Use of bacillus thuringiensis y9 in reducing palladium ions

Abstract

The application belongs to the technical field of environmental governance, and discloses an application of bacillus thuringiensis Y9 in reducing palladium ions, wherein the bacillus thuringiensis Y9 strain has been preserved in the China Center for Type Culture Collection on September 17, 2021, and the preservation number is CCTCC NO.M 20211195. The strain is an aerobic bacterium, which can be rapidly cultured under aerobic conditions, and can reduce Pd 2+ in water by using an endogenous electron donor, and Pd 0 (111) single-crystal surface Pd-NPs can be synthesized under mild room temperature conditions, and the method is environmentally friendly, simple and inexpensive.

Description

Technical Field

[0001] This invention belongs to the field of environmental remediation technology and relates to the application of Bacillus thuringiensis Y9 in the reduction of palladium ions. Background Technology

[0002] Many are used to reduce Pd 2+ The microorganisms involved are all anaerobic bacteria, such as sulfate-reducing bacteria (Desulfovibriodesulfuricans) and iron-reducing bacteria (Geobacter sulfurreducens), but they require absolutely anaerobic conditions to reduce Pd. 2+ Bacterial growth is slow, anaerobic processes are complex, and costs are high. Furthermore, microbial reduction of Pd... 2+ Both require an external electron donor. Therefore, it is necessary to screen aerobic microorganisms to reduce Pd under aerobic and non-external electron donor conditions. 2+ . Summary of the Invention

[0003] The purpose of this invention is to provide an application of Bacillus thuringiensis Y9 in the reduction of palladium ions, which can reduce Pd in ​​wastewater. 2+ It also involves the recycling of palladium resources, which is environmentally friendly, simple, and inexpensive.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] The 16S rDNA sequence of Bacillus thuringiensis Y9 provided by this invention has been included in GenBank, accession number MN420977; this Bacillus thuringiensis Y9 strain was deposited at the China Center for Type Culture Collection (CCTCC, located at Wuhan University Collection Center, Luojia Mountain, Wuchang, Wuhan, Hubei Province) on September 17, 2021, with accession number CCTCC NO.M 20211195Y9.

[0006] This invention also provides the application of Bacillus thuringiensis Y9 in the reduction of palladium ions.

[0007] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0008] The present invention describes how Bacillus thuringiensis Y9 can reduce Pd in ​​water under mild room temperature conditions. 2+ Pd can be synthesized 0 (111) Single-crystal Pd-NPs have an environmentally friendly and simple preparation process.

[0009] This invention utilizes transcriptomics technology to analyze the reduction of Pd by Bacillus thuringiensis Y9. 2+The underlying mechanism was discovered: Bacillus thuringiensis Y9 possesses qcrB and qcrC cytochrome c reductase genes, and cytochrome c reductase can directly reduce Pd. 2+ Bacillus thuringiensis Y9 also has many genes that can produce NADH and NADPH. In the absence of external electron donors, the bacteria can utilize endogenous electron donors NADH and NADPH to reduce Pd. 2+ Furthermore, Bacillus thuringiensis Y9 contains a large number of endogenous electron mediators capable of generating quinone rings (Q-rings) and riboflavin genes (ribE), which can accelerate electron transfer processes, thereby increasing Pd. 2+ The efficiency of restoration.

[0010] Hydrogenases can also reduce Pd 2+ The hydrogenase activity of live bacteria under N2 conditions is higher than that under O2 conditions. O2 inhibits hydrogenase activity. Figure 6 O2 will also react with Pd 2+ Competition for electrons, thereby reducing Pd under O2 conditions. 2+ The amount is even less. It is worth noting that some hydrogenases in live bacteria remain insensitive to O2 ( Figure 6 These O2-insensitive hydrogenases may reduce Pd. 2+ . Attached Figure Description

[0011] Figure 1 This is the phylogenetic tree of Bacillus thuringiensis Y9 of the present invention.

[0012] Figure 2 To test the activity of live bacteria against Pd under aerobic and anaerobic conditions 2+ Removal rate.

[0013] Figure 3 To reduce Pd by live bacteria under aerobic and anaerobic conditions 2+ XRD.

[0014] Figure 4 To reduce Pd by live bacteria under aerobic conditions 2+ TEM image of bacterial section.

[0015] Figure 5 As a live bacteria control, live bacteria reduced Pd under aerobic and anaerobic conditions. 2+ Response gene expression in transcriptomic analysis of the samples.

[0016] Figure 6 The hydrogenase activity of live and dead bacteria under aerobic and anaerobic conditions was measured. Detailed Implementation

[0017] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the test methods in the following embodiments are conventional methods.

[0018] Example 1: Isolation and Identification of Bacillus thuringiensis Y9

[0019] Bacillus thuringiensis Y9 was isolated from sludge from an electronic waste plant in Guangzhou.

[0020] Weigh 10.0g of sludge from an electronic waste treatment plant into a 250mL Erlenmeyer flask, add 100mL of fresh LB medium, and simultaneously add 0.2mL of 5000mg L... -1 Pd 2+ (Prepared with sodium tetrachloropalladium), sealed with air-sealing film, and placed in a shaker at 150 rpm and 30°C for acclimatization and enrichment culture. Initially, 10 mg L... -1 Pd 2+ Then acclimatize for three days, followed by doses of 10 mg L. -1 Increment the concentration gradually, allowing each concentration to be acclimated for three days, until finally reaching Pd. 2+ Concentration of 100 mg / L -1 The acclimatization process takes approximately 30 days. Take 1 mL of the acclimatized mixed bacterial solution and adjust the concentration by 10... -1 ...10 -7 Dilute the sample, then take 100 μL and spread it evenly on a plate. Incubate the plate at 30°C for 12 h. Pick a single colony and culture it in liquid again. Dilute and spread the sample again for further isolation and purification until the colony morphology is consistent. Then preserve the sample in glycerol.

[0021] Then, the reduction of Pd by each bacterium was examined. 2+ The effect of restoring Pd 2+ The bacteria that showed good results were further purified and cultured on a larger scale, and then the strains were identified.

[0022] Based on the 16S rDNA sequencing results of the screened strains, the sequences were entered into GenBank for sequence alignment, and a phylogenetic tree of the strains was constructed using MEGA 4.1 software, as follows: Figure 1 As shown. From Figure 1 It can be seen that this strain has 100% similarity to MF526967.1 Bacillus thuringiensis strain UF GS2. This strain is named Bacillus thuringiensis Y9, and its Gen-Bank accession number is MN420977.

[0023] Example 2: Application of Bacillus thuringiensis Y9 in the reduction of palladium ions

[0024] 2.1 Live bacteria reduce palladium ions

[0025] All solutions and glassware used in the experiment were sterilized in an autoclave at 121°C for 20 minutes to maintain cleanliness and sterility. To investigate the effect of oxygen, 679 mg / L of oxygen was used without an external electron donor. -1 Live bacteria reduced 100 mg L under both air and anaerobic conditions. -1 Pd 2+ All other conditions were maintained at 30°C and pH 7.0. After 12 hours of reaction, the remaining Pd was determined using flame atomic absorption spectrometry (FAAS). 2+ Concentration was determined to account for the adsorption and reduction of Pd by Bacillus thuringiensis Y9 under different conditions. 2+ The amount. FAAS operating conditions: hollow cathode lamp wavelength 247.6nm, acetylene pressure 0.075MPa, air pressure 0.28MPa, slit width 0.2mm, burner head height 10mm. First, prepare 0, 1, 2, 5, 10, 20mg L. -1 concentration of Pd 2+ The solution, used as the test solution for the standard curve, is tested before the standard sample. The bio-palladium nanoparticle solution sample after the above reaction is centrifuged at 4000 rpm. -1 Centrifuge at 5 min at a speed of [speed missing], then filter the supernatant through a 0.22 μm filter membrane to separate the precipitate and prevent clogging of the testing instrument. Subsequently, determine the Pd concentration in the solution using a flame atomic absorption spectrophotometer. 2+ The sample is drawn into an air-acetylene lean-burn flame from the tube, where it dissociates into corresponding excited-state atoms at high temperature. The characteristic spectrum at a wavelength of 247.6 nm of the hollow cathode lamp absorbs proportionally to this, and this proportion can be used to analyze Pd. 2+ Quantitative determination.

[0026] 1. The crystal structure of microbially synthesized zero-valent palladium nanoparticles was determined using X-ray diffraction (XRD). The test conditions were: CuKα ray source, ray wavelength λ = 0.154178 nm, energy 1.6 kW (voltage 40 kV, current 40 mA), scanning range 2θ = 30°–90°, scanning step size 0.02°, and scanning speed 1°min. -1 The sample preparation steps were as follows: After microbial synthesis of Pd(0)-NPs, the solution was heated at 4000 rpm. -1Centrifuge for 5 min, discard the supernatant, freeze the concentrate at -20℃ for 2 h, and then freeze-dry for 12 h. Grind the dried Pd(0)-NPs into powder using an agate mortar and pestle, and package for analysis. Peak analysis was performed using Jade software.

[0027] 2. The adsorption and reduction of Pd by Bacillus thuringiensis Y9 under air conditions were characterized using transmission electron microscopy (TEM). 2+ The bacterial sections were then examined to observe the morphology and distribution of Pd(0)-NPs synthesized by Bacillus thuringiensis Y9.

[0028] The process of preparing bacterial sections is as follows:

[0029] (1) The bacterial suspension after exposure to the virus was heated at 4000 rpm. -1 After centrifuging for 5 minutes, discard the supernatant and pick out a small piece of the bacterial clump from the bottom.

[0030] (2) Pre-fixation: The selected sample was quickly transferred to the fixative (4% paraformaldehyde + 2.5% glutaraldehyde), and after vacuuming, it was fixed at 4°C for 12 hours.

[0031] (3) Rinsing: Discard the fixative and rinse with phosphate buffer (0.1 mol / L). -1 Rinse the sample three times (pH 7.2) for 10 minutes each time.

[0032] (4) Post-fixation: Discard the rinsing solution and fix different bacterial samples with 1% osmium tetroxide for 1-2 hours.

[0033] (5) Rinse: Discard the fixative and rinse with phosphate buffer (0.1 mol / L). -1 Rinse the sample three times (pH 7.2) for 10 minutes each time.

[0034] (6) Dehydration: Discard the rinsing solution and dehydrate the bacterial sample with ethanol at concentration gradients of 30%, 50%, 70%, 80%, 90% and 95%, once for each gradient and 10 min each time, and then twice with 100% ethanol for 15 min each time.

[0035] (7) Transition: Finally, transition to acetone treatment twice, 15 min each time.

[0036] (8) Infiltration: Different bacterial samples were treated with a mixture of embedding agent and acetone at a volume ratio of 1:3 for 2-4 hours; then treated with a mixture of embedding agent and acetone (V / V = 1 / 1) for 2-4 hours; then treated with a mixture of embedding agent and acetone at a volume ratio of 3:1 for 3-12 hours; the bacterial samples were treated with pure embedding agent for 12-24 hours, and then the pure resin and tubes were changed.

[0037] (9) Polymerization: The infiltration-treated bacterial sample is embedded in a mold and polymerized at 45°C for 24 hours, and then polymerized at 60°C for 24 hours to obtain the embedded bacterial sample. It is then removed and stored in a desiccator for later use in sectioning.

[0038] (10) Ultrathin sectioning and staining: The embedded blocks were trimmed to an appropriate size and cut surface by a trimmer, and bacterial sections with a thickness of 70-100 nm were obtained by an ultrathin sectioner. The sections were stained with uranium acetate solution and lead citrate solution, dried, and then placed on a copper grid for observation under a transmission electron microscope.

[0039] Live bacteria under aerobic and anaerobic conditions for Pd 2+ Removal rate such as Figure 2 As shown. From Figure 2 It can be seen that live bacteria can inhibit Pd under both aerobic and anaerobic conditions. 2+ The removal rates were 40.73% and 62.85%, respectively.

[0040] Live bacteria reduce Pd under aerobic and anaerobic conditions 2+ XRD such as Figure 3 As shown. From Figure 3 It can be seen that the palladium nanoparticles (Pd-NPs) synthesized by live bacteria under aerobic conditions are mainly Pd 0 (111) Crystal plane, this result indicates that the bacterium can synthesize Pd-NPs using endogenous electron donors under aerobic conditions. Simultaneously, XRD results show that the live bacteria can further enhance Pd synthesis under N2 conditions. 2+ The restoration.

[0041] Live bacteria reduce Pd under aerobic conditions 2+ TEM results of bacterial sections as follows Figure 4 As shown. Figure 4 This indicates that palladium nanoparticles are present both intracellularly and extracellularly in this bacterium, further demonstrating that Bacillus thuringiensis Y9 can reduce Pd using endogenous electron donors under aerobic conditions. 2+ To synthesize palladium nanoparticles (Pd-NPs).

[0042] This invention is the first to discover that Bacillus thuringiensis Y9 can reduce Pd using endogenous electron donors. 2+ .

[0043] 2.2, Strain reduction of Pd 2+ Transcriptome analysis

[0044] Total RNA extraction and transcriptome analysis:

[0045] Pd was reduced from pure live bacteria (control group) and live bacteria under aerobic and anaerobic conditions, respectively. 2+ Total RNA was extracted from the samples. The concentration and quality of total RNA were measured using a NanoDrop 2000 (Thermo Scientific, USA). The isolated RNA was used for further transcriptome analysis and reverse transcription to synthesize cDNA. Transcriptome sequences were obtained from an Illumina HiSeq 2000 sequencer. HISAT2 (version 2.1.0) was used to align clean reads with a reference genome and was run with default parameters. RSeQC (version 2.6.1) was used to statistically analyze the alignment results, analyzing the distribution of redundant sequences and inserts. BEDTools (version 2.26.0) was used for statistical analysis of gene coverage and the distribution of sequencing sequences on the chromosome.

[0046] The concentration and purity of total RNA are shown in Table 1. The results showed that the RNA concentration in all three samples was greater than 150 ng / μl. -1 The sample amounts were all greater than 4 μg, fully meeting the requirements for RNA-SEQ library construction. RIN values ​​were between 7 and 10, indicating high RNA integrity. These results demonstrate that the total RNA concentration and purity were both high, with good integrity, providing a solid experimental foundation for transcriptome sequencing.

[0047] Table 1. Concentration and purity of total RNA

[0048]

[0049] Table 2 lists the Illumina sequencing data details for the three samples. The sequence numbers for the three samples range from 28,632,040 to 34,900,500. Of all RNA sequence reads, 95.24%–97.87% were localized to the Bacillus thuringiensis genome (Table 2), indicating that the sequencing data are completely reliable.

[0050] Table 2 Illumina sequencing data

[0051]

[0052]

[0053] Live bacteria reduce Pd under aerobic and anaerobic conditions 2+ The transcriptome analysis of the samples showed the expression results of the response genes. Figure 5 As shown. From Figure 5It can be seen that Bacillus thuringiensis Y9 possesses the qcrB and qcrC cytochrome c reductase genes, and cytochrome c reductase can directly reduce Pd. 2+ .

[0054] Bacillus thuringiensis Y9 also has many genes that can produce NADH and NADPH. In the absence of external electron donors, the bacteria can utilize endogenous electron donors NADH and NADPH to reduce Pd. 2+ .

[0055] Furthermore, Bacillus thuringiensis Y9 contains a large number of endogenous electron mediators capable of generating quinone rings (Q-rings) and riboflavin genes (ribE), which can accelerate electron transfer processes and thus enhance Pd. 2+ The efficiency of restoration.

[0056] The hydrogenase activities of live and dead bacteria were determined spectrophotometrically under 5 mM methyl viologen conditions. The hydrogenase activities of live and dead bacteria under aerobic and anaerobic conditions were as follows: Figure 6 As shown. From Figure 6 It can be seen that the hydrogenase activity of live bacteria under N2 conditions is higher than that under oxygen conditions, indicating that O2 inhibits enzyme activity; O2 inhibits hydrogenase activity, and O2 and Pd 2+ It also competes for electrons, leading to Pd 2+ Fewer electrons are gained under O2, thus reducing Pd. 2+ The reduction of Pd. It is noteworthy that some hydrogenases in live bacteria remain insensitive to O2; these O2-insensitive hydrogenases may reduce Pd. 2+ .

[0057] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. The application of Bacillus thuringiensis Y9 in the reduction of palladium ions, characterized in that, The Bacillus thuringiensis ( Bacillus thuringiensis Y9 was deposited at the China Center for Type Culture Collection on September 17, 2021, with accession number CCTCC NO:M 20211195. It can reduce palladium ions using its own endogenous electron donor.

2. The application of Bacillus thuringiensis Y9 in the reduction of palladium ions according to claim 1, characterized in that, The Bacillus thuringiensis Y9 strain synthesized palladium ions by reducing them with an endogenous electron donor under aerobic conditions. Single-crystal palladium nanoparticles.

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