KR20040048334A - Solution containing diatom, diatom and method of culturing diatom - Google Patents

Abstract

PURPOSE: Liquid containing diatom, diatom and a method of culturing diatom are provided, thereby culturing Chaetoceros sp. at a high cell concentration with stability, and improving availability of diatom as feed for marine organisms. CONSTITUTION: The liquid for culturing diatom comprises nitrogen, silicon and phosphorus, wherein a value of silicon/nitrogen which represents the ratio of concentration by mass of silicon to nitrogen is set to be a value of 0.06 or more; a value of phosphorus/nitrogen which represents the ratio of concentration by mass of phosphorus to nitrogen is set to be a value of 0.18 or more; the pH of the culture liquid is set in the range of 6.4 to 8.4; the cell concentration of diatom cultured is greater than 4.0x1013 cells/m3; and the diatom is Chaetoceros sp. or Phaeodactylum sp.

Description

SOLUTION CONTAINING DIATOM, DIATOM AND METHOD OF CULTURING DIATOM}

The present invention relates to the diatoms of microalgae used as feed for fish seedlings, such as shellfish such as oysters and pearl shells, crustaceans such as middle and lower seashells, kelp, sea cucumbers and sea urchins, More specifically, the present invention relates to a liquid containing the diatom and a method of culturing the diatom.

As a feed for aquatic seedlings such as shellfish, crustaceans and echinoderm, the diatoms of microalgae (for example, chitoceras of the genus Chitoceras), which are recently classified into the genus chitoceros and the pheodactylum. Attention is drawn to Calcitoceros calcitrans, Chitoceras glacilis, and the like.

For example, Chitoceras calcitence has four long horns and one long flagella each. Since the diameter (total length) of a chitoceras calcitic lance is about 3-6 micrometers, it is especially suitable for the feed of the fish seedling larvae.

Diatoms, a type of microalgae, are superior in mineral balance and vitamin balance compared to other phytoplankton. Diatoms also accumulate useful fatty acids such as EPA (eicosapentaenoic acid; polyunsaturated fatty acids) and DHA (docosahexaenoic acid; polyunsaturated fatty acids). For these reasons, diatoms of microalgae are highly evaluated as feed.

However, it is very difficult to stably culture microalgae of microalgae. (1) Fish breeding technology test, (1) irrigation of seedling production, cultivation of biological feed (hereinafter referred to as Document 1) and `` Mass Culture Technology Research Report for Microalgae in 4 Years of Special R & D Promotion Project '' , Document 2), it is reported that the upper limit of the cell concentration of chitoceras calcitlance (hereinafter abbreviated as Chitoceras) obtained by a conventional culture method is about 3.0 × 10 ¹³ cells / m 3. Here, the cell concentration represents the cell number per 1 m 3 of the culture solution.

In the conventional culture method, the upper limit of the cell concentration of chitoceras is about 3.0 × 10 ¹³ cells / m 3, so that a sufficient culture amount could not be obtained. For this reason, there is a need for a liquid (culture medium, medium) and a culture method capable of culturing chitoceras stably at high concentrations.

The present invention has been made in view of the above-described circumstances, and provides a method for culturing liquid and diatoms containing diatoms capable of stably culturing diatoms, which is one type of microalgae at high concentration, and also providing diatoms cultivated by the above method. It aims to do it.

1 shows the components of the culture solution used in this embodiment.

Figure 2 shows the cell concentration change, nitrogen consumption, phosphorus consumption and silicon consumption of chitoceras produced in each culture medium of Samples 1 to 11.

3A shows the relationship between nitrogen consumption and cell concentration change.

3B shows the relationship between phosphorus consumption and cell concentration change.

3C shows the relationship between silicon consumption and cell concentration change.

4 shows the component composition of the culture solutions 1 to 6 used in the present embodiment, and the component composition of the conventional culture drug 7 or 8, respectively.

FIG. 5 shows the mass concentration of nitrogen, the mass concentration of phosphorus and the mass concentration of silicon in each culture solution shown in FIG. 4.

6 shows the relationship between the pH value of the culture solution 4 and the amount of hydrolysis.

7 is a side view of the flat culture bottle used in the present embodiment.

Fig. 8 shows the relationship between the Ph value and the cell concentration change of the culture solution 1 when the sodium bicarbonate was added to the culture medium 1 and the sodium hydrogen carbonate was not added to the culture medium 1, respectively.

Fig. 9 shows the time-dependent change in cell concentration in culture medium 1 in which the mass concentration of sodium hydrogen carbonate was prepared at 0.02 × 10 ³ ppm, 0.14 × 10 ³ ppm, and 1.0 × 10 ³ ppm, respectively.

Fig. 10 shows the time change of the pH value in the culture medium 1 prepared with a mass concentration of sodium hydrogen carbonate at 0.02 × 10 ³ ppm, 0.14 × 10 ³ ppm, and 1.0 × 10 ³ ppm, respectively.

FIG. 11 shows time-dependent changes in cell concentration in culture medium 1 prepared with a mass concentration of sodium hydrogen carbonate at 0.07 × 10 ³ ppm, 0.14 × 10 ³ ppm, 0.28 × 10 ³ ppm, 1.0 × 10 ³ ppm.

Fig. 12 shows the time-dependent change in pH value in the culture medium 1 prepared with a mass concentration of sodium hydrogen carbonate at 0.07 × 10 ³ ppm, 0.14 × 10 ³ ppm, 0.28 × 10 ³ ppm, 1.0 × 10 ³ ppm, respectively.

FIG. 13 shows the time-dependent change in cell concentration in the culture medium 1 prepared by preparing the mass concentration of sodium hydrogen carbonate at 0.28 × 10 ³ ppm, 0.56 × 10 ³ ppm, 1.0 × 10 ³ ppm, and 2.0 × 10 ³ ppm.

Fig. 14 shows the time-dependent change in pH value in the culture 1 prepared with the mass concentration of sodium hydrogen carbonate at 0.28 × 10 ³ ppm, 0.56 × 10 ³ ppm, 1.0 × 10 ³ ppm, and 2.0 × 10 ³ ppm, respectively.

Fig. 15 shows the change in time of the cell concentration in the culture medium 1 prepared by preparing the mass concentration of sodium hydrogen carbonate at 1.0 × 10 ³ ppm and 7.0 × 10 ³ ppm, respectively.

Fig. 16 shows the time change of the pH value in the culture solution 1 prepared with a mass concentration of sodium hydrogen carbonate at 1.0 × 10 ³ ppm and 7.0 × 10 ³ ppm, respectively.

Fig. 17 shows the time change of the cell concentrations of the culture solutions 1 to 4 in Examples 1 to 4, respectively.

Fig. 18 shows time changes of the pH values of the culture solutions 1 to 4 in Examples 1 to 4, respectively.

Fig. 19 shows the time change of the cell concentrations of the culture solutions 1 to 4 in the reproducing experiment, respectively.

20 shows the time change of the pH values of the culture solutions 1 to 4 in the reproducing experiment, respectively.

Fig. 21 shows changes in the cell concentrations of the culture solutions 2, 4 and 5 in Examples 5 to 7, respectively.

Fig. 22 shows time changes of the pH values of the culture solutions 2, 4 and 5 in Examples 5 to 7, respectively.

Fig. 23 shows time changes of the cell concentrations of the culture solutions 2, 4 and 5 in the culture experiment.

Fig. 24 shows time changes of pH values of the culture solutions 2, 4, and 5 in the culture experiment.

25 is a flowchart of half batch continuous culture used in Example 8. FIG.

Fig. 26 shows time changes of the cell concentrations of the culture solutions 4 and 6 in Example 8 and Example 9, respectively.

Fig. 27 shows time changes of the pH values of the culture solutions 4 and 6 in Example 8 and Example 9, respectively.

Fig. 28 shows the ratio of total phosphorus consumption and total nitrogen consumption per Chitoceras 1.0 × 10 ⁴ cells in Examples 8 and 9, and the ratio of total silicon consumption and total nitrogen consumption.

Fig. 29 shows the time change of the cell concentration of the culture medium 4 in Example 10.

FIG. 30 shows time change of the pH value of the culture solution 4 in Example 10. FIG.

Fig. 31 shows the ratio of total phosphorus consumption and total nitrogen consumption per Chitoceras 1.0 × 10 ⁴ cells in Example 10, and the ratio of total silicon consumption and total nitrogen consumption.

32 is a flowchart of a modification of half batch continuous culture.

33 is a flowchart of another variant of half-batch continuous culture.

Fig. 34 shows the composition of components of the culture solutions 10 and 11 used in the present embodiment, respectively.

Fig. 35 shows time changes of the cell concentrations of the culture solutions 10 and 11 in Example 11 and Comparative Example, respectively.

Fig. 36 shows time variations of the pH values of the culture solutions 10 and 11 in Example 11 and Comparative Example, respectively.

In order to achieve the above object, the present invention is a liquid for culturing diatom, the silicon / nitrogen containing the silicon, silicon and nitrogen, the silicon / nitrogen representing the mass concentration ratio of the silicon and the nitrogen is set to a value of more than 0.18 It provides a liquid containing a diatom, characterized in that.

In order to achieve the above object, the present invention is characterized by culturing silicon / nitrogen containing silicon and nitrogen and showing the mass concentration ratio of silicon and nitrogen in a liquid set to a value exceeding 0.18. Provide diatoms.

According to the present invention, since the silicon / nitrogen which represents the mass concentration ratio of silicon and nitrogen contained in the liquid for culturing diatom is set to a value exceeding 0.18, diatom can be cultured stably at high concentration.

In order to achieve the above object, the present invention is a liquid for culturing diatom, phosphorus / nitrogen containing the diatom, phosphorus, silicon, and nitrogen, and represents the mass concentration ratio of the phosphorus and the nitrogen, and the silicon and the nitrogen A silicon-containing liquid is provided, wherein the silicon / nitrogen representing the mass concentration ratio of is set to a value of at least 0.1.

In order to achieve the above object, the present invention provides phosphorus / nitrogen containing phosphorus, nitrogen and silicon, and also showing silicon / nitrogen showing the mass concentration ratio of phosphorus and nitrogen and silicon / nitrogen showing the mass concentration ratio of silicon and nitrogen, respectively. Diatom is provided by culturing in a liquid set to 0.1 or more.

According to the present invention, since phosphorus / nitrogen showing the mass concentration ratio of phosphorus and nitrogen and silicon / nitrogen showing the mass concentration ratio of silicon and nitrogen contained in the liquid for culturing diatom are respectively set to a value of 0.1 or more, It can be cultured stably at high concentrations.

In order to achieve the above object, the present invention provides a liquid for culturing diatom, containing diatom, phosphorus, silicon, and nitrogen, the liquid containing diatom characterized in that the pH is in the range of 6.4-8.4. do.

In order to achieve the above object, the present invention provides a diatom characterized by culturing in a liquid containing phosphorus, nitrogen and silicon, and having a pH set in the range of 6.4 to 8.4.

According to the present invention, since the pH of the liquid for culturing diatom is set in the range of 6.4-8.4, the diatom can be cultured stably at a high concentration.

In order to achieve the above object, the present invention is a method for culturing diatom using a diatom culture liquid containing nitrogen and silicon, the initial concentration of nitrogen and the initial concentration of silicon contained in the liquid, respectively A pre-measuring step of measuring, an inoculation step of inoculating the diatom in the liquid, a pre-measurement measuring step of measuring the pre-concentration concentration of the nitrogen and the pre-concentration concentration of the silicon, and the initial concentration and the pre-supplement of the nitrogen A concentration difference measurement step of obtaining a nitrogen concentration difference representing a difference in concentration, and a silicon concentration difference representing a difference between the initial concentration of silicon and the concentration before pre-supply, and coinciding with the ratio of the initial concentration of nitrogen to the initial concentration of silicon; The amount of nitrogen and the amount of silicon to be supplied to the liquid are respectively determined based on the nitrogen concentration difference and the silicon concentration difference, and they are supplied to the liquid. Provides a culture of the diatom characterized in that it comprises a diffusion step.

According to the present invention, the initial concentration of nitrogen and the initial concentration of silicon are measured before inoculation of diatoms, and the concentration before nitrogen supplementation and the concentration before silicon supplementation are measured before nitrogen and silicon are supplemented. Then, based on the nitrogen concentration difference indicating the difference between the initial concentration of nitrogen and the concentration before the replenishment and the silicon concentration difference indicating the difference between the initial concentration of the silicon and the concentration before the replenishment, the amount of nitrogen and the amount of silicon to be supplied to the liquid is determined. Therefore, since the ratio of the mass concentration of nitrogen and the mass concentration of silicon is always kept constant, the diatom can be cultured stably at a high concentration.

In order to achieve the above object, the present invention is a method for culturing diatoms using a diatom culture liquid containing nitrogen and silicon, the diatom cultivation method, the concentration of the nitrogen pre-supplement and the concentration of the pre-supplement of silicon, respectively A concentration difference measuring step of obtaining a preconcentration measurement step, a nitrogen concentration difference indicating a difference between the predetermined concentration of nitrogen and a preconcentration concentration, and a silicon concentration difference indicating a difference between the predetermined concentration of silicon and the preconcentration concentration; The amount of nitrogen to be supplied to the liquid and the amount of silicon to be supplied to the liquid are respectively determined based on the nitrogen concentration difference and the silicon concentration difference to coincide with the ratio of the predetermined concentration of the nitrogen to the predetermined concentration of the silicon, and the supply to supply them to the liquid. It provides a method of culturing diatoms characterized in that it comprises a step.

According to the present invention, before replenishing nitrogen and silicon, after measuring the pre-supplied concentration of nitrogen and the pre-supplied concentration of silicon, the nitrogen concentration difference indicating the difference between the predetermined concentration of nitrogen and the pre-supplied concentration and the predetermined concentration of silicon The amount of nitrogen and the amount of silicon to be supplied to the liquid are determined based on the difference in silicon concentration indicating the difference between the concentrations before and the supply. Therefore, since the ratio of the mass concentration of nitrogen and the mass concentration of silicon is always kept constant, the diatom can be cultured stably at a high concentration.

In order to achieve the above object, the present invention provides a method for culturing diatoms using a diatom culture liquid containing nitrogen and silicon, the initial concentration of nitrogen and silicon contained in the inoculated liquid A pre-measuring step of measuring the initial concentration, respectively, a pre-measuring measurement step of measuring the pre-concentration concentration of nitrogen and the pre-concentration concentration of silicon, and a nitrogen concentration difference indicating a difference between the initial concentration of the nitrogen and the pre-supplementation concentration. And a concentration difference measuring step of obtaining a silicon concentration difference representing a difference between the initial concentration of silicon and the concentration before the replenishment, and the nitrogen concentration difference and the so as to coincide with the ratio of the initial concentration of nitrogen and the initial concentration of silicon. And a replenishment step of determining the amount of nitrogen and the amount of silicon to be supplied to the liquid based on the difference in silicon concentration and supplying them to the liquid, respectively. Provides a method of culturing diatoms with gong.

According to the present invention, immediately after inoculation of diatoms, the initial concentration of nitrogen and the initial concentration of silicon are measured, and the concentration before nitrogen replenishment and the concentration before silicon replenishment are measured before nitrogen and silicon are replenished. Then, based on the nitrogen concentration difference indicating the difference between the initial concentration of nitrogen and the concentration before the replenishment and the silicon concentration difference indicating the difference between the initial concentration of the silicon and the concentration before the replenishment, the amount of nitrogen and the amount of silicon to be supplied to the liquid is determined. . Therefore, since the ratio of the mass concentration of nitrogen and the mass concentration of silicon is always kept constant, the diatom can be cultured stably at a high concentration.

In order to achieve the above object, the present invention is a cultivation method of diatoms in which diatoms are cultivated using a culture liquid containing nitrogen and silicon, the consumption measurement liquid inoculated with the diatoms, consumed by the diatoms A pre-measuring step of measuring nitrogen consumption and silicon consumption respectively, an inoculation step of inoculating the diatom in the culture liquid, a measuring step of measuring the cell concentration of the diatom, and the cell concentration exceeding a predetermined amount And a preparation step of preparing an additional cost liquid, and a replenishment step of replenishing the prepared additional cost liquid to the culture liquid such that the ratio of nitrogen and silicon is approximately equal to the ratio of the nitrogen consumption and the silicon consumption. It provides a method for culturing diatoms characterized in that.

According to the present invention, in the liquid for consumption measurement, after measuring the nitrogen consumption and silicon consumption consumed by the diatom, respectively, if the cell concentration of the diatom exceeds a predetermined amount when culturing the diatom with the culture liquid, nitrogen and The ratio of silicon is determined to approximately match the ratio of nitrogen consumption to silicon consumption. Therefore, since the ratio of the mass concentration of nitrogen and the mass concentration of silicon is always kept constant, the diatom can be cultured stably at a high concentration.

In this embodiment, five experiments and analysis were first performed as a preliminary investigation, and the conditions for stably culturing diatoms at high concentration were calculated | required. Next, on the basis of these conditions, a method of stably culturing diatoms at high concentration was obtained.

(Preliminary investigation)

First, the relationship between the change (increase) of the culture amount of chitoceras inoculated in a liquid (culture medium) and the consumption amount of the components constituting the culture solution is described.

Eleven samples having the culture medium components shown in FIG. 1 and differing in composition ratios were prepared. Each sample is placed in an outdoor (natural light) hermetic reactor having a predetermined capacity (e.g., about 65 x 10 ^-3 m ³ ). The volume of the culture solution in each sample is constant. Then, chitoceras of a predetermined cell number is inoculated into each reactor. Next, after a predetermined time has elapsed, the change in cell concentration (culture cell concentration-initial cell concentration, unit: cell / m 3), nitrogen consumption, phosphorus consumption, and silicon consumption are measured for each sample. Nitrogen (N), phosphorus (P), and silicon (Si) are important constituents in the culture of chitoceras.

Sea Life (manufactured by Marine Tech Co., Ltd.) shown in FIG. 1 is dissolved in water to form artificial seawater. Moreover, as long as it is a component which melt | dissolves in water and becomes an artificial seawater, it is also possible to use components other than sealife. It is also possible to use not only artificial seawater, but also natural seawater sterilized by autoclave, filtration sterilization, or chemicals, or unsterilized natural seawater.

Nitrogen consumption is represented by the mass concentration consumed (mass concentration of nitrogen in the initial culture-mass concentration of remaining nitrogen) because the capacity of each culture is constant. The unit of mass concentration of nitrogen is ppm.

Phosphorus consumption is represented by the mass concentration consumed (mass concentration of phosphorus in the initial culture-mass concentration of remaining phosphorus) since the capacity of each culture is constant. The unit of mass concentration of phosphorus is ppm.

Silicon consumption is represented by the mass concentration consumed (mass concentration of silicon in the initial culture-the mass concentration of remaining silicon) because the capacity of each culture is constant. The unit of mass concentration of silicon is ppm.

Fig. 2 shows changes in cell concentration (cells / m 3), nitrogen consumption, phosphorus consumption and silicon consumption in each of the cultures in 11 samples.

Based on this measurement result, the relationship between the nitrogen consumption and the cell concentration change of the chitoceras, the phosphorus consumption and the change of the cell concentration of the chitoceras, and the relationship between the silicon consumption and the cell concentration change of the chitoceras are shown in Figs. And FIG. 3C.

A proportional relationship is established between the respective consumption amounts of nitrogen, phosphorus and silicon and the cell concentration change amount (proliferation amount) as indicated by the double-dotted lines shown in Figs.

Second, the value of P / N which shows the mass concentration ratio of phosphorus and nitrogen in a culture liquid component, and the value of Si / N which shows the mass concentration ratio of silicon and nitrogen are described.

Documents 1 and 2, which disclose conventional culture methods, describe the respective mass concentrations of nitrogen, phosphorus, and silicon in chitoceras culture. The mass concentration of nitrogen, the mass concentration of phosphorus amount, and the mass concentration of silicon amount in the culture solution of Document 1 are 13.8 ppm, 1.4 ppm, and 0.15 ppm, respectively. The mass concentration of nitrogen, the mass concentration of phosphorus amount, and the mass concentration of silicon amount in the culture solution of Document 2 were 102 ppm, 1.4 ppm, and 8.0 ppm, respectively.

P / N and Si / N in the culture solution of Document 1 are 0.10 and 0.011, respectively. P / N and Si / N in the culture solution of Document 2 are 0.014 and 0.078, respectively.

As shown in Figs. 3A, 3B, and 3C, since a proportional relationship is established between each consumption of nitrogen, phosphorus, and silicon and the change in cell concentration, P / N and Si / N are calculated from Literature 1 and Literature 2; It is assumed that the use of a culture medium set to a value or more, that is, at least 0.1 or more, increases the amount of chitoceras proliferated compared to the conventional culture method.

3A, 3B, and 3C, the mass concentration of nitrogen is about 160 ppm or more and the mass concentration of phosphorus is about to make the concentration of chitoceras at about 6.0 × 10 ¹³ cells / m 3 or more. It is speculated that it is preferable to use a culture solution prepared with a concentration of 20 ppm or more and a silicon concentration of about 60 ppm or more.

Thus, in order to verify the above two conjectures, the culture solutions 1 to 6 having the composition of compositions shown in FIG. 4 were prepared. The composition of the culture solution 7 is that of the culture solution described in Document 1, and the composition of the culture solution 8 is that of the culture solution described in Document 2. The mass concentration of nitrogen, the mass concentration of phosphorus, the mass concentration of silicon, P / N and Si / N in each culture solution are shown in FIG. 5, respectively.

The culture solution 1 has a lower mass concentration of phosphorus and a higher mass concentration of silicon than the estimated value. In the culture solution 1, the mass concentration of nitrogen, the mass concentration of phosphorus, and the mass concentration of silicon were 166 ppm, 10 ppm, and 30 ppm, respectively. P / N and Si / N are 0.06 and 0.18, respectively.

The culture solution 2 is prepared so that the mass concentration of phosphorus becomes twice (mass concentration of phosphorus = 20 ppm, P / N = 0.12) with respect to the composition of the culture medium 1.

The culture solution 3 is prepared so that the mass concentration of silicon is doubled (mass concentration of silicon = 60 ppm, Si / N = 0.33) with respect to the composition of the culture solution 1.

In the culture medium 4, the mass concentration of phosphorus and the mass concentration of silicon were doubled with respect to the composition of the culture medium 1 (mass concentration of phosphorus = 20 ppm, P / N = 0.12, mass concentration of silicon = 60 ppm, Si / N = 0.36).

The culture medium 5 is prepared so that the mass concentration of silicon is three times (mass concentration of silicon mass = 90 ppm, Si / N = 0.54) with respect to the composition of the culture medium 2.

The culture solution 6 is prepared so that the mass concentration of silicon is four times (mass concentration of silicon amount = 120 ppm, Si / N = 0.72) with respect to the composition of the culture solution 2.

The mass concentration of phosphorus in the culture solutions 2 to 6, the mass concentration of silicon, P / N, and Si / N are the mass concentrations of phosphorus, the mass concentration of silicon, and P / N in the culture solutions 7, 8 having conventional compositional components. , All have a high value as compared to Si / N.

Third, the relationship between the pH value and the amount of hydrolysis in the culture medium having a high concentration of phosphorus and a high concentration of silicon is described.

The mass concentration of sodium hydrogencarbonate (NaHCO ₃ ), which is one of the culture components, is adjusted to change the pH of the culture solution 4 between 7.0 and 9.0. At this time, the amount of hydrolysis generated in the culture solution 4 (mass (kg) of hydrolyzate precipitated per 1 m 3 of solution) was measured. In addition, the culture medium 4 is aerated by aeration while supplying the culture medium 4 with air containing 3% carbon dioxide gas, which is a component necessary for maintaining raw noodles in the culture medium, as the air for culture medium agitation.

6 is a graph showing this measurement result. The solid line and the broken line of FIG. 6 showed the change of the amount of hydrolysis obtained on the other experiment day, respectively, and the substantially matched result was obtained.

From this measurement result, when the pH of the culture solution 4 exceeded 8.5, the change in the amount of hydrolysis suddenly increased, and the amount of hydrolysis (precipitation amount) increased. When the pH of the culture solution 4 exceeded 8.5, chitoceras did not grow because chitoceras were contained in the precipitate.

Fourthly, the upper limit of the pH of the culture solution is determined. The pH of the culture solution is adjusted using air in which sodium hydrogen carbonate, which is one of the culture solution components, and 3% carbon dioxide gas are mixed. In general, as the amount of sodium bicarbonate increases, the pH of the solution increases, and as the concentration of carbon dioxide increases, the pH value of the solution decreases. Sodium hydrogencarbonate and carbon dioxide play a role as a carbon source in chitoceras photosynthesis and as a pH adjuster of the culture solution.

The culture solution 1 (110 × 10 ⁻³ m ³ ) was placed in an airtight reactor installed outdoors and the temperature was maintained at about 25 ° C., and the natural solar light having an intensity of about 1200 μmol / m 2 / s was obtained. We check in 1. In this state, two measurements were performed. First, the culture medium 1 was agitated by air mixed with 3% carbon dioxide gas, and the time change of the cell concentration and the time change of pH in the culture solution 1 were measured, respectively. Second, sodium hydrogencarbonate (7.0 × 10 ³ ppm) is added, and the culture medium 1 is aerated and agitated by air to measure the time change of the cell concentration of Chitoceras and the time change of pH in the culture solution 1, respectively. .

8 is a graph showing this measurement result. The solid line in FIG. 8 shows the first measurement result, and shows the time change of the pH of the culture solution 1 in the case where the aeration was carried out with air mixed with 3% carbon dioxide gas without adding sodium hydrogencarbonate. The dashed line of FIG. 8 shows the 2nd measurement result, and shows the time change of the pH of the culture liquid 1 in the case of adding sodium hydrogencarbonate and carrying out the air stirring.

From this measurement result, when the culture medium 1 was aerated and agitated with air and sodium hydrogencarbonate was added, precipitation occurred when the pH of the culture medium 1 exceeded 8.5. At this time, chitoceras precipitated together with the precipitate. In addition, when aeration and aeration were carried out with air mixed with 3% carbon dioxide gas, and sodium hydrogencarbonate was not added, the pH of the culture solution 1 gradually decreased.

From the third and fourth measurement results, when sodium hydrogen carbonate is added to the culture solution, the pH can be set to 8.4 or less by aeration of the culture solution with air mixed with 3% carbon dioxide gas. As a result, occurrence of precipitation and accompanying precipitation of chitoceras can be prevented, respectively.

In addition, the components of the culture solution 1 are the same as the components of the culture solutions 2 to 6 except for P / N and / or Si / N, and the other components are the same. Therefore, the solubility of the culture medium 1 is approximately the same as the solubility of the other culture medium, so that even if another culture solution is added with sodium hydrogen carbonate to the culture medium, precipitation is caused by aeration of the culture solution with air mixed with carbon dioxide gas, and precipitation of chitoceras is accompanied. It is assumed that precipitation can be prevented respectively.

Fifthly, the lower limit of the pH of the culture and the range of the appropriate addition amount of sodium bicarbonate are described.

By adjusting the mass concentration of sodium hydrogen carbonate, which is one of the culture components, the following eight culture solutions 1 were prepared, and each culture solution 1 was aerated and agitated with air containing 3% carbon dioxide gas; [1] 0.02 × 10 ³ ppm, [2] 0.07 × 10 ³ ppm, [3] 0.14 × 10 ³ ppm, [4] 0.28 × 10 ³ ppm, [5] 0.56 × 10 ³ ppm, [6] 1.0 × 10 ³ ppm, [7] 2.0 × 10 ³ ppm, [8] 7.0 × 10 ³ ppm. For each culture solution 1 thus prepared, a culture experiment of chitoceras was performed to measure the time change of cell concentration and the time change of pH. In addition, the experiment was conducted four times with different dates in order to ensure reproducibility.

9 and 10 show results of the first measurement. In the first culture experiment, culture medium 1 having a mass concentration of three sodium hydrogen carbonates shown below was used; [1] 0.02 × 10 ³ ppm, [3] 0.14 × 10 ³ ppm, [6] 1.0 × 10 ³ ppm. X1, X2, X3 shown in Figure 9 is the time change of the cell concentration of chitoceras cultured in [1] 0.02 × 10 ³ ppm, [3] 0.14 × 10 ³ ppm, [6] 1.0 × 10 ³ ppm Respectively. XA1, XA2, and XA3 shown in Fig. 10 show time-dependent changes in pH of the culture solution 1 at [1] 0.02 × 10 ³ ppm, [3] 0.14 × 10 ³ ppm, and [6] 1.0 × 10 ³ ppm, respectively.

11 and 12 show results of the second measurement. In the second culture experiment, culture medium 1 having a mass concentration of four sodium hydrogen carbonates shown below was used; [2] 0.07 × 10 ³ ppm, [3] 0.14 × 10 ³ ppm, [4] 0.28 × 10 ³ ppm, [6] 1.0 × 10 ³ ppm. Y1, Y2, Y3 and Y4 shown in FIG. 11 are [2] 0.07 × 10 ³ ppm, [3] 0.14 × 10 ³ ppm, [4] 0.28 × 10 ³ ppm, [6] 1.0 × 10 ³ ppm shows the time change of the cell concentration of the culture Quito seraseu in each. shown in Fig. 12 YA1, YA2, YA3, YA4 is ^{[2] 0.07 × 10 3 ppm} , [3] 0.14 × 10 3 ppm, [4] 0.28 × The time change of the pH of the culture solution 1 of 10 ³ ppm and [6] 1.0 × 10 ³ ppm is shown, respectively.

13 and 14 show results of the third measurement. In the third culture experiment, culture medium 1 having a mass concentration of four sodium hydrogen carbonates shown below was used; [4] 0.28 × 10 ³ ppm, [5] 0.56 × 10 ³ ppm, [6] 1.0 × 10 ³ ppm, [7] 2.0 × 10 ³ ppm. Z1, Z2, Z3, and Z4 shown in FIG. 13 are [4] 0.28 × 10 ³ ppm, [5] 0.56 × 10 ³ ppm, [6] 1.0 × 10 ³ ppm, and [7] 2.0 × 10 ³ ppm. It shows the time change of the cell concentration of chitoceras cultured at, respectively. ZA1, ZA2, ZA3, and ZA4 shown in FIG. 14 are [4] 0.28 × 10 ³ ppm, [5] 0.56 × 10 ³ ppm, [6] 1.0 × 10 ³ ppm, [7] 2.0 × 10 ³ ppm Each time change of pH is shown.

15 and 16 show results of the fourth measurement. In the fourth culture experiment, a culture solution 1 having a mass concentration of two sodium hydrogen carbonates shown below was used; [6] 1.0 × 10 ³ ppm, [8] 7.0 × 10 ³ ppm. W1 and W2 shown in FIG. 15 show the time-dependent changes of the cell concentrations of chitoceras cultured in the culture solution 1 of [6] 1.0 × 10 ³ ppm and [8] 7.0 × 10 ³ ppm, respectively. WA1 and WA2 shown in FIG. 16 show the time-dependent change of pH of the culture solution 1 of [6] 1.0 × 10 ³ ppm and [8] 7.0 × 10 ³ ppm, respectively.

The following can be said from this measurement result.

[1] In the culture solution 1 of 0.02 × 10 ³ ppm, the pH value immediately decreased after the start of the experiment, and after about 25 hours, the pH value was lower than 6.4, and the cell concentration did not reach 3 × 10 ¹³ cells / m 3.

[2] In the culture solution 1 of 0.07 × 10 ³ ppm, the pH value was maintained in the range of about 6.4 to 7.0, and the cell concentration reached 3 × 10 ¹³ cells / m 3.

[3] In 0.14 × 10 ³ ppm of Culture Solution 1, the pH value was maintained in the range of about 6.4 to 7.4, and the cell concentration exceeded 4 × 10 ¹³ cells / m 3.

[4] In 0.28 × 10 ³ ppm of the culture solution 1, the pH value was maintained in the range of about 6.4 to 7.6, and the cell concentration exceeded 4 × 10 ¹³ cells / m 3.

[5] In the culture solution 1 of 0.56 × 10 ³ ppm, the pH value was maintained in the range of about 7.0 to 7.6, and the cell concentration exceeded 4 × 10 ¹³ cells / m 3.

[6] In the culture solution 1 of 1.0 × 10 ³ ppm, the pH value was maintained in the range of about 6.4 to 7.9, and the cell concentration exceeded 6 × 10 ¹³ cells / m 3.

[7] In the culture solution 1 of 2.0 × 10 ³ ppm, the pH value was maintained in the range of about 7.0 to 8.1, and the cell concentration exceeded 5 × 10 ¹³ cells / m 3.

[8] In the culture solution 1 of 7.0 × 10 ³ ppm, the pH value was maintained in the range of about 6.7 to 8.1, and the cell concentration exceeded 3 × 10 ¹³ cells / m 3.

Therefore, after preparing a culture solution 1 having a mass concentration of sodium hydrogen carbonate higher than 0.02 × 10 ³ ppm, and then stirring the culture solution 1 with air mixed with 3% carbon dioxide gas, the pH of the culture solution 1 is set to 6.4 or more. The cell concentration of chitoceras exceeded the upper limit (3 × 10 ¹³ cells / m 3) of the cell concentration of chitoceras in the conventional culture method.

In addition, the components of the culture solution 1 are the same as the components of the culture solutions 2 to 6, except for P / N and / or Si / N, and the other composition components are the same. Therefore, in the same manner as in the other culture medium, culture medium 1 having a mass concentration of sodium hydrogen carbonate of 0.02 × 10 ³ ppm or more is prepared, and then agitated the culture medium 1 with air containing 3% carbon dioxide gas, and the pH of the culture medium 1 is 6.4 or more. By setting to, the cell concentration of chitoceras exceeded the upper limit (3 × 10 ¹³ cells / m 3) of the cell concentration of chitoceras in the conventional culture method.

From the above four measurement results, when culturing chitoceras using the culture solutions 1 to 6, chitoceras can be stably cultured by setting the pH of the culture solution to be in the range of 6.4 to 8.4. In order to satisfy this condition, for example, a culture medium having a mass concentration of sodium hydrogen carbonate higher than 0.02 × 10 ³ ppm is prepared, and the culture medium is aerated and agitated with air mixed with 3% carbon dioxide gas.

By setting the pH of the culture solutions 1 to 6 or more, the upper limit of the cell concentration of chitoceras in the conventional culture method can be exceeded. In addition, by setting the pH of the culture solutions 1 to 6 or less, it is possible to suppress the occurrence of co-settlement of chitoceras due to the hydrolyzate produced by the pH increase, thereby preventing the reduction of the growth of chitoceras.

Next, from Examples 1 to 11, on the basis of these conditions, a method of stably culturing diatoms at a high concentration is obtained.

(Example 1)

As shown in FIG. 7, culture medium 1 (1.5 × 10 ⁻³ m ³ ) was placed in a flat culture bottle 11 (capacity 1.5 × 10 ⁻³ m ³ ) prepared in a laboratory, and the mass concentration of sodium hydrogencarbonate was 1.0 × 10 ^3. Sodium bicarbonate was added to the broth 1 to make it ppm. Then, the culture solution 1 was inoculated with a predetermined amount of chitoceras. Next, while maintaining the temperature of the culture solution at about 25 ° C to about 35 ° C and irradiating the culture medium 1 with light having an intensity of about 200 μmol / m 2 / s of light quantum quantity using a fluorescent lamp or the like, Air mixed with carbon dioxide gas was injected into the culture medium 1 through the glass tube 13 penetrating the stopper 12 of the bottle 11, and cultured experiments of Chitoceras were carried out while aeration and aeration of the culture solution 1 were carried out. The time change of the cell concentration of chitoceras and the time change of pH in the culture solution 1 were measured, respectively.

(Example 2)

In a flat culture bottle 11 prepared in the laboratory, culture medium 2 (1.5 × 10 ⁻³ m ³ ) was added, and sodium hydrogen carbonate was added so that the mass concentration of sodium hydrogen carbonate was 1.0 × 10 ³ ppm. Then, a predetermined amount (the same amount as Example 1) was inoculated into the culture solution 2, and under substantially the same conditions as in Example 1 (temperature of the culture solution: from about 25 ° C to about 35 ° C, and light quantity: about 200 µm). A culture experiment of chitoceras was carried out by aeration stirring with air containing mol / m 2 / s and 3% carbon dioxide gas. The time change of the cell concentration of chitoceras and the time change of pH in the culture solution 2 were measured, respectively.

(Example 3)

In a flat culture bottle 11 prepared in a laboratory, culture solution 3 (1.5 × 10 ⁻³ m ³ ) was added, and sodium hydrogen carbonate was added so that the mass concentration of sodium hydrogen carbonate was 1.0 × 10 ³ ppm. Then, the culture medium 3 was inoculated with a predetermined amount of chitoceras (the same amount as in Example 1), and under substantially the same conditions as in Example 1 (temperature of the culture medium: from about 25 ° C to about 35 ° C, and light quantity: about 200 µm). A culture experiment of chitoceras was carried out by aeration stirring with air containing mol / m 2 / s and 3% carbon dioxide gas. The time change of the cell concentration of chitoceras and the time change of pH in the culture solution 3 were measured, respectively.

(Example 4)

To the flat culture bottle 11 prepared in the laboratory, culture medium 4 (1.5 × 10 ⁻³ m ³ ) was added, and sodium hydrogen carbonate was added so that the mass concentration of sodium hydrogen carbonate was 1.0 × 10 ³ ppm. Then, the culture medium 4 was inoculated with a predetermined amount of chitoceras (the same amount as in Example 1), and under substantially the same conditions as in Example 1 (temperature of the culture solution: from about 25 ° C to about 35 ° C, and light quantum quantity: about 200μ) A culture experiment of chitoceras was carried out by aeration stirring with air containing mol / m 2 / s and 3% carbon dioxide gas. The time change of the cell concentration of chitoceras and the time change of pH in the culture solution 4 were measured, respectively.

Fig. 17 is a graph showing the time variation of the cell concentration of chitoceras in Examples 1 to 4; C1, C2, C3, and C4 shown in FIG. 17 represent time-dependent changes in cell concentrations of chitoceras cultured in culture solutions 1, 2, 3, and 4, respectively. In addition, A shown in FIG. 17 represents time at which the fertilizer component (nitrogen, phosphorus, silicon, vitamins, minerals contained in the culture solution) was supplied to each culture solution.

FIG. 18 is a graph showing a time change of pH in Examples 1 to 4. FIG. D1, D2, D3, and D4 shown in FIG. 18 represent time changes of pH of the culture solutions 1, 2, 3, and 4, respectively.

The following can be said from this measurement result.

About culture medium 1 (P / N = 0.06, Si / N = 0.18), culture medium 3 (P / N = 0.06, Si / N = 0.36) having doubled the amount of silicon was obtained from A cell concentration (about 8.0 × 10 ¹³ cells / m 3) higher than the cell concentration (about 6.0 × 10 ¹³ cells / m 3) was obtained.

With respect to culture medium 1 (P / N = 0.06, Si / N = 0.18), culture medium 4 (P / N = 0.12, Si / N = 0.36) in which the amount of phosphorus and silicon were doubled, respectively, A cell concentration (about 7.0 × 10 ¹³ cells / m 3) higher than that of Chitoceras (about 6.0 × 10 ¹³ cells / m 3) was obtained.

Each pH of the cultures 1 to 4 was maintained in the range of 6.4 to 8.5, more specifically in the range of about 7.5 to 8.5.

In addition, the measurement of the cell concentration of the culture liquid 4 in Example 4 may be insufficient supply of air mixed with carbon dioxide during the culture experiment, and may be slightly different from the actual one.

In order to examine the reproducibility of the measurement results of Examples 1 to 4, the time concentration and pH of chitoceras cell concentration in culture medium 1 to 4 were again not supplied to the fertilizer components under the same conditions as in Examples 1 to 4. The time change of was measured, respectively.

Fig. 19 is a graph showing the time change of the cell concentration of chitoceras in the culture solutions 1 to 4 used in the reproducing experiment. E1, E2, E3 and E4 shown in FIG. 19 show the time-dependent change of the cell concentration of chitocera cultivated in the culture medium 1, 2, 3, 4, respectively.

20 is a graph showing the time-dependent change in pH in cultures 1 to 4 used in the reproducing experiment. F1, F2, F3, and F4 shown in FIG. 20 represent pHs of the culture solutions 1, 2, 3, and 4, respectively.

The following can be said from this measurement result.

The culture solution 2 (P / N = 0.12, Si / N = 0.18) in which the amount of phosphorus was doubled with respect to the culture solution 1 (P / N = 0.06 and Si / N = 0.18) is the cell of Chitoceras in the culture solution 1. Cell concentrations higher than the concentrations were obtained.

About culture medium 1 (P / N = 0.06, Si / N = 0.18), culture medium 3 (P / N = 0.06, Si / N = 0.36) having doubled the amount of silicon was obtained from A cell concentration higher than the cell concentration was obtained.

With respect to culture medium 1 (P / N = 0.06, Si / N = 0.18), culture medium 4 (P / N = 0.12, Si / N = 0.36) in which the amount of phosphorus and silicon were doubled, respectively, Cell concentrations higher than those of Chitoceras were obtained.

In culture medium 4, a cell concentration higher than that of chitoceras in culture solutions 2 and 3 was obtained.

Each pH of the cultures 1 to 4 was maintained in the range of 6.4 to 8.5, more specifically in the range of 7.4 to 8.0.

From the results of Examples 1 to 4, when chitoceras is cultivated with a culture solution containing 2 times or more of phosphorus amount to culture medium 1 and / or 2 times or more silicon amount to culture medium 1, the cell concentration of chitoceras is obtained. Is estimated to be about 6.0 × 10 ¹³ cells / m 3 or more.

To demonstrate this conjecture, culture 2 containing 2 times the amount of phosphorus 1 for culture 1, culture 4 containing 2 times the amount of phosphorus and culture 2 times the amount of silicon, 2 times the amount of culture 1 and Chitocera was incubated with a culture solution 5 containing three times the amount of silicon, respectively.

(Example 5)

In the same manner as in Example 2, culture solution 2 (1.5 × 10 ⁻³ m ³ ) was added to the flat culture bottle 11, and sodium hydrogen carbonate was added so that the mass concentration of sodium hydrogen carbonate was 1.0 × 10 ³ ppm. Then, the culture medium 2 was inoculated with a predetermined amount (the same amount as Example 2) of chitoceras, and under substantially the same conditions as Example 2 (temperature of the culture solution: from about 25 ° C to about 35 ° C, and light quantity: about 200 µ) A culture experiment of chitoceras was carried out by aeration stirring with air containing mol / m 2 / s and 3% carbon dioxide gas. The time change of the cell concentration of chitoceras and the time change of pH in the culture solution 2 were measured, respectively.

(Example 6)

In the same manner as in Example 4, culture medium 4 (1.5 × 10 ⁻³ m ³ ) was added to the flat culture bottle 11, and sodium hydrogen carbonate was added so that the mass concentration of sodium hydrogen carbonate was 1.0 × 10 ³ ppm. Then, the culture medium 4 was inoculated with a predetermined amount of chitoceras (the same amount as Example 5), and under substantially the same conditions as in Example 5 (temperature of the culture solution: from about 25 ° C to about 35 ° C, and light quantity: about 200 µm). A culture experiment of chitoceras was carried out by aeration stirring with air containing mol / m 2 / s and 3% carbon dioxide gas. The time change of the cell concentration of chitoceras and the time change of pH in the culture solution 4 were measured, respectively.

(Example 7)

To the flat culture bottle 11, culture solution 5 (1.5 × 10 ⁻³ m ³ ) was added, and sodium hydrogen carbonate was added so that the mass concentration of sodium hydrogen carbonate was 1.0 × 10 ³ ppm. Then, the culture medium 5 was inoculated with a predetermined amount of chitoceras (the same amount as Example 5), and under substantially the same conditions as in Example 5 (temperature of the culture solution: from about 25 ° C to about 35 ° C, and light quantum quantity: about 200μ) A culture experiment of chitoceras was carried out by aeration stirring with air containing mol / m 2 / s and 3% carbon dioxide gas. The time change of the cell concentration of chitoceras and the time change of pH in the culture solution 5 were measured, respectively.

Fig. 21 is a graph showing the time variation of the cell concentration of chitoceras in Example 5-7. G1, G2, and G3 shown in FIG. 21 represent time changes of the cell concentrations of chitoceras cultured in the culture solutions 2, 4, and 5, respectively. A shown in FIG. 21 represents the time at which the fertilizer component (nitrogen, phosphorus, silicon, vitamins and minerals contained in the culture solution) was supplied to each culture solution.

FIG. 22 is a graph showing the time-dependent change in pH in Examples 5 to 7. FIG. H1, H2 and H3 shown in FIG. 22 represent time-dependent changes in pH of the culture solutions 2, 4, and 5, respectively. In addition, H4 shown in FIG. 22 shows the time change of the room temperature in a laboratory, and H5 shows the time change of the temperature of a culture liquid.

The following can be said from this measurement result.

About culture medium 2 (P / N = 0.12, Si / N = 0.18), culture medium 4 (P / N = 0.12, Si / N = 0.36) which set the amount of silicon twice was the value of chitoceras in culture medium 2. A cell concentration higher than the cell concentration (about 9.0 x 10 ¹³ cells / m 3) was obtained.

With respect to culture medium 2 (P / N = 0.12, Si / N = 0.18), culture medium 5 (P / N = 0.12, Si / N = 0.54) in which the amount of silicon was tripled is determined by the amount of chitoceras in culture medium 2. A cell concentration higher than the cell concentration (about 9.0 x 10 ¹³ cells / m 3) was obtained.

The cell concentration of chitoceras in the culture medium 4 was not significantly different as compared with the cell concentration of chitoceras in the culture solution 5.

Each pH of the cultures 2, 4 and 5 was maintained in the range of 6.4 to 8.5, more specifically in the range of about 7.4 to 8.2.

From the above experimental results, when chitoceras is cultivated with a culture medium having a silicon content of 2 times or more with respect to the culture medium 2, the cell concentration of chitoceras is estimated to be about 8.0 × 10 ¹³ cells / m 3 or more.

In order to prove this conjecture, the culture experiment was carried out again under the same conditions as in Example 5 using the culture solution 6 in addition to the culture solutions 2, 4 and 5, and the cell concentration of chitoceras in the culture solutions 2, 4, 5 and 6 The time change of and the time change of pH were measured, respectively.

Fig. 23 is a graph showing the time change of the cell concentration of chitoceras in the culture experiment. I1, I2, I3 and I4 shown in FIG. 23 show the time-dependent change of the cell concentration of chitoceras cultured in the cultures 2, 4, 5, and 6, respectively.

24 is a graph showing a time change of the pH in the culture experiment. J1, J2, J3, J4 shown in FIG. 24 represent the pH of the culture solutions 2, 4, 5, 6, respectively. In addition, J5 shown in FIG. 24 shows the time change of the room temperature in a laboratory, and J6 shows the time change of the temperature of a culture liquid.

The following can be said from this measurement result.

For culture medium 2 (P / N = 0.12, Si / N = 0.18), the culture solution 4 (Si / N = 0.36) having twice the amount of silicon had a cell concentration higher than that of chitoceras in culture medium 2. (About 1.0 × 10 ¹⁴ cells / m 3) was obtained.

For culture medium 2 (P / N = 0.12, Si / N = 0.18), culture solution 5 (Si / N = 0.54) having three times the amount of silicon was higher than the cell concentration of chitoceras in culture medium 2. (About 9.0 x 10 ¹³ cells / m 3) was obtained.

For culture medium 2 (P / N = 0.12, Si / N = 0.18), culture medium 6 (Si / N = 0.72) having four times the amount of silicon had a cell concentration higher than that of chitoceras in culture medium 2. (About 1.0 × 10 ¹⁴ cells / m 3) was obtained.

The cell concentration of chitoceras in the culture solution 4 was not significantly different from the cell concentrations of chitoceras in the culture solutions 5 and 6.

Each pH of the culture solutions 2, 4, 5, 6 was maintained in the range of 6.4 to 8.5, more specifically in the range of 7.3 to 8.1.

From the results of the experiments in Examples 5 to 7, the cell concentration of chitoceras is increased to about 8.0 × 10 ¹³ cells / m 3 or more by culturing chitoceras using one of the culture solutions 4 to 6. . In other words, by culturing chitoceras using a culture solution having a P / N of 0.12 or more and a Si / N of 0.36 or more, the cell concentration of chitoceras is increased to about 8.0 × 10 ¹³ cells / m 3 or more.

Next, in order to examine whether or not the culture conditions (P / N ≧ 0.12 and Si / N ≧ 0.36) were established indoors and outdoors during continuous culture, the culture solutions 4 and 6 were used in Examples 8 to 10. Based on the experiment.

(Example 8)

In a flat culture bottle 11 prepared in a laboratory, in a culture solution 4 (1.5 × 10 ⁻³ m ³ ) containing nitrogen (166 ppm), phosphorus (20 ppm) and silicon (60 ppm), the mass concentration of sodium hydrogen carbonate was 1.0 × 10 ^3. Sodium hydrogen carbonate was added to ppm. Then, a predetermined amount of chitoceras was inoculated into the culture solution 4, and under substantially the same conditions as in Example 1 (temperature of the culture solution: from about 25 ° C. to about 35 ° C., photoquantity: about 200 μmol / m 2 / s, 3%) Aeration experiment with aeration of air mixed with carbonic acid gas of The time change of the cell concentration of chitoceras and the time change of pH in the culture solution 4 were measured, respectively.

In the measurement process, when the cell concentration of chitoceras reaches about 6.0 × 10 ¹³ cells / m 3 or more, about 2/3 of the total solution is removed from the culture medium 4. Then, artificial seawater in which the sealife is dissolved is prepared by the amount removed from the culture solution 4. By dissolving the fertilizer component containing nitrogen, phosphorus, silicon, vitamins, and the like in the artificial seawater, a supplementary culture medium is prepared. This additional cost culture medium is supplied to culture medium 4 in the flat culture bottle 11. This is called half batch continuous culture.

In the half-batch continuous culture, the nitrogen content, the phosphorus amount, The amount of silicon is measured respectively. Then, the difference between the nitrogen amount, the phosphorus amount and the silicon amount contained in the preliminary culture medium 4a, and the nitrogen amount, the phosphorus amount and the silicon amount contained in the culture medium (initial culture solution) 4 before inoculating chitoceras is determined, respectively. In addition, the nitrogen amount, phosphorus amount, and silicon amount in the initial culture solution 4 are 166 ppm, 20 ppm, and 60 ppm, respectively.

Based on the difference in the amount of nitrogen, phosphorus and silicon, P / N and Si / N in the culture medium 4a before the addition were P / N (= 0.12) and Si / N (= 0.36) in the initial culture solution 4. ), The additional cost culture medium in which the nitrogen amount, phosphorus amount and silicon amount of the fertilizer component are adjusted is replenished into the culture medium before fertilization 4a. By this operation, P / N and Si / N in the culture solution 4 are constant before and after replenishment (see FIG. 25).

Repeating the half-batch continuous culture, the time change of the cell concentration and the time change of pH in the culture solution 4 were measured, respectively, and nitrogen consumption, phosphorus consumption, and silicon consumption per 1 x 10 ⁴ cell were determined.

(Example 9)

In a flat culture bottle 11 prepared in the laboratory, in a culture solution 6 (1.5 × 10 ⁻³ m ³ ) containing nitrogen (166 ppm), phosphorus (20 ppm) and silicon (120 ppm), the mass concentration of sodium hydrogen carbonate was 1.0 × 10 ^3. Sodium hydrogen carbonate was added to ppm. Then, a predetermined amount (same as Example 8) was inoculated into the culture solution 6, and under substantially the same conditions as in Example 8 (temperature of the culture solution: from about 25 ° C to about 35 ° C, and light quantity: about 200 µmol). Permeation agitation with air mixed with carbon dioxide gas (m 2 / s / m 2 / s)), and a culture experiment of chitoceras (half batch culture continuous culture experiment) was performed. The time change of the cell concentration of Chitoceras and the time change of pH in the culture solution 6 were measured, respectively, and nitrogen consumption, phosphorus consumption, and silicon consumption per 1 × 10 ⁴ cells of Chitoceras were determined.

FIG. 26 is a graph showing the time-dependent change in cell concentration of chitoceras in Example 8 and Example 9. FIG. K1 and K2 shown in FIG. 26 represent cell concentrations of chitoceras cultured in culture solutions 4 and 6. FIG. A1 shown in FIG. 26 shows the time when the fertilizer component (nitrogen, silicon, vitamins, minerals) was first replenished to each culture. At this time, the cell concentration of chitoceras did not reach 6.0x10 ¹³ cells / m 3, and thus, about 2/3 of the total solution was not removed from each culture. In A2 to A7 shown in Fig. 26, when the cell concentration of chitoceras exceeds 6.0x10 ¹³ cells / m 3, approximately 2/3 of the total solution was removed from each culture solution, and the additional cost culture solution was supplied to each culture solution. Each time is shown.

FIG. 27 is a graph showing a time-dependent change in pH in Examples 8 and 9. FIG. L1 and L2 shown in FIG. 27 represent pHs of the culture solutions 4 and 6. FIG.

The nitrogen consumption of chitoceras, the total nitrogen consumption of chitoceras, and the total nitrogen consumption of chitoceras per unit cell in each of the replenishment times in the culture solutions 4 and 6 can be determined by the following equations, respectively.

NCOM _A1 = NFIR-NREM _A1

NCOM _A2 = NFIR + NADD _A1 -NREM _A2

NCOM _A3 = NREM _A2 + NADD _A2- NREM _A3

NCOM _A4 = NREM _A3 + NADD _A3- NREM _A4

NCOM _A5 = NREM _A4 + NADD _A4- NREM _A5

NCOM _A6 = NREM _A5 + NADD _A5- NREM _A6

NCOM _A7 = NREM _A6 + NADD _A6- NREM _A7

N _ALL = Σ _1≤i≤7 (NCOM _Ai / CELL _Ai )

Here, NCOM _Ai , NFIR, NREM _Ai , NADD _Ai , NCOM _UNIT , CELL _Ai , and N _ALL represent the following amounts, respectively. NCOM _Ai represents the amount of nitrogen (ppm) consumed by chitoceras between the propagation time A (i-1) to _Ai . NFIR represents the amount of nitrogen (ppm) in the initial culture liquid. NREM _Ai represents the amount of nitrogen (ppm) remaining in the culture at the replenishment time Ai. NADD _Ai represents the amount of nitrogen (ppm) contained in the supplementary culture medium supplemented at the propagation time Ai. N _ALL represents the total amount of nitrogen (ppm) consumed by chitoceras per unit cell. CELL _Ai is the number of unit cells of chitoceras in the supply time Ai, and the unit cell number can be obtained from the cell concentration of chitoceras in the supply time Ai.

By the same formula, the total phosphorus consumption (P _ALL ) and the total silicon consumption (Si _ALL ) consumed by chitoceras per unit cell in the culture solutions 4 and 6 can be obtained.

28 shows the mass concentration of nitrogen, the mass concentration of phosphorus, the mass concentration of silicon, P / N, and Si / N in the initial culture liquids of Examples 8 and 9. FIG. 28 shows the total phosphorus consumption obtained from the total nitrogen consumption (ppm), the total phosphorus consumption (ppm), and the total silicon consumption (ppm) consumed by Chitoceras per 1.0 × 10 ⁴ cells in Example 8. And the ratio of total nitrogen consumption (P _ALL / N _ALL ) and the ratio of total silicon consumption to total nitrogen consumption (Si _ALL / N _ALL ). In addition, Example 9 showed the same result.

The following can be said from this measurement result.

Chitoceras can be cultured continuously and stably in the vicinity of about 6.0 × 10 ¹³ cells / m 3.

The pH of the cultures 4 and 6 is maintained in the range of 6.4 to 8.5, more specifically 7.5 to 8.5.

P _ALL / N _ALL and Si _ALL / N _ALL take values close to P / N and Si / N of the initial culture solution 4. In addition, P _ALL / N _ALL takes a value close to P / N of the initial culture solution 6, and Si _ALL / N _ALL takes a value smaller than Si / N of the initial culture solution 6. Therefore, when the culture solution 4 or 6 is put into the culture bottle 11 prepared in the laboratory, and chitoceras is cultured, chitoceras can be continuously stably cultured at high cell concentration. In addition, in the case of the culture solution 4, it is possible to culture at the optimum component ratio without adding excess silicon.

(Example 10)

In a closed reactor installed outdoors, hydrogen carbonate so that the mass concentration of sodium hydrogen carbonate is 1.0 × 10 ³ ppm in culture medium 4 (65 × 10 ⁻³ m ³ ) containing nitrogen (166 ppm), phosphorus (20 ppm) and silicon (60 ppm). Sodium was added. Then, the culture medium 4 was inoculated with a predetermined amount of chitoceras. Next, the culture medium temperature is maintained in the range of about 15 ° C to about 35 ° C, preferably at 25 ° C, and natural sunlight (light quantum amount: about 0μmol / m 2 / s to 1200μmol / m 2 / s) ) Under the condition of irradiating the culture medium 4, air culture containing 3% carbon dioxide gas was injected into the reactor, and the culture experiment of Chitoceras was carried out while the culture medium 4 was agitated. The time change of the cell concentration of Chitoceras and the time change of pH in the culture medium 4 of the outdoor were measured, respectively.

In the measurement, when the cell concentration of chitoceras reaches about 5.0 × 10 ¹³ cells / m 3 or more, about half (33 × 10 ⁻³ m ³ ) of the solution is removed from the culture medium 4. Then, the cited seawater dissolved in the sealife is prepared by the amount removed from the culture solution 4. By dissolving the fertilizer component containing nitrogen, phosphorus, silicon, vitamins, and the like in the artificial seawater, a supplementary culture medium is prepared. This extra cost culture liquid is replenished in culture medium 4 in the reactor to carry out half batch continuous culture.

In the half-batch continuous culture, after removing about half of the solution from the culture medium 4, the nitrogen amount, phosphorus amount and silicon amount of the culture medium 4a before addition are measured, respectively. Then, the difference between the amount of nitrogen, the amount of phosphorus and the amount of silicon contained in the culture medium 4a before the weight ratio, and the amount of the amount of nitrogen, the amount of phosphorus and the amount of silicon contained in the initial culture solution 4 is determined, respectively. In addition, the nitrogen amount, phosphorus amount, and silicon amount in the initial culture solution 4 are 166 ppm, 20 ppm, and 60 ppm, respectively.

Based on the difference between the nitrogen amount, phosphorus amount and silicon amount, P / N and Si / N in the culture medium 4a before the addition were divided into P / N (= 0.12) and Si / N (= 0.36) in the initial culture solution 4. The additional cost culture medium in which the nitrogen amount, phosphorus amount and silicon amount of the fertilizer component are adjusted so as to coincide with each other is replenished in the culture medium 4a before the harvest. By this operation, immediately after replenishment, P / N and Si / N of the culture solution 4 are respectively constant.

By repeating the half batch continuous culture, the time change of the cell concentration and the time change of pH of the chitoceras in the culture medium 4 were measured, respectively, and the nitrogen consumption, phosphorus consumption, and silicon consumption per 1 x 10 ⁴ cells of the chitoceras were determined. .

FIG. 29 is a graph showing the time-dependent change in cell concentration of chitoceras in Example 10. FIG. A11 shown in FIG. 29 shows the time when the fertilizer component was supplied to culture medium 4 for the first time. At this time, since the cell concentration of chitoceras did not reach 5.0 × 10 ¹³ cells / m 3, about half of the solution from the culture medium 4 was not removed. A12 to A17 shown in Fig. 29 shows the time when the half concentration of the total solution was removed from the culture medium 4 when the cell concentration of chitoceras exceeded 5.0 x 10 ¹³ cells / m 3, and the supplementary culture medium was supplied to the culture medium 4. Represent each. 30 is a graph showing a time change of pH in Example 10. FIG.

In Example 10, the total nitrogen consumption (N _ALL ) and phosphorus total consumption (P _ALL ) consumed by chitoceras per unit cell (for example, 1.0 × 10 ⁴ cells) in the culture medium 4 in the same manner as in Example 8. ) And the total silicon consumption (Si _ALL ) are obtained.

31 shows the mass concentration of nitrogen, the mass concentration of phosphorus, the mass concentration of silicon, P / N, and Si / N in the initial culture solution of Example 10. FIG. 31 shows the total phosphorus consumption obtained from the total nitrogen consumption (ppm), the total phosphorus consumption (ppm), and the total silicon consumption (ppm) consumed by Chitoceras per 1.0 × 10 ⁴ cells in Example 10. And the ratio of total nitrogen consumption (P _ALL / N _ALL ) and the ratio of total silicon consumption to total nitrogen consumption (Si _ALL / N _ALL ).

The following can be said from this measurement result.

Chitoceras can be cultured continuously and stably in the vicinity of about 5.0 × 10 ¹³ cells / m 3.

The pH of the culture solution 4 is maintained in the range of 6.4 to 8.5, more specifically 7.4 to 8.2.

P _ALL / N _ALL and Si _ALL / N _ALL take values close to P / N and Si / N of the initial culture solution 4. Therefore, when culture medium 4 is put in a closed reactor prepared outdoors, and chitoceras is cultured, chitoceras can be continuously cultured stably at a high cell concentration.

From the experimental results in Examples 8 to 10, the culture conditions (P / N ≧ 0.12 and Si / N ≧ 0.36) were established both indoors and outdoors during continuous culture. Therefore, compared to the conventional culture method, chitoceras can be continuously cultured stably at a high cell concentration of about 6.0 × 10 ¹³ cells / m 3 or more in the room and 5.0 × 10 ¹³ cells / m 3 or more outdoors in the continuous culture. . In addition, the pH of the culture solution 4 is maintained in the range of 6.4 to 8.5 indoors and outdoors.

In addition, since the infusion rate of nitrogen and silicon by chitoceras is slow, in Examples 8 to 10, the initial concentration measurement of the culture solutions 4 and 6 is not limited to the inoculation of chitoceras, for example, inoculation of chitoceras. It may be performed immediately after (see FIG. 32) or after a lapse of one day.

In Examples 8 to 10, nitrogen consumption, phosphorus consumption, and silicon consumption were measured in real time for the experimental culture for actually culturing chitoceras, but the culture solution for consumption measurement having the same composition as the experimental culture was used. As a result, chitoceras may be inoculated to measure nitrogen consumption, phosphorus consumption, and silicon consumption in advance (see FIG. 33).

The reason for this is as follows. The values of PCOM / NCOM and SiCOM / NCOM in the actual culture experiment are always constant for the amount of nitrogen (NCOM), phosphorus (PCOM) and silicon (SiCOM) consumed per unit time by chitoceras per unit cell. This can be seen from this experiment and interpretation. Therefore, nitrogen consumption, phosphorus consumption and silicon consumption are measured in advance from the consumption measurement culture medium, and P _ALL / N _ALL and Si _ALL / N _ALL of the diatoms to be cultured are obtained. Even if the consumption amount, phosphorus consumption and silicon consumption are not measured, the supplementary culture medium in which P / N and Si / N are adjusted so as to correspond to P _ALL / N _ALL and Si _ALL / N _ALL obtained in advance is supplied. It can be simplified and can be added with an optimal amount of culture composition.

For example, when the amount of chitoceras inoculated in the culture solution is the same as the amount inoculated in culture medium 4 in Example 10, the values of P _ALL / N _ALL and Si _ALL / N _ALL are equal to 0.143 and 0.402, respectively (FIG. 27). It is sufficient to match the values of P / N and Si / N of the additional culture medium to 0.143 and 0.402, respectively.

In Examples 8 to 10, when the cell concentration of chitoceras exceeded about 6.0 × 10 ¹³ cells / m 3, the supplementary culture medium was replenished in the culture solution 4, but the cell concentration of chitoceras was not limited thereto. It can be set arbitrarily.

Advantages of the culture method of the present invention are as follows.

Since the culture amount of chitocera per unit volume increases, the workload, culture space, storage space, and transportation cost are reduced as compared with the conventional culture method. For example, with regard to the reduction in the amount of work, the amount of the culture solution of chitoceras introduced into a tank having a fish seedling with chitoceras can be reduced.

Since the cell concentration at the start of the culture can be adjusted by changing the inoculation amount of chitoceras at the start of the culture, it is possible to estimate the time taken to reach a predetermined amount of cell concentration.

As mentioned above, although the diatom inject | poured into a culture liquid was made into chitoceras, it is not limited to this, The diatom classified into another genus may be sufficient.

For example, in the case of culturing diatom (hereinafter, referred to as peodactil) classified into the genus Feodactilium, a culture solution 10 having the composition of components shown in FIG. 34 is prepared. In addition, the component of the culture liquid 10 is the same as that of the culture liquid 4, and P / N = 0.12 and Si / N = 0.36. The composition of the culture solution 11 is that of a known culture solution used for culturing peodactil, and P / N = 1.29 and Si / N = 0 (ie no silicon).

(Example 11)

Flat culture bottle prepared in the laboratory (11) such that the addition of sodium hydrogen carbonate (1.5 × 10 ^-3 ㎥), the culture solution 10 (1.5 × 10 ^-3 ㎥) were dissolved carbon dioxide is the mass concentration of the sodium hydrogen 1.0 × 10 ³ ppm It was. Thereafter, the culture solution 10 was inoculated with a predetermined amount of feodactilium. Next, under substantially the same conditions as in Example 1 (temperature of culture medium: about 25 ° C to about 35 ° C, light quantum quantity: about 200 μmol / m 2 / s, air aeration and agitation by mixing 3% carbon dioxide gas) Phodactilum was incubated at. The time change of the cell concentration and the time change of pH in the culture solution 10 were measured, respectively.

(Comparative Example)

Culture medium 11 (1.5 × 10 ⁻³ m ³ ) was added to the flat culture bottle 11, and sodium hydrogen carbonate was added so that the mass concentration of sodium hydrogen carbonate was 1.0 × 10 ³ ppm. Thereafter, the culture medium 11 was inoculated with the same amount of phodactyllium as in Example 11. Next, under substantially the same conditions as in Example 1 (temperature of culture medium: about 25 ° C to about 35 ° C, light quantum quantity: about 200 μmol / m 2 / s, air aeration and agitation by mixing 3% carbon dioxide gas) Phodactilum was incubated at. The time change of the cell concentration and the time change of pH in the culture solution 11 were measured, respectively.

FIG. 35 is a graph showing the time-dependent change in cell concentration of feodactyllium in Example 11 and Comparative Example. FIG. M1 and M2 shown in FIG. 35 represent the cell concentrations of feodactyllium cultured in the culture solutions 10 and 11.

36 is a graph showing the time-based change in pH in Example 11 and Comparative Example. N1 and N2 shown in FIG. 36 represent pHs of the culture solutions 10 and 11.

The following can be said from the said measurement result.

The cell concentration of feodactilium in the culture solution 10 exceeds about 1.2 × 10 ¹⁴ cells / m 3. The cell concentration of peodactilium in the comparative example did not reach 4.0x10 ¹³ cells / m 3.

The pH in the culture solution 10 is maintained in the range of 6.4 to 8.5. PH in a comparative example is also maintained in the range of 6.4-8.5.

From the results of the experiment in Example 11, by culturing the feodactilium using the culture solution 10, it is possible to stably cultivate the peodactilum at a high cell concentration compared with the conventional orientation solution 11.

Therefore, as in the case of Chitoceras, the workload, the culture space, the storage space, and the transportation cost are reduced as compared with the conventional culture method.

In Examples 1 to 11, sodium hydrogencarbonate was added as a pH adjuster of the culture solution. However, in addition to sodium hydrogen carbonate, hydrogen carbonate containing monovalent alkali metals except for sodium (Li (lithium), K (potassium), Rb (rubidium), Cs (cesium), etc.) is added to form a hydrogen carbonate in the culture solution. You may make it contain. In addition, a divalent alkaline earth metal may be added as the pH adjusting agent, and the hydrogen carbonate alkali metal salt may be contained in the culture solution.

In Examples 1 to 11, air mixed with 3% carbon dioxide gas was used as the gas to be agitated and stirred in the culture medium. However, if components necessary for life support in the culture medium were added, a predetermined amount of components such as oxygen and carbon dioxide were added. It may be a gas.

In addition, in this embodiment, although P / N and Si / N in the diatom culture medium were prepared, respectively, this invention is not limited to this structure, What is necessary is just to prepare Si / N at least.

This invention is not limited to the said embodiment and Example, It can change and implement suitably within the range which does not deviate from the summary of this invention.

As described above, according to the cultivation method of the liquid, diatom and diatom containing the diatom according to the present invention, diatoms classified into the genus Chitoceras, Peodactilium and the like are highly concentrated, for example, 5.0 × 10 ¹³ cells / It can be cultured stably at a high concentration of m 3 or more, and the practicality of diatoms as feed for fish seedlings can be improved.

Claims (41)

As a liquid for culturing diatoms, Contains diatoms, silicon, and nitrogen, A silicon-containing liquid, wherein the silicon / nitrogen representing the mass concentration ratio of silicon and nitrogen is set to a value exceeding 0.18. The diatom-containing liquid according to claim 1, wherein the pH is 6.4 or more. The liquid containing diatom of Claim 1 containing hydrogen carbonate. The liquid containing diatom according to claim 1, wherein the silicon / nitrogen in the solution before inoculation of the diatom is determined to substantially coincide with the ratio of silicon consumption and nitrogen consumption of the diatom measured in advance. The liquid containing diatom according to claim 1, wherein the cell concentration of the cultured diatom is 4.0 × 10 ¹³ cells / m 3 or more. The diatom-containing liquid according to claim 1, wherein the diatom is classified into the genus Chitoceras. 2. A liquid containing diatom according to claim 1, wherein the diatom is classified into the genus Peodactil. As a liquid for culturing diatoms, Contains diatoms, phosphorus, silicon, and nitrogen, A phosphor containing diatom, characterized in that phosphorus / nitrogen representing the mass concentration ratio of phosphorus and nitrogen and silicon / nitrogen representing the mass concentration ratio of silicon and nitrogen are each set to a value of 0.1 or more. 9. A diatom-containing liquid according to claim 8, wherein the pH is at least 6.4. 9. A diatom-containing liquid according to claim 8, which contains hydrogen carbonate. The liquid containing diatom according to claim 8, wherein the silicon / nitrogen in the solution before inoculation of the diatom is determined to substantially coincide with the ratio of silicon consumption and nitrogen consumption of the diatom measured in advance. The liquid containing diatom according to claim 8, wherein the phosphorus / nitrogen in the solution before inoculating the diatom is determined so as to substantially coincide with the ratio of the phosphorus consumption and the nitrogen consumption of the diatom measured in advance. 9. A liquid containing diatom according to claim 8, wherein the cell concentration of the cultured diatom is at least 4.0x10 ¹³ cells / m 3. The liquid containing diatoms according to claim 8, wherein the diatoms are classified into the genus Chitoceras. The liquid containing diatoms according to claim 8, wherein the diatoms are classified into the genus Peodactil. As a liquid for culturing diatoms, Contains diatoms, phosphorus, silicon, and nitrogen, Diatom-containing liquid, characterized in that the pH is 6.4 or more and 8.4 or less. 17. The diatom-containing diaphragm according to claim 16, wherein phosphorus / nitrogen representing the mass concentration ratio of phosphorus and nitrogen is set to at least 0.06, and silicon / nitrogen representing the mass concentration ratio of silicon and the nitrogen is set to 0.18 or higher, respectively. Liquid. The diatom-containing liquid according to claim 16, comprising hydrogen carbonate. 17. The liquid containing diatom according to claim 16, wherein the silicon / nitrogen in the solution before inoculating the diatom is determined to substantially coincide with the ratio of silicon consumption and nitrogen consumption of the diatom measured in advance. 17. The liquid containing diatom according to claim 16, wherein the phosphorus / nitrogen in the solution before inoculation of the diatom is determined to approximately coincide with the ratio of the phosphorus consumption and the nitrogen consumption of the diatom measured in advance. 17. The liquid containing diatom according to claim 16, wherein said diatom is classified as Chitoceras. 17. The liquid containing diatom according to claim 16, wherein said diatom is classified into the genus Peodactil. A silicon / nitrogen containing silicon and nitrogen, wherein the silicon / nitrogen showing the mass concentration ratio of the silicon and the nitrogen is incubated in a liquid having a value exceeding 0.18. Phosphorus / nitrogen containing phosphorus, nitrogen and silicon, and representing the mass concentration ratio of phosphorus and nitrogen, and silicon / nitrogen representing the mass concentration ratio of silicon and nitrogen, respectively, were cultured in a liquid set at 0.1 or more. Diatom to be made. A diatom characterized by culturing in a liquid containing phosphorus, nitrogen and silicon and having a pH set in the range of 6.4 to 8.4. As a method of culturing diatoms, using a diatom culture liquid containing nitrogen and silicon to cultivate diatoms, A pre-measuring step of measuring the initial concentration of nitrogen and the initial concentration of silicon contained in the liquid, respectively; An inoculation step of inoculating the diatom in the liquid; A pre-supplement measurement step of measuring the pre-concentration concentration of the nitrogen and the pre-concentration concentration of the silicon, respectively; A concentration difference measuring step of obtaining a nitrogen concentration difference representing the difference between the initial concentration of nitrogen and the concentration before the replenishment, and a silicon concentration difference representing the difference between the initial concentration of the silicon and the concentration before the replenishment; And The amount of nitrogen to be supplied to the liquid and the amount of silicon to be supplied to the liquid are determined based on the nitrogen concentration difference and the silicon concentration difference so as to coincide with the ratio of the initial concentration of nitrogen to the initial concentration of silicon, and the supply to supply them to the liquid. Diatom cultivation method characterized in that it comprises a step. As a method of culturing diatoms, using a diatom culture liquid containing nitrogen and silicon to cultivate diatoms, A pre-supplement measurement step of measuring the pre-concentration concentration of the nitrogen and the pre-concentration concentration of the silicon, respectively; A concentration difference measuring step of obtaining a nitrogen concentration difference representing the difference between the predetermined concentration of nitrogen and the concentration before the replenishment, and a silicon concentration difference representing the difference between the predetermined concentration of the silicon and the concentration before the replenishment; And The amount of nitrogen to be supplied to the liquid and the amount of silicon to be supplied to the liquid are respectively determined based on the nitrogen concentration difference and the silicon concentration difference to coincide with the ratio of the predetermined concentration of the nitrogen to the predetermined concentration of the silicon, and the supply to supply them to the liquid. Diatom cultivation method characterized in that it comprises a step. The method of claim 27, wherein the liquid further contains phosphorus, In the pre-replenishment measuring step, the concentration of the pre-replenishment of phosphorus is measured, In the concentration difference measurement step, obtaining a phosphorus concentration difference representing the difference between the predetermined concentration of phosphorus and the concentration before replenishment, In the replenishment step, cultivation of diatoms characterized in that the amount of phosphorus to be supplied to the liquid is determined based on the difference in the concentration of phosphorus so as to match the ratio of the predetermined concentration of the nitrogen and the predetermined concentration of the phosphorus. Way. 28. The method for culturing diatoms according to claim 27, wherein the diatom cultivation is performed while venting a gas into the liquid. The method for culturing diatoms according to claim 27, wherein the silicon / nitrogen in the solution before inoculation of the diatoms is determined to be approximately equal to the ratio of silicon consumption and nitrogen consumption of the diatom measured beforehand. 29. The method for culturing diatoms according to claim 28, wherein the phosphorus / nitrogen in the solution before inoculation of the diatoms is determined to be approximately equal to the ratio of the phosphorus consumption and the nitrogen consumption of the diatom measured in advance. As a method of culturing diatoms, using a diatom culture liquid containing nitrogen and silicon to cultivate diatoms, A pre-measuring step of measuring the initial concentration of nitrogen and the initial concentration of silicon contained in the diatomized liquid, respectively; A pre-supplement measurement step of measuring the pre-concentration concentration of the nitrogen and the pre-concentration concentration of the silicon, respectively; A concentration difference measuring step of obtaining a nitrogen concentration difference representing the difference between the initial concentration of nitrogen and the concentration before the replenishment, and a silicon concentration difference representing the difference between the initial concentration of the silicon and the concentration before the replenishment; And The amount of nitrogen to be supplied to the liquid and the amount of silicon to be supplied to the liquid are determined based on the nitrogen concentration difference and the silicon concentration difference so as to coincide with the ratio of the initial concentration of nitrogen to the initial concentration of silicon, and the supply to supply them to the liquid. Diatom cultivation method characterized in that it comprises a step. 33. The method of claim 32, wherein the liquid further contains phosphorus, In the pre-measuring step, the initial concentration of the phosphorus contained in the liquid is measured, In the pre-replenishment measuring step, the pre-replenishment concentration of the phosphorus is measured, In the step of measuring the concentration difference, the phosphorus concentration difference representing the difference between the initial concentration of phosphorus and the concentration before replenishment is obtained, In the replenishment step, cultivation of diatoms characterized in that the amount of phosphorus to be supplied to the liquid is determined based on the difference in the concentration of phosphorus so as to match the ratio of the initial concentration of the nitrogen and the predetermined concentration of the phosphorus. Way. 33. The method for culturing diatoms according to claim 32, wherein the cultivation of the diatoms is performed while venting a gas into the liquid. 33. The method of culturing diatoms according to claim 32, wherein the silicon / nitrogen in the solution before inoculating the diatoms is determined to be approximately equal to the ratio of silicon consumption and nitrogen consumption of the diatom measured in advance. The method of culturing diatoms according to claim 33, wherein the phosphorus / nitrogen in the solution before inoculation of the diatoms is determined to be approximately equal to the ratio of the phosphorus consumption and the nitrogen consumption of the diatom measured in advance. As a method of culturing diatoms, in which diatoms are cultivated using a culture liquid containing nitrogen and silicon, A consumption measuring liquid inoculated with the diatom, the pre-measuring step of measuring nitrogen consumption and silicon consumption respectively consumed by the diatom; Inoculation step of inoculating the diatom in the culture liquid; A measuring step of measuring a cell concentration of the diatom; When the cell concentration exceeds a predetermined amount, preparing an extra cost liquid such that the ratio of nitrogen and silicon is approximately equal to the ratio of nitrogen consumption and silicon consumption; And And a replenishment step of replenishing the prepared extra cost liquid to the culture liquid. The liquid of claim 37, wherein the culture liquid further contains phosphorus, In the pre-measuring step, the phosphorus consumption consumed by the diatom in the liquid for consumption measurement is measured, In the preparing step, a supplementary cost liquid is prepared so that the ratio of nitrogen and phosphorus is approximately equal to the ratio of nitrogen consumption and phosphorus consumption. 38. The method for culturing diatoms according to claim 37, wherein the cultivation of the diatoms is performed while venting a gas into the liquid. 38. The method of culturing diatoms according to claim 37, wherein the silicon / nitrogen in the solution before inoculating the diatoms is determined to be approximately equal to the ratio of silicon consumption and nitrogen consumption of the diatom measured beforehand. The method of culturing diatoms according to claim 38, wherein the phosphorus / nitrogen in the solution before inoculating the diatoms is determined so as to substantially match the ratio of the phosphorus consumption and the nitrogen consumption of the diatom measured in advance.