CN117721041A - Bacterial strain with heavy metal resistance and capable of producing organic acid and application thereof - Google Patents
Bacterial strain with heavy metal resistance and capable of producing organic acid and application thereof Download PDFInfo
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- CN117721041A CN117721041A CN202311729187.4A CN202311729187A CN117721041A CN 117721041 A CN117721041 A CN 117721041A CN 202311729187 A CN202311729187 A CN 202311729187A CN 117721041 A CN117721041 A CN 117721041A
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- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 77
- 150000007524 organic acids Chemical class 0.000 title claims abstract description 33
- 230000001580 bacterial effect Effects 0.000 title claims description 16
- 239000002689 soil Substances 0.000 claims abstract description 62
- 241000168225 Pseudomonas alcaligenes Species 0.000 claims abstract description 41
- 238000005067 remediation Methods 0.000 claims abstract description 4
- 238000000855 fermentation Methods 0.000 claims description 17
- 230000004151 fermentation Effects 0.000 claims description 17
- 235000005985 organic acids Nutrition 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 12
- 229910052793 cadmium Inorganic materials 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 6
- 235000015278 beef Nutrition 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
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- 239000002054 inoculum Substances 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
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- 238000000746 purification Methods 0.000 description 4
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- 229910052725 zinc Inorganic materials 0.000 description 4
- 239000011701 zinc Substances 0.000 description 4
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 2
- 235000013878 L-cysteine Nutrition 0.000 description 2
- 239000004201 L-cysteine Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 2
- 235000011054 acetic acid Nutrition 0.000 description 2
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- 239000011651 chromium Substances 0.000 description 2
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- 238000012258 culturing Methods 0.000 description 2
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- 230000012010 growth Effects 0.000 description 2
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- 238000003199 nucleic acid amplification method Methods 0.000 description 2
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- 239000001384 succinic acid Substances 0.000 description 2
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- 239000011975 tartaric acid Substances 0.000 description 2
- 235000002906 tartaric acid Nutrition 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- 231100000693 bioaccumulation Toxicity 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to the technical fields of environmental engineering and microbial engineering. It discloses a strain which has heavy metal tolerance and can generate organic acid and application thereof in repairing heavy metal contaminated soil. The strain is Pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: m20211665. The pseudomonas alcaligenes JM7 disclosed by the invention can resist heavy metals in the environment, can activate the heavy metals in an acid-producing mode, and can be combined with electric or plant restoration to efficiently remove the heavy metals in the soil. The strain obtained by separation has good application prospect in the remediation of heavy metal contaminated soil.
Description
Technical Field
The invention belongs to the technical fields of environmental engineering and microbial engineering. It discloses a strain which has heavy metal tolerance and can generate organic acid and application thereof in repairing heavy metal contaminated soil.
Background
In recent years, with the continuous development of urban and industrial, soil pollution is becoming more serious. Investigation has shown that many contaminated soils exhibit a tendency to become contaminated with heavy metals. Soil is a major reservoir of contaminants in the environment, and heavy metals released into the environment via various routes eventually enter the soil.
Heavy Metals (HMs) are typical inorganic contaminants, and are typically cadmium (Cd), copper (Cu), lead (Pb), zinc (Zn), chromium (Cr), arsenic (As), etc., and are widely used in industrial production and daily life, and are common in both point source and non-point source contamination. Heavy metal pollution has durability, is easy to adsorb and deposit, and heavy metal and derivatives thereof have the characteristics of residue, bioaccumulation and the like, can continuously migrate and enrich into animals and human bodies through food chains, cause larger damage, and draw great attention of people.
The biotoxicity of heavy metal ions to microbial flora is not ignored, when the content of heavy metal ions in soil is high, the physicochemical property of the soil is changed, the physiological structure of microorganisms is destroyed, and the growth and metabolism of the soil microorganisms are further influenced, so that the functions of decomposing organic matters and repairing the soil are thoroughly lost. It is therefore of great interest to obtain a strain resistant to heavy metals with the ability to produce organic acids.
Disclosure of Invention
Aiming at the defect that the heavy metal resistant strain does not produce organic acid in the prior art, the invention provides a strain which has heavy metal resistance and can produce organic acid and application thereof in repairing heavy metal contaminated soil.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a strain which has heavy metal tolerance and can generate organic acid is Pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7, and is preserved in China center for type culture Collection (CCTCC NO) at the year 2021, 12 and 23: m20211665.
The strain is obtained by separating and screening heavy metal polluted soil near a Shenyang coking plant, and can realize the action of producing organic acid. It was determined to be Pseudomonas alcaligenes by physiological biochemical identification and 16S rRNA sequencing analysis.
The Pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 has biological characteristics of gram negative bacteria; bacillus with diameter of 0.5-1.0X12-3 μm; polar monoflagellum; the bacterial colony is round, the edge is neat, the surface is convex, smooth and moist, and opaque; the optimal growth temperature is 35 ℃; and (5) specificity and aerobics.
Use of a strain having heavy metal tolerance and being capable of producing organic acids, said strain being used in the tolerance of heavy metals or in the production of organic acids.
The heavy metal is one or more than one of Cd, cu, pb, zn, as.
Strains which are resistant to heavy metals and can produce organic acids and their use in the remediation of heavy metal contaminated soils.
The use of said strain as a formulation for tolerating heavy metals and/or dissolving bound heavy metals in soil.
A preparation for repairing heavy metal contaminated soil contains Pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7.
The preparation contains a fermentation culture, a fermentation culture bacterial suspension or a fermentation liquid of the strain.
The method for repairing heavy metal contaminated soil comprises the steps of adding pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 or the preparation into the contaminated soil to be repaired, planting enriched plants in the soil until the mature period, and further removing heavy metals in the soil.
Inoculating Pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 into beef extract peptone liquid culture medium for propagation culture, stopping culture after logarithmic growth phase, centrifuging to collect thallus, and preparing OD with sterile distilled water 600 Bacterial suspension=1.0; then adding the mixture into soil to be repaired to ensure that the JM7 concentration in the soil is more than 1.0X10 8 CFU/g dry soil.
The invention has the advantages that:
the pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 with heavy metal resistance and organic acid production capability provided by the invention has good heavy metal tolerance, can activate the combined heavy metal in the soil, and can remarkably improve the removal efficiency of the heavy metal in the soil in combination with phytoremediation.
Drawings
FIG. 1 is a photograph showing the morphology of Pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 provided in the examples of the present invention.
FIG. 2 is a phylogenetic tree of the 16SrRNA gene sequence of Pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 provided by the embodiment of the invention.
FIG. 3 shows the effect of heavy metals of different concentrations on the acid producing ability of Pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 provided in the examples of the present invention.
Detailed Description
The technical scheme of the present invention will be further specifically described by the following specific examples, but the present invention is not limited to these examples.
The invention utilizes organic acid produced by the metabolism to influence heavy metal pollutants under the condition of not influencing the metabolism of microorganisms. First, a strain having heavy metal resistance and an ability to produce organic acids is isolated and purified from heavy metal contaminated soil. And then, carrying out functional analysis on the strain, further exploring the capability of the strain for generating organic acid to activate heavy metal, and providing a theoretical basis for the application of the strain in the remediation of heavy metal contaminated soil.
EXAMPLE 1 screening and cultivation of Strain
Preparation of a low-phosphorus sucrose liquid medium containing heavy metals: sucrose 10g/L, mgSO 4 ·7H 2 O 0.5g/L，K 2 HPO 4 0.2g/L，NaCl 0.1g/L，(NH 4 ) 2 SO 4 1.0g/L, yeast extract 0.5g/L, distilled water, pH adjustment to 7.2-7.4, sterilization at 121 ℃ for 30min, and preparing into low-phosphorus sucrose liquid culture medium, and adding heavy metals under aseptic operation, wherein the final concentration of each heavy metal Cd, cu, pb, zn, as is 50mg/L.
Preparing a separation and purification solid culture medium: adding 2% agar powder before wet heat sterilization of low-phosphorus sucrose liquid culture medium, adjusting pH to 7.2-7.4, sterilizing at 121deg.C for 30min, adding heavy metals under aseptic operation, and mixing, wherein the final concentration of each heavy metal Cd, cu, pb, zn, as is 50mg/L. Then pour the plate into sterilized petri dishes for later use.
Preparation of beef extract peptone solid medium: beef extract 5g/L, peptone 10g/L, naCl 10g/L, 2% agar powder, distilled water, pH 7.0, sterilizing at 121deg.C for 30min, and pouring into a sterilized culture dish.
Isolation of strains: weighing 10g of heavy metal contaminated soil (contaminated soil around a certain smeltery in Jilin), wherein the total content of Cd, cu and Zn in the soil is 23.24mg/kg, 134.13mg/kg and 164.57mg/kg, and the effective concentration of Cd, cu and Zn is 5.38mg/kg, 21.75mg/kg and 25.15mg/kg. 100mL of sterile water and 0.5g of sterilized glass beads are added, the mixture is shaken for 5 hours in a 180r shaking table at 35 ℃, and after standing for 40 minutes, 5mL of supernatant is transferred into 50mL of sterilized low-phosphorus sucrose liquid medium containing heavy metals, and the culture is continued for 3 days at 35 ℃ and 180 rpm. After continuous transfer enrichment culture for 3 times, a certain amount of culture solution is diluted and coated on the surface of the separation and purification solid culture medium, single colonies with different forms are selected after culture for 5d at 35 ℃ and are subjected to plate streak purification on the beef extract peptone solid plate culture medium.
Example 2 identification of acid producing Capacity of Strain
Preparation of an acidogenic bacteria identification solid culture medium: glucose 8.0g/L, caCl 2 0.1g/L, peptone 1.5g/L, yeast extract 1.5g/L, K 2 HPO 4 0.4g/L，NaCl 3.0g/L，(NH 4 ) 2 SO 4 0.5g/L, L-cysteine 0.5g/L, mgSO 4 ·7H 2 O 0.05g/L，FeCl 3 0.01g/L，MgCl 2 0.1g/L, 0.04% bromocresol purple and 2% agar powder are added, the pH is adjusted to 6.8, sterilization is carried out for 30min at 121 ℃, and the plates are poured into sterilized culture dishes for standby.
Identification of acid production capacity of the strain: inoculating the single strain obtained after separation and purification on an acidogenic bacteria identification solid culture medium, culturing for 2d at 35 ℃, observing that the bacterial colony surrounding the acidogenic bacteria identification solid culture medium turns yellow from purple to indicate that the strain has acidogenic capacity, if no change occurs, indicating that the strain does not have acidogenic capacity, and selecting the single strain with acidogenic capacity for biological identification.
Example 3 identification of strains
The biological characteristics of the strain are gram-negative bacteria; the bacterial colony is round, the edge is neat, the surface is convex, smooth and moist, and opaque; and (5) specificity and aerobics. The 16S rRNA gene segment is obtained through PCR amplification, the obtained segment is sequenced by the division of biological engineering (Shanghai) and the sequencing result is compared on GeneBank, and the strain is identified as Pseudomonas alcaligenes (Pseudomonas alcaligenes). The 16SrRNA amplification sequencing result of the strain is drawn into a phylogenetic tree as shown in figure 2, and the amplification sequencing result is as follows:
GGTATGGGGCGGGCGGGCTAACACATGCAAGTCGAGCGGATGAGTGG
AGCTTGCTCCATGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAAT
CTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGC
ATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATC
AGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAAAGGCTCACC
AAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGG
AACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATA
TTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAG
AAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTA
AGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCT
AACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAAT
CGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGA
TGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTACTGA
GCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAA
TGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGA
CTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTA
GATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGA
TCCTTGAGATCTTAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGG
GGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCG
CACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACC
TTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCC
TTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGT
CGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTA
GTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACA
AACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGG
CCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAA
CCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCA
GTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATC
AGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC
ACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCGCAAG
GGGGACGGTACCACGGAGATTCCGG
further, the microbial strain is preserved in China general microbiological culture collection center (CCTCC) with the preservation number of CCTCC NO2 in the year 2021, 12 and 23: 0211665.
experimental example 4 evaluation of acid producing ability of Strain
(1) Evaluation of acid producing ability of Strain
To evaluate the acid-producing ability of JM7 strain, JM7 was inoculated into a fermentation medium, and the Optical Density (OD) of the fermentation broth at a wavelength of 600nm was measured 600 ) When OD 600 At 1.0, JM7 suspension was inoculated into 250mL of fermentation medium. The fermentation broth was further subjected to shaking culture at 30℃and 180rpm for 3d, the type and concentration of the organic acid in the fermentation broth were determined, and the experiment was repeated three times to ensure reliable results.
The fermentation medium comprises the following culture medium components: glucose8.0g/L，CaCl 2 0.1g/L, peptone 1.5g/L, yeast extract 1.5g/L, K 2 HPO 4 0.4g/L，NaCl 3.0g/L，(NH 4 ) 2 SO 4 0.5g/L, L-cysteine 0.5g/L, mgSO 4 ·7H 2 O 0.05g/L，FeCl 3 0.01g/L，MgCl 2 0.1g/L, constant volume to 1L with distilled water, pH 6.8, and sterilizing at 121deg.C for 30 min.
Analysis of the organic acids in the medium was performed as described in Chen et al (2014). The supernatant was centrifuged and then membrane filtered using a 0.45 μm filter and stored at 4 ℃ for subsequent analysis. To establish a standard mixture for organic acid analysis, four organic acids were selected: tartaric acid, formic acid, acetic acid and succinic acid. These organic acids were accurately weighed and dissolved in a 50mL volumetric flask. The original solution is gradually diluted to prepare five standard samples with different concentrations, and the standard samples are stored at 4 ℃. The organic acid analysis was performed by High Performance Liquid Chromatography (HPLC) and was modified according to the literature of Wangguo Pan et al (2022). The specific chromatographic conditions are as follows: c18 column, particle size 5 μm, size 150X 4.6mm, sample introduction amount 20. Mu.L, mobile phase acetonitrile and ultrapure water solution (pH 2.2). Phosphoric acid with a mass fraction of 8.5%) was used. Organic acid detection was performed using an isocratic elution procedure at a flow rate of 0.8mL/min (acetonitrile: water=5:95), a wavelength of 210nm, a column temperature of 26 ℃, and external standard quantification using four acid mixed standard solutions.
The determination result shows that the concentrations of tartaric acid, formic acid, acetic acid and succinic acid in the fermentation liquor of the JM7 pseudomonas alcaligenes are respectively 4.83mg/L, 14.18mg/L, 68.99mg/L and 5.18mg/L, so that the JM7 pseudomonas alcaligenes has good organic acid production capability.
(2) Influence of heavy metals with different concentrations on acid production capacity of strain
JM7 strain was tested for its effect on the acid production capacity of the strain for different concentrations of heavy metals. OD prepared as described above will be at 1% inoculum size 600 The pseudomonas alcaligenes JM7 bacterial suspension=1.0 was inoculated sequentially into 100mL of inorganic salt liquid medium with final concentration of 0.5, 5, 50, 500mg/L of heavy metal Cd. After shaking culture for 5d at 35℃in a 180rpm shaker, the organic substances were assayed at different heavy metal concentration gradientsThe amount of acid produced was 3 replicates per treatment, and 3 replicates per treatment were measured, with the non-sterile treatment serving as a blank. Strain in Cd 2+ The four organic acid concentrations at 0.5, 5, 50, 500mg/L are shown in FIG. 3, with total organic acid concentrations of 43.18mg/L,38.22mg/L,33.24mg/L and 13.51mg/kg, respectively. The strain has stronger Cd tolerance, and can still maintain certain acid production capacity under the stress of high-concentration Cd.
(3) Optimization of acid producing capacity conditions of bacterial strains
The screened pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 is subjected to propagation culture and then centrifuged (5000 r.min) -1 ) Collecting thallus, preparing OD with sterile distilled water 600 Bacterial suspension=1.0. The bacterial suspension is inoculated into a fermentation medium containing Cd with the concentration of 20mg/kg, and the influence of pH value, temperature and inoculum size on the acid production capacity of the strain JM7 is examined through a multi-factor experiment. The pH, temperature, inoculum size were set as follows: (1) The pH value of the initial culture medium is set to be 5.0, 6.0, 7.0, 8.0 and 9.0; (2) The fermentation culture temperature is set at 20 ℃, 25 ℃, 30 ℃,35 ℃ and 40 ℃; (2) The inoculum size was set to 0.5%, 1.0%, 1.2% and 1.5%. Each treatment was repeated three times to ensure statistical stability.
The experimental result shows that JM7 has the strongest acid production capacity when the pH is 6.0, the culture temperature is 35 ℃ and the inoculation amount is 1.2%, and the total amount of four organic acids reaches 58.75mg/L.
Experimental example 5: analysis of Strain activated heavy metal ability
20 grams of contaminated soil from example 1 were weighed and transferred to a 250mL Erlenmeyer flask. Subsequently, 50mL OD 600 JM7 bacterial suspension=1.0 was added to the flask. The flasks were then shake-cultured at 35℃on a 180rpm shaker for 7d. A blank sample without microbial fermentation broth was used as a control. Three times to ensure reliability and reproducibility. After the incubation period is completed, the contents of Cd, cu and Zn in the soil in effective state are determined by Inductively Coupled Plasma (ICP) analysis.
The results show that after the experiment is finished, the contents of the effective Cd, cu and Zn in the soil added with the microbial fermentation broth are respectively increased by 1.05mg/kg, 3.68mg/kg and 4.74mg/kg, and the blank sample has no obvious change.
Therefore, the addition of the pseudomonas alcaligenes JM7 can effectively activate the heavy metals in the soil and increase the content of the heavy metals in the soil in an effective state.
Example 6 promotion of Strain on phytoremediation of heavy Metal contaminated soil
The soil used in the experiment was the contaminated soil of example 1. Experiments total 2 treatments were designed, respectively a separate plant treatment and a combined microorganism-plant treatment, incorporating the OD described in example 4 into contaminated soil 600 =1 pseudomonas alcaligenes JM7 suspension, JM7 concentration in soil 7.5×10 8 CFU/g dry soil, JM1 bacterial suspension was not added to the plant treated soil alone. The enriched plant is ryegrass belonging to the genus ryegrass of the family Gramineae, and the seeds are purchased at seed companies. Loading the contaminated soil with or without the bacterial suspension into a plant repairing experimental device, then planting ryegrass, culturing for 60 days at the temperature of 25 ℃ under the condition of natural light, collecting plant and soil samples after the experiment is finished, and measuring the heavy metal content in ryegrass and soil.
The results showed that after the end of the experiment, the total removal rate of three heavy metals in the soil treated with the microorganism-plant was significantly higher than that of the soil treated with the plant alone, and the total removal rates of Cd, cu, zn in the soil treated with the microorganism-plant were 18.05%, 23.64% and 22.98%, respectively, higher than that of the soil treated with the plant alone. The content of Cd, cu, zn in the root portion of ryegrass in the microbial-plant treatment was 19.36%, 28.67% and 21.19% higher than that in ryegrass alone, while the aerial portion was 16.98%, 19.57% and 14.37% higher.
Therefore, the addition of the pseudomonas alcaligenes JM7 can effectively promote the restoration effect of the plants on the heavy metal contaminated soil.
To sum up, the Pseudomonas alcaligenes JM7 provided by the present patent provides a potential application prospect for engineering restoration of heavy metal contaminated soil. While the invention has been described in detail in this specification with reference to the general description and the specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (9)
1. A strain having heavy metal tolerance and capable of producing organic acid, characterized in that the strain is pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7, which has been preserved in the China center for type culture collection (CCTCC NO) at 12/23 of 2021: m20211665.
2. Use of a strain having heavy metal tolerance and capable of producing organic acids according to claim 1, wherein: the strain is applied to tolerance of heavy metals or production of organic acids.
3. Use of a strain having heavy metal tolerance and capable of producing organic acids according to claim 2, wherein: the heavy metal is one or more than one of Cd, cu, pb, zn, as.
4. The strain which has heavy metal tolerance and can produce organic acid and the application thereof as claimed in claim 1, wherein the strain is applied to the remediation of heavy metal contaminated soil.
5. Use of a strain having resistance to heavy metals and the ability to produce organic acids according to claim 1, characterized in that: the use of said strain as a formulation for tolerating heavy metals and/or dissolving bound heavy metals in soil.
6. A preparation for repairing heavy metal contaminated soil, which is characterized in that: comprising Pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 of claim 1.
7. The formulation for restoring heavy metal contaminated soil according to claim 5, wherein: the preparation contains a fermentation culture, a fermentation culture bacterial suspension or a fermentation liquid of the strain.
8. A method for repairing heavy metal contaminated soil is characterized by comprising the following steps: adding the formulation comprising pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 of claim 1 or claim 6 to the contaminated soil to be remediated, and planting the super-enriched plants in the soil until the maturity stage, thereby removing the heavy metals in the soil.
9. The repair method of claim 7, wherein: inoculating the pseudomonas alcaligenes (Pseudomonas alcaligenes) JM7 into a beef extract peptone liquid culture medium for propagation culture, stopping culture after the logarithmic growth phase is reached, centrifugally collecting thalli, washing, and preparing a bacterial suspension with OD600 = 1.0 by using sterile distilled water; then adding the mixture into soil to be repaired to ensure that the JM7 concentration in the soil is more than 1.0X10 8 CFU/g dry soil.
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