CN114657089A - Chromium reducing bacillus and method for restoring chromium-polluted soil by using same - Google Patents
Chromium reducing bacillus and method for restoring chromium-polluted soil by using same Download PDFInfo
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- CN114657089A CN114657089A CN202210203831.3A CN202210203831A CN114657089A CN 114657089 A CN114657089 A CN 114657089A CN 202210203831 A CN202210203831 A CN 202210203831A CN 114657089 A CN114657089 A CN 114657089A
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Abstract
The invention provides a chromium reducing bacillus and a method for restoring chromium-polluted soil by using the same. The classification of the chromium reducing bacilli is named as: leucobacter chloririudescens GRINML SWG2, the preservation unit is: china center for type culture Collection, addresses: the preservation date of Wuhan university in Wuhan, China is as follows: 2021, 8/6, with a deposit number: CCTCC NO: m2021991. The removal rate of the contaminated soil with the repaired chromium redissolved by the chromium-reducing bacillus domesticated is as high as more than 70%.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to a microorganism and a polluted soil remediation technology using the microorganism, and particularly relates to chromium reducing bacillus and a method for remediating chromium-polluted soil using the chromium reducing bacillus.
Background
The chromium slag is mainly generated in the sodium dichromate production process of the chromium salt industry and a few chromium metal enterprises, particularly the chromium salt production enterprises adopting the calcium roasting production process generate the largest amount of chromium slag. The chromium compound is one of the main series products in inorganic chemical industry, and is widely applied to the industries of chemical industry, light industry, metallurgy, textile, machinery and the like.
Chromium pollution usually causes allergic dermatitis or eczema, inhalation, irritation and respiratory tract corrosion, pharyngitis, bronchitis and other diseases, and hexavalent chromium compounds cannot be naturally degraded, can accumulate and enrich in organisms and human bodies for a long time, and also belong to a heavy environmental pollution substance. The method for treating the chromium slag by the microbiological method has the advantages of thorough detoxification, no secondary pollution, economy, high efficiency and the like, but has the problems of unstable detoxification effect and the like caused by the externally introduced strains and the problem of low tolerance of the strains to chromium.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide a chromium-reducing bacterium which can be used for repairing soil-contaminated soil with chromium and stably solidifying hexavalent chromium for a long period of time.
The second object of the present invention is to provide a medium for scale-up culture of the above-mentioned chromium-reducing bacterium.
The third purpose of the invention is to provide a method for restoring chromium-contaminated soil by using chromium-reducing bacillus, which fixes and reduces hexavalent chromium by direct action and indirect action of the chromium-reducing bacillus, and finally realizes long-acting stable solidification of hexavalent chromium in the chromium slag-contaminated soil.
In order to achieve the above object, the present invention provides a chromium-reducing bacterium, which is classified and named as: leucobacter chloririudescens GRINML SWG2, the preservation unit is: china center for type culture Collection, addresses: the preservation date of Wuhan university in Wuhan, China is as follows: 2021, 8/6, with a deposit number: CCTCC NO: m2021991.
The invention also provides a culture medium for the amplified culture of the chromium-reduced bacillus, and the formula of the culture medium is as follows: 8-10g of tryptone, 3-5g of yeast extract, 10-13g of sodium chloride, 900mL of distilled water of 700-.
The invention also provides a method for restoring chromium-contaminated soil by using chromium-reducing bacillus, which comprises the following steps:
1) inoculating the chromium reducing bacillus in the culture medium, and culturing for 7-10 days in an air bath constant temperature oscillator at 20-30 ℃ to obtain a bacterial liquid of the chromium reducing bacillus; wherein 0.5mol/L HNO is adopted before the culture medium is inoculated3Or NaOH is used for adjusting the initial pH value to 7.0-8.0;
2) placing the polluted soil in a conical flask, inoculating the bacterial liquid of the chromium-reducing bacillus cultured in the step 1) into the conical flask, and culturing the conical flask in an air bath constant-temperature oscillator at the temperature of 20-35 ℃ for 10-15 days to restore the soil.
Preferably, the inoculation volume of the strain in the step 1) is 8-15% of the volume of the culture medium, and the bacterial concentration of the bacterial liquid initially used for inoculation is 8 multiplied by 107～9.1×107one/mL.
Preferably, the inoculation volume of the chromium-reducing bacillus in the step 2) is 10-12% of the volume of the culture medium, and the bacterial concentration of the bacterial liquid of the chromium-reducing bacillus initially supplied for inoculation is 9.0 x 107-9.7×107One per mL.
Preferably, the Cr (VI) concentration gradient in the step 2) is increased to 200mg/L of 100-.
Preferably, the adding amount of the bacterial liquid in the step 3) is that the mass-to-volume ratio (g: mL) of the soil to the bacterial liquid is 1:1-2: 1.
During the culture, the medium was added every 2 days to supplement the natural and microbial consumption of the medium during the reaction.
After inoculation, when the concentration of bacteria in the medium is 8X 107～9.1×107When the chromium content is 1600mL/L, the chromium removal rate can reach 70 percent.
Through analysis and detection, chromium is combined with microbial metabolites to generate a chromium chelate, and the chromium chelate can stably exist in soil and has weak mobility and bioavailability.
The chromium-reducing bacillus used in the invention is domesticated step by step from the initial concentration of Cr (VI) of 200mg/L to the initial concentration of Cr (VI) of 1600 mg/L; after 65 days of repair, the removal rate of 1600mg/L Cr (VI) is gradually increased and reaches more than 70 percent.
The invention has the beneficial effects that:
the invention provides a chromium reducing bacillus and a method for restoring chromium-polluted soil by utilizing the chromium reducing bacillus.
Drawings
FIG. 1 is a photograph showing colonies of the chromium-reducing bacterium provided by the present invention.
FIG. 2A is a graph showing the trend of pH and Eh changes in a remediation group for remediating chromium-contaminated soil by using the chromium-reducing bacillus provided by the present invention.
FIG. 2B is a graph showing the trend of pH and Eh changes in a control group for repairing chromium-contaminated soil by using the bacteria of the present invention.
FIG. 3 is a statistical chart of the reduction rate of Cr (VI) by Crassibacillus in the control group and the repair group provided by the present invention.
Detailed Description
The following detailed and complete description of the embodiments of the present invention is provided to enable those skilled in the art to more easily understand the advantages and features of the present invention, and to clearly and clearly define the scope of the present invention.
The invention relates to chromium reducing bacillus for restoring polluted soil, which is classified and named as follows: leucobacter chloririudescens GRINML SWG2, the preservation unit is: china center for type culture Collection, address: the preservation date of Wuhan university in Wuhan, China is as follows: 2021, 8/6, with a deposit number: CCTCC NO: m2021991.
Example 1 isolation, culture and acclimatization of chromium-reducing bacteria
1) Adding 10g of soil sample of a chromium-polluted site collected from a north chemical plant of the original sea of Qinghai province into 90mL of deionized water, placing the mixture in a shaker at 25 ℃ and 120rpm for shaking for 30 minutes, taking out the mixture, standing for 60 minutes, and collecting supernatant;
2) adding 1mL of the supernatant into 10mL of culture medium, and culturing for 10 days at 25 ℃ under the condition of 120rpm shaking table; the formula of the culture medium is as follows: 5.0g/L yeast extract, 10.0g/L peptone, 10.0g/L NaCl, K2Cr2O 7100 mg/L and pH 7;
3) adding 1mL of the above cultured bacterial solution into 10mL of sterile water, and shaking to obtain 10-1Diluting the gradient dilution solution according to gradient 10-2To 10-5Evenly coating on a separation culture medium flat plate, carrying out inverted culture at 25 ℃ for 10 days, and then picking up a single colony on the flat plate for four-line three-zone streaking culture; the formula of the separation culture medium is as follows: 5.0g/L yeast extract, 10.0g/L peptone, 10.0g/L NaCl, K2Cr2O7Sterilizing 100mg/L agar powder with pH of 7 at 121 deg.C for 15min, pouring out, and cooling to obtain separation culture medium plate;
4) multiple transfers and isolation and purification were performed using the isolation medium plates to obtain single colonies with consistent colony characteristics, as shown in FIG. 1. As can be seen from FIG. 1, the colony is yellow translucent, round, smooth in surface, regular in edges, and slightly convex in the center.
5) Extracting genome DNA from colonies, performing PCR amplification by using a primer 27F/1492R, then cutting a gel, purifying and recovering PCR products, sequencing by using the primer 27F/1492R, and performing Blast comparison homology on sequencing result sequences at NCBI. Through identification and analysis, the strain is determined to have the closest relationship with the Leucobacter chlorireiducens and is named as Leucobacter chlorireiducens GRINML SWG 2.
6) Inoculating identified Leucobacter chlorireiducuens GRINML SWG2 into the culture medium in step 2), and culturing the strain liquid in a shaking table at 25 ℃ and 120rpm until the bacterial concentration is 9.0 multiplied by 107-9.7×107Per mL; then adding the cultured bacteria liquid into a 250-300mL conical flask filled with 80-100mL of culture medium according to the volume ratio of 10-12%, then adjusting the initial Cr (VI) concentration in the culture medium to be 200-1600mg/L by using 0.2mol/L potassium dichromate, gradually increasing the Cr (VI) concentration in the conical flask by a gradient of 80-100mg/L, sequentially acclimating the chromium-reducing bacillus, and culturing the conical flask in an air bath constant-temperature oscillator at the temperature of 20-30 ℃ for 7-10 days to obtain the bacteria liquid of the high-tolerance and high-efficiency chromium-reducing bacillus.
And (4) performing biological preservation on the domesticated Leucobacter chlorireiducuens GRINML SWG 2.
Example 2 microbial remediation of chromium contaminated soil remediation
Detoxification treatment is carried out on chromium-polluted soil in the Qinghai region by a microbiological method, and three sampling points of the original Cr-polluted soil, a green planting region and a rock region are taken as research objects. The pH values of samples of Cr-contaminated soil as such, green planting areas and rock areas in a 1mol/L KCl solution (according to the soil pH determination method (NY-T1377-2007)) were 10.62, 8.67 and 7.48, respectively. Different geographical locations are affected by multiple factors such as groundwater and exhibit different pH. The chromium content of the three samples was measured by ICP and the results showed that the Cr content of the samples contaminated with Cr, in the green areas and in the rock areas was 2.67%, 0.19% and 0.56%, respectively, which far exceeded the soil environmental quality standards (the total chromium content of the class III standard was less than 300 mg/kg).
The components of the culture medium used for repairing are as follows: 9g of tryptone, 4g of yeast extract, 10g of sodium chloride and 700mL of distilled water, 0.2g of potassium dichromate is added, the pH value is adjusted to 7 by using 1% nitric acid, and finally the volume is adjusted to 1000mL by using a volumetric flask.
Samples of chromium-contaminated soil, green plant areas and rock areas are taken and set as a restoration group, a culture medium control group and 200g of samples of each group are respectively placed in 3000mL conical flasks. The repairing group is added with 100mL of culture medium, 10mL of bacterial liquid is inoculated, and the culture medium contrast group is only added with 100mL of culture medium. Then, all the test groups were placed in a constant temperature incubator and subjected to static culture at 25 ℃ for 10 days to determine the microbial remediation effect.
1) Comparing the Eh and the pH change before and after the repair, the Eh of the repair group is reduced from 310mV, which is 250 + initially, to-380 mV-250 mV, which obviously shows a reduction trend, and the Eh of the culture medium control group tends to be stable (320 mV, 260-); the pH of the test group repaired by the chromium-reducing bacillus is stabilized in a region of 7.3-8.7, and the stable form of chromium in the pH and Eh range is chromium chelate.
2) Repairing effect of microorganism
As shown in fig. 3, compared with the control group of culture medium, the test group of chromium-reducing bacteria has significant effect on repairing chromium-contaminated soil, and after 7-10 days of microbial remediation, when the original chromium concentration is 200mg/L, the Cr (VI) reduction fixation rate reaches over 99.2%; when the chromium concentration is 1600mg/L, the reduction removal rate of chromium reaches more than 70%, and the precipitated product is detected as chromium chelate.
It can be seen from the above embodiments that the chromium-reducing bacillus and the method for repairing chromium-contaminated soil by the chromium-reducing bacillus provided by the invention can fix and reduce hexavalent chromium in the chromium-contaminated soil, thereby preventing chromium from being redissolved and realizing long-acting stable solidification of hexavalent chromium in the chromium slag-contaminated soil.
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. A chromium-reducing bacterium, characterized in that the classification of said chromium-reducing bacterium is: leucobacter chloririudescens GRINML SWG2, the preservation unit is: china center for type culture Collection, addresses: wuhan university in Wuhan, China, the preservation date is: 2021, 8/6, with a deposit number: CCTCC NO: m2021991.
2. A medium for scale-up culture of the chromium-reducing bacterium of claim 1, wherein the medium is formulated as: 8-10g of tryptone, 3-5g of yeast extract, 10-13g of sodium chloride and 900mL of 700-mL distilled water, then adjusting the pH to 7 by using nitric acid with the mass percentage concentration of 1%, then adding 0.1-0.3 g of potassium dichromate, and fixing the volume to 1000mL by using the distilled water.
3. A method for restoring chromium-contaminated soil by using chromium-reducing bacillus is characterized by comprising the following steps:
1) inoculating the chromium-reducing bacillus of claim 1 in the culture medium of claim 2, and culturing for 7-10 days in an air bath constant temperature oscillator at 20-30 ℃ to obtain a bacterial liquid of the chromium-reducing bacillus; wherein 3-5% HNO is adopted before the culture medium is inoculated3Or NaOH is used for adjusting the initial pH value to 7.0-8.0;
2) placing the polluted soil in a conical flask, inoculating the bacterial liquid of the chromium reducing bacillus cultured in the step 1) in the conical flask, and placing the conical flask in an air bath constant-temperature oscillator at the temperature of 20-35 ℃ for culturing for 10-15 days, so that the soil is repaired.
4. The method according to claim 3, wherein the inoculation volume of the chromium-reducing bacteria in step 1) is 8-15% of the culture medium volume, and the bacteria concentration of the bacterial solution of the chromium-reducing bacteria initially inoculated is 8 x 107～9.1×107One per mL.
5. The method according to claim 3, wherein the amount of the bacterial liquid added in the step 3) is 1:1-2:1 in a mass-to-volume ratio g: mL of soil to bacterial liquid.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114606159A (en) * | 2022-03-02 | 2022-06-10 | 有研资源环境技术研究院(北京)有限公司 | Chromium reduction mixed bacteria and Fe synergy thereof3+Method for restoring chromium-polluted soil |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114606159A (en) * | 2022-03-02 | 2022-06-10 | 有研资源环境技术研究院(北京)有限公司 | Chromium reduction mixed bacteria and Fe synergy thereof3+Method for restoring chromium-polluted soil |
| CN114606159B (en) * | 2022-03-02 | 2023-06-20 | 有研资源环境技术研究院(北京)有限公司 | Chromium reduction mixed bacteria and synergistic Fe thereof 3+ Method for repairing chromium-polluted soil |
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