Microorganism for promoting microalgae growth, microalgae growth promoter, microalgae cultivation method, and microorganism screening method

Abstract

To promote the growth of microalgae.SOLUTION: The present invention provides a microorganism belonging to any genus selected from Rhodococcus, Xanthobacter, Ancylobacter, Shewanella, or Aeromonas. The microorganism may be Rhodococcus cerastii of the genus Rhodococcus, Xanthobacter flavus of the genus Xanthobacter, or Ancylobacter rudongensis of the genus Ancylobacter. The microorganism may be intended for promoting the growth of cyanobacteria, green algae, gray algae or euglenoids.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to a microorganism for promoting the growth of microalgae, a microalgae growth promoter, a method for culturing microalgae using the microorganism for promoting the growth of microalgae, and a screening method for isolating a microorganism capable of promoting the growth of microalgae.

Background Art

[0002] Microalgae are used, for example, in the industrial production of useful substances such as astaxanthin, which is a kind of carotenoid pigment exhibiting a red color derived from natural products. Useful substances are extracted from microalgae and used as raw materials for foods, pharmaceuticals, feeds, fertilizers, and the like. Recently, attention has been paid to the production of petroleum and bioethanol using carbohydrates and lipids accumulated in microalgae cells as useful substances. In addition, Euglena, which is one of the microalgae, is itself industrially produced as a food raw material. Microalgae can fix carbon dioxide by their photosynthetic ability. Therefore, culturing microalgae is also useful as a measure against global warming.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] The growth rate of microalgae is lower than that of general heterotrophic microorganisms. For example, Patent Document 1 describes that in order to improve the economic efficiency of the production of useful substances and the like using microalgae, the growth rate of microalgae is increased by increasing the levels of nutrients such as nitrogen and phosphorus. However, the method described in Patent Document 1 has a problem that the growth rate of microalgae cannot be sufficiently increased.

[0005] Therefore, the present invention has been made in view of these points, and an object thereof is to provide a microorganism for promoting the growth of microalgae, a microalgae growth promoter containing the microorganism, a method for culturing microalgae, and a screening method for separating a microorganism capable of promoting the growth of microalgae.

Means for Solving the Problems

[0006] The microorganism for promoting the growth of microalgae according to the first aspect of the present invention is a microorganism belonging to the genus Rhodococcus, the genus Xanthobacter, the genus Anoxybacter, the genus Shewanella, or the genus Aeromonas.

[0007] The microorganism may be Rhodococcus cerylicus of the genus Rhodococcus, Xanthobacter flavus of the genus Xanthobacter, or Anoxybacter ruddensis of the genus Anoxybacter. Further, the microorganism may be Rhodococcus cerylicus of the genus Rhodococcus, Aeromonas salmonicida or Aeromonas piscicola of the genus Aeromonas. The microorganism may be for promoting the growth of cyanobacteria, green algae, gray algae or Euglena algae. The microorganism may be for promoting the growth of organisms belonging to the phylum Cyanobacteria or the phylum Euglenophyta.

[0008] The microalgae growth promoter according to the second aspect of the present invention contains the microorganism for promoting the growth of microalgae.

[0009] The method for culturing microalgae according to the third aspect of the present invention is characterized in that one or more microorganisms belonging to the genus Rhodococcus, the genus Xanthobacter, the genus Anoxybacter, the genus Shewanella, or the genus Aeromonas are co-cultured with microalgae.

[0010] The method for screening microorganisms according to the fourth aspect of the present invention comprises the steps of co-culturing a microorganism to be screened together with microalgae, and quantifying chlorophyll contained in the medium after co-culturing. The step of quantifying the chlorophyll may be a step of measuring the fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 680 nm to 720 nm. When the microalgae is PCC7942, it is preferable to measure the fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 683 nm. When the microalgae is NIES-2173, it is preferable to measure the fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 685 nm. When the microalgae is NIES-48, it is preferable to measure the fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 700 nm.

Advantages of the Invention

[0011] According to the present invention, it has the effect of promoting the growth of microalgae.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] [Microorganisms for promoting the growth of microalgae] The microorganism for promoting the growth of microalgae of the present invention is a microorganism belonging to the genus Rhodococcus, genus Xanthobacter, genus Shewanella, genus Ancylobacter, genus Bacillus, or genus Aeromonas. These microorganisms are obtained by screening using a fermented cattle urine (FCU) obtained by treating cattle urine with microorganisms and a microalgae growth promoter produced from FCU. However, the microorganism for promoting the growth of microalgae of the present invention is not limited to those isolated from FCU as long as it is a microorganism belonging to any of the above genera and having the action of promoting the growth of microalgae. Preferably, the microorganism for promoting the growth of microalgae is Rhodococcus cerastii of the genus Rhodococcus, Xanthobacter flavus of the genus Xanthobacter, Ancylobacter rudongensis of the genus Ancylobacter, or Shewanella sp.

[0014] Preferably, the microorganism for promoting the growth of microalgae is Bacillus licheniformis, Bacillus pumilus, Bacillus zhangzhouensis, Peribacillus acanthi, Bacillus australimaris, Bacillus safensis of the genus Bacillus, Rhodococcus cerastii of the genus Rhodococcus, or Aeromonas salmonicida, Aeromonas piscicola of the genus Aeromonas. The microorganism for promoting the growth of microalgae of the present invention can promote the growth of microalgae by co-culturing with microalgae or by adding or mixing the medium for culturing the microorganism for promoting the growth of microalgae into the medium for microalgae.

[0015] [Microalgae] The microorganism for promoting the growth of microalgae of the present invention promotes the growth of microalgae. Microalgae are among photosynthetic organisms that require a microscope for individual identification.

[0016] Microalgae are, for example, biological species belonging to the phylum Cyanobacteria (e.g., cyanobacteria), Heterokontophyta, Euglenophyta (e.g., Euglena), Cryptophyta, Haptophyta, Cercozoa, Glaucophyta (e.g., glaucophytes), Rhodophyta, Chlorophyta (e.g., green algae), or Streptophyta. As microalgae, organisms belonging to the phylum Cyanobacteria such as cyanobacteria or the phylum Euglenophyta such as Euglena are preferred.

[0017] Microalgae include, for example, species belonging to the orders Chroococcales, Oscillatoriales, Nostocales, and Stigonematales of the phylum Cyanobacteria. Specifically, examples include Chroococcus sp., Microcystis aeruginosa, Oscillataria sp., Microcoleus sp., Nostoc sp., Cylindrospermum, Stigonema, or Synecochoccus elongatus.

[0018] Microalgae may also be, for example, species belonging to the orders Ochromonadales or Mallomonadales of the class Chrysophyceae of the phylum Heterokontophyta. Specifically, examples include Uroglenopsis americana, Uroglena volvox, Mallomonas, or Synura sp. Microalgae may also be, for example, species belonging to the class Bacillariophyceae of the phylum Heterokontophyta. Specifically, examples include Coscinodiscus sp. or Diatoma.

[0019] Microalgae may also be, for example, species belonging to the class Xanthophyceae of the phylum Heterokontophyta. Specifically, examples include Pseudostaurastrum sp. or Characiopsis sp. Microalgae may also be, for example, species belonging to the class Dictyochophyceae of the phylum Heterokontophyta. Specifically, an example is Dictyocha sp. Microalgae may also be, for example, species belonging to the class Dinophyceae of the phylum Heterokontophyta. Specifically, examples include Peridinium sp. or Scrippsiella trochoidea.

[0020] Microalgae may be, for example, biological species belonging to the Euglenophyceae class of the Euglenophyta phylum. Specifically, examples include Euglena sp. or Phacus sp. Microalgae may be, for example, biological species belonging to the Cryptophyceae class of the Cryptophyta phylum. Specifically, examples include Cryptomonas sp. or Rhodomonas sp.

[0021] Microalgae may be, for example, biological species belonging to the Haptophyceae class of the Haptophyta phylum. Specifically, examples include Coronosphaera sp. or Gephyrocapsa sp. Microalgae may be, for example, biological species belonging to the Testate Filose Amoebae class of the Cercozoa phylum. Specifically, an example is Paulinella chromatophora. Microalgae may be, for example, biological species belonging to the Glaucophyceae class of the Glaucophyta phylum. Specifically, an example is Glaucocystis sp.

[0022] Microalgae may be, for example, biological species belonging to the Rhodophyceae class of the Rhodophyta phylum. Specifically, examples include Cyanidium sp. or Galdieria sp. Microalgae may be, for example, biological species belonging to the Chlorophyceae class of the Chlorophyta phylum. Specifically, examples include Pediastrum duplex, Volvox sp., Chlamydomonas sp., Asterococcus sp., or Chlorella. Microalgae may be, for example, biological species belonging to the Trebouxiophyceae class of the Chlorophyta phylum. Specifically, they may be unicellular green algae belonging to the genus Chlorella. The unicellular green algae belonging to the genus Chlorella may be, for example, Chlorella sorokiniana.

[0023] The microalgae may be, for example, biological species belonging to the Mesostigmatophyceae class of the Streptophyta phylum, and specifically, Mesostigma sp. may be mentioned. The microalgae may be, for example, biological species belonging to the Zygnematophyceae class of the Streptophyta phylum, and specifically, Zygnema sp. may be mentioned. As an example, the microalgae include Synecochoccus elongatus, a kind of so-called cyanobacteria which is a type of blue-green algae.

[0024] [Microalgae growth promoter] The microalgae growth promoter containing the microorganism for promoting microalgae growth of the present invention is not limited in dosage form and may be any of liquid, solid, slurry, etc., but is preferably liquid. Further, the microalgae growth promoter of the present invention may contain auxiliary components according to the dosage form, such as diluents, stabilizers, thickeners, granulating agents, etc. The microalgae growth promoter can be used, for example, by adding it to a medium for culturing microalgae.

[0025] The microalgae growth promoter of the present invention contains the above-mentioned microorganism for promoting microalgae growth. The microalgae growth promoter may be used as it is, or the microorganism may be removed before use. The method for removing the microorganism is not particularly limited, and examples include removal by a filter, removal by centrifugation, sterilization by an autoclave, and sterilization by ultraviolet irradiation. By adding the microalgae growth promoter of the present invention, the growth of microalgae can be promoted.

[0026] The method for culturing microalgae of the present invention co-cultures the above-mentioned microorganism for promoting microalgae growth together with the microalgae. For example, the microorganism for promoting microalgae growth is co-cultured with the microalgae in an FCU medium. In this way, the growth of microalgae can be promoted.

[0027] [Screening method] The screening method of the present invention is a method for screening microorganisms capable of promoting the growth of microalgae, and includes the following steps (1) and (2). (1) Step of co-culturing the microorganism to be screened together with microalgae. (2) Step of quantifying the chlorophyll contained in the medium after co-culture.

[0028] (1) The method for preparing the microorganism to be screened is not particularly limited. For example, it is possible to isolate candidate microorganisms from a naturally-derived liquid capable of promoting the growth of microalgae, such as Fermented Cattle Urine (FCU) obtained by fermenting cattle urine through aeration. Instead of cattle urine, microorganisms contained in fermented livestock urine obtained by fermenting urine of other livestock such as horses and pigs may be used as the screening target. When using FCU, a diluted solution of this FCU is added to the Ormerod medium and cultured. Figure 1 shows the concentration of each medium component added to the Ormerod medium. By repeating the operation of separating and culturing the colonies formed in the Ormerod medium in another Ormerod medium, a plurality of single colonies are obtained. Each of the formed plurality of single colonies is co-cultured in a plurality of media containing microalgae.

[0029] Also, the microorganism to be screened may be cultured in the FCU medium instead of the Ormerod medium or the JCM520 medium. In this case, the FCU may be autoclaved at 121°C for 20 minutes, or may be sterilized by passing through a 0.22 μm filter. By mixing FCU and sterilized water, the amount of FCU is adjusted so that the concentration of FCU in the completed FCU medium is 20% or 10%. To the FCU medium, gellan gum adjusted so that the concentration in the completed FCU medium is 0.8%, or agar adjusted so that the concentration in the completed FCU medium is 2% is added.

[0030] As shown in (2) above, by quantifying the chlorophyll contained in microalgae after co-culturing the microorganism to be screened and microalgae, it is possible to evaluate to what extent this microorganism exerts an effect of promoting the growth of microalgae. In the step of quantifying chlorophyll, using a fluorescence spectrophotometer, the fluorescence intensity is measured at an excitation wavelength of 488 nm and a fluorescence wavelength of 683 nm to 720 nm. Also, it is possible to change the wavelength depending on the type of microalgae. For example, in the case of PCC7942, it is preferable to measure the fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 683 nm, in the case of NIES-2173 at an excitation wavelength of 488 nm and a fluorescence wavelength of 685 nm, and in the case of NIES-48 at an excitation wavelength of 488 nm and a fluorescence wavelength of 700 nm. In this way, by measuring the light of the wavelength corresponding to the fluorescence of chlorophyll, it is possible to accurately evaluate to what extent the microorganism promotes the growth of microalgae.

[0031] The DNA sequence corresponding to 16S rRNA etc. in the microorganism genome of the identified colony is decoded by sequencing. As an example, a known microorganism having a DNA sequence with the highest degree of identity to this DNA sequence is identified as the microorganism corresponding to this colony or a candidate for the microorganism corresponding to this colony. Also, a known microorganism having a DNA sequence with a degree of identity to the DNA sequence corresponding to 16S rRNA etc. in the microorganism genome of the identified colony that is equal to or higher than a threshold value may be identified as the microorganism corresponding to this colony. The threshold value is, for example, a value within the range of 97% to 98%.

Example

[0032] [Method for Separating and Culturing Microorganism Promoting Microalgae Growth] Using an Omerod medium, a microorganism having an effect of promoting microalgae growth was separated. In addition to each medium component shown in FIG. 1, 2% agar was added, and an Omerod agar medium was prepared by performing an autoclave at 121 ° C for 20 minutes. Using the fermented FCU as a separation source, the FCU of the separation source was diluted from 10-fold to 10 5A solution diluted within a double range was prepared. 100 μL of the diluted FCU was seeded onto the solidified Omerod medium in each petri dish, inoculated with a spreader stick, covered, and cultured until colony formation in an environment with aerobic conditions, room temperature (24 °C), 115 - 120 μmol / m 2 / s, and irradiated with light for only 12 hours out of 24 hours. The formed colonies were picked up with a disposable loop and subcultured several times in the same medium as the medium used for separation to obtain single colonies.

[0033] [Outline of Screening for Microalgae Growth-Promoting Bacteria by High-Throughput Co-Culture Evaluation System] (1) Cyanobacteria It was used to evaluate the microalgae growth-promoting effect of Synechococcus elongatus PCC7942 (hereinafter also simply referred to as PCC7942), which is a model organism of cyanobacteria. In the case of single culture of PCC7942, as a preculture, flask culture was carried out at 30 °C, 120 rpm, and a photosynthetic photon flux density of 115 - 120 μmol / m 2 / s for 7 days with continuous light irradiation for 24 hours. The photosynthetic photon flux density represents the intensity of light.

[0034] Figure 2 shows the concentrations of each medium component of the co-culture medium. The trace elements in Figure 2 are 0.25 g of CuSO4·5H2O, 0.37 g of (NH4)6Mo7O 24 ·4H2O, 2.47 g of H3BO3, 0.29 g of ZnSO4·7H2O, and 1.58 g of MnCl2·4H2O in 100 mL of solution, respectively. PCC7942 was inoculated into the co-culture medium without mixing with the isolates, and main culture was carried out at 30 °C, 120 rpm, and a photosynthetic photon flux density of 115 - 120 μmol / m 2 / s for 7 days with continuous light irradiation for 24 hours. The chlorophyll a concentration (μg / mL) was measured every 24 hours.

[0035] When co-culturing the isolates separated from FCU and PCC7942, for the preculture for co-culturing, using a 100 mL flask, 500 μL of the strain glycerol stock solution was inoculated into 40 mL of the co-culture medium and carried out at 30 °C and 160 rpm. At the time of main culture, 40 mL of the co-culture medium was dispensed into a 100 mL flask, and the amount of PCC7942 was adjusted so that the absorbance A of the light at a wavelength of 600 nm of PCC7942 600 would become 0.05 after mixing with the isolate, and the absorbance A of the light at a wavelength of 600 nm of each isolate separated from FCU 600 was adjusted so that it would become 0.01, 0.05 or 0.08 etc. after mixing with PCC7942.

[0036] After mixing PCC7942 and the isolate, it was inoculated into the co-culture medium and main cultured at 30 °C, 120 rpm, and a light quantum flux density of 115 - 120 μmol / m 2 / s for 7 days with continuous light irradiation for 24 hours. The chlorophyll a concentration (μg / mL) was quantified every 24 hours. For each isolate, it was confirmed that it did not have the same chlorophyll fluorescence as PCC7942. From this, it can be seen that each isolate does not affect the measurement result of the chlorophyll fluorescence intensity derived from PCC7942 during co-culture with PCC7942.

[0037] <Chlorophyll Quantification> 1 mL of the sample after co-culture was collected and centrifuged at 15,000×g for 7 minutes. The supernatant fraction was removed, and the cells recovered as a precipitate were resuspended in 1 mL of cold 100% (vol / vol) methanol. To extract the pigment from the cells, the sample was placed in the dark at 4.0 °C for 1 hour and incubated. After incubation, the sample was centrifuged at 15,000×g for 10 minutes at 4.0 °C, and the chlorophyll a contained in the supernatant was quantified by spectrophotometry. Calibrated using methanol as a blank, the absorbances at 665 nm and 720 nm were measured. The chlorophyll a concentration was determined by the formula (chlorophyll a concentration (μg / mL) = 12.9447×(A 665 -A 720 ).

[0038] <Flow cytometry analysis> Using a flow cytometer Cube8, the cell count and chlorophyll fluorescence per cell were measured. 1 mL of each culture solution diluted 1000-fold was prepared as a sample for measurement. The voltages of forward scatter (FSC-H), side scatter (SSC-H), and chlorophyll fluorescence (FL2-H) were set to 200.0 V, 275.0 V, 525.0 V, and 675.0 V, respectively.

[0039] (2) Chlorella <Culture method> As another example of microalgae, the microalgae growth promoting effect was evaluated using Chlorella sorokiniana NIES-2173 (hereinafter also simply referred to as NIES-2173). Pre-culture was carried out in a modified BG11 medium supplemented with 5 g / L glucose at 30 °C, 125 rpm, PPFD, 133 μmol / m 2 / s, with a light-dark cycle of 24 h / 0 h in a flask culture for 3 days. In the case of isolates, in the pre-culture, using a 100 mL flask, 1 mL of the strain glycerol solution was inoculated into 40 mL of the co-culture medium and carried out at 30 °C and 168 rpm.

[0040] At the time of main culture, 40 mL of the modified BG11 medium supplemented with 5 g / L glucose was dispensed into a 100 mL flask, and Chlorella sorokiniana NIES-2173 was adjusted to A 750 : 0.025, and each strain was adjusted to A 600 : 0.005, A 600 : 0.025, A 600 : 0.04, etc., and inoculated into the modified BG11 medium supplemented with 5 g / L glucose, and main-cultured at 30 °C, 125 rpm, PPFD, 133 μmol / m 2 / s, with a light-dark cycle of 24 h / 0 h for 4 days. (n = 3).

[0041] <Chlorophyll quantification> 1 mL of the culture solution was collected in a 1.5 mL tube and centrifuged at 8,000 rpm for 10 min at 4°C. After removing the supernatant, 1.5 mL of pure methanol was added, and the mixture was allowed to stand in the dark at 4°C for 24 h to immerse the cell mass in methanol. After 24 h, it was stirred with a vortex mixer and centrifuged again at 8,000 rpm for 10 min at 4°C. The supernatant was transferred to a glass cuvette, and the absorbances at 653 nm, 666 nm, and 750 nm were measured. The amount of chlorophyll a pigment was calculated by the formula (1) ChlA (mg / L) = 15.65 (Abs666 - Abs750) - 7.34 (Abs653 - Abs750). In formula (1), ChlA is the abbreviation of chlorophyll a. Abs653, Abs666, and Abs750 are the absorbances at wavelengths 653 nm, 666 nm, and 750 nm, respectively. The amount of chlorophyll b pigment was calculated by the formula (2) ChlB (mg / L) = 27.05 (Abs653 - Abs750) - 11.21 (Abs666 - Abs750) (2). In formula (2), ChlB is the abbreviation of chlorophyll b.

[0042] <Flow Cytometry Analysis> Using a flow cytometer Cube8, the absolute number of bacteria and chlorophyll fluorescence per cell were measured. Each 100-fold diluted culture solution was adjusted to 1 mL and used as a measurement sample. The voltages of forward scatter (FSC-H), side scatter (SSC-H), and chlorophyll fluorescence (FL2-H) were set to 125.0 V, 180.0 V, 525.0 V, and 400.0 V, respectively.

[0043] [Genomic Extraction of Microalgae Growth-Promoting Strains] Each isolate was cultured pure and subjected to genomic extraction. The tube containing the bacterial cells was centrifuged at 10,000 rpm and 4 °C for 5 minutes using a cooling centrifuge, and the cell pellet (precipitate) was collected. Using a pipettor, the supernatant was discarded into the waste liquid reservoir. 560 μL of TE buffer was added to the cell pellet and stirred well to suspend the cells. 30 μL of 10% SDS and 10 μL of proteinase K solution were added, mixed well, and then incubated at 37 °C for 1 hour. 100 μL of 5 M NaCl was added and mixed well. 80 μL of CTAB / NaCl solution was added, mixed well, and incubated at 65 °C for 10 minutes. 0.7 mL of chloroform / isoamyl alcohol was added, the tube cap was closed, and the tube was inverted 5 - 6 times up and down, stirred well, and then centrifuged at 15,000 rpm and 4 °C for 5 minutes using a cooling centrifuge.

[0044] 0.5 - 0.6 mL of the upper layer liquid was taken and transferred to a new 1.5 mL microtube. An equal volume of phenol / chloroform / isoamyl alcohol as the transferred liquid volume was added, stirred well, and then centrifuged at 15,000 rpm and 4 °C for 5 minutes using a cooling centrifuge. 0.5 - 0.6 mL of the supernatant was taken and transferred to a new 1.5 mL microtube. 0.6 times the volume of isopropanol of the transferred solution was added to precipitate the DNA, and then centrifuged at 15,000 rpm and 4 °C for 5 minutes using a cooling centrifuge. The supernatant was gently discarded by pipetting, 1 mL of 70% ethanol was added, and centrifuged again at 15,000 rpm and 4 °C for 5 minutes using a cooling centrifuge. The supernatant was discarded, and the tube cap was left open and dried for about 10 minutes. The precipitate was dissolved in 100 μL of TE buffer.

[0045] 19 μL of a PCR reaction solution containing 27F primer, 1492R primer, TaKaRa LA Taq polymerase, etc. was added with 1 μL of a DNA sample in which the precipitate was dissolved in TE buffer. Figure 3 shows the primers used in the PCR reaction. In Figure 3, the sequence number, primer name, and oligonucleotide sequence of the primers used in the PCR reaction are shown. The oligonucleotide sequence of the 27F primer is shown in the first row from the top of Figure 3, and the oligonucleotide sequence of the 1492R primer is shown in the second row from the top of Figure 3. The tube was spun down with a simple centrifuge, inserted into the thermostatic block of the thermal cycler, and subjected to 30 cycles of thermal cycle reaction.

[0046] [Column purification] A membrane binding solution equal in volume to the DNA sample was added. The SV mini-column was inserted into the collection tube (also referred to as column assembly). The entire solution was transferred to the SV mini-column and left at room temperature for about 1 minute. This column assembly was set in a cooling centrifuge and centrifuged at 16,000×g for 1 minute at 4°C. 500 μL of the membrane wash solution was put into the SV mini-column and centrifuged at 16,000×g for 1 minute at 4°C.

[0047] The liquid in the collection tube was discarded into the waste liquid reservoir, and the SV mini-column was inserted into the collection tube again. 500 μL of the membrane wash solution was put into the SV mini-column and centrifuged at 16,000×g for 5 minutes at 4°C. The liquid in the collection tube was discarded into the waste liquid reservoir, and the column assembly with the SV mini-column inserted into the collection tube again was centrifuged at 16,000×g for 1 minute at 4°C. The SV mini-column was inserted into a new 1.5 mL microtube. 50 μL of sterilized water was put into the SV mini-column, left at room temperature for about 1 minute, and then centrifuged at 16,000×g for 1 minute at 4°C to elute the DNA.

[0048] [Cycle sequencing] A sequencing reaction solution containing 27F primer etc. was prepared, 8 μL of this was mixed with 2 μL of the sample, and the reaction was carried out using a thermal cycler (initial denaturation: 96 °C for 1 minute, [denaturation: 96 °C for 10 seconds, annealing: 50 °C for 5 seconds, extension: 60 °C for 4 seconds] × 29 cycles, final extension: 4 °C with no time limit). Any one of the primers from SEQ ID NO: 1 to SEQ ID NO: 8 shown in FIG. 3 was used for the sequencing reaction solution. After the thermal cycler, 5 μL of 125 mM EDTA and 60 μL of 99.5% EtOH were added, mixed by inversion, wrapped with aluminum foil, and left for 15 minutes. Then, centrifugation was carried out at 3,750 × g for 30 minutes, and while still inverted, centrifugation was carried out at 185 × g for 10 seconds.

[0049] 60 μL of 70% EtOH was added, centrifuged at 3,750 × g for 5 minutes, and further centrifuged at 185 × g for 10 seconds. 15 μL of HiDi formamide was added, vortexed for 2 minutes, heat shocked at 95 °C for 2 minutes and at 4 °C for 2 minutes, and capillary sequencing was performed. The raw data of the DNA sequencing was analyzed for ATGC data using GENETYX's ATGC software, and the analyzed data was subjected to BLAST analysis at NCBI. Then, using the GENETYX software, FASTA data of related strains was obtained using the NCBI database, and a phylogenetic tree of each isolate was created.

[0050] [Selection of microalgae growth-promoting bacteria by co-culture evaluation system] FIG. 4 and FIG. 5 show isolates with a chlorophyll fluorescence intensity magnification exceeding 1.0 on the 6th day of co-culture. The fifth column from the left in FIGS. 4 and 5 shows the magnification of the chlorophyll fluorescence intensity compared to the case of culturing with PCC7942 alone. The average, standard deviation, magnification, and standard deviation of the chlorophyll fluorescence intensity when culturing with PCC7942 alone are shown in the second row from the bottom (PCC7942A in FIG. 5) and the first row (PCC7942B in FIG. 5) of FIG. 5.

[0051] Those marked with "a" in the magnification comparison criteria in the first column from the right in FIGS. 4 and 5 indicate negative controls. For those marked with "b" in the magnification comparison criteria in the first column from the right in FIGS. 4 and 5, the magnification of the chlorophyll fluorescence intensity compared with PCC7942A in FIG. 5 is shown in the magnification column in the fifth column from the left in FIGS. 4 and 5. For those marked with "c" in the magnification comparison criteria in the first column from the right in FIGS. 4 and 5, the magnification of the chlorophyll fluorescence intensity compared with PCC7942B in FIG. 5 is shown in the magnification column in the fifth column from the left in FIGS. 4 and 5.

[0052] Among the 144 isolates from the FCU, the chlorophyll fluorescence intensity of 34 isolates was 1.0 times or more. FIGS. 4 and 5 show these 34 isolates in descending order of magnification. The isolate "AF2108" in the first row from the top in FIG. 4 showed the highest magnification of 7.5 times.

[0053] Hereinafter, isolates with a chlorophyll fluorescence intensity magnification exceeding 3.0 (AF2108 strain, GA1226 strain, AF2111 strain, OR151 strain) were selected, and the PCC7942 growth promotion effect and the NIES-2173 growth promotion effect were confirmed using flask co-culture, respectively.

[0054] [AF2108 isolate] FIGS. 6(a) and 6(b) show the measurement results of chlorophyll a after co-culture of the AF2108 isolate and PCC7942. The first and second columns from the left in FIG. 6(a) show the average value and standard deviation of the chlorophyll a concentration during the single culture of PCC7942 every 24 hours. In the example of the first and second columns from the left in FIG. 6, the amount of PCC7942 was adjusted so that the absorbance at a wavelength of 730 nm at the start of the single culture of PCC7942 was 0.05.

[0055] The third and fourth columns from the left in Fig. 6 show the average value and standard deviation of the chlorophyll a concentration after co-culture of PCC7942 and the AF2108 isolate every 24 hours. In the examples of the third and fourth columns from the left in Fig. 6, the amount of PCC7942 was adjusted so that the absorbance at a wavelength of 730 nm of the PCC7942 component at the start of co-culture in the co-culture medium in which PCC7942 and the AF2108 isolate were mixed was 0.05.

[0056] In this co-culture medium, the amount of the AF2108 isolate was adjusted so that the absorbance at a wavelength of 600 nm of the AF2108 isolate component at the start of co-culture was 0.05. After 168 hours of single culture of PCC7942, chlorophyll a increased to 9.079 μg / mL. On the other hand, after 168 hours of co-culture of PCC7942 and the AF2108 isolate, chlorophyll a increased to 64.249 μg / mL.

[0057] Fig. 6(b) shows the magnification obtained by dividing the chlorophyll a concentration during the co-culture of PCC7942 and the AF2108 isolate by the chlorophyll a concentration during the single culture of PCC7942. As shown in the second row from the top in Fig. 6(b), the magnification of the chlorophyll a concentration during the co-culture of PCC7942 and the AF2108 isolate compared to the single culture of PCC7942 was maximally 15.65 ± 0.72 times at 48 hours and minimally 7.08 ± 4.90 times at 168 hours.

[0058] Fig. 7 shows the results of measuring the cell number, cell size, and chlorophyll fluorescence intensity of PCC7942 by flow cytometry. The first row from the top in Fig. 7 shows the cell number etc. of PCC7942 when the single culture of PCC7942 was continued for 168 hours. The second row from the top in Fig. 7 shows the cell number etc. of PCC7942 when the co-culture of PCC7942 and the AF2108 isolate was continued for 168 hours. The cell size was measured by forward scatter (FSC-H) of flow cytometry. After 168 hours of co-culture of PCC7942 and the AF2108 isolate, the cell size and chlorophyll fluorescence intensity were 2.40 ± 0.12 times and 3.53 ± 0.41 times, respectively, compared to the culture with PCC7942 alone.

[0059] Figures 8(a) to 8(g) show the time-course of co-culture of the PCC7942 and AF2108 isolates in flasks. Samples during co-culture after 24 hours of PCC7942 and AF2108 isolates (the first to the third from the left) were confirmed to be much greener than samples during the monoculture of PCC7942 (the fourth to the sixth from the left).

[0060] Figures 9(a) and 9(b) show the measurement results of the total concentration of chlorophyll a and chlorophyll b (hereinafter also referred to as chlorophyll a+b concentration) during co-culture of the AF2108 isolate and NIES-2173. The first and second columns from the left in Fig. 9(a) show the average value and standard deviation of the chlorophyll a+b concentration during the monoculture of Chlorella NIES-2173 every 24 hours. In the example of the first and second columns from the left in Fig. 9, the amount of NIES-2173 was adjusted so that the absorbance at a wavelength of 750 nm at the start of the monoculture of NIES-2173 was 0.025.

[0061] The third and fourth columns from the left in Fig. 9 show the average value and standard deviation of the chlorophyll a+b concentration during co-culture of NIES-2173 and the AF2108 isolate every 24 hours. In the example of the third and fourth columns from the left in Fig. 9, the amount of AF2108 was adjusted so that the absorbance at a wavelength of 600 nm of the AF2108 component at the start of co-culture was 0.04 in the co-culture medium in which NIES-2173 and the AF2108 isolate were mixed.

[0062] The chlorophyll a+b concentration increased to 10.22 μg / mL after 120 hours of monoculture of NIES-2173. On the other hand, the chlorophyll a+b concentration increased to 17.60 μg / mL after 120 hours of co-culture of NIES-2173 and the AF2108 isolate.

[0063] Figure 9(b) shows the magnification obtained by dividing the chlorophyll a+b concentration during the co-culture of NIES-2173 and the AF2108 isolate by the chlorophyll a+b concentration during the monoculture of NIES-2173. As shown in Figure 9(b), the magnification of the chlorophyll a+b concentration during the co-culture of NIES-2173 and the AF2108 isolate compared to that during the monoculture of NIES-2173 was maximally 2.09 ± 0.08-fold at 96 hours and minimally 1.25 ± 0.09-fold at 24 hours. Therefore, it was confirmed that the AF2108 isolate promoted the growth of NIES-2173.

[0064] Figure 10 shows the results of measuring the cell number, cell size, and chlorophyll fluorescence intensity of NIES-2173 by flow cytometry. The first row from the top in Figure 10 shows the cell number, etc. of NIES-2173 after monoculture of NIES-2173. The second row from the top in Figure 10 shows the cell number, etc. of NIES-2173 after co-culture of NIES-2173 and the AF2108 isolate. The cell size was measured by forward scatter (FSC-H) of flow cytometry. The cell size and chlorophyll fluorescence intensity after co-culture of NIES-2173 and the AF2108 isolate were 0.8 ± 0.1-fold and 1.0 ± 0.1-fold, respectively, compared to the culture with NIES-2173 alone.

[0065] Figure 11 shows the phylogenetic tree of the genus Rhodococcus. The reference strain Pseudonocardia dioxanivorans CP1190 was used as the outgroup. The numerical values in Figure 11 are bootstrap values indicating the reliability of the shape of the phylogenetic tree. Only values with a bootstrap value of 50 or more are shown. The scale bar indicates the scale of the number of amino acid substitutions. From this phylogenetic tree, the AF2108 strain was closest to Rhodococcus cerastii. The AF2108 strain was identical to Rhodococcus cerastii with 100% homology and was identified as Rhodococcus cerastii (accession number: NITE P-03678).

[0066] [AF2111 isolate] Figures 12(a) and 12(b) show the measurement results of chlorophyll a during the co-culture of PCC7942 and AF2111 isolates. In the first and second columns from the left in Figure 12(a), the average value and standard deviation of the chlorophyll a concentration during the monoculture of PCC7942 are shown every 24 hours. In the example of the monoculture of PCC7942 shown in Figure 12, the amount of PCC7942 was adjusted so that the absorbance at a wavelength of 730 nm at the start of the monoculture of PCC7942 was 0.05.

[0067] The third and fourth columns from the left in Figure 12 show the average value and standard deviation of the chlorophyll a concentration during the co-culture of PCC7942 and AF2111 isolates every 24 hours. In the example of the co-culture of PCC7942 and AF2111 isolates in Figure 12, in the co-culture medium in which PCC7942 and AF2111 isolates were mixed, the amount of PCC7942 was adjusted so that the absorbance at a wavelength of 730 nm at the start of the co-culture of the PCC7942 component was 0.05, and in the co-culture medium in which PCC7942 and AF2111 isolates were mixed, the amount of the AF2108 isolate was adjusted so that the absorbance at a wavelength of 600 nm at the start of the co-culture of the AF2108 isolate component was 0.01.

[0068] As shown in the first row from the bottom of Figure 12(a), in the monoculture of PCC7942, chlorophyll a increased to 9.079 μg / mL after 168 hours of culture. On the other hand, in the co-culture of PCC7942 and AF2111 isolates, chlorophyll a increased to 33.699 μg / mL after 168 hours of culture.

[0069] Figure 12(b) shows the magnification obtained by dividing the chlorophyll a concentration during the co-culture of PCC7942 and AF2111 isolates by the chlorophyll a concentration during the monoculture of PCC7942. This magnification was maximally 6.92 ± 0.48 times at 24 hours and minimally 3.24 ± 0.99 times at 144 hours.

[0070] Figure 13 shows the results of measuring the cell count, cell size, and chlorophyll fluorescence intensity of PCC7942 by flow cytometry. The first row from the top in Figure 13 shows the cell count etc. of PCC7942 at the time when the monoculture of PCC7942 was continued for 168 hours. The second row from the top in Figure 13 shows the cell count etc. of PCC7942 at the time when the co-culture of PCC794 and the AF2111 isolate was continued for 168 hours. As shown in the second row from the top in Figure 13, the cell size and chlorophyll fluorescence intensity after the co-culture of PCC7942 and the AF2111 isolate were 2.19 ± 0.11 times and 1.98 ± 0.18 times, respectively, compared to the cell size etc. after the monoculture of PCC7942.

[0071] Figures 14(a) to 14(g) show the state of the co-culture of PCC7942 and the AF2111 isolate in flasks in chronological order. It was confirmed that the samples (the first to the third from the left) during the co-culture of PCC7942 and the AF2111 isolate after 24 hours were much greener than the samples (the fourth to the sixth from the left) during the monoculture of PCC7942.

[0072] Figures 15(a) and 15(b) show the measurement results of the chlorophyll a + b concentration during the co-culture of the AF2111 isolate and NIES-2173. The first and second columns from the left in Figure 15(a) show the average value and standard deviation of the chlorophyll a + b concentration during the monoculture of Chlorella NIES-2173 every 24 hours, and also show the average value etc. of the chlorophyll a + b concentration after 84 hours. In the example of the first and second columns from the left in Figure 15, the amount of NIES-2173 was adjusted so that the absorbance at a wavelength of 750 nm at the start of the monoculture of NIES-2173 was 0.025.

[0073] The third and fourth columns from the left in Figure 15 show the average value and standard deviation of the chlorophyll a + b concentration during the co-culture of NIES-2173 and the AF2111 isolate. In the example of the third and fourth columns from the left in Figure 15, the amount of AF2111 was adjusted so that the absorbance at a wavelength of 600 nm of the AF2111 component at the start of the co-culture was 0.005 in the co-culture medium in which NIES-2173 and the AF2111 isolate were mixed.

[0074] After 96 hours of monoculture of NIES-2173, the chlorophyll a+b concentration increased to 8.44 μg / mL. On the other hand, after 96 hours of co-culture of NIES-2173 and the AF2111 isolate, the chlorophyll a+b concentration increased to 8.77 μg / mL.

[0075] Figure 15(b) shows the ratio of the chlorophyll a+b concentration during the co-culture of NIES-2173 and the AF2111 isolate divided by the chlorophyll a+b concentration during the monoculture of NIES-2173. As shown in Figure 15(b), the ratio of the chlorophyll a+b concentration during the co-culture of NIES-2173 and the AF2111 isolate compared to that during the monoculture of NIES-2173 was 1.86 ± 0.15 times at 0 hours, excluding this value, it reached a maximum of 1.04 ± 0.06 times at 96 hours and a minimum of 0.88 ± 0.10 times at 48 hours.

[0076] Figure 16 shows the results of measuring the cell number, cell size, and chlorophyll fluorescence intensity of NIES-2173 by flow cytometry. The first row from the top in Figure 16 shows the cell number, etc. of NIES-2173 after monoculture of NIES-2173. The second row from the top in Figure 16 shows the cell number, etc. of NIES-2173 after co-culture of NIES-2173 and the AF2111 isolate. The cell size was measured by the forward scatter (FSC-H) of flow cytometry. After co-culture of NIES-2173 and the AF2111 isolate, the cell size and chlorophyll fluorescence intensity were 0.9 ± 0.0 times and 0.9 ± 0.2 times, respectively, compared to the culture of NIES-2173 alone.

[0077] Figure 17 shows the phylogenetic tree of the genus Xanthobacter. The reference strain Blastochloris gulmargensis JA248 was used as the outgroup. From this phylogenetic tree, strain AF2111 is closest to Xanthobacter flavus. Strain AF2111 showed 100% homology with Xanthobacter flavus and was identified as Xanthobacter flavus (Accession number: NITE P-03679). Therefore, it was shown that Xanthobacter flavus has a growth promoting effect on microalgae.

[0078] [GA1226 isolate] Figures 18(a) and 18(b) show the measurement results of chlorophyll a during the co-culture of PCC7942 and the GA1226 isolate. The first and second columns from the left in Figure 18(a) show the average value and standard deviation of the chlorophyll a concentration during the monoculture of PCC7942 every 24 hours. In the monoculture of PCC7942, the absorbance at a wavelength of 730 nm at the start of the culture of PCC7942 was adjusted to 0.05.

[0079] The third and fourth columns from the left in Figure 18 show the average value and standard deviation of the chlorophyll a concentration during the co-culture of PCC7942 and the GA1226 isolate every 24 hours. In the co-culture of PCC7942 and the GA1226 isolate, in the mixed co-culture medium of PCC7942 and the GA1226 isolate, the amount of PCC7942 was adjusted so that the absorbance at a wavelength of 730 nm at the start of the co-culture of the PCC7942 component was 0.05. In this co-culture medium, the amount of the GA1226 isolate was adjusted so that the absorbance at a wavelength of 600 nm at the start of the co-culture of the GA1226 isolate component was 0.05. As shown in the first row from the bottom of Figure 18(a), in the monoculture of PCC7942, chlorophyll a increased to 9.079 μg / mL after 168 hours of culture. On the other hand, in the co-culture of PCC7942 and the GA1226 isolate, chlorophyll a increased to 29.73 μg / mL after 168 hours of culture.

[0080] Figure 18(b) shows the magnification obtained by dividing the chlorophyll a concentration after co-culturing the PCC7942 and GA1226 isolates by the chlorophyll a concentration after culturing PCC7942 alone. This magnification was up to 3.31 ± 1.83-fold (120 hours) at maximum and at least 1.56 ± 0.06-fold (24 hours) at minimum.

[0081] Figure 19 shows the results of measuring the cell count, cell size, and chlorophyll fluorescence intensity of PCC7942 by flow cytometry. The first row from the top in Figure 19 shows the cell count etc. of PCC7942 when the single culture of PCC7942 was continued for 168 hours. The second row from the top in Figure 19 shows the cell count etc. of PCC7942 when the co-culture of PCC7942 and the GA1226 isolate was continued for 168 hours. The cell size and chlorophyll fluorescence intensity after co-culturing the PCC7942 and GA1226 isolates became 1.25 ± 0.16-fold and 1.49 ± 0-fold, respectively, compared to the cell size etc. after single culturing of PCC7942.

[0082] Figures 20(a) to 20(g) show the states of co-culturing the PCC7942 and GA1226 isolates in flasks in chronological order. Samples during co-culturing of the PCC7942 and GA1226 isolates after 48 hours (the first to the third from the left) were confirmed to be much greener than samples during single culturing of PCC7942 (the fourth to the sixth from the left).

[0083] Figures 21(a) and 21(b) show the measurement results of the chlorophyll a + b concentration during co-culturing of the GA1226 isolate and NIES-2173. The first and second columns from the left in Figure 21(a) show the average value and standard deviation of the chlorophyll a + b concentration during single culturing of Chlorella NIES-2173 every 24 hours, and also show the average value etc. of the chlorophyll a + b concentration after 84 hours. In the example of the first and second columns from the left in Figure 21, the amount of NIES-2173 was adjusted so that the absorbance at a wavelength of 750 nm at the start of single culturing of NIES-2173 became 0.025.

[0084] In the third and fourth columns from the left in Fig. 21, the average values and standard deviations of the chlorophyll a + b concentrations during the co - culture of the NIES - 2173 and GA1226 isolates were shown. In the examples of the third and fourth columns from the left in Fig. 21, the amount of GA1226 was adjusted so that the absorbance at a wavelength of 600 nm of the GA1226 component at the start of co - culture was 0.025 in the co - culture medium in which the NIES - 2173 and GA1226 isolates were mixed. After 96 - hour single - culture of NIES - 2173, the chlorophyll a + b concentration increased to 8.44 μg / mL. On the other hand, after 96 - hour co - culture of NIES - 2173 and the GA1226 isolate, the chlorophyll a + b concentration increased to 8.05 μg / mL.

[0085] Fig. 21(b) shows the magnification obtained by dividing the chlorophyll a + b concentration during the co - culture of the NIES - 2173 and GA1226 isolates by the chlorophyll a + b concentration during the single - culture of NIES - 2173. As shown in Fig. 21(b), the magnification of the chlorophyll a + b concentration during the co - culture of the NIES - 2173 and GA1226 isolates compared to that during the single - culture of NIES - 2173 became 0.97±0.00 times at the maximum at 84 hours and 0.94±0.11 times at the minimum at 72 hours, excluding 1.60±0.28 times at 0 hour.

[0086] Fig. 22 shows the results of measuring the cell number, cell size, and chlorophyll fluorescence intensity of NIES - 2173 by flow cytometry. The first row from the top in Fig. 22 shows the cell number, etc. of NIES - 2173 after single - culture of NIES - 2173. The second row from the top in Fig. 21 shows the cell number, etc. of NIES - 2173 after co - culture of NIES - 2173 and the GA1226 isolate. The cell size was measured by the forward scatter (FSC - H) of flow cytometry. After co - culture of the NIES - 2173 and GA1226 isolates, the cell size and chlorophyll fluorescence intensity became 0.9±0.0 times and 0.7±0.0 times, respectively, compared to the culture with NIES - 2173 alone.

[0087] Figure 23 shows the phylogenetic tree of the genus Ancylobacter. The reference strain Blastochloris gulmargensis JA248T was used as the outgroup. From this phylogenetic tree, the GA1226 isolate was found to be closest to Ancylobacter rudongensis. The GA1226 isolate showed 99.42% homology with Ancylobacter rudongensis and was identified as Ancylobacter rudongensis (accession number: NITE P-03779). Therefore, it was shown that Ancylobacter rudongensis has the effect of promoting the growth of microalgae.

[0088] [OR151 isolate] Figures 24(a) and 24(b) show the measurement results of chlorophyll a during the co-culture of PCC7942 and the OR151 isolate. The first and second columns from the left in Figure 24(a) show the average value and standard deviation of the chlorophyll a concentration during the monoculture of PCC7942 every 24 hours. In the monoculture of PCC7942, the amount of PCC7942 was adjusted so that the absorbance at a wavelength of 730 nm at the start of the culture of PCC7942 was 0.05.

[0089] The first and second columns from the left in Figure 24 show the average value and standard deviation of the chlorophyll a concentration during the co-culture of PCC7942 and the OR151 isolate every 24 hours. In the co-culture of PCC7942 and the OR151 isolate, in the mixed co-culture medium of PCC7942 and the OR151 isolate, the amount of PCC7942 was adjusted so that the absorbance at a wavelength of 730 nm at the start of the culture of the PCC7942 component was 0.05. In this co-culture medium, the amount of the OR151 isolate was adjusted so that the absorbance at a wavelength of 600 nm at the start of the culture of the OR151 isolate component was 0.05. As shown in the first row from the bottom in Figure 24(a), in the monoculture of PCC7942, chlorophyll a increased to 7.21 μg / mL after 168 hours of culture, while in the co-culture of PCC7942 and the OR151 isolate, chlorophyll a increased to 8.958 μg / mL after 168 hours of culture.

[0090] Figure 24(b) shows the magnification obtained by dividing the chlorophyll a concentration during the co-culture of PCC7942 and the OR151 isolate by the chlorophyll a concentration during the monoculture of PCC7942. This magnification was maximally 2.36 ± 0.14-fold (48 hours) and 2.36 ± 0.08-fold (72 hours), and minimally 1.24 ± 0.02-fold (168 hours).

[0091] Figure 25 shows the results of measuring the cell count, cell size, and chlorophyll fluorescence intensity of PCC7942 by flow cytometry. The first row from the top in Figure 25 shows the cell count etc. of PCC7942 at the time when the monoculture of PCC7942 was continued for 168 hours. The second row from the top in Figure 25 shows the cell count etc. of PCC7942 at the time when the co-culture of PCC7942 and the OR151 isolate was continued for 168 hours. The cell size and chlorophyll fluorescence intensity after 168 hours of co-culture of PCC7942 and the OR151 isolate became 2.04 ± 0.09-fold and 2.44 ± 0-fold, respectively, compared to the cell size etc. after the monoculture of PCC 7942.

[0092] Figures 26(a) to 26(g) show the state of the co-culture of PCC7942 and the OR151 isolate in flasks in chronological order. It was confirmed that the samples (the first to the third from the left) during the co-culture of PCC7942 and the OR151 isolate after 24 hours were much greener than the samples (the fourth to the sixth from the left) during the monoculture of PCC7942.

[0093] Figures 27(a) and 27(b) show the measurement results of the chlorophyll a + b concentration during the co-culture of the OR151 isolate and NIES-2173. The first and second columns from the left in Figure 27(a) show the average value and standard deviation of the chlorophyll a + b concentration during the monoculture of Chlorella NIES-2173 every 24 hours, and also show the average value etc. of the chlorophyll a + b concentration after 84 hours. In the example of the first and second columns from the left in Figure 27, the amount of NIES-2173 was adjusted so that the absorbance at a wavelength of 750 nm at the start of the monoculture of NIES-2173 became 0.025.

[0094] In the third and fourth columns from the left in Fig. 27, the average values and standard deviations of the chlorophyll a + b concentrations during the co - culture of NIES - 2173 and the OR151 isolate are shown. In the example of the third and fourth columns from the left in Fig. 21, the amount of OR151 was adjusted so that the absorbance at a wavelength of 600 nm of the OR151 component at the start of co - culture was 0.04 in the co - culture medium in which NIES - 2173 and the OR151 isolate were mixed. After 96 - hour single - culture of NIES - 2173, the chlorophyll a + b concentration increased to 8.44 μg / mL. On the other hand, after 96 - hour co - culture of NIES - 2173 and the OR151 isolate, the chlorophyll a + b concentration increased to 13.06 μg / mL.

[0095] Fig. 27(b) shows the magnification obtained by dividing the chlorophyll a + b concentration during the co - culture of NIES - 2173 and the OR151 isolate by the chlorophyll a + b concentration during the single - culture of NIES - 2173. As shown in Fig. 27(b), the magnification of the chlorophyll a + b concentration during the co - culture of NIES - 2173 and the OR151 isolate compared to the single - culture of NIES - 2173 was maximally 1.55 ± 0.07 times at 96 hours, and became minimally 1.10 ± 0.06 times at 48 hours, excluding 1.31 ± 0.31 times at 0 hours. Therefore, it was found that the OR151 isolate brought about a growth - promoting effect on NIES - 2173.

[0096] Fig. 28 shows the results of measuring the cell number, cell size, and chlorophyll fluorescence intensity of NIES - 2173 by flow cytometry. The first row from the top in Fig. 28 shows the cell number, etc. of NIES - 2173 after single - culture of NIES - 2173. The second row from the top in Fig. 28 shows the cell number, etc. of NIES - 2173 after co - culture of NIES - 2173 and the OR151 isolate. The cell size was measured by the forward scatter (FSC - H) of flow cytometry. The cell size and chlorophyll fluorescence intensity after co - culture of NIES - 2173 and the OR151 isolate were 1.0 ± 0.1 times and 0.8 ± 0.1 times, respectively, compared to the culture with NIES - 2173 alone.

[0097] Figure 29 shows the phylogenetic tree of the genus Shewanella. Psychromonas antarctica star-1 reference strain was used as an outgroup. From this phylogenetic tree, the OR151 isolate is relatively close to the species Shewanella oneidensis. However, the homology between the OR151 isolate and Shewanella oneidensis is lower than the homology between the OR151 isolate and Shewanella putrefaciens, or the homology between the OR151 isolate and Shewanella profunda. The OR151 isolate is known to be a microorganism classified in the genus Shewanella, but its species name has not been specified (accession number: NITE P-03682). The OR151 isolate has thus been shown to have the effect of promoting the growth of microalgae by microorganisms in the genus Shewanella.

[0098] [Method for separating and culturing microorganisms promoting microalgae growth] Using JCM520 medium, microorganisms having a microalgae growth promoting effect were separated. The components of JCM520 medium are shown in Figure 30. In addition to each medium component shown in Figure 30, agar was added to a concentration of 2%, and JCM520 agar medium was prepared by performing autoclaving at 121 °C for 20 minutes. Using fermented FCU as a separation source, a solution diluted in the range of 10 times to 10 5 times of the separation source FCU was prepared. 100 μL of the diluted FCU was inoculated onto the JCM520 medium solidified in each petri dish, inoculated with a spreader, covered, and cultured until colony formation in an environment of aerobic conditions, room temperature (24 °C), 115 - 120 μmol / m 2 / s, and irradiated with light for 12 hours out of 24 hours. The formed colonies were picked up with a disposable loop and subcultured several times in the same medium as the medium used for separation to obtain single colonies.

[0099] [Outline of screening for microalgae growth promoting bacteria by high-throughput co-culture evaluation system] (1) Euglena It was used to evaluate the microalgae growth promoting effect of Euglena gracilis NIES-48 (hereinafter also simply referred to as "NIES-48"), which is a model organism of Euglena algae. In the case of monoculture of NIES-48, as a preculture, modified CM medium was used for flask culture at 25 °C, 100 rpm, photon flux density of 150 μmol / m 2 / s, with continuous light irradiation for 24 hours for 7 days. The photon flux density represents the intensity of light.

[0100] It was inoculated into the modified CM medium (composition shown in Fig. 31) without mixing the isolates with NIES-48. The composition of the modified CM medium is shown in Fig. 31. After inoculating NIES-48 into the modified CM medium, main culture was carried out at 25 °C, 100 rpm, photon flux density of 150 μmol / m 2 / s, with continuous light irradiation for 24 hours for 7 days. The chlorophyll a concentration (μg / mL) and chlorophyll b concentration (μg / mL) were measured every 24 hours.

[0101] When co-culturing the isolates separated from FCU and NIES-48, as a preculture for co-culture, a 100 mL flask was used, and 2 mL of the strain glycerol stock solution was inoculated into 40 mL of the modified CM medium, and cultured at 30 °C, 168 rpm for 2 days. At the time of main culture, 40 mL of the co-culture medium was dispensed into a 100 mL flask, and the amount of NIES-48 was adjusted so that the absorbance A730 of the light with a wavelength of 730 nm of NIES-48 became 0.05 after mixing with the isolates, and the amount of each isolate separated from FCU was adjusted so that the absorbance A600 of the light with a wavelength of 600 nm became 0.01 or 0.05, etc. after mixing with NIES-48.

[0102] After mixing NIES-48 and the isolates, it was inoculated into the modified CM medium, and cultured at 25 °C, 100 rpm, photon flux density of 150 μmol / m 2 / s, and the main culture was carried out for 7 days with continuous light irradiation for 24 hours. The chlorophyll a concentration (mg / L) and chlorophyll b concentration (mg / L) were quantified every 24 hours. For each isolate, it was confirmed that they did not have the same chlorophyll fluorescence as NIES-48. From this, it can be seen that each isolate does not affect the measurement results of the chlorophyll fluorescence intensity derived from NIES-48 during co-culture with NIES-48.

[0103] <Chlorophyll Quantification> 1 mL of the sample after co-culture was collected and centrifuged at 4°C and 6,000×g for 10 minutes. The supernatant fraction was removed, and the cells recovered as a precipitate were resuspended in an 80% (vol / vol) aqueous acetone solution. After standing at 4°C for 1 hour, it was centrifuged at 4°C and 6,000×g for 10 minutes, and the supernatant was collected in a 1.5 mL tube as a pigment extract. To prevent dew condensation on the surface of the cuvette, after returning the extract to room temperature, it was transferred to a glass cuvette, and the absorbances at 646 nm, 663 nm, and 750 nm were measured. In addition, to correct for the effects of turbidity and colored compounds, 750 nm was set as the zero point in the spectrophotometer.

[0104] The amount of each pigment was calculated based on the following calculation formulas. ChlA is chlorophyll A, and ChlB is chlorophyll B. ChlA (mg / L) = 12.21 (Abs663 - Abs750) - 2.81 (Abs646 - Abs750) ChlB (mg / L) = 20.13 (Abs646 - Abs750) - 5.03 (Abs663 - Abs750) When measuring the absorbance at each wavelength, a blank was measured with an 80% (v / v) aqueous acetone solution, and then the measurement was carried out.

[0105] <Flow Cytometry Analysis> Using a flow cytometer Cube8, the cell count and chlorophyll fluorescence per cell were measured. 1 mL of each 20-fold diluted culture solution was prepared as a sample for measurement. The voltages of forward scatter (FSC-H), side scatter (SSC-H), and chlorophyll fluorescence (FL2-H) were set to 125.0 V, 180.0 V, 525.0 V, and 350.0 V, respectively.

[0106] [Genomic Extraction of Microalgae Growth-Promoting Strains] Each isolate was cultured pure and subjected to genomic extraction. The tube containing the cells was centrifuged at 10,000 rpm and 4 °C for 5 minutes using a cooling centrifuge, and the cell pellet (precipitate) was collected. Using a pipettor, the supernatant was discarded into the waste liquid reservoir. 560 μL of TE buffer was added to the cell pellet and stirred well to suspend the cells. 30 μL of 10% SDS and 10 μL of proteinase K solution were added, mixed well, and then incubated at 37 °C for 1 hour. 100 μL of 5 M NaCl was added and mixed well. 80 μL of CTAB / NaCl solution was added, mixed well, and incubated at 65 °C for 10 minutes. 0.7 mL of chloroform / isoamyl alcohol was added, the tube cap was closed, and the tube was inverted 5 - 6 times up and down, stirred well, and then centrifuged at 15,000 rpm and 4 °C for 5 minutes using a cooling centrifuge.

[0107] 0.5 - 0.6 mL of the upper layer liquid was taken and transferred to a new 1.5 mL microtube. An equal volume of phenol / chloroform / isoamyl alcohol to the transferred liquid volume was added, stirred well, and then centrifuged at 15,000 rpm and 4 °C for 5 minutes using a cooling centrifuge. 0.5 - 0.6 mL of the supernatant was taken and transferred to a new 1.5 mL microtube. 0.6 times the volume of isopropanol of the transferred solution was added to precipitate the DNA, and then centrifuged at 15,000 rpm and 4 °C for 5 minutes using a cooling centrifuge. The supernatant was gently discarded by pipetting, 1 mL of 70% ethanol was added, and centrifuged again at 15,000 rpm and 4 °C for 5 minutes using a cooling centrifuge. The supernatant was discarded, and the tube was left open and dried for about 10 minutes. The precipitate was dissolved in 100 μL of TE buffer.

[0108] 19 μL of a PCR reaction solution containing 27F primer, 1492R primer, TaKaRa LA Taq polymerase, etc. was added with 1 μL of a DNA sample in which the precipitate was dissolved in TE buffer. Figure 3 shows the primers used in the PCR reaction. In Figure 3, the sequence numbers, primer names, and oligonucleotide sequences of the primers used in the PCR reaction are shown. The oligonucleotide sequence of the 27F primer (sequence number 1) is shown in the first row from the top of Figure 3, and the oligonucleotide sequence of the 1492R primer (sequence number 2) is shown in the second row from the top of Figure 3. The tube was spun down with a microcentrifuge, inserted into the thermal block of the thermal cycler, and subjected to 30 cycles of thermal cycle reaction.

[0109] [Column Purification] An equal volume of membrane binding solution was added to the DNA sample. The SV mini-column was inserted into the collection tube (also referred to as column assembly). The entire solution was transferred to the SV mini-column and left at room temperature for about 1 minute. This column assembly was set in a cooling centrifuge and centrifuged at 16,000×g for 1 minute at 4°C. 500 μL of membrane wash solution was put into the SV mini-column and centrifuged at 16,000×g for 1 minute at 4°C.

[0110] The liquid in the collection tube was discarded into the waste liquid reservoir, and the SV mini-column was inserted into the collection tube again. 500 μL of membrane wash solution was put into the SV mini-column and centrifuged at 16,000×g for 5 minutes at 4°C. The liquid in the collection tube was discarded into the waste liquid reservoir, and the column assembly with the SV mini-column inserted into the collection tube again was centrifuged at 16,000×g for 1 minute at 4°C. The SV mini-column was inserted into a new 1.5 mL microtube. 50 μL of sterilized water was put into the SV mini-column, left at room temperature for about 1 minute, and then centrifuged at 16,000×g for 1 minute at 4°C to elute the DNA.

[0111] [Cycle Sequencing] A sequencing reaction solution containing 27F primer etc. was prepared, 8 μL of this was mixed with 2 μL of the sample, and it was reacted using a thermal cycler (initial denaturation: 96 °C for 1 minute, [denaturation: 96 °C for 10 seconds, annealing: 50 °C for 5 seconds, extension: 60 °C for 4 seconds] × 29 times, final extension: 4 °C with no time limit). Any one of the primers from SEQ ID NO: 1 to SEQ ID NO: 8 shown in Figure 3 was used for the sequencing reaction solution. After the thermal cycler, 5 μL of 125 mM EDTA and 60 μL of 99.5% EtOH were added, mixed by inversion, wrapped with aluminum foil, and left for 15 minutes. Then, it was centrifuged at 3,750 × g for 30 minutes and then centrifuged at 185 × g for 10 seconds while inverted.

[0112] 60 μL of 70% EtOH was added, centrifuged at 3,750 × g for 5 minutes, and further centrifuged at 185 × g for 10 seconds. 15 μL of HiDi formamide was added, vortexed for 2 minutes, heat shocked at 95 °C for 2 minutes and at 4 °C for 2 minutes, and capillary sequencing was performed. The raw data of the DNA sequencing was analyzed for ATGC data using GENETYX's ATGC software, and the analyzed data was subjected to BLAST analysis at NCBI. Then, using GENETYX software, FASTA data of related strains was obtained using the NCBI database, and a phylogenetic tree of each isolate was created.

[0113] [Selection of microalgae growth-promoting bacteria by co-culture evaluation system] Figure 32 shows the isolates among those with a chlorophyll fluorescence intensity magnification exceeding 1.0 at 168 hours of co-culture, arranged in descending order of the growth magnification from those with a high growth magnification. The isolate names were arbitrarily given by the inventors. The "magnification" in Figure 32 indicates the magnification of the chlorophyll fluorescence intensity compared to the case of culturing only with NIES-48.

[0114] The chlorophyll fluorescence intensity shown in Fig. 32 represents the average value of the measurement results of the chlorophyll fluorescence intensity for multiple times. Those with "a" attached to the comparison target column indicate the magnification compared to NIES-48(1) shown third from the bottom in Fig. 32. Those with "b" attached to the comparison target column indicate the magnification compared to NIES-48(2) shown second from the bottom in Fig. 32. Those with "c" attached to the comparison target column indicate the magnification compared to NIES-48(3) shown first from the bottom in Fig. 32.

[0115] Among the 144 isolates from FCU, the chlorophyll fluorescence intensity of 21 isolates showed a magnification of 5.6 times or more.

[0116] Hereinafter, among the isolates with a chlorophyll fluorescence intensity magnification exceeding 5.6, the JM311 strain, JM321 strain, AF2108 strain, and JM202 strain were selected, and the NIES-48 growth promotion effect was confirmed using co-culture in flasks.

[0117] [JM311 isolate] Fig. 33 shows the measurement results of chlorophyll a+b after co-culture of the JM311 isolate and NIES-48. In Fig. 33, the * mark indicates a significant difference from the single culture of NIES-48 (p < 0.05). By co-culturing NIES-48 and the JM311 strain, the chlorophyll amount reached 30.47 mg / L at 168 hours of culture, which was 3.20 times higher than 9.52 mg / L during single culture.

[0118] Figure 34 shows the results of measuring the cell count, cell size, and chlorophyll fluorescence intensity of NIES-48 during single culture or co-culture with JM311 by flow cytometry. The cell size was measured by forward scatter (FSC-H) of flow cytometry. In Figure 34, the * mark indicates a significant difference from the single culture of NIES-48 (p<0.05). The cell count after 168 hours of co-culture of NIES-48 and the JM311 isolate increased 3.74-fold compared to the culture of NIES-48 alone. There was no change in the chlorophyll fluorescence intensity per cell, which was 0.78-fold. Forward light scatter decreased slightly to 0.88-fold. From this, it became clear that co-culturing NIES-48 and JM311 promoted growth and increased the cell count.

[0119] The nearly complete 16S rRNA sequence of the JM311 strain was determined by sequencing (1383 bp). As a result of sequence analysis by Brast n, the top 5 strains with high homology were Bacillus pumilus ATCC7061, Bacillus pumilus NBRC12092, Bacillus zhangzhouensis MCCC1A08372, Peribacillus acanthi L28, and Bacillus australimaris MCCC1A05787, with 99.86%, 99.86%, 99.78%, 99.78%, and 99.71% homology, respectively. The phylogenetic tree of the Bacillus genus closely related to JM311 is shown in Figure 35. The phylogenetic tree shown in Figure 35 was created based on the 16S rRNA sequence of JM311. Bootstrap values of 50 or more are shown. The Streptococcus intermedius 1877 reference strain was used as the outgroup. JM311 was deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (Deposit number: NITE P-03919).

[0120] [JM321 isolate] Figure 36 shows the measurement results of chlorophyll a+b after co-culture of JM321 isolate and NIES-48. In Figure 36, the * mark indicates a significant difference from the monoculture of NIES-48 (p<0.05). By co-culturing NIES-48 and JM321 strain, the chlorophyll amount reached 34.22 mg / L at 168 hours of culture, which was 3.78 times higher than 9.06 mg / L in the monoculture.

[0121] Figure 37 shows the results of measuring the cell number, cell size and chlorophyll fluorescence intensity of NIES-48 by flow cytometry during monoculture of NIES-48 or co-culture with JM321. The cell size was measured by the forward scatter (FSC-H) of flow cytometry. In Figure 37, the * mark indicates a significant difference from the monoculture of NIES-48 (p<0.05). After 168 hours of co-culture of NIES-48 and JM321 isolate, the cell number and chlorophyll fluorescence intensity were 3.48 times and 1.16 times, respectively, compared with the culture of NIES-48 alone. The forward light scatter was 0.95 times. From this, it became clear that co-culturing NIES-48 and JM321 slightly increased the chlorophyll amount per cell, promoted growth, and increased the cell number.

[0122] The nearly complete 16S rRNA sequence of JM321 strain (1483 bp) was determined by sequencing. As a result of sequence analysis by Blastn, the top 5 strains with high homology were Bacillus licheniformis DSM13, Bacillus licheniformis BCRC11702, Bacillus paralicheniformis KJ-16, Bacillus licheniformis NRRL B-41327, and Bacillus licheniformis ATCC14580, with 99.85%, 99.85%, 99.76%, 99.69%, and 99.691% homology respectively. The phylogenetic tree of the Bacillus genus closely related to JM321 is shown in Figure 38. The phylogenetic tree shown in Figure 38 was created based on the 16S rRNA gene sequence of JM321. Bootstrap values of 50 or more are shown. Streptococcus intermedius 1877 reference strain was used as the Outgoup. JM321 was identified as Bacillus licheniformis. JM321 was deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Accession No.: NITE P-03920).

[0123] [AF2108 isolate] Figure 39 shows the measurement results of chlorophyll a+b after co-culturing the AF2108 isolate and NIES-48. In Figure 39, the * mark indicates a significant difference from the monoculture of NIES-48 (p < 0.05). By co-culturing NIES-48 and the AF2108 strain, the chlorophyll content reached 32.66 mg / L at 168 hours of culture, which was 3.36 times higher than 9.32 mg / L in the monoculture.

[0124] Figure 40 shows the results of measuring the cell count, cell size, and chlorophyll fluorescence intensity of NIES-48 during single culture or co-culture with AF2108 by flow cytometry. The cell size was measured by forward scatter (FSC-H) of flow cytometry. In Figure 40, the * mark indicates a significant difference from the single culture of NIES-48 (p<0.05). The cell count after 168 hours of co-culture of NIES-48 and AF2108 isolates increased 2.93-fold compared to the culture of NIES-48 alone. The chlorophyll fluorescence intensity did not change, being 1.12-fold. The forward light scatter decreased to 0.92-fold. From this, it became clear that co-culturing NIES-48 and AF2108 promoted growth and increased the cell count.

[0125] The almost complete 16S rRNA sequence of the AF2018 strain was determined by sequencing (1394 bp). As a result of sequence analysis by Brast n, the top 5 strains with high homology were Rhodococcus cerastii C5, Rhodococcus cercidiphylli YIM65003, Rhodococcus yunnanensis YIM 70056, Rhodococcus fascians ATCC 12974, and Rhodococcus fascians CF17, which were 100.00%, 99.58%, 99.31%, 99.16%, and 99.15% respectively. The phylogenetic tree of the Rhodococcus genus closely related to AF2108 is shown in Figure 41. The phylogenetic tree shown in Figure 41 was created based on the 16S rRNA gene sequence of AE2108. Bootstrap values of 50 or more are shown. As the Outgroup, the Pseudonocardia dioxanivorans CB1190 reference strain was used. AF2108 was identified as Rhodococcus cerastii.

[0126] [JM202 isolate] Figure 42 shows the measurement results of chlorophyll a+b after co-culture of JM202 isolate and NIES-48. In Figure 42, the * mark indicates a significant difference from the monoculture of NIES-48 (p<0.05). By co-culturing NIES-48 and JM202 strain, the chlorophyll amount reached 26.38 mg / L at 168 hours of culture, which was 2.91 times higher than 9.06 mg / L in the monoculture, an increase of 2.91 times.

[0127] Figure 43 shows the results of measuring the cell number, cell size, and chlorophyll fluorescence intensity of NIES-48 by flow cytometry during monoculture of NIES-48 or co-culture with JM202. The cell size was measured by forward scatter (FSC-H) of flow cytometry. In Figure 43, the * mark indicates a significant difference from the monoculture of NIES-48 (p<0.05). After 168 hours of co-culture of NIES-48 and JM202 isolate, the cell number and chlorophyll fluorescence intensity were 3.17 times and 0.88 times, respectively, compared with the culture of NIES-48 alone. There was no change in forward light scatter, which was 1.01 times. From this, it became clear that co-culturing NIES-48 and JM202 promoted growth and increased the cell number, although the chlorophyll amount per cell decreased slightly.

[0128] The nearly complete 16S rRNA sequence of JM202 strain (1458 bp) was determined by sequencing. As a result of sequence analysis by Brast n, the top 5 strains with high homology were Aeromonas salmonicida CECT 894, Aeromonas salmonicida ATCC 33658, Aeromonas salmonicida NCIMB 1102, Aeromonas salmonicida ATCC 33658, Aeromonas salmonicida subsp. masoucida NBRC 13784, and their homology was 99.86%, 99.84%, 99.79%, 99.79%, and 99.79%, respectively. The phylogenetic tree of the genus Aeromonas closely related to JM202 is shown in Fig. 44. The phylogenetic tree shown in Fig. 44 was created based on the 16S rRNA gene sequence of JM202. Bootstrap values of 50 or more are shown. As the outgroup, the reference strain Succinatimonas hippei YIT 12066 was used. JM202 was deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Accession No.: NITE P-03835).

[0129] [Effects of the present invention] The microorganism for promoting the growth of microalgae belonging to the genus Rhodococcus, Xanthobacter, Anoxybacillus, Shewanella, Bacillus, or Aeromonas of the present invention can promote the growth of microalgae.

[0130] As described above, the present invention has been described using embodiments. However, the technical scope of the present invention is not limited to the scope described in the above embodiments, and various modifications and changes are possible within the scope of the gist. For example, all or part of the device can be functionally or physically distributed and integrated in any unit. Also, new embodiments resulting from any combination of a plurality of embodiments are included in the embodiments of the present invention. The effects of the new embodiments resulting from the combination have the effects of the original embodiments combined.

Claims

1. A microorganism for promoting the growth of microalgae, characterized in that it is a microorganism belonging to the genus Rhodococcus, Xanthobacter, Ancylobacter, Shewanella, or Aeromonas.

2. The microorganism for promoting the growth of microalgae according to claim 1, wherein the microorganism is Rhodococcus cerasii of the genus Rhodococcus, Xanthobacter flavus of the genus Xanthobacter, or Ancylobacter rudogensis of the genus Ancylobacter.

3. The microorganism for promoting the growth of microalgae according to claim 1, wherein the microorganism is Rhodococcus cerasii of the genus Rhodococcus, Aeromonas salmonicida or Aeromonas pisicola of the genus Aeromonas.

4. The microorganism for promoting the growth of microalgae according to any one of claims 1 to 3, for promoting the growth of cyanobacteria, green algae, glaucoalgae or Euglena algae.

5. The microorganism for promoting the growth of microalgae according to any one of claims 1 to 3, for promoting the growth of organisms belonging to the phylum Cyanobacteria or Euglenophyta.

6. A microalgae growth promoter comprising the microorganism for promoting microalgae growth according to any one of claims 1 to 3.

7. A method for culturing microalgae, comprising co-culturing one or more microorganisms belonging to the genus Rhodococcus, Xanthobacter, Ancylobacter, Shewanella, or Aeromonas together with the microalgae.

8. A step of co-culturing a microorganism to be screened with microalgae; Quantifying the amount of chlorophyll contained in the medium after co-culture; A method for screening microorganisms comprising:

9. 9. The method for screening microorganisms according to claim 8, wherein the step of quantifying chlorophyll is a step of measuring fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 680 to 720 nm.

10. The step of quantifying chlorophyll is a step of measuring fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 683 nm when the microalgae is PCC7942, at an excitation wavelength of 488 nm and a fluorescence wavelength of 685 nm when the microalgae is NIES-2173, and at an excitation wavelength of 488 nm and a fluorescence wavelength of 700 nm when the microalgae is NIES-48. The method for screening microorganisms according to claim 8.

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