CN113122463A - Bacillus megaterium and method for restoring chromium-contaminated soil by using same - Google Patents

Abstract

The invention provides a bacillus megaterium strain, which is named as follows: bacillus megaterium (Bacillus megaterium) GRINML5, the collection unit is: china center for type culture Collection, Address: the preservation date of Wuhan university in Wuhan, China is as follows: year 2018, 10 month 15, deposit accession number: CCTCC NO: m2018672. The strain can be used for efficiently removing soluble hexavalent chromium ions under the alkaline condition, and provides experimental technology and theoretical basis for applying the microbial solidification technology to chromium-contaminated soil remediation.

Description

Bacillus megaterium and method for restoring chromium-contaminated soil by using same

Technical Field

The invention relates to the technical field of microorganisms. In particular to a Bacillus megaterium (Bacillus megaterium) bacterium, a culture method thereof and a method for restoring chromium-polluted soil by using the bacterium.

Background

The soil is not only a natural resource on which human beings rely for survival, but also a material basis for industrial and agricultural development. In recent years, with the rapid development of industrialization, soil heavy metal pollution is more and more serious, wherein the harm of chromium pollution in soil to natural environment and biological health is particularly prominent. According to the data statistics of the comprehensive treatment scheme for chromium slag pollution in China, about 670 million tons of chromium slag are generated in a national accumulation manner, the chromium content of large-scale soil exceeds the standard due to long-term disordered stacking of the chromium slag, the serious threat to the safety of surrounding residents and the ecological environment is formed, and the problem of treatment and repair of the chromium pollution in the soil is very urgent. The appearance of the microbial remediation technology provides a new scheme for selecting a chromium pollution treatment process which is green, environment-friendly, economical, efficient and free of secondary pollution.

Disclosure of Invention

The first purpose of the invention is to provide a high-efficiency bacillus megaterium strain which can effectively remove soluble hexavalent chromium ions under a slightly alkaline condition (pH is 8-10).

The second purpose of the invention is to provide a culture medium formula which can enrich, screen and separate and culture the bacteria.

The third purpose of the invention is to provide a method for restoring chromium-contaminated soil by using the bacterium.

The invention provides a bacillus megaterium, and the strain name is as follows: bacillus megaterium GRINML5 (short for: Bacillus megaterium GRINML5), the collection unit is China center for type culture Collection, address: the preservation date of Wuhan university in Wuhan, China is as follows: year 2018, 10 month 15, deposit accession number: CCTCC NO: m2018672.

The invention also provides a culture medium formula (abbreviated as enrichment culture medium) for enriching, screening and culturing the bacillus megaterium, wherein the formula comprises the following components: peptone 8.0g/L, glucose 3.0g/L, yeast extract 3.0g/L, NaCl 10g/L, K₂Cr₂O₇100mg/L, pH8, 1000mL of deionized water and 15-20 g/L of agar powder.

The invention also provides a culture medium formula (abbreviated as separation culture medium) for separating and culturing the bacillus megaterium, and the preparation method is used for preparing the culture medium formulaThe method comprises the following steps: 1.8g/L yeast extract powder, 1.2g/L glucose peptone 3.0g/L, K₂HPO₄ 0.5g/L，MgSO₄·7H₂O 0.5g/L,K₂Cr₂O₇1000mg/L, 0.1mL/L of microelement mother liquor, 1000mL of deionized water, pH8 and 15-20 g/L of agar powder.

The microelement mother liquor comprises the following components: h₃BO₃ 6g/L，CoCl₂·6H₂O 4g/L，ZnSO₄·7H₂O 2g/L，MnCl₂·4H₂O 0.6g/L，Na₂MoO₄·7H₂O 0.6g/L，NiCl₂·6H₂O 0.4g/L，CuCl₂·2H₂O 0.2g/L。

The preparation method comprises mixing the above materials, adjusting pH to 8, and sterilizing at 121 deg.C for 25 min.

In the enrichment culture method of the bacillus megaterium, the bacillus megaterium strain is inoculated into an enrichment culture medium and is subjected to shake cultivation for 7-10 days at 140rpm and at the culture temperature of 30 ℃ until the bacterium concentration in a culture solution reaches OD₆₀₀0.4 to 0.6.

The invention also provides a method for restoring chromium-contaminated soil by microorganism in-situ solidification,

1) weighing 150g of chromium-polluted soil with the pH of 8-10 in 300mL triangular flasks respectively; respectively setting a control group and a repair group; wherein, 150mL of deionized water is added into the control group, and the equal volume of culture solution is added into the restoration group.

2) Respectively inoculating the bacillus megaterium of claim 1 into a chromium-contaminated soil remediation group after the bacillus megaterium is subjected to enrichment culture by the method of claim 3;

3) and (3) sampling at regular time to detect the concentration of the residual hexavalent chromium in the solution, wherein the Eh in the solution changes until the reduction speed of the concentration of the hexavalent chromium is obviously slowed down, and meanwhile, the Eh is lower than 0, so that a stable reduction state is maintained.

Further, the inoculation amount of the bacillus megaterium in the step 2) is 10%.

The invention has the beneficial effects that:

the invention provides a bacillus megaterium strain GRINML5, which is used for removing soluble hexavalent chromium ions in chromium-polluted soil under a slightly alkaline condition (the pH is 8-10), so that the migration and conversion capacity of high-toxicity hexavalent chromium in the soil is limited, the expansion of a chromium pollution range is effectively prevented, and the aim of effectively detoxifying the chromium-polluted soil is fulfilled.

Drawings

FIG. 1 is a scanning electron micrograph of the strain Bacillus megaterium GRINML5 of the present invention.

FIG. 2 is a photograph showing the change in environmental potential of the strain Bacillus megaterium GRINML5 of the present invention.

FIG. 3 shows the removal rate of free chromium from Bacillus megaterium GRINML5 of the present invention.

Detailed Description

The present invention is further illustrated by the following examples.

The Bacillus megaterium related by the invention is preserved, the strain name is Bacillus megaterium (Bacillus megaterium) GRINML5, the preservation unit is China center for type culture Collection, address: the preservation date of Wuhan university in Wuhan, China is as follows: year 2018, 10 month 15, deposit accession number: CCTCC NO: m2018672.

The bacillus megaterium is separated from chromium slag yard soil of a Zhongxing chemical plant in Qinghai.

The culture medium for isolated culture of the bacillus megaterium (abbreviated as isolated culture medium) has the following formula:

1.8g/L yeast extract powder, 1.2g/L glucose, 3.0g/L peptone and K₂HPO₄ 0.5g/L，MgSO₄·7H₂O 0.5g/L，K₂Cr₂O₇1000mg/L, 0.1mL/L of microelement mother liquor, 1000mL of deionized water, pH8, 20g/L of agar powder, sterilizing at 121 ℃ for 25 minutes, and pouring into a flat plate.

The microelement mother liquor comprises the following components: h₃BO₃ 6g/L，CoCl₂·6H₂O 4g/L，ZnSO₄·7H₂O 2g/L，MnCl₂·4H₂O 0.6g/L，Na₂MoO₄·7H₂O 0.6g/L，NiCl₂·6H₂O 0.4g/L，CuCl₂·2H₂O 0.2g/L。

The preparation method comprises mixing the components except agar powder, adding agar powder, adjusting pH to 8, and sterilizing at 121 deg.C for 25 min. 100mL of the medium was prepared in the above ratio, and one plate was poured per 20 mL.

The culture medium for enrichment culture of the bacillus megaterium (abbreviated as enrichment culture medium) has the following formula: peptone 8.0g/L, glucose 3.0g/L, yeast extract 3.0g/L, NaCl 10g/L, K₂Cr₂O₇100mg/L, 1000mL of deionized water and 20g/L of agar powder.

The preparation method comprises mixing the above materials, adjusting pH to 8, and sterilizing at 121 deg.C for 25 min.

Example 1 Bacillus megaterium acquisition and identification

The method for obtaining the bacillus megaterium comprises the following steps:

1) 5g of the soil sample containing the bacillus megaterium is added into 95mL of deionized water, placed on a shaking table at 100rpm and gently shaken for 15 minutes, and then taken out and stood for 30 minutes.

2) Removing 1mL of the supernatant in a screening enrichment medium, shaking for 7 days at 30 ℃ and 140rpm of a shaking table, and detecting and judging the growth condition of bacteria by using a microscope; when the bacterial concentration reaches OD₆₀₀When the value is 0.4, the shaking is stopped and the sample is taken out for standby.

3) The enrichment medium liquid is diluted by 1, 2, 3, 4 and 5 (respectively corresponding to 10)^-1，10^-2，10^-3，10^-4，10^-5) Each 100. mu.L of the dilution was applied to a plate prepared in a separate medium and cultured at 30 ℃ for 7 days. And (4) selecting single colonies, further carrying out plate streaking, and separating to obtain single colonies. After the single colony is picked, the single colony is transferred to a new solid medium plate, and the separation culture is continued by using a plate streaking separation method until the single colony is obtained.

4) The culture medium used in the enrichment, screening and separation process is adjusted to pH8, sterilized at 121 ℃ for 25 minutes, and all operations in the screening and purification process are carried out under aseptic conditions.

The cell morphology was observed by scanning electron microscopy and is shown in FIG. 1.

The 16S rDNA clone library technology is used for analyzing and identifying the strains. Centrifuging 1mL of bacterial liquid to obtain bacterial sludge, extracting total DNA, and amplifying a 16S rDNA fragment by using a PCR technology and a prokaryotic general primer 27f and a 1492 r. The PCR product was purified and ligated with a T-easy vector of Promega to transform E.coli DH 5. alpha. The white colonies picked were confirmed to be negative by colony PCR, and 4 clones were sequenced by enzyme digestion and typing. The obtained sequence is shown by Blast comparison, the strain is a Bacillus bacterium and is named as Bacillus megaterium GRINML 5.

Example 2 Bacillus megaterium for microbial curing in remediation of chromium-contaminated soil

1) Respectively putting 150g of chromium-contaminated soil in 300mL triangular flasks from a Michelia micrantha chemical plant in Qinghai; respectively setting a control group and a repair group; wherein, 150mL of deionized water is added into the control group, and the equal volume of culture solution is added into the restoration group.

The basic parameters of the waste residue are as follows: pH 8.8; cr (chromium) component²⁺: 248mg/L, total iron ion concentration: 5370 mg/L.

2) Inoculating identified Bacillus megaterium strain in enrichment medium, and culturing at 30 deg.C with shaking table at 140rpm until the bacterial concentration reaches OD₆₀₀Is 0.4; then inoculating the bacterial liquid into a 300mL triangular flask, wherein the inoculation amount is 10%.

3) And in the repairing process, sampling and detecting the concentration of the residual hexavalent chromium in the solution at regular intervals every 6 days, wherein the Eh in the solution is changed, and the whole process is continuously operated for 60 days. As shown in fig. 2, Eh continues to be below 0, being in a stable reduced state.

And taking out the solution in the experimental system, and calculating the removal rate of the soluble chromium ions removed by the microorganisms in the process.

The results are shown in FIG. 3, K₂Cr₂O₇When the initial concentration of the hexavalent chromium is 1000mg/L, the removal rate is as high as 32 percent within 60 days.

According to the method for repairing chromium-polluted soil by microorganism in-situ solidification, hexavalent chromium ions in the solution are effectively limited and detoxified under the removing action of the bacillus megaterium strain, a basis is provided for further selecting a green, environment-friendly, economical and efficient repairing technology without secondary pollution, and active and effective reference is provided for developing the treatment work of chromium pollution in soil in the future.

Claims (7)

1. A bacillus megaterium is characterized in that the strain is classified and named as: bacillus megaterium (Bacillus megaterium) GRINML5, the collection unit is: china center for type culture Collection, Address: the preservation date of Wuhan university in Wuhan, China is as follows: year 2018, 10 month 15, deposit accession number: CCTCC NO: m2018672.

2. A medium for enriching and screening Bacillus megaterium as claimed in claim 1, wherein the formulation is: peptone 8.0g/L, glucose 3.0g/L, yeast extract 3.0g/L, NaCl 10g/L, K₂Cr₂O₇100mg/L, pH8, 1000mL of deionized water and 15-20 g/L of agar powder.

3. A medium for isolating bacillus megaterium according to claim 1, wherein the formulation is: 1.8g/L yeast extract powder, 1.2g/L glucose, 3.0g/L peptone and K₂HPO₄ 0.5g/L，MgSO₄·7H₂O 0.5g/L，K₂Cr₂O₇1000mg/L, 0.1mL/L of microelement mother liquor, 1000mL of deionized water, pH8 and 15-20 g/L of agar powder.

4. The culture medium of claim 3, wherein the microelement mother liquor comprises: h₃BO₃ 6g/L，CoCl₂·6H₂O 4g/L，ZnSO₄·7H₂O 2g/L，MnCl₂·4H₂O 0.6g/L，Na₂MoO₄·7H₂O 0.6g/L，NiCl₂·6H₂O 0.4g/L，CuCl₂·2H₂O 0.2g/L。

5. The method for culturing Bacillus megaterium in an enriched state according to claim 1, wherein the Bacillus megaterium of claim 1 is inoculated into the medium of claim 2, and the mixture is subjected to shake cultivation at 140rpm at a cultivation temperature of 30 to 33 ℃ for 7 to 10 days until the concentration of the bacteria in the culture solution reaches a value ofOD₆₀₀0.4 to 0.6.

6. The method for restoring the chromium-contaminated soil is characterized by comprising the following steps of:

1) weighing 150g of chromium-polluted soil with the pH of 8-10 in 300mL triangular flasks respectively; respectively setting a control group and a repair group; wherein, 150mL of deionized water is added into the control group, and the equal volume of culture solution is added into the restoration group.

2) Respectively inoculating the bacillus megaterium of claim 1 into a chromium-contaminated soil remediation group after the bacillus megaterium is subjected to enrichment culture by the method of claim 3;

3) and (3) sampling at regular time to detect the concentration of the residual hexavalent chromium in the solution, wherein the Eh in the solution changes until the reduction speed of the concentration of the hexavalent chromium is obviously slowed down, and meanwhile, the Eh is lower than 0, so that a stable reduction state is maintained.

7. The method for remediating chromium-contaminated soil as recited in claim 6, wherein the amount of Bacillus megaterium inoculated into the remediated group in step 2) is 10%.

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