Method for determining the culture state of microalgae

Abstract

In a separation step of a microalgal culture state determination method, a culture liquid, in which a microalga is cultured, is separated into the microalga and a separation liquid. In a transmittance measurement step, the separation liquid is irradiated with light and the transmittance of light to the separation liquid is measured to obtain the transmittance measurement value. In a determination step, the culture state is determined on the basis of the transmittance measurement value.

Description

[Technical field]

[0001] The present invention relates to a method for determining the culture state of microalgae. [Background technology]

[0002] For example, JP 2008-283946 A proposes a method for determining the proliferation activity of microalgae as a culture state of the microalgae. In this method, a culture solution of the microalgae is sampled as a test sample. The test sample containing the microalgae is irradiated with light. The intensity of the fluorescence emitted from the test sample is measured. The fluorescence intensity obtained by this measurement is compared with data obtained in advance. This data indicates the relationship between the intensity of the fluorescence and the proliferation activity. As a result, it becomes possible to quantitatively determine the proliferation activity of the microalgae contained in the culture solution. Summary of the Invention

[0003] In the above-mentioned determination method, the sampled test sample contains both living microalgae and dead microalgae (dead microalgae before decomposition). It is difficult to distinguish between dead microalgae and living microalgae until a certain time has passed since the microalgae died. For this reason, there is a concern that dead microalgae will emit fluorescence of the same intensity as living microalgae. As a result, there is a concern that the fluorescence emitted by dead microalgae will reduce the accuracy of determining the proliferation activity of living microalgae. In other words, there is a concern that the above-mentioned determination method cannot determine the culture state of microalgae with high accuracy.

[0004] The present invention aims to solve the above-mentioned problems.

[0005] One aspect of the present invention is a method for determining the culture state of microalgae, which determines the culture state of microalgae, and includes a separation process for separating a culture solution in which the microalgae are cultured into the microalgae and a separation solution, a transmittance measurement process for irradiating the separation solution with light and measuring the transmittance of the light through the separation solution to obtain a transmittance measurement value, and a determination process for determining the culture state based on the transmittance measurement value.

[0006] In this culture state determination method, the culture state of the microalgae can be quantitatively determined based on the transmittance measurement value of the separation liquid that does not contain the microalgae. In this case, for example, it is possible to prevent the microalgae in the culture liquid from reducing the accuracy of the determination of the culture state. Therefore, it is possible to quantitatively and highly accurately determine the culture state of the microalgae. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 is a process diagram illustrating a method for determining a culture state of microalgae according to an embodiment of the present invention. [Diagram 2] FIG. 2 shows the transmittance spectrum of the first separation liquid for each culture day. [Diagram 3] FIG. 3 shows the transmittance spectrum of the second separation liquid for each culture day. [Figure 4] FIG. 4 is a graph showing the relationship between the number of culture days for each of microalgae A to D and the light transmittance of the third separation liquid. [Diagram 5] FIG. 5 is a graph showing the relationship between the number of culture days and the absorbance of the third separation liquid for each of microalgae A to D. [Figure 6] FIG. 6 is a graph showing the relationship between the number of days of culture for each of microalgae A to D and the concentration of the entire microalgae in the fourth sampling liquid. [Figure 7] FIG. 7 is a graph illustrating the start transmission spectrum and the end transmission spectrum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The culture state determination method according to the present embodiment determines the culture state of microalgae cultured in a culture solution. The culture solution is, for example, contained in a culture tank (not shown) together with microalgae. The culture solution preferably contains water and nutrients necessary for culturing the microalgae. The nutrients are, for example, nitrogen, phosphorus, potassium, etc. The culture tank is placed in an environment capable of receiving light of a wavelength (e.g., 340 to 700 nm) necessary for the growth of the microalgae. One example of such an environment is outdoors where at least one of sunlight and artificial light can be received. Another example of the environment is indoors where at least one of sunlight and artificial light can be received.

[0009] Microalgae whose culture state can be determined by the culture state determination method are not particularly limited. However, when a biofuel such as ethanol is produced using cultured microalgae, the microalgae are preferably classified into the class Chlorophyceae (e.g., Chlamydomonas or Chlorella), Prasinophyceae, Cryptophyceae, or Cyanobacteria (e.g., Spirulina).

[0010] In the culture state determination method according to the present embodiment, the proliferation activity of microalgae is determined as the culture state. In the following, an example will be described in which the culture state determination method according to the present embodiment is applied to select microalgae to be used as seed algae when culturing and producing microalgae. That is, in the culture state determination method, the proliferation activity of the microalgae to be used as seed algae is determined. In this case, for example, a proliferation activity that allows the microalgae to proliferate well and a predetermined production amount to be obtained when the microalgae are cultured as seed algae is determined as a judgment reference value through a prior experiment or the like.

[0011] As shown in FIG. 1, the culture state determination method includes a separation step. In this embodiment, microalgae are cultured in a culture solution in a culture tank until a predetermined culture time (e.g., 4 days) appropriate for culturing seed algae has elapsed. After the predetermined culture time has elapsed, a part of the culture solution in the culture tank is sampled to obtain a first sampling solution. In the separation step, the microalgae are separated from the first sampling solution, for example, by centrifugation. As a result, the first sampling solution is separated into the microalgae and a separation solution not containing the microalgae.

[0012] In this embodiment, a concentration measurement step is performed in addition to the separation step. In the concentration measurement step, a part of the culture solution in the culture tank is sampled to obtain a second sampling solution at the same timing as when the first sampling solution is obtained. The total concentration of the microalgae contained in this second sampling solution is obtained. The total concentration of the microalgae contained in the second sampling solution can be treated as the same value as the total concentration of the microalgae contained in the culture solution in the culture tank after a predetermined culture time has elapsed.

[0013] Here, the culture solution in the culture tank (as well as the second sampling solution) contains both living microalgae and dead microalgae before decomposition. Therefore, the concentration of the total microalgae is the concentration of both living microalgae and dead microalgae that are difficult to distinguish from the living microalgae in the culture solution.

[0014] As a specific method for determining the total concentration of microalgae contained in the second sampling liquid, first, the mass of the second sampling liquid is measured. Next, the second sampling liquid is, for example, filtered to obtain microalgae separated from the culture liquid. The dry mass of this microalgae is measured. The dry mass is the mass of the microalgae in a state in which the water has been removed by drying. The total concentration of microalgae in the second sampling liquid is determined from the mass of the second sampling liquid measured as described above and the dry mass of the microalgae. That is, the total concentration of microalgae contained in the culture liquid in the culture tank after a predetermined culture time has elapsed is determined.

[0015] In addition, in the concentration measurement step, in addition to the total concentration of microalgae contained in the culture solution in the culture tank after a predetermined culture time has elapsed, the total concentration of microalgae contained in the culture solution in the culture tank at the start of culture may be determined in advance. The total concentration of microalgae contained in the culture solution in the culture tank at the start of culture can also be determined in the same manner as the total concentration of microalgae contained in the culture solution in the culture tank after a predetermined culture time has elapsed.

[0016] After the separation step and the concentration measurement step are completed as described above, a transmittance measurement step is performed. In the transmittance measurement step, for example, a spectrophotometer is used to irradiate the separated liquid with light and measure the light transmittance of the separated liquid. This provides a transmittance measurement value.

[0017] FIG. 2 shows the transmission spectrum of the first separated liquid obtained by separating microalgae from a culture solution in which microalgae with a proliferation activity equal to or greater than the judgment reference value are cultured. The transmission spectrum in FIG. 2 is measured as follows. The microalgae (seed algae) are cultured for 7 days. From the start of the culture (day 0) to the fourth day of the culture, the culture solution in the culture tank is sampled every day to obtain a first separated liquid for five days. In addition, the culture solution in the culture tank on the seventh day of the culture is sampled to obtain a first separated liquid. For each of these first separated liquids, the transmission spectrum in the wavelength range of 190 to 900 nm was measured.

[0018] On the other hand, the transmission spectrum of the second separated liquid obtained by separating the microalgae from the culture liquid in which the microalgae having a proliferation activity lower than the judgment reference value are cultured is shown in Fig. 3. The transmission spectrum of the second separated liquid in Fig. 3 is also measured in the same manner as the transmission spectrum of the first separated liquid in Fig. 2.

[0019] As shown in Figures 2 and 3, the transmittance of both the first and second separation liquids tends to decrease as the number of culture days increases. The amount of decrease in transmittance is greater in the second separation liquid than in the first separation liquid. In other words, the second separation liquid obtained by culturing microalgae whose proliferation activity is less than the judgment reference value exhibits a greater decrease in transmittance as the number of culture days increases, compared to the first separation liquid obtained by culturing microalgae whose proliferation activity is equal to or greater than the judgment reference value. This is thought to be due to the fact that the amount of decrease in transmittance is proportional to the dead algae concentration. The dead algae concentration is the concentration of dead microalgae.

[0020] When the proliferation activity is smaller than the judgment reference value, the rate at which the microalgae die is likely to be large relative to the rate at which the microalgae proliferate. As a result, the amount of dead microalgae in the culture solution increases, and the dead algae concentration increases. It is believed that the transmittance decreases in proportion to the increase in the dead algae concentration. As a result, the amount of decrease in the transmittance in the second separation liquid is greater than the amount of decrease in the transmittance in the first separation liquid.

[0021] In other words, there is a correlation between the transmittance of a separation liquid obtained by separating microalgae from a culture liquid in which the microalgae are cultured, and the proliferation activity of the microalgae. Based on this correlation, for example, a minimum value of the transmittance of a separation liquid having a proliferation activity at a reference value is obtained by a prior experiment or the like, and the minimum value is set as the transmittance reference value. An example of the transmittance reference value can be 70%, but is not particularly limited to this.

[0022] In the transmittance measurement step, the wavelength of light irradiated onto the separation liquid to obtain a transmittance measurement value is preferably selected from, for example, a range of 230 to 500 nm. From Figs. 2 and 3, when the separation liquid is irradiated with light in a wavelength range of 500 nm or less, a noticeable decrease in transmittance is observed with the passage of the number of days of culture. Furthermore, when the separation liquid is irradiated with light in a wavelength range of 230 nm or more, a decrease in transmittance with the passage of the number of days of culture can be observed within a range where the influence of error is small. Therefore, by selecting the wavelength of light irradiated onto the separation liquid from a range of 230 to 500 nm, it becomes possible to determine the culture state of the microalgae with high accuracy. In the transmittance measurement step of this embodiment, the separation liquid is irradiated with light of a wavelength of 250 nm to obtain a transmittance measurement value.

[0023] Next, as shown in FIG. 1, a determination step is performed. In the determination step, it is determined whether or not the proliferation activity of the microalgae is equal to or greater than a reference value. Specifically, in the determination step, the transmittance measurement value obtained in the transmittance measurement step is compared with the transmittance reference value. As described above, by determining the transmittance reference value, when the transmittance measurement value is equal to or greater than the transmittance reference value, it is possible to determine that the proliferation activity of the microalgae is equal to or greater than the reference value. On the other hand, when the transmittance measurement value is smaller than the transmittance reference value, it is possible to determine that the proliferation activity of the microalgae is smaller than the reference value.

[0024] In addition, in the determination step, when determining the proliferation activity of the microalgae, the concentration of the entire microalgae contained in the culture solution in the culture tank obtained in the concentration measurement step may be further taken into consideration. That is, for example, when the concentration of the entire microalgae contained in the culture solution in the culture tank after a predetermined culture time has elapsed is equal to or lower than the concentration of the entire microalgae contained in the culture solution in the culture tank at the start of the culture, the proliferation activity of the microalgae may be determined to be smaller than the judgment reference value.

[0025] Also, for example, when the concentration of the whole microalgae contained in the culture solution in the culture tank after a predetermined culture time has elapsed is equal to or greater than the concentration reference value and the measured transmittance value is equal to or greater than the transmittance reference value, it may be determined that the proliferation activity of the microalgae is equal to or greater than the judgment reference value. Furthermore, for example, when the concentration of the whole microalgae contained in the culture solution in the culture tank after a predetermined culture time has elapsed is equal to or greater than the concentration reference value and the measured transmittance value is smaller than the transmittance reference value, it may be determined that the proliferation activity of the microalgae is smaller than the judgment reference value.

[0026] The concentration reference value can be set, for example, by a prior experiment, etc. The concentration reference value can be, for example, the minimum concentration of the whole microalgae at which it is possible to determine that the proliferation activity of the microalgae is equal to or greater than the determination reference value when the microalgae are cultured for a predetermined culture time.

[0027] As described above, in the culture state determination method according to the present embodiment, the culture state of the microalgae can be quantitatively determined based on the transmittance measurement value of the separation liquid that does not contain the microalgae. In this case, for example, it is possible to prevent the microalgae in the culture liquid from reducing the accuracy of the determination of the culture state. Therefore, it is possible to quantitatively and highly accurately determine the culture state of the microalgae.

[0028] In the determination step according to the embodiment described above, the culture state is determined by determining whether or not the proliferation activity of the microalgae is equal to or greater than a reference value.

[0029] In the determination step of the above embodiment, the measured transmittance value is compared with the reference transmittance value, and if the measured transmittance value is equal to or greater than the reference transmittance value, the proliferation activity is determined to be equal to or greater than the determination reference value.

[0030] However, in the determination step, the method of determining whether the proliferation activity of microalgae is equal to or greater than the determination standard value based on the transmittance measurement value is not limited to comparing the transmittance measurement value with the transmittance standard value.

[0031] For example, in the determination step, the dead algae concentration may be determined from the measured transmittance value. As described above, the amount of decrease in the transmittance of the separation liquid over the number of days of culture is considered to be proportional to the dead algae concentration. Therefore, there is a correlation between the transmittance of the separation liquid and the dead algae concentration. Therefore, for example, the relationship between the transmittance of the separation liquid and the dead algae concentration is determined by preliminary experiments or the like as follows.

[0032] Four types of microalgae with different proliferation activities (microalgae A, B, C, D) are cultured for seven days. For each microalgae, the culture solution in the culture tank is sampled every day from the start of culture (day 0) to the fourth day of culture to obtain a third sampling solution for five days. In addition, the culture solution on the seventh day of culture is sampled to obtain a third sampling solution. In other words, a total of six days' worth of third sampling solution is obtained for each of microalgae A to D.

[0033] The microalgae contained in each third sampling liquid are separated, for example, by centrifugation, to obtain a third separation liquid that does not contain microalgae. Each of these third separation liquids is irradiated with light of a wavelength of 250 nm, for example, using a spectrophotometer, to measure the light transmittance of each third separation liquid. Figure 4 shows the relationship between the number of days of culture and the measured light transmittance of each third separation liquid. In addition, the light transmittance of each third separation liquid was logarithmically converted to determine the absorbance. Figure 5 shows the relationship between the number of days of culture and the absorbance of each third separation liquid.

[0034] At the same time as obtaining the third sampling liquid, a part of the culture liquid in the culture tank is sampled to obtain a fourth sampling liquid. That is, for each of the microalgae A to D, a total of six days' worth of the fourth sampling liquid is obtained.

[0035] The total concentration of microalgae contained in each of the fourth sampling solutions was measured. The relationship between the number of days of culture for each of microalgae A to D and the measured total concentration of microalgae is shown in FIG. 6. It can be seen from FIG. 6 that the rate of increase in the total concentration of microalgae decreases as the number of days of culture increases. It is considered that the microalgae are ideally growing during the number of days of culture before the rate of increase decreases. Therefore, the rate of increase in the total concentration of microalgae during the number of days of culture before the rate of increase decreases (the slope of the graph in FIG. 6) was taken as the ideal rate of increase.

[0036] In FIG. 6, the ideal concentration of all microalgae when microalgae A grows at the ideal growth rate is shown by the thick solid line Ai. The ideal concentration of all microalgae when microalgae B grows at the ideal growth rate is shown by the thin solid line Bi. The ideal concentration of all microalgae when microalgae C grows at the ideal growth rate is shown by the thick dashed line Ci. The ideal concentration of all microalgae when microalgae D grows at the ideal growth rate is shown by the thin dashed line Di.

[0037] The difference between the ideal concentration of the whole microalgae according to the ideal growth rate of the microalgae and the actually measured concentration of the whole microalgae is considered to be caused by the death of the microalgae during the culture. Therefore, the difference between the ideal concentration of the whole microalgae and the actually measured concentration of the whole microalgae is taken as the dead algae concentration. That is, as shown in FIG. 6, for example, for microalgae A, the difference between the ideal concentration of the whole microalgae on the thick solid line Ai and the actually measured concentration of the whole microalgae (indicated by x in FIG. 6) becomes large after the fourth day of culture. The difference between the ideal concentration of the whole microalgae on this thick solid line Ai and the actually measured concentration of the whole microalgae is taken as the dead algae concentration of microalgae A.

[0038] A conversion coefficient (proportionality constant) for converting absorbance into dead algae concentration can be obtained from the absorbance of each third separation liquid in Figure 5 and the dead algae concentration in Figure 6. By determining the conversion coefficient in advance in this manner, the dead algae concentration can be determined in the determination step from the transmittance measurement value acquired in the transmittance measurement step.

[0039] In the determination step, the ratio of the dead algae concentration to the total concentration of microalgae contained in the culture solution in the culture tank obtained in the concentration measurement step is determined. This ratio of the dead algae concentration to the total concentration of microalgae is compared with a ratio reference value. When the ratio of the dead algae concentration to the total concentration of microalgae is smaller than the ratio reference value, it can be determined that the proliferation activity of the microalgae is equal to or greater than the determination reference value. On the other hand, when the ratio of the dead algae concentration to the total concentration of microalgae is equal to or greater than the ratio reference value, it can be determined that the proliferation activity of the microalgae is smaller than the determination reference value.

[0040] The ratio reference value can be set, for example, by a prior experiment, etc. The ratio reference value can be, for example, the minimum value of the ratio of the concentration of dead algae to the concentration of the whole microalgae at which it is possible to determine that the proliferation activity of the microalgae is equal to or greater than the determination reference value when the microalgae are cultured for a predetermined culture time.

[0041] That is, in the determination step according to the above-mentioned modified example, the dead algae concentration, which is the concentration of dead microalgae in the culture solution, is calculated from the transmittance measurement value, and when the ratio of the dead algae concentration to the total concentration of microalgae in the culture solution is smaller than a ratio reference value, it is determined that the proliferation activity is equal to or greater than the determination reference value. Even in this case, the culture state of the microalgae can be quantitatively and highly accurately determined based on the transmittance measurement value of the separation solution that does not contain microalgae.

[0042] As described above, the culture solution in the culture tank contains both living and dead microalgae. The dead microalgae include those that are difficult to distinguish from living microalgae. For this reason, there is a concern that the proliferation activity of the microalgae cannot be accurately determined from a measurement value that simply measures the concentration of the entire microalgae contained in the culture solution. However, in the determination step according to the modified example of this embodiment, the dead algae concentration can be obtained from the transmittance measurement value of the separation solution. For this reason, the culture state of the microalgae can be determined with high accuracy regardless of whether dead microalgae are contained in the culture solution.

[0043] In the separation step according to the above embodiment, a separation liquid is obtained from the culture liquid in which the microalgae have been cultured until a predetermined culture time has elapsed, and in the determination step, when the proliferation activity is equal to or greater than a judgment reference value, the microalgae are determined to be suitable as seed algae. In this case, when it is determined in the determination step that the proliferation activity is equal to or greater than the judgment reference value, it can be determined that the microalgae cultured until a predetermined culture time has elapsed are suitable as seed algae. By culturing the seed algae thus determined, it is possible to improve the production amount of microalgae by culture. However, the microalgae whose proliferation activity is determined in the determination step are not limited to seed algae, and may be, for example, microalgae being cultivated and produced.

[0044] The culture state determined in the determination step is not limited to the proliferation activity of the microalgae. For example, in the determination step, the culture state may be determined by determining whether or not the survival rate of the microalgae in the culture solution is equal to or greater than a reference value.

[0045] That is, in the determination process, the dead algae concentration, which is the concentration of dead microalgae in the culture solution, is calculated from the transmittance measurement value, and the survival rate, which is the proportion of surviving microalgae to the total microalgae in the culture solution, is calculated from the total concentration of microalgae in the culture solution and the dead algae concentration, and the culture state is determined by determining whether the survival rate is above a reference value.

[0046] The concentration of surviving microalgae can be calculated from the difference between the total concentration of microalgae in the culture solution and the concentration of dead microalgae. Therefore, the survival rate of microalgae in the culture solution can be calculated. For example, when the survival rate of microalgae is equal to or greater than the judgment reference value, it can be determined that the culture solution contains a sufficient amount of surviving microalgae, and therefore the microalgae are well cultured. On the other hand, when the survival rate of microalgae is smaller than the judgment reference value, it can be determined that there is a concern that there is a problem with the culture state of the microalgae, since there are many dead microalgae in the culture solution.

[0047] In the method for determining the culture state of microalgae according to the above embodiment, the wavelength of light irradiated to the separation liquid in the transmittance measurement step is preferably 230 nm or more. In this case, as shown in Figures 2 and 3, the decrease in transmittance over the number of culture days can be observed within a range where the influence of error is small. As a result, it becomes possible to determine the culture state of microalgae with high accuracy.

[0048] In the method for determining the culture state of microalgae according to the above embodiment, the wavelength of light irradiated to the separation liquid in the transmittance measurement step is preferably 500 nm or less. In this case, as shown in Figures 2 and 3, the decrease in transmittance over the number of culture days can be observed in a range where the decrease in transmittance is noticeable. As a result, it becomes possible to determine the culture state of microalgae with high accuracy.

[0049] The wavelength of the light irradiated to the separation liquid in the transmittance measurement step may be determined by performing a wavelength determination step before the transmittance measurement step. In this case, the method for determining the culture state of microalgae includes a wavelength determination step of culturing the detection microalgae in a detection culture liquid and determining the wavelength of the light irradiated to the separation liquid in the transmittance measurement step before the transmittance measurement step, and the wavelength determination step includes a first separation step of separating the detection culture liquid at the start of the culture of the detection microalgae in the detection culture liquid into the detection microalgae and a start separation liquid, a second separation step of separating the detection culture liquid at the end of the culture of the detection microalgae in the detection culture liquid into the detection microalgae and a finish separation liquid, a first acquisition step of acquiring a start transmission spectrum from the transmittance obtained by irradiating the start separation liquid with light in a predetermined wavelength range, and a finish transmission spectrum from the transmittance obtained by irradiating the finish separation liquid with light in a predetermined wavelength range. the selection step includes a step of dividing the start transmission spectrum into a first region where the transmittance increases rapidly with increasing wavelength and a second region where the transmittance increases more slowly with increasing wavelength compared to the first region, and identifying a first wavelength which is the boundary between the first and second regions; a step of dividing the end transmission spectrum into a third region where the transmittance increases rapidly with increasing wavelength and a fourth region where the transmittance increases more slowly with increasing wavelength compared to the third region, and identifying a second wavelength which is the boundary between the third and fourth regions; and a step of selecting a wavelength of light to be irradiated to the separation liquid from the wavelength range between the first and second wavelengths.

[0050] Specifically, a wavelength determination step is performed before the transmittance measurement step. In the wavelength determination step, the detection microalgae are cultured in the detection culture solution for a predetermined number of days (e.g., 7 days). In the first separation step, the culture solution at the start of the culture is sampled and separated into the detection microalgae and a start-time separation solution. In the second separation step, the culture solution at the end of the culture is sampled and separated into the detection microalgae and a end-time separation solution.

[0051] In the first acquisition step, a start transmission spectrum (FIG. 7) is acquired from the transmittance obtained by irradiating the start separation liquid with light in a predetermined wavelength range (e.g., 190-900 nm). In the second acquisition step, a finish transmission spectrum (FIG. 7) is acquired from the transmittance obtained by irradiating the end separation liquid with light in a predetermined wavelength range (e.g., 190-900 nm).

[0052] In the selection step, the wavelength of light to be irradiated to the separation liquid in the transmittance measurement step is selected using the start transmission spectrum and end transmission spectrum acquired as described above. Specifically, in the selection step, the start transmission spectrum is divided into a first region and a second region as shown in FIG. 7. In the first region, the transmittance increases more rapidly with increasing wavelength than in the second region. In the second region, the transmittance increases more gently with increasing wavelength than in the first region. The wavelength at the boundary point between the first region and the second region, shown by a white circle in FIG. 7, is identified as the first wavelength.

[0053] In the selection process, the final transmission spectrum is divided into a third region and a fourth region. The third region has a steeper increase in transmittance with respect to an increase in wavelength than the fourth region. The fourth region has a gentler increase in transmittance with respect to an increase in wavelength than the third region. The wavelength at the boundary between the third region and the fourth region, shown by a black circle in FIG. 7, is identified as the second wavelength.

[0054] The wavelength of light to be irradiated to the separation liquid in the transmittance measurement step is selected from the wavelength range between the first wavelength and the second wavelength specified as described above. In this way, by selecting the wavelength of light to be irradiated to the separation liquid in the transmittance measurement step, the decrease in transmittance over the number of days of culture can be observed in a range where the effect of error is small. Furthermore, the decrease in transmittance over the number of days of culture can be observed in a range where the decrease in transmittance is noticeable. As a result, it becomes possible to determine the culture state of the microalgae with high accuracy.

[0055] The present invention is not limited to the above disclosure, and various configurations can be adopted without departing from the gist of the present invention.

Claims

1. A method for determining a culture state of microalgae, comprising: A separation step of separating the culture solution for culturing the microalgae into the microalgae and a separation solution; a transmittance measuring step of irradiating the separation liquid with light having a predetermined wavelength selected from a wavelength range of 230 nm to 500 nm and measuring the transmittance of the light through the separation liquid to obtain a transmittance measurement value; A determination step of determining the culture state based on the transmittance measurement value; having In the determination step, the transmittance measurement value is compared with a predetermined transmittance reference value, and when the transmittance measurement value is equal to or greater than the transmittance reference value, the proliferation activity of the microalgae is determined to be equal to or greater than the determination reference value; A method for determining the culture state of microalgae, wherein the transmittance reference value is a transmittance measurement value obtained by irradiating light onto a reference separation liquid obtained by removing the microalgae from a culture liquid in which the microalgae can grow, and measuring the transmittance of the light for the reference separation liquid.

2. The method for determining a culture state of microalgae according to claim 1, In the determination process, the dead algae concentration, which is the concentration of dead microalgae in the culture solution, is calculated from the transmittance measurement value, and if the ratio of the dead algae concentration to the total concentration of the microalgae in the culture solution is smaller than a ratio standard value, the method for determining the culture state of microalgae determines that the proliferation activity is equal to or greater than the determination standard value.

3. The method for determining a culture state of microalgae according to claim 1, In the separation step, the separation liquid is obtained from the culture liquid in which the microalgae have been cultured until a predetermined culture time has elapsed, In the determination step, the microalgae are determined to be suitable as seed algae when the proliferation activity is equal to or greater than the determination reference value.

4. A method for determining a culture state of microalgae, comprising: A separation step of separating the culture solution for culturing the microalgae into the microalgae and a separation solution; a transmittance measuring step of irradiating the separation liquid with light having a predetermined wavelength selected from a wavelength range of 230 nm to 500 nm and measuring the transmittance of the light through the separation liquid to obtain a transmittance measurement value; A determination step of determining the culture state based on the transmittance measurement value; having In the determination process, the dead algae concentration, which is the concentration of dead microalgae in the culture solution, is calculated from the transmittance measurement value, and the survival rate, which is the proportion of surviving microalgae to the total microalgae in the culture solution, is calculated from the total concentration of the microalgae in the culture solution and the dead algae concentration, and the culture state is determined by determining whether the survival rate is equal to or greater than a determination standard value.

5. The method for determining a culture state of microalgae according to any one of claims 1 to 4, A wavelength determination step of culturing detection microalgae in a detection culture solution prior to the transmittance measurement step and determining the wavelength of the light to be irradiated to the separation solution in the transmittance measurement step, The wavelength determination step includes: A first separation step of separating the detection culture solution into the detection microalgae and a start-up separation solution when starting to culture the detection microalgae in the detection culture solution; A second separation step of separating the detection culture solution into the detection microalgae and a termination separation solution when the culture of the detection microalgae in the detection culture solution is terminated; a first acquisition step of acquiring an initial transmission spectrum from a transmittance obtained by irradiating the initial separation liquid with light in a predetermined wavelength range; a second acquisition step of acquiring a final transmission spectrum from a transmittance obtained by irradiating the final separation liquid with light of a predetermined wavelength range; A selection step of selecting a wavelength of the light to be irradiated onto the separation liquid in the transmittance measurement step, The selection step includes: dividing the initial transmission spectrum into a first region in which the transmittance increases rapidly with increasing wavelength and a second region in which the transmittance increases more slowly with increasing wavelength than the first region, and identifying a first wavelength that is a boundary between the first region and the second region; dividing the end transmission spectrum into a third region in which the transmittance increases rapidly with increasing wavelength and a fourth region in which the transmittance increases more slowly with increasing wavelength than the third region, and identifying a second wavelength that is a boundary between the third region and the fourth region; and selecting the wavelength of the light to be irradiated to the separation liquid from a wavelength range between the first wavelength and the second wavelength.

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