A method for simultaneously treating heavy metals and organic pollutants

Abstract

The present invention discloses a method for simultaneously treating heavy metals and organic pollutants. The method uses the white rot fungus Pleurotus ostreatus GEMB-PO1 with a deposit number of CCTCCNO: M 2023358 to jointly repair heavy metals and organic pollutants. In the present invention, when the white rot fungus Pleurotus ostreatus GEMB-PO1 is used to jointly repair heavy metals and organic pollutants, the heavy metals and organic pollutants can be removed simultaneously, and the removal of organic pollutants in the presence of heavy metals is more conducive to the removal of organic pollutants. The method has the advantages of simple process, convenient operation, high treatment efficiency, good removal effect, etc., and is of great significance for effectively repairing heavy metal-organic composite pollution and effectively solving the environmental pollution and ecological imbalance problems caused by heavy metals and organic matter.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a method for simultaneously treating heavy metals and organic pollutants, and particularly relates to a method for simultaneously treating heavy metals and organic pollutants by utilizing white rot fungi. Background Art

[0002] Heavy metal and organic pollutants are widely distributed in the environment, and under practical conditions, they often coexist in the system. For example, large amounts of copper and various organic synthetic dyes are commonly found in wastewater from the printing and dyeing industry; copper often coexists with various aromatic and phenolic compounds in wastewater from the pesticide and paint industries; copper often forms complex pollution with antibiotics in the livestock industry; and in real-world ecosystems, copper-contaminated soil and water bodies often also contain organic pollution. Therefore, it is crucial to develop a method that can simultaneously treat heavy metal and organic pollutants.

[0003] Traditional physical and chemical remediation methods have disadvantages such as high operating costs, many secondary by-products, and easy formation of secondary pollution. Bioremediation methods represented by microbial treatment have the advantages of relatively low cost and being green and pollution-free, and are currently the more popular environmental remediation strategies. However, under the conditions of the simultaneous presence of heavy metal and organic pollution, the stress and impact of the two types of pollutants on the microorganisms themselves will interfere with the bioremediation of microorganisms to varying degrees. On the one hand, heavy metals may affect the metabolic processes in microorganisms, such as ATP production and the activity of biological enzymes, thereby affecting the remediation and degradation of organic pollutants. On the other hand, the metabolism of organic pollution by microorganisms will also have additional effects on cells, thereby affecting the tolerance and remediation of heavy metals. At present, some research focuses on the microbial remediation of a single type of pollution, but there is little progress in the use of microorganisms for the simultaneous remediation of heavy metal-organic composite pollution.

[0004] White rot fungi are a class of microbial resources with strong environmental application potential. They are widely used in the degradation and remediation of organic pollutants such as industrial wastewater, polycyclic aromatic hydrocarbons, pesticides and antibiotics. These include various phenolic compounds including phenol and o-nitrophenol, various synthetic dye simulated wastewater and actual printing and dyeing wastewater, various difficult-to-degrade pharmaceutical wastewaters, various antibiotics, and various organic environmental pollutants such as polycyclic aromatic hydrocarbons including phenanthrene and fluoranthene. Moreover, based on the different types of white rot fungi, their removal capabilities for triphenylmethane dyes, sulfonamide antibiotics and phenolic organic pollution are also different. However, in the early stage research of the present inventors, it was found that, as shown in Table 1, when various white rot fungi were used for the remediation of organic pollution, there were still defects such as long degradation time, poor degradation effect and poor adaptability to high concentrations of organic pollutants. Meanwhile, as shown in Table 2, the existing known white rot fungi have poor tolerance to heavy metals. Therefore, under the condition of heavy metal presence, these microorganisms are difficult to reproduce rapidly or even die, thus being difficult to achieve effective remediation of heavy metal pollution in the environment. In addition, the research on the use of white rot fungi to repair heavy metal-organic composite pollution is relatively scarce. Therefore, obtaining a method for treating heavy metal and organic pollutants simultaneously with simple process, convenient operation, high treatment efficiency and good removal effect is of great significance for effectively repairing heavy metal-organic composite pollution and effectively reducing environmental pollution and ecological imbalance caused by heavy metals and organic matter.

[0005] Table 1 Removal effect of known white rot fungi on organic pollutants

[0006]

[0007] Table 2 Common white rot fungi tolerance to copper

[0008] Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for simultaneously treating heavy metals and organic pollutants with simple process, convenient operation, high treatment efficiency and good removal effect.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0011] A method for simultaneously treating heavy metals and organic pollutants, using the white rot fungus Pleurotus ostreatus GEMB-PO1 to jointly repair heavy metals and organic pollutants; the white rot fungus Pleurotus ostreatus GEMB-PO1 was deposited in the China Center for Type Culture Collection on March 17, 2023, with the deposit number CCTCC NO: M2023358.

[0012] The above method is further improved, and when the white rot fungus Pleurotus ostreatus GEMB-PO1 is used to jointly repair heavy metals and organic pollutants, the method includes the following steps: mixing heavy metal-organic composite pollutants with the white rot fungus Pleurotus ostreatus GEMB-PO1, cultivating the white rot fungus Pleurotus ostreatus GEMB-PO1, and completing the joint repair of heavy metals and organic pollutants.

[0013] The above method is further improved in that during the culture process, the initial concentration of heavy metals in the control system is ≤8 mM, and the initial concentration of organic pollutants is ≤100 mg / L.

[0014] The above method is further improved in that the temperature of the control system during the culture process is 25°C to 30°C; the culture is carried out at a rotation speed of 150rpm to 200rpm; and the culture time is 1 day to 7 days.

[0015] The above method is further improved, and when the white rot fungus Pleurotus ostreatus GEMB-PO1 is used to jointly repair heavy metals and organic pollutants, the method includes the following steps: mixing heavy metals, organic pollutants and the white rot fungus Pleurotus ostreatus GEMB-PO1, and culturing the white rot fungus Pleurotus ostreatus GEMB-PO1 to complete the joint repair of heavy metals and organic pollutants.

[0016] The above method is further improved in that during the culture process, the initial concentration of heavy metals in the control system is ≤8 mM, and the initial concentration of organic pollutants is ≤100 mg / L.

[0017] The above method is further improved in that the temperature of the control system during the culture process is 25°C to 30°C; the culture is carried out at a rotation speed of 150rpm to 200rpm; and the culture time is 1 day to 7 days.

[0018] The above method is further improved, wherein the white rot fungus Pleurotus ostreatus GEMB-PO1 further includes an expansion culture treatment before use, comprising the following steps:

[0019] (1) The white rot fungus Pleurotus ostreatus GEMB-PO1 was inoculated into PDA medium for subculture and expansion;

[0020] (2) Mycelium on the PDA medium was inoculated into GYP liquid medium for primary culture to obtain a primary liquid strain;

[0021] (3) The primary liquid strain was inoculated into GYP liquid culture medium for secondary culture to obtain the white rot fungus Pleurotus ostreatus GEMB-PO1 for simultaneous treatment of heavy metals and organic pollutants.

[0022] The above method is further improved in that in step (1), the PDA culture medium includes 200 g / L of potatoes, 20 g / L of glucose, 15 g / L of agar, and the rest is water; and the subculture expansion culture is carried out at a temperature of 28°C.

[0023] The above method is further improved, in step (2), the GYP liquid culture medium includes 20g / L glucose, 5g / L yeast extract, 5g / L peptone, 1g / L magnesium sulfate heptahydrate, and the rest is water; the temperature of the control system during the primary culture process is 25°C to 30°C; the primary culture is carried out at a rotation speed of 150rpm to 200rpm; and the primary culture time is 6 days to 8 days.

[0024] The above method is further improved, in step (3), the volume ratio of the primary liquid bacteria to the GYP liquid culture medium is 1:50-200; the GYP liquid culture medium comprises 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L magnesium sulfate heptahydrate, and the remainder is water; the temperature of the control system during the secondary culture process is 25°C to 30°C; the secondary culture is carried out at a rotation speed of 150 rpm to 200 rpm; and the secondary culture time is 2 to 4 days.

[0025] The above method is further improved, wherein the heavy metal is copper ion and / or cadmium ion; the organic pollutant is at least one of phenolic compounds, dyes, antibiotics, and polycyclic aromatic hydrocarbons; the phenolic compound includes at least one of bisphenol A and tetrabromobisphenol A; the dye is at least one of crystal violet, malachite green, methyl blue, and basic fuchsin; the antibiotic is at least one of sulfonamide, sulfathiazole, sulfamethoxazole, and sulfadimethoxine; and the polycyclic aromatic hydrocarbons are at least one of phenanthrene, fluoranthene, fluorene, and pyrene.

[0026] Compared with the prior art, the advantages of the present invention are:

[0027] In response to the defects of existing white rot fungi such as poor tolerance, difficulty in adapting to complex pollution environments, and the resulting difficulty in efficiently degrading organic pollutants and simultaneously and effectively repairing heavy metal-organic complex pollutants, the present invention creatively proposes a method for simultaneously treating heavy metals and organic pollutants, using the white rot fungus Pleurotus ostreatus GEMB-PO1 to jointly repair heavy metals and organic pollutants. The white rot fungus Pleurotus ostreatus GEMB-PO1 has been deposited in the China Center for Type Culture Collection on March 17, 2023, at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC NO: M 2023358. Compared with conventional white rot fungi, the white rot fungi Pleurotus ostreatus GEMB-PO1 used in the present invention has excellent heavy metal tolerance, with an absolute lethal concentration of up to 8mM, while the conventional white rot fungi Pleurotus ostreatus is only 3mM, and can efficiently express lignin degrading enzymes (such as laccase, type II peroxidase, dye decolorizing peroxidase). Therefore, when the white rot fungi Pleurotus ostreatus GEMB-PO1 is used for the co-remediation of heavy metals and organic pollutants, the white rot fungi Pleurotus ostreatus GEMB-PO1 can survive and reproduce in a complex environment of heavy metals and organic pollutants. During this process, organic pollutants can serve as a carbon source for its growth, thereby converting organic pollutants into non-toxic or less toxic metabolic components during its growth. It can also eliminate organic pollution in the environment through intracellular absorption and degradation, extracellular enzyme catalysis, and biological free radical degradation. At the same time, it can also remove heavy metals from the environment through complexation, adsorption, chelation, and enrichment. In particular, in the presence of heavy metals, heavy metals can also stimulate the rapid and massive reproduction of the white-rot fungus Pleurotus ostreatus GEMB-PO1. For example, transcriptomic data analysis shows that the presence of copper ions can significantly induce the expression of enzymes related to the xenobiotic degradation enzyme system of P. ostreatus GEMB-PO1, such as further promoting the efficient expression of laccase, type II peroxidase, and dye decolorization peroxidase. Therefore, it can further promote the removal efficiency of organic pollutants by the white-rot fungus Pleurotus ostreatus GEMB-PO1, thereby achieving rapid and efficient removal of organic pollutants and heavy metals.The method of the present invention utilizes the white rot fungus Pleurotus ostreatus GEMB-PO1 to perform co-remediation of heavy metals and organic pollutants, thereby achieving simultaneous removal of heavy metals and organic pollutants. The method has the advantages of simple process, convenient operation, high treatment efficiency, and good removal effect. It is of great significance for effectively remediating heavy metal-organic composite pollution and effectively solving environmental pollution and ecological imbalance problems caused by heavy metals and organic matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0029] Figure 1 This is a graph showing the growth of the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 1 of the present invention on GYP plates containing different concentrations of copper ions.

[0030] Figure 2 This is a diagram showing the removal effect of sulfanilamide in sulfanilamide and copper complex pollution by the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 5 of the present invention.

[0031] Figure 3 This is a diagram showing the removal effect of sulfathiazole in sulfathiazole and copper composite pollution by the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 5 of the present invention.

[0032] Figure 4 This is a diagram showing the removal effect of crystal violet in crystal violet and copper composite pollution by the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 5 of the present invention.

[0033] Figure 5 This is a diagram showing the removal effect of bisphenol A from bisphenol A and copper composite pollution by the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 5 of the present invention.

[0034] Figure 6 This is a diagram showing the removal effect of copper ions in sulfonamide and copper complex pollution by the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 5 of the present invention.

[0035] Figure 7 This is a diagram showing the removal effect of copper ions from sulfathiazole and copper composite pollution by the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 5 of the present invention.

[0036] Figure 8 This is a diagram showing the removal effect of copper ions from crystal violet and copper composite pollution by the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 5 of the present invention.

[0037] Figure 9 This is a diagram showing the removal effect of copper ions from bisphenol A and copper composite pollution by the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 5 of the present invention.

[0038] Figure 10 This is a graph showing the differential expression results of lignin degradation-related enzymes in the white-rot fungus Pleurotus ostreatus GEMB-PO1 under heavy metal conditions in Example 5 of the present invention. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but the scope of protection of the present invention is not limited thereby. The materials and instruments used in the following examples are all commercially available.

[0040] In the present invention, the white rot fungus Pleurotus ostreatus GEMB-PO1 used, abbreviated as P.ostreatusGEMB-PO1, is classified and named Pleurotus ostreatus. It was deposited in the China Center for Type Culture Collection (CCTCC) on March 17, 2023, at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: M 2023358.

[0041] Example 1

[0042] Investigating the tolerance of white rot fungus Pleurotus ostreatus GEMB-PO1 to heavy metals

[0043] (1) The white rot fungus P. ostreatus GEMB-PO1 was inoculated into a PDA medium and cultured in a 28°C incubator to allow the white rot fungus P. ostreatus GEMB-PO1 to grow. In this step, the PDA medium contained 200 g / L potato, 20 g / L glucose, 15 g / L agar, and the remainder water.

[0044] (2) Copper sulfate mother liquor was added to the sterilized GYP solid culture medium to adjust the initial concentration of copper ions in the system to 0, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, and 8 mM, respectively. The medium was then poured into sterile culture dishes with a diameter of 90 mm. Each 100 mL of culture medium was divided into three plates. Each culture medium contained the following ingredients: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, and 1 g / L magnesium sulfate heptahydrate.

[0045] (3) Use a hole puncher to punch holes at a distance from the inoculation point on the plate. Place the inoculated block with the mycelium side facing down and inoculate it in the center of the plate. Set up three parallel groups for each copper ion concentration, take photos and record them every day, and use the software Digimizer (https: / / www.digimizer.com / ) to measure the area. The results are as follows: Figure 1 shown.

[0046] Figure 1 The figure shows the growth of the white rot fungus Pleurotus ostreatus GEMB-PO1 in Example 1 of the present invention on GYP plates containing different concentrations of copper ions. Figure 1 As shown in the data, when the copper ion concentration is 2 mM and below, the growth area of the white rot fungus Pleurotus ostreatus GEMB-PO1 is not weaker than or even better than the 0 mM control group. Concentrations above 4 mM will inhibit the growth of the white rot fungus, which shows that P. ostreatus GEMB-PO1 has good tolerance to copper; in particular, when the concentration is 8 mM, the white rot fungus Pleurotus ostreatus GEMB-PO1 basically has no growth, that is, the absolute lethal concentration of the white rot fungus Pleurotus ostreatus GEMB-PO1 in the copper ion system is 8 mM, which is significantly higher than that of conventional white rot fungi.

[0047] Example 2

[0048] Removal capacity of 1 mM copper ions by different white rot fungi

[0049] (1) P. ostreatus GEMB-PO1, Physisporinus vitreus GEMB-PV1, Echinodontium taxodii GEMB-ET1, Trametes versicolor GEMB-TV1, and Ganoderma lucidum GEMB-GL1 were inoculated into PDA medium and cultured in a 28°C incubator to allow the white rot fungi to grow. In this step, the PDA medium contained 200 g / L potato, 20 g / L glucose, 15 g / L agar, and the remainder was water.

[0050] (2) After full mycelium growth, a piece of white rot fungus mycelium was cut and inoculated into GYP liquid medium. The culture was incubated at 28°C and 180 rpm in a shaker for 7 days to obtain a primary liquid culture. In this step, the GYP liquid medium was prepared by the following method: 20 g glucose, 5 g yeast extract, 5 g peptone, and 1 g magnesium sulfate heptahydrate were dissolved in water and the volume was adjusted to 1 L with water.

[0051] (3) Pipette 1 mL of the primary liquid seed into a conical flask containing GYP liquid medium and incubate at 28°C, 180 rpm, for 3 days. At this point, the pellets are uniform in size and are the secondary liquid seed. In this step, GYP liquid medium is prepared by dissolving 20 g of glucose, 5 g of yeast extract, 5 g of peptone, and 1 g of magnesium sulfate heptahydrate in water and then adding water to make the volume 1 L.

[0052] (4) Take 5 groups of GYP liquid culture media obtained in step (3) and add copper sulfate mother solution to make the initial concentration of copper ions in each culture medium be 1 mM.

[0053] Determination of the residual concentration of copper ions in different samples:

[0054] (a) Preparation of standard curve: Use 2% (v / v) nitric acid solution to adjust the copper standard solution (1 g / L) to the concentrations of 2, 4, 6, 8, and 10 mg / L.

[0055] (b) Atomic flame absorption spectrometer parameter settings:

[0056] Light Setting: Select Cu light.

[0057] Establish the method: Enter "1" in the "ID" of "Calibration Blank", enter the concentration of the prepared standard sample 1 (2 mg / L) in the "Concentration" of "Standard Sample 1", and enter the concentrations of "Standard Sample 2", "Standard Sample 3", "Standard Sample 4", "Standard Sample 5" and so on as 4, 6, 8, and 10 mg / L respectively. After setting, save the method, then measure the blank and standard samples in turn, and draw the standard curve.

[0058] (c) Sample treatment: Take 250 μL of the copper ion solution treated with white rot fungi (sample to be tested) and filter it through a 0.22 μm filter.

[0059] The sample was diluted with 2% (v / v) dilute nitric acid to the concentration range of the standard curve, and the copper ion concentration was detected on an atomic flame absorption spectrometer.

[0060] The calculation formula for copper ion removal efficiency is shown in formula (1):

[0061]

[0062] Wherein, in formula (1), e is the removal rate of copper ions by white rot fungi, in %; c0 is the initial concentration of copper ions in the sample, in mM; c n is the residual concentration of copper ions in the sample, in mM.

[0063] The results showed that after 7 days of cultivation in a system with an initial concentration of 1 mM, the removal rates of copper ions by P.ostreatus GEMB-PO1, Physisporinus vitreus GEMB-PV1, Echinodontium taxodii GEMB-ET1, Trametesversicolor GEMB-TV1, and Ganoderma lucidum GEMB-GL1 were 47.08%, 45.93%, 19.00%, 18.63%, and 10.38%, respectively, among which P.ostreatus GEMB-PO1 had the highest removal rate.

[0064] Example 3

[0065] Investigating the ability of white rot fungus Pleurotus ostreatus GEMB-PO1 to repair heavy metal copper

[0066] (1) The white rot fungus P. ostreatus GEMB-PO1 was inoculated into a PDA medium and cultured in a 28°C incubator to allow the white rot fungus P. ostreatus GEMB-PO1 to grow. In this step, the PDA medium contained 200 g / L potato, 20 g / L glucose, 15 g / L agar, and the remainder water.

[0067] (2) After full mycelium growth, a piece of white rot fungus mycelium was cut and inoculated into GYP liquid medium. The culture was incubated at 28°C and 180 rpm in a shaker for 7 days to obtain a primary liquid culture. In this step, the GYP liquid medium was prepared by the following method: 20 g glucose, 5 g yeast extract, 5 g peptone, and 1 g magnesium sulfate heptahydrate were dissolved in water and the volume was adjusted to 1 L with water.

[0068] (3) Pipette 1 mL of the primary liquid seed into a conical flask containing GYP liquid medium and incubate at 28°C, 180 rpm, for 3 days. At this point, the pellets are uniform in size and are the secondary liquid seed. In this step, GYP liquid medium is prepared by dissolving 20 g of glucose, 5 g of yeast extract, 5 g of peptone, and 1 g of magnesium sulfate heptahydrate in water and then adding water to make the volume 1 L.

[0069] (4) Take 6 groups of GYP liquid culture media obtained in step (3), add copper sulfate mother solution to make the initial concentration of copper ions in each culture medium 0.5mM, 1mM, 2mM, 4mM, 6mM, and 8mM, and culture in a shaker at 28°C and 180rpm for 7 days.

[0070] The residual concentration of copper ions in different samples was determined using the method in Example 2:

[0071] The results showed that the highest removal rates of copper ions by white rot fungus P.ostreatus GEMB-PO1 in the systems with initial concentrations of 0.5mM, 1mM, 2mM, 4mM, 6mM and 8mM were 64.56%, 48.51%, 46.89%, 24.89%, 24.22% and 22.90%, respectively, which was consistent with the results of the previous study. Figure 1 The growth trend results of the intermediate white rot fungus P. ostreatus GEMB-PO1 were consistent.

[0072] Example 4

[0073] Investigating the repair ability of white rot fungus P.ostreatus GEMB-PO1 on organic matter pollution

[0074] (1) The white rot fungus P. ostreatus GEMB-PO1 was inoculated into a PDA medium and cultured in a 28°C incubator to allow the white rot fungus P. ostreatus GEMB-PO1 to grow. In this step, the PDA medium contained 200 g / L potato, 20 g / L glucose, 15 g / L agar, and the remainder water.

[0075] (2) After full mycelium growth, a piece of white rot fungus mycelium was cut and inoculated into GYP liquid medium. The culture was incubated at 28°C and 180 rpm in a shaker for 7 days to obtain a primary liquid culture. In this step, the GYP liquid medium was prepared by the following method: 20 g glucose, 5 g yeast extract, 5 g peptone, and 1 g magnesium sulfate heptahydrate were dissolved in water and the volume was adjusted to 1 L with water.

[0076] (3) Pipette 1 mL of the primary liquid seed into a conical flask containing GYP liquid medium and incubate at 28°C, 180 rpm, for 3 days. At this point, the pellets are uniform in size and are the secondary liquid seed. In this step, GYP liquid medium is prepared by dissolving 20 g of glucose, 5 g of yeast extract, 5 g of peptone, and 1 g of magnesium sulfate heptahydrate in water and then adding water to make the volume 1 L.

[0077] (4) Take four groups of GYP liquid culture media obtained in step (3), add sulfonamide, sulfathiazole, crystal violet and bisphenol A respectively, so that the initial concentrations of sulfonamide, sulfathiazole, crystal violet and bisphenol A in the corresponding culture medium are 100 mg / L, 100 mg / L, 100 mg / L and 50 mg / L, and culture in a shaker at 28°C and 180 rpm for 7 days.

[0078] Determination of residual concentrations of organic pollutants in different samples:

[0079] Crystal violet was appropriately diluted and its absorbance was measured at 584 nm using a UV spectrophotometer.

[0080] Sulfonamide, sulfathiazole, and bisphenol A were determined using high performance liquid chromatography (HPLC) under the following conditions:

[0081] The mobile phase used for sulfonamide determination was acetonitrile:water = 20:80; the wavelength was 270 nm, the column oven temperature was set to 40°C, the flow rate was 1 mL / min, and the peak elution time was approximately 2.7 min;

[0082] Sulfathiazole: The mobile phase was acetonitrile: 0.1% (v / v) formic acid aqueous solution = 30:70; the wavelength was 268 nm, the column oven temperature was set to 40°C, the flow rate was 1 mL / min, and the peak elution time was approximately 2.7 min;

[0083] Bisphenol A: The mobile phase was acetonitrile: water = 50:50; the wavelength was 226 nm, the column oven temperature was set to 40°C, the flow rate was 1 mL / min, and the peak elution time was approximately 4.9 min.

[0084] First, measure the concentration of the standard sample, fit it into a standard curve, substitute the area measured by the sample into the corresponding standard curve, and calculate the actual concentration of the sample at this time. The concentration measured by sampling on day n is C n , after adding pollutants to the secondary species, the concentration of the sample taken out is C0, then the degradation rate on the nth day is:

[0085]

[0086] Wherein, in formula (2), e is the removal rate of organic pollutants by white rot fungus P.ostreatus GEMB-PO1, in %; c0 is the initial concentration of organic pollutants in the sample, in mg / L; c n It is the residual concentration of organic pollutants in the sample, in mg / L.

[0087] The results showed that after seven days of cultivation, the removal rates of P.ostreatus GEMB-PO1 for crystal violet, sulfonamide, sulfathiazole, and bisphenol A were 77.42%, 56.05%, 98.60%, and 51.79%, respectively. This indicates that the white rot fungus P.ostreatus GEMB-PO1 of the present invention can quickly remove organic pollutants in the system.

[0088] Example 5

[0089] A method for simultaneously treating heavy metals and organic pollutants, specifically using the white rot fungus Pleurotusostreatus GEMB-PO1 to jointly repair heavy metals and organic pollutants, comprising the following steps:

[0090] (1) The white rot fungus P. ostreatus GEMB-PO1 was inoculated into a PDA medium for cultivation and placed in a 28°C incubator to allow the white rot fungus P. ostreatus GEMB-PO1 to grow. In this step, the PDA medium contained 200 g / L of potato, 20 g / L of glucose, 15 g / L of agar, and the remainder was water.

[0091] (2) After full mycelium growth, a piece of white rot fungus mycelium was cut and inoculated into GYP liquid medium. The culture was incubated at 28°C and 180 rpm in a shaker for 7 days to obtain a primary liquid culture. In this step, the GYP liquid medium was prepared by the following method: 20 g glucose, 5 g yeast extract, 5 g peptone, and 1 g magnesium sulfate heptahydrate were dissolved in water and the volume was adjusted to 1 L with water.

[0092] (3) Pipette 1 mL of the primary liquid seed into a conical flask containing GYP liquid medium and incubate at 28°C, 180 rpm, for 3 days. At this point, the pellets are uniform in size and are the secondary liquid seed. In this step, GYP liquid medium is prepared by dissolving 20 g of glucose, 5 g of yeast extract, 5 g of peptone, and 1 g of magnesium sulfate heptahydrate in water and then adding water to make the volume 1 L.

[0093] (4) According to the initial concentration of copper ions in the system being 0.5 mM, and the initial concentrations of sulfonamide, sulfathiazole, crystal violet and bisphenol A being 100 mg / L, 100 mg / L, 100 mg / L and 50 mg / L, four groups of GYP liquid culture media obtained in step (3) were taken, copper sulfate mother liquor was first added, and then sulfonamide, sulfathiazole, crystal violet and bisphenol A were added respectively, and cultured at 28°C and 180 rpm in a shaker for 7 days.

[0094] During the cultivation process, samples were taken every day to test the residual concentrations of copper ions and organic pollutants in the samples, and the co-remediation effect of the white rot fungus Pleurotus ostreatus GEMB-PO1 on heavy metals and organic pollutants was calculated according to formula (1) and formula (2). The results are as follows: Figure 2-9 shown.

[0095] Depend on Figure 2-5 It can be seen that in the composite pollution system of copper and sulfonamide, sulfathiazole, crystal violet and bisphenol A, after one day of culture, the removal rate of crystal violet by P.ostreatus GEMB-PO1 was 81.73%; after three days of culture, the removal rates of sulfathiazole and bisphenol A by P.ostreatus GEMB-PO1 were 98.42% and 75.33% respectively; after seven days of culture, the removal rates of crystal violet, sulfonamide, sulfathiazole and bisphenol A by P.ostreatus GEMB-PO1 were 89.07%, 92.72%, 99.25% and 100% respectively. Among them, the degradation process was significantly accelerated compared with the single degradation system.

[0096] Depend on Figure 6-9 It can be seen that in the composite pollution system of copper and sulfonamide, sulfathiazole, crystal violet and bisphenol A, after 7 days of cultivation, the highest removal rate of copper in different composite pollution by P. ostreatus GEMB-PO1 was 35.48%, 30.94%, 19.13% and 19.44%, respectively.

[0097] By comparing the results in Example 4 and Example 5, it can be seen that the presence of heavy metals is more conducive to the effective removal of organic pollutants. In the presence of heavy metals, the removal rate of crystal violet by the white rot fungus Pleurotus ostreatus GEMB-PO1 increased from 77.42% to 89.07%, the removal rate of sulfonamide increased from 56.05% to 92.72%, and the removal rate of bisphenol A increased from 51.79% to 100%. At the same time, the degradation process of sulfonamide was significantly accelerated from the original five days to three days. This is because the presence of copper ions can significantly induce the expression of enzymes related to the xenobiotic degradation enzyme system of P. ostreatus GEMB-PO1, such as laccase, type II peroxidase, and dye decolorization peroxidase (the results are shown in FIG). Figure 10 As shown), the white rot fungus Pleurotus ostreatus GEMB-PO1 can promote the improvement of the removal effect of organic pollutants, thereby achieving rapid and efficient removal of organic pollutants in the presence of heavy metals. At the same time, the heavy metals are also removed accordingly. This means that the white rot fungus Pleurotus ostreatus GEMB-PO1 of the present invention can achieve synergistic remediation of heavy metal copper and organic pollutants.

[0098] Example 6

[0099] A method for simultaneously treating heavy metal and organic pollutants, specifically using the white rot fungus Pleurotusostreatus GEMB-PO1 to repair heavy metal-organic complex pollutants, comprising the following steps:

[0100] (1) The white rot fungus P. ostreatus GEMB-PO1 was inoculated into a PDA medium and cultured in a 28°C incubator to allow the white rot fungus P. ostreatus GEMB-PO1 to grow. In this step, the PDA medium contained 200 g / L potato, 20 g / L glucose, 15 g / L agar, and the remainder water.

[0101] (2) After full mycelium growth, a piece of white rot fungus mycelium was cut and inoculated into GYP liquid medium. The culture was incubated at 28°C and 180 rpm in a shaker for 7 days to obtain a primary liquid culture. In this step, the GYP liquid medium was prepared by the following method: 20 g glucose, 5 g yeast extract, 5 g peptone, and 1 g magnesium sulfate heptahydrate were dissolved in water and the volume was adjusted to 1 L with water.

[0102] (3) Pipette 1 mL of the primary liquid seed into a conical flask containing GYP liquid medium and incubate at 28°C, 180 rpm, for 3 days. At this point, the pellets are uniform in size and are the secondary liquid seed. In this step, GYP liquid medium is prepared by dissolving 20 g of glucose, 5 g of yeast extract, 5 g of peptone, and 1 g of magnesium sulfate heptahydrate in water and then adding water to make the volume 1 L.

[0103] (4) Take 4 groups of GYP liquid culture medium obtained in step (3), add heavy metal-organic composite pollutants containing heavy metals and organic pollutants respectively, at this time, the initial concentration of copper ions in the system is 0.5 mM, the initial concentration of sulfathiazole is 100 mg / L, and culture in a shaker at 28°C and 180 rpm for 7 days.

[0104] The results showed that after seven days of cultivation, the white rot fungus Pleurotus ostreatus GEMB-PO1 of the present invention had a removal rate of 26.52% for copper ions in heavy metal-organic complex pollutants and a removal rate of 99.25% for sulfathiazole. This indicates that the white rot fungus Pleurotus ostreatus GEMB-PO1 of the present invention can be directly used to remediate complex pollutants containing heavy metals and organic pollutants, and can simultaneously remove heavy metals and organic pollutants in the complex pollutants, and can more effectively remove organic pollutants in the presence of heavy metals.

[0105] In summary, the white rot fungus Pleurotus ostreatus GEMB-PO1 used in the present invention has excellent heavy metal tolerance, with an absolute lethal concentration of up to 8mM, while the conventional white rot fungus Pleurotus ostreatus is only 3mM, and can efficiently express lignin degrading enzymes (such as laccase, type II peroxidase, dye decolorizing peroxidase). Therefore, when the white rot fungus Pleurotus ostreatus GEMB-PO1 is used for the co-remediation of heavy metals and organic pollutants, the white rot fungus Pleurotus ostreatus GEMB-PO1 can survive and reproduce in a complex environment of heavy metals and organic pollutants. During this process, organic pollutants can serve as carbon sources for its growth, thereby converting organic pollutants into non-toxic or less toxic metabolic components during its growth. At the same time, it can also eliminate organic pollution in the environment through intracellular absorption and degradation, extracellular enzyme catalysis, and biological free radical degradation. At the same time, it can also remove heavy metals in the environment through complexation, adsorption, chelation, and enrichment. In particular, in the presence of heavy metals, heavy metals can also stimulate the rapid and massive reproduction of the white rot fungus Pleurotus ostreatus GEMB-PO1. Therefore, when using the white rot fungus Pleurotus ostreatus GEMB-PO1 to jointly remediate heavy metals and organic pollutants, it can achieve simultaneous removal of heavy metals and organic pollutants, and the presence of heavy metals is more conducive to the removal of organic pollutants. It has the advantages of simple process, convenient operation, high treatment efficiency, and good removal effect. It is of great significance for effectively remediating heavy metal-organic complex pollution and effectively solving environmental pollution and ecological imbalance problems caused by heavy metals and organic matter.

[0106] The above description is only a preferred embodiment of the present invention and does not constitute any formal limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for simultaneously treating heavy metals and organic pollutants, characterized in that: White rot fungi Pleurotus ostreatus GEMB-PO1 performs co-remediation of heavy metals and organic pollutants, including the following steps: combining heavy metal-organic composite pollutants with white rot fungi Pleurotus ostreatus GEMB-PO1 mixture, against white rot fungi Pleurotus ostreatus GEMB-PO1 is cultured to complete the joint remediation of heavy metals and organic pollutants; during the culture process, the initial concentration of heavy metals in the control system is ≤8 mM, and the initial concentration of organic pollutants is ≤100 mg / L; the heavy metals are copper ions and / or cadmium ions; the organic pollutants are at least one of phenolic compounds, dyes, antibiotics, and polycyclic aromatic hydrocarbons; the white rot fungi Pleurotus ostreatus GEMB-PO1 was deposited in China Center for Type Culture Collection on March 17, 2023, with the accession number CCTCCNO: M2023358; the white rot fungus Pleurotus ostreatus GEMB-PO1 also includes expansion culture processing before use, including the following steps: (1) White rot fungi Pleurotus ostreatus GEMB-PO1 was inoculated into PDA medium for subculture and expansion; (2) Take the mycelium on the PDA medium and inoculate it into the GYP liquid medium for primary culture to obtain the primary liquid strain; (3) The first-level liquid strain was inoculated into GYP liquid culture medium for secondary culture to obtain white rot fungi for simultaneous treatment of heavy metals and organic pollutants. Pleurotus ostreatus GEMB-PO1.

2. The method according to claim 1, wherein During the culture process, the temperature of the control system is 25° C. to 30° C.; the culture is carried out at a rotation speed of 150 rpm to 200 rpm; and the culture time is 1 day to 7 days.

3. The method according to claim 1, wherein White rot fungi Pleurotus ostreatus GEMB-PO1's co-remediation of heavy metals and organic pollutants includes the following steps: Pleurotus ostreatus GEMB-PO1 mixture, against white rot fungi Pleurotus ostreatus GEMB-PO1 was cultivated to complete the joint remediation of heavy metals and organic pollutants.

4. The method according to any one of claims 1 to 3, characterized in that In step (1), the PDA culture medium includes 200 g / L of potato, 20 g / L of glucose, 15 g / L of agar, and the remainder is water; the subculture expansion culture is carried out at a temperature of 28°C; In step (2), the GYP liquid culture medium comprises 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L magnesium sulfate heptahydrate, and the remainder is water; the temperature of the control system during the primary culture process is 25°C to 30°C; the primary culture is carried out at a rotation speed of 150 rpm to 200 rpm; and the primary culture lasts for 6 to 8 days; In step (3), the volume ratio of the primary liquid culture to the GYP liquid culture medium is 1:50-200; the GYP liquid culture medium includes 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L magnesium sulfate heptahydrate, and the rest is water; the temperature of the control system during the secondary culture process is 25°C-30°C; the secondary culture is carried out at a rotation speed of 150 rpm-200 rpm; and the duration of the secondary culture is 2 days-4 days.

5. The method according to any one of claims 1 to 3, characterized in that The phenolic compound includes at least one of bisphenol A and tetrabromobisphenol A; the dye is at least one of crystal violet, malachite green, methyl blue, and basic fuchsin; the antibiotic is at least one of sulfonamide, sulfathiazole, sulfamethoxazole, and sulfadimethoxine; the polycyclic aromatic hydrocarbon pollutant is at least one of phenanthrene, fluoranthene, fluorene, and pyrene.

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