JP6115199B2 - Determination method of fat-soluble component content of microalgae and culture method of microalgae - Google Patents

Description

  The present invention relates to a method for determining the content of fat-soluble components of microalgae for determining the culture state of microalgae. The present invention also relates to a method for culturing microalgae using this determination method.

  Microalgae are single-cell organisms with a size of several μm to several tens of μm. Among them, those that carry out photosynthesis efficiently use solar energy as lipids and wax components such as fatty acids and oils and fat-soluble components such as triterpene hydrocarbons. In addition, because it contains various minerals and unsaturated fatty acids at high concentrations, fat-soluble components such as lipids and hydrocarbons produced in the body are used as alternative fuels for oil such as jet fuel and diesel fuel. Various uses have been proposed, such as using it as a health food as represented by chlorella, or extracting functional lipids such as unsaturated fatty acids into supplements and functional feeds. In particular, the use as biofuel has attracted attention in recent years.

  When such microalgae are produced by a predetermined growth process for the purpose of producing fuel, it is necessary to separate the microalgae from the culture solution and to extract fat-soluble components such as fatty acids and fats and oils from the microalgae. . As a method of extracting the fat-soluble component from the microalgae and measuring the extracted fat-soluble component content, a method of measuring the weight by extracting the fat-soluble component with a solvent, extracting the fat-soluble component with a solvent, and then extracting the fat-soluble component by gas chromatography. A method for measuring the weight or the like of a soluble component is generally known.

  Although these methods have high measurement accuracy of fat-soluble component content, they require many steps such as solid-liquid separation, drying, extraction and analysis, and it takes a long time to complete the analysis. There is a problem that collection (harvesting) cannot be performed at the optimum timing. In culturing microalgae, it is important to be able to quickly determine the harvest time because microalgae may be killed in a short time due to contamination with protozoa and bacteria in the culture pond.

  Thus, as a method for quickly judging the harvest time of microalgae, various methods for staining fat-soluble components in microalgae cells with fluorescent dyes and estimating the fat-soluble component from the fluorescence intensity using a fluorescence detector are disclosed. Has been. For example, Patent Document 1, Non-Patent Document 1 and Non-Patent Document 2 each stain a fat-soluble component in microalgae cells with Nile Red, and determine the amount of the fat-soluble component from the size of the stained site with a fluorescence microscope. A method has been proposed. Non-Patent Document 3 proposes a method of staining a fat-soluble component in microalgae cells with BODIPY and determining fluorescence intensity with a fluorescence plate reader. These methods have the advantage that the measurement accuracy of the fat-soluble component content is relatively inferior to the above-described method of extracting the fat-soluble component with a solvent and measuring the weight, but it is relatively high and requires a simple apparatus.

JP 05-268993 A

“Determination of Algal Cell Lipids Using Nile Red Using Microplates to Monitor Neutral Lipids in Chlorella Vulgaris” Raymond et al. , Application Note, Bio Tek Instrument, Inc. , AN072111_08, Rev07 / 12/11, 2011 Q. “A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae”. Hu et al. , Journal of Microbiological Methods, Vol. 77, no. 1, pp. 41-47, 2009 “Visualizing“ green oil ”in live algae cells” A. Cattolico et al. , Journal of Bioscience and Bioengineering, Vol. 109, no. 2, pp. 198-201, 2010

  However, the methods described in Patent Literature 1, Non-Patent Literature 1, Non-Patent Literature 2 and Non-Patent Literature 3 have a problem that they are unsuitable for on-site automatic analysis because of complicated operations. Therefore, it is conceivable to construct a system for completely automating on-site analysis, but this involves a problem that the equipment cost is very high.

  In the process of producing fuel from microalgae, in order to perform stable fuel production, it is important that the microalgae collect (crop) at a timing when the fat-soluble components are sufficiently accumulated. Here, in order to reduce the culture cost, microalgae are often cultured in an open-air culture pond called an open pond. In this open pond, the culture conditions of microalgae are affected by environmental factors such as temperature and solar radiation, so the culture period until microalgae accumulate the maximum fat-soluble component suitable for recovery varies. Therefore, if it collects | recovers after a fixed culture | cultivation period, variation will arise in the fat-soluble component content which can be extracted. However, when analyzing the content of the fat-soluble component by various methods as described above and measuring the optimal timing for collecting the microalgae, it takes time and cost and is not practical. Furthermore, the open pound at the time of practical use is expected to be on the order of several thousand ha, and a method for efficiently monitoring fat-soluble components accumulated in a wide range of microalgae is required, but such a monitoring technique has not been available.

  This invention is made | formed in view of the said subject, and it aims at providing the method for monitoring the culture state of a micro algae efficiently and judging the fat-soluble component content of the micro algae. Another object of the present invention is to provide a method for culturing microalgae using this determination method.

  In order to solve the above-mentioned problems, first, the present invention is a method for determining the content of fat-soluble components of microalgae. From the color of the culture solution containing microalgae, blue light (450 to 490 nm), green light ( 500 to 570 nm) and red light (620 to 740 nm) are detected by decomposing at least two or more wavelength regions, and the amount of fat-soluble component content of microalgae is judged based on the light intensity of the two or more light wavelengths. A method for judging the content of fat-soluble components of microalgae is provided (Invention 1).

  According to this invention (Invention 1), in order to decompose the color of the culture solution containing microalgae into blue light, green light, and red light, it is only necessary to detect the chromaticity of visible light separately for RGB, which is inexpensive. A simple color sensor can be applied. By applying the color sensor, online measurement is possible, and the measurement time can be greatly shortened and the measurement can be simplified. Furthermore, this color sensor can sense changes in a wide range of color tone using only visible light and using satellite photographs. By these, it becomes possible to monitor the culture state of a microalgae efficiently and judge the fat-soluble component content of the microalgae.

  In the said invention (invention 1), the said green light (500-570 nm) and the said red light (620-740 nm) are decomposed | disassembled and detected, the light intensity of the wavelength of green light, and the light intensity of the wavelength of red light, From the above, it is preferable to determine the amount of fat-soluble component content of microalgae (Invention 2). In particular, the microalgae is calculated by calculating the absorbance of the red light and the absorbance of the green light from the light intensity of the wavelength of the green light and the light intensity of the wavelength range of the red light, and dividing the absorbance of the green light by the absorbance of the red light. It is preferable to determine the amount of the fat-soluble component content (Invention 3).

  According to the inventions (Inventions 2 and 3), the light intensity ratio between the absorbance of red light and the absorbance of green light is correlated with the fat-soluble component content of the green microalga that performs photosynthesis. Since the absorbance ratio decreases as the content increases, if this absorbance ratio falls below a predetermined value according to the type of microalgae, it is possible to determine that the fat-soluble component content retained by the microalgae is sufficient. .

  In the said invention (invention 1-3), it calculates by contrasting the light intensity of each wavelength which the culture solution containing the said micro algae spectrally divided with the light intensity of each wavelength which isolate | separated white light, respectively. It is preferable to determine the amount of the fat-soluble component content (Invention 4). In the said invention (invention 4), it is preferable that the said white light is the white light which permeate | transmitted clear water, or the reflected light from the white substance immersed in clear water (invention 5).

  According to the inventions (Inventions 4 and 5), the light intensity of the blue light, the green light and the red light of the culture solution containing microalgae is compared with the light intensity of each wavelength obtained by spectrally dividing the white light. By calculating the absorbance, the fat-soluble component content retained by the microalgae can be easily determined.

  Secondly, the present invention detects the color of a culture solution containing microalgae by decomposing it into green light (500 to 570 nm) and red light (620 to 740 nm), and the absorbance of the red light is measured with green light. A method of cultivating microalgae is provided, wherein a value divided by absorbance is calculated, and when the value falls below a predetermined threshold, all or part of the culture solution is collected (Invention 6).

  According to this invention (Invention 6), the color of the culture solution containing microalgae is detected by decomposing it into green light and red light, the light intensity of the green light and red light is calculated, and the absorbance of this red light is calculated. The value (absorbance ratio) divided by the absorbance of green light is monitored. At this time, the green microalgae that carry out photosynthesis decreases the absorbance ratio when the content of the fat-soluble component held increases, so if this absorbance ratio falls below a predetermined threshold, the content of the fat-soluble component held by the microalgae is sufficient. By judging and recovering a part or the whole amount, the microalgae can be efficiently cultured.

  Furthermore, thirdly, the present invention detects the color of a culture solution containing microalgae by decomposing it into green light (500 to 570 nm) and red light (620 to 740 nm), and the absorbance of the red light is measured with green light. Provided is a method for culturing microalgae, wherein a value divided by absorbance is calculated, and if the value shows a tendency to decrease with time, all or part of the culture solution is recovered (Invention 7).

  According to this invention (Invention 7), the color of the culture solution containing microalgae is detected by decomposing it into green light and red light, and the value (absorbance ratio) obtained by dividing the absorbance of red light by the absorbance of green light is obtained. Monitor while calculating. At this time, the green microalgae that perform photosynthesis will decrease in the absorbance ratio when the content of the fat-soluble component held increases, so if the absorbance ratio tends to decrease over time, the content of the fat-soluble component held in the microalgae By judging that it has become sufficient and recovering a part or all of it, microalgae can be cultured efficiently.

  According to the present invention, the color of a culture solution containing microalgae is detected by decomposing into blue light, green light and red light, and the lipid solubility retained by the microalgae by comparing the light intensity in these wavelength ranges. Since the component content is determined, the on-line measurement of the fat-soluble component content of microalgae can be performed, and the measurement time can be greatly shortened and the measurement can be simplified. This makes it possible to efficiently monitor the culture state of the microalgae and determine the fat-soluble component content of the microalgae.

  In particular, the color of a culture solution containing green microalgae that performs photosynthesis is detected by decomposing green light and red light, and the absorbance of red light is determined from the light intensity of the green light wavelength and the light intensity of the red light wavelength. Is calculated by dividing the absorbance by the absorbance of green light (absorbance ratio). If the ratio of these absorbances falls below a predetermined value, it can be determined that the fat-soluble component content retained by the microalgae is sufficient.

It is the schematic which shows the apparatus which can implement the judgment method of the fat-soluble component content of the micro algae concerning 1st embodiment of this invention. It is the schematic which shows the apparatus which can implement the judgment method of the fat-soluble component content of the micro algae which concerns on 2nd embodiment of this invention. It is a graph which shows the relationship between the fat-soluble component content and the light absorbency in the judgment method of the fat-soluble component content of the micro algae (Ikadamo) in Example 1. It is a graph which shows the relationship between the fat-soluble component content and the light absorbency in the judgment method of the fat-soluble component content of the micro algae (Chlorella) in Example 2.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. However, this embodiment is only an example, and the present invention is not limited to this.

  The present invention decomposes the color (chromaticity) of a culture solution containing microalgae into at least two wavelength ranges of blue light (450 to 490 nm), green light (500 to 570 nm), and red light (620 to 740 nm). The amount of the fat-soluble component content of the microalgae is judged based on the light intensity in the wavelength range of these two or more lights.

  Specifically, in the case of green microalgae that undergo photosynthesis, detection is performed by decomposing into green light (500 to 570 nm) and red light (620 to 740 nm) from the hue (chromaticity) of the culture solution containing microalgae. The fat-soluble component content accumulated in the microalgae is determined from the light intensity in the wavelength range of green light and the light intensity in the wavelength range of red light. Here, as the green microalgae, those having a high maximum fat-soluble component content, that is, those having an excellent ability to produce a fat-soluble component are preferable.

  As a means for detecting the chromaticity of the culture solution, it is preferable to use a color sensor because it is inexpensive and can separately detect green light, red light and blue light. This color sensor has a mechanism in which a measured color is decomposed into RGB components by a color filter, and the light intensity of each color component is detected by a photodiode or the like. This color sensor can sense changes in a wide range of color tone using only visible light and using satellite photographs.

  Specifically, the content of fat-soluble components of microalgae is determined using a color sensor as follows. That is, first, white light is irradiated to transparent water (for example, pure water) that does not absorb light, and the transmitted light is detected by a color sensor. Since the white light is separated into RGB components by the color filter of the color sensor and received, the respective light intensities of the red band light (green light) R1 and the green band light (green light) G1 are obtained. measure.

  Next, the culture solution containing microalgae is irradiated with white light in the same manner using the same color sensor, and the transmitted light is detected by the color sensor. Since this transmitted light is separated into RGB components by the color filter of the color sensor and received, the respective light intensities of the red band light (red light) R2 and the green band light (green light) G2 at this time are obtained. measure.

  The red band light (red light) and the green band light (green light) can be measured using, for example, a transmissive color sensor 1 as shown in FIG. 1 described in Japanese Patent Application Laid-Open No. 2010-151605. it can. The transmissive color sensor 1 includes a light emitting unit 2 and a light receiving unit 3 including a color filter (not shown). The light that has been irradiated with white light from the light emitting unit 2 and transmitted through the culture solution 4. Is received by the light receiving unit 3, and the light intensity of each of the red band light (green light) and the green band light (green light) is calculated by a control mechanism (not shown).

  A reflective color sensor 11 as shown in FIG. 2 described in JP 2010-181150 can also be used. The reflective color sensor 11 includes a light-emitting unit 12, a light-receiving unit 13 including a color filter (not shown), and a reflective plate 14. The light transmitted through the culture medium 15 is received by the light receiving unit 13, and the light intensity of each of the red band light (green light) and the green band light (green light) is calculated by a control mechanism (not shown).

After measuring the red light and green light absorption intensities of the culture solution containing transparent water and microalgae in this way, the red light absorbance and the green light absorbance and the ratio (absorbance ratio) of the red light and the green light are calculated according to the following formula. Calculate each.
Red band light absorbance: A _R = −log (R2 / R1)
Green band light absorbance: A _G = −log (G2 / G1)
Absorbance ratio: X = _AR / _AG

  On the other hand, in the initial stage, the fat-soluble component content of microalgae in the culture solution is measured. Extraction of a fat-soluble component from microalgae may be performed by a Soxhlet extraction method using an organic solvent such as n-hexane as an extraction solvent.

  According to the inventor's research, it has been found that a high correlation is observed between the absorbance ratio of red light and green light and the content of fat-soluble components of microalgae. Therefore, as a result of analyzing this correlation, when the absorbance ratio (X) (the value obtained by dividing the absorbance of red light by the absorbance of green light) falls below a predetermined threshold, the accumulation state (content) of the fat-soluble component is sufficient. However, even if it is further cultured, the efficiency of increasing the fat-soluble component is reduced and the culture efficiency is not preferable. Therefore, by collecting (harvesting) a part or all of the microalgae, the microalgae can be efficiently recovered. It can be cultured. And since this threshold value changes with the kind of micro algae, a micro algae should be cultured beforehand and a threshold value should just be set from the relationship with a fat-soluble component content.

  Furthermore, if this correlation is applied, it is judged that the fat-soluble component has increased when the absorbance ratio (X) shows a tendency to decrease over time, and a part or all of the microalgae is recovered (harvested). By doing so, you may make it culture | cultivate a micro algae efficiently.

  As mentioned above, although this invention has been demonstrated based on the said embodiment, this invention is not restricted to the said embodiment, A various change implementation is possible. For example, in the present embodiment, for green microalgae, the culture state of microalgae is determined based on green light (500 to 570 nm) and red light (620 to 740 nm). In the case of red microalgae, absorbance data of blue light (450 to 490 nm) can also be used.

The present invention will be described in more detail based on the following specific examples and comparative examples, but the present invention is not limited to the following examples.
(Examples 1 and 2)

Table 1 in which Ikadamo (NIES-96 strain: Example 1) and Chlorella (NIES-2170 strain: Example 2) distributed from the National Institute for Environmental Studies microbial strain preservation facility were adjusted to pH 6.5 to 7.5 Using C medium with the composition shown in Table 2, this C medium was aerated with 3% by volume of industrial CO ₂ added to the air, and cultured under fluorescent lamp illumination (bright / dark = 12 hr / 12 hr) Went.

  The fat-soluble component content of the microalgae was judged as follows using the color sensor shown in FIG. That is, pure water produced by a pure water production apparatus “WG270” manufactured by Yamato Scientific Co., Ltd. is used as transparent water that does not absorb light, and the pure water is irradiated with white light, and the transmitted light is detected by a color sensor. The light intensities of the red band light (red light) R1 and the green band light (green light) G1 were measured.

  Next, using the same color sensor, the culture medium of Example 1 and Example 2 described above is irradiated with white light in the same manner, and the transmitted light is detected by the color sensor, and red band light (red light) R2 and green are detected. Each light intensity with the band light (green light) G2 was measured. The red light absorbance and green light absorbance were calculated from the red light intensity and the green light intensity, and the absorbance ratio was calculated from both absorbance values.

  On the other hand, a fat-soluble component was extracted from squid damo and chlorella in the culture solutions of Example 1 and Example 2, and the content of the fat-soluble component was measured and compared. Extraction was performed by Soxhlet extraction method using n-hexane as an extraction solvent, and the weight of the extract was measured.

  In addition, culture | cultivation by the said culture solution was each performed on two conditions shown in Table 3, and the relationship between the light absorbency ratio X and a fat-soluble component content was observed intermittently. The results are shown in FIGS.

  As is clear from FIGS. 3 and 4, a high correlation was observed between the fat-soluble component content and the absorbance ratio (X) of red light and green light regardless of the culture conditions, and the absorbance ratio (X) of the fat-soluble component was It was confirmed that it could be an indicator of accumulation status. In addition, the correlation between the fat-soluble component content and the absorbance ratio (X) is different between Ikadamo (Example 1) and Chlorella (Example 2), and the correlation is confirmed in advance for each type of microalgae to be cultured. I knew that I needed to do that.

  According to the method for determining the content of fat-soluble components of microalgae and the method for culturing microalgae of the present invention as described above, an inexpensive color sensor can be applied, and by applying this color sensor, online Measurement with this is possible. Thereby, the measurement time can be greatly shortened, the measurement can be simplified, and the measurement cost can be reduced. Furthermore, this color sensor can sense changes in a wide range of color tone using only visible light and using satellite photographs. By these, it becomes possible to monitor the culture state of microalgae efficiently and inexpensively, and to judge the fat-soluble component content of the microalgae. As a result, it is possible to determine the optimum timing for collecting (harvesting) the microalgae, and to realize stable production of the microalgae as the fuel raw material.

DESCRIPTION OF SYMBOLS 1 ... Transmission type color sensor 2 ... Light-emitting part 3 ... Light-receiving part 4 ... Culture solution 11 ... Reflection type color sensor 12 ... Light-emitting part 13 ... Light-receiving part 14 ... Reflecting plate 15 ... Culture solution

Claims (7)

A method for determining the content of fat-soluble components of microalgae,
From the color of the culture solution containing microalgae, decompose and detect at least two wavelength ranges of blue light (450 to 490 nm), green light (500 to 570 nm) and red light (620 to 740 nm),
A method for determining the content of a fat-soluble component of a microalgae, comprising determining the amount of the fat-soluble component content of the microalgae based on the light intensity at a wavelength of two or more light.   Decompose and detect the green light (500 to 570 nm) and the red light (620 to 740 nm), and the fat-soluble component content of microalgae from the light intensity of the green light wavelength and the light intensity of the red light wavelength range The method for judging the content of fat-soluble components of microalgae according to claim 1, wherein the amount of fat is determined.   From the light intensity of the green light wavelength and the light intensity of the red light wavelength range, the red light absorbance and the green light absorbance are calculated, and the value of the microalgae is calculated by dividing the green light absorbance by the red light absorbance. The method for determining the content of a fat-soluble component in microalgae according to claim 2, wherein the amount of the fat-soluble component is determined.   Calculating the light intensity of each wavelength of the culture solution containing the microalgae by comparing with the light intensity of each wavelength obtained by spectrally separating white light, and determining the amount of the fat-soluble component content of the microalgae. The method for judging the fat-soluble component content of microalgae according to any one of claims 1 to 3. The said white light is the white light which permeate | transmitted clear water, or the reflected light from the white substance immersed in clear water, The determination method of the fat-soluble component content of the micro algae of Claim 4 characterized by the above-mentioned.   The color of the culture solution containing microalgae is detected by decomposing it into green light (500-570 nm) and red light (620-740 nm), and the value obtained by dividing the absorbance of the red light by the absorbance of the green light is calculated, When the value falls below a predetermined threshold value, a method for culturing microalgae, comprising collecting all or part of the culture solution.   The color of the culture solution containing microalgae is detected by decomposing it into green light (500-570 nm) and red light (620-740 nm), and the value obtained by dividing the absorbance of the red light by the absorbance of the green light is calculated, A method for culturing microalgae, wherein if the value shows a tendency to decrease with time, all or part of the culture solution is recovered.

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