Monascus microalgae GLY-1 and application thereof

Abstract

The invention discloses monolith microalgae GLY-1 and application thereof, and belongs to the technical field of microalgae biology. According to the invention, a microalga GLY-1 in the genus of monolith is separated from a fresh water river, is determined to be a new species of the genus of monolith through morphological and molecular biological identification, is named as Coelastra haikouensis. GLY-1 in taxonomy, is preserved in China Center for Type Culture Collection on March 24, 2025, and has a preservation number of CCTCC No: M2025579. According to the method, the algae fertilizer prepared by using the microalgae GLY-1 is applied when water spinach seedlings grow, so that water spinach plants can grow higher and thicker, and the yield and quality of the water spinach are remarkably improved. The microalgae GLY-1 is proved to have application potential in the aspects of promoting the growth of crops and increasing the yield and quality of the crops.

Description

Technical Field

[0001] The present invention relates to the field of microalgae biotechnology, and particularly to a microalgae of the genus Monostroma GLY-1 and its application. Background Art

[0002] Food production is closely related to the fertility level of the soil to a great extent. The level of soil fertility directly determines the growth and development of crops, nutrient absorption, and ultimately the yield and quality. In recent years, biofertilizers, as a green and efficient means of soil improvement and fertility enhancement, have received wide attention. Biofertilizers contain live or dead cells of beneficial microorganisms. After being applied to the soil system, they can quickly invade the rhizosphere of crops and convert difficult-to-obtain mineral forms into nutrients required by crops through mechanisms such as nitrogen fixation, mineralization, and improvement of phosphate solubility, thereby promoting the growth and development of crops. When applied to agricultural production, biofertilizers can significantly improve the chemical and biological characteristics of the soil, restore soil fertility, and promote crop growth. They are an environmentally friendly green fertilizer.

[0003] Algae are the most unique organisms on Earth and have potential agricultural applications such as biofertilizers and soil conditioners to improve soil fertility. Microalgae, which are tiny algae widely distributed in marine, freshwater, and terrestrial environments, have characteristics such as high photosynthetic efficiency, rich nutrition, and rapid growth. As a new type of biological resource, microalgae show great application potential in multiple fields such as food, feed, cosmetics, medicine, and energy. In the agricultural field, microalgae fertilizer, as a green and efficient biofertilizer, has achieved remarkable application effects. The core advantage of microalgae fertilizer lies in its promotion of soil fertility and crop growth. Microalgae cells are rich in nutrients such as nitrogen, phosphorus, and potassium, as well as bioactive substances such as proteins, polysaccharides, polyunsaturated fatty acids, and plant hormones, which can effectively promote crop growth, increase yield and quality. In addition, microalgae have the abilities of carbon fixation, nitrogen fixation, and phosphorus solubilization, can improve soil structure, enhance the water and fertilizer retention performance of the soil, and repair degraded soil.

[0004] In agricultural engineering, the application prospect of microalgae resources is broad. The cultivation methods of microalgae are diverse, with a short growth cycle, and there is no need to compete with traditional food crops for limited arable land. The nutrients in wastewater can be utilized during the cultivation process to achieve pollutant removal and resource recycling. For example, microalgae biofertilizer has been successfully applied to field cultivation, significantly improving the tolerance and growth performance of crops such as millet under nitrogen and phosphorus stress conditions. As a multifunctional biological resource, microalgae fertilizer has great development value in the agricultural field, and its green and efficient characteristics make it an important force in promoting the sustainable development of agriculture.

[0005] Coelastrella belongs to Chlorophyta and Trebouxiophyceae, and is a type of unicellular or colonial microalgae. Its cell wall often has complex patterns (such as ridge-like protrusions or reticular structures), and the intracellular chloroplasts are star-shaped, which is a typical taxonomic identifier for this genus. Coelastrella species show unique ecological adaptability. Most of its groups are obligately distributed in terrestrial or semi-terrestrial ecosystems (such as humus layers, karst substrates, building interfaces, and bark epiphytic microenvironments), and facultatively distributed in freshwater aquatic ecosystems. This genus has a pantropical distribution characteristic, and its ecological amplitude covers from the extreme cold zone (Arctic Circle) to tropical climate regions. It is worth noting that some germplasm resources can initiate the lipid hyperaccumulation mechanism at the end of the natural growth period or under stress conditions (including high irradiance, osmotic pressure fluctuations, and nutrient limitation conditions). A typical representative is the Coelastrella sp. D3-1 strain, which can achieve a total lipid content of 20%-44% (w / w) of the cell dry weight in a nitrogen- and phosphorus-deficient medium. This characteristic makes it one of the most competitive microalgae candidates for the production of third-generation biodiesel. In addition, many species in this genus show advantages in the diversity of secondary metabolites. Their carotenoid components include high-value-added bioactive substances such as astaxanthin, canthaxanthin, lutein, and adonixanthin, which have important development value. Therefore, in-depth development of Coelastrella strains with biological growth-promoting functions is of great significance for promoting the sustainable development of agriculture and enriching the application of microalgae resources. Summary of the Invention

[0006] The object of the present invention is to provide a Coelastrella microalgae GLY-1 and its application to solve the problems existing in the above-mentioned prior art. A new species of Coelastrella, Coelastrellahaikouensis, was isolated from a freshwater river in the present invention. The microalgae cells were broken and extracted to prepare algal fertilizer, which has application potential in promoting the growth of crops, increasing the yield and quality of crops.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a Coelastrella microalgae (Coelastrella haikouensis.) GLY-1, which was deposited with the China Center for Type Culture Collection on March 24, 2025, and the deposit number is CCTCC No: M2025579.

[0009] The present invention also provides the application of the Coelastrella microalgae GLY-1 in the preparation of algal fertilizer.

[0010] The present invention also provides an algal fertilizer, which is prepared from the extract of the microalgae GLY-1 of the genus Coelastrella.

[0011] Optionally, the preparation method of the algal fertilizer includes the following steps:

[0012] The microalgae GLY-1 of the genus Coelastrella are expanded in culture to obtain a pure microalgae liquid;

[0013] The pure microalgae liquid is centrifuged to obtain a microalgae cell mud;

[0014] The microalgae cell mud is freeze-dried to obtain an algal powder; the algal powder is dissolved and then ultrasonically disrupted, and then centrifuged to obtain a supernatant, and the supernatant is diluted to obtain the algal fertilizer.

[0015] Optionally, the conditions for the expansion culture are light of 50 μmol / (m 2 ·s), a temperature of 25 °C, and a culture time of 15 d.

[0016] Optionally, the conditions for the ultrasonic disruption are an ultrasonic power of 400 W, ice-bath disruption for 15 min, with an interval of 4 s for every 6 s of ultrasonic treatment.

[0017] The present invention also provides the application of the microalgae GLY-1 of the genus Coelastrella or the algal fertilizer in promoting the growth of crops.

[0018] The present invention also provides the application of the microalgae GLY-1 of the genus Coelastrella or the algal fertilizer in improving the yield and quality of crops.

[0019] Optionally, the crop is water spinach.

[0020] Optionally, the improvement of the yield and quality of the crop includes increasing the plant height and the thickness of the stem of the crop.

[0021] The present invention discloses the following technical effects:

[0022] A microalgae GLY-1 of the genus Coelastrella is isolated from a freshwater river. Through morphological and molecular biological identification, it is determined to be a new species of the genus Coelastrella, and its taxonomic name is Coelastrella haikouensis GLY-1. The algal fertilizer can be prepared by crushing and extracting the microalgae cells.

[0023] The algal fertilizer prepared by using the microalgae GLY-1 of the present invention is applied during the seedling stage of water spinach, which can make the water spinach plants grow taller and thicker, and significantly improve the yield and quality of water spinach. It is proved that the microalgae GLY-1 has application potential in promoting the growth of crops, increasing the yield and quality of crops. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is a streak plate isolation diagram of microalgae GLY-1;

[0026] Figure 2 It is a liquid culture diagram of microalgae GLY-1;

[0027] Figure 3 It is an optical microscopic observation diagram of microalgae GLY-1;

[0028] Figure 4 It is a scanning electron microscope diagram of microalgae GLY-1;

[0029] Figure 5 It is a transmission electron microscope diagram of microalgae GLY-1;

[0030] Figure 6 It is a phylogenetic tree constructed based on the 18S rRNA gene of microalgae GLY-1;

[0031] Figure 7 It is a physical diagram of microalgae extracts (algae fertilizers) with different concentrations;

[0032] Figure 8 It is a phenotypic picture of water spinach plants in each group. Detailed implementation manners

[0033] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or range of values, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0036] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0037] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0038] Example 1 Isolation, Purification and Identification of Microalgae

[0039] 1. Isolation and Purification of Microalgae

[0040] The original algal species were collected from the river near Guilin Yang Campus of Hainan Normal University, Meilan District, Haikou, Hainan. The original algal species were inoculated into BG11 liquid medium (HaiBo Biotechnology; product number HB8793) that had been autoclaved at 121 °C for 20 min to obtain the original algal liquid. The original algal liquid was examined under a microscope to determine that the original algal liquid contained free single-celled algae.

[0041] The plate coating method was used for the isolation and purification of microalgae. The above-mentioned original algal liquid was spread and inoculated on BG11 solid medium (agar powder content 0.8 wt%) and cultured on a light culture rack for 2 - 3 weeks. The culture conditions were light intensity 50 μmol / (m 2 ·s), temperature 25 °C. After algal colonies grew on the plate, the target single algal colony was picked out with an inoculation loop and spread and inoculated on a new BG11 solid plate for purification culture for 2 - 3 weeks with the same culture conditions. After repeating 3 - 4 times, it was examined under a microscope to confirm that purified single algal colonies were obtained, as Figure 1 shown. The microalgae obtained by purification were named GLY-1.

[0042] 2. Scale-up Culture of Microalgae

[0043] The BG11 liquid medium sterilized at 121°C for 20 min was dispensed into cell culture flasks, with 25 mL of BG11 liquid medium added to each flask. The pure algal strain after microscopic examination was transferred into the cell culture flasks for cultivation to obtain pure freshwater algal seeds. After culturing the pure algal strain in the cell culture flasks for 15 d, it was inoculated into a photobioreactor for scale-up cultivation. The cultivation conditions were: light intensity 50 μmol / (m 2 ·s), temperature 25°C, as Figure 2 shown.

[0044] 3. Identification of microalgae

[0045] 1. Morphological identification

[0046] After culturing in the photobioreactor for 15 d, 1 mL of the freshwater algal solution was taken and observed and photographed under an optical microscope (Nikon, ECLIPSE Ni-E), as Figure 3 shown.

[0047] The freshwater algae in the logarithmic growth phase were collected. 50 mL of the algal solution was centrifuged (horizontal rotor 1500×g, 10 min) to collect the cells, which were washed twice with 0.1 mol / L PBS (pH 7.4), 10 min each time. The cell precipitate was collected by centrifugation, and the precipitate was required to be at least the size of a mung bean. Glutaraldehyde-polyformaldehyde (2.0%-3.0%) (G1102-10ML, Wuhan Sevier Biotechnology Co., Ltd.) was added, and it was fixed at room temperature in the dark for 2 h and then stored at 4°C for later use. The fixed samples were rinsed 3 times with 0.1 M phosphate buffer PB (pH 7.4), 15 min each time. 1% osmium tetroxide was prepared with 0.1 M phosphate buffer PBS (pH 7.4) and fixed at room temperature in the dark for 1-2 h. The samples were rinsed 3 times with 0.1 M phosphate buffer PBS (pH 7.4), 15 min each time. Then the cells were successively placed in 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% alcohol for 15 min each time, and isopentyl acetate was added for 15 min. The samples were placed in a critical point dryer for drying. The samples were closely attached to the conductive carbon film double-sided tape and placed on the sample stage of an ion sputtering instrument for sputtering gold for about 30 s. Observation was carried out under a scanning electron microscope, as Figure 4 shown.

[0048] Collect freshwater algae in the logarithmic growth phase. Take 50 mL of the algal solution and centrifuge it (horizontal rotor, 1500×g, 10 min) to collect the cells. Wash the cells twice with 0.1 mol / L PBS (pH 7.4), 10 min each time. Centrifuge to collect the cell pellet, and the pellet should be at least the size of a mung bean. Add glutaraldehyde - paraformaldehyde (2.0% - 3.0%) (G1102 - 10ML, Wuhan Sevier Biotechnology Co., Ltd.), fix it at room temperature in the dark for 2 h, and then store it at 4 °C for later use. Take the fixed cells and rinse them 3 times with PBS, 15 min each time. Prepare 1% agarose solution by heating and dissolving it in advance. After slightly cooling, add it into an EP tube. Before the agarose solidifies, pick up the pellet with forceps and suspend it in the agarose. Fix it in the dark at room temperature for 2 h with 1% osmium tetroxide prepared with 0.1 M phosphate buffer PBS (pH 7.4). Rinse it 3 times with 0.1 M phosphate buffer PB (pH 7.4), 15 min each time. Dehydrate the sample through an ethanol gradient (30%, 50%, 70%, 80%, 95% and twice 100%, 20 min each time), and embed it with 812 embedding medium (SPI). Acetone:812 embedding medium = 1:1 (V / V), 37 °C for 2 - 4 h, acetone:812 embedding medium = 1:2 (V / V), infiltrate overnight at 37 °C, and pure 812 embedding medium at 37 °C for 5 - 8 h. Pour the pure 812 embedding medium into the embedding plate, insert the sample into the embedding plate, and then place it in an oven at 37 °C overnight. Place the embedding plate in an oven at 60 °C for polymerization for 48 h, and take out the resin block for later use. Cut the embedded block with an EM UC7 ultramicrotome (Leica), and pick up the sections with a 150 - mesh Fanghua membrane copper grid. Stain the copper grid with a saturated alcohol solution of 2% uranyl acetate in the dark for 8 min; wash it 3 times with 70% alcohol; wash it 3 times with ultrapure water; stain it with a 2.6% lead citrate solution in the dark to avoid carbon dioxide for 8 min; wash it 3 times with ultrapure water, and gently blot it with filter paper. Finally, observe and take pictures with an HT7800 transmission electron microscope (HITACHI). The transmission electron micrograph of the microalgae is as Figure 5 shown.

[0049] From the above morphological identification results, it can be seen that the microalgae GLY - 1 is irregularly spherical or ellipsoidal (diameter 8 - 15 μm), and its surface is covered with nanoscale concave - convex textures.

[0050] 2. Molecular identification

[0051] To conduct the molecular identification of freshwater algae, centrifuge the monoclonal cell algal solution at 5000 rpm for 10 min to collect it. Extract the genomic DNA of the purified microalgae GLY - 1 according to the operation instructions in the plant DNA extraction kit (OMEGA Plant DNA Kit). Detect the DNA quality and purity with Qubit4 and Nannoview. Use the extracted DNA as the template for polymerase chain reaction (PCR) to amplify the 18S rDNA sequence for phylogenetic analysis and molecular identification.

[0052] The 18S rDNA gene fragment was amplified using primers ss5 (5'-CCTGGTTGATCCTGCCAG-3′, SEQ ID NO.1) and ss3 (5'-TTGATCCTTCTGCAGGTTCA-3′, SEQ ID NO.2). The PCR reaction was carried out in a 50 μL reaction system, containing approximately 50 ng of DNA, 20 μL of polymerase chain reaction mixture (TaKaRa TaqTM Version 2.0 plus dye), 2 μL each of the upstream and downstream primers (10 μmol / L), and ddH2O. The reaction program was: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 58 °C for 45 s, extension at 72 °C for 1 min, for 30 cycles; final extension at 72 °C for 8 min. The PCR products were detected by 1% agarose gel electrophoresis and sequenced.

[0053] The DNA sequence obtained by amplification is shown as SEQ ID NO.3:

[0054]

[0055] After obtaining the DNA sequence, use the gene database in NCBI (National Center for Biotechnology Information) for Blast alignment to obtain the sequence with the highest similarity to the measured DNA and its related biological species information. According to the obtained related biological species information, use MEGA software to construct a phylogenetic tree.

[0056] From the constructed phylogenetic tree ( Figure 6 ), it can be seen that the microalgae GLY-1 has the closest genetic relationship with two sequences (AB037087.1, LC477068.1) of Pseudodidymocystis planctonica. However, the characteristics such as cell size, morphology, and its community structure of GLY-1 are significantly inconsistent with those of Pseudodidymocystis planctonica with the closest genetic relationship. Instead, it is more similar to Coelastrella sp. KGU-D002 with a relatively closer genetic relationship. Therefore, the microalgae GLY-1 was identified as a new species and named Coelastrella haikouensis GLY-1. This algal strain was deposited in the China Center for Type Culture Collection on March 24, 2025. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC No: M2025579.

[0057] Example 2 Preparation of algal fertilizer

[0058] Inoculate microalgae GLY-1 into a photobioreactor and culture it in BG11 medium for 15 days under the conditions of light intensity 50 μmol / (m 2 ·s) and temperature 25 °C to obtain a pure freshwater algal solution with good growth conditions.

[0059] Centrifuge the algal solution with good growth state (10 min, 4000 r·min -1 ), take the lower-layer algal cell precipitate, resuspend the algal cell precipitate with distilled water and centrifuge again (10 min, 4000 r·min -1 ). Repeat 2 - 3 times to obtain pure algal cell mud (algal mud).

[0060] Freeze-dry the pure algal mud in a freeze dryer for 48 h to make algal powder. Dissolve 1 g of dry algal powder in 50 mL of distilled water, put it into an ultrasonic crusher, and perform ultrasonic treatment under the condition of ultrasonic power 400 W. Ice-bath crush for 15 min (ultrasonic for 6 s, interval for 4 s). Shake the crushed solution for 1 h, and then centrifuge (15 min, 8000 r·min -1)。The supernatant after centrifugation is the mother liquor of microalgae extract, which is the mother liquor of algal fertilizer, and its concentration is 0.02 g·mL -1 。Dilute the microalgae extract with deionized water to different concentrations (100, 200 mg·L -1 ), and store it at 4 °C for later use ( Figure 7 )。

[0061] Example 3 Growth-promoting effect on crops

[0062] Select water spinach seeds of the same size and plumpness, soak them in 0.5% NaClO for 30 min for disinfection, and rinse them 3 times with sterile water to wash off the disinfectant on the seed surface. Subsequently, under the condition of 25 °C in the dark, soak the seeds in distilled water and incubate them in a petri dish for 2 d. Select seeds with consistent growth and put them into a germination tray containing the same substrate.

[0063] When the water spinach grows 2 - 3 cm seedlings, add different concentrations of microalgae extract (100, 200 mg·L -1 ) in groups. Use distilled water added as a control. Set 4 replicates for each treatment. Pour the microalgae extract or distilled water once every 3 days, 25 mL each time. All water spinach plants are cultured under the conditions of light intensity 100 μmol / (m 2 ·s), temperature 25 °C, and photoperiod of 12 h:12 h. After 27 d, the experiment ends, harvest all plants and conduct observation and measurement.

[0064] The results are as Figure 8 shown. The plant height and stem thickness of the water spinach plants treated with this algal fertilizer are significantly higher than those of the water spinach plants without applying this algal fertilizer, and the effect of the experimental group applying the algal fertilizer at a concentration of 200 mg·L -1 is more obvious. It shows that the algal fertilizer prepared from microalgae GLY-1 has application potential in promoting the growth of crops, increasing the yield and quality of crops.

[0065] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A kind of Coelastrella haikouensis microalgae GLY-1, characterized in that, The microalgae GLY-1 of the genus Monostroma was deposited at the China Center for Type Culture Collection on March 24, 2025, with the deposit number CCTCC No: M2025579.

2. Use of the microalgae GLY-1 of the genus Monostroma according to claim 1 in the preparation of algal fertilizer.

3. An algal fertilizer, characterized in that, Obtained by preparing an extract of the microalgae GLY-1 of the genus Monostroma according to claim 1.

4. The algal fertilizer according to claim 3, wherein The preparation method of the algal fertilizer comprises the following steps: The microalgae GLY-1 of the genus Monostroma is subjected to enlarged culture to obtain a pure microalgae liquid; The pure microalgae liquid is centrifuged to obtain microalgae cell mud; The microalgae cell mud is freeze-dried to obtain algal powder; the algal powder is dissolved and then ultrasonically disrupted, and then centrifuged to obtain a supernatant, and the supernatant is diluted to obtain the algal fertilizer.

5. The algal fertilizer according to claim 4, wherein, The conditions for the enlarged culture are light of 50 μmol / (m 2 ·s), a temperature of 25 °C, and culturing for 15 days.

6. The algal fertilizer according to claim 4, wherein The conditions for the ultrasonic disruption are an ultrasonic power of 400 W and ice-bath disruption for 15 min, with an interval of 4 s for every 6 s of ultrasonic treatment.

7. Use of the microalgae GLY-1 of the genus Monostroma according to claim 1 or the algal fertilizer according to any one of claims 3 to 6 in promoting crop growth.

8. Use of the microalgae GLY-1 of the genus Monostroma according to claim 1 or the algal fertilizer according to any one of claims 3 to 6 in improving crop yield and quality.

9. The application according to claim 7 or 8, characterized in that, The crop is water spinach.

10. The application according to claim 8, wherein The improvement of crop yield and quality includes increasing the plant height and the thickness of the stem of the crop.

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