JP6047300B2 - Microalgae culture method, biofilm formed on the liquid surface by the culture method, oil production method, biofilm recovery method, and biomass fuel production method - Google Patents

Description

  The present invention relates to a method for culturing microalgae, a biofilm formed on a liquid surface by the culturing method, biomass and oil obtained from the biofilm, a method for recovering the biofilm, and a method for producing biomass fuel. According to the method for culturing microalgae according to the present invention, a biofilm can be formed on the liquid surface, and this culturing method is useful in the energy field.

  In recent years, with the development of industrial activities, global warming has progressed due to soaring fuel prices due to the use of large amounts of fossil fuels and the greenhouse effect of carbon dioxide released into the atmosphere by using fossil fuels. It is a problem to do. As means for solving such a problem, there is an increasing expectation for the use of microalgae having the ability to fix carbon dioxide with light energy and convert it into hydrocarbon compounds, biodiesel (triglycerides) and the like. For example, various studies have already been conducted to cultivate microalgae to immobilize carbon dioxide using light energy and produce biomass such as biodiesel and hydrocarbon compounds.

  By the way, in the presence form of microorganisms such as microalgae, for example, in order to protect themselves from external attacks, viscous secretions are produced and higher-order structures, so-called biofilms (microorganisms) There is a presence form that forms an aggregate or a microbial membrane), and has recently attracted a great deal of attention in fields such as medicine and the environment. It is known that when a biofilm is formed, microorganisms behave differently from individual properties and become aggregated. A biofilm is usually formed on the surface of a solid substrate such as a stone or a plant surface, and no specific report has been confirmed that microalgae form a biofilm on the liquid surface.

  There are various problems in the production of biomass using microalgae, and an efficient method for culturing microalgae, a method for collecting microalgae, and a method for extracting biomass such as oil have not been developed. Due to the high cost, production on a commercial scale is not carried out. One of the biggest causes is the lack of an efficient method for collecting microalgae. Specifically, since microalgae usually grow while floating in the liquid, in order to use microalgae as biomass, a very dilute concentration of microalgae must be recovered from a large amount of liquid. . In addition, since light energy is required for the growth of microalgae, the concentration of microalgae present in the liquid cannot be excessively increased in order to ensure sufficient light irradiation. As a result, in order to collect the microalgae floating in the liquid, it was necessary to filter a large amount of water. Also, the size of microalgae is generally small and filtration is not easy. As a study of a recovery method for solving such problems, a method using a precipitant, a method using a centrifuge, a method of recovering the large organism after feeding microalgae to a larger organism, etc. However, none of these methods led to a fundamental solution.

For the reasons described above, in order to recover microalgae efficiently, simply and at low cost, it is desired to grow microalgae on the liquid surface.
Regarding the floating of microalgae on the liquid surface, there are naturally occurring blue-tailed sea bream, botriococcus blooming (mass breeding), and red tide generated in the ocean. However, since these are all phenomena that occur in nature, there are many kinds of microalgae on the liquid surface that are mixed together with a large amount of impurities, and whether the microalgae are purely growing on the liquid surface. Not sure. Aokko is a microalga that has not been purely sterilized, that is, it is composed of a variety of microalgae, floats on the surface of the liquid, and is mainly agglomerated in the form of powdered microalgae as indicated by blue powder. It means a thing and is different from the biofilm according to the present invention.
Regarding culturing Botriococcus, Non-Patent Documents 1 and 2 show that when Botriococcus accumulates oil, it has a floating property on the liquid surface. Further, Patent Document 1 describes in the abstract that Botriococcus floats on the liquid surface. However, in Patent Document 1, Botriococcus is cultured on the surface of a hygroscopic cloth based on the phenomenon that the accumulation rate of hydrocarbon compounds is improved by direct contact between carbon dioxide and algae. Yes, there is no description of an example in which the botulinum is floating directly on the liquid surface.
Non-Patent Document 3 discloses Botryococcus sp. As Botriococcus sudeticus. Although the UTEX-2629 strain is disclosed, it has a higher specific gravity than the Botriococcus braunii, and there is no description about floating on the liquid surface.
As described above, although there is a suggestion that the bottling coccus floats on the liquid level in the conventional technology, there is no description of a specific mode in which the botulococcus actually floats on the liquid level, and the liquid There is no description of forming a biofilm on the surface.
Non-Patent Document 4 describes that a dinoflagellate algae (C. cohni) floats on the liquid surface by culturing, but there is no description about forming a biofilm on the liquid surface. .

US Patent Application Publication No. 2009/0087889

The Ecology of Cyanobacteria. Their Diversity in Time and Space, B.E. A. Whiton & M. Potts, Eds, Kluwer Academic (1999), pp. 160 Oceanological Studies, 1998, Vol. 27, No. 1, Publisher: Index Corporateus p17 Melis et al. , J apply Physol (2010) “Growth Inhibition of Dinoflagellate Algae in Shake Flasks: Not Due to Shear This Time”, Biotechnol. Prog. , 2010, Vol. 26, no. 1, pages 79-87 Journal of Agricultural Civil Engineering pp 971-975, Vol. 56 (10), 1988

Although culturing microorganisms such as microalgae and taking out biomass such as biodiesel and hydrocarbon compounds from the cultured microorganisms has attracted much attention, production on a commercial scale has not yet been performed. One reason is that production costs are still too high compared to fossil-derived energy resources obtained from oil fields. The main cause of this high production cost is the high recovery cost for collecting small-sized microalgae dispersed in the liquid from the liquid, and generally a vast area is required to produce biomass, For this large area, there is no method for introducing carbon dioxide uniformly and at low cost, the piping is very long and the installation cost is high, and the production volume of algal biomass per light receiving surface is low. In addition, an apparatus for stirring the liquid medium in which algae are dispersed and an operating cost are high.
In addition, in the conventional culture of microalgae, it is necessary to handle a large amount of liquid medium, which requires a large amount of energy. Furthermore, in order to supply a large amount of liquid medium, a large amount of water was required.
Furthermore, according to Non-Patent Document 5, it is reported that the recovery rate of the sea bream propagated from the water on the surface of the water is about 80%, and the water content of the separated concentrated sea lion is 97% on average. In order to lower the production cost, a higher recovery rate and a lower moisture content of the recovered product were required.

In order to produce a biomass product at a low cost by culturing microorganisms such as microalgae on a commercial scale, there are various problems described above. Among the above, the present invention facilitates the recovery of small microalgae, reduces the recovery cost, reduces the supply cost of carbon dioxide (that is, reduces the cost of installing carbon dioxide piping), the liquid surface area To eliminate the need to use a large amount of water, eliminate the need to handle a large amount of liquid medium, increase the recovery rate of microalgae, and reduce the moisture content of recovered microalgae Let it be an issue.
In other words, an object of the present invention is to provide a method for culturing microalgae that cultivate microalgae capable of forming a biofilm on a liquid surface so as to form a biofilm on the liquid surface of a liquid medium.
Another object of the present invention is to provide a biofilm formed on the liquid surface by the culture method according to the present invention, biomass and oil obtained from the biofilm, a method for recovering the biofilm, and a method for producing biomass fuel. Is to provide.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that certain microalgae grow while forming a biofilm on the liquid surface by culturing under certain conditions. Moreover, it discovered that the biofilm formed in this way was easily collectable using substrates, such as a slide glass and a plastic film. Moreover, it discovered that the biofilm formed on the liquid level was recoverable with a high recovery rate, and the moisture content was low. Furthermore, it discovered that oil could be obtained from the biofilm collect | recovered in this way, and discovered that the biofilm which concerns on this invention was useful as biomass. The present invention has been completed based on these findings and has the following configuration.

<1>
A method for culturing microalgae, comprising culturing microalgae capable of forming a biofilm on a liquid surface so as to form a biofilm on the liquid surface of a liquid medium,
The microalgae is Botriococcus sudeticus AVFF007 strain (Accession No. FERM BP-11420),
The liquid medium contains calcium;
The culture temperature is 20 ° C. or higher and lower than 40 ° C.,
Under light irradiation of 2600 to 90800 lux,
A method for culturing microalgae, comprising stationary culture.
<2>
The method for culturing microalgae according to <1>, wherein the calcium concentration in the liquid medium is 0.3 mM or more.
<3>
The method for cultivating microalgae according to <1> or <2>, wherein the carbon dioxide concentration in the gas phase is not less than the carbon dioxide concentration in the atmosphere and less than 20% by volume.
<4>
The method for culturing microalgae according to any one of <1> to <3>, wherein the pH of the liquid medium immediately after the start of culturing is in the range of 2 to 11.
<5>
The method for culturing microalgae according to any one of <1> to <4>, wherein the water depth of the liquid medium is 0.4 cm or more.
<6>
The method for culturing microalgae according to <5>, wherein the water depth of the liquid medium is 2.0 cm to 1 m.
<7>
The method for culturing microalgae according to any one of <1> to <6>, wherein the water content of the biofilm is 95% by mass or less.
<8>
A biofilm formed on a liquid surface by the method for culturing microalgae according to any one of <1> to <7> .
<9 >
A method for producing oil, wherein the oil is obtained from the biofilm according to <8>.
< 10 >
A biofilm collection method, wherein the biofilm formed on the liquid surface according to <8> is collected by being deposited on a substrate.
< 11 >
A biofilm recovery method, wherein the biofilm formed on the liquid surface according to <8> is transferred to a substrate and recovered.
< 12 >
The biofilm recovery method according to < 10 > or < 11 >, wherein 70% or more of the biofilm formed on the liquid surface is recovered.
< 13 >
The biofilm recovery method according to < 12 >, wherein 80% or more of the biofilm formed on the liquid surface is recovered.
< 14 >
The biofilm recovery method according to < 12 > or < 13 >, wherein 90% or more of the biofilm formed on the liquid surface is recovered.
< 15 >
< 10 >-< 14 > The manufacturing method of biomass fuel which uses the biofilm collect | recovered by the biofilm collection | recovery method as described in any one of < 14 > as a fuel.
The present invention is an invention according to the above <1> to < 15 >, but other matters (for example, the following [1] to [21]) are also described below.
[1]
A method for culturing microalgae, comprising culturing microalgae capable of forming a biofilm on a liquid surface so as to form a biofilm on the liquid surface of a liquid medium.
[2]
The method for culturing microalgae according to [1] above, comprising stationary culture of microalgae capable of forming a biofilm on the liquid surface.
[3]
The method for culturing microalgae according to [1] or [2] above, wherein the liquid medium contains calcium.
[4]
The method for culturing microalgae according to [3] above, wherein the calcium concentration in the liquid medium is 0.3 mM or more.
[5]
The method for culturing microalgae according to any one of [1] to [4] above, wherein the carbon dioxide concentration in the gas phase is not less than 20% by volume but not less than the carbon dioxide concentration in the atmosphere.
[6]
The method for culturing microalgae according to any one of [1] to [5] above, wherein the pH of the liquid medium immediately after the start of culturing is in the range of 2 to 11.
[7]
The cultivation method of the micro algae of any one of said [1]-[6] whose culture | cultivation temperature is 20 degreeC or more and less than 40 degreeC.
[8]
The method for culturing microalgae according to any one of the above [1] to [7], wherein the water depth of the liquid medium is 0.4 cm or more.
[9]
The method for culturing microalgae according to [8] above, wherein the water depth of the liquid medium is 2.0 cm to 1 m.
[10]
Among the base sequences encoding the 18S rRNA gene region, the microalgae have a homology of 95.0% or more and 99.9% in a partial region with a base sequence corresponding to Botryococcus sudeticus. The method for culturing microalgae according to any one of [1] to [9] above, which is not more than%.
[11]
The method for culturing microalgae according to [10] above, wherein the microalgae is Botryococcus sudeticus AVFF007 strain (Accession No. FERM BP-11420).
[12]
The method for culturing microalgae according to any one of [1] to [11] above, wherein the water content of the biofilm is 95% by mass or less.
[13]
A biofilm formed on a liquid surface by the method for culturing microalgae according to any one of [1] to [12].
[14]
Biomass obtained from the biofilm according to [13] above.
[15]
Oil obtained from the biofilm as described in [13] above.
[16]
A biofilm recovery method, wherein the biofilm formed on the liquid surface according to the above [13] is collected by being deposited on a substrate.
[17]
A biofilm recovery method, wherein the biofilm formed on the liquid surface according to the above [13] is transferred to a substrate and recovered.
[18]
The biofilm recovery method according to the above [16] or [17], wherein 70% or more of the biofilm formed on the liquid surface is recovered.
[19]
The biofilm collection method according to [18], wherein 80% or more of the biofilm formed on the liquid surface is collected.
[20]
The biofilm recovery method according to [18] or [19] above, wherein 90% or more of the biofilm formed on the liquid surface is recovered.
[21]
The manufacturing method of biomass fuel which uses the biofilm collect | recovered by the collection method of the biofilm as described in any one of said [16]-[20] as a fuel.

  According to the method for culturing microalgae according to the present invention, it is possible to form a biofilm on the liquid surface, and it becomes extremely easy to collect microalgae compared to conventional suspension culture, and it is fine with a high recovery rate. Algae can be collected. That is, in the present invention, at the stage of collecting microalgae, a biofilm composed of aggregates of microalgae floats on the liquid surface, and the biofilm is a collection target. Since there is no need to collect microalgae from the medium and only the biofilm on the liquid surface and the water contained in the biofilm are to be collected, the collection cost can be greatly reduced. In addition, the water content of the recovered biofilm can be reduced. Furthermore, there is no need to handle a large amount of liquid medium and there is no need to use a large amount of water.

C medium composition. The composition of the CSi medium. It is a figure which shows the microscope picture (40-times multiplication factor) of AVFF007 stock | strain. It is the base sequence (SEQ ID NO: 1) of AVFF007 strain used for BLAST analysis. It is a systematic diagram of the microalga Botriococcus sudeticus AVFF007 strain. The composition of IMK medium. It is a figure which shows the microscope picture (40-times multiplication factor) of ASFF001 strain. It is the base sequence (SEQ ID NO: 2) of ASFF001 strain used for BLAST analysis. It is a figure which shows the microscope picture (40-times multiplication factor) of AVFF004 stock | strain. It is the base sequence (SEQ ID NO: 3) of the AVFF004 strain used for BLAST analysis. It is a schematic diagram which shows an example of the cultivation method of the micro algae of this invention. It is a state that the biofilm (film-like structure in FIG. 12) formed on the liquid surface and composed of microalgae is collected using the second substrate (glass substrate). FIG. 13 shows a state of a biofilm deposited on a second substrate (glass substrate) after the biofilm formed on the liquid surface in FIG. 12 is collected. It is a figure which shows the microscope picture of the process in which AVFF007 strain | stump | stock forms a film-form structure on a liquid surface (left side: 4 times magnification, right side: 40 times magnification). In addition, the numerical value described on the right side shows the number of culture days. The composition of CSiFF04 medium. It is a figure which shows the photograph of the biofilm (three-dimensional structure) formed on the liquid level in Example 2. FIG. It is a figure which shows the relationship between the liquid surface floating culture days in Example 2, and the dry algal body amount of the biofilm in each liquid surface floating culture days. It is a figure which shows the relationship between the liquid surface floating culture days in Example 2, and the maximum height (cm) of the biofilm in each liquid surface floating culture days. It is a figure which shows the relationship between the liquid surface floating culture days in Example 2, and the moisture content of the biofilm in each liquid surface floating culture day. 4 is an analysis result of oil components obtained from the biofilm formed in Example 4. FIG. In Example 5, it is a figure which shows the mode (FIG. 21 (a)) of the biofilm formed on the liquid level, and the mode (FIG. 21 (b)) of the liquid level after collection | recovery of a biofilm. . It is a composition of CSiFF01 medium. The liquid level floating culture temperature in Example 6 and the relationship between the dry algal body amount ratio of the biofilm at each liquid surface floating culture temperature (based on the dry alga body amount of the biofilm by temperature-controlled culture at a temperature of 23 ° C.) are shown. FIG. It is a figure which shows the relationship between the water depth of a culture medium, and the dry algal body amount of the biofilm in the culture medium of each water depth. It is a figure which shows the relationship between the culture | cultivation days in each culture medium density | concentration and light quantity, and the dry algal body amount of a biofilm. It is a figure which shows the relationship between the light quantity in each culture medium density | concentration, and the dry alga body amount of a biofilm. It is a figure which shows the relationship between the various nitric acid sources and the dry algal body amount of a biofilm in the culture | cultivation days for 7 days and 14 days. It is a figure which shows the relationship between the calcium concentration in the culture | cultivation days of 7 days and 14 days, and the dry algal body amount of the biofilm formed on the liquid level. It is a composition of CSiFF03 medium. It is a figure which shows the relationship between pH and the dry algal body amount of the biofilm formed on the liquid level. And the CO ₂ concentration in the gas phase is a diagram showing the relationship between algal number of biofilm formed on the liquid surface. It is the result of having observed the culture of the micro algae on the liquid surface at the time of using various microalgal species. This is a result of microscopic observation (4 times magnification) of the culture on the liquid surface in the case of AVFF004 strain. Example of liquid surface suspension culture ++. It is the result of having observed the culture on the liquid level in the case of NIES-2249 strain | microscope observation (4 times magnification). Example of liquid surface suspension culture ++. This is a result of microscopic observation (4 times magnification) of the culture on the liquid surface in the case of AVFF007 strain. Example of liquid surface floating culture +++. It is a figure which shows the relationship between a light quantity and the dry algal body amount of the biofilm formed on the liquid level. It is a figure which shows the influence which shaking has on liquid level floating culture.

Hereinafter, the method for culturing microalgae of the present invention will be described in detail.
In the method for culturing microalgae of the present invention, microalgae capable of forming a biofilm on the liquid surface are cultured so as to form a biofilm on the liquid surface of the liquid medium.
According to the present invention, unlike the conventional method of collecting microalgae having a very small size dispersed in a liquid medium, the biofilm formed on the liquid surface is targeted for collection. Its size is very large compared to the former, making it easy to collect. Furthermore, in the case of suspension culture in a conventional liquid medium, a large amount of water remains even after the microalgae are collected, and in order to remove such water, cost such as centrifugation and evaporation is high. Although it is necessary to perform operations, in the method of the present invention, biofilms having a low water content are collected because they are formed on the liquid surface as compared with the above method. Compared with this method, it can be unnecessary or very simple.
Moreover, in the method of this invention, since it culture | cultivates on the gas-liquid surface, there are few deaths by drying of an algal body.

[Microalgae capable of forming a biofilm on the liquid surface]
The microalgae capable of forming a biofilm on the liquid surface in the present invention has a biofilm forming ability on the liquid surface.
The microalgae referred to in the present invention refers to a microalgae whose individual presence cannot be identified with the human naked eye. The classification of microalgae is not particularly limited as long as it has the ability to form a biofilm on the liquid surface, and may be either prokaryotic or eukaryotic.

  The microalgae is not particularly limited and may be appropriately selected depending on the intended purpose.For example, indigo plant gate, gray plant gate, red plant gate, green plant gate, cryptophyte gate, haptophyte gate, Examples thereof include isophyllous plant gates, dinoflagellate plant gates, Euglena plant gates, and chloracarnion plant gates. These may be used alone or in combination of two or more. Among these, as the microalgae, diatoms and green plant gates of unequal algae plant are preferable, and the genus Botryococcus is more preferable in terms of producing biomass.

  The method for obtaining the microalgae is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include a method of collecting from the natural world, a method of using a commercial product, a method of obtaining from a storage organization and a depository organization, etc. Is mentioned.

  The biofilm referred to in the present invention refers to a film-like structure composed of microorganisms or a three-dimensional three-dimensional structure described later, and is usually a microbial structure attached to a rock or plastic surface. In the present invention, in addition to these, in the present invention, a film-like structure composed of microorganisms that are present on a fluid surface such as a liquid surface. A structure or a three-dimensional structure described later is also referred to as a biofilm. In general, in particular, biofilms in nature contain garbage and plant fragments in addition to microorganisms, but these may be included in the present invention. However, for example, in an open environment such as outdoors, it is impossible to avoid contamination other than the target microorganism. Therefore, in the present invention, samples intentionally containing these are not targeted. However, from the viewpoint of the efficiency of the recovery of microalgae, it is preferable that the biofilm does not contain impurities such as garbage and plant debris. Ideally, the biofilm is secreted during the growth of the microalgae according to the present invention and the microalgae. More preferably, it is composed only of a substance such as an intercellular matrix. In the present invention, the biofilm preferably has a structure in which individual microalgae adhere to each other directly or via a substance such as an intercellular matrix.

  The microalgae capable of forming a biofilm on the liquid surface in the present invention is preferably the microalga Botriococcus sudeticus. Among the base sequences encoding the 18S rRNA gene region of the microalgae according to the present invention, the homology of some regions with a base sequence corresponding to Botriococcus dentics is 95.0% or more and 99.9% or less. It is more preferable that The “partial region” mentioned here means a region of 1000 base sequences or more. When testing homology, homology tests using the entire nucleotide sequence are the most reliable, but determining the entire nucleotide sequence is technically and costly except for a very small number of species. In addition, only a specific part (specifically, the vicinity of the base sequence corresponding to the base sequence of the AVFF007 strain, which will be described later) has been disclosed. Furthermore, it is generally said that attribution is possible if about 1000 base sequences are read. From the above, in the present invention, homology was tested by comparing base sequences of “partial regions”, but the reliability is considered to be sufficiently high.

The microalgae according to the present invention preferably has a high growth rate on the liquid surface. Specifically, the growth rate of the microalgae on the liquid surface in the logarithmic growth phase (that is, one day during the logarithmic growth period) Average growth rate) is preferably 0.1 g / m ² / day or more in terms of dry weight, more preferably 1 g / m ² / day or more, and 5 g / m ² / day or more. Is more preferable, and most preferably 10 g / m ² / day or more. The growth rate in the logarithmic growth phase of microalgae on the liquid surface is generally 1000 g / m ² / day or less in terms of dry weight.
In particular, culturing on the liquid surface and recoverability from the liquid surface is good, it has a high growth rate, it contains a high content of oil, and at least there is no odor during culturing, and it is toxic The microalgae according to the present invention is more preferably the microalga Botriococcus dtex AVFF007 strain (hereinafter abbreviated as AVFF007 strain) from the viewpoint that the generation of substances has not been confirmed.

  The microalgae, AVFF007 strain used in the examples of the present specification, has the accession number FERM BP-11420 on September 28, 2011 (National Institute of Advanced Industrial Science and Technology, Japan). It has been deposited internationally under the Butabest Treaty at Tsukuba City, Ibaraki Prefecture, 1-chome Higashi 1-chome 1 Center 6).

The AVFF007 strain is a novel strain of freshwater microalgae belonging to the Botriococcus genus Sudetics species isolated from a pond in Kyoto Prefecture by the present inventors.
Hereinafter, the method for isolating the microalgae (hereinafter, also referred to as “pure sterilization”) and the process of determining the AVFF007 strain of the microalgae as a new strain will be described.

(Purification of microalgae AVFF007 strain)
Natural fresh water was collected from a pond in Kyoto Prefecture by placing it in a 5 mL homogenizing tube (Tommy Seiko Co., Ltd., TM-655S). In a 24-well plate (As One Co., Ltd., microorganism culture plate 1-8355-02) containing 1.9 mL of a 1: 1 mixed (volume ratio) medium of C medium shown in FIG. 1 and CSi medium shown in FIG. 100 μL of the collected natural fresh water was added and installed in plant bioshelf tissue culture (Rika Ikeda, AV152261-12-2), and cultured at 23 ° C. under continuous light irradiation of 4000 lux. About one month later, yellow aggregates were formed in the wells of the 24-well plate. When observed with an optical microscope, the presence of many microorganisms was confirmed.
1 g of agarose (invitrogen, UltraPure ^™ Agarose) was weighed, and 200 mL of a 1: 1 mixed (volume ratio) medium of C medium and CSi medium was placed in a 500 mL Erlenmeyer flask. This was autoclaved at 121 ° C. for 10 minutes, and agarose gel was prepared by putting about 20 mL each in an Aznole Petri dish (As One Co., Ltd., 1-8549-04) in a clean bench before cooling and solidifying.
By diluting the solution containing microalgae in the 24-well plate, attaching the solution to the loop portion of the disposable (As One Co., Ltd., 1-4633-12), and applying the solution on the agarose gel prepared above, A petri dish with microalgae applied on the gel was prepared.
This petri dish was set up for plant bioshelf tissue culture and cultured at 23 ° C. under continuous light irradiation of 4000 lux. After about 2 weeks, a green colony appeared on the agarose gel. Using a sterilized bamboo skewer (As One Co., Ltd., 1-5980-01), the colony was attached to the tip, and C medium and CSi medium were Of 1: 1 mixed (volume ratio) medium was suspended in wells of a 24-well plate containing 2 mL. A 24-well plate containing microalgae prepared in this way was installed for plant bioshelf tissue culture, and cultured at 23 ° C. under continuous light irradiation of 4000 lux. After about 2 weeks, the aqueous solution in the well becomes green, so a small amount of solution is collected from all wells, and the microalgae is observed using an optical microscope. If there is only a single microalgae, Pure bacteria were obtained by finding a possible well.
The composition of C medium and CSi medium is as shown in FIG. 1 and FIG. 2, and both were subjected to autoclaving of 900 mL of distilled water at 121 ° C. for 10 minutes to obtain 10 times concentration of C medium or CSi medium. After preparing 100 mL, it prepared by mixing with the solution which sterilized with the filter of the pore size 0.45 micrometer.
Moreover, the figure which shows the microscope picture in 40 time of AVFF007 strain | stump | stock was shown in FIG.

(Morphological properties)
-If you wait for a while after performing the dispersion process, everything will sink to the bottom.
・ If the culture is continued for a while, some floating material appears on the liquid surface. Therefore, it is divided into what is sinking on the bottom and what is floating on the liquid level. When the culture is further continued, a film-like structure appears on the liquid surface. When further culturing is performed, a three-dimensional structure appears.
-Those floating on the liquid surface have a larger diameter of individual microalgae, the average particle size is 22.1 μm, those sinking on the bottom are smaller than those floating on the liquid surface, the average The particle size is 7.8 μm.
-Both the liquid surface and the bottom surface are spherical in shape, and the sizes are not constant but have a distribution.
・ It has cohesiveness and forms huge colonies.
-The color is green and changes to yellow as the culture progresses.
・ Although there is almost no odor in the culture and recovered material, it may feel like raw vegetables. What removed the solvent from the recovered substance has a smell like sulfur.

(Culture properties)
・ Growth in fresh water, proliferation in seawater is extremely slow. Even if only a few percent of seawater is mixed, the growth rate is affected.
-During cell proliferation, it proliferates with zoospores. From one cell, several to tens of zoospores are generated.
・ Photoautotrophic culture by photosynthesis is possible.
-Nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, manganese, and iron are essential for growth. In addition, if zinc, cobalt, molybdenum, and boron are contained, it grows suitably. The addition of vitamins also promotes growth.

(Physiological properties)
-Growth temperature is 37 degrees C or less. The higher the temperature, the better the growth.
・ Although it hardly grows at 40 ° C, it can withstand at least several hours even in a 40 ° C environment.
-Growth pH is 5-9. Depending on the type of medium, the pH after growth may be 8 or more, for example, the pH may be 10.5.
・ When light or heat is applied, carotenoids are easily generated.
-Accumulate oil in the fungus body and accumulate 15wt% to 30wt% in dry weight ratio.
-Algae floating on the liquid surface has a higher oil content than those on the bottom.
・ Oil is mainly composed of hydrocarbon compounds and fatty acids. Fatty acids are mainly C16: 0, C16: 1, C18: 1, and C18: 2. The hydrocarbon compounds are mainly C17 and C21.
-Observation of Nile red-stained AVFF007 strain with a fluorescence microscope confirms the presence of oil colored with Nile red as a bright fluorescent colored region in the algae in the fluorescent field. The oil can accumulate in large areas within algal cells.
-When a culture solution containing AVFF007 strain is dropped on a slide glass, and covered with a cover glass and observed under a microscope, oily oil droplets are released from the AVFF007 strain.
-Algae floating on the liquid surface is lighter in specific gravity than alga bodies sinking on the bottom, but heavier than water.
Light intensity is 200~800μmol ^{/ m} 2 ^/ s is a quantity which can be suitably grown, at about 40μmol ^{/ m} 2 ^/ s, grown in about half the growth rate also preferred amount of about 1500μmol ^{/ m} 2 ^/ s is Is possible.

Furthermore, the AVFF007 strain was identified according to the following method.
(Identification of microalgae AVFF007 strain)
The AVFF007 strain was cultured by introducing 50 mL of CSi medium into a 100 mL Erlenmeyer flask, adding 0.5 mL of 1000 × 10 ⁴ / mL AVFF007 strain solution, and shaking culture under light irradiation at 25 ° C. for 14 times. Went for days.
In order to obtain a dry powder of AVFF007 strain, 40 mL of the medium containing AVFF007 strain obtained as described above was centrifuged at 6000 × g at 4 ° C. for 10 minutes using MX-300 (Tomy Seiko). After removing the supernatant, the solid was frozen in a container using liquid nitrogen, transferred to a mortar previously cooled with liquid nitrogen, and a pestle previously cooled with liquid nitrogen was removed. Used to grind.
DNA was extracted from microalgae using DNeasy Plant Mini Kit (manufactured by Qiagen) according to the manual described. The DNA after extraction was measured for purity and quantity using e-spect (manufactured by malcom). The extracted DNA achieved A _{260 nm} / A _{280 nm} = 1.8 or more, which is an index of the degree of purification, and it was confirmed that about 5 ng / μL of DNA was obtained.
Since there was no problem with the purity of the DNA after extraction, a sample for PCR was prepared by diluting 10 ⁴ times with ultrapure water. As a sample for PCR, an 18S rRNA gene region (rDNA region) was used. For PCR, GeneAmp PCR System 9700 (manufactured by Applied Biosystems) was used, and a cycle of 98 ° C. for 10 seconds, 60 ° C. for 50 seconds, and 72 ° C. for 10 seconds was performed 30 times. The enzyme used was Prime Star Max (manufactured by Takara Bio). The obtained PCR product was confirmed to be a single band by 1% agarose electrophoresis.
The PCR product was purified using a PCR purification kit (manufactured by Qiagen). The method was performed according to the method described in the manual. In order to confirm whether the PCR reaction was sufficient and to confirm the degree of purification, e-spect was used to measure the purity and quantity, and A _260nm / A _280nm = 1.8 or more I decided not to.

Next, using the purified product as a template, cycle sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing kit (manufactured by Applied Biosystems). Conditions followed the manual. The base sequence of the obtained reaction product was decoded using ABI PRISM 3100-Avant Genetic Analyzer (manufactured by Applied Biosystems).
This was subjected to a homologous analysis by BLAST (Basic Local Alignment Search Tool). The method is a BLAST search of all the nucleotide sequence information on the data of the National Center for Biotechnology Information (NCBI), and the most homologous species is identified as a related species of the AVFF007 strain. did. Only the base sequence to be compared (1111 bases, SEQ ID NO: 1) is shown in FIG. Specifically, several bases at both ends of the decoded base sequence were not shown in FIG. 4 because they were not compared by BLAST analysis. The upper left of the base sequence shown in FIG. 4 is the 5 ′ end, and the lower right is the 3 ′ end.
As a result of homology analysis, Botryococcus sp. UTEX2629 strain and Botryococcus sp. Among the 1118 bases on the UTEX2629 strain side, there was homology (ie, 99% homology) with the 1109 bases on the AVFF007 strain side. Therefore, the AVFF007 strain is Botryococcus sp. It was classified as a microalgae closely related to the UTEX2629 strain.
A system diagram obtained as a result of the above analysis is shown in FIG. The AVFF007 strain is Characiopodium sp. It is also closely related to Mary 9/21 T-3w, and the AVFF007 strain may be renamed to the genus Characiopodium in the future. In the present invention, when the name of Botriococcus Deticis is changed, Similarly, the AVFF007 strain will be renamed as well. Further, it is assumed that the same treatment can be performed when the name is changed to a name other than the genus Characiopodium.

(Density measurement of microalgae AVFF007 strain)
10 mM ethylenediaminetetraacetic acid (EDTA, Ethylenediamine-N, N, N ′, N′-tetraacetic acid), 5 mM HEPES (4- (2-hydroxyethyl) -1-piperazine etheric acid) in a solution of KOH (pH 7.5) By dissolving cesium, a solution with a cesium chloride concentration of 35 to 105% (w / v) is prepared for every 10% of cesium chloride concentration, and the tube tip from the tube tip to the liquid surface portion in a Polyallomer tube (manufactured by Hitachi Koki) A concentration gradient was created so that the concentration decreased toward the surface.
5 × 10 ⁶ cells / mL AVFF007 strain was applied to the upper surface of the tube, and centrifuged at 20000 × g and 4 ° C. for 30 minutes using a centrifuge.
The cell density of the algal cells floating on the liquid surface is 1.26 g / mL, and Botryococcus sp. It was lighter than the cell density of UTEX-2629 strain, 1.34 g / mL.
From the above, from the viewpoint of density, Botryococcus sp. It is not completely the same as the UTEX-2629 strain, but Botryococcus sp. It was judged to be a microalgae close to the UTEX-2629 strain.
Furthermore, Botryococcus sp. The fact that the microalgae closely related to the UTEX-2629 strain indicates that they are almost the same when comparing FIG. 3 of Non-Patent Document 3 with the diagram showing the micrograph of the AVFF007 strain shown in FIG. I understand that there is.

In addition, according to the culture method of the present invention, it is considered that even a microalga other than the AVFF007 strain can form a biofilm on the liquid surface by setting the culture conditions to be suitable conditions.
Examples of such microalgae include the ASFF001 strain, AVFF004 strain, and NIES-2249 strain described below, and the ASFF001 strain and the NIES-2249 strain are preferable from the viewpoint of biofilm forming ability.

(Purification of microalgae ASFF001 strain)
The biofilm adhering to the rocks of the footbath in Shizuoka Prefecture was peeled off and collected by placing it in a 5 mL homogenizing tube (Tommy Seiko Co., Ltd., TM-655S). Pure sterilization was performed by the same method as AVFF007 strain. As a medium, a 1: 1 mixture (volume ratio) of IMK medium shown in FIG. 6 and CSi medium shown in FIG. 2 was used. The IMK medium is a mixture with artificial seawater, and marine art SF-1 (Tonda Pharmaceutical Co., Ltd.) was used as the artificial seawater. In addition, unlike a general IMK medium, the present invention is an IMK medium that does not contain selenium.
Moreover, the figure which shows the microscope picture in 40 time of ASFF001 strain | stump | stock was shown in FIG. The properties of ASFF001 strain are described below.
-Only Chlorella saccharophila (Chlorella saccharophila) has 1725 bases of homology (base sequence (1727 bases, SEQ ID NO: 2) to be compared in BLAST analysis) is shown in FIG. Several bases at both ends of the decoded base sequence were not shown in Fig. 8 because they were not compared by BLAST analysis, and the upper left of the base sequence shown in Fig. 8 is the 5 'end. Below is the 3 'end.)
・ Photoautotrophic culture by photosynthesis is possible. The form is oval.
・ Green algae ・ Size is 5μm to 20μm. It exists as a mixture of large and small.
-Shows the property of floating on the liquid surface.

(Purification of microalgae AVFF004 strain)
Natural fresh water was collected from a pond in Kyoto Prefecture by placing it in a 5 mL homogenizing tube (Tommy Seiko Co., Ltd., TM-655S). Pure sterilization was performed by the same method as AVFF007 strain. As the medium, a 1: 1 mixture (volume ratio) of C medium and CSi medium was used.
Moreover, the figure which shows the microscope picture in 40 time of AVFF004 strain | stump | stock was shown in FIG. The properties of the AVFF004 strain are described below.
-Only the base sequence (1201 bases, SEQ ID NO: 3) to be compared in the BLAST analysis is shown in Fig. 10. Specifically, it is shown in Fig. 10. Several bases at both ends of the base sequence were not compared in the BLAST analysis and thus were not shown in Fig. 10. The upper left of the base sequence shown in Fig. 10 is the 5 'end, and the lower right is 3 ′ end.)
・ Photoautotrophic culture by photosynthesis is possible. The form is oval or water droplets.
・ Green algae ・ Size is 1μm to 5μm.
-Shows the property of floating on the liquid surface.

  The microalgae, AVFF004 strain used in the examples of the present specification, has the accession number FERM BP-11444 as of December 21, 2011 (National Institute of Advanced Industrial Science and Technology, Japan). It has been deposited internationally under the Butabest Treaty at Tsukuba City, Ibaraki Prefecture, 1-chome Higashi 1-chome 1 Center 6).

  The NIES-2249 strain is Chlorococcum echinozygotum Starr purchased from the National Institute for Environmental Studies.

[Method of the present invention]
A basic configuration of the culture method of the present invention is shown in FIG. In addition, since this schematic diagram is for demonstrating this invention, there exists a part currently described simplified.
As shown in FIG. 11A, first, a suspension or dispersion solution of microalgae is prepared. Next, when the culture vessel is left stationary, the microalgae usually sink to the bottom in several seconds to several tens of minutes depending on the type of microalgae as shown in FIG. In this state, when the microalgae are cultured for a while, a biofilm composed of the microalgae is formed on the liquid surface as shown in FIG. Normally, as shown in FIG. 11 (c), microalgae are also present on the bottom surface of the culture vessel, and although not shown in the figure, they are also present on the side surface of the culture vessel.
In the present invention, it is preferable to leave the culture vessel stationary during the culture, but as long as the culture is not vigorously stirred, the same culture as in the present invention can be performed even if shake culture is performed. However, since the number of algal bodies in the biofilm formed on the liquid surface increases, culture in a stationary state is preferable. The phrase “microalgae sinks to the bottom surface of the culture vessel” means that most of the microalgae sinks to the bottom surface, and does not mean a state in which the microalgae are completely absent from the liquid surface or in the liquid. For example, when shake culture is performed, liquid surface floating algae is 1/6 of static culture, and the algal bodies in the medium are 3.5 times of static culture, on the bottom surface, compared to the case of static culture. The algae body becomes 1.4 times that of stationary culture, and by shaking culture, the proportion of algal bodies present on the liquid surface decreases, and the proportion of algal bodies present in the medium and on the bottom surface increases.

When the first substrate is brought into contact with the biofilm formed on the liquid surface as shown in FIG. 11 (d), the biofilm adheres to the surface of the first substrate ((( e)). The biofilm (mainly algal bodies) can be recovered by peeling the attached biofilm from the first substrate (hereinafter also referred to as “collection by transfer”). In the figure, the substrate is in contact with the entire liquid surface of the culture vessel. However, the substrate may be partially contacted, or the entire surface or partial contact may be repeated a plurality of times. Thus, the collection efficiency of the microalgae on the liquid surface is improved by contacting them a plurality of times. In the above, a film-like structure or a three-dimensional structure can be transferred so as to overlap the surface of the first substrate. Among these, in the case of a culture area of less than 1 m ² , it is preferable to carry out a single transfer, and in the case of a culture area of 1 m ² or more, it is preferable to carry out a plurality of transcriptions. The details of the film-like structure and the three-dimensional structure are as described later.

Hereinafter, collection by transfer will be described in more detail.
[Recovery by transfer of biofilm on liquid surface]
First, a transfer material to which a biofilm on a liquid surface is attached is prepared as a first substrate. Here, the first substrate is used to transfer and collect a film-like structure or a three-dimensional structure composed of microalgae on the liquid surface used in FIG. It is a substrate. In the figure, the first substrate is a substrate that covers the entire surface of the culture vessel. However, a substrate that covers the entire culture vessel may be used in this way, or only a part of the culture vessel may be covered. A first substrate that can be used may be used.
As the transfer material, glass, polyethylene, polypropylene, nylon, polystyrene, vinyl chloride, polyester and the like can be used, but are not limited thereto. It is desirable that the transfer material can be cut with scissors or the like as necessary so as to be smaller than the area of the culture vessel. For example, when the culture container is a 6-hole plate, it is preferable to cut the transfer material into a circular shape having a diameter of about 3.5 cm. The cut transfer material is preferably washed to remove dust on the surface. In addition, when using the extract | collected biofilm for the following culture | cultivation, it is preferable to use after further immersing in ethanol for disinfection and drying the surface of a transcription | transfer material.

Next, the cut transfer material is gently inserted so as to be at an angle parallel to or close to the liquid surface formed by the culture vessel, and the microalgae on the liquid surface are attached to the cut transfer material. When inserting, the cut transfer material is inserted slightly obliquely with respect to the liquid surface and finally made parallel to the liquid surface. It is preferable because it can be recovered. By gently pulling up the cut transfer material to which the biofilm on the liquid surface is attached, the biofilm can be transferred from the liquid surface of the culture vessel to the transfer material cut. The transfer of the biofilm on the liquid surface by the first substrate may be performed a plurality of times. This is because the transfer rate is further improved by performing the treatment a plurality of times.
As a method for recovering the biofilm from the first substrate (for example, the cut transfer material), any method can be used as long as it can peel microalgae from the substrate, but a water flow is added. Or ultrasonically treat the container with the cut transfer material, close the lid of the container with the cut transfer material, shake vigorously, perform high-speed shaking treatment, or something like a cell scraper By using it, the biofilm can be peeled off from the transfer material. Among these, a method of stripping the biofilm from the substrate using a jig using a material that does not damage the first substrate, such as a cell scraper, is preferable. The first substrate may be reused any number of times.

  Depending on the state of the culture, the microalgal biofilm growing on the liquid surface in the culture vessel may grow from a film shape into a pleated shape in the culture medium. In this case, it is also possible to collect a pleated biofilm using a pipette.

Next, a collection method different from collection by transfer will be described.
As shown in FIG. 11 (f), it is possible to collect the biofilm on the liquid surface so as to be collected using the second substrate. Here, the second substrate is a substrate used for recovering a film-like structure or a three-dimensional structure composed of microalgae on the liquid surface used in FIG. It is.
In the figure, the substrate is moved from the right side to the left side of the figure. The direction of movement of the second substrate may be reversed (that is, movement of the substrate from the left side to the right side of the drawing) or may be collected multiple times. This is because the collection rate is improved by performing the collection multiple times. When collecting multiple times, the second substrate with the biofilm attached may be used, or the substrate after the biofilm has been completely or partially removed from the surface of the first substrate described above. It may be used as the second substrate or a new substrate may be used. Further, although only one second substrate is shown in FIG. 11, a plurality of second substrates may be used simultaneously. Thereby, a recovery rate improves. In addition, as long as the strength of the second substrate allows in this, after removing the collected biofilm using one second substrate, it is possible to resume the collection using the same second substrate, This is preferable from the viewpoint of the installation cost of the recovery device. The size of the second substrate, the angle of the second substrate with respect to the liquid surface, the moving speed, and the like can be freely set according to the purpose. In addition, (g) of FIG. 11 is the state by which the biofilm was collect | recovered on the 2nd board | substrate.

Hereinafter, recovery by the second substrate will be described in more detail.
[Recovery of biofilm on liquid surface by second substrate]
FIG. 12 shows a film-like structure composed of microalgae floating on the liquid surface after a biofilm (film-like structure in the example of the figure) is formed on the liquid surface. It is a mode that it collect | recovers using the board | substrate (slide glass (76x26 mm)).
In FIG. 12, the long side of the slide glass is inserted diagonally with respect to the biofilm on the liquid surface, and the biofilm on the liquid surface is deposited on the slide glass while proceeding to the left as it is. The film was collected. The left side of the figure represents the liquid level formed by the biofilm before collection, and the right side of the figure represents the liquid level after the biofilm was collected.

  FIG. 13 shows a state in which the biofilm is deposited on the slide glass so as to be folded. The collection by the second substrate corresponds to the process from (f) to (g) in FIG. In addition to the slide glass, other substrates such as a nylon film can be used as the second substrate. The size of the second substrate can be appropriately changed according to the size of the culture vessel, but it is preferable to use a substrate smaller than the surface area of the culture vessel as the second substrate.

Although it is the timing to collect the biofilm on the liquid surface, it is possible to collect it while the liquid surface in the culture vessel is partially covered with biofilm, but the amount of microalgae is large. Therefore, it is preferable to collect the liquid after the entire liquid surface in the culture vessel is covered with the biofilm. In addition, after the biofilm covers the entire liquid surface, the culture may be continued for a while and then recovered.
In particular, it is preferable to recover after a three-dimensional structure to be described later is formed on the liquid surface. A three-dimensional structure is generally a structure that can be seen when the growth of microalgae in a film-like structure further progresses and can be recovered compared to a two-dimensional film-like structure. This is because the amount of microalgae is large and the water content is lower.

  The state after collecting the biofilm on the liquid surface is (h) in FIG. On the bottom surface of the culture vessel, microalgae are attached or deposited. In the schematic diagram of the present invention, the supply of microalgae to the liquid surface is described as being performed from the bottom surface, but in practice, the microalgae are also in a medium other than the liquid surface and the bottom surface while the concentration is low. Existing. Moreover, even if the microalgae are supplied into the liquid from the liquid surface and the bottom surface, the present invention describes that the microalgae are supplied from the bottom surface of the culture vessel to the liquid surface. In addition, the supply of microalgae from the bottom of the culture vessel to the liquid surface means that the microalgae actually move from the bottom to the liquid surface when they move on the liquid surface without the growth of microalgae on the bottom surface. There are both cases of proliferating.

  The culture vessel may be an open system or a closed system, but it can prevent the introduction of unintended microorganisms and dust from the outside, the destruction of liquid algae by wind, the drying of liquid algae, the prevention of water evaporation, etc. From the viewpoint, culture in a closed system is more preferable.

Moreover, only the biofilm on the liquid surface may be collected, or both the biofilm on the liquid surface and the microalgae on the bottom surface may be collected. This is because both the liquid and bottom microalgae can be used as biomass.
Further, depending on the state of culture, the microalgal biofilm growing on the liquid surface in the culture vessel may grow from a film shape into a pleated shape in the culture medium. In this case, a fold-like biofilm can be collected by increasing the insertion depth of the second substrate into the liquid.
In the present invention, the culture method for culturing microalgae on the liquid surface as shown in FIG. 11 (c) is called liquid surface floating culture. That is, the culture method for culturing microalgae in only one or both of the liquid and the bottom of the liquid is not included in the liquid surface floating culture.
The liquid surface in the present invention is typically the liquid surface of a liquid medium described later, and is usually an interface between the liquid medium and air.
Further, in the present invention, the liquid surface suspension culture as in the state of FIG. 11 (c) is performed in a stationary state, which is called liquid surface suspension culture by stationary culture.

  Any method can be used to recover the biofilm from the second substrate as long as the biofilm can be peeled off from the substrate, but a jig using a material that does not damage the second substrate is used. For example, it is preferable to use a cell scraper. The second substrate may be reused any number of times.

  According to an example of an embodiment of the present invention, a suspension or dispersion solution containing microalgae is prepared by dispersing microalgae obtained through the purification process in a liquid medium containing an artificial culture medium. By culturing in a culture apparatus, a microalgae film-like structure or a three-dimensional structure is formed on the liquid surface of the liquid medium, and the microalgae grown from the entire culture tank or the liquid surface It can be recovered.

  The above recovery method preferably recovers 70% or more of the biofilm formed on the liquid surface, more preferably 80% or more, and more preferably 90% or more. Yes, most preferably 100% recovery. The recovery rate of the biofilm formed on the liquid surface can be confirmed visually, for example.

[Pure sterilization process]
In the case of microalgae in a natural state, the purification process as referred to in the present invention refers to a state in which various types of organisms and garbage coexist with microalgae and the target microalgae. The process of isolation only. In reality, such complete isolation is difficult, and it is defined in the present invention that if the desired microalgae can be obtained mainly, it can be purified. The term “mainly obtained in the present invention” means that microalgae other than the target could not be confirmed in several visual fields in observation under a microscope.
As a method for purifying bacteria, after developing a diluted suspension solution containing microalgae on an agar medium, culturing and forming colonies, the colonies are collected, or collected one by one under a microscope. The method etc. are mentioned.

[Pre-culture process]
The pre-culturing step referred to in the present invention generally preserves microalgae obtained after completion of the sterilization step, but microalgae are grown until the preserved microalgae are grown and main culture can be performed. It is a process of increasing the number of. Any known culture method can be used as the culture method in the pre-culture step. It is also possible to perform the liquid surface suspension culture of the present invention. In addition, in the pre-culture process, several pre-culture processes may be performed in order to grow microalgae to a scale that allows main culture.
In general, a culture tank having a surface area of 1 cm ^{2 to} 1 m ² or less can be used, and the culture can be performed both indoors and outdoors, but indoors are preferred.

[Main culture process]
The main culturing step referred to in the present invention is a culturing step after performing the pre-culturing step, and means a culturing step up to immediately before performing the final recovery step. The main culture process may be performed a plurality of times.
In general, a culture tank having a surface area of 100 cm ² or more can be used, and the culture can be performed both indoors and outdoors, but outdoor culture is preferred.

[Use of microalgae]
As the microalgae used for the preculture and the main culture, those obtained by any known culture method such as suspension culture, liquid surface suspension culture, and adherent culture may be used. However, in liquid surface suspension culture, it is preferable to perform a suspension treatment.

[Suspension treatment]
In the present invention, the suspension treatment can include any treatment method for making the aggregate of microalgae into a smaller aggregate or a single microalgae. For example, pipetting, shaking a microalgae solution in a container by hand, weak processing such as processing with a stirrer chip or stir bar, strong processing such as ultrasonic processing or high-speed shaking processing, adhesive substances such as intercellular matrix Including a method of using a substance such as an enzyme that decomposes.
By suspending the microalgae into smaller aggregates or single microalgae, the smaller the size of the microalgae aggregate compared to the larger size of the microalgae aggregate, the more the medium and This increases the contact area, and as a result, the growth rate of microalgae may be improved, which is preferable.

Sonication is a method characterized by directly applying a vibration wave having a high frequency that cannot be heard by human ears to a microalgae solution or a container holding a microalgae solution. In this method, the container does not necessarily need to be a sealed container, and the solution containing microalgae does not leave the bottom surface of the container.
In high-speed shaking treatment, a solution containing agglomerates of microalgae is placed in a shaking container so that an air layer can be formed. A processing method that leaves or contacts the inner wall of a closed container.

[Suspension solution]
The suspension solution referred to in the present invention refers to a solution subjected to suspension treatment.

[Suspension solution of microalgae]
The microalga suspension solution refers to a solution obtained by suspending microalgae. Specifically, this is the solution used in FIG. 11 (a), and the microalgae may be a suspension culture in a liquid, or a microalgae attached and cultured on a substrate. May be used after removing microalgae from the substrate surface. Moreover, you may use the micro algae obtained by the liquid surface floating culture. Moreover, you may use the mixture of the micro algae derived from these at least 2 or more culture forms. For example, a mixture of the microalgae derived from the liquid surface suspension culture and the microalgae on the bottom surface of the culture vessel at the time of the liquid surface culture can be used.

By performing the suspension treatment, it is possible to make a smaller microalgae aggregate or a single microalgae, and more uniformly deposit the microalgae on the bottom of the culture vessel, making effective use of these surfaces In addition, since the microalgae can easily obtain light and nutrients necessary for the growth, the growth rate of the microalgae is improved, and as a result, the growth rate of the liquid surface floating culture is also improved. Furthermore, when the suspension treatment is performed, the film structure of the film-like structure composed of microalgae on the liquid surface becomes uniform and the surface unevenness is reduced, so that the transfer to the first substrate becomes easy. The efficiency is also improved.
Further, in both the pre-culture and the main culture, a suspension treatment may be performed in order to confirm the growth state of microalgae. This is because the number of microalgae can be easily counted by this treatment.

[Distributed processing]
In the present invention, the dispersion treatment is a treatment method for making a microalgae aggregate into a smaller aggregate or a single microalgae. For example, pipetting or shaking a microalgae solution in a container by hand. This refers to suspension treatment excluding weak treatments such as treatment, stirrer chips and stirring rods. That is, the dispersion treatment includes a strong treatment such as ultrasonic treatment and high-speed shaking treatment, and a method using a substance such as an enzyme that decomposes an adhesive substance such as an intercellular matrix.

[Dispersion solution]
The dispersion solution referred to in the present invention refers to a solution that has been subjected to a dispersion treatment such as ultrasonic treatment or high-speed shaking treatment, and includes pipetting and hand shaking a microalgae solution in a container, a stirrer chip, It is not said that the solution has been subjected to a weak suspension treatment such as a treatment with a stir bar.

[Dispersion solution of microalgae]
The microalgae dispersion solution refers to a solution obtained by dispersing microalgae. Specifically, this is the solution used in FIG. 11 (a), and the microalgae may be a suspension culture in a liquid, or a microalgae attached and cultured on a substrate. May be used after peeling off the microalgae from the substrate surface, or microalgae peeled off from the substrate surface simultaneously with the suspension treatment may be used. Moreover, you may use the micro algae obtained by the liquid surface floating culture. A mixture of microalgae derived from at least two of these culture forms may be used. For example, a mixture of the microalgae derived from the liquid surface suspension culture and the microalgae on the bottom surface of the culture vessel at the time of the liquid surface culture can be used.

  By carrying out the dispersion treatment, it is possible to make a smaller microalgae aggregate or a single microalgae, and more uniformly deposit the microalgae on the bottom surface of the culture vessel, making effective use of these surfaces. In addition, since the microalgae can easily obtain light and nutrients necessary for growth, it is considered that the growth rate of the microalgae is improved, and as a result, the growth rate of the liquid surface floating culture is also improved. Furthermore, when the dispersion treatment is performed, the film structure of the film-like structure composed of microalgae on the liquid surface becomes uniform and the surface unevenness is reduced, so that the transfer to the first substrate is facilitated. Efficiency also increases.

  Further, in both pre-culture and main culture, dispersion treatment may be performed in order to confirm the growth state of microalgae. This is because the number of microalgae can be easily counted by this treatment.

[Deposition]
The term “deposition” as used in the present invention refers to a state in which microalgae are present in the vicinity of, or attached to, the bottom of the culture vessel, or a state in which both states are mixed. The state in which microalgae are present in the vicinity means a state in which microalgae easily move from the substrate or the bottom surface of the culture vessel with a slight movement of the liquid medium. In addition, the deposition includes adhesion. In the present invention, as shown in FIG. 11, it is desirable to temporarily deposit microalgae. This is because by depositing, a film-like structure composed of microalgae formed by growing on the liquid surface becomes uniform, and the efficiency of transfer to the first substrate increases.

[Adhesion]
The adhesion referred to in the present invention refers to a state in which microalgae is directly attached to the bottom surface of the substrate or the culture vessel. Say that you are. In the present invention, a state in which a biofilm (a film-like structure or a three-dimensional structure) composed of microalgae formed on the liquid surface of the liquid medium is floating on the liquid surface is defined as the liquid surface. It may be described as adhering.

[Deposition process]
The microalgae deposition step of FIGS. 11A to 11B is a step of allowing microalgae to deposit on the bottom surface of the culture vessel by allowing the suspension or dispersion solution of microalgae to stand still. In addition, as shown in FIG. 11 (c), after a biofilm (a film-like structure or a three-dimensional structure) made of microalgae is partially formed on the liquid surface, the biofilm is cultured from this biofilm. Although there is a possibility that microalgae may settle on the surface of the bottom surface of the container, in the present invention, these are not included in the deposition step. In addition, depending on the type of microalgae, it usually takes several days to two weeks until the biofilm composed of microalgae can be visually confirmed at least on the liquid surface after deposition. This process is not a deposition process but is included in the culture process. In this step, although the process proceeds even in a shaking state, the number of microalgae deposited in the stationary state increases, so that the stationary state is preferable. Further, in the present invention, almost all the microalgae are deposited as long as they are visually observed. However, when observation is performed using an instrument such as a microscope, the microalgae are on the liquid surface or in the liquid, although the number is very small. However, in this invention, even in such a case, since most of the microalgae are sinking on the bottom surface, it is described as being deposited.

  In the deposition step, the microalgae settle on the surface of the bottom of the culture vessel in 1 second to 1 day depending on the type. When the deposition process is accompanied by adhesion, it takes a longer time and may overlap with the culture process.

[Culture process]
It refers to the process from the step after depositing the microalgae to just before collecting the biofilm (film-like structure or three-dimensional structure) composed of the microalgae on the liquid surface. That is, the process from (b) to (c) in FIG. 11 or the process from (h) to (c) in FIG.
The culturing step is a step in which at least a part of the deposited microalgae emerges toward the liquid surface and forms a biofilm composed of the microalgae on the liquid surface. In order to form a biofilm composed of microalgae, the growth of microalgae is necessary. This is because when the deposited microalgae are grown and then supplied on the liquid surface, We believe that there are either or both of the cases where floating microalgae grow on the liquid surface.
The culture step includes a step of further proliferating these structures after forming a biofilm on the liquid surface. In general, after a film-like structure is formed, a three-dimensional structure is formed. These processes are also included in the culture process.
In addition, in the culture | cultivation process in this invention, the smell accompanying culture | cultivation is hardly felt.

In the culturing step, shaking culture may be performed. This is because although the number of microalgae is smaller than that of stationary culture, formation of a film-like structure or a three-dimensional structure composed of microalgae on the liquid surface is possible even by shaking culture. However, it is preferable to perform stationary culture for the purpose of increasing the number of biofilms composed of microalgae on the liquid surface. In addition, unlike the conventional suspension culture, stationary culture does not require power such as shaking and stirring, and does not require power energy and a power generation device, which is preferable because the cost can be significantly reduced.
That is, the method for culturing microalgae of the present invention preferably includes stationary culture of microalgae capable of forming a biofilm on the liquid surface.

[Distribution of microalgae]
In the culture of microalgae using the culture method of the present invention, the distribution state of microalgae immediately before performing transfer using the first substrate or biofilm recovery using the second substrate is the microalgae in the liquid. The number of microalgae is smaller than the number of microalgae on the liquid surface or the number of microalgae deposited on the bottom of the culture vessel. Moreover, in the distribution state at this time, it is preferable that the number of microalgae on the liquid surface is larger than the microalgae deposited on the bottom surface of the culture vessel, but this is not necessarily limited thereto. Note that the number of microalgae mentioned here does not include zoospores generated from cells of microalgae that are difficult to observe with a microscope.

  The number in the liquid refers to the number of microalgae counted in the vicinity of the midpoint between the liquid level and the bottom surface of the culture vessel, and refers to the number of microalgae per cubic centimeter. In addition, the number of microalgae on the liquid surface or in the vicinity of the bottom surface of the culture vessel refers to the number of microalgae per square centimeter. The number of microalgae can be replaced by a method capable of quantifying microalgae, such as the weight of microalgae, dry weight, and turbidity.

[Static culture]
The stationary culture referred to in the present invention refers to a culture method in which microalgae are cultured without being intentionally moved. That is, for example, the culture medium convects with a local change in the culture medium temperature, and the microalgae may move by the flow, but this is because the microalgae is not intentionally moved. Including the case, in the present invention, it is referred to as static culture.

  In this invention, it is preferable to culture | cultivate by a stationary state in all the processes (namely, stationary culture | cultivation). This is to prevent the biofilm (film-like structure or three-dimensional structure) composed of microalgae on the liquid surface from being destroyed. If a biofilm composed of microalgae is destroyed, the efficiency of recovery using the second substrate will be reduced, and the comb in the liquid may move to the bottom of the culture vessel It is. However, even if it is not in a stationary state, it is possible to form a biofilm composed of microalgae on the liquid surface, although the number of microalgae is reduced. Culture is preferred, but not limited to this.

  In the present invention, as a method for creating a state that is not in a stationary state, a method of shaking the entire culture vessel, a method of stirring the culture vessel with a stirrer such as a stirrer chip or a stirring rod, a gas containing air or high-concentration carbon dioxide is used. Examples thereof include a method of bubbling and a method of flowing a suspension of microalgae.

[Adherent culture]
The adhesion culture as referred to in the present invention refers to culturing in a state in which microalgae are attached to the substrate surface or the wall surface of the culture vessel (for example, the bottom surface or the side surface of the culture vessel).
In addition, in order to promote adhesion of microalgae, it may be once adhered on the substrate using a medium having a different medium composition. For example, a medium supplemented with calcium that may promote the adhesion of microalgae can be used. Calcium is involved in the adhesion of microalgae, especially diatoms, see, for example, Plant. Physiol. (1980) 65, 129-131. It is described in.

[Floating culture]
In the present invention, culturing microalgae in a liquid (specifically, in a liquid medium described later) is called floating culture. In the present invention, a state in which microalgae are suspended while being suspended in a liquid other than the liquid surface, the side surface of the culture vessel, and the bottom surface is called floating culture.

[Liquid surface suspension culture]
In the present invention, the culture method for culturing microalgae on the liquid surface is called liquid surface floating culture. In addition, even if microalgae are simultaneously present in the bottom of the culture vessel or in the culture medium, it is called liquid surface floating culture. Moreover, since it can also be considered that the liquid surface floating culture is attached to the liquid surface and cultured, in the present invention, it may be treated as a kind of adherent culture.
In addition, when liquid surface suspension culture is performed in the present invention, a phenomenon in which aggregates of microalgae are leached from the biofilm (film-like structure or three-dimensional structure) on the liquid surface into the liquid. May be seen. In the present invention, the culture in such a situation is also included in the liquid surface suspension culture.
In the present invention, liquid surface suspension culture can be performed, and one or both of the above-described suspension culture and adherent culture can be performed simultaneously.
As described above, according to the present invention, the names of the culture methods are distinguished as described above according to the location in the liquid medium in which the microalgae are cultured. However, as described above, these can be performed simultaneously. That is, for example, when microalgae are cultured on the liquid surface and also in a state of being dispersed in the liquid, it is said that the liquid surface floating culture and the floating culture are performed simultaneously.

As the step of culturing the microalgae of the present invention by liquid surface suspension culture, suspension culture and adhesion culture, for example, a known method capable of culturing microalgae belonging to the genus Botriococcus can be applied. For example, a microalgae may be inoculated into a culture vessel such as an Erlenmeyer flask containing a liquid medium, and aeration culture may be performed under light irradiation while standing.
Next, a liquid medium that can be used in liquid surface suspension culture, suspension culture, and adherent culture will be described.

[Medium (liquid medium)]
In the present invention, any known medium (liquid medium) can be used as long as microalgae can be cultured. The medium is preferably selected according to the type of microalgae to be cultured. For example, since the AVFF007 strain described above grows in fresh water, the medium is preferably fresh water. Known media include AF-6 medium, Allen medium, BBM medium, C medium, CA medium, CAM medium, CB medium, CC medium, CHU medium, CSi 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, 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 these, those that are fresh water are AF-6 medium, Allen medium, BBM medium, C medium, CA medium, CAM medium, CB medium, CC medium, CHU 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, P35 medium, URO medium, VT medium, VTAC medium, VTYT medium, W medium, SW medium, SOT medium is there. As the medium for culturing the AVFF007 strain described above, C medium, CSi medium, CHU medium, and a mixture of these mediums are preferable, and C medium, CSi medium, and a mixture of these mediums are more preferable.
The medium may or may not be UV sterilized, autoclaved, or filter sterilized.

  In the present invention, the liquid medium preferably contains calcium. That is, it is desirable to add calcium to the medium. This is because when calcium is contained in the medium, the growth rate of microalgae is improved, and the formation of a biofilm (film-like structure or three-dimensional structure) on the liquid surface is facilitated. Although the calcium concentration in a liquid culture medium is not specifically limited, Preferably it is 0.3 mM or more, More preferably, it is 0.5 mM or more. Although the upper limit of the calcium concentration in a liquid culture medium is not specifically limited, Usually, it is 100 mM or less, Preferably it is 50 mM or less, More preferably, it is 5 mM or less.

[Water depth]
Although the water depth of the liquid culture medium used by this invention is not specifically limited, The one where the water depth is shallow is preferable. This is because the amount of water used decreases as the water depth decreases. Moreover, it is because the complexity and the amount of energy used for handling water movement are reduced. Furthermore, it is because the manufacturing cost of a culture container also falls. The water depth is preferably 0.4 cm or more, more preferably 1.0 cm to 10 m, further preferably 2.0 cm to 1 m, and most preferably 5.0 cm to 30 cm. When the water depth is 0.4 cm or more, it becomes possible to form a biofilm, and when the water depth is 1.0 cm or more, the water evaporates before the biofilm is formed on the liquid surface, thereby culturing microalgae. In addition, it is avoided that the suspension containing the culture medium and microalgae becomes too difficult when the water depth is 10 m or less, and the absorption of light into the liquid is sufficiently avoided. It is because it can suppress that the utilization efficiency of light falls. When the water depth is 5.0 cm to 30 cm, there is little evaporation of water in the medium until the biofilm is formed, and handling of the suspension containing the medium and microalgae is facilitated. Light utilization efficiency is improved because the absorption of light by the microalgae present in the trace amount is minimized. In addition, the shallower the water, the easier the supply of carbon dioxide and the easier the release of oxygen generated by photosynthesis into the atmosphere.

According to the present invention, basically, the liquid mainly plays a role of supplying nutrients for culturing, and the role as a space used for growing and growing microalgae is small. As a result, the depth of the liquid medium can be reduced, and the amount of the medium solution can be greatly reduced. This not only significantly reduces water and medium costs, but also facilitates handling for moving the liquid. In addition, the processing cost of the spent medium is easy because the amount of liquid medium is small. Furthermore, the shallow depth is advantageous for the diffusion of carbon dioxide (CO ₂ ) in the gas phase throughout the liquid medium tank. Further, as the water depth of the culture pond increases, the amount of light is absorbed by the culture medium. However, since the depth is shallow, a decrease in the amount of light due to absorption is minimal.

[carbon dioxide]
In the present invention, it is preferable to culture without intentionally supplying carbon dioxide into the medium. That is, it is preferable not to use a method of supplying a gas containing carbon dioxide into the culture medium by bubbling. This is to prevent the biofilm (film-like structure or three-dimensional structure) made of microalgae on the liquid surface from being destroyed by bubbling. However, if the destruction is partial, carbon dioxide may be supplied by bubbling. Further, methods for floating microalgae on the liquid surface by bubbling have been disclosed in JP 2001-340847, JP 2007-160178, JP 62-213892, JP 1-113111, etc. Thus, it is possible to employ means for forcibly floating microalgae on the liquid surface and supplying carbon dioxide. In the method without bubbling, it is not necessary to install a gas pipe for supplying carbon dioxide, and it is generally difficult to secure a carbon dioxide supply source such as a thermal power plant or a steelworks. Because it is very difficult to control the supply of carbon dioxide evenly over a large area, the ability to use carbon dioxide in the gas phase directly leads to significant cost reductions. Is advantageous.

  In the present invention, it is preferable not to use means for intentionally supplying carbon dioxide into the medium. However, carbon dioxide in the gas phase is a region where microalgae or microalgae are not present on the liquid surface. When carbon dioxide is supplied into the medium via the route, it is defined as not corresponding to the above means. In addition, before the film-like structure composed of microalgae is formed on the liquid surface, there are many portions where the liquid surface of the medium and the gas containing carbon dioxide are in direct contact, In this case, carbon dioxide dissolves into the medium through the liquid surface, but this does not mean that carbon dioxide is intentionally supplied. In addition, even after a film-like structure or a three-dimensional structure is formed on the liquid surface, carbon dioxide dissolves in the medium via the film-like structure or the three-dimensional structure. In this case, however, carbon dioxide is not intentionally supplied into the medium.

In the present invention, it is desirable to use carbon dioxide in the atmosphere because it is advantageous in terms of cost, but it is possible to use carbon dioxide having a concentration higher than the concentration in the atmosphere. In this case, in order to prevent the loss of carbon dioxide due to diffusion, it is desirable to culture in a closed culture vessel. The concentration of carbon dioxide in the gas phase in this case is not particularly limited as long as the effect of the present invention can be achieved, but is preferably not less than 20% by volume and preferably 0.1 to 15% by volume in the atmosphere. %, More preferably 0.1 to 10% by volume.
The carbon dioxide concentration in the atmosphere is generally said to be about 0.04% by volume.

That is, according to the present invention, there is an advantage that carbon dioxide in the atmosphere can be used. This is because the microalgae that are desired to grow greatly are on the liquid surface, so that carbon dioxide in the gas phase is easily taken up. In that case, the piping and bubbling of carbon dioxide are unnecessary, and the cost of algal body production can be reduced.
Moreover, in the method of this invention, since it culture | cultivates on the gas-liquid surface, there are few deaths by drying of an algal body.

[Other culture conditions]
The pH of the liquid medium immediately after the start of the culture (hereinafter, the liquid medium is also referred to as a culture solution) is preferably in the range of 2 to 11, more preferably in the range of 5 to 9. The pH is most preferably in the range of 5 to 7. This can suitably increase the growth rate of microalgae, and a medium that tends to generate precipitates at a high pH, such as CSiFF03 medium, may generate some precipitates on the weak alkaline side. This is because when the acidity is weak, almost no precipitate is generated. In addition, since the number of algae on the liquid surface is increased as compared with the algae on the bottom surface, it is desirable to perform the culture under mildly acidic conditions. Moreover, since suitable pH changes depending on the kind of micro algae, it is preferable to select pH according to the kind of micro algae. The pH of the liquid medium is the pH at the start of culture. In addition, since the pH immediately after the start of the culture and the pH after the start of the culture may change as the microalgae grow, the pH of the liquid medium immediately after the start of the culture may be different from the pH at the time of the recovery of the microalgae. In the present invention, it is good.

  The culture temperature can be selected according to the type of microalgae, but is preferably 0 ° C. or higher and 90 ° C. or lower. More preferably, it is 15 degreeC or more and 50 degrees C or less, Most preferably, it is 20 degreeC or more and less than 40 degreeC. When the culture temperature is 20 ° C. or higher and lower than 40 ° C., the growth rate of microalgae is sufficiently fast, and in particular, the culture at 37 ° C. has the fastest growth rate.

The minimum initial algal body concentration of microalgae is not particularly limited because it is possible to grow if there is only one alga body in the culture solution, but it is preferably at least 1 / mL. More preferably 1000 / mL or more, and even more preferably 1 × 10 ⁴ / mL or more. The upper limit initial algal body concentration of microalgae can grow at any high concentration, so there is no particular limitation, but the higher the algal body concentration is above a certain concentration, the higher the number of input alga bodies And the ratio of the number of alga bodies after growth is preferably 10,000 × 10 ⁴ cells / mL or less, more preferably 1000 × 10 ⁴ cells / mL or less, and even more preferably 500 × 10 ⁴ cells / mL or less.

  In the present invention, a medium once used for culture can be mixed with a newly prepared medium and used. By doing in this way, the amount of microalgae grown may be reduced, but the amount of water used can be reduced. In addition, as a method for suppressing a decrease in the growth amount of the above-mentioned microalgae, a method of adding a high concentration medium is conceivable, and these methods can be used in the present invention.

In the present invention, the preculture period in the case of liquid surface suspension culture is preferably 1 day to 300 days, more preferably 3 days to 100 days, and further preferably 7 days to 50 days.
As the period of the liquid surface floating culture, the culture can be continued as long as the microalgae grow. Usually, the culture is preferably performed for 1 to 100 days, more preferably 7 to 50 days, and more preferably 10 to 30. More preferably, it is performed in days.

  In the present invention, it is also possible to add to the medium a substance having a buffering action that keeps the pH in the medium constant. In general, it is known that microalgae release various substances to the outside of the cells with survival and growth, but depending on the substance to be released, the pH in the medium can be changed, and the culture of microalgae It is also possible to change to an environment that is not suitably performed. In order to avoid such a phenomenon, it is preferable to add a substance having a buffering action. Furthermore, although microalgae grow using carbon dioxide as a carbon source, the pH of the medium decreases as the carbon dioxide dissolves in the medium, and the environment in which microalgae is not suitably cultured It may be possible to change. In order to avoid such a phenomenon, it is preferable to add a substance having a buffering action. As the substance having a buffering action, a known substance can be used, and its use is not limited, but 4- (2-hydroxyethyl) -1-piperazine etheric acid (HEPES), sodium phosphate buffer, A potassium phosphate buffer or the like can be preferably used. The concentration and type of these buffer substances can be determined according to the culture environment of microalgae.

[Culture container]
As the shape of the culture vessel (culture pond) that can be used in the present invention, any known type of culture vessel can be used as long as it can hold a suspension solution of microalgae. For example, an indefinite shape such as a columnar shape, a square shape, a spherical shape, a plate shape, a tube shape, or a plastic bag can be used. Moreover, the culture container which can be used by this invention can use various well-known systems, such as an open pond (open pond) type, a raceway type, and a tube type (J. Biotechnol., 92, 113, 2001). For example, Journal of Biotechnology 70 (1999) 313-321, Eng. Life Sci. 9, 165-177 (2009). And the culture vessel described in (1). Among these, it is preferable from the viewpoint of cost to use an open pond type or a raceway type.

  The culture vessel that can be used in the present invention can be either an open type or a closed type. However, it is possible to prevent contamination of microorganisms and dust other than the purpose of culture, to suppress evaporation of the culture medium, In order to prevent destruction and movement, and to prevent diffusion of carbon dioxide outside the culture vessel when carbon dioxide having a concentration of carbon dioxide in the atmosphere or higher is used, a closed culture vessel can be preferably used.

[Light source and light intensity]
As the light source that can be used in the light irradiation, any light source can be used, and sunlight, LED light, fluorescent lamp, incandescent bulb, xenon lamp light, halogen lamp, and the like can be used. It is preferable to use sunlight, an LED with good luminous efficiency, or a fluorescent lamp that can be used easily.

  The amount of light is preferably from 100 lux to 1 million lux, and more preferably from 300 lux to 500,000 lux. The most preferable light amount is 1000 lux or more and 200,000 lux or less. The greater the amount of light, the better the growth rate of the microalgae, which is preferable. However, if the light amount is 1000 lux or more, the growth rate of the microalgae is sufficiently high, and if it is 200,000 lux or less, the occurrence of light damage is suppressed. Therefore, the growth rate of microalgae can be reduced or the increase in the rate of death can be sufficiently suppressed.

  The light may be either continuous irradiation or a method of repeating irradiation and non-irradiation at a certain time interval. However, since it is close to the natural state, it is preferable to turn the light on and off at intervals of 12 hours. From the results of the experiment, it is preferable that the liquid surface suspension culture is continuously irradiated immediately after the start of the culture, and is irradiated with light ON / OFF at intervals of 12 hours from the middle stage to the late stage of the culture.

  The wavelength of light is not particularly limited, but any wavelength can be used as long as photosynthesis is possible. Although a preferable wavelength is sunlight or a wavelength similar to sunlight, an example in which the growth rate of photosynthetic organisms is improved by irradiating a single wavelength has been reported. In the present invention, such an irradiation method is also used. It is preferable to use it. On the other hand, from the viewpoint of cost, irradiation using light that does not control the wavelength is more advantageous than irradiation using light of a single wavelength, and using sunlight is most advantageous from the viewpoint of cost. is there.

[Biofilm formed on the liquid surface]
The present invention also relates to a biofilm formed on a liquid surface by the method for culturing microalgae of the present invention.
A biofilm is usually a film-like structure, and refers to a state in which microalgae are connected to each other to form a film-like structure. In order for microalgae to be connected to each other, for example, a substance such as an intercellular matrix (such as a polysaccharide) is released from the microalgae, and the microalgae are linked together by their chemical action. In other words, it means a state where they are coupled so that they are not separated from each other by the movement of a weak water flow. In general, such a film-like structure is often referred to as a biofilm.
The biofilm according to the present invention is preferably a film-like structure or a three-dimensional three-dimensional structure described later, which is formed by the microalgae AVFF007.

The biofilm according to the present invention may be a uniform film-like structure having no breaks in the microalgae aggregates over the entire surface of the culture vessel, but a part of such a film-like structure is a bubble. It may be a three-dimensional three-dimensional structure formed so as to rise. In addition, a part of such a three-dimensional three-dimensional structure may be a complicated structure that is further formed in a bubble shape. A phenomenon in which a part of the film-like structure swells in the form of bubbles is observed as the microalgae grow. In addition, it is presumed that the phenomenon that rises in the form of bubbles is due to oxygen discharged by fixing CO ₂ during the growth of microalgae. Further, when a three-dimensional structure is formed, a structure in which a lot of microalgae are present near the light source may be used.
There may be many three-dimensional three-dimensional structures formed in the form of bubbles in the culture vessel, and the sizes thereof may be different.

  As the three-dimensional structure develops, the location of the microalgae moves away from the liquid surface and becomes closer to the light source. This means that the supply of moisture from the liquid surface is reduced, and it becomes difficult for heat to diffuse due to light irradiation. As a result, the moisture content of the microalgae present at a location away from the liquid surface decreases. The reduction in water content enables simplification of the dehydration process when performing the oil extraction process after the recovery process, and is advantageous for reducing the cost of biomass production using microalgae. In general, a centrifuge is used to lower the moisture content of the microalgae during the recovery process. In the recovery method using the culture method of the present invention, the microalgae obtained by the centrifuge is used. It is also possible to make the moisture content lower than that of algae.

In addition, a wrinkle-like structure may appear in the film-like structure as the microalgae grow, but the film-like structure may have such a structure.
Furthermore, as the growth of microalgae progresses, a pleat-like or curtain-like structure may be formed in the medium in a film-like structure or a three-dimensional three-dimensional structure. Such a structure or a three-dimensional structure may be accompanied by such a structure.
As described above, a film-like structure or a three-dimensional three-dimensional structure may be accompanied by a wrinkled, pleated, or curtain-like structure, or a film-like structure is a bubble-like structure. It may be a three-dimensional three-dimensional structure formed along with, and such a structure is preferable because the amount of algal bodies per unit area increases.

The area of the film-like structure is an area that does not allow the fragments of the film-like structure existing on the liquid surface to escape from the substrate through the liquid surface when collecting using the substrate. It is preferable that there is no break in the film-like structure over the entire culture container. For example, such an area can be 1 cm ² or more, and preferably 10 cm ² or more. Most preferably, it is 100 cm ² or more. The upper limit of such an area is not particularly limited as long as it is not more than the area of the liquid surface of the culture vessel.

The thickness of the film-like structure is usually in the range of 1 μm to 10000 μm, preferably in the range of 1 μm to 1000 μm, and more preferably in the range of 10 μm to 1000 μm.
When the biofilm according to the present invention is a three-dimensional three-dimensional structure formed in a bubble shape in a part or a plurality of parts of the film-like structure, the biofilm is based on the liquid level of the medium. The height of the three-dimensional structure is usually in the range of 0.01 mm to 100 mm, preferably in the range of 0.1 mm to 20 mm, and more preferably in the range of 5 mm to 20 mm.

The dry algal 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. It is particularly preferred. Most preferably, it is 5 mg / cm ² or more. This is because it is expected that the amount of biomass such as oil to be collected increases as the dry alga body weight per unit area increases. The dry alga body weight per unit area of the biofilm is usually 100 mg / cm ² or less.

The film density per the microalgae, unit area in the biofilm of the present invention is preferably 100,000 / cm ² or more, more preferably 1,000,000 / cm ² or more, 10 million It is particularly preferable that the number of particles / cm ² or more. When the film density per unit area of microalgae in the biofilm is less than 100,000 / cm ² , biofilm formation cannot be confirmed on the liquid surface, and the microalgae recoverability becomes poor. On the other hand, if the membrane density per unit area of the microalgae in the biofilm is 10 million pieces / cm ² or more, the membrane density is high and a strong biofilm is formed on the liquid surface, thereby improving the recoverability. . The upper limit of the film density per unit area of microalgae in the biofilm is not particularly limited because it is preferably as large as possible, but is usually 10 billion pieces / cm ² or less.

  The biofilm according to the present invention preferably has a low moisture content from the viewpoint of labor saving in the process of removing moisture and cost reduction. Specifically, the water content of the biofilm is preferably 95% by mass or less, more preferably 90% by mass or less, particularly preferably 80% by mass or less, and most preferably 70% by mass or less. . The water content of the biofilm is usually 50% by mass or more. The water content is the value obtained by subtracting the weight of the algal body after drying from the weight of the algal body before drying and dividing by the weight of the algal body before drying and multiplying by 100.

  The biofilm according to the present invention preferably has a high oil content from the viewpoint of 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 alga body of the biofilm is usually 80% by mass or less.

  As the microalgae of the present invention, a microfilm capable of forming on the liquid surface a biofilm having the above structure, the area in the above range, the thickness, the dry alga body weight per unit area, the water content, and the oil content. Algae is preferable for the same reason as described above.

[Recovery process]
As shown in FIGS. 11 (d) to 11 (e), the recovery process refers to the biofilm (film-like structure or three-dimensional structure) composed of microalgae on the liquid surface. The step of peeling the biofilm from the first substrate after the transfer to the substrate, or the biofilm composed of microalgae on the liquid surface (see (f) to (g) of FIG. 11) It is a step of recovering a film-like structure or a three-dimensional structure) using the second substrate.
The present invention also relates to a biofilm recovery method in which a biofilm formed on a liquid surface is transferred to a substrate (first substrate) and recovered.
The present invention also relates to a biofilm recovery method in which a biofilm formed on a liquid surface is deposited on a substrate (second substrate) and recovered.

For recovering the biofilm from the first substrate, any known method can be used as long as the biofilm can be peeled from the first substrate. For example, a method of stripping a biofilm from a substrate using a cell scraper, a method of using a water flow, a method of using an ultrasonic wave, and the like can be given, and a method using a cell scraper is preferable. This is because in other methods, the biofilm is diluted with a medium or the like and may need to be concentrated again, which is inefficient.
For recovering the biofilm from the second substrate, any known method can be used as long as the biofilm can be peeled from the second substrate. For example, a method using gravity, a method of peeling a biofilm from a substrate using a cell scraper, a method using a water flow, a method using ultrasonic waves, etc. Alternatively, a method using a cell scraper is preferable. This is because in other methods, the biofilm is diluted with a medium or the like and may need to be concentrated again, which is inefficient. Moreover, after recovering a biofilm using natural fall by gravity, algae remaining on the second substrate can also be recovered using a cell scraper or the like.

[Total volume recovery]
Collecting the total amount means collecting all the microalgae in the culture vessel. That is, all the biofilms (film-like structures or three-dimensional structures) composed of the deposited microalgae and the microalgae on the liquid surface are collected. Such a recovery method can be performed when culturing is completed, when another microalgae is cultured, or when the medium in the culture vessel is replaced. After the biofilm composed of microalgae on the liquid surface is transferred using the first substrate or recovered using the second substrate, the medium is removed, and the remaining microalgae on the bottom of the culture vessel are removed. It may be collected. Furthermore, you may collect | recover all the micro algae in a culture container by a well-known method. Examples of the known method include collection with a filter and collection with a flocculant.

For example, after collecting the microalgae biofilm on the liquid surface, the microalgae on the bottom surface of the culture vessel can be recovered.
Alternatively, after collecting the biofilm composed of microalgae on the liquid surface, the culture solution in the culture vessel may be removed, and the microalgae on the bottom surface of the culture vessel or the surface of the first substrate may be recovered.

[Biofilm transfer]
The transfer referred to in the present invention is a kind of adhesion and is adhesion that does not substantially accompany proliferation. In the present invention, an operation of transferring a biofilm formed on a liquid surface to the surface of the first substrate in a substantially intact form using the first substrate.

  The transfer of the biofilm is a process of transferring the biofilm formed on the liquid surface onto the surface of the substrate using the first substrate as shown in FIG. In the figure, a biofilm is formed on the entire surface of the culture vessel, and the recovery process is performed in such a state. However, the recovery may be performed in such a state, or a biofilm made of microalgae. In the present invention, the recovery step can be carried out even when there is a state in which is not partially present. Further, when culturing is performed by the method of the present invention, the biofilm formed on the liquid surface is wrinkled or folded, and the film is composed of pleated microalgae. In some cases, the structure may grow in liquid like an aurora (curtain shape). In the present invention, recovery can be performed even in such a state, and recovery methods and culture methods by such methods can also be included in the present invention.

[substrate]
The substrate referred to in the present invention is a substrate used for transferring or collecting a biofilm composed of microalgae on the liquid surface used in FIG. 11 (d) or FIG. 11 (f). Say that.

  The surface of the first substrate and the surface of the second substrate refer to all surfaces 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 are attached to these surfaces, they are in contact with the culture vessel or the like, and microalgae may come into the culture solution from the layer formed between the substrate and the surface of the culture vessel or the like. 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, corrugated, etc., but it is easy to attach, deposit, transfer, and collect microalgae from the substrate. It is preferable that it is a film form or a plate form from easiness of care.
The first substrate and the second substrate may have the same shape, or may have different shapes. The areas of the first substrate and the second substrate are preferably smaller than the area of the culture solution liquid surface in the culture vessel.

[Material]
The materials for the culture vessel, the first substrate, and the second substrate that can be used in the present invention are not particularly limited, and known materials can be used. For example, a material composed of an organic polymer compound, an inorganic compound, or a composite thereof can be used. It is also possible to use a mixture thereof.

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 derivatives, polyacrylonitrile derivatives, polyvinyl alcohol derivatives, polyethersulfone derivatives, polyarylate derivatives, allyl diglycol carbonate derivatives, ethylene-vinyl acetate copolymer derivatives, fluororesin derivatives, polylactic acid derivatives, acrylic resin derivatives, An ethylene-vinyl alcohol copolymer, an ethylene-methacrylic acid copolymer, or the like can be used.
As the inorganic compound, glass, ceramics, concrete, or the like can be used.
As the metal compound, an alloy such as iron, aluminum, copper, and stainless steel can be used.

  Among the above, a part of the material of the first substrate, the second substrate, or the culture vessel is composed of at least one selected from glass, polyethylene, polypropylene, nylon, polystyrene, vinyl chloride, and polyester. Preferably it is.

Moreover, the materials of the culture vessel, the first substrate, and the second substrate may be the same or different.
When a closed culture vessel is used, the light receiving surface is preferably a material that transmits light, and more preferably a transparent material.

[Substrate surface irregularities]
In the present invention, irregularities can be formed on the surface of the substrate. By forming irregularities on the surface of the substrate, the adhesion surface area may increase, and the adhesion, transfer and recovery stability of algal bodies may be improved.

[Bottom]
The bottom surface referred to in the present invention may include a portion other than the bottom surface of the culture vessel. For example, the side surface of the culture vessel or the surface of a sensor immersed in a liquid can be raised. The influence of the side surface increases as the culture vessel becomes smaller.

[Biomass and oil]
The present invention also relates to biomass and oil obtained from a biofilm formed on the liquid surface by the microalgae of the present invention.
In the present invention, “biomass” refers to organic resources derived from renewable organisms excluding fossil resources, and examples include biological materials, foods, materials, fuels, resources, and the like.
In the present invention, “oil” means a combustible fluid substance, which is a compound mainly composed of carbon and hydrogen, and in some cases, a substance containing oxygen, nitrogen and the like. is there. Oil is generally a mixture and is a substance that is extracted using a low polarity solvent such as hexane or acetone. Its composition is composed of hydrocarbon compounds, fatty acids, triglycerides and the like. Some are esterified and used as biodiesel.

The method for collecting biomass and oil contained in the biofilm according to the present invention is not particularly limited as long as the effects of the present invention are not impaired.
As a general method for recovering oil, which is an example of biomass, a biofilm is dried by heating to obtain a dried alga body, and then cell disruption is performed as necessary, and the oil is extracted using an organic solvent. Since the extracted oil generally contains impurities such as chlorophyll, it needs to be purified. Purification includes silica gel column chromatography and distillation (for example, the distillation method described in JP-T 2010-539300).
In addition, after preparing a high-concentration microalgae solution, the microalgae are crushed by ultrasonic treatment, or the microalgae are crushed by proteases or enzymes, and then the oil in the algal bodies is extracted using an organic solvent. There is also a method (for example, the method described in JP-T-2010-530741).

  Thus, the biofilm according to the present invention is useful as a biomass fuel. That is, this invention relates also to the manufacturing method of biomass fuel which uses the biofilm collect | recovered by the biofilm recovery method which concerns on this invention as a fuel.

[Dry algae]
The dry alga body in the present invention is obtained by drying the biofilm according to the present invention.
The method for drying the biofilm is not particularly limited as long as it is a method capable of removing moisture in the biofilm. For example, a method of drying the biofilm in the sun, a method of heating and drying the biofilm, a method of freeze-drying (freeze drying) the biofilm, a method of spraying dry air on the biofilm, and the like can be mentioned. Among these, from the viewpoint of suppressing the decomposition of the components contained in the biofilm, the freeze drying method is preferable, and the heat drying method is preferable from the viewpoint of efficient drying in a short time.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples.

[Example 1]
<Formation of biofilm (film-like structure) on the liquid surface by AVFF007 strain>
Put the total amount of the solution containing the purified AVFF007 strain into a 5 mL homogenizing tube (Tommy Seiko Co., Ltd., TM-655S) and set it in a bead-type cell crusher (MS-100, Tommy Seiko Co., Ltd.) A suspension of AVFF007 strain was obtained by performing homogenization treatment at 4200 rpm for 20 seconds three times.
The entire amount of this solution was placed in an Erlenmeyer flask containing 40 mL of CSi medium, and statically cultured (hereinafter also referred to as preculture) in a desiccator under a 5% CO ₂ atmosphere. The culture was performed at a temperature of 23 ° C. and a light intensity of 2000 lux.
A solution containing microalgae was collected from the Erlenmeyer flask, placed in a 5 mL homogenizing tube, set in a bead-type cell crusher, and homogenized at 4200 rpm for 20 seconds three times. Here, no cell disruption beads are used.
50 μL of microalgae suspension is collected from this solution, placed in a homogenizing microtube to which 950 μL of CSi medium has been added in advance, set in a bead-type cell crusher, and homogenized for 20 seconds at 5500 rpm. went. In this case, no cell disruption beads are used. About 10 μL of this solution was collected and counted on a hemocytometer, and the algal body concentration in the 5 mL homogenizing tube was 695 × 10 ⁴ cells / mL.

Add 50 mL of CSi medium to a 50 mL centrifuge tube (MS-57500, Sumitomo Bakelite Co., Ltd.), add 720 μL of the microalgae suspension in the 5 mL tube, stir this well, and then add 8 mL each to the 6-well plate. did.
The culture is performed using plant bioshelf tissue culture (Ikeda Rika Co., Ltd., AV152261-12-2), and the 6-well plate is placed in a vacuum desiccator (As One Co., Ltd., 1-070-01). went. Under conditions of 4000 lux, room temperature (23 ° C.), light irradiation ON / OFF switching every 12 hours (hereinafter also referred to as “12 hours ON-OFF irradiation”, “12 hours ON-OFF control”) Incubation was performed. The CO ₂ concentration in the vacuum desiccator is 10%.

During the liquid surface suspension culture, the 6-well plate was removed from the vacuum desiccator and directly observed on the liquid surface using an optical microscope. The observation results are shown in FIG. The left side of FIG. 14 is the result of observation at a magnification of 4 times with an optical microscope, and the right side of FIG. 14 is the result of observation at a magnification of 40 times with an optical microscope. Until the first day of liquid surface suspension culture, there is almost no AVFF007 strain on the liquid surface, and there is a slight presence on the second day of culture. AVFF007 stock was completely covered. On day 7 of culture, it can be visually confirmed that a solid film-like structure is formed on the liquid surface. On day 12 of culture, AVFF007 strain on the liquid surface is proliferated. In order to gain surface area, it grew to a wrinkled state.
As is clear from the above results, the AVFF007 strain, which is the microalgae of the present invention, can form a biofilm on the liquid surface.

[Example 2]
<Formation of biofilm (three-dimensional three-dimensional structure formed in a bubble shape in a part or a plurality of parts of a film-like structure) on the liquid surface by AVFF007 strain>
Microalgae AVFF007 was precultured for 28 days in the same manner as in Example 1. After the pre-culture, the AVFF007 strain on the liquid surface was collected and put in a 5 mL tube for homogenization to prepare a suspension solution of 3 mL of AVFF007 strain. The alga body concentration of the prepared suspension of AVFF007 strain was counted in the same manner as in Example 1 and found to be 32389 × 10 ⁴ cells / mL.

By adding 350 mL of CSiFF04 medium shown in FIG. 15 and 540 μL of the suspension solution of the algae AVFF007 strain to a 500 mL glass Erlenmeyer flask, a solution having a concentration of 50 × 10 ⁴ cells / mL of the AVFF007 strain was prepared.
Three solutions of each 40 mL (water depth 1.5 cm) of this solution were prepared in each case of PS case 28 type (As One Co., Ltd., material polystyrene). All the PS case 28 type lids were put in the vacuum desiccator without the lid, and the atmosphere was kept in a 5% CO ₂ atmosphere, and the vacuum desiccator door was closed. Carbon dioxide was newly prepared every evaluation day.
This was installed in a plant bioshelf, and liquid surface floating culture by stationary culture was performed under the condition of switching 15000 lux, 23 ° C., light irradiation ON and OFF every 12 hours.
After the liquid surface suspension culture for 4, 7, and 10 days, the biofilm on the liquid surface was recovered by the same method as described with reference to FIG. 12, and the dry weight was measured by heating and drying the biofilm. As shown in FIG. 16, the biofilm on the liquid surface after 14 days of liquid surface floating culture has a three-dimensional structure formed by overlapping three-dimensional structures formed as bubbles. Was forming. In addition, the microalgae at a position distant from the liquid surface seemed to have a reduced water content, and further changed color from yellow to light brown. Oil accumulation algae such as Botriococcus are known to color from green to brown, yellow, red, etc. when oil is accumulated. From these, the AVFF007 strain in the biofilm accumulates oil. It was thought that there was.

FIG. 17 shows the relationship between the number of days of liquid surface floating culture and the amount of dry alga body of the biofilm in each number of days of liquid surface floating culture. Ten days after the start of the liquid surface suspension culture, the dry alga body weight reached 12.6 mg / cm ² . Between 10 days from the start of liquid surface suspension culture, 13 g / m ² / day, between 4 days and 10 days, 20 g / m ² / day, between 7 days and 10 days, 32 g A growth rate of / m ² / day was reached. As is clear from FIG. 17, the logarithmic growth phase of the AVFF007 strain on the liquid surface in Example 2 is between the 4th and 10th days.
Further, FIG. 18 shows the relationship between the liquid surface floating culture days and the maximum height (cm) of the biofilm in each liquid surface floating culture days. The maximum height of the biofilm is a value obtained by measuring the height of the biofilm at the position where the height from the liquid surface is maximum in the biofilm formed on the liquid surface. As can be seen from FIG. 18, the maximum height increased sharply after the fourth day, and during this time, the structure changed from a film-like structure to a three-dimensional structure. .

  Furthermore, in the formed biofilm, the microalgae that existed at a position away from the liquid level seemed to have a reduced moisture content, so the water content of the biofilm in Example 2 is considered low, The water content was calculated. The water content of the biofilm is the value obtained by subtracting the weight of the algal body after drying from the weight of the algal body before drying, and multiplying by 100 by the weight of the algal body before drying.

FIG. 19 shows the relationship between the liquid surface floating culture days and the water content of the biofilm in each liquid surface floating culture days. In general, after culturing in suspension culture, microalgae are settled on the bottom of the culture vessel with a flocculant, and after collecting this, the moisture content of the algal bodies that can be recovered by a centrifuge is about 90% by mass. is there. In the case of the present microalga AVFF007 strain, the moisture content is about 90% by mass only by performing recovery using the substrate. As the liquid surface floating culture further progresses, a three-dimensional structure on the liquid surface develops, and moisture in the microalgae at a position away from the liquid surface begins to evaporate. The water content was further reduced.
Thus, the water content of the biofilm recovered by the liquid surface suspension culture method in the present invention is higher than the water content of the microalgae recovered by the centrifuge (usually water content of about 90% by mass). Can be lowered. In other words, the biofilm formed on the liquid surface by the microalgae of the present invention is not only easy to recover, but also has a low moisture content, which enables labor saving in the process of removing moisture, and the subsequent oil extraction process ( Cost reduction in processes such as a drying process) can be expected.

[Example 3]
<Oil content when liquid surface culture is performed>
The solution containing the purified strain AVFF007 was put into a 100 mL Erlenmeyer flask that had been sterilized by dry heat and contained 40 mL of a mixed medium of C medium and CSi medium (volume ratio 1: 1), and plant bioshelf It was installed for tissue culture and static culture (hereinafter also referred to as pre-culture) was performed at 23 ° C. under continuous light irradiation of 4000 lux.
A solution containing microalgae is collected from the Erlenmeyer flask, placed in a 5 mL homogenizing tube, set in a bead-type cell crusher, and homogenized for 20 seconds at 4200 rpm three times without using the cell crushing beads. went.
50 μL of microalgae suspension is collected from this solution, placed in a homogenizing microtube to which 950 μL of CSi medium has been added in advance, set in a bead-type cell crusher, and homogenized for 20 seconds at 5500 rpm. This was carried out without using the beads for cell disruption. About 10 μL of this solution was collected and counted on a hemocytometer, and the algal cell concentration in the 5 mL homogenizing tube was 4975 × 10 ⁴ cells / mL.

  Add 50 mL of CSi medium to a 50 mL centrifuge tube, add 361.8 μL of microalgae suspension in the 5 mL tube, stir this well, add 8 mL each to a 6-well plate, and use for plant bioshelf tissue culture Above, culture was performed under continuous light irradiation with a 4000 lux fluorescent lamp. In addition, room temperature is set to 23 degreeC and culture | cultivation is stationary culture.

After 30 days of culture, the culture was stopped and the biofilm on the liquid surface was recovered using a nylon film.
The biofilm obtained by the above method was placed in a 50 mL centrifuge tube, set in a spin dryer centrifuge (VC-96R, Taitec Co., Ltd.), and centrifuged at 2000 r / min (440 × g). . The entire amount of the resulting precipitate was put into a 2 mL glass sample bottle that had been weighed in advance, and this was centrifuged again using the centrifuge, and then the supernatant was removed and 100 ° C. It dried in the drying oven of the ventilation constant temperature thermostat set to (DKM600, Yamato Scientific Co., Ltd.).

  After drying, weigh the sample bottle containing the dried microalgae and subtract the weight of the empty sample bottle from the weight of the solid component in the medium diluted 1/10 calculated from the amount of solvent, The dry weight of microalgae was used. In addition, the amount of solvent was estimated from the weight difference before and behind heat drying.

1 mL of 5% hydrochloric acid-methanol mixed solution was added to the sample bottle, and the reaction was carried out using a blast incubator set at 100 ° C.
After cooling to room temperature, the entire amount was transferred to a 10 mL sample bottle, and 1.5 mL of hexane and 0.5 mL of distilled water were further added. After vigorously stirring using a vortex mixer, the mixture was allowed to stand to separate into two phases, and the hexane phase was placed in a 2 mL sample bottle that had been weighed in advance.

The 2 mL sample bottle was set in a spin dryer centrifuge, and the organic solvent was evaporated under reduced pressure while centrifuging at 2000 r / min (440 × g).
After evaporation, the sample is heated in a constant-temperature incubator set at 55 ° C. and cooled to room temperature. The weight of the 2 mL sample bottle is measured, and the difference from the weight when no sample is put is determined as the oil weight. did. Moreover, the oil content per dry alga body was calculated by dividing the oil weight by the dry alga body weight.
In the case of the AVFF007 strain, the oil content was 11.72% by mass.

[Example 4]
<Results of analysis of oil components>
CFF medium prepared by purifying sterilized AVFF007 strain with 1 × 10 ⁴ cells / mL was placed in 5 L, 60 cm wide, 45 cm deep, 45 cm high water tank, temperature 25 ° C., light irradiation intensity 65 μmol / m Liquid surface suspension culture by stationary culture was performed under the condition of ² / s.
30 days after the start of the culture, the biofilm on the liquid surface was attached to the parafilm and recovered (that is, the recovery method using the first substrate), and the recovered material was collected at 6000 × g for 10 minutes, and further at 8000 × g for 10 minutes. Centrifugation was performed for minutes, and the supernatant was removed, followed by lyophilization.
The obtained dried product was crushed using a mortar and pestle, 4 mL of hexane was added, the whole amount was transferred to a centrifuge tube, and centrifuged at 1500 × g for 10 minutes. The supernatant hexane phase was collected and placed in a separate container. A 5% hydrochloric acid-methanol solution was added to the solid, heat-treated at 100 ° C. for 1 hour, cooled to room temperature, and hexane and distilled water were added. After stirring this solution, centrifugation was performed at 8500 × g for 5 minutes to remove the aqueous phase, and then distilled water was added again to the remaining hexane phase. After stirring, the solution was stirred at 8500 × g for 5 minutes. Centrifugation was performed. The hexane phase of the supernatant was mixed with the hexane phase collected earlier and analyzed by GC-MS.
The GC-MS analysis conditions were as follows: Rtx-5MS (manufactured by Restek) was used as a column, held at 90 ° C. for 1 minute, and then heated at 10 ° C./min.
The results are shown in FIG. It was revealed that C16 and C21 were mainly contained as oil components.
Moreover, the solvent of the obtained hexane phase was removed, and the oil content calculated from the amount of dry algal bodies used was 25% by mass.

[Example 5]
<Recovery of biofilm formed on the liquid surface>
Except for using CSiFF04 medium as a medium, a solution with an alga body concentration of 50 × 10 ⁴ cells / mL was prepared by dispersing AVFF007 strain in 220 mL of CSiFF04 medium in the same manner as in Example 1. Was all put into a styrene square case 8 type.
This was put in a vacuum desiccator with the case lid removed, and installed for plant bioshelf tissue culture, 15000 lux (12 hours ON-OFF irradiation) with a fluorescent lamp in a 5% CO ₂ atmosphere, 23 ° C. The stationary liquid surface suspension culture was carried out. The culture was performed for 21 days, and the styrene square case 8 was removed from the vacuum desiccator. The state of the biofilm formed on the liquid surface is shown in FIG. As shown to (a) of FIG. 21, the micro algae grew so that it might rise on the liquid surface, and the three-dimensional three-dimensional structure was formed.
Next, using a nylon 6 film having the same length as the short side of the case, a biofilm (three-dimensional three-dimensional structure) on the liquid surface was collected by the method shown in FIG. The state of the liquid level after recovery is shown in FIG. As shown in FIG. 21 (b), the microalgae on the liquid level were not observed at all. Furthermore, recovery of microalgae on the liquid surface was attempted by transfer using a polyethylene film, but the recovery amount was 0 g and could not be recovered at all. Although microalgae are present on the bottom and side surfaces of the culture vessel, they are not targeted for collection.

[Example 6]
<Temperature dependence of liquid surface suspension culture>
In the same manner as in Example 1, the microalgae AVFF007 was precultured.
In the same manner as in Example 1, the AVFF007 strain on the liquid surface of the 6-well plate was collected, and all of the AVFF007 strain was put into a 5 mL tube for homogenization to prepare about 3 mL of a suspension solution of AVFF007 strain.
A 50 mL centrifuge tube is added with 50 mL of the CSiFF01 medium shown in FIG. 22 and a suspension solution of the algae AVFF007 strain, so that a concentration of the AVFF007 strain (initial algal body concentration) is 10 × 10 ⁴ cells / mL. Prepared. After thoroughly stirring this solution, 8 mL of a suspension solution of AVFF007 strain was placed in a 6-well plate. This was placed in a vacuum desiccator and cultured.

The culture was divided into room temperature culture or temperature-controlled culture, and was performed simultaneously at each culture temperature.
For room temperature culture, a 6-well plate placed in a vacuum desiccator was installed for plant bioshelf tissue culture, the CO ₂ concentration was 5%, the vacuum desiccator lid was locked, and liquid surface suspension culture was started. The culture conditions are liquid surface floating culture by stationary culture at a light intensity of 4000 lux (on-off control for 12 hours) and a temperature of 23 ° C.
For temperature-controlled culture, a 6-well plate placed in a vacuum desiccator is placed on a shaking culture machine (RGS-20RL, Sanki Seiki Co., Ltd.), the CO ₂ concentration is 5%, the vacuum desiccator lid is locked, Surface suspension culture was started. The culture conditions were a light intensity of 4000 lux (continuous light), static at each set temperature (specifically, 20 ° C., 25 ° C., 30 ° C., 35 ° C., 37 ° C., 38 ° C., 39 ° C. and 40 ° C.). It is a liquid surface floating culture by stationary culture.
Liquid surface suspension culture was performed for 7 days. The biofilm on the liquid surface was collected by the same method as the collecting method (collecting method by transfer) using the first substrate in FIG. The measurement of the dry algal mass of the biofilm was performed by the method in Example 2.
FIG. 23 shows the relationship between the liquid surface suspension culture temperature and the ratio of dry alga bodies of the biofilm at each liquid surface suspension culture temperature (based on the dry alga body amount of the biofilm obtained by room temperature culture at a temperature of 23 ° C.). When the liquid surface suspension culture temperature is up to 37 ° C, the higher the temperature, the higher the dry alga mass ratio. At 37 ° C, the dry alga mass ratio is approximately three times that of room temperature (23 ° C). It was. However, at 40 ° C., no biofilm was formed on the liquid surface. From the above, it was found that in the case of liquid surface suspension culture using AVFF007 strain, it is preferable to perform the growth at a culture temperature of 38 ° C. or lower.

[Example 7]
<Effect of medium depth on liquid surface suspension culture>
In the same manner as in Example 1, a suspension solution of AVFF007 strain having an alga body concentration of AVFF007 strain of 9726 × 10 ⁴ cells / mL was prepared.

A CSiFF01 medium was added in the following amounts to waist-high petri dishes having a height of 45, 60, and 90 mm (Part No. 1-4402, As One Co., Ltd.) to prepare media having different water depths.
Waist height petri dish with a height of 45 mm: 9.5 mL (water depth 0.4 cm), 19 mL (water depth 0.8 cm), 47.5 mL (water depth 2 cm), 71.3 mL (water depth 3 cm)
Waist height petri dish of height 60mm: 107mL (water depth 4.5cm)
Waist height petri dish of height 90mm: 178mL (water depth 7.5cm)

To each glass waist petri dish, 195 μL of the suspension solution of the AVFF007 strain was added and stirred (that is, the algae bodies of the added AVFF007 strain were kept constant). In addition, the amount of the suspension solution of the AVFF007 strain is the same although the medium volume is different, but the total number of algal bodies in each waist-high petri dish is the same, but the algal body concentration is different and the water depth is deep. The alga body concentration becomes thinner. It was put in the vacuum desiccator without the lid of the waist high petri dish. The CO ₂ concentration was set to 5%.

Next, the waist-high petri dish containing the suspension of AVFF007 strain was moved to plant bioshelf tissue culture, and cultured under light irradiation with a 4000 lux fluorescent lamp. In addition, room temperature is set to 23 degreeC and culture | cultivation is a liquid surface floating culture by stationary culture. The light was on-off controlled for 12 hours. The culture days are 7 days and 14 days.
The liquid level biofilm was collected in the same manner as in Example 3.

FIG. 24 shows the relationship between the water depth of the medium and the amount of dry alga bodies of the biofilm in the medium at each water depth. In the figure, white circles are the results for 7 days of culture, and black circles are the results for 14 days of culture. The deeper the water in the medium, the greater the dry algal mass of the biofilm on the liquid surface. When the water depth increased from 0.8 cm to 2 cm, the amount of dry alga bodies increased rapidly, and even when the water depth was increased thereafter, the amount of dry alga bodies only slightly increased.
From the above, in the liquid surface suspension culture using the AVFF007 strain, the culture can be performed if the water depth of the medium is 0.4 cm or more, and if the water depth is 2 cm or more, the growth rate is increased and the dried algae of the recovered biofilm. I found that my body weight increased.

[Example 8]
<Effects of light intensity, culture concentration, and number of culture days on liquid surface suspension culture>
In the same manner as in Example 1, a suspension solution of AVFF007 strain having an algal body concentration of 16.7 × 10 ⁴ cells / mL of AVFF007 strain was prepared.

As a liquid medium, 2 × CSiFF01 medium and 5 × CSiFF01 medium were prepared in addition to the CSiFF01 medium (1000 mL) shown in FIG. The 2 × CSiFF01 medium and the 5 × CSiFF01 medium mean that the concentration is 2 times and 5 times that of the 1 × CSiFF01 medium, respectively.
After the preparation, autoclaving at 121 ° C. for 10 minutes was performed. 200 mL of the suspension solution of the AVFF007 strain having a concentration of 16.7 × 10 ⁴ cells / mL prepared above was placed in a 6-well plate, 8 mL per well. In addition, two wells were used for one experimental condition, and a total of 54 wells were used. The 6-well plate was not covered and placed in a vacuum desiccator, and the culture was started at a CO ₂ concentration of 5%.

Next, it moved to plant bioshelf tissue culture | cultivation, and it culture | cultivated under the light irradiation by the fluorescent lamp of 4000 lux, 8000 lux, or 16000 lux. In addition, room temperature is set to 23 degreeC and culture | cultivation is a liquid surface floating culture by stationary culture. The light was on-off controlled for 12 hours.
The biofilm on the liquid surface was collected in the same manner as in Example 3.
FIG. 25 shows the relationship between the number of days of culture at each medium concentration and light amount, and the amount of dry algal bodies of the biofilm. Moreover, the relationship between the light quantity in each culture medium density | concentration on the culture | cultivation days 14 days and the dry alga body amount of a biofilm was shown in FIG. In FIG. 26, 1, 2, and 5 on the horizontal axis mean the results when using 1 × CSiFF01 medium, 2 × CSiFF01 medium, and 5 × CSiFF01 medium, respectively, and the numerical values described above mean the amount of light. . The greater the amount of light and the higher the medium concentration, the faster the growth rate, and the amount of dried alga bodies in the recovered biofilm tended to increase.

[Example 9]
<Effect of calcium on liquid surface suspension culture>
A nitrate source (specifically, Ca (NO ₃ ) ₂ .4H ₂ O and KNO ₃ in CSiFF01 medium) in CSiFF01 medium (1000 mL) shown in FIG. Four types of media changed in such a manner were prepared.
In the same manner as in Example 1, 50 mL of a suspension solution of AVFF007 strain with an alga body concentration of AVFF007 strain of 10 × 10 ⁴ cells / mL was prepared. 8 mL of this solution was placed in each well of a 6-well plate, and liquid surface floating culture by static culture was started. The 6-well plate was put in a vacuum desiccator without a lid, and CO ₂ gas was adjusted to 5% in the vacuum desiccator to start culture. The amount of light used was 4000 lux. The culture was performed for 7 days and 14 days.
The biofilm on the liquid surface was collected in the same manner as in Example 3.

FIG. 27 shows the relationship between various nitric acid sources and the amount of dry alga bodies of the biofilm formed on the liquid surface in the culture days of 7 days and 14 days. The nitric acid source of the standard CSiFF01 medium is a mixture of Ca (NO ₃ ) ₂ .4H ₂ O and KNO ₃ . Compared with the case where the nitric acid source was NaNO ₃ , KNO ₃ , and NH ₄ NO ₃ , the amount of dry algae after 14 days was significantly increased when Ca (NO ₃ ) ₂ .4H ₂ O was contained. This result indicates that the presence of a calcium source in the medium is important for liquid surface suspension culture.

[Example 10]
<Effect of calcium concentration on growth rate>
To a medium without the addition of _{Ca (NO 3) 2 · 4H} 2 O from in CSiFF01 medium shown in FIG. 22, the CaCl ₂ · 2H ₂ O 0,0.32,0.64,1.27,3 Five media with different calcium concentrations were prepared to 18 mM.
In the same manner as in Example 1, a suspension solution of AVFF007 strain having an alga body concentration of AVFF007 strain of 10 × 10 ⁴ cells / mL was prepared. 8 mL of this solution was placed in each well of a 6-well plate, and liquid surface floating culture by static culture was started. The 6-well plate was put in a vacuum desiccator without a lid, and CO ₂ gas was adjusted to 5% in the vacuum desiccator to start culture. The amount of light used was 4000 lux. The culture was performed for 7 days and 14 days.
The biofilm on the liquid surface was collected in the same manner as in Example 3.

  FIG. 28 shows the relationship between the calcium concentration and the amount of dry alga body of the biofilm formed on the liquid surface in the culture days of 7 days and 14 days. When the calcium concentration was 0, there was almost no amount of microalgae on the liquid surface, whereas at a concentration of 0.32 mM or more, formation of a biofilm composed of microalgae was observed on the liquid surface. It was. These results indicate that the presence of calcium is important for the formation of a biofilm on the liquid surface.

[Example 11]
<Effect of pH on liquid surface suspension culture>
In the same manner as in Example 1, a suspension solution of AVFF007 strain having an alga body concentration of 19713 × 10 ⁴ cells / mL of AVFF007 strain was prepared.
29. Each of the CSiFF03 media shown in FIG. 29 having a pH adjusted to 5 to 9 was placed in 50 mL and 50 mL centrifuge tubes, and 25.4 μL of the suspension solution of the AVFF007 strain was added. A suspension of ⁴ / mL AVFF007 strain was prepared. After stirring well, 8 mL of the suspension solution of AVFF007 strain was put into a 6-well plate.
The 6-well plate was placed in a vacuum desiccator, installed for plant bioshelf tissue culture, the lid of the vacuum desiccator was locked, and the culture was started in a 5% CO ₂ atmosphere. The culture conditions are liquid surface floating culture by stationary culture at a light intensity of 4000 lux (on-off control for 12 hours) and a temperature of 23 ° C.

Incubation was carried out for 14 days. Biofilms were collected by the same method as described in Example 3. The dry alga mass was measured by the method in Example 2.
FIG. 30 shows the relationship between the pH and the amount of dry algal bodies of the biofilm formed on the liquid surface. In the conventional culturing method, culturing at pH 7 is generally performed, but it has been found that culturing at a lower pH is more preferable.
From the above, it was found that when the AVFF007 strain was used, the pH of the medium for liquid surface suspension culture was preferably 7 or less.

[Example 12]
<Effect of the amount of carbon dioxide in the atmosphere (gas phase) on liquid surface suspension culture>
The solution containing the purified strain AVFF007 was put into a 100 mL Erlenmeyer flask containing 40 mL of a mixed medium of C medium and CSi medium (volume ratio 1: 1) and installed for plant bioshelf tissue culture. Culturing was performed at 23 ° C. under continuous light irradiation of 4000 lux. In addition, the 100 mL Erlenmeyer flask used what was dry-heat-sterilized beforehand at 180 degreeC for 10 minute (s). The culture is stationary culture.
A biofilm on the liquid surface was collected in the same manner as in Example 4 except that polyethylene film was used instead of parafilm, and a microalga suspension solution was prepared in the same manner as in Example 1. Since the algal count was 13900 × 10 ⁴ cells / mL, in order to prepare 250 mL of a microalgae dispersion having a concentration of 10 × 10 ⁴ cells / mL, 179.9 μL of the microalgae suspension solution was prepared. used.
8 mL of the suspension solution of AVFF007 strain having a concentration of 10 × 10 ⁴ cells / mL prepared in the above was placed in a 6-well plate. In addition, two wells were used for one experimental condition, and a total of 30 wells were used. The 6-hole plate was covered.

Next, the 6-well plate containing the suspension solution of microalgae was moved to plant bioshelf tissue culture and cultured under light irradiation with a fluorescent light of 4000 lux. In addition, room temperature is set to 23 degreeC and culture | cultivation is a liquid surface floating culture by stationary culture. The light was on-off controlled for 12 hours. Moreover, for culture under CO ₂ atmosphere, in a vacuum desiccator, CO ₂ generating agent, placed 6-well plate containing microalgae above, together to generate CO ₂ at a concentration of CO ₂ in the object The lid of the vacuum desiccator was closed and the culture was started. The culture was performed for 7 days, 14 days or 21 days.
The liquid surface biofilm was collected by the same method as in Example 4 except that a polyethylene film was used instead of the parafilm, and the number of algal bodies was counted by the same method as in Example 1.

FIG. 31 shows the relationship between the CO ₂ concentration (% by volume) in the gas phase and the number of algal bodies of the biofilm formed on the liquid surface.
As described above, in the liquid level floating culture using AVFF007 strain, CO ₂ concentration or more in the atmosphere, it was found that it is possible cultured in a CO ₂ concentration of less than 20% by volume.

The CO ₂ concentration of 0% by volume means that the main body of the 6-hole plate and the lid are sealed with parafilm. On the other hand, the CO ₂ concentration of 0.04% by volume is the CO ₂ concentration in the atmosphere, and the main body of the 6-hole plate and the lid are not sealed with parafilm. The former two were cultured in a vacuum desiccator but did not use a CO ₂ generator.
Further, the CO ₂ concentration in this example is considered that the AVFF007 strain grew using CO ₂ in the atmosphere because CO ₂ was not bubbled into the medium.

[Example 13]
<Liquid surface floating culture of other microalgae>
Using the four types of microalgae shown in FIG. 32 and the medium, a search was made for microalgae that can be subjected to liquid surface suspension culture in addition to the AVFF007 strain. When the medium shown in FIG. 32 is a mixed medium, the mixed medium is a mixture of each medium at a liquid ratio of 1: 1.

  Each microalgae that had been subcultured was placed in a clean bench and left for a while to sink the microalgae in the Erlenmeyer flask to the bottom. In the subculture, most of the microalgae did not exist on the liquid surface. After the microalgae has settled on the bottom of the Erlenmeyer flask, the solution containing the microalgae is collected using a pipette, placed in a 5 mL homogenizing tube (TM-655S), and set in a bead-type cell disrupter (MS-100). The homogenization treatment for 20 seconds at 4200 rpm was performed 3 times.

Next, 20 mL of the medium shown in FIG. 32 was placed in a 50 mL centrifuge tube, and the above-mentioned microalga suspension solution was added to prepare a microalga suspension solution for culture.
After stirring the above solution, 8 mL was added per well of a 6-well plate. Two wells were used for one experimental condition.

  Next, the 6-well plate containing the microalgae suspension is transferred to plant bioshelf tissue culture, the 6-well plate is placed in a vacuum desiccator, and cultured for 7 days under light irradiation with a 4000 lux fluorescent lamp. went. In addition, room temperature is set to 23 degreeC and culture | cultivation is stationary culture. The light irradiation was turned on and off at 12-hour intervals.

Whether or not microalgae could be cultured on the liquid surface was judged by microscopic observation (magnification 4 times).
In FIG. 32, when + is almost no microalgae on the liquid surface, ++ is slightly microalgae on the liquid surface, ++ is when a considerable number of microalgae are present, +++ is the liquid surface This is the case when a clear biofilm is formed on the top. As a representative example of ++, FIG. 33 shows an example of the AVFF004 strain, FIG. 34 shows an example of the NIES-2249 strain as a representative example of ++, and FIG. 35 shows an example of the AVFF007 strain as a representative example of ++++.
The density per unit area of microalgae on the liquid surface was measured by directly counting micrographs. The results are shown below together with the evaluation of + to +++ described above.
ASFF001 stock 630,000 / cm ² (+++)
AVFF004 strain 1400 / cm ² (++)
AVFF007 stock 1 million pieces / cm ² (++++)
NIES-2249 500,000 pieces / cm ² (+++)

  The NIES-2249 strain is Chlorococcum etinozygotum Starr purchased from the National Institute for Environmental Studies. AS and AV series were collected from fresh water in Shizuoka Prefecture and Kyoto Prefecture and purified.

  In the present invention, microalgae determined to be ++ and +++ devise culture conditions based on the knowledge described in the present application, and by forming suitable culture conditions, a clear biofilm is formed on the liquid surface as in +++ It is considered possible.

[Example 14]
<Effect of light intensity on liquid surface suspension culture>
Microalgae were recovered by transferring the AVFF007 strain that had been pre-cultured using the CSiFF03 medium shown in FIG. 29 on a 6-well plate using the first substrate. The total amount is added to a 5 mL homogenizing tube containing CSiFF03 medium, and as a result of high-speed shaking treatment and turbidity measurement using a spectrophotometer, the algal body concentration is 19816 × 10 ⁴ pieces. found.
In a container in which 52 mL of CSiFF04 medium shown in FIG. 15 was previously added, 131 μL of the dispersion solution of the AVFF007 strain was added and stirred well to prepare a solution of 50 × 10 ⁴ cells / mL.
Ten cases were prepared by putting 5 mL each of this solution in PS Case No. 23 (As One Co., Ltd., 4-5605-04, outer size 30 × 30 mm).
Using Lumina Ace (As One Co., Ltd., 1-7373-01, halogen lamp) as a light source, light of 2600 lux to 90800 lux was irradiated with ON / OFF control at 12-hour intervals, and after 14 days, the second Using the substrate, the film-like structure on the liquid surface was recovered. Other experimental conditions are a temperature of 23 ° C., static culture, a CO ₂ concentration of atmospheric concentration, and a liquid medium water depth of 0.8 cm. In addition, two sample containers were prepared for one light amount, and the light amount was made the same as much as possible by adjusting the light amount setting of the Lumina Ace main body and the distance between the light source and the sample.

  Evaluation was performed by measuring dry weight. The microalgae obtained using the second substrate, that is, the nylon film, was transferred in its entirety onto a cover glass that had been weighed in advance using a cell scraper, and heated and dried at 120 ° C. for 30 minutes. The dry weight was calculated from the weight difference. The average value of two samples was used as the dry weight.

  The results are shown in FIG. In the culture of microalgae under high light intensity such as 100,000 lux, which corresponds to the amount of light under the clear sky of sunlight, a decrease in the growth rate generally considered to be due to light damage is seen, and depending on the type of microalgae, In some cases, it may not grow. As for the growth rate of the AVFF007 strain, 22900 lux is the maximum growth rate, and 90800 lux can secure about half the growth rate with respect to the maximum growth rate and can be cultured under high light intensity. Indicated.

[Example 15]
[Static culture and shaking culture]
A suspension with an algal body concentration of 1 × 10 ⁴ cells / mL was prepared in the same manner as in Example 12. In addition, since the count number became 3350 × 10 ⁴ pieces / mL, in order to prepare 170 mL of the microalgae dispersion solution having a concentration of 10 × 10 ⁴ pieces / mL, 507.5 μL of the microalgae dispersion solution was used.

20 mL of the microalgae dispersion solution having a concentration of 10 × 10 ⁴ / mL prepared above was placed in four 100 mL glass Erlenmeyer flasks. Note that two Erlenmeyer flasks were used for one experimental condition. The Erlenmeyer flask was stoppered with silicosene (As One Co., Ltd.).

When performing static culture, the Erlenmeyer flask containing the microalgae suspension was moved to plant bioshelf tissue culture and cultured under light irradiation with a 4000 lux fluorescent lamp. The room temperature was set to 23 ° C. The light is continuous light.
When shaking culture is performed, the Erlenmeyer flask containing the microalgae suspension is moved to a shaking culture machine (RGS-20RL, Sanki Seiki Co., Ltd.), and shaken at 100 rpm under light irradiation with a 4000 lux fluorescent lamp. The culture was performed at a rate. The temperature in the culture vessel was set to 23 ° C. The light is continuous light.

The culture period is 1 week in all cases.
The recovery of microalgae on the liquid surface was performed in the same manner as in Example 4. The microalgae in the liquid were collected by collecting the medium around the liquid surface and the bottom using a pipette after collecting the microalgae on the liquid surface. These counts were performed in the same manner as in Example 1. For collecting and counting the microalgae on the bottom surface, the microalgae on the liquid surface were collected, the medium was removed, a new medium was added and dispersed, and then counting was performed in the same manner as in Example 1. However, a polyethylene film was used instead of parafilm.
The results are shown in FIG. The number of algal bodies on the liquid surface was 788 × 10 ⁴ cells / mL for stationary culture and 127 × 10 ⁴ cells / mL for shaking culture, and the stationary culture was significantly larger. Therefore, in the present invention, it is preferable to perform static culture, but it is possible to perform shaking culture because the algal biofilm is formed on the liquid surface even in shaking culture. Note that the biofilm on the liquid surface can be clearly seen even in the shaking culture of this experiment. In the case of shaking culture, the number of microalgae in the liquid and on the bottom surface is larger than that in stationary culture. The ratio of the number of microalgae in the liquid and on the bottom surface is considered to change depending on the time from the stop of shaking until the sample is collected for counting.
Moreover, in static culture, it is thought that the fact that the number of algal bodies has increased significantly compared to the previous results is due to the use of atmospheric CO ₂ by silicosene and the material of the culture vessel.
As is clear from FIG. 37, when static culture was performed, most of the microalgae were present on the liquid surface and the bottom surface.
It is known that there is a considerable amount of aquatic growth even at a depth of about 3 m from the surface of the water, and it is clear that it is different from the growth of microalgae in the stationary culture of the present invention.

  The method for culturing microalgae according to the present invention can form a biofilm on the liquid surface, and can significantly reduce the recovery cost compared to the recovery of conventional microalgae. Moreover, the biofilm formed on the liquid surface can be recovered at a high recovery rate, and the water content is low. Furthermore, oil can be obtained from the collected biofilm, and the biofilm can be expected as biomass aimed at preventing global warming.

Claims (15)

A method for culturing microalgae, comprising culturing microalgae capable of forming a biofilm on a liquid surface so as to form a biofilm on the liquid surface of a liquid medium,
The microalgae is Botriococcus sudeticus AVFF007 strain (Accession No. FERM BP-11420),
The liquid medium contains calcium;
The culture temperature is 20 ° C. or higher and lower than 40 ° C.,
Under light irradiation of 2600 to 90800 lux,
A method for culturing microalgae, comprising stationary culture.   The method for culturing microalgae according to claim 1, wherein the calcium concentration in the liquid medium is 0.3 mM or more.   The method for culturing microalgae according to claim 1 or 2, wherein the concentration of carbon dioxide in the gas phase is not less than 20% by volume but not less than the concentration of carbon dioxide in the atmosphere.   The method for culturing microalgae according to any one of claims 1 to 3, wherein the pH of the liquid medium immediately after the start of culturing is in the range of 2 to 11.   The method for culturing microalgae according to any one of claims 1 to 4, wherein the liquid medium has a water depth of 0.4 cm or more.   The method for culturing microalgae according to claim 5, wherein the water depth of the liquid medium is 2.0 cm to 1 m.   The method for culturing microalgae according to any one of claims 1 to 6, wherein the water content of the biofilm is 95% by mass or less.   A biofilm formed on a liquid surface by the method for culturing microalgae according to any one of claims 1 to 7.   The manufacturing method of oil which obtains oil from the biofilm of Claim 8.   A biofilm recovery method, wherein the biofilm formed on the liquid surface according to claim 8 is collected by being deposited on a substrate.   A biofilm recovery method, wherein the biofilm formed on the liquid surface according to claim 8 is transferred to a substrate and recovered. The biofilm recovery method according to claim 10 or 11 , wherein 70% or more of the biofilm formed on the liquid surface is recovered. The biofilm recovery method according to claim 12 , wherein 80% or more of the biofilm formed on the liquid surface is recovered. The biofilm recovery method according to claim 12 or 13 , wherein 90% or more of the biofilm formed on the liquid surface is recovered. It is used as fuel biofilm recovered by the recovery process of bio film according to any one of claims 10-14, production process of the biomass fuel.