A strain of arsenic accumulating and arsenic oxidizing tetraselmis subcordiformis and its application

Abstract

The application discloses a strain of arsenic enrichment and arsenic oxidation tetraselmis suecica A2, which can enrich arsenic and oxidize As(III). The tetraselmis suecica A2 can oxidize As(III) into As(V), and the cell surface adsorption capacity of the tetraselmis suecica A2 to arsenic is also very strong. The tetraselmis suecica A2 can be used for treating arsenic contaminated soil, reducing the toxic effect of arsenic in the soil, and especially applied to oxidation of As(III) in the arsenic contaminated soil under the condition of flooding.

Description

Technical Field

[0001] This invention relates to the field of microalgae technology, and in particular to an arsenic-enriching and arsenic-oxidizing algae and its applications. Background Technology

[0002] Arsenic (As) is a toxic metalloid. Due to human activities such as mining and the application of pesticides and fertilizers, the concentration of arsenic in soil is increasing, causing varying degrees of soil arsenic pollution worldwide. The toxicity, mobility, and fate of arsenic in the environment depend on its form, and changes in arsenic form are usually driven by microbial processes in the environment. In flooded paddy field soils, the reductive dissolution of iron oxides and the reduction of pentavalent arsenic (As(V)) to As(III) promote the release of arsenic from the soil solid phase into pore water, increasing the bioavailability of arsenic. Therefore, rice is more susceptible to arsenic contamination than other crops.

[0003] In response to the severe environmental problem of soil arsenic pollution, several remediation methods have been developed both domestically and internationally. Currently, the main methods used for soil arsenic pollution remediation are traditional physicochemical methods and bioremediation methods. Physicochemical methods are expensive and not suitable for large-scale purification and promotion. Bioremediation utilizes the metabolic activities of organisms and their metabolic products to passivate, reduce toxicity, or remove arsenic from the soil through enrichment and transformation. Because bioremediation is low-cost, can be carried out directly in situ, and does not cause secondary pollution, it is increasingly attracting the attention of researchers. In flooded soils, strategies to reduce the effectiveness of arsenic include promoting the bioaccumulation of arsenic and promoting the oxidation of As(III) to reduce its toxicity and activity.

[0004] Algae are a fundamental component of terrestrial and aquatic ecosystems, characterized by rapid growth and strong environmental adaptability. Some microalgae, such as Synechocystis and Chlorella, possess strong arsenic adsorption and accumulation capabilities, removing more than 51% of inorganic arsenic from water. Therefore, isolating and screening microalgae with highly efficient arsenic accumulation and oxidation capabilities can promote the oxidation and fixation of As(III) in the rice rhizosphere, thereby reducing the availability of arsenic and the accumulation of arsenic in rice grains, which is of great significance for the remediation of arsenic-contaminated soil. Summary of the Invention

[0005] The first aspect of this invention provides a strain of *Tetracyclis obliqueis* A2, which is an algal strain deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024171. It may be a variant strain that has essentially the same characteristics as the algal strain with accession number CCTCC NO: M 2024171.

[0006] The *Tetrahaemonopteris* A2 in this invention was isolated from paddy field soil in South China. Based on morphological and 18S rRNA sequence analysis, A2 belongs to the genus *Tetrahaemonopteris*. *Tetrahaemonopteris* A2 possesses excellent arsenic enrichment and As(III) oxidation capabilities. Through efficient arsenic enrichment and As(III) oxidation, it can reduce the dissolved arsenic concentration in its growth environment and can be used for the remediation of arsenic-contaminated water and / or soil.

[0007] The second aspect of this invention provides a method for preparing a culture of *Tetracyclis obliquus* A2, comprising: Provide liquid culture systems; The first aspect of the *Tetrahaemolyticus* A2 was inoculated into the liquid culture system for cultivation.

[0008] In one or more embodiments of the present invention, the liquid culture system contains nutrients, including nitrogen, phosphorus, and trace metal elements. For example, the nutrients can be provided in the form of a liquid culture medium selected from either BG-11 medium (BG-11 Medium for Blue Green Algae) or BBM medium (Bold's Basal Medium).

[0009] BG-11 medium, also known as blue-green medium or blue-green algae medium, is used to culture freshwater algae and protozoa, and is one of the best media for culturing cyanobacteria such as Synechococcus elongata.

[0010] In one or more embodiments of the present invention, the pH of the liquid culture system is 6.5-7.5 during the culture process.

[0011] In one or more embodiments of the present invention, the culture temperature is 20-30°C, preferably 25°C.

[0012] In one or more embodiments of the present invention, the culture humidity is 50%-70%, preferably 60%.

[0013] In one or more embodiments of the present invention, the cultivation light intensity is 1500-2500 lux, preferably 2000 lux.

[0014] In one or more embodiments of the present invention, the culture light-dark cycle is from 12 h / 12 ​​h to 24 h / 0 h, such as 12 h / 12 ​​h, 13 h / 11 h, 14 h / 10 h, 15 h / 9 h, 16 h / 8 h, 17 h / 7 h, 18 h / 6 h, 19 h / 5 h, 20 h / 4 h, 21 h / 3 h, 22 h / 2 h, 23 h / 1 h, or 24 h / 0 h.

[0015] In one or more embodiments of the present invention, the inoculation cell density of the *Tetracyclis obliquus* A2 is 1 × 10⁻⁶. 3 -1×10 7 Cells / mL, preferably 1×10⁻⁶ 4 -1×10 6 per mL.

[0016] The third aspect of the present invention provides a culture of *Tetracyclis obliqueensis* A2, which is prepared by the preparation method of the second aspect.

[0017] The fourth aspect of the present invention provides a microalgae preparation comprising either the first aspect of *Tetracyclis aquaticus* A2 or the third aspect of *Tetracyclis aquaticus* A2 culture.

[0018] In one or more embodiments of the present invention, the microalgae preparation further comprises a metal oxide selected from at least one of iron oxides (hydrothermalite, goethite, ferrihydrite, hematite), manganese oxides (sodium manganite, pyrolusite, and pyrolusite), and aluminum oxides (gibbsite, boehmite, diaspore).

[0019] In one or more embodiments of the present invention, the microalgae preparation further comprises an adjuvant selected from at least one of a extender, solvent, diluent, emulsifier, dispersant, binder, fixative, wetting agent, dye, pigment, defoamer, preservative, secondary thickener, and adhesive.

[0020] The fifth aspect of the present invention provides the application of any one of the following aspects of the first aspect of *Tetrahays obliquus* A2, the third aspect of *Tetrahays obliquus* A2 culture, and the fourth aspect of microalgae preparation: (1) enriching arsenic; (2) preparing products enriched with arsenic; (3) oxidizing arsenic; (4) preparing products oxidized with arsenic; (5) treating arsenic pollution; (6) preparing products for treating arsenic pollution.

[0021] In one or more embodiments of the present invention, the enrichment of arsenic refers to the adsorption of arsenic by the algal cells of the oblique tetra-chain algae A2, wherein the adsorbed arsenic includes As(III) and As(V).

[0022] In one or more embodiments of the present invention, the arsenic oxide refers to the oxidation of As(Ⅲ) to As(Ⅴ).

[0023] In one or more embodiments of the present invention, the arsenic pollution includes arsenic pollution in water bodies and arsenic pollution in soil.

[0024] In one or more embodiments of the present invention, the soil is waterlogged soil (such as paddy field soil).

[0025] In one or more embodiments of the present invention, the treatment of arsenic pollution refers to the treatment of arsenic pollution by enriching arsenic and oxidizing arsenic.

[0026] In one or more embodiments of the present invention, the enriched arsenic, the arsenic oxide, and the arsenic in the arsenic pollution include inorganic and organic forms of arsenic. The inorganic forms of arsenic include arsenates and arsenites, and the organic forms of arsenic include dimethylarsenic acid (DMAA) and monomethylarsenic acid (MMAA). The enriched arsenic, the arsenic oxide, and the arsenic in the arsenic pollution specifically refer to inorganic forms of arsenic.

[0027] The sixth aspect of the present invention provides a method for treating arsenic pollution, comprising treating arsenic-polluted water and / or soil using any one of the following: *Tetracyclis aquaticus* A2 of the first aspect, *Tetracyclis aquaticus* A2 culture of the third aspect, and microalgae preparation of the fourth aspect.

[0028] In one or more embodiments of the present invention, the method includes inoculating the *Tetracyclis obliquus* A2 into the water body and / or soil that needs to be treated.

[0029] The beneficial effects of this invention are: This invention provides a strain of *Tetracyclis obliqueis* A2 isolated from paddy field soil in South China, capable of accumulating arsenic and oxidizing arsenic. *Tetracyclis obliqueis* A2 can oxidize As(III) to As(V), and its cell surface has a strong adsorption capacity for both As(III) and As(V). It can be used for the remediation of arsenic-contaminated soil, reducing the bioavailability and toxicity of arsenic in the soil, especially for the oxidation and fixation of As(III) in arsenic-contaminated soil under flooded conditions. Attached Figure Description

[0030] Figure 1 These are fluorescence micrographs of arsenic-enriched and arsenic-oxidizing algal strain A2.

[0031] Figure 2 This is a phylogenetic analysis of arsenic enrichment and arsenic-oxidizing algal strain A2.

[0032] Figure 3 The curves show the arsenic enrichment and oxidation of As(III) by algal strain A2.

[0033] Figure 4 This is the growth curve of arsenic-enriched and oxidized algal strain A2.

[0034] Figure 5 The images show the intracellular (a) and extracellular (b) arsenic forms of arsenic enrichment and oxidation in algal strain A2.

[0035] Figure 6These are adsorption isotherms of arsenic enrichment and oxidation algae A2, where (a) is the Langmuir and Freundlich model fitting the isothermal adsorption of As(III) by algae, and (b) is the Langmuir and Freundlich model fitting the isothermal adsorption of As(V) by algae.

[0036] Figure 7 The arsenic forms in the overlying water (a) and pore water (b) of paddy soil are shown. Detailed Implementation

[0037] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0038] Example 1: Isolation and purification of arsenic-enriched and oxidizing microalgae from paddy field soil (1) Sample collection Samples were collected from the topsoil and surface water of paddy fields in Shaoguan City, Guangdong Province. The surface soil was collected at a depth of 0-20 cm, sealed in sterile self-sealing bags, placed on ice, and transported back to the laboratory for algae screening.

[0039] (2) Enrichment culture of As(III) tolerant microalgae Weigh 1.0 g of fresh paddy soil and place it in 100 mL of sterilized BG-11 liquid culture medium, shaking overnight in a shaker. Take 1 mL of soil leachate or 1 mL of top dressing water and add it to 100 mL of sterilized BG-11 liquid culture medium. Place the mixture in an artificial climate incubator under the following conditions: temperature 25℃, light intensity 2000 lux, and light / dark cycle 12 h / 12 ​​h. The final concentration of sodium arsenite in the BG-11 liquid culture medium was 0.1 mg / L. During the experiment, shake the conical flask twice daily (09:00 and 17:00), randomly changing the position of the flask to ensure even light exposure. Strict aseptic technique was followed during soil inoculation and culture.

[0040] Arsenic-resistant monoalgae were isolated using a serial dilution plate spread method. Sodium arsenite was added to BG-11 solid medium to achieve a final As(III) concentration of 0.1 mg / L. In a clean bench, 1 mL of enrichment culture was taken and serially diluted with BG-11 liquid medium. 100 μL of the diluted solution was then placed on a sterilized BG-11 solid medium plate and spread evenly over the entire plate using a spreader. The plate was incubated upside down in a 25°C light incubator at a light intensity of 2000 lux and a light / dark cycle of 12 h / 12 ​​h.

[0041] In a clean bench, single algal colonies of varying sizes and shapes (dark green or yellowish-green) with good growth were picked using an inoculation loop and purified by streak plating. These colonies were then cultured further in BG-11 solid medium containing As(III). The streak plating process was repeated until single algal colonies were obtained. The microstructure of the algae was observed using a colony counter and microscope to confirm the absence of contaminating bacteria or algae. Single-clone algal strains were then inoculated into 100 mL of sterilized BG-11 liquid medium for scale-up culture to obtain an algal suspension. The scale-up culture conditions were as follows: temperature 25℃, light intensity 2000 lux, and light / dark cycle 12 h / 12 ​​h.

[0042] The BG-11 liquid culture medium used for the enrichment culture and isolation and purification of algae in the overlying water and soil is prepared as shown in Tables 1 and 2.

[0043] Table 1. Formulation of BG-11 liquid culture medium

[0044] Table 2 Formula for A5 Trace Element Solution

[0045] The difference between BG-11 solid medium and BG-11 liquid medium is that BG-11 also contains 15 g / L of agar powder.

[0046] Example 2: Classification and identification of arsenic-enriched and arsenic-oxidizing algal strains (1) Morphological observation The pure algae isolated in Example 1 were inoculated into sterile BG-11 liquid medium and cultured in a 25°C light incubator until the logarithmic growth phase, with a light intensity of 2000 lux and a light / dark cycle of 12 h / 12 ​​h. Two mL of the microalgae culture medium in the logarithmic growth phase was observed under a fluorescence microscope, and the results are as follows. Figure 1 As shown, the microalgal colonies selected are round in shape, and the algal cells are spindle-shaped.

[0047] Table 3 Morphology of Algal Strains A2

[0048] Based on the above morphological characteristics, this algal species belongs to the phylum Chlorophyta ( Chlorophyta ), Chlorophyta ( Chlorophyceae ), Cyclophyceales ( Sphaeropleales ), Scenedesmus family ( Scenedesmaceae ), Tetrachainella ( Tetradesmus ).

[0049] (2) 18S rRNA sequence and phylogenetic tree analysis Genomic DNA was extracted from algal strain A2 using the Ezup column-based fungal genomic DNA extraction kit, and the 18S rRNA gene fragment was amplified. The 18S rRNA of algal strain A2 was amplified using universal algal 18S rRNA primers 18S-F (5'-ACCTGGTTGATCCTGCCAGT-3') (SEQ ID NO:1) and 18S-R (5'-TCACCTACGGAAACCT TGT-3') (SEQ ID NO:2). The amplified products were subjected to agarose gel electrophoresis. A clear band appeared around 1700 bp for algal strain A2. The PCR products were recovered and sequenced. A phylum of Scenedesmus (Sclerotium spp.) was constructed using the MEGA5 maximum parsimony method. Scenedesmaceae The phylogenetic tree of ) is shown in the following figure. Figure 2 As shown.

[0050] Based on the above morphological and 18S rRNA gene sequence analysis results, this algal strain should belong to the genus Tetracycla (Tetracycla). Tetradesmus ), named *Tetracyclis obliqueis* ( Tetradesmus obliquus A2, taxonomically named Tetradesmus obliquus A2. It was deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on January 22, 2024, with accession number CCTCC NO: M 2024171.

[0051] The 18S rRNA sequence of *Tetracyclis obliqueis* A2 is as follows:

[0052] Example 3: Identification of the arsenic oxidation characteristics of *Tetracyclis obliquus* A2 The culture medium formulation was the same as in Example 1. An algal suspension of *Tetracyclis obliqueis* A2 was inoculated into sterilized BG-11 liquid medium to achieve a cell count of 6.6 × 10⁻⁶. 4 The culture medium was incubated with 1 mg / L and 5 mg / L As(III) at a concentration of 1 / mL, and then placed in a 25°C light incubator with a light intensity of 2000 lux, humidity of 60%, and a light / dark cycle of 12 h / 12 ​​h. A control group without inoculation was also included, and each treatment was performed in triplicate. Samples were taken on days 0, 2, 4, 7, and 10, and the concentrations of As(III) and As(V) in the culture medium were determined by liquid chromatography-atomic fluorescence spectrometry.

[0053] The test results are as follows Figure 3 As shown. From Figure 3 It can be seen that under light conditions, algal strain A2 can oxidize 48% of 1 mg / L As(III) and 27% of 5 mg / L As(III) within 10 days. Excluding As(III) oxidation caused by abiotic processes, algal strain A2 can oxidize 25-92 μg / L of As(III) to As(V) per day.

[0054] Example 4: As(III) tolerance test of *Tetracyclis obliqueis* A2 Using BG-11 liquid medium as the basal medium, sodium arsenite was added to adjust the As(III) concentration in the BG-11 medium to 1 and 5 mg / L, respectively. Algal suspension of *Tetracyclis obliqueensis* A2 was inoculated to achieve a cell count of 6.6 × 10⁻⁶. 4 Cells / mL were collected and cultured in a 25℃ light incubator for 10 days under a light intensity of 2000 lux and a humidity of 60%, with a light / dark cycle of 12 h / 12 ​​h. Samples were taken every 24 h to measure the algal strain at a wavelength of 680 nm (OD). 680 nm The absorbance value at ) was calculated. A control group was set up without the addition of sodium arsenite, and three replicates were set up for each treatment.

[0055] The results showed that *Tetracyclis obliqueis* A2 was able to grow in media containing 1 and 5 mg / L As(III) (e.g., Figure 4 As shown), and compared with the control group without As(III), the OD of *Tetracyclis obliqueis* A2 on day 10 was... 680 nm The reduction of only 5% indicates that Alternaria obliqueiformis A2 has excellent tolerance to As(III).

[0056] Example 5: Arsenic Adsorption Capacity Test of Alternaria obliqueiformis A2 Inoculate a suspension of *Tetracyclis obliqueis* A2 into BG-11 liquid medium until the algal cell count reaches 6.6 × 10⁻⁶. 4 Algae were inoculated at a concentration of 1 mg / L and 5 mg / L of As(III) into BG-11 medium. The medium was then incubated at 25°C under a light intensity of 2000 lux and a relative humidity of 60%, with a 12 h / 12 ​​h light / dark cycle. A control group without inoculation was also included, and each treatment was repeated in triplicate. On day 10, the algal suspension was collected, and the collected algal cells were centrifuged. Arsenic bound to the algal cell surface was extracted using PBS buffer. The eluent was collected, and the arsenic speciation was determined using liquid chromatography-atomic fluorescence spectrometry. Algal cells were collected, freeze-dried, and then extracted with dilute nitric acid at 90°C for 10 min. The algal cells and extract were collected by centrifugation, and the extraction was repeated three times. All supernatants were collected, filtered, and the arsenic speciation (intracellular arsenic speciation) was determined.

[0057] The results showed that *Tetracyclis obliqueensis* A2 could accumulate arsenic concentrations as high as 190 mg / kg under 5 mg / L As(III) stress. Extracellular adsorption of arsenic accounted for 97% of the total arsenic accumulation in the algal cells, indicating that extracellular adsorption was the main method of arsenic accumulation in *Tetracyclis obliqueensis* A2 algal cells. Figure 5 In the extracellular adsorption of algal cells, As(V) accounts for approximately 54%-64% of the total arsenic, indicating that *Tetracyclis obliqueensis* A2 is a good adsorbent for both As(III) and As(V). These results suggest that *Tetracyclis obliqueensis* A2 can simultaneously oxidize As(III) and adsorb arsenic during its growth.

[0058] Example 6: Adsorption isotherm of arsenic by *Tetracyclis obliquus* A2 The algal strain isolated and purified in Example 1 was inoculated into sterile BG-11 liquid medium and cultured in a 25°C light incubator until the logarithmic growth phase, with a light intensity of 2000 lux and a light / dark cycle of 12 h / 12 ​​h. The algal suspension in the logarithmic phase was collected, centrifuged at 4°C and 4000 rpm for 10 min, the supernatant was discarded, and the algal cells were resuspended in ultrapure water. The absorbance at 680 nm was measured using 0.2 mL of the algal suspension and adjusted to OD0.05. 680 =0.3. Subsequently, algal suspension and As(III) or As(V) solutions of different concentration gradients were added to centrifuge tubes, and the pH was adjusted to 7.0. The concentrations of As(III) or As(V) in the adsorption reaction system were 0, 0.5, 1, 2.5, 5, 7.5, 10 and 15 mg / L, respectively.

[0059] The reaction system was placed in a constant-temperature shaker at 25 ℃ (260 rpm) for adsorption experiments. Samples of 2 mL were taken at 0 h and 24 h, filtered, and the total arsenic content in the solution was determined. Finally, the adsorption processes of As(III) and As(V) by the algae were fitted and analyzed using Langmuir and Freundlich isotherm adsorption models, respectively.

[0060] The isothermal adsorption curve of arsenic for *Tetracyclis obliqueis* A2 is as follows: Figure 6 As shown in the figure. (a) The Langmuir and Freundlich models fit the isothermal adsorption of As(III) by the algae, and (b) The Langmuir and Freundlich models fit the isothermal adsorption of As(V) by the algae. The results show that the maximum adsorption capacity (Q) of *Tetracyclis obliquus* A2 for As(III) and As(V) is... max The concentrations of As(V) and As(III) were 3.42 mg / g and 3.68 mg / g, respectively, indicating that the adsorption capacity of As(V) by *Tetracyclis obliquus* A2 was slightly higher than that of As(III).

[0061] Example 7: Test on the ability of *Tetrahays obliqueii* A2 to transform arsenic speciation in flooded soil 500 g of paddy soil (arsenic concentration 15.5 mg / kg) and 700 mL of ultrapure water were mixed in a 2 L glass beaker and pre-cultured for 7 days to promote the release and reduction of arsenic in the paddy soil. The experiment was divided into three groups: the first group was paddy soil cultured at 25℃ under dark conditions (Soil+dark); the second group was paddy soil cultured in a 25℃ artificial climate chamber with a 12 h / 12 ​​h light-dark cycle (Soil); and the third group was inoculated with 100 mL of fresh *Tetracyclis obliquus* A2 algal suspension and cultured in a 25℃ artificial climate chamber for 30 days with a 12 h / 12 ​​h light-dark cycle (Soil+A2) and a light intensity of 2000 lux. Each treatment had three biological replicates. Overlying water was collected using a syringe on days 0, 10, 20 and 30 of cultivation. Soil pore water was collected using a pore water collector and filtered through a 0.22 μm aqueous filter membrane. The concentration of As(III) in the overlying water and pore water was determined using atomic fluorescence-liquid chromatography. Arsenic speciation in paddy soil was determined using a sequential extraction method. 2.0 g of moist soil was weighed and placed in a 50 mL centrifuge tube. The following solutions were added sequentially: 8 mL of 1 mol / L magnesium chloride, 15 mL of 1 mol / L sodium acetate, 40 mL of 0.2 mol / L ammonium oxalate, 40 mL of 4 mol / L nitric acid, 40 mL of 0.2 mol / L ammonium oxalate (containing 0.2 mol / L ascorbic acid), 20 mL of 2 mol / L sodium pyrophosphate (pH 1.0), 20 mL of 2 mol / L sodium pyrophosphate (pH 10.0), and 19 mL of a nitric acid-perchloric acid-hydrofluoric acid mixture. After each extraction step, the supernatant was collected by centrifugation, and the arsenic content was determined by atomic fluorescence spectrometry.

[0062] The test results are as follows Figure 7 As shown. From Figure 7 It was found that in soil inoculated with A2 algae, the As(III) concentration in the overlying water was 0 μg / L on day 30, and the As(III) concentration in the pore water was 45% and 38% of that in the Soil+dark and Soil treatment groups, respectively. This indicates that inoculating the soil with *Tetracyclis obliquus* A2 can significantly reduce the As(III) concentration in both the overlying water and the pore water. In soil inoculated with *Tetracyclis obliquus* A2, the amount of arsenic bound to iron and aluminum oxides (including amorphous and crystalline iron oxides) was significantly increased compared to the Soil+dark and Soil treatment groups, indicating that inoculation with *Tetracyclis obliquus* A2 can also promote the adsorption and fixation of arsenic by metal oxides in the soil.

[0063] Table 4. Changes in the speciation of arsenic in soil after inoculation with *Tetraodon obliquus* A2 under flooded conditions.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. *Tetrachainia obliqueis* ( Tetradesmus obliquus A2, the oblique tetra-chain algae A2 is an algal strain preserved at the China Center for Type Culture Collection, with accession number CCTCC NO: M2024171.

2. A method for preparing the A2 culture of *Tetracyclis obliqueis*, including: Provide liquid culture systems; The *Tetrahaemophyta* A2 of claim 1 is inoculated into the liquid culture system for cultivation.

3. The preparation method according to claim 2, characterized in that, The liquid culture system contains nutrients, including nitrogen, phosphorus, and trace metal elements.

4. The preparation method according to claim 2, characterized in that, The culture temperature is 20-30℃.

5. The preparation method according to claim 2, characterized in that, The culture humidity is 50%-70%.

6. The preparation method according to claim 2, characterized in that, The cultivation light intensity is 1500-2500 lux.

7. A culture of *Tetracyclis obliqueensis* A2, wherein the *Tetracyclis obliqueensis* A2 culture is prepared by the preparation method according to any one of claims 2-6.

8. A microalgae preparation comprising the *Tetraodon obliquus* A2 of claim 1 or the *Tetraodon obliquus* A2 culture of claim 7.

9. The microalgae preparation according to claim 8, characterized in that, The microalgae preparation also contains a metal oxide, which is selected from at least one of iron oxide, manganese oxide, and aluminum oxide.

10. The use of any one of the following in any of the following aspects: (1) enriching arsenic; (2) preparing products enriched with arsenic; (3) oxidizing arsenic; (4) preparing products oxidized with arsenic; (5) treating arsenic pollution; (6) preparing products for treating arsenic pollution.

11. The application according to claim 10, characterized in that, The arsenic oxide mentioned refers to the oxidation of trivalent arsenic to pentavalent arsenic.

12. The application according to claim 10, wherein the arsenic pollution includes arsenic pollution in water bodies and arsenic pollution in soil.

13. A method for treating arsenic pollution, comprising treating arsenic-polluted water and / or soil using any one of the following: the *Tetraodon asiaticus* A2 of claim 1, the *Tetraodon asiaticus* A2 culture of claim 7, or the microalgae preparation of claim 8 or 9.

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