Low-temperature chlorella and application thereof

Abstract

The invention relates to the technical field of biological water treatment, and discloses chlorella capable of resisting low temperature of 5-15 DEG C. Even in winter with lower temperature, the chlorella can grow and proliferate at a certain speed. Meanwhile, the chlorella cs-8 can reduce various pollutants in the water body to a lower level, and specifically, the chlorella cs-8 can reduce the ammonia nitrogen content to 0.01 mg / L, the total phosphorus content to 0.13 mg / L and the sulfide content to 0.16 [mu] g / L. The chlorella cs-8 has high-efficiency degradation capability on the various pollutants, and has remarkable advantages and application potential in the aspects of water body pollution treatment and environment restoration. The laccase mutant is designed through mutation at the same time, so that the laccase mutant still keeps high enzyme activity in a low-temperature environment. According to the invention, the chlorella and the laccase mutant are subjected to composite treatment and can be applied to water pollution treatment in winter on a large scale.

Description

Technical Field

[0001] This invention relates to the field of biological water treatment technology, and more specifically to a low-temperature Chlorella and its applications. Background Technology

[0002] United Nations statistics show that freshwater accounts for only 2.6% of the total water volume on Earth, and the proportion of surface freshwater is less than 1%, making freshwater resources extremely precious. In recent years, my country has made significant progress in water pollution control, and many rivers and their surrounding environments have improved. However, some rivers, especially those with poor flow, still exhibit blackening and foul odors after treatment, mainly due to excessively high ammonia nitrogen levels. According to relevant research, water bodies are considered polluted when nitrogen levels exceed 0.5 mg / L. Since Zhejiang Province implemented the "Five Waters Co-Governance" campaign, black and smelly rivers and ditches have gradually disappeared, the environment has significantly improved, and public attention and demands for river water quality have continuously increased. Field investigations of surrounding rivers revealed that although most rivers have beautiful landscape designs and relatively clear water appearances, rapid testing showed that ammonia nitrogen levels in these water bodies remained high. Therefore, effectively reducing ammonia nitrogen levels in rivers will greatly improve the water quality of urban rivers and prevent further deterioration.

[0003] In biological water pollution treatment technologies, Chlorella and laccase have become research hotspots due to their high efficiency, environmental friendliness, and sustainability. Existing research has confirmed that Chlorella plays multiple roles in regulating nitrogen and phosphorus levels in water, inhibiting bacterial growth, and improving river water quality. Chlorella-based wastewater treatment technologies are gradually becoming a new direction for advanced water treatment and resource utilization. However, factors such as temperature, pH, and light intensity significantly affect the growth efficiency of Chlorella. Temperature is a key external factor affecting Chlorella growth and metabolism; high temperatures can accelerate the metabolic rate but slow down biomass accumulation, while low temperatures may inhibit growth. The optimal growth temperature range for most Chlorella species is 20 to 25°C, but in winter, the drop in water temperature poses a challenge to their growth and distribution. Laccase can remove phenolic compounds from water. Laccase can convert phenolic substances (such as degradation products of certain dyes and surfactants) into non-toxic quinones or other products that can be further degraded through oxidation reactions. This effectively removes toxic pollutants from water. However, the activity of laccase decreases significantly at low temperatures, and its purification capacity cannot meet the water purification needs in winter. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, one of the objectives of this invention is to provide a Chlorella strain that can withstand low temperatures of 15°C and reduce the content of ammonia nitrogen, total phosphorus and sulfides in water.

[0005] A low-temperature Chlorella species, wherein the Chlorella species has the preservation number: CCTCC NO: M 2025509; and the strain name is:

[0006] Chlorella Vulgari cs-8; deposited at the China Center for Type Culture Collection on March 17, 2025; deposited at Wuhan University. The described Chlorella can withstand low temperatures of 10–15°C.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The second objective of this invention is to provide a method for treating water bodies polluted by ammonia nitrogen, total phosphorus, sulfides and phenolic compounds.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a composite Chlorella sphere, comprising a sphere wall 3 and a cavity 1, wherein the cavity 1 is located within the sphere wall 3, and the sphere wall 3 is provided with multiple flow holes 2, the cavity 1 communicating with the outside of the sphere wall 3 through the flow holes 2, the cavity 1 containing a composite material, the composite material comprising activated carbon, the Chlorella described in claim 1, and a laccase mutant. The laccase mutant uses the wild-type laccase described in SEQ ID NO:1 as the parent material, and undergoes sequence modification. The wild-type laccase sequence is obtained from the protein database NCBI: GenBank: KAG7430786.1. The laccase mutant uses the wild-type laccase described in SEQ ID NO:1 as the parent material and undergoes the following mutation: P50K+V115Q+A191S+I245Y.

[0010] Preferably, the laccase mutant is based on the wild-type laccase described in SEQ ID NO:1 and undergoes the following mutation: P50K+V115Q+A191S+I245Y+E256G+L396S.

[0011] Preferably, the laccase mutant is based on the wild-type laccase described in SEQ ID NO:1 and undergoes the following mutation: P50K+V115Q+A191S+I245Y+E256G+W466D.

[0012] Preferably, the laccase mutant is based on the wild-type laccase described in SEQ ID NO:1 and undergoes the following mutation: P50K+V115Q+A191S+I245Y+E256G+F540P.

[0013] Preferably, the laccase mutant is based on the wild-type laccase described in SEQ ID NO:1 and undergoes the following mutation: P50K+V115Q+A191S+I245Y+E256G+L396S+W466D+F540P.

[0014] Preferably, the preparation steps of the composite material include: culturing the Chlorella in a liquid culture medium, then adding laccase mutant, mixing evenly, adding activated carbon, mixing evenly, culturing while stirring, and then centrifuging to obtain the composite material.

[0015] Preferably, the volume of the activated carbon is 3 to 8 times the total volume of Chlorella and the laccase mutant.

[0016] Preferably, the volume of the laccase mutant is 1 to 3 times the volume of Chlorella.

[0017] Preferably, the composite chlorella balls are used to treat eutrophic water bodies and / or water bodies polluted by sulfides and / or water bodies polluted by phenolic compounds.

[0018] The beneficial effects of this invention are as follows: This invention screened a Chlorella species that can withstand low temperatures of 5–15℃. Even in the cold winter, this Chlorella can maintain a certain growth and proliferation rate. Simultaneously, Chlorella CS-8 can reduce various pollutants in water to low levels. Specifically, Chlorella CS-8 can reduce ammonia nitrogen content to 0.01 mg / L, total phosphorus content to 0.13 mg / L, and sulfide content to 0.16 μg / L. The highly efficient degradation capacity of Chlorella CS-8 for these pollutants demonstrates its significant advantages and application potential in water pollution control and environmental remediation. Furthermore, the laccase mutant maintains an enzyme activity of over 10 U / mg in low-temperature environments of 5–15℃, enabling efficient degradation of phenolic compounds in water during winter. Chlorella can serve as a substrate environment for laccase enrichment, and laccase degradation products can serve as a carbon source for Chlorella, forming a synergistic cycle. Attached Figure Description

[0019] Figure 1 Photo of Chlorella CS-8 cultured in conical flasks;

[0020] Figure 2 This is a diagram of the morphology of a solid flat plate.

[0021] Figure 3 A streak plate purification graph;

[0022] Figure 4 Light microscopic image of Chlorella CS-8;

[0023] Figure 5 This is an observation image of the Chlorella CS-8 electric well.

[0024] Figure 6 Close-up photo of Chlorella CS-8 conical flask culture;

[0025] Figure 7 This is a growth curve diagram of Chlorella CS-8.

[0026] Figure 8 This is a cross-sectional view of the composite microalgal sphere.

[0027] Explanation of reference numerals in the attached diagram: 1. Cavity; 2. Flow hole; 3. Ball wall. Detailed Implementation

[0028] It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terminology used in this application's specification is for the purpose of describing specific embodiments only and is not intended to limit the application. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments described below are for illustrative purposes only and are not intended to limit the invention.

[0030] Example 1:

[0031] 1. Chlorella screening

[0032] Water samples were collected from different sections of the Jianqiao River in Shangcheng District, Hangzhou City. Multiple water samples were filtered through gauze, sterilized, and the pH was adjusted to 7.5. 200 mL samples were then transferred to Erlenmeyer flasks and placed in a light incubator for aseptic aeration. The light intensity was 3000 Lux, the temperature was 25℃, and the aeration rate was 1 L / min. Figure 1 (As shown). Once the culture medium turned a distinct green, it was transferred to BG-11 medium and incubated at 15℃ under light. After further greening, it was streaked in BG-11 solid medium for separation and incubated in a light incubator for 7 days. Single algal colonies were then picked, inoculated into BG-11 liquid medium, cultured for 7 days, and streaked again to obtain pure *Chlorella vulgaris*. LB medium was used to identify any contamination. The BG-11 medium was purchased from Haibo Biotechnology Co., Ltd. LB liquid medium consisted of 0.5% yeast extract, 1% peptone, and 1% sodium chloride.

[0033] Through repeated plate picking and separation, it was found that a typical green algal patch grew on a certain initial plate. Figure 2 This indicates that Chlorella, which is adapted to a low-temperature environment, has already grown. Further selection of algal patches followed by streaking is then performed. Figure 3 As shown, by Figure 3 It can be seen that the selected Chlorella grew well at low temperatures and was named cs-8.

[0034] 2. Observation of Chlorella cell morphology

[0035] Purified CS-8 Chlorella cells were selected and observed and photographed under an optical microscope to record changes in cell morphology. Simultaneously, another sample was sent to an electron microscope laboratory for photographing to observe the internal morphology and structure of the Chlorella.

[0036] Under an optical microscope, Chlorella typically appears pale green, and its cells are oval in shape. These cells tend to form colonies, although some cells exist singly. When observed more closely under an electron microscope, individual Chlorella CS-8 cells are found to be actually elliptical in shape, with a diameter of 5–6 μm. Mature Chlorella CS-8 cells possess a protein nucleus or starch grain structure, exhibiting strong refractive properties. Simultaneously, during vigorous growth, the endoplasmic reticulum structure is well-developed, and the cell nucleus is not prominent (e.g., ...). Figure 4 and Figure 5 (As shown).

[0037] 3. Screening of Chlorella in liquid culture under low temperature conditions

[0038] River water samples were filtered through gauze, sterilized at high temperature, and then poured into Erlenmeyer flasks in 300 mL increments. Initial inoculation with Chlorella vulgaris was performed at OD500. 680 The pH was adjusted to 7.5, and the cells were placed in a light incubator for aeration culture at a light intensity of 3000 Lux, a temperature of 15℃, and an aeration rate of 1 L / min for 14 days. Samples were taken every 2 days to determine the growth of Chlorella under low-temperature conditions, expressed as OD. 680 express.

[0039] The growth of Chlorella in river water is as follows: Figure 6 and Figure 7 As shown, by Figure 7 It can be seen that CS-8 still grows relatively quickly under low-temperature conditions. Two days after inoculation, *Chlorella CS-8* begins to enter a slow-growth phase. However, from day 4 onwards, the growth rate of *Chlorella CS-8* cells significantly accelerates, entering a rapid logarithmic growth phase. Under laboratory culture conditions, the maximum OD value of this algae can reach approximately 1.2. Observation in conical flasks shows that the water is initially pale green after inoculation, indicating a low number of *Chlorella* cells. After 14 days of culture, the number of *Chlorella* cells significantly increases, as evidenced by a darker color in the culture medium, indicating that *Chlorella* has adapted to the low-temperature growth environment.

[0040] Example 2: Biomass accumulation of different Chlorella species

[0041] The control algae were GY-D19 Chlorella and GY-D28 Chlorella pyrenoidosa ZF strains, purchased from Shanghai Guangyu Biotechnology Co., Ltd.

[0042] GY-D19 Chlorella is a ubiquitous single-celled green alga belonging to the Chlorella genus of the Chlorophyta phylum. It is a spherical, single-celled freshwater alga, 3–8 micrometers in diameter, and is one of the earliest life forms on Earth, appearing over 2 billion years ago. Its genes have remained unchanged throughout its life, making it a highly efficient photosynthetic plant that grows and reproduces through photosynthetic autotrophy. It is widely distributed. Chlorella has the ability to adsorb heavy metals and toxins.

[0043] GY-D28 is a freshwater Chlorella pyrenoidosa ZF strain. This Chlorella pyrenoidosa contains a unique bioactive growth factor—CGF—which promotes cell proliferation and stimulates cell growth. Therefore, Chlorella pyrenoidosa grows relatively quickly.

[0044] River water samples were filtered through gauze, sterilized at high temperature, and then poured into Erlenmeyer flasks in 300 mL systems. The *Chlorella vulgaris* of this invention and the aforementioned control *Chlorella vulgaris* were inoculated into different Erlenmeyer flask systems under the same conditions. The initial inoculum size of *Chlorella vulgaris* was 1%. The pH was adjusted to 7.5, and the flasks were placed in a light incubator for aeration culture at a light intensity of 3000 Lux, a temperature of 15℃, and an aeration rate of 1 L / min for 14 days. After the culture period, the *Chlorella vulgaris* were precipitated by centrifugation at 8000 r / min, and then placed in a desiccator to dry to constant weight. The weight of the dried *Chlorella vulgaris* was measured, and the results are shown in Table 1.

[0045] Table 1. Biomass accumulation of different Chlorella species after 14 days of culture.

[0046]

[0047] As shown in Table 1, the Chlorella CS-8 strain of this invention accumulates biomass significantly faster in river water than the two control Chlorella strains. Chlorella with rapid biomass accumulation is an ideal raw material for biodiesel production. The oil yield of Chlorella is at least seven times that of oil crops such as soybeans, sunflowers, and corn, and its fatty acid composition is highly suitable for biodiesel production. This helps reduce the production cost of biofuels and improve their market competitiveness. Chlorella can also serve as a high-quality protein source for animal feed, with a protein content far exceeding that of common foods such as soybeans and eggs. This not only improves the nutritional value of the feed but also reduces feed costs.

[0048] Example 3: Effects of different Chlorella species on ammonia nitrogen content in water bodies

[0049] River water samples heavily polluted with organic matter were collected. The samples were filtered through gauze, sterilized at high temperature, and the ammonia nitrogen content was determined using the salicylic acid spectrophotometric method (GB7481-87). The ammonia nitrogen content in the river water was determined to be 2.5 mg / L. 300 mL of the river water was added to several identical clean conical flasks. The *Chlorella vulgaris* of this invention and the aforementioned control *Chlorella vulgaris* were inoculated into the conical flask systems, with an initial inoculation amount of 1% for each. The pH was adjusted to 7.5, and the flasks were placed in a light incubator for aeration culture at a light intensity of 3000 Lux, a temperature of 15℃, and an aeration rate of 1 L / min for 14 days. After the incubation period, the *Chlorella vulgaris* were precipitated by centrifugation at 8000 r / min, and the supernatant was used to determine the ammonia nitrogen content. The results are shown in Table 2.

[0050] Table 2. Residual ammonia nitrogen in water after 14 days of culture with different Chlorella species.

[0051]

[0052] According to the "Surface Water Environmental Quality Standard" (GB 3838-2002), the limits for ammonia nitrogen content in water are as follows: When the ammonia nitrogen content in water is below 0.01 mg / L, the water body is not polluted. Class I water quality: ammonia nitrogen standard concentration ≤0.15 mg / L; Class II water quality: ammonia nitrogen standard concentration ≤0.5 mg / L; Class III water quality: ammonia nitrogen standard concentration ≤1.0 mg / L; Class IV water quality: ammonia nitrogen standard concentration ≤1.5 mg / L; Class V water quality: ammonia nitrogen standard concentration ≤2.0 mg / L.

[0053] Table 2 shows that Chlorella CS-8 achieved a 99.6% degradation rate of ammonia nitrogen in the water, resulting in water quality reaching Class I. Class I water quality is primarily suitable for source water and national nature reserves. This type of water can be used as drinking water after simple treatment and disinfection. For river water, reaching Class I or Class II water quality is considered a successful water treatment. Even among the two control Chlorella species, the pyrenoidosa, which has a relatively good capacity for degrading organic matter, still showed an ammonia nitrogen content of 1.547 mg / L after 14 days of degradation, exceeding 1.5 mg / L. Class IV water quality is mainly suitable for general industrial water use and recreational water use where there is no direct human contact, and is not considered high-quality water for fish reproduction.

[0054] Example 4: Effects of different Chlorella species on total phosphorus content in water.

[0055] River water samples heavily polluted with organic matter were collected. The samples were filtered through gauze, sterilized at high temperature, and the total phosphorus content was determined using the ammonium molybdate spectrophotometric method (GB 11893-89). The total phosphorus content in the river water was determined to be 0.55 mg / L. 300 mL of the river water was added to several identical clean conical flasks. The *Chlorella vulgaris* of this invention and the aforementioned control *Chlorella vulgaris* were inoculated into the conical flask systems, with an initial inoculation amount of 1% for each. The pH was adjusted to 7.5, and the flasks were placed in a light incubator for aeration culture at a light intensity of 3000 Lux, a temperature of 15℃, and an aeration rate of 1 L / min for 14 days. After the incubation period, the *Chlorella vulgaris* were precipitated by centrifugation at 8000 r / min, and the supernatant was used to determine the total phosphorus content. The results are shown in Table 3.

[0056] Table 3. Total phosphorus residues in water after 14 days of culture with different Chlorella species.

[0057]

[0058] According to the "Surface Water Environmental Quality Standard" (GB 3838-2002), the limits for total phosphorus content in water are as follows: When the total phosphorus content in water is below 0.01 mg / L, the water body is not polluted. Class I water quality: ≤0.02 mg / L; Class II water quality: ≤0.1 mg / L; Class III water quality: ≤0.2 mg / L; Class IV water quality: ≤0.3 mg / L; Class V water quality: ≤0.4 mg / L.

[0059] Table 3 shows that Chlorella CS-8 achieved a total phosphorus degradation rate of up to 76.4% in the water, and the total phosphorus content was significantly lower than 0.1 mg / L, classifying it as Class III water quality. Class III water quality is mainly applicable to secondary protection zones of centralized drinking water surface water sources, overwintering grounds for fish and shrimp, migration channels, aquaculture areas, and swimming areas. For river water, achieving Class III water quality is considered a successful water treatment. Even among the two control Chlorella species, the pyrenoidosa, which has a better capacity for organic matter degradation, after 14 days of degradation, the total phosphorus content in the water was still 0.39 mg / L, exceeding 0.3 mg / L, classifying this water quality as Class IV. Class IV water quality is mainly applicable to general industrial water areas and recreational water areas where there is no direct human contact, and is not considered high-quality water quality conducive to fish reproduction.

[0060] Example 5: Effects of different Chlorella species on sulfide content in water bodies

[0061] River water samples heavily polluted with sulfides were collected. The samples were filtered through gauze, sterilized at high temperature, and the sulfide content was determined using the methylene blue spectrophotometric method (GB / T 16489-1996). The sulfide content in the river water was determined to be 0.82 mg / L. 300 mL of the river water was added to several identical clean conical flasks. The *Chlorella vulgaris* of this invention and the aforementioned control *Chlorella vulgaris* were inoculated into the conical flask systems, with an initial inoculation amount of 1% for each. The pH was adjusted to 7.5, and the flasks were placed in a light incubator for aeration culture at a light intensity of 3000 Lux, a temperature of 15℃, and an aeration rate of 1 L / min for 14 days. After the incubation period, the *Chlorella vulgaris* were centrifuged at 8000 r / min to precipitate the precipitate, and the supernatant was used to determine the sulfide content. The results are shown in Table 4.

[0062] Table 4. Sulfide residues in water after 14 days of culture with different Chlorella species.

[0063]

[0064] According to the "Surface Water Environmental Quality Standard" (GB 3838-2002), the limits for sulfide content in water are as follows: Class I water quality: ≤0.05 mg / L; Class II water quality: ≤0.1 mg / L; Class III water quality: ≤0.2 mg / L; Class IV water quality: ≤0.5 mg / L; Class V water quality: ≤1 mg / L.

[0065] Table 4 shows that *Chlorella CS-8* achieved a sulfide degradation rate of up to 80.5% in the water, and the sulfide content was significantly lower than 0.2 mg / L, classifying it as Class III water quality. Class III water quality is mainly applicable to secondary protection zones of centralized drinking water surface water sources, overwintering grounds for fish and shrimp, migration channels, aquaculture areas, and swimming areas. For river water, achieving Class III water quality is considered a successful water treatment. Both control *Chlorella* species showed poor sulfide degradation capabilities; after 14 days, the sulfide content in the water remained above 0.5 mg / L, classifying this water quality as Class V. Class V water quality is mainly applicable to agricultural water areas and waters with general landscape requirements, and is not considered high-quality water conducive to fish reproduction.

[0066] Example 6: Low-temperature tolerance of Chlorella CS-8

[0067] Pour 300 mL of BG-11 liquid into an Erlenmeyer flask, sterilize, and inoculate with Chlorella CS-8. Initially, the Chlorella is inoculated with OD. 680The pH was adjusted to 7.5, and the cells were placed in a light incubator with aeration at a light intensity of 3000 Lux and an aeration rate of 1 L / min for 14 days. The only difference between the different systems was the culture temperature; all other conditions were the same. Samples were taken every 2 days to measure the growth of *Chlorella vulgaris* under low-temperature conditions, expressed as OD0.1. 680 express.

[0068] Table 5. OD of Chlorella CS-8 after 14 days of cultivation under different low-temperature conditions. 680

[0069]

[0070]

[0071] As shown in Table 5, Chlorella CS-8 cannot adapt to a low temperature environment of 0℃ and can hardly grow. Chlorella CS-8 can maintain relatively slow growth at 5℃ and can adapt very well to an environment of 10-15℃.

[0072] Example 6: Tolerance of Chlorella CS-8 to ammonia nitrogen concentration

[0073] Different amounts of ammonium chloride were added to multiple portions of BG-11 liquid culture medium, which were then sterilized. The mixture was then poured into Erlenmeyer flasks in 300 mL portions. Initial inoculation of Chlorella with OD was performed. 680 The pH was adjusted to 7.5, and the cells were placed in a light incubator for aeration culture at a light intensity of 3000 Lux, a temperature of 15℃, and an aeration rate of 1 L / min for 14 days. The only difference between the different systems was the ammonia nitrogen content; all other conditions were the same. Samples were taken every 2 days to determine the growth of *Chlorella vulgaris* under high ammonia nitrogen conditions, expressed as ODg. 680 This indicates that the measured OD 680 The IC50 value was determined. Three parallel experiments showed that the IC50 value of Chlorella CS-8 for ammonia nitrogen was 103.17 mg-FA / L.

[0074] Example 7: Tolerance of Chlorella CS-8 to Total Phosphorus Concentration

[0075] Different amounts of sodium phosphate were added to multiple portions of BG-11 liquid culture medium, which were then sterilized and poured into Erlenmeyer flasks in 300 mL portions. Initial inoculation of Chlorella vulgaris with OD... 680 The pH was adjusted to 7.5, and the cells were placed in a light incubator for aeration culture at a light intensity of 3000 Lux, a temperature of 15℃, and an aeration rate of 1 L / min for 14 days. The only difference between the different systems was the total phosphorus content; all other conditions were the same. Samples were taken every 2 days to determine the growth of *Chlorella vulgaris* under high total phosphorus conditions, expressed as OD0.05. 680 This indicates that the measured OD 680The IC50 value was determined. Three parallel experiments showed that the IC50 value of Chlorella CS-8 for total phosphorus was 43.46 mg-FA / L.

[0076] Example 8: Tolerance of Chlorella CS-8 to Lead Ion Concentration

[0077] Different amounts of lead nitrate were added to multiple portions of BG-11 liquid culture medium, which were then sterilized and poured into Erlenmeyer flasks in 300 mL portions. Initial inoculation of Chlorella with OD was performed. 680 The pH was adjusted to 7.5, and the cells were placed in a light incubator for aeration culture at a light intensity of 3000 Lux, a temperature of 15℃, and an aeration rate of 1 L / min for 14 days. The only difference between the different systems was the lead nitrate content; all other conditions were the same. Samples were taken every 2 days to measure the growth of *Chlorella vulgaris* in a high lead ion environment, expressed as OD0.05. 680 This indicates that the measured OD 680 The IC50 value was determined. Three parallel experiments showed that the IC50 value of Chlorella CS-8 for lead ions was 513 μg-FA / L.

[0078] Example 9: Tolerance of Chlorella CS-8 to Sulfide Concentration

[0079] Different amounts of sodium thiosulfate were added to multiple portions of BG-11 liquid culture medium, which were then sterilized. The mixture was then poured into Erlenmeyer flasks in 300 mL portions. Initial inoculation of Chlorella with OD was performed. 680 The pH was adjusted to 7.5, and the cells were placed in a light incubator for aeration culture at a light intensity of 3000 Lux, a temperature of 15℃, and an aeration rate of 1 L / min for 14 days. The only difference between the different systems was the sodium thiosulfate content; all other conditions were the same. Samples were taken every 2 days to measure the growth of *Chlorella vulgaris* in the high-sulfur environment, expressed as OD0.05. 680 This indicates that the measured OD 680 The IC50 value was determined. Three parallel experiments showed that the IC50 value of Chlorella CS-8 for sulfides was 3.83 mg-FA / L.

[0080] Example 10: Enzyme activity of laccase mutants

[0081] 1. Preparation of laccase:

[0082] The nucleotide sequences corresponding to wild-type laccase and laccase mutants were synthesized and inserted into expression systems to obtain the producing bacteria. Specifically, *E. coli* pET-28a (T7 promoter + His-tag) and yeast pPICZαA (containing AOX1 promoter + α-factor signal peptide) can be selected. Many existing expression systems can express the wild-type laccase and laccase mutants of this invention. The producing bacteria were inoculated into liquid culture medium and cultured in a shaker at 37°C, followed by IPTG induction culture. After overnight culture, the bacterial cells were collected by centrifugation, and after adding buffer, the cells were sonicated and then centrifuged again to obtain the crude enzyme solution. The crude enzyme solution was purified through multiple steps and dried at low temperature to obtain wild-type laccase / laccase mutant powder.

[0083] 2. Enzyme activity assay:

[0084] 2.1 Reaction system: The total reaction volume of 10 mL contains 1.1 mol / L of 3-ethylbenzothiazole-6-sulfonic acid (ABTS), 0.1 mol / L of acetate buffer, and 0.02 g / L of laccase. The pH of the system is 5.0.

[0085] 2.2 Methods for determining the activity of different laccases:

[0086] The reaction was carried out according to the reaction system in 2.1, and the reaction temperature was 5–15 °C. Wild-type laccase and laccase mutant were added to the same reaction system, respectively. After being acted upon by laccase, ABTS formed ABTS free radicals. At 420 nm, the absorbance coefficient of ABTS free radicals was much greater than that of the substrate ABTS. As the concentration of ABTS free radicals increased, the absorbance value increased. Therefore, the enzyme activity of wild-type laccase or laccase mutant could be obtained by detecting the change in OD value at 420 nm.

[0087] Enzyme activity is defined as the increase in absorbance (ΔOD) caused by oxidizing substrate (ABTS) with 1 mg of laccase per minute under the above conditions (5℃, pH 5.0). 420 / (mg.min)). The enzyme activities of wild-type laccase and laccase variants were determined and are shown in Table 6:

[0088] Table 6 Enzyme activities of wild-type laccase and laccase mutants

[0089] Laccase Enzyme activity (U / mg) QM-1 (SEQ ID NO:1) 1.052 QM-2(P50K+V115Q+A191S+I245Y) 11.23 QM-3(P50K+V115Q+A191S+I245Y+E256G+L396S) 13.87 QM-4(P50K+V115Q+A191S+I245Y+E256G+W466D) 12.49 QM-5(P50K+V115Q+A191S+I245Y+E256G+F540P) 13.22 QM-6(P50K+V115Q+A191S+I245Y+E256G+L396S+W466D+F540P) 14.71

[0090] Enzyme activity is defined as the increase in absorbance (ΔOD) caused by 1 mg of laccase per minute oxidizing substrate (ABTS) under the above conditions (10℃, pH 5.0). 420 / (mg.min)). The enzyme activities of wild-type laccase and laccase variants were determined and are shown in Table 7:

[0091] Table 7 Enzyme activities of wild-type laccase and laccase mutants

[0092] Laccase Enzyme activity (U / mg) QM-1 (SEQ ID NO:1) 1.843 QM-2(P50K+V115Q+A191S+I245Y) 13.45 QM-3(P50K+V115Q+A191S+I245Y+E256G+L396S) 13.84 QM-4(P50K+V115Q+A191S+I245Y+E256G+W466D) 15.81 QM-5(P50K+V115Q+A191S+I245Y+E256G+F540P) 15.02 QM-6(P50K+V115Q+A191S+I245Y+E256G+L396S+W466D+F540P) 16.36

[0093] Enzyme activity is defined as the increase in absorbance (ΔOD) caused by oxidizing substrate (ABTS) with 1 mg of laccase per minute under the above conditions (15℃, pH 5.0). 420 / (mg.min)). The enzyme activities of wild-type laccase and laccase variants were determined and are shown in Table 8:

[0094] Table 8 Enzyme activities of wild-type laccase and laccase mutants

[0095] Laccase Enzyme activity (U / mg) QM-1 (SEQ ID NO:1) 3.633 QM-2(P50K+V115Q+A191S+I245Y) 11.77 QM-3(P50K+V115Q+A191S+I245Y+E256G+L396S) 12.46 QM-4(P50K+V115Q+A191S+I245Y+E256G+W466D) 14.81 QM-5(P50K+V115Q+A191S+I245Y+E256G+F540P) 11.58 QM-6(P50K+V115Q+A191S+I245Y+E256G+L396S+W466D+F540P) 14.24

[0096] As shown in 6-8, the enzyme activity of the laccase mutant is significantly better than that of the wild-type laccase in the 5-15℃ environment. The enzyme activity of the wild-type laccase increases with the increase of temperature in the 5-15℃ environment, while the optimal temperature of the laccase mutant is 10℃, and the enzyme activity is maintained above 10U / mg, which is suitable for water purification in winter.

[0097] Example 11:

[0098] 5g of Chlorella CS-8 was cultured in liquid culture medium, and then 10g of laccase mutants QM-2 to QM-6 were added separately. After mixing thoroughly, 60g of activated carbon was added and mixed thoroughly. The mixture was then cultured for 3 days with stirring, and then centrifuged to obtain the composite material. The composite material was placed in cavity 1 to obtain composite Chlorella balls.

[0099] Example 12:

[0100] 5g of Chlorella CS-8 was cultured in liquid culture medium, then 6g of laccase mutant QM-2-6 was added and mixed thoroughly. 35g of activated carbon was then added and mixed again. The mixture was cultured for 3 days with stirring, followed by centrifugation to obtain the composite material. The composite material was placed in cavity 1 to obtain composite Chlorella balls.

[0101] Example 13:

[0102] 5g of Chlorella CS-8 was cultured in liquid culture medium, then 15g of laccase mutant QM-2-6 was added and mixed thoroughly. 160g of activated carbon was then added and mixed thoroughly again. The mixture was cultured for 3 days with stirring, followed by centrifugation to obtain the composite material. The composite material was placed in cavity 1 to obtain composite Chlorella spheres.

[0103] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A type of low-temperature Chlorella, characterized in that... It contains Chlorella, whose preservation number is CCTCC NO:M2025509; the preservation unit is China Center for Type Culture Collection, and the Chlorella can withstand low temperatures of 5-15℃.

2. A composite microalgae sphere, characterized in that... The composite Chlorella sphere includes a sphere wall (3) and a cavity (1). The cavity (1) is located inside the sphere wall (3). The sphere wall (3) is provided with multiple flow holes (2). The cavity (1) communicates with the outside of the sphere wall (3) through the flow holes (2). The cavity (1) contains a composite material, which includes activated carbon, Chlorella as described in claim 1, and a laccase mutant. The laccase mutant uses the wild-type laccase described in SEQ ID NO:1 as the parent material and undergoes the following mutation: P50K+V115Q+A191S+I245Y.

3. The composite microalgae sphere according to claim 2, characterized in that... The laccase mutant uses the wild-type laccase described in SEQ ID NO:1 as the parent material, and the sequence modification includes not only the sequence modification described in claim 2, but also the following mutation set: E256G+L396S.

4. The composite microalgae sphere according to claim 2, characterized in that... The laccase mutant uses the wild-type laccase described in SEQ ID NO:1 as the parent material, and the sequence modification includes not only the sequence modification described in claim 2, but also the following mutation set: E256G+W466D.

5. The composite microalgae sphere according to claim 2, characterized in that... The laccase mutant uses the wild-type laccase described in SEQ ID NO:1 as the parent material, and the sequence modification includes not only the sequence modification described in claim 2, but also the following mutation set: E256G+F540P.

6. The composite microalgae sphere according to claim 2, characterized in that... The laccase mutant uses the wild-type laccase described in SEQ ID NO:1 as the parent material, and the sequence modification includes the sequence modification described in claim 2, as well as the following mutation set: E256G+L396S+W466D+F540P.

7. The composite microalgae sphere according to claim 2, characterized in that... The preparation steps of the composite material include: placing the Chlorella in a liquid culture medium for cultivation, then adding laccase mutant, mixing evenly, adding activated carbon, mixing evenly, culturing while stirring, and then centrifuging to obtain the composite material.

8. The composite microalgae sphere according to claim 7, characterized in that... The volume of the activated carbon is 3 to 8 times the total volume of Chlorella and the laccase mutant.

9. A composite microalgae sphere according to claim 7, characterized in that... The volume of the laccase mutant is 1 to 3 times that of Chlorella.

10. A composite microalgal sphere according to any one of claims 2 to 9, characterized in that... The composite chlorella balls are used to treat eutrophic water bodies and / or water bodies polluted by sulfides and / or water bodies polluted by phenolic compounds.

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