JP2013085534A - Method for culturing microalgae - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an extensive large scale method for culturing microalgae, having stability and a high yield, to market by minimizing the effect of an outside environmental temperature to a culturing temperature, in addition avoiding the effects of rainfall to prevent the effluence of cultured material and also surely keeping the supply of nutrient salts in facing the problem of reduction of yield caused by the effects of culturing temperature and rainfall in the outdoor large scale culture of the microalgae under a climate condition in our country.SOLUTION: This stable and high yield extensive large scale culture of the microalgae implemented by using many long tubular containers having a multilayered structure consisting of film layers set by soft water-impermeable polymer film and space layers is provided by maintaining the lowest temperature in the culture as within 8°C from the optimum temperature of chlorophyll fluorescence yield of the microalgae.

Description

  The present invention relates to a method for culturing microalgae. In particular, the present invention relates to a method for large-scale cultivation of microalgae that is carried out outdoors with a simple configuration and operation in a climatic environment where there is a cold season and abundant rainfall.

  Microalgae have been used for alternative foods (microbial proteins), functional foods, aquaculture feeds, water treatment, etc., and open pounds used for large-scale outdoor cultivation from indoor bioreactors depending on the application. Various culture systems have been developed up to the system and raceway system. In recent years, the use of a new microalgae called a fuel crop has been found, and the development of a large-scale and low-cost culture system has become an urgent task.

  In general, the cultivation of microalgae for the purpose of fuel production requires large-scale cultivation on a hectare scale with a sufficiently low production cost. So far, large-scale culture has been developed mainly overseas, and rough release culture (Non-patent Document 1) using open artificial ponds such as the open pond system and raceway system has been the mainstream. However, it was difficult to simply adopt the large-scale culture method.

This is because, as in the Japanese climate, the large-scale cultivation in a natural environment rich in rainfall with four seasons is cultivated due to a decrease in the environmental temperature in the cold season, the temperature difference of the personality, and concentrated rain such as typhoons and heavy rains. It was necessary to fully consider the negative effects of rough outdoor culture, such as dilution and spilling of substances / liquids.
Furthermore, when cultivating microalgae for the purpose of fuel production, in order to achieve an inexpensive unit price of fuel, a broad-scale large-scale culture method with a lower installation cost than the open pound method or raceway method is desirable. It was where

  However, many microalgae culture devices found in conventional Japanese patent literature are bioreactor systems (patents) that are frequently used in fermentation production, etc., mainly consisting of a transparent and solid light-receiving container and an environmental control device. Documents 1 to 5) are the mainstream, and these conventional techniques have a problem that it is extremely difficult to scale up to large-scale culture from the viewpoint of the complexity of the structure of the enclosure and the cost of manufacturing the apparatus.

Moreover, although it is a small number, the outdoor culture system was also proposed like the prior art shown by patent documents 6-9. For example, technology for placing transparent roof sheets on small raceway culture equipment (Patent Documents 6 and 7), technology for installing floating rods and microalgae culture vessels in water (Patent Document 8), slope of terrain like rice terraces There was a culture technique (Patent Document 9) using
However, as with the bioreactor method, there are problems in terms of cost related to scale-up to large-scale culture, and there are problems such as setting a suitable culture temperature and avoiding the effects of rainfall. It was.

  On the other hand, in recent years, an invention has been proposed in which a large-scale culturing in a wide area of water is performed using a culture instrument having a tube structure as in Patent Documents 10 and 11. These conventional techniques using a culture device having a tube structure are based on the fact that a tube made of a polymer film is used as a culture container instead of constructing a water shielding sheet curing used in an open pond system or the like that is a conventional large-scale culture means. As a culture of microalgae.

Japanese Patent No. 2743316 Japanese Patent No. 3035153 Japanese Patent No. 3600250 Japanese Patent No. 4389500 JP 2007-319039 A Japanese Patent No. 3061467 JP-A-5-184347 JP-A-5-236935 Japanese Patent Laid-Open No. 5-284958 JP 2004-81157 A JP 2007-330215 A

Borowitzka, M.A., “Chapter 14: Culturing Microalgae in Outdoor Ponds” in Algal Culturing Techniques, R. Anderson, Editor, AcademicPress, Burlington, MA p. 205-218 (2005).

  However, the conventional techniques (Patent Documents 10 and 11) using the tube method described above are intended to reduce and remove water pollutants such as nitrogen and phosphorus in an aqueous environment. Since the production of was only a secondary in the water treatment, there were various inconveniences when it was carried out mainly for large-scale cultivation of microalgae.

That is, the culture temperature in the prior arts of Patent Documents 10 and 11 is the same as the ambient temperature, and has a problem that mass culture in the cold season is extremely difficult.
In addition, the supply of nutrients depends on the inflow from the installed water system through the functional membrane into the tube. Therefore, if the nutrient concentration in the external environment decreases due to rainfall, etc., the nutrients are naturally supplied to the culture apparatus. However, there is an inevitable problem that becomes a rate-limiting factor for culture and proliferation.

  In general, the culture techniques of Patent Documents 10 and 11 are microalgae culture methods using an inexpensive tube method. However, as in the case of the conventional loose-pond culture techniques such as the open pond method and the raceway method, Sufficient consideration has not been given to the effects of rainfall dilution such as nutrient salts, and it has not been a technology to solve various problems derived from the climate in extensive large-scale culture in Japan.

  The purpose of the present invention is to minimize the influence of the external environment temperature on the culture temperature, in relation to the problems related to the decrease in yield due to the influence of the cultivation temperature and rainfall in outdoor large-scale cultivation of microalgae under the climatic conditions in Japan, In addition, it is to provide a stable and high-yield large-scale culturing method with a stable and high yield that avoids the influence of rainfall and prevents the outflow of the culture and reliably supplies nutrients.

  That is, with an inexpensive culture vessel made of a tubular film that is impermeable to moisture and nutrients, and a simple temperature control system for the purpose of controlling the temperature of the culture vessel, avoiding rain dilution and appropriate control of the culture temperature In addition to providing a low-cost, large-scale, large-scale culturing method characterized by the above, the details of the relationship between the culture temperature of microalgae and the photoenergy yield of photosynthesis in relation to the temperature control are clarified, and high light energy The purpose is to provide the market with a temperature control method at the time of culturing in which a high yield of the target product can be expected under the yield conditions.

The gist of the present invention for achieving the object described above resides in the inventions of the following items.
First, the present invention according to claim 1 is a part of a tubular container having a multilayer structure composed of a film layer and a space layer, and having a multilayer structure of at least three layers, which is set with a flexible water-impermeable polymer film. A method for culturing microalgae through a culture solution in a space layer of the microalgae, which is obtained from a variable fluorescence value (Fv) obtained by subtracting a minimum fluorescence analysis value (Fo) from a maximum fluorescence analysis value (Fm) of chlorophyll in the microalgae. The optimum temperature at which the chlorophyll fluorescence yield (Fv / Fm) is maximized is obtained, and the minimum temperature at the time of control is set as the optimum temperature for the chlorophyll fluorescence yield of the microalgae as the culture control temperature during photosynthesis. On the other hand, it is a method for cultivating microalgae characterized by maintaining within 8 degrees Celsius.

  Moreover, the present invention according to claim 2 is the method for culturing microalgae according to claim 1, wherein the space layer through which the culture solution passes is maintained in a thin flat structure.

  Further, the present invention according to claim 3 is characterized in that the temperature adjustment fluid is passed around the space layer through which the culture solution passes, and the temperature of the space layer through the culture solution is adjusted. It is the culture method of the described microalgae.

  In addition, the present invention according to claim 4 is characterized in that the culture of the microalgae is carried out by combining not only photoautotrophic culture but also heterotrophic culture using organic matter. The method for culturing microalgae according to 1, 2 or 3.

  According to the method for culturing microalgae according to claim 1 of the present invention, by using a tubular container having a multilayer structure composed of a film layer and a space layer, which is set with a flexible water-impermeable polymer film. In addition, it is possible to prevent inadvertent dilution of the culture solution and the concentration of microalgae due to rainfall and the like during outdoor rough cultivation.

  The avoidance of the above-mentioned rainfall effect is due to the function of the water-impermeable outermost layer film, but as a secondary function of the other outermost layer film, it is due to inadvertent breakage of the tube that partitions the space layer through which the culture solution passes. Prevention of outflow of culture solution and microalgae, protection thereof, and the like can be mentioned, and in combination with these secondary functions, more stable growth of microalgae can be achieved.

In particular, when microalgae are cultured as fuel crops to produce oils and fats, hydrocarbons, etc., leakage of these fuel components and nitrogen and phosphorus in the medium to the environment is not only an economic loss in culture, but also public water areas It is also necessary to consider environmental impacts such as contamination and soil / groundwater contamination.
Similarly, when the microalgae to be used is a gene recombinant, etc., it is necessary to consider the environmental impact against the leakage of the recombinant. Furthermore, the multilayer structure set by the flexible water-impermeable polymer film according to the present invention for puncture breakage or structural destruction of culture vessels due to birds and beasts or flying objects, which is often a problem in practice, It is possible to extremely effectively protect the encapsulated algal culture.

  In general, by using a tube-shaped container having a multilayer structure composed of a film layer and a space layer, which is set by a flexible, water-impermeable polymer film according to the present invention, the environmental influences in such extensive culture can be multiplied. It is possible to prevent and ensure stable microalgae culture and production of the target substance.

  In addition, as described above, in the cultivation of microalgae using a tube-shaped container having a multilayer structure, the minimum temperature is maintained within 8 degrees Celsius with respect to the optimum temperature for chlorophyll fluorescence yield as the culture temperature during photosynthesis. By doing so, a high yield of the target product can be achieved.

Here, the idea about culture management using chlorophyll fluorescence yield is shown.
For example, the purpose of cultivating microalgae as a fuel crop is primarily to produce organic substances such as lipids, hydrocarbons, and carbohydrates (via hydrogen / ethanol) as target products. In general, the target product can significantly improve the intracellular content of the target product by transferring the microalgae under nitrogen-deficient conditions and reducing the reproduction of the microalgae.
This is nothing but a translocation control operation in which the energy that has been oriented to reproductive growth is more oriented to the production of organic matter as the target product and the intracellular content is increased while maintaining the light energy yield. In other words, rather than paying attention to commonly used reproductive growth indicators (cell number change, growth rate, etc.), culture setting / management focusing on light energy yield (chlorophyll fluorescence yield, etc.) is intended. Therefore, a high yield of the target product can be expected.

  In the present invention, focusing on the relationship between the light energy yield and the culture temperature, by clarifying the details, the temperature setting at the time of culture at which a high yield of the target product can be expected under high light energy yield conditions. And came to provide a temperature control method.

  According to the method for culturing microalgae according to claim 2 of the present invention, the microalgae are cultured at high density under light autotrophic conditions by keeping the space layer through which the culture solution passes in a thin flat structure. can do.

  Microalgae grow by photosynthesis under photoautotrophic conditions, but when reaching a certain culture density, further photoautotrophic growth becomes difficult due to mutual light shielding between the grown alga bodies. In other words, since the upper limit of the biomass amount of the microalgae that can be grown in a certain light irradiation area is naturally limited, the thinner the culture container with a shorter optical path length, the higher the culture density.

  This high-density culture can sufficiently reduce the amount of culture solution without reducing the production volume of alga bodies, so that alga bodies can be collected more efficiently, the amount of drainage can be sufficiently reduced, etc., and alga bodies with good cost performance Production can be carried out.

  In addition, the above-described high-density culture has an advantage of facilitating the operation of suppressing the growth of protozoa or the like as predators of microalgae. That is, many of the protozoa are aerobic, require oxygen for their growth and activity, and die under anaerobic conditions that are anoxic. On the other hand, many microalgae require oxygen for growth and activity like protozoa, but show resistance under anaerobic conditions. In the culturing process, by setting anaerobic conditions in part, the growth of protozoa that are predators of microalgae can be significantly reduced.

According to the method for culturing microalgae according to claim 2, it is possible to culture the microalgae with high density, and this microalgae high-density culture easily sets anaerobic conditions in the culture process. can do.
That is, microalgae under dark conditions cannot perform photosynthesis that generates oxygen, but only breathe that consumes oxygen. If the microalgae concentration is high here, oxygen in the culture solution is consumed according to the concentration, so the higher the microalgae concentration in the culture solution, the easier the transition to the anaerobic state becomes easier. .
In addition, the higher the concentration of microalgae in the culture solution, the easier it is to consolidate the culture, and the simple operation of shifting to dark conditions, i.e. recovery at night or in a light-shading collection container, etc. And the proliferation of protozoa can be effectively reduced.

By the way, the microalgae culture maintained in an anaerobic state can fermentatively produce fuel raw materials such as hydrogen and ethanol depending on the culture species. Even when the purpose is to produce these fuel raw materials, high-density culture is preferable because the fuel raw materials can be easily recovered and anaerobic conditions can be easily set.
During the daytime, a large amount of organic matter is accumulated in the cells by photosynthesis, and after completion of the daytime culture, the high-density culture is accumulated in a specific container, and quickly transferred to an anaerobic state, so that fuel raw materials such as hydrogen and ethanol are used. It can be fermented.

  According to the method for culturing microalgae according to claim 3 of the present invention, the temperature of the culture solution can be adjusted by passing a temperature-controlled fluid around the space layer through which the culture solution passes.

  By this temperature control mechanism, as described in claim 1, by maintaining the minimum culture temperature at the time of photosynthesis within 8 degrees Celsius with respect to the optimum temperature of the chlorophyll fluorescence yield, High yield of the target product can be expected. In addition, it is possible to minimize damage to the culture apparatus due to freezing due to a decrease in temperature during the cold season.

The temperature control fluid may be either gas or liquid. However, in the case of gas, unlike the drainage, the exhaust is not costly. Good algal production can be carried out.
Further, when the temperature control gas is passed through the space layer in contact with the outermost layer film, even if a slight crack damage occurs in a part of the outermost layer film, the outermost layer film has the supply pressure of the temperature control gas. It is also possible to maintain the tube structure, and it is possible to adjust the temperature of the culture having both durability and maintainability of the structure.

  According to the method for culturing microalgae according to claim 4 of the present invention, the culture of the microalgae is not limited to photoautotrophic culture but also mixotroph that combines heterotrophic culture using organic matter. By carrying out the culture, algal bodies can be produced more efficiently and at a high concentration.

In particular, as described in claim 2 of the present invention, when the space layer through which the culture solution passes is kept in a thin flat structure and high-density large-scale culture of microalgae is performed under light irradiation, the microalgae to be produced The culture concentration can be maintained at a significantly higher concentration than the heterotrophic microorganism concentration as a contamination.
If we shift to heterotrophic culture using organic matter on this assumption, most of the supplied organic matter will be consumed by microalgae and will ultimately contribute greatly to the increase in the yield of the target algal bodies. In general, the addition of organic matter is particularly effective.

  In the conventional microalgae culture using organic matter, the order of heterotrophic aseptic tank culture in the previous stage and photoautotrophic culture in the subsequent stage has been favored, especially for large-scale culture. In the culture described above, it is obvious that it is difficult to operate a system with a complicated aseptic operation as in the prior art from both the initial / running costs.

  As in the present invention, in culture aimed at large-scale culture, high-density culture under photoautotrophic is performed as the previous stage, and heterotrophic culture is performed as the subsequent stage, so that the orientation of organic substance supply can be changed to bacteria, etc. Therefore, it is possible to achieve large-scale culture with a high yield that is sufficiently directed to the target microalgae side, not to the contamination side.

It is drawing which shows the concept of the culture system which is this invention. It is drawing which shows the example of embodiment of the tubular container which is this invention. It is a graph which shows the 1st pilot verification test result. It is a graph which shows the 2nd pilot verification test result.

  Hereinafter, embodiments representative of the present invention will be described.

  The method for culturing microalgae according to the present embodiment is characterized by using a tubular container having a multilayer structure composed of a film layer and a space layer, which is set with a flexible water-impermeable polymer film.

  It is desirable that the tubular containers having the multilayer structure are aligned in the horizontal direction, for example, and microalgae are cultured under efficient sunlight receiving conditions. In this case, the diameter, length, and the like of the tube-like container are arbitrary, and a continuous long body is aligned and installed at a predetermined place so as to obtain a desired culture volume.

The concept of the culture system in the present invention is shown in FIG. 1, and the details of the tubular container are shown in FIG.
This culture system has a unit structure in which one end of a tube-shaped container (group) 1 having a multilayer structure made of a flexible impermeable polymer film is connected to the supply device 2 and the other end is connected to the recovery device 3. Furthermore, it is based on the structure which can culture | cultivate a micro algae in this tubular container 1 via the transfer power 4, such as a pump.
In addition, according to culture | cultivation, it is good also as a multistage system by connecting several units via the transfer power 4. FIG. The supply unit 2 of the first stage unit and the recovery unit 3 of the final unit can be connected via a transfer power 4, and the system can be implemented as a loop (/ mixed) culture with a loop structure. It is good also as a plug flow type culture, without making it a circulation loop structure.
That is, the tubular container (group) 1 having a multi-layer structure has a unit structure in which one end communicates with the feeder 2 and the other end communicates with the recovery device 3, and further via transfer power 4 such as a pump. As long as the system is configured so as to enable large-scale cultivation of microalgae in the tubular container 1, the shape and combination thereof are not questioned.

Further, in the present invention, the temperature control of the culture, which is particularly important, may be performed by adjusting the temperature of the culture solution through a temperature control fluid around the space layer through which the culture solution according to claim 3 passes ( You may implement temperature control via the simple water tank etc. which were used in the below-mentioned Example of A and B) of FIG. More simply, a closed water area such as a paddy field can be used like a temperature-controlled water tank.
Furthermore, if the site can use waste heat abundantly, a sufficient temperature-controlled fluid can be passed around the space layer through which the culture solution passes, so the culture system can be used in sea areas, river areas, lake areas. It can also be installed in open waters where temperature control is generally difficult.

  In addition, when installing in closed system waters, such as the above-mentioned paddy field, the tubular container 1 is sent by supplying air to the space layer through which the culture solution in the tubular container 1 having a multilayer structure passes or to another space layer. Is kept floating on the water surface 11 like a float, so that the microalgae culture solution present in the space layer through which the culture solution passes can be held at a position with good photosynthesis yield suitable for sunlight reception. (C in FIG. 2).

  Furthermore, when the temperature-controlled water around the tube-shaped container 1 is clear, the function of a part of the space layer required for ascent is abolished without particularly considering ascent for the purpose of optimizing sunlight reception. Instead, a multilayer film suitable for clear water can be obtained by using a polymer film having a surface adhesion preventing function such as aquatic organisms as the outermost layer. At this time, the outermost layer polymer film and the polymer film used as a partition of the space layer through which the culture solution is passed are adhered and laminated like a laminate film (a three-layer structure (the outermost layer film layer and the space layer). A tube-shaped container having a three-layer structure of a partition layer and a space layer through which a culture solution passes may be used (D in FIG. 2).

  By the way, as an incidental operation of the culture system according to the present invention, ventilation is performed not only in the tube-shaped container (group) 1 but also in the supply device 2 or the recovery device 3 to degas the dissolved oxygen gas in the culture solution. Carbon dioxide gas can be dissolved. Moreover, it is good also as a substitute of this ventilation | gas_flowing by using what has a gas exchange function as a polymer film used as a partition of the space layer which lets a culture solution pass. Furthermore, if necessary, supplementation according to the consumption of nutrients can be carried out through a part of the culture system.

  In addition, if the installation place of the culture system of the micro algae of this invention is on the ground, a flat ground may be sufficient and an inclined ground may be sufficient. In the case of an inclined land, a gentle thin layer of the culture fluid flow can be easily formed by the inclination, so that high-density culture of microalgae can be easily achieved.

Moreover, the flexible water-impermeable tube-like container 1 according to the present invention is a film-like material in which an organic polymer is stretched, cooled, and heat-set in the longitudinal direction and / or the width direction as necessary. It is manufactured by a melt extrusion molding method or the like.
Examples of the organic polymer include polyethylene, polypropylene, polyethylene terephthalate, polyethylene-2, 6-naphthalate, nylon 6, nylon 4, nylon 66, nylon 12, polyvinyl chloride, polyvinylidene chloride, polyvinyl alcohol, all Aromatic polyamide, polyamideimide, polyimide, polyetherimide, polysulfone, polyphenylene sulfide, polyphenylene oxide, nylon, fluororesins and the like can be mentioned. Alternatively, an organic polymer film obtained by copolymerizing or blending the organic polymer with another organic polymer in a small amount, or laminating by a co-extrusion method or a laminating method may be used.

  By the way, the organic polymer may be added with known additives such as ultraviolet absorbers, antistatic agents, plasticizers, lubricants, colorants, pigments having a wavelength conversion function, etc. Those having a transmittance of at least 60% or more are preferred.

In addition, the tube-shaped film used in the present invention is subjected to corona discharge treatment, glow discharge treatment, and other surface roughening treatment prior to the lamination of the thin film layer, unless the object of the present invention is impaired. Moreover, anchor coating treatment, printing, and decoration may be performed.
In particular, when the incident light is effectively used for culturing microalgae, it is preferable to use a mirror film sticking decoration or white printing that promotes light reflection on a part of the tubular film. In addition, a sticking decoration of a white heat insulating material such as expandable polyethylene for the purpose of enhancing heat retention and light reflection during culture is more preferable.

  In addition, the tube-like film used in the present invention preferably has a thickness in the range of 50 to 500 μm, more preferably in the range of 50 to 200 μm. And a film having impact, tear, and puncture strength.

  By the way, the relative positional relationship between the respective tube-like films constituting the above-mentioned multilayer may be in a state where all of them are in close contact like a single layer as in a laminate structure, or each of them is accompanied by a space layer. The part may be in contact. As long as it has a multilayer structure of at least three layers consisting of a film layer and a space layer set by a flexible water-impermeable polymer film, its shape and positional relationship are not questioned.

  Here, as the microalgae that can be used in the present invention, “photosynthesis that generates oxygen”, which is the definition of algae prescribed in Mitsuo Chihara “Biology of Algal Diversity” / Yuhuabo / (1999). Among all the remaining organisms excluding moss plants, fern plants, and seed plants from among the organisms that perform " Examples include microorganisms having a size of several μm to 200 μm. For example, chlorella, spirulina, etc. are microalgae that are generally known, but these microalgae contain part of eukaryotes represented by chlorella and part of prokaryotes represented by spirulina. Taxonomically, it is a broadly spread category.

In addition, the waste heat source is present in steel mills, non-ferrous metal factories, chemical factories, paper mills, automobile factories, food factories, pharmaceutical factories, refineries, power plants, glass factories, cement factories, spinning factories, hot spring facilities, etc. Boiler waste heat, industrial furnace waste heat, waste incinerator waste heat, engine / turbine waste heat, combustion exhaust gas waste heat, geothermal power generation waste heat, hot spring waste heat, and the like.
These waste heat sources may be used as they are, or another fluid may be heated through a heat exchanger or the like to secondary use of the heat source. Needless to say, even if it is not the waste heat source listed above, it can be utilized in the present invention if there is a heat source that can be used at low cost.

  By the way, as an organic substance source used for heterotrophic growth of microalgae, organic waste liquid, saccharides derived from unused biomass, and the like can be used.

This organic waste liquid is a waste liquid containing an organic substance treated by a normal aerobic biological treatment method, but may contain a non-biodegradable organic substance or an inorganic substance. Examples of such organic waste liquid include sewage, human waste, food factory wastewater, and other industrial waste liquids.
Moreover, as an unused biomass as a raw material of saccharides produced through acid hydrolysis technology, specifically, for example, plant-generated materials (weeds, crop herbs, bark, thinned chips, rice husks, sawdust, etc.), derived from food production Vegetable oil lees (oil lees, rice lees, bran and corn steep liquor, soybean oil lees, corn oil lees, sesame oil lees, legume oil lees, rice lees oil lees, waste molasses, etc.), fermentation residues (soy sauce lees, sake lees, beer lees, shochu lees etc.), Organic geology (peat, lignite, lignite, peat, etc.) can be mentioned.

  In addition, the above-mentioned organic substance source is not restricted to organic waste liquid or saccharides derived from unused biomass. Needless to say, if the supply cost and the target product yield of microalgae are appropriate, the present invention can be used.

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

In this experiment, we clarified the details of the relationship between the culture temperature of microalgae and the photoenergy yield of photosynthesis, and examined the preset temperature during the culture where high yields of the target product can be expected under high photoenergy yield conditions. .
In addition, all the microalgae groups of the test contain chlorophyll a as a reaction center for photosynthesis, but the diversity of antenna pigments is given, and the culture temperature and the photoenergy yield of photosynthesis under a wide classification system of microalgae. In relation to the relationship (in the experiment, the chlorophyll fluorescence yield was measured and used as an index of the photoenergy yield of photosynthesis), the evaluation on the interclass distribution was also performed.

The test procedure is shown below.
(A) As test microalgae, diatom (Ketoceras), haptoalgae (Isolysis), prasinoalgae (Tetracermis), true eye spot algae (Nannochloropsis), red algae (Porphyridium), green algae (Nanochloris), Eight kinds of axle algae (Sticococcus) and cyanobacteria (Cinecocystis) were used.

(B) Preliminary culture was performed by aeration culture using a 0.5 L flat flask at a photon flux density of 1.2 mmol · m ⁻² · s ⁻¹ under irradiation with a metal halide lamp. As the medium, IMK medium (manufactured by Wako Pure Chemical Industries) was used, and in the case of marine algae, Daigo artificial seawater SP (manufactured by Wako Pure Chemical Industries) was used as the base of the medium. The pre-culture temperature was the optimum temperature for the chlorophyll fluorescence yield of each microalgae.

(C) Subsequently, the precultured microalgae broth is collected at the later stage of the logarithmic growth phase, and the collected culture broth is divided into 5 parts. From the optimum temperature of each microalgae, 0 degrees Celsius, 5 degrees, 8 degrees In a constant temperature culture tank set to a temperature reduced by 10 degrees and 15 degrees, the light quantum flux density was adjusted to 1.2 mmol · m ^-2 · s ^-1 under irradiation with a metal halide lamp. After 2 hours of acclimatization, samples acclimated for 30 minutes or more under the optimum temperature / dark conditions for each chlorophyll fluorescence yield were used as test samples for chlorophyll fluorescence yield measurement.

  (D) The chlorophyll fluorescence yield (Fv / Fm = (Fm-Fo) / Fm) was measured using PAM-2000 (manufactured by Waltz) and suspension probe KS-101 (manufactured by Waltz). Fo and Fm were measured and calculated.

The experimental results are shown in Table 2.

  As is clear from Table 2, all the microalgae showed a tendency that the decrease in the fluorescence yield became more remarkable as the exposed low temperature level was larger. In a low-temperature environment where the chlorophyll fluorescence yield is reduced by 5 degrees Celsius from the optimum temperature, the chlorophyll fluorescence yield ratio remains at about 90% of the original, and in a low-temperature environment where the chlorophyll fluorescence yield is reduced beyond 8 degrees Celsius, However, it has been found that there is an obstacle that drops to about 70% or less of the initial value.

The daily temperature difference in clear weather in Japan often exceeds 10 degrees Celsius (Note that the environmental temperature in this case is not just mere temperature, but also includes ground temperature, water temperature, direct sunlight temperature, etc.) Refers to the culture environment temperature). It was found that, in such a large-scale outdoor large-scale culture under an environment having such a temperature difference, precise control of the culture temperature contributes significantly to algal body production. In addition, it was found that culture with excellent light energy yield is possible by setting the temperature adjustment range within about 8 degrees Celsius, preferably within 5 degrees Celsius with respect to the low temperature side.
This phenomenon is not limited to a specific microalgal taxonomic group, but has universality in which all the test microalgae including at least chlorophyll a as a reaction center have a uniform tendency. Special mention.

  Subsequently, the effectiveness of this culture method on a pilot scale was verified by dividing it into two experiments. The outline of the first test is shown as Example 2, and the outline of the second test is shown as Example 3 below.

In this experiment, we verified the effectiveness of temperature control and thin layer gradient culture.
(A) A gutter-like container with a width of 60 cm, a depth of 60 cm, a length of about 11 m, and a water collecting mass on both sides using a commercially available U-shaped side gutter and a white waterproof sheet on a 10 m x 15 m outdoor site Four lines were created. The side groove-like container was water-filled according to the conditions of the experimental section and used according to the conditions of each test section, such as when used as a thermostatic bath or when water filling was not performed. Moreover, the culture apparatus installed the supply device and the collection | recovery device at the both ends of a 10-m tube-shaped container, respectively, connected each supply / collection device with the pump piping, and built the circulation culture system for every system | strain.

  (B) The test tube-shaped container has a four-layer structure in which a cylindrical polyethylene tube of 30 cmφ is encapsulated in a cylindrical polyethylene tube of 50 cmφ and has two film layers and two space layers. When temperature control is performed using the tube, the culture medium in the 30 cmφ tube is passed through the temperature control water or temperature control air using the space layer formed between the tubes as a temperature control jacket. The setting was kept at ± 4 degrees. The detailed conditions of the test section using each water tank are as follows.

(Test Zone 1): No tilting of the bottom of the water tank / no water tank water tension / space layer temperature control (Test Zone 2): 2% of the bottom of the water tank / no water tank water tension / space layer temperature control (Test Zone 3): Water tank Bottom slope 4% / No water tank water tension / Spatial temperature control (Test area 4): No water tank bottom tilt / Water tank water tension / No temperature control

  (C) As a microalgae to be tested, green algae (chlorella sp.) From hot springs having an optimum temperature of chlorophyll fluorescence yield around 30 degrees Celsius was used, and MC medium was used as the medium. In addition, as the culture progresses, the concentration of nitric acid / phosphoric acid / potassium in the culture solution is confirmed using positive / anion chromatography, and when each component is reduced, potassium nitrate and phosphate are added to the culture. Added.

(D) The above-mentioned pre-culture of microalgae used for the outdoor culture test was performed indoors. Pre-culture is aeration culture using a suspended cylindrical polyethylene tube with a length of 1.5 m and 30 cmφ under irradiation with a metal halide lamp at a photon flux density of 0.8 mmol · m ⁻² · s ⁻¹ . Went. The pre-culture temperature was set to 25 degrees Celsius in the laboratory and kept constant.

  (E) Using a pre-culture that reached the latter half of the linear phase, each test section was inoculated with microalgae to a dry weight of 0.1 g per liter of culture solution, and an outdoor culture test was conducted. . In addition, the total amount of culture in the test sections 2 and 3 having an inclination was 150 L, and the total amount was 700 L in other cultures. The aeration was performed at 1 vvm with respect to the amount of culture solution in the supply / collector with air containing about 5% (v / v) carbon dioxide concentration.

  (F) The culture was performed by fed-batch culture. That is, at night, 50% of the culture solution was extracted from each test section every 5 days, the algal bodies were collected by centrifugation, and the same amount of fresh medium as the extracted culture solution was supplied to the culture. A 90-day outdoor culture test was conducted.

(Notices)
The tube-shaped container having a multi-layer structure was a cylindrical polyethylene tube, and the temperature of the culture solution was controlled through temperature-controlled water in the space layer formed between both tubes, but within one week after the start. In addition, several portions of the outermost polyethylene tube were damaged, and water of temperature-controlled water leaked. In addition, any damage location was to the outermost layer, and damage was not seen by the inner tube filled with the culture solution. Judging from the state of injury and the situation at the time of the injury, it was judged that there was a high possibility of puncture damage due to crows or the like.
As a result, it was judged that temperature-controlled air was easier to handle than temperature-controlled water in temperature control of the culture solution using the space layer formed between both tubes. We decided to carry out.
Even after changing to temperature-controlled air, puncture / scratch damage, which seems to be caused by crows or small animals, was occasionally observed, but it did not hinder the temperature control of the culture solution by temperature-controlled air, We were able to complete the first and second outdoor culture tests for a total of 180 days using this method. (In addition, there was one puncture accident that could be attributed to a flying object in a strong wind. Only the outermost polyethylene tube was damaged.)

  The experimental results are shown in FIG. As is clear from FIG. 3, from the comparison of test groups 1 and 4, stable algal body production was observed in the test system in which the temperature of test group 1 was controlled. In test group 4, the concentration of microalgae in the culture solution decreased immediately after the start of the test, and stable production was not observed throughout the test period.

  Moreover, in the comparison between the test groups 1 and 2 and 3, the thin layer culture system in which the bottoms of the water tanks of the test groups 2 and 3 are inclined has a higher density of algal body production than the test group 1. Observed.

  In addition, in test plots 2 and 3 in which good productivity was observed, algal cells reaching a higher concentration in test plot 3 having a slope of 4% than test plot 2 having a slope of 2% Production was observed. It was found that the concentration reached in high-density culture changes with the inclination angle.

Next, the second pilot demonstration test procedure is shown below. In this test, verification of the external temperature control method and comparison between the present invention and the prior art using a culture instrument having a tube structure were performed. The preliminary culture, algae used, medium type, nutrient salt addition method, inoculation method, culture solution extraction method, alga body collection method, temperature control method, and the like were carried out according to the first test.
Details different from the first test are shown below together with the conditions of each test section.

(Test section 5): No tilting of water tank bottom / No water tank water tension / Space layer temperature control (Test section 6): Water tank water tension / Polyethylene double tube used / Floating type / Water tank temperature control (Test area 7) : Aquarium water tension / Polyethylene single tube / No temperature control

  The test conditions for the test group 5 were the same as those for the first test group 1. In the test group 6, water for temperature adjustment was filled in the side groove-like water tank, and the tube-shaped container was floated on the water surface like a floating ring by ventilating the space layer of the tube-shaped container. In addition, although the test group 7 imitates the prior arts of Patent Documents 10 and 11, it sets the conditions for supplying nutrients without excess or deficiency using an impermeable tube, and more than the original prior art. Also, more favorable culture conditions were set.

  The experimental results are shown in FIG. As is clear from FIG. 4, from the comparison of test groups 5 to 7, stable algal body production was observed in the test systems of test groups 5 and 6 according to the present invention, while test group 7 was the first test. As in test group 4 in FIG. 4, the culture solution concentration decreased immediately after the start of the test, and stable production was not observed throughout the test period.

  In addition, based on the results of the test sections 5 and 6 that showed good productivity, in these temperature control methods, even if the temperature control air is passed through the space layer of the tube-shaped container, the temperature-controlled water is generated from the periphery of the tube-shaped container. It was suggested that the method of adjustment using the

  In this experiment, we examined the productivity improvement effect by combining not only photoautotrophic culture but also heterotrophic culture using organic matter in microalgae culture.

The test procedure is shown below.
(A) As a source of microalgae, a marine integrated culture that has been subcultured by photoautotrophic was used. This enriched culture is a single algae culture mainly composed of green algae chlorella species as microalgae, but has not been sterilized, and protozoan species existed in addition to bacteria.

(B) Preliminary culture was performed by aeration culture using a 0.5 L flat flask at a photon flux density of 1.2 mmol · m ⁻² · s ⁻¹ under irradiation with a metal halide lamp. The medium used is IMK medium (manufactured by Wako Pure Chemical Industries) and Daigo artificial seawater SP (manufactured by Wako Pure Chemical Industries), air aerated with 5% carbon dioxide at 1 vvm, and cultured in a constant temperature water bath at 25 ° C. Went.

  (C) Subsequently, the pre-cultured microalgae were harvested in the first half of the linear phase, centrifuged and then inoculated into a predetermined incubator so that the concentration was about 1 ml per liter (PCV concentration). Two types of thin-layered flat flask-like culture containers (hereinafter abbreviated as thin-layer culture containers) prepared by laminating two acrylic plates with an interval of 5 mm or 35 mm were prepared as culture containers.

  (D) The following test sections were set using the two types of thin-layer culture vessels.

(Test section 8): 5 mm thin layer culture vessel (added organic matter later)
(Test section 9): 35 mm thin layer culture vessel

In the post-addition of the organic substance, after confirming that the growth of microalgae in the test section 8 was stopped in the preceding photoautotrophic culture, the molasses was added to the medium so as to be 5 g per liter. .
In addition, as the culture progresses, the concentration of nitric acid / phosphoric acid / potassium in the culture solution is confirmed using positive / anion chromatography, and when each component is reduced, potassium nitrate and phosphate are added to the culture. Added. Furthermore, the consumption of saccharides in the medium was confirmed using the phenol sulfuric acid method, and waste molasses was added according to the consumption.

  As a growth index of microalgae and the like in the above culture, the dry weight concentration is measured in the culture under the photoautotrophic condition, and the fluorescence of the chlorophyll and the nucleic acid staining reagent DAPI is observed in the fluorescence under the heterotrophic condition. The microalgae and other contaminating microorganisms were distinguished by direct microscopic observation using, and each was counted.

  The experimental results are shown in Table 3.

As is clear from Table 3, from the comparison of test groups 8 and 9, growth of microalgae up to high density was observed in test group 8 using a 5 mm thin-layer culture vessel with a short optical path length. However, the algal body production was almost the same in both test sections. Results It was found that the final algal body production was almost the same in both test groups if the irradiation light intensity was the same under the photoautotrophic condition.
It should be noted that, under a certain amount of irradiation light, if the algal body production is substantially the same, the electrical energy required for circulation and centrifugation of the culture solution, and the amount of drainage is generated according to the culture volume, It was suggested that the smaller the culture volume, the more efficient large-scale culture can be performed.

  In addition, Table 4 shows the number of microalgae after addition of the organic matter in Test Zone 8 and the number of other contaminating microorganisms (bacteria, protozoa, etc.). Until the second day after the addition of organic matter, the number of microalgae and the number of contaminating microorganisms showed an increasing tendency, but thereafter, the increase in the number of contaminating microorganisms slowed down and was repeated. On the other hand, only the number of microalgae showed a steady increase, reaching a final concentration of 183 ml (PCV concentration) per liter.

In addition, although the verification using various microalgae similar to the above Example 4 and the verification of the effect of adding organic substances for each growth stage have been eagerly advanced, there is a certain tendency with respect to the behavior of contamination microorganisms in each case. It was found that there was a problem in reproducibility when considering the behavior of the entire microbial community (data not shown).
However, it should be noted that, as a tendency common to all the related tests that have been verified, when organic substances are added, the yield of microalgae is significantly improved compared to culture under photoautotrophic conditions.

  The embodiments of the present invention have been described above, but the specific configuration is not limited to the above-described embodiments, and modifications and additions within the scope of the present invention are included in the present invention. Needless to say.

  In the large-scale cultivation of microalgae outdoors, the present invention minimizes the influence of the external environment temperature on the cultivation temperature, and additionally prevents the influence of rainfall to prevent the outflow of the culture and ensures the supply of nutrient salts. It is particularly applicable when carrying out a stable and high-yield crude large-scale culture method.

1 ... Tube-shaped container (group)
2 ... Supplyer 3 ... Recovery device 4 ... Transfer power 5 ... Film layer (outermost film layer)
6 ... Spatial layer (space layer through temperature control fluid)
7 ... Film layer (film layer that partitions the space layer)
8 ... Spatial layer (spatial layer through the culture medium)
9 ... Thermal insulation sheet 10 ... Ground 11 ... Water surface 12 ... Spatial layer (space layer that fills the gas and aims to float on the water surface)
13 ... film double layer (laminate film-like double layer)
14 ... Water bottom

Claims (4)

A microalgae to be implemented through a culture solution in a part of a spatial layer in a tube-shaped container having a multilayer structure of at least three layers, which is composed of a film layer and a spatial layer, which is set with a flexible impermeable polymer film A culture method of
The optimum temperature at which the chlorophyll fluorescence yield (Fv / Fm) is maximized, which is obtained from the fluctuation fluorescence value (Fv) obtained by subtracting the minimum fluorescence analysis value (Fo) from the maximum fluorescence analysis value (Fm) of chlorophyll in the microalgae. Seeking
Furthermore, a method for cultivating microalgae, characterized in that, as a culture control temperature during photosynthesis, the minimum temperature during control is maintained within 8 degrees Celsius with respect to the optimum temperature for the chlorophyll fluorescence yield of the microalgae.   The method for culturing microalgae according to claim 1, wherein the space layer through which the culture solution passes is maintained in a thin flat structure.   The method for cultivating microalgae according to claim 1 or 2, wherein the temperature of the space layer through which the culture solution is passed is adjusted by passing a temperature-controlled fluid around the space layer through which the culture solution is passed.   The culture of microalgae according to claim 1, 2 or 3, wherein the culture of microalgae is carried out in combination with not only photoautotrophic culture but also heterotrophic culture using organic matter. Method.

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