CN114042748A - Application of toxophila agglomerans JB19 in remediation of heavy metal contaminated soil - Google Patents
Application of toxophila agglomerans JB19 in remediation of heavy metal contaminated soil Download PDFInfo
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Abstract
The invention provides application of archea agglomerans JB19 in repairing heavy metal contaminated soil, and relates to the field of strain functions and the technical field of application. The strain JB19 is separated and screened from lead-cadmium contaminated soil in the life science and agriculture and forestry institute environmental remediation laboratory of Qizihaer university, Qizihaer, Heilongjiang province, and JB19 can contain 200mg/L Pb2+、200mg/L Cd2+Survival in the culture medium, when the culture is carried out on an LB culture medium plate containing lead and cadmium, and the lead-cadmium composite concentration is 200/20mg/L, JB19 can still survive, so the lead-cadmium resistance is better, the starch and maltose can be used as carbon sources for growth, cellulose can not be hydrolyzed, the starch and the maltose are shown as negative in methyl red and gelatin liquefaction experiments, and the ITS sequence is identified as Enterobacter agglomerans (Ensifer adhaerens), so the microorganism can provide bacteria for repairing heavy metal polluted soilAnd (4) planting the resources.
Description
Technical Field
The invention belongs to the technical field of strain functions and application, and particularly relates to application of archea clinopodioides JB19 in remediation of heavy metal contaminated soil.
Background
With the development of industrial technology, heavy metal pollution is one of the main environmental pollutions. Heavy metals are very difficult to degrade due to long residence time and high toxicity in the environment, and cause serious harm to human health through food chain enrichment. If a large amount of heavy metals exist in the soil, the activity of the soil is reduced, the normal growth of plants is influenced, and the yield of grains is reduced. Therefore, how to effectively treat the heavy metal pollution also becomes a research hotspot of scholars at home and abroad, and the microorganism has wide application prospect in repairing the heavy metal pollution.
Disclosure of Invention
In view of the above, the invention aims to provide an application of archea agglomerans JB19 in repairing heavy metal contaminated soil, and JB19 has good lead and cadmium resistance, and can provide strain resources for microorganisms to repair heavy metal contaminated soil.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of Enterobacter agglutinans JB19 in repairing heavy metal contaminated soil, wherein the preservation number of the Enterobacter agglutinans JB19 is CGMCC NO. 22962.
Preferably, the ITS gene sequence accession number of JB19 is MZ 157028.
Preferably, the heavy metal comprises lead or cadmium.
Preferably, when the JB19 is used for repairing the lead-polluted soil, the concentration of lead in a solution of the soil is not higher than 200 mg/L;
when the JB19 is used for repairing cadmium-polluted soil, the concentration of cadmium in the solution of the soil is not higher than 200 mg/L;
when the JB19 is used for repairing the lead and/or cadmium polluted soil, the concentration of lead in the solution of the soil is not higher than 200mg/L, and the concentration of cadmium in the solution of the soil is not higher than 20 mg/L.
The invention also provides a microbial inoculum for repairing the lead and/or cadmium polluted soil, and the active component of the microbial inoculum comprises the arrow-adhered bacterium JB 19.
The invention provides application of Clerodendrum Aoshimi JB19 in repairing heavy metal contaminated soil, wherein JB19 is separated and screened from lead-cadmium contaminated soil in the life science and agriculture and forestry institute environmental repair laboratory of Qiihale university in Qiihale, Black Dragon Jiang province, and JB19 can contain 200mg/L Pb through ladder screening2+、200mg/L Cd2+The bacillus subtilis survives in a culture medium, has better lead-cadmium resistance, can grow by using starch and maltose as carbon sources, cannot hydrolyze cellulose, shows negative in both methyl red and gelatin liquefaction experiments, and is identified as Enterobacter agglomerans (Ensifer adhaerens) by an ITS sequence. In the embodiment of the invention, when the JB19 is cultured on an LB plate culture medium containing lead, the minimum inhibitory concentration of lead ions is 200 mg/L; when the culture is carried out on LB plate culture medium containing cadmium, the minimum inhibitory concentration of cadmium ions is 200 mg/L; when the strain is cultured on LB medium plates containing lead and cadmium, the strain JB19 can still survive when the lead-cadmium complex concentration is 200/20 mg/L. The JB19 is proved to have better lead and cadmium resistance and can provide strain resources for the microorganisms to repair the heavy metal polluted soil.
Biological preservation information
The toxophilum adherens (Ensifer adhaerens) JB19 is deposited at China general microbiological culture Collection center (CGMCC), with the specific address of CGMCC No.22962, the collection date of 26 days at 2021 year 7 and 26 days at No. 3 Zhongxi Lu 1 institute of Ministry of China, North Kyoho, Beijing.
Drawings
FIG. 1 shows an evolution tree constructed using ITS sequences.
Detailed Description
The invention provides application of Enterobacter agglutinans JB19 in repairing heavy metal contaminated soil, wherein the preservation number of the Enterobacter agglutinans JB19 is CGMCC NO. 22962.
The ITS gene sequence accession number of JB19 is preferably MZ157028, and the ITS gene sequence is preferably shown in SEQ ID NO. 1:
GTTTGATCATGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGC CCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCTTTTCTACGG AATAACGCAGGGAAACTTGTGCTAATACCGTATACGCCCTTCGGGGGAAAGATTTATCGG GAAAGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGA CGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAA CTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCA TGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATG ACGGTAACCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAA GGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGACATTTAAGT CAGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTTGATACTGGGTGTCTAGAGT ATGGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAA CACCAGTGGCGAAGGCGGCTCACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGG GGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGT CGGGCAGTTTACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTAC GGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATG TGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATCCCGATCGCGGATT ACGGAGACGTTTTCCTTCAGTTCGGCTGGATCGGAGACAGGTGCTGCATGGCTGTCGTC AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGT TGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGT GGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGG TGGTGACAGTGGGCAGCGAGACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCTCAGT TCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCAGATCA GCATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAG TTGGTTCTACCCGAAGGTAGTGCGCTAACCGCAAGGAGGCAGCTAACCACGGTAGGGTC AGCGACTGGGGTGAAGTCGTAACAAAGG are provided. The ITS sequence and the existing ITS sequence in GenBank are utilized to carry out BLAST homology analysis, software MEGA X is utilized to construct an evolutionary tree, as shown in figure 1, the similarity of the strain and the Enterobacter agglomerans (Ensifer adhaerens) is higher, the separated strain is determined to be the Enterobacter agglomerans with the number of JB19, the sequence is submitted to the GENBANK database of NCBI, and the accession number of the gene sequence of the strain is MZ 157028.
The JB19 is preferably separated and screened from lead-cadmium contaminated soil in the life sciences and agriculture and forestry institute environmental remediation laboratory of the university of zizahal, Heilongjiang province, and the method for separating and screening is not particularly limited, and preferably comprises the following steps: will 10-5g/mL、10-6g/mL、10-70.1mL of soil suspension with the g/mL concentration is inoculated to an LB culture medium plate with the lead concentration of 200mg/L and the cadmium concentration of 20mg/L, the soil suspension is cultured for 3 to 5 days in a constant temperature incubator at 30 ℃, whether a bacterial colony grows out or not is observed, and if a bacterial strain grows out, the concentrations of lead and cadmium in the LB culture medium are continuously increased, so that the minimum inhibition concentrations of lead and cadmium ions are determined; because the stress of the concentration of the composite lead and cadmium is higher than that of single lead and cadmium, the screening is carried out by an LB culture medium containing the concentration of the composite lead and cadmium. The heavy metals of the present invention preferably include lead and/or cadmium. In the invention, when JB19 is used for repairing lead-polluted soil, the concentration of lead in a solution of the soil is preferably not higher than 200 mg/L; when the JB19 is used for repairing cadmium-polluted soil, the concentration of cadmium in a solution of the soil is preferably not higher than 200 mg/L; when JB19 is used for repairing lead and cadmium contaminated soil, the concentration of lead in the solution of the soil is preferably not higher than 200mg/L, and the concentration of cadmium in the solution of the soil is preferably not higher than 20 mg/L.
The invention also provides a microbial inoculum for repairing the lead and/or cadmium polluted soil, and the active component of the microbial inoculum comprises JB 19.
The application of the archea agglomerans JB19 in repairing heavy metal contaminated soil according to the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Isolation and characterization of Arthrobacter adhesina (Ensifer adhaerens)
The JB19 is a resistant strain screened from lead-cadmium contaminated soil in the life science and agriculture and forestry institute environmental remediation laboratory of the university of Qizahal, Heilongjiang, and specifically comprises the following steps:
1. preparation of culture Medium
LB culture medium: 3g of beef extract, 10g of peptone, 20g of agar, 1000mL of distilled water, 5g of sodium chloride and 7.4 of pH value. The specific preparation process of the LB culture medium comprises the following steps: putting 3g of beef extract, 10g of peptone and 5g of sodium chloride into a beaker, adding a small amount of distilled water, stirring and dissolving by using a glass rod, adding 20g of agar powder, stirring uniformly, replenishing water to 1000mL, subpackaging in 250mL triangular bottles, and setting a sterilization pot at 121 ℃ for 20 min.
2. Screening culture
Weighing 10g of soil sample, placing the soil sample in a conical flask, adding 90mL of sterile water to prepare a soil suspension, placing a proper amount of glass beads, sealing, transferring the soil suspension into a constant temperature shaking table, treating at 180r/min and 30 ℃ for 2h, and standing for 30 min. Adding 1mL of the soil suspension into 9mL of sterile water to obtain 10-1Continuously diluting the soil bacterium suspension to obtain 10-2、10-3、10-4、10-5、10-6、10-7And 10-8The concentration of (3) is soil bacterium suspension. Will 10-5、10-6、10-70.1mL of the bacterial suspension with the concentration is inoculated on an LB culture medium plate with the lead concentration of 200mg/L and the cadmium concentration of 20mg/L, the bacterial suspension is cultured in a constant temperature incubator for 3-5 days at the temperature of 30 ℃, whether bacterial colonies grow out or not is observed, if bacterial strains grow out, the concentration of lead and cadmium in the LB culture medium is continuously increased, and therefore the minimum inhibition concentration of lead and cadmium ions is determined. Because the stress of the concentration of the composite lead and cadmium is higher than that of single lead and cadmium, the screening is carried out by an LB culture medium containing the concentration of the composite lead and cadmium. Determining the minimum inhibitory concentration of lead ions to be 200 mg/L; when the minimum inhibitory concentration of cadmium ions is 200mg/L and the lead-cadmium complex concentration is 200/20mg/L, JB19 can still survive.
Secondly, classification and identification of JB19 by ITS gene sequencing method
ITS gene PCR amplification primers are as follows:
ITS1(7F)：5‘-CAGAGTTTGATCCTGGCT-3’；
ITS2(1540R)：5‘-AGGAGGTGATCCAGCCGCA-3’。
ITS3(27F)：5‘-AGTTTGATCMTGGCTCAG-3’；
ITS4(1492R)：5‘-GGTTACCTTGTTACGACTT-3’。
2. the amplification procedure was: pre-denaturation at 95 ℃ for 300 s; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 90s, and repeating for 30 times; the repair extension is 600s at 72 ℃.
After the gene amplification product is purified, sequencing is carried out by a company, BLAST homology analysis is carried out on the obtained sequence and the existing ITS sequence in GenBank, and the software MEGA X is utilized to construct an evolutionary tree. As shown in FIG. 1, the strain has high similarity to the Enterobacter agglomerans (Ensifer adhaerens), and the isolated strain is determined to be the Enterobacter agglomerans, which is numbered JB19, the sequence of which is submitted to the GenBank database of NCBI, and the sequence of the strain is registered as MZ 157028.
Physiological and biochemical test of JB19
1. Methyl Red test
(1) Glucose peptone medium: 7.5g of peptone, 7.5g of glucose, 7.5g of dipotassium hydrogen phosphate, to a volume of 1500 mL, and pH 7.
(2) The method comprises the following steps: inoculating JB19 into a glucose peptone culture medium, sealing with a sterilized cotton plug, culturing at 30 ℃, dropwise adding 1-2 drops of methyl red reagent into the glucose peptone culture medium after 24 hours, and observing color change. When the color changed to red, it was positive if the medium contained acidic substances, and it is indicated by "+"; if the color changed to yellow, it was negative if the medium contained no acidic substance, and it is represented by "-".
2. Liquefaction test of gelatin
(1) Gelatin liquefaction culture medium: 5g of peptone, 200g of gelatin, 20g of glucose and 1000mL of distilled water.
(2) The method comprises the following steps: JB19 was inoculated onto gelatin surface, cultured at 28 deg.C in dark, and observed every 5 days. The tube is first cooled at low temperature, and the gelatin in the non-inoculated CK tube is observed and recorded after being solidified. If the gelatin does not liquefy after cooling, no proteolytic action is obtained, otherwise, proteolytic action is obtained. Hydrolysis positive is indicated by "+" and non-hydrolysis negative is indicated by "-".
3. Starch hydrolysis test
(1) Starch hydrolysis culture medium: 10g of dipotassium hydrogen phosphate, 0.3g of monopotassium phosphate, 0.5g of sodium chloride, 1g of magnesium carbonate, 1g of potassium nitrate, 2.0g of soluble starch, 15g of agar, pH 7.2 and 1000mL of distilled water.
(2) The method comprises the following steps: pouring the culture medium into a culture dish, inoculating by adopting a point grafting method after the culture medium is solidified, performing dark culture at 28 ℃ for 10-20 days, and pouring the prepared iodine solution onto the surface of the culture medium. If amylase is produced, the periphery of the colony does not turn blue, otherwise, the colony turns blue, indicating that amylase is produced. The formed transparent ring is used for detecting whether amylase is produced. Positive amylase hydrolysis is indicated by a "+" and negative amylase hydrolysis is indicated by a "-".
4. Hydrolysis test of cellulose
(1) Cellulose hydrolysis medium: 0.5g of magnesium sulfate, 0.5g of dipotassium hydrogen phosphate, 0.5g of sodium chloride, 1g of potassium nitrate, filter paper strips and 1000mL of distilled water.
(2) The method comprises the following steps: placing the filter paper strip in a culture medium of a test tube, immersing half of the filter paper strip in the culture medium, immersing half of the filter paper strip on the culture medium, inoculating the strain to be tested on the filter paper strip in the sterilized test tube, culturing at 28 ℃ for 30d, and observing whether the strain can grow on the filter paper strip. The sign of positive hydrolysis of cellulose is "+", and the sign of negative non-hydrolysis of cellulose is "-".
5. Maltose utilization assay
(1) 200g of potatoes, 20g of maltose, 20g of agar and 1000mL of distilled water.
(2) And observing whether the strain can grow normally. Positive growth was indicated by "+" and abnormal growth was indicated by "-".
6. The test results are shown in Table 1
Physiological and biochemical properties of JB19 in Table 1
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. The application of the Enterobacter adherens JB19 in repairing heavy metal contaminated soil is characterized in that the preservation number of the Enterobacter adherens JB19 is CGMCC NO. 22962.
2. The use of claim 1, wherein the ITS gene sequence accession number of JB19 is MZ 157028.
3. Use according to claim 1, wherein the heavy metals comprise lead and cadmium.
4. The use according to claim 3, wherein when JB19 is used for repairing lead-contaminated soil, the concentration of lead in the solution of the soil is not higher than 200 mg/L;
when the JB19 is used for repairing cadmium-polluted soil, the concentration of cadmium in the solution of the soil is not higher than 200 mg/L;
when the JB19 is used for repairing the soil polluted by lead and cadmium, the concentration of lead in the solution of the soil is not higher than 200mg/L, and the concentration of cadmium in the solution of the soil is not higher than 20 mg/L.
5. A microbial inoculum for remedying lead and/or cadmium contaminated soil, which is characterized in that the active component of the microbial inoculum comprises the arrow-adhered bacterium JB19 in any one of claims 1 to 4.
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| CN114292797A (en) * | 2022-03-08 | 2022-04-08 | 佛山市玉凰生态环境科技有限公司 | Physarum viscosum and application of microbial flocculant thereof in sewage treatment |
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