Strain for heavy metal pollution control and application thereof

Abstract

The invention belongs to the field of pollution prevention and control, and particularly relates to a strain for heavy metal pollution prevention and control, which is Cupriavidus sp.Zn6 preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCCNO.32288. The invention further discloses a preparation method of the strain for heavy metal pollution prevention and control. The Cupriavidus sp. Zn6 has a good adsorption effect on heavy metal cadmium, and can tolerate toxicity of a plurality of other heavy metals such as Zn, Cu and Pb, so that the Cupriavidus sp. Zn6 can adapt to a complex natural environment and can be practically applied to treatment of heavy metal pollution in the environment. The strain Zn6 is screened from an environment in a completely natural state, has no ecological invasion threat to the environment, and has practical popularization and application values.

Description

Technical Field

[0001] The invention belongs to the field of pollution prevention and control, and specifically relates to a strain for heavy metal pollution prevention and control and an application thereof. Background Art

[0002] With the development of modern society in my country, the population growth, industrialization and urbanization are accelerating, and the heavy metal industry is maintaining a strong growth momentum. The heavy metal pollution brought about by this will inevitably become a topic of the times. Studies have shown that there is a significant accumulation of heavy metals in the soil of most provinces in China, among which cadmium and mercury are the most common heavy metal pollution. Heavy metals existing in the environment will not decompose naturally due to their inorganic nature. They will stay in the soil and water for a long time, and will undergo bioaccumulation and bioenhancement along the food chain, causing serious harm to animals, plants and humans.

[0003] Traditional methods for treating heavy metal pollution include chemical precipitation, chemical oxidation-reduction, ion exchange, filtration, electrochemical treatment, reverse osmosis, membrane technology, etc. These treatment methods are suitable for treating heavy metal pollution, but they have little effect on heavy metal pollution with a concentration range of 10-100 mg / L. Compared with traditional physical and chemical remediation, bioremediation has the characteristics of low cost, less environmental damage, recyclability, and public acceptance. Among them, the use of microorganisms as adsorbents to treat heavy metal pollution has attracted the attention of scholars in the industry due to its advantages of economy, environmental protection, safety, high efficiency, and no secondary pollution, and has achieved ideal adsorption effects.

[0004] Patent CN106367364A discloses an alkaliphilic copper-loving bacteria that can efficiently remove cadmium pollution, which can completely remove 1000mg / L and below of cadmium in sewage. Due to the complex types of heavy metals in sewage in the natural environment, heavy metal ions (such as mercury, cadmium, lead, etc.) can interfere with their normal functions by binding to enzymes and proteins in cells. The alkaliphilic copper-loving bacteria that remove cadmium pollution cannot tolerate the toxicity of multiple heavy metals, and its practical application value will be greatly reduced. Therefore, it is necessary to provide a strain that can efficiently remove cadmium and tolerate the toxicity of multiple heavy metals at the same time, so as to improve the effect of actual microbial treatment of heavy metal pollution. Summary of the invention

[0005] To achieve the above purpose, the present invention provides a strain for the prevention and treatment of heavy metal pollution, which is the copper-greedy bacteria Cupriavidus sp. Zn6 deposited in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC NO.32288.

[0006] The Cupriavidus sp.Zn6 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on October 21, 2024, and was classified and named: Cupriavidus sp., and the storage address is: Building 1, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides a use of the aforementioned strain in preparing a detergent for preventing and treating heavy metal pollution.

[0008] Further, the detergent comprises a biosorbent.

[0009] Furthermore, the biosorbent has the function of adsorbing heavy metals, including Zn, Cu, Cd and / or Pb.

[0010] Furthermore, the heavy metal is Cd.

[0011] Furthermore, the biosorbent is a preparation made of Cupriavidus sp. Zn6 as an active ingredient and acceptable excipients.

[0012] Furthermore, the preparation includes solution, powder, granule and spray.

[0013] The bacterial amount of Cupriavidus sp. Zn6 in the biosorbent preparation is 1.0-3.0 g / L.

[0014] The invention also provides a detergent for preventing and treating heavy metal pollution, which comprises the aforementioned strain.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The copper-greedy bacteria Zn6 of the present invention has a good adsorption effect on heavy metal cadmium, and can tolerate the toxicity of multiple other heavy metals, such as Zn, Cu, Pb, etc., and therefore can adapt to complex natural environments and can be actually applied to the treatment of heavy metal pollution in the environment. The strain Zn6 is screened from a completely natural environment, does not pose an ecological invasion threat to the environment, and has practical promotion and application value.

[0017] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0018] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The colony morphology of the Zn6 strain in the nutrient agar medium of the embodiment of the present invention is shown;

[0020] Figure 2 This is the Gram staining result of the Zn6 strain of the present invention (oil immersion lens 10×100);

[0021] Figure 3 This is a cluster analysis diagram of the Zn6 strain of the embodiment of the present invention;

[0022] Figure 4 This is a picture of the Zn6 strain of the present invention before adsorbing Cd under an electron microscope;

[0023] Figure 5 This is a picture of the Zn6 strain of the present invention after it adsorbs Cd under an electron microscope. DETAILED DESCRIPTION

[0024] The raw materials, reagents and equipment used in the specific embodiments of the present invention are all purchased from the market.

[0025] Example 1 Preparation of the detergent of the present invention

[0026] Cupriavidus sp. Zn6 was inoculated into nutrient broth medium and cultured at 150 rpm and 25 °C for 24 h. The cells were collected by centrifugation at 8000 rpm and 4 °C for 10 min and then washed with ddH 2 O, rinse three times, freeze in a -20℃ refrigerator, freeze-dry and grind into bacterial powder using a freeze dryer, and add acceptable auxiliary materials.

[0027] The beneficial effects of the present invention are further illustrated by test examples below:

[0028] Test Example 1 Screening and efficacy experiment of heavy metal adsorbing bacteria

[0029] 1. Strain Screening

[0030] 1.1 Soil collection

[0031] Soil samples were collected from a tailings soil in Panzhihua. The topsoil of the top 5 to 10 cm was removed, and soil samples were collected using the plum blossom five-point sampling method and placed in a 4°C refrigerator for later use.

[0032] 1.2 Culture medium

[0033] Nutrient agar medium: peptone 10.0 g / L, beef extract powder 5.0 g / L, sodium chloride 5.0 g / L, agar 12.0 g / L, adjust pH to 7.3±0.1.

[0034] Isolation medium: Nutrient agar medium as the basic medium, add sterilized 1 mol / L Cd(NO 3 ) 2 Solution makes Cd 2+ The concentration is 1mg / L.

[0035] Screening medium: Nutrient agar medium was used as the basic medium, and a certain amount of Cd (NO 3 ) 2 solution to achieve the Cd required by the specific experiment 2+ concentration.

[0036] Nutrient broth medium: peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, adjust pH to 7.4±0.2.

[0037] 1.3 Isolation and screening of strains

[0038] Weigh 10 g of soil from a mine tailing in Panzhihua and place it in 90 mL of saline solution containing glass beads. Mix well by oscillation and perform 10-fold gradient dilution to obtain 10 -1 ~10 -5 Sample diluent; select 10 -3 ~10 -5 For each of the three dilutions, 0.1 mL of the sample dilution was spread on the separation medium and cultured at 37 °C for 48 h;

[0039] Pick a single colony from the isolation medium and streak it to Cd 2+ The strain was cultured at 37°C for 48 hours on screening plates with concentrations of 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, and 700 mg / L. The growth was observed and a strain with good cadmium resistance was finally screened out and named Zn6.

[0040] 2. Strain Identification

[0041] 2.1 Morphological characteristics

[0042] The screened strain Zn6 was inoculated on a nutrient agar plate and cultured, and the colony morphology was observed.

[0043] 2.2 Analysis of 16S rDNA sequences of strains

[0044] The genomic DNA of strain Zn6 was extracted by boiling. The universal primer 27F (SEQ ID NO.1:5 , -AGAGTTTGATCMTGGCTCAG-3 , ) and 1492R (SEQ ID NO. 2: 5 , -GGCTACCTTGTTACGACTT-3 , ) were used as primers to amplify the 16SrDNA sequence of strain Zn6. PCR amplification reaction system: 5μL 10×buffer, 4μL dNTP, 0.25μL TaqPCRmasterMix, 2μL template DNA, 2μL 27F primer, 2μL 1492R reverse primer, 34.75μL ddH2O. PCR amplification program: pre-denaturation (94℃, 5min), denaturation (94℃, 30s), annealing (56℃, 30s), extension (72℃, 2min), 32 cycles, 72℃ extension for 10min. The PCR amplification product was entrusted to Chengdu Qingke Zixi Biotechnology Co., Ltd., and the PCR primers were used as sequencing primers. The forward and reverse strands were sequenced respectively, and the sequences were spliced. The nucleotide BLAST search tool was used to compare with the 16SrDNA sequence in Genebank (http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi). The neighbor-joining method of MEGA version 5.0 was used to construct a phylogenetic tree and analyze the evolutionary relationship between the strains and existing strains in the database.

[0045] 3.Metal endurance test

[0046] Strain Zn6 was streaked on nutrient agar plates with Zn, Cu, Cd and Pb concentrations of 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L and 700 mg / L, respectively, and cultured at 37°C for 48 h. The growth was observed to obtain the maximum tolerance concentration of strain Zn6 to the four heavy metals.

[0047] 4. Adsorption characteristics

[0048] 4.1 Preparation of bacterial powder

[0049] Add 100 mL of nutrient broth medium to a 250 mL conical flask, inoculate strain Zn6, and culture at 150 rpm and 25 °C for 24 h. Collect the cells by centrifugation at 8000 rpm and 4 °C for 10 min, and then rinse with ddH 2 O for three times, freeze in a -20℃ refrigerator, freeze-dry and grind into bacterial powder for later use.

[0050] 4.2 Effect of bacterial dosage on the adsorption of Zn6

[0051] Add 100 mg / L Cd into a 50 mL conical flask. 2+ 20 mL of solution was added with Zn6 powder at the dosage of 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, and 3.0 g / L, respectively. The mixture was cultured at 25 °C and 150 rpm for 24 h. The supernatant was collected by centrifugation at 8000 rpm for 10 min. After filtering through a 0.45 μm filter membrane, the Cd content in the supernatant was determined by flame atomic absorption spectrometry. 2+ Concentration, Cd is calculated by formula (1) 2+ Adsorption rate (R):

[0052]

[0053] (Where: C 0 and C R Cd in the supernatant 2+ Initial concentration and residual concentration, mg / L. )

[0054] 4.3 Initial Cd 2+ Effect of concentration on the adsorption of Zn6 by strains

[0055] Add different concentrations of Cd into a 50 mL conical flask. 2+ Solution 20mL, Cd 2+ The concentrations were 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L, respectively. Zn6 powder was added at a dosage of 2.5 g / L. The mixture was cultured at 25 °C and 150 rpm for 24 h. The supernatant was collected by centrifugation at 8000 rpm for 10 min. After filtering through a 0.45 μm filter membrane, the Cd content in the supernatant was determined by a flame atomic absorption spectrometer. 2+ Concentration, Cd is calculated by formula (1) 2+ Adsorption rate.

[0056] 4.4 Effect of adsorption time on the adsorption effect of strain Zn6

[0057] Add Cd into the conical flask 2+ solution and Zn6 bacterial powder, cultured at 25℃, 150rpm, with a bacterial dosage of 2.5g / L, Cd 2+ The concentration was 20 mg / L, the culture time was 3 h, 6 h, 9 h, 12 h, and 15 h, and the supernatant was collected by centrifugation at 8000 rpm for 10 min. After filtering through a 0.45 μm filter membrane, the Cd content in the supernatant was determined by flame atomic absorption spectrometry. 2+ Concentration, use formula (1) to calculate Cd 2+ Adsorption rate.

[0058] 5. Adsorption mechanism

[0059] 5.1 Scanning electron microscopy (SEM) analysis

[0060] Add 100 mL of nutrient broth medium to a 250 mL conical flask, inoculate strain Zn6, and culture at 150 rpm and 25 °C for 24 h. Centrifuge at 8000 rpm and 4 °C for 10 min to collect the cells, transfer them to a 1.5 mL conical bottom centrifuge tube, and suspend them in ddH 2 After washing three times with O, 3% glutaraldehyde fixative was added to obtain the bacteria before adsorption of Cd. Zn6 bacterial powder was added at a concentration of 20 mg / L at a bacterial addition rate of 2.5 g / L. 2+ After 6 h of adsorption, the cells were collected and transferred to a 1.5 mL conical bottom centrifuge tube with ddHO. 2 After washing three times with 0, 3% glutaraldehyde fixative was added to obtain the bacteria after Cd adsorption. The morphological changes of bacteria before and after Cd adsorption were observed using a scanning electron microscope (model: JSM-IT700HR).

[0061] 5.2 Energy spectrum analysis

[0062] Add 2.5 g / L Zn6 powder to 20 mg / L Cd 2+ After 6 h of adsorption, ddH 2 O was rinsed three times, the bacteria were collected, freeze-dried and ground into powder, which was used as the bacteria after Cd adsorption. The element composition before and after Cd adsorption was analyzed using an energy spectrometer (model: Hitachi Regulus 8100, Japan).

[0063] 6. Results

[0064] 6.1 Identification of strain Zn6

[0065] Zn6 colonies are approximately round and light yellow ( Figure 1 ). Observed under an optical microscope, the bacteria are small rods, without flagella, and scattered; Gram staining of the bacteria is red, which is G - bacteria( Figure 2 ). Strain Zn6 was PCR amplified and sequenced, and the product size was 1432 bp. The species level identification standard was defined as 16SrDNA sequence similarity greater than 99%. The phylogenetic tree was constructed using the neighbor-joining method of MEGA5.0 version, and the evolutionary relationship between the strain and the existing strains in the database was analyzed. Figure 3 ,Combining the results of morphology, Gram staining, molecular identification and phylogenetic tree analysis, strain Zn6 was identified as belonging to the genus Cupriavidus.

[0066] The strain Zn6 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on October 21, 2024, with the deposit number CGMCC NO.32288, and the classification name: Cupriavidus sp., and the deposit address is: Building 1, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0067] 6.2 Tolerance of strain Zn6 to heavy metals

[0068] Strain Zn6 has a certain tolerance to Zn, Cu, Cd, and Pb. Its tolerance to Cu is the weakest, with the highest tolerance concentration being 300 mg / L. It has the strongest tolerance to Cd, with the highest tolerance concentration reaching 700 mg / L. The highest tolerance concentrations to Zn and Pb are 600 mg / L and 500 mg / L, respectively. The results are shown in Table 1.

[0069] Table 1 Tolerance of heavy metals

[0070]

[0071] 6.3 Adsorption characteristics

[0072] 6.3.1 Effect of bacterial dosage on Zn6 adsorption

[0073] Initial Cd 2+ The concentration was 100 mg / L, and the amount of bacteria added was 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, and 3.0 g / L, respectively. The culture was carried out at 25°C and 150 rpm for 24 h. When the amount of bacteria added was 1.0 g / L-2.5 g / L, the effect of Zn6 on Cd 2+ The removal rate gradually increased, reaching the highest at 2.5g / L, with an adsorption rate of 26.39%. When the added bacteria amount was 3.0g / L, the adsorption rate decreased slightly to 26.01%.

[0074] 6.3.2 Initial Cd 2+ Effect of concentration on the adsorption of Zn6 by strains

[0075] Add different concentrations of Cd into a 50 mL conical flask. 2+ Solution 20mL, initial Cd 2+ The concentrations were 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L, respectively. The amount of bacteria added was 2.5 g / L. The culture was carried out at 25°C and 150 rpm for 24 hours. The removal rate reached the highest at a concentration of 20 mg / L, which was 58.83%. When the concentration was 20 mg / L-50 mg / L, the removal rate gradually decreased with the increase of concentration.

[0076] 6.3.3 Effect of adsorption time on the adsorption effect of strain Zn6

[0077] The amount of bacteria added was 2.5 g / L, and the initial Cd 2+ The concentration was 20 mg / L, 25°C, and 150 rpm for 3 h, 6 h, 9 h, 12 h, and 15 h. The removal rate gradually increased with time, reaching 70.3% at 6 h, reaching the highest at 70.5% at 9 h, and then decreased with time. Since the removal rates at 6 h and 9 h were close, 6 h was selected as the subsequent adsorption time to shorten the adsorption time.

[0078] 6.4 Adsorption mechanism

[0079] 6.4.1 Scanning electron microscopy (SEM) analysis

[0080] Effect of Zn6 strain on Cd 2+ The changes in bacterial morphology before and after adsorption are as follows Figure 4 , Figure 5 As shown in the figure, before adsorption, the cells were intact in morphology, with clear outlines, full surfaces, and sparse distribution. After adsorption, the cells deformed, shrank, and the cell walls broke, and the distribution was dense, forming a complex. This proves that strain Zn6 reacted with Cd 2+ Combined to achieve a good adsorption effect.

[0081] 6.4.2 Energy spectrum analysis

[0082] Effect of Zn6 strain on Cd 2+ Comparison of element contents before and after adsorption. The presence of Cd element after strain adsorption indicates that strain Zn6 may react with Cd during the adsorption process. 2+ Combine to form a metal complex, thus achieving a good adsorption effect.

[0083] In summary, the copper-greedy bacteria Zn6 of the present invention has a good adsorption effect on heavy metal cadmium, and can tolerate the toxicity of various other heavy metals, such as Zn, Cu, Pb, etc. Therefore, it can adapt to the complex natural environment and can be actually applied to the control of heavy metal pollution in the environment.

Claims

1. A strain for the prevention and treatment of heavy metal pollution, characterized in that: It is Cupriavidus sp. Zn6 which is deposited in China General Microbiological Culture Collection Center (CGMCC) with the deposit number of CGMCC NO.32288.

2. Use of the strain according to claim 1 in preparing a detergent for preventing and treating heavy metal pollution.

3. The use according to claim 2, characterized in that: The detergent comprises a biosorbent.

4. The use according to claim 3, characterized in that: The biosorbent has the function of adsorbing heavy metals, including Zn, Cu, Cd and / or Pb.

5. The use according to claim 4, characterized in that: The heavy metal is Cd.

6. The use according to any one of claims 2 to 5, characterized in that: The detergent is a preparation made of Cupriavidus sp. Zn6 as an active ingredient and acceptable auxiliary materials.

7. The use according to claim 6, characterized in that: The preparations include solutions, powders, granules and sprays.

8. The use according to claim 6, characterized in that: The bacterial amount of Cupriavidus sp. Zn6 in the preparation is 1.0-3.0 g / L.

9. A detergent for preventing and treating heavy metal pollution, characterized in that: It comprises the strain described in claim 1.

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