CN113976619B - Screening and application of lead-cadmium pollution resistant bacteria - Google Patents

Screening and application of lead-cadmium pollution resistant bacteria Download PDF

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CN113976619B
CN113976619B CN202111001835.5A CN202111001835A CN113976619B CN 113976619 B CN113976619 B CN 113976619B CN 202111001835 A CN202111001835 A CN 202111001835A CN 113976619 B CN113976619 B CN 113976619B
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lead
cadmium
soil
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CN113976619A (en
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陈阳
金一锋
谭策
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Qiqihar University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

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Abstract

The invention provides a radiation resistant methyl groupThe application of bacillus JB18 in repairing heavy metal contaminated soil relates to the field of strain functions and application technology. The strain JB18 in the invention is used for separating and screening lead-cadmium polluted soil from the environmental repair laboratory of the university of Qihar students, qihar, miq-haar, heilongjiang province, and JB18 can contain 200mg/L Pb 2+ 、400mg/L Cd 2+ When the culture medium is cultured on LB culture medium plates containing lead and cadmium, JB18 can still survive when the lead-cadmium compound concentration is 200/20mg/L, so that the culture medium has better lead-cadmium resistance, can grow by using starch and maltose as carbon sources, can not hydrolyze cellulose, is negative in methyl red and gelatin liquefaction experiments, and is identified as radiation-resistant methyl bacillus (Methylobacterium radiotolerans) by ITS sequences, thereby providing strain resources for repairing heavy metal polluted soil by microorganisms.

Description

Screening and application of lead-cadmium pollution resistant bacteria
Technical Field
The invention belongs to the technical field of functions and application of strains, and particularly relates to application of radiation-resistant methylobacterium JB18 in repairing heavy metal contaminated soil.
Background
The development of human activities and industrialization continuously releases toxic and harmful substances into soil, water and the atmosphere, which deteriorates ecological destruction and environmental quality. Heavy metals are one of the pollutants with great harm, and the influence on the whole human living environment is more and more prominent. Heavy metal contaminated plants enter the food chain through daily intake by humans and animals, resulting in accumulation of toxic metals in the animals and human bodies, to which many carcinogens and other deleterious effects are associated.
Disclosure of Invention
Therefore, the invention aims to provide the application of the radiation-resistant methylobacterium JB18 in the remediation of heavy metal contaminated soil, and the JB18 has better lead and cadmium resistance and can provide strain resources for the microorganism remediation of heavy metal contaminated soil.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of radiation-resistant methyl bacillus (Methylobacterium radiotolerans) JB18 in repairing heavy metal contaminated soil, wherein the preservation number of the radiation-resistant methyl bacillus (Methylobacterium radiotolerans) JB18 is CGMCC NO.22961.
Preferably, the ITS gene sequence accession number of JB18 is MZ723900.
Preferably, the heavy metal comprises lead or cadmium.
Preferably, when the JB18 is used for repairing lead-polluted soil, the concentration of lead in the solution of the soil is not higher than 200mg/L;
when the JB18 is used for repairing the cadmium-polluted soil, the concentration of cadmium in the solution of the soil is not higher than 400mg/L;
when the JB18 is used for repairing 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 is not higher than 20mg/L.
The invention also provides a microbial inoculum for repairing lead and/or cadmium polluted soil, wherein the active ingredients of the microbial inoculum comprise the radiation-resistant methylobacterium JB18.
The invention provides an application of radiation-resistant methyl bacillus JB18 in repairing heavy metal contaminated soil, wherein JB18 is separated and screened from lead-cadmium contaminated soil of a university of Qihar student life science and agriculture and forestry college environment repair laboratory of Qihar city of Qihar, heilongjiang province, and JB18 can contain 200mg/L Pb after stepwise screening 2+ 、400mg/L Cd 2+ The strain survives in the culture medium, has better lead and cadmium resistance, can grow by using starch and maltose as carbon sources, can not hydrolyze cellulose, is negative in methyl red and gelatin liquefaction experiments, and is identified as radiation-resistant methyl bacillus (Methylobacterium radiotolerans). In the embodiment of the invention, when the JB18 is cultured on a LB plate medium containing lead, the minimum inhibition concentration of lead ions is 200mg/L; when the culture is carried out on an LB plate culture medium containing cadmium, the minimum inhibition concentration of cadmium ions is 400mg/L; when the strain JB18 is cultured on LB medium plates containing lead and cadmium, the compound concentration of lead and cadmium is 200/20mg/L, and the strain JB18 can still survive. The JB18 has better lead and cadmium resistance, and can provide strain resources for repairing heavy metal polluted soil by microorganisms.
Biological preservation information
The radiation resistant methyl bacillus (Methylobacterium radiotolerans) JB18 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), the specific address is China academy of sciences of China including national academy of sciences of China No. 3, north Chen West Lu 1, korea, beijing, the city, the preservation number is CGMCC NO.22961, and the preservation date is 2021, 7 and 26.
Drawings
FIG. 1 is a tree constructed using ITS sequences.
Detailed Description
The invention provides application of radiation-resistant methyl bacillus (Methylobacterium radiotolerans) JB18 in repairing heavy metal contaminated soil, wherein the preservation number of the radiation-resistant methyl bacillus (Methylobacterium radiotolerans) JB18 is CGMCC NO.22961.
The accession number of the ITS gene sequence of JB18 is preferably MZ723900, and the ITS gene sequence is preferably shown in SEQ ID NO. 1:
GGCTCAGAGCGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGGCCCTTCGG GGTCAGCGGCGGACGGGTGAGTAACGCGTGGGAACGTGCCTTCTGGTTCGGAATAACC CTGGGAAACTAGGGCTAATACCGGATACGCCCTTTTGGGGAAAGGTTTACTGCCGGAAG ATCGGCCCGCGTCTGATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCAG TAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTA CGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCG CGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTTATCCGGGACGATAATGACGGTA CCGGAGGAATAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGC TAGCGTTGCTCGGAATCACTGGGCGTAAAGGGCGCGTAGGCGGCGTTTTAAGTCGGGGG TGAAAGCCTGTGGCTCAACCACAGAATGGCCTTCGATACTGGGACGCTTGAGTATGGTA GAGGTTGGTGGAACTGCGAGTGTAGAGGTGAAATTCGTAGATATTCGCAAGAACACCGG TGGCGAAGGCGGCCAACTGGACCATTACTGACGCTGAGGCGCGAAAGCGTGGGGAGCA AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCCAGCTGTTGGGGT GCTTGCACCGCAGTAGCGCAGCTAACGCTTTGAGCATTCCGCCTGGGGAGTACGGTCGC AAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT AATTCGAAGCAACGCGCAGAACCTTACCATCCTTTGACATGGCGTGTTACCCAGAGAGA TCTGGGGTCCCCTTCGGGGGCGCGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGT CGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCACGTCCTTAGTTGCCATCAT TCAGTTGGGCACTCTAGGGAGACTGCCGGTGATAAGCCGCGAGGAAGGTGTGGATGAC GTCAAGTCCTCATGGCCCTTACGGGATGGGCTACACACGTGCTACAATGGCGGTGACAG TGGGAGGCGAAGGAGCGATCTGGAGCAAATCCCCAAAAGCCGTCTCAGTTCGGATTGC ACTCTGCAACTCGAGTGCATGAAGGCGGAATCGCTAGTAATCGTGGATCAGCATGCCAC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTCTTA CCCGACGGCGCTGCGCCAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACT GGGGTGAAGTCGTAA. The invention uses the ITS sequence to carry out BLAST homology analysis with the existing ITS sequence in GenBank, and uses software MEGA X to construct a evolutionary tree, as shown in figure 1, the similarity between the strain and the radiation-resistant methyl bacillus (Methylobacterium radiotolerans) is higher, the isolated strain is determined to be the radiation-resistant methyl bacillus, the number is JB18, the sequence is submitted to the GENBANK database of NCBI, and the gene sequence accession number of the strain is MZ723900.
The JB18 is preferably used for separating and screening the lead-cadmium polluted soil from the environment repair laboratory of the university of Qihar students, qihar, heilongjiang province, and the method is not particularly limited and preferably comprises the following steps: will 10 -5 g/mL、10 -6 g/mL、10 -7 Inoculating 0.1mL of the g/mL soil suspension to an LB culture medium flat plate with the lead concentration of 200mg/L and the cadmium concentration of 20mg/L, culturing for 3-5 days in a constant temperature incubator at 30 ℃, observing whether bacterial colonies grow out, and if bacterial strains grow out, continuously increasing the lead and cadmium concentrations in the LB culture medium so as to determine the minimum inhibition concentration of lead and cadmium ions; because the stress of the compound lead-cadmium concentration is larger than that of single lead and cadmium, the compound lead-cadmium concentration is screened by an LB culture medium containing the compound lead-cadmium concentration. The heavy metals of the invention preferably include lead and/or cadmium. In the present invention, when the JB18 is used for repairing lead-polluted soil, the concentration of lead in the solution of the soil is preferably not higher than 200mg/L; when the JB18 is utilized to repair the cadmium-polluted soil, the concentration of cadmium in the solution of the soil is preferably not higher than 400mg/L; when the JB18 is used for repairing lead and cadmium polluted soil, the concentration of lead in the solution of the soil is preferably not higher than 200mg/L, and the concentration of cadmium is preferably not higher than 20mg/L.
The invention also provides a microbial inoculum for repairing lead and/or cadmium polluted soil, wherein the active ingredients of the microbial inoculum comprise JB18.
The application of the methylobacterium radiodurans JB18 provided by the invention in the remediation of heavy metal contaminated soil is described in detail below with reference to examples, but they should not be construed as limiting the scope of the invention.
Example 1
Isolation and characterization of Methylobacillus radiodurans (Methylobacterium radiotolerans)
1. JB18 is a resistant strain screened from lead-cadmium polluted soil in the university of Qihar student, science and agriculture and forestry college environmental remediation laboratory, and specifically comprises the following steps:
1. preparing culture medium
LB medium: beef extract 3g, peptone 10g, agar 20g, distilled water 1000mL, sodium chloride 5g and pH 7.4. The preparation process of the LB culture medium comprises the following steps: 3g of beef extract, 10g of peptone and 5g of sodium chloride are placed in a beaker, a small amount of distilled water is added, 20g of agar powder is added after stirring and dissolving by a glass rod, stirring is uniform, water is added to 1000mL, the mixture is divided into 250mL triangular flasks, and a sterilizing pot is set at 121 ℃ for 20min.
2. Screening culture
10g of soil sample is weighed and placed in an conical flask, 90mL of sterile water is added to prepare soil suspension, a proper amount of glass beads are placed in the conical flask, the mixture is sealed and transferred into a constant temperature shaking table, the mixture is treated for 2 hours at 180r/min and 30 ℃, and the mixture is kept stand for 30 minutes. 1mL of the soil suspension was taken and added to 9mL of sterile water to give 10 -1 Continuously diluting to obtain 10 in turn -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 And 10 -8 Is a suspension of soil bacteria at a concentration. Will 10 -5 、10 -6 、10 -7 Inoculating 0.1mL of the concentrated bacterial suspension to an LB culture medium flat plate with the lead concentration of 200mg/L and the cadmium concentration of 20mg/L, culturing for 3-5 days at the temperature of 30 ℃ in a constant temperature incubator, observing whether bacterial colonies grow out, and if bacterial strains grow out, continuously increasing the lead and cadmium concentrations in the LB culture medium, thereby determining the minimum inhibition concentration of lead and cadmium ions. Because the stress of the compound lead-cadmium concentration is larger than that of single lead and cadmium, the compound lead-cadmium concentration is screened by an LB culture medium containing the compound lead-cadmium concentration. Determining the minimum inhibition concentration of lead ions to be 200mg/L; the minimum inhibition concentration of cadmium ions is 400mg/L, and JB18 can still survive when the lead-cadmium composite concentration is 200/20 mg/L.
2. Classified identification of JB18 by ITS gene sequencing method
The 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 300s; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 90s, and repetition for 30 times; repair extension at 72℃for 600s.
After the gene amplification product is purified, sequencing is carried out by a manufacturer company, BLAST homology analysis is carried out on the obtained sequence and ITS sequences existing in GenBank, and a evolutionary tree is constructed by utilizing software MEGA X. As shown in FIG. 1, the strain has high similarity with the radiation-resistant methyl bacillus (Methylobacterium radiotolerans), the isolated strain is determined to be the radiation-resistant methyl bacillus, the number is JB18, the sequence is submitted to the GenBank database of NCBI, and the gene sequence of the strain is registered with the accession number of MZ723900.
3. Physiological biochemical test of JB18
1. Methyl Red test
(1) Glucose peptone medium: 7.5g peptone, 7.5g glucose, 7.5g dipotassium hydrogen phosphate, and pH 7 to 1500 mL.
(2) The steps are as follows: JB18 is inoculated into glucose peptone culture medium, a sterilized cotton plug is sealed, after the culture temperature is 30 ℃,24 and h, 1 to 2 drops of methyl red reagent are dripped into the glucose peptone culture medium, and the color change is observed. If the color of the culture medium is changed to red, the culture medium contains acidic substances, and the culture medium is positive and is indicated by "+"; a change to yellow indicates that the medium does not contain acidic substances, and is negative, indicated by "-".
2. Gelatin liquefaction test
(1) Gelatin liquefaction medium: peptone 5g, gelatin 200g, glucose 20g, distilled water 1000mL.
(2) The steps are as follows: JB18 was inoculated on the gelatin surface, and the culture was performed at 28℃in dark, and observed every 5 d. The tube was first cooled down and the record was observed after gelatin in the unvaccinated CK tube solidified. If the gelatin is not liquefied after cooling, no protease hydrolysis is performed, otherwise, the protease hydrolysis is indicated. Hydrolysis to positive is indicated by "+" and non-hydrolysis to negative is indicated by "-".
3. Starch hydrolysis test
(1) Starch hydrolysis medium: 10g of dipotassium hydrogen phosphate, 0.3g of potassium dihydrogen phosphate, 0.5g of sodium chloride, 1g of magnesium carbonate, 1g of potassium nitrate, 2.0g of soluble starch, 15g of agar, pH of 7.2 and 1000mL of distilled water.
(2) The steps are as follows: pouring the culture medium into a culture dish, inoculating by a spot grafting method after the culture medium is solidified, and pouring the prepared iodine solution into the surface of the culture medium after dark culture for 10-20 d at 28 ℃. If amylase is produced, the surrounding of the colony will not turn blue, whereas the surrounding will turn blue, indicating amylase production. The transparent ring formed was used to detect whether amylase was produced. The positive hydrolysis of amylase is indicated by "+" and the negative non-hydrolysis of amylase is indicated by "-".
4. Cellulose hydrolysis test
(1) Cellulose hydrolysis medium: magnesium sulfate 0.5g, dipotassium hydrogen phosphate 0.5g, sodium chloride 0.5g, potassium nitrate 1g, filter paper strips and distilled water 1000mL.
(2) The steps are as follows: the filter paper strips were placed in the medium of the test tube, half of the filter paper strips were immersed in the medium and half of the filter paper strips were inoculated on the test tube after sterilization, and after 30d incubation at 28 ℃, it was observed whether the test strains could grow on the filter paper strips. Hydrolysis of cellulose to positive is indicated by "+" and non-hydrolysis of cellulose to negative is indicated by "-".
5. Maltose utilization assay
(1) 200g of potato, 20g of maltose, 20g of agar and 1000mL of distilled water.
(2) The strain was observed for normal growth. Positive ones are indicated by "+" if normal growth is possible, and "-" if normal growth is impossible.
6. The test results are shown in Table 1
TABLE 1 physiological and biochemical Properties of JB18
Figure BDA0003235740750000061
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (5)

1. The application of the radiation-resistant methylobacterium (Methylobacterium radiotolerans) JB18 in the remediation of heavy metal contaminated soil is characterized in that the preservation number of the radiation-resistant methylobacterium (Methylobacterium radiotolerans) JB18 is CGMCC NO.22961.
2. The use according to claim 1, wherein the ITS gene sequence accession number of JB18 is MZ723900.
3. The use according to claim 1, wherein the heavy metals comprise lead and cadmium.
4. Use according to claim 3, wherein when repairing lead contaminated soil with the JB18, the concentration of lead in the solution of the soil is not higher than 200mg/L;
when the JB18 is used for repairing the cadmium-polluted soil, the concentration of cadmium in the solution of the soil is not higher than 400mg/L;
when the JB18 is used for repairing lead and cadmium polluted soil, the concentration of lead in the solution of the soil is not higher than 200mg/L, and the concentration of cadmium is not higher than 20mg/L.
5. A microbial inoculum for repairing lead and/or cadmium contaminated soil, characterized in that the active ingredient of the microbial inoculum comprises the radiation resistant methylobacterium JB18 according to any one of claims 1 to 4.
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JPH09271749A (en) * 1996-04-09 1997-10-21 Canon Inc Biological restoration method of polluted soil by microorganism using suitable solute
CN1216521A (en) * 1996-04-22 1999-05-12 盐野义制药株式会社 Novel terphenyl compounds and medicines containing the same
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