Microorganism for promoting growth of microalgae, microalga growth promoter, method for culturing microalgae, and method for screening microorganisms

Abstract

The present invention relates to a microorganism belonging to the genus Rhodococcus, the genus Xanthobacter, the genus Ancylobacter, the genus Shewanella, or the genus Aeromonas, and a method for producing the same, and more specifically, to a microorganism belonging to the genus Rhodococcus, the genus Xanthobacter, the genus Ancylobacter, the genus Shewanella, or the genus Aeromonas. The microorganism can be Rhodococcus hedyotidis (Rhodococcus cerevisiae) of the genus Rhodococcus, Xanthobacter flavus (Xanthobacter flavus) of the genus Xanthobacter, or Ancylobacter rudongensis of the genus Ancylobacter, and the microorganism can be a microorganism of the genus Ancylobacter, or a microorganism of the genus Ancylobacter, or a microorganism of the genus Ancylobacter, or a microorganism of the genus Ancylobacter and a microorganism of the genus Ancylobacter. The microorganism can be used for promoting the growth of blue algae, green algae, grey algae or euglena. The microorganism can be used to promote the growth of an organism belonging to the phylum cyanobacteria, unequal flagellum, euglenophyta, cryptophyta, rhodophyta, chlorophyta or chalaphyta.

Description

Technical Field

[0001] This invention relates to microalgae growth-promoting microorganisms, microalgae growth promoters, microalgae cultivation methods using said microalgae growth-promoting microorganisms, and screening methods for isolating microorganisms capable of promoting microalgae growth. Background of the Invention

[0002] For example, microalgae are used industrially to produce useful substances such as astaxanthin, a naturally occurring carotenoid pigment that exhibits a red color. These useful substances are extracted from microalgae and used as raw materials for food, pharmaceuticals, feed, fertilizers, etc. Recently, the use of sugars and lipids accumulated within microalgal cells as useful substances for the production of petroleum and bioethanol has attracted attention. Furthermore, Euglena, as a type of microalgae, is itself industrially produced as a food ingredient. Microalgae can fix carbon dioxide through their photosynthetic capacity. Therefore, cultivating microalgae is also useful as a countermeasure against global warming. Existing technology Patent documents

[0003] Patent Document 1: Japanese Patent Publication No. 2014-509188. Summary of the Invention The problem to be solved by the present invention

[0004] Microalgae grow at a slower rate than typical heterotrophic microorganisms. For example, Patent Document 1 discloses increasing the growth rate of microalgae by increasing the levels of nutrients such as nitrogen and phosphorus, in order to improve the economic efficiency of using microalgae to produce useful substances. However, the method described in Patent Document 1 has the problem of not being able to sufficiently increase the growth rate of microalgae.

[0005] The present invention was made in view of these problems, and its object is to provide microalgae growth promoting microorganisms that can promote microalgae growth, microalgae growth promoters containing said microorganisms, microalgae culture methods, and screening methods for isolating microorganisms that can promote microalgae growth. Problem-solving methods

[0006] The microalgae growth-promoting microorganisms of the first aspect of the present invention are microorganisms belonging to the genera Rhodococcus, Flavobacterium, Trichobacterium, Shewanella, Bacillus, or Aeromonas.

[0007] The microorganisms may be *Rhodococcus cerastii* (of the genus *Rhodococcus*), *Xanthobacter flavus* (of the genus *Xanthobacter*), or *Ancylobacter rudongensis* (of the genus *Ancylobacter*). Alternatively, the microorganisms may be *Bacillus licheniformis*, *Bacillus pumilus*, *Bacillus zhangzhouensis*, *Bacillus australimaris*, *Bacillus safensis*, or *Peribacillus acanthi* (of the genus *Bacillus*), *Rhodococcus cerastii* (of the genus *Rhodococcus*), or *Aeromonas salmonicida* or *Aeromonas piscicola* (of the genus *Aeromonas*). The microorganisms described herein can be used to promote the growth of cyanobacteria, green algae, gray algae, or euglenoids. Specifically, they can promote the growth of organisms belonging to the phylum Cyanobacteria, Phylum Euglena, Cryptophyta, Phylum Cercozoa, Phylum Glaucophyta, Phylum Rhodophyta, Phylum Chlorophyta, or Phylum Streptophyta. Furthermore, they can promote the growth of organisms belonging to the phylum Cyanobacteria or Phylum Euglena.

[0008] The microalgae growth promoter of the second aspect of the present invention contains the microalgae growth promoting microorganisms.

[0009] The microalgae cultivation method of the third aspect of the present invention includes co-culturing one or more microorganisms selected from the genera Rhodococcus, Flavobacterium, Trichoderma, Shewanella, Bacillus, or Aeromonas with microalgae.

[0010] The microbial screening method of the fourth aspect of the present invention includes the steps of co-culturing the microorganism to be screened with microalgae; and quantifying the chlorophyll contained in the culture medium after co-culturing. The chlorophyll quantification step may be a step of measuring fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 680 nm to 720 nm, and preferably, if the microalgae is PCC7972, the fluorescence intensity is measured at an excitation wavelength of 488 nm and a fluorescence wavelength of 683 nm; if the microalgae is NIES-2173, the fluorescence intensity is measured at an excitation wavelength of 488 nm and a fluorescence wavelength of 685 nm; and if the microalgae is NIES-48, the fluorescence intensity is measured at an excitation wavelength of 488 nm and a fluorescence wavelength of 700 nm. The screening method preferably further includes a step of evaluating the degree of microalgal growth-promoting effect exhibited by the microorganism to be screened using the quantitative results of the chlorophyll contained in the culture medium after co-culturing. Effects of the present invention

[0011] This invention promotes the growth of microalgae. Brief description of the attached figures

[0012] Figure 1 The concentration of each medium component added to Ormerod medium is shown. Figure 2 The concentration of each medium component in the co-culture medium is shown. Figure 3 The primers used in the PCR reaction are shown. Figure 4 Isolates with a fold increase of more than 1.0 in chlorophyll fluorescence intensity on day 6 of co-culture are shown. Figure 5 Isolates with a fold increase of more than 1.0 in chlorophyll fluorescence intensity on day 6 of co-culture are shown. Figure 6 The changes in chlorophyll a over time are shown during pure culture of PCC7942 (hereinafter also referred to as "isolated culture") and during co-culture with strain AF2108. Figure 7 The results of flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity at 168 hours of culture time are shown during pure culture of PCC7942 and during co-culture with strain AF2108. Figure 8 The images show, in chronological order, the individual cultures of PCC7942 and its co-culture with strain AF2108 in flasks. Figure 9The changes in chlorophyll a+b over time are shown during pure culture of NIES-2173 and during co-culture with strain AF2108. Figure 10 The results of flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity at 120 hours of culture time are shown during pure culture of NIES-2173 and during co-culture with strain AF2108. Figure 11 A phylogenetic tree of the genus Rhodococcus is shown. Figure 12 The changes in chlorophyll a over time are shown during pure culture of PCC7942 and during co-culture with strain AF2111. Figure 13 The results of flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity at 168 hours of culture time are shown during pure culture of PCC7942 and during co-culture with strain AF2111. Figure 14 The images show, in chronological order, the individual cultures of PCC7942 and its co-culture with strain AF2111 in flasks. Figure 15 The changes in chlorophyll a+b over time are shown during pure culture of NIES-2173 and during co-culture with strain AF2111. Figure 16 The results of flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity at 120 hours of culture time are shown during pure culture of NIES-2173 and during co-culture with AF2111. Figure 17 A phylogenetic tree of the genus Xanthobacter is shown. Figure 18 The changes in chlorophyll a over time are shown during pure culture of PCC7942 and during co-culture with strain GA1226. Figure 19 The results of flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity at 168 hours of culture time are shown during pure culture of PCC7942 and during co-culture with strain GA1226. Figure 20 The pure culture of PCC7942 and its co-culture with strain GA1226 in flasks are shown in chronological order. Figure 21 The changes in chlorophyll a+b over time are shown during pure culture of NIES-2173 and during co-culture with strain GA1226. Figure 22 The results of flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity at 120 hours of culture time are shown during pure culture of NIES-2173 and during co-culture with strain GA1226. Figure 23 A phylogenetic tree of the genus Ancylobacter is shown. Figure 24 The changes in chlorophyll a over time are shown during pure culture of PCC7942 and during co-culture with strain OR151. Figure 25 The results of flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity at 168 hours of culture time are shown during pure culture of PCC7942 and during co-culture with strain OR121. Figure 26 The pure culture of PCC7942 and its co-culture with OR151 strain in flasks are shown in chronological order. Figure 27 The changes in chlorophyll a+b concentrations over time are shown during pure culture of NIES-2173 and during co-culture with strain OR151. Figure 28 The results of flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity at 120 hours of culture time are shown during pure culture of NIES-2173 and during co-culture with OR151 strain. Figure 29 A phylogenetic tree of the genus Shewanella is shown. Figure 30 The composition of JCM medium is shown. Figure 31 The composition of the modified CM medium is shown. Figure 32 Some of the isolates obtained in the examples are shown. Figure 33 The changes in chlorophyll content over time are shown during individual or co-culture. Figure 34 The results of flow cytometry are shown. Figure 35 A phylogenetic tree of the genus Bacillus associated with JM311 is shown. Figure 36 The changes in chlorophyll content over time are shown during individual or co-culture. Figure 37 The results of flow cytometry are shown. Figure 38A phylogenetic tree of the Bacillus genus associated with JM321 is shown. Figure 39 The changes in chlorophyll content over time are shown during individual or co-culture. Figure 40 The results of flow cytometry are shown. Figure 41 A phylogenetic tree of the genus *Rhodococcus* associated with AF2108 is shown. Figure 42 The changes in chlorophyll content over time are shown during individual or co-culture. Figure 43 The results of flow cytometry are shown. Figure 44 A phylogenetic tree of the genus Aeromonas associated with JM202 is shown. Detailed Implementation

[0013] Microalgae growth-promoting microorganisms The microalgae growth-promoting microorganisms in this embodiment belong to the genera *Rhodococcus*, *Xanthobacter*, *Shewanella*, *Ancylobacter*, *Bacillus*, or *Aeromonas*. These microorganisms are obtained through screening using fermented cow urine (FCU) and a microalgae growth promoter produced from FCU. The fermented cow urine is obtained by treating cow urine with microorganisms. Furthermore, the microalgae growth-promoting microorganisms in this embodiment are not limited to those isolated from FCU; any microalgae growth-promoting microorganism belonging to any of the above genera and acting on microalgae to promote their growth is acceptable. Preferably, the microalgae growth-promoting microorganisms are Rhodococcus cerastii (of the genus Rhodococcus), Xanthobacter flavus (of the genus Xanthobacter), Ancylobacter rudongensis (of the genus Ancylobacter), or Shewanella sp.

[0014] Furthermore, preferably, the microalgae growth-promoting microorganisms are Bacillus licheniformis, Bacillus pumilus, Bacillus zhangzhouensis, Peribacillus acanthi, Bacillus australimaris, or Bacillus safensis (all belonging to the genus Bacillus); Rhodococcus cerastii (belonging to the genus Rhodococcus); or Aeromonas salmonicida or Aeromonas piscicola (belonging to the genus Aeromonas). The microalgae growth-promoting microorganisms of this embodiment can promote microalgae growth by co-culturing with microalgae or by adding or mixing the culture medium containing the microalgae growth-promoting microorganisms into the microalgae culture medium.

[0015] Microalgae The microalgae growth-promoting microorganisms in this embodiment promote the growth of microalgae. Microalgae are photosynthetic organisms that require a microscope to identify individual individuals.

[0016] For example, microalgae are organisms belonging to the phylum Cyanobacteria (e.g., cyanobacteria), Anisochondria, Euglenophyta (e.g., Euglenophyta), Cryptophyta, Diplocophyta, Uropoda, Gyrophyta (e.g., Gyrophyta), Rhodophyta, Chlorophyta (e.g., Chlorophyta), or Chain Plantae (hereinafter "species"). Microalgae are preferably organisms belonging to the phylum Cyanobacteria (e.g., cyanobacteria) or the phylum Euglenophyta (e.g., Euglenophyta).

[0017] Examples of microalgae include species belonging to the phylum Chroococcales, Oscillatoriales, Nostocales, or Stigonemales, and specific examples include the genera *Chroococcus* sp., *Microcystis aeruginosa*, *Oscillataria* sp., *Microcoleus* sp., *Nostoc* sp., *Cylindrospermum*, *Stigonema*, and *Synecochoccus elongatus*.

[0018] For example, microalgae can be species belonging to the order Phaeodinales or Mallomonadales within the class Chrysophyceae of the phylum Anisochodinia, and specific examples include *Uroglenopsisamericana*, *Uroglena volvox*, *Mallomonas*, and *Synterasp.*. Similarly, microalgae can be species belonging to the class Bacillus within the phylum Anisochodinia, and specific examples include *Coscinodiscus* sp. and *Diatoma*.

[0019] For example, microalgae can be species belonging to the class Xanthophyceae of the phylum Anisochodinia, and specific examples include the genera *Pseudostaurastrum* and *Characiopsis*. For example, microalgae can be species belonging to the class Dictyocha of the phylum Anisochodinia, and specific examples include the genus *Dictyocha*. For example, microalgae can be species belonging to the class Dinophyceae of the phylum Anisochodinia, and specific examples include the genera *Peridinium* and *Scrippsiella trochoidea*.

[0020] For example, microalgae can be species belonging to the class Euglenophyceae within the phylum Euglenophyta, and specific examples include the genera *Euglena* and *Phacus*. Similarly, microalgae can be species belonging to the class Cryptophyceae within the phylum Cryptophyta, and specific examples include the genera *Cryptomonas* and *Rhodomonas*.

[0021] For example, microalgae can be species belonging to the class Chordata within the phylum Chordata, and specific examples include the genera *Coronosphaera* and *Gephyrocapsa*. For example, microalgae can be species belonging to the class *testate filose amoebae* within the phylum Leptostomes, and specific examples include *Paulinella chromoophora*. For example, microalgae can be species belonging to the class Glaucocystis within the phylum Glaucophyta, and specific examples include the genus *Glaucocystis*.

[0022] For example, microalgae can be species belonging to the class Rhodophyta within the phylum Rhodophyta, and specific examples include the genera *Cyanidium* and *Galdieria*. For example, microalgae can be species belonging to the class Chlorophyta within the phylum Chlorophyta, and specific examples include *Pediastrum duplex*, *Volvox*, *Chlamydomonas*, *Asterococcus*, and *Chlorella*. For example, microalgae can be species belonging to the class Chlorophyta within the phylum Chlorophyta, and specifically, can be single-celled green algae belonging to the genus *Chlorella*. For example, a single-celled green alga belonging to the genus *Chlorella* could be *Chlorella sorokiniana*.

[0023] For example, microalgae can be species belonging to the class Mesostigma within the phylum Chainidae, and specific examples include the genus *Mesostigma*. For example, microalgae can be species belonging to the class Conjugata within the phylum Chainidae, and specific examples include the genus *Zygnema*. For example, examples of microalgae include *Synecochoccus elongatus*, a type of so-called cyanobacteria, belonging to the phylum Cyanobacteria.

[0024] [Microalgae growth promoter] The microalgae growth promoter containing the microalgae growth-promoting microorganisms of this embodiment is not limited in form. The microalgae growth promoter can be liquid, solid, slurry, etc., and is preferably liquid. Furthermore, the microalgae growth promoter of this embodiment may contain byproducts, such as diluents, stabilizers, thickeners, and granulators, depending on its form. For example, the microalgae growth promoter can be used by adding it to the culture medium for cultivating microalgae.

[0025] The microalgae growth promoter of this embodiment contains the microalgae growth-promoting microorganisms mentioned above. The microalgae growth promoter can be used directly as is, or the microorganisms can be removed from it before use. There are no particular limitations on the method of removing the microorganisms, and examples include, for instance, removal by filtration, removal by centrifugation, sterilization using an autoclave, and sterilization by ultraviolet irradiation. The microalgae growth promoter of this embodiment can promote the growth of microalgae by adding it to the culture medium for culturing microalgae.

[0026] The microalgae cultivation method of this embodiment includes co-culturing microalgae with the microalgae-promoting microorganisms mentioned above. For example, the microalgae cultivation method of this embodiment includes co-culturing microalgae with microalgae in FCU medium. In this way, the microalgae cultivation method of this embodiment can promote the growth of microalgae.

[0027] [Filtering Method] The screening method of this embodiment is a method for screening microorganisms that can promote the growth of microalgae, and includes the following steps (1) and (2). (1) The step of co-culturing the microorganisms and microalgae that are to be screened. (2) A step of quantifying the chlorophyll content in the culture medium after co-culture.

[0028] There are no particular limitations on the method for preparing the microorganisms to be screened in (1). For example, candidate microorganisms can be isolated from liquids of natural sources that can promote the growth of microalgae, such as fermented cattle urine broth (FCU) obtained by fermenting cattle urine with aeration. The screening method of this embodiment can treat microorganisms contained in fermented animal urine broth obtained by fermenting urine from other livestock such as horses or pigs, rather than cattle urine, as screening targets. When using FCU, the screening method of this embodiment includes culturing Ormerod medium by adding a diluted solution obtained by diluting the FCU. Figure 1 The concentration of each culture medium component added to the Ormerod medium is shown. The screening method according to this embodiment includes repeatedly isolating colonies formed in Ormerod medium into other Ormerod mediums for cultivation to obtain multiple single colonies. The screening method of this embodiment includes co-culturing each of the multiple single colonies that have been formed in multiple culture media containing microalgae.

[0029] Furthermore, the screening method of this embodiment may include culturing the microorganisms to be screened in FCU medium instead of Ormerod medium or JCM520 medium. In this case, the screening method of this embodiment may use FCU sterilized by autoclaving at 121°C for 20 minutes, or FCU sterilized by passing it through a 0.22 μm filter. The screening method of this embodiment includes mixing FCU with sterile water to adjust the amount of FCU such that the concentration of FCU in the finished FCU medium is 20% or 10%. The screening method of this embodiment includes adding gellan gum or agar to the FCU medium, wherein the gellan gum concentration in the finished FCU medium is adjusted to 0.8%, and the agar concentration in the finished FCU medium is adjusted to 2%.

[0030] The chlorophyll quantification steps described in (2) above include quantifying the chlorophyll contained in the microalgae after co-culturing the microorganisms and microalgae used as screening targets, so as to evaluate the extent to which the microorganisms exhibit their growth-promoting effect on the microalgae. The chlorophyll quantification steps include measuring the fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength from 683 nm to 720 nm using a fluorescence spectrophotometer. Furthermore, the chlorophyll quantification steps may include changing the wavelength according to the type of microalgae. For example, the chlorophyll quantification steps preferably include the following steps: measuring the fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 683 nm when the microalgae is PCC7972, measuring the fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 685 nm when the microalgae is NIES-2173, and measuring the fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 700 nm when the microalgae is NIES-48. In this way, by measuring light at wavelengths corresponding to chlorophyll fluorescence, the quantitative step of chlorophyll measurement can accurately evaluate the extent to which microorganisms promote microalgal growth.

[0031] The screening method of this embodiment includes decoding the DNA sequence corresponding to 16S rRNA or the like in the microbial genome of the identified colony by sequencing. For example, the screening method of this embodiment includes identifying a known microorganism having a DNA sequence with the highest consistency with the decoded DNA sequence as the microorganism corresponding to the colony or a candidate microorganism corresponding to the colony. Furthermore, the screening method of this embodiment may include identifying a known microorganism as the microorganism corresponding to the colony, wherein the DNA sequence of the known microorganism has a consistency equal to or higher than a threshold for the decoded DNA sequence corresponding to 16S rRNA or the like in the microbial genome of the identified colony. For example, the threshold is a value ranging from 97% to 98%. Example

[0032] [Methods for Isolating and Cultivating Microorganisms that Promote Microalgae Growth] To examine the screening method of this embodiment, the inventors of this application used Ormerod medium to isolate microorganisms that promote microalgae growth. In addition to Figure 1 In addition to each of the culture medium components shown, the inventors of this application added 2% agar and autoclaved at 121°C for 20 minutes to prepare Ormerod agar medium. The inventors of this application used fermented FCU as the isolation source and prepared a liquid in which the isolation source FCU was diluted 10-fold to 10-fold. 5The inventors of this application spread 100 μL of diluted FCU onto solidified Ormerod medium in each culture dish and inoculated it using a Conradi stick. The lid was placed on the culture dish, and the dish was irradiated under aerobic conditions at room temperature (24°C) at a concentration of 115 to 120 μmol / m³ for only 12 hours every 24 hours. 2 Microorganisms are cultured in a light-filled environment until colonies form. The inventors of this application use a disposable inoculation loop to select the formed colonies, subculture the colonies several times in the same medium used for isolation, and obtain single colonies.

[0033] [Summary of screening microalgal growth-promoting bacteria using a high-throughput co-culture evaluation system] (1) Cyanobacteria The inventors of this application used the cyanobacterial model organism *Synechococcus elongatus* PCC7942 (hereinafter referred to as PCC7942) to evaluate its microalgal growth-promoting effect. As a pre-culture, in the case of PCC7942 culture alone, the inventors used a co-culture medium at 30°C and 120 rpm with a photon flux density of 115 to 120 μmol / m³. 2 The samples were cultured in flasks for 7 days under continuous 24-hour light irradiation. Photon flux density represents the intensity of light.

[0034] Figure 2 The concentration of each component in the co-culture medium is shown. The concentration contained in 100 mL of solution is... Figure 2 The trace elements in the sample are 0.25g of CuSO4·5H2O and 0.37g of (NH4)6Mo7O. 24 The ingredients included 4H₂O, 2.47 g of H₃BO₃, 0.29 g of ZnSO₄·7H₂O, and 1.58 g of MnCl₂·4H₂O. The inventors of this application inoculated PCC7942 onto a co-culture medium without mixing it with the isolate, and incubated it at 30°C and 120 rpm with a photon flux density of 115 to 120 μmol / m². 2 The cells were cultured for 7 days under continuous 24-hour light exposure. The inventors of this application measured the chlorophyll a concentration (μg / mL) at 24-hour intervals.

[0035] In the case of co-culturing isolates from FCU with PCC7942, as a pre-culture for co-culture, the inventors of this application inoculated 500 μL of bacterial glycerol stock solution into 40 mL of co-culture medium in a 100 mL flask and co-cultured the microorganisms at 30 °C and 160 rpm. During the main culture, the inventors of this application dispensed 40 mL of co-culture medium into 100 mL flasks and adjusted the amount of PC7942 so that the absorbance A of PCC7942 at 600 nm wavelength after mixing with the isolates was [missing information]. 600 The value was changed to 0.05, and the amount of each isolate from the FCU was adjusted so that the absorbance A of the isolate at 600 nm wavelength after mixing with PCC7942 was... 600 It becomes 0.01, 0.05, or 0.08, etc.

[0036] The inventors of this application, after mixing PCC7942 and the isolate, inoculated PCC7942 onto a co-culture medium and cultured it at 30°C and 120 rpm with a photon flux density of 115 to 120 μmol / m 2 The isolates were cultured for 7 days under continuous 24-hour light irradiation. The inventors quantified the chlorophyll a concentration (μg / mL) at 24-hour intervals. The inventors confirmed that none of the isolates exhibited chlorophyll fluorescence similar to that of PCC7942. This indicates that co-culturing each isolate with PCC7942 did not affect the measurement results of chlorophyll fluorescence intensity derived from PCC7942.

[0037] <Quantitative Chlorophyll> The inventors of this application recovered 1 mL of sample after co-culturing and centrifuged the sample at 15000×g for 7 minutes. The supernatant was removed, and the cells recovered as a precipitate were resuspended in 1 mL of cooled 100% (vol / vol) methanol. To extract pigment from the cells, the inventors placed the sample in a dark environment at 4.0°C for 1 hour and incubated the sample. After incubation, the inventors centrifuged the sample at 15000×g for 10 minutes at 4.0°C and quantified the chlorophyll a content in the supernatant by spectrophotometry. The inventors used methanol as a blank for calibration and measured the absorbance at 665 nm and 720 nm. The inventors used the formula "chlorophyll a concentration (μg / mL) = 12.9447 × (A)" to calculate the chlorophyll a concentration. 665 -A 720 The concentration of chlorophyll a was determined.

[0038] <Flow Cytometry Analysis> The inventors of this application used a Cube8 flow cytometer to measure cell number and chlorophyll fluorescence per cell. They prepared 1 mL of each culture medium diluted 1000 times and used these as measurement samples. The inventors set the voltages for forward scattering (FSC-H), side scattering (SSC-H), and chlorophyll fluorescence (FL2-H) to 200.0 V, 275.0 V, 525.0 V, and 675.0 V, respectively.

[0039] (2) Chlorella <Cultivation Methods> The inventors of this application evaluated the growth-promoting effect of microalgae using *Chlorella sorokiniana* NIES-2173 (hereinafter also referred to as NIES-2173) as another example of microalgae. The inventors used flasks for cultivation, wherein the culture was conducted in a modified BG11 medium supplemented with 5 g / L glucose at 30°C, 125 rpm, PPFD, and 133 μmol / m³. 2 Pre-culture was performed for 3 days under a light / dark cycle (light / dark) of 24h / 0h. For the isolates, the inventors of this application inoculated 1 mL of the strain's glycerol stock solution into 40 mL of co-culture medium in a 100 mL flask and cultured it at 30°C and 168 rpm.

[0040] During the main culture process, the inventors of this application injected 40 mL of modified BG11 medium supplemented with 5 g / L glucose into 100 mL flasks to transform Chlorella nIES-2173 into A. 750 :0.025 and caused each strain to become A 600 0.005, A 600 0.025, A 600 Adjust the concentration using methods such as 0.04 g / L glucose, and inoculate into modified BG11 medium supplemented with 5 g / L glucose. Incubate at 30°C, 125 rpm, PPFD, and 133 μmol / L glucose. 2 Master culture was performed for 4 days under a 24h / 0h light / dark cycle (n=3).

[0041] <Quantitative Chlorophyll> The inventors of this application recovered 1 mL of culture solution into a 1.5 mL tube and centrifuged the culture solution at 8000 rpm for 10 minutes at 4°C. After removing the supernatant, the inventors added 1.5 mL of pure methanol and let it stand in the dark at 4°C for 24 hours to allow the cell pellet to be immersed in the methanol. After 24 hours, the inventors used a vortex mixer to shake the cell pellet and centrifuged it again at 8000 rpm for 10 minutes at 4°C. The inventors transferred the supernatant to a glass cuvette and measured the absorbance at 653 nm, 666 nm, and 750 nm. The inventors used the calculation formula (1) "ChlA (mg / L) = 15.65(Abs666-Abs750) - 7.34(Abs653-Abs750)" to calculate the pigment content of chlorophyll a. In the calculation formula (1), ChlA is an abbreviation for chlorophyll a. Abs653, Abs666, and Abs750 are the absorbances at wavelengths of 653 nm, 666 nm, and 750 nm, respectively. The inventors of this application calculated the pigment content of chlorophyll b using formula (2): “ChlB (mg / L) = 27.05(Abs653-Abs750) - 11.21(Abs666-Abs750) (2)”. In formula (2), Chlb is an abbreviation for chlorophyll b.

[0042] <Flow Cytometry Analysis> The inventors of this application used a Cube8 flow cytometer to measure the absolute number of bacteria and the chlorophyll fluorescence per cell. The inventors prepared 1 mL samples from various culture solutions diluted 100 times as measurement samples. The inventors set the voltages for forward scattering (FSC-H), side scattering (SSC-H), and chlorophyll fluorescence (FL2-H) to 125.0 V, 180.0 V, 525.0 V, and 400.0 V, respectively.

[0043] [Genomic extraction of microalgae growth-promoting strains] The inventors of this application performed pure culture (isolated culture) of various isolates and extracted their genomes. The inventors centrifuged tubes containing bacterial cells at 10,000 rpm and 4°C for 5 minutes in a refrigerated centrifuge and recovered the cell pellet (precipitate). The inventors discarded the supernatant in a waste container using a pipette. The inventors added 560 μL of TE buffer to the cell pellet, shook the mixture thoroughly, and resuspended the cells. The inventors added 30 μL of 10% SDS and 10 μL of proteinase K solution, mixed thoroughly, and then incubated the mixture at 37°C for 1 hour. The inventors added 100 μL of 5M NaCl and mixed thoroughly. The inventors added 80 μL of CTAB / NaCl solution, mixed thoroughly, and incubated the mixture at 65°C for 10 minutes. The inventors of this application added 0.7 mL of chloroform / isoamyl alcohol, closed the tube cap, inverted the tube 5 to 6 times, shook the mixture thoroughly, and then centrifuged the mixture at 15,000 rpm for 5 minutes at 4°C in a refrigerated centrifuge.

[0044] The inventors of this application collected 0.5 to 0.6 mL of the supernatant and transferred it to a new 1.5 mL microtube. They added an equal volume of phenol / chloroform / isoamyl alcohol to the transferred liquid, shook the mixture thoroughly, and then centrifuged it at 15,000 rpm for 5 minutes at 4°C in a refrigerated centrifuge. The inventors collected 0.5 to 0.6 mL of the supernatant and transferred it to a new 1.5 mL microtube. They added 0.6 times the volume of the transferred solution of isopropanol to precipitate the DNA, and then centrifuged the mixture at 15,000 rpm for 5 minutes at 4°C in a refrigerated centrifuge. The inventors gently discarded the supernatant using a pipette, added 1 mL of 70% ethanol, and centrifuged the mixture again at 15,000 rpm for 5 minutes at 4°C in a refrigerated centrifuge. Finally, they discarded the supernatant and allowed the capped tube to dry for approximately 10 minutes. Dissolve the precipitate in 100 μL of TE buffer.

[0045] The inventors of this application prepared a PCR reaction solution containing 27F primer, 1492R primer, TaKaRa LA Taq polymerase, etc., and added 1 μL of DNA sample (which was obtained by dissolving the precipitate in TE buffer) to 19 μL of the PCR reaction solution. Figure 3 The primers used in the PCR reaction are shown. Figure 3 The sequence numbers, primer names, and oligonucleotide sequences of the primers used in the PCR reaction are shown. Figure 3 The first line from the top represents the oligonucleotide sequence of the 27F primer, and Figure 3The second line from the top represents the oligonucleotide sequence of primer 1492R. The inventors of this application used a simple centrifuge to spin-down the tube, inserted the tube into the thermostat module of a thermal cycler, and performed 30 cycles of thermal cycling.

[0046] [Column Purification] The inventors of this application added a membrane binding solution equal in volume to the DNA sample. The inventors of this application inserted an SV microcolumn into a recovery tube (also called a column assembly). The inventors of this application transferred the entire solution to the SV microcolumn and allowed it to stand at room temperature for approximately 1 minute. The inventors of this application placed the column assembly in a refrigerated centrifuge and centrifuged the column assembly at 16000×g for 1 minute at 4°C. The inventors of this application added 500 μL of membrane washing solution to the SV microcolumn and centrifuged the mixture at 16000×g for 1 minute at 4°C.

[0047] The inventors of this application discarded the liquid in the recovery tube into a waste liquid tank and reinserted the SV microcolumn into the recovery tube. They added 500 μL of membrane washing solution to the SV microcolumn and centrifuged the mixture at 16000 × g for 5 minutes at 4°C. They then discarded the liquid in the recovery tube into a waste liquid tank and reinserted the SV microcolumn into the column assembly in the recovery tube, centrifuging again at 16000 × g for 1 minute at 4°C. They then inserted the SV microcolumn into a new 1.5 mL microtube. Finally, they added 50 μL of sterile water to the SV microcolumn, allowed it to stand at room temperature for approximately 1 minute, then centrifuged the SV microcolumn at 16000 × g for 1 minute at 4°C, and eluted the DNA.

[0048] [Cyclic Sequencing] The inventors of this application prepared a sequencing reaction solution containing 27F primers, etc., mixed 8 μL of the sequencing reaction solution with 2 μL of sample, and carried out the reaction in 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). The inventors of this application used […] in the sequencing reaction solution. Figure 3 One of the primers listed as serial numbers 1 to 8. After the process in a thermal cycler, the inventors of this application added 5 μL of 125 mM EDTA and 60 μL of 99.5% EtOH and mixed them inverted. The mixture was then wrapped in aluminum foil and allowed to stand for 15 minutes. Subsequently, the inventors of this application centrifuged the mixture at 3750 × g for 30 minutes and then centrifuged it at 185 × g for 10 seconds while keeping it inverted.

[0049] The inventors of this application added 60 μL of 70% EtOH, centrifuged the mixture at 3750 × g for 5 minutes, and then further centrifuged it at 185 × g for 10 seconds. They then added 15 μL of HiDi formamide, vortexed the mixture for 2 minutes, subjected it to a heat shock at 95°C for 2 minutes and then at 4°C for 2 minutes, followed by capillary sequencing. The inventors used GENETYX's ATGC software to analyze the raw DNA sequencing data via ATGC data analysis, and performed BLAST analysis on the analyzed data using NCBI. Subsequently, the inventors used GENETYX software and the NCBI database to obtain FASTA data for the relevant strains and constructed a phylogenetic tree for each isolate.

[0050] [Selecting bacteria that promote microalgae growth through a co-culture evaluation system] Figure 4 and Figure 5 Isolates with chlorophyll fluorescence intensity exceeding 1.0 fold on day 6 of co-culture are shown. Figure 4 and Figure 5 The fifth column from the left represents the fold increase in chlorophyll fluorescence intensity compared to the case of culture with PCC7942 alone. In the case of culture with PCC7942 alone, the mean, standard deviation, fold increase, and standard deviation of chlorophyll fluorescence intensity are... Figure 5 The second line from the bottom ( Figure 5 PCC7942A and Figure 5 The first line from the bottom ( Figure 5 The PCC7942B in the text represents this.

[0051] exist Figure 4 and Figure 5 In the first column from the right, the items marked with "a" in the ratio comparison standards represent negative controls. For... Figure 4 and Figure 5 The items marked with "b" in the first column from the right in the ratio comparison standard are compared with... Figure 5 The variability in chlorophyll fluorescence intensity compared to PCC7942A was determined by... Figure 4 and Figure 5 The multiplier is indicated in the fifth column from the left. For in Figure 4 and Figure 5 The items marked with "c" in the first column from the right in the ratio comparison standard are compared with... Figure 5 The variability in chlorophyll fluorescence intensity of PCC7942B compared to PCC7942B was determined by... Figure 4 and Figure 5 The multiplier is indicated in the fifth column from the left.

[0052] Of the 144 isolates from FCU, 34 strains had a fold of chlorophyll fluorescence intensity equal to or greater than 1.0. Figure 4 and Figure 5 The 34 strains are shown in descending order of fold size. Figure 4 The isolate “AF2108” in the first row from the top showed the highest multiple of 7.5.

[0053] As described below, the inventors of this application selected isolates with a chlorophyll fluorescence intensity ratio exceeding 3.0 (AF2108 strain, GA1226 strain, AF2111 strain, OR151 strain) and examined the growth-promoting effects of PCC7942 and NIES-2173 respectively by co-culturing them in flasks.

[0054] [AF2108 isolate] Figure 6 (a) and Figure 6 (b) shows the chlorophyll a measurement results of AF2108 isolate after co-culturing with PCC7942. Figure 6 In (a), the first and second columns from the left represent the mean and standard deviation of chlorophyll a concentration at 24-hour intervals during individual cultivation of PCC7942. Figure 6 In the examples in the first and second columns from the left, the inventors of this application adjusted the amount of PCC7942 such that the absorbance at a wavelength of 730 nm became 0.05 at the start of individual cultivation of PCC7942.

[0055] Figure 6 The third and fourth columns from the left in the middle represent the mean and standard deviation of chlorophyll a concentration at 24-hour intervals after co-culturing PCC7942 and AF2108 isolates. Figure 6 In the examples in the third and fourth columns from the left, the inventors of this application adjusted the amount of PCC7942 in the co-culture medium of PCC7942 and AF2108 isolates so that the absorbance of the PCC7942 component at a wavelength of 730 nm became 0.05 at the beginning of co-culture.

[0056] The inventors of this application adjusted the amount of AF2108 isolate in the co-culture medium so that the absorbance of the AF2108 isolate components at a wavelength of 600 nm became 0.05 at the start of co-culture. After 168 hours of culture alone in PCC7942, chlorophyll a increased to 9.079 μg / mL. On the other hand, after 168 hours of co-culture of PCC7942 and AF2108 isolate, chlorophyll a increased to 64.249 μg / mL.

[0057] Figure 6(b) shows the folding obtained by dividing the chlorophyll a concentration during co-culture of PCC7942 with AF2108 isolates by the chlorophyll a concentration during PCC7942 culture alone. Figure 6 As shown in the second row from the top in (b), the fold increase of chlorophyll a concentration during co-culture of PCC7942 and AF2108 isolates compared to the chlorophyll a concentration during PCC7942 culture alone was the maximum value of 15.65 ± 0.72 at 48 hours and the minimum value of 7.08 ± 4.90 at 168 hours.

[0058] Figure 7 The flow cytometry results of cell number, cell size, and chlorophyll fluorescence intensity of PCC7942 are shown. Figure 7 The first line from the top indicates the number of PCC7942 cells after 168 hours of individual culture. Figure 7 The second line from the top indicates the cell number of PCC7942 isolates after co-culturing with AF2108 isolates for 168 hours. The inventors of this application used forward scattering (FSC-H) in flow cytometry to measure cell size. Compared to PCC7942 culture alone, the cell size and chlorophyll fluorescence intensity of PCC7942 after co-culturing with AF2108 isolates for 168 hours increased by 2.40 ± 0.12 times and 3.53 ± 0.41 times, respectively.

[0059] Figure 8 (a) to Figure 8 (g) shows the co-culture status of PCC7942 and AF2108 isolates in flasks in chronological order. It was confirmed that the samples after 24 hours of co-culture of PCC7942 and AF2108 isolates (first to third samples from the left) were greener than the samples during PCC7942 culture alone (fourth to sixth samples from the left).

[0060] Figure 9 (a) and Figure 9 (b) shows the measurement results of the sum of chlorophyll a and chlorophyll b concentrations (hereinafter also referred to as chlorophyll a+b concentration) during the co-culture of AF2108 isolate with NIES-2173. Figure 9 In (a), the first and second columns from the left represent the mean and standard deviation of chlorophyll a+b concentrations at 24-hour intervals during individual culture of Chlorella NIES-2173. Figure 9 In the examples in the first and second columns from the left, the inventors of this application adjusted the amount of NIES-2173 so that the absorbance at a wavelength of 750 nm became 0.025 at the start of individual cultivation of NIES-2173.

[0061] Figure 9 The third and fourth columns from the left represent the mean and standard deviation of chlorophyll a+b concentrations at 24-hour intervals during the co-culture of NIES-2173 and AF2108 isolates. Figure 9 In the examples in the third and fourth columns from the left, the inventors of this application adjusted the amount of AF2108 in the co-culture medium of NIES-2173 and AF2108 isolates so that the absorbance of the AF2108 component at a wavelength of 600 nm became 0.04 at the start of co-culture.

[0062] After culturing NIES-2173 alone for 120 hours, the chlorophyll a+b concentration increased to 10.22 μg / mL. On the other hand, after co-culturing NIES-2173 with AF2108 isolate for 120 hours, the chlorophyll a+b concentration increased to 17.60 μg / mL.

[0063] Figure 9 (b) shows the folding obtained by dividing the chlorophyll a+b concentration during the co-culture of NIES-2173 with AF2108 isolates by the chlorophyll a+b concentration during the individual culture of NIES-2173. Figure 9 As shown in (b), the fold increase in chlorophyll a+b concentration during co-culture of NIES-2173 and AF2108 isolates compared to the chlorophyll a+b concentration during NIES-2173 culture alone reached a maximum of 2.09 ± 0.08 after 96 hours and a minimum of 1.25 ± 0.09 after 24 hours. Therefore, the inventors of this application have demonstrated that the AF2108 isolate promotes the growth of NIES-2173.

[0064] Figure 10 The flow cytometry results of cell number, cell size, and chlorophyll fluorescence intensity of NIES-2173 are shown. Figure 10 The first line from the top indicates the number of NIES-2173 cells after NIES-2173 was cultured alone. Figure 10 The second row from the top indicates the cell number of NIES-2173 after co-culturing with the AF2108 isolate. The inventors of this application used forward scattering (FSC-H) in flow cytometry to measure cell size. Compared with NIES-2173 cultured alone, the cell size and chlorophyll fluorescence intensity of NIES-2173 after co-culturing with the AF2108 isolate increased by 0.8 ± 0.1 times and 1.0 ± 0.1 times, respectively.

[0065] Figure 11A phylogenetic tree of the genus Rhodococcus is shown. The standard strain (type strain) of Pseudonocardia dioxanivorans CP1190 is used as an outgroup. Figure 11 The values ​​shown are Bootstrap values ​​indicating the reliability of the phylogenetic tree representation. Only Bootstrap values ​​above 50 are shown. The scale bar represents the scale of the number of amino acid substitutions. According to the phylogenetic tree, strain AF2108 is most related to *Rhodococcus cerastii*. Strain AF2108 matches *Rhodococcus cerastii* with 100% homology and was identified as *Rhodococcus cerastii* (accession number: NITE P-03678).

[0066] [AF2111 isolate] Figure 12 (a) and Figure 12 (b) shows the chlorophyll a measurement results during co-culture of PCC7942 and AF2111 isolates. Figure 12 In (a), the first and second columns from the left represent the mean and standard deviation of chlorophyll a concentration at 24-hour intervals during individual cultivation of PCC7942. Figure 12 In the example of individual cultivation of PCC7942 described in the present application, the inventors of this application adjusted the amount of PCC7942 such that the absorbance at a wavelength of 730 nm became 0.05 at the start of individual cultivation of PCC7942.

[0067] Figure 12 The third and fourth columns from the left in the middle represent the mean and standard deviation of chlorophyll a concentration at 24-hour intervals during the co-culture of PCC7942 and AF2111 isolates. Figure 12 In the example of co-culturing PCC7942 and AF2111 isolates, the inventors of this application adjusted the amount of PCC7942 in the mixed co-culture medium of PCC7942 and AF2111 isolates so that the absorbance of PCC7942 at a wavelength of 730 nm became 0.05 at the beginning of co-culture, and adjusted the amount of AF2108 isolate in the mixed co-culture medium of PCC7942 and AF2111 isolates so that the absorbance of AF2108 isolate at a wavelength of 600 nm was 0.01 at the beginning of co-culture.

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

[0069] Figure 12 (b) shows the folding ratio obtained by dividing the chlorophyll a concentration during co-culture of PCC7942 with AF2111 isolates by the chlorophyll a concentration during PCC7942 culture alone. This folding ratio reached a maximum of 6.92 ± 0.48 at 24 hours and a minimum of 3.24 ± 0.99 at 144 hours.

[0070] Figure 13 The flow cytometry results of cell number, cell size, and chlorophyll fluorescence intensity of PCC7942 are shown. Figure 13 The first line from the top indicates the number of PCC7942 cells after 168 hours of individual culture. Figure 13 The second line from the top indicates the cell count of PCC7942 after co-culturing PCC794 and AF2111 isolates for 168 hours. (Example) Figure 13 As shown in the second row from the top, compared with the cell size after PCC7942 was cultured alone, the cell size and chlorophyll fluorescence intensity after co-culturing PCC7942 with AF2111 isolate were 2.19±0.11 times and 1.98±0.18 times, respectively.

[0071] Figure 14 (a) to Figure 14 (g) shows the co-culture status of PCC7942 and AF2111 isolates in flasks in chronological order. It was confirmed that the samples after 24 hours of co-culture of PCC7942 and AF2111 isolates (first to third samples from the left) were greener than the samples during PCC7942 culture alone (fourth to sixth samples from the left).

[0072] Figure 15 (a) and Figure 15 (b) shows the results of chlorophyll a+b concentration measurements during co-culture of AF2111 isolate with NIES-2173. Figure 15 In (a), the first and second columns from the left represent the mean and standard deviation of chlorophyll a+b concentrations at 24-hour intervals during individual culture of Chlorella NIES-2173, and also represent the mean and standard deviation of chlorophyll a+b concentrations after 84 hours. Figure 15In the examples in the first and second columns from the left, the inventors of this application adjusted the amount of NIES-2173 so that the absorbance at a wavelength of 750 nm became 0.025 at the start of individual cultivation of NIES-2173.

[0073] Figure 15 The third and fourth columns from the left represent the mean and standard deviation of chlorophyll a+b concentrations during the co-culture of NIES-2173 and AF2111 isolates. Figure 15 In the examples in the third and fourth columns from the left, the inventors of this application adjusted the amount of AF2111 in the co-culture medium of NIES-2173 and AF2111 isolates so that the absorbance of the AF2111 component at a wavelength of 600 nm became 0.005 at the start of co-culture.

[0074] After culturing NIES-2173 alone for 96 hours, the chlorophyll a+b concentration increased to 8.44 μg / mL. On the other hand, after co-culturing NIES-2173 with AF2111 isolate for 96 hours, the chlorophyll a+b concentration increased to 8.77 μg / mL.

[0075] Figure 15 (b) shows the folding obtained by dividing the chlorophyll a+b concentration during co-culture of NIES-2173 with AF2111 isolates by the chlorophyll a+b concentration during NIES-2173 culture alone. Figure 15 As shown in (b), the fold increase of chlorophyll a+b concentration during co-culture of NIES-2173 with AF2111 isolates compared to that during NIES-2173 culture alone was the largest at 96 hours (1.04 ± 0.06) and the smallest at 48 hours (excluding 1.86 ± 0.15 at 0 hours).

[0076] Figure 16 The flow cytometry results of cell number, cell size, and chlorophyll fluorescence intensity of NIES-2173 are shown. Figure 16 The first line from the top indicates the number of NIES-2173 cells after NIES-2173 was cultured alone. Figure 16 The second row from the top indicates the cell number of NIES-2173 after co-culturing with the AF2111 isolate. The inventors of this application used forward scattering (FSC-H) in flow cytometry to measure cell size. Compared with NIES-2173 cultured alone, the cell size and chlorophyll fluorescence intensity of NIES-2173 after co-culturing with the AF2111 isolate increased by 0.9 ± 0.0 times and 0.9 ± 0.2 times, respectively.

[0077] Figure 17 A phylogenetic tree of the genus *Xanthobacter* is shown. The inventors of this application used the standard strain *Blastochloris gulmargensis* JA248 as an outgroup. According to the phylogenetic tree, strain AF2111 is most related to *Xanthobacter flavus*. Strain AF2111 matched *Xanthobacter flavus* with 100% homology and was identified as *Xanthobacter flavus* (accession number: NITE P-03679). Therefore, it is demonstrated that *Xanthobacter flavus* has a microalgae growth-promoting effect.

[0078] [GA1226 isolate] Figure 18 (a) and Figure 18 (b) shows the results of chlorophyll a measurement during co-culture of PCC7942 and GA1226 isolates. Figure 18 In (a), the first and second columns from the left represent the mean and standard deviation of chlorophyll a concentration at 24-hour intervals during the individual cultivation of PCC7942. The inventors of this application adjusted the individual cultivation of PCC7942 such that the absorbance at a wavelength of 730 nm was 0.05 at the start of the PCC7942 cultivation.

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

[0080] Figure 18(b) shows the folding ratio obtained by dividing the chlorophyll a concentration after co-culturing PCC7942 with GA1226 isolates by the chlorophyll a concentration after PCC7942 was cultured alone. The maximum value of this folding ratio became 3.31 ± 1.83 (120 h), and even at its minimum, it was 1.56 ± 0.06 (24 h).

[0081] Figure 19 The flow cytometry results of cell number, cell size, and chlorophyll fluorescence intensity of PCC7942 are shown. Figure 19 The first line from the top indicates the number of PCC7942 cells after 168 hours of individual culture. Figure 19 The second row from the top indicates the cell number of PCC7942 after co-culturing PCC794 and GA1226 isolates for 168 hours. Compared with the cell size after PCC7942 was cultured alone, the cell size and chlorophyll fluorescence intensity of PCC7942 after co-culturing with GA1226 isolates changed by 1.25±0.16 times and 1.49±0 times, respectively.

[0082] Figure 20 (a) to Figure 20 (g) shows the co-culture status of PCC7942 and GA1226 isolates in flasks in chronological order. It was confirmed that the samples after 48 hours of co-culture of PCC7942 and GA1226 isolates (first to third samples from the left) were greener than the samples during PCC7942 culture alone (fourth to sixth samples from the left).

[0083] Figure 21 (a) and Figure 21 (b) shows the results of chlorophyll a+b concentration measurements during co-culture of GA1226 isolate with NIES-2173. Figure 21 In (a), the first and second columns from the left represent the mean and standard deviation of chlorophyll a+b concentrations at 24-hour intervals during the individual culture of Chlorella NIES-2173, and also represent the mean of chlorophyll a+b concentrations after 84 hours, etc. Figure 21 In the examples in the first and second columns from the left, the inventors of this application adjusted the amount of NIES-2173 so that the absorbance at a wavelength of 750 nm became 0.025 at the start of individual cultivation of NIES-2173.

[0084] Figure 21 The third and fourth columns from the left represent the mean and standard deviation of chlorophyll a+b concentrations during the co-culture of NIES-2173 and GA1226 isolates. Figure 21In the examples in the third and fourth columns from the left, the inventors of this application adjusted the amount of GA1226 in the co-culture medium of NIES-2173 and GA1226 isolates so that the absorbance of the GA1226 component at a wavelength of 600 nm became 0.025 at the start of co-culture. After 96 hours of culture of NIES-2173 alone, 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 GA1226 isolates, the chlorophyll a+b concentration increased to 8.05 μg / mL.

[0085] Figure 21 (b) shows the folding obtained by dividing the chlorophyll a+b concentration during co-culture of NIES-2173 with GA1226 isolates by the chlorophyll a+b concentration during individual culture of NIES-2173. Figure 21 As shown in (b), the fold increase in chlorophyll a+b concentration during co-culture of NIES-2173 and GA1226 isolates compared to that during NIES-2173 culture alone reached a maximum of 0.97 ± 0.00 at 84 hours and a minimum of 0.94 ± 0.11 at 72 hours (excluding 1.60 ± 0.28 at 0 hours).

[0086] Figure 22 The flow cytometry results of cell number, cell size, and chlorophyll fluorescence intensity of NIES-2173 are shown. Figure 22 The first line from the top indicates the number of NIES-2173 cells after NIES-2173 was cultured alone. Figure 21 The second row from the top indicates the cell number of NIES-2173 isolates after co-culturing with GA1226 isolates. The inventors of this application used forward scattering (FSC-H) in flow cytometry to measure cell size. Compared to NIES-2173 cultured alone, the cell size and chlorophyll fluorescence intensity of NIES-2173 co-cultured with GA1226 isolates increased by 0.9 ± 0.0 times and 0.7 ± 0.0 times, respectively.

[0087] Figure 23 A phylogenetic tree of the genus *Ancylobacter* is shown. The inventors of this application used the *Ancylobacter buddingus* JA248T standard strain as an outgroup. According to the phylogenetic tree, isolate GA1226 was most related to *Ancylobacter rudongensis*. Isolate GA1226 matched *Ancylobacter rudongensis* with 99.42% homology and was identified as *Ancylobacter rudongensis* (accession number: NITE P-03779). Therefore, the microalgae growth-promoting effect of *Ancylobacter rudongensis* has been demonstrated.

[0088] [OR151 isolate] Figure 24 (a) and Figure 24 (b) shows the results of chlorophyll a measurements during co-culture of PCC7942 and OR151 isolates. Figure 24 In (a), the first and second columns from the left represent the mean and standard deviation of chlorophyll a concentration at 24-hour intervals during the individual culture of PCC7942. The inventors of this application adjusted the amount of PCC7942 in the individual culture of PCC7942 so that the absorbance at a wavelength of 730 nm was 0.05 at the start of the PCC7942 culture.

[0089] Figure 24 The first and second columns from the left represent the mean and standard deviation of chlorophyll a concentration at 24-hour intervals during the co-culture of PCC7942 and OR151 isolates. The inventors of this application adjusted the amount of PCC7942 in the co-culture of PCC7942 and OR151 isolates such that the absorbance at 730 nm at the start of the PCC7942 component culture in the mixed co-culture medium was 0.05. The inventors of this application adjusted the amount of OR151 isolate in the co-culture medium such that the absorbance at 600 nm at the start of the OR151 isolate component culture was 0.05. The inventors of this application demonstrated that... Figure 24 As shown in the first row from the bottom in (a), although chlorophyll a increased to 7.21 μg / mL after 168 hours of culture when PCC7942 was cultured alone, chlorophyll a increased to 8.958 μg / mL after 168 hours of co-culture of PCC7942 with OR151 isolate.

[0090] Figure 24 (b) shows the folding ratio obtained by dividing the chlorophyll a concentration during co-culture of PCC7942 with OR151 isolates by the chlorophyll a concentration during PCC7942 culture alone. The maximum value of this folding ratio becomes 2.36 ± 0.14 (48 h) and 2.36 ± 0.08 (72 h), and the minimum value becomes 1.24 ± 0.02 (168 h).

[0091] Figure 25 The flow cytometry results of cell number, cell size, and chlorophyll fluorescence intensity of PCC7942 are shown. Figure 25 The first line from the top indicates the number of PCC7942 cells after 168 hours of individual culture. Figure 25The second row from the top indicates the cell number, etc., of PCC7942 after co-culturing PCC794 and OR151 isolates for 168 hours. Compared with the cell size after PCC7942 was cultured alone, the cell size and chlorophyll fluorescence intensity of PCC7942 after co-culturing with OR151 isolates for 168 hours changed by 2.04±0.09 times and 2.44±0 times, respectively.

[0092] Figure 26 (a) to Figure 26 (g) shows the co-culture status of PCC7942 and OR151 isolates in flasks in chronological order. It was confirmed that the samples after 24 hours of co-culture of PCC7942 and OR151 isolates (first to third samples from the left) were greener than the samples during PCC7942 culture alone (fourth to sixth samples from the left).

[0093] Figure 27 (a) and Figure 27 (b) shows the results of chlorophyll a+b concentration measurements during co-culture of OR151 isolate with NIES-2173. Figure 27 In (a), the first and second columns from the left represent the mean and standard deviation of chlorophyll a+b concentrations at 24-hour intervals during individual culture of Chlorella NIES-2173, and also represent the mean and standard deviation of chlorophyll a+b concentrations after 84 hours. Figure 27 In the examples in the first and second columns from the left, the inventors of this application adjusted the amount of NIES-2173 so that the absorbance at a wavelength of 750 nm became 0.025 at the start of individual cultivation of NIES-2173.

[0094] Figure 27 The third and fourth columns from the left represent the mean and standard deviation of chlorophyll a+b concentrations during the co-culture of NIES-2173 and OR151 isolates. Figure 21 In the examples in the third and fourth columns from the left, the inventors of this application adjusted the amount of OR151 in the co-culture medium of NIES-2173 and OR151 isolates so that the absorbance of the OR151 component at a wavelength of 600 nm became 0.04 at the start of co-culture. After 96 hours of culture of NIES-2173 alone, 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 OR151 isolates, the chlorophyll a+b concentration increased to 13.06 μg / mL.

[0095] Figure 27(b) shows the folding obtained by dividing the chlorophyll a+b concentration during co-culture of NIES-2173 with OR151 isolates by the chlorophyll a+b concentration during NIES-2173 culture alone. Figure 27 As shown in (b), the fold increase in chlorophyll a+b concentration between NIES-2173 co-cultured with OR151 isolates and NIES-2173 cultured alone reached a maximum of 1.55 ± 0.07 at 96 hours and a minimum of 1.10 ± 0.06 at 48 hours (excluding 1.31 ± 0.31 at 0 hours). Therefore, the growth-promoting effect of OR151 isolates on NIES-2173 has been demonstrated.

[0096] Figure 28 The flow cytometry results of cell number, cell size, and chlorophyll fluorescence intensity of NIES-2173 are shown. Figure 28 The first line from the top indicates the number of NIES-2173 cells after NIES-2173 was cultured alone. Figure 28 The second row from the top indicates the cell number of NIES-2173 after co-culturing with OR151 isolates. The inventors of this application used forward scattering (FSC-H) in flow cytometry to measure cell size. Compared with NIES-2173 culture alone, the cell size and chlorophyll fluorescence intensity of NIES-2173 after co-culturing with OR151 isolates increased by 1.0 ± 0.1 times and 0.8 ± 0.1 times, respectively.

[0097] Figure 29 A phylogenetic tree of the genus *Shewanella* is shown. The inventors of this application used the *Psychromonas antarctica* Star-1 standard strain as an outgroup. According to this phylogenetic tree, the OR151 isolate is relatively related 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* and the OR151 isolate and *Shewanella profunda*. The OR151 isolate is known to be a microorganism classified as belonging to the genus *Shewanella*, but its species name has not yet been identified (accession number: NITE P-03682). The OR151 isolate (i.e., a microorganism of the genus *Shewanella*) has been shown to promote microalgal growth.

[0098] [Methods for Isolating and Cultivating Microorganisms that Promote Microalgae Growth] The inventors of this application isolated microorganisms with microalgae growth-promoting effects using JCM520 culture medium. The components of JCM520 culture medium are as follows: Figure 30 As shown in the image. Besides... Figure 30 In addition to each of the culture medium components shown, the inventors of this application added 2% agar and autoclaved at 121°C for 20 minutes to prepare JCM520 agar medium. The inventors of this application used fermented FCU as the isolation source and prepared a liquid in which the isolation source FCU was diluted 10-fold to 10-fold. 5 The inventors of this application spread 100 μL of diluted FCU onto solidified JCM520 medium in various culture dishes, inoculated the diluted FCU using a Conradi stick, placed the lid on the culture dish, and incubated under aerobic conditions at room temperature (24°C) at a rate of 115 to 120 μmol / m³ for 12 hours every 24 hours. 2 Microorganisms are cultured in an environment irradiated with light until colonies form. The inventors of this application use a disposable inoculation loop to select the formed colonies, and then subculture the formed colonies several times on the same medium used for isolation to obtain single colonies.

[0099] [Summary of screening microalgal growth-promoting bacteria using a high-throughput co-culture evaluation system] (1) Euglena The inventors of this application used the model organism *Euglena gracilis* NIES-48 (hereinafter referred to as "NIES-48") to evaluate its microalgal growth-promoting effect. As a pre-culture, with NIES-48 cultured alone, the inventors used a modified CM medium at 25°C and 100 rpm with a photon flux density of 150 μmol / m². 2 The samples were cultured in flasks for 7 days under continuous 24-hour light irradiation. Photon flux density represents the intensity of light.

[0100] The inventors of this application inoculated NIES-48 into a modified CM medium without mixing it with isolates. Figure 31 The composition is described in the text. Figure 31 The composition of the modified CM medium is shown. After inoculating NIES-48 onto the modified CM medium, the inventors of this application used a photon flux density of 150 μmol / m at 25°C and 100 rpm. 2 The cells were cultured for 7 days under continuous 24-hour light irradiation. The inventors of this application measured the concentrations of chlorophyll a (μg / mL) and chlorophyll b (μg / mL) at 24-hour intervals.

[0101] In the case of co-culturing isolates from FCU with NIES-48, as a pre-culture for co-culture, the inventors of this application used a 100 mL flask, inoculated 2 mL of bacterial glycerol stock solution into 40 mL of modified CM medium, and cultured the microorganisms at 30°C and 168 rpm for two days. During the main culture, the inventors of this application distributed 40 mL of co-culture medium into the 100 mL flask, adjusted the amount of NIES-48 so that the absorbance A730 of NIES-48 at 730 nm wavelength became 0.05 after mixing with the isolates, and adjusted the amount of each isolate from FCU so that the absorbance A600 of the isolates at 600 nm wavelength became 0.01, 0.05, etc. after mixing with NIES-48.

[0102] The inventors of this application mixed NIES-48 with the isolate, then inoculated NIES-48 onto a modified CM medium, and applied it at 25°C and 100 rpm with a photon flux density of 150 μmol / m². 2 The isolates were cultured for 7 days under continuous 24-hour light irradiation. The inventors quantified the concentrations of chlorophyll a (mg / L) and chlorophyll b (mg / L) at 24-hour intervals. The inventors confirmed that none of the isolates exhibited chlorophyll fluorescence similar to that of NIES-48. This indicates that co-culturing the various isolates with NIES-48 did not affect the measurement results of chlorophyll fluorescence intensity derived from NIES-48.

[0103] <Quantitative Chlorophyll> The inventors of this application recovered 1 mL of sample after co-culturing and centrifuged the sample at 4°C and 6000×g for 10 minutes. The supernatant was removed, and the cells recovered as a precipitate were resuspended in an 80% (vol / vol) aqueous acetone solution. The mixture was allowed to stand at 4°C for 1 hour, then centrifuged at 4°C and 6000×g for 10 minutes, and the supernatant was recovered in a 1.5 mL tube to obtain the pigment extract. To prevent condensation on the cuvette surface, the extract was returned to room temperature, then transferred to a glass cuvette, and the absorbance was measured at 646 nm, 663 nm, and 750 nm. Note that to calibrate for the effects of turbidity and colored compounds, the inventors set 750 nm as the zero point on the spectrophotometer.

[0104] The inventors of this application calculated the content of each pigment based on the following formula. ChlA represents chlorophyll A, and ChlB represents 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 absorbance at various wavelengths, the inventors of this application performed the measurements after measuring a blank using an 80% (v / v) aqueous acetone solution.

[0105] <Flow Cytometry Analysis> The inventors of this application used a Cube8 flow cytometer to measure cell number and chlorophyll fluorescence per cell. They prepared 1 mL of various culture solutions diluted 20 times and used this as the measurement sample. The inventors set the voltages for forward scattering (FSC-H), side scattering (SSC-H), and chlorophyll fluorescence (FL2-H) to 125.0 V, 180.0 V, 525.0 V, and 350.0 V, respectively.

[0106] [Genomic extraction of microalgae growth-promoting strains] The inventors of this application cultured various isolates individually and extracted their genomes. They centrifuged tubes containing bacterial cells at 10,000 rpm and 4°C for 5 minutes in a refrigerated centrifuge and recovered the cell pellet (precipitate). The inventors discarded the supernatant in a waste container using a pipette. They added 560 μL of TE buffer to the cell pellet, agitated the mixture thoroughly, and resuspended the cells. They added 30 μL of 10% SDS and 10 μL of proteinase K solution, mixed thoroughly, and then incubated the mixture at 37°C for 1 hour. They added 100 μL of 5M NaCl and mixed thoroughly. They added 80 μL of CTAB / NaCl solution, mixed thoroughly, and incubated the mixture at 65°C for 10 minutes. The inventors of this application added 0.7 mL of chloroform / isoamyl alcohol, closed the tube cap, inverted the tube 5 to 6 times, shook the mixture thoroughly, and then centrifuged the mixture at 15,000 rpm for 5 minutes at 4°C in a refrigerated centrifuge.

[0107] The inventors of this application collected 0.5 to 0.6 mL of the supernatant and transferred it to a new 1.5 mL microtube. They added an equal volume of phenol / chloroform / isoamyl alcohol to the transferred liquid, shook the mixture thoroughly, and then centrifuged it at 15,000 rpm for 5 minutes at 4°C in a refrigerated centrifuge. The inventors collected 0.5 to 0.6 mL of the supernatant and transferred it to a new 1.5 mL microtube. They added 0.6 times the volume of the transferred solution of isopropanol to precipitate the DNA, and then centrifuged the mixture at 15,000 rpm for 5 minutes at 4°C in a refrigerated centrifuge. The inventors gently discarded the supernatant using a pipette, added 1 mL of 70% ethanol, and centrifuged the mixture again at 15,000 rpm for 5 minutes at 4°C in a refrigerated centrifuge. Finally, they discarded the supernatant and allowed the capped tube to dry for approximately 10 minutes. The inventors of this application dissolved the precipitate in 100 μL of TE buffer.

[0108] The inventors of this application prepared a PCR reaction solution containing 27F primer, 1492R primer, TaKaRa LA Taq polymerase, etc., and added 1 μL of DNA sample (which was obtained by dissolving the precipitate in TE buffer) to 19 μL of the PCR reaction solution. Figure 3 The primers used in the PCR reaction are shown. Figure 3 The sequence numbers, primer names, and oligonucleotide sequences of the primers used in the PCR reaction are shown. Figure 3 The first line from the top represents the oligonucleotide sequence of the 27F primer (sequence number 1), and Figure 3 The second line from the top represents the oligonucleotide sequence (Sequence No. 2) of primer 1492R. The inventors of this application centrifuged the tube using a simple centrifuge, inserted the tube into the thermostat module of a thermal cycler, and performed 30 cycles of thermal cycling.

[0109] [Column Purification] The inventors of this application added a membrane binding solution equal in volume to the DNA sample. The inventors of this application inserted an SV microcolumn into a recovery tube (also called a column assembly). The inventors of this application transferred the entire solution to the SV microcolumn and allowed it to stand at room temperature for approximately 1 minute. The inventors of this application placed the column assembly in a refrigerated centrifuge and centrifuged the column assembly at 16000×g for 1 minute at 4°C. The inventors of this application added 500 μL of membrane washing solution to the SV microcolumn and centrifuged the mixture at 16000×g for 1 minute at 4°C.

[0110] The inventors of this application discarded the liquid in the recovery tube into a waste liquid tank and reinserted the SV microcolumn into the recovery tube. They added 500 μL of membrane washing solution to the SV microcolumn and centrifuged the mixture at 16000 × g for 5 minutes at 4°C. They then discarded the liquid in the recovery tube into a waste liquid tank and reinserted the SV microcolumn into the column assembly in the recovery tube, centrifuging again at 16000 × g for 1 minute at 4°C. They then inserted the SV microcolumn into a new 1.5 mL microtube. Finally, they added 50 μL of sterile water to the SV microcolumn, allowed it to stand at room temperature for approximately 1 minute, then centrifuged the SV microcolumn at 16000 × g for 1 minute at 4°C, and eluted the DNA.

[0111] [Cyclic Sequencing] The inventors of this application prepared a sequencing reaction solution containing 27F primers, etc., mixed 8 μL of the sequencing reaction solution with 2 μL of sample, and carried out the reaction in 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). The inventors of this application used […] in the sequencing reaction solution. Figure 3 One of the primers listed as serial numbers 1 to 8. After the process in a thermal cycler, the inventors of this application added 5 μL of 125 mM EDTA and 60 μL of 99.5% EtOH and mixed them inverted. The mixture was then wrapped in aluminum foil and allowed to stand for 15 minutes. Subsequently, the inventors of this application centrifuged the mixture at 3750 × g for 30 minutes and then centrifuged it at 185 × g for 10 seconds while keeping it inverted.

[0112] The inventors of this application added 60 μL of 70% EtOH, centrifuged the mixture at 3750 × g for 5 minutes, and then further centrifuged it at 185 × g for 10 seconds. They then added 15 μL of HiDi formamide, vortexed the mixture for 2 minutes, subjected it to a heat shock at 95°C for 2 minutes and then at 4°C for 2 minutes, followed by capillary sequencing. The inventors used GENETYX's ATGC software to analyze the raw DNA sequencing data via ATGC data analysis, and performed BLAST analysis on the analyzed data using NCBI. Subsequently, the inventors used GENETYX software and the NCBI database to obtain FASTA data for the relevant strains and constructed a phylogenetic tree for each isolate.

[0113] [Selecting bacteria that promote microalgae growth through a co-culture evaluation system] Figure 32The isolates with a chlorophyll fluorescence intensity fold of over 1.0 are described in descending order of fold increase, and those whose chlorophyll fluorescence intensity fold changes to 5.6 or higher after 168 hours of co-culture are described. For convenience, the inventors of this application have given the names of the isolates. Figure 32 The “multiplier” in the text refers to the multiplier of chlorophyll fluorescence intensity compared to the case of cultured with NIES-48 alone.

[0114] Figure 32 The chlorophyll fluorescence intensity shown represents the average of multiple measurements of chlorophyll fluorescence intensity. Items marked with "a" in the comparison column indicate those compared to those measured by... Figure 32 The third line from the bottom indicates the multiplier for NIES-48(1). Items marked with "b" in the comparison object column indicate the comparison with... Figure 32 The second line from the bottom indicates the multiplier for the NIES-48(2) comparison. Items marked with "c" in the comparison object column indicate the comparison with... Figure 32 The first line from the bottom indicates the multiplier of the NIES-48(3) comparison.

[0115] Of the 144 isolates from FCU, 21 strains had a 5.6-fold or greater increase in chlorophyll fluorescence intensity.

[0116] In the following cases, the inventors of this application selected strains JM311, JM321, AF2108, and JM202 from isolates with a chlorophyll fluorescence intensity ratio exceeding 5.6, and examined the growth-promoting effect of NIES-48 by co-culturing them in flasks.

[0117] [JM311 isolate] Figure 33 The results of chlorophyll a+b measurement are shown after JM311 isolate was co-cultured with NIES-48. Figure 33 The asterisk (*) indicates a significant difference compared to NIES-48 culture alone (p<0.05). Co-culture of NIES-48 with strain JM311 resulted in a chlorophyll content of 30.47 mg / L at 168 hours of culture, representing a 3.20-fold increase compared to 9.52 mg / L during culture alone.

[0118] Figure 34 Flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity of NIES-48 cells during solitary culture or co-culture with JM311 are shown. The inventors of this application used forward scattering (FSC-H) in flow cytometry to measure cell size. Figure 34The asterisk (*) indicates a significant difference compared to NIES-48 culture alone (p<0.05). Compared to NIES-48 culture alone, the cell number increased 3.74-fold after co-culturing NIES-48 with JM311 isolates for 168 hours. Chlorophyll fluorescence intensity per cell remained unchanged at 0.78-fold. Forward light scattering decreased slightly at 0.88-fold. This indicates that co-culturing NIES-48 with JM311 promoted growth and increased cell number.

[0119] The inventors of this application determined the almost complete 16S rRNA sequence (1383 bp) of strain JM311 through sequencing. According to sequence analysis using Brast n, the top five strains with high homology were *Bacillus pumilus* ATCC7061, *Bacillus pumilus* NBRC12092, *Bacillus zhangzhouensis* MCCC1A08372, *Bacillus leucocephala* L28, and *Bacillus spp.* MCCC1A05787, with homology of 99.86%, 99.86%, 99.78%, 99.78%, and 99.71%, respectively. Figure 35 A phylogenetic tree of the Bacillus genus associated with JM311 is shown. The inventors of this application constructed a phylogenetic tree based on the 16S rRNA sequence of JM311. Figure 35 The phylogenetic tree shown. Bootstrap values ​​above 50 are shown. The inventors of this application used the standard strain *Streptococcus intermedius* 1877 as the outgroup. JM311 is deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (NITE P-03919) in Japan.

[0120] [JM321 isolate] Figure 36 The results of chlorophyll a+b measurement are shown after JM321 isolate was co-cultured with NIES-48. Figure 36 The asterisk (*) indicates a significant difference compared to NIES-48 culture alone (p<0.05). Co-culture of NIES-48 and JM321 resulted in a chlorophyll content of 34.22 mg / L at 168 hours of culture, representing a 3.78-fold increase compared to 9.06 mg / L during culture alone.

[0121] Figure 37This application describes flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity of NIES-48 cells during solitary culture or co-culture with JM321. The inventors of this application used forward scattering (FSC-H) in flow cytometry to measure cell size. Figure 37 The asterisk (*) indicates a significant difference compared to NIES-48 culture alone (p<0.05). Compared to NIES-48 culture alone, the cell number and chlorophyll fluorescence intensity of NIES-48 co-cultured with JM321 isolates for 168 hours were 3.48-fold and 1.16-fold, respectively. Forward light scattering changed to 0.95-fold. This indicates that co-culturing NIES-48 with JM321 slightly increased the chlorophyll content per cell, promoted growth, and increased cell number.

[0122] The inventors of this application determined the almost complete 16S rRNA sequence (1483 bp) of strain JM321 through sequencing. According to sequence analysis using Brast n, the top five 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 homology of 99.85%, 99.85%, 99.76%, 99.69%, and 99.691%, respectively. Figure 38 A phylogenetic tree of the Bacillus genus associated with JM321 is shown. The inventors of this application constructed a phylogenetic tree based on the 16S rRNA gene sequence of JM321. Figure 38 The phylogenetic tree shown. Bootstrap values ​​above 50 are shown. The inventors of this application used the intermediate-type Streptococcus 1877 standard strain as an outgroup. The inventors of this application identified JM321 as Bacillus licheniformis. JM321 is deposited at the Patent Microbiology Collection Center of the National Institute of Technology and Evaluation, Japan (Accession No.: NITE P-03920).

[0123] [AF2108 isolate] Figure 39 The results of chlorophyll a+b measurement are shown after co-culturing AF2108 isolate with NIES-48. Figure 39 The asterisk (*) indicates a significant difference compared to NIES-48 culture alone (p<0.05). Co-culture of NIES-48 with AF2108 resulted in a chlorophyll content of 32.66 mg / L at 168 hours of culture, representing a 3.36-fold increase compared to 9.32 mg / L during culture alone.

[0124] Figure 40Flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity of NIES-48 cells during individual culture or co-culture with AF2108 are shown. The inventors of this application used forward scattering (FSC-H) in flow cytometry to measure cell size. Figure 40 The asterisk (*) indicates a significant difference compared to NIES-48 culture alone (p<0.05). Compared to NIES-48 culture alone, the cell number increased 2.93-fold after co-culturing NIES-48 with AF2108 isolates for 168 hours. Chlorophyll fluorescence intensity remained unchanged at 1.12-fold. Forward light scattering decreased to 0.92-fold. This indicates that co-culturing NIES-48 with AF2108 promoted growth and increased cell number.

[0125] The inventors of this application determined the almost complete 16S rRNA sequence (1394 bp) of strain AF2018 through sequencing. According to the sequence analysis using Brast n, the top 5 strains with high homology were Rhodococcus cercidiphylli C5, Rhodococcus cercidiphylli YIM65003, Rhodococcus yunnanensis YIM70056, Rhodococcus fascians ATCC 12974, and Rhodococcus fascians CF17, with homology of 100.00%, 99.58%, 99.31%, 99.16%, and 99.15%, respectively. Figure 41 A phylogenetic tree of the genus *Rhodococcus* associated with AF2108 is shown. The inventors of this application constructed a phylogenetic tree based on the 16S rRNA gene sequence of AF2108. Figure 41 The phylogenetic tree is shown. Bootstrap values ​​above 50 are shown. *Pseudomonas dioxadensis* CB1190 standard strain was used as an outgroup. The inventors of this application identified AF2108 as *Rhodococcus aureus*.

[0126] [JM202 isolate] Figure 42 The results of chlorophyll a+b measurement are shown after JM202 isolate was co-cultured with NIES-48. Figure 42 The asterisk (*) indicates a significant difference compared to NIES-48 culture alone (p<0.05). The inventors of this application, through co-culturing NIES-48 with strain JM202, achieved a chlorophyll content of 26.38 mg / L at 168 hours of culture, representing a 2.91-fold increase compared to 9.06 mg / L during the single-culture period.

[0127] Figure 43Flow cytometry measurements of cell number, cell size, and chlorophyll fluorescence intensity of NIES-48 cells during individual culture or co-culture with JM202 are shown. The inventors of this application used forward scattering (FSC-H) in flow cytometry to measure cell size. Figure 43 The asterisk (*) indicates a significant difference compared to NIES-48 culture alone (p<0.05). Compared to NIES-48 culture alone, the cell number and chlorophyll fluorescence intensity of NIES-48 co-cultured with JM202 isolates for 168 hours were 3.17-fold and 0.88-fold, respectively. Forward light scattering remained unchanged at 1.01-fold. This indicates that co-culturing NIES-48 with JM202 slightly reduced the chlorophyll content per cell but promoted growth and increased cell number.

[0128] The inventors of this application determined the almost complete 16S rRNA sequence (1458 bp) of strain JM202 through sequencing. Based on the sequence analysis using Brast n, the top five strains with high homology were *Aeromonas salmonicida* CECT894, *Aeromonas salmonicida* ATCC 33658, *Aeromonas salmonicida* NCIMB 1102, *Aeromonas salmonicida* ATCC 33658, and *Aeromonas salmonicida* subsp. *masoucida* NBRC 13784, with homology of 99.86%, 99.84%, 99.79%, 99.79%, and 99.79%, respectively. Figure 44 A phylogenetic tree of the Aeromonas genus associated with JM202 is shown. The inventors of this application constructed a phylogenetic tree based on the 16S rRNA gene sequence of JM202. Figure 44 The phylogenetic tree shown. Bootstrap values ​​above 50 are shown. The inventors of this application used the standard strain *Succinatimonas hippei* YIT 12066 as an outgroup. JM202 has been deposited at the Patent Microbiology Collection Center of the National Institute of Technology and Evaluation, Japan (Accession No.: NITE P-03835).

[0129] [Effects of the Invention] The microalgae growth-promoting microorganisms belonging to the genera Rhodococcus, Flavobacterium, Trichobacterium, Shewanella, Bacillus, or Aeromonas in this embodiment can promote the growth of microalgae.

[0130] Although the present invention has been explained using embodiments as described above, the technical scope of this embodiment is not limited to the scope described in the above embodiments, and various modifications and changes can be made within the scope of the spirit of the invention. For example, all or part of the device can be configured by functionally or physically distributing or integrating arbitrary units (elements). Furthermore, new embodiments resulting from any combination of multiple embodiments are also included in the embodiments of this embodiment. The effects of new embodiments resulting from combinations include the effects of the original embodiments.

[0131]

Claims

1. A microalgal growth promoting microorganism, characterized in that, It is a microorganism belonging to the genus Rhodococcus, Xanthobacter, Ancylobacter, Shewanella, Bacillus, or Aeromonas.

2. The microalga growth-promoting microorganism according to claim 1, wherein the microorganism is Rhodococcus cerastii of the genus Rhodococcus, Xanthobacter flavus of the genus Xanthobacter, or Ancylobacter rudongensis of the genus Ancylobacter.

3. The microalga growth-promoting microorganism according to claim 1, wherein the microorganism is Bacillus licheniformis, Bacillus pumilus, Bacillus zhangzhouensis, Bacillus australimaris, Bacillus safensis, or Peribacillus acanthi of the genus Bacillus, R. cerastii of the genus Rhodococcus, or Aeromonas salmonicida or Aeromonas piscicola of the genus Aeromonas.

4. The microalga growth-promoting microorganism according to any one of claims 1 to 3, wherein the microorganism is used for promoting the growth of a cyanobacterium, a green alga, a haptophyte, or a euglenid.

5. The microalga growth-promoting microorganism according to any one of claims 1 to 3, wherein the microorganism is used for promoting the growth of an organism belonging to the phylum Cyanobacteria, Heterokontophyta, Euglena, Cryptophyta, Haptophyta, Cercozoa, Glaucophyta, Rhodophyta, Chlorophyta, or Streptophyta.

6. The microalga growth-promoting microorganism according to any one of claims 1 to 3, wherein the microorganism is used for promoting the growth of an organism belonging to the phylum Cyanobacteria or Euglena.

7. A microalga growth-promoting agent comprising the microalga growth-promoting microorganism according to any one of claims 1 to 3.

8. A method for culturing microalgae, characterized by, More than one microorganism selected from the group consisting of microorganisms belonging to the genus Rhodococcus, Xanthobacter, Ancylobacter, Shewanella, Bacillus, or Aeromonas is co-cultured with a microalga.

9. A microorganism screening method comprising: a step of co-culturing a microorganism to be screened together with a microalga; and a step of quantifying chlorophyll contained in a culture medium after the co-culturing.

10. The microbial screening method of claim 8, wherein, The quantification step of the chlorophyll includes a step of measuring fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 680 nm to 720 nm.

11. The microorganism screening method according to claim 8, wherein the quantification step of the chlorophyll includes a step of measuring fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 683 nm in the case where the microalga is PCC7972, measuring fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 685 nm in the case where the microalga is NIES-2173, and measuring fluorescence intensity at an excitation wavelength of 488 nm and a fluorescence wavelength of 700 nm in the case where the microalga is NIES-48.

12. The microorganism screening method according to claim 8, further comprising: a step of evaluating the degree of microalga growth promotion exhibited by the microorganism to be screened using the quantification result of the chlorophyll contained in the culture medium after the co-culturing.

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