TWI294913B - Liquid containing diatom and method for culturing diatom - Google Patents

Description

1294913 玖, the invention description: [Technical Field] The present invention relates to the use of material filling, pearls and other shellfish, prawns, grass shrimp and other crustaceans In the second, the algae, in detail, is a method of cultivating the liquid and the algae containing the algae. ^ [Prior Art] Among the feeds for aquaculture seedlings such as shellfish, crustaceans, and echinoderms, they have recently been classified as the algae of the microalgae such as Chaetoceros and Phaeodactylum. Calcareous horny horns (ca/c/ir(10)s), Chaetoceros gracilis (gMd/b), etc. are highly attractive. For example, C. oxysporum (C. (3) Chemicals (10) illusion has a long flagella in its four corners, and because the diameter (length) of the horned algae is only about 3 to 6, it is quite suitable for the aquatic seedlings. The feed. The microalgae, the trace minerals and vitamins contained in the algae, are superior to other plant plankton and are balanced. In addition, the algae have ΕρΑ (廿 pentadecene: a polyvalent unsaturated fatty acid) and Useful fatty acids such as DHA (dihexamethylenedicarboxylic acid; a polyvalent unsaturated fatty acid). For the above reasons, microalgae, algae are a highly evaluated feed. However, the work of stably cultivating microalgae is very Difficulties. For example, "Study on the breeding technology of the competition, (1) research on the production of salt water seedlings, cultivation of biological feeds (hereinafter referred to as the literature 1), and large-scale cultivation techniques of microalgae in the specific research and development promotion of the Heisei 4 years (1992). According to the report of the Development Research Report (with τ $ for the literature 2), the upper limit of the cell concentration of the genus horned horn and the vegetable 1294913 (C, like cerevisiae) based on the previous culture method is about 3 ·〇χ 1 0 13 cells/m3. The cell concentration refers to the number of cells per 1 m3 of the culture solution. Since the upper limit of the cell concentration of C. cerevisiae is about 3·〇χ10π cells/m3' according to the previous culture method, It is not ideal, so there is a great need for a liquid (culture medium, culture solution) and culture method for cultivating Chaetoceros in a stable and high concentration. SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and its object is to provide an energy source. A kind of microalgae, a high concentration and stable culture of the algae-containing liquid and a second culture method, and providing the crushed algae cultured by the above method. To achieve the above object, the present invention provides a cultured algae. a liquid containing a hard algae liquid containing > 5 algae, minced and nitrogen, and having the above-mentioned qualities of the above-mentioned nitrogen and the ratio of the nitrogen to the cerium/nitrogen of 〇·18 or more. The present invention provides a pulverized algae, which is characterized in that it contains zephyr and nitrogen, and the sputum is cultured in a liquid having a mass concentration ratio of 氮/nitrogen of 氮·18 or more. The ratio of the mass concentration of cerium to nitrogen contained in the liquid for the algae is set at 0.18 or more, so that the algae can be cultured at a high concentration and stably. To achieve the above object, the present invention provides a liquid for cultivating algae. Specifically, it is characterized in that it contains second algae, phosphorus, antimony and nitrogen, and the mass concentration ratio of the phosphorus to the nitrogen is phosphorus/nitrogen, and the mass concentration ratio of the niobium to the nitrogen and the niobium/nitrogen system are respectively set to 〇· 1 or more of the algae-containing liquid. 1294913 In order to achieve the above object, the present invention provides an algae, characterized in that the algae are contained in phosphorus, nitrogen and strontium, and the mass concentration ratio of the phosphorus to the nitrogen is % / nitrogen. And the above-mentioned cesium is cultured in a liquid having a mass concentration ratio 矽/nitrogen of 〇_1 or more, respectively. According to the present invention, the concentration of phosphorus and nitrogen contained in the liquid for cultivating diatoms is higher than that of phosphorus/nitrogen and The ratio of the mass concentration of cerium to nitrogen is set to 〇1 or more, respectively, so that the local concentration is stabilized and the culture is thin. In order to achieve the above object, the present invention provides a liquid containing diatoms which is characterized by cultivating a liquid for diatoms and containing diatoms, phosphorus, strontium and nitrogen, and having a pH of 6.4 or more and 8.4 or less. In order to achieve the above object, the present invention provides a diatom bacterium which is cultured in a liquid containing ¥, nitrogen and hydrazine and having a pH of 64 or more and 8.4 or less. According to the present invention, the pH of the liquid for cultivating the algae is set to be in the range of 6.4 or more and 8.4 or less, so that the algae can be cultured at a high concentration and stably. In order to achieve the above object, the present invention provides a method for cultivating a diatom bacterium, which is characterized in that a method for cultivating diatoms in a liquid for cultivating algae containing nitrogen and strontium is used, and the method for cultivating the larvae is to preliminarily determine the initial concentration of nitrogen contained in the liquid. And the pre-measurement step of the initial concentration of the initial sense, the above-mentioned diatoms are inoculated into the inoculation step of the liquid, and the measurement of the concentration of the nitrogen before the replenishment of the nitrogen and the concentration of the recharge of the sputum are measured, and the calculation is performed. a concentration difference measurement step performed by the difference between the initial concentration of nitrogen and the concentration of nitrogen before the concentration, and the concentration difference between the initial concentration of the enthalpy and the enthalpy before and after the replenishment, and the difference between the nitrogen concentration and the enthalpy concentration The difference is determined by the amount of nitrogen and the amount of nitrogen supplied to the liquid 1294913, respectively, so that the initial concentration of the nitrogen is equal to the initial concentration ratio of the above-mentioned enthalpy, and the supply step of the liquid is replenished. According to the present invention, the initial concentration of nitrogen and the initial concentration of strontium are measured before inoculation of diatoms, and the pre-recharge concentration of nitrogen and the pre-recharge concentration of hydrazine are measured before nitrogen and strontium supplementation. Then, based on the difference between the initial concentration of nitrogen and the concentration of nitrogen before recharge, and the difference between the initial concentration of strontium and the concentration before recharge, the amount of nitrogen supplied to the liquid and the amount of strontium are determined. Therefore, it is possible to maintain a constant ratio of the mass concentration of nitrogen to the mass concentration of strontium, thereby enabling high concentration and stable cultivation of rare algae. In order to achieve the above object, the present invention provides a method for cultivating a diatom algae, which is a method for culturing a second algae using a liquid containing nitrogen and a liquid for cultivation of Shigata sinensis, characterized in that the method is performed by separately measuring the supply of nitrogen before the replenishment. The concentration and the pre-recharge concentration of the above-mentioned hydrazine are measured before the replenishment, and the difference between the nitrogen concentration of the predetermined concentration and the pre-recharge concentration, and the concentration difference between the sputum concentration of the sputum and the pre-supplement concentration are respectively determined. And determining, according to the difference in nitrogen concentration and the concentration of ruthenium, the amount of nitrogen supplied to the liquid and the amount of ruthenium, respectively, such that the ratio of the predetermined concentration of the nitrogen and the predetermined concentration of the enthalpy are equalized to supply the liquid to the replenishing step. . According to the present invention, before the nitrogen supply and the strontium are measured, the pre-recharge concentration of nitrogen and the pre-recharge concentration of strontium are determined, and the difference between the nitrogen concentration of the predetermined concentration and the pre-recharge concentration and the enthalpy concentration difference between the sputum concentration and the pre-supplement concentration are determined. The amount of nitrogen and the amount of helium to be supplied to the liquid are determined. Therefore, the ratio of the mass concentration of nitrogen to the mass concentration of strontium is often maintained at a constant level, so that a high concentration and stable culture can be achieved. In order to achieve the above object, the present invention provides a method for cultivating diatoms, which is a method for cultivating sand algae using a nitrogen-containing and second-time culture liquid, which is characterized by separately measuring nitrogen contained in the liquid of the above-mentioned Japanese algae. The pre-measurement step of the initial concentration of the initial concentration cut, and the pre-replenishment step of the concentration of the nitrogen supply and the pre-recharge concentration of the dream are respectively measured, and the difference between the initial concentration of the nitrogen and the concentration of the nitrogen before the recharge is discarded. a concentration difference measuring step of the difference between the initial concentration of the stone stalk and the concentration before the replenishment concentration, and determining the amount of nitrogen and the amount of second supplied to the present body based on the difference in the concentration of nitrogen and the difference in the second concentration to make the ratio When the initial concentration of nitrogen and the initial degree of the upper cut are the same, it is supplied to the replenishing step of the liquid. According to the present invention, the initial concentration of nitrogen and the initial concentration of the nitrogen are measured in the (4) algae money, and the nitrogen concentration and the pre-recharge concentration before the recharge and the pre-recharge concentration before the recharge are measured. Then, based on the difference between the initial concentration of nitrogen and the pre-recharge concentration, the difference between the initial concentration of the second and the concentration before the recharge, the amount of nitrogen applied to the liquid is determined. Therefore, the concentration of nitrogen (4) is only a constant degree, and the ratio is often kept constant to achieve high concentration and stable cultivation. In order to achieve the above object, the present invention provides a method for cultivating diatoms, which is a method for cultivating diatoms using a culture liquid containing nitrogen and strontium, which is characterized in that: The pre-measurement step of the above-mentioned rare nitrogen consumption and the amount of the raw material, the inoculation step of inoculating the rare algae in the culture liquid, and measuring the cell concentration of the above-mentioned algae 2 determination step 'when the concentration of the above cells exceeds a prescribed amount The topdressing liquid is prepared by blending the liquid for top dressing so that the nitrogen/rhenium ratio is slightly different from the above-mentioned nitrogen consumption amount and the above-mentioned consumption ratio 1294913. For the above-mentioned culture liquid for use in the above-mentioned culture liquid, according to the present invention, the nitrogen consumption and the consumption amount of the consumption amount are respectively measured (4), and the cell concentration of the algae is more than the standard (4) In the case of algae, the nitrogen consumption and the nitrogen content of the feed amount are adjusted to be slightly different from the mass concentration system, which is often maintained at -== thus the 'nitrogen mass concentration and the algae. , thereby achieving high concentration and stable culture

[Embodiment] In the present embodiment, first, five kinds of real buckles and knife analysis were carried out as a preliminary investigation, and it was found that the conditions of stable and high concentration culture of 矽菱浼 were raised. Then, a method of cultivating the algae at a high concentration in a stable manner was examined based on the conditions. (Pre-investigation) First, the relationship between the change in the amount of cultured Chaetoceros inoculated in the liquid (culture medium) (the amount of reproduction) and the consumption of the components constituting the culture solution will be described. Eleven samples having the culture components as shown in Fig. 1 and having different composition ratios were prepared. Each sample is placed in a fixed capacity (for example, about 65 x 1 (r3 m3) of households - external (natural light) closed type reactor. The volume of the culture solution in each sample is constant. Then, the specified number of cells is inoculated into each Reactor. After a certain period of time, determine the change in cell concentration of each sample [(culture cell concentration Μ initial cell concentration)]; unit · · cells / m3), nitrogen consumption, phosphorus consumption and consumption the amount. Nitrogen (N), phosphorus (P) and strontium (Si) are important constituent elements for the cultivation of Chaetoceros. SEALIFE (manufactured by MARINETECH Co., Ltd.) shown in Fig. 1 is dissolved in water and -11 - 1294913. As long as it dissolves in water, the ingredients. &quot; 4 water, other than SEAUER or drug sterilization: two 'can not be used by high pressure reduction, filtration sterilization nitrogen consumption!: seawater or unsterilized natural seawater. Concentration [(the first two raises:: the capacity of the culture hard - set, the quality of the available hair is also used because of the capacity of each culture solution - concentration [(the initial quality of phosphorus in the culture solution is concentrated 2: the quality of consumption) Shishi: The unit of mass concentration of 蹲 is (2;: the concentration of phosphorus remaining) is thicker than each meter mm, and can be used for the quality of the consumption. Please check the mass concentration of the nutrient solution) (residual scale mass concentration)] 2 indicates the cells of each culture solution of π kinds of samples/m3) 'nitrogen consumption amount, phosphorus consumption amount and Wei dosage. &amp; f (cell, nitrogen consumption according to the measurement result and Chaetoceros Changes in cell concentration: relationship between the amount of consumption and the change of cell concentration of Chaetoceros chinensis. The relationship between the amount of c and the change of cell concentration of Chaetoceros sinensis is shown in Figure 3A, Figure 3B and Figure 3A, Figure 3B, respectively. And the dotted line of Fig. 3C shows that there is a proportional relationship between the consumption of nitrogen, phosphorus and strontium and the amount of change in cell growth (the amount of reproduction). The first step is to record the mass concentration ratio of phosphorus to nitrogen in the composition of the culture solution. The P/N value and the mass concentration ratio of cerium to nitrogen are Si/N values. The literatures 1 and 2 revealing the previous culture methods all describe the angle. The concentration of nitrogen, phosphorus and strontium in the algae culture solution. The concentration of nitrogen in the culture solution described in the literature 1 - 1294913 concentration and the mass concentration of strontium are 13.8 (ppm), 1.4 ( Ppm)' 0.15 (ppm). The mass concentration of nitrogen in the culture solution described in the literature 2, phosphorus berry; the end of the 'secondary mass concentration' are 1 4 2 (ppm), 1 · 4 (ppm), 8.0 ( Pp/N, Si/N in the culture solution of Document 1 is 0.10 and 0.011, respectively, and in the culture solution of Document 2, 8丨/1^ are respectively 0.014 and 0.078. As shown in Fig. 3A, As shown in 3B and 3C, there is a proportional relationship between the amount of nitrogen, phosphorus and strontium and the amount of change in cell concentration. Since P/N and Si/N are calculated according to literature 1 and literature 2, as long as the ratio is used, For culture medium above 0·1, the amount of cultured Chaetoceros should be larger than that of the previous culture method. In addition, as shown by the dotted lines in Figures 3A, 3B and 3C, if the cell concentration of Chaetoceros sp. is raised to about 6_〇 Χ1013 cells/m3 or more, it seems appropriate to adjust the nitrogen concentration to about 160 ppm or more, and adjust the phosphorus concentration to about 20 PPm or more and adjust the tannin concentration. In order to check the above two inferences, the culture solution 1 to the culture solution 6 having the component composition of Fig. 4 are prepared. The composition of the culture solution 7 is the culture solution described in the literature 1. The component composition, and the component composition of the culture solution 8 is the component composition of the culture solution described in Document 2. The nitrogen concentration, the phosphorus concentration, the barium mass concentration, P/N and Si/N in each culture solution are respectively expressed in Fig. 5. The mass concentration of phosphorus and the concentration of strontium in the culture solution 1 are smaller than the above-mentioned inferential values. The concentration of nitrogen in the culture solution 1, the mass concentration of phosphorus, and the concentration of strontium are respectively 166 (ppm), l (ppm), 30 (ppm), while p/N, Si/N are 0.06 and 0.18, respectively. The phosphorus concentration of the culture solution 2 was adjusted to twice the concentration of the culture solution 1 (phosphorus mass concentration 1294913 degrees = 20 (ppm), p/n = 〇 12). The mass concentration of the culture solution 3 was adjusted to twice the concentration of the culture solution (矽 mass concentration = 60 (ppm), si/N = 0.36). The phosphorus concentration and the strontium mass concentration of the culture solution 4 were respectively adjusted to twice the culture solution 1 (phosphorus mass concentration = 20 (ppm), Ρ / Ν = 0.12, 矽 mass concentration = 60 (ppm), Si / N=〇.36). The mass concentration of the culture solution 5 was adjusted to 3 times that of the culture solution 2 (矽 mass concentration = 90 (ppm), si/N = 0.54). The mass concentration of the culture solution 6 was adjusted to 4 times that of the culture solution 2 (矽 mass concentration = 120 (ppm), si/N = 0.72). The mass concentration of phosphorus in culture medium 2-6, the mass concentration of strontium, p/N& Si/N, the mass concentration of phosphorus in the culture liquids 7 and 8 of the previous composition, the mass concentration of the mass, P/N, Si/ N is high. The third step is the relationship between the pH value and the amount of hydrolysis in the culture solution having a higher phosphorus concentration and a higher mass concentration. The concentration of sodium hydrogencarbonate (NaHc〇3), which is one of the components of the culture solution, was increased by 1 to change the pH of the culture solution 4 between 7.0 and 9.0. At the same time, the amount of hydrolysis produced in the culture solution 4 (the mass (kg) of the hydrolyzate produced per im3 of the solution was measured). During the period, the culture solution 4 was supplied with a component which was necessary to maintain life in the culture solution, and 3°/. The air of carbonation gas is used as a cultivating solution 4 for aeration and agitation. The results of the child test are shown in Figure 6. The solid line and the broken line in Fig. 6 show the change in the amount of hydrolysis obtained on different experimental days, respectively, and the results are slightly consistent. As the measurement was learned, when the pH of the culture solution 4 exceeded 8·5, the change in the amount of hydrolysis accelerated, and the amount of hydrolysis (precipitate amount) increased. The mouth of the culture liquid is more than 8 after 1294913: the sediment contains Chaetoceros, and the Chaetoceros no longer breeds. The fourth step is to determine the upper limit of the positive value of the culture solution. In this experiment, the pH of the sodium hydride and the 3% carbonation gas were adjusted, and the amount of σ & acid hydrogen was increased, and the pH of the solution was also increased, while the carbonation time was increased. ^ Plus 'Luo liquid heart pH is reduced. The carbon source and the carbonic acid gas supply to the Chaetoceros are used as a pH adjuster for the culture solution. The culture solution 1 (11 〇xl〇-3m3) is placed in a closed reactor set outdoors: the temperature is maintained below about 25 ° C, and the quantum of light is about 12 〇 0_ol/mVs · intensity <self &amp; sunlight Irradiated on the culture solution. In this state, the first measurement was carried out by mixing the air culture agitated culture solution i with 3% carbonation gas, and the change in the concentration of the cells of the Chaetoceros in the culture medium 1 and the time. The second item was the addition of sodium bicarbonate (7 0xl03 and then air ventilated to see the mixed culture solution 1 to determine the time change of the concentration of the cells in the culture liquid i and the time change of the pH. The test results are shown in Fig. 8. The solid line in Fig. 8 is the result of the first measurement, that is, the time change of the pH of the culture solution 1 in the case of mixing with 3% carbonation gas under the action of sodium hydrogencarbonate. The dotted line is the second measurement, which means the time change of the pH of the culture solution 1 under the addition of sodium bicarbonate, only when air is ventilated. It is learned from the above measurement results that the culture medium is ventilated by air. Stirring, and adding sodium bicarbonate, when the kpH of the culture solution exceeds 8.5, the sinking hall is produced. At this time, the dinoflagellate is co-precipitated with the precipitate, and is agitated with air mixed with 3% carbonic acid gas without adding sodium bicarbonate. At the same time, the pH of the culture solution 1 gradually decreases. -15- 1294913 The third and fourth items are determined by the sister ^ ^. Also, when adding sodium bicarbonate to the culture solution, 'right* is mixed with 3% citrate. The stirring culture solution with , sa, and two can be set to a pH of 8.4 or less. It is necessary to prevent the precipitation from occurring and the co-precipitation of Chaetoceros. - As for the composition of the culture solution 1, the components other than the P/N or Si/N are the same as the components of the culture solution 2 to 6. Therefore, The solubility of the culture is the same as the solubility of other culture solutions. When other culture solutions are used, if the culture solution is agitated with air mixed with carbonation gas in the state of adding bicarbonate to the culture solution, the sink should be prevented. The fifth step is about the lower limit of p H of the culture solution and the appropriate addition and addition range of sodium bicarbonate. The quality of sodium bicarbonate which is one of the components of the culture medium is changed. Concentration, adjust the following 8 kinds of culture solution 1 to mix 3% carbonation air, agitate and stir the culture solution J. [l]0.02xl03 ppm^ [2]0.07xl〇3 ppm , [3]〇.14xl〇3 ppm , [4] 〇.28 xlO3 ppm, [5]0·56χ103 ppm, [6]1.OxlO3 ppm, [7]2〇χ1〇3 ppm, [8]7·〇χ 1〇3 ppm. Each culture medium ι prepared is subjected to a culture experiment of Chaetoceros, and the time change of the cell concentration and the time change of the pH are measured. · In order to ensure reproducibility, the above experiments are different. It is carried out four times a day. Figures 9 and 10 are the results of the first measurement. The first culture experiment uses a culture solution having the following three concentrations of sodium bicarbonate: [ι]〇·〇2χΐ〇3 ppm, [ 3] 0.14xl03 ppm, [6] l. 〇 xl03 ppm. Figure XI shows that XI, X2 and X3 are represented by [1]0·02χ103 ppm, [3]0·14χ103 ppm,[6]1·〇 Time variation of cell concentration of Chaetoceros cultivarum cultured in χ103 ppm of culture medium. Figure ι 〇 ΧΑ ΧΑ ΧΑ 2 and ΧΑ 3 respectively indicate [1] 〇 · 〇 2 χ 103 ppm, [3] 〇 · 14 χ 1 〇 3 ppm, [6] 1 · 〇χ 103 ppm of the pH of the culture solution 1 Variety. -16 - 1294913 Figures 11 and 12 are the results of the second measurement. The second culture experiment used a culture solution having the following four concentrations of sodium bicarbonate: [2] 〇〇 7xl 〇 3 ppm, [3] 0·14 χ 103 ppm, [4] 0·28 χ 103 ppm and [6] 1·〇χ1〇3 ppm. Y, Y2, Y3, and Y4 of Fig. u are represented by [2] 〇.〇7xi〇3 ppm, [3]〇.ΐ4 χΙΟ3 ppm, [4]0.28xl03 ppm and [6]1·〇χ1〇3 ppm The culture solution is changed in time of cell concentration of C. albicans. In Fig. 12, yab YA2, YA3, and YA4 respectively represent [2]0.07&gt;&lt;10 ppm '[3]0·14χ103 ppm, [4]0·28 xlO3 ppm and [6]l.〇xl03 ppm The time of the pH of the culture solution 1 changes. Figures 13 and 14 show the results of the third measurement. The third culture test used four culture solutions with the following concentrations of hydrogen carbonate: [4] 〇.28χ103 ppm, [5]0.56xl03 ppm, [6]l.〇xl〇3 ppm and [7]2 ·〇χ1〇3 ppm. Z1, Z2, Z3, and Z4 shown in Fig. 13 are represented by [4] 〇 · 28 χ 103 ppm, [5] 0 · 56 x lO3 ppm, [6] l. 〇 xl03 ppm and [7] 2. 〇 xl 〇 3 ppm The time change of the concentration of the cells of the cultured Chaetoceros cultured in the culture solution 1. On the other hand, ZA1, ZA2, ZA3, and ZA4 in Fig. 14 represent [4]0·28χ103 ppm, [5]0.56x 103 ppm, [6]1·0 xlO3 ppm, and [7]2.0 xlO3 ppm of culture solution 1 The time of pH changes. Figures 15 and 16 are the results of the fourth measurement. The fourth culture experiment used a culture solution containing the following two concentrations of sodium bicarbonate: [6] 1.0x1 〇 3 ppm, [8] 7 · 〇χ 103 ppm. Wl and W2 of Fig. 15 show the time-dependent changes in the concentration of Chaetoceros cells cultured in the culture solution 1 of [6]1·〇χ1〇3 ppm, [8]7.〇xl〇3 ppm, respectively. On the other hand, WA1 and WA2 in Fig. 16 indicate the time change of [6] l_〇xl 〇 3 ppm, [8] 7·0 x lO3 ppm culture solution 1, respectively. The following results were obtained from the above measurement results. [1] In 0.02X 103 ppm of culture solution 1, the pH value decreased immediately after the start of the experiment, 1294913, and decreased to below 6.4 after about 25 hours, and the cell concentration was less than 3 cells/m3. [2] In the culture solution of 0.07X1G3 ppm, the pH is maintained above about 7.0 and below the range, and the cell concentration is up to 3χ1〇η cells~3. [3] In the culture of 〇.14xH)3 ppm, the pH value is maintained above about u and below about 7.4, while the cell concentration exceeds 4χ1〇π cells^3. [4] In 0.28X1G3 ppm of culture! The _ direct line is maintained at about 7.6 or less, and the cell concentration is above 4 χΐ〇 13 cells. [5] In the culture solution 1 of 0.56 Χ 103 ppm, the ρ Η value is maintained above about Μ and about 7.6 or less, and the cell concentration thereof is 4 &lt; 1 〇 13 cells / ^ or more. [6] In l.〇xl〇3ppmi culture solution 1, which? The value of 1^ is maintained in the range of about 6.4 or more and about 7.9 or less, and the cell concentration thereof is 6χ1〇13 cells/melon 3 or more. [7] In the culture solution of 2.0x10ppm, the pH is maintained at about 7〇 or more and about 8.1 or less, and the cell concentration is 5χ1〇13 cells/m3 or more. [8] Medium in 7.0xl03ppm! The pH is maintained at about 6. 7 or more and about 8.1 or less, and the cell concentration is 3 χ 1 〇 13 cells/m 3 or more. Therefore, after preparing a culture solution 1 having a mass concentration of sodium hydrogencarbonate higher than 〇〇2xl〇3ppm or more, the mixture is aerated and stirred with air mixed with 3% carbonation gas, and the pH of the culture solution 1 is set to 6.4 or more, and the angle can be made. The cell concentration of Chaetoceros exceeds the upper limit of the concentration of Chaetoceros cells reached by the previous culture method (3χΐ〇π cells/m3).

As for the component of the culture solution 1, the components other than the components of the culture solution 2 to 6 were the same except for p/N -18-1294913 and/or S i/N. Therefore, when using another culture solution, it is possible to prepare a culture solution 1 containing a mass concentration of sodium bicarbonate of 0. 02 χ 103 ppm or more, and then agitating the culture solution with a mixture of 3% carbonation gas to adjust the pH of the culture solution 1. When the value is set to 6.4 or more, the upper limit of the concentration of Chaetoceros cells (3 X 1〇13 cells/m3) or more which is achieved by the previous culture method can be achieved. From the results of the above-mentioned four measurements, it was found that when the culture liquid i to 6 was used to culture the Chaetoceros, the pH of the culture solution was set to 6.4 or more and 8.4 or less. In order to meet this condition, one of the feasible methods is to prepare a culture solution having a sodium bicarbonate mass concentration higher than 0. 02 χ 103 ppm, and mixing the culture solution with a mixture of 3% carbonation gas. When the pH of the culture solution 1 to 6 is set to 6.4 or more, the cell concentration of Chaetoceros can be made higher than the upper limit when cultured by the prior culture method. Further, by setting the pH of the culture liquids 1 to 6 to 8.4 or less, it is possible to suppress the co-precipitation of the Chaetoceros caused by the water angle f due to the increase in pH, thereby preventing the growth of Chaetoceros from decreasing. [Embodiment] Hereinafter, the algae cultivation method which is stabilized at a high concentration will be described in detail in the examples i-U in accordance with the above conditions. (Example 1) The flat culture flask 11 (capacity 丨·5χ 1〇-3 m3) for the experiment as shown in Fig. 7 was placed in the culture solution 1 (1·5χ10·3 m3), and sodium hydrogencarbonate was added to make carbonic acid. The mass concentration of sodium hydrogen is 1.0 OxlO3 ppm. Then, a predetermined amount of Chaetoceros was inoculated to the culture solution 1. The temperature of the culture solution is maintained at about 25t to about 351, and the culture solution 1 is irradiated with light having a light quantum amount of about 200 pm〇i/m2/s by a fluorescent lamp. In this state, the plug 12 is passed through the bottle. The glass tube 13 is fed with a mixture of 1294913 3% 3⁄43⁄4 of air in the culture solution 1 'C. The time change of the concentration of Chaetoceros cells in the culture solution 1 and the time change of the pH were measured. (Example 2) The culture solution 2 (1.5XI (Γ3 m3) was placed in a flat culture flask u in a laboratory, and sodium hydrogencarbonate was added so that the mass concentration of sodium hydrogencarbonate was 1 〇 x 1 〇 3 ppm. Then The culture solution 2 was inoculated with a predetermined amount (the same amount as in Example 1) to have the same conditions as in Example 1 (culture temperature: about 25 ° C to about 35 ° C, photoquantity: about 200 pmol/m 2 ) /s, the air culture was mixed with 3% carbonation gas to carry out a culture experiment, and the time change of the concentration of the cells of the cultured liquid 2 and the time change of the pH were respectively measured. (Example 3) The culture liquid 3 (1.5) X1 (T3 m3) is placed in the flat culture flask 11 in the laboratory, sodium bicarbonate is added, so that the mass concentration of sodium bicarbonate is 1.0 OxlO3 ppm. Then, the culture solution 3 is inoculated with a prescribed amount (the same amount as the embodiment) ) C. elegans, in the same conditions as in Example 1 (culture temperature: about 25 ° C ~ about 35 ° C, quantum of light: about 200 pni〇l / m2 / s, mixed with 3% carbon dioxide air The aeration experiment was carried out, and the time change of the concentration of the cells of the cultured liquid 3 and the time change of the pH were measured. The culture solution 4 (1 · 5XI (T3 is placed in a flat culture flask in the laboratory, sodium bicarbonate is added, so that the mass concentration of sodium bicarbonate is 1.0 OxlO3 ppm. Then the culture solution 4 is inoculated with a prescribed amount ( The same amount as in Example 1 was carried out under the same conditions as in Example 1 (culture temperature: about 25 ° C to about 35 ° C, photon 1294913 f · about 200 gmol / m 2 / s, to mix 3% carbonation gas air ventilating) For the culture experiment of C. cerevisiae, the C. albicans cells of the culture solution 4 were measured separately; the time change of the time and the time change of the pH. Figure 1 7 shows Example 1 The time change of the concentration of Chaetoceros cells in ~4. The C 1 'C2 'C3 'C4 in Figure 1 7 shows the time change of the cell concentration of Chaetoceros sinensis cultured in culture medium 1, 2, 3, 4, respectively. In the case of Fig. 7, A is the time for supplying the topdressing component (nitrogen, phosphorus, strontium, vitamins, and trace minerals contained in the culture solution) to each culture solution. Fig. 18 shows the results of Examples 1 to 4. The time change diagram of pH. D1 D2 'D3 ' D4 of Fig. 18 indicates the time change of the pH of the culture solution 1, 2, 3, *, respectively. The results were ascertained: For the culture solution 1 (P/N=0.06, Si/N=0.18), the cell concentration of the culture solution 3 (P/N=0.06, Si/N=〇_36) was set to 2 times. About 8〇xl〇13 cells ~3) Ask about the concentration of C. elegans cells in culture medium 1 (about 6 〇xl〇i3 cells/m3). For culture medium 1 (P/N=0.06, Si/N=0.18) The amount of phosphorus and the amount of strontium were respectively set to 2 times the culture solution 4 (P/N:=(M2, Si/N=〇·36), and the cell concentration (about 7 cells/m) was immersed in the culture solution i. Cell concentration of Chaetoceros (about 6 〇 x 1〇u) cells/m3. The pH of each of the culture solutions 1-4 is maintained in the range of 6.4 or more and 8.5 or less, i.e., maintained at a range of about 7.5 or more and about 8.5 or less. As for the measurement of the cell concentration of the culture solution 4 of Experiment 4, since the supply of the air of the mixed gas in the experiment may not be sufficient, the data may be slightly different from the experiment. -21 - 1294913, and the reproducibility of the measurement results of Example 1-4 was examined in the same manner as in Example 1-4 'Measurement of the culture medium 1 - 4, and the conditions were not raised under the top dressing ingredients. , measuring the time change of the concentration of the cells of the hairy algae and the time change of the pH, and the culture medium i 1 to 4 in the reproducibility experiment in FIG.

pH. It is known from the above measurement results that the phosphorus T is set to 2 times the culture night of the culture solution 1 (ρ/Ν=〇·〇6, si/N=0.18) 2 (Ρ/Ν=0·12 'Si/N=角.l8) The concentration of horn cells and algae cells is higher than that of culture medium 1 . Set the amount of sputum to the horn of the culture solution 3 (P/N=G.G6, Si/N'6) twice the culture solution 1 (Ρ/Ν=〇·〇6, Si/N=〇18) The algal cell concentration is higher than the C. elegans cell concentration in the culture solution 1. The amount of phosphorus and the amount of strontium were respectively set to the culture solution 1 (1&gt;/1^=〇.〇6, Si/N=〇i8) Ai 2 times the culture solution 4 (P/N==〇12, Si/N = 〇.36) The concentration of Chaetoceros cells in the culture medium 1 is the concentration of Chaetoceros cells. The concentration of C. albicans cells in the culture solution 4 was higher than the cell concentration of the culture solution 2, 3. The pH of each of the culture solutions 1-4 is maintained at 6.4 or more, and is in the range of 8.5 or less, i.e., maintained in the range of about 7.4 or more to about 8 Å or less. From the results of the above Example 1 _4, it can be inferred that when the culture medium containing more than 2 times of the culture solution 1 and/or 2 times or more of the culture solution 1 is used as the top dressing 1294913, the horn hair The cell concentration of the algae will be above about 6 〇xl〇n cells/m3. In order to confirm the above inference, the culture solution 2 containing 2 times the amount of phosphorus in the culture solution 1 and the culture solution 4 containing 2 times the amount of phosphorus and 2 times the amount of the solution, and 2 % times the volume of the medium containing the solution F and 3 times the amount of the culture solution 5 as a top dressing, respectively cultured Chaetoceros. (Example 5) As in Example 2, the culture solution 2 (1.5×10 −3 m 3 ) was placed in a flat culture flask 1 1 ' After adding sodium hydrogen hydride, the mass concentration of sodium hydrogencarbonate was 1 〇 x 10 3 ppm. Then, the culture solution 2 was inoculated with a predetermined amount (the same amount as in Example 2) of C. elegans in the same condition as #Example 2 (culture temperature: about ~5 χ:, quantum amount of light: about 200 (amol) /mVs, aerated with a mixture of 3% carbonation gas, aerated) The Chaetoceros culture experiment was carried out, and the time change of the concentration of Chaetoceros cells in the culture solution 2 and the time change of the pH were respectively measured. (Example 6) 4. Place the culture solution 4 (15 &gt;&lt;1〇-31113) in a flat culture flask, and add sodium bicarbonate so that the mass concentration of sodium hydrogencarbonate is 1·〇χ1〇3 ppm. Then, the culture solution 4 Inoculation of a specified amount (the same amount as in Example 5) of Chaetoceros sinensis, in the same condition as in the scent of Example 5 (the temperature of the culture solution was about 25 Ό to about 35 〇 c, and the quantum of light was about 200 Kmol/m 2 /s). The mixture was ventilated with air mixed with 3% carbonic acid gas to carry out a culture experiment of Chaetoceros sinensis, and the time change of the concentration of Chaetoceros cells in the culture solution 4 and the time change of the pH were respectively determined. (Example 7) The culture solution 5 ( 1·5χ1 (Γ3 m3) put into the flat culture bottle!〗, add the hydrogen carbonate surface, so that the mass concentration of sodium bicarbonate is l. Xl 〇 3 ppm. Then, the culture solution 5 -23-1294913 was inoculated with a prescribed amount (the same amount as in Example 5) of Chaetoceros, with the same conditions as in Example 5 (culture temperature: about 25 it ~ about 35 t, The quantum of light: about 2 〇 〇 ' / ^ carried out the culture of Chaetoceros ' and the time change of the concentration of Chaetoceros cells in culture medium 5 and the time change of pH. Figure 21 is the angular hair of Example 5-7 The time change of the algae cell concentration. The G1 G2 G3 knife force in Figure 21 does not change the cell concentration of C. elegans cultured in culture medium 2, 4, and 5. The eighth in Figure 21 is for each culture solution. Replenishing the top dressing component (the time of the nitrogen methane and the trace mineral contained in the culture solution. / Figure 22 is the time change diagram of the PH of the example. The HI, H2, H3 in Fig. 22 are the culture solution 2, 4 respectively. The time of the yang of 5 changes, while the figure η represents the time change of the room temperature in the laboratory. H5 represents the time change of the temperature of the culture solution. It is known from the above measurement results: the sputum is set as the culture solution 2 ( ρ / ν = 〇 · 12, Si / N = 0.18) twice the culture solution, P / N = 〇. U, Si / N = 0.36) Chaetoceros cell concentration (9. 〇χ丨〇u cells/m3) are higher than the concentration of Chaetoceros cells in the culture solution 2. The cultivar is set to culture 3 times the culture solution 2 (ρ/Ν=〇·ΐ2, Si/N=0.18) The concentration of horns and algae cells (about 9·〇χ1〇13 cells/m3) of liquid 5 (Υ/Ν==0_12, Si/N=0·54) was higher than that of culture medium 2. The difference between the concentration of the cultured liquid 4 and the culture liquid 5 is not large. The pH of each of the private liquids 2, 4, and 5 is maintained at 6.4 or higher and 8.5 or less, that is, maintained at about 7.4 or higher. , about 8.2 or less. It is inferred from the above experimental results that the cell concentration of C. cerevisiae can reach about 8·〇χ1〇ΐ3 cells/m3 or more when the cultured C. elegans is cultured with the culture medium containing 2 times or more of the broth 2-24. . In order to confirm the inference, the culture liquids 2, 4, 5 and the culture liquid 6 were used, and the culture experiment was again carried out under the same conditions as in Example 5, and the time change of the concentration of the cells of the cultured liquid 2, 4, 5, and 6 was measured. And the time of pH changes. Figure 23 is a graph showing the time change of the concentration of Chaetoceros cells in the re-culture experiment. II, 12, 13, and 14 of Fig. 23 show temporal changes in cell concentration of Chaetoceros cultured in culture medium 2, 4, 5, and 6. Figure 24 is a graph showing the time change of pH at the time of the re-culture experiment. Ji, J2, J3' J4 of Fig. 24 indicate the pH of the culture liquids 2, 4, 5, and 6. J5 of Fig. 24 indicates the time change of the room temperature in the laboratory, and J 6 indicates the time change of the temperature of the culture solution. From the above measurement results, it was found that the concentration of sputum was determined by setting the amount of sputum to the culture solution 4 (Si/N = 0.36) twice the culture solution 2 (p/n = 0.12, Si/N = 0.18). (About ι·〇χ1〇η cells/claw 3) The concentration of Chaetoceros cells obtained from the culture solution 2 was higher. Set the amount of sputum to the concentration of Chaetoceros cells obtained by the culture solution 5 (Si/N = 0.54) which is 3 times the culture solution 2 (Ρ/Ν=0·12, Si/N=0.18) (about 9, 〇xl〇i3 cells/m3) is higher than the concentration of Chaetoceros cells obtained in the culture solution 2. Set the amount of sputum to the concentration of Chaetoceros cells obtained from the culture solution 2 (Ρ/Ν=0·12, Si/N==(U8) 4 times the culture solution 6 (Si/N=0.72). 1·〇χ1〇14 cells/m3) The concentration of Chaetoceros cells obtained from the culture solution 2. The concentration of Chaetoceros cells in the culture solution 4 was not significantly different from that of the culture solutions 5 and 6. Culture medium 2 The pH of each of 4, 5, and 6 is maintained above 6.4, and the range of 8.5 or less is -25 - 1294913. It is maintained at about 7.3 or above, and is below 8.1. The above-mentioned shell examples 5-7 As a result of the experiment, it can be seen that if the culture medium of the culture medium 4_6 is used to culture the Chaetoceros, the cell concentration of the Chaetoceros can be increased to be more than 8·10 1 cells/m3. In other words, the Ρ/Ν is used. 0.12 or more and '/N is 0.36 or more. When the culture medium is cultured, the concentration of horn cells will increase to about 8.OxlO13 cells/m3 or more. In order to review the above culture conditions (Ρ/ΝΜ·12&amp; 8 ί / ΝΜ · 36) Whether it can be established indoors or outdoors during continuous culture, using the culture solution 4 and the culture solution 6, the experiment of the following Example 84 is carried out. Lu (Example 8), for Han Wan, Shi ^ In the type of culture flask u, put nitrogen (166 叩 melon), phosphorus (20 卯 and 矽 (60 卯 111) broth 4 (1.5 &gt;&lt; 1 〇 _31113), add carbon 8 hydrogen Na's the mass concentration of the bicarbonate steel is UxW ppm. Then the soil nutrient solution 4 is inoculated with a specified amount of Chaetoceros, in the same condition as in Example 1 (cultivation temperature: about 2 points ~ about hunger, light quantum Amount: about 2 〇〇μιη〇ι/πι2/3, ❸ mixed 3% carbonation gas air aeration and stirring) to carry out the culture of C. cerevisiae, and determine the time change of the concentration of C. elegans cells in culture medium 4 and Change of time.

During the measurement, when the cell concentration of C. cerevisiae was about 6 〇χΐ〇 13 cell site 3 or more, about 2/3 of the solution of the culture solution 4 was taken out, and it was replaced with artificial seawater which dissolved seaufe. The artificial seawater dissolves the topdressing component containing nitrogen, phosphorus, strontium, vitamins, and the like into a dressing culture liquid, and is used as a culture liquid 4 which is supplied to the flat culture bottle u. This operation is called semi-batch continuous culture. In the course of a half batch of continuous culture, about 2/3 of the solution -26-1294913 is extracted from the culture solution 4, and the culture solution 4 is measured before the culture solution for the top dressing is not added (hereinafter referred to as the pre-dressing culture solution 4a). The amount of nitrogen, the amount of phosphorus and the amount of strontium are then calculated. The nitrogen content, the amount of phosphorus and the amount of strontium before the cultivation of the pre-fertilized culture solution and the nitrogen content and phosphorus of the culture medium (initial culture solution) before the inoculation of Chaetoceros sinensis are respectively calculated. The difference between quantity and quantity. As for the amount of nitrogen, phosphorus and strontium in the initial culture solution 4, 166 ppm, 20 ppm, and 60 Q ppm, respectively. The difference in nitrogen, phosphorus and strontium obtained from the above calculations will be adjusted for nitrogen and phosphorus. The cultivating solution for topdressing is supplemented with pre-dressing culture solution 4a, so that P/N and Si/N in the culture solution 4a before top dressing and 初期/Ν (=0·12) and Si/N in the initial culture solution 4 ( = 〇_36). After the operation, the enthalpy/Ν and Si/N of the culture solution 4 can be kept constant (see Fig. 25). In the repeated half-batch continuous culture, the time change of the concentration of Chaetoceros cells in the culture solution 4 and the time change of the pH were measured, respectively, and the amount of nitrogen, phosphorus and strontium consumed by each ixl04 cell of Chaetoceros gracilis was calculated. (Example 9) For the flat culture flask 11 in the laboratory, a culture solution 6 (1·5χ1 (Τ3 m3) containing nitrogen (166 ppnl), phosphorus (20 ppm) and Shi Xi (120 PPm) was placed. The hydrogen carbonate steel was added to make the mass concentration of sodium hydrogencarbonate 1·〇χ1〇3 ρριη. Then, the culture solution 6 was inoculated with a predetermined amount (the same as Experimental Example 8), to the same conditions as in Example 8 ( The temperature of the culture solution: about 25 ° C to about 35 ° C, the quantum of light: about 200 pmol / m 2 / s, agitated with air mixed with 3% carbonation gas) to carry out the culture of Chaetoceros (continuous culture of half batch culture). The time change of the concentration of Chaetoceros cells in the culture solution 6 and the time change of the pH were measured in the middle, and the amount of nitrogen, the amount of scale, and the amount of nitrogen consumed by each of the 1&gt;1〇4 cells were calculated. 1294913 Fig. 26 is a graph showing the time change of the concentration of Chaetoceros cells in Examples 8 and 9. K1 and K2 in Fig. 26 indicate the concentration of Chaetoceros cells cultured in the culture solutions 4 and 6. A1 of Fig. 26 indicates the respective culture solutions. The time to replenish the topdressing ingredients (nitrogen, phosphorus, antimony, vitamins and minerals) for the first time. At this time, the cell concentration of Chaetoceros has not reached 6.0x10. 13 cells/m3, therefore, the amount extracted from each culture solution is less than about 2/3 of the total amount. A2 to 7 of Fig. 26 respectively indicate that the cell concentration of C. cerevisiae is 6.OxlO13 cells/m3, respectively, from each culture solution. A total of about 2/3 of the solution was taken and the time for the dressing culture solution was replenished. Fig. 27 is a graph showing the time change of the pH of Examples 8 and 9. LI and L2 of Fig. 27 indicate the pH of the culture solutions 4 and 6. The nitrogen consumption of the algae in the replenishment time of 4 and 6 , the total nitrogen consumption of the horned algae and the total nitrogen consumption of the algae of the single cell are calculated according to the following formula: NCOMai=NFIR- NREMAi NCOMA2=NFIR+NADDai-NREMa2 NCOMa3=NREMa2+NADDa2-NREMA3 NCOMa4=NREMa3+NADDA3-NREMa4 ncomA5=nremA4+naddA4-nremA5 NCOMa6=NREMa5+NADDa5-NREMa6 NCOMa7=NREMa6+NADDa6-NREMa7

Nall4 丨化 7 (NCOMAi/CELLAi)

NCOMAi, NFIR, NREMAi, NADDAi, NCOMunit, CELLAi and NALL in the above various formulas represent the following amounts, respectively. The amount of nitrogen (ppm) consumed by NCOMAi in the supply time A (il) ~ Ai, NFIR 1294913 is the amount of nitrogen in the initial culture solution (ppm), and the NREMAi is in the replenishment time ^, remaining in the culture The amount of nitrogen in the liquid (ppm), the amount of nitrogen contained in the culture solution for the top dressing when the supply time is μ, and the amount of nitrogen in the fine-grained algae in the unit, the total amount of nitrogen consumed (ppm) . CELLAi is the number of cells in the early stage of the supply of A. cerevisiae. The unit cell number is calculated from the cell concentration of C. albicans at the time of replenishment. The above calculation formula is also applicable to the calculation of the total phosphorus consumption PALL and the total consumption of strontium used by the culture medium 4 and 6. Figure 28 is a graph showing the nitrogen concentration, phosphorus concentration, mass concentration, P/N and Si/N of the initial culture solutions of Examples 8 and 9. Figure 28 also shows the total phosphorus consumption (ppm), total phosphorus consumption (ppm) and total strontium consumption (ppm) of the total amount of phosphorus consumed per 1.0X104 cells of Example 8 in Example 8. The ratio of consumption to total nitrogen consumption (pall/nall) and the ratio of total consumption to total nitrogen consumption (SiALL/NALL). Example 9 also shows the same result. It is known from the above measurement results; Chaetoceros can be continuously and stably cultured at around 6·〇χ 1〇13 cells/m3. ^ The pH of the culture medium 4, 6 is maintained at 6.4 or higher and 8.5 or lower, that is, maintained at 7.5 or higher and 8.5 or lower.

The Pall/Nall and Siall/Nall values are close to the p/N and Si/N values of the initial culture solution. The pALL/NALL value is close to the p/N value of the initial culture solution 6, but the siALL/NALL value is smaller than the Si/N value of the initial culture solution 6. Therefore, in the culture flask 11 of the experiment, the culture of the cultured liquid 4 or 6 can be used to continuously and stably culture the Chaetoceros under high cell length. The culture solution 4 can be used to culture the horns at the optimum composition ratio without adding -29-1294913. (Example 10) For a closed type reactor installed outdoors, a culture solution 4 (65 x 1 (r3 m3) containing nitrogen (166p (tetra), _ - (20 PPm), and cesium (60 ppm)) was added, and sodium hydrogencarbonate was added. The mass concentration of sodium bicarbonate is i.OxiO3 ppm. Then, the culture solution 4 is inoculated with a predetermined amount of Chaetoceros. Secondly, the temperature of the culture solution is maintained in the range of about 15 ° C to about 35 ° C (should be maintained at 25). 〇, and under irradiation of natural solar light (quantity of light: about 0 to 1200 nm 〇l / m2 / S, the air is mixed with 3% carbonation gas, the culture solution 4 is stirred, and the culture of Chaetoceros is carried out. The time change and pHi time of the C. elegans cell concentration in the outdoor culture solution 4 were measured respectively. During the measurement, the cell concentration of C. cerevisiae was about 5〇xl〇i3 cells/m3, and it was taken out from the culture solution 4. About half (33xl〇-3m3) of the whole amount of the solution, and then prepare the artificial seawater prepared by dissolving SEALIFE in the same amount as the culture solution 4 taken out. The topdressing component containing nitrogen, phosphorus, strontium and vitamins is dissolved in the artificial seawater. The culture solution for top dressing. The culture solution for top dressing is supplied to the culture solution 4 in the reactor. A half batch of continuous culture is carried out. In a half batch of continuous culture, about half of the total amount of the solution is taken out from the culture solution 4, and then the nitrogen amount, the phosphorus amount and the amount of strontium before the pre-dressing culture solution 4a are separately determined, and the pre-fertilization culture solution is calculated. The amount of nitrogen, phosphorus and attack in 4a is different from the amount of nitrogen, phosphorus and amount in the initial culture solution 4. The nitrogen, phosphorus and amount of the initial culture solution 4 are 166 ppm, respectively. 20 ppm and 60 ppm. According to the difference between the amount of nitrogen, the amount of phosphorus and the amount of strontium, the pre-dressing culture solution 4a is supplemented with a dressing culture medium for modulating the amount of nitrogen, phosphorus and strontium, so that the topdressing -30-1294913初期/Ν (=〇·12) of the initial culture solution 4 and Ρ/Ν of the culture solution 4 before and after replenishment. P/Ν and Si/N and Si/N (-0·36) of the culture solution 4a are used. According to this operation and Si/N. In the repeated semi-batch continuous culture, the time change of the concentration of Chaetoceros cells in the culture solution 4 and the time change of the sputum were measured, and the unit gixi〇4 cells were calculated &lt;虱 consumption, phosphorus consumption and consumption consumption. The figure shows the time change of the cell concentration of the horns of the cockroach. Figure 29 The time when the liquid 4 first replenished the top dressing component. At this time, since the cell concentration of the Chaetoceros has not reached 5·0Χ10&quot; cells/m3, because "the pumping solution 4: about-half solution. The A12~Al7 cell in Fig. 29 = up to 5·〇χ10&quot; When the cell/m3 or more, the total amount of the solution is extracted from the culture solution*, and the time for the culture solution 4 is supplemented with the culture solution for the top dressing. Fig. Μ is the time of the pH of the embodiment 10 Variation Example. Example 1 〇, when calculating the culture solution 4 in the same calculation formula as in Example 8, the total nitrogen consumption of the horn horn in the cells (for example, red (three) cells): ΐ (NALL), total phosphorus Consumption (Pall) and total consumption. Fig. 31 is a view showing the mass concentration of nitrogen contained in the initial culture solution of Example 10, the factory concentration '矽 mass concentration' Ρ/Ν and Si/Ne, and Fig. 31 shows: Example 10 &lt;1.〇Χ1〇4 Total nitrogen consumption of C. elegans consumption = (ppm) Ratio of total phosphorus consumption (ppm) and total consumption of phosphorus (ppm) to total phosphorus consumption (Pall/Nall) and the ratio of total consumption and consumption T (SiALL/NALL). It is known from the above measurement results that the cornea can be continuously and stably cultured in the vicinity of about 5·〇XI〇&quot; cells/m3. -31 - 1294913 The pH of the culture solution 4 is maintained at 6.4 or more and 8.5 or less, that is, maintained at 7.4 or more and 8, 2 or less.

Pall/NAll and SIall/Nall maintain the P/N and Si/N values close to the initial culture solution 4. Therefore, when the outdoor closed reactor is placed in the culture solution 4 to culture Chaetoceros, a high concentration, continuous and stable angular hair growth culture can be carried out. From the experimental results of the above Examples 8 to 10, it was found that the culture conditions (0.12 for p/N, Si/NSO.3 6) can be applied to continuous culture indoors and outdoors. Therefore, compared with the previous culture method, even if continuous culture is used, the indoor can be above about 6.〇xl〇13 cells/m3, and the outdoor is at a high cell concentration of about 5·〇χΐ〇13 cells/μ. The pH of the culture solution 4 is maintained at a pH of 6.4 or more and 8.5 or less, both indoors and outdoors. However, since the rate of nitrogen and strontium intake by Char. cerevisiae is slow, in the case of Example 8~1, the initial concentration of the culture solution 4, 6 is not necessarily determined before the inoculation of Chaetoceros', or immediately after inoculation ( See Figure 3 2) or after half a day after inoculation. In the examples 8 to 10, the experimental culture medium for the actual cultivation of Chaetoceros was used to measure the nitrogen consumption, the phosphorus consumption and the consumption amount in real time, but it may also have the same composition as the experimental culture solution. For the consumption of the culture solution for measuring the consumption, the seedlings were inoculated, and the nitrogen consumption, the phosphorus consumption, and the consumption amount were measured in advance (see Fig. 33). Its basis is as follows. According to the experiment and analysis, the amount of nitrogen (NCOM), the amount of phosphorus (pc〇M) and the amount of cut (SiCOM) used per unit time of the unit cell, the pc〇M/ in the actual culture experiment. NCOM and SiCOM/NCOM values are always fixed. Therefore, the PALL/NALL and SiALL/NALL of the algae to be cultured can be calculated by pre-determining the nitrogen consumption, the phosphorus consumption and the consumption of the culture medium 1294913. The nitrogen consumption, the phosphorus consumption and the consumption of the experimental culture solution are measured at the time of replenishment, and the topdressing according to the p/N and Si/N ratios is respectively prepared according to the pre-calculated PALL/NALL and SiALL/NALL. By replenishing with the culture solution, the cultivation process can be simplified, and the top dressing can be carried out with the optimum culture liquid composition. For example, when the amount of Chaetoceros inoculated in the culture solution is equal to the amount inoculated in the culture solution 4 in Example 1, the PALL/NALL&amp;SiALL/NALL values are 〇143 and 0.402, respectively (refer to Fig. 27), and therefore, It is sufficient to set the dressing culture solution to 0.143 and 0.402, respectively. Further, in Example 8-10, when the cell concentration of C. cerevisiae is about 6 〇χΐ〇 π cells/m3 or more, the culture solution 4 is supplemented with the culture solution for top dressing, but the actual operation is not necessarily the case, the C. cerevisiae cell The concentration can be set arbitrarily. The advantages of the culture method of the present invention are as follows. Compared with the prior culture method, the amount of cultured C. elegans per unit volume of the present invention is increased, so that the amount of work, the cultivation space, the storage space, and the transportation cost can be reduced. For example, it is possible to reduce the amount of Chaetoceros culture liquid in the sink of the aquatic seedlings which are fed with the horned algae as a feed, thereby reducing the amount of work. When the culture is started, the amount of the inoculum can be changed to adjust the cell concentration at the start of the culture, thereby predicting the time when the concentration of the cells in the culture solution reaches the desired amount. In the above embodiment, Chaetoceros is used as the algae inoculated in the culture solution, but the present invention is not limited thereto. For example, a culture liquid 10 having a composition as shown in Fig. 34 is used for the culture of the algae of the genus Phaeodactylum (hereinafter referred to as P. euphorbia - 33 - 1294913 (Phueodactylum). The composition and culture of the culture solution 10 The liquid 4 is the same, and P/N = 012, S i / N 0 · 3 6. The composition of the culture solution 11 is composed of a triangular brown finger and a composition of a conventional culture solution for algae culture, and ρ/Ν=ι ·29, Si/N = 0 (ie, no 矽 added). (Example 11) Put the culture flask 10 (1.5×1〇3 m3) in the laboratory into the culture medium 10 (1·5χ1〇_ 3 m3), sodium bicarbonate was added so that the mass concentration of sodium bicarbonate was 1·〇χ1〇3 ppm. Then, the culture solution was inoculated with a predetermined amount of Phaeodactylum tricornutum, which was slightly the same as the condition of Example 1 ( Culture medium temperature: about 25 ° C ~ about 35 ° C, photoquantity: about 200 pmol / mVs, mixed with 3% carbonation gas air aeration and stirring) cultured Trichophytonus tricornutum, respectively, determination of culture medium 1 〇 triangle brown finger Time change of algal cell concentration and time change of pH. (Comparative example) Put the culture medium 11 (1.5xl0-3 m3) into the flat culture flask 11 and add sodium bicarbonate to make sodium bicarbonate The mass concentration was 1 · 〇 X 1 〇 3 ppm. Then, the culture solution 11 was inoculated with the same amount of Phaeodactylum tricornutum as in Example 11. The conditions were similar to those of the examples (culture temperature: about 25 ° C~ Approximately 35 ° C, the quantum of light: about 200 pmol / m2 / s, mixed with 3% carbonation gas air ventilating), cultured Trichophytonus tricornutum, respectively, determine the time change of cell concentration of Phaeodactylum tricornutum And the time change of pH. Fig. 35 shows the time change of the cell concentration of Phaeodactylum tricornutum in Example 11 and Comparative Example. Ml, M2 of Fig. 35 indicates the cell concentration of Phaeodactylum tricornutum cultured in the culture solution 10, 11. - 1294913 Fig. 36 shows the time change of the pH of the examples u and the comparative examples. Fig. 2: Kawagawa N2 indicates the pH of the culture solution 10, and the concentration of the culture medium 10 is determined by the above measurement results. Up to Μ·2χ1014 cells/m3 or more, and the concentration of triangular brown finger cells in the comparative example is less than 4 〇丨3 cells/m3 〇' The pH of the culture solution 10 is maintained above 6.4 or above, below 8·5. The pH of the range is also maintained above 6.4, and below 8.5.

From the experimental results of the above Example 1!, it was found that when the cultured liquid cultivar was cultured with the culture medium, the cell concentration of the culture medium 114 was higher than that of the culture medium 114. ^ Bu, like the corner hair thinner, can reduce the amount of work, the cultivation space, the storage space and the transportation cost compared to the previous culture method. As for the examples i to U', hydrogen carbonate (tetra) was added as a medium conditioner for the culture solution. However, in addition to sodium bicarbonate, bicarbonate such as i (tetra) metal (such as face (Li) 'potassium (K) 'face _ '铯 (Cs)) may be added to make the culture solution contain bicarbonate. . Further, a method of adding a bivalent soil-measuring metal as a hydrazine pH adjusting agent to the alkali metal hydrogencarbonate in the culture solution may be added. Further, in the embodiment (1), the mixture is ventilated with air mixed with 3% carbonic acid gas. However, as long as the component of the life in the culture solution is maintained, a gas containing a suitable amount of oxygen, carbon dioxide or the like may be used. Further, in the present embodiment, P/N and (4) of the culture solution for cultivating algae are separately adjusted. However, the present invention is not limited to this configuration, and at least _ may be adjusted. The present invention is not limited to the above-described embodiments and examples, and the present invention may be modified as appropriate within the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a component of a culture solution used in the present embodiment. Fig. 2 shows the change in the concentration of the cells of the Chaetoceros produced by each culture solution of the sample, the amount of nitrogen consumption, the amount of phosphorus consumed, and the amount of consumption. Figure 3 shows the relationship between nitrogen consumption and cell concentration. Figure 3B shows the relationship between phosphorus consumption and cell concentration. Figure 3C shows the relationship between the consumption of Shi Xi and the change of cell concentration. Fig. 4 is a composition of the composition of the culture solution 丨 to 6 used in the present embodiment and the composition of the previous culture liquids 7 to 8. Fig. 5 is a graph showing the nitrogen concentration, the phosphorus mass concentration, and the rhodium mass concentration of each of the culture solutions shown in Fig. 4. Fig. 6 shows the relationship between the pH of the culture solution 4 and the amount of hydrolysis. Fig. 7 is a side view showing a flat type culture bottle used in the embodiment. Fig. 8 is a graph showing the relationship between the pH value of the culture solution 1 and the change in the cell concentration when sodium hydrogencarbonate is added to the culture solution 1, and when the culture solution is not added with sodium hydrogencarbonate. Fig. 9 is a graph showing the time change of the concentration of sodium bicarbonate to the cell concentration of the culture solution 1 of 〇 2xl 〇 3 pprn, 〇 14 x 103 ppm and 1·〇χ1 〇 3 ppm. Figure 10 is a graph showing the time change of the pH of the culture solution 1 of 〇.〇2xl〇3 ppm, O·14x 1〇3 PPm and 1_〇χίο3 ppm. Figure 11 is a graph showing the change in the cell concentration of the sodium bicarbonate to the cell concentration of the culture solution 1 of 〇.〇7xl〇3 ppm, 〇·14χ 10 Ppm, 0·28χ103 ppm and 1.0χ103 ppm. 1294913 Figure 12 is a time-dependent change in the mass concentration of sodium bicarbonate to 培养 〇 7χ1〇3 ρρηι, 0·14χ 103 ppm, 0.28xl〇3 ppm, and ΐ·〇χ1〇3 ppm. Figure 13 is a graph showing the time change of the concentration of sodium bicarbonate to a cell concentration of 培养·28χ1〇3 pprn, 0·56χ 103ppm, i.〇xi〇3ppm and 2〇xl〇3ppm. Fig. 14 is a period in which the mass concentration of sodium hydrogencarbonate is adjusted to 阳 28χ1〇3, 0·56χ 103 ppm, ι·〇χ1〇3 ppm, and 2 〇χ1〇3 of the culture solution at a positive time. Figure 15 is a medium in which the mass concentration of sodium bicarbonate is adjusted to 1〇χ1〇3 ppm&amp;7 〇x 103 ppm! The time of cell concentration changes. Fig. 16 is a graph showing the time change of the pH of the sodium hydrogencarbonate to a pH of 1 〇χΐ〇 3 卯 (5) and (10) 10 ppm of the culture solution 1. Fig. 1 is a graph showing the time change of the cell concentration of the culture solution 丨 to 4 of Examples 丨 to 4, respectively. Figure I8 shows the culture solutions of Examples 丨 to 4, respectively, to 4? The time of the 11 value changes. The time of the cell concentration of the culture solutions 1 to 4 in the solid-state reproducibility experiment was changed. Fig. 20 is a graph showing the time change of the pH of the culture solutions 1 to 4 at the time of the reproducibility experiment. Fig. 21 is a graph showing the time change of the cell rolling degree of the culture solutions 2, 4 and 5 of Examples 5 to 7, respectively. Fig. 22 shows the positive values of the culture solutions 2, 4 and 5 of Examples 5 to 7, respectively. 1294913 Fig. 23 shows the time change of the cell concentration of the culture solutions 2, 4 and 5 at the time of the re-culture experiment. Fig. 24 is a graph showing the time change of the pH values of the culture solutions 2, 4 and 5 at the time of the re-culture experiment. Figure 25 is a flow chart showing the half batch continuous culture method used in Example 8. Fig. 2 is a graph showing temporal changes in cell concentrations of the culture solutions 4 and 6 of Examples 8 to 9, respectively. Fig. 27 is a graph showing the time variation of the pH values of the culture solutions 4 and 6 of Examples 8 to 9, respectively. Figure 28 is a graph showing the ratio of total phosphorus consumption to total nitrogen consumption per 1.0 x 10 O4 cells of Examples 8 to 9 and the ratio of consumption to total nitrogen consumption. Figure 29 is a graph showing the time change of the cell concentration of the culture solution 4 of Example 1. Figure 30 is a graph showing the time change of the pH of the culture solution 4 of Example 1. Fig. 3 1 is the ratio of the total phosphorus consumption to the total nitrogen consumption per 1 · 〇χ 1 〇 4 cells and the ratio of the total strontium consumption to the total nitrogen consumption. Figure 32 is a flow chart showing a modification of the semi-batch continuous culture method. Figure 3 is a flow chart showing another variation of the semi-batch continuous culture method. Fig. 3 is a composition of the composition of the culture solution 1 π and π used in the present embodiment. Fig. 3 is a time change of the cell concentration of the culture solutions 1 and 11 of Example 11 and Comparative Example. Fig. 3 is a time course of pH values of the culture solutions 1 and 11 of the examples and the comparative examples.

Claims (1)

1294913 Pick up, apply for patent scope: A liquid containing diatoms, which is characterized in that it is a liquid for cultivating diatoms, and contains diarrhea, scorpion and nitrogen, wherein the mass concentration of the above-mentioned scales is l〇PPm or more, and the quality of the sputum is contained. The concentration is 3 〇 ppm or more, and the mass concentration ratio of the phosphorus to the nitrogen is set to 〇〇6 or more, and the mass concentration ratio 矽/nitrogen of the 矽 and the nitrogen is set to 〇·丨8 or more. The pH value of the liquid is 6.4 or more and 8.4 or less. 2. If the application is patented||The liquid containing the diarrhea in the first item, the bismuth contains the chlorinated carbonate. 3. If the patent application is the first item The liquid containing (4), wherein the above-mentioned liquid before inoculation of the above-mentioned wonderful algae means that the mass ratio of the nitrogen of the above-mentioned technology is the ratio of the amount of the nitrogen/nitrogen system and the above-mentioned measured stone consumption and the nitrogen consumption amount. The ratio is slightly the same. 4. If the liquid of the cut-off algae is applied in the patent, the phosphorus/nitrogen system of the above-mentioned liquid indicating the mass concentration ratio of the phosphorus and the nitrogen is determined before the inoculation of the algae. (4) The ratio of phosphorus consumption to nitrogen consumption is consistent 5. 6. The scope of patent ^ 4 "depth of liquid silicon, wherein the concentration of the silicon-based cultured cells of algae cells in the above item 4. 〇χ1〇ΐ3 the first 1 / m3. For example, apply for the first item of the patent scope. The scorpion of the scorpion is classified into the genus Chaetoceros. For example, in the first paragraph of the patent application, the sarcophagus, the hexagram and the amp; the deep-footed hard body, wherein the diatom genus is classified into the genus Phaeodactylum. a method for cultivating a smashing culture, which is characterized in that it is used to culture diatoms using a liquid containing phosphorus, strontium, and arboric acid, and the concentration of the phosphorus is more than 10 ppm, and the strontium is contained in the liquid. The mass concentration is 30 ppm or more, the phosphorus/nitrogen ratio of the phosphorus to the nitrogen is set to 0.06 or more, and the mass concentration ratio 矽/nitrogen of the hydrazine to the nitrogen is set to 0.18 or more, and the pH of the liquid is at 6.4 or more and 8.4 or less. 9. The method of culturing a second algae according to claim 8, comprising the steps of: separately measuring a pre-recharge concentration of the nitrogen before replenishment and a pre-recharge concentration of the strontium; and calculating a concentration of the nitrogen and a pre-recharge concentration, respectively. a concentration difference measuring step of a difference in the difference between the nitrogen concentration of the difference and the difference between the predetermined concentration of the enthalpy and the concentration before the replenishment; and determining the amount of nitrogen and the amount of hydrazine supplied to the liquid based on the difference in the concentration of nitrogen and the difference in cesium concentration And the replenishing step of the liquid is replenished to a ratio of the predetermined concentration of the nitrogen to the predetermined concentration of the niobium, and the rehydration step of the liquid is as described in the eighth aspect of the patent application, wherein the pre-replenishment measurement step The concentration of the phosphorus before the replenishment is measured, and the difference in phosphorus concentration between the predetermined concentration of the phosphorus and the concentration before the replenishment is calculated in the concentration difference measuring step, and the amount of phosphorus supplied to the liquid is determined according to the difference in the scale concentration in the replenishing step. The ratio of the specified concentration of nitrogen to the specified concentration of phosphorus described above is replenished to the above liquid. 1L = the eighth method of the patented range of algae cultivation methods, wherein the above algae. The line is carried out under the above-mentioned liquid internal gas. 1294913 12. The method for cultivating algae in the eighth item of the patent scope of claim 4, wherein the sputum liquid on the sputum and the sputum in the sputum and the sputum : The ratio of mass concentration to quantity is the same. In the evening, the consumption and the nitrogen consumption are as follows: (4) The culture method of the eighth item, wherein the phosphorus/nitrogen system of the above-mentioned scale 1 has the mass concentration ratio of the nitrogen in the liquid before the inoculation of the second algae. It is slightly consistent with the ratio of phosphorus consumption and nitrogen consumption of the above-mentioned diatoms measured in advance.