CN111334435A - Separation and identification method of acidophilic fungus with biological induction mineralization effect - Google Patents

Separation and identification method of acidophilic fungus with biological induction mineralization effect Download PDF

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CN111334435A
CN111334435A CN202010075074.7A CN202010075074A CN111334435A CN 111334435 A CN111334435 A CN 111334435A CN 202010075074 A CN202010075074 A CN 202010075074A CN 111334435 A CN111334435 A CN 111334435A
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欧舒宁
江晓敏
梁洁良
贾璞
束文圣
李金天
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Abstract

The invention provides a method for separating and identifying acidophilic fungi with biological induction mineralization effect, which comprises the following steps: s1 obtaining pure cultured acidophilic fungi from the acid bottom mud of the lead zinc mine tailings, identifying the colony morphology and determining the taxonomic status of the strains; s2 culturing acidophilic fungus with the acid waste water of lead-zinc ore tailings as culture liquid, and analyzing the metabolism potential of acidophilic fungus in acid environment. The separation and identification method can effectively separate and identify the induced mineralization capability of the acidophilic fungi separated from the acid bottom mud of the tailings of the lead and zinc ores.

Description

Separation and identification method of acidophilic fungus with biological induction mineralization effect
Technical Field
The invention belongs to the technical field of microorganism separation, and particularly relates to a method for separating and identifying acidophilic fungi with a biological induction mineralization effect.
Background
The mine acidic environment mainly includes mine acidic wastewater (AMD), sediment (segment), biofilm (biofilm), and the like, and is generated in production activities such as mining and the like of human beings. Large amounts of unusable residues from mining activities, i.e. tailings (tails), and ores with a relatively low mineral content (lean ores), etc., are often stored in open-air heaps or are treated by surface casing (or flooding); thus, by spontaneous or microbial-mediated chemical processes, it is possible to create a wide variety of mine acidic environments.
AMD is mainly composed of pyrite (FeS, pyrite)2) And other metal sulfides are generated after being exposed to the atmosphere and water for oxidation and dissolution. AMD and its associated environment has extremely low pH, high heavy metal concentration and high sulfate concentration characteristics, in AMD environment microbial research is favorable for the discovery of microorganisms in the AMD process of role, so as to find suitable strategy to inhibit or reduce the AMD production. Some beneficial microorganisms that can raise pH and immobilize heavy metal ions are also present in the AMD environment, and their research and utilization contribute to our development of bioremediation and management strategies for AMD contamination.
The acidic environment of mine is a representative of extreme environment, wherein the microbial community has low diversity, the interrelation between organisms is relatively simple, and the microbial community gradually forms some unique mechanisms in the long-term evolution process so as to respond to various extreme environment stresses such as low pH value and high heavy metal content. Such a well-functioning and simple microbial system is considered as a model system for studying the ecology and evolution of microorganisms. By revealing the diversity and functional characteristics of prokaryotic, eukaryotic microorganisms and viruses among others, we have helped to understand the (co-) evolutionary history of the various components of the community as a whole and their interactions. In addition, the AMD environment is very similar to the conditions in the early stages of the formation of the earth, which can help people to understand the behavior mode in the early stage of life. Therefore, the research on the acid environment of the mine has important theoretical research value.
Despite the various environmental stresses, as with microbial communities of other habitats, there are different functional groups of microorganisms in the acidic environment of mines that not only drive the major biogeochemical cycle processes, but also biodiversity is crucial to the function and stability of the AMD ecosystem. In the past decades, based on technologies such as microorganism isolation culture, SSU rRNA/ITS molecular marker gene sequencing and metagenome analysis, the species and diversity of microorganisms in the acidic environment of a mine are deeply known. But there is little research on fungi compared to bacterial and archaeal prokaryotes. Like prokaryotes, there are many fungi in the acidic environment of mines, but few species.
The most studied cases of obtaining pure cultures of fungi in the acidic environment of mines were isolated by researchers in the AMD waters and nearby acidic soils of Ohio and Virginia, USA, to 189 fungi, including yeast-type and filamentous fungi. Fungi such as Candida, Rhodotorula, and Trichosporon are often isolated from AMD waters.
Generally, fungi can adapt to a wide range of pH (0-11) and their presence has been monitored in many acidic environments such as volcanic hot springs, AMD or industrially contaminated acidic wastewater, but many of them are acid-tolerant fungi, and few pure cultured acidophilic fungi have been obtained from acidic environments as of Starkey and Waksman since the last 40 th generation successfully isolated two extreme acidophilic and heavy metal tolerant fungi Scytalidium acidophilum and Acontium velatum from water containing 4% copper sulfate, the former even growing under pH0. researchers later isolated acidophilic fungi from several places such as soil (pH 1.4-3.5) adjacent to sulfur dumps, acidic wastewater (pH 0.8-1.38), lignite rich in humic and fulvic acids (pH0.6), acidic hot springs et al 2002, Amaral Zettler et al, using 18S gene cloning methods, first studied Western nucleotide sequencing libraries (pH 2), revealed that most of the bacterial strains are highly characterized by the presence of a high pH-10. A. can be a. by sequencing of fungi, sequencing of bacteria, sequencing of strains of bacteria, a variety of bacteria belonging to a high pH, a species belonging to a high strain belonging to a family of a variety of bacteria belonging to a high pH, a family of a. a family of bacteria, a variety of a family of bacteria, a family of bacteria, a family of bacteria, a family of a.
Recently, Mesa et al have studied the diversity of bacteria, archaea and eukaryotes in three different microenvironments, including AMD water, sediment and biofilm, in a waste mercury mine in the southwest of Spain, using high throughput 16S/18S rRNA sequencing technology. Despite some progress, the current understanding of the diversity of AMD eukaryotic microorganisms and their ecological distribution patterns is still very limited.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for separating and identifying acidophilic fungi with biological induction mineralization effect.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for separating and identifying acidophilic fungi with biological induction mineralization effect comprises:
s1 obtaining pure cultured acidophilic fungi from the acid bottom mud of the lead zinc mine tailings, identifying the colony morphology and determining the taxonomic status of the strains;
s2 culturing acidophilic fungus with the acid waste water of lead-zinc ore tailings as culture liquid, and analyzing the metabolism potential of acidophilic fungus in acid environment.
Further, the step S1 is to obtain pure cultured acidophilic fungi by the following steps: coating diluted lead-zinc ore tailing acid bottom mud in an optimized 9K plate culture medium, and culturing the plate at 25 ℃ for 1 month; single colonies on the plates were picked and inoculated into 9K liquid medium to obtain pure cultures.
Further, the preparation method of the 9K culture medium comprises the following steps: A. sterilizing solution C at 121 deg.C for 20min, filtering solution B with 0.2 μm filter membrane to remove mixed bacteria, and mixing solutions A, B, C to obtain 9K culture medium; wherein the formula of the solution A is as follows: (NH)4)2SO43g，KCl 0.1g，K2HPO40.5g，MgSO4·7H2O 0.5g，Ca(NO3)2·H20.01g of O and 500ml of distilled water, wherein the pH value is 2.5; the formula of the solution B is as follows: FeSO4·7H2O33 g; 300ml of distilled water; the pH was 2.5; the formula of the solution C is as follows: agarose 15g, distilled water 200 ml.
Further, the step S1 of determining the taxonomic status of the strains includes the following steps: extracting genome DNA of acidophilic fungi to perform PCR amplification, sequencing the whole genome, performing blast comparison on the obtained sequence on NCBI to obtain a known sequence with higher homology with the sequence, and determining the taxonomic status of strains.
Further, the primers used for the PCR amplification are ITS 1: 5'
-TCCGTAGGTGAACCTGCGG-3’；ITS4：5’-TCCTCCGCTTATTGATATGC-3’。
Further, the step S2 of analyzing the metabolic potential of the acidophilic fungus in an acidic environment specifically includes the following steps: adding glucose liquid into the acid wastewater of the lead-zinc ore tailings, inoculating acidophilic fungi, culturing, detecting the cultured precipitate, and performing element analysis on the precipitate.
Further, the element analysis is to detect S, K, C, O, N, Fe and Ca elements.
Further, the elemental analysis is analyzed by an X-ray energy spectrometer.
Further, the step S2 further includes identifying the pH adaptation range of the acidophilic fungus.
Further, said step of identifying the pH adaptation range of the acidophilic fungus comprises the steps of: the culture medium is set with 10 pH gradients of 1-10 respectively, and the acidophilic fungus is cultured at 25 deg.C for 7 days with an inoculum size of 1%. The growth of the cells was observed, and the dry weight of the cells was determined by centrifugal air-drying.
The invention obtains 31 strains of fungi from pure culture of AMD bottom mud of the tailings of the minite. Based on ITS data, one fungal strain that yielded the most (20 strains) in our culture was found to belong to the Acidiella bohemica, which is classified as a dothideomyces, Capnodiales, Teratosphaeriaceae. The colony morphology observation shows that the colony of the filamentous fungus, namely A. bohemica (Acidiella bohemica) grows slowly and is relatively flat on the whole; the colony is stacked in the middle, is very compact, and has a flat edge and a little velvet shape. In the acid 9k medium, olive green is in a black color. The mycelium consists of isolated colorless or light colored hyphae, which form conidia by fission, with oval, swollen arthrospores. The invention selects Acidiella bohemica SYSU C17045 as a representative to carry out subsequent experiments.
The results of experiments in pH range showed that Acidiella bohemica SYSU C17045 can grow in pH range 2-8, wherein the growth is best at pH 3, and growth is inhibited at pH 1, pH 9, and pH 10. Bohemica was used in simulation experiments to understand the role a. bohemica may play in AMD habitat. After the A.bohemica is inoculated by adding glucose liquid into an AMD water sample of the plumbum-zincum tailings, early-stage fungi grow and propagate in a better trend, and the surface of a bacterial colony is dark brown and black; in addition to the agglomerated colonies in the liquid medium, a part of the cells adhered to the wall of the flask for growth. After 10 days, yellow substances can be seen on the surfaces of the colonies, and no obvious growth and reproduction phenomenon of the colonies is observed; after 25 days, more yellow substances can be obviously observed to be attached to the surface of the bacterial colony, and part of brownish black bacteria are still exposed outside and are not wrapped; by 50 days of culture, it was seen that the colonies were tightly packed with yellow material. Part of the precipitate was taken out and could not be broken up with a bamboo stick, the yellow precipitate and the cells were very tightly bound together, and the precipitate was not dissolved under acidic conditions.
Detecting the yellow precipitate, wherein some particles are on the surface of hypha as shown in scanning electron microscope (FIG. 1); needle-shaped substances are attached to the surfaces of the hyphae; a large amount of regularly arranged fusiform substances are observed in some areas; some regions accumulate a large amount of needle-like substances. The results of analysis on a scanning electron microscope by combining an X-ray energy spectrometer show (figure 2) that the substances contain high content of iron element and S, K element, and the elements are exactly the composition of jarosite which is a common mineral in an AMD system; while a high proportion of C, O, N is primarily a constituent element of the organism. Thus indicating that Acidiella bohemica SYSU C17045 has the biological induction mineralization effect.
By observing with a transmission electron microscope (FIG. 3) using the ultrathin section, we can see that there are needle-like substances around the cell surface and that there are a lot of needle-like substances in the precipitate.
In the heavy metal ion utilization experiment, the physicochemical analysis of the lead-zinc mine tailing acid wastewater culture solution shows that compared with a blank control group, after the A.bohemica is cultured in a PDA culture medium with the optimum pH value of 3 at 25 ℃ for 50 days, the Fe element in the culture solution is greatly reduced, the total iron precipitation rate reaches 20.7%, and the Fe precipitation rate reaches 20.7%2+The oxidation rate of the catalyst is as high as 46.3%, and S, Ca elements are all obviously reduced. Under the condition, the yield of jarosite precipitate obtained after the culture is finished is 12.49g/L after the jarosite precipitate is washed and dried, and the yield reaches 46.17%. From this, we can determine that a. bohemica has an important role in the bio-induced mineralization (biologicalinduction) process.
TABLE 1 partial element content (unit mg/L) of the culture broth
Figure BDA0002378280150000051
Therefore, the biological induction mineralization function of the acidophilic fungi can be effectively identified by the separation and identification method.
Compared with the prior art, the invention has the following advantages:
1. the invention successfully separates and cultures 20 acidophilic fungi Acidiella bohemica from the acidic bottom mud of the Guangdong Van Kong lead zinc ore tailings, utilizes the acidic mine wastewater to culture and observe, and simultaneously utilizes the third generation single molecule real-time sequencing to carry out sequencing analysis on the whole genome, thereby exploring the adaptation mechanism and the influence of the acidophilic fungi Acidiella bohemica on the acid environment of the mine.
2. The biological induction mineralization capability of the A.bohemica is identified through a pH adaptability experiment and a heavy metal ion utilization experiment. Meanwhile, the transcriptome of the gene is combined to carry out gene prediction, 10985 genes are obtained in total and used for reconstructing C, N, S and Fe related metabolic pathways of the gene, and the complete glycolysis and TCA pathways are annotated in A.bohemica, and nitrate reduction and sulfate reduction are assimilated.
3. Phylogenetic analyses based on the whole proteome gave 5966 families of homologous genes shared by a. bohemica, baudoisonia panameriana, dotistaria septosporum and Acidomyces richmonodens and 162 families unique to a. bohemica. Bohemica has a genomic co-linearity of only 20.82% with Baudoinia panamericaa. Bohemica and its related species are widely distributed in extreme environments, and future more genome sequencing may help explain the molecular systems of these extreme microorganisms to be problematic.
Drawings
FIG. 1 is a scanning electron microscope result chart of the present invention.
FIG. 2 is a schematic diagram of the results of an X-ray spectrometer analysis.
FIG. 3 is a transmission electron micrograph.
Fig. 4 is a colony morphology and microscopic observation morphology of an acidophilic fungus a. bohemica plate of the present invention.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
1. Strain screening
AMD bottom mud samples are collected from the yellow sublevel reservoir area of the minium lead-zinc ore tailing reservoir, collected by adopting a sterile 50ml centrifugal tube, transported back to a laboratory in an ice bath mode and stored at 4 ℃. AMD water samples were collected in 25L buckets and transported back to the laboratory for immediate subsequent treatment.
The pH value of a sediment sample is 2.2, PBS is added into the sediment for treatment, and then Phosphate buffer solution (PBS for short) is coated on an optimized 9K plate culture medium for culture, wherein the formula of the culture medium is as follows:
solution A: (NH)4)2SO43g；KCl 0.1g；K2HPO40.5g；MgSO4·7H2O 0.5g；Ca(NO3)2·H2O0.01g；Distilled H2O 500ml；pH(H2SO4)2.5。
And B, liquid B: FeSO4·7H2O 33g；Distilled H2O 300ml；pH(H2SO4)2.5。
And C, liquid C: agarose 15 g; distilled H2O 200ml。
A. Sterilizing solution C at 121 deg.C for 20min, filtering solution B with 0.2 μm filter membrane to remove mixed bacteria, mixing with A, B, C, transferring, and inoculating. Culturing at 25 deg.C for 1 month to obtain fungus, and purifying.
2. Identification and functional explanation of strains
1) Morphological characteristics and physiological and biochemical characteristics identification:
colony morphology can be visually observed as follows: the bacterial colony grows slowly, and the whole body is relatively flat; the colony is stacked in the middle, is very compact, and has a flat edge and a little velvet shape. In the acid 9k medium, olive green is in a black color. The mycelium was observed by electron microscopy to consist of isolated colorless or light colored hyphae, which were merozoized to form conidia with oval swollen arthrospores, as shown in fig. 4.
And (3) culturing by using a potato juice-glucose liquid culture medium, and identifying the pH adaptation range of the fungus. We used 10 pH gradients, 1-10 respectively. In culture medium, concentrated H with pH of 1-32SO4Adjusting pH to 4-5 with citric acid and trisodium citrate, and adjusting pH to 6-8 with KH2PO4Adjusting with NaOH, and NaHCO at pH 9-103And Na2CO3And (6) adjusting. The cells were cultured at 25 ℃ for 7 days in an inoculum size of 1%, the growth of the cells was observed, and the dry weight of the cells was determined by centrifugal air-drying. The results of pH adaptation experiments show that Acidiella bohemica SYSU C17045 can grow in a pH range of 2-8, wherein the growth is best at pH 3, and the growth is inhibited at pH 1, pH 9 and pH 10.
Taking an AMD water sample of the minium lead-zinc tailing pond, vertically filtering the AMD water sample by a filter membrane with the aperture of 0.8 mu m and 0.22 mu m, and then tangentially filtering the AMD water sample by a filter membrane with the aperture of 1000kDa and 30kDa to obtain a filtrate. 3% glucose was added to the filtrate to culture the fungus. 3 blank controls were set up without inoculation of the strain. The precipitate cultured for 30 days was washed thoroughly with 0.1M PBS solution 3 times to remove the culture solution and other impurities. 2.5% pentanediol for 4 h; washing with PBS for 3 times to remove residual pentanediol; performing gradient dehydration by using 10%, 30%, 50%, 70%, 90% and 100% (3 times) ethanol at room temperature in sequence; then replacing ethanol with 100% tert-butanol, repeating for 3 times, and freeze drying; and (4) manufacturing a scanning electron microscope sample, observing by using a thermal field emission scanning electron microscope, and analyzing by combining an X-ray energy spectrometer. Fixing an ultrathin slice sample for transmission electron microscope observation by using 4% paraformaldehyde and 2.5% glutaraldehyde, carrying out processes of rinsing, post-fixing, rinsing, gradient dehydration, soaking and the like, then embedding and fixing, then manufacturing an ultrathin slice, and observing by using a transmission electron microscope.
And (3) taking the precipitate, washing the precipitate with sterile water, removing culture solution and other impurities, washing with 5% SDS, performing ultrasonic treatment, centrifuging and removing most cell debris. After freeze-drying, the powder was ground through a 300 mesh sieve and analyzed by an X-ray powder diffractometer. The measurement angle range (2. theta.) was 10 to 80 ℃ and the scanning speed was 10/min, and the X-ray diffraction pattern was analyzed by Jade 5.0 software. Will be provided withAnd (4) after removing precipitates from the culture solution, measuring the element content in the culture solution. Taking 1ml of water sample into a digestion tube, adding 6ml of concentrated hydrochloric acid and 3ml of concentrated nitric acid, and uniformly mixing. Microwave digesting at 180 deg.C for 20min, cooling, diluting to 50ml, and measuring metal element and S element by inductively coupled plasma emission spectrometer. Ferric ion (Fe)3+) And ferrous ion (Fe)2+) The concentration of (A) is determined by using a 1, 10-phenanthroline ultraviolet colorimetric method.
2) Whole genome assay
In order to more clearly understand the extreme environment survival and adaptation mechanism of Acidiella bohemica and the important role thereof in the geochemical cycle of iron element, the whole genome sequencing is carried out. First, the genomic DNA of Acidiella bohemimica was extracted, and the DNA was randomly fragmented into 500bp and 800bp by Covaris M220, followed by use
Figure BDA0002378280150000081
UltraTMII DNA Library Prep Kit for
Figure BDA0002378280150000082
Constructing an insert library of 500bp and 800bp, and performing double-end 250bp sequencing by using an Illumina Miseq sequencer to obtain 9.17Gb data. Using SMRTbellTMThe 20kbp SMRT long library was constructed using the template Kit (Pacific Bioscience, # 100-. Extracting genome RNA of Acidiella bohemica, enriching mRNA by poly-A selection of total RNA, constructing a cDNA library by reverse transcription, and performing double-end 150bp sequencing on a Hiseq X Ten platform to obtain transcriptome data. Based on illumina sequencing data, using GCE software to estimate genome conditions, selecting kmer length as 17 to analyze, and based on Pacbio sequential data, using Falcon and Falcon-unzip to assemble genome. And finally, using a driver to optimize, and obtaining a genome with high splicing quality. Then, the gene prediction is carried out on the spliced genome by using GeneMark-ET and Augustus of software BRAKER, the prediction results of the above methods are integrated by using EVM software (EVidenecodeler), the integration result is optimized by using PASA software, and finally R is usedThe SEM software calculates the predicted expression level of the gene in combination with genome and transcriptome data. And comparing the predicted gene with NCBI-nr, KOG, KEGG and Pfam database blast, and annotating with the standard that e-value is less than or equal to 10-5. The metabolic pathway is constructed through response mechanisms to basic metabolism (metabolism of C, N, S, Fe and the like) and resistance (including heavy metals, pH resistance, oxidative stress and the like) of Acidiella bohemimica.
Kmer analysis used illumina sequencing sequences, and based on the statistical results of the generated 17-mers, it was shown that a. bohemica is a heterozygous diploid, and the genome size and number of a. bohemica are high in homologous species due to the diploid nature. Bohemica and baudoianamelicana are closest among the species of published genomes, reaching 100% confidence. According to the functional classification abundance of KEGG metabolic pathway statistics 20, the genes related to pathways such as cysteine and methionine (sulfur-containing amino acid) metabolism, cell circulation, meiosis, RNA degradation, proteolysis, glycolysis/gluconeogenesis and the like in the metabolic pathway have higher abundance in the metabolic pathway, which indicates that the A.bohemica can improve the self viability through continuous and rapid anabolism and reproduction, and the energy obtained through various metabolisms is used as survival reserve. In the aspect of basic metabolic characteristics, the A.bohemica has complete genes related to coding assimilation sulfate reduction pathways, SO that the purpose of reducing SO in the nature is achieved4 2-Is reduced to S2-And into the amino acid. In addition to the genes predicted to encode reductive uptake pathways in the a. bohemica genome, the presence of these genes was also found in transcriptome data, a process involving iron oxidation and reduction reactions. Bohemica, a species growing in AMD sediment, also encodes many other resistance mechanisms associated proteins to combat extreme environmental stresses, such as transporters of various heavy metals and some proteins associated with detoxification reactions. In addition, against low pH stress, a. bohemica maintains intracellular pH homeostasis mainly in the following way: (1) k+Positive potential within the cell membrane due to influx; (2) a highly impermeable cell membrane; (3) passing excess protons in the cell through the ATPaseAntiporter, cotransporter transport to the outside of the cell; (4) cytoplasmic buffering (e.g., glutamate and phosphate); (5) degradation of organic acids. Due to the above heavy metal resistance mechanism and the existence of various pH stress response mechanisms, a. bohemica has the functions of survival under the conditions of low pH and high concentration of heavy metal ions and heavy metal transport and detoxification. Therefore, the A.bohemica can be better applied to mines and other industrial wastewater on the basis of knowing the life habits and functions of the A.bohemica in detail, and toxic and harmful elements such as S, Fe, Cr and the like can be removed.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A method for separating and identifying acidophilic fungi with biological induction mineralization effect is characterized by comprising the following steps:
s1 obtaining pure cultured acidophilic fungi from the acid bottom mud of the lead zinc mine tailings, identifying the colony morphology and determining the taxonomic status of the strains;
s2 culturing acidophilic fungus with the acid waste water of lead-zinc ore tailings as culture liquid, and analyzing the metabolism potential of acidophilic fungus in acid environment.
2. The method of claim 1, wherein the step S1 of obtaining a pure culture of the acidophilic fungus is as follows: coating diluted lead-zinc ore tailing acid bottom mud in an optimized 9K plate culture medium, and culturing the plate at 25 ℃ for 1 month; single colonies on the plates were picked and inoculated into 9K liquid medium to obtain pure cultures.
3. The method of claim 2, wherein the 9K culture is performedThe preparation method of the nutrient medium comprises the following steps: A. sterilizing solution C at 121 deg.C for 20min, filtering solution B with 0.2 μm filter membrane to remove mixed bacteria, and mixing solutions A, B, C to obtain 9K culture medium; wherein the formula of the solution A is as follows: (NH)4)2SO43g，KCl 0.1g，K2HPO40.5g，MgSO4·7H2O 0.5g，Ca(NO3)2·H2O0.01g, 500ml of distilled water and pH of 2.5; the formula of the solution B is as follows: FeSO4·7H2O33 g; 300ml of distilled water; the pH was 2.5; the formula of the solution C is as follows: agarose 15g, distilled water 200 ml.
4. The method of claim 1, wherein the step S1 of determining the taxonomic status of the bacterial species comprises the following steps: extracting genome DNA of acidophilic fungi to perform PCR amplification, sequencing the whole genome, performing blast comparison on the obtained sequence on NCBI to obtain a known sequence with higher homology with the sequence, and determining the taxonomic status of strains.
5. The method of claim 5, wherein the PCR amplification uses primers that are:
ITS1：5’-TCCGTAGGTGAACCTGCGG-3’；
ITS4：5’-TCCTCCGCTTATTGATATGC-3’。
6. the method according to claim 1, wherein said step S2 of analyzing the metabolic potential of the acidophilic fungus in an acidic environment comprises the following steps: adding glucose liquid into the acid wastewater of the lead-zinc ore tailings, inoculating acidophilic fungi, culturing, detecting the cultured precipitate, and performing element analysis on the precipitate.
7. The method according to claim 6, wherein the elemental analysis is in particular the detection of S, K, C, O, N, Fe, Ca elements.
8. The method of claim 6, wherein the elemental analysis is analyzed using an X-ray energy spectrometer.
9. The method of claim 1, wherein step S2 further comprises identifying the pH adaptation range of the acidophilic fungus.
10. The method of claim 9, wherein said step of identifying the pH adaptation range of the acidophilic fungus comprises the steps of: the culture medium is set with 10 pH gradients of 1-10 respectively, and the acidophilic fungus is cultured at 25 deg.C for 7 days with an inoculum size of 1%.
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