Aspergillus niger with heavy metal lead adsorption capacity and application thereof

Abstract

The invention belongs to the technical field of environmental microorganisms, and particularly discloses aspergillus niger with heavy metal lead adsorption capacity and application of the aspergillus niger. According to the invention, 33 parts of water and sediments at the Henan section of the Yellow River basin are subjected to microbial separation to screen out a fungus strain with high lead tolerance and adsorption, the fungus strain is named as Aspergillus niger AN-2 through morphology and ITS molecular identification, and the fungus strain is preserved in Guangdong Microbial Culture Collection Center (GDMCC) with the preservation number of GDMCC NO: 63697. Compared with other isolated strains, the Aspergillus niger AN-2 has the strongest tolerance to Pb < 2 + >, and the MIC of Pb < 2 + > tolerance is as high as 3500-3800mg / L. After being prepared into biomass, the strain has a good adsorption effect on heavy metal lead, can be used for preparing a heavy metal pollution bioremediation preparation, and has a wide application prospect and a relatively high economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and particularly relates to an Aspergillus niger with heavy metal lead adsorption capability and application thereof. Background Art

[0002] In recent years, heavy metal pollution in the environment has become increasingly serious and has become a global environmental problem. Heavy metal elements and their compounds in the natural environment can only undergo changes in form and valence, and are not easily degraded. They are a type of harmful substance with persistent and cumulative toxicity. Among them, lead is a highly toxic heavy metal. Its toxicity manifests itself in causing poor cardiovascular function, affecting nerve cell differentiation, causing a decrease in isoenzyme activity, etc., thereby causing irreversible damage to the human cardiovascular system, nervous system, skeletal system, reproductive system, etc.

[0003] At present, the situation of lead pollution in rivers, lakes and other water bodies is not optimistic, and the technology for removing heavy metals from water bodies has been greatly developed. Compared with chemical precipitation, redox, electrocoagulation, ion exchange, membrane separation and physical and chemical adsorption, which are costly and prone to secondary pollution, microbial removal has the advantages of simple operation and thorough removal of heavy metals, especially for wastewater with heavy metal concentrations of <10mg / L or >100mg / L, it has good heavy metal adsorption capacity.

[0004] Among many environmental microorganisms, fungi, as a type of microorganism with potential value for heavy metal pollution remediation, have irreplaceable advantages in treating heavy metal pollution in water bodies. So far, researchers have isolated and purified a variety of heavy metal-tolerant fungi from heavy metal-contaminated water bodies, sediments, soil and other environments, such as Penicillium canescens, Penicillium chrysogenum, Penicillium oxalicum, Saccharomyces cerevzsiae, Aspergillus niger, and Aspergillus flavus. These microorganisms can be made into heavy metal adsorption materials alone or mixed with materials such as biochar, and are a research hotspot for ecological remediation of heavy metal pollution in soil and water bodies.

[0005] Chinese patent CN109182136B (Nanjing Agricultural University) discloses a phosphate-dissolving bacterium with heavy metal pollution repair ability, which is classified and named as Aspergillus niger JXZ01 and the deposit number is CGMCC No: 15994. The strain has good colonization ability and phosphate-dissolving ability under soil culture conditions, and is also effective in Pb 2+ , Cu 2+It has high tolerance and adsorption capacity for heavy metals such as lead and can be used to prepare soil heavy metal pollution remediation preparations. However, the strain has insufficient tolerance to heavy metal lead, and high Pb 2+ At the concentration (1000mg / L), Aspergillus niger JXZ01 had a 2+ The adsorption removal rate is only 68.21%. Therefore, it is urgent to develop a new microbial strain that can effectively repair high lead pollution. Summary of the invention

[0006] The technical problem to be solved by the invention is to provide an Aspergillus niger with heavy metal lead adsorption capacity.

[0007] Secondly, the invention also provides an application of Aspergillus niger having heavy metal lead adsorption capacity.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The invention discloses an Aspergillus niger with heavy metal lead adsorption capability. The Aspergillus niger is deposited as Aspergillus niger AN-2 (Aspergillus niger AN-2) and has a deposit number of GDMCC NO: 63697.

[0010] The Aspergillus niger AN-2 provided by the present invention is preserved in Guangdong Provincial Microbiological Culture Collection Center (GDMCC), and the preservation date is July 31, 2023.

[0011] An application of Aspergillus niger having heavy metal lead adsorption capacity includes but is not limited to:

[0012] (1) Application in adsorption and removal of heavy metal lead;

[0013] (2) Application in remediation of heavy metal lead pollution.

[0014] As a preferred embodiment of the present invention, the remediation includes one or more of soil remediation, water remediation, sediment remediation, etc.

[0015] An application of Aspergillus niger having heavy metal lead adsorption capacity includes but is not limited to:

[0016] (1) Application in the preparation of preparations for adsorption and removal of heavy metal lead;

[0017] (2) Application in the preparation of bioremediation preparations for heavy metal lead pollution.

[0018] As a preferred embodiment of the present invention, the application is: culturing Aspergillus niger AN-2 to prepare a spore suspension, which is used as a preparation for adsorbing and removing heavy metal lead.

[0019] A preparation for adsorbing and removing heavy metal lead, or a bioremediation preparation for heavy metal lead pollution, wherein the preparation contains an effective amount of Aspergillus niger AN-2.

[0020] As a preferred embodiment of the present invention, the preparation method of the preparation for adsorbing and removing heavy metal lead is: culturing Aspergillus nigerAN-2 (Aspergillus nigerAN-2) to prepare a spore suspension.

[0021] Specifically, the concentration of the spore suspension is 1-100×10 6 / mL, and the inoculation amount is 0.5%-5%.

[0022] As a preferred embodiment of the present invention, the content of Aspergillus niger AN-2 in the heavy metal lead pollution bioremediation preparation is 0.1wt%-50wt%.

[0023] As a preferred embodiment of the present invention, the preparation also includes other components that can assist Aspergillus niger AN-2 in adsorbing and removing heavy metal lead or repairing heavy metal lead pollution.

[0024] As a preferred embodiment of the present invention, the preparation further includes a pharmaceutically acceptable carrier or auxiliary material, including but not limited to one or more of a solvent, an excipient, a salt for adjusting osmotic pressure, a buffer, a stabilizer, and the like.

[0025] As a preferred embodiment of the present invention, the dosage form of the preparation includes but is not limited to one or more of solution, dispersion, powder, spray and the like.

[0026] Beneficial effects of the present invention:

[0027] The present invention screened out a fungal strain that tolerates and absorbs high lead by separating microorganisms from 33 samples of water and sediments in the Henan section of the Yellow River Basin. After morphological identification and ITS molecular identification, it was identified as Aspergillus nigerAN-2, and it was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) with the preservation number GDMCC NO: 63697. Compared with other isolated strains, Aspergillus niger AN-2 has a high tolerance to Pb. 2+ The tolerance is the strongest, and it can tolerate Pb 2+ The MIC is as high as 3500-3800 mg / L. After the strain was made into biomass, it was found that it had a good adsorption and removal effect on heavy metal lead, and can be used to prepare heavy metal pollution bioremediation preparations, with broad application prospects and high economic value.

[0028] Collection Information

[0029] Deposit name: Aspergillus niger AN-2.

[0030] Deposit number: GDMCC NO:63697.

[0031] Depository: Guangdong Microbiological Culture Collection Center (GDMCC), address: Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences.

[0032] Date of preservation: July 31, 2023. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the phylogenetic tree of the ITS region sequences of Aspergillus niger AN-2.

[0034] Figure 2 Aspergillus niger AN-2 in different Pb 2+ Colony morphology after culture at 28℃ for 5 days at the same concentration.

[0035] Figure 3 Pb of Aspergillus niger AN-2 2+ Tolerance test results.

[0036] Figure 4 is the initial Pb 2+ Effect of concentration on the removal of Pb by Aspergillus niger AN-2 2+ impact.

[0037] Figure 5 The initial pH value for the removal of Pb by Aspergillus niger AN-2 2+ impact.

[0038] Figure 6 The raw biomass (control) and chemically modified biomass residues were 2+ Performance of Pb biosorption under the conditions of 10 mg / L concentration and pH 6.0.

[0039] Figure 7 Aspergillus niger AN-2 at 500mg / LPb 2+ Expression of key antioxidant enzyme genes under stress.

[0040] Note: The untreated expression level was set as "1", the internal reference gene was ACTIN, the data in the figure are the average of 3 biological replicates, the error bars are standard errors, and the t-test was used for significance analysis. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.005.

[0041] Figure 8Aspergillus niger AN-2 in 500mg / L Pb 2+ Colony morphology and microscopic morphology of the cultured cells after 3 days of culture with the addition of 5 mM cAMP under stress.

[0042] Fig. 9 K-mer distribution assembled for the whole genome sequencing of Aspergillus niger AN-2.

[0043] Fig.10 This is the gene length distribution of the whole genome of Aspergillus niger AN-2.

[0044] Fig.11 These are the results of G+C content analysis of the whole genome of Aspergillus niger AN-2.

[0045] Fig.12 This is the NR data alignment of the whole genome of Aspergillus niger AN-2.

[0046] Fig.13 This is the GO annotation result of the whole genome of Aspergillus niger AN-2.

[0047] Fig.14 This is the KOG annotation result of the whole genome of Aspergillus niger AN-2.

[0048] Fig.15 This is the KEGG annotation result of the whole genome of Aspergillus niger AN-2.

[0049] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the above drawings obtained in the experimental examples are briefly introduced. It should be understood that the above drawings only show some experimental results of the present invention and should not be regarded as any limitation on the protection scope of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work. DETAILED DESCRIPTION

[0050] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. It should be understood by those skilled in the art that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, other technical solutions obtained by ordinary technicians in the field without paying creative work, such as technical solutions obtained after modification, deformation or simple replacement, all belong to the scope of protection of the present invention.

[0051] Unless otherwise specified, the experimental methods used in the embodiments and experimental examples are conventional methods in the art.

[0052] Unless otherwise specified, the raw materials, reagents, instruments, etc. used in the embodiments and experimental examples are commonly used in the art and are available to the public (for example, purchased through commercial channels).

[0053] Unless otherwise specified, the terms, abbreviations, etc. involved in the embodiments and experimental examples have the conventional meanings in the art.

[0054] Example 1

[0055] The present embodiment provides an Aspergillus niger having the ability to adsorb heavy metal lead, the preservation name is Aspergillus niger AN-2 (Aspergillus nigerAN-2), the preservation number is GDMCC NO: 63697, the preservation unit is Guangdong Microbiological Culture Collection Center (GDMCC), and the preservation date is July 31, 2023.

[0056] Example 2

[0057] This embodiment provides an application of Aspergillus niger having heavy metal lead adsorption capability, including but not limited to:

[0058] (1) Application of Aspergillus niger AN-2 in the adsorption and removal of heavy metal lead;

[0059] (2) Application of Aspergillus niger AN-2 in the remediation of heavy metal lead pollution;

[0060] (3) Application of Aspergillus niger AN-2 in the preparation of preparations for adsorption and removal of heavy metal lead;

[0061] (4) Application of Aspergillus niger AN-2 in the preparation of bioremediation preparations for heavy metal lead pollution.

[0062] This embodiment also provides a preparation for adsorbing and removing heavy metal lead, including: an effective amount of Aspergillus niger AN-2 (Aspergillus niger AN-2), and a pharmaceutically acceptable carrier or excipient (such as water), which is used to prepare Aspergillus niger AN-2 into a dispersion dosage form (such as a spore suspension).

[0063] This embodiment also provides a heavy metal lead pollution bioremediation preparation, including: an effective amount of Aspergillus niger AN-2 (Aspergillus niger AN-2), and a pharmaceutically acceptable carrier or excipient, which is used to prepare Aspergillus niger AN-2 into a powder dosage form.

[0064] In other embodiments of the present invention, the preparation for adsorbing and removing heavy metal lead also includes other components that can cooperate with Aspergillus niger AN-2 to adsorb and remove heavy metal lead, as well as other pharmaceutically acceptable carriers or excipients, which are used to prepare Aspergillus niger AN-2 into a spray dosage form.

[0065] Experimental example

[0066] 1. Experimental Methods

[0067] By isolating microorganisms from 33 samples of water and sediments from the Henan section of the Yellow River Basin, several fungal strains with high Pb tolerance and adsorption were screened out.

[0068] 1. Identification of bacterial species

[0069] 1.1 Morphological identification

[0070] The selected strain AN-2 was inoculated into PDA medium and cultured at 28°C for 3 days. The colony morphology was recorded, and the hyphae were picked and microscopic morphology was observed and measured using sterile water as a floating medium.

[0071] 1.2 Molecular identification

[0072] (1) DNA extraction

[0073] The genomic DNA of strain AN-2 was extracted by SDS alkaline lysis method. The detailed operation steps are as follows:

[0074] AN-2 was cultured in PDA medium at 28°C, and the cells were collected after 3 days and freeze-dried with liquid nitrogen;

[0075] 1) Weigh 0.1-0.5g of freeze-dried bacteria and add it into a mortar that has been pre-cooled with liquid nitrogen. Use a pestle to crush the bacteria and grind them into powder without particles. Immediately transfer the ground bacteria to a 2mL centrifuge tube and add 800μL of DNA extraction solution containing SDS. Mix by shaking. Heat in a 65℃ water bath for 1h, shaking every 15min.

[0076] 2) Add 800 μL of an equal volume of phenol-chloroform-isoamyl alcohol (1:24:25) in a fume hood, mix by inversion several times, and centrifuge at 12,000 rpm at room temperature for 10 min.

[0077] 3) Slowly aspirate the supernatant into another new 1.5 mL centrifuge tube (do not aspirate the turbid material at the stratification point), add isopropanol of the same volume as the aspirated supernatant, gently invert to mix, place in a -20°C refrigerator to cool for 30 minutes, centrifuge at 12000 rpm at room temperature for 10 minutes, and discard the supernatant.

[0078] 4) Wash the precipitate twice with 75% ethanol solution, centrifuge at 7500 rpm for 5 min at room temperature, discard the supernatant, use a pipette to absorb the remaining ethanol solution, then use sterile filter paper to absorb the droplets at the tube mouth, and place it in a ventilated kitchen to air dry.

[0079] 5) Add 600 μL of sterile water and 5 μL of RNase A to dissolve the precipitated DNA, mix well with a pipette, and place in a 37°C water bath for 30 min.

[0080] 6) Add 600 μL of an equal volume of phenol chloroform isoamyl alcohol in a fume hood, repeatedly invert and mix several times, centrifuge at 12000 rpm at room temperature for 10 min, slowly aspirate the supernatant into another clean 1.5 mL centrifuge tube, add 2 times the volume of anhydrous ethanol solution and 1 / 10 volume of 3M NaAc, slowly invert and mix, cool in a -20°C refrigerator for 30 min, and centrifuge at 12000 rpm at room temperature for 10 min.

[0081] 7) Slowly discard the supernatant and wash the precipitate twice with 75% ethanol solution, centrifuge at 7500 rpm at room temperature for 5 minutes, discard the supernatant, use a pipette to absorb the remaining ethanol solution, use sterilized filter paper to absorb the droplets at the tube mouth, and place it in a ventilated kitchen to air dry.

[0082] 8) Add 200 μL sterile water to dissolve the DNA precipitate, place it at 37°C to dissolve, and store it in a refrigerator at -20°C after it is completely dissolved. The genomic DNA is used for ITS sequence amplification.

[0083] (2) ITS sequence amplification

[0084] 1) Using wild-type AN-2 DNA as a template, design universal primers ITS4 / ITS5, and use Vazyme's Phanta Max Super-Fidelity DNA Polymerase to prepare a 25 μL reaction system according to Table 1. The PCR reaction program is: 94°C for 3 min; 94°C for 15 sec; 60°C for 15 sec; 72°C for 30 sec / kb; 72°C for 7 min.

[0085] Table 1 Amplification reaction system

[0086]

[0087] 2) After the PCR is completed, the PCR product is placed in a 4°C refrigerator for storage. After the PCR product is detected by electrophoresis, the PCR product is purified.

[0088] A. Separation of PCR products by electrophoresis

[0089] 1) Gel preparation: Prepare 1% agarose gel with 1×TBA buffer and agarose powder, heat to boiling to dissolve all agarose, cool to room temperature, add gold view nucleic acid dye when the temperature drops to about 50°C, mix well and slowly pour onto the electrophoresis plate with a comb inserted, leave at room temperature for 20 minutes, and use it after it solidifies.

[0090] 2) Sample loading: Place the gel plate horizontally in the electrophoresis tank with one end of the sample loading well close to the cathode. Inject an appropriate amount of 1×TBA buffer into the electrophoresis tank, take 5μL of PCR product and mix with 1μL of 6×Loading buffer, and then add it to the sample loading well.

[0091] 3) Electrophoresis: voltage 120 V, electrophoresis time 25 min.

[0092] 4) Gel imaging: Place the gel after electrophoresis into a gel imager and take a photo.

[0093] B. Purification of single band PCR product

[0094] The Cycle Pure Kit provided by OMEGA was used to recover DNA fragments from specific bands. The detailed steps are as follows:

[0095] 1) Add 4-5 times of Buffer CP reagent to the PCR product and mix well.

[0096] 2) Slowly transfer the mixed liquid to the adsorption column (no more than 750 μL each time) and centrifuge at 12000 rpm at room temperature for 1 min.

[0097] 3) Discard the liquid at the bottom of the adsorption column, add 500 μL Wash Buffer to the collection column, place at room temperature for 1 min, centrifuge at 12000 rpm for 1 min at room temperature, and repeat the washing twice.

[0098] 4) Discard the filtrate and centrifuge the empty column at 12000 rpm for 2 min at room temperature (to dry the collection column).

[0099] 5) Add 30-50 μL ddH2O to the adsorption column, place at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and send to BGI for sequencing.

[0100] C. Sequence alignment and phylogenetic tree construction

[0101] The ITS sequence obtained by sequencing was compared with the online blastn in NCBI, and the result showed that the similarity with Aspergillus niger was 100%. The top ten fungal ITS sequences with similarity were obtained in NCBI. The boostrap NJ tree was built using MEGA to obtain the phylogenetic relationship of the AN-2 strain ITS.

[0102] 2. Optimization of Pb adsorption conditions

[0103] (1) Optimization of initial adsorption concentration and pH

[0104] Setting Pb 2+The initial concentrations were 500, 1000, 1500 and 2000 mg / L, respectively. Aspergillus niger AN-2 was cultured with PD culture medium at 28°C with shaking for 72 h. The mycelium obtained from the culture was determined by atomic absorption spectrophotometer.

[0105] Set Pb in PD culture medium 2+ The initial concentration was 1000 mg / L, and the initial pH values ​​of the PD culture medium were adjusted to 2, 3, 4, 5, 6, 7, and 8, respectively. Aspergillus niger AN-2 was cultured with shaking at 28°C for 72 h, and the mycelium obtained from the culture was determined by atomic absorption spectrophotometer.

[0106] (2) Atomic absorption spectrophotometer

[0107] 1) Take 1 g of dried mycelium, add 50 mL of EDTA-2Na (0.1 M) solution, shake for 30 min, centrifuge at 12000 rpm for 5 min, and collect the mycelium and supernatant separately.

[0108] 2) Dilute the supernatant 10 times with deionized water and determine the Pb content using an atomic absorption spectrometer. 2+ concentration.

[0109] 3) Use a mixture of nitric acid and perchloric acid (4:1) to boil the mycelium at 400°C until the boiled liquid is transparent and clear. After cooling to room temperature, measure the Pb content in the boiled liquid. 2+ The digestion process must be carried out on a constant temperature digestion furnace in a fume hood.

[0110] 4) Pb in supernatant 2+ It is the Pb adsorbed on the surface of mycelium and sclerotium, and the Pb in the digestion liquid 2+ That is, the Pb enriched in microbial cells.

[0111] 3. Preparation of Pb adsorbed biomass

[0112] Aspergillus niger AN-2 was used to prepare Pb-adsorbed biomass, and the operation steps were as follows:

[0113] 1) The growth medium was filtered through a 150 μm mesh to separate the mycelium and spores of Aspergillus niger AN-2 and washed with a large amount of deionized water.

[0114] 2) Take 40-50 g (wet weight) and boil it in 0.5 M NaOH (400-500 mL) for 15 min. Wash the alkali-treated biomass thoroughly with deionized water until the pH value of the washing solution is close to neutral.

[0115] 3) The biomass was dried and powdered using a mortar and pestle.

[0116] 4) 2 g of biomass was dispersed in anhydrous methanol, concentrated hydrochloric acid was added to the dispersion, and the obtained biomass residue was called M1.

[0117] 5) 1 g of biomass was mixed with formaldehyde (HCHO) and formic acid for reaction, and the reaction mixture was shaken on a rotary shaker. The obtained biomass residue was called M2.

[0118] 6) 1 g of biomass was mixed with triethyl phosphite and nitromethane under reflux conditions, and the biomass residue obtained from this chemical treatment was called M3.

[0119] 7) 1 g of biomass was treated with benzene or acetone under reflux conditions, and the biomass residues obtained after the benzene and acetone treatments were called M4 and M5, respectively.

[0120] 8) Testing of raw biomass and chemically modified biomass residues for Pb 2+ The performance of Pb adsorption under the conditions of concentration 10 mg / L and pH 6.0.

[0121] 4. Expression of genes related to oxidative stress

[0122] (1) Extraction of total RNA

[0123] Total RNA was extracted using the Trizol method. The steps are as follows:

[0124] 1) Prepare the necessary instruments and medicines: sterilize the mortar, pestle, medicine spoon, gun tip, etc. under high temperature and high pressure.

[0125] 2) Grinding: Place the bacteria in liquid nitrogen and grind quickly until they are ground into fine powder.

[0126] 3) Lysis: Add a small amount of evenly ground sample into Trizol lysis buffer, mix thoroughly, and centrifuge at 4°C, 12,000 rpm for 15 min.

[0127] 4) Removal of impurities: aspirate the supernatant, add 200 μL of chloroform, shake vigorously for 20 seconds, let stand for 5 minutes, and then centrifuge at 4°C and 12,000 rpm for 10 minutes.

[0128] 5) Precipitation: Transfer the upper aqueous phase to another clean centrifuge tube, add 500 μL of isopropanol, let stand for 10 min, and centrifuge at 4°C and 12,000 rpm for 10 min.

[0129] 6) Washing: remove the supernatant, add 800 μL 75% ethanol, centrifuge at 7500 rpm for 5 min, and wash the RNA precipitate twice.

[0130] 7) Dissolution: Dry in air for 10 min, add 30-50 μL DEPC water.

[0131] (2) cDNA synthesis

[0132] The extracted RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix kit from TAKARA. The specific steps are as follows:

[0133] 1) Prepare RT reaction solution according to Table 2 below (the entire preparation process should be performed on ice).

[0134] Table 2 Reverse transcription reaction system

[0135]

[0136] 2) After the reaction solution is mixed, reverse transcription reaction is carried out under the following reaction conditions: 37°C for 15 min; 85°C for 5 sec (inactivation of reverse transcriptase); 4°C (cooling storage).

[0137] (3) Real-time fluorescence quantitative PCR and expression analysis

[0138] Takara chimeric Real Time PCR was used to detect the expression of the target gene. The specific operation is as follows:

[0139] 1) Prepare PCR reaction solution according to Table 3 below (the entire preparation process should be performed on ice).

[0140] Table 3 Reverse transcription reaction system

[0141]

[0142] 2) Real Time PCR was performed with the following reaction conditions: 95°C for 5 sec; 60°C for 30 sec; 35 cycles.

[0143] 3) After the reaction is completed, confirm the melting curve and amplification curve of Real Time PCR, and use a quantitative instrument to create a standard curve.

[0144] 4) By calculating formula 2 -ΔΔCt Calculate the relative expression of the target gene.

[0145] 5. Effect of cAMP addition on the adsorption of heavy metal Pb

[0146] cAMP is a second messenger widely present in cells. Its intracellular content can greatly affect cell proliferation, differentiation and stress response to adversity and has a wide range of biological activities. In order to detect the effect of cAMP on the adsorption of heavy metal Pb by Aspergillus niger AN-2, Aspergillus niger AN-2 strain was activated on PDA plates for 3 days, spores were washed with sterile water and counted with a hemocytometer. The conidia concentration was adjusted to 10 7 / mL, add 5mM concentration of cAMP to the culture medium, and add Pb 2+ The culture was carried out in 500 mg / L PDA for 3 days, and the plate without cAMP was used as the control. The morphology of hyphae and spores was observed under a microscope to detect the effect of cAMP on the adsorption of Pb by Aspergillus niger AN-2.

[0147] 6. Genome sequencing, assembly and gene prediction of Aspergillus niger AN-2

[0148] 6.1 Genome Sequencing

[0149] (1) Extraction of genomic DNA

[0150] The genomic DNA was extracted by SDS method, and the concentration and purity were detected by agarose gel electrophoresis and Qubit 2.0 fluorometer (Thermo Fisher).

[0151] (2) Database Construction

[0152] 1 μg of DNA was taken as input material for sample preparation. Sequencing libraries were generated using NEBNext Ultra DNA Library. Illumina's Prep Kit was added to each sample according to the manufacturer's recommendations and index code. In brief, the DNA samples were ultrasonically fragmented to 350 bp in size, and then the DNA fragments were end-trimmed and A-treated and ligated with full-length adapters for Illumina sequencing and further PCR amplification. Finally, the PCR products were purified with magnetic beads (AMPure XP system), and the fragment size distribution of the library was analyzed with an Agilent 2100 analyzer and quantified with real-time quantitative PCR.

[0153] (3) Sequencing

[0154] Beijing Novartis Bioinformatics Co., Ltd. was commissioned to sequence the whole genome of Aspergillus niger AN-2 using IlluminaNova Seq PE150.

[0155] 6.2 Genome assembly and gene prediction

[0156] (1) Sequencing quality test

[0157] The raw data obtained by sequencing contains a certain proportion of low-quality data. In order to ensure the accuracy and reliability of the subsequent information analysis results, the raw data must be screened to obtain valid data (Clean Data). The specific processing steps include:

[0158] 1) Remove (default 40%) reads containing low-quality bases (quality value ≤ 20).

[0159] 2) Remove N from reads that exceed a certain proportion (the default is 10%).

[0160] 3) Remove reads whose overlap with the adapter exceeds a certain threshold (the default is 15bp) and there are less than 3 mismatches between them.

[0161] 4) If the sample is contaminated by the host, a Blast comparison is required.

[0162] (2) Genome assembly

[0163] Use Clean Data for genome assembly. The specific processing steps include:

[0164] 1) Use SOAP denovo software for assembly: select different K-mers (the default values ​​are 95, 107, and 119, respectively) for assembly, select the optimal K-mer according to the project type, further adjust other parameters and the minimum scaffold as the preliminary assembly result.

[0165] 2) Assemble using spades software: Select different K-mers (the default values ​​are 99 and 127) for assembly, and obtain the assembly result with the optimal kmer and the least scaffold according to the project type.

[0166] 3) Assemble using abyss software: Select K-mer 64 for assembly and obtain the assembly result.

[0167] 4) Integrate the assembly results of the three software with the CISA software respectively.

[0168] Perl 5.14.2 (http: / / www.perl.org / get.html) was used to count the size, N50 value, number of contigs and N90 value of the whole genome assembled with different K-mer values ​​in the assembly results of the three software, and the sequencing depth (sequencing depth = sequencing data / assembled genome size) was calculated. The whole genome with the largest N50 was selected for the next gene prediction.

[0169] Under the Linux operating system, the de novo gene prediction software AUGUSTUS (http: / / bioinf.uni-greifswald.de / augustus / binaries / ) was used to perform de novo gene prediction on the selected whole genome by searching for important signal sites in the genome.

[0170] 7. Annotation of the Aspergillus niger AN-2 genome

[0171] Seven databases were used to predict gene functions, namely GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), NR (Non-Redundant Protein Database), KOG (Eukaryotic Orthologous Groups), Pfam and Swiss-Prot. Whole genome Blast search (E value less than 1e -5 , similarity percentage is greater than 40%).

[0172] 3. Experimental Results

[0173] 1. Isolate and obtain a Pb 2+ Aspergillus niger

[0174] By isolating microorganisms from 33 samples of water and sediments in the Henan section of the Yellow River Basin, we screened out fungal strains with high Pb tolerance and adsorption. After morphological identification and ITS molecular identification, we obtained Aspergillus niger AN-2 (phylogenetic tree of ITS regional sequences as shown in Figure 2). Figure 1 As shown in the figure), A. flavus and P. chrysogenum. Among them, A. niger AN-2 was cultured on PDA at 28℃ for 3 days, with a diameter of about 68mm, a carbon black colony surface, flat or slightly convex in the center, and a velvety texture. Microscopic observation showed that the diameter of the apical capsule was 40-70μm, brown; the spore-producing structure was dark brown; the phialide was (8-10)μm×(2.5-3)μm, and the conidia were dark brown-black, spherical, and 3.5-5.5μm in diameter.

[0175] Compared with other strains, Aspergillus niger AN-2 had a higher sensitivity to Pb 2+ The tolerance of Aspergillus niger AN-2 was the strongest. 2+ The strain was cultured in PDA medium at 28°C for 5 days, the colony diameter was measured, the minimum inhibitory concentration (MIC) was determined, and the half inhibitory concentration (EC50) was calculated. Figure 2 , Figure 3 As shown, Aspergillus niger AN-2 was exposed to different concentrations of Pb 2+ Under the action of Pb 2+ When the concentration was 3500 mg / L, the inhibition rate was 89.9%. 2+ The half effective concentration EC50 under the action of Pb is 1482.67 mg / L. 2+ The regression equation between concentration and inhibition rate is as follows:

[0176] y=0.0003x+0.0552,R 2 =0.9264.

[0177] Based on the above experimental results, Aspergillus nigerAN-2 was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with the deposit number GDMCC NO: 63697 and the deposit date of July 31, 2023.

[0178] The ITS sequence of Aspergillus niger AN-2 is shown below:

[0179] (SEQ ID NO: 1).

[0180] 2. Optimization of adsorption conditions for Aspergillus niger AN-2

[0181] Initial Pb 2+ The concentration value is closely related to the heavy metal adsorption of microorganisms. Therefore, this experiment was conducted to investigate the bioavailability of Aspergillus niger AN-2 at different initial Pb 2+ Pb at concentration value 2+ The removal efficiency was studied and the results are as follows Figure 4 As shown, when the initial Pb 2+When the concentration is 500 mg / L, the Pb in the solution 2+ The removal efficiency is the highest, and when the initial Pb 2+ When the concentration is 2000 mg / L, the concentration of Pb2+ in the solution decreases slowly and the removal effect is poor. 2+ The concentration was 500 mg / L, and all subsequent experiments used this Pb 2+ Concentration conditions.

[0182] The initial pH value is directly related to the growth and metabolism of microorganisms and the adsorption and analysis of heavy metals. Therefore, this experiment was conducted to investigate the Pb 2+ The removal efficiency was studied and the results are as follows Figure 5 As shown, in Pb 2+ Under the condition of 1000mg / L concentration, when the initial pH value is 3-7, the Pb 2+ can be removed quickly, and the removal efficiency is highest when the initial pH is 6. When the initial pH is 2 and 8, Pb in the solution 2+ The concentration decreased slowly and fluctuated significantly, which may be due to the fact that too low pH inhibited the growth and metabolism of microorganisms, while higher pH 2+ Re-extracted from unstable Pb compounds, resulting in Pb in the solution 2+ It can be seen that the optimal initial pH value is 6, and this pH value condition was used in subsequent experiments.

[0183] 3. Biomass adsorption effect of Aspergillus niger AN-2

[0184] The product obtained after the biomass is contacted with deionized water is the raw biomass (ie, the product of step 1)). Figure 6 The raw biomass (control) and chemically modified biomass residues were 2+ The performance of Pb biosorption under the conditions of 10 mg / L concentration and pH 6.0. The analysis shows that when the carboxyl group is esterified, the biosorption of Pb is severely inhibited, which indicates that the carboxyl group plays an important role in the biosorption of heavy metal Pb. For biomass with methylated amino groups, the biosorption of heavy metal Pb is also reduced accordingly. However, the contribution of amino groups further depends on its protonation degree at pH 6.0. The biosorption of Pb is more sensitive to the modification of carboxyl groups than to the modification of amine groups, while the biosorption of cadmium and copper is more sensitive to the modification of amino groups than to the modification of carboxyl groups. When lipids were extracted from biomass, a slight decrease in the biosorption of Pb was observed. The above experimental results indicate that the reduction in the biosorption of heavy metal Pb may be the result of lipid extraction, or it may be due to some structural changes caused by the harsh conditions of the biomass during lipid extraction. The biosorption of Pb by biomass treated with triethyl phosphite and nitromethane decreased slightly.

[0185] 4. Analysis of the expression of oxidase-related genes in Aspergillus niger AN-2 under Pb stress

[0186] Aspergillus niger AN-2 pure culture in the initial Pb 2+ After treatment at a concentration of 500 mg / L for 72 h, total RNA was extracted and reverse transcribed, and the relative expression of oxidase was detected by qPCR, such as Figure 7 As shown in the figure, the genes GPX2 and GPX4 of catalase CAT1, copper-thioprotein CRS5, key enzyme of high osmotic pressure pathway HOG1, glutathione peroxidase 2 and glutathione peroxidase 4 were significantly upregulated after Pb oxidative stress (p < 0.05), indicating that these genes can respond rapidly when heavy metal stress occurs.

[0187] 5. The synergistic effect of Aspergillus niger AN-2 on low concentration cAMP under Pb stress

[0188] 5 mM cAMP was added to the culture medium of Aspergillus niger AN-2, and Pb was added 2+ The cells were cultured in 500 mg / L PDA for 3 days. Figure 8 As shown, compared with the case without adding Pb 2+ Compared with the control, the average colony diameter increased by 3.3 cm, and the spore production decreased significantly compared with the control. It is not difficult to see from microscopic observation and spore concentration counting that the mycelium with 5mM cAMP is thicker and the spore production is reduced by about 42.8%, indicating that 5mM cAMP can tolerate 500mg / LPb to Aspergillus niger AN-2. 2+ There is a synergistic effect. Analysis shows that Aspergillus niger AN-2 may improve its resistance to Pb by reducing spore production.

[0189] 6. Genome characteristics and gene prediction of Aspergillus niger AN-2

[0190] Aspergillus niger AN-2 has potential adsorption capacity for heavy metal Pb in wastewater, so it is necessary to study its mechanism of Pb adsorption. The whole genome of Aspergillus niger AN-2 was sequenced using Illumina sequencing technology, and the sequencing adapters and low-quality reads were removed using Fastp software (https: / / github.com / OpenGene / fastp). Aspergillus niger AN-2 was assembled into 2095 contigs, of which 293 contigs were longer than 500bp, the N50 size was 245.9kb, the draft genome size was 35.5Mb, and the G+C content was 48.96%. After the genome assembly, a total of 6831 protein-coding genes were predicted with a total length of 9,192,098bp. The detailed genomic characteristics are shown in Table 4. The K-mer distribution of the whole genome sequencing assembly of Aspergillus niger AN-2 is shown in Table 4. Fig. 9 The distribution of gene length in the whole genome is shown in Fig.10 The results of the whole genome G+C content analysis are shown in Fig.11 shown.

[0191] Table 4 Genome characteristics of Aspergillus niger AN-2

[0192]

[0193] 7. Annotation results of Aspergillus niger AN-2 genome

[0194] like Fig.12 As shown in the Non-Redundant protein sequence (NR) database, Aspergillus niger AN-2 was annotated with a total of 6502 genes, which exceeded 95% of the total number of genes predicted in the whole genome. It is worth noting that a fairly high proportion of orthologous genes are shared with known Aspergillus species. The three species with the highest matching degree with Aspergillus niger AN-2 are Aspergillus tubingensis (41.0%), Aspergillus niger (13.7%) and Aspergillus costaricanus (11.9%), indicating that Aspergillus niger AN-2 has a high degree of species homology with other Aspergillus species. In the Swiss Prot and Pfam protein family databases, Aspergillus niger AN-2 was annotated with 3116 and 4873 genes, respectively. As Fig.13 As shown in the Gene Ontology (GO) database, a total of 4873 genes were annotated in Aspergillus niger AN-2, which exceeded 75% of the predicted number of genes in the whole genome. These genes annotated in the GO database constitute three main categories: Cellular Components (CC), Molecular Functions (MF) and Biological Processes (BP). The biological process category of Aspergillus niger AN-2 contains the largest number of gene annotations, with a total of 7756 hits, and the top three functions are metabolic process, cellular process and localization. For the molecular function (MF) category, a total of 5770 hits were obtained, and the top three functions were catalytic activity, binding and transporter activity. The cellular component (CC) category generated 3200 alignments, and the highest function was cellular anatomical entity, followed by metabolic process, cellular process and localization, protein-containing entity and viral particle part. As Fig.14 As shown in the Eukaryotic Orthologous Groups (KOG) database, a total of 1967 genes were annotated for Aspergillus niger AN-2, which is more than 30% of the predicted genes in the whole genome. In summary, Aspergillus niger AN-2 has a good annotation performance in KOG, GO, NR and KEGG (the annotation results are shown in Fig.15Most genes can be annotated in at least two databases, and the annotation features are shown in Table 5.

[0195] Table 5 Genome annotation features of Aspergillus niger AN-2

[0196]

[0197] Although the technical solution of the present invention has been described in detail above with general descriptions, specific implementation methods and test examples, it should be noted that the embodiments and test examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the protection scope of the present invention. Simple deformation, modification or improvement based on the technical concept of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An Aspergillus niger having heavy metal lead adsorption capacity, characterized in that: The deposited name of the Aspergillus niger is Aspergillus niger AN-2, and the deposited number is GDMCC NO:63697.

2. An application of Aspergillus niger having heavy metal lead adsorption capacity as claimed in claim 1, characterized in that: The applications include but are not limited to: (1) Application in adsorption removal of heavy metal lead; (2) Application in remediation of heavy metal lead pollution.

3. The use according to claim 2, characterized in that: The remediation includes one or more of soil remediation, water remediation, and sediment remediation.

4. An application of the Aspergillus niger having heavy metal lead adsorption capacity as claimed in claim 1, characterized in that: The applications include but are not limited to: (1) Application in the preparation of preparations for adsorption and removal of heavy metal lead; (2) Application in the preparation of bioremediation preparations for heavy metal lead pollution.

5. The use according to claim 4, characterized in that: The remediation includes one or more of soil remediation, water remediation, and sediment remediation.

6. A preparation for adsorbing and removing heavy metal lead, or a preparation for bioremediation of heavy metal lead pollution, characterized in that: The preparation contains an effective amount of the Aspergillus niger having heavy metal lead adsorption capacity according to claim 1.

7. The preparation according to claim 6, characterized in that: The preparation method of the preparation for adsorbing and removing heavy metal lead is as follows: culturing Aspergillus niger AN-2 to prepare a spore suspension; And / or, the content of Aspergillus niger AN-2 in the heavy metal lead pollution bioremediation preparation is 0.1wt%-50wt%.

8. The preparation according to claim 6, characterized in that: The preparation also includes other components that can assist Aspergillus niger AN-2 in adsorbing and removing heavy metal lead or repairing heavy metal lead pollution.

9. The preparation according to claim 6, characterized in that: The preparation also includes a pharmaceutically acceptable carrier or auxiliary material, including but not limited to one or more of a solvent, an excipient, a salt for adjusting osmotic pressure, a buffer, and a stabilizer.

10. The preparation according to claim 6, characterized in that: The dosage form of the preparation includes, but is not limited to, one or more of a solution, a dispersion, a powder, and a spray.

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