Heteromorphic alga RXH-ZY24 and its applications

Abstract

The present disclosure relates to the field of microbial technology and provides a Dysmorphococcus globosus RXH-ZY24, which was deposited at the China Center for Type Culture Collection on April 7, 2024, with the deposit number CCTCC NO: M 2024627. The present disclosure also provides the application of the above-mentioned Dysmorphococcus globosus RXH-ZY24, including using the above-mentioned Dysmorphococcus globosus RXH-ZY24 to enrich at least one of zinc and carotenoids in a zinc ion stress environment. The Dysmorphococcus globosus RXH-ZY24 provided by the present disclosure has a certain tolerance to zinc ion stress, can not only enrich zinc in a zinc ion stress environment, but also promote the accumulation of carotenoids, and has the effect of synergistically enriching zinc and carotenoids.

Description

Technical Field

[0001] This disclosure relates to the field of microbial technology, for example to a heteromorphic alga RXH-ZY24 and its applications. Background Technology

[0002] Microalgae are high-quality raw materials for biofuels, cosmetics, pharmaceuticals, nutrition, food additives, and aquaculture. They can also be used in agriculture as biostimulants, biofertilizers, and bioremediation agents. Some microalgae possess important high-value pigment resources, such as carotenoids. Carotenoids not only offer numerous health benefits, such as anti-diabetic, anti-inflammatory, nutritional and pharmaceutical applications, and prevention of cardiovascular disease, certain types of cancer, and some immune system disorders, but also possess strong antioxidant capabilities, preventing premature aging, UV radiation, and photo-oxidation. Due to the various health benefits of carotenoids, market demand is rapidly growing. Microalgae associated with carotenoid accumulation, due to their rapid growth, active metabolism, and balanced biochemical precursors / pathways, are expected to meet the growing market demand for value-added bioproducts. Microalgae that accumulate secondary carotenoids under environmental stress may be an excellent source of natural carotenoids. The development of novel microalgae strains for the production of natural carotenoids for industrial applications is encouraged worldwide.

[0003] Studies have found that microalgae are rich in various nutrients, such as astaxanthin, β-carotene, protein, and trace elements. Currently, research on zinc-rich algae includes Spirulina spp., Pavlova viridid, and Caulerpa lentillifera. In addition, Chinese patent document CN201110448718.3 discloses a Scenedesmus sp. strain ENN2201A, which can grow over a wide temperature range, grows rapidly, is easy to collect, and effectively produces fatty acids and carotenoids. However, current research has not yet addressed algal strains capable of accumulating zinc and carotenoids under zinc ion stress. Summary of the Invention

[0004] The purpose of this disclosure is to overcome the shortcomings of the prior art and provide a heteromorphic alga RXH-ZY24 and its application. This heteromorphic alga RXH-ZY24 has a certain tolerance to zinc ion stress. It can not only enrich zinc in the zinc ion stress environment, but also promote the accumulation of carotenoids, and has the effect of synergistic enrichment of zinc and carotenoids.

[0005] On the one hand, a heteromorphic alga (Dysmorphococcus globosus) RXH-ZY24 is provided. The heteromorphic alga RXH-ZY24 was deposited at the China Center for Type Culture Collection on April 7, 2024, with accession number CCTCC NO:M2024627.

[0006] On the other hand, an application of the heteromorphic algae RXH-ZY24 as described in the above embodiments is provided. The application includes using the heteromorphic algae RXH-ZY24 to enrich at least one of zinc and carotenoids under zinc ion stress.

[0007] In some embodiments, the application includes using the heteromorphic algae RXH-ZY24 to enrich zinc under zinc ion stress.

[0008] In some embodiments, the application includes: heterotrophic or co-trophic culture of the *Heterotrophic algae* RXH-ZY24 when it is used to enrich zinc under zinc ion stress.

[0009] In some examples, the conditions for heterotrophic culture include: no light, a culture temperature of 22–28°C, a culture rotation speed of 120–180 rpm, and a culture time of 2–7 days.

[0010] For example, the conditions for heterotrophic culture include: no light, a culture temperature of 24-26°C, a culture rotation speed of 150-180 rpm, and a culture time of 7 days.

[0011] In some examples, the conditions for the commensal culture include: a light intensity of 200–600 μmol / m². -2 / s -1 The culture temperature is 22–28℃, the culture speed is 120–180 rpm, and the culture time is 2–7 days.

[0012] For example, the conditions for the commensal culture include: a light intensity of 600 μmol / m². -2 / s -1 The culture temperature was 24–26℃, the culture speed was 150–180 rpm, and the culture time was 7 days.

[0013] In some embodiments, the application includes using the heteromorphic algae RXH-ZY24 to synergistically enrich zinc and carotenoids under zinc ion stress.

[0014] In some embodiments, when the *Heterotrophic algae* RXH-ZY24 is used to synergistically enrich zinc and carotenoids under zinc ion stress, the *Heterotrophic algae* RXH-ZY24 is subjected to a multitrophic culture.

[0015] In some examples, the conditions for the commensal culture include: a light intensity of 200–600 μmol / m². -2 / s -1 The culture temperature is 22–28℃, the culture speed is 120–180 rpm, and the culture time is 2–7 days.

[0016] For example, the conditions for the commensal culture include: a light intensity of 600 μmol / m². -2 / s -1 The culture temperature was 24–26℃, the culture speed was 150–180 rpm, and the culture time was 7 days.

[0017] In some embodiments, the carotenoids include at least one of β-cryptoxanthin, zeaxanthin, canthaxanthin, and astaxanthin.

[0018] In some embodiments, the concentration of zinc ions in the zinc ion stress environment is 100–1600 mg / L.

[0019] In some examples, the concentration of zinc ions in the zinc ion stress environment is 100–800 mg / L.

[0020] For example, in the zinc ion stress environment, the concentration of zinc ions is 200 to 800 mg / L, for example, 200 mg / L.

[0021] Furthermore, an application of the heteromorphic algae RXH-ZY24 as described in the above embodiments is provided. The application includes using the heteromorphic algae RXH-ZY24 in the preparation of at least one of the raw materials and additives for feed.

[0022] In some embodiments, the feed includes aquaculture feed.

[0023] It is worth noting that carotenoids (e.g., astaxanthin) can not only enhance the antioxidant capacity and immunity of animals (e.g., aquatic animals), but also improve their growth and color. The *Heteromorpha* RXH-ZY24 disclosed herein can promote the accumulation of carotenoids (e.g., astaxanthin) while enriching zinc ions within algal cells. Using it as a feed ingredient and / or additive can effectively improve symptoms of malnutrition in animals (e.g., aquatic animals).

[0024] The beneficial effects of this disclosure are:

[0025] The disclosed algae (Dysmorphococcus globosus) RXH-ZY24 exhibits a certain tolerance to zinc ion stress. It can not only accumulate zinc in zinc ion stress environment, but also promote the accumulation of carotenoids, thus having a synergistic effect of accumulating zinc and carotenoids.

[0026] Biological Preservation

[0027] The heteromorphic algae (Dysmorphococcus globosus) RXH-ZY24 disclosed herein was deposited on April 7, 2024, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO:M 2024627. The depositary address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the morphology of algal cells from the algal strain isolated in Part 1 of Example 3; wherein, Figure 1 A illustrates the morphological changes in algal cells. Figure 1 B illustrates the process of algal cell division and change;

[0029] Figure 2 The transmission electron microscope ultrastructure of algal cells from the algal strain isolated in Part 1 of Example 3 is shown.

[0030] 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.

[0031] Figure 4 This is a BLAST sequence alignment result of the psaB gene sequence of the algal strain isolated in Part 2 of Example 3 in the NCBI database.

[0032] Figure 5 The image shows the BLAST sequence alignment results of the ITS gene sequence of the algal strain isolated in Part 2 of Example 3 in the NCBI database.

[0033] Figure 6 The neighbor-joining tree constructed based on the 18S rRNA gene sequence of the isolated algal strain in Part 2 of Example 3;

[0034] Figure 7 This is the neighbor-joining tree constructed based on the psaB gene sequence of the isolated algal strain in Part 2 of Example 3;

[0035] Figure 8 This refers to the neighbor-joining tree constructed based on the ITS gene sequence of the isolated algal strain in Part 2 of Example 3;

[0036] Figure 9 This is a graph showing the effects of different zinc concentrations on the phenotype and cells of ZY24 heterotrophic culture, as described in Example 4. Figure 9 A shows the effect on the phenotype of ZY24 heterotrophic culture. Figure 9 B shows the effect on ZY24 heterotrophic cultured cells;

[0037] Figure 10 The figure shows the effect of different zinc concentrations on the optical density of ZY24 heterotrophic cells in Example 4.

[0038] Figure 11 This is a figure showing the effect of different zinc concentrations on chlorophyll fluorescence in heterotrophic ZY24 cells, as described in Example 4. Figure 11 A shows the changes in chlorophyll fluorescence in ZY24 cells; Figure 11 B shows the variation in the maximum photosynthetic efficiency of ZY24; Figure 11 C shows the variation in the actual photosynthetic efficiency of ZY24;

[0039] Figure 12 The figure shows the effect of different zinc concentrations on the total zinc content in ZY24 heterotrophic cells in Example 4.

[0040] Figure 13 The figure shows the effect of different zinc concentrations on the macroscopic phenotype of ZY24 ditrophic culture in Example 5.

[0041] Figure 14 This is a graph showing the effect of different zinc concentrations on the microscopic cells of ZY24 multitrophic culture in Example 5.

[0042] Figure 15 This is a graph showing the effect of different zinc concentrations on the optical density of ZY24 cells in Example 5.

[0043] Figure 16 This is a graph showing the effect of different zinc concentrations on chlorophyll fluorescence imaging of ZY24 ditrophic culture in Example 5.

[0044] Figure 17 This is a graph showing the effect of different zinc concentrations on chlorophyll fluorescence parameters in ZY24 ditrophic culture, as described in Example 5; where, Figure 17 A shows the variation in the maximum photosynthetic efficiency of ZY24; Figure 17 B shows the variation in the actual photosynthetic efficiency of ZY24;

[0045] Figure 18 This is a graph showing the effect of different zinc concentrations on the total zinc content in ZY24 multitrophic cultured cells in Example 5.

[0046] Figure 19 This is a graph showing the effect of different zinc concentrations on carotenoid content in ZY24 multitrophic cultured cells in Example 5; where, Figure 19 A shows the effect on lutein content; Figure 19 B shows the effect on β-carotene content; Figure 19 C shows the effect on β-cryptoxanthin content; Figure 19 D shows the effect on the xanthine content of maize; Figure 19 E shows the effect on canthaxanthin content; Figure 19 F shows the effect on astaxanthin content. Detailed Implementation

[0047] 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.

[0048] 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.

[0049] 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.

[0050] Example 1 Culture medium

[0051] 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; pH value is 6.8~7.2.

[0052] 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.

[0053] 3. Modify the BG11 liquid culture medium with the following formula: sodium acetate 1.0–3.0 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; pH value is 6.8~7.2.

[0054] Example 2: Collection, activation, isolation, culture, and purification of algal strains

[0055] 1. Collection of algal strains

[0056] Water samples were collected on March 25, 2023, from Qianhe Village, Donghe Town, Ziyang County, Ankang City, Shaanxi Province (108°60′92″E, 32°49′31″N) using a phytoplankton net with a pore size of 64μm (200 mesh).

[0057] 2. Algal strain activation

[0058] 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.

[0059] 3. Isolation and culture of algal strains

[0060] 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 BG11 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.

[0061] 4. Purification of algal strains

[0062] 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 BG11 liquid medium (containing Amp) 100+ Kan 50+ Cef 100+ Mix thoroughly by suction and whisk, then serially dilute 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.

[0063] Example 3: Identification and Preservation of Algal Strains

[0064] 1. Morphological observation of algal strains

[0065] 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.

[0066] The morphology of the isolated microalgae was observed using optical and electron microscopy, and the results are as follows: Figures 1-2 As shown.

[0067] The isolated algal strains were single-celled green microalgae, some exhibiting cell aggregation. The microalgae exhibited diverse morphologies, including round, spherical, and elliptical shapes, with cell sizes ranging from 10 to 50 μm. Cellular characteristics included a pair of flagella of equal length, a single green cell surrounded by a broad, ovoid thalassium, and possibly elongated or striped eyespots or pigment dots. The cell center contained one to two distinct, round pyrenoids surrounded by a starch sheath and a urn-shaped or wall-like chloroplast that almost filled the entire cell space. These green cell aggregates were enclosed by the sporangium wall or the parental thalassium.

[0068] In the early stages of cell growth, a distinct periplasmic space is visible between the cell wall and the protoplast. As the cells grow, their color gradually changes to greenish-brown (reddish in the center and green at the edges), and their shape tends to become spherical. With continued culture, most cells form new cell walls and transform into non-motile spherical cells with a diameter of 20–50 μm. During this process, not only does the cell wall thicken, but the periplasmic space also decreases, and the internal pigment changes from greenish-brown to red or orange.

[0069] Asexual reproduction results showed that a single mother cell could divide to produce 2–16 daughter cells; among them, Figure 1 B specifically demonstrates the process by which a mother cell divides into 8 daughter cells.

[0070] 2. Molecular identification of algal strains

[0071] 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.

[0072] The 50 μL PCR reaction system consisted of 25 ng of genomic DNA from the RXH-ZY24 algal strain. 10 μL Reaction Buffer, 1 μL 10 mM dNTPs, 2.5 μL each of 10 μM forward and reverse primers. 0.5 μL of High-Fidelity DNA Polymerase was added, and the volume was made up to 50 μL with double-distilled water.

[0073] PCR was used to identify three genes: 18S ribosomal RNA (18S rRNA), photosystem IP700 chlorophyll a apoprotein A2 (psaB), and internal transcribed spacer (ITS). The reaction conditions were: 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.

[0074] Among them, there are three pairs of primers for molecular identification, namely:

[0075] 1) Amplify the 18S rRNA region using the upstream primer 5'-AACCTGGTTGATCCTGCCAGT-3' and the downstream primer 5'-TGATCCTTCTGCAGGTTCACCTAC-3';

[0076] 2) Amplify the psaB region using the upstream primer 5'-GGTGGTTTTCATCCACAAACTC-3' and the downstream primer 5'-GAACCACGTGCATCTAAAGCACCT-3';

[0077] 3) Amplify the upstream primer 5'-TGGTGCCAGCAGCCGCGGTA-3' and the downstream primer 5'-CTCAGTAAGCTTGATCCTTCCGCAGGTTCACC-3' of the ITS region.

[0078] The products obtained from PCR amplification of the 18S rRNA region, psaB region, and ITS region 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: Figures 3-5 As shown, the gene sequencing results of the products corresponding to the 18S rRNA region, the psaB region, and the ITS region have a query coverage of 100%, 98%, and 98% respectively, and a per-identity of 95.99%, 95.18%, and 96.33% respectively, with an E value of 0 for all of them.

[0079] The gene sequences of 18S rRNA, psaB, and ITS of this algal strain are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively. The phylogenetic trees of the gene sequences of 18S rRNA, psaB, and ITS of this algal strain using the joint neighbor-joining method are shown in the figures below. Figures 6-8 As shown, the NCBI accession numbers for the gene sequences of 18S rRNA, psaB, and ITS of this algal strain are PQ002471, PQ046780, and PQ035102, respectively.

[0080] Analysis showed that this algal strain belongs to the same evolutionary branch as Dysmorphococcus globosus.

[0081] 3. Preservation of algal strains

[0082] Based on the combined results of morphological observation and molecular identification, the algal strain obtained in this embodiment belongs to the genus *Dysmorphococcus globosus*. This strain is named *Dysmorphococcus globosus* RXH-ZY24 and was deposited on April 7, 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 2024627.

[0083] Example 4: Zinc tolerance test of Heteromorpha RXH-ZY24

[0084] 1. Experimental Materials

[0085] The heteromorphic algae RXH-ZY24 (i.e., ZY24) from Example 3 was used in the treatment group of this example.

[0086] 2. Experimental Methods

[0087] 2.1 Treatment with different zinc concentrations

[0088] ZY24 cells were cultured to the late logarithmic growth phase, then replaced with fresh modified BG11 liquid culture medium, and the initial algal cell concentration was adjusted to OD680 = 0.5. The cells were then dispensed into 300 mL Erlenmeyer flasks for subsequent treatment experiments.

[0089] Different concentrations of zinc were used for treatment with ZnSO4·7H2O, with concentrations of 0 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 800 mg / L and 1600 mg / L, respectively. The culture temperature was 25±1℃, the culture speed was 150~180 rpm, and the heterotrophic culture was carried out continuously for 7 days without light.

[0090] 2.2 Determination of microalgal growth curves

[0091] A certain amount of fresh algal solution was taken, and the optical density value of ZY24 cells at 680nm was measured using an ELISA reader (Metash, UV-9000, China), and a growth curve was plotted.

[0092] 2.3 Chlorophyll Fluorescence Analysis

[0093] 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.

[0094] 2.4 Determination of total zinc content

[0095] Weigh 100±1 mg of freeze-dried algae powder and determine and calculate its content using the first method of inductively coupled plasma mass spectrometry (ICP-MS) according to the national standard (GB 5009.268-2016).

[0096] 3. Experimental Results

[0097] 3.1 ZY24 exhibits a certain degree of tolerance to zinc ion stress.

[0098] This example investigated the effects of different zinc concentrations (ZnSO4·7H2O, at concentrations of 0 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 800 mg / L, and 1600 mg / L) on the growth characteristics of ZY24 (treatment group) algal cells under heterotrophic, light-free continuous culture for 7 days. The results are as follows: Figures 9-12 As shown. Specifically, compared to the 0 mg / L ZnSO4·7H2O treatment, the color of the culture medium of ZY24 changed from green to brownish-green after treatment with 200–1600 mg / L ZnSO4·7H2O (e.g., ...). Figure 9 As shown in A), the color of the algal cells changed from green to light brown (as shown in A). Figure 9 As shown in B), cell optical density values ​​(e.g.) Figure 10 (as shown), maximum photosynthetic efficiency Fv / Fm (as shown) Figure 11 (as shown in B) and the actual photosynthetic efficiency Y(II) (as shown in B) Figure 11 As shown in C, chlorophyll fluorescence gradually decreased with increasing treatment time (e.g., as shown in Figure C). Figure 11 (As shown in A) also changed from blue-green to orange-red; slightly different, after treatment with 100 mg / L ZnSO4·7H2O, the ZY24 culture medium remained green, but the color of its algal cells also changed to light brown. The cell optical density value, maximum photosynthetic efficiency Fv / Fm and actual photosynthetic efficiency Y(II) also gradually decreased, and the degree of decrease in some indicators was not as significant as the results after treatment with 200-1600 mg / L ZnSO4·7H2O.

[0099] Overall, ZY24 exhibits a certain degree of tolerance to 100–1600 mg / L ZnSO4·7H2O.

[0100] 3.2ZY24 has a high zinc enrichment capacity.

[0101] This embodiment also investigated the zinc enrichment capacity of ZY24 after continuous dark incubation for 7 days at concentrations of 0 mg / L ZnSO4·7H2O, 200 mg / L ZnSO4·7H2O, and 1600 mg / L ZnSO4·7H2O, respectively. The results are as follows: Figure 12 As shown in the figure, specifically, ZY24 was cultured for 7 days at 200 mg / L ZnSO4·7H2O and 1600 mg / L ZnSO4·7H2O, and the total zinc accumulation was 1.519 g / kg and 22.453 g / kg, respectively.

[0102] Therefore, it can be seen that ZY24 has a high zinc enrichment capacity.

[0103] Example 5: Test on the synergistic enrichment effect of heteromorphic algae RXH-ZY24 on zinc and carotenoids.

[0104] 1. Experimental Methods

[0105] 1.1 Treatment with different zinc concentrations

[0106] The heteromorphic algae RXH-ZY24 (i.e., ZY24) in Example 3 was cultured to the late logarithmic growth phase, replaced with fresh modified BG11 liquid culture medium, and the initial concentration of algal cells was adjusted to OD680 = 0.5. The cells were then dispensed into 300 mL Erlenmeyer flasks for subsequent treatment experiments.

[0107] Different concentrations of zinc were used for treatment with ZnSO4·7H2O at concentrations of 0 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, and 800 mg / L. The incubation temperature was 25 ± 1℃, the incubation speed was 150–180 rpm, and the incubation volume was 600 μmol / m³. -2 / s -1 Cultured under continuous light for 7 days.

[0108] 1.2 Determination of Carotenoid Content

[0109] Determination of carotenoid content: 50±5 mg of freeze-dried algal powder was weighed, and astaxanthin and other carotenoids were extracted according to the national standard (GB / T311520-2015). Carotenoid content was detected by HPLC-DAD and LC-QTOF-MS, and quantification was performed using reference standards. The standards were purchased from Sigma-Aldrich.

[0110] It should be noted that the other experimental methods involved in this embodiment are the same as those in Embodiment 4, and will not be repeated here.

[0111] 2. Experimental Results

[0112] 2.1 Effects of different zinc concentrations on the growth of ZY24 under illumination

[0113] The initial concentration of ZY24 cells was OD680 = 0.5, 600 μmol / m³. -2 / s -1 After 7 days of continuous light cultivation, the results showed that treatments with 200 mg / L ZnSO4·7H2O, 400 mg / L ZnSO4·7H2O, and 800 mg / L ZnSO4·7H2O all altered the macroscopic phenotype and microscopic cellular color of the microalgae to an orange-red color (e.g., ...). Figures 13-14 As shown), the cell optical density value decreased significantly (e.g. Figure 15 (As shown); In contrast, the color changes of microalgal cells were not obvious under treatments of 0 mg / L ZnSO4·7H2O and 100 mg / L ZnSO4·7H2O (as shown). Figures 13-14 As shown), the cell optical density value increased slightly (e.g. Figure 15 (As shown).

[0114] The above results indicate that treatment with 200–800 mg / L ZnSO4·7H2O promotes the accumulation of colored pigments in ZY24 algal cells and limits their cell growth.

[0115] 2.2 Analysis of the effect of different zinc concentrations on chlorophyll fluorescence in ZY24 chlorophyll under light irradiation

[0116] The results are as follows Figures 16-17 As shown, under different concentrations of zinc under illumination, the maximum photosynthetic efficiency Fv / Fm and the actual photosynthetic efficiency Y(II) of ZY24 generally showed a decreasing trend. Compared with the 0–100 mg / L ZnSO4·7H2O treatment, the decrease was more significant with 200–800 mg / L ZnSO4·7H2O, indicating that the 200–800 mg / L ZnSO4·7H2O treatment had a significant stress effect on microalgal photosynthesis.

[0117] 2.3 Effects of different zinc concentrations under light on the enrichment of zinc and carotenoids in ZY24

[0118] The results are as follows Figure 18 As shown, under illumination, the total zinc content enriched by microalgae increased with the increase of zinc ion treatment concentration, indicating that ZY24 has a good effect on enriching zinc ions.

[0119] At the same time, such as Figure 19 The detection results of carotenoids such as astaxanthin in ZY24 showed that it contained carotenoids such as β-carotene, β-cryptoxanthin, zeaxanthin, astaxanthin, canthaxanthin, and lutein. With the increase of zinc ion treatment concentration, the precursor β-carotene gradually synthesized more high-value pigments such as β-cryptoxanthin, zeaxanthin, canthaxanthin, and astaxanthin. Therefore, the content of β-carotene gradually decreased, while the contents of β-cryptoxanthin, zeaxanthin, canthaxanthin, and astaxanthin gradually increased. The treatment with 200 mg / L ZnSO4·7H2O was the dividing concentration point for pigment accumulation. When the concentration was 400-800 mg / L, the astaxanthin content began to decrease, possibly because the high concentration of zinc ions caused some cells to die, thereby reducing the production of astaxanthin.

[0120] In conclusion, the algae RXH-ZY24 can promote the accumulation of carotenoids such as astaxanthin while enriching zinc, and the treatment with 200 mg / L ZnSO4·7H2O is the optimal concentration for its overall quality.

Claims

1. Dysmorphococcus globosus RXH-ZY24, characterized in that The heteromorphic alga RXH-ZY24 was deposited at the China Center for Type Culture Collection on April 7, 2024, and its deposit number is CCTCC NO: M2024627.

2. Use of the heteromorphic alga RXH-ZY24 as described in claim 1, characterized in that, Use the heteromorphic alga RXH-ZY24 to enrich at least one of zinc and carotenoids in a zinc ion stress environment; Among them, in the zinc ion stress environment, the zinc ion concentration is 100-1600 mg / L; The carotenoid is at least one of β-cryptoxanthin, zeaxanthin, canthaxanthin, and astaxanthin.

3. The application according to claim 2, characterized in that Including: Use the heteromorphic alga RXH-ZY24 to enrich zinc in a zinc ion stress environment.

4. The application according to claim 3, wherein Including: When using the heteromorphic alga RXH-ZY24 to enrich zinc in a zinc ion stress environment, perform heterotrophic culture or mixotrophic culture on the heteromorphic alga RXH-ZY24.

5. The application according to claim 2, characterized in that, Including: Use the heteromorphic alga RXH-ZY24 to co-enrich zinc and carotenoids in a zinc ion stress environment.

6. The application according to claim 5, wherein Including: When using the heteromorphic alga RXH-ZY24 to co-enrich zinc and carotenoids in a zinc ion stress environment, perform mixotrophic culture on the heteromorphic alga RXH-ZY24.

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