Chlorella sorojin and application thereof

Abstract

The invention discloses chlorella sorojin and application thereof, and belongs to the technical field of microalgae biological fertilizers. The Chlorella sorokiniana is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.46635, the preservation date is September 15, 2025, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, the postal code is 100101, and the Chlorella sorokiniana is classified and named as Chlorella sorokiniana. The screened chlorella sorojin can release substances capable of improving the growth quality of crops, and also has the effects of regulating soil microflora and releasing IAA (Indoleacetic Acid). The chlorella sorojin biological fertilizer can increase the nutrient content of soil, increase the oxygen content in the soil and promote the growth of beneficial microorganisms in the soil; the chemical fertilizer consumption is reduced; the crop growth quality is improved; and no pollution is caused to the environment.

Description

Technical Field

[0001] This invention relates to the field of microalgae biofertilizer technology, specifically to a type of Chlorella sorokin and its applications. Background Technology

[0002] Long-term reliance on inorganic fertilizers has led to soil acidification, organic matter loss, and salinization, severely hindering the healthy growth of chili peppers. Excessive application of traditional chemical fertilizers (especially nitrogen fertilizers) can easily cause an imbalance of essential elements such as phosphorus, potassium, and calcium in chili peppers, impeding their growth and development and reducing yield. Although organic compound fertilizers are widely used, their fixed nutrient ratios are difficult to match the stage-specific needs of chili peppers, and they still have limitations in terms of declining growth quality. Therefore, the development of new fertilizers that combine high production efficiency with environmental sustainability is urgently needed.

[0003] Microalgae biofertilizers, due to their unique biological activity, can promote soil nutrient transformation, enhance fertility, and optimize microbial community structure through metabolic activities, making them an important direction for the green transformation of agriculture. Summary of the Invention

[0004] The purpose of this invention is to provide a *Chlorella sorokinense* and its specific applications to overcome the shortcomings of the prior art.

[0005] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0006] A species of Chlorella sorokiniana, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46635, deposited on September 15, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 100101, is classified as Chlorella sorokiniana, with the designation LXL-01.

[0007] The growth characteristics of the *Chlorella sorokinica* were as follows: light conditions of 4000 Lux (light-dark cycle of 12 hours:12 hours), pH of 8.20, and culture temperature of 25℃.

[0008] The application of *Chlorella sorokinense* in the preparation of microalgae biofertilizer.

[0009] The application of *Chlorella sorokinense* in the preparation of microalgae biofertilizer that promotes pepper growth.

[0010] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0011] The *Chlorella sorokinense* strain screened in this invention can release substances that improve crop growth quality, and also regulates soil microbial communities and releases IAA (inorganic acid). *Chlorella sorokinense* bio-fertilizer can increase soil nutrient content, increase soil oxygen content, promote the growth of beneficial soil microorganisms, reduce the amount of chemical fertilizer used, improve crop growth quality, and is environmentally friendly.

[0012] Furthermore, practical experience has proven that Sorokin Chlorella bio-fertilizer can significantly improve the growth quality of chili plants before the fruiting period. Attached Figure Description

[0013] Figure 1 This is a morphological observation of the functional microalgae used in the examples.

[0014] Figure 2 This is the functional microalgae evolutionary tree used in the examples.

[0015] Figure 3 The chili pepper plants in each treatment at the end of the experiment in the example are shown.

[0016] Figure 4 The height of each treatment in the examples is shown.

[0017] Figure 5 The stem diameter for each treatment in the examples is shown.

[0018] Figure 6 The width of each processed leaf in the example is shown.

[0019] Figure 7 The leaf lengths for each treatment in the examples are shown.

[0020] Figure 8 The SPADs used in the examples are shown.

[0021] Figure 9 The root activity of each treatment plant in the example is shown. Detailed Implementation

[0022] The present invention will be further described below through specific embodiments and accompanying drawings.

[0023] Example 1:

[0024] This embodiment conducts an experiment on a method for the efficient resource utilization and treatment of biogas slurry pollution from large-scale livestock and poultry farms. The biogas slurry used in the experiment was collected from a large-scale pig farm in Hetou Village, Changle County, Weifang City, Shandong Province (119°05'E, 26°53'N).

[0025] (1) Livestock biogas slurry was collected from a large-scale pig farm in Changle County, Weifang City, Shandong Province. After preliminary filtration through four layers of gauze, 20 mL of the filtrate was taken and diluted 50 times with BG11 liquid culture medium and placed in an Erlenmeyer flask. The Erlenmeyer flask was placed in a light incubator and enriched under the conditions of 25℃, 4000 Lux light intensity, and a light-dark cycle of 12h:12h until the culture medium turned a distinct green color.

[0026] (2) Take 100 μL of the enriched culture medium from step (1) and dilute it with sterile water to a final concentration of 10 μL. 2 10 3 10 4 Take bacterial suspensions of each dilution gradient and spread them onto the surface of BG11 solid medium plates. Incubate the plates under the same conditions as above until single algal colonies grow on the plates. Select plump, uniform green single algal colonies and purify them three times using the streak plate method until two pure single algal colonies are obtained, which are preliminarily named algal strain A and algal strain B, respectively.

[0027] (3) The purified algal colonies obtained in step (2) were inoculated into BG11 liquid culture medium for expansion culture. The algal solution in the logarithmic growth phase was taken and observed under an optical microscope (Nikon Eclipse E100). It was observed that the algal cells of strain A were unicellular spherical or nearly spherical, with a diameter between 2.5 and 5.0 μm. The cell walls were smooth, possessing typical cup-shaped chloroplasts and a central protein nucleus structure, without a gelatinous sheath, and mainly existing in unicellular form. This morphological characteristic is consistent with the typical morphology of the genus *Chlorella*. Figure 1 a); Algal strain B is oval to ellipsoidal (4.0–7.0 μm × 8.0–12.0 μm), with cells enclosed in a transparent gelatinous sac. Typically, cells cluster in groups of 2–4, containing 1–2 plate-like chloroplasts and lacking a pyrenoid. Reproduction occurs through the release of spore-like structures from within the gelatinous sac. Figure 1 b)

[0028] (4) Collect the algal cells expanded from step (3) and send them to Sangon Biotech (Shanghai) Co., Ltd. for 18S rRNA gene sequence analysis. PCR amplification was performed using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The obtained PCR products were sequenced, and the sequences were compared for homology (BLAST) in the National Center for Biotechnology Information (NCBI) database. A phylogenetic tree was constructed using MEGA-X software based on the maximum likelihood method (Bootstrap = 1000). Figure 2 ).

[0029] (5) Molecular identification results showed that the 18S rRNA gene sequence of the isolated algal strain LXL-01 was 100% homologous to the reference strain of Chlorella sorokiniana, and it clustered with Chlorella sorokiniana isolate IS118 on the phylogenetic tree, forming an independent cluster.

[0030] Based on morphological observation and molecular biological identification results, the isolated algal strain LXL-01 was finally identified as *Chlorella sorokiniana*. This strain was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 46635.

[0031] Example 2:

[0032] This embodiment prepares a microalgae biofertilizer containing Chlorella sorokinense through the following steps:

[0033] (1) Collect biogas slurry from pig farms, dilute it with 50 times ultrapure water, pretreat it for 10 hours with synthetic microbial communities (including synthetic microorganisms such as Bacillus subtilis and Rhodococcus), and then disinfect it with H2O2 to obtain pretreated biogas slurry;

[0034] (2) Chlorella sorokiniana was inoculated into pretreated biogas slurry and cultured for 15 days at 25℃, 4000 Lux light (12-hour light-dark cycle), and 60 L / h aeration until the biomass reached 1.12 × 10⁻⁶. 7 cells / mL.

[0035] (3) Dilute the microalgae obtained in step 2 by a factor of 10 to a microalgae density of 1.12 × 10⁻⁶. 6 The microalgae bio-fertilizer used in the experiment was obtained by measuring cells / mL.

[0036] The amount of microalgae bio-fertilizer obtained is equivalent to 1L / acre. When applying, dilute the obtained microalgae bio-fertilizer with irrigation water and apply it once a week.

[0037] The experimental group's situation is shown in Table 1:

[0038] To verify the growth-promoting effect of the prepared microalgae bio-fertilizer on plants, a small potted plant experiment with chili peppers was conducted. The plants were continuously cultured for 49 days. Chili pepper seeds were used as experimental materials, and ordinary original soil was used as the substrate soil for the chili peppers to create the potted plant system, which served as the basic culture device for this experiment.

[0039] The experiment consisted of seven treatments: C: conventional fertilization + ultrapure water; W(B): conventional fertilization + culture medium of pretreated and microbial-treated biogas slurry; W(BS): conventional fertilization + culture medium of pretreated and microbial-treated *Chlorella vulgaris*; W: conventional fertilization + pretreated biogas slurry; W(S): conventional fertilization + culture medium of pretreated biogas slurry containing *Chlorella vulgaris*; BG11: conventional fertilization + BG11 culture medium; BG11(S): conventional fertilization + BG11 *Chlorella vulgaris* culture medium. Each treatment had eight replicates, for a total of 56 replicates, with four plants per replicate. The pepper seeds were placed in petri dishes, covered with a moistened paper towel for germination, and watered frequently with a spray bottle to maintain seed moisture. Once more than 90% of the seeds showed signs of sprouting, they were sown in seedling trays containing sufficient substrate soil and watered promptly. Apply compound fertilizer to the potting system and mix it with the substrate soil. When the seedlings have grown to 3-4 true leaves, transplant them into the potting system, with 2 seedlings per pot. When the peppers have grown to 2-4 leaves, use a pipette to take 1 mL of algae culture solution treated with bacteria and apply it to the roots for one week. The control group is treated with an equal amount of distilled water as a control. Then apply it once every 7 days.

[0040] Table 1 Experimental group settings

[0041]

[0042]

[0043] As shown in Table 2, compared with the BG11(S) group, the levels of triglycerides, soluble protein, total sugar, and IAA in *Chlorella sorokinense* cultured in the W(BS) group were significantly increased by 13.4%, 488.2%, 53.8%, and 107.2%, respectively. Compared with the BG11(S) group, the W(S) group showed a 29.2% decrease in triglycerides, while soluble protein, total sugar, and IAA increased by 2.6 times, 44.9%, and 27.1%, respectively.

[0044] Table 2. Determination of nutrient composition of Chlorella sorokinense

[0045]

[0046] After 49 days of cultivation, such as Figure 3 As shown, compared with the control group, BG11 group, BG11 group, and W(B) group, the peppers in the W(BS) group grew the best.

[0047] like Figure 3-8As shown, the W(BS) group exhibited the best plant height, stem diameter, leaf length, leaf width, and SPAD growth. Compared to the control group, the W(B) group showed changes of -14.5%, 5.7%, -0.7%, -5.2%, and 6.3%, respectively; the W(BS) group showed increases of 26.4%, 12.3%, 6.0%, 7.4%, and 12.3%; the W group showed changes of -7.27%, 4.2%, -1%, -5.9%, and 9.1%, respectively; the W(S) group showed changes of 5.48%, 4.28%, 1.8%, -3.7%, and 4.7%, respectively; the BG11 group showed changes of -9.6%, 2.2%, -0.3%, -5.2%, and 3.8%; and the BG11 group showed increases of -2.2%, 4.8%, 0.7%, -0.7%, and 8.9%, respectively.

[0048] from Figure 9 As shown in Table 3, at the end of the experiment, compared with the control group, the root activity and total root length of group W(B) increased by 52.4% and 23.8%, respectively, while the dry weight and fresh weight decreased by 6.5% and 13.0%, respectively. Group W(BS) showed increases of 320.6%, 44.8%, 73.6%, and 24.9%, respectively. Root activity, total root length, and dry weight of group W increased by 3.2 times, 9.3%, and 7.0%, respectively, while fresh weight decreased by 2.3%. Group W(S) showed increases of 2.2 times, 30.2%, 6.7%, and 33.0%, respectively. Group BG11 showed decreases of 6.5%, 18.4%, and 17.0%, respectively, while total root length increased by 36.0%. Group BG11(S) showed increases of 93.9% and 33.7%, respectively, while dry weight and fresh weight decreased by 5.6% and 14.1%, respectively.

[0049] Table 3 Characteristics of the whole chili plant

[0050]

[0051] As shown in Table 4, at the end of the experiment, compared with the control group, the chlorophyll a, chlorophyll b, chlorophyll a / b, and total chlorophyll content of leaves in group W(B) increased by 12.5%, 15.7%, 2.3%, and 13.3%, respectively; those in group W(BS) increased by 59.3%, 54.3%, 4.5%, and 58.3%, respectively; the chlorophyll a, chlorophyll b, and total chlorophyll content of group W decreased by 20.95%, 23.73%, and 21.55%, respectively; and the chlorophyll a / b increased by 4.33%. In group W(S), the chlorophyll a, chlorophyll b, and total chlorophyll content increased by 39.5%, 61.4%, and 44.4%, respectively; and the chlorophyll a / b decreased by 13.0%. In the BG11 group, the chlorophyll content decreased by 23.1%, 11.5%, 13.3%, and 20.5%, respectively. In the BG11(S) group, the chlorophyll a, chlorophyll b, and total chlorophyll content increased by 6.0%, 26.7%, and 10.6%, respectively, while the chlorophyll a / b ratio decreased by 15.7%.

[0052] Table 4 Chlorophyll content of pepper leaves

[0053]

[0054] As shown in Table 5, at the end of the experiment, compared with the control group, the average maximum photosynthetic efficiency of the W(B) group increased by 0.3% and the average absolute electron transport rate decreased by 12.9%. The average maximum photosynthetic efficiency and average absolute electron transport rate of the W(BS) group increased by 18.1% and 1.6%, respectively, while those of the W group decreased by 10.34% and 16.27%, respectively. The average absolute electron transport rate of the W(S) group decreased by 3.97% and 49.96%, respectively. The average absolute electron transport rate of the BG11 group decreased by 12.2% and 31.7%, respectively, while those of the BG11(S) group decreased by 4.0% and 26.0%, respectively.

[0055] Table 5. Photosynthetic effect of leaves in each group

[0056]

[0057] As shown in Table 6, at the end of the experiment, compared with the control group, the available nitrogen, available phosphorus, available potassium, and organic matter in the soil of the W(BS) group increased by 38.89%, 4.00 times, 73.09%, and 23.54 times, respectively; those of the W(B) group increased by 16.67%, 1.67 times, 40.63%, and 1.46 times, respectively; the available nitrogen, available phosphorus, and organic matter in the W group increased by 5.56%, 1.11 times, and 1.03 times, respectively, while available potassium decreased by 15.67%; those of the W(S) group increased by 22.22%, 4.00 times, 68.98%, and 90.08%, respectively; those of the BG11 group increased by 22.22%, 2.44 times, 7.746%, and 91.06%, respectively; and those of the BG11(S) group increased by 33.33%, 2.89 times, 58.22%, and 1.20 times, respectively.

[0058] Table 6 Soil composition of each group

[0059]

[0060] The experimental data above show that microalgae bio-fertilizer containing Sorokin Chlorella can effectively improve the growth-promoting effect on plants.

[0061] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of Chlorella sorokinensis, characterized in that, This *Chlorella sorokinense* species is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46635, deposited on September 15, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, 100101, China. It is classified and named as follows: Chlorella sorokiniana .

2. The application of *Chlorella sorokinense* as described in claim 1 in the preparation of microalgae biofertilizer.

3. The application of the Chlorella sorokinense according to claim 1 in the preparation of microalgae bio-fertilizer that promotes the growth of chili peppers.

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