drainage hole.

The support may also have grooves or the like for alignment with the first film.

Here, the storage member may be configured to include the second film in which a plurality of through holes are formed on the outermost surface on the side in contact with the culture solution M. Culture apparatus 10g shown in FIG. 8 is a schematic cross-sectional view of another example of the culture apparatus according to the invention, except that storage apparatus 12g is provided with container 16a and second film 24g instead of storage member 12a. Since the configuration is the same as that of the culture apparatus 10a of FIG. 2, the same reference numerals are given to the same parts, and different parts will mainly be described in the following description. The second film 24g has two surfaces, the front side (the side closer to the liquid surface of the culture solution M) and the back side (the side closer to the first film).

The storage member 12g of the culture apparatus 10g includes a container 16a and a second film 24g in which a plurality of through holes are formed.
The second film 24g is formed larger than the open surface of the container 16a, and is disposed in the container 16a with its end locked to the edge of the open surface.
The second film 24g may have the property of permeating the culture solution M and having difficulty in permeating microalgae. For example, mesh (wire mesh, plastic mesh, cloth, yarn, string, grass, fibers, etc.) And those having a large number of holes in the film, and the like.

Thus, when the second film 24 g which permeates the culture fluid M and hardly permeates the microalgae is disposed on the culture fluid M side, the liquid volume of the culture fluid M is reduced in the liquid volume reduction step described later. Since the cultured microalgae P remains on the second film 24g, the culture solution M can be more easily reduced. Also, as in the case of the first film described above, after the liquid volume of the culture solution M is reduced in the liquid volume reduction step and the cultured microalgae P is brought into contact with the second film 24g, in the recovery step, The microalgae P can be easily recovered by moving (detaching) the second film 24g from the container 16a.

In addition, when using the second film 24g which permeates the culture solution M and does not easily permeate the microalgae P, the second microalgae P, particularly a biofilm obtained by the culture, is collected. It is very efficient because it can remove moisture at the same time as recovering 24 g of film.

Here, in the case of using the microalga which forms a biofilm on the liquid surface, to which the present invention is suitably applied, the cultured microalga forms a solid in the form of a film; It is hard to penetrate the hole. Therefore, even if the size of the through hole formed in the second film 24g is relatively large, the microalgae P remains on the second film 24g, so that the water can be removed more easily, which is preferable. is there.

In the example shown in FIG. 8, the container 16a for storing the culture fluid M is combined with the second film 24g in which a plurality of through holes are formed, but the present invention is not limited thereto. The second film 24g may be combined with the culture apparatus 10b shown, the culture apparatus 10f shown in FIG. 6, or the culture apparatus 10h shown in FIG.

The culture apparatus according to the present invention preferably comprises a first film moving member for moving the first film. In this case, the culture solution of the cultured microalga is brought into contact with the first film by reducing the volume of the culture solution in the volume reduction step, and the first film is removed from the support in the recovery step. It can be moved to recover cultures of microalgae. For example, in the culture apparatus 10f shown in FIG. 6, as the first film 22f is disposed on the outermost surface of the storage member in contact with the culture solution M, the first film 22f is used as the first film moving member The microalgae P can be recovered by moving the first film 22 f using not shown. However, the member disposed on the outermost surface of the storage member in contact with the culture solution M is not limited to the first film, and is generally removable from the film-like structure having flexibility and further the outermost surface of the storage member. Any structure may be used, and the structure may be desorbed to recover the microalgae P together with the structure.

The culture apparatus according to the present invention preferably comprises a second film moving member for moving the second film. In this case, the culture solution of the cultured microalga is brought into contact with the second film by reducing the volume of the culture solution in the volume reduction step, and the second film is used as the storage member in the recovery step. The microalgal culture can be recovered by moving from the outermost surface in contact with the culture solution. Also, in this case, in the liquid volume reduction step, the second film is moved from the outermost surface of the storage member on the side in contact with the culture solution to a position from the liquid surface of the culture solution to Reducing the liquid volume to bring the cultured microalgal culture into contact with the second film, and in the recovery step moving the second film from its position to recover the microalgal culture You can also. Furthermore, in this case, in the liquid volume reduction step, the liquid volume of the culture solution is reduced to such an extent that the culture of the cultured microalga does not substantially contact the second film, and the second film is The microalgal culture can also be recovered by moving it from the outermost surface of the storage member in contact with the culture solution. For example, in the culture apparatus 10g shown in FIG. 8, the second film 24g is used as the second film moving member in the recovery step as a configuration in which the second film 24g is disposed on the outermost surface of the storage member in contact with the culture solution M. The microalgae P can be recovered by moving the second film 24 g by using (not shown). However, the member positioned on the outermost surface of the storage member in contact with the culture solution M is not limited to the second film, and is generally removable from the film-like structure having flexibility and further the outermost surface of the storage member Any structure may be used, and the structure may be desorbed to recover the microalgae P together with the structure.

A flexible film-like structure represented by the first film or the second film may be folded or folded as required, or may be wound into a roll, in addition to lifting and pulling. It is preferable because various recovery methods can be selected according to the situation, such as taking.

The culture apparatus according to the present invention preferably comprises a first winder for winding the first film in a roll. In this case, in the recovery step, the first winding machine can recover the microalgal culture by winding the first film together with the microalgal culture in a roll. In addition, the culture apparatus according to the present invention preferably further comprises a first recovery member for peeling the culture of the microalga from the first film. When the culture apparatus according to the present invention includes the first winding machine and the first recovery member, the first winding machine winds the first film into a roll in the recovery step. The recovery member of the present invention can peel off the microalgal culture from the first film to recover the microalgal culture.

Moreover, it is preferable that the culture apparatus according to the present invention comprises a second winding machine for winding the second film in a roll. In this case, in the recovery step, the second winding machine can recover the microalgal culture by rolling up the second film together with the microalgal culture in a roll. In addition, the culture apparatus according to the present invention preferably further comprises a second recovery member that peels the culture of the microalga from the second film. When the culture apparatus according to the present invention includes the second winding machine and the second recovery member, the second winding machine winds the second film in a roll in the recovery step, The recovery member of the present invention can peel off the microalgal culture from the second film to recover the microalgal culture.

The thickness, strength, etc. of the first film 22f or the second film 24g are not particularly limited, but in the case of collecting microalgae using the first film 22f or the second film 24g in the recovery step It is desirable to have sufficient strength not to break at the time of recovery. On the other hand, light weight is also important for large area work. Moreover, when winding up in roll shape, the thinner one is preferable.
From the above point of view, the thickness of the first film 22f and the second film 24g is preferably 1 μm to 10000 μm, more preferably 5 μm to 1000 μm, and most preferably 10 to 500 μm, although it depends on the material. If the thickness is 10 μm or more, the first film or the second film can be handled without breakage, and if the thickness is 500 μm or less, the problem due to weight can be avoided.

In addition, in the culture apparatus 10f shown in FIG. 6, the storage member 12f is configured to include the first film 22f and the support 20f, but is not limited thereto, and the first film 22f and the support 20f are not limited thereto. Between the two, one or more third films may be provided. FIG. 9 shows a schematic cross-sectional view of another example of the culture apparatus used in the culture and recovery method of the present invention. Here, the culture apparatus 10j shown in FIG. 9 is provided with a storage member 12j provided with a third film 26j between the first film 22f and the support 20h, instead of the storage member 12h of FIG. The configuration is the same as that of the culture apparatus 10h shown in FIG. The third film 26 j has two surfaces, the front side (the side closer to the culture solution M) and the rear side (the side farther from the culture solution M).

The storage member 12j of the culture apparatus 10j includes a first film 22f, a support 20h for supporting the first film, and a third film 26j disposed between the first film 22f and the support 20h. Prepare.
The third film 26 j is not particularly limited, but the same film as the first film 22 f can be used.

Thus, by arranging one or more third films 26j between the first film 22f and the support 20h, the cultured microalgae P is recovered together with the first film 22f, and then the outermost surface side is obtained. Since the third film 26j of the second film 26j can be used as the first film, it becomes possible to reuse without re-installing the collected first film 22f, and it is possible to culture the microalga continuously. , Can improve the manufacturing efficiency.
Note that the third film used as the first film is regarded as the first film.

When the support is formed of soil, clay or the like, or when the first film 22f is in contact with the installation surface (in particular, the soil surface) of the apparatus as in the culture apparatus 10h shown in FIG. The back side of the first film 22f is contaminated by microorganisms and the like present in the installation surface and the soil, and the surface side (culture medium M side) is also contaminated when the first film 22f is wound up, etc. The microorganisms etc. may be mixed in the cultured microalgae P.
On the other hand, disposing the third film 26 j between the first film 22 f and the support (installation surface) prevents the first film 22 f from being in direct contact with the support or the installation surface. It is preferable because it can prevent the mixing of microorganisms and the like present in the installation surface and in the soil.
In addition, when installing a 3rd film in order to prevent mixing of a microbe etc., in order to prevent that a 3rd film moves at the time of a movement of a 1st film, a 3rd film Is preferably fixed to a support or the like.

From the above viewpoint, when the microalga is continuously cultured using the third film as a new first film after recovery of the first film, the first film may be used as the third film. It is preferred to use the same film as the film.
On the other hand, in the case where the third film is installed to prevent the contamination of microorganisms and the like, it is preferable that the third film is hardly soiled by the above-mentioned microorganisms and the like, hardly decomposed and capable of maintaining strength for a long time.

In addition, when the storage member is configured to include at least one of the first film and the second film, and the first film or the second film is moved to recover the microalgae in the recovery step, It is preferable to perform a treatment to reduce the friction between the first film or the second film and a member in contact with the films. Specifically, a method of applying a substance that reduces the frictional force to the surface of the first film or the surface of the second film, a method of surface treating the surface, or a substance that reduces the frictional force between these members The method to make it exist is illustrated. When both the first film and the second film are provided, at least one of the surface of the first film and the surface of the second film is preferably treated to reduce friction. Moreover, when providing a 3rd film, it is preferable to perform the process which reduces friction on the surface of a 3rd film.
Thereby, the frictional force at the time of moving a film by winding-up etc. reduces, Loads, such as winding-up, can be reduced and the required energy amount can be reduced and it is preferable.
Substances that reduce friction can include lubricating oils, greases, waxes, and granules with uniform particle sizes, such as flour and starch powder. These substances may decrease with the number of times the film is used, and can be used additionally as needed.

Further, in the illustrated example, the storage member is configured to include one liquid holding unit, but is not limited thereto, and may be divided into a plurality of sections by one or more partitions. In this case, each compartment is preferably capable of holding the culture solution for a predetermined period.

Further, as described above, the storage member may be provided with a drain pipe, a valve or the like for the purpose of draining in order to reduce the amount of culture solution. In the liquid volume reduction step, when draining to reduce the liquid volume of the culture solution, the bottom surface of the liquid holding portion of the storage member is preferably inclined so as to become lower toward the drainage pipe side.
It is possible to drain easily by changing the water depth so that drainage can be performed from the lower side by tilting the bottom surface of the liquid holding portion.
The method of inclining the bottom surface of the liquid holding portion is not particularly limited, and the bottom surface of the liquid holding portion may be inclined relative to the bottom surface of the storage member, or as shown in FIG. The storage member 12a may be installed on the inclined installation surface G. Alternatively, as shown in FIG. 11, the bottom surface of the liquid holding portion may be inclined by sandwiching the bulking member 30 between one end side of the bottom surface of the storage member 12a and the installation surface G. Alternatively, as in a culture apparatus 10t shown in FIG. 12, the shape of the storage member 12t may be a shape having a step with a deep part of the liquid holding portion, and may be installed on the installation surface G having a step. Alternatively, as in the storage member 12 u of the culture device 10 u illustrated in FIG. 13, only a part of the liquid holding unit may be inclined.
In addition, it is preferable to make the largest height difference of the liquid holding | maintenance part which culture | cultivates into less than the same as the water depth at the time of culturing.
Further, when the culture solution is drained to reduce the amount of liquid, it is preferable to provide a water channel for the liquid drained from the drain pipe around the storage member. For example, piping such as a gutter may be disposed, and as shown in FIG. 14, a groove H may be formed in the installation surface G to serve as a water channel for liquid drained from the drain pipe 18.

Further, the storage member may be disposed in the recess of the installation surface.
For example, as shown in FIG. 15, the storage member 12 a may be disposed in the recess Gb of the installation surface G. Alternatively, as shown in FIG. 16, a convex portion Gd may be formed on the installation surface G, and the storage member 12 a may be arranged in a region surrounded by the convex portion Gd.
Thus, by disposing the storage member in the recess of the installation surface, the temperature change of the culture solution can be suppressed, which is preferable.
Of course, the installation surface G may be indoor or outdoor.

Here, as described above, soil, clay or the like can be used as the material of the container or the support that constitutes the storage member.
The configuration in which a container or support made of such soil or clay is disposed in a recess on the installation surface as shown in FIGS. 15 and 16 is formed using soil or clay on the ground which is the installation surface. Can. Such a configuration can be said to have formed a bowl on the ground.

For example, the culture apparatus 10r shown in FIG. 17 is a container in which the container 16r is formed by solidifying the soil on the ground which is the installation surface to form a recess. In the illustrated example, the second film 24g is disposed in the liquid holding portion of the container 16r to configure the storage member 12r.
Moreover, the culture apparatus 10n shown in FIG. 18 solidifies the soil of the ground which is an installation surface, and forms the support body 20n by forming a recessed part. The storage member 12n is configured by the support 20n and the first film 22f.
Although such a container 16r or a support 20n is integrated with the ground which is the installation surface, the region where the soil is hardened to form a recess is the container 16r or a support 20n.

When the container 16r or the support 20n is formed into a bowl shape, combustion ash, concrete, cement, plastic, pottery, wood, grass, in addition to ground components such as soil, clay, sand, annual ash, volcanic ash, etc. Solids such as metals; Liquids such as water; Gases such as air and gas; Supercritical fluids such as supercritical carbon dioxide; etc. may be combined.
In the case where the container 16r or the support 20n is made of soil, clay or the like, it is not limited to the structure using soil or clay derived from the ground to be installed, but using soil or clay obtained from other places It is also good.
Soil, clay, sand, burning ash and volcanic ash are low cost and readily available, and in some cases may be discarded as waste, and may be available at the culture site. Moreover, these materials can ensure both shape stability and thermal conductivity by adjusting the water content. In addition, the adjustment of the moisture content and the selection of the particle size can reduce the surface irregularities. When the first film is used, the unevenness on the inner side of the liquid holding portion can be reduced by reducing the unevenness of the support, so the water depth of the liquid holding portion can be made shallower, and the amount of water used There is an advantage of being able to reduce drying and of facilitating drying.

In addition, when processing the ground to form a container or support, the container or support may be formed by digging the ground or forming a bowl on the ground. Is preferred.
Moreover, such a container or support is preferably formed using a paddy field, a fallow field, agriculture, an abandoned cultivation site or the like, from the viewpoint that a large area suitable for culture can be obtained inexpensively. In particular, a paddy field or a fallow field is suitable also from the point which originally has the function of maintaining, replenishing and draining water in advance.
However, it may be changed to a form suitable for culture of microalgae. For example, if a road is installed, the asphalt may be removed, the soil may be exposed, and then the container or support may be formed by digging a hole so that the culture solution can be held.

Also, depending on the land topography, the land may be smoothed or divided into small sections. In addition, it is preferable that the surface in contact with the culture solution be hydrophilic so that a small amount of culture solution can be easily spread over a large area.
The wall surface of the container or support may be shared with the adjacent container or support. By doing this, it is possible to install more culture devices in a certain area, and it is possible to reduce the time and effort for forming the wall surface. For example, a trough formed of soil may be shared with an adjacent container or support.

<< Culture fluid >>
The culture solution is retained in the reservoir of the reservoir member. The culture solution is a liquid medium. Details of the culture solution will be described later.

«Shielding member»
The culture apparatus used in the present invention has a shielding member disposed above the storage member so as to cover at least a part of the liquid surface of the culture solution held by the storage member when viewed from above in the vertical direction. It is preferable to have.
The shielding member preferably covers at least a part of the liquid surface of the culture solution, and if there is no restriction, more preferably covers 50% or more of the liquid surface area of the culture solution, and further covers 90% or more It is more preferable to cover 95% or more, and it is particularly preferable to cover 100%, but according to securing of work space, installation of other members, restrictions on topography, etc., a ratio to cover the liquid surface of culture fluid Can be set as appropriate.
Preferably, the shielding member has a region through which light of at least a part of the wavelength can be transmitted. Here, it is preferable that at least a part of the wavelength includes the wavelength of light necessary for the culture of the microalga.

The shielding member protects the culture solution and the cultured microalga from factors that inhibit culture due to natural conditions such as rain, wind, and dryness, and also prevents or suppresses contamination of foreign matter or foreign organisms from the outside. is there. In addition, the shielding member is to prevent and suppress the natural evaporation of the culture solution.

For example, the shielding member 14a of the culture device 10a shown in FIG. 1 and FIG. is set up.
In addition, the shielding member 14a has light transmissivity for transmitting light of a predetermined wavelength.

Moreover, as shown in FIG. 2, the air layer N exists between the shielding member 14a and the culture solution M (microalga P which culture | cultivated).
By forming such an air layer N, carbon dioxide necessary for the culture of the microalgae P can be supplied during the culture.

The material constituting the shielding member 14 is not particularly limited as long as at least a part of the material can transmit light, but, for example, plastic, glass, soil, concrete, cement, wood, metal, clay, pottery, Among these, combinations of two or more may be mentioned. Further, the shape of the shielding member 14 is, for example, a material that can be deformed, such as a film, a sheet, a cloth, a net, a knitted yarn, etc. even if it is a rigid structure such as a wall or a plate. Or any combination of these.

Moreover, as a material which comprises the shielding member 14, it is preferable to use the raw material comprised from an organic polymer compound, an inorganic compound, a metal, and those complexes, for example. It is also possible to use mixtures thereof.
Examples of organic polymer compounds include polyethylene derivatives, polyvinyl chloride derivatives, polyester derivatives, polyamide derivatives, polystyrene derivatives, polypropylene derivatives, polyacryl derivatives, polyethylene terephthalate derivatives, polybutylene terephthalate derivatives, nylon derivatives, polyethylene naphthalate derivatives, polycarbonate derivatives , Polyvinylidene chloride derivative, polyacrylonitrile derivative, polyvinyl alcohol derivative, polyether sulfone derivative, polyarylate derivative, allyl diglycol carbonate derivative, ethylene-vinyl acetate copolymer derivative, fluorine resin derivative, polylactic acid derivative, acrylic resin derivative, Ethylene-vinyl alcohol copolymer, ethylene-methacrylic acid copolymer, etc. can be used.
Glass, ceramics, concrete or the like can be used as the inorganic compound. As the metal compound, alloys such as iron, aluminum, copper and stainless steel can be used.

Above all, as a material constituting the shielding member 14a, it has light transmissivity, and it is lightweight while having the workability of cultivation and recovery, necessary strength, and is installed in a shape according to the shape of the storage member Plastics which are easy to do are preferred. Examples of plastics include, for example, polyolefins such as polyethylene and polypropylene, polyesters such as vinyl chlorides and polyethylene terephthalate, polylactic acids, acetyl celluloses, polyurethanes, polyamides, silicone resins, fluorine resins, Composite films etc. which combined two or more of these, etc. are mentioned. Further, it is also preferable to use glass (inorganic glass) in particular from the viewpoint of transparency and strength.

In the example shown in FIG. 2, the shielding member 14a has a plate shape and is disposed in close contact with the storage member 12a. However, the present invention is not limited to this. The shape and position of the shielding member is the upper side in the vertical direction. If it is possible to cover at least the liquid surface of the culture solution M and to allow an air layer to be present between the liquid surface of the culture solution M and the second structure when viewed from the point of view, there is no particular limitation. I will not.

That is, the shape of the shielding member may be, for example, flat, inclined, uneven, irregular, or a combination of at least one of these. Among these, it is preferable that it is an inclined shape or a convex shape which can be quickly removed since rain, snow, or the like hardly accumulates on the shielding member.
Also, the shielding member may be disposed apart from the storage member.

For example, the shielding member 14c of the culture device 10c shown in FIG. 19 is larger than the storage member 12a and has an arch-like curved shape, and the storage member 12a is disposed inside the arch of the shielding member 14c.
Thus, when the shielding member 14c is disposed above the storage member 12a so as to cover the liquid surface of the culture fluid M held by the storage member 12a when viewed from above in the vertical direction, the storage member 12a It does not have to be in contact with

Further, as in a shielding member 14d of a culture apparatus 10d shown in FIG.
Alternatively, as in a culture apparatus 10e shown in FIG. 21, a support 28e may be provided to support the shielding member 14a so as to be disposed above the storage member 12a. At that time, it is preferable to support the shielding member 14a by inclining with respect to the horizontal plane.
Alternatively, as in a culture apparatus 10i shown in FIG. 22, a support for supporting a shielding member 14d having a convex shape combining two inclined surfaces inclined with respect to a horizontal surface to be disposed above the storage member 12a The body 28 may be provided.
The support 28 is not particularly limited as long as it can support the shielding member 14a, and any structure such as a column or a wall may be used.

In addition, the shielding member preferably has a strength sufficient to be used for a long period of time in a culture environment. Therefore, it is also preferable to reinforce the shielding member by using walls, columns, beams, etc., and pipes, wires, etc. which are found in agricultural houses.

In addition, the shielding member may form a closed space alone or together with the storage member, but it is possible to easily open or remove a part of the area, take up, etc., when necessary. It is also preferable to make it openable. In addition, a space, an air vent or the like can be provided in advance as long as there is no hindrance to culture and the like. That is, it is preferable to keep the air layer N in communication with the atmosphere or in a communication state.
Thus, keeping the air layer N in communication with the atmosphere is preferable in that it becomes possible to take in carbon dioxide from the open air during culture.
In addition, the vent may be provided with a ventilating device such as a fan.

Further, it is preferable that at least a part of the shielding member is openable, removable, capable of being wounded, and the like. By this, the culture solution, the input operation of the seed algae which is the source of the culture, the observation of the culture condition, the addition of the culture solution or the seed algae, the input operation, the operation of reducing at least a part of the culture solution, the operation of recovering the culture There is an advantage that various tasks such as can be easily performed.

In addition, when the shielding member is used for a long time in various environments indoors and outdoors, rain, snow, soil, dust, dust, microorganisms, or other various dirt causing substances fly, and the shielding member However, it may cause problems such as dirt and light transmission. Therefore, it is preferable that the shielding member is hard to adhere to those components, and that even if it adheres, it can be easily removed by rain or a simple water washing operation. For example, as a material, a fluorine resin or silicone resin to which dirt does not easily adhere may be used, or the surface may be subjected to a hydrophobization treatment, a hydrophilization treatment, an antifouling treatment, or the like. Moreover, you may coat the substance which suppresses the adhesion of microorganisms.

In addition, the internal water may grow as a water droplet on the inner surface (surface on the storage member side) of the shielding member during culture, and this may drop to the liquid surface of the culture solution to inhibit the growth of the biofilm on the liquid surface. is there. In order to prevent such a phenomenon, the material of the shielding member may be a material on which droplets do not easily grow, or a droplet may not easily drop.
Specifically, a glass plate, a light transmitting plastic plate, a plastic film (PE, PP, PET, fluorocarbon resin, acetyl cellulose, PLA), in particular, a glass or a film which has been treated so that water droplets are not easily attached to the inner surface. (For example, Takinas C. I. Kasei Co., Ltd., thickness 0.15 mm) and hydrophilic films (acetyl cellulose etc.) are preferable.

Alternatively, the inner surface of the shielding member may be provided with a structure in which water droplets are likely to fall, for example, a place where the structure is lowered, a portion which is a projection (such as a screw) of the structure, etc. If it is larger than the portion being carried out, it is preferable to provide an area in which the water droplet is likely to fall to the outside of the portion being cultured or to flow directly to the ground or the like. In addition, even when dropping to a portion where culture is being performed, it is preferable to set the area of the drop prone region of the water droplet formed inside the shielding member to 20% or less of the area of the portion where culture is being performed. It is more preferable to set it so that it becomes% or less.
In addition, a structure for receiving the water droplets may be provided under a place where the water droplets are particularly easily accumulated. The obtained water can be reused as distilled water or the like.

In addition, when the temperature and the liquid temperature are high for the purpose of controlling at least one of the air temperature of the air layer and the liquid temperature of the culture solution to a temperature range suitable for the culture of the microalga during the culture A film that suppresses the transmission of heat rays during fine weather, etc., and a film that maintains the temperature when the temperature or liquid temperature is low (especially in winter, when the amount of sunlight is low and the temperature is low, etc.) You may have as.
As an example of the former, there is a method of using an infrared ray reflective film, an infrared ray absorbing film, and in the case of the latter, two or more sheets of films stacked.

Moreover, in order to control the culture state of microalgae, according to the microalga to be cultured, a film having a characteristic that the transmittance of wavelength effective for growth is high, or conversely, the transmittance of wavelength unnecessary or harmful for growth Films having low properties can be used. Specifically, an ultraviolet reflective film, an ultraviolet absorbing film, a wavelength selective transmission film, a wavelength selective reflective film, a light scattering film or the like may be used as the shielding member.
The wavelength effective for growth is preferably about 360 nm to about 830 nm, more preferably about 380 nm to about 760 nm, and still more preferably about 400 nm to about 700 nm.
Wavelengths harmful to growth are generally less than about 360 nm. Electromagnetic waves having a wavelength of less than 360 nm may damage microalgal cells. In addition, electromagnetic waves having a wavelength of more than about 830 nm are not effective for the growth of microalgae. The light in the infrared region having a wavelength of more than about 830 nm is likely to increase at least one of the air temperature and the liquid temperature at the time of culture, which may inhibit the growth of microalgae. However, when the culture temperature is low for the culture of the microalga to be cultured, light in an infrared region having a wavelength of more than about 830 nm may be used to raise the liquid temperature or the air temperature of the culture solution.

In addition, in the case of a film, a sheet, or the like, at least a part of the shielding member may be burnt as a fuel together with the dried algal cells. In this case, as the shielding member, a film (PE, polyester such as PP, PET, polyurethane, polyamide, etc.) which does not generate toxic substances such as chlorine even when burned together with the dried algal cells is preferable, and further environmental load (warming prevention) From the viewpoint of), biomass-derived films (bioPE, biopolyester, polylactic acid, acetyl cellulose, etc.) are preferred.

Further, in the example shown in FIG. 2, the shielding member 14 a is configured to have the light transmitting property over the entire surface, but is not limited thereto, and has a region capable of transmitting light of at least a part of wavelengths. It is good also as composition.
For example, as in a shielding member 14k of the culture device 10k shown in FIG. 23, the region T disposed vertically above the liquid surface of the culture solution may be configured to transmit light of at least a part of the wavelength. In this case, the region T is formed of a light transmissive material.

Here, light of at least a part of wavelength refers to light of a wavelength that microalgae can utilize for photosynthesis, generally, about 360 nm to about 830 nm is preferable, about 380 nm to about 760 nm is more preferable, and about 400 nm Further preferred is about -700 nm. Within this range, light of a wavelength that the microalgae can effectively use for photosynthesis is included.

Although the wavelength of light necessary for microalgae to carry out photosynthesis varies depending on the type and combination of photosynthetic pigments that microalgae has, microalgae usually has one or more photosynthetic pigments in addition to chlorophyll a, and Since the photosynthetic pigment absorbs light of a wavelength which can not be absorbed by chlorophyll a and passes energy to chlorophyll a, the light including the absorption wavelength of chlorophyll a is not absolutely required.
The main photosynthetic pigments possessed by microalgae include chlorophyll a, chlorophyll b, chlorophyll c, phycobilin pigments and carotenoid pigments. In general, microalgae that perform photosynthesis often contain one or more of chlorophyll b, chlorophyll c, phycobilin-based pigment and carotenoid-based pigment in addition to chlorophyll a as a photosynthetic pigment. For example, the green algae such as Chlamydomonas (Clamydomonas), Chloroccoccum, Botryococcus, Tetracystis, Characium, Protochiphon, Protosiphon, Haematococcus, etc. Have chlorophyll b in addition to chlorophyll a, gray algae and red algae have phycobilin based pigments in addition to chlorophyll a, chlora lacunion algae and euglena have chlorophyll b in addition to chlorophyll a, and crypt algae have chlorophyll In addition to a, it has chlorophyll c and phycobilin-based pigments, and haptoalgae has chlorophyll c and caroteno in addition to chlorophyll a. Inorganic dyes such as brown algae, golden algae, rafido algae, yellow green algae, diatoms, etc. have chlorophyll a in addition to chlorophyll a, and dinoflagellates have chlorophyll b and chlorophyll c in addition to chlorophyll a. And phycobilin-based pigments, and one or more of carotenoid-based pigments, chromella algae have chlorophyll a, and cyanobacteria have chlorophyll b or chlorophyll d in addition to chlorophyll a.
The absorption peak wavelength of chlorophyll is, for example, 90% acetone-water mixed solvent, chlorophyll a is about 430 nm and about 664 nm, chlorophyll b is about 460 nm and about 647 nm, chlorophyll c ₁ is about 442 nm and about 630 nm, chlorophyll c ₂ Are about 444 nm and about 630 nm, and chlorophyll d is about 401 nm, about 455 nm and about 696 nm.

Furthermore, the shielding member may be integrated with a member constituting the storage member, for example, the first film. For example, the culture apparatus 90 shown in FIG. 24 is a tubular (tube-like) member 92 formed by integrating the shielding member and the first film, and a support 94 for supporting the member 92. The culture solution M is held inside a member 92 formed in a tubular shape.

Further, in the example shown in FIG. 1, one shielding member 14a is configured to cover one storage member 12a. However, the present invention is not limited to this, and one shielding member may be configured to cover two or more storage members. Good.
For example, the culture apparatus 100 shown in FIG. 25 has a configuration in which one shielding member 14a is disposed so as to cover two storage members 12f consisting of a first film 22f and a support 20f. As shown in the figure, the shielding member 14a is supported vertically above the storage member 12f by two supports 28e which are spaced apart so as to sandwich the two storage members 12f.

<< Temperature control part / liquid temperature control part >>
The culture apparatus preferably includes at least one of an air temperature control unit that controls the temperature of the air layer N and a liquid temperature control unit that controls the temperature of the culture solution M.
By providing the temperature control unit, it is possible to control the temperature of the air layer N within the temperature range suitable for the culture of the microalga during the culture. Further, in the recovery step, the temperature of the air layer N can be controlled to a temperature higher than the temperature range suitable for the culture of the microalga, and the moisture content of the culture can be reduced to accelerate the drying.
By providing the liquid temperature control unit, the temperature of the culture solution M can be controlled within the temperature range suitable for the culture of the microalga during the culture. In addition, in the recovery step, the temperature of the culture solution M can be controlled to a temperature higher than the temperature range suitable for the culture of the microalga, and the moisture content of the culture can be reduced to accelerate the drying.

The range of the air temperature of the air layer N suitable for the culture of microalgae and the liquid temperature of the culture solution M varies depending on the type of microalgae to be cultured, but it is generally about 10 to about 50 ° C, preferably about 15 to about 45 ° C It is near.

The culture temperature can be selected according to the type of microalgae, but the liquid temperature of the culture solution is preferably 0 ° C. or more and 90 ° C. or less. The temperature is more preferably 15 ° C. or more and 50 ° C. or less, and particularly preferably 20 ° C. or more and less than 40 ° C. When the culture temperature is 20 ° C. or more and less than 40 ° C., the growth rate of microalgae is sufficiently fast.

As the air temperature control unit or the liquid temperature control unit, for example, a configuration in which a member such as a liquid such as water or a gas such as air is caused to flow as a heat transfer medium may be provided around the storage member.

For example, the culture apparatus 50 shown in FIG. 26 includes a second container 52 having an opening larger than the outer shape of the storage member 12a. In the second container 52, the storage member 12a is installed, and water W, which is a heat transfer medium, is placed in the second container 52. A part of the storage member 12a is immersed in the water W from the bottom surface side and installed on the support protrusion 54.
Such a configuration can suppress changes in liquid temperature and air temperature. Moreover, the storage member 12a is simply immersed in the water W not only to suppress changes in the liquid temperature and the air temperature, but also by controlling the temperature of the water W, the temperature of the culture solution M or the air layer N is a microalga. It may be controlled to a temperature range suitable for culture of Preferably, the water W in the second container 52 can be circulated.

Further, the culture device 60 shown in FIG. 27 includes a storage member 62, and a heat transfer pipe 64 which communicates the storage member 62 from the outside to the inside (liquid holding portion). The heat transfer pipe 64 is a pipe through which the heat transfer medium can flow.
With such a configuration, by flowing the heat transfer medium into the heat transfer pipe 64, it is possible to control the temperature of the culture solution M and the air layer N.

Moreover, you may provide the ventilation part connected to an air layer as an air temperature control part or a liquid temperature control part. Specifically, it is also preferable that at least a part of the area between the storage member and the shielding member can be easily opened, removed, taken up, etc., and can be opened when necessary. In addition, a space, an air vent, or the like can be provided in advance without bringing the storage member and the shielding member into close contact with each other as long as there is no hindrance to culture and the like. Also, by changing the height of the shielding member continuously and providing a structure that can be ventilated to at least the high side, a gas with a temperature higher than the outside temperature in the air layer N using natural convection. Can also be configured to be able to be released from the higher side of the shielding member to the outside. By setting it as such a structure, the temperature rise of the culture solution M can also be suppressed especially. The passage of gas in the aeration unit may be natural ventilation, or the passage of gas in the aeration unit may be controlled by the aeration control unit. Examples of means for controlling ventilation include forced ventilation means such as a fan.

巻取 Winding machine》
The culture apparatus according to the present invention further comprises a winding machine (first winding machine) for winding the first film and a winding machine (second winding machine for winding the second film). It is preferable to provide both or one of these. Here, the first winding machine and the second winding machine may be separate winding machines, or one winding machine, and the film to be wound is the first film. Sometimes it may be a first winder, and it may be a second winder when the film to be taken up is a second film. The details will be described in the description of the recovery step.

<< Collection member >>
The culture apparatus according to the present invention further recovers the collection member (first collection member) for collecting the culture of microalgae on the first film and the culture of microalgae on the second film It is preferable to provide both or one of these recovery members (second recovery members). Here, the first recovery member and the second recovery member may be separate recovery members, or one recovery member, and the microalgal culture to be recovered is on the first film. When it is a thing, it is good also as a 2nd collection | recovery member, when it is set as a 1st collection | recovery member and culture | cultivation of the micro algae to collect | recover is 2nd film-like. The recovery member will be described in detail in the description of the recovery process.

<< Related to Other Culture Equipments >>
In order to prevent the culture apparatus from natural disasters such as typhoons, storms, gusts and the like, windproof equipment such as a windproof wall may be installed around the culture apparatus separately from the elements constituting the culture apparatus. Even when a windproof facility is installed, it is desirable to have a structure and position that does not significantly block the light necessary for the microalgal culture.
Moreover, you may have an apparatus which supplies the carbon dioxide required for culture | cultivation of micro algae, and a light source etc. which supply light required for culture of micro algae.

<Fine algae>
The microalgae used in the culture and recovery method of the present invention is not particularly limited, and can be appropriately selected according to the purpose. Here, microalgae refers to microalgae whose individual existence can not be recognized by the naked eye of a person. The microalgae may be either prokaryote or eukaryote.

Examples of microalgae used in the culture and recovery method of the present invention include green algae (Chlorophyta), gray algae (Glaucophyta), red algae (Rhodophyta), chlora lacunion algae (Chlorarachniophyta), euglena (Euglenophya), cryptoalga ( Microalgae belonging to any of Cryptophyta), Brown algae (Phaeophyta), Haptophyta (Haptophyta), Heterophyta (Heterokontophyta), Dinophyta (Dinophyta), Chromella algae (Chromerida), Cyanobacteria, etc. Can be mentioned. The microalgae may have an undefined taxonomic group, and more preferably molecular phylogenetically it has been shown to be in or closely related to these taxa.

In the culture and recovery method of the present invention, microalgae can be used singly or in combination of two or more. If it is in a symbiotic relationship with another organism, it may be used with that organism.

Moreover, as a microalga used by the culture | cultivation and collection | recovery method of this invention, what will be in the state which floated substantially in the liquid surface before a collection process is preferable. Here, the state of floating on the liquid surface refers to a state in which about 50% or more of the algal biomass present in the culture liquid is present on the liquid surface, regardless of whether a biofilm is formed on the culture liquid surface or not. .

Moreover, as a microalga used by the culture | cultivation and collection | recovery method of this invention, what can form a biofilm in the liquid surface of a culture solution is preferable. The biofilm will be described later.

As microalgae capable of forming a biofilm on the liquid surface of the culture solution, green algae (green algae) or diatoms (non-uniform algae) are preferable.

As microalgae capable of forming a biofilm on the liquid surface of the culture solution, Botryococcus sp. Botryosphaerella sp. , Chlamydomonas sp. , Chloroccum sp, Chlamydomonadaceae sp. , Tetracystis sp. , Characium sp. Proto Chiffon (Protosiphon) sp. Or Haematococcus (Haematococcus) sp. Those belonging to are more preferred.

As microalgae capable of forming a biofilm on the liquid surface of the culture solution, Botryococcus sudeticus (Botryococcus sudeticus) or Chlorococcum sp. Those belonging to are more preferred.

As a microalga capable of forming a biofilm on the liquid surface of the culture solution, a microalga strain or chlorococum having the same taxonomical properties as Botryococcus sudeticus FERM BP-11420 strain or its microalgal strain (Chlorococcum) sp. Even more preferable is a microalgal strain having the same taxonomical properties as the FERM BP-22262 strain or its microalgal strain, and the 73rd nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 1 among the nucleotide sequences of the gene region encoding 18S rRNA The nucleotide sequence of the portion corresponding to the nucleotide sequence of nucleotides 1 to 1722, and the nucleotide sequence of nucleotides 73 to 1722 of the nucleotide sequence shown in SEQ ID NO: 1, at least 99.94% (rounded off to the third decimal place) Still more preferred is a microalga having a sequence identity of Especially preferred are microalgae having the same taxonomical properties as the FERM BP-22262 strain or its microalgae strain.

There is no restriction | limiting in particular as a method to obtain a microalga, According to the objective, it can select suitably, For example, the method of extract | collecting from nature, the method of using a commercial item, the method of acquiring from a preservation organization or a depository It can be mentioned.

<< Chloroccocum sp. Taxonomical properties of FERM BP-22262 strain (FFG039 strain)
Chloroccum sp. The taxonomical properties of the FERM BP-22262 strain are shown below.
(Morphological properties)
It is circular. When static culture is performed, a film-like structure is formed on the liquid surface. Produce oil.
(Cultural properties)
Culture medium: CSiFF04 medium or CSi modified medium (CSiFF04 medium is a modification of CSi medium. The composition is shown in Table 1. The pH is adjusted to 6.0 and the latter to 7.0 with NaOH or HCl, respectively. The culture solution can be autoclaved under conditions of 121 ° C. and 10 min.)

Culture temperature: It can be cultured at 15-25 ° C.
Culture time: 2 to 4 weeks Culture method: static culture is suitable.
Light requirement: Necessary. Illuminance 4000 lx-22000 lx, light-dark cycle: 12 hours light period / 12 hours dark period.

Among the microalga having the same taxonomical properties as the FERM BP-22262 strain are microalgae belonging to the genus Chlorococcum, whose 18S rRNA gene comprises a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 Included are those having at least 99.94% sequence identity.

In addition, SEQ ID NO: 1 is Chlorococcum sp. SEQ ID NO: 2 is a part of the nucleotide sequence of the 18S rRNA gene of RK261 strain (Japan DNA Data Bank accession number = AB490286), and is a sequence of Chlorococcum sp. A part of base sequence of 18S rRNA gene of FFG039 strain (FERM BP-22262 strain) is shown, respectively.

Taxonomical properties of Botryococcus sudetics FERM BP-11420 (AVFF 007)
The taxonomical properties of Botryococcus sudeticus FERM BP-11420 are shown below.
Botryococcus sudeticus (Botryococcus sudeticus) is a bassonim (basic synonym) of Botryosphaerella sudetica (Botryosphaerella sudetica).

(Morphological properties)
It has a green circle shape. It is floating and can grow on the liquid and bottom surfaces. It grows on the liquid surface to form a film-like structure. The size is approximately 4 to 100 μm. Also, algal bodies on the liquid surface are relatively large, and algal bodies on the bottom are relatively small. During the growth, bubbles are generated in the film-like structure on the liquid surface, and the foam-like structure including a structure in which they are combined, coalesced, or three-dimensionally overlapped, as well as when they are present alone. Form a body.

(Cultural properties)
Culture solution: CSiFF 04 medium (improved in CSi medium, the composition is shown in Table 2. Adjust pH to 6.0 with NaOH or HCl. Culture medium should be autoclaved under conditions of 121 ° C., 10 min. Can)

Culture temperature: The preferred temperature is 23 ° C., and culture can be carried out at 37 ° C. or less.
Culture period (generally, a period until reaching the stationary phase): 2 weeks to 1 month, depending on the amount of algal cells used initially. Usually, culture can be performed at 1 × 10 ⁵ cells / mL.
Culture method: static culture is suitable.
・ Light requirement: Necessary. Illuminance 4000 lx to 15000 lx. Light-dark cycle: 12 hours specified time / 12 hours dark period. In the case of subculture, it is possible to culture at 4000 lx.

The strain having taxonomically identical properties to the FERM BP-11420 strain is a microalga, wherein the 18S rRNA gene is at least 95.0%, preferably at least 95.0%, with a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 Included are those with 98.0%, more preferably 99.0%, still more preferably 99.5%, most preferably 99.9% sequence identity.

In the present invention, the term "identity" for the base sequence means the percentage of the number of matched bases shared between the two two sequences in the aligned region when the two sequences are aligned pairwise in an optimal manner. Do. That is, it can be calculated by identity = (number of matched bases / total number of bases) × 100, and can be calculated using a commercially available or generally published algorithm. The search and analysis regarding the identity of the base sequence can be performed by an algorithm or program well known to those skilled in the art. The parameters for using the program can be appropriately set by those skilled in the art, and may use the default parameters of each program. Specific procedures of these analysis methods are also well known to those skilled in the art.

In the sequence listing, SEQ ID NO: 3 represents a part of the nucleotide sequence of 18S rRNA gene of Botryococcus sudeticus AVFF 007 strain (FERM BP-11420 strain).

Biofilm generally refers to a film-like microbial structure (microbial assembly or membrane) composed of microorganisms and extracellular matrix attached to the surface of rock, plastic or other solid, but in the present invention, In addition to these, film-like or three-dimensional (foam-like, scaly-like, etc.) microbial structures (microbe-aggregated, etc.) composed of microorganisms and extracellular matrix etc. suspended on a fluid surface such as liquid surface Body or microbial membranes) are also referred to as biofilms.

In a preferred embodiment, the biofilm on the liquid surface has a three-dimensional form such as a foam-like structure, a scaly structure, etc. which were not conventionally found in biofilms attached to rocks, plastics or other solid surfaces Structures may be formed. In addition, by leaving the culture solution stationary, it becomes easier to form a biofilm on the liquid surface.
The foamy structure is considered to be formed from a gas such as carbon dioxide or oxygen generated by the metabolism of microalgae and the extracellular matrix. The diameter is generally about 2-3 mm to about 5-10 cm in size, and the shape is not particularly defined except that it is foamy. Also, the foam-like structure may be a single foam-like structure isolated from other foam-like structures, or a plurality of foam-like structures are aggregated and superimposed in two or three dimensions. May include structure.
The rod-like structure is considered to be formed by the formation of a portion invading the culture solution in the film-like biofilm or a portion protruding in the air as the algae grows. Biofilm invaginated in the culture solution may be partially torn down to the bottom of the culture solution. When the biofilm protruding into the air can not support its own weight, it is folded and overlaps the biofilm on the liquid surface.

Generally, in addition to microorganisms, in particular, biofilms in the natural world include debris and debris of plants, but they may be included in the present invention as well. However, in an open environment such as, for example, outdoors, the avoidance of contamination other than the target microorganism is practically impossible, so the present invention does not target samples intentionally containing these. However, from the viewpoint of the efficiency of recovery of microalgae, it is preferable that the biofilm does not contain impurities such as trash and plant fragments, and ideally, between the microalgae according to the present invention and the cells secreted during its growth. More preferably, it is composed only of a substance such as a matrix. Furthermore, in the present invention, the biofilm is preferably a structure in which individual microalgae are attached to each other directly or through a substance such as an intercellular matrix.

Next, the culture and recovery method of the culture of microalga of the present invention will be described in detail.
The culture and recovery method of the present invention is a culture and recovery method of a culture of microalgae using the above-described culture apparatus,
A main culture step of forming an air layer between the liquid surface of the culture solution and the shielding member to culture a culture of the microalga with the culture solution;
After the main culturing step, a fluid volume reducing step of reducing the volume of the culture fluid,
And a recovery step of recovering a culture of microalgae after the liquid volume reduction step, and a culture and recovery method of the microalgal culture.

<culture>
In the culture of microalgae, microalgae are cultured in a culture solution while an air layer is present above the culture solution. The culture solution and microalgae are as described above.

Pre-culture process
In the present invention, the pre-culture step is a step of growing the number of microalgae until main culture can be performed by growing the storage microalgae obtained after the purification step is completed. The culture method for pre-culture can be selected by any known culture method. For example, it is also possible to carry out the dispersion culture method, the adhesion culture method, and the liquid surface suspension culture. In addition, in order to grow microalgae to a scale that can be subjected to main culture, pre-culture may be performed several times. In the pre-culture, stationary culture may be performed depending on the purpose, or non-static culture such as shaking culture may be performed. In addition, the pre-culture may be performed multiple times in order to grow the microalgae to a scale at which the main culture can be performed. In general, a culture vessel (storage member) having a surface area of 1 cm ² to 1 m ² or less is used, and culture can be performed indoors or outside.

Main culture process
In the present invention, the main culturing step is a step of culturing after pre-culturing, and refers to the culturing step up to immediately before the final recovery step when the following post-culturing is not performed. In the case of performing the post-culture described later, it refers to the culture step before the post-culture. The main culture can be ended when a sufficient amount of microalga algae is formed. In particular, in the case of microalgae which forms a biofilm on the liquid surface, it can be ended when a sufficient amount of biofilm is formed on the liquid surface. The main culture can be completed, for example, in several days to several weeks, and more specifically, in five days to four weeks. Also, main culture may be performed multiple times.
Generally, an incubator (storage member) having a surface area of 100 cm ² or more is used, and culture can be carried out indoors or outdoors, but culture outdoors is preferable.

<Post-culture process>
In the present invention, the post-culture step is a step of culturing after performing a liquid volume reduction step. The device for performing the post-culture step may be the same as the device for performing the main culture step, or another culture device may be prepared for use in the subsequent step. In the case of the process of drying the culture medium and discharging the culture solution, the former is preferable in the liquid volume reduction process described later. On the other hand, the liquid volume reduction step described later may be the former or the latter in the case of a step of skimming algal cells together with the culture medium, but from the culture tank of the main culture It is also preferable to use a shallow culture tank. Further, the area of the culture tank in the post-culture is not particularly limited, but is preferably 10 times to 1/10 times the area of the culture tank in the main culture, more preferably 5 times to 1/5 And more preferably 2 to 1 to 4 times. If the area of the culture tank in the post-culture is 10 times to 1/10 times the area of the culture tank in the main culture, the depth of the culture solution is shallow and photosynthesis at the bottom of the culture tank becomes easy to progress, and the area is not too large. Therefore, algal body productivity and oil productivity are improved.

The reason why algal productivity and / or oil productivity can be improved by further culturing microalgae in the coexistence of medium solution components after the liquid volume reduction step has not been clarified, but, for example, the medium amount decreases. It is presumed that photosynthesis is promoted by the influence of the fact that the temperature of the culture medium water easily rises under photosynthetic conditions (light irradiation state) or that light can sufficiently reach the bottom of the water. Alternatively, the oil content may be improved as a result of heat stress condition that the temperature of the culture medium rises and stress condition of the nutrient depleting state as the culture medium components decrease and nutrient components decrease. Sex is also considered.

Here, productivity means the yield per unit area of a product and per unit day. Specifically, algal productivity is a value obtained by dividing the obtained algal weight (for example, g) by the culture area (for example, m ² ) and the number of culture days (day) (g / m ² / day) It is defined. In addition, oil productivity is defined as a value (g / m ² / day) obtained by multiplying the algal body productivity (g / m ² / day) by the oil content rate (mass%) in the algal body.

The conditions for post-culture are described below.
The carbon dioxide concentration is not particularly limited, and may be a normal atmospheric carbon dioxide concentration, but is preferably less than 20% by volume, more preferably from the atmospheric concentration to 15% by volume, and still more preferably 0.1 to It is 10% by volume. Within this range, culture can be further advanced.
・ Culture (medium)
The medium is not particularly limited, and may be mere water, but a medium usable for pre-culture or main culture is preferable, and using the medium used for the main culture as it is is particularly preferable from the viewpoint of process simplification. preferable.

<< Suspension culture, stationary culture, liquid surface suspension culture >>
(Suspension culture)
In the present invention, culturing the microalga in a dispersed state in a culture medium is called suspension culture. In the present invention, culture on the liquid surface is not referred to as suspension culture. Suspension culture can be used depending on the purpose, whether it is main culture or pre-culture.

(Static culture)
In the main culture in the present invention, it is preferable to perform stationary culture. Stationary culture is a culture method in which the medium or the like is not intentionally moved during the culture.

(Liquid suspension culture)
In the present invention, the culture method of culturing microalgae on liquid surface is called liquid surface floating culture. In addition, even if microalgae are simultaneously present on the bottom, sides, and other surfaces of the incubator (storage member), in the culture medium, etc., when the main purpose is culture on the liquid surface, it is called liquid surface floating culture. . In addition to the biofilm, many foams may be present on the liquid surface, the position of the liquid surface may not always be clear, and the biofilm may be slightly sunk below the liquid surface by its own weight. The term "above the liquid level" includes not only the complete liquid level but also such cases. However, culture methods in which microalgae are cultured only in one or both of the bottom of a culture vessel (storage member) or both in a liquid are not included in liquid surface suspension culture.
The liquid level in the present invention is typically the liquid level of the liquid culture medium described later, and is usually the interface between the liquid culture medium and air. Also, when water is the main component, it means the water surface. In addition, when the liquid surface floating culture in the present invention is performed, a phenomenon may be observed in which a fold-like structure intrudes into the liquid from the on-liquid surface biofilm. In the present invention, culture in such a situation is also included in liquid surface floating culture.
As seed algae for performing liquid surface suspension culture, after performing suspension processing, it may be added to a culture vessel (storage member), and after addition of seed algae, in order to promote mixing with a liquid medium Stirring may be performed. Alternatively, the microalgal biofilm may be added to the liquid surface of the incubator (storage member), and the culture may be started in a floating state, or the floating of the microalgal biofilm from the liquid surface is minimal after floating. The microalgal biofilm may be divided on the liquid surface so as not to sink as much as possible so as to be limited, and may be further stirred so as to be dispersed on the liquid surface of the incubator (storage member).

"Seed algae"
The term "seed algae" in the present invention refers to microalgae used at the start of pre-culture or main culture, and refers to microalgae which is the source of culture of microalgae in pre-culture or main culture. In addition, the culture can be started in a state in which the microalgal biofilm is floated on the liquid surface or in a state in which the microalgae is present on the bottom, and also in these cases, these microalgae are used as seed algae. Can. Further, microalgae attached to the bottom surface, other places of the incubator (storage member), other jigs constituting the culture, etc. can also be used as seed algae. Moreover, after the recovery step, the culture can be resumed by using microalgae remaining on the liquid surface as a seed algae.

In the present invention, culture can also be carried out using a microalgal biofilm on the liquid surface as a seed algae. It is a method of leaving a part of the microalgal biofilm on the liquid surface. Moreover, it is also possible to start culture by collecting a part of microalgal biofilm and floating it on the liquid surface. Furthermore, it is also possible to start the culture by performing the dividing treatment while floating the biofilm on the liquid surface as much as possible to the liquid surface. By doing this, the liquid level of the incubator (storage member) can be effectively used, and the growth rate can be improved because it can be made to exist even in the microalgal absence region. Because there are many. Moreover, it is also possible to start culture from the state which left a part of micro algae biofilm on a bottom face and a liquid surface.

The bottom algae refers to microalgae present near the bottom of the incubator (storage member). Among these, there are non-adherent bottom algae which adhere to the bottom surface and do not peel off when the liquid flow is light, or nonadherent bottom algae which exists near the bottom and moves even when the light flow is around. In addition, in the present invention, non-adherent bottom algae can also include liquid surface algae that are separated from the microalgal biofilm by the recovery operation and sink to the vicinity of the bottom. In the case where microalgae are present at a low concentration even in the culture medium other than the liquid level and the bottom, these may be a source of seed algae supply. In addition, the supply of microalgae from the bottom of the incubator (storage member) to the liquid surface means that the microalgae moves from the bottom to the liquid surface when it moves onto the liquid surface without growth of the microalgae on the bottom. There are both cases of proliferation while moving.

In the present invention, microalgae on the bottom can be used as seed algae and culture can be continued. As long as the nutrient components remain in the medium, the used medium may be used as it is to continue the culture, or part of the used medium may be discarded and fresh medium may be added. The amount of fresh culture medium added may be equal to or less than the amount discarded. In addition, it is more preferable to add a new culture medium from the viewpoint of being able to improve the growth rate of microalgae in the subsequent main culture. When the microalgae on the bottom is used as a seed algae, part of the bottom algae may be peeled off and dispersed in the medium. By doing this, it is possible to contact microalgae in a state in which only a part of the algal cells can be in contact with the medium with more medium, and it is possible to suitably improve the growth rate. is there.

Nonadherent microalgae present on the bottom surface may be removed. This is because the unnecessary presence of microalgae on the bottom surface causes a decrease in growth rate which is considered to be caused by unnecessary consumption of nutrient components. Also, the abundance of bottom algae used as seed algae may be adjusted. This is because it is possible to perform appropriate culture. The amount of microalgae present on the bottom of the culture is preferably 0.001 μg / cm ² or more and 100 mg / cm ² or less, more preferably 0.1 μg / cm ² or more and 10 mg / cm ² or more, and 1 mg / cm 2 ² or 5 mg / cm ² being most preferred. If it is 0.1 μg / cm ² or more, the ratio of the amount of microalgae before and after cultivation can be increased in a short time, which is preferable.

In the present invention, a suspension-treated microalgal sample may be used as a seed algae. By performing the suspension process, the microalgae in the solution is made uniform, and as a result, the film thickness after culture is made uniform, so the amount of microalgae per culture area may increase. As the suspension treatment, any known method can be used, but pipetting, shaking treatment of the microalgal solution put in the container by hand, weak treatment such as treatment with a stirrer tip or a stirring rod, ultrasonic treatment or the like Examples thereof include strong treatment such as high-speed shaking treatment, and a method using a substance such as an enzyme that degrades an adhesive substance such as an intercellular matrix.

In addition, when carrying out culture on a large area outdoors etc., unlike experiments in a laboratory, the input of seed algae as a source of culture is almost uniformly applied throughout the incubator (storage member) It becomes difficult to do. Therefore, in the present invention, for example, it is also preferable to disperse the microalga to be the seed algae in advance in a water-absorbent substance (for example, seratin etc.) and appropriately charge it.

<< Culture fluid (medium) >>
In the present invention, as the medium used for culture, any known medium (liquid medium) can be used as long as microorganisms can be cultured. As a known medium, AF-6 medium, Allen medium, BBM medium, C medium, CA medium, CAM medium, CB medium, CC medium, CHU medium, CSi medium, CT medium, CT medium, CYT medium, D medium, ESM medium, f / 2 medium, HUT medium, M-11 medium, MA medium, MAF-6 medium, MF medium, MDM medium, MG medium, MGM medium, MKM medium, MNK medium, MNK medium, MW medium, P35 medium, URO medium, VT medium, Examples include VTAC medium, VTYT medium, W medium, WESM medium, SW medium, SOT medium and the like. Among them, freshwater ones are AF-6 medium, Allen medium, BBM medium, C medium, CA medium, CAM medium, CB medium, CC medium, CHU medium, CSi medium, CSi medium, CT medium, CYT medium, D medium, HUT medium M-11 medium, MA medium, MAF-6 medium, MDM medium, MG medium, MGM medium, MW medium, MW medium, P35 medium, URO medium, VT medium, VTAC medium, VTYT medium, W medium, SW medium, SOT medium is there. As a culture medium which culture | cultivates the above-mentioned AVFF007 strain | stump | stock, C culture medium, CSi culture medium, CHU culture medium, and the mixture of these culture media are preferable. The culture medium is preferably selected according to the type of microorganism to be cultured.

The medium may or may not be UV sterilized, autoclaved, filter sterilized. As the medium, the same type of medium may be used in the pre-culture and the main culture, or different types of medium may be used. Also, different culture media may be used during the culture.

Also, when performing culture on a large area outdoors, etc., unlike the experiment in the laboratory, if the medium components are diluted to the concentration used in advance, the volume of the solution will be large, so to the culture place Inefficient from the aspect of transport and input work. Therefore, it is efficient to keep the necessary medium components in a concentrated state (hereinafter referred to as concentrated culture solution) and dilute with water at the culture site. In addition, since the culture medium components vary, when it is going to adjust a concentration culture medium in this way, depending on the combination of the components, precipitation may occur. In such a case, it is preferable to divide the concentrated medium as two or more concentrated mediums, and separate the components that cause precipitation into separate concentrated medium.

"carbon dioxide"
Many microalgal cultures require the provision of carbon dioxide.
When dispersion culture is performed by pre-culture, carbon dioxide may be supplied into the medium by bubbling as in the conventional method, but when liquid surface suspension culture is performed, carbon dioxide is extracted from the air layer. It is preferable to supply it. This is because the structure of the microalgal biofilm on the liquid surface is destroyed when carbon dioxide is supplied into the medium by bubbling or the like method, and algal mass is generated, and the biofilm recovery efficiency on the substrate in the recovery step And the amount of recovered algal bodies may decrease.

In the present invention, although carbon dioxide in the atmosphere can be used, carbon dioxide in a concentration higher than the atmospheric concentration can also be used. In this case, in order to prevent the loss of carbon dioxide due to diffusion, it is desirable to culture in a culture apparatus covered with a closed culture apparatus or a coating such as an agricultural film. The concentration of carbon dioxide in this case is not particularly limited as long as the effects of the present invention can be achieved, but is preferably not less than atmospheric concentration and less than 20% by volume, preferably 0.01 to 15% by volume, more preferably 0. 1 to 10% by volume. Also, the carbon dioxide may be carbon dioxide discharged by the combustion device. Alternatively, carbon dioxide may be generated by the reagent.

In the culture, carbon dioxide is preferably supplied to the entire culture apparatus. Carbon dioxide can also be used in the culture solution culture medium and carbon dioxide in the air layer, but in order to obtain a sufficient amount of biofilm, it is desirable to supply more carbon dioxide.

As a carbon dioxide supply method, it is possible to open the part of the shielding member and take in the outside air, but in addition, carbon dioxide gas may be introduced from a cylinder or the like by piping. Moreover, the method of throwing in a solid substance in a culture apparatus as a carbon dioxide source can also be utilized. As a solid carbon dioxide source, carbonates (for example, sodium hydrogen carbonate, sodium carbonate, calcium carbonate and the like) and the like can be mentioned. Moreover, it is also preferable to use a commercially available CO ₂ tablet (CO ₂ tablet) or the like.

The generation of carbon dioxide is not particularly limited as long as it is a solid state at normal temperature and pressure and can generate carbon dioxide by a chemical reaction. For example, the generation of carbon dioxide can be performed by the reaction of a soluble acid source and a carbonate source. The reaction is promoted by contact with water. Soluble acid sources include citric acid, malic acid, fumaric acid, adipic acid, succinic acid, ascorbic acid, maleic acid, maleic acid, boric acid, tartaric acid, mandelic acid, malonic acid, pyruvic acid, glutaric acid, aspartic acid, oxalic acid, Salicylic acid, lactic acid, hydrochloric acid, acetic acid, benzoic acid, hydroxybenzoic acid, methoxybenzoic acid, sodium dihydrogenphosphate, disodium dihydrogen pyrophosphate, potassium bitartrate, primary sodium phosphate, primary sodium citrate, primary tartaric acid Sodium can be used, and as a carbonate source, potassium carbonate, magnesium carbonate, L-lysine carbonate, arginine carbonate, ammonium carbonate, sodium carbonate, glycine carbonate, calcium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, sodium bicarbonate , Potassium bicarbonate, sodium sesquicarbonate, gum Emissions sodium carbonate, potassium dihydrogen citrate, or the like can be used amorphous calcium carbonate.

The binder for forming the tablet may be any binder that can achieve the purpose, for example, gum arabic, gelatin, tragacanth gum, starch, cellulose substances such as methyl cellulose, alginic acid and salts thereof, polyethylene glycol, Guar gum, polysaccharide acid, bentonite, saccharides and the like can be used.

In order to promote the culture efficiently, the relationship between the carbon dioxide supply time and the light irradiation time during which photosynthesis is actually performed is also important, and the carbon dioxide supply time and the light irradiation time match at least 50% or more. It is preferable to do. Moreover, as a supply time of carbon dioxide, it is preferable to supply within 2 hours after actual light irradiation is started. In addition, when supplying carbon dioxide, pH in the culture solution is also an important factor, and it should be performed when the pH in the culture solution is at least one lower than the optimum pH of the microalga to be cultured. Is preferred.

<< Light source and illuminance >>
As a light source which can be used in the present invention, any known light source can be used. Among these, it is preferable to use sunlight which is natural energy, an LED with high luminous efficiency, and a fluorescent lamp which can be easily used.

The illuminance (unit: lux (lx)) is preferably 100 lux or more and 1,000,000 lux or less, and more preferably 300 lux or more and 500,000 lux or less. The most preferable lux is at least 1000 lux and at most 200,000 lux. When the illuminance is 1000 lux or more, microalgae can be cultured, and when it is 200,000 lux or less, the adverse effect on the culture due to light damage is small. The illumination preferably refers to the illumination of the liquid surface of the culture solution.

When light is irradiated with an artificial light source, light may be either continuous irradiation or a method of setting an irradiation cycle in which irradiation and non-irradiation are repeated at a certain time interval, for example, in view of ordinary sunshine conditions. Preferably, the light is turned on and off at intervals of about 12 hours. In addition, irradiation conditions may be changed during the culture, such as continuous irradiation for several days (for example, 3 to 5 days) from the start of the culture, and then turning on and off light at intervals of 12 hours. In the pre-culture, continuous light may be used positively. In the method of setting the irradiation cycle, the period in which the light is on may be referred to as a “bright period”, and the period in which the light is off may be referred to as a “dark period”.

The wavelength of light may be any wavelength as long as photosynthesis can be performed, and the wavelength is not limited, but a preferred wavelength is sunlight or a wavelength similar to sunlight. An example has also been reported in which the growth rate of photosynthetic organisms is improved by irradiation with a single wavelength, and such an irradiation method can also be used in the present invention.

<< Other culture conditions >>
In the present invention, the pH of the culture solution (liquid medium) used in the pre-culture or the main culture is preferably in the range of 1 to 13, more preferably in the range of 3 to 11, and 5 to 9 It is further preferable to be within the range of, and it is most preferable to be within the range of 6 to 8. Moreover, since suitable pH changes according to the kind of microalga, it is preferable to select pH according to the kind of microalga. The pH of the liquid medium is the pH at the start of culture. In addition, since the pH during culture may change with the culture, the pH may change during the culture.

In the present invention, a substance having a buffer action to keep the pH in the medium constant can be added to the medium. As a result, it may be possible to suppress the problem that the pH in the medium changes as the microalgal culture progresses, or to suppress the phenomenon that the pH changes due to the supply of carbon dioxide into the medium. As the substance having a buffer action, known substances can be used, and there is no limitation on its use, but 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES), phosphoric acid Sodium buffer, potassium phosphate buffer and the like can be suitably used. The concentration and type of the buffer substance can be determined according to the type of microalga and the culture environment.

When the liquid culture medium in the case of dispersion culture is deep, the light does not reach and there is a problem that the stirring efficiency is deteriorated, and there is a limit. However, in the case of liquid surface floating culture, since microalgae are grown at high density on the liquid surface, there is no need to supply light to the deep part of the incubator (storage member), basically Since the stirring is not performed, the water depth can be shallow. Thus, it is preferable to reduce the water depth because the amount of water used is small and the handling efficiency is improved. The depth of water is preferably 0.4 cm or more, more preferably 1 cm to 10 m, still more preferably 2 cm to 1 m, and most preferably 4 cm to 30 cm. When the water depth is 0.4 cm or more, formation of a biofilm is possible, and when the water depth is 10 m or less, handling is easy. When the water depth is 4 cm to 30 cm, the influence of evaporation of water is minimal, and the handling of the solution containing the culture medium and microalgae is easy.

The culture temperature can be selected according to the type of microalgae and is not particularly limited, but is preferably 0 ° C. or more and 90 ° C. or less, more preferably 15 ° C. or more and 50 ° C. or less, 20 ° C. or more and 40 ° C. Less than is most preferred. When the culture temperature is 20 ° C. or more and less than 40 ° C., microalgae can be suitably grown.

The lower limit input amount of microalga, ie, the amount of microalgae used at the start of culture, is not particularly limited because it is possible to proliferate if there is only one in the culture range, as long as it takes time. , Preferably 1 / cm ³ or more, more preferably 1000 / cm ³ or more, and still more preferably 1 × 10 ⁴ / cm ³ or more. The upper limit input microalgae amount of microalgae basically can be grown at any high concentration, so there is no particular limitation, but the more the microalgae is, the more the amount of microalgae is, Since the ratio of the amount of input microalgae to the amount of microalgae after growth decreases, 1 × 10 ⁹ cells / cm ³ or less is preferable, 1 × 10 ⁸ cells / cm ³ or less is more preferable, and 5 × 10 ⁷ cells / cm ^3. More preferably, cm ³ or less.

The pre-culture period and the main culture period in the present invention can be selected according to the type of microalgae and is not particularly limited, but is preferably 1 day to 300 days and more preferably 3 days to 100 days. 7 days to 50 days are more preferable. In addition, when culture | cultivation period was defined with days, it may be called "culture days."

In the present invention, after collecting the microalgal biofilm, as long as a nutrient component for growth remains in the medium, the microalgae remaining on the bottom surface or in other parts are recultured as seed algae. , Can be done again and again. However, if the concentration is too low, the growth rate is likely to be slow, and in such a case, the medium is newly added, or at least a part of the medium is replaced, or solid-like And high concentration of nutrient components can be added to the medium.

Preferably the size of the microalgae biofilm is 0.1 cm ² or more, more preferably 1 cm ² or more, more preferably 10 cm ² or more, and most preferably equal to the liquid surface area of the incubator (storage member). If it is 0.1 cm ² or more, it is preferable because the ratio of the amount of microalgae at the end of culture to the amount of microalgae at the start of culture can be increased in a short time. Also, a plurality of microalgal biofilms may be present in the culture region. The thickness of the microalgal biofilm is preferably in the range of 1 μm to 10000 μm, more preferably in the range of 1 μm to 1000 μm, and most preferably in the range of 10 μm to 1000 μm. If it is in the range of 10 μm to 1000 μm, sufficient strength can be obtained to harvest a sufficient amount of biofilm.

When the biofilm according to the present invention forms a three-dimensional structure, particularly a foam, the general height of the foam relative to the liquid surface of the culture medium is 0.01 mm to 100 mm. The range of 0.1 mm to 40 mm is more preferable, and the range of 3 mm to 30 mm is most preferable. When the height of the foam structure from the liquid surface is in the range of 3 mm to 30 mm, the moisture content can be sufficiently lowered, and the height of the incubator (storage member) can be suppressed to a low level. The microalgae according to the present invention preferably has a high growth rate on the liquid surface, and the growth rate in the logarithmic growth phase (that is, the average growth rate per day in the logarithmic growth phase) is 0 in dry weight. is preferably .1g ^/ m 2 ^/ day or more, more preferably 0.5g ^/ m 2 ^/ day or more, further preferably ^1g / m 2 / day or more, ^3g / m 2 / day It is most preferable that it is more than. The growth rate in the logarithmic growth phase of microalgae is generally 1000 g / m ² / day or less in dry weight.

The dry alga body weight per unit area of the biofilm according to the present invention is preferably 0.001 mg / cm ² or more, more preferably 0.1 mg / cm ² or more, and 1 mg / cm ² or more. Being particularly preferred. Most preferably, it is 5 mg / cm ² or more. It is because it is anticipated that the quantity of biomass, such as oil obtained, will become large, so that the dry alga body weight per unit area is large. The dry algal body weight per unit area of the biofilm is usually 100 mg / cm ² or less.
Moreover, as the microalga of the present invention, a microalga capable of forming a biofilm having the above-mentioned structure, area, thickness, height, growth rate and dry algal body weight per unit area in the above range on a liquid surface Is preferred for the same reasons as described above.

Liquid volume reduction process
The volume reduction step is a step of reducing the volume of the culture solution. In the culture and recovery method of the microalga culture of the present invention, the liquid volume reduction step may be performed after the main culture step or may be performed in parallel with the main culture step. Moreover, when performing a post-culture process, a post-culture process is performed after a liquid volume reduction process.
The method for reducing the culture solution volume is not particularly limited, but it is important to reduce the culture solution coexisting with the microalgal culture. Specific methods include, for example, drying the culture solution, discharging the culture solution, skimming the algal cells with the culture medium, or a combination thereof. By any method, the amount of medium coexisting with algal cells can be reduced. Also, by drying or discharging the culture solution, the culture solution present in the region between the liquid surface and the bottom of the culture solution can be reduced, and the culture of microalgae can be efficiently recovered It becomes easy.

Examples of the method for drying the culture solution include natural drying, air drying, heat drying, lyophilization, a combination thereof and the like. In the case of natural drying, natural drying can be performed, for example, by opening at least a part of the shielding member. This drying may be carried out in the presence of light or substantially in the absence of light. In the case of using a microalga which forms a biofilm on the liquid surface, to which the culture and recovery method of the present invention is suitably applied, it is possible even if the required culture solution is very shallow compared to the conventional method. Because of this, such natural drying can be easily dried. Therefore, it consumes less energy and contributes to cost reduction of algal biofuel.

As a method of discharging the culture solution, as shown in FIG. 2 described above, a drainage pipe, a flow path, a water channel, etc. are provided in advance, and the method is opened, or a method of suction and drainage with a pump, etc. The combination of these etc. is mentioned.

The discharge of the culture solution may be performed from any part, but discharging from a position where the amount of microalga is present is preferable from the viewpoint of suppressing the outflow of microalga. When using the microalga which forms a biofilm on the liquid surface to which the manufacturing method of the present invention is suitably applied, the microalgal biofilm is formed on the liquid surface, so the bottom or bottom of the liquid holding portion It is preferable to discharge from the side of the vicinity. Alternatively, microalgae forming a biofilm on the liquid surface may form a microalgal biofilm also on the bottom of the liquid holding unit. In such a case, it is preferable to discharge from the intermediate position because the amount of microalgae is small in the intermediate region excluding the liquid surface and the bottom portion. That is, discharge of the culture solution can be performed so as to remove at least a part of the culture medium present in the region between the liquid surface and the bottom, and in particular, the microalgae in the region between the liquid surface and the bottom It is preferable to carry out so as to remove at least a part of the culture solution present in the region substantially absent or not visually observed. Although it is expected that zoospores are present in the area where the microalgae is substantially absent or not visually observed, the zoospores are not visually observed because they are much smaller than the algal cells. .

Moreover, although the discharged culture solution may be discarded, it is also possible to reuse it as a culture solution. Moreover, the culture solution which remained in the storage member after the recovery process mentioned later can be reused similarly. That is, if nutrient components remain in the culture solution, the used culture solution may be used as it is to continue the culture solution, or a part of the used culture solution may be discarded and a new culture solution may be added. You may The amount of fresh culture medium added may be equal to or less than the amount discarded. In addition, it is more preferable to add a new culture solution from the viewpoint of being able to improve the growth rate of the microalga in the main culture in the latter stage.

When a fresh culture solution is added, the nutrient component may be added in the state of being dissolved in the liquid, or may be added as a solid content. When it is added as a solid, it may be necessary to stir the culture solution, so it is more preferable to add it in a liquid state.

For the addition of the culture solution, a culture solution of the same component as the culture solution used for the main culture in the previous stage may be used, or a culture solution having different components may be added.

In addition, a fresh medium having a high concentration of a specific component can be added to the culture solution used for the culture. The specific component is, for example, a compound including, but not limited to, any one selected from the group consisting of nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, and iron. When a fresh medium having a high concentration of a specific component is added to the culture solution used for the culture, the concentration is preferably 1.01 times to 100,000 times the normal case, and 10 It is more preferably twice or more and 10000 times or less, and most preferably 50 times or more and 5000 times or less. When the concentration of a specific component of the fresh medium is less than 1.01, more water is required for its production, and the distance between the fresh medium preparation place and the incubator (storage member) installation place increases. In order to reduce the transportability and introduce a large amount of culture medium into the incubator (storage member), various devices such as supplying fresh culture medium to a storage container for the fresh culture medium and the incubator (storage member) There are various problems, such as the need for Furthermore, it was assumed that the medium component in the culture solution in the culture apparatus was almost zero when the medium volume in the culture apparatus increased, for example, when a concentrate of 1.01 times was added to the culture apparatus In the case, it reduces to about 1/2 concentration. These things are likely to reduce the growth rate and amount of growth. When the specific component capacity concentration of the culture medium is more than 100,000 times, the volume of the culture solution becomes very small and the transportability and the introduction efficiency of the concentrated medium into the culture apparatus are improved, but depending on the type of medium component, the solubility Is worse. Furthermore, unnecessary precipitate may be generated upon introduction into the culture apparatus, and may not dissolve even after being diluted by stirring in the culture apparatus. As a result, the growth rate and growth amount of microalgae may be reduced.

Although the component composition of the fresh medium may be the same as the composition of the medium component in the culture solution in the culture apparatus, a fresh medium composed of different components may be added. Furthermore, the medium and components in the culture solution may be partially the same. In addition, it is also possible to use a fresh medium which is prepared so that the components of the medium in the culture solution and the final concentration after addition differ greatly.

Recovery process
The recovery step is a step of recovering the culture of microalgae after the liquid volume reduction step.
The recovery method is not particularly limited, but for example, the obtained culture (biofilm, algal cells) is skimmed from a culture vessel (storage member) using tools of various forms such as a spatula-like, film-like member, Or the method of suctioning using a pump etc. is mentioned.

As a method of skimming the culture (biofilm, algal cells) together with the medium, a method using a filter, filter paper or the like which allows water to permeate but not algal cells can be used, or a centrifugation method can be used.
When using a step of skimming algal cells with the culture solution, the microalgae may be dispersed in the culture solution, but microalgal algae having the property of floating on the liquid surface by standing as described above, air, etc. It is preferable to apply the present invention to microalgae in which microalgae is floated on the liquid surface by floating by bubbling treatment, oil accumulation, or addition of a standing step, and microalgae capable of standing culture. Above all, it is particularly preferable to use for microalga which can form a biofilm on the liquid surface. In this case, since the algal cells form a biofilm on the liquid surface of the culture medium, it is easy to transfer this biofilm (algal cells) to another substrate or deposit it using another substrate for recovery. (For details, refer to Japanese Patent Application Laid-Open No. 2013-226063 etc.).
In this case, not only algal cells present on the liquid surface but also algal bodies present in the water and bottom of the water may be recovered. In particular, when the medium is drained and dried, the algal cells are skimmed together with the medium since it is easy to simultaneously scoop algal bodies present in the water and the bottom of the water.
These recovery methods are briefly described below.

For example, as shown in FIG. 46, a method of transferring a biofilm (algal cells) to another substrate for recovery is a biofilm (algal cells) 903a composed of microalgae on the liquid surface of the culture tank 901, as shown in FIG. After transferring (a film-like structure or a three-dimensional structure) to the first substrate 902, a biofilm (alga) 903b is recovered from the first substrate 902.
Also, a method of depositing and collecting a biofilm (algal cells) using another substrate is, for example, a biofilm (algal cells) 903a composed of microalgae on the liquid surface, as shown in FIG. After depositing (a film-like structure or a three-dimensional structure) on the second substrate 904, a biofilm (alga) 903b is recovered from the second substrate 904.

For recovery of the biofilm (algal cells) from the first substrate, any known method can be used as long as the biofilm (algal cells) can be peeled from the first substrate. For example, a method such as peeling a biofilm from a substrate using a cell scraper, a method using a water flow, a method using ultrasonic waves, etc. can be mentioned, but a method using a cell scraper is preferable. This is because, in another method, the biofilm (algal cells) will be diluted with a medium or the like, which may require concentration again, which is inefficient.
Recovery of the biofilm (algal cells) from the second substrate can be carried out by any known method as long as the biofilm (algal cells) can be peeled off from the second substrate. For example, a method by gravity, a method of peeling a biofilm from a substrate using a cell scraper, a method of using a water flow, a method of using ultrasonic waves, etc. can be mentioned, but a method utilizing a free fall by gravity. Or, a method using a cell scraper or the like is preferable. This is because, in another method, the biofilm (algal cells) will be diluted with a medium or the like, which may require concentration again, which is inefficient. Alternatively, after the biofilm (algal cells) is recovered using gravity-induced free fall, algal cells remaining on the second substrate can be recovered using a cell scraper or the like.

All of the deposited microalgae and the biofilm (film-like structure or three-dimensional structure) composed of microalgae on the liquid surface may be recovered. Such a recovery method can be carried out even when the culture is terminated, another microalgae is cultured, or the medium in the culture tank is replaced. After the biofilm (algal cells) composed of microalgae on the liquid surface is transferred using the first substrate or recovered by the second substrate, the culture medium is removed, and the bottom of the culture vessel remains Microalgae may be recovered. Furthermore, all the microalgae in the culture tank may be recovered by a known method. Examples of the known method include recovery by a filter, recovery by a coagulant, and the like.

In addition, for example, after collecting the biofilm (algal body) of the microalga on the liquid surface, the microalga on the bottom of the culture tank can be collected.
Furthermore, after the biofilm consisting of microalgae on the liquid surface is recovered, the culture solution in the culture vessel may be removed to recover the microalgae on the bottom of the culture vessel or on the surface of the first substrate.

"Transfer" when transferring a biofilm (algal cells) to a substrate is one type of adhesion, and adhesion substantially without growth. In the present invention, an operation of transferring the biofilm (algal cells) formed on the liquid surface to the surface of the first substrate substantially as it is using the first substrate is referred to.

The transfer of the biofilm (algal cells) is a step of transferring the biofilm (algal cells) formed on the liquid surface to the surface of the substrate using the first substrate, as shown in FIG. In FIG. 46, a biofilm (algal body) is formed on the entire surface in the culture tank 901, and the recovery step is performed in such a state, but recovery in such a state may be performed, In the present invention, the recovery step can be performed even when there is a state in which a biofilm (algal body) consisting of microalgae is partially absent. In addition, when culturing is carried out by the method as in the present invention, the biofilm (algal cells) formed on the liquid surface may be wrinkled or folded, and from the folded microalgae. The constructed film-like structure may grow in the liquid like aurora (curtain-like).

The first substrate and the second substrate are substrates used to transfer or recover a biofilm (algal cells) composed of microalgae on the liquid surface used in FIG. 46 or 47.

The surface of the first substrate and the surface of the second substrate refer to any surface of the substrate, and refer to the top surface of the substrate, the bottom surface of the substrate, and the side surface of the substrate. In addition, even if microalgae adheres to these surfaces, it is in contact with a culture tank etc., and microalgae may come out to a culture solution from the layer formed between a substrate and the surface of a culture tank etc. If not, it is not the surface of the present invention.

The shape of the substrate may be any shape such as film, plate, fiber, porous, convex, or wavy, but the adhesion, deposition, easiness of transfer, etc., and recovery of microalgae from the substrate It is preferable that it is a film form or plate shape from the ease of carrying out.
The first substrate and the second substrate may have the same shape or different shapes. The area of the first substrate and the second substrate is preferably smaller than the area of the culture solution liquid surface in the culture vessel.

By the way, in order to improve algal cell productivity and / or oil productivity, it is important not to isolate only algal cells in the recovery step but to maintain a state in which a culture solution (medium) is present. In this case, the mass ratio of the algal cells to the culture solution (medium) is {mass of algal cells / mass of culture solution (medium)}, preferably 20/80 to 0.1 / 99.9, more preferably Is from 15/85 to 0.2 / 99.8, more preferably from 10/90 to 0.4 / 99.6.
When the mass of the algal cells is in the range of 0.1 to 20% by mass of the total mass of the algal cells and the culture solution (medium), the light is easily transmitted during the culture, and the culture is easily progressed. As it is not too much, the reduction effect of the load at the time of collection becomes large.

In the present invention, when culture and recovery of microalgae are performed using a culture apparatus having a configuration in which a film is disposed on the surface of the liquid holding unit in contact with the culture solution, the storage member is used in the liquid volume reduction step. It is also preferable to recover the microalgae by reducing the liquid volume of the culture solution in the medium and bringing the microalgae (biofilm) into contact with the film to move the film.

As a method of bringing a film into contact with a culture of a microalga (biofilm, algal cells) as a pre-step for collecting microalgae, the first film or the second one in the above-described liquid volume reduction step A method of reducing the liquid volume of the culture solution until the film and the culture of microalgae come in contact, or lifting the second film to a position between the bottom and the liquid surface, the culture of the second film and the microalgae In the liquid volume reduction step, such as a method of reducing the culture liquid until the protein comes into contact, in addition to the method of contacting the film with the culture of microalgae, the second liquid film of the culture liquid volume in the liquid volume reduction step Or the like, and the second film is lifted in the recovery step to be brought into contact with the microalga, and the like, and the like.
As such a culture apparatus, specifically, a culture apparatus 10f provided with the first film 22f described above, or a culture apparatus 10g provided with the second film 24g can be used.
In the following description, the first film and the second film used for the recovery of the microalgae are collectively referred to as a recovery film.

As a collection method in the case of collecting microalgae using a collecting film, after lifting, pulling out, winding up the collecting film to which the microalga is attached, the collecting film is inclined or turned upside down For example, it is possible to recover the microalga naturally by dropping the microalga or scraping the microalga from the film for recovery using a member such as a spatula, plate or blade.
Among them, it is preferable to wind up and recover a recovery film.

Here, an example of the recovery step in the manufacturing method of the present invention will be described with reference to FIGS. 28 to 32 and 33 to 37.
FIG. 28 is a schematic cross-sectional view showing an example of the culture apparatus 10 p after the main culturing step, FIG. 29 is a schematic cross-sectional view showing the state of the culture device after the liquid amount reduction step, and FIG. FIG. 31 is a schematic cross-sectional view showing the state of the culture apparatus in the recovery step, and FIG. 32 is a schematic view showing the state in which the recovery film is being re-installed after the recovery step. FIG.
33 is a top view of the culture apparatus of FIG. 29, FIGS. 34 and 35 are top views of the culture apparatus of FIG. 31, and FIGS. 36 and 37 are recovery steps. It is a figure for demonstrating reinstallation of the film for subsequent collection | recovery.
In FIG. 29 to FIG. 32, the illustration of the shielding member is omitted. Further, in FIG. 33 to FIG. 37, illustration of some members is omitted for the sake of explanation.

The culture apparatus 10p shown in FIG. 28 covers the storage member 12n in which the third film 26j and the first film 22f are laminated in this order on the concave support 20n, and the liquid holding portion of the storage member 12n. And an arched shielding member 14c disposed on the
The culture apparatus 10p shown in FIG. 28 shows the state after the main culture step, and the microalgae P is cultured on the liquid surface of the culture solution M held in the storage member 12n to form a biofilm.
Next, as shown in FIGS. 29 and 33, in the liquid volume reduction step, the culture fluid M is dried, drained from a drain pipe (not shown), or drained from a drying and drain pipe (not shown). . When the culture solution M is dried, discharged, or dried and discharged, the microalgae P is deposited on the first film 22f.

Next, the first film 22f is moved to recover the microalgae P.
Here, as shown in FIG. 30, as a means for moving the first film 22f, it has a first winding machine 202 provided with a winding roller, and as a first recovery member for recovering the microalgae P, A spatula 204 and a recovery container 206 are provided.
As shown in FIGS. 31, 34, and 35, the first film 22f is wound around the winding roller while the first film 22f to which the microalgae P is attached is wound by the first winder 202. At the position, the spatula 204 scrapes off the microalgae P on the first film 22 f and drops it to recover the microalgae P in the collection container 206.
The winding roller of the first winder 202 may be rotated by a drive unit (not shown) or may be rotated manually.

When the first film 22f is reused after recovery of the microalgae, as shown in FIGS. 36 and 37, the film 210 for reinstallation wound around the installation roller 208 is unwound. The film 22f is moved to the top of the support 20n by connecting the end of the first film 22f by adhesion or the like and winding the film 210 for repositioning by the setting roller 208. The film 22f of 1 can be reused.
By repeatedly using the recovery film in this manner, costs can be reduced, which is preferable.

Here, one set of the first winding machine 202 and the first recovery member is not limited to the configuration provided corresponding to one storage member, and the one set of the first winding machine 202 and the first collection member The first recovery member may be configured to perform recovery with a plurality of storage members.
For example, as shown in FIG. 38, a plurality of storage members 12 are arranged in a direction orthogonal to the longitudinal direction of the storage member 12 and installed, and one set of the first winding machine 202a and the installation roller 208 are It is good also as composition arranged so that movement is possible in the direction which intersects perpendicularly with the longitudinal direction of storage member 12, respectively in the end part side of the longitudinal direction 12 of storage member. According to this configuration, one set of the first winding machine 202a and the installation roller 208 are moved in the vertical direction in the drawing to wind up the recovery film of each storage member 12 and the microalgae from each storage member 12 It can be recovered.

Further, as shown in FIG. 38, a storage member group 220 including a plurality of storage members 12 arranged in a direction orthogonal to the longitudinal direction of the storage members 12 is arranged and installed in the longitudinal direction of the storage member 12 The first winder 202 may be disposed between the storage member group 220. In this case, the first winding machine used as the installation roller 208 in the storage member group 220 on the left in the drawing may be used as the first winding machine 202 b in the storage member group 220 in the middle of the drawing. At that time, the first winding machine 202c disposed adjacent to the right of the storage member group 220 in the middle in the figure can be used as an installation roller 208 which forms a pair with the first winding machine 202b.

In addition, in the example of illustration, although it was set as the structure which peels and collect | recovers the obtained micro algae P from the film for collection | recovery, it is not limited to this, The micro algae P is collect | recovered with the film for collection, It can also be used as

The culture (biofilm, algal cells) thus obtained is then transported or used as a fuel thereafter, and extraction and purification of the active ingredient in the culture (biofilm, algal cells) In order to make the process easy, a drying process may be performed to further reduce the water content. The water content of the finally obtained culture (biofilm, algae) is preferably 80% by weight or less, more preferably 50% by weight or less, particularly preferably 20% by weight or less .

The integral product of the culture (biofilm, algal cells) and film thus obtained can also be used as a fuel as it is. At this time, it is desirable that the culture (biofilm, algal cells) be sufficiently dried.

In addition, once the culture (biofilm, algal cells) is obtained by reducing at least a part of the culture solution, the culture solution is collected again, and if necessary, the culture seed algae and the like without collecting it. It is also possible to obtain a larger amount of culture (thick biofilm, algal cells) by repeating the culture and recovery steps by adding such microalgae. This operation may be repeated once or a plurality of times. Performing such a process is efficient because the amount of algal biomass obtained in one recovery process increases.

(Water content)
The moisture content in the present invention is the weight of the moisture contained in the recovered product divided by the weight of the recovered product and multiplied by 100. The upper limit of the water content of the microalgal biofilm in the present invention is not particularly limited, but is preferably less than 98% by mass, more preferably 95% by mass or less, and particularly preferably 88% by mass or less.

The moisture content of the microalgal biofilm cultured according to the culture and recovery method of the present invention is usually about 95 to 80% by mass even in the state where the algal cells on the water surface are recovered by the aforementioned recovery method. Furthermore, it is possible to make it about 90-75 mass% by processing with a centrifuge. In the case of culture by dispersion culture, most of the algal cells are present in water, and thus recovery is almost impossible with the above-mentioned recovery method. Therefore, methods such as recovering microalgae using a centrifuge with a culture solution are used, but even in that case, the water content is generally 90% by mass or more, and it is obtained by the culture method of the present invention. The moisture content of the above-liquid surface biofilm is lower than that and superior to conventional methods. In addition, the moisture content is lower in the three-dimensional structure including the foam structure, the scissor structure and the like than the film structure. It is presumed that this is because the three-dimensional structure is more distant from the liquid surface and closer to the light source, and the drying progresses to some extent. Also, it is estimated that the moisture content varies depending on the site of the biofilm. For example, it is considered that the water content of the portion in contact with the liquid surface is high, and the water content of the portion not in contact with the liquid surface is low. The water content is the case where the biofilm on the liquid surface is recovered, and the area of the recovered matter is at least 10 cm ² or more. Also, it is different from the moisture content after drying of the present invention.

[Method for producing algal biomass and algal biomass]
Algal biomass can be produced by using the above-described culture and recovery method of microalgae.
The algal biomass in the present invention means organic resources derived from renewable organisms excluding fossil resources, and examples thereof include biological materials, foods, materials, fuels, resources and the like. The algal biomass includes microalga itself (which may be in the form of a biofilm), microalgal residue after collecting useful substances.
The useful substance in the present invention is a kind of biomass derived from microalgae, which is a generic term for substances useful for the industry obtained from the biomass through steps such as an extraction step and a purification step. As such substances, final products and intermediates and raw materials such as pharmaceuticals, cosmetics and health foods, raw materials of chemical compounds, intermediates and final products, hydrocarbon compounds, further oils, alcohol compounds, hydrogen and methane And energy substitutes such as enzymes, proteins, nucleic acids, sugars and lipid compounds such as DHA, astaxanthin etc. The useful substance can also be accumulated in the microalga by the useful substance accumulation step.

[Method for producing algal biofuel and algal biofuel]
Algal biofuels can be manufactured by using the above-described culture and recovery method of microalgae culture.
The algal biofuel in the present invention is a microalgal dry matter, particularly one containing a flammable oil component inside, or a flammable flowable substance (oil) obtained from the microalgal dry matter. , A compound mainly composed of carbon and hydrogen, and in some cases, a substance containing an oxygen atom, a nitrogen atom and the like. An oil is generally a mixture and is a substance that is extracted using a low polarity solvent such as hexane or acetone. The composition may be composed of a hydrocarbon compound, fatty acid, triglyceride or the like, or may be composed of a plurality of compositions selected from these. It can also be esterified and used as biodiesel.

The method for collecting the oil contained in the microalgae recovery is not particularly limited as long as it does not impair the effect of the present invention. In a general extraction method of oil, the final collected product is dried by heating to obtain a dried algal cell, then the cells are disrupted and the oil is extracted using an organic solvent. The extracted oil is generally purified because it contains impurities such as chlorophyll. Purification may be by silica gel column chromatography, by distillation (for example, by the distillation method described in JP-A-2010-539300). Such a method can also be used in the present invention. In addition, there is also a method of extracting the oil in the algal body using an organic solvent after crushing the microalga by sonication or dissolving the microalga with a protease, an enzyme or the like (for example, JP-A-2010-530741) The method described in). Such a method can also be used in the present invention. Moreover, it is preferable that the biofilm which concerns on this invention has high oil content from a viewpoint of the usefulness as biomass. Specifically, the oil content per dry algal body of the biofilm is preferably 5% by mass or more, more preferably 10% by mass or more, and particularly preferably 15% by mass or more. The oil content per dry algal body of the biofilm is usually 80% by mass or less.

The present invention will be more specifically described by the following examples, but the present invention is not limited by the following examples.

Example 1
(First pre-culture)
As a first pre-culture, PS (polystyrene; polystyrene) (No. 28, As One Corp .; outer dimensions 63 mm × 50 mm × 25.5 mm; product number 4-5605-05) (in this example, case 28 made of PS) 40 mL of CSiFF 04 medium (the medium composition is shown in Table 3 below) and the microalga Chlorococcum sp. A mixture with the FFG 039 strain (FERM BP-22262 strain) (alga concentration 0.032 mg / mL) was added. The PS case 28 containing the culture mixture is placed in a vacuum desiccator (VS type, As One Co., Ltd .; outer dimensions 300 mm × 300 mm × 170 mm; product number 1-070-01), temperature 23 ° C., carbon dioxide concentration 5% by volume Under the conditions, stationary cultures of microalgae were performed using a day white fluorescent lamp, at a light intensity of 15000 lx, a light period of 12 hours, and a dark period of 12 hours with a proof cycle of 15000 lx of the medium surface (first preculture). The culture temperature was controlled using an air conditioner set at 23 ° C. The number of days of culture for the first preculture was 14 days. Fourteen days after the start of culture, the PS case No. 28 was taken out of the vacuum desiccator, and a nylon film (thickness 1 mm) having the same length as the short diameter of the PS case No. 28 was used. The algal biofilm was recovered. Put the collected microalgal biofilm into a 5 mL tube for homogenization (Tomy Seiko Co., Ltd., TM-655) together with a small amount of CSiFF04 medium, set it in a bead cell disrupter MS-100 (Tomy Seiko Co., Ltd.), and use 4200 rpm The homogenization treatment for 20 seconds was performed three times to obtain a microalgal suspension. However, beads were not used.

(Second Pre-Culture) A second pre-culture was performed using the suspension of microalgae obtained in the first pre-culture in the same manner as the first pre-culture. However, using Seal Boy (No. 3, As One Co., Ltd .; external dimensions 319 mm × 230 mm × 113 mm; product number 4-5613-03) for the culture vessel, stationary culture of microalgae using 900 mL algal cell dispersion Done (second preculture). A vacuum desiccator is not used. The number of culture days for the second preculture was 14 days. Fourteen days after the start of culture, the microalgal biofilm formed on the liquid surface of the culture medium was recovered, and the same treatment as in the first pre-culture was performed to obtain a microalgal suspension. However, algal cells were dispersed manually using a 100 mL plastic container.

(Third pre-culture) Put 27L CSiFF 04 culture medium (culture medium) in plastic tub jumbo 180 installed on the installation surface on the metal rack installed in the room in the reinforced concrete building (water depth 5cm), The seed algae prepared by the second pre-culture was placed in a water tank so that the number of cells was 5 × 10 ⁵ cells / mL. Attach a double-sided tape to the ridge of the upper part of the water tank, and cover the top opening of the water tank with a tangy pear (second structure, agricultural film, Cai Kasei Co., Ltd., water droplet formation prevention film) Storage member). Liquid surface floating culture was started using a fluorescent lamp as a light source. The culture temperature was controlled using an air conditioner set at 23 ° C. Carbon dioxide is introduced into the air layer between the liquid surface of the culture solution in the incubator (storage member) and the groundnut after mixing the gas to a 1% concentration using a compressor and a carbon dioxide cylinder did. After 14 days, when the agricultural film was removed, formation of a film-like structure consisting of microalgae was observed on the liquid surface. The recovery was carried out in the same manner as in the first pre-culture, and a part thereof was used as an input algal body for the next main culture.

(Main culture) Form a weir (concave support 20n) on the ground, lay an agricultural film (third film 26j) there, and stack it there to form an agricultural film (first film 22f) A rectangular storage member 27 having a long side of 5 m and a short side of 1 m was formed.
Next, a tunnel-like (arch-like) shielding member 14c covering the water tank using semicircular columns installed at intervals of 1 m parallel to the short sides of the storage member 27 so as to straddle the storage member 27 Were placed to prepare a culture apparatus 10 p (see FIG. 28).
250 L of CSiFF 04 medium (culture fluid) was placed in the liquid holding portion of the storage member 12 p. An air layer N was present between the shielding member 14 c in the culture apparatus 10 p and the liquid surface of the culture solution M.
The culture medium was precultured with Chlorococcum sp. The culture seed algae of the FFG 039 strain (FERM BP-22262 strain) was added so that the cell number would be 5 × 10 ⁴ cells / mL. After the seed algae was added, stirring was performed to disperse the algae almost uniformly. After the algal cells were dispersed, the cells were cultured in a stationary state without stirring. As in the third pre-incubation of this example, carbon dioxide is mixed with gas using a compressor and a carbon dioxide bomb so as to have a concentration of 1%, and then the tunnel-like structure and the medium (culture medium) It was introduced into the air layer between the liquid level. In addition, distilled water was added as needed to prevent the loss of water in the culture solution accompanying evaporation.
In addition, when preparing CSiFF04 medium, after preparing 6 kinds of high concentration culture broth of N compound, two kinds of P compounds, Mg compound, EDTA compound, and other compounds, add one by one in this order, if necessary Stirring was performed to avoid promoting formation of unwanted precipitates.
In this state, the culture was carried out for 14 days, and the majority of the culture solution was removed by a pump from the region with few algal cells between the liquid surface of the culture solution and the bottom of the water tank. The remaining culture solution was allowed to dry naturally during the day by opening a part of the shielding member 14c (see FIGS. 29 and 30). The state of the first film after drying is shown in FIG.
After drying, one of the agricultural films constituting the bottom of the water tank, which was in contact with the culture solution, is wound using the first winder 202, and at the same time, the dried algal cells on the film are stripped off. The dried algal cells were collected (see FIG. 31).

Example 2
(First Preculture, Second Preculture, and Third Preculture) In the same manner as in Example 1, the first preculture, the second preculture, and the third preculture were performed.
(Main culture) A storage member was formed by laying one plastic film (Tekinasi) as a first film on a rectangular concrete water tank having a long side of 3 m and a short side of 1.2 m, which was installed outdoors. The concrete water tank was used for breeding goldfish, and the water tank was sufficiently cleaned before producing the storage member.
Put 200L of CSiFF04 medium (sometimes simply referred to as "culture solution" in this example) in the storage member, cover the plastic film cover as a shielding member so as to cover the entire top opening of the storage member, Configured. An air layer was present between the liquid surface of the culture solution in the culture apparatus and the plastic film cover.
The culture medium was precultured with Chlorococcum sp. The culture seed algae of the FFG 039 strain (FERM BP-22262 strain) was added so that the cell number would be 5 × 10 ⁴ cells / mL, and agitation was performed to disperse the algae bodies almost uniformly. Thereafter, the main culture is carried out for 14 days in the same manner as in Example 1, and the dried algal cells are removed by removing the culture solution, naturally drying, winding the first film and stripping the dried algal cells from the first film. I was able to get

Comparative Example 1
(First Preculture, Second Preculture, and Third Preculture) In the same manner as in Example 1, the first preculture, the second preculture, and the third preculture were performed.
(Main culture) Main culture was performed in the same manner as in Example 1. Two weeks after the start of culture, a biofilm consisting of microalgae was formed on the liquid surface, so the culture solution is contacted with the agricultural film constituting the bottom of the culture tank without reducing the volume of the culture solution. I pulled out the one who was doing it. As a result, it was possible to recover microalgae deposited on the bottom of the culture tank and a very small amount of microalgal biofilm on the liquid surface of the culture solution, but most of the microalgae on the liquid surface were recovered I could not do that.

[Example 3]
(First Preculture, Second Preculture, and Third Preculture) In the same manner as in Example 1, the first preculture, the second preculture, and the third preculture were performed.
(First Main Culture) The main culture was performed in the same manner as in Example 1 except that only one agricultural film was used when producing the culture apparatus. Two weeks after the start of culture, a biofilm consisting of microalgae was formed on the liquid surface of the culture solution. Therefore, after reducing the liquid volume of the culture solution by the same method as in Example 1, the culture solution volume is further increased by evaporation. I reduced it. In this state, the agricultural film which constituted the bottom of the culture tank was wound up with a first winder and was simultaneously recovered by depriving the microalga on the agricultural film.
(Second time of main culture) Next, using an installation roller, a film for agriculture is spread again on the ground on which the ridges are formed to produce a culture apparatus (FIG. 32), and culture is carried out in the same manner. After the reduction, the agricultural film constituting the storage member was taken up by the first winding machine and recovered by simultaneously depriving the microalga on the agricultural film.

The calorific value per unit mass of the dried algal cells obtained by the first main culture was compared with the calorific value per unit mass of the dried algal bodies obtained by the second main culture. As a result, the calorific value per unit mass of the dried algal cells obtained by the second main culture was lower than the calorific value per unit mass of the dry algal bodies obtained by the first main culture.
By observing and comparing the dried algal cells obtained by the first main culture and the dried algal bodies obtained by the second main culture, the dried algal bodies obtained by the second main culture are contaminated with soil. The It was found that due to the mixed soil, the calorific value per unit mass decreased. Moreover, although the soil had adhered to the lower part of the film for agriculture which wound up after the 1st main culture, the contamination of the soil wound the film for agriculture after collect | recovering the micro algae which exists in the upper part Although the soil did not contaminate the recovered product, the side that was in contact with the culture solution of the agricultural film at the time of winding was in contact with the side that was in contact with the ground and was in contact with the culture solution. When a part of the soil adheres to the side where the soil was attached, and an agricultural film is laid on the ground forming a bowl-like structure in this state to make a culture device, the inside of the storage member, ie, the culture solution of the agricultural film contacts The soil intruded into the soil, and in this state, soil was mixed in the recovered product because the main culture, partial removal of the culture solution, drying, stripping of the dried algal cells from the agricultural film, and recovery were performed. .

Example 4
First Preculture and Second Preculture In the same manner as in Example 1, the first preculture and the second preculture were performed.
(Main culture) Plastic bowl jumbo 180 (plastic, inner size 92.5 cm × 59.1 cm) (may be simply referred to as “culture tank” in this example) was placed on the ground (soil).
In this culture vessel, 27 L of CSiFF04 medium (sometimes referred to simply as “culture solution” in this example) is placed (water depth 5 cm), and the seed algae prepared by pre-culture is 5 × 10 ⁵ cells. It was added to the culture solution in the culture tank so as to be / mL.
A double-sided tape was attached to the top of the culture vessel, and a culture apparatus was produced by covering the upper surface of the culture vessel with texas.
Liquid surface suspension culture using sunlight as a light source was started (main culture). Distilled water was added as needed to prevent a decrease in liquid volume due to evaporation of water in the culture solution.
In addition, when preparing CSiFF04 medium, after preparing 6 kinds of high concentration culture broth of N compound, two kinds of P compounds, Mg compound, EDTA compound, and other compounds, add one by one in this order, if necessary Stirring was performed to avoid promoting formation of unwanted precipitates.
In addition, carbon dioxide was supplied in the morning of the second, fourth, sixth, ninth, eleventh and thirteenth days of culture so that the concentration in the air layer was 5%. After 14 days of culture, when the agricultural film covering the culture tank was removed, the formation of a film-like structure consisting of microalgae was observed on the liquid surface of the culture solution.
The culture solution between the liquid surface of the culture solution and the bottom of the culture vessel was removed using a pump so as not to aspirate microalgae as much as possible. The microalgae in the culture tank was dried by leaving it in this state for 6 hours. That is, it was naturally dried during the day. The temperature was 31 ° C. and the humidity was 64%.
The dried algal cells adhering to the inside of the culture vessel were collected by scraping using a spatula. The moisture content was 24%. Therefore, it was further dried using a drier at 105 ° C. overnight. The weight after drying was 25.4 g.

[Example 5]
A 120 cm × 80 cm texas film is placed under plastic jumbo 180 (which may simply be referred to as “incubation tank” in this example), except that the cultivation tank is not in direct contact with the soil. In the same manner as in Example 4, pre-culture and main culture were performed.
In the same manner as in Example 4, the culture, partial dehydration of the culture solution, and recovery of algal cells were able to be performed, and no rebound of the soil was observed when it rained on the outer side surface of the culture tank .
On the other hand, in Example 4, since there was a day when it rained during the culture period, adhesion of soil due to rebound of water droplets was observed on the outer side surface of the culture tank after the culture. Moreover, when the culture tank was moved and two or more were piled up, soil penetrated the inside of a culture tank.

[Example 6]
A tekina film was laid inside the plastic tub jumbo 180 (it may be simply referred to as "water tank" in this example) to prepare a culture tank.
After culture, partial culture solution removal, and natural drying were carried out in the same manner as in Example 4 except that the culture tank prepared as described above was used in place of the plastic mulberry jumbo 180, the vegan film was removed. It was removed from the water tank and deprived of dried algal cells on the texas film.

[Example 7]
A tekina film was laid inside the plastic tub jumbo 180 (it may be simply referred to as "water tank" in this example) to prepare a culture tank.
In the same manner as in Example 4, culture and partial culture fluid removal were carried out in the same manner as in Example 4 except that the culture tank prepared as described above was used instead of the plastic tub jumbo 180, and Most of the algal cells on the texas film did not move even if they were moved. At the same time as moving texas film to a place different from the water tank and further drying, another texas film was laid in the water tank and culture was started. By doing this, drying time was saved. The dried algal cells were obtained by depriving the dried algal cells on the first texas film.

[Example 8]
The point which floated the plastic bowl jumbo 180 (in this example, it may only be called "the culture tank") in the water tank which filled the water of 10 cm depth with a rectangular shape of 2 m long side x 1.2 m short side Except for the above, culture, partial dehydration of the culture solution, and recovery of algal cells were performed in the same manner as in Example 4. In addition, in order to prevent shaking of the culture tank, holes were made at the corners of the culture tank and the water tank and fixed with a rope so as not to move.

[Example 9]
As an algal strain, Chlorococcum sp. The culture is performed in the same manner as in Example 1 except that 28 kinds of microalgae described in Table 4 are used instead of the FFG039 strain (FERM BP-22262 strain), and a biofilm consisting of microalgae is recovered did.

[Example 10]
As an algal strain, Chlorococcum sp. The culture was carried out in the same manner as in Example 1 except that the FFG055 strain and the FFG061 strain described in Table 4 were used instead of the FFG 039 strain (FERM BP-22262 strain). Although both strains do not float on the liquid surface, they are algal strains that can be statically cultured.
After the culture, the culture solution was removed from the liquid surface. Since the algae strain used is only partially attached to the agricultural film that constitutes the storage member, care was taken not to aspirate and remove the algae when removing the culture solution using a pump. Only about 20% of the solution could be removed. This is considered to be due to the release of a water-absorbing substance into algal bodies.
After partially removing the culture solution, natural drying was performed outdoors, but it took more time than in Example 1.
It was possible to wind up the film in contact with the culture solution among the two agricultural films constituting the storage member, and to recover the dried algal bodies deposited thereon. Thus, even the algae not floating on the liquid surface could be cultured by the culture and recovery method of the present invention.

[Example 11]
Culture was performed in the same manner as in Example 1. However, as shown in FIG. 15, as the installation surface, one in which the ground is dug down and a bowl-like object is installed at a site at the height of the ground, or Using the above, two agricultural films were laid on each installation surface to produce a culture apparatus. As a result, in the case of the former, the rise of the temperature of the air layer and the temperature of the liquid phase in the culture apparatus when the outside temperature is high can be suppressed, and the growth of microalgae can be appropriately performed. Workability has improved. After the culture, the incubator (storage member) was removed, the soil was returned to the original state, and spinach was grown, but it could be grown without any problem.

[Example 12]
Culture was performed in the same manner as in Example 1. However, by applying clay on red soil, volcanic ash collected in the vicinity of Sakurajima to water to make it into a clay shape, or sand, it is applied to the surface of the support constituting the storage member, thereby eliminating new irregularities as much as possible. The body was prepared, and an agricultural film was placed thereon to prepare a storage member. Moreover, the storage member was produced using the support which did not perform these processes, and it culture | cultivated similarly. In either case, it was possible to culture the microalgae, but if processing to eliminate the unevenness was not performed, the film on the side that contacts the culture solution, out of the two agricultural films that constitute the storage member There were several places where a part of the substance jumped out of the liquid surface of the culture solution into the air layer, and as a result, the phenomenon that the amount of algal cells decreased was observed. In addition, when a support that is made as uneven as possible by mixing clay with a clay made of red soil or volcanic ash collected in the vicinity of Sakurajima is prepared, the heat accumulated in the culture apparatus can be used successfully. Since the escape was possible, the culture was in a better condition as compared to the case of preparing a support with as little unevenness as possible using sand.

[Example 13]
Culture was performed in the same manner as in Example 1. However, the film for agriculture for producing a storage member was used not in two sheets but in five sheets, and two films for agriculture of the shielding member were used. In addition, as a comparison, culture was performed with the agricultural film of the shielding member remaining as one sheet.
When two-ply agricultural films were used, the proliferation was improved when the outside air temperature was low, as compared to the case where only one agricultural film was used. This is considered to be the effect of heat retention. In addition, recovery was performed only about the film for agriculture which is in direct contact with the culture solution among the five films for agriculture. The agricultural film to which the algal cells are attached was wound and burned as it was. That is, it was used as a solid fuel.
In addition, the culture solution could be added to the remaining four films and cultured again. Thereby, the effort which installs the film for agriculture in a incubator (storage member) one by one was able to be reduced significantly.

Example 14
Culture was performed in the same manner as in Example 1. However, instead of the agricultural film (first film) on the front side, a mesh (a second film) with an opening of 4 cm was used. As a comparative example, an agricultural film, that is, a film without an opening was used as the film on the surface side. Further, a groove of 5 cm in width and 5 cm in depth was dug in the bottom of the installation surface in the major axis direction of the incubator (storage member).
After completion of the culture, when most of the culture solution was removed, the network appeared to be covered with the microalgal biofilm on the liquid surface. Drying was carried out in this state, and algal cells remained alive at the bottom grooves, but algal cells on the reticulum could produce a dry state.
After winding the network and desorbing algal cells, the network was again placed in the incubator (storage member), and a new culture solution was placed in the culture apparatus to start culture. That is, the microalgae which remained in the groove part can be cultured as a seed algae, and the culture can be performed without using the seed algae newly procured from another culture apparatus.

[Example 15]
Culture was performed in the same manner as in Example 1. However, glass beads having a diameter of 0.177 to 0.250 (As One Ltd., 6-257-04) mm were spread between the two agricultural films. The culture proceeded without any problem, and the film could be wound smoothly.
In addition, a rod-like structure is previously installed between two agricultural films, and when winding up one of the two agricultural films that was in contact with the culture solution, the rod-like structure is vertically upward. By lifting, the frictional force between the two agricultural films could be reduced, and it was possible to smoothly roll up the two agricultural films which were in contact with the culture solution. The culture also proceeded without problems.

[Example 16]
Culture was performed in the same manner as in Example 1. However, two storage members were installed in one shielding member. Also, the start of culture in each storage member was delayed for 7 days. Culture in any of the reservoirs proceeded without problems. In addition, after collecting the algal cells in one of the storage members, the alveolar structure is partially broken by collapsing the ridge-like structure of the partition with the storage member in which the culture is being performed. It was also possible to introduce it and start the culture again. As a result, it was possible to greatly reduce the time and effort of feeding the seed algae at the start of culture.

[Example 17]
The microalga was cultured in the same manner as in Example 1. However, the bottom of the support portion of the storage member was inclined, and the difference in depth between the shallow side and the deep side of the bottom was set to 5 cm. The culture proceeded without problems. This facilitated the removal of the culture solution.

[Example 18]
Culture was performed in the same manner as in Example 1. However, any of the functional films listed in Table 5 was used in place of the agricultural film for forming the tunnel-like structure of the shielding member. Both were able to culture without problems.

[Example 19]
Culture was performed in the same manner as in Example 1. However, the shape of the tunnel-like structure was modified to the shape shown in FIG. 19 or 21.
The tunnel-like structure having the shape shown in FIG. 19 was excellent in the measures against rain, the prevention of water drops, and the wind resistance.
The tunnel-like structure having the shape shown in FIG. 21 is excellent in the measures against rain, the prevention of water drops, and the wind resistance.

[Example 20]
Culture was performed in the same manner as in Example 1. However, when a vinyl for waist winding is installed on the side of the tunnel-like structure, which is a shielding member, and the temperature rises 7 ° C. lower than the high temperature side of the microalga culture temperature range, a part of the vinyl for waist winding Temperature control was performed by opening it. When the temperature was not adjusted, the weight of the obtained algal cells was reduced as compared to the one performed, but the algae could be harvested without any problem.

[Example 21]
Culture was performed in the same manner as in Example 1. However, the temperature in the culture apparatus could be lowered by about 2 to 6 ° C. by placing the cryostat on the outside of the tunnel-like structure which is a shielding member. Although the weight of the obtained algal cells was lower than that of the one without the main measures, it could be harvested without any problem.

Example 22
Culture was performed in the same manner as in Example 1. However, the temperature in the culture apparatus could be lowered by about 3 to 7 ° C. by spraying cooling water to the outside of the tunnel-like structure which is the shielding member. Although the weight of the obtained algal cells was lower than that of the one without the main measures, it could be harvested without any problem.

[Example 23]
Culture was performed in the same manner as in Example 1. However, by flowing cooling water between the two agricultural films that make up the storage member and between the agricultural film that is not in contact with the culture solution and the ground, the temperature in the culture apparatus can be reduced. The temperature could be lowered by about 3 to 7 ° C. Although the weight of the obtained algal cells was lower than that of the one without the main measures, it could be harvested without any problem.

[Example 24]
Culture was performed in the same manner as in Example 1. However, the temperature in the culture apparatus could be lowered by about 3 to 8 ° C. by spraying particulate water droplets on the air layer in the culture apparatus. Although the weight of the obtained algal cells was lower than that of the one without the main measures, it could be harvested without any problem.

[Example 25]
Culture was performed in the same manner as in Example 1. However, a network-like material through which gas can pass is installed at a low position and a high position in the inner space of the tunnel-like structure constituted by the columns and the agricultural film, and the high temperature air flows upward. The temperature in the culture apparatus could be lowered by about 2 to 6 ° C. Although the weight of the obtained algal cells was lower than that of the one without the main measures, it could be harvested without any problem.

[Example 26]
Culture was performed in the same manner as in Example 1. However, without collecting the algal cells on the agricultural film, a new culture solution and seed algae were added and cultured again. This is repeated five times to dry the algal cells on the agricultural film that has been in direct contact with the culture solution, and at the same time winding the film, the algal cells are stripped from the film to obtain a dry algal body. The This made it possible to reduce the process of winding up the film and removing algal cells from the film.

[Example 27]
Culture was performed in the same manner as in Example 1. However, carbon dioxide was supplied by CO ₂ tablets. As a supply method, the thing put in the culture tank which is culturing microalga, the small water tank which is not culturing the algae body is installed in the air layer in the culture apparatus, and the CO ₂ tablet is placed in the water tank Those used for generating carbon dioxide were used. In the case of the former, although part of the water surface algae was destroyed, the ratio to the whole was a part, and there was almost no influence. In any case, the culture could be done without problems.

[Example 28]
Culture was performed in the same manner as in Example 1. However, natural drying of algal bodies was performed at night. By this, the daytime required for culture could be used effectively, and the dry algal body production amount in a fixed period could be improved.

[Example 29]
The dried algal cells obtained in Example 1 were subjected to hexane extraction to obtain an oil. The oil content was 35% of the dry weight.

[Example 30]
Pellets were prepared from the dried algal cells obtained in Example 1. The heat of combustion of the obtained pellets was 6000 cal / g.

[Example 31]
The culture was carried out in the same manner as in Example 1, and after removing most of the medium with a pump, the wet algal cells were placed in a 1 L beaker and homogenized by thoroughly stirring. This was divided into 12 and each was applied onto the materials listed in the "material" column of Table 6. Next, they were placed in an oven and dried by heating to 130 ° C.
After drying, the adhesion or peelability of the dried product was evaluated from the material. The results are shown in the "adhesion or peelability" column of Table 6. The dried algal cells were firmly attached to the cardboard, the aluminum bat and the aluminum foil (matted surface, glossy surface), but the dried algal cells were easily exfoliated from the glass and various main polymers only by tilting.
In FIG. 40, for each material of aluminum bat, aluminum foil (matted surface) and fluorocarbon resin, only the material is wet, and the algal cells are dried and dried, and the dried algal cells are removed from the material. The state of each after peeling is shown.

[Example 32]
The culture was carried out in the same manner as in Example 1 to form a biofilm on the liquid surface.
When the biofilm formed on the liquid surface was collected from a plurality of places and observed with an optical microscope, the microscopic image schematically represented in FIG. 41 or FIG. 42, or the ratio of algal bodies 601 and algal aggregate 603 A microscope image of an intermediate state of the microscope images schematically represented in FIGS. 41 and 42 can be obtained.
FIG. 41 schematically illustrates the state in which the algal bodies 601 and the extracellular matrix 602 form a biofilm. FIG. 42 schematically shows a state in which the algal bodies 601 and the algal body aggregate 603 and the extracellular matrix 602 form a biofilm. The aggregate of algal bodies 603 is considered to be one where algal bodies 601 are bound by extracellular polysaccharide.
On the other hand, when the microalgae suspended in the culture solution were observed with an optical microscope, a microscopic image schematically represented in FIG. 43 was obtained.
The biofilm formed on the liquid surface was a three-dimensional structure, and as shown in FIGS. 44 and 45, a foam-like structure and a scaly structure were formed.
A biofilm 701 was formed on the liquid surface of the culture solution 802 placed in the culture tank (storage member) 801. In the biofilm 701, a foam-like structure 703 and a cocoon-like structure 702 were also observed.
The foam-like structure 703 varied in size and shape, but was often several millimeters to several centimeters large, and the foam film could be easily broken. The gas inside the foam structure 703 is presumed to contain carbon dioxide or oxygen which has not been confirmed, but is generated by metabolism.
The rod-shaped structure 702 was found to be invaginated in the culture solution 802. The rod-shaped structure 702 is presumed to be invaginated in the culture solution 802 by the force from the lateral direction of the rod-shaped structure 702 along with the extension of the biofilm 701.

[Example 33]
(First pre-culture) PS (polystyrene; polystyrene) case (No. 28, As One Corporation; outer dimensions 63 mm × 50 mm × 25.5 mm; part number 4-5605-05) (in this example, “PS product made in the following manner In the case of No. 28), 40 mL of CSiFF 04 medium (the medium composition is shown in the above-mentioned Table 3) and the microalga Chlorococcum sp. A mixture of the FFG 039 strain (FERM BP-22262 strain) with 1 mL of a CSiFF 04 medium dispersion (algal concentration 1.2 mg / mL) (hereinafter sometimes referred to as "culture mixture") was added. Using a No. 28 PS case containing a culture mixture, using a white fluorescent lamp under conditions of a temperature of 23 ° C. and a carbon dioxide concentration of 5% by volume, the illuminance of the liquid surface of the medium is 15000 lx, the light period 12 hours, the dark period 12 Stationary culture (liquid surface suspension culture) of the microalga was carried out in the irradiation cycle of time (first pre-culture). The culture temperature was controlled using an air conditioner set at 23 ° C. The number of days of culture for the first preculture was 14 days. Fourteen days after the start of culture, a microalgal biofilm formed on the liquid surface of the medium was recovered using a nylon film (thickness 1 mm) having the same length as the short diameter of Case 28 made of PS.

(Second pre-culture) A second pre-culture was performed using the microalgal biofilm obtained in the first pre-culture. Specifically, using a plastic vat (30 cm × 23 cm × 7 cm) in the culture tank, add half amount (mass base) of the microalgal biofilm obtained in the first pre-culture to 2800 mL of CSiFF04 medium, Stationary culture (liquid surface suspension culture) of the microalga was performed under the same culture conditions as the first pre-culture (second pre-culture). The number of days of culture for the second preculture was 14 days. Fourteen days after the start of culture, the microalgal biofilm formed on the liquid surface of the medium was recovered. The collected microalgal biofilm was transferred to a 500 mL plastic container containing 300 mL of CSiFF04 medium. After shaking this plastic container by hand, ultrasonication was carried out for 5 minutes to obtain a microalga CSiFF 04 medium dispersion (hereinafter sometimes referred to as “seed algae dispersion” in this example). 5 mL was collected from the obtained seed algal dispersion, followed by filtration and drying (130 ° C., 1 hour), and the dry algal body weight of the microalga obtained by the pre-culture was determined. Furthermore, when the algal concentration in the seed algal dispersion (hereinafter sometimes referred to as "seed algal concentration" in the present example) (mg / mL) is calculated from the dry algal weight obtained, it is 10.7 mg. It was / ml.

(Main culture) A plastic cylindrical container (diameter 13.4 cm, height 21 cm) is used for the culture tank, and the improved culture medium (the composition of the culture medium is shown in Table 7 below) and the depth of the culture medium are used for this culture tank. Was put so that it would be 20 cm. Next, a seed algal dispersion was added to the improved culture medium in the culture tank so that the algal body concentration was 4.0 g / m ² . Using a culture vessel containing this medium and seed algal dispersion, at a temperature of 23 ° C. and a carbon dioxide concentration of 5% by volume, using a daylight white fluorescent lamp, the illuminance on the medium liquid surface is 22000 lx, and the light period is 12 hours. Dark period A stationary culture (liquid surface suspension culture) of microalgae was performed (main culture) in an irradiation cycle of 12 hours. The culture days of the main culture were 9 days.

(Media volume reduction) The media surface was exposed by gently pushing away the microalgal biofilm formed on the entire surface of the culture medium using a plastic plate, taking care not to cause sedimentation. Using a pump, 95% by volume of the culture medium at the end of the main culture was discharged so as not to discharge the microalgal biofilm and algal cells formed on the liquid surface of the culture medium and the algal cells at the bottom of the culture tank (medium Medium volume discharged at volume reduction (95% by volume). The medium depth after draining was 1 cm. By removing the plastic plate, the microalgal biofilm spread again all over the medium surface. In addition, a part of the microalgal biofilm is collected and dried at 130 ° C. in a constant temperature dryer for 3 hours, and the weight before and after the drying is measured to determine the algal body solid content of the microalgal biofilm (this example) In the above, “the solid content of algal bodies after discharging the medium” was determined (mass%). The results are shown in Table 8.

(Post-Culture) Using the culture tank after volume reduction of medium volume, temperature 23 ° C., carbon dioxide concentration 5% by volume, using white-white fluorescent light, illuminance on medium level 22000 lx, light period 12 hours, Dark period Still culture (liquid surface suspension culture) of microalgae was further continued (post culture) in a 12-hour irradiation cycle. The number of days for post-culture was 5 days.

(Recovery) The total amount of the microalgal biofilm formed on the liquid surface of the culture medium and the algal cells precipitated on the bottom of the culture vessel was scraped and collected. The collected microalgal biofilm and algal cells were dried at 130 ° C. in a constant temperature dryer for 3 hours to obtain dry algal bodies (hereinafter sometimes referred to as “dry algal bodies” in this example). The weight of the dried algal cells was measured to obtain the dried algal body weight (g). The value obtained by dividing dry algal body weight by culture area (container bottom area) (m ² ) is used as algal body yield (g / m ² ), and value obtained by dividing algal body yield (g / m ² ) by culture days is algal Body productivity (g / m ² / day).

(Oil content and oil productivity) About 50 mg of the obtained dried algal cells were precisely weighed and thoroughly ground using a mortar and a pestle for 5 minutes to obtain a crushed material. The resulting pulverized product was washed with methanol (twice with 1 mL) and chloroform (once with 4 mL), the solvent-soluble part was collected, and centrifugation (1000 rcf, 5 minutes) was performed. The solvent was evaporated from the supernatant after centrifugation, the weight of the residue was precisely weighed, and the oil content was determined by the following equation.
Oil content (mass%) = (weight of residue (g) / weight of dried algal body (g)) × 100
The oil productivity (g / m ² / day) was calculated by multiplying the algal cell productivity (g / m ² / day) previously obtained by the oil content rate. The results are shown in Table 8.

[Example 34]
The culture and recovery of the microalga are carried out in the same manner as in Example 33 except that the culture days of the main culture are changed from 9 days to 11 days, and further, the culture days of the post culture are changed from 5 days to 3 days. Algal cell yield, algal cell productivity, oil content and oil productivity were calculated. The results are shown in Table 8.

[Example 35]
The amount of medium discharged at the time of reduction of the amount of medium fluid was changed from 95% by volume to 75% by volume of the amount of medium fluid at the end of the main culture (the amount of medium discharged at the time of volume reduction of medium 75% by volume). The microalgae was cultured and recovered in the same manner as 33, and algal body yield, algal body productivity, oil content and oil productivity were calculated. The results are shown in Table 8.

Comparative Example 2
Cultivation and recovery of microalgae were carried out in the same manner as in Example 33 except that the number of days of culture of the main culture was changed from 9 days to 14 days, and post-culture was not carried out, algal body yield, algal body productivity , Oil content and oil productivity were calculated. The results are shown in Table 8.

In Table 8, "total culture days (day)" represents the total number of main culture days and post culture days.

[Example 36]
(First Pre-Culturing and Second Pre-Culturing) In the same manner as in Example 33, the first pre-culturing and the second pre-culturing were performed to obtain a seed algal dispersion.

(Main culture) A plastic cylindrical container (diameter 13.4 cm, height 21 cm) is used for the culture tank, and the CSiFF04 medium (the medium composition is shown in the above-mentioned Table 3) is used for this culture tank, and the depth of the medium. Was put so that it would be 20 cm. Next, a seed algal dispersion was added to the CSiFF04 medium in the culture tank so that the algal body concentration was 4.0 g / m ² . Using a culture vessel containing a culture medium and a seed algae dispersion, using a white-white fluorescent lamp under conditions of a temperature 23 ° C. and a carbon dioxide concentration of 5% by volume, an illuminance of 22000 lx at the liquid surface of the medium, a light period 12 hours dark Stationary culture (liquid surface suspension culture) of microalgae was performed in an irradiation cycle of 12 hours. The culture days of the main culture were 14 days.

(Mold volume reduction) Prepare a plastic plate adapted to the size of the culture vessel, insert this plastic plate from the end of the culture vessel to the bottom of the microalgal biofilm, and move the plastic plate to the other side of the container Thus, the microalgal biofilm was deposited on the plastic plate, and the whole microalgal biofilm formed on the medium surface was skimmed off. A portion of the scooped microalgal biofilm is collected, dried in a constant temperature dryer at 130 ° C. for 3 hours, and the weight before and after the drying is measured to determine the solid content of algal bodies in the microalgal biofilm (this practice) In the example, "the solid content of algal bodies after discharging the medium" (mass%) was determined. The results are shown in Table 9.

(Post-Culture) A plastic cylindrical container (diameter 13.4 cm, height 6 cm) was prepared in a culture vessel, and the microalgal biofilm scooped on a plastic plate was spread on the bottom of the culture vessel. The collected microalgal biofilm was wet, and the culture medium coexisted. Using this culture vessel, temperature 23 ° C., carbon dioxide concentration 5% by volume, using a white-white fluorescent lamp, the microalgae biofilm surface illuminance spread at the bottom of the culture vessel 22000 lx, 12 hours light, dark Cultivation of microalgae was further continued with an irradiation cycle of 12 hours (post culture). The number of days for post-culture was 3 days.

(Recovery) The whole amount of microalgal biofilm and algal cells obtained by culture was recovered. The collected microalgal biofilm and algal cells were dried at 130 ° C. in a constant temperature dryer for 3 hours to obtain dry algal bodies (hereinafter sometimes referred to as “dry algal bodies” in this example). The weight of the dried algal cells was measured to obtain the dried algal body weight (g). The value obtained by dividing dry algal body weight by culture area (container bottom area) (m ² ) is used as algal body yield (g / m ² ), and value obtained by dividing algal body yield (g / m ² ) by culture days is algal Body productivity (g / m ² / day).

(Oil content and oil productivity) In the same manner as in Example 33, the oil content (% by mass) and the oil productivity (g / m ² / day) were calculated. The results are shown in Table 9.

[Example 37]
Cultivation and recovery of microalgae were carried out in the same manner as in Example 36, except that the post-culture was changed to the atmosphere under a carbon dioxide concentration of 5% by volume, and algal yield, algal productivity, oil content and oil productivity Was calculated. The results are shown in Table 9.

Comparative Example 3
The first preculture, the second preculture, and the main culture were performed as in Example 36. Thereafter, all the microalgal biofilm and algal cells obtained by the culture are collected, suction-filtered, and the medium components are removed by washing with water (100 mL × 2 times) to obtain a microalgal culture. The To the obtained culture, 5.0 mL of water was added and stirred to obtain an aqueous dispersion (algaloid dispersion) of fine algae. The solid content of algal bodies in this algal dispersion was determined to be 8.34%. Thus, microalgae was post-cultured and recovered in the same manner as in Example 36 except that the medium was replaced with water, and algal body yield, algal body productivity, oil content, and oil productivity were calculated. The results are shown in Table 9.

Comparative Example 4
The first preculture, the second preculture, and the main culture were performed as in Example 36. Thereafter, the entire amount of the microalgal biofilm and algal cells obtained by the culture was collected and suction-filtered to obtain a culture of the microalgae. The obtained culture was dried at 130 ° C. in a constant temperature dryer for 3 hours, and its weight was measured to determine the dry algal body weight (g). In the same manner as in Example 36, algal cell yield (g / m ² ), algal cell productivity (g / m ² / day), oil content (mass%) and oil productivity (g / m ² / day) are calculated. did. The results are shown in Table 9.

Comparative Example 5
The first preculture, the second preculture, and the main culture were performed in the same manner as in Example 36 except that the culture days of the main culture were changed from 14 days to 17 days. Thereafter, the entire amount of the microalgal biofilm and algal cells obtained by the culture was collected and suction-filtered to obtain a culture of the microalgae. The resulting culture was dried at 130 ° C. in a constant temperature dryer for 3 hours, and its weight was measured to determine the amount of algal bodies (g). The algal cell yield, algal cell productivity, oil content and oil productivity were calculated in the same manner as in Example 36. The results are shown in Table 9.

In Table 9, "total culture days (day)" represents the total of main culture days and post culture days.

[Example 38]
(First pre-culture) PS (polystyrene; polystyrene) case (No. 28, As One Corporation; outer dimensions 63 mm × 50 mm × 25.5 mm; part number 4-5605-05) (in this example, “PS product made in the following manner In the case 28), 40 mL of CSiFF04 medium (the composition is shown in Table 3 above) and a dispersion of CSiFF04 medium of the microalga Botryococcus sudeticus AVFF 007 strain (FERM BP-11420 strain) A mixture with 1 mL (algal concentration 1.2 mg / mL) (hereinafter sometimes referred to as "culture mixture" in this example) was added. Using a PS case 28 containing a culture mixture, temperature 23 ° C., carbon dioxide concentration 5% by volume, using a white fluorescent lamp, the illuminance of the liquid surface of the culture medium 15000 lx, light period 12 hours, dark period 12 Stationary culture (liquid surface suspension culture) of the microalga was carried out in the irradiation cycle of time (first pre-culture). The culture temperature was controlled using an air conditioner set at 23 ° C. The number of days of culture for the first preculture was 14 days. Fourteen days after the start of culture, a microalgal biofilm formed on the liquid surface of the medium was recovered using a nylon film (thickness 1 mm) having the same length as the short diameter of Case 28 made of PS.

(Second pre-culture) A second pre-culture was performed using the microalgal biofilm obtained in the first pre-culture. Specifically, using a plastic vat (30 cm × 23 cm × 7 cm) in the culture tank, add half amount (mass base) of the microalgal biofilm obtained in the first pre-culture to 2800 mL of CSiFF04 medium, Stationary culture (liquid surface suspension culture) of the microalga was performed under the same culture conditions as the first pre-culture (second pre-culture). The number of days of culture for the second preculture was 14 days. Fourteen days after the start of culture, the microalgal biofilm formed on the liquid surface of the medium was recovered. The collected microalgal biofilm was transferred to a 500 mL plastic container containing 300 mL of CSiFF04 medium. After shaking this plastic container by hand, ultrasonication was carried out for 5 minutes to obtain a microalga CSiFF 04 medium dispersion (hereinafter referred to as "seed algae dispersion" in this example). 5 mL was collected from the obtained seed algal dispersion, followed by filtration and drying (130 ° C., 1 hour), and the dry algal body weight of the microalga obtained by the pre-culture was determined. Furthermore, when the algal concentration in the seed algal dispersion (sometimes referred to as "seed algal concentration" in this example) (mg / mL) was calculated from the dry algal weight obtained, it was 10.7 mg / ml. Met.

(Main culture) Using TPX-stained bat (vertical type, As One Co., Ltd .; size 94 mm × 46 mm × 96 mm; product number 2-3029-01) in the culture tank, the CSiFF 04 medium (composition described in Table 3 above) was used in this culture tank. ) Is placed so that the depth of culture medium is 5 cm. Next, a seed algal dispersion was added to the CSiFF04 medium in the culture tank so that the algal body concentration was 0.67 g / m ² . Using a culture vessel containing this medium and seed algal dispersion, using a white-white fluorescent lamp under conditions of temperature 23 ° C. and carbon dioxide concentration 5% by volume, illuminance 15,000 lx at the medium liquid surface, light period 12 hours, Dark period A stationary culture (liquid surface suspension culture) of microalgae was performed (main culture) in an irradiation cycle of 12 hours. The culture days of the main culture were 14 days.

(Mold volume reduction) Prepare a plastic plate adapted to the size of the culture vessel, insert this plastic plate from the end of the culture vessel to the bottom of the microalgal biofilm, and move the plastic plate to the other side of the container Thus, the microalgal biofilm was deposited on the plastic plate, and the whole microalgal biofilm formed on the medium surface was skimmed off. A portion of the scooped microalgal biofilm is collected and dried at 130 ° C. in a constant temperature dryer for 3 hours, and the weight before and after drying is measured to determine the solid content of algal bodies in the microalgal biofilm (this practice) In the example, "the solid content of algal bodies after discharging the medium" (mass%) was determined. The results are shown in Table 10.

(Post-Culture) PS Case No. 28 was prepared in a culture vessel, and the microalgal biofilm scooped on a plastic plate was spread on the bottom of this culture vessel. Using this culture vessel, the temperature on a microalgal biofilm surface spread to the bottom of the culture vessel using a day-white fluorescent lamp under conditions of a temperature of 23 ° C. and a carbon dioxide concentration of 5 vol. In the dark, a 12-hour irradiation cycle, the microalgae culture was further continued (post culture). The number of days for post-culture was 3 days.

(Oil content and oil productivity) In the same manner as in Example 33, the oil content (% by mass) and the oil productivity (g / m ² / day) were calculated. These results are shown in Table 10.

Comparative Example 6
The first preculture, the second preculture, and the main culture were performed in the same manner as in Example 38. Thereafter, the entire amount of the microalgal biofilm and algal cells obtained by the culture was collected and suction-filtered to obtain a culture of the microalgae. The obtained culture was dried at 130 ° C. in a constant temperature dryer for 3 hours, and its weight was measured to determine the dry algal body weight (g). In the same manner as in Example 38, algal yield (g / m ² ), algal productivity (g / m ² / day), oil content (mass%) and oil productivity (g / m ² / day) were produced. did. The results are shown in Table 10.

Comparative Example 7
The first preculture, the second preculture, and the main culture were performed in the same manner as in Example 38 except that the culture days of the main culture were changed from 14 days to 17 days. Thereafter, the entire amount of the microalgal biofilm and algal cells obtained by the culture was collected and suction-filtered to obtain a culture of the microalgae. The resulting culture was dried at 130 ° C. in a constant temperature dryer for 3 hours, and its weight was measured to determine the amount of algal bodies (g). Algal body yield, algal body productivity, oil content and oil productivity were produced in the same manner as in Example 38. The results are shown in Table 10.

In Table 10, "total culture days (day)" represents the total of main culture days and post culture days.

10a, 10b, 10c, 10e, 10e, 10g, 10h, 10i, 10k, 10n, 10r, 10t, 10u

Culture apparatus

12, 12a, 12b, 12f, 12g, 12h, 12j, 12n, 12t, 12t ,

12u Storage members

14a, 14b, 14c, 14d,

14k Shielding members

16a, 16r Containers 17 Valves 18

Drains

20f, 20h, 20n, 28, 28e Support 22f First film 24g Second film 26j Third film 27 Storage member 30

Bulking member

50, 60

Culture device

52, 62 Storage member 64 Heat transfer piping 90 Culture device 92 Member formed in a tubular shape (tube shape)

Support body

100, 200

Culture device

202, 202a, 202b, 202c First winding machine 204 for installation of spatula 206 collection container 208 Over La 210 film 220 storage member group 601 algal cells 602 extracellular matrix 603 algae assemblage 701 biofilm 702 pleated structure 703 foam structure 800 culture apparatus 801 culture tank for repositioning (storage member)
802 culture solution 901 culture tank 902 first substrate 903a biofilm (alga)
903b Biofilm (Alga)
904 Second substrate G Installation surface Gb Concave part Gd Convex part M Culture solution N Air layer P, P ₂ , P ₃ Microalgae T Region W Water

Claims

A culture and recovery method of a microalga culture using a culture apparatus including a storage member for holding a culture solution,
A main culture step of culturing a microalga in the culture solution while maintaining an air layer above the culture solution;
A volume reduction step of reducing the volume of the culture solution;
And a recovery step of recovering the culture of the microalga after the liquid volume reduction step.
After the liquid volume reduction step and before the recovery step,
The culture | cultivation and collection | recovery method of the culture of the microalga of Claim 1 including the post-culture process which culture | cultivates the said microalga in presence of a culture solution.
3. The method according to claim 2, wherein in the post-culturing step, the mass ratio of the microalga algal cells to the culture solution is: mass of algal cells / mass of culture solution = 20/80 to 0.1 / 99.9. Method for culturing and recovering cultures of microalgae.
The culture device is further disposed above the storage member so as to cover at least a part of the liquid surface of the culture solution when viewed in the vertical direction, and can transmit light of at least a part of wavelengths The culture of the culture of the microalga according to any one of claims 1 to 3, further comprising a shielding member, wherein the air layer is formed between the liquid surface of the culture solution and the shielding member. Recovery method.
The culture apparatus further includes at least one of an air temperature control unit that controls the temperature of the air layer and a liquid temperature control unit that controls the temperature of the culture solution,
The microalga according to any one of claims 1 to 4, wherein, in the main culture step, at least one of the temperature of the air layer and the temperature of the culture solution is controlled to a temperature range suitable for culturing the microalga. Method for culturing and recovering a culture of
The method for cultivating and recovering a culture of microalga according to any one of claims 1 to 5, wherein the culture apparatus further comprises an aeration unit in communication with the air layer.
The culture and recovery method of a culture of microalga according to claim 6, wherein the culture apparatus further comprises an aeration control unit that controls passage of gas in the aeration unit.
The storage member includes a flexible first film and a support for supporting the first film.
The culture of the microalga according to any one of claims 1 to 7, wherein the support supports the first film to form a pool-like recess, and the culture solution is held by the recess. Method of culture and recovery of
The culture apparatus has a first film moving member for moving the first film,
In the liquid volume reduction step, the culture volume of the culture solution is reduced to bring the culture of the cultured microalga into contact with the first film;
The culture and recovery method of a culture of microalga according to claim 8, wherein the first film is moved from the support in the recovery step, and the culture of the microalga is recovered.
The culture apparatus includes a first winding machine which winds the first film in a roll.
10. The method according to claim 8, wherein in the recovery step, the first winder recovers the culture of the microalga by rolling up the first film together with the culture of the microalgae in a roll. The culture | cultivation and collection | recovery method of the culture of micro algae as described in 4.
The culture apparatus includes a first winding machine which winds the first film in a roll, and a first recovery member which peels the culture of the microalga from the first film.
In the recovery step, the first recovery member peels the culture of the microalgae from the first film while the first winder winds the first film into a roll, The culture | cultivation and collection | recovery method of the culture of the micro algae of Claim 8 or 9 which collect | recovers the culture of micro algae.
The culture apparatus of a microalga according to any one of claims 8 to 11, wherein the culture apparatus comprises one or more third films between the first film and the support. And recovery methods.
The culture of the microalga according to any one of claims 1 to 12, wherein the storage member comprises a second film having a plurality of through holes formed on the outermost surface in contact with the culture solution. Culture and recovery method.
The culture apparatus has a second film moving member for moving the second film,
The method for cultivating and collecting a microalgal culture according to claim 13, wherein in the collecting step, the second film is moved to collect the microalgal culture.
The culture apparatus includes a second winding machine which winds the second film in a roll.
In the recovery step, the second winding machine recovers the culture of the microalga by rolling up the second film together with the culture of the microalgae in a roll shape. The culture | cultivation and collection | recovery method of the culture of micro algae as described in 4.
The culture apparatus has a second winding machine for winding the second film in a roll, and a second recovery member for peeling the culture of the microalga from the second film.
In the recovery step, the second recovery member exfoliates the culture of the microalgae from the second film while the second winding machine winds the second film in a roll shape. The culture | cultivation and collection | recovery method of the culture of microalga of Claim 13 or 14 which collect | recovers the culture of microalga.
The culture and recovery method of a culture of microalga according to any one of claims 1 to 16, wherein the microalga is a microalga capable of forming a biofilm on the liquid surface of the culture solution.