Fungus hdef-01 with multiple heavy metal resistance and application thereof

Abstract

The application relates to a fungus HDEF-01 with multiple heavy metal resistance and an application thereof, and relates to the field of microorganisms. The fungus HDEF-01 is Verticillium sp HDEF-01, which is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC No: M 20232129, a preservation address of No. 299, Bayingyi Road, Wuchang District, Wuhan City, Hubei Province, and a preservation date of November 3, 2023. The strain has excellent multiple heavy metal tolerance, and can normally survive and grow under the conditions of high concentrations of manganese, lead, cadmium and chromium. The fungus has extremely high manganese tolerance, and can survive and grow under the condition of a maximum of 80000 mg / L of Mn 2+ ion concentration. The fungus has high lead tolerance, cadmium tolerance and chromium tolerance.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology, specifically relating to a fungus HDEF-01 with multiple heavy metal resistance and its applications. Background Technology

[0002] Heavy metal pollution has long been a global environmental concern. Heavy metal pollution refers to environmental pollution caused by metals such as lead, cadmium, and chromium, or their compounds, primarily due to anthropogenic factors such as mining, wastewater discharge, wastewater irrigation, and the use of products exceeding heavy metal limits. More than 50% of the over ten million contaminated sites worldwide are caused by heavy metal pollution. Heavy metals are non-degradable and bioaccumulate, posing a severe challenge to the ecological environment and human survival. Remediation methods for heavy metal pollution mainly include physical remediation, chemical remediation, phytoremediation, microbial remediation, and combined remediation. Among these, bioremediation technologies are widely considered one of the most feasible technologies for correcting and restoring environmental stability in contaminated areas. Compared to physical and chemical remediation methods, it has advantages such as flexibility, durability, low pollution, and cost-effectiveness. Microorganisms, with their unique characteristics, have become a focus of research in heavy metal pollution remediation technologies. Methods such as chemical precipitation, adsorption, electrolysis, and reverse osmosis used in heavy metal wastewater treatment generally suffer from high investment and operating costs, complex operation and management, incomplete heavy metal removal, and the potential for secondary pollution. Microorganisms utilize their bioactivity to adsorb or transform heavy metals, altering their form in soil or water, thereby mitigating environmental pollution caused by heavy metals. This approach offers advantages such as being green, environmentally friendly, highly efficient, and low-cost. Metal ions can adversely affect the growth of fungal cells, ultimately inhibiting or killing them. Filamentous fungi are among the most economical and bio-friendly adsorbents available today, exhibiting strong environmental adaptability and a well-developed porous structure, making them promising candidates for biological water pollution treatment technologies. Summary of the Invention

[0003] The purpose of this invention is to provide a fungus, HDEF-01, with resistance to multiple heavy metals and its application, so as to improve the ability of fungal agents to treat heavy metals.

[0004] The present invention relates to a fungus HDEF-01 with multiple heavy metal resistance, which is Verticillium sp. HDEF-01, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No: M 20232129, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 3, 2023.

[0005] The present invention relates to the application of a fungus HDEF-01 with multiple heavy metal resistance, specifically the application of Verticillium sp. HDEF-01 in the preparation of heavy metal resistant fungal agents.

[0006] Furthermore, the preparation method of the aforementioned anti-heavy metal bacterial agent is as follows:

[0007] The heavy metal resistant agent was prepared by inoculating Verticillium sp. HDEF-01 into PDA medium or Martin medium.

[0008] Furthermore, the culture conditions are pH = 5.5-6.5 and temperature 28-30℃.

[0009] Furthermore, the PDA culture medium contains potato at a mass concentration of 100-300 g / L, glucose at a mass concentration of 10-30 g / L, and agar powder at a mass concentration of 10-30 g / L.

[0010] Furthermore, the Martin medium contains peptone at a mass concentration of 2-7 g / L, yeast extract at a mass concentration of 1-3 g / L, glucose at a mass concentration of 10-30 g / L, dipotassium hydrogen phosphate at a mass concentration of 0.5-2 g / L, magnesium sulfate at a mass concentration of 0.1-1 g / L, and agar powder at a mass concentration of 10-30 g / L.

[0011] Furthermore, the heavy metal is Mn. 2+ Pb 2+ Cd 2+ or Cr 6+ .

[0012] Furthermore, the Mn 2+ The mass concentration is 1000–80000 mg / L, Pb 2+ The mass concentration is 250–2000 mg / L, Cd 2+ The mass concentration is 250–2000 mg / L, Cr 6+ The mass concentration is 25–200 mg / L.

[0013] Furthermore, the application of Verticillium sp. HDEF-01 in the preparation of bioremediation agents for treating heavy metal contaminated environments; the heavy metal contaminated environment is a soil or water environment.

[0014] A fungal agent containing HDEF-01, a fungus with multiple heavy metal resistance as described in this invention.

[0015] The present invention has the following beneficial effects:

[0016] The strain of this invention exhibits excellent tolerance to multiple heavy metals, and can tolerate high concentrations of manganese (Mn). 2+ ), lead (Pb) 2+ ), cadmium (Cd 2+ ), chromium (Cr) 6+ It can survive and grow normally under certain conditions.

[0017] The fungus exhibits extremely high manganese resistance, tolerating concentrations up to 80,000 mg / L of Mn. 2+ Survival and growth under ion concentration.

[0018] The fungus also exhibits high resistance to lead (Pb). 2+ ), resistant to cadmium (Cd 2+ ) and chromium resistant (Cr 6+ Performance, capable of handling Pb concentrations up to 1500 mg / L 2+ 2000 mg / L of Cd 2+ 200 mg / L Cr 6+ It can survive and grow in solution. Attached Figure Description

[0019] Figure 1 Phylogenetic tree diagram of HDEF-1;

[0020] Figure 2 Colony morphology of HDEF-1 on Martin solid medium;

[0021] Figure 3 HDEF-1 at different concentrations of Mn 2+ Growth diagram on PDA medium;

[0022] Figure 4 HDEF-1 at different Pb concentrations 2+ Growth diagram on PDA medium;

[0023] Figure 5 HDEF-1 at different Cd concentrations 2+ Growth diagram on PDA medium;

[0024] Figure 6 HDEF-1 at different concentrations of Cr 6+ Growth diagram on PDA medium;

[0025] Figure 7 HDEF-1 at different concentrations of Mn 2+ Tolerance index (IT) graph on PDA medium;

[0026] Figure 8 HDEF-1 at different Pb concentrations 2+ Tolerance index (IT) graph on PDA medium;

[0027] Figure 9 HDEF-1 at different Cd concentrations 2+ Tolerance index (IT) graph on PDA medium;

[0028] Figure 10 HDEF-1 at different concentrations of Cr 6+ The tolerance index (IT) graph on PDA medium. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0030] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0031] Example 1

[0032] The fungus HDEF-01 with multiple heavy metal resistance in this embodiment is Verticillium sp. HDEF-01, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M20232129, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 3, 2023.

[0033] Screening, classification and identification of Verticillium sp. HDEF-01

[0034] 1) Screening process for Verticillium sp. HDEF-01:

[0035] Weigh 5g of collected Zhaodong saline-alkali soil, mix thoroughly with 100mL of sterile water, let stand for 1 hour, and then use a sterile pipette to add 1mL of the supernatant to a solution containing Mn. 2+ The culture medium was spread on PDA solid medium at concentrations of 10000 mg / L, 20000 mg / L, 30000 mg / L, 40000 mg / L, and 50000 mg / L, and incubated at 28°C for 20 days. Single colonies with good growth were then punched into mycelial cakes using a sterilized punch (5 mm diameter) in a clean bench. This process was then gradually repeated to higher concentrations of Mn. 2+The culture was further acclimatized on PDA plates at concentrations (60000 mg / L, 70000 mg / L, 80000 mg / L). Finally, the obtained single colonies were transferred more than three times for strain purification and stored in a refrigerator at 4°C. The isolated fungus was named HDEF-01.

[0036] 2) Classification and identification of Verticillium sp. HDEF-01:

[0037] 2.1 Morphological characteristics of Verticillium sp. HDEF-01: This strain is a filamentous fungus. Colonies are round with relatively regular edges. The hyphae grow vigorously, are white, and have a relatively thick hyphal layer. The aerial hyphae are dense, fluffy, and cottony, and are colorless and transparent. It is an aerobic bacterium.

[0038] 2.2 Molecular Identification

[0039] Fresh cells of *Verticillium sp.* HDEF-01 were collected, and total DNA was extracted using the CTAB method. Specifically, the dried bacterial cells were rapidly ground into powder using an appropriate amount of liquid nitrogen and placed in a sterile 1.5 mL centrifuge tube. 800 μL of preheated (65°C) CTAB extraction buffer was added and mixed thoroughly. The centrifuge tube was sealed and incubated at 65°C for 1 hour, inverting and mixing every 10-15 minutes. After incubation, 800 μL of phenol:chloroform:isopropanol (25:24:1) was added to the centrifuge tube, inverted and mixed, and then centrifuged at 12000 rpm for 10 minutes. Transfer the upper aqueous phase to a new 1.5 mL centrifuge tube, add an equal volume of chilled chloroform:isopropanol (24:1), invert and mix well, centrifuge at 12000 rpm for 10 min, collect the supernatant, add approximately 0.6 times the volume of the supernatant to pre-chilled isopropanol at -20℃, mix well, and incubate at -20℃ for 30 min to precipitate. Centrifuge at 12000 rpm for 10 min, discard the supernatant, wash twice with 75% ethanol, blot off excess ethanol with filter paper, and air dry in a clean bench. After drying, dissolve in 50 μL of distilled water, pipette and mix well, take 5 μL for 1.2% agarose gel electrophoresis detection, recover the bacterial DNA sample and store at -20℃. Total extracted DNA was used as a template for PCR amplification. Universal primers for fungal 18S rDNA were selected: NS1: 5'-GTAGTCATATGCTTGTCTC-3'; NS8: 5'-TCCGCAGGTTCACCTACGGA-3' to amplify the 18S rDNA sequence of *Verticillium sp.*. The conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec; 55℃ annealing for 30 sec; 72℃ extension for 1 min; a total of 35 cycles; final extension at 72℃ for 10 min; and storage at 4℃. The PCR products were recovered by gel extraction using the TaKaRaAgarose Gel DNA Purification Kit Ver.2.0, and the purified PCR products were sequenced by Shanghai Lifei Biotechnology Co., Ltd. The 18S rDNA sequencing results were compared with Nucleotide Blast on NCBI. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 11.0 software. The phylogenetic tree constructed by MEGA 11.0 software showed that this strain clustered with Verticillium sp ZLXH-10 (accession number KM886275.1) in one branch, with a branch support rate of 85%. Based on molecular biological phylogenetic analysis, this strain was identified as an unknown species of the genus Verticillium.

[0040] Example 2

[0041] The application of Verticillium sp. HDEF-01 in heavy metals in this embodiment is as follows:

[0042] Detection of different heavy metal tolerance levels by Verticillium sp. HDEF-01

[0043] After activating Verticillium sp. HDEF-01 on a PDA plate, mycelial cakes were created in a clean bench using a sterilized punch (5 mm diameter). These cakes were then inoculated onto plates containing different heavy metal ions (Mn). 2+ Pb 2+ Cd 2+ Cr 6+ On culture media with varying concentrations, the mycelial surface was brought into contact with the medium using sterile forceps, and the media were incubated upside down at 28°C for at least 14 days. Growth on PDA plates free of heavy metals served as a control. Three replicates were set up for each group to conduct different heavy metal tolerance tests. Mn in the medium was adjusted with MnSO4·H2O. 2+ Concentration, preparation of Mn 2+ PDA plate culture media with concentrations of 1000 mg / L, 4000 mg / L, 8000 mg / L, 16000 mg / L, 32000 mg / L, 40000 mg / L, 50000 mg / L, 60000 mg / L, 70000 mg / L, and 80000 mg / L; Pb content of the culture medium adjusted with (CH3COO)2Pb·3H2O. 2+ Concentration, preparation of Pb 2+ PDA plate culture media with concentrations of 250 mg / L, 500 mg / L, 750 mg / L, 1000 mg / L, 1125 mg / L, 1250 mg / L, 1375 mg / L, 1500 mg / L, and 2000 mg / L; the Cd concentration in the culture medium was adjusted with CdCl2·5 / 2H2O. 2+ Concentration, preparation of Cd 2+ PDA plate culture media with concentrations of 250 mg / L, 500 mg / L, 750 mg / L, 1000 mg / L, 1500 mg / L, and 2000 mg / L; Cr content of the culture medium was adjusted with K2Cr2O7. 6+ Concentration, preparation of Cr 6+ PDA plates with stock solution concentrations of 25 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L were used. The growth status of the strain on the culture medium was observed, and the diameter (mm) of Verticillium sp. HDEF-01 on PDA plates with different heavy metal concentrations was measured and recorded.

[0044] The tolerance indices (IT) of Verticillium sp. HDEF-01 to different heavy metals are as follows: Figure 7 , Figure 8 , Figure 9 , Figure 10 As shown, the tolerance index IT = diameter of colonies on a plate containing heavy metals / diameter of colonies on a blank plate. Verticillium sp. HDEF-01 is shown to be effective against manganese (Mn). 2+ ), lead (Pb) 2+ ), cadmium (Cd 2+ ), chromium (Cr) 6+ It exhibits high tolerance to high concentrations of Mn. 2+ Under stress of 70,000 mg / L or less, the tolerance index was greater than or equal to 0.36; under high concentrations of Pb... 2+ Under stress of 1500 mg / L or less, the tolerance index was greater than or equal to 0.59; under high concentrations of Cd... 2+ Under stress of 1500 mg / L or less, the tolerance index was greater than or equal to 0.21; under high concentrations of Cr... 6+ Under stress of 200 mg / L or less, the tolerance index is greater than or equal to 0.60. This confirms that the *Verticillium sp.* HDEF-01 provided by this invention is resistant to manganese (Mn) 2+ ), lead (Pb) 2+ ), cadmium (Cd 2+ ), chromium (Cr) 6+ They all exhibit good resistance, with particularly outstanding resistance to manganese. 2+ At a concentration of 80,000 mg / L, the tolerance index was still as high as 0.14.

[0045] Table 1 HDEF-1 at different concentrations of Mn 2+ Pb 2+ Cd 2+ Cr 6+ Growth comparison

[0046]

Claims

1. A fungus, HDEF-01, exhibiting resistance to multiple heavy metals, characterized in that... It is Verticillium ( Verticillium sp HDEF-01, deposited at the China Center for Type Culture Collection, accession number CCTCC No: M 20232129, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 3, 2023.

2. The application of the fungus HDEF-01 with multiple heavy metal resistance as described in claim 1, characterized in that... The aforementioned Verticillium ( Verticillium sp Application of HDEF-01 in the preparation of anti-heavy metal bacterial agents; the heavy metal is Mn. 2+ Pb 2+ Cd 2+ or Cr 6+ .

3. The application according to claim 2, characterized in that... The preparation method of the anti-heavy metal bacterial agent is as follows: Verticillium ( Verticillium sp HDEF-01 was inoculated into PDA medium or Martin medium for culture to obtain an anti-heavy metal bacterial agent.

4. The application according to claim 3, characterized in that... The culture conditions are pH 5.5-6.5 and temperature 28-30℃.

5. The application according to claim 3, characterized in that... The PDA culture medium contains 100-300 g / L potato, 10-30 g / L glucose, and 10-30 g / L agar powder.

6. The application according to claim 3, characterized in that... The Martin medium contains peptone at a mass concentration of 2-7 g / L, yeast extract at a mass concentration of 1-3 g / L, glucose at a mass concentration of 10-30 g / L, dipotassium hydrogen phosphate at a mass concentration of 0.5-2 g / L, magnesium sulfate at a mass concentration of 0.1-1 g / L, and agar powder at a mass concentration of 10-30 g / L.

7. The application according to claim 2, characterized in that... The Mn 2+ The mass concentration is 1000-80000 mg / L, Pb 2+ The mass concentration is 250-2000 mg / L, Cd 2+ The mass concentration is 250-2000 mg / L, Cr 6+ The mass concentration is 25-200 mg / L.

8. The application according to claim 2, characterized in that... Verticillium ( Verticillium sp The application of HDEF-01 in the preparation of bioremediation agents for treating heavy metal contaminated environments; the heavy metal contaminated environment is a soil or water environment.

9. A fungal agent containing HDEF-01, a fungus with multiple heavy metal resistance as described in claim 1.

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