WO2013005799A1 - Culture medium for microalgae - Google Patents

Abstract

The purpose of the present invention is to increase the flexibility in the selection of nutrient components in a culture medium for use in culturing in the production of various biomass materials, to reduce the cost for the culturing, and to enable the utilization of resources dispersed in different parts in the world, whereby it becomes possible to grow microalgae with high efficiency even in an aqueous-solution-like culture medium having high turbidity, and to achieve the efficient production of various biomass materials in a stabilized manner and at a reduced cost. The present invention relates to: a culture medium for microalgae, which is characterized by having a clearness degree ranging from 15 cm (15 degrees) to 0.5 cm (0.5 degree), preferably from 10 cm (10 degrees) to 1 cm (1 degree), more preferably from 8 cm (8 degrees) to 1.5 cm (1.5 degrees); a method for culturing a microalga using the culture medium for microalgae; a microalga produced by the method; a method for removing and collecting a poorly water-soluble compound from the culture solution during or after the completion of the culturing by the culturing method; and others.

Description

Medium for microalgae

The present invention is a medium (culture solution) containing water as a main component for growing microalgae growing in water or on land artificially and efficiently by the action of photosynthesis of the algae itself, and in particular, the main component is water. The present invention relates to a medium for microalgae that contains a compound having low solubility in water as a nutrient and has high turbidity and precipitation.

Among the organisms that perform photosynthesis that generate oxygen, excluding moss plants, fern plants, and seed plants, microalgae are plants that cannot be observed in their individual forms when grown using a microscope. Many of these are phytoplankton communities in the ocean, rivers and lakes, and are attached to benthic and other macroalgae at the bottom of the hydrosphere, or corals and some mollusks. Some of them grow symbioticly. On land, there are many that grow on the soil surface and those that live together with fungi. Some grow on the surface of structures such as other higher plants, animals, buildings or walls. Microalgae that carry out photosynthesis are seen on the earth in the range where indirect light and artificial visible light reach even with direct sunlight and weak light, and some moisture is secured from the polar region to the temperate zone, desert region, and the tropics. As long as there is a place and period, it grows by adapting, evolving to various environments.

Microalgae are so diverse that their types are estimated to be tens of thousands. For this reason, there are many types of biomass produced by different microalgae. In fact, proteins, amino acids, carbohydrates, lipids, nucleic acids, and vitamins are components that all microalgae have during growth, but the composition of the alga body varies greatly depending on the type of microalgae and the growth conditions. . In addition, there are types that contain hydrocarbons as raw materials for biofuels in the alga bodies or are discharged out of the alga bodies during growth. By already culturing such microalgae, Research and development aimed at producing raw materials for heavy oil, light oil, or jet fuel have also begun. Furthermore, many new substances have been discovered from microalgae, and particularly compounds having toxicity are being studied for application as pharmaceuticals such as anticancer agents. There are also microalgae that contain substances necessary for maintaining our health, such as astaxanthin, β-carotene, and docosahexaenoic acid. These microalgae are mass-cultured and sold as health functional foods. ing. Furthermore, mass cultivation of microalgae is effective for increasing feed for livestock and seafood.

Nitrogen, phosphorus, potassium, sulfur, magnesium, calcium, silicon (included in diatoms) and various trace elements, which are elements in the algal bodies of microalgae, are also necessary for higher plants that are crop plants. Attempts have been made to utilize living microalgae, fermented alga bodies, or residues after extracting the various useful biomass described above as fertilizers. In fact, when the composition of elements contained in the body of many microalgae is expressed in molar ratio, when the phosphorus atom is 1 mol, nitrogen is about 16 mol, potassium is less than 2 mol, sulfur is about 1.5 mol, Magnesium and calcium are about 0.5 moles (described in Limnol. Oceanogr., Vol.33, 796-822 (1988), Proc. R. Soc. B, 278, 526-534 (2011)). It is also known that in the case of microalgae such as chlorella that can accumulate polyphosphate in cells, the molar ratio of phosphorus in the alga body is extremely high compared to other elements if there is excess phosphoric acid in the growing water area. It has been.

Furthermore, microalgae that concentrate useful elements such as rare earth elements, harmful heavy metal elements such as lead and cadmium, and nuclear fuel elements such as uranium into algal bodies, and microalgae that can adsorb these to extracellular polysaccharides and phosphate residues Is also known. Attempts to use microalgae have also begun to specifically recover these special elements.

In order to utilize these microalgae, which are very useful for human beings, as new biomass, or to utilize their ability to treat sewage and concentrate special elements, phytoplankton, which is a collection of microalgae, is used. It is necessary to culture as a group, to isolate each microalgae from the place where they originally grew, or to transfer them after receiving a strain of microalgae from Culture か ら collection. Furthermore, the target biomass can be produced in large quantities by mass-culturing specific microalgae. In particular, in order to cultivate phytoplankton and specific microalgae by their own photosynthesis, in many cases, visible light, commonly referred to as "medium", containing nutrients mainly composed of inorganic substances in water. A transparent aqueous solution (transparent culture solution) is used.

By the way, the range in which microalgae of Minamata can grow by photosynthesis is called Mahiko layer, but the deepest of this is about 2.5 times the transparency required by the Seky disk method (Marine photosynthesis: with special emphasis on the ecological aspects. Elsevier, (1975) Described in）). Furthermore, the transparency is 1.5 ~ 2 times the transparency used for the measurement in the present invention (California Agriculture 巻 Vol.58, 149-153 (2004)). Therefore, if the transparency is 1 cm, the bottom is fine. The estimated depth at which algae can grow by photosynthesis is 4 to 5 cm.

When visible light such as sunlight is irradiated on plants such as microalgae, the energy of the light is converted into transmission, scattering, heat and fluorescence in addition to being used for photosynthesis of plants. In particular, in photosynthesis studies of aquatic plants that contain the majority of microalgae, the measurement in a medium with less scattering makes it easier to accurately measure the characteristics of photosynthesis from the comparison of incident light and transmitted light in the experiment. Medium (medium with high transparency) has been used. Furthermore, there are various types of microalgae that grow on land in non-water areas and that grow well in a medium with high transparency developed for microalgae in chickenpox. Also, since research on photosynthesis using microalgae has been conducted before the usefulness of microalgae biomass has attracted attention, transparent media are also used in production sites of useful biomass by microalgae. In addition, when a necessary nutrient is precipitated without dissolving at all in a medium with high turbidity or a lot of precipitation (medium with low transparency), it is expected that microalgae do not grow.

For this reason, in the prior art, a medium free from turbidity and precipitation (a medium with high transparency) is regarded as essential for the photosynthetic culture of microalgae. Therefore, when cultivating microalgae by photosynthesis, compounds that are highly soluble in water have been used as the various inorganic nutrient compounds used in the medium. However, even if it is highly soluble in water, depending on the combination with another compound, a precipitate may be formed after mixing or after aseptic processing. For example, when high concentration calcium salt or magnesium salt and phosphate are mixed under the condition of specific pH. In addition, precipitation may be slow at room temperature, but precipitation is accelerated during high-temperature treatment in high-pressure steam sterilization frequently used during aseptic culture. In these cases, phosphoric acid is indispensable for the growth of all microalgae and therefore cannot be removed from the medium components. Therefore, the medium should have a pH value that does not cause precipitation, or a complex that precipitates with phosphoric acid in an aqueous solution. The type of calcium salt used was not used in the medium or was used at low concentrations (conditions such as less than 1 millimol per liter). Furthermore, most of the culture media have a composition to which expensive chelating agents such as EDTA and citric acid are added to prevent precipitation of insoluble salts.

Incidentally, elements such as nitrogen, phosphorus, potassium, sulfur, and magnesium are main components of a medium used for culturing microalgae. Furthermore, each element such as sodium, calcium, iron, boron, zinc, copper, cobalt, and molybdenum is known as a trace element necessary for cultivation of general microalgae. In addition, there are diatoms in which silicon is essential, and microalgae containing strontium, cadmium, nickel, and arsenic as constituents of algal bodies.

JP-A-10-155478

Algae experiment method, (editors: Hiroshi Hirota, Atsushi Watanabe), 4th edition, Nanedo, 1975

However, in such a conventional medium (medium with high transparency) constituted by a prescription using only salts having high solubility in water, various resources on the earth cannot be fully utilized. In fact, many salts and compounds are hardly soluble. For the purpose of producing various biomass with various microalgae, it is expected that there will be various types of media required for it, and from a global perspective, it is soluble in water that has been used in the past. Only high salts and compounds have the disadvantage that the cultivation of microalgae is directly affected by fluctuations in supply and costs that reflect the production and production volume of each compound and the political situation.

As another problem, the phosphoric acid component in the phosphorus fertilizer for crops sprayed on the soil is changed to aluminum phosphate in volcanic ash soil and acidic soil, iron phosphate in red soil and acidic soil, calcium phosphate in alkaline, and the like. These phosphate compounds are called non-available phosphate and remain in the soil without being absorbed by the crop. Examples of potassium compounds that are difficult to use as fertilizers include potash feldspar, amphibole, and mica as minerals. Importantly, most of the rocks in the crust are of the feldspar group, and potash feldspar accounts for about 10% of the feldspar group. Amphibole and mica are also high in the crust.

Accordingly, the main object of the present invention is to efficiently grow microalgae and phytoplankton even in a medium with water as a main component and high turbidity, and provide stabilization and cost reduction of efficient production of various biomass. There is.

In order to solve the above problems, the present inventor has intensively studied the amount of elements suitable for the growth of microalgae added to the medium and the availability thereof, and as a result, the turbidity and precipitation are clearly mixed into the aqueous solution. Even if it occurs, it has been found that the growth rate of microalgae equivalent to that described in conventional patents and papers, or at least sufficient to achieve the above-mentioned object can be obtained, and the present invention has been achieved.

That is, the present invention relates to a medium (liquid medium) for cultivating microalgae, and the medium has low transparency compared to a conventional medium, that is, has a certain turbidity or precipitation. It is characterized by.

More specifically, in the present invention, the transparency is 15 cm (15 degrees) to 0.5 cm (0.5 degrees), preferably 10 cm (10 degrees) to 1 cm (1 degree), more preferably 8 cm. The present invention relates to a medium for microalgae characterized by being in the range of (8 degrees) to 1.5 cm (1.5 degrees).

In the culture medium of the present invention, there is no particular limitation on the means for obtaining the above-mentioned transparency, but for example, a compound that is sparingly soluble in water as any one or more kinds of compounds contained as nutrients, or other The transparency in the above specific range can be obtained by a specific method such as the use of two or more kinds of compounds that form a complex with the above compound to form a hardly soluble precipitate.

In particular, a nitrogen source, a phosphorus source, a potassium source, a sulfur source, a magnesium source, and at least one compound that is hardly soluble in water as a calcium source, or a nitrogen source, a phosphorus source, a sulfur source, a magnesium source, In addition, use a compound that is sparingly soluble in water for all of the calcium source, or use a sparingly soluble compound for all of the nitrogen source, phosphorus source, potassium source, sulfur source, magnesium source, and calcium source. Therefore, it is preferable to obtain the transparency in the specific range. As each nutrient source, in addition to a compound that is hardly soluble in water, a conventionally used compound having high solubility in water may be used in combination.

The present invention further includes a method for culturing microalgae comprising using such a medium for microalgae, microalgae obtained by the method, and water contained as a nutrient in the microalgae medium of the present invention. By removing and collecting hardly soluble inorganic compounds from the medium, the present invention also relates to reuse of these inorganic compounds and the like.

According to the present invention, it has become possible to use a medium (having low transparency) having turbidity and precipitation that has not been actively used for liquid culture of microalgae until now. In this invention, the freedom of selection of inorganic compounds as nutrients in the medium used in culture in various biomass production, removal / recovery from the medium, and reuse was greatly increased. It can be expected to reduce costs and use unused resources distributed throughout the world.

The type of microalgae that can be used in the medium of the present invention is not particularly limited, and can be used for culturing any microalgae known to those skilled in the art. Representative examples of such microalgae are freshwater or brackish waters, green algae plants growing in seas, salt lakes or soils, red plants, crypt plants, non-hairy plants, hapto plants, dinoflagellates, gray plants. , Euglena plants and cyanobacteria (also known as cyanobacteria) can be used. If these microalgae are illustrated by classification nets and genera, the green algae include Ankistrodesmus, Botryococcus, Chlamydomonas, Chlorella, Chlorococcum, Dunaliella, Eudorina, Haematococus, Monoraphidium, Scenedesmus Genus Cyrentium, Galdieria, Hildenbrandia and Porphyridium as red plants, Chroomonas genus, Cryptomonas genus and Rhodomonas genus as crypt plants, , Rafido algae net, yellow green algae net, true eyed point algae net, Pinguo algae, especially among the diatom net, Chaetoceros, Cyclotella, Cylindrotheca, Phaeodactylum, Skeletonema, Tetraselmis, Thalassiosira, True An example is the genus Nannochloropsis in the ocular algae network. In addition, Cryptomonas genus, Dicrateria genus, Isochrysis genus, Pavlova genus as hapto plants, Ceratium genus, Peridinium genus as dinoflagellate plants, Cyanophora genus, Glaucocystis genus as gray plants, Eugena genus as Euglena plants, Examples of cyanobacteria include those contained in the genus Anabaena, Arthrospira, Microcoleus, Nostoc, Oscillatoria, Planktothrix, Schizothrix, Scytonema, Synochococcus, Synechocystis, and Tolypothrix. More specifically, there can be mentioned microalga belonging to the genus Chlorella, Botryococcus, Dunaliella, Porphyridium, Nostoc or Tolypothrix as described in Examples. Furthermore, as an object of the present invention, phytoplankton, which is a group composed of many types of microalgae, can be mentioned without being limited to the cultivation of a specific type of microalgae.

As described above, in order to obtain the transparency of the culture medium of the present invention, one or more compounds (including inorganic salts and minerals) that are sparingly soluble in water are included as optional nutrients, or are combined with other compounds. The turbidity and precipitation of this medium can be caused (reduced transparency) by using two or more kinds of compounds that form a body and form a hardly soluble precipitate. The preferred range of weight in the case of adding each compound that is hardly soluble in water is shown below. The weight of the nitrogen compound hardly soluble in water is 200 g or less per 1 kg of the total weight of the medium, and in the case of cyanobacteria that fix nitrogen, the nitrogen compound may not be contained. In addition, the soil containing a phosphorus compound that is hardly soluble in water or non-available phosphoric acid added to the medium is 200 to 10 mg per kg of the total weight of the medium. Minerals such as mica, hornblende, potash feldspar, and other minerals containing potassium that is sparingly soluble in water, and their pulverized particles are 400 g or less 0.1 kg or more per 1 kg of the total weight of the medium. The total weight of the magnesium-containing minerals such as magnesium compound or dolomite, which is hardly soluble in water, is 100 μg or less and 10 μg or more per 1 kg of the total weight of the medium. Furthermore, unless a chelating agent is added, it is essential to add a water-soluble iron compound to the medium. It is a medium characterized by the weight of calcium compounds that are sparingly soluble in water, bone fragments containing calcium, minerals (such as limestone), coral sand, and the like, with a total weight of 100 kg or less per kg of the medium. Furthermore, it is preferable that the water content ratio of the medium (the value obtained by dividing the weight of water by the total weight of the solute and insoluble substance) is greater than 0.8. The water content is calculated using the definition formula in soil physics (Soil Chemical Analysis, (Supervision: Japan Society of Soil Fertilizer), 2nd edition, Hirotomo, 2000).

Further, the mineral used in the culture medium of the present invention, the compound hardly soluble in water, or the soil may be in the form of crystals, crushed, non-crushed, plate-like, or a pool formed by bonding with other materials. Any combination may be used. When crushed, the particle size of the particles may be any size from, for example, boulders of 256 millimeters or more to gravels of various sizes, sand and silt, and clays of 0.004 millimeters or less.

In addition, since the main nutrients contained in the culture medium of the present invention are compounds that are hardly soluble in water, they can be removed and recovered from the culture medium using any means known to those skilled in the art using the characteristics. In particular, using a means such as a plastic or metal sieve having a sieve size of 0.1 mm or more, filter paper, filter cloth, etc., it can be removed and collected with great ease, low cost and energy saving. Therefore, it is desirable to use minerals, compounds that are sparingly soluble in water, or soil, which has a shape suitable for such removal and recovery methods.

Furthermore, typical examples of compounds added as nutrients to the medium of the present invention are shown below.

As the nitrogen source added to the medium, high urea solubility in _{water, Ca (NO 3) 2 .4H} 2 O, Ca (NO 3) 2 .nH 2 O, KNO 3, Mg (NO 3) 2 .6H _{2 O, Mg (NO 3)} 2 .nH 2 O, NaNH 4 HPO 4 .4H 2 O, NaNO 3, NH 4 Cl, NH 4 NO 3, (NH 4) 2 SO 4, NH 4 HCO 3, (NH ₄ ) Nitrogen (7,9-dihydro-1H-purine-2,6,8 (3H) -trione) is an insoluble nitrogen compound added to inorganic salts such as ₂ CO ₃ and nitric acid or as an alternative. ) And MgNH ₄ PO ₄ .6H ₂ O.

The phosphorus source added to the medium, the solubility in water is high _{Ca (H 2 PO 4) 2} .H 2 O, KH 2 PO 4, K 2 HPO 4, K 2 HPO 4 .3H 2 O, K 3 PO _{4, NaH 2 PO 4, Na} 2 HPO 4, Na 3 PO 4, Na 4 P 2 O 4, Na 4 P 2 O 7 .10H 2 O, NH 4 H 2 PO 4, (NH 4) 2 HPO 4, H ₄ O ₇ P ₂ , phosphoric acid compounds such as polyphosphoric acid, phosphates containing nitrogen, acid forms such as phosphoric acid and polyphosphoric acid, phosphoperphosphate (Ca (H ₂ PO ₄ ) ₂ · H ₂ O + 2CaSO ₄ ) or heavy superphosphate lime (Ca (H ₂ PO ₄ ) ₂ · H ₂ O), or as an alternative, a water-insoluble phosphorus compound may be AlPO ₄ , Ba ₃ (PO _{4) 2, Ca (PO 3} ) 2, CaHPO 4 .2H 2 O, Ca 2 P 2 O 7, Ca 3 (PO 4) 2, 3Ca 3 (PO 4) 2 .Ca (OH) 2, Ca 4 ( _{PO 4) 2 O, Ca 8} H 2 (PO 4) 6 .5H 2 O, Ca 10 (PO 4) 6. (OH) 2, Co 3 (PO 4) 2, Cu 3 (PO 4) 2, FePO _{4 .2H 2 O, MgHPO 4 .3H} 2 O, MgKPO 4, MgKPO 4 .6H 2 O, Mg 2 P 2 O 7, Mg 3 (PO 4) 2, Mg 3 (PO 4) 2 .8H 2 O, Mn ₃ (PO ₄ ) ₂ , Zn ₂ P ₂ O ₇ , monoalkyl phosphoric acid, heat-treated or calcined animal bone, and soil containing either AlPO ₄ , CaHPO ₄ or FePO ₄ .

Potassium sources to be added to the medium include the above-mentioned potassium nitrate and potassium phosphate conventionally used for culturing microalgae, and KCl, K ₂ CO ₃ , KHCO ₃ , and KHSO ₄ that have high solubility in water. In addition, minerals such as muscovite, biotite, amphibole, potash feldspar, etc. that are sparingly soluble in water to be added or alternatively added as plant-derived plant ash, pulverized particles thereof, or synthetic mica are exemplified.

The sulfur source to be added to the medium, high solubility in water _{K 2 SO 4, MgSO 4,} MgSO 4 .7H 2 O, MgSO 4 .nH 2 O, Na 2 SO 4, sulfur-containing inorganic salts or sulfuric acid etc. There are, or added to, as a sparingly soluble sulfur compounds in water to be added as an _{alternative, BaSO 4, CaSO 4, CaSO} 4 .0.5H 2 O, it is CaSO ₄ .2H ₂ O and the like.

The magnesium source to be added to the medium, the solubility in water is higher _{MgCl 2, MgCl 2 .6H 2 O} , MgSO 4, MgSO 4 .7H 2 O, there is MgSO ₄ .nH ₂ O, but require further neutralization , there are MgO, or added to, as a sparingly soluble magnesium compound in water to be added as an _{alternative, MgCO 3, MgCO 3 .3H 2} O, MgCO 3 .5H 2 O, Mg (OH) 2 or minerals, Dolomite is given.

Regardless of whether the solubility is high or low, the calcium source added to the medium forms a complex with phosphoric acid to form a slightly soluble precipitate in water, such as CaCl ₂ and CaCl ₂ . There are 2H ₂ O, Ca (NO ₃ ) ₂ and neutralization is necessary, but there is CaO, Ca (OH) ₂ , and calcium compounds that are hardly soluble in water added to these or as an alternative are as follows: Although overlapping the sparingly soluble salts in water as described _{above, CaCO 3, Ca (PO 3} ) 2, CaHPO 4 .2H 2 O, Ca 2 P 2 O 7, Ca 3 (PO 4) 2, 3Ca 3 (PO 4 _{) 2 .Ca (OH) 2,} Ca 4 (PO 4) 2 O, Ca 8 H 2 (PO 4) 6 .5H 2 O, Ca 10 (PO 4) 6. (OH) 2, CaSO 4, CaSO 4 .0.5H ₂ O, CaSO ₄ .2H ₂ O.

Iron, although a minor component, the precipitated iron compound, in order to not easily be utilized in microalgae, a soluble salt in water which is conventionally _{used, FeCl 2 .4H 2 O, FeCl} 3, Fe ( III) -EDTA, Fe (NO ₃ ) ₂ , Fe (NO ₃ ) ₃ , FeSO ₄ , Fe ₂ (SO ₄ ) _3, etc. need to be added.

In addition, among other trace elements such as sodium, boron, zinc, copper, manganese, cobalt, and molybdenum, an appropriate amount may be added according to the type of microalgae for the purpose of culture.

The medium of the present invention can be prepared by any method known to those skilled in the art. For example, when the medium needs to be sterilized, the method includes, for example, inorganic salts and minerals as described above so that a compound containing nitrogen, phosphorus, potassium, sulfur, magnesium, calcium is added within a specified weight range. Weigh the water and add it to purified water, tap water, river or well water, brackish water, seawater or salt lake water, and then adjust the pH to about 4 to 9 by adding acid or base as necessary. . Next, it is desirable to sterilize it with high pressure steam (120 ° C to 130 ° C, 10 ° C to 20 ° C for 20 minutes).

Alternatively, as a sterilization method, a sodium hypochlorite solution, a sardine solution, or a sardine powder is added to a medium that does not contain iron compounds, trace elements or ammonia compounds, and then sterilized, and then a sodium thiosulfate solution is added, or the medium It is desirable to remove hypochlorous acid by irradiating it with sunlight, and then add an iron compound, trace elements and ammonia compound as necessary to prepare a medium in which microalgae can be mixed immediately .

Furthermore, as another sterilization means, purified lime water (CaO) firewood or firewood MgO or a mixture thereof is added to purified water, tap water, river water, well water, brackish water, seawater or salt lake water in a reactor vessel or culture pool. It is also possible to prepare a medium by adding the above-mentioned measured salts and compounds after neutralization with diluted nitric acid, sulfuric acid, or hydrochloric acid, and then adding a weighed salt and compound.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited at all by the Example.

Origin of microalgae: Chlorella UK001 strain was isolated by research at MBI Corporation's Marine Biotechnology Research Institute (MBI Laboratories) under the project of the Japan Environmental Technology Research Organization, namely the Global Environment Industry Technology Research and Development Project. It was. The Chlorella UK001 strain used was managed by the NPO Regional Promotion Support Center after obtaining permission from MBI Lab. Chlorella regularis var. Minima UTEX1807 strain (UTEX Number 1807) was purchased and used at Texas State University. Botryococcus braunii strain (NIES Number 2199), Dunaliella salina strain (NIES Number 2257) and Tolypothrix tenuis strain (NIES Number 2135) are purchased and used at the National Institute for Environmental Studies in Japan. did. Porphyridium cruentum strain (IAM-R1, which is the same species as NIES Number 2138), was invented by the microbial strain storage facility of the University of Tokyo (former) Applied Microbiology Research Institute in Japan. However, it was obtained at the time of the research using the medium of Aizawa et al.
Nostoc flagelliforme used the Chinese food ingredients that were sold in Chinatown in Japan (English: Fat choy), which the inventors isolated to algae in a liquid medium.

Measurement of medium transparency: Measurement was performed by a standard method using a commercially available fluorometer (ASONE ST-100 or ST-30). In other words, this measurement is performed in a bright room with a transparent meter consisting of a long transparent tube. This was done by finding the maximum value (cm) of the water layer that the cross could identify. It should be noted that determination of a numerical value with a transparency degree of 70 cm (70 degrees) or more is not performed because it is unnecessary for the present invention.

Cultivation method: Each 250 g or 50 g medium weighed with two-digit accuracy was placed in a 500 ml or 100 ml Pyrex (registered trademark) IWAKI conical flask with a screw cap. The screw cap, once rotated 180 degrees from the fully closed position, was loosened to secure the inflow of the gas phase surrounding the Erlenmeyer flask using the space between the cap thread and the flask thread.

In the pre-culture prior to the implementation of this example, Chlorella UK001 strain and Chlorellaularregularis var. Minima UTEX1807 strain are Kanazawa et al. Medium (Table 3), Botryococcus braunii strain is Chu13 medium (Table 9), and Nostoc flagelliforme strain is the present invention. Medium 5 (Table 11), Dunaliella salina and Porphyridium cruentum strains were from Aizawa et al. (Table 13), and Tolypothrix tenuis strain was MT medium (Table 16). The temperature and light conditions during the pre-culture were the same as in each example. After confirming that the growth of the algal cells in the pre-culture has progressed in the linear growth phase, put an appropriate amount of the algal suspension in a sterilized large centrifuge tube (approximately 400 ml capacity) and algal cells using an angle rotor centrifuge. And the supernatant was discarded. Here, in the case of Chlorella UK001, Chlorella regularis var. Minima UTEX1807, Botryococcus braunii, Nostoc flagelliforme, and Tolypothrix tenuis, the same amount of sterile distilled water, Dunaliella salina and Porentidiumali and Porentidium In this case, after thoroughly suspending in sterile distilled water containing the same amount of 0.5 M NaCl as the discarded supernatant, the same volume of suspension is removed from this suspension by several sterile small centrifuge tubes (about 15 The alga bodies were precipitated using a swing centrifuge, and the supernatant was discarded. Add the culture medium shown in each example to each alga body precipitated and collected in several centrifuge tubes, suspend it well, transfer it to a container for culture experiments, and add the necessary additional amount of each medium. The culture experiment was started in such a way that the algae concentration at the start was the same between different media. The algae centrifugation, precipitation recovery, and suspension operations from pre-culture to experiment in the examples were all performed under aseptic conditions at room temperature. In addition, all the cultures were performed aseptically except for the examples in the medium 2 of the present invention (all the rightmost column data in Table 7).

Culture light: Irradiation with sunlight at the window that passed through a glass window made of lamelex BG of template glass, or using a white fluorescent lamp (HAKUBA, Light Viewer 7000PRO) in a dark room Irradiation was performed by providing continuous light with no dark period from the lower surface, or the light period (white fluorescent lamp irradiation period) and dark period (non-light irradiation period) shown in the title of each example. In the former case, 62 to 65% of sunlight was irradiated. In the latter case, the amount of irradiation light was 2,900-3,100 lux. These were performed in a transparent desiccator made of acrylic resin (inner dimensions (mm): 485 x 275 x 285) in a room kept at 24-26 degrees Celsius. In this desiccator, an Erlenmeyer flask containing the culture solution and microalgae was allowed to stand, and CO ₂ gas having a final concentration of 2% (v / v) was sealed in the air and cultured in a sealed state. In addition, CO ₂ gas was periodically replenished so that the CO ₂ concentration in the desiccator was 1.4% or more and 2% or less.

Measurement of growth: It was carried out by measuring the absorbance at 750 nm using a spectrophotometer (Shimadzu U-1800). The bandpass at this time was 4 nm. The cells dispersed in the solution in the flask during the culture were well suspended, and several milliliters were collected from the flask and mixed well with a 100 millimolar EDTA-Na2 solution per 4 volumes of this liter. Thereafter, an appropriate amount of the diluted solution was transferred to a plastic cuvette (light path 1 cm), and turbidity was measured.

In addition, when mineral debris such as potassium feldspar was added to the culture medium, the turbidity of the culture medium during culture was drastically changed during the spectroscopic measurement and was not determined. Therefore, the cells dispersed in the solution in the flask in culture were well suspended, and several milliliters were collected from the flask into a test tube, and centrifuged to separate the precipitate fraction containing the supernatant and alga bodies. . A certain amount of methanol solution was mixed into the remaining precipitate fraction after discarding the supernatant, and the test tube was capped and stirred, and then chlorophyll was extracted into the methanol solution by overheating at 65 degrees Celsius for 20 minutes. This test tube is centrifuged again to remove algal bodies and insoluble substances as precipitates, and the total chlorophyll concentration recovered in the supernatant methanol fraction is calculated using the existing calculation method (Biochem. Biophys. Res. Comm. 49 1616- 1623 (1972)), the absorbance was measured at 665 nm and 650 nm.

Photosynthesis multiplication by sunlight through the window glass of Chlorella UK 001 <br/> Comparison of the turbidity of microalgae during growth in 250 ml each of the medium 1 of the present invention (transparency 5 to 8 cm) and the medium of Tsujimoto et al. did. Tables 1, 2 and 3 show the compositions of the medium of the present invention and the medium of Enomoto et al. Used in the test. The medium 1 of the present invention was turbid and showed a value equivalent to the growth in the medium of Enomoto et al. (Table 4) despite the transparency of 5 to 8 cm (Table 1). In addition, the degree of transparency after storing an aqueous solution made only of Ca salt and phosphate in the components of the medium 1 of the present invention at room temperature for several days or after high-pressure steam sterilization is 10-12 cm, The main cause of the low transparency of the culture medium 1 of the present invention was due to precipitation formed by mixing these two kinds of salts.

Photosynthesis multiplication by continuous light irradiation of Chlorella UK 001 fluorescent lamp <br/> The turbidity of microalgae during growth was determined by the medium 1 of the present invention, the medium of Enomoto et al., MC medium (permeability 23-45 cm), Kanazawa These media (permeability of 70 cm or more) were compared at 50 ml each. Tables 1, 2 and 3 show the compositions of the medium of the present invention and the medium of Kanazawa et al. Used in the test. The medium 1 of the present invention is turbid and exhibits a value equivalent to or higher than that of the medium of Enomoto et al., MC medium, Kanazawa et al. (Table 5).

Photosynthesis multiplication by sunlight through window glass of Chlorella regularis var. Minima UTEX 1807 The turbidity of microalgae during the growth was compared with each of 250 ml of the medium 1 of the present invention and the medium of Kanazawa et al. Tables 1, 2 and 3 show the compositions of the medium of the present invention and the medium of Kanazawa et al. Used in the test. The medium 1 of the present invention was turbid and showed a value equivalent to the growth in the medium of Kanazawa et al. (Table 6) despite the transparency of 5 to 8 cm (Table 1).

Photosynthesis multiplication by sunlight through window glass of Chlorella regularis var. Minima UTEX 1807 As shown in Tables 4, 5 and 6, the culture medium 1 of the present invention is equal to or more than the conventional culture medium. It has been found that it causes the growth of Therefore, this time, uric acid, a nitrogen source that is sparingly soluble in water, Ca ₃ (PO ₄ ) ₂ , a phosphoric acid source that is sparingly soluble in water, Mg (OH) ₂ , a magnesium source that is sparingly soluble in water, and sparingly soluble in water. The medium 2 of the present invention containing BaSO ₄ as a sulfur source was compared with 250 ml of the medium 1 of the present invention. Tables 1 and 2 show the compositions of the media 1 and 2 of the present invention used in the test. As a result, it was shown that the medium 2 of the present invention containing a salt hardly soluble in water and having a transparency of 7 cm to 10 cm is a medium that causes sufficient growth (Table 7).

Photosynthesis growth by irradiation of Chlorella UK 001 fluorescent lamp (14 hours light period / 10 hours dark period per day) Furthermore, this time, uric acid, a nitrogen source that is hardly soluble in water, is hardly soluble in water. Phosphoric acid source AlPO ₄ , Phosphoric acid source that is sparingly soluble in water, Potassium feldspar debris powder that is sparingly soluble in water, Mg (OH) ₂ that is sparingly soluble in water, Magnesium source that is sparingly soluble in water The culture medium 3 of the present invention containing BaSO ₄ as a sulfur source was compared with 250 ml of the culture medium 1 of the present invention. Tables 1 and 2 show the compositions of the media 1 and 3 of the present invention used in the test. As a result, it was shown that even the medium 3 of the present invention containing a hardly soluble salt in water and having a transparency of 1.5 cm to 3 cm was sufficiently turbid (Table 8). .

Photosynthesis growth of Botryococcus braunii by fluorescent lamp irradiation (14 hours light period / 10 hours dark period per day) For the green alga Botryococcus producing biofuel, Chu13 medium used as standard medium for this microalgae , Uric acid, a nitrogen source that is sparingly soluble in water, Ca ₃ (PO ₄ ) ₂ , a phosphoric acid source that is sparingly soluble in water, The medium 4 of the present invention containing Mg (OH) _{2 as} a magnesium source and BaSO ₄ as a sulfur source hardly soluble in water and a potassium-deficient medium obtained by removing potassium feldspar from the medium 4 were compared at 250 ml each. Tables 2 and 9 show the compositions of these media used in the test. As a result, even if the present microalga contains a salt that is hardly soluble in water and has a transparency of 5 cm to 8 cm, the microalgae can grow sufficiently even when the microalgae stop growing when potassium is deficient. (Table 10).

Photosynthetic growth by irradiation of fluorescent lamps of Nostoc flagelliforme (12 hours light period / 12 hours dark period per day) Grown in the desert, known as a valuable Chinese food, and further contains cyanobacteria containing antiviral components Nostoc flagelliforme can be prepared at a lower cost than the BG11 medium (described in Bacteriol. Reviews Volume 35, 171-205 (1971)) used as the standard medium for the microalgae, and the titration operation of pH is unnecessary. From the medium 6 and the medium 6 according to the present invention, which can be prepared at a lower cost than the BG11 medium, and does not require a pH titration operation, and contains potassium calcite debris as a potassium source that is hardly soluble in water. Each of the potassium-deficient media excluding potassium feldspar was compared at 250 ml. Tables 2 and 11 show the compositions of these media used in the test. As a result, since the medium 5 of the present invention is alkaline with a pH of 8.4 to 8.6, any of the water-insoluble compounds such as MgCO ₃ , MgHPO ₄ , MgKPO ₄ , Mg (OH) ₂ , and Mg ₃ (PO ₄ ) ₂ Is formed during autoclaving, grows in spite of being cloudy and has a transparency of 10 to 15 cm (Table 11), and the growth of the microalgae is stopped in a potassium-deficient medium without the addition of potassium compounds Nevertheless, the medium 6 of the present invention containing potassium feldspar which is sparingly soluble in water and having a transparency of 6 cm to 8 cm (Table 11) was shown to be a medium that provides sufficient growth (Table 12). In addition, at the stage of the preliminary experiment, it was confirmed that the growth rate in the linear phase of Nostoc flagelliforme was equivalent to that of the medium 5 of the present invention and the BG11 medium as the standard medium.

Photosynthetic growth by irradiation of fluorescent lamps of Dunaliella salina (14 hours light period / 10 hours dark period per day). Produces salt-resistant green algae that can grow in salt lakes, seawater and brackish water, and has promising carotenoid production About Dunaliella salina, the medium of Aizawa et al. Reported as a medium for several types of Dunaliella and Porphyridium cruentum (described in Plant Cell Physiol. 26, 1199-1203 (1985)), and cheaper than this It can be prepared and pH titration operation is not required. Furthermore, uric acid, a nitrogen source that is sparingly soluble in water, phosphoric acid that is sparingly soluble in water, a phosphoric acid source that is sparingly soluble in water, a phosphoric acid source that is sparingly soluble in water A book containing AlPO ₄ , PO ₄ , potassium calcite debris, a poorly soluble potassium source, Mg (OH) ₂ , a poorly soluble magnesium source, and BaSO ₄ , a poorly soluble sulfur source. Inventive medium 7 was compared at 250 ml each. Tables 2 and 13 show the compositions of these media used in the test. As a result, it was shown that the culture medium 7 of the present invention is a turbid medium that provides sufficient growth despite the transparency of 2 to 3 cm (Table 13) (Table 14).

Photosynthesis growth by irradiation with fluorescent lamps of Porphyridium cruentum R1 (14 hours light period / 10 hours dark period per day) <br/> Single cell red algae that can grow in seawater, brackish water and sewage, and is also promising for sewage treatment Porphyridium cruentum R1 is a medium of Aizawa et al. Reported as a medium for several types of Dunaliella and Porphyridium cruentum (described in Plant Cell Physiol. 26, 1199-1203 (1985)), and from this Alpo ₄ , PO ₄ , which is a low-solubility phosphoric acid source, can be prepared at low cost, and does not require pH titration. 250 for each of the medium 7 of the present invention containing potassium calcite debris, a potassium source that is sparingly soluble in water, Mg (OH) ₂ that is a sparingly soluble magnesium source, and BaSO ₄ that is a sparingly soluble sulfur source. Comparison in ml. Tables 2 and 13 show the compositions of these media used in the test. As a result, it was shown that the medium 7 of the present invention is turbid and has a growth equivalent to that of Aizawa et al. Despite the transparency of 2 cm to 3 cm (Table 13) (Table 15). .

Photosynthesis multiplication by irradiation of fluorescent lamps of Tolypothrix tenuis (per day, 12 hours light period / 12 hours dark period) <br/> It grows on the surface of tropical and subtropical soils, and it is used as a source of biofertilizer such as rice Cyanobacteria Tolypothrix tenuis is expected to be cheaper than MT medium (described in J. Gen. Appl. Microbiol., Vol. 6, 283-292 (1960)) The present invention medium 8 containing potassium feldspar debris powder, which is soluble in water and does not require pH titration, and potassium deficient medium obtained by removing potassium feldspar from medium 8 at 250 ml each. Compared. Tables 2 and 16 show the compositions of these media used in the test. As a result, the potassium-deficient medium without the addition of potassium compounds contains potassium feldspar debris that is sparingly soluble in water, even though the growth of the microalgae stops and cell death begins. It was shown that the medium 8 of the present invention, which is 5 cm to 8 cm (Table 16), is a medium that provides sufficient growth (Table 17).

By this invention, it becomes possible to greatly increase the degree of freedom in selecting nutrient components in the medium used for culturing in the production of various biomass, and it is possible to reduce the cost of culturing and to reduce unused resources distributed throughout the world. Use is expected.

Claims (11)

A medium for microalgae characterized by having a transparency in a range of 15 cm (15 degrees) to 0.5 cm (0.5 degrees). The medium for microalgae according to claim 1, wherein the degree of transparency is in the range of 10 cm2 (10 degrees) to 1 cm2 (1 degree). The medium for microalgae according to claim 2, wherein the transparency is in the range of 8 cm2 (8 degrees) to 1.5 cm2 (1.5 degrees). The medium for microalgae according to any one of claims 1 to 3, wherein at least one compound that is hardly soluble in water is used as a nitrogen source, a phosphorus source, a potassium source, a sulfur source, a magnesium source, and a calcium source. . The medium for microalgae according to claim 4, wherein a compound hardly soluble in water is used for all of the nitrogen source, phosphorus source, sulfur source, magnesium source, and calcium source. The medium for microalgae according to claim 4, wherein a compound hardly soluble in water is used for all of a nitrogen source, a phosphorus source, a potassium source, a sulfur source, a magnesium source, and a calcium source. The medium for microalgae according to any one of claims 1 to 6, wherein the water content is greater than 0.8. A method for culturing microalgae, comprising using the medium for microalgae according to any one of claims 1 to 7. The culture method according to claim 8, wherein the microalgae belongs to any one genus selected from the group consisting of Chlorella genus, Botryococcus genus, Dunaliella genus, Porphyridium genus, Nostoc genus and Tolypothrix genus. A microalga obtained by the method according to claim 9. A method for removing a compound which is hardly soluble in water from the medium during or after the cultivation by the method for culturing microalgae according to claim 9 and recovering the compound.