WO2017169713A1

WO2017169713A1 - Method for culturing microalga and method for producing algal biomass

Info

Publication number: WO2017169713A1
Application number: PCT/JP2017/010090
Authority: WO
Inventors: 沙織松山; 若田　裕一
Original assignee: 富士フイルム株式会社
Priority date: 2016-03-29
Filing date: 2017-03-14
Publication date: 2017-10-05
Also published as: JPWO2017169713A1

Abstract

The present invention addresses the problem of conducting more efficient culture by optimizing medium components. A method for culturing a microalga capable of producing a useful substance, said method comprising a step for culturing the microalga in a culture container using a culture medium, wherein the molar concentration ratio of nitrate ion to phosphate ion (N/P ratio) is 0.7-4.0 and the nitrate ion content is not less than 0.2 mol per m² of the culture area, to thereby allow the microalga to form a biofilm on the liquid surface of the culture medium.

Description

Method for culturing microalgae and method for producing algal biomass

The present invention relates to a method for culturing useful algae-producing microalgae.

Production of useful substances such as oils, fats, lipids, and fuels by microalgae is expected from the viewpoints of reducing the amount of CO ₂ generated from petrochemical fuels and the associated prevention of global warming. There is a problem that the cost is high compared with other petrochemical fuels. One of the causes of the high cost is that there is no efficient method for collecting microalgae. Specifically, since microalgae usually grow while floating in the liquid, in order to use the microalgae as biomass, a very dilute concentration of microalgae must be recovered from a large amount of liquid. In addition, the growth of microalgae requires light energy, and there is a limit to the culture density of microalgae in order to ensure sufficient light irradiation for microalgae present in the liquid. As a result, in order to collect microalgae in the liquid to obtain useful substances, it is necessary to filter a large amount of water, but the size of microalgae is generally small and the filtration operation itself 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 using microalgae as food for a larger organism, etc. However, none of these methods led to a fundamental solution.

In order to collect microalgae simply and at low cost, it has been proposed to form a biofilm by floating culture of microalgae having productivity of useful substances on the liquid surface (for example, Patent Document 1). According to this culturing method, in the stage of collecting microalgae, a biofilm composed of aggregates of microalgae floats on the liquid surface, and the biofilm is targeted for recovery. Is easy to separate. In addition, it is not necessary to collect microalgae dispersed in a large amount of medium as in the past, and since the water content of the biofilm on the liquid surface is relatively low, it is not necessary to handle a large amount of liquid, and it is also collected. The cost of drying items can also be reduced.

On the other hand, in order to efficiently produce useful substances, studies on culture conditions have been made. For example, Patent Document 2 discloses a first step of aerobically cultivating microalgae Euglena, a second step of further culturing a medium in which the microalgae Euglena is cultured in a nitrogen-starved state, and anaerobic cells. A method for producing a wax ester-rich Euglena comprising a third step held below is proposed. In this method, a nitrogen starvation condition is created by replacing the medium with a nitrogen-deficient medium.

Patent Document 3 is a method for culturing microalgae that is useful substance-productive, comprising culturing microalgae in a culture medium in a culture vessel and forming a biofilm on the liquid surface of the culture medium; and A method for culturing microalgae is proposed, which comprises a step of changing the concentration of at least one component and increasing the useful substance produced by the microalgae by changing the concentration of the component. In this method, the step of changing the concentration of at least one component contained in the medium is preferably by reducing the concentration of the component containing nitrogen or phosphorus.

JP 2013-226063 A JP 2012-23977 A JP2015-192647A

The medium used for culturing microalgae usually contains nitrate ions (nitrogen components), but the oil content in microalgae can be improved by reducing the amount of nitrate ions. However, even though the reduction of nitrate ions can improve the oil content, algal body productivity is reduced. Therefore, the improvement of oil productivity (that is, algal body productivity x oil content) is not immediate. There is a problem that it is not connected.

Further, the method of Patent Document 2 includes switching from an aerobic condition to an anaerobic condition, and in addition to the complexity of the original culturing process, the method is further replaced with a nitrogen-deficient medium during the culturing. Requires operation. And even if it goes through such a process and operation, the improvement factor of oil productivity is less than 1.2 times.

Furthermore, in the method of Patent Document 3, in order to carry out the step of changing the concentration of at least one component contained in the culture medium, while taking care not to affect the algal bodies that are being produced during the culture, A special operation of replacing at least a part or adding a low nutrient medium is required. Even if such a special operation is carried out, especially the concentration of the component containing nitrogen is reduced, the improvement factor of the oil productivity is 1.2 times or less.

It is an object of the present invention to provide a method for culturing microalgae, and a method for producing algal biomass using the same, in which higher oil productivity can be obtained than in the above-described prior art methods or the operation is simpler. To do.

The present inventors have intensively studied liquid surface suspension culture of microalgae. And now, focusing on not only nitrate ions but also phosphate ions among the medium components, the molar concentration ratio (N / P ratio) of nitrate ions to phosphate ions is 0.7 to 4.0, and Since the amount of nitrate ions per culture area is 0.2 mol / m ² or more, high useful substance productivity is maintained without the need for complicated steps such as medium replacement and addition during culture. A possible medium composition was found. By using the above medium components. It has also been found that oil productivity can be improved at a higher magnification than the prior art that focuses only on the reduction of nitrate ions without requiring special operations. Based on these findings, the present invention has been completed.

The present invention provides the following.
[1] A method for culturing microalgae that is useful substance productivity,
Microalgae in a medium in which the molar concentration ratio of nitrate ion to phosphate ion (N / P ratio) is 0.7 to 4.0 and the amount of nitrate ion per culture area is 0.2 mol / m ² or more. A method for culturing microalgae, comprising a step of cultivating and forming a biofilm on a liquid surface of a medium.
[2] The culture according to [1], wherein the N / P ratio is 1.0 to 4.0 and the nitrate ion amount per culture area is 0.2 mol / m ² to 1.5 mol / m ^2. Method.
[3] The culture method according to [1] or [2], wherein the culture is performed by static culture.
[4] The culture method according to any one of [1] to [3], wherein the biofilm has a three-dimensional structure.
[5] The culture method according to any one of [1] to [4], wherein the medium is not exchanged.
[6] The culture method according to any one of [1] to [5], wherein the microalgae are green algae.
[7] The microalgae is Botryococcus sp. Chlamydomonas sp. , Chlorococcum sp, Chlamydomonad sp. Tetracystis sp. Characium sp. Or Protosiphon sp. The culture method according to any one of [1] to [6], which belongs to the above.
[8] The microalgae is Botryococcus sudueticus FERM BP-11420, or Chlorococcus sp. The culture method according to any one of [1] to [7], which belongs to the same species as FERM BP-22262.
[9] A culture step including the culture method according to any one of [1] to [8]; and a step of recovering the formed biofilm;
A method for producing algal biomass.
[10] The production method according to [9], wherein the algal biomass is oil.

According to the present invention, it is possible to provide a method for culturing microalgae, which can provide high oil productivity or make operation simpler, and a method for producing algal biomass using the same.

It is a schematic diagram which shows an example of the cultivation method of a micro algae. Graph plotting _{^{^{- (mol / m 2 NO 3}}} ) molar ratio of nitric acid ions and phosphoric acid ions to (N / P ratio), nitrate ion per culture area in Examples and Comparative Examples. The oil productivity for each comparison object is as follows: white rhombus: 1.4 times or more, white circle: 1.3 times or more and less than 1.4 times, white triangle: 1.2 times or more and less than 1.3 times, black triangle: 1. 1 times or more and less than 1.2 times, X mark: less than 1.1 times

Hereinafter, preferred embodiments of the method for culturing microalgae according to the present invention will be described in detail. The numerical range expressed using “to” means a range including numerical values described before and after “to” as the lower limit value and the upper limit value.

[Culture method]
The basic culture method of the present invention is shown in FIG. In addition, since this schematic diagram is for demonstrating this invention, there exists the part simplified and described.
(culture)
As shown in FIG. 1 (a), 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 microalgae are cultured for a while, a biofilm composed of microalgae is formed on the liquid surface as shown in FIG. Normally, as shown in FIG. 1 (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 shaking culture is performed, as compared with the case of performing stationary culture, although depending on the strength of shaking conditions, as an example, liquid surface floating algae is about 1/6 of stationary culture, Algae body is about 3.5 times that of stationary culture, alga body on the bottom surface is about 1.4 times that of stationary culture, and by shaking culture, the proportion of alga bodies existing on the liquid surface is reduced, The percentage of algal bodies present in the middle and bottom is increased.

(Recovery)
As shown in FIG. 1D, the first substrate is brought into contact with the biofilm formed on the liquid surface, and the biofilm is adhered to the surface of the first substrate (( e)) can be recovered (hereinafter also referred to as “collection by transfer”). In FIG. 1, the substrate is brought into 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 one 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.
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 liquid level of the culture medium in the culture vessel. However, a substrate that covers the entire liquid level of the culture medium in the culture vessel may be used as described above. You may use the 1st board | substrate which can cover only a part of medium culture liquid surface.
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 preferable that the transfer material can be cut with scissors or the like, if necessary, so as to be smaller than the liquid surface area of the culture vessel. For example, when the culture vessel is a 6-hole plate, it is preferable to cut the material into a circular shape having a diameter of about 3.5 cm. The cut transfer material is preferably used as the first substrate after washing to remove dust on the surface. When the collected biofilm is used for the next culture, it is preferable that the transfer material is further immersed in disinfecting ethanol and the surface is dried to be used as the first substrate.

Next, the first substrate is gently inserted so that the first substrate is parallel to or close to the liquid surface formed by the culture vessel, and the microalgae on the liquid surface are attached to the first substrate. When inserting, if the first substrate is inserted slightly obliquely with respect to the liquid surface and finally made parallel to the liquid surface, many liquid surface biofilms can be transferred with a small number of transfer times. It is preferable because it can be recovered. By gently pulling up the first substrate to which the biofilm on the liquid surface is attached, the biofilm can be transferred to the first substrate from the liquid surface of the culture vessel. 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 a plurality of times.

Depending on the state of culture, the microalgae 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. 1 (f), it is also possible to collect the biofilm on the liquid surface so as to be collected using the second substrate (hereinafter also referred to as “recovery by deposition”). 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. In FIG. 1, only one second substrate is shown, but 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. 1 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.
The collection by the second substrate corresponds to the process from (f) to (g) in FIG. In addition to the slide glass, other materials 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. Thereby, while moving the second substrate, unnecessary contact with the inner wall of the culture vessel can be avoided, and the microalgae biofilm on the liquid surface is separated from the culture vessel and the second substrate. This is because a recovery leakage due to passing through the gap between the two is less likely to occur.

Depending on the culture state, 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.

(H) of FIG. 1 is a state after the biofilm on the liquid surface is collected. 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.

In the present invention, the culture method for culturing microalgae on the liquid surface as shown in FIG. 1 (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 (c) of FIG. 1 is performed in a stationary state, which is called liquid surface suspension culture by stationary culture.

(Desorption)
In the schematic diagram of FIG. 1, the microalgae biofilm is detached after the substrate is taken out of the culture vessel. However, it may be detached in the culture vessel.
As a method for desorbing the microalgae biofilm on the substrate, any method can be used as long as the microalgae can be peeled off from the substrate. After processing or closing the lid of the container containing the substrate, the microalgal biofilm can be peeled off the substrate by shaking vigorously, performing high-speed shaking treatment, or using something like a cell scraper it can. Among these, a method of stripping the microalgal biofilm from the substrate using a jig using a material that does not damage the substrate, such as a cell scraper, is preferable. Furthermore, the microalgal biofilm can be peeled off the substrate simply by tilting the substrate. Since this method is simple, it is the most preferable method. The substrate may be reused any number of times.

[Medium (liquid medium)]
The medium (liquid medium) that can be used in the present invention is capable of cultivating microalgae, and has a molar concentration ratio of nitrate ion to phosphate ion (N / P ratio) of 0.7 to 4.0 as described later. There is no particular limitation as long as the amount of nitrate ions per culture area in the culture vessel is 0.2 mol / m ² or more. The medium used in the present invention can be prepared by increasing or decreasing predetermined components in a conventional medium. Conventional 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 a medium for culturing the AVFF007 strain, a C medium, a CSi medium, a CHU medium, and a mixture of these mediums are preferable. The medium is preferably selected according to the type of microalgae to be cultured.

The medium may or may not be sterilized by ultraviolet light, autoclaving, or filter sterilization.

As the medium, different media may be used in the pre-culture process and the main culture process. Moreover, you may change to a different culture medium in the middle of a culture | cultivation process.

(Molar concentration ratio of nitrate ion to phosphate ion (N / P ratio))
In the present invention, a culture medium is used in which the molar concentration ratio of nitrate ions to phosphate ions is set in an effective range from the viewpoint of improving the productivity of useful substances. The N / P ratio is determined by the formula: (molar concentration of nitrate ions in the medium) / (molar concentration of phosphate ions in the medium). The nitrate ion refers to a component represented by NO ₃ ⁻ contained in the medium. Nitrate ions are derived from potassium nitrate, sodium nitrate, calcium nitrate and the like among the medium components. The phosphoric acid ion, refers to H ₂ PO ₄ over, HPO ₄ ^2-and PO ₄ ^3- represented by the components contained in the medium. The number of moles of phosphate ions refers to the total number of moles of these ions. Among the medium components, phosphate ions are dipotassium hydrogen phosphate (K ₂ HPO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ), potassium dihydrogen phosphate (H ₂ KPO ₄ ), and dihydrogen phosphate. Derived from sodium (H ₂ NaPO ₄ ) and the like.

The N / P ratio is preferably 0.7 or more. This is because the productivity improvement rate of the useful substance can be 1.1 or more if it is at least this value. The N / P ratio is more preferably 1.0 or more. This is because the productivity improvement rate of the useful substance can be 1.2 or more if the value is equal to or greater than this value. From the viewpoint of further increasing the productivity improvement rate of useful substances, the N / P ratio is more preferably 1.5 or more. The upper limit value of the N / P ratio is preferably 4.0 or less regardless of the lower limit value of the N / P ratio. If this value is exceeded, the algal bodies will grow and the algal body productivity will be improved, but the content of useful substances per alga body will be reduced and the productivity of useful substances will be inferior.

(Nitrate ion amount per culture area)
In the present invention, a medium is used in which not only the N / P ratio but also the nitrate ion amount (mol / m ² ) per culture area is set in an effective range from the viewpoint of improving the productivity of useful substances. The amount of nitrate ions per culture area is determined by the formula: (mol of nitrate ions in the medium) / (culture area). The culture area is an area of the liquid surface in the culture system, and is one of the important parameters in the culture of the present invention in which a biofilm is formed on the liquid surface.

The amount of nitrate ions per culture area is preferably 0.2 mol / m ² or more. This is because the productivity improvement rate of the useful substance can be 1.1 or more if it is at least this value. The nitrate ion amount per culture area is more preferably 0.26 mol / m ² or more. This is because the productivity improvement rate of useful substances can be set to 1.2 or more if the value is equal to or greater than this value. From the viewpoint of further increasing the productivity improvement rate of useful substances, the nitrate ion amount per culture area is more preferably 0.28 mol / m ² or more. Upper limit of nitrate ion per culture area is preferably the lower limit of nitrate ion per culture area is a 1.5 mol / m ² even less in each case, 1.2 mol / m ² or less It is more preferable that If this value is exceeded, the growth of algal bodies will progress and algal body productivity will improve, but the content of useful substances per alga will decrease and the productivity of useful substances will be poor. .

(Other ingredients)
The culture medium may contain a sugar that can be assimilated by microalgae as a carbon source. By using a medium containing sugar, it may be possible to suitably improve the growth rate as compared with the case where light and carbon dioxide are used. Also, the oil content tends to be high.
The sugar that can be assimilated by microalgae that can be used in the present invention includes at least one of monosaccharide, disaccharide, trisaccharide, and polysaccharide. Any known monosaccharide can be used, but galactose, mannose, talose, ribose, xylose, arabinose, erythrose, threose, glyceraldehyde, fructose, xylulose, erythrulose, and the like can be used. Any known disaccharide can be used, but trehalose, cordobiose, nigerose, maltose, isomaltose and the like can be used. In addition, any of tricarbon sugar, tetracarbon sugar, pentose sugar, hexose sugar and heptose sugar can be used. As the polysaccharide, starch, amylose, glycohegen, cellulose and the like can be used, and as the oligosaccharide, galactooligosaccharide, deoxyribose, glucuronic acid, glucosamine, glycerin, xylitol and the like can be used.
The concentration of sugar in the medium is preferably 0.1 μg / mL or more, more preferably 0.1 mg / mL or more, and most preferably 1 mg / mL or more. It is preferable for it to be 0.1 μg / mL or more because the growth rate of microalgae can be suitably improved. Moreover, although there is no upper limit in particular, it is preferably not more than solubility, more preferably not more than half of solubility, and still more preferably 1/10 concentration of solubility. More specifically, when glucose is used as the sugar, it can be 30 mg / mL or less, preferably 10 mg / mL or less, and more preferably 5 mg / mL or less. The sugar concentration is a concentration (initial concentration) immediately before the start of culture, and the concentration of sugar during the culture often changes continuously.
As the sugar, a single kind of sugar may be used, or two or more kinds of sugars may be used.

[Change medium]
In this invention, culture | cultivation operation becomes simpler by prescribing | regulating the N / P ratio in a culture medium, and the nitrate ion amount per culture | cultivation area. The simplification of the culturing operation includes the fact that the medium need not be changed during the culturing. Not performing medium exchange includes not replacing all or part of the medium, not adding or removing any selected from the group consisting of medium, medium components and water. Usually, medium exchange is performed while observing and measuring the growth state of algal bodies that are being produced during culture, and taking into account the growth medium that does not affect the algal bodies that are being produced. Although necessary, according to the present invention, there is a merit that cannot be achieved by the prior art that an operation requiring such consideration is not required. Further, according to the present invention, switching from an aerobic condition to an anaerobic condition is not required.

[Productivity of useful substances]
In the present invention, the productivity of useful substances can be improved by defining the N / P ratio in the medium and the amount of nitrate ions per culture area. The productivity (g / m ² · day) of the useful substance is determined by multiplying the previously obtained algal body productivity (g / m ² · day) by the content of the useful substance per algal body weight. The improvement rate of the useful substance productivity is calculated as the ratio of the useful substance productivity in the target culture system to the productivity of the useful substance in the comparative culture system. The comparative culture system is a culture system in which the amount of nitrate ions and phosphate ions in the medium is different from the target culture system, but the other culture conditions are the same. Other culture conditions include the amount of algal bodies at the start of culture (sometimes referred to as initial algal body amount), the number of culture days, the culture area, the depth of the medium (water depth), the amount of light, and the like.

In the present invention, by defining the N / P ratio in the culture medium and the amount of nitrate ions per culture area, the productivity improvement rate of useful substances is comparable to that of the prior art without replacing the culture medium. That is, it can be at least 1.1 times. In the preferable aspect of this invention, the improvement rate of productivity of a useful substance can be 1.2 times or more. Such a high improvement rate could not be achieved under the conventional culture conditions focusing only on nitrate ions (nitrogen components). Further, in a more preferred embodiment of the present invention, the productivity improvement rate of useful substances can be 1.3 times or more, and in a more preferred embodiment, the productivity improvement rate of useful materials is 1.4 times. This can be done.

[Microalgae]
The microalgae refers to microalgae whose individual presence cannot be identified with the human eye. The microalgae of the present invention is not particularly limited as long as it is useful substance productivity and has the ability to form a biofilm on the liquid surface.

The microalgae used in the present invention are useful substance productivity, in particular, intermediates and final products of pharmaceuticals, cosmetics, health foods, raw materials used in synthetic chemistry, oils such as hydrocarbon compounds, triglycerides and fatty acid compounds. Preferred are microalgae that generate a gas, gas such as hydrogen. Microalgae also have good culturing on the liquid surface and recovery from the liquid surface, have a high growth rate, can have a high content of useful substances, have at least little odor during culturing, It is preferable to satisfy any one of the cases where generation of toxic substances has not been confirmed.

In the present invention, microalgae capable of forming a biofilm on the liquid surface is used. Such microalgae are, for example, indigo plant gate, gray plant gate, red plant gate, green plant gate, cryptophyte gate, haptophyte gate, unequal hair plant gate, dinoflagellate plant gate, Euglena plant gate, chlora It may be a microalga belonging to Lacción planta. Among these, those belonging to the green plant gate are preferable, and green algae are more preferable.

From the viewpoint of high productivity of biomass, any of the genus Haematococcus sp., Chlamydomonas sp., Chlorococcum sp., And Botriococcus sp. Those belonging to are preferred. More preferred examples are those belonging to the genus Botriococcus sudeticus or Chlorococcum. Specific examples include Botriococcus Sudetics AVFF007 strain (hereinafter abbreviated as AVFF007 strain) and FFG039 strain.

[AVFF007 strain]
The AVFF007 strain, under the accession number FERM BP-11420, dated September 28, 2011, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st East, 1st Street, Tsukuba, Ibaraki, Japan, 1st Central 6) is deposited internationally by Fujifilm Corporation (2-30-30 Nishiazabu, Minato-ku, Tokyo, Japan) under the Butabest Convention. The National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center has been in operation since April 1, 2012. The National Institute of Technology and Evaluation, Patent Biological Depositary Center (Kisarazu City, Chiba Prefecture, Japan) Kazusa Kamashika 2-5-8 Room 120).

AVFF007 strain is a novel strain of freshwater microalgae isolated by the present inventors from a freshwater pond in Kyoto, Japan. AVFF007 strain was analyzed by BLAST based on the data of the National Center for Biotechnology Information (NCBI) of a part of the base sequence of the 18S rRNA gene. As a result, Botryococcus sp. It was identified as a microalgae closely related to the UTEX2629 (Botryococcus sudeticus) strain (1109 bases on the AVFF007 strain side were the same among 1118 bases on the UTEX2629 strain side). The AVFF007 strain is Characiopodium sp. Mary 9/21 is a closely related microalgae with T-3w and may be changed to the genus Characiopodium in the future. In this case, the name of the AVFF007 strain is changed, and the name of the AVFF007 strain is also changed when the name is changed to other than the genus Characiopodium.

In the present invention, a strain having the same taxonomic properties as the AVFF007 strain can be used. The taxonomic properties of AVFF007 strain are shown below.

Taxonomic properties of AVFF007 strain Morphological properties It has a green circle shape. It is free-floating and can grow on the liquid and bottom surfaces. The size is 4-30 μm (the one on the liquid surface is relatively large and the one on the bottom surface is relatively small). It grows on the liquid surface and forms a film-like structure. Along with the growth, bubbles are generated on the liquid surface, and they overlap to form a three-dimensional structure on the liquid surface. It also produces oil.
2. Culture characteristics (culture method)
(1) Medium: CSiFF04 (improved CSi medium) Adjust to pH 6.0 with NaOH or HCl. The medium can be sterilized at 121 ° C. for 10 minutes. )
(2) Culture temperature: The preferred temperature is 23 ° C., and culture is possible at 37 ° C. or less.
(3) The culture period (approximately the period until reaching the stationary phase) is 2 weeks to 1 month depending on the amount of algal bodies used initially. Usually, it can be cultured at 1 × 10 ⁵ cells / mL.
(4) Culture method: Aerobic culture and stationary culture are suitable.
(5) Optical requirement: Necessary. Light intensity: 4000 to 15000 lux, light / dark cycle: light period 12 hours / dark period 12 hours. When subcultured, it can be cultured at 4000 lux.

The AVFF007 strain can be stored by subculture according to the above culture properties (culture method). Planting can be performed by collecting microalgae floating on the liquid surface, dispersing by pipetting, etc., and then dispersing in a new medium. In addition, although it is sinking in the bottom of a culture container immediately after subculture, it begins to form a biofilm on the liquid surface in about one week. Even if it is present on the liquid surface immediately after passage, it can grow. The planting interval is about one month. If it becomes yellowish, pass it on.

The strain having the same taxonomic characteristics as the AVFF007 strain is a microalgae, the 18S rRNA gene of which is at least 95.0% with the polynucleotide comprising the base sequence of SEQ ID NO: 1 of the aforementioned Patent Document 3, Preferably, those having a sequence identity of 98.0%, more preferably 99.0%, even more preferably 99.5%, most preferably 99.9% are included.

In the present invention, the term “sequence identity” for a base sequence means the percentage of the number of matched bases shared between the two sequences in the aligned region when the two sequences are aligned in an optimal manner. That is, it can be calculated by identity = (number of matched bases / total number of bases) × 100, and can be calculated using commercially available or publicly available algorithms. Search / analysis regarding the identity of base sequences can be performed by algorithms or programs well known to those skilled in the art. Those skilled in the art can appropriately set parameters when using a program, and the default parameters of each program may be used. Specific techniques for these analysis methods are also well known to those skilled in the art.

[FFG039 strain]
The microalgae FFG039 used in the examples of the present specification was collected by the present inventors in Nara Prefecture, Japan. Compared with AVFF007 strain, it has good growth and oil productivity. In addition, the biofilm structure is not easily broken and is easy to collect.

The FFG039 strain has the accession number FERM BP-22262 on February 6, 2014, the National Institute for Product Evaluation Technology Patent Biological Depositary Center (2-5 Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan) 8 Room 120) is deposited internationally by FUJIFILM Corporation (2-30-30 Nishiazabu, Minato-ku, Tokyo, Japan) under the Butabest Convention.

In the present invention, a strain having the same taxonomic characteristics as the FFG039 strain can be used. The taxonomic properties of the FFG039 strain are shown below.

Taxonomic properties of FFG039 strain Morphological properties It is circular. When static culture is performed, a film-like structure is formed on the liquid surface. Produce oil.
2. Culture characteristics (culture method)
(1) Medium: CSiFF04 medium or CSi modified medium (Ca (NO ₃ ) ₂ .4H ₂ O 150 mg / L, KNO ₃ 100 mg / L, K ₂ HPO ₄ 28.4 mg / L, KH ₂ PO ₄ 22 0.2 mg / L, MgSO ₄ .7H ₂ O 40 mg / L, FeCl ₃ .6H ₂ O 588 ug / L, MnCl ₂ .4H ₂ O 108 ug / L, ZnSO ₄ .7H ₂ O 66 ug / L, CoCl ₂ · 6H ₂ O 12 ug / L, Na ₂ MoO ₄ · 2H ₂ O 7.5 ug / L, Na ₂ EDTA · 2H ₂ O 3 mg / L, Vitamin B12 0.1 ug / L, Biotin 0. 1 ug / L, thiamine · HCl 10 ug / L, pH 7.0)
(2) Culture temperature: can be cultured at 15 to 25 ° C.
(3) Culture period: 2 to 4 weeks (4) Culture method: Stationary culture is suitable.
(5) Optical requirement: Necessary. Light intensity: 4000 to 15000 lux, light / dark cycle: light period 12 hours / dark period 12 hours.
Microalgae that have taxonomically identical properties to the FFG039 strain include Chlorococcum sp. The microalgae belonging to the genus include those whose 18S rRNA gene has at least 99.94% sequence identity with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 of Patent Document 3 described above.

The method for obtaining microalgae is not particularly limited and may be appropriately selected according to the purpose. Examples thereof include a method of collecting from the natural world, a method of using a commercially available product, a method of obtaining from a storage organization and a depository organization, and the like. can give. In the present invention, one type of microalgae or a plurality of types of microalgae may be used. Moreover, you may use the micro algae which passed through the purification process. A purification process is a process performed for the purpose of making microalgae into a single kind, and does not necessarily mean that only a single microalgae is made completely.

[Presence of microalgae]
In the present invention, microalgae that are useful substance productivity and can form a biofilm on the liquid surface, and when cultured in a medium in a culture vessel, the following (1) to (8) Microalgae having at least one characteristic selected from the group may be used. By using such microalgae, operations such as medium replacement, biofilm recovery, and resumption of culture become easier, and the implementation of the present invention at a lower cost can be expected.
(1) The sum of the algal bodies of microalgae present on the liquid surface and in the region from 1 cm to the liquid surface below the liquid surface and the algal bodies of the microalgae on the bottom surface of the culture vessel is other than that in the culture vessel 10 times or more, preferably 20 times or more, more preferably 30 times or more the amount of algal bodies present in the region. The other region in the culture container referred to here refers to the region on the liquid surface and in the vicinity of the liquid surface, that is, the region from 1 cm below the liquid surface to the liquid surface and the region excluding the bottom surface. Although microalgae may adhere to the side of the culture vessel and the surface of various structures installed in the culture vessel such as a sensor for monitoring culture, such microalgae may not be included in either area . The amount of algal bodies can be expressed as the weight of algal bodies per bottom area of the culture vessel.
(2) The specific gravity of the microalgae on the liquid surface is smaller than the specific gravity of the microalgae on the bottom surface of the culture vessel. The specific gravity of microalgae can be determined by a known method such as a concentration gradient method. The specific gravity of the microalgae on the liquid surface when the specific gravity of the microalgae on the bottom surface is 1, although depending on the type of microalgae, is, for example, 0.99 or less, preferably 0.98 or less, and more Preferably it is 0.96 or less. Although there is no restriction | limiting in particular in a lower limit, Even if an upper limit is any case, it is 0.75 or more, for example, Preferably it is 0.77 or more, More preferably, it is 0.79 or more.
(3) The specific gravity of microalgae on the liquid surface is greater than the specific gravity of water.
(4) The oil content of the microalgae on the liquid surface is higher than the oil content of the microalgae on the bottom surface. When the oil content of the microalgae on the bottom surface is 1, the oil content of the microalgae on the liquid surface is, for example, 1.1 or more, preferably 1.2 or more, more preferably 1. 3 or more. Although there is no restriction | limiting in particular in an upper limit, Even if a lower limit is any case, it is 3.0 or less, for example, Preferably it is 2.5 or less, More preferably, it is 2.0 or less.
(5) The size (diameter) of the microalgae on the liquid surface is larger than the size of the microalgae on the bottom surface. The size of the microalgae can be determined by a known method. When the size of the microalgae on the bottom surface is 1, the size of the microalgae on the liquid surface is, for example, 1.5 or more, preferably 1.8 or more, more preferably 2.0 or more. . Although there is no restriction | limiting in particular in an upper limit, Even if a lower limit is any case, it is 4.0 or less, for example, Preferably it is 3.5 or less, More preferably, it is 3.0 or less.
(6) The biofilm to be formed includes a film-like outer layer and an inner layer having a plurality of foam-like structures, and the outer layer is thicker than the inner layer. The thickness of the layer can be determined by a known method. When the thickness of the inner layer is 1, the thickness of the outer layer is, for example, 2.0 or more, preferably 3.0 or more, more preferably 5.0 or more. Although there is no restriction | limiting in particular in an upper limit, Even if a lower limit is any case, it is 18.0 or less, for example, Preferably it is 14.0 or less, More preferably, it is 10.0 or less. The biofilm formed may also be just the outer layer. Therefore, it is also one of the characteristics of the microalgae of the present invention that the formed biofilm has either a film-like outer layer or an inner layer having a plurality of foam-like structures. be able to.
(7) A part of the formed biofilm has a pleated structure in the medium.
(8) When the microalgae obtained by collecting and suspending the formed biofilm is seeded on the liquid surface of the medium, it can settle in the medium. Usually, the biofilm formed on the liquid surface can be floated on the liquid surface by carefully applying it onto the liquid surface without being subjected to a suspending treatment after the collection. However, the suspension treatment makes it difficult to float on the liquid surface, resulting in frequent sedimentation.
The microalgae capable of forming a biofilm on the liquid surface as used in the present invention, and at least selected from the group consisting of the above (1) to (8) when cultured in a medium in a culture vessel A microalgae having one characteristic can be distinguished from a group of other algae by at least one characteristic selected from the group consisting of the above (1) to (8), and the at least one characteristic is A collection of microalgae that can be kept and propagated. Regarding (3), (4), and (5) above, the average specific gravity, oil content, or size of the target microalgae can be determined and determined.

[Biofilm]
The biofilm generally seen is a thin film (substantially two-dimensional structure) formed by attaching microorganisms to a solid surface such as a rock, but the biofilm formed by microalgae in the present invention is a general biofilm. Unlike a film, it does not need to be attached to the surface of a solid, and can exist while maintaining a film-like structure on a fluid surface such as a liquid surface. Moreover, the film-like structure formed with microalgae may have a three-dimensional structure with a certain thickness.

More specifically, the biofilm formed from the microalgae in the present invention may have a gas barrier property such that gas generated during the culturing process (oxygen or the like generated as a result of photosynthesis) cannot permeate. Can be held inside as bubbles. The bubble size is on a mm scale and can be confirmed visually. In addition, the biofilm formed from the microalgae in the present invention has a certain degree of strength, can hold bubbles without being broken by the expectation generated as described above, and the microalgae are separated from each other with a weak water flow. It is hard to become. The area of the biofilm can be greater than the cm ² scale and is different from a mere colony.

In addition, the biofilm formed from microalgae in the present invention may form a wrinkled structure or a curtain-shaped structure as the culture proceeds. The formation of such a structure indicates that the biofilm grows while being bent by the growth of microalgae even after the biofilm is formed to fill the limited liquid surface of the culture vessel. .

[Static culture]
In the main culture step in the present invention, it is preferable to perform stationary culture. Static culture is a culture method in which the medium is not intentionally stirred or shaken during the culture.

[Liquid surface suspension culture]
The culture method for culturing microalgae on the liquid surface is called liquid surface floating culture. In addition, even when microalgae are simultaneously present on the bottom surface, side surface, other surface of the culture vessel, or in the culture medium, when the main purpose is culture on the liquid surface, it is called liquid surface floating culture. In addition, a lot of foam is present on the liquid surface along with the biofilm, and the position of the liquid surface may not always be clear, and the biofilm may be submerged slightly below the liquid surface due to its own weight. The term “on the liquid surface” includes such a case as well as a complete liquid surface. However, the culture method of culturing microalgae in the liquid, only one or both of the bottom surfaces of the culture vessel is not included in the liquid surface floating culture.

In addition, the liquid level in the present invention is typically the liquid level of a liquid medium described later, and is usually an interface between the liquid medium and air. Moreover, when water becomes a main component, it is a water surface. In addition, when liquid surface suspension culture is performed in the present invention, a phenomenon that a pleated structure enters a liquid from a biofilm on the liquid surface may be seen. In the present invention, the culture in such a situation is also included in the liquid surface suspension culture.

[Pre-culture process]
Before performing the culture of the present invention, a preculture step may be performed. The pre-culture process is a process of increasing the number of microalgae until the microalgae for storage are grown and main culture can be performed. The culture method of the pre-culture process can be selected by any known culture method. For example, a dispersion culture method, an adhesion culture method, a liquid surface floating culture developed by the present inventors, a culture method of the present invention, and the like can be performed. Moreover, in order to grow microalgae to a scale that allows main culture, pre-culture may be performed several times. In the pre-culture step, static culture may be performed according to the purpose, or non-static culture such as shaking culture may be performed.
In general, a culture vessel having a surface area of 1 cm ² to 1 m ² or less is used, and the culture can be performed both indoors and outdoors.

[Main culture process]
The main culturing step refers to a culturing step for the purpose of producing useful substances after performing the pre-culturing step. The main culturing step can be completed when a sufficient amount of biofilm is formed on the liquid surface. The main culturing step can be completed in, for example, several days to several weeks, more specifically, 5 days to 4 weeks. Further, the main culturing step may be performed a plurality of times.

In addition, the main culturing step is generally performed by using a culture vessel having a surface area of 100 cm ² or more (in the case of a larger scale outdoors, the culture vessel may be referred to as a culture vessel). It can be performed on a large scale and can be performed both indoors and outdoors.

[Seed algae]
The seed algae in the present invention refers to the microalgae used at the start of the preculture process or the main culture process, and refers to the microalgae that are the source of the culture of the microalgae in the preculture process or the main culture process. .
In addition, the culture can be started with the microalgae biofilm floating on the liquid surface or with the microalgae present on the bottom surface. In these cases, these microalgae should be used as seed algae. Can do. Furthermore, microalgae attached to the bottom surface, other places of the culture container, other jigs constituting the culture, and the like can also be used as seed algae.
Moreover, culture | cultivation can also be restarted using the micro algae which remain | survives on the liquid level as a seed algae after a collection | recovery process.

[Suspension treatment]
In the present invention, a microalgae sample subjected to suspension treatment may be used as a seed algae. This is because by performing the suspension treatment, the microalgae in the solution become uniform and the film thickness after the culture becomes uniform, and as a result, the amount of microalgae per culture area may increase. As the suspension treatment, any known method can be used, but weak treatment such as pipetting, shaking the microalgae solution in the container by hand, treatment with a stirrer chip or a stir bar, ultrasonic treatment, Examples include a strong treatment such as a high-speed shaking treatment, and a method using a substance such as an enzyme that decomposes an adhesive substance such as an intercellular matrix.

[Culture container]
As the shape of the culture vessel (culture tank) that can be used in the present invention, any known shape can be used as long as the medium can be retained. 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. Various known methods such as an open pond (open pond) type, a raceway type, and a tube type (J. Biotechnol., 92, 113, 2001) can be used. Shapes that can be used as culture vessels are described, for example, in 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, but it prevents diffusion of carbon dioxide outside the culture vessel when using a higher carbon dioxide concentration than in the atmosphere. Therefore, it is preferable to use a closed culture vessel. By using a closed type culture vessel, it is possible to minimize the contamination of microorganisms other than the culture purpose and dust, the suppression of the evaporation of the culture medium, and the influence of the wind on the biofilm structure. However, when commercial production is performed, culture in an open system is preferable from the viewpoint of low construction costs. Also, in order to achieve both reduction of these external influences and reduction of construction costs, at least the medium liquid is used for the purpose of protecting the biofilm formed on the medium liquid surface, although not completely closed, from external influences such as wind and rain. It is more preferable to provide a cover in the vertical direction of a part of the surface.

[substrate]
The substrate in the present invention is a solid material used in (d) and (f) of FIG. The shape of the substrate may be any shape such as film, plate, fiber, porous, convex, corrugated, but it is easy to transfer and easy to collect microalgae from the substrate. The film shape or the plate shape is preferable.
[Material]
The materials for the culture vessel and the 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, a metal, 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 or stainless steel can be used.

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

In addition, the materials of the culture vessel, the substrate, and the penetrating structure may be the same or different.

In the case of using a closed culture vessel, at least a part of the light receiving surface is preferably a material that transmits at least a part of the light, and more preferably a transparent material.

[carbon dioxide]
For the cultivation of many microalgae, it is necessary to supply carbon dioxide.

When dispersed culture is performed in the pre-culture process, carbon dioxide may be supplied to the medium by bubbling as in the conventional method. However, when liquid surface suspension culture is performed, carbon dioxide is It is preferable to supply from This is because when carbon dioxide is supplied into the medium by a method such as bubbling, the structure of the microalgae biofilm on the liquid surface is destroyed, resulting in spots of algal mass, and the efficiency of biofilm recovery onto the substrate during the recovery process This is because there is a possibility that the amount of recovered alga bodies may be reduced.

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

[Light source and light intensity]
As the light source that can be used in the present invention, any known 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, which is energy, an LED with good luminous efficiency, or a fluorescent lamp that can be used easily.

The amount of light is preferably 1000 lux or more and 300,000 lux or less, and more preferably 2000 lux or more and 150,000 lux or less. The most preferable amount of light is 10,000 lux or more and 100,000 lux or less. If the light intensity is 2000 lux or more, microalgae can be cultured at a sufficient rate, and if it is 150,000 lux or less, there is little adverse effect on the culture due to light damage.

The light may be either continuous irradiation or a method of repeating irradiation and non-irradiation at a certain time interval. However, according to the culture with sunlight outdoors, for example, the light is emitted at intervals of 12 hours within one day. It is preferable to turn on and off.

As long as the wavelength of light can be used for photosynthesis, any wavelength can be used, and there is no limitation. However, 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, and such an irradiation method can also be used in the present invention.
[Other culture conditions]
In the present invention, the pH of the liquid medium used in the pre-culture process and the main culture process (hereinafter, the liquid medium is also referred to as a medium) is preferably in the range of 1 to 13, preferably in the range of 3 to 11. More preferably, it is more preferably in the range of 5 to 9, and most preferably in the range of 6 to 8.

Further, since a suitable pH varies depending on the type of microalgae, it is preferable to select a pH corresponding to the type of microalgae. The pH of the liquid medium is the pH at the start of culture. Moreover, since the pH in the culture process may change with the culture, the pH may change in the culture process.

In the present invention, a substance having a buffering action for keeping the pH in the medium constant can be added to the medium. Thereby, the problem that the pH in the medium changes with the progress of the culture of microalgae can be suppressed, and the phenomenon that the pH changes due to the supply of carbon dioxide into the medium can be suppressed. 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 type of microalgae and the culture environment.

When performing dispersion culture, if the liquid medium has a deep water depth, there is a problem in that light does not reach and stirring efficiency deteriorates. However, in the case of liquid surface suspension culture, since microalgae grow on the liquid surface with high density, there is no need to supply light to the deep part of the culture vessel, and basically no agitation is performed. For this reason, the water depth can be reduced. Thereby, since the amount of water used is small and handling efficiency is improved, it is preferable to reduce the water depth. The water depth is preferably 0.4 cm or more, more preferably 1 cm to 10 m, further preferably 2 cm to 1 m, and most preferably 4 cm to 30 cm. When the water depth is 0.4 cm or more, a biofilm can be formed, and when the water depth is 10 m or less, handling is easy. When the water depth is 4 cm to 30 cm, the influence of water evaporation is minimal, and handling of a solution containing a medium and microalgae is easy.

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

The amount of microalgae used at the start of the culture is not particularly limited because the number of cells is one in the culture range and can be grown as long as time is spent. ³ or more, more preferably 1000 pieces / cm ³ or more, and further preferably 1 × 10 ⁴ pieces / cm ³ or more. Alternatively, the amount used can be defined by the weight of microalgae per unit area. In this case also possible proliferation if only over time is not particularly limited as well, preferably in view of productivity and at 0.05 g / m ² or more, more preferably 0.1 g / m ² Or more, more preferably 1 g / m ² or more.

The pre-culture period and the main culture period in the present invention can be selected according to the type of microalgae and are not particularly limited, but are preferably 1 day or more and 100 days or less, more preferably 3 days or more and 50 days or less. 7 days or more and 31 days or less are more preferable.

[Size and growth rate of microalgal biofilm formed on the liquid surface]
Preferably the size of the microalgae biofilm is 0.1 cm ² or more, more preferably 1 cm ² or more, more preferably 10 cm ² or more, and most preferably equal to the liquid surface area of the culture vessel. If it is 0.1 cm ² or more, the ratio of the amount of microalgae at the end of culture to the amount of microalgae at the start of culture can be increased in a short time.
A plurality of microalgal biofilms may exist in the culture region.
The thickness of the microalgal biofilm is preferably in the range of 1 μm to 10000 μm, more preferably in the range of 1 μm to 1000 μm, and most preferably in the range of 10 μm to 1000 μm. When the thickness is in the range of 10 μm to 1000 μm, the strength is high and a sufficient amount of biofilm can be harvested.

When the biofilm according to the present invention is a three-dimensional three-dimensional structure formed by rising in a bubble shape in a part or a plurality of parts of the film-like structure, the liquid film of the medium is used as a reference. The general height of the three-dimensional structure is preferably in the range of 0.01 mm to 100 mm, more preferably in the range of 0.1 mm to 20 mm, and most preferably in the range of 5 mm to 20 mm. preferable. When the thickness is in the range of 5 mm to 20 mm, the water content can be sufficiently lowered, and the height of the culture vessel can be kept low.
The microalgae according to the present invention preferably has a high growth rate on the liquid surface, and the growth rate in the logarithmic growth phase (that is, the average growth rate per day during the logarithmic growth phase) is 0 by dry weight. is preferably .1g / m ² / day or more, more preferably 0.5 g / m ² / day or more, more preferably 1 g / m ² / day or more, 3 g / m ² / day The above is most preferable. The growth rate of microalgae in the logarithmic growth phase is generally 10 to 30 g / m ² / day or less in terms of dry weight.

The dry algal body weight per unit area of the biofilm according to the present invention is preferably 0.01 g / m ² or more, more preferably 1 g / m ² or more, and 10 g / m ² or more. Is particularly preferred. Most preferably, it is 50 g / m ² or more. This is because it is expected that the amount of biomass such as oil obtained is larger when the dry alga body weight per unit area is larger. The dry alga body weight per unit area of the biofilm is usually 20 to 300 g / m ² .

Further, as the microalgae of the present invention, the microalgae capable of forming a biofilm having the above structure, the above-mentioned area, thickness, height, growth rate, and dry alga body weight per unit area on the liquid surface. It is preferable for the same reason as described above.

[Recovery]
The microalgae biofilm on the liquid surface can be collected in a state where the liquid surface in the culture vessel is partially covered with the biofilm, but the amount of microalgae is large. It is preferable to carry out after the liquid level in the culture vessel is entirely 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 is formed on the liquid surface. The three-dimensional structure is a structure that is seen when the film-like structure further grows. Compared with the two-dimensional film-like structure, the amount of microalgae that can be recovered is large, and the moisture content is high. It is preferable because it is lower. Further, only the biofilm on the liquid surface may be collected, or at least a part of the biofilm on the liquid surface and the microalgae on the bottom surface may be collected. This is because microalgae on the liquid surface and microalgae on the bottom surface can be used as biomass. However, in general, when oil is considered as a useful substance, the oil content of the microalgae on the liquid surface is higher than that of the microalgae on the bottom surface. Therefore, it is better to avoid collecting bottom algae as much as possible.

The above-described collection method preferably collects 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.

[Dry algae]
The dry alga body in the present invention is obtained by drying the collected microalgae obtained by the present invention.
As a method for drying the microalgae collection product, any known method can be used as long as it can reduce the moisture in the microalgae collection product, and is not particularly limited. For example, a method of drying the microalgae collected in the sun, a method of heating and drying the microalgae recovered, a method of freeze-drying (freeze drying) the microalgae recovered, a method of blowing dry air on the microalgae recovered, etc. It is done. Among these, freeze drying is preferable from the viewpoint of suppressing decomposition of components contained in the microalgae collection, and heat drying or sun drying is preferable from the viewpoint of efficient drying in a short time.

[Moisture content]
The water content in the present invention is obtained by dividing the weight of water contained in the recovered material by the weight of the recovered material and multiplying by 100. The water content of the microalgal biofilm in the present invention is preferably 99 to 60%, more preferably 95 to 80%, and most preferably 90 to 85%. However, this does not apply when culturing using a penetrating structure.
The water content when the microalgae are collected by culturing in a dispersed culture and using a centrifuge is generally about 90%, and the water content of the biofilm on the liquid surface obtained by the culture method of the present invention Is lower than that and is superior to the conventional method. Note that the water content of the three-dimensional structure is lower than that of the film-like structure. This is presumed to be caused by the fact that the three-dimensional structure is farther from the liquid surface, closer to the light source, and a certain degree of drying has progressed.

[Useful substances]
The useful substance in the present invention is a kind of biomass derived from microalgae, and is a general term for substances useful for industries obtained from biomass through an extraction process, a purification process, and the like. Such substances include final products, intermediates and raw materials such as pharmaceuticals, cosmetics and health foods, raw materials for chemical compounds, intermediates and final products, hydrocarbon compounds, oils, alcohol compounds, hydrogen and methane. Energy substitute substances such as enzymes, proteins, nucleic acids, sugars and lipid compounds such as DHA, astaxanthin and the like. The useful substance can be accumulated in the microalgae by the useful substance accumulation process.

[Biomass and oil]
Biomass in the present invention refers to organic resources derived from renewable organisms excluding fossil resources, and examples thereof include biological substances, foods, materials, fuels, resources, and the like. The algal biomass includes microalgae itself (may be in the form of a biofilm) and microalgae residue after collecting useful substances.
The oil in the present invention is a combustible fluid substance, which is a compound mainly composed of carbon and hydrogen, and in some cases, a substance containing oxygen atoms, nitrogen atoms, etc. is there. Oil is generally a mixture and is a substance that is extracted using a low polarity solvent such as hexane or acetone. The composition may be composed of a hydrocarbon compound, a fatty acid, a triglyceride or the like, or may be composed of a plurality of kinds of compositions selected from these. It can also be esterified and used as biodiesel.

The method for collecting useful substances and oil contained in the microalgae collection is not particularly limited as long as the effects of the present invention are not impaired.

In general oil extraction methods, the final recovered material is dried by heating to obtain dried alga bodies, followed by cell disruption and extraction of the oil using an organic solvent. The extracted oil is generally refined because it contains impurities such as chlorophyll. Purification includes silica gel column chromatography and distillation (for example, the distillation method described in JP-T 2010-539300). Such a method can also be used in the present invention.

In addition, there is a method in which microalgae are crushed by ultrasonic treatment, or microalgae are dissolved by protease, enzyme, or the like, and then the oil in the algal bodies is extracted using an organic solvent (for example, JP 2010-530741 A). Method). Such a method can also be used in the present invention.

In addition, 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.

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

[Example 1]
(First preculture)
As the first pre-culture, CSiFF04 medium (composition is shown in the table below) in PS Case No. 28 (As One Co., Ltd., 4-5605-05). In this example, hereinafter, it may be simply referred to as “medium”. ) 40 mL and Chlorococcum sp. Put a mixture with FFG039 strain (FERMBP-22262 strain) (1 mL of dispersion liquid with algal body concentration of 1.2 mg / mL), under irradiation of fluorescent light of 15000 lux (light irradiation ON-OFF every 12 hours), 23 ° C., Static culture was performed at a carbon dioxide concentration of 5%. The culture temperature was controlled using an air conditioner set at 23 ° C. Fourteen days later, the microalgae biofilm on the liquid surface was collected using a nylon film (thickness 1 mm) having the same length as the short diameter of PS Case 28.

(Second preculture)
A second preculture was performed in the same manner as the first preculture using the microalgae obtained in the first preculture. However, as the culture vessel, a plastic bat of 30 cm × 23 cm × 7 cm was used, and the second culture was performed by adding half of the algae recovered in the first culture to 2800 mL of the medium. After 14 days, the microalgae biofilm on the liquid surface was collected, transferred to a 500 ml plastic container containing 300 ml of CSiFF04 medium, shaken by the plastic container by hand, and then subjected to sonication for 5 minutes to obtain a seed algae dispersion. Obtained. 5 ml of this seed algae dispersion was filtered and dried (130 ° C., 1 hour) to determine the alga body weight, and the seed algae concentration (mg / ml) in the dispersion was calculated therefrom.

(Main culture)
Medium (b) (the composition is shown in the table below) in which the NO ₃ ion concentration of 2.8 L of CSiFF04 medium was halved was placed in the plastic vat so that the water depth was 5 cm. The seed algae dispersion prepared by the second preculture was added so that the amount of algal bodies was 0.17 g / m ² . Static culture was performed at 23 ° C. and a carbon dioxide concentration of 5% under irradiation of a fluorescent lamp of 15000 lux (light irradiation ON / OFF every 12 hours). The number of culture days was 31 days. In addition, the algal body amount (g / m ² ) at the start of the main culture is referred to as the initial algal body amount (the same applies to the following examples and comparative examples).

(Recovery of biofilm)
As a second substrate in the process of FIG. 1, prepare a plastic plate that matches the size of the container, insert this plastic plate from the end of the culture container to the bottom of the biofilm, and bring the plastic plate to the opposite side of the container The biofilm formed by depositing was deposited on the second substrate, thereby scooping up all the biofilm formed on the surface of the culture medium. The biofilm was dried at 130 ° C. for 3 hours in a constant temperature dryer, and the weight was measured to obtain the algal mass (g). The value obtained by dividing the weight by the container area (m ² ) and the number of culture days was defined as algal body productivity (g / m ² · day).

(Oil content / oil productivity)
About 50 mg of dried alga bodies obtained in the collecting step were precisely weighed, and this was sufficiently ground using a mortar and pestle for 5 minutes. This was washed with methanol (1 ml twice) and chloroform (4 ml once), and this was collected and centrifuged. The solvent of the obtained supernatant was volatilized, the weight of the residue was precisely weighed, and the oil content (g / g dry weight dry%) was determined from this and the dry alga body weight before treatment.

The oil productivity (g / m ² · day) was calculated by multiplying the previously obtained algal body productivity (g / m ² · day) by the oil content.

(Evaluation)
Since oil productivity also changes depending on culture conditions, oil productivity is compared with comparative examples under the same culture conditions (the initial algal mass, culture days, water depth, and light intensity are the same). did. Compared to the comparative example, the oil productivity is less than 1.1 times (E), 1.1 times to less than 1.2 times (D), 1.2 times to less than 1.3 times The product was evaluated as (C), the product of 1.3 times to less than 1.4 times as (B), and the product of 1.4 times or more as (A).

Compared with Comparative Example 1 under the same culture conditions, a remarkable improvement effect was observed in algal body productivity, oil content, and oil productivity.

[Example 2]
A first preculture and a second preculture similar to those in Example 1 were performed.

(Main culture)
A cylindrical plastic container having a diameter of 13.4 cm and a height of 21 cm is charged with a medium (c) in which the NO ₃ ion concentration of 2.7 L of CSiFF04 medium is 1/4 times (water depth 20 cm), and the algal mass is 4 g / m. ^The seed algae dispersion prepared by the second pre-culture was added so as to be 2. Static culture was performed at 2 ° C. under a carbon dioxide concentration of 5% under irradiation of a fluorescent lamp of 22,000 lux (light irradiation ON / OFF every 12 hours). The culture days were 14 days. This was cultured, recovered and evaluated in the same manner as in Example 1.

[Example 3]
Culture was performed in the same manner as in Example 2 except that the medium for main culture in Example 2 was changed to a medium (b) in which the NO ₃ ion concentration of CSiFF04 medium was halved.

Example 2 and Example 3 were significantly improved in algal body productivity, oil content, and oil productivity as compared with Comparative Example 6 under the same culture conditions.

[Example 4]
In the main culture of Example 2, the initial algal mass was changed to ² g / m ² , the NO ₃ ion concentration of the CSiFF04 medium was changed to ¼ times, and the phosphate ion concentration was halved as the medium. The same culture as in Example 2 was performed except that the changed one was used and the light intensity was changed to 19000 lux.
Compared to Comparative Examples 7 to 10 under the same culture conditions, an improvement effect was observed in algal body productivity, oil content, and oil productivity.

[Example 5]
In the main culture of Example 4, the same culture as in Example 4 was performed except that the initial algal body amount was changed to 4 g / m ² , the light intensity was changed to 20000 lux, and the culture days were changed to 21 days.

[Example 6]
In the main culture of Example 5, the same culture as in Example 5 was performed except that the medium in which the NO ₃ ion concentration of the CSiFF04 medium was changed to 0.11 times was used.

[Example 7]
In the main culture of Example 5, the same culture as that of Example 5 was performed except that the medium (c) in which the NO ₃ ion concentration of the CSiFF04 medium was changed to 1/4 times was used as the medium.

[Example 8]
In the main culture of Example 5, the same culture as in Example 5 was performed except that the medium (b) in which the NO ₃ ion concentration of the CSiFF04 medium was changed to ½ was used as the medium.

In Examples 5, 7 and 8, significant improvement effects were observed in algal body productivity, oil content, and oil productivity compared to Comparative Example 14 and Comparative Example 15 under the same culture conditions.
In Example 6, as compared with Comparative Example 14 and Comparative Example 15 under the same culture conditions, a large improvement effect in the oil content was recognized.

[Comparative Example 1]
Cultivation / collection / evaluation was performed in the same manner as in Example 1, except that the main culture medium of Example 1 was changed to CSiFF04 medium (a).

[Comparative Example 2]
In the main culture of Example 1, the initial amount of algal bodies was changed to 4 g / m ² , the medium was changed to a CSiFF04 medium in which the NO ₃ ion concentration was 0.85 times, and the light intensity was changed to 13000 lux. In addition, the same culture, recovery and evaluation as in Example 1 were performed except that the culture days were changed to 14 days.

[Comparative Example 3]
In the main culture of Example 1, the initial amount of algal bodies was changed to 4 g / m ² , the medium was changed to a CSiFF04 medium in which the NO ₃ ion concentration was 0.85 times, and the light intensity was changed to 13000 lux. In addition, the same culture, recovery and evaluation as in Example 1 were performed except that the culture days were changed to 14 days.

[Comparative Example 4]
In the main culture of Example 2, the medium was changed to one in which the NO ₃ ion concentration of the CSiFF04 medium was 0.7 times, the water depth was changed to 15 cm, and the light intensity was changed to 20000 lux. Were cultured, collected and evaluated.

In Comparative Examples 2 to 4, the oil productivity was lower than those in Examples 1, 3 and 4 under similar conditions.

[Comparative Example 5]
In the main culture of Example 2, the same culture, collection, and evaluation as in Example 2 were performed except that the medium was changed to CSiFF04 medium (a), the water depth was changed to 15 cm, and the light intensity was changed to 20000 lux.
[Comparative Example 6]
Culture was performed in the same manner as in Example 2 except that the main culture medium in Example 2 was changed to CSiFF04 medium (a).

[Comparative Example 7]
In Example 4, the same culture, collection, and evaluation as in Example 4 were performed, except that the main culture medium was changed to a CSiFF04 medium in which the NO ₃ ion concentration was 0.85 times.

[Comparative Example 8]
In Example 4, the same culture, recovery and evaluation as in Example 4 were performed, except that the main culture medium was changed to a CSiFF04 medium in which the NO ₃ ion concentration was 0.7 times.
[Comparative Example 9]
In Example 4, the culture / recovery / recovery method of Example 4 was changed except that the main culture medium was changed to a CSiFF04 medium with a NO ₃ ion concentration of 0.45 times and a phosphate ion concentration of 4.5 times. Evaluation was performed.

[Comparative Example 10]
In Example 4, the same culture, collection and evaluation as in Example 4 were performed except that the medium for main culture was changed to CSiFF04 medium (a).

[Comparative Example 11]
In the main culture of Example 5, the culture medium was changed to one in which the NO ₃ ion concentration of the CSiFF04 medium was 1/8 times, and the culture period was the same as in Example 5, except that the culture period was 14 days. Went.
[Comparative Example 12]
In the main culture of Example 5, the medium was changed to a CSiFF04 medium in which the NO ₃ ion concentration was changed to 1/8 times and the phosphate ion concentration was set to 1/4 times, and the culture days were changed to 14 days. The same culture, collection and evaluation as in Example 5 were performed.

[Comparative Example 13]
In the main culture of Example 5, the same culture, collection, and evaluation as in Example 5 were performed except that the medium was changed to CSiFF04 medium (a) and the number of culture days was 14 days.

[Comparative Example 14]
In Example 5, the same culture, collection, and evaluation as in Example 5 were performed, except that the main culture medium was changed to a CSiFF04 medium in which the NO ₃ ion concentration was 0.7 times.

[Comparative Example 15]
In Example 5, the same culture, collection and evaluation as in Example 5 were performed except that the medium for main culture was changed to CSiFF04 medium (a).

The results of Examples and Comparative Examples are shown in the table below and FIG.

Claims

A method for culturing microalgae that is useful substance productivity,
Microalgae in a medium in which the molar concentration ratio of nitrate ion to phosphate ion (N / P ratio) is 0.7 to 4.0 and the amount of nitrate ion per culture area is 0.2 mol / m 2 or more. A method for culturing microalgae, comprising a step of cultivating and forming a biofilm on a liquid surface of a medium.
The N / P ratio is 1.0 to 4.0, nitrate ion per the culture area is 0.2mol / m 2 ~ 1.5mol / m 2, the culture method of claim 1.
The culture method according to claim 1 or 2, wherein the culture is performed by stationary culture.
The culture method according to any one of claims 1 to 3, wherein the biofilm has a three-dimensional structure.
The culture method according to any one of claims 1 to 4, wherein the medium is not exchanged.
The culture method according to any one of claims 1 to 5, wherein the microalgae are green algae.
The microalgae is Botryococcus sp. , Chlamydomonas sp. , Chlorococcum sp, Chlamydomonad sp. Tetracystis sp. , Characium sp. Or Protosiphon sp. The culture method according to any one of claims 1 to 6, which belongs to the above.
The microalgae is Botryococcus sudueticus FERM BP-11420, or Chlorococcus sp. The culture method according to any one of claims 1 to 7, which belongs to the same species as FERM BP-22262.
A culture step comprising the culture method according to any one of claims 1 to 8; and a step of recovering the formed biofilm;
A method for producing algal biomass.
The production method according to claim 9, wherein the algal biomass is oil.