Thiobacillus thiooxidans resistant to toxicity of Ni and Cu and application of thiobacillus thiooxidans
By screening out the thiobacillus thiooxidans QH2C, which is resistant to Ni and Cu toxicity, the problem of low treatment efficiency of existing bioleaching strains under high metal concentrations has been solved, and a highly efficient solid waste metal leaching effect has been achieved.
Patent Information
- Application Number
- CN202410795053.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-19
- Publication Date
- 2025-12-19
AI Technical Summary
Existing bioleaching strains are unable to tolerate high concentrations of metal toxicity, resulting in low efficiency in biological treatment of solid waste, especially in the difficulty of effectively leaching metals under high slurry density.
A strain of *Acidithiobacillus thiooxidans* QH2C, resistant to Ni and Cu toxicity, was provided. It was obtained through plasma mutagenesis and directed screening and can grow under high concentrations of Ni and Cu. It was prepared as a bacterial agent and mixed with solid waste for bioleaching.
It significantly improves the efficiency of bioleaching treatment, enabling the processing of metal-containing solid waste with higher slurry density, and increasing the leaching rate and treatment efficiency of metals.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of solid waste biological treatment, and particularly relates to a strain of Acidithiobacillus thiooxidans resistant to Ni and Cu toxicity and application thereof. BACKGROUND
[0002] Solid wastes such as waste catalysts, waste batteries and printed circuit boards contain metal components. If not properly disposed, the solid wastes will not only seriously pollute the ecological environment, but also cause great resource waste. Therefore, the resource utilization of metal in solid wastes not only can alleviate the current situation of resource shortage, but also can reduce environmental hazards, and has high economic and environmental benefits.
[0003] Thermal treatment method, hydrometallurgical method and bioleaching method are commonly used methods for recycling metals in solid wastes. However, the thermal treatment method and the hydrometallurgical method have the disadvantages of complex process, high energy consumption and serious secondary pollution. The bioleaching method has become a research hotspot due to its advantages of simple process, low energy consumption, mild conditions and environmental friendliness. The most commonly used microorganisms for leaching metals from solid wastes include Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans and other acidophilic bacteria.
[0004] The current acidophilic bacteria have the problems of low leaching rate and long leaching period in the process of leaching metals, which restricts their industrial application. Moreover, due to the high toxicity of metals such as Ni and Cu in solid wastes to microorganisms, it is difficult for the single batch of bioleaching technology to treat solid wastes with higher ore slurry density, which also leads to low processing efficiency. Therefore, it is necessary to develop acidophilic bacteria that can tolerate high concentration of metal toxicity and have faster growth rate for efficient leaching of metals in solid wastes. SUMMARY
[0005] The purpose of the present application is to overcome the problem that the bioleaching strain in the prior art is difficult to tolerate high concentration of metal toxicity, which affects the biological treatment efficiency of solid wastes. The present application provides a strain of Acidithiobacillus thiooxidans resistant to Ni and Cu toxicity and application thereof. The strain has high tolerance to Ni and Cu toxicity, and can significantly remove heavy metals in solid wastes under higher ore slurry density, thereby greatly improving the disposal efficiency of bioleaching.
[0006] In order to achieve the above purpose, the present application provides a strain of Acidithiobacillus thiooxidans resistant to Ni and Cu toxicity. The Acidithiobacillus thiooxidans is Acidithiobacillus thiooxidans QH2C, and the preservation number is GDMCC No: 63521.
[0007] Preferably, the Acidithiobacillus thiooxidans can tolerate Ni with a concentration of not higher than 3000 mg / L.
[0008] Preferably, the Acidithiobacillus thiooxidans can tolerate Cu at a concentration of no more than 60 g / L.
[0009] The second aspect of the present application provides a microbial agent containing the Acidithiobacillus thiooxidans as described above.
[0010] Preferably, the microbial agent further contains a carrier.
[0011] Preferably, the carrier is selected from one or more than two of sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol, corn flour, starch, clay, talc, kaolin, zeolite and biochar.
[0012] Preferably, the dosage form of the microbial agent is a liquid, an emulsion, a suspension, a powder or a granule.
[0013] The third aspect of the present application provides the use of the Acidithiobacillus thiooxidans as described above or the microbial agent as described above in treating metal-containing solid waste.
[0014] Preferably, the metal-containing solid waste contains Ni and / or Cu.
[0015] Preferably, the metal-containing solid waste is waste printed circuit boards, waste catalysts or waste batteries.
[0016] The fourth aspect of the present application provides a method for treating metal-containing solid waste, which comprises mixing the Acidithiobacillus thiooxidans as described above or the microbial agent as described above with the metal-containing solid waste and leaching the metal in the metal-containing solid waste.
[0017] Preferably, the metal-containing solid waste contains Ni and / or Cu.
[0018] Preferably, the mixing conditions include a temperature of 30-40℃ and a time of 20-120h.
[0019] The present application provides a new strain of Acidithiobacillus thiooxidans QH2C, the strain has a preservation number of GDMCC No: 63521, and the strain has high tolerance to Ni and Cu, so that the strain can treat higher slurry density of metal-containing solid waste in batches, improve the efficiency of biological leaching disposal, and has good application prospect.
[0020] Biological preservation
[0021] The strain provided by the present application is deposited on June 2, 2023, the deposit number is GDMCC No: 63521, the classification name is Acidithiobacillus thiooxidans QH2C, the deposit unit is Guangdong Microbial Culture Collection Center (GDMCC), the deposit address is 5th floor, No. 59 Building, Academy of Microbiology, Guangdong Province, Institute of Microbiology, 100, Martyrs' Road, Guangzhou, Postcode: 510070. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 is the growth curve of the Acidithiobacillus thiooxidans QH2C under different Ni concentrations provided by the present application;
[0023] Figure 2 is the growth curve of the Acidithiobacillus thiooxidans QH2C under different Cu concentrations provided by the present application;
[0024] Figure 3 is the GST enzyme activity of the Acidithiobacillus thiooxidans QH2C and the mixed bacterial liquid (original strain) before mutagenesis under different metal exposure provided by the present application;
[0025] Figure 4 is the leaching effect of Cu in waste circuit boards by the Acidithiobacillus thiooxidans QH2C provided by the present application;
[0026] Figure 5 is the leaching effect of Ni in FCC waste catalysts by the Acidithiobacillus thiooxidans QH2C provided by the present application;
[0027] Figure 6 is the growth curve of the mixed bacterial liquid before mutagenesis under different Ni concentrations;
[0028] Figure 7 is the growth curve of the mixed bacterial liquid before mutagenesis under different Cu concentrations. DETAILED DESCRIPTION
[0029] The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.
[0030] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and the values are approximate values and should be understood to include values approximately near these ranges and values within these ranges. For ranges of values, the endpoints of the ranges are combined with the individual points to form new ranges within the scope of the present application.
[0031] Biological leaching technology is an effective disposal method for solid waste rich in heavy metals such as Ni and Cu. However, the current biological leaching strains are difficult to tolerate high concentration of metal toxicity, which affects the efficiency of biological treatment of solid waste.
[0032] Therefore, the application provides an Acidithiobacillus thiooxidans strain resistant to Ni and Cu toxicity, which is Acidithiobacillus thiooxidans QH2C and has a preservation number of GDMCC No: 63521.
[0033] In the application, the 16S rRNA gene sequencing result of the Acidithiobacillus thiooxidans is shown in SEQ ID NO: 1.
[0034] The Acidithiobacillus thiooxidans provided by the application is separated from activated sludge of a sewage treatment plant of Qilu Petrochemical (Sinopec Qilu Branch), and is obtained through plasma mutagenesis and directional screening. Compared with strains directly separated from the existing environment, the Acidithiobacillus thiooxidans QH2C has higher resistance to Ni and Cu toxicity.
[0035] The Acidithiobacillus thiooxidans provided by the application can grow normally under the condition of high concentration of Ni and Cu and can effectively remove metals in solids. The strain has high toxicity resistance and can treat higher-density metal-containing solid waste in a single batch, thereby improving the efficiency of biological leaching disposal.
[0036] Specifically, the Acidithiobacillus thiooxidans can tolerate Ni with a concentration of not higher than 3000 mg / L and has high resistance to Ni toxicity. Therefore, the Acidithiobacillus thiooxidans can realize biological leaching of solid waste rich in Ni under high-density ore slurry.
[0037] Specifically, the Acidithiobacillus thiooxidans can tolerate Cu with a concentration of not higher than 60 g / L and has high resistance to Cu toxicity. Therefore, the Acidithiobacillus thiooxidans can realize biological leaching of solid waste rich in Cu under high-density ore slurry.
[0038] In the application, the screening method of the Acidithiobacillus thiooxidans comprises the following steps:
[0039] (1) mixing (NH4)2SO4 2-3 g / L, KH2PO4 2-4 g / L, MgSO4·7H2O 0.3-0.7 g / L, CaCl2 0.2-0.3 g / L, FeSO4 0.005-0.02 g / L and sulfur powder 8-15 g / L, and then adjusting the pH of the biological leaching liquid culture medium to 2.5-3.5 by using a sulfuric acid solution to obtain a biological leaching liquid culture medium;
[0040] (2) from the activated sludge sample collected from the sewage treatment plant of Qilu Petrochemical, inoculate the activated sludge sample in the biological leaching liquid medium at an amount of 5-10wt%, and obtain a culture solution containing a biological leaching strain after constant temperature shaking bed culture for 3-7 days; remove 5%-10% volume of the culture solution to a new batch of biological leaching liquid medium, and culture again for 3-7 days; repeat the above process for 4-6 times, and finally obtain a mixed bacterial solution after expansion culture;
[0041] (3) perform mutagenesis treatment on the mixed bacterial solution after expansion culture obtained in step (2), and the mutagenesis intensity is to make the mortality rate of microorganisms in the mixed bacterial solution reach 65%-95%;
[0042] (4) add Ni to the biological leaching liquid medium in step (1) to obtain a screening medium, inoculate the remaining surviving bacteria in the mixed bacterial solution after mutagenesis in step (3) into the screening medium, and culture for 2-5 days; the bacteria that can survive and grow normally are the target biological leaching culture solution with high metal toxicity tolerance;
[0043] (5) perform coating dilution streaking separation culture on the biological leaching culture solution, and thus the sulfur-oxidizing bacteria with Ni and Cu toxicity tolerance are obtained.
[0044] In the screening method, the metal toxicity tolerance of the bacteria is improved by performing mutagenesis treatment on the mixed bacterial solution obtained in step (2). The mutagenesis treatment technology can be ultraviolet mutagenesis, microwave mutagenesis, plasma mutagenesis, etc., and the plasma mutagenesis is preferred.
[0045] In the screening method, the mutagenesis treatment in step (3) can be repeated until the mortality rate of microorganisms in the mixed bacterial solution reaches 65%-95%.
[0046] Preferably, in step (3), the mutagenesis intensity is to make the mortality rate of microorganisms in the mixed bacterial solution reach 75%-85%.
[0047] In the screening method, step (5) specifically includes:
[0048] Dilute the biological leaching culture solution obtained in step (4) into 10 4 , 10 6 , and 10 -8 using the biological leaching liquid medium in step (1), respectively; after 150μL liquid of each concentration gradient is taken and coated on a solid culture medium plate, at least 3 parallel plates are prepared for each gradient; after a certain time of culture, appropriate single colonies are picked up with an inoculation loop and cultured in the biological leaching liquid medium; after the bacteria are cultured to the exponential growth phase, the bacteria are coated on a solid culture medium plate again, and the above process is repeated for 2-3 times; and thus the pure bacteria obtained are the target strain (sulfur-oxidizing bacteria with Ni and Cu toxicity tolerance);
[0049] The solid culture medium is prepared by the following process: mixing A liquid and B liquid when cooled to 80 DEG C, then adding C liquid and mixing, pouring into a culture dish, and preparing the solid culture medium after cooling; wherein, A liquid: 100 mL distilled water containing 0.4-0.8 g (NH4)2SO4, 0.4-0.8 g KH2PO4, 0.05-0.15 g MgSO4·7H2O, 0.03-0.08 g CaCl2·2H2O, high-pressure sterilization at 115-120 DEG C for 20-40 min; B liquid: 100 mL distilled water containing 1.5-2.5 g agar powder, high-pressure sterilization at 115-120 DEG C for 20-40 min; C liquid: 10 mL sterile water containing 1-3 g Na2S2O3, 0.005-0.01 g FeSO4·7H2O, filtering and sterilizing.
[0050] In the present application, the bacteria solution separated from the activated sludge sample is subjected to mutagenesis treatment, then subjected to directional screening through the Ni-containing screening medium, and then separated and purified to obtain the Thiobacillus thioxidans capable of tolerating high-concentration Ni. Cu is a common metal in solid waste, and it is found through detection that the Thiobacillus thioxidans also has high tolerance to Cu.
[0051] The present application also provides a bacterial agent containing the Thiobacillus thioxidans as described above.
[0052] The present application does not limit the specific components in the bacterial agent, and only needs to contain the bacterial suspension, fermentation liquor or culture concentrate of the Thiobacillus thioxidans as described above. It can be understood that the bacterial agent can also contain other microorganisms compounded with the Thiobacillus thioxidans for improving the leaching effect of the bacterial agent on Ni and / or Cu.
[0053] In the preferred embodiments, the bacterial agent contains a carrier in addition to the active ingredients described above. The carrier can be a carrier commonly used in the field of bacterial agents and biologically inert.
[0054] In the preferred embodiments, the carrier is selected from one or two or more of sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol, corn flour, starch, clay, talc, kaolin, zeolite and biochar. The present application does not have special limitations on the source of the carrier, and commercially available goods can be used, or the carrier can be prepared by the method known to those skilled in the art.
[0055] The present application also does not limit the specific dosage form of the bacterial agent, and the dosage form can be selected according to the actual needs. Specifically, it can be liquid agent, emulsion, suspension, powder or granules.
[0056] The present application also provides the use of the Thiobacillus thioxidans as described above or the bacterial agent as described above in the treatment of metal-containing solid waste.
[0057] Specifically, the application is in leaching metals in the metal-containing solid waste.
[0058] In the application, the metal-containing solid waste contains Ni and / or Cu. Since the Acidithiobacillus thiooxidans can tolerate high concentrations of Ni and Cu, the Acidithiobacillus thiooxidans or the microbial inoculum containing the Acidithiobacillus thiooxidans as an active component can realize bioleaching of the solid waste rich in Ni and / or Cu at high pulp density, thereby efficiently leaching metals in the metal-containing solid waste.
[0059] The application does not limit the specific type of the metal-containing solid waste. Specifically, the metal-containing solid waste can be, for example, waste printed circuit boards, waste catalysts or waste batteries containing Ni or Cu.
[0060] The application further provides a method for treating metal-containing solid waste, which comprises mixing the Acidithiobacillus thiooxidans or the microbial inoculum as described above with the metal-containing solid waste, and leaching metals in the metal-containing solid waste.
[0061] In a specific embodiment, the step of mixing the Acidithiobacillus thiooxidans with the metal-containing solid waste comprises:
[0062] A1, mixing (NH4)2SO4 2-3 g / L, KH2PO4 2-4 g / L, MgSO4·7H2O 0.3-0.7 g / L, CaCl2 0.2-0.3 g / L, FeSO4 0.005-0.02 g / L and sulfur powder 8-15 g / L, and then adjusting the pH to 2.5-3.5 with a sulfuric acid solution to obtain a bioleaching liquid medium;
[0063] A2, inoculating the Acidithiobacillus thiooxidans into the bioleaching liquid medium for expansion culture to obtain a bioleaching bacterial solution;
[0064] A3, adding the metal-containing solid waste to the bioleaching bacterial solution and continuing to culture for 3-5 days, so that the metals in the metal-containing solid waste are dissolved in the bioleaching bacterial solution under the action of the Acidithiobacillus thiooxidans.
[0065] In a specific embodiment, in step A2, the expansion culture is performed by shake flask culture, wherein the conditions of the shake flask culture include a temperature of 30-40°C, a rotation speed of 120-135 rpm and a time of 24-96 h.
[0066] When a large amount of bacteria is needed, after the shake flask culture, the method further comprises: performing reactor culture on the culture solution obtained by the shake flask culture, wherein the conditions of the reactor culture include a temperature of 30-40°C, an aeration rate of 0.1 vvm and a stirring rate of 30-90 rpm.
[0067] In a specific embodiment, in step A3, the metal-containing solid waste contains Ni and / or Cu.
[0068] In a specific embodiment, in step A3, the metal-containing solid waste is waste printed circuit board, waste catalyst or waste battery.
[0069] In order to make the treatment of the metal-containing solid waste more efficient, in a preferred embodiment, in step A3, the concentration of the metal-containing solid waste added needs to ensure that the concentration of Ni in the bioleaching bacterial solution after the reaction is completed is not more than 3000 mg / L, and the concentration of Cu is not more than 60 g / L.
[0070] The application will be described in detail below through examples, but the protection scope of the application is not limited thereto. In the following examples, the experimental methods are conventional methods in the art unless otherwise specified. The experimental materials used in the following examples are commercially available unless otherwise specified.
[0071] In the following examples, the culture medium used includes:
[0072] Bioleaching liquid medium: (NH4)2SO4 3 g / L, KH2PO4 3 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.25 g / L, FeSO4 0.01 g / L and sulfur powder 10 g / L, pH adjusted to 3;
[0073] Screening medium: 35 mM of NiCl2 (the content of Ni ions is 2055 mg / L) is added to the bioleaching liquid medium;
[0074] Solid medium: A liquid and B liquid are mixed when cooled to 80°C, and then C liquid is added and mixed, poured into a culture dish, and after cooling, the solid medium is prepared; wherein, A liquid: 100 mL of distilled water contains 0.5 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.05 g of MgSO4·7H2O, 0.03 g of CaCl2·2H2O, 120°C high pressure sterilization for 20 min; B liquid: 100 mL of distilled water contains 2.0 g of agar powder, 115°C high pressure sterilization for 30 min; C liquid: 10 mL of sterile water contains 1 g of Na2S2O3, 0.005 g of FeSO4·7H2O, filtered and sterilized.
[0075] Example 1
[0076] This example is used to illustrate the screening of Thiobacillus thioxidans QH2C described in the application.
[0077] (1) Collect activated sludge samples from the wastewater treatment plant of Qilu Petrochemical. Place the activated sludge samples in a bioleaching liquid culture medium with an inoculum of 5wt%. After culturing in a constant temperature shaker at 35℃ and 130rpm for 5 days, a culture medium containing bioleaching strains is obtained.
[0078] (2) Transfer 10% of the culture medium to a new biological leaching liquid culture medium and culture it again for 5 days; repeat this process 4 times to finally obtain the expanded mixed bacterial culture.
[0079] (3) The mixed bacterial solution obtained in step (2) was subjected to mutagenesis using an ARTP plasma mutagenesis instrument under the following conditions: power 120w, gas flow rate 10SLM, and mutagenesis time 5s; the lethality rate of bacteria in the mixed bacterial solution after mutagenesis reached 82%.
[0080] (4) Add the remaining surviving bacteria in the mixed bacterial solution after mutagenesis in step (3) to the screening medium (containing 35mM NiCl2). After culturing for 5 days, the bacteria that can survive and grow normally are the target high-toxicity resistant biological leaching culture.
[0081] (5) The bioleaching culture medium obtained in step (4) is subjected to plating, dilution, streaking, and separation culture. The specific operation is as follows: dilute the bioleaching culture medium to 10. -4 10 -6 , and 10 -8 For each concentration gradient, 150 μL of liquid was aspirated and spread onto a solid medium plate, with three replicates for each gradient. After incubation for a certain period, a suitable single colony was picked up with an inoculation loop and cultured in a bioleaching liquid medium until the exponential growth phase was reached. The colony was then spread onto a solid medium plate again. This process was repeated twice. The resulting pure colony was the target strain, designated QH2C.
[0082] Example 2
[0083] This embodiment is used to illustrate the molecular biological identification of the thiobacillus thiooxidans QH2C described in this invention.
[0084] (1) The strain QH2C obtained in Example 1 was streaked in solid medium. After culturing, its whole genome was extracted using a DNA kit, and PCR amplification was performed using primers 27-F and 1492-R. The PCR reaction system was 25 μL: 1 μL each of forward and reverse primers (10 μmol / L), 1 μL of DNA template (10 ng / μL), 12.5 μL of 2×Taq Master Mix, and ultrapure water to a final volume of 25 μL. The PCR reaction conditions were: 94℃ for 5 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min 30 s, 35 cycles; final extension at 72℃ for 7 min.
[0085] (2) The obtained PCR amplification product was entrusted to Shanghai Shengong Bioengineering Co., Ltd. for sequencing, and the obtained gene sequence was subjected to homology comparison through GenBank to determine its taxonomic status.
[0086] The 16S rRNA gene sequencing result of the strain QH2C is shown as SEQ ID NO: 1, and through database comparison analysis, the strain QH2C is identified as Acidithiobacillus thiooxidans, which is named Acidithiobacillus thiooxidans QH2C, and the strain is preserved in the Guangdong Microbial Culture Collection Center on June 2, 2023, with the preservation number of GDMCC No: 63521.
[0087] The sequence of SEQ ID NO: 1 is as follows:
[0088] CAGATTGAACGCTGGCGGCATGCCTAACACATGCAAGTCGAACGG
[0089] CAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGGACGGGTGAGTAA
[0090] TGCGTAGGAATCTGTCCAATAGTTTGGGACAACCCAGGGAAACTTGGG
[0091] CTAATACCGGATACGTCCTGAGGGAGAAAGCGGGGGATCTTCGGACCT
[0092] CGTGCTATTGGAGGGGCCTACGTTCGATTAGCTAGTTGGCAGGGTAAG
[0093] GGCCTACCAAGGCGACGATCGATAGCTGGTCTGAGAGGACGATCAGCC
[0094] ACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTG
[0095] GGGAATTTTTCGCAATGGGGGCAACCCTGACGAAGCAATGCCGCGTGA
[0096] ATGAAGAAGGCCTTCGGGTTGTAAAGTTCTTTCGTGGGAGACGAAAA
[0097] GGTAATCGCTAATATCGGTTACTGTTGACGTGAACCCAAGAAGAAGCA
[0098] CCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGC
[0099] GTTAATCGGAATCACTGGGCGTAAAGGGCGCGTAGGCGGTGGGTTACG
[0100] TCTGCCGTGAAATCCCCGGGCTCAACCTGGGAATGGCGGTGGAAACG
[0101] GGCTGACTAGAGTATGGGAGAGGGTGATGGAATTCCAGGTGTAGCGGT
[0102] GAAATGCGTAGAGATCTGGAGGAACATCAGTGGCGAAGGCGGTCACC
[0103] TGGCCCAATACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGG
[0104] ATTAGATACCCTGGTAGTCCACGCCCTAAACGATGGATACTAGATGTTT
[0105] GGTGCCTTAGGTGCTGAGTGTCGTAGCTAACGTGATAAGTATCCCGCCT
[0106] GGGAAGTACGGCCGCAAGGTTAAAACTCAAAGGAATTGACGGGGGCC
[0107] CGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGCAGAAC
[0108] CTTACCTGGGCTTGACATCCAGAGAATCCTGCAGAGATGTGGGAGTGC
[0109] CTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGT
[0110] CGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCTA
[0111] GTTGCCAGCGGTTCGGCCGGGCACTCTAGGGAGACTGCCGGTGACAA
[0112] ACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCTTTATGTCC
[0113] AGGGCTACACACGTGCTACAATGGCGCGTACAGAGGGAAGCGAGACC
[0114] GCGAGGTGGAGCAGACCCCAGAAAGCGCGCCGTAGTTCGGATTGCAG
[0115] TCTGCAACTCGACTGCATGAAGTCGGAATCGCTAGTAATCGCGGATCA
[0116] GCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCA
[0117] CACCATGGGAGTGGATTGTACCAGAAGCAGCTAGCCTAACCTTCGGGG
[0118] GGGGCGGTTACCACGGTATGGGTTCATGAC.
[0119] Example 3
[0120] This embodiment is used to illustrate the Ni toxicity tolerance test of the *Thiobacillus thiooxidans* QH2C described in this invention.
[0121] Add different concentrations of Ni to 250 mL Erlenmeyer flasks sealed with rubber stoppers, then add Thiobacillus thiooxidans QH2C and adjust the bacterial concentration to 5 × 10⁻⁶. 8 The bacterial count was measured at 37°C and 130 rpm in a constant-temperature shaker. Samples were taken every 3 days to determine the bacterial concentration. Results are as follows: Figure 1 As shown.
[0122] Depend on Figure 1 It can be seen that the thiobacillus thiooxidans QH2C provided by this invention can tolerate up to 3000 mg / L of Ni.
[0123] Example 4
[0124] This example is used to illustrate the Cu toxicity tolerance determination of Acidithiobacillus thiooxidans QH2C according to the present application.
[0125] A rubber plug-sealed 250 mL triangular flask was added with a bioleaching liquid medium containing different Cu concentrations, then Acidithiobacillus thiooxidans QH2C was added, and the bacterial concentration was adjusted to 5×10 8 Individuals / mL, and incubated in a constant temperature shaker at a temperature of 37℃ and a rotation speed of 130 rpm, and the bacterial concentration was detected every 3 days, and the results are shown in Figure 2
[0126] It can be seen that the Acidithiobacillus thiooxidans QH2C provided by the present application can tolerate up to 60 g / L of Cu. Figure 2
[0127] Example 5
[0128] This example is used to illustrate the glutathione S-transferase (GST) activity of Acidithiobacillus thiooxidans QH2C according to the present application.
[0129] The glutathione S-transferase activity of Acidithiobacillus thiooxidans QH2C under the exposure of 40 g / L of Cu and 2000 mg / L of Ni was determined.
[0130] Test method: (1) A rubber plug-sealed 250 mL triangular flask was added with 200 mL of a bioleaching liquid medium containing 2000 mg / L of Ni, then Acidithiobacillus thiooxidans QH2C was added, and the bacterial concentration was adjusted to 5×10 8 Individuals / mL, and incubated in a constant temperature shaker at a temperature of 37℃ and a rotation speed of 130 rpm for 5 days;
[0131] (2) A rubber plug-sealed 250 mL triangular flask was added with 200 mL of a bioleaching liquid medium containing 40 g / L of Cu, then Acidithiobacillus thiooxidans QH2C was added, and the bacterial concentration was adjusted to 5×10 8 Individuals / mL, and incubated in a constant temperature shaker at a temperature of 37℃ and a rotation speed of 130 rpm for 5 days;
[0132] (3) The bacterial solution obtained in steps (1) and (2) was centrifuged (10000 g, 10 min, 4℃), then resuspended with PBS buffer solution, and then broken by an ultrasonic disruptor, and used for enzyme activity determination, wherein the enzyme activity was determined by using a GST kit (brand: mlBio / Enzyme-linked Bio; product number: mlsw_E0469).
[0133] The test results are shown in Figure 3 Figure 3 In the present application, the mutagenized strain refers to Thiobacillus thioxidans QH2C, and the starting strain refers to the mixed bacterial solution before mutagenesis.
[0134] By Figure 3 It can be seen that, compared with the control group, the exposure of Ni and Cu can significantly enhance the GST activity in the cells of Thiobacillus thioxidans QH2C, indicating that the Thiobacillus thioxidans QH2C provided by the present application has high metal detoxification metabolic capacity.
[0135] Example 6
[0136] The present example is used to illustrate the method for treating metal-containing solid waste provided by the present application.
[0137] The bioleaching of Cu in waste circuit boards by Thiobacillus thioxidans QH2C was determined, wherein the Cu content in the waste circuit boards was 17.5wt%.
[0138] Test method: Thiobacillus thioxidans QH2C was inoculated in the bioleaching liquid medium for culture, and the culture conditions were as follows: temperature was 37℃, rotation speed was 120rpm, and the culture was continued until the bacterial concentration of the bioleaching bacterial solution reached 5×10 8 / mL; 300mL of the bioleaching bacterial solution was added in a 500mL shake flask, 90g of waste circuit boards was added, and the culture was continued at 37℃ and 120rpm, and the leaching concentration of Cu in the bacterial solution was detected every 8h.
[0139] The leaching curve of Cu in waste circuit boards with the reaction time (h) under the action of Thiobacillus thioxidans QH2C is shown in Figure 4 The results show that, after 32h of bioleaching reaction, the leaching concentration of Cu in the waste circuit boards reached 48.8g / L, and the leaching rate can reach 48.8×0.3 / (90×17.5%)=92.95%.
[0140] Example 7
[0141] The present example is used to illustrate the method for treating metal-containing solid waste provided by the present application.
[0142] The bioleaching of Ni in FCC waste catalyst by Thiobacillus thioxidans QH2C was determined, wherein the Ni content in the FCC waste catalyst was 3.8mg / g.
[0143] Test method: Thiobacillus thioxidans QH2C was inoculated in the bioleaching liquid medium for culture, and the culture conditions were as follows: temperature was 37℃, rotation speed was 120rpm, and the culture was continued until the bacterial concentration of the bioleaching bacterial solution reached 5×10 8The bioleaching bacterial solution was prepared by adding 300 mL of the bioleaching bacterial solution to a 500 mL shake flask and adding 60 g of FCC waste catalyst. The mixture was then cultured at 37 °C and 120 rpm. The leaching concentration of Ni in the bacterial solution was measured every 1 day.
[0144] The leaching curve of Ni from FCC waste catalyst under the action of Thiobacillus thiooxidans QH2C is shown below. Figure 5 As shown in the figure. The results showed that after 5 days of bioleaching reaction, the leaching concentration of Ni in the FCC waste catalyst was 415 mg / L, and the leaching rate reached 0.415 × 0.3 / (60 × 0.38%) = 54.6%.
[0145] Comparative Example 1
[0146] This comparative example is used to illustrate the Ni toxicity tolerance effect of the mixed bacterial culture (starting strain) before mutagenesis.
[0147] Add biological leaching liquid culture medium containing different Ni concentrations to 250mL Erlenmeyer flasks sealed with rubber stoppers, then add the expanded mixed bacterial solution obtained in step (1) of Example 1, and adjust the bacterial concentration to 5×10⁻⁶. 8 The bacterial count was measured at 37°C and 130 rpm in a constant-temperature shaker. Samples were taken every 3 days to determine the bacterial concentration. Results are as follows: Figure 6 As shown.
[0148] Depend on Figure 6 It can be seen that the starting strain of Thiobacillus thiooxidans QH2C can tolerate up to 500 mg / L Ni, but cannot grow under exposure to 1000 mg / L Ni, and its tolerance is much lower than that of Thiobacillus thiooxidans QH2C.
[0149] Comparative Example 2
[0150] This comparative example was used to determine the Cu toxicity tolerance effect of the mixed bacterial culture (starting strain) before mutagenesis.
[0151] Add biological leaching liquid culture medium containing different Cu concentrations to 250mL Erlenmeyer flasks sealed with rubber stoppers, then add the expanded mixed bacterial solution obtained in step (1) of Example 1, and adjust the bacterial concentration to 5×10⁻⁶. 8 The bacterial count was measured at 37°C and 130 rpm in a constant-temperature shaker. Samples were taken every 3 days to determine the bacterial concentration. Results are as follows: Figure 7 As shown.
[0152] Depend on Figure 7 It can be seen that the starting strain of Thiobacillus thiooxidans QH2C can tolerate a maximum of 40 g / L of Cu, which is lower than that of Thiobacillus thiooxidans QH2C.
[0153] Comparative Example 3
[0154] The comparative example is used to determine the glutathione S-transferase (GST) activity of the mixed bacterial solution before mutagenesis (the starting strain).
[0155] The glutathione S-transferase activity of the mixed bacterial solution before mutagenesis (the starting strain) is determined under the exposure of 10 g / L Cu and 500 mg / L Ni.
[0156] Test method: (1) 200 mL of bioleaching liquid medium containing 500 mg / L of Ni is added to a 250 mL triangular flask sealed with a rubber plug, then the starting strain is added, and the bacterial concentration is adjusted to 5 x 10 8 individuals / mL, and incubated in a constant temperature shaker at 37 DEG C and a rotation speed of 130 rpm for 5 days;
[0157] (2) 200 mL of bioleaching liquid medium containing 10 g / L of Cu is added to a 250 mL triangular flask sealed with a rubber plug, then the starting strain is added, and the bacterial concentration is adjusted to 5 x 10 8 individuals / mL, and incubated in a constant temperature shaker at 37 DEG C and a rotation speed of 130 rpm for 5 days;
[0158] (3) The bacterial solution obtained in steps (1) and (2) is centrifuged (10000 g, 10 min, 4 DEG C), then resuspended with PBS buffer solution, and then broken by an ultrasonic disrupter for enzyme activity determination, wherein the enzyme activity is determined by using a GST kit (brand: mlBio / Enzyme-linked biological; product number: mlsw_E0469).
[0159] The test results are shown in Table 1. Figure 3 Figure 3 Among them, the mutagenized strain refers to Thiobacillus thioxidans QH2C, and the starting strain refers to the mixed bacterial solution before mutagenesis.
[0160] As can be seen from Table 1, Figure 3 compared with the control group or Thiobacillus thioxidans QH2C, the GST activity of the starting strain is significantly reduced under the exposure of Ni and Cu.
[0161] The above describes the preferred embodiments of the present application in detail, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the present application, and all fall within the protection scope of the present application.
Claims
1. A strain of *Thiobacillus thiooxidans* resistant to Ni and Cu toxicity, characterized in that, The thiooxidizing bacillus is Acidithiobacillus thiooxidans QH2C, with accession number GDMCC No: 63521.
2. The *Thiobacillus thiooxidans* according to claim 1, characterized in that, The thiobacillus thiooxidans can tolerate Ni concentrations not exceeding 3000 mg / L.
3. The *Thiobacillus thiooxidans* according to claim 1, characterized in that, The thiobacillus thiooxidans can tolerate Cu concentrations not exceeding 60 g / L.
4. A microbial agent, characterized in that, The bacterial agent contains *Thiobacillus thiooxidans* as described in any one of claims 1-3.
5. The microbial agent according to claim 4, characterized in that, The bacterial agent also contains a carrier.
6. The microbial agent according to claim 5, characterized in that, The carrier is selected from one or more of sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol, corn flour, starch, clay, talc, kaolin, zeolite and biochar.
7. The microbial agent according to claim 4, characterized in that, The formulation of the microbial agent is liquid, emulsion, suspension, powder or granules.
8. The application of the Thiobacillus thiooxidans according to any one of claims 1-3 or the bacterial agent according to any one of claims 4-7 in the treatment of metal-containing solid waste.
9. The application according to claim 8, characterized in that, The metal-containing solid waste contains Ni and / or Cu.
10. The application according to claim 8 or 9, characterized in that, The metal-containing solid waste includes waste circuit boards, waste catalysts, or waste batteries.
11. A method for treating metal-containing solid waste, characterized in that, The method includes: mixing the *Thiobacillus thiooxidans* according to any one of claims 1-3 or the bacterial agent according to any one of claims 4-7 with metal-containing solid waste, and leaching the metal from the metal-containing solid waste.
12. The method according to claim 11, characterized in that, The metal-containing solid waste contains Ni and / or Cu.
13. The method according to claim 11, characterized in that, The mixing conditions include a temperature of 30–40°C and a time of 20–120 h.