Single-cell green alga and application thereof

Abstract

The invention discloses a single-cell green alga and application thereof. The name of the single-cell green alga is Coelastrus sp. BS01, the preservation number is CCTCC NO: M 20251671, the preservation date is July 22, 2025, and the preservation unit is China Center for Type Culture Collection in Wuhan University, Wuhan, China. The single-cell green alga can grow in a selenium-polluted water body with relatively high concentration, can efficiently remove selenium in the water body and is enriched in alga cells, so that the single-cell green alga has the application potential of enriching and converting selenium.

Description

Technical Field

[0001] This invention belongs to the fields of microbiology and biological treatment of pollutants, and specifically relates to a single-celled green algae and its applications. Background Technology

[0002] Selenium is an important component of more than 30 selenoproteins and selenoenases, playing a vital role in antioxidation, anti-cancer activity, and enhancing human immunity. Selenium deficiency in the human body can easily lead to premature aging, heart failure, Keshan disease, and Kashin-Beck disease, while excessive selenium intake can cause poisoning and even death. Currently, at least 1 billion people worldwide are selenium deficient, and approximately 500 million people in China have insufficient selenium intake. However, inorganic selenium compounds have a narrow safe range of beneficial concentrations for organisms and are highly toxic, while organic selenium has higher safety and bioavailability. Consuming selenium-enriched biological products is considered the safest and most effective way to supplement selenium.

[0003] Some algae can absorb inorganic selenides in water during their growth and convert them into organic selenium. Compared to traditional chemical or physical treatment techniques, microalgae removal / conversion of selenium has advantages such as low cost, sustainability, and eco-friendliness. However, research on the toxic effects of different microalgae on selenium and their removal and conversion of selenium at different concentrations remains limited. Obtaining algal strains that can tolerate high concentrations of selenium and remove it remains a research hotspot. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a single-celled green algae that can tolerate high concentrations of selenium.

[0005] Another object of the present invention is to provide applications of the above-mentioned single-celled green algae.

[0006] The objective of this invention is achieved through the following technical solution: a single-celled green alga, named Coelastrella sp. BS01, with accession number CCTCC NO: M 20251671, accession date July 22, 2025, and deposited at the China Center for Type Culture Collection located at Wuhan University, Wuhan, China.

[0007] The application of the above-mentioned single-celled green algae in removing selenium from water or enriching and converting selenium includes the following steps: adding the above-mentioned single-celled green algae strain or algal solution to selenium-polluted water for cultivation.

[0008] The algal solution is an algal solution cultured to the logarithmic growth phase; preferably, it is obtained by the following steps: inoculating the above-mentioned single-celled green algae species into a culture medium and culturing it to the logarithmic growth phase or the stationary phase.

[0009] The culture is preferably carried out at 25-30℃, light intensity of 9000-11000 lux, and light-dark time of 14-18h:6-10h; more preferably, it is carried out at 28±0.5℃, light intensity of 10000±1000 lux, and light-dark time of 16h:8h.

[0010] The preferred culture medium is a liquid culture medium obtained by adding glucose as a carbon source to TAP medium, which is simply referred to as TAP+C medium.

[0011] The composition of TAP+C medium is as follows: 2.42 g / L tris(hydroxymethyl)aminomethane, 25 mL / L TAP salt solution, 1 mL / L phosphate solution, 1 mL / L trace element solution, 1 mL / L CH3COOH, 10 g / L glucose, and water as the solvent.

[0012] The composition of the TAP salt solution is as follows: NH4Cl 15g / L, MgSO4·7H2O 4g / L, CaCl2·2H2O 2g / L, with water as the solvent.

[0013] The phosphate solution has the following composition: 28.8 g / 100 mL K₂HPO₄, 14.4 g / 100 mL KH₂PO₄, and water as the solvent.

[0014] The composition of the trace element solution is as follows: Na₂EDTA·2H₂O 5.00g / 100mL, ZnSO₄·7H₂O 2.2g / 100mL, H₃BO₃ 1.14g / 100mL, MnCl₂·4H₂O 0.5g / 100mL, FeSO₄·7H₂O 0.5g / 100mL, CoCl₂·6H₂O 0.16g / 100mL, CuSO₄·5H₂O 0.16g / 100mL, (NH₄)₆Mo₇O 24 ·4H2O 0.11g / 100mL, solvent is water.

[0015] In this invention, the water in the culture medium is preferably deionized water or purified water.

[0016] The selenium in the selenium-polluted water is inorganic selenium; preferably, it is derived from compounds of tetravalent selenium, such as sodium selenite.

[0017] The selenium concentration in the selenium-polluted water body is below 160 mg / L; preferably below 40 mg / L; more preferably 10–40 mg / L.

[0018] The amount of algal seed or algal solution added is based on the OD of algal cells in the water. 680 Calculated as 0.1 to 0.2; more preferably based on the OD of algal strains in the water. 680 Calculated as 0.1.

[0019] The present invention has the following advantages and effects compared with the prior art:

[0020] This invention discovers that a single-celled green algae, Coelastrella sp. BS01, can tolerate high concentrations of selenium and can efficiently remove selenium from water and accumulate it within the algal cells. Therefore, Coelastrella sp. BS01 has the potential to enrich and transform selenium. Attached Figure Description

[0021] Figure 1 These are experimental photographs of four single-celled green algae, BS01, HH01, HS02, and GX03, cultured in TAP+C medium containing 160 mg / L selenium on day 4.

[0022] Figure 2 These are 400x microscopic images of four single-celled green algae species, BS01, HH01, HS02, and GX03, cultured in TAP+C medium containing 160 mg / L selenium on day 4.

[0023] Figure 3 These are experimental photographs of BS01 cultured in TAP+C medium containing different concentrations of selenium for 0, 2, 4, 6, and 8 days.

[0024] Figure 4 The OD values ​​of BS01 cultured in TAP+C medium containing different concentrations of selenium for 0, 2, 4, 6, and 8 days are... 680 Result image.

[0025] Figure 5 The graph shows the chlorophyll content of BS01 cultured in TAP+C medium containing different concentrations of selenium for 0, 4, and 8 days. Among them, (a) is the chlorophyll a content, (b) is the chlorophyll b content, and (c) is the carotenoid content.

[0026] Figure 6 The graph shows the changes in two chlorophyll fluorescence parameters of BS01 after culturing in TAP+C medium containing different concentrations of selenium for 0, 4, and 8 days; where (a) represents Fv / Fm and (b) represents Pi_abs.

[0027] Figure 7 This is a graph showing the selenium removal effect of BS01 on day 8 of culture in TAP+C medium containing different concentrations of selenium.

[0028] Figure 8 This is a morphological image of BS01; from left to right are a photograph of a TAP-agar solid plate and a photograph under a 400x optical microscope. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0030] The composition of TAP liquid culture medium is as follows: 2.42 g / L tris(hydroxymethyl)aminomethane, 25 mL / L TAP salt solution, 1 mL / L phosphate solution, 1 mL / L trace element solution, 1 mL / L CH3COOH, and deionized water as the solvent. Solid culture medium is simply liquid culture medium with 15-20 g / L agar added.

[0031] The composition of TAP+C medium is as follows: 2.42 g / L tris(hydroxymethyl)aminomethane, 25 mL / L TAP salt solution, 1 mL / L phosphate solution, 1 mL / L trace element solution, 1 mL / L CH3COOH, 10 g / L glucose, and deionized water as the solvent.

[0032] The composition of the TAP salt solution is as follows: NH4Cl 15g / L, MgSO4·7H2O 4g / L, CaCl2·2H2O 2g / L, and deionized water as the solvent.

[0033] The phosphate solution has the following composition: 28.8 g / 100 mL K₂HPO₄, 14.4 g / 100 mL KH₂PO₄, and deionized water as the solvent.

[0034] The composition of the trace element solution is as follows: Na₂EDTA·2H₂O 5.00g / 100mL, ZnSO₄·7H₂O 2.2g / 100mL, H₃BO₃ 1.14g / 100mL, MnCl₂·4H₂O 0.5g / 100mL, FeSO₄·7H₂O 0.5g / 100mL, CoCl₂·6H₂O 0.16g / 100mL, CuSO₄·5H₂O 0.16g / 100mL, (NH₄)₆Mo₇O 24 ·4H2O 0.11g / 100mL, solvent is deionized water.

[0035] Chlorella sp. HH01 and Chlorella sp. HS02 have been published in the literature “He HZ, et al. Treatment of non-sterile biogas slurry from pig farm using microalgae isolated from activated sludge of sewage plants. Microbiology Spectrum, 2025, DOI 10.1128 / spectrum.00688-25”.

[0036] The accession number for *Coelastrum* sp. GX03 is CCTCC NO: M20241314, the accession date is June 20, 2024, and the depositary institution is the China Center for Type Culture Collection, Wuhan University, Wuhan, China. This algal strain has been disclosed in Chinese invention patent CN118685274B-A microalga capable of tolerating high concentrations of carbon dioxide and its applications.

[0037] Example 1: Microalgae Isolation and Purification

[0038] Algal isolation and purification: Observable green microalgae were scraped from the surface layer of soil in Baishuizhai, Zengcheng District, Guangzhou City, Guangdong Province. 20 mL of TAP liquid medium was added, and the mixture was pre-cultured in a light incubator at 28.5℃, 4000 lux light intensity, and a light-dark ratio of 12 h:12 h. After 10 days of incubation, the mixture was shaken well, and 0.1 mL of the culture solution was spread onto five 9 cm diameter petri dishes containing sterile TAP-agar solid medium. These dishes were then incubated upside down in the incubator under the same conditions. Once visible algae appeared, they were carefully removed, re-plated, and examined under a microscope. This process was repeated multiple times to obtain monocultures of microalgae. Finally, the algae were treated with a combination of five antibiotics (chloramphenicol, gentamicin, kanamycin, penicillin hydrochloride, and streptomycin) and actinomycin ketone. The specific steps were as follows: Before use, stock solutions of all six antibiotics were prepared and filtered through a sterile disposable 0.22 μm microporous membrane (Millipore). These solutions were then added to sterile water to achieve a concentration of 10 μg / mL for each antibiotic, resulting in sterile water containing the antibiotics. A certain amount of algae was carefully selected from the single-agar culture plates isolated in the above steps and added to the aforementioned sterile water containing the antibiotics. After thorough shaking, centrifugation was performed, and the supernatant was discarded. This step was repeated twice. Then, sterile TAP liquid medium was added, and the algal cells were washed and centrifuged twice. The algal cells were then inoculated onto TAP solid medium containing the six antibiotics (each antibiotic concentration was 0.1 μg / mL) and cultured for 6 days. Finally, streaks were applied to 9 cm diameter planes on TAP solid agar, PDA, and NA media, and the cells were cultured for 2 days to check for bacterial contamination. If bacteria were present, the purification steps were repeated until sterility was achieved.

[0039] Experiment Example 2: Screening for microalgae tolerant to high concentrations of sodium selenite

[0040] Chlorella sp. HH01, Chlorella sp. HS02, Coelastrum sp. GX03, and the algal strain BS01 isolated and purified from Example 1 were inoculated into a medium containing a high concentration of sodium selenite to screen for microalgae that can tolerate high concentrations of selenium. This experiment involved adding sodium selenite to TAP+C liquid medium to simulate selenium-containing wastewater, followed by the addition of algal strains for cultivation. The specific steps are as follows:

[0041] (1) The TAP+C liquid culture medium was autoclaved at 121℃ for 16 minutes. After cooling, 20 mL was poured into 50 mL glass Erlenmeyer flasks, and 12 flasks were divided into 4 experimental groups, with 3 replicates for each group.

[0042] (2) Add sodium selenite stock solution to the sterilized TAP+C liquid medium of the experimental group to make the final selenium concentration of the medium 160 mg / L. Inoculate with algal species BS01, HH01, HS02, and GX03 respectively, setting the initial algal concentration to OD. 680 =0.1, incubated at an incubator temperature of 28.0±0.5℃, light intensity of 10000±1000 lux, day-night ratio of 16h:8h, and shaken 2-3 times a day.

[0043] (3) Samples were taken on the 4th day of culture to measure physiological indicators. Figure 1 The photograph shows the experiment on day 4. The culture media of HH01 and HSO2 appeared orange and turbid, presumably due to the formation of nano-selenium caused by high selenium stress. OD values ​​for each experimental group. 680 The chlorophyll fluorescence parameters are shown in Table 1. The results show that although the OD value of HH01 was significantly higher than other experimental groups, its chlorophyll fluorescence parameter was approximately 0, indicating that photosystem II was severely inhibited. The OD value of BS01 was second only to HH01, and its Fv / Fm and Pi_abs were significantly higher than those of HH01 and HSO2, indicating that BS01 still retained some physiological capacity under high selenium stress. Further observation under a 400x microscope (…) Figure 2 At the same multiple, BS showed significantly better cell number and cell size than other algal strains, indicating that BS01 has a better ability to tolerate high concentrations of selenium than HH01, HS02, and GX03.

[0044] Table 1. Physiological indicators of BS01, HH01, HS02, and GX03 under high selenium treatment

[0045] Note: Different letters in the same column indicate significant differences in the relevant parameter values ​​of different algae (p<0.05); Fv / Fm represents the maximum photochemical quantum yield of PSII, and Pi_abs represents the performance index of PII.

[0046] Example 3: Evaluation of the toxicity of sodium selenite to this algae

[0047] The growth of BS01 in sodium selenite-containing medium was evaluated. This experiment involved adding sodium selenite to TAP+C liquid medium to simulate selenium-containing wastewater, followed by the addition of algal inoculum for cultivation. The specific steps are as follows:

[0048] (1) The TAP+C liquid culture medium was autoclaved at 121℃ for 16 minutes. After cooling, 200 mL was poured into 500 mL glass Erlenmeyer flasks, and 12 flasks were divided into 1 control group and 3 experimental groups, with 3 replicates for each group.

[0049] (2) Sodium selenite stock solution was added to the sterilized TAP+C liquid medium in the experimental group to achieve final selenium concentrations of 10, 40, and 160 mg / L, respectively. The BS01 algal strain was inoculated, with an initial algal concentration of OD0. 680 =0.1, incubated at an incubator temperature of 28.0±0.5℃, light intensity of 10000±1000 lux, day-night ratio of 16h:8h, and shaken 2-3 times a day; at the same time, a control group without sodium selenite stock solution was set up.

[0050] (3) Samples were taken on days 0, 2, 4, 6 and 8 of the experiment to determine the growth index of BS01. Figure 3 Photos of the experimental process. OD of BS01. 680 The results are as follows Figure 4 The results show that a selenium concentration of 10 mg / L has no significant effect on BS01 cell density; however, as the selenium concentration increases, the BS01 cell density decreases, and BS01 growth is inhibited. On day 8 of culture, the OD values ​​of each experimental group... 680 Compared with the control group, the chlorophyll content decreased by 4.77%, 17.3%, and 41.5%, respectively. Following the method described in the reference (Lichtenthaler, H. and Wellburn, A. (1983) Determinations of Total Carotenoids and Chlorophylls a and b of Leaf Extracts in Different Solvents. Biochemical Society Transactions, 603, 591-592.), the chlorophyll content of BS01 under different selenium treatments was determined, and the contents of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Cars) were measured as follows: Figure 5 As shown, on day 4 of culture, chlorophyll content decreased with increasing selenium concentration; on day 8, treatments with 10 mg / L and 40 mg / L selenium significantly increased the accumulation of chlorophyll a and chlorophyll b in BS01, with increases ranging from 37.5% to 48.8%. Treatment with 160 mg / L selenium significantly decreased chlorophyll content. The Fv / Fm and Pi_abs results for BS01 on days 4 and 8 of culture are shown below. Figure 6 As shown, with increasing culture time, the chlorophyll fluorescence parameters of the experimental group were significantly lower than those of the control group, and the performance of photosystem II was inhibited.

[0051] (4) On day 8 of the experiment, the algal solutions of the control group and the experimental group were freeze-dried into algal powder samples, and the biomass yield and lipid yield of BS01 were determined. The results of the analysis of biomass yield and lipid yield of BS01 under different selenium treatments are shown in Table 2. With the increase of selenium concentration, the biomass yield and lipid yield of BS01 decreased, and the highest concentration of 160 mg / L selenium treatment decreased by 24.6% and 30.4% respectively compared with the control.

[0052] Table 2. Biomass and lipid production capacity of BS01 under different treatments.

[0053]

[0054] Note: Different letters in the same column indicate significant differences (p<0.05), and the same applies below.

[0055] In summary, appropriate concentrations of selenium (10–40 mg / L) promoted the accumulation of chlorophyll a and chlorophyll b in BS01, with an increase of up to 48.8%. However, the addition of selenium significantly affected photosystem II in BS01, with both Fv / Fm and Pi_abs showing significant decreases. As the selenium concentration increased, the biomass and lipid yield of BS01 decreased, but the decrease was only 24.6% and 30.4% at the highest concentration, indicating that BS01 still possesses a strong bioenergy production capacity under high selenium concentration treatment and has the potential to tolerate even higher selenium concentrations. Therefore, it has promising application prospects.

[0056] Example 4: Evaluation of the sodium selenite removal capacity of this algae

[0057] This study evaluated the ability of the microalga BS01, isolated and purified in our laboratory, to remove and transform inorganic selenium in a simulated sodium selenite-containing medium. The experiment involved adding sodium selenite to TAP+C liquid medium to simulate selenium-polluted water, followed by the addition of the algal strain for cultivation.

[0058] (1) The TAP+C liquid culture medium was autoclaved at 121℃ for 16 minutes. After cooling, 200 mL was poured into 500 mL glass Erlenmeyer flasks, and 12 flasks were divided into 1 control group and 3 experimental groups, with 3 replicates for each group.

[0059] (2) Sodium selenite stock solution was added to the sterilized TAP+C liquid medium in the experimental group to achieve final selenium concentrations of 10, 40, and 160 mg / L, respectively. The BS01 algal strain was inoculated, with an initial algal concentration of OD. 680 =0.1, incubated at an incubator temperature of 28.0±0.5℃, light intensity of 10000±1000 lux, day-night ratio of 16h:8h, and shaken 2-3 times a day; at the same time, a control group without sodium selenite stock solution was set up.

[0060] (3) Samples were taken on day 8 of the experiment, and the concentration of selenium in the culture medium was determined by inductively coupled plasma mass spectrometry according to HJ 700-2014. The removal effect of BS01 on sodium selenite on day 8 was as follows: Figure 7 As shown in the figure, BS01 exhibits optimal selenium removal at a concentration of 40 mg / L, removing 89.1% of the selenium in the aquatic environment. Conversely, concentrations of 10 mg / L and 160 mg / L show significantly lower removal rates than the 40 mg / L treatment, with removal rates of 65.3% and 41.7%, respectively. Therefore, the optimal concentration for selenium removal / conversion is below 40 mg / L, more preferably 10–40 mg / L. Furthermore, this algae may have the potential to convert selenium into organic selenium for the production of organic selenium supplements. The algal culture was collected after 8 days of culture, freeze-dried, and then digested to determine the total intracellular selenium content of BS01. The results are shown in Table 2. The 40 mg / L selenium treatment resulted in the highest selenium content and conversion rate in the algal powder, significantly outperforming other treatments.

[0061] Table 3. Enrichment capacity of BS01 under different selenium treatments

[0062]

[0063] Example 5: Algal Strain Identification

[0064] Through the above experiments, a microalga BS01 was selected that can tolerate high concentrations of selenium and has a strong enrichment and transformation capacity. The morphology of microalga BS01 is as follows: Figure 8 As shown, this is a single-celled green algae that can grow rapidly in TAP+C medium.

[0065] Molecular identification: PCR amplification was performed using the ITS primer pair shown below, followed by BLAST alignment using GenBank. The ITS results showed a 99.54% similarity to the known algal strain *Coelastrella yingshanensis* (Table 4), indicating that the algae belongs to the genus *Coelastrella*. Therefore, it was named *Coelastrella sp. BS01*, with accession number CCTCCNO:M 20251671, accession date July 22, 2025, and deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China.

[0066] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3';

[0067] ITS4: 5'-TCCTCCGCTTATTGATATGC3'.

[0068] ITS sequence:

[0069] .

[0070] Table 4. Blast analysis results of ITS of the isolates

[0071]

[0072] The preferred embodiments of this invention have been described in detail, but it should be understood that the technical solutions of this invention are not limited to the specific implementation details described above. Under the guidance of the technical concept of this invention, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the protection scope of this invention.

Claims

1. A single-celled green algae, characterized by: The single-celled green algae is named Coelastrella sp. BS01, with accession number CCTCC NO: M 20251671, accession date July 22, 2025, and depositary institution China Center for Type Culture Collection located at Wuhan University, Wuhan, China.

2. The application of the single-celled green algae as described in claim 1 in the removal or enrichment of selenium in water.

3. The application according to claim 2, characterized in that... The method includes the following steps: adding the single-celled green algae strain or algal solution as described in claim 1 to a selenium-polluted water body for cultivation.

4. The application according to claim 3, characterized in that: The algal solution is obtained by the following steps: inoculating the single-celled green algae strain described in claim 1 into a culture medium and culturing it to the logarithmic growth phase or the stationary phase.

5. The application according to claim 4, characterized in that: The culture was carried out at 25–30°C, with a light intensity of 9000–11000 lux and a light-dark period of 14–18 h: 6–10 h.

6. The application according to claim 5, characterized in that: The culture medium is TAP+C medium; The composition of TAP+C medium is as follows: 2.42 g / L tris(hydroxymethyl)aminomethane, 25 mL / L TAP salt solution, 1 mL / L phosphate solution, 1 mL / L trace element solution, 1 mL / L CH3COOH, 10 g / L glucose, and water as the solvent; The composition of the TAP salt solution is as follows: NH4Cl 15g / L, MgSO4·7H2O 4g / L, CaCl2·2H2O 2g / L, with water as the solvent; The phosphate solution has the following composition: K₂HPO₄ 28.8 g / 100 mL, KH₂PO₄ 14.4 g / 100 mL, and water as the solvent; The composition of the trace element solution is as follows: Na₂EDTA·2H₂O 5.00g / 100mL, ZnSO₄·7H₂O 2.2g / 100mL, H₃BO₃ 1.14g / 100mL, MnCl₂·4H₂O 0.5g / 100mL, FeSO₄·7H₂O 0.5g / 100mL, CoCl₂·6H₂O 0.16g / 100mL, CuSO₄·5H₂O 0.16g / 100mL, (NH₄)₆Mo₇O 24 ·4H2O 0.11g / 100mL, solvent is water.

7. The application according to claim 3, characterized in that: The selenium in the selenium-polluted water is inorganic selenium.

8. The application according to claim 7, characterized in that: The selenium in the aforementioned selenium-polluted water body originates from sodium selenite.

9. The application according to any one of claims 3 to 8, characterized in that: The selenium concentration in the selenium-polluted water body is below 160 mg / L; The amount of algal seed or algal solution added is based on the OD of algal cells in the water. 680 Calculated for values ​​between 0.1 and 0.2.

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