Acid-resistant fungus hdmf-1 with high manganese resistance and application thereof

Abstract

This invention relates to an acid-resistant fungus, HDMF-1, with high manganese resistance and its applications. The strain is *Acidomyces sp.* HDMF-1, deposited at the China Center for Type Culture Collection (CCTCC), accession number M 20232128, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 3, 2023. This strain exhibits extremely high acid resistance, surviving and growing normally under acidic conditions (pH 0.5–4.0) and alkaline conditions (pH 8–10). It can withstand manganese concentrations up to 1.5 mol / L. 2+ It survives and grows under ion concentrations. The fungus also exhibits high resistance to zinc, copper, and lithium, and can survive in concentrations of approximately 1 g / L Zn. 2+ Cu 2+ Li + It survives and grows in solutions containing Mn. The fungus has a high Mn content. 2+ Adsorption capacity.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology, specifically relating to an acid-resistant fungus HDMF-1 with high manganese resistance and its applications. Background Technology

[0002] With the rapid development of the economy, my country's demand for mineral resources is increasing. However, industrial activities such as metal mining, beneficiation and smelting, machining, electroplating, and detoxification of heavy metal-containing solid waste generate large amounts of acidic heavy metal wastewater. This type of wastewater is characterized by low pH (pH < 5.0) and high concentrations of heavy metal ions, polluting water bodies and soil, and seriously threatening human health and the ecological environment. Methods such as chemical precipitation, adsorption, electrolysis, and reverse osmosis used in the treatment of acidic heavy metal wastewater generally suffer from high investment and operating costs, complex operation and management, incomplete heavy metal removal, and the potential for secondary pollution. Metal ions can adversely affect the growth process of fungal cells, ultimately leading to inhibited or dead fungi. Acid-resistant fungi possess physiological characteristics that tolerate low pH levels, exhibit strong environmental adaptability, and have a well-developed porous structure, making them one of the most economical and bio-friendly adsorbents with broad application prospects in the biological treatment of acidic wastewater pollution.

[0003] pH affects microbial activity as a result of the interaction between hydrogen ions and enzymes in the cell membrane, and also affects the activity of enzymes on the cell wall. pH is an important factor affecting the growth and reproduction of microorganisms. The optimal pH for the growth and reproduction of most microorganisms is 5-9, and most microorganisms cannot survive in low pH environments.

[0004] Manganese is an essential trace metal element for living organisms and one of the most abundant minerals in the Earth's crust, possessing diverse chemical properties and wide applications. In recent years, with increased human activity, environmental manganese levels have exceeded standards, leading to pollution and posing potential health threats to both organisms and humans. While trace amounts of manganese can stimulate microbial activity, excessive intake can lead to competition with other essential metals for binding sites of sulfur compounds and oxygen, damaging nucleic acid and protein structures, interfering with oxidative phosphorylation and osmotic pressure balance, resulting in tissue damage and manganese poisoning symptoms in organisms and even humans. This threat to biodiversity has attracted widespread social attention. Summary of the Invention

[0005] This invention provides an acid-resistant fungus, HDMF-1, with high manganese resistance and its application, to improve the ability of fungal agents to treat heavy metals, acids, and alkalis.

[0006] The present invention relates to an acid-resistant fungus, HDMF-1, with high manganese resistance, which is an acidophilic fungus (HDMF-1). Acidomyces spHDMF-1, deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20232128, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 3, 2023.

[0007] The present invention relates to the application of an acid-resistant fungus, HDMF-1, with high manganese resistance, in the preparation of acid-resistant, alkali-resistant, and / or heavy metal-resistant fungal agents.

[0008] Furthermore, acidophilic fungi ( Acidomyces sp HDMF-1 was inoculated into PDA medium or Martin medium and cultured to obtain the bacterial agent.

[0009] Furthermore, the culture conditions are pH=1.0-4.0 and temperature 28-30℃.

[0010] 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.

[0011] 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.

[0012] Furthermore, the heavy metal is Mn. 2+ Zn 2+ Cu 2+ Or Li + .

[0013] Furthermore, the molar concentration of manganese is 0.2-1.5 mol / L, and Zn 2+ The mass concentration is 0.5-1.5 g / L, Cu 2+ The mass concentration is 0.5-1.5 mol / L, Li + The mass concentration is 0.5-1.5 mol / L.

[0014] Furthermore, the alkali-resistant pH is 8-10.

[0015] The present invention has the following beneficial effects: The strain of this invention exhibits extremely high acid resistance, surviving and growing normally under acidic conditions of pH 0.5–4.0, and simultaneously surviving and growing normally under alkaline conditions of pH 8–10. The fungus also exhibits extremely high manganese resistance, able to survive at concentrations up to 1.5 mol / L of Mn.2+ It survives and grows at ion concentrations. The fungus also exhibits high resistance to zinc, copper, and lithium, and can survive in concentrations of approximately 1 g / L Zn. 2+ Cu 2+ Li + It survives and grows in solutions containing Mn. The fungus has a high Mn content. 2+ Adsorption capacity. Attached Figure Description

[0016] Figure 1 This is a phylogenetic tree diagram of HDMF-1 of the present invention; Figure 2 Image showing the growth morphology and hyphal morphology of HDMF-1 on PDA solid medium; Figure 3 Image of HDMF-1 hyphal morphology under a scanning electron microscope; Figure 4 Figures showing the growth status of HDMF-1 on solid Martin medium at different pH values; Figure 5 This is a diagram showing the growth of HDMF-1 in solutions of different heavy metal ions. Figure 6 HDMF-1 in different Mn 2+ Growth diagrams at different concentrations; Figure 7 For no Mn 2+ X-ray energy distribution maps of HDMF-1 under the specified conditions; (a) EDS layered image (Mn element); (b) spot scan; (c) line scan; (d) (e) elemental distribution map; Figure 8 0.5 mol / L Mn 2+ X-ray energy distribution maps of HDMF-1 under the specified conditions; where (a) EDS layered image (Mn element); (b) spot scan; (c) line scan; (d) (e) elemental distribution map; Figure 9 1.25 mol / L Mn 2+ X-ray energy spectrum of HDMF-1 under the specified conditions; where (a) EDS layered image (Mn element); (b) spot scan; (c) line scan; (d) (e) elemental distribution map. Detailed Implementation

[0017] 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.

[0018] 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. Example

[0019] This embodiment describes an acid-resistant fungus, HDMF-1, with high manganese resistance. It is an acidophilic fungus (HDMF-1). Acidomyces sp HDMF-1, deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20232128, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 3, 2023.

[0020] The acidophilic fungi ( Acidomyces sp HDMF-1 screening, classification and identification 1) Acidophilic fungi ( Acidomyces sp The HDMF-1 screening process: Weigh 5 g of oil shale sample collected from the Yilan Basin in Heilongjiang Province and grind it into powder. Mix thoroughly with 100 mL of sterile water and let stand for 1 h. Use a sterile pipette to add 1 mL of the supernatant to 50 mL of potato broth (PDB) and incubate at 28 °C and 120 r·min. -1 Enrichment culture was carried out under the specified conditions for 72 h, with subculturing every 72 h, for a total of 2 subculturings before use. Isolation and purification of acid-tolerant fungi: HCl solution was added to potato broth (PDB) to adjust the pH to 0.5, and then 1 mL of the subcultured and enriched bacterial solution was added. The culture was then incubated at 28℃ and 120 r·min. -1 The bacterial culture was subjected to acid-resistant enrichment culture in a constant temperature shaking incubator for 14 days. 0.2 mL of the acid-resistant cultured bacterial solution was aseptically spread onto potato dextrose agar plates and incubated upside down in a 28°C constant temperature incubator for 2-3 days until visible colonies appeared. Typical single colonies growing on the plates were selected and repeatedly streaked until the bacterial morphology was uniform under a microscope, at which point streaking was stopped for isolation. The obtained pure colonies were stored as a starter culture at 4°C, and then transferred to a -80°C freezer after one day.

[0021] 2) Acidophilic fungi ( Acidomyces sp Classification and identification of HDMF-1: 2.1 Acidophilic fungi ( Acidomyces sp Morphological characteristics of HDMF-1: This strain is a filamentous fungus with vigorous hyphal growth, septate hyphae, and elliptical spores. The colony surface is grayish-black with wrinkles; it is an aerobic bacterium. The acidophilic fungus of this invention (… Acidomyces sp The hyphal morphology of HDMF-1 under a scanning electron microscope is as follows: Figure 3 As shown.

[0022] 2.2 Molecular Identification Fresh mycelium was collected, and acidophilic fungi were extracted using the CTAB chemical method. Acidomyces The total DNA of sp.) HDMF-1 was extracted using the following method: Dry bacterial cells were rapidly ground into powder using an appropriate amount of liquid nitrogen. The powder was then placed in a sterile 1.5 mL centrifuge tube, and 800 μL of preheated CTAB extraction buffer (65°C) was added and mixed thoroughly. The centrifuge tube was sealed and placed in a 65°C water bath for 1 hour, inverting and mixing every 10-15 minutes. After completion, 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 to mix, and centrifuge at 12000 rpm for 10 min. Take the supernatant and add approximately 0.6 times the volume of pre-chilled isopropanol (at -20°C), mix well, and incubate at -20°C 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 to mix, and take 5 μL for 1.2% agarose gel electrophoresis. Recover the bacterial DNA sample and store at -20°C. 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 acidophilic fungi ( Acidomyces The 18S rDNA sequence of *S.* sp. was obtained. The conditions were as follows: 94℃ pre-denaturation, 7 min; 94℃ denaturation, 1 min; 56.5℃ annealing, 30 s; 72℃ extension, 1 min; a total of 35 cycles; final extension at 72℃, 7 min; storage at 4℃. The PCR products were gel-cleaved and purified using the TaKaRa Agarose Gel DNA Purification Kit Ver.2.0. The purified PCR products were sequenced by Shanghai Lifei Biotechnology Co., Ltd. The 18S rDNA sequencing results were aligned using Nucleotide Blast on NCBI, and a phylogenetic tree was constructed using neighbor-joining in MEGA 11.0 software. The phylogenetic tree constructed using MEGA 11.0 software showed that this strain was related to acidophilic fungi (…). Acidomyces sp. AK72 / 03 (accession number FJ430711.1) clustered together, with a branching support of 79%. Based on molecular biological phylogenetic analysis, this strain was identified as an acidophilic fungus. Acidomyces An unknown species. Example

[0023] This embodiment of acidophilic fungi ( Acidomyces sp The applications of HDMF-1 in acid (alkali) resistance and heavy metal resistance are as follows: 1) Resistant to acids (alkalis) pH gradient plate preparation: Prepare 50 ml of Martin liquid medium at pH 0.1, pH 0.5, pH 1, pH 2, pH 3, and pH 4 respectively (adjust the pH of the medium using concentrated HCl). At the same time, prepare agar solution (20 g / L). Autoclave each medium separately (121℃, 20 min). After sterilization, under aseptic conditions, mix the Martin liquid medium of each pH value with different volumes of melted agar solution (20 g / L), then pour the mixture into petri dishes. Place the petri dishes horizontally and allow them to solidify. This will give you the pH gradient plates of pH 0.5, pH 1, pH 2, pH 3, and pH 4. In addition, prepare 100 ml of each of Martin liquid culture medium at pH 5, pH 6, pH 7, pH 8, pH 9, and pH 10 (adjust the pH of the culture medium using concentrated HCl and solid NaOH), add 2 g of agar to each medium, and autoclave at 121°C for 20 min. After sterilization, under aseptic conditions, pour the mixture into petri dishes, place the petri dishes horizontally, and allow them to stand and solidify to form gradient plates at pH 5, pH 6, pH 7, pH 8, pH 9, and pH 10.

[0024] Acidophilic fungi ( Acidomyces sp HDMF-1 was inoculated onto the above pH gradient plates, with inoculum blocks of 5 mm diameter (using a sterilized 5 mm diameter punch). The incubation temperature was 28-30℃, and the acidophilic fungi were cultured for 14 days. Acidomyces sp HDMF-1 strain status as follows Figure 4 As shown, Figure 4 The growth status of this strain on solid Martin medium at different pH values ​​is shown by... Figure 4 Acidophilic fungi ( ) can be identified Acidomyces sp HDMF-1 survives and grows normally under conditions of pH 0.5 to pH 10, and is an acidophilic fungus in the pH range of pH 1 to pH 4. Acidomyces sp HDMF-1 grows rapidly and has a large colony diameter, indicating that its optimal growth pH range is pH 1 - 4.

[0025] 2) Resistant to heavy metals Preparation of liquid culture medium for metal ion resistance screening: Appropriate amounts of MnCl2, ZnCl2, CuCl2, LiCl, NiCl, CdCl2, K2Cr2O7, and (CH3COO)2Pb were added to 100 mL of prepared PDB liquid culture medium to prepare solutions with different heavy metal ion concentrations (MnCl2, ZnCl2, CuCl2, LiCl, NiCl, CdCl2, K2Cr2O7, and (CH3COO)2Pb) at 1 g / L. 2+ Zn 2+ Cu 2+ Li + Ni + Cd 2+ Cr 6+ Pb 2+ PDB liquid culture medium was prepared. The medium was autoclaved (121°C, 20 min). Acidophilic fungi (…) were then added. Acidomyces sp HDMF-1 was inoculated into the above-mentioned culture medium using 5mm diameter mycelial blocks (using a sterilized 5mm diameter punch), and cultured at 28-30℃. After 14 days of culture, the acidophilic fungi ( Acidomyces sp The growth status of HDMF-1 in solutions of different heavy metal ions is as follows: Figure 5 As shown, by Figure 5 It can be seen that HDMF-1 can be used in the presence of Mn 2+ Zn 2+ Cu 2+ Li + Growth in culture media containing heavy metal ions. Table 1. Acidophilic fungi ( Acidomyces sp The tolerance of HDMF-1 to different heavy metal ions is shown in Table 1. Table 1 shows that HDMF-1 tolerates various heavy metal ions (Mn2, 22-24-25 ... 2+ Zn 2 + Cu 2+ Li + It has resistance.

[0026] Table 1. Tolerance of HDMF-1 to different heavy metal ions HDMF-1 (sp.) +++ +++ ++ + - - - - Note: + indicates growth status; the more +s, the better the growth status; - indicates no growth.

[0027] 3) Resistant to heavy metal Mn Preparation of gradient liquid culture media containing heavy metal Mn: Add appropriate amounts of MnCl2 to 100 mL of prepared Martin liquid medium to prepare media containing different amounts of Mn. 2+Initial concentrations of Martin liquid broth (0, 200, 400, 600, 800, 1000, 1250, 1500 mmol / L) were prepared, and the pH of the broth was adjusted to pH 3 using HCl solution. The broth was then autoclaved (121°C, 20 min). Acidophilic fungi (…) Acidomyces sp HDMF-1 was inoculated into the above-mentioned culture medium using 5 mm diameter mycelial blocks (using a sterilized 5 mm diameter punch), and cultured at 28-30℃. After 14 days of culture, the acidophilic fungi ( Acidomyces sp HDMF-1 in different Mn 2+ Growth at concentrations such as Figure 6 As shown, by Figure 6 It can be concluded that, with Mn 2+ Increased concentration of acidophilic fungi ( Acidomyces sp HDMF-1 showed a trend of first increasing and then decreasing at high concentrations of Mn. 2+ It still grows well under stress, even at 1500 mmol / L Mn. 2+ It can grow even at low concentrations, indicating that it is resistant to the heavy metal Mn. 2+ It exhibits very high resistance, far exceeding that of other strains reported in the literature to Mn. 2+ Tolerance. Figure 7 For no Mn 2+ acidophilic fungi under certain conditions ( Acidomyces sp X-ray energy dispersive spectroscopy of HDMF-1 and line scan results of HDMF-1 hyphae show that the mass percentage and atomic percentage of Mn are only 0.12% and 0.03%, respectively. Figure 8 0.5 mol / L Mn 2+ acidophilic fungi under certain conditions ( Acidomyces sp X-ray energy dispersive spectroscopy of HDMF-1. Linear scanning results of HDMF-1 hyphae show that the mass percentage and atomic percentage of Mn are 3.45% and 0.86%, respectively. Figure 9 1.25 mol / LMn 2+ acidophilic fungi under certain conditions ( Acidomyces sp X-ray energy dispersive spectroscopy (EDS) of HDMF-1 and line scanning analysis of HDMF-1 hyphae showed that the mass percentage and atomic percentage of Mn were 19.09% and 5.27%, respectively. (Summary) Figure 7 , Figure 8 , Figure 9 Based on the data in Table 2, it can be seen that as Mn... 2+ As concentration increases, acidophilic fungi ( Acidomyces sp HDMF-1 can effectively adsorb Mn 2 + .

[0028] Table 2 Mn at different concentrations 2+ Elemental changes on the surface of HDMF-1 mycelia under certain conditions Note: Lowercase letters represent different Mn 2+ The difference in the percentage of Mn on the bacterial cell surface (Wt%) between concentration treatments, where uppercase letters represent different Mn concentrations. 2+ Differences in the percentage of Mn atoms (At%) on the bacterial cell surface between concentration treatments ( P <0.05).

Claims

1. A strain of acidophilic fungus ( Acidomyces sp HDMF-1, characterized in that, The acidophilic fungi ( Acidomyces sp HDMF-1, deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20232128, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 3, 2023.

2. The acidophilic fungus of claim 1 ( Acidomyces sp The application of HDMF-1 in the preparation of acidophilic, alkali-resistant and / or metal-resistant microbial agents, characterized in that the metal is Mn. 2+ Zn 2+ Cu 2+ Or Li + The Mn 2+ The molar concentration of Zn is 0.2-1.5 mol / L. 2+ The mass concentration of Cu is 0.5-1.5 g / L. 2+ The molar concentration of the Li is 0.5-1.5 mol / L. + The molar concentration is 0.5-1.5 mol / L, the acid resistance pH is 1-4.0, and the alkali resistance pH is 8-10.

3. The application according to claim 2, characterized in that, The acidophilic fungus HDMF-1 was inoculated into PDA medium or Martin medium and cultured to prepare the inoculum.

4. The application according to claim 3, characterized in that, The culture conditions are pH=1.0-4.0 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.

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