Salt-tolerant monostar algae KM2-2 and its applications

Abstract

This disclosure relates to the field of microbial technology, providing a salt- and alkali-tolerant *Coelastrella saipanensis* KM2-2, which was deposited at the China Center for Type Culture Collection on July 31, 2024, with accession number CCTCC NO: M 20241700. This disclosure also provides applications of the aforementioned *Coelastrella saipanensis* KM2-2. The *Coelastrella saipanensis* KM2-2 provided by this disclosure exhibits good salt and alkali tolerance, enabling it to grow and be cultured in saline- and alkali environments, and thus improving these environments, thereby achieving comprehensive utilization and management of saline- and alkali-tolerant environments.

Description

Technical Field

[0001] This disclosure relates to the field of microbial technology, for example to a salt-tolerant monostellatium KM2-2 and its applications. Background Technology

[0002] Currently, land and water salinization has become a global problem. Therefore, the comprehensive utilization and management of saline-alkali environments is not only a necessary measure to promote economic development and ecological restoration, but also an important way to ensure national food security.

[0003] In recent years, the use of saline-alkali water for aquaculture of shrimp, tilapia, and other aquatic animals has yielded significant improvements in economic, social, and ecological benefits. This demonstrates that saline-alkali water aquaculture is not only an important way to increase the supply of aquatic products and arable land resources, but also a crucial measure for implementing the "Greater Food Concept," playing a vital role in expanding the development space of fisheries and ensuring food security. However, there are no reports on the use of saline-alkali environments for microalgae cultivation.

[0004] Microalgae are tiny, single-celled algae that can not only cooperate with other organisms in the soil to improve soil structure, but also modify soil pH and increase soil permeability through secretions, thereby improving land water use efficiency and promoting plant growth. Overall, microalgae can leverage their growth advantages in the soil to improve the soil environment, promote plant growth, and lay a solid foundation for comprehensive land improvement. However, the high salinity and alkalinity of saline-alkali environments are unfavorable for microalgae growth, thus limiting their application in the comprehensive utilization and management of saline-alkali environments.

[0005] In conclusion, there is an urgent need for a microalgae with good salt and alkali tolerance, capable of growing and cultivating in saline-alkali environments, thereby enabling comprehensive utilization and management of saline-alkali environments and allowing microalgae management technology to play a greater role in agricultural and environmental protection fields. Summary of the Invention

[0006] The purpose of this disclosure is to overcome the shortcomings of the prior art and provide a salt- and alkali-tolerant algae KM2-2 and its application. This algae KM2-2 has good salt and alkali tolerance, can be grown and cultivated in saline-alkali environments, and can improve saline-alkali environments, thereby realizing the comprehensive utilization and comprehensive management of saline-alkali environments.

[0007] The above objective is achieved through the following technical solution:

[0008] On the one hand, a salt-tolerant single-star algae (Coelastrella saipanensis) KM2-2 is provided. This single-star algae KM2-2 was deposited at the China Center for Type Culture Collection on July 31, 2024, with accession number CCTCC NO: M20241700.

[0009] It is worth noting that the single-star algae KM2-2 disclosed herein has good salt and alkali tolerance.

[0010] On the other hand, we provide an application of the monostar algae KM2-2 as described in any of the above embodiments in growth and cultivation using a saline-alkali environment.

[0011] In some embodiments, the saline-alkali environment includes at least one of saline-alkali soil and saline-alkali water.

[0012] In some examples, the salt concentration of the saline-alkali environment is 10–30 g / L.

[0013] In some examples, the pH of the saline-alkali environment is greater than 7 and less than or equal to 9.5.

[0014] In some embodiments, the growth and culture conditions include: a light-dark cycle of 8–16 h / 8–16 h and a light intensity of 30–70 μmol·m⁻². -2 ·s -1 The incubation temperature is 24–26℃, and the incubation speed is 150–180 rpm.

[0015] In some examples, the light-dark cycle is 12h / 12h.

[0016] In some embodiments, the growth culture includes at least one of fasciotrophic growth and autotrophic growth.

[0017] It should be noted that, since the single-star algae KM2-2 provided in this disclosure has good salt and alkali tolerance, it can be grown and cultivated in a saline-alkali environment, thus realizing the comprehensive utilization of the saline-alkali environment.

[0018] On another aspect, we provide an application of the monostar algae KM2-2 as described in any of the above embodiments in improving saline-alkali environments.

[0019] In some embodiments, the saline-alkali environment includes at least one of saline-alkali soil and saline-alkali water.

[0020] In some examples, the salt concentration of the saline-alkali environment is 10–30 g / L.

[0021] In some examples, the pH of the saline-alkali environment is greater than 7 and less than or equal to 9.5.

[0022] It should be noted that microalgae can not only cooperate with other organisms in the soil to improve soil structure, but also improve soil pH and increase soil permeability through secretions, thereby improving land water use efficiency and promoting plant growth. On this basis, since the single-star algae KM2-2 provided in this disclosure has good salt and alkali tolerance, it can grow and be cultivated in saline-alkali environments and improve saline-alkali environments, thereby achieving comprehensive management of saline-alkali environments.

[0023] In another aspect, the application of the monostar algae KM2-2 as described in any of the above embodiments is provided in the development of genes for salt and / or alkali tolerance.

[0024] In some embodiments, when the single-star algae KM2-2 is used only for the development of genes for salt tolerance, the salt concentration for salt tolerance is 10-50 g / L, for example, 10-30 g / L.

[0025] In other embodiments, when the single-star algae KM2-2 is used only for gene development targeting alkali tolerance, the alkali tolerance is a pH greater than 7 and less than or equal to 9.5.

[0026] In some other embodiments, when the single-star algae KM2-2 is used in the development of genes for salt and alkali tolerance, the salt concentration for salt tolerance is 10-30 g / L; and the pH for alkali tolerance is greater than 7 and less than or equal to 9.5.

[0027] In another aspect, the use of the monostar algae KM2-2 as described in any of the above embodiments in the preparation of products with high salt and / or high alkali is provided.

[0028] In some embodiments, the article includes at least one of feed and food.

[0029] In another aspect, we provide an application of the monostar algae KM2-2 as described in any of the above embodiments in promoting plant growth under salt and / or alkali stress.

[0030] It should be noted that microalgae can not only cooperate with other organisms in the soil to improve soil structure, but also improve soil pH and increase soil permeability through secretions, thereby improving land water use efficiency and promoting plant growth. On this basis, since the single-star algae KM2-2 provided in this disclosure has good salt and alkali tolerance, it can promote plant growth under salt stress and / or alkali stress.

[0031] The beneficial effects of this disclosure are:

[0032] 1. The monostar algae KM2-2 disclosed herein has good salt and alkali resistance.

[0033] 2. The single-star algae KM2-2 disclosed herein can grow and be cultivated in a saline-alkali environment and improve the saline-alkali environment, thereby realizing the comprehensive utilization and comprehensive management of the saline-alkali environment.

[0034] 3. The monostar algae KM2-2 disclosed herein can promote plant growth under salt stress and / or alkali stress.

[0035] Biological Preservation

[0036] The single-star algae (Coelastrella saipanensis) KM2-2 disclosed herein was deposited at the China Center for Type Culture Collection (CCTCC) on July 31, 2024, with accession number CCTCC NO:M 20241700. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the morphology of algal cells from the algal strain isolated in Part 1 of Example 3;

[0038] Figure 2 The transmission electron microscope ultrastructure of algal cells from the algal strain isolated in Part 1 of Example 3 is shown.

[0039] Figure 3 The image shows the BLAST sequence alignment results of the 18S rRNA gene sequence of the algal strain isolated in Part 2 of Example 3 in the NCBI database.

[0040] Figure 4 The image shows the BLAST sequence alignment results of the tufA gene sequence of the algal strain isolated in Part 2 of Example 3 in the NCBI database.

[0041] Figure 5 The neighbor-joining tree constructed based on the gene sequence of the 18S rRNA of the isolated algal strain in Part 2 of Example 3;

[0042] Figure 6 This refers to the neighbor-joining tree constructed based on the gene sequence of tufA from the isolated algal strain in Part 2 of Example 3;

[0043] Figure 7 The figure shows the phenotypic effects of different salinity and alkalinity treatments on the growth dynamics of KM2-2 in Example 4.

[0044] Figure 8 The figure shows the effect of different salinity and alkalinity treatments on the cell state of KM2-2 cells after 7 days in Example 4.

[0045] Figure 9 The graph shows the effect of different salinity and alkalinity treatments for 7 days on the optical density of KM2-2 cells in Example 4.

[0046] Figure 10 The figure shows the effect of different salinity and alkalinity treatments for 7 days on the maximum quantum efficiency of KM2-2 in Example 4; where, Figure 10 A shows the change in the maximum photon efficiency of chlorophyll fluorescence in KM2-2 cells; Figure 10 B shows the variation in the maximum quantum efficiency of KM2-2;

[0047] Figure 11 This is a graph showing the effect of different salinity and alkalinity treatments for 7 days on the actual photon yield of KM2-2 in Example 4; where, Figure 11 A shows the changes in the actual photon yield of chlorophyll fluorescence in KM2-2 cells; Figure 11 B shows the variation in the actual photon yield of KM2-2;

[0048] Figure 12 The figure shows the effect of different salinity and alkalinity treatments for 7 days on the pH and salinity of the KM2-2 cell culture medium in Example 4; where, Figure 12 A shows the pH changes in the cell culture medium of KM2-2; Figure 12 B shows the changes in salinity of the cell culture medium for KM2-2;

[0049] Figure 13 The figure shows the phenotypic effects of different sources of saline-alkali water treatment on the growth dynamics of KM2-2 in Example 5.

[0050] Figure 14 The figure shows the effect of 7 days of treatment with saline-alkali water from different sources on the cell state of KM2-2 cells in Example 5; where, Figure 14 A shows the effect of saline water from different sources without the addition of modified BG11 medium on the cell state of KM2-2. Figure 14 B shows the effect of adding modified BG11 medium to saline water from different sources on the cell state of KM2-2;

[0051] Figure 15 The graph shows the effect of 7 days of treatment with saline-alkali water from different sources on the optical density of KM2-2 cells in Example 5.

[0052] Figure 16The figure shows the effect of 7 days of treatment with saline-alkali water from different sources on the chlorophyll fluorescence of KM2-2 in Example 5.

[0053] Figure 17 This is a graph showing the effect of 7 days of treatment with saline-alkali water from different sources on the maximum quantum efficiency and actual quantum yield of KM2-2 in Example 5; where, Figure 17 A shows the variation in the maximum quantum efficiency of KM2-2; Figure 17 B shows the variation in the actual photon yield of KM2-2;

[0054] Figure 18 The figure shows the effect of 7 days of treatment with saline-alkali water from different sources on the pH and salinity of the KM2-2 cell culture medium in Example 5; where, Figure 18 A shows the pH changes in the cell culture medium of KM2-2; Figure 18 B shows the changes in salinity of the cell culture medium for KM2-2. Detailed Implementation

[0055] The technical solutions in some embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments provided in this disclosure, all other embodiments obtained by those skilled in the art are within the scope of protection of this disclosure.

[0056] Unless the context otherwise requires, throughout the specification and claims, the term "comprising" is interpreted as open and encompassing, that is, "including, but not limited to".

[0057] In describing some embodiments, the expression "A and / or B" may be used. It is readily understood that "A and / or B" includes the following three combinations: A only, B only, and a combination of A and B.

[0058] In describing some embodiments, the expressions "at least one of A, B and C" and "at least one of A, B or C" may be used, both of which have the same meaning and include the following combinations of A, B and C: only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, and a combination of A, B and C.

[0059] Example 1 Culture medium

[0060] 1. BG11 liquid culture medium, its formula is as follows: sodium nitrate 1.4-1.6 g / L, K2HPO4·3H2O 0.03-0.05 g / L, MgSO4·7H2O 0.065-0.085 g / L, CaCl2·2H2O 0.026-0.046 g / L, citric acid 0.005-0.007 g / L, ferric ammonium citrate 0.005-0.007 g / L, EDTA 0.0005-0.0015 g / L, sodium carbonate 0.015-0.025 g / L, boric acid 0.0027-0.0029 g / L, MnCl2·H2O 0.0017-0.0019 g / L, ZnSO4·7H2O 0.00021~0.00023g / L, CuSO4·5H2O 0.00007~0.00009g / L, Na2MoO4·2H2O 0.0003~0.0005g / L, and Co(NO3)2·6H2O 0.00004~0.00006g / L, with ddH2O as the solvent; pH value is 6.8~7.2.

[0061] 2. BG11 solid plate, with the following formula: agar powder 12-17 g / L, sodium nitrate 1.4-1.6 g / L, K2HPO4·3H2O 0.03-0.05 g / L, MgSO4·7H2O 0.065-0.085 g / L, CaCl2·2H2O 0.026-0.046 g / L, citric acid 0.005-0.007 g / L, ferric ammonium citrate 0.005-0.007 g / L, EDTA 0.0005-0.0015 g / L, sodium carbonate 0.015-0.025 g / L, boric acid 0.0027-0.0029 g / L, MnCl2·H2O 0.0017~0.0019g / L, ZnSO4·7H2O 0.00021~0.00023g / L, CuSO4·5H2O 0.00007~0.00009g / L, Na2MoO4·2H2O 0.0003~0.0005g / L, and Co(NO3)2·6H2O 0.00004~0.00006g / L; pH value is 6.8~7.2.

[0062] 3. Modify the BG11 liquid culture medium with the following formula: glucose 10.0–30.0 g / L, sodium nitrate 1.4–1.6 g / L, K₂HPO₄·3H₂O 0.03–0.05 g / L, MgSO₄·7H₂O 0.065–0.085 g / L, CaCl₂·2H₂O 0.026–0.046 g / L, citric acid 0.005–0.007 g / L, ferric ammonium citrate 0.005–0.007 g / L, EDTA 0.0005–0.0015 g / L, sodium carbonate 0.015–0.025 g / L, boric acid 0.0027–0.0029 g / L, MnCl₂·H₂O 0.0017~0.0019g / L, ZnSO4·7H2O 0.00021~0.00023g / L, CuSO4·5H2O 0.00007~0.00009g / L, Na2MoO4·2H2O 0.0003~0.0005g / L, and Co(NO3)2·6H2O 0.00004~0.00006g / L, with ddH2O as the solvent; pH value is 6.8~7.2.

[0063] Example 2: Collection, activation, isolation, culture, and purification of algal strains

[0064] 1. Collection of algal strains

[0065] Water samples were collected on January 12, 2024, from the Shilin Scenic Area (103°32′76″E, 24°81′61″N) in Shilin Yi Autonomous County, Kunming City, Yunnan Province, using a phytoplankton net with a pore size of 64 μm (200 mesh).

[0066] 2. Algal Activation

[0067] Transfer the collected water sample to a 15mL centrifuge tube and let it stand for 1-2 hours. Gently remove most of the supernatant, leaving 2-3mL of liquid at the bottom. Mix thoroughly and then incubate on a shaker at 25±1℃ and 150-180rpm for 6 hours to fully activate the sample.

[0068] 3. Isolation and culture of algal strains

[0069] 10 μL of the activated sample was aspirated and dropped onto a glass slide. After confirming the presence of suspected target algal cells under a microscope, a single cell was picked up under the microscope using a capillary siphon method. This process of aspiration, microscopic examination, and dilution was repeated until only a single target algal cell was contained in the water droplet. The sample was then transferred to a 96-well plate containing 100 μL of Glucono-Potassium 11 liquid medium (without antibiotics) and incubated statically at a light / dark cycle of 12 h / 12 ​​h and a light intensity of 20–30 μmol / m². -2 / s -1The culture temperature is 25±1℃. During the static culture period, BG11 liquid culture medium should be added to about 100μL as needed.

[0070] 4. Purification of algal strains

[0071] After statically culturing individual target algal cells for 20–30 days, the growth of the algal species was examined under a microscope. If the algal cells grew well, they were serially diluted to 10-1. 0 10 -1 10 -2 and 10 -3 The concentrations were thoroughly mixed, and 200 μL of each gradient dilution was applied to a solution containing 50 mg / L ampicillin (Amp). 50+ ), 50 mg / L kanamycin (Kan 50+ ) and 100 mg / L cephalosporin (Cef 100 + Place the plates on BG11 solid plates and invert them in a constant temperature and light incubator. The light / dark cycle is 12h / 12h, and the light intensity is 10–30 μmol / m². -2 / s -1 The culture temperature was 25±1℃, and the plates were inverted for 7–15 days until single algal colonies appeared. The growth of single algal colonies and other microorganisms on the plates was then observed. If obvious microorganisms were present on the plates, single algal colonies were transferred to 200 μL of BG11 liquid medium (containing Amp) 100+ Kan 50+ Cef 100+ Mix thoroughly by suction and whisk, then dilute serially to 10⁻⁶. -1 and 10 -2 The concentration was thoroughly mixed and recoated onto a BG11 solid plate (which also contains Amp). 100+ Kan 50+ Cef 100+ The algae were cultured on a plate. The algal strain was purified through multiple platings until no obvious contaminating bacteria were observed on either the plate or individual algal colonies. Then, individual algal colonies were transferred to a modified BG11 liquid medium (containing Amp) for further purification. 25+ Cef 50+ Inoculate at a ratio of 1:10 and scale up the culture stepwise. The culture temperature is 25±1℃, the rotation speed is 150–180 rpm, the light / dark cycle is 12h / 12h, and the light intensity is 30–50 μmol / m². -2 / s -1 This serves as a reserve of algal cells for subsequent steps.

[0072] Example 3: Identification and Preservation of Algal Strains

[0073] 1. Morphological observation of algal strains

[0074] A small amount of purified fresh algal cells were taken and their morphology was observed under a Leica DM5000 inverted microscope (Leica, Germany). Images were acquired using a Leica DFC320 CCD and subjected to differential interference contrast (DIC) and phase contrast (PH) photography. Simultaneously, a small amount of fresh algal cells were pre-fixed with 3% glutaraldehyde, re-fixed with 1% osmium tetroxide, dehydrated stepwise with acetone, and embedded in Epon 812. Semi-thin sections were stained with toluidine blue for optical localization, ultrathin sections were prepared using a diamond scalpel, stained with uranium acetate and lead citrate, and the ultrastructure of the cells was observed using a JEM-1400FLASH transmission electron microscope.

[0075] The morphology of the isolated microalgae was observed using optical and electron microscopy, and the results are as follows: Figures 1-2 As shown.

[0076] The isolated algal strains are single-celled green microalgae. The microalgal cells are spherical, ellipsoidal or spindle-shaped with blunt and rounded ends. The size of the young cells is about (6-9) μm × (5-7) μm, and the size of the mature cells is about 8-14 μm. The cell walls are relatively thick.

[0077] 2. Molecular identification of algal strains

[0078] The purified algal strain was centrifuged at 5000 rpm for 5 min to collect the algal cells. The cells were repeatedly resuspended in sterile water to remove the liquid culture medium, and then collected for further molecular identification. The Ezup column-based plant tissue genomic DNA extraction kit from Sangon Biotech Co., Ltd. was used to extract the algal genome according to the manufacturer's instructions. New England Biolabs... High-Fidelity DNA Polymerase was used for PCR amplification.

[0079] The 50 μL PCR reaction system consisted of 25 ng of genomic DNA from the KM2-2 algal strain. Reaction Buffer 10 μL, 10 mM dNTP 1 μL, 10 μM forward and reverse primers 2.5 μL each. High-Fidelity DNA Polymerase 0.5 μL, made up to 50 μL with double-distilled water.

[0080] Two genes, 18S ribosomal RNA (18S rRNA) and translation elongation factor Tu (tufA), were used for PCR molecular identification. The reaction conditions were as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 55℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; 72℃ extension for 5 min.

[0081] Among them, there are two pairs of primers for molecular identification, namely:

[0082] 1) Amplify the 18S rRNA region using the upstream primer 5'-AACCTGGTTGATCCTGCCAGT-3' and the downstream primer 5'-TGATCCTTCTGCAGGTTCACCTAC-3';

[0083] 2) Amplify the tufA region using upstream primer 5'-TGAAACAGAAMAWCGTCATTATGC-3' and downstream primer 5'-CCTTCNCGAATMGCRAAWCGC-3'.

[0084] The products obtained from PCR amplification of the 18S rRNA and tufA regions were recovered and purified using a Takara gel extraction kit and sent to the Chengdu branch of Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were submitted to the National Center for Biotechnology Information (NCBI) database for Nucleotide BLSAT sequence alignment. The results are as follows: Figure 3 and Figure 4 As shown, the gene sequencing results of the products corresponding to the 18S rRNA region and the tufA region have a query coverage of 98% and 98% respectively, a per-identity of 99.77% and 96.08% respectively, and an E value of 0 for both.

[0085] The gene sequences of the 18S rRNA and tufA of this algal strain are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. The phylogenetic trees of the 18S rRNA and tufA gene sequences of this algal strain using the joint neighbor-joining method are shown in... Figure 5 and Figure 6As shown, the NCBI accession numbers for the 18S rRNA and tufA gene sequences of this algal strain are PQ108912.1 and PQ127014.1, respectively.

[0086] Analysis showed that this algal strain belongs to the same evolutionary branch as Coelastrella saipanensis.

[0087] 3. Preservation of algal strains

[0088] Based on the combined results of morphological observation and molecular identification, the algal strain obtained in this embodiment is *Coelastrella saipanensis*, named *Coelastrella saipanensis* KM2-2. It was deposited on July 31, 2024, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China, with accession number CCTCC NO:M 20241700.

[0089] Example 4: Salt and alkali tolerance test of *Single-star algae* KM2-2

[0090] 1. Experimental Materials

[0091] The single-star algae KM2-2 (i.e., KM2-2) from Example 3 is used in this example.

[0092] 2. Experimental Methods

[0093] 2.1 Treatment with different salinity and alkalinity

[0094] KM2-2 cells were cultured under the conditions described in Part 4 of Example 2 until the late logarithmic growth phase. Fresh modified BG11 liquid culture medium was then used, and the initial concentration of algal cells was adjusted to OD680 = 0.5. The cells were then dispensed into 50 mL Erlenmeyer flasks for subsequent treatment experiments.

[0095] Different alkalinity treatments were performed at pH 7.0 and pH 9.5; different salinity treatments were performed using NaCl at concentrations of 0 g / L, 10 g / L, 30 g / L, and 50 g / L, corresponding to salinities of 0%, 1%, 3%, and 5%; and different alkalinity and salinity treatments were combined in combination; the culture temperature was 25 ± 1℃, the culture rotation speed was 150–180 rpm, and the light intensity was 30–70 μmol·m⁻¹. -2 ·s -1 The light-dark cycle was 12h / 12h, and the culture was carried out continuously for 7 days.

[0096] 2.2 Determination of microalgal growth curves

[0097] A certain amount of fresh algal solution was taken, and the optical density value of KM2-2 cells at 680nm was measured using an ELISA reader (Metash, UV-9000, China), and a growth curve was plotted.

[0098] 2.3 Chlorophyll Fluorescence Analysis

[0099] Chlorophyll fluorescence was measured using MAXI-IMAGING-PAM (WALZ, Germany). After mixing the samples, 200 μL was pipetted into each black 96-well plate, and chlorophyll fluorescence parameters were measured after 5 min of dark adaptation.

[0100] 2.4 Determination of pH and Salinity

[0101] pH was measured using a pH meter, and salinity was measured using a salinity meter.

[0102] 3. Experimental Results

[0103] This example investigated the effects of different alkalinities (initial pH 7.0 and 9.5) and different salinities (using NaCl at concentrations of 0 g / L, 10 g / L, 30 g / L, and 50 g / L, corresponding to salinities of 0%, 1%, 3%, and 5%) on the growth status of KM2-2 algal cells after continuous culture for 7 days. The results are as follows: Figures 7-12 As shown.

[0104] 3.1 Test of KM2-2 tolerance to salt ion stress

[0105] When the initial pH of the culture medium is 7.0, the specific behavior is as follows:

[0106] With prolonged cultivation time, under 1% NaCl treatment, the color of the algal culture medium gradually changed from light green to dark green within 0–7 days (e.g., ...). Figure 7 (As shown); After 7 days of continuous culture, compared with the NaCl treatment with a salinity of 0%, the number of cells in the NaCl treatment with a salinity of 1% increased significantly (as shown). Figure 8 As shown), the cell optical density value was maintained at 0.8–0.9 (as shown). Figure 9 As shown), the maximum quantum efficiency Fv / Fm and the actual quantum yield Y(II) are slightly reduced (as shown). Figures 10-11 (As shown in the figure). This indicates that when the initial pH of the culture medium is 7.0, treatment with 1% NaCl promotes the growth of KM2-2.

[0107] With prolonged cultivation time, under 3% NaCl treatment, the color of the algal culture medium changed from light green to dark green in the first 0-5 days, and from dark green to light green in the second 5-7 days (e.g., ...). Figure 7 (As shown); After 7 days of continuous culture, the number of cells increased slightly compared to the NaCl treatment with a salinity of 0 (as shown). Figure 8 As shown), the cell optical density value was maintained at 0.5–0.6 (as shown). Figure 9 As shown), the maximum quantum efficiency Fv / Fm and the actual quantum yield Y(II) are reduced (e.g. Figures 10-11 (As shown in the figure). This indicates that when the initial pH of the culture medium is 7.0, treatment with 3% NaCl promotes the growth of KM2-2 within 0 to 5 days, but if the culture time is too long, it can no longer promote its growth.

[0108] With prolonged cultivation time, under 5% NaCl treatment, the color of the algal culture medium changed from light green to brownish-green within 0–7 days (e.g., ...). Figure 7 (As shown); after 7 days of continuous culture, the number of cells was significantly reduced compared to NaCl treatment with a salinity of 0 (as shown). Figure 8 As shown), the cell optical density value was maintained at 0.4–0.5 (as shown). Figure 9 As shown), the maximum quantum efficiency Fv / Fm and the actual quantum yield Y(II) are significantly reduced (e.g. Figures 10-11 (As shown in the figure). This indicates that when the initial pH of the culture medium is 7.0, treatment with 5% NaCl can only maintain the survival of KM2-2 cells, but cannot promote their cell growth.

[0109] When the initial pH of the culture medium was 7.0, after 7 days of continuous culture with NaCl at salinities of 0%, 1%, 3%, and 5%, the pH changes were 9.53, 9.32, 8.93, and 8.70, respectively. Figure 12 As shown in A), its salinity remains basically at the initial added concentration (e.g., Figure 12 (as shown in B).

[0110] In summary, compared with NaCl treatments with salinity of 1%–5%, KM2-2 showed the fastest growth rate and best growth condition under NaCl treatment with a salinity of 0%. In addition, when the initial pH of the culture medium was 7.0, KM2-2 exhibited good tolerance to NaCl with a salinity of 1%–3% and some tolerance to NaCl with a salinity of 5%.

[0111] 3.2KM2-2 tolerance test to severe alkaline water stress

[0112] When the initial pH of the culture medium is 9.5, the specific behavior is as follows:

[0113] With prolonged cultivation time, under 1% NaCl treatment, the color of the algal culture medium gradually changed from light green to dark green within 0–7 days (e.g., ...). Figure 7 (As shown); After 7 days of continuous culture, the number of cells increased significantly compared to the NaCl treatment with a salinity of 0 (as shown). Figure 8 As shown), the cell optical density value was maintained at 0.8–0.9 (as shown). Figure 9 As shown), the maximum quantum efficiency Fv / Fm and the actual quantum yield Y(II) are slightly reduced (as shown). Figures 10-11 (As shown in the figure). This indicates that when the initial pH of the culture medium is 9.5, treatment with 1% NaCl promotes the growth of KM2-2.

[0114] With prolonged culture time, under 3% NaCl treatment, the color of the algal culture medium changed from light green to brownish-green within 0–7 days (e.g., ...). Figure 7 (As shown); after 7 days of continuous culture, the number of cells was significantly reduced compared to NaCl treatment with a salinity of 0 (as shown). Figure 8 As shown), the cell optical density value was maintained at 0.4–0.5 (as shown). Figure 9 As shown), the maximum quantum efficiency Fv / Fm and the actual quantum yield Y(II) are significantly reduced (e.g. Figures 10-11 (As shown in the figure). This indicates that when the initial pH of the culture medium is 9.5, treatment with 3% NaCl can only maintain the survival of KM2-2 cells, but cannot promote their cell growth.

[0115] With prolonged culture time, under 5% NaCl treatment, the color of the algal culture medium changed from light green to brownish-yellow within 0–7 days (e.g., ...). Figure 7 (As shown); after 7 days of continuous culture, the number of cells was significantly reduced compared to NaCl treatment with a salinity of 0 (as shown). Figure 8 As shown), the cell optical density value was maintained at 0.3–0.4 (as shown). Figure 9 As shown), the maximum quantum efficiency Fv / Fm and the actual quantum yield Y(II) are almost undetectable (as shown). Figures 10-11 (As shown in the figure). This indicates that when the initial pH of the culture medium is 9.5, treatment with 5% NaCl inhibits the growth of KM2-2 cells, leading to gradual cell death.

[0116] When the initial pH of the culture medium was 9.5, after 7 days of continuous culture with NaCl at salinities of 0%, 1%, 3%, and 5%, the pH changes were 9.49, 9.33, 8.83, and 7.85, respectively. Figure 12 As shown in A), its salinity remains basically at the initial added concentration (e.g., Figure 12 (as shown in B).

[0117] In summary, compared to NaCl treatments with salinity of 1%–5%, KM2-2 showed the fastest growth rate and best growth condition under NaCl treatment with a salinity of 0%. This indicates that when the initial pH of the culture medium is 9.5, NaCl treatment with a salinity of 0 does not affect the growth of KM2-2 at all. In addition, when the initial pH of the culture medium is 9.5, KM2-2 has good tolerance to NaCl with a salinity of 1%, some tolerance to NaCl with a salinity of 3%, and poor tolerance to NaCl with a salinity of 5%.

[0118] Example 5: Study on the effect of using saline-alkali water for the growth and cultivation of *Singula simulans* KM2-2.

[0119] 1. Experimental Materials

[0120] The single-star algae KM2-2 (i.e., KM2-2) from Example 3 is used in this example.

[0121] 2. Experimental Methods

[0122] 2.1 Treatment of saline water from different sources

[0123] Samples A, B, and C used in the saline-alkali water treatment were all collected in Artush City, Kizilsu Kyrgyz Autonomous Prefecture, Xinjiang Uygur Autonomous Region. Specifically, samples A, B, and C were collected in Kumusak Village, Azak Town (76°15′E, 39°40′N), Xiaoerkule Salt Lake, Ang'ezi Village, Halajun Township (75°31′E, 39°39′N), and Langgan Village, Azak Town (76°3′E, 39°40′N), respectively.

[0124] Treatment 1: KM2-2 cells were cultured to the late logarithmic growth phase under the conditions described in Part 4 of Example 2, and then transferred to samples A, B, and C for treatment, resulting in Treatment 1-1, Treatment 1-2, and Treatment 1-3, respectively. ddH2O was used as Control 1. The initial algal cell concentration in each group was adjusted to OD680 = 0.5, and then dispensed into 50mL Erlenmeyer flasks for subsequent treatment experiments. Specific information about the culture medium before treatment is shown in Table 1.

[0125] Table 1. Statistical table of information of each group of culture medium before treatment 1.

[0126] Comparison 1 Process 1-1 Process 1-2 Process 1-3 Sample source <![CDATA[ddH2O]]> Sample A Sample B Sample C pH 7.00 8.50 8.00 8.55 salinity(%) 0 2.0 27.3 2.1

[0127] Treatment 2: Modified BG11 medium was added to samples A, B, and C respectively, and the samples were autoclaved at 121°C for 15 minutes. KM2-2 cells were cultured to the late logarithmic growth phase according to the culture conditions in Part 4 of Example 2, and then transferred to samples A + modified BG11 medium, B + modified BG11 medium, and C + modified BG11 medium respectively, to obtain Treatment 2-1, Treatment 2-2, and Treatment 2-3. Modified BG11 medium using ddH2O as solvent was used as Control 2. The initial algal cell concentration in each group was adjusted to OD680 = 0.5, and then dispensed into 50mL Erlenmeyer flasks for subsequent treatment experiments. Specific information about the culture media before treatment is shown in Table 2.

[0128] Table 2. Statistical table of information of each group of culture medium before treatment 2.

[0129]

[0130]

[0131] It should be noted that the difference between the "modified BG11 culture medium" mentioned above and the "modified BG11 liquid culture medium" in the embodiments of this disclosure is that it does not contain the solvent ddH2O. Therefore, the "adding modified BG11 culture medium to samples A, B, and C respectively" mentioned above can also be understood as "replacing the solvent ddH2O in the modified BG11 liquid culture medium with samples A, B, and C respectively"; similarly, the "modified BG11 culture medium using ddH2O as the solvent" mentioned above is the "modified BG11 liquid culture medium" in the embodiments of this disclosure.

[0132] During treatments 1 and 2, the KM2-2 was cultured at a temperature of 25±1℃, a rotation speed of 150–180 rpm, and a light intensity of 30–70 μmol·m⁻². -2 ·s -1 The light-dark cycle was 12h / 12h, and the culture was carried out continuously for 7 days.

[0133] Other experimental methods are the same as in Example 4, and will not be repeated here.

[0134] 3. Experimental Results

[0135] 3.1 Study on the effects of autotrophic growth using saline water from different sources on KM2-2

[0136] As shown in Table 1, the pH values ​​of the culture media for treatments 1-1, 1-2, and 1-3 before treatment were 8.50, 8.00, and 8.55, respectively, and the salinities were 2.0%, 27.3%, and 2.1%, respectively; while the pH value of the culture media for control 1 before treatment was 7.00 and the salinity was 0.

[0137] This embodiment compares the effects of natural saline water samples A, B, and C from different sources on the autotrophic growth status of KM2-2 algal cells. The results are as follows: Figures 13-18 As shown. When saline solution is used directly as the culture medium, the specific behavior is as follows:

[0138] As the culture time increased, the algal culture medium for treatments 1-1 and 1-3 maintained a dark green color during the 0-7 day period (e.g., Figure 13 (As shown); After 7 days of continuous culture, the number of cells in treatments 1-1 and 1-3 was slightly increased compared to control 1 (as shown). Figure 14 As shown in Figure A), the cell optical density value was maintained at 0.5–0.6 (as shown in Figure A). Figure 15 As shown), there was no significant difference in chlorophyll fluorescence parameters (e.g. Figure 16 (As shown in the figure). This indicates that the saline solution in samples A and C can maintain the activity of KM2-2 cells.

[0139] As the culture time increased, from 0 to 7 days, the color of the algal culture medium for treatments 1-2 changed from dark green to brownish-green (e.g., Figure 13 (As shown); After 7 days of continuous culture, compared with control 1, treatment 1-1 and treatment 1-3, the number of cells in treatment 1-2 was slightly reduced (as shown). Figure 14 As shown in A), the cell optical density value decreased slightly (as shown in A). Figure 15 As shown), chlorophyll fluorescence imaging, maximum photon efficiency Fv / Fm, and actual photon yield Y(II) are almost undetectable (e.g. Figures 16-17 (As shown). This indicates that the salinity of sample B, at 27.3%, hindered normal photosynthesis in KM2-2 cells, affecting their normal growth and reproduction.

[0140] After 7 days of continuous culture, the pH and salinity of the cell culture medium for KM2-2 in treatments 1-1, 1-2, and 1-3 remained essentially unchanged (e.g., Figure 18 (As shown).

[0141] In summary, KM2-2 exhibits good tolerance to saline-alkali water samples A and C, and can utilize them for normal autotrophic growth, but shows poor tolerance to saline-alkali water sample B.

[0142] 3.2 Research on the application of KM2-2 for commensal growth in saline-alkali water from different sources

[0143] After high-temperature and high-pressure sterilization, the culture media of treatments 2-1, 2-2, and 2-3 turned caramel-colored, with a decrease in pH and an increase in salinity. Table 2 shows that the pH of the culture media before treatment 2-1, 2-2, and 2-3 was 6.42, 5.32, and 6.91, respectively, and the salinities were 3.1%, above 28%, and 3.2%, respectively; while the pH of the culture media of control 2 before treatment was 6.54, and the salinity was 1.1.

[0144] This embodiment compares the effects of natural saline water samples A, B, and C from different sources on the eutrophic growth status of KM2-2 algal cells. The results are as follows: Figures 13-18 As shown. When using saline solution + modified BG11 medium as the culture medium, the specific performance is as follows:

[0145] As the culture time increased, from 0 to 7 days, the color of the algal culture medium for treatments 2-1 and 2-3 changed from green to dark green (e.g., Figure 13 (As shown); After 7 days of continuous culture, the number of cells in treatments 2-1 and 2-3 was comparable to that in control 2 (as shown). Figure 14 As shown in B), the cell optical density values ​​were 2.16 and 1.22, respectively (as shown in B). Figure 15 As shown), there was no significant difference in chlorophyll fluorescence parameters (e.g. Figure 16 (As shown in the figure). This indicates that the saline solution of samples A and C, after the addition of modified BG11 medium, can promote the growth of KM2-2 cells.

[0146] As the culture time increased, from 0 to 7 days, the color of the algal culture medium for treatment 2-2 changed from dark green to dark brown (e.g., Figure 13 (As shown); After 7 days of continuous culture, compared with control 2, treatment 2-1, and treatment 2-3, the number of cells in treatment 2-2 was reduced (as shown). Figure 14 As shown in B), the cell optical density value decreases (e.g., as shown in B). Figure 15 As shown), chlorophyll fluorescence imaging, maximum photon efficiency Fv / Fm, and actual photon yield Y(II) are almost undetectable (e.g. Figures 16-17 (As shown). This indicates that even with the addition of modified BG11 medium, the salinity of sample B at 27.3% hindered normal photosynthesis in KM2-2 cells, affecting their normal growth and reproduction.

[0147] After 7 days of continuous culture, the pH and salinity of the cell culture medium for KM2-2 in treatments 2-1, 2-2, and 2-3 remained essentially unchanged (e.g., Figure 18 (As shown).

[0148] In conclusion, the addition of modified BG11 medium can enhance the tolerance of KM2-2 cells to natural saline water and promote their multitrophic growth.

Claims

1. A salt-tolerant monostellar alga ( Coelastrella saipanensis KM2-2, characterized in that, The single-star algae KM2-2 was deposited at the China Center for Type Culture Collection on July 31, 2024, with accession number CCTCC NO: M20241700.

2. The application of the single-star algae KM2-2 as described in claim 1 in the growth and cultivation of algae in a saline-alkali environment.

3. The application according to claim 2, characterized in that, The saline-alkali environment includes at least one of saline-alkali soil and saline-alkali water.

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