Aspergillus niger with lead adsorption capacity and its application

Abstract

This invention belongs to the field of environmental microbiology technology, specifically disclosing a *Aspergillus niger* strain with lead adsorption capacity and its applications. This invention involved the isolation of microorganisms from 33 samples of water and sediment from the Henan section of the Yellow River basin. A fungal strain with high lead tolerance and adsorption was screened out, and after morphological and ITS molecular identification, it was named *Aspergillus niger* AN-2 and deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC NO: 63697. Compared with other isolated strains, *Aspergillus niger* AN-2 exhibits high lead tolerance and adsorption capacity for Pb. 2+ It has the strongest tolerance and is most tolerant to Pb. 2+ The MIC is as high as 3500-3800 mg / L. When this strain is processed into biomass, it exhibits excellent adsorption performance for lead, making it suitable for preparing bioremediation agents for heavy metal pollution. It has broad application prospects and high economic value.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to Aspergillus niger with the ability to adsorb heavy metal lead and its applications. Background Technology

[0002] In recent years, heavy metal pollution in the environment has become increasingly serious, posing 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, making them a class of harmful substances with persistent and cumulative toxicity. Lead, in particular, is a highly toxic heavy metal. Its toxicity manifests as impaired cardiovascular function, impaired nerve cell differentiation, and decreased activity of heterologous enzymes, thereby causing irreversible damage to the human cardiovascular, nervous, skeletal, and reproductive systems.

[0003] Currently, lead pollution in rivers, lakes, and other water bodies is a serious concern, and heavy metal removal technologies for water have seen significant development. Compared to costly and potentially polluting methods such as chemical precipitation, oxidation-reduction, electrocoagulation, ion exchange, membrane separation, and physicochemical adsorption, microbial removal offers advantages such as ease of operation and thorough heavy metal removal, especially for wastewater with heavy metal concentrations <10 mg / L or >100 mg / L, demonstrating excellent heavy metal adsorption capacity.

[0004] Among numerous environmental microorganisms, fungi, as a class of microorganisms with potential value in the remediation of heavy metal pollution, possess irreplaceable advantages in treating heavy metal pollution in water bodies. To date, researchers have isolated and purified various heavy metal-tolerant fungi from environments such as heavy metal-polluted water bodies, sediments, and soil, including *Penicillium canescens*, *Penicillium chrysogenum*, *Penicillium oxalicum*, *Saccharomyces cerevzsiae*, *Aspergillus niger*, and *Aspergillus flavus*. These microorganisms can be used alone or mixed with materials such as biochar to prepare heavy metal adsorbents, making them a research hotspot for the ecological remediation of heavy metal pollution in soil and water.

[0005] Chinese patent CN109182136B (Nanjing Agricultural University) discloses a phosphate-solubilizing bacterium with heavy metal pollution remediation capabilities, classified as *Aspergillus niger* JXZ01, with accession number CGMCCNo: 15994. This strain exhibits good colonization and phosphate-solubilizing abilities under soil culture conditions, and also shows resistance to Pb. 2+ Cu 2+This strain exhibits high tolerance and adsorption capacity for heavy metals, making it suitable for preparing soil heavy metal pollution remediation agents. However, it lacks tolerance to lead and exhibits high Pb content. 2+ At a concentration (1000 mg / L), Aspergillus niger JXZ01 inhibits Pb 2+ The adsorption and removal rate was only 68.21%. Therefore, there is an urgent need to develop a new microbial strain that can effectively remediate high lead pollution. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a type of Aspergillus niger with the ability to adsorb heavy metal lead.

[0007] Secondly, this invention also provides an application of Aspergillus niger with the ability to adsorb heavy metal lead.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0009] A type of Aspergillus niger with the ability to adsorb heavy metal lead, the preservation name of which is Aspergillus niger AN-2, and the preservation number is GDMCC NO: 63697.

[0010] The Aspergillus nigerAN-2 provided by this invention is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on July 31, 2023.

[0011] Applications of Aspergillus niger with the ability to adsorb heavy metal lead include, but are not limited to:

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

[0013] (2) Application in the 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, and sediment remediation.

[0015] Applications of Aspergillus niger with the ability to adsorb heavy metal lead include, but are not limited to:

[0016] (1) Application in the preparation of formulations for adsorbing and removing heavy metal lead;

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

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

[0019] An adsorption and removal agent for heavy metal lead, or a bioremediation agent for heavy metal lead pollution, wherein the agent contains an effective amount of Aspergillus niger AN-2.

[0020] As a preferred embodiment of the present invention, the preparation method of the agent for adsorbing and removing heavy metal lead is as follows: after culturing Aspergillus nigerAN-2, a spore suspension is prepared, which is then obtained.

[0021] Specifically, the concentration of the spore suspension is 1-100×10⁻⁶. 6 The inoculum density is 0.5%-5% per mL.

[0022] In a preferred embodiment of the present invention, the content of Aspergillus niger AN-2 in the bioremediation agent for lead pollution is 0.1wt%-50wt%.

[0023] As a preferred embodiment of the present invention, the formulation also includes other components that can synergistically adsorb and remove heavy metal lead or remediate heavy metal lead pollution by Aspergillus niger AN-2.

[0024] As a preferred embodiment of the present invention, the formulation further includes a pharmaceutically acceptable carrier or excipient, including but not limited to one or more of solvents, excipients, salts for adjusting osmotic pressure, buffers, and stabilizers.

[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 the following: solution, dispersion, powder, spray.

[0026] The beneficial effects of this invention are:

[0027] This invention involved the isolation of microorganisms from 33 water and sediment samples from the Henan section of the Yellow River basin. A fungal strain exhibiting high lead tolerance and adsorption was screened, and after morphological and ITS molecular identification, it was confirmed to be *Aspergillus niger* AN-2. This strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC NO: 63697. Compared to other isolated strains, *Aspergillus niger* AN-2 exhibits high Pb tolerance. 2+ It has the strongest tolerance and is most tolerant to Pb. 2+ The MIC is as high as 3500-3800 mg / L. After being processed into biomass, this strain was found to have excellent adsorption and removal effects on lead, making it suitable for preparing bioremediation agents for heavy metal pollution. It has broad application prospects and high economic value.

[0028] Preservation Information

[0029] Preservation name: Aspergillus nigerAN-2.

[0030] Accession number: GDMCC NO: 63697.

[0031] Preservation institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), Address: Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences.

[0032] Date of preservation: July 31, 2023. Attached Figure Description

[0033] Figure 1 Phylogenetic tree of the ITS region sequence of Aspergillus niger AN-2.

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

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

[0036] Figure 4 For the initial Pb 2+ Concentration of Pb removal by Aspergillus niger AN-2 2+ impact.

[0037] Figure 5 To determine the initial pH value for Pb removal from Aspergillus niger AN-2 2+ impact.

[0038] Figure 6 For raw biomass (control) and chemically modified biomass residues in Pb 2+ Performance of biosorption of Pb at a concentration of 10 mg / L and pH 6.0.

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

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

[0041] Figure 8 Aspergillus niger AN-2 at 500 mg / L Pb 2+ Colony morphology and microstructure after culturing with 5 mM cAMP for 3 days under stress.

[0042] Figure 9 K-mer distribution was assembled from the whole genome sequence of Aspergillus niger AN-2.

[0043] Figure 10 The gene length distribution of the whole genome of Aspergillus niger AN-2.

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

[0045] Figure 12 This is a comparison of the whole genome NR data of Aspergillus niger AN-2.

[0046] Figure 13 The results of GO annotation of the whole genome of Aspergillus niger AN-2.

[0047] Figure 14 KOG annotation results for the whole genome of Aspergillus niger AN-2.

[0048] Figure 15 The results of KEGG annotation of the whole genome of Aspergillus niger AN-2.

[0049] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings obtained in the experimental examples have been briefly described above. It should be understood that the above drawings only illustrate certain experimental results of the present invention and should not be considered as any limitation on the scope of protection of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without any creative effort. Detailed Implementation

[0050] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments and experimental examples. Those skilled in the art should understand that the embodiments are only used to illustrate the technical solutions of the present invention and should not be considered as limiting the scope of protection of the present invention. Other technical solutions obtained by those skilled in the art based on the following embodiments without creative effort, such as technical solutions obtained after modification, variation, or simple substitution, all fall within 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 examples and experimental cases are all commonly used in the art and are publicly available (e.g., commercially available).

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

[0054] Example 1

[0055] This embodiment provides a *Aspergillus niger* strain with the ability to adsorb heavy metal lead. Its preservation name is *Aspergillus niger* AN-2, its preservation number is GDMCC NO: 63697, its depositary is Guangdong Provincial Microbial Culture Collection Center (GDMCC), and its preservation date is July 31, 2023.

[0056] Example 2

[0057] This embodiment provides an application of Aspergillus niger with the ability to adsorb heavy metal lead, 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 formulations for adsorbing and removing heavy metal lead;

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

[0062] This embodiment also provides a formulation for adsorbing and removing heavy metal lead, comprising: an effective amount of Aspergillus niger AN-2, and a pharmaceutically acceptable carrier or excipient (such as water) for preparing Aspergillus niger AN-2 into a dispersion formulation (such as a spore suspension).

[0063] This embodiment also provides a bioremediation preparation for heavy metal lead pollution, comprising: an effective amount of Aspergillus niger AN-2, and a pharmaceutically acceptable carrier or excipient, for preparing Aspergillus niger AN-2 into a powder dosage form.

[0064] In other embodiments of the present invention, the formulation for adsorbing and removing heavy metal lead also includes other components that can synergistically adsorb and remove heavy metal lead with Aspergillus niger AN-2, as well as other pharmaceutically acceptable carriers or excipients, for formulating Aspergillus niger AN-2 into a spray formulation.

[0065] Experimental example

[0066] I. Experimental Methods

[0067] Microbial isolation was performed on 33 samples of water and sediment from the Henan section of the Yellow River Basin, and several fungal strains with high Pb tolerance and adsorption were screened out.

[0068] 1. Strain identification

[0069] 1.1 Morphological identification

[0070] The selected strain AN-2 was inoculated in PDA medium and cultured at 28°C for 3 days. Colony morphology was recorded, and hyphae were picked and observed and measured under a microscope using sterile water as a carrier.

[0071] 1.2 Molecular identification

[0072] (1) DNA extraction

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

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

[0075] 1) Weigh 0.1-0.5g of freeze-dried bacterial cells, add them to a mortar that has been pre-cooled with liquid nitrogen, crush the bacterial cells with a mortar and pestle until the powder is free of particles, immediately transfer the crushed bacterial cells to a 2mL centrifuge tube, add 800μL of DNA extraction solution containing SDS, shake to mix, heat in a 65℃ water bath for 1h, and shake once every 15min.

[0076] 2) Add 800 μL of phenol-chloroform-isoamyl alcohol (1:24:25) to the fume hood, mix by inverting several times, and centrifuge at 12000 rpm at room temperature for 10 min.

[0077] 3) Slowly transfer the supernatant to another new 1.5 mL centrifuge tube (do not aspirate the turbidity at the separation point), add an equal volume of isopropanol to the supernatant, gently invert to mix, and place in a -20°C refrigerator to cool for 30 min. Centrifuge at 12000 rpm at room temperature for 10 min, and discard the supernatant.

[0078] 4) Wash the precipitate twice with 75% ethanol solution, centrifuge at 7500 rpm at room temperature for 5 min, discard the supernatant, use a pipette to absorb the remaining ethanol solution, then use sterile filter paper to absorb the droplets at the tube opening, and place it in a fume hood 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 phenol-chloroform-isoamyl alcohol to a fume hood, mix by inverting several times, centrifuge at 12000 rpm at room temperature for 10 min, slowly transfer the supernatant to another clean 1.5 mL centrifuge tube, add 2 volumes of anhydrous ethanol solution and 1 / 10 volume of 3M NaAc, mix by inverting slowly, cool in a -20℃ 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 for 5 min at room temperature, discard the supernatant, use a pipette to absorb the remaining ethanol solution, use sterile filter paper to absorb the droplets at the tube opening, and place it in a fume hood to air dry.

[0082] 8) Add 200 μL of sterile water to dissolve the DNA precipitate, place it at 37°C to dissolve, and after complete dissolution, store it at -20°C. 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, universal primers ITS4 / ITS5 were designed. A 25 μL reaction system was prepared using Vazyme's Phanta Max Super-Fidelity DNA Polymerase according to Table 1 below. The PCR reaction program was: 94℃ for 3 min; 94℃ for 15 sec; 60℃ for 15 sec; 72℃ for 30 sec / kb; 72℃ for 7 min.

[0085] Table 1 Amplification reaction system

[0086]

[0087] 2) After PCR is completed, store the PCR products at 4°C for later use. After electrophoresis detection, the PCR products are then purified.

[0088] A. Electrophoretic separation of PCR products

[0089] 1) Gel preparation: Prepare a 1% agarose gel using 1×TBA buffer and agarose powder. Heat to boiling to dissolve all the agarose. Cool to room temperature. When the temperature drops to about 50°C, add Goldview nucleic acid dye, mix well, and slowly pour into an electrophoresis plate with a comb inserted. Let it stand at room temperature for 20 minutes until it solidifies before use.

[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 it with 1μL of 6×Loading buffer, and then add it to the sample loading well.

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

[0092] 4) Gel imaging: After electrophoresis, the gel is placed in a gel imaging system to take pictures and save them.

[0093] B. Purification of single-band PCR products

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

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

[0096] 2) Slowly transfer the well-mixed liquid onto 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 of Wash Buffer to the collection column, incubate at room temperature for 1 min, centrifuge at 12000 rpm at room temperature for 1 min, and repeat the washing process twice.

[0098] 4) Discard the filtrate and centrifuge the empty column at 12,000 rpm for 2 minutes at room temperature (to dry the collection column).

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

[0100] C. Sequence alignment and phylogenetic tree construction

[0101] The ITS sequences obtained from sequencing were aligned online using NCBI's BLASTN database, showing 100% similarity to Aspergillus niger. The top ten fungal ITS sequences with the highest similarity were then retrieved from NCBI. A MEGA tree was used to construct a bootstrap NJ tree, revealing the phylogenetic relationships of the AN-2 strain's ITS sequences.

[0102] 2. Optimization of Pb adsorption conditions

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

[0104] Set Pb 2+The initial concentrations were 500, 1000, 1500 and 2000 mg / L, respectively. Aspergillus niger AN-2 was cultured in PD medium at 28℃ with shaking for 72 h. The mycelia obtained from the culture were used for atomic absorption spectrophotometry.

[0105] Set Pb in PD culture medium 2+ The initial concentration of the PD culture medium was 1000 mg / L. 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 at 28℃ with shaking for 72 h. The mycelium obtained from the culture was used for atomic absorption spectrophotometry.

[0106] (2) Atomic absorption spectrophotometry determination

[0107] 1) Take 1g of dried mycelium, add 50mL of EDTA-2Na (0.1M) solution, shake for 30min, centrifuge at 12000rpm for 5min, 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) The mycelium was digested at 400°C with a mixture of nitric acid and perchloric acid (4:1) until the digestion solution was clear and transparent. After cooling to room temperature, the Pb content in the digestion solution was measured. 2+ The concentration and digestion process must be carried out in a constant-temperature digestion oven in a fume hood.

[0110] 4) Pb in the supernatant 2+ This refers to Pb adsorbed on the surface of mycelium and sclerotia, and Pb in the digestion solution. 2+ This refers to Pb enriched within microbial cells.

[0111] 3. Preparation of biomass by Pb adsorption

[0112] The following are the steps for preparing Pb-adsorbed biomass using Aspergillus niger AN-2:

[0113] 1) Isolate Aspergillus niger AN-2 mycelia and spores by filtering the growth medium through a 150 μm sieve and washing with plenty of deionized water.

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

[0115] 3) Dry the biomass and pulverize it using a mortar and pestle.

[0116] 4) Disperse 2g of biomass in anhydrous methanol, add concentrated hydrochloric acid to the dispersion, and the resulting biomass residue is called M1.

[0117] 5) Mix 1g of biomass with formaldehyde (HCHO) and formic acid and react them. Shake the reaction mixture on a rotary vibrator. The resulting biomass residue is called M2.

[0118] 6) Mix 1g of biomass with triethyl phosphite and nitromethane under reflux conditions. The biomass residue obtained from this chemical treatment is called M3.

[0119] 7) Treat 1g of biomass with benzene or acetone under reflux conditions. The biomass residues obtained after benzene and acetone treatment are called M4 and M5, respectively.

[0120] 8) Test the Pb content of raw biomass and chemically modified biomass residues. 2+ Adsorption performance of Pb at a concentration of 10 mg / L and a pH of 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, and the procedure is as follows:

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

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

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

[0127] 4) Remove impurities: Take the supernatant, add 200 μL of chloroform, shake vigorously for 20 seconds, let stand for 5 minutes, and then centrifuge at 4℃ and 12000 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℃ and 12000 rpm for 10 min.

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

[0130] 7) Dissolve: Dry in air for 10 minutes, then add 30-50 μL of DEPC water.

[0131] (2) cDNA synthesis

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

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

[0134] Table 2 Reverse Transcription Reaction System

[0135]

[0136] 2) After mixing the reaction solution, perform reverse transcription reaction under the following conditions: 37℃ for 15 min; 85℃ for 5 sec (reverse transcriptase inactivation); 4℃ (cooling and storage).

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

[0138] The expression of the target gene was detected using Takara chimeric Real-Time PCR. The specific procedure is as follows:

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

[0140] Table 3 Real-time quantitative PCR reaction system

[0141]

[0142] 2) Perform Real-Time PCR under the following conditions: 95℃ for 5 seconds; 60℃ for 30 seconds; 35 cycles.

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

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

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

[0146] cAMP is a widely distributed second messenger within cells, and its intracellular content can significantly influence cell proliferation, differentiation, and stress responses, exhibiting broad biological activity. To investigate the effect of cAMP on the adsorption of heavy metal Pb by *Aspergillus niger* AN-2 strain, *Aspergillus niger* AN-2 strain was activated on PDA plates for 3 days. Spores were collected after sterile washing with water and counted using a hemocytometer. The conidial concentration was adjusted to 102. 7cAMP was added at a concentration of 5 mM to the culture medium, followed by the addition of Pb. 2+ The bacteria were cultured in 500 mg / L PDA for 3 days, with plates without cAMP serving as a control. Hyphae and spore morphology were observed under a microscope to detect the effect of cAMP on Pb adsorption by Aspergillus niger AN-2.

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

[0148] 6.1 Genome sequencing

[0149] (1) Extraction of genomic DNA

[0150] Genomic DNA was extracted using the SDS method, and its concentration and purity were detected by agarose gel electrophoresis and then measured using a Qubit 2.0 fluorometer (Thermo Fisher Scientific).

[0151] (2) Database creation

[0152] 1 μg of DNA was used as input material for sample preparation. Sequencing libraries were generated using the NEBNext Ultra DNA Library. Following the manufacturer's recommendations and index codes, an Illumina Prep Kit was added to each sample. In short, the DNA samples were sonicated to a size of 350 bp, then the DNA fragments were subjected to final trimming and A-addition, and ligated with full-length adapters for Illumina sequencing and further PCR amplification. Finally, the PCR products were purified using magnetic beads (AMPure XP system), the fragment size distribution of the library was analyzed using an Agilent 2100 analyzer, and quantification was performed using real-time quantitative PCR.

[0153] (3) Sequencing

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

[0155] 6.2 Genome Assembly and Gene Prediction

[0156] (1) Sequencing quality testing

[0157] The raw data obtained from sequencing contains a certain proportion of low-quality data. To ensure the accuracy and reliability of subsequent information analysis results, the raw data must be screened to obtain clean data. 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 percentage (default is 10%).

[0160] 3) Remove reads that overlap with the Adapter by more than a certain threshold (default is 15bp) and have fewer 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] Genome assembly using Clean Data involves the following processing steps:

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

[0165] 2) Assemble using spades software: Select different K-mers (default values ​​are 99 and 127) for assembly, and obtain the assembly result with the optimal kmer and the fewest scaffolds 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 programs with the CISA software respectively.

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

[0169] Under the Linux operating system, the de novo gene prediction software AUGUSTUS (http: / / bioinf.uni-greifswald.de / augustus / binaries / ) is used to perform de novo gene prediction on selected whole genomes 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 function: GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), NR (Non-Redundant Protein Database), KOG (Eukaryotic Orthologous Groups), Pfam, and Swiss-Prot. Genome-wide BLAST searches were performed on six of these databases (E-value less than 1 e^(-1 / 2)). -5 (Similarity percentage greater than 40%).

[0172] III. Experimental Results

[0173] 1. A Pb strain was isolated. 2+ Highly tolerant strain identified as Aspergillus niger

[0174] Microbial isolation was performed on 33 water and sediment samples from the Henan section of the Yellow River basin. Fungal strains with high Pb tolerance and adsorption were screened. After morphological identification and ITS molecular identification, a phylogenetic tree of the ITS regional sequence of Aspergillus niger AN-2 was obtained. Figure 1 The strains shown are Aspergillus flavus and Penicillium chrysogenum. Among them, Aspergillus niger AN-2, after being cultured on a PDA at 28℃ for 3 days, reached a diameter of approximately 68 mm. The colony surface was charcoal black, flat or slightly convex in the center, and had a velvety texture. Microscopic observation revealed that the vesicle diameter was 40-70 μm, brown; the sporulation structure was dark brown; the phialid was (8-10) μm × (2.5-3) μm, and the conidia were dark brownish-black, spherical, and 3.5-5.5 μm in diameter.

[0175] Compared with other strains, Aspergillus niger AN-2 is more resistant to Pb. 2+ It exhibits the strongest tolerance. Aspergillus niger AN-2 was subjected to different concentrations of Pb. 2+ The cells were cultured in PDA medium at 28°C for 5 days. Colony diameters were measured to determine the minimum inhibitory concentration (MIC) and the half-maximal inhibitory concentration (EC50) was calculated. Figure 2 , Figure 3 As shown, Aspergillus niger AN-2 at different concentrations of Pb 2+ Under the influence of Pb, the MIC value is between 3500-3800 mg / L. 2+ At a concentration of 3500 mg / L, the inhibition rate was 89.9%. Furthermore, Aspergillus niger AN-2 showed inhibition at Pb... 2+ The half-maximal effective concentration (EC50) under the action was 1482.67 mg / L, Pb 2+ The regression equation for concentration versus 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 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC NO: 63697 and 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 investigated the adsorption of heavy metals by Aspergillus niger AN-2 under different initial Pb values. 2+ Pb concentration values 2+ The removal efficiency was studied, and the results are as follows: Figure 4 As shown, when the initial Pb 2+At a concentration of 500 mg / L, the Pb in the solution 2+ The removal efficiency is the highest when the initial Pb is... 2+ At a concentration of 2000 mg / L, the concentration of Pb²⁺ in the solution decreased relatively slowly, indicating poor removal efficiency. Therefore, the optimal initial Pb concentration is... 2+ The concentration was 500 mg / L, and this Pb was used in all subsequent experiments. 2+ Concentration conditions.

[0182] Initial pH value is directly related to the growth and metabolism of microorganisms and the adsorption and desorption of heavy metals. Therefore, this experiment investigated the Pb content of Aspergillus niger AN-2 at different initial pH values. 2+ The removal efficiency was studied, and the results are as follows: Figure 5 As shown, in Pb 2+ Under conditions of a concentration of 1000 mg / L, and with an initial pH of 3-7, the concentration of Pb in the solution... 2+ It can be removed rapidly, with the highest removal efficiency at an initial pH of 6. However, when the initial pH is 2 and 8, the removal efficiency of Pb in the solution is significantly lower. 2+ The concentration decreased relatively slowly and fluctuated significantly, possibly because excessively low pH values ​​inhibited microbial growth and metabolism, while higher pH values ​​increased Pb levels. 2+ The Pb is re-eluted from the unstable Pb compound, resulting in Pb in the solution. 2+ The concentration fluctuated. Therefore, the optimal initial pH value was 6, and all subsequent experiments used this pH condition.

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

[0184] The product obtained after biomass is contacted with deionized water is the product of the raw biomass (i.e., step 1). Figure 6 For raw biomass (control) and chemically modified biomass residues in Pb 2+ The biosorption of Pb at a concentration of 10 mg / L and a pH of 6.0 was investigated. Analysis showed that Pb biosorption was severely inhibited when the carboxyl group was esterified, indicating that the carboxyl group plays a crucial role in the biosorption of heavy metal Pb. For biomass with methylated amino groups, the biosorption of heavy metal Pb also decreased accordingly. However, the contribution of amino groups further depends on their protonation degree at pH 6.0. Pb biosorption was more sensitive to carboxyl group modification than to amino group modification, while the biosorption of cadmium and copper was more sensitive to amino group modification than to carboxyl group modification. A slight decrease in Pb biosorption was observed when lipids were extracted from biomass. These experimental results suggest that the reduction in heavy metal Pb biosorption may be a result of lipid extraction or due to structural changes caused by the harsh conditions encountered by the biomass during lipid extraction. Furthermore, the biosorption of Pb by biomass treated with triethyl phosphite and nitromethane showed a slight decrease.

[0185] 4. Expression analysis of oxidase-related genes in Aspergillus niger AN-2 under Pb stress

[0186] Aspergillus niger AN-2 pure culture at initial Pb 2+ After treatment at a concentration of 500 mg / L for 72 h, total RNA was extracted and reverse transcribed. The relative expression levels of oxidases were detected by qPCR. Figure 7 As shown, the genes GPX2 and GPX4 of catalase CAT1, copper-sulfur protein CRS5, HOG1 (a key enzyme in the hyperosmolar pathway), glutathione peroxidase 2, and glutathione peroxidase 4 were significantly upregulated after Pb oxidative stress (p0.05). ＜ The result (0.05) indicates that these genes can respond rapidly when heavy metal stress occurs.

[0187] 5. Synergistic effect of Aspergillus niger AN-2 on low concentrations of cAMP under Pb stress

[0188] Add 5 mM cAMP to the culture medium of Aspergillus niger AN-2, and then add Pb. 2+ After culturing in PDA at a concentration of 500 mg / L for 3 days, the results are as follows: Figure 8 As shown, compared with the control without cAMP, the average colony diameter increased by 3.3 cm, and the sporulation rate decreased significantly. Microscopic observation and spore count clearly show that the hyphae with 5 mM cAMP were thicker, and the sporulation rate decreased by approximately 42.8%, indicating that 5 mM cAMP is resistant to Aspergillus niger AN-2 at 500 mg / L. 2+ It has a synergistic effect; analysis suggests that Aspergillus niger AN-2 may enhance 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 exhibits potential adsorption capacity for heavy metal Pb in wastewater, thus necessitating an investigation into its adsorption mechanism. The whole genome of Aspergillus niger AN-2 was sequenced using Illumina sequencing technology, and 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 were longer than 500 bp. The N50 size was 245.9 kb, the genome draft size was 35.5 Mb, and the G+C content was 48.96%. A total of 6831 protein-coding genes were predicted after genome assembly, with a total length of 9,192,098 bp. Detailed genomic characteristics are shown in Table 4. The K-mer distribution of the Aspergillus niger AN-2 whole genome sequencing assembly is shown in Table 4. Figure 9 As shown, the distribution of gene lengths across the entire genome is as follows: Figure 10 As shown, the results of the whole-genome G+C content analysis are as follows: Figure 11 As shown.

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

[0192]

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

[0194] like Figure 12 As shown, in the non-redundant protein sequence (NR) database, *Aspergillus niger* AN-2 has 6502 genes annotated, exceeding 95% of the total predicted genes for the whole genome. Notably, a significant proportion of orthologous genes are shared with known *Aspergillus* species. The three species with the highest matching degree to *Aspergillus niger* AN-2 are *Aspergillus tabbinensis* (41.0%), *Aspergillus neoniger* (13.7%), and *Aspergillus costa camara* (11.9%), indicating high species homology between *Aspergillus niger* AN-2 and other *Aspergillus* species. In the Swiss Prot and Pfam protein family databases, *Aspergillus niger* AN-2 has 3116 and 4873 genes annotated, respectively. Figure 13 As shown, in the Gene Ontology (GO) database, 4873 genes from *Aspergillus niger* AN-2 were annotated, exceeding 75% of the predicted whole-genome genes. These annotated genes in the GO database constitute three main categories: Cellular Components (CC), Molecular Functions (MF), and Biological Processes (BP). The Biological Processes category of *Aspergillus niger* AN-2 contained the most gene annotations, with 7756 hits. The top three functions were metabolic processes, cellular processes, and localization. For the Molecular Functions (MF) category, 5770 hits were obtained, with the top three functions being catalytic activity, binding and transporting protein activity. The Cellular Components (CC) category generated 3200 matches, with the highest number of functions being cellular anatomical entities, followed by metabolic processes, cellular processes and localization, protein-containing entities, and viral particle components. Figure 14 As shown, in the Eukaryotic Orthologous Groups (KOG) database, a total of 1967 genes of Aspergillus niger AN-2 were annotated, exceeding 30% of the predicted number of genes in the whole genome. In summary, Aspergillus niger AN-2 has significant genetic diversity in KOG, GO, NR, and KEGG (annotation results are shown in Figure 1). Figure 15In databases such as (as shown in Table 5), most genes can be annotated in at least two databases, and the annotation characteristics are shown in Table 5.

[0195] Table 5. Genomic 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 embodiments, and experimental examples, it should be noted that the embodiments and experimental 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 scope of protection of the present invention. Simple modifications, alterations, or improvements made based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. cAMP enhances the activity of Aspergillus niger AN-2 ( Aspergillus niger The application of AN-2 in lead resistance is characterized by: The preservation name of the Aspergillus niger is Aspergillus niger AN-2 ( Aspergillus niger AN-2), with accession number GDMCCNO: 63697.

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