CN113122463B - Bacillus megaterium and method for restoring chromium-contaminated soil by using same - Google Patents
Bacillus megaterium and method for restoring chromium-contaminated soil by using same Download PDFInfo
- Publication number
- CN113122463B CN113122463B CN201911407441.2A CN201911407441A CN113122463B CN 113122463 B CN113122463 B CN 113122463B CN 201911407441 A CN201911407441 A CN 201911407441A CN 113122463 B CN113122463 B CN 113122463B
- Authority
- CN
- China
- Prior art keywords
- bacillus megaterium
- chromium
- contaminated soil
- group
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Environmental & Geological Engineering (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Soil Sciences (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Processing Of Solid Wastes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 an experimental technology and a theoretical basis are provided for applying a microbial curing technology to chromium-contaminated soil remediation.
Description
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) with the collection unit of China center for type culture collection, address: wuhan university in Wuhan, China, the preservation date is: 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 deionized water and 15-20 g/L agar powder.
The invention also provides a culture medium formula (abbreviated as separation culture medium) for separating and culturing the bacillus megaterium, wherein the formula comprises the following components: 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 repairing chromium-contaminated soil by microorganism in-situ solidification,
1) weighing 150g of chromium-contaminated soil with the pH value of 8-10 in 300mL triangular flasks respectively; respectively setting a contrast 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 percent.
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-contaminated soil under a slightly alkaline condition (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 contamination range is effectively prevented, and the purpose of effectively detoxifying the chromium-contaminated soil is realized.
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 to the invention is preserved, the name of the strain is Bacillus megaterium (Bacillus megaterium) GRINML5, the preservation unit is China center for type culture Collection, the address is as follows: wuhan university in Wuhan, China, the preservation date is: 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 deionized water and 20g/L 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 prokaryote universal 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 Blast comparison of the obtained sequence shows that the strain belongs to the genus Bacillus and is named as Bacillus megaterium GRINML 5.
Example 2 Bacillus megaterium for microbial curing in chromium-contaminated soil remediation
1) Respectively taking 150g of chromium-contaminated soil in a 300mL triangular flask from the chromium-contaminated soil taken back by the Qinghai Zhongxing chemical plant; respectively setting a control group and a repair group; wherein, 150mL of deionized water is added into the control group, and equal volume of culture solution is added into the restoration group.
The basic parameters of the waste residue are as follows: the pH value is 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, the bacterial liquid was inoculated into a 300mL triangular flask, with the inoculum size being 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 microorganisms for removing the soluble chromium ions 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-contaminated soil by microorganism in-situ solidification, hexavalent chromium ions in the solution are effectively limited and detoxified under the removing action of bacillus megaterium strains, a basis is provided for further selecting a green, environment-friendly, economic and efficient repairing technology without secondary pollution, and a positive and effective reference is provided for developing the treatment work of chromium contamination in soil in the future.
Claims (4)
1. A bacillus megaterium is characterized in that the strain is classified and named as: bacillus megaterium (B.) (B.megaterium)Bacillus megaterium) GRINML5, deposited as: china center for type culture Collection, Address: wuhan university in Wuhan, China, the preservation date is: 10/15/2018, deposited onMarking: CCTCC NO: m2018672.
2. A method for culturing Bacillus megaterium in an enriched state according to claim 1, wherein the Bacillus megaterium of claim 1 is inoculated into a culture medium and shake-cultured at 140rpm at a culture temperature of 30 to 33 ℃ for 7 to 10 days until the bacterial concentration in the culture medium reaches OD6000.4 to 0.6;
wherein, the formula of the culture medium is as follows: 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. The method for restoring chromium-contaminated soil is characterized by comprising the following steps:
1) weighing 150g of chromium-polluted soil with the pH of 8-10 in 300mL triangular flasks respectively; respectively setting a contrast group and a repair group; wherein 150mL of deionized water was added to the control group;
2) after the bacillus megaterium is subjected to enrichment culture by the method as claimed in claim 2, the bacillus megaterium is inoculated into a chromium-contaminated soil remediation group;
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.
4. The method for remediating chromium-contaminated soil as recited in claim 3, wherein the inoculation amount of Bacillus megaterium for the remediating group in step 2) is 10%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911407441.2A CN113122463B (en) | 2019-12-31 | 2019-12-31 | Bacillus megaterium and method for restoring chromium-contaminated soil by using same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911407441.2A CN113122463B (en) | 2019-12-31 | 2019-12-31 | Bacillus megaterium and method for restoring chromium-contaminated soil by using same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113122463A CN113122463A (en) | 2021-07-16 |
| CN113122463B true CN113122463B (en) | 2022-07-19 |
Family
ID=76769286
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911407441.2A Active CN113122463B (en) | 2019-12-31 | 2019-12-31 | Bacillus megaterium and method for restoring chromium-contaminated soil by using same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113122463B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113481128B (en) * | 2021-08-01 | 2023-06-13 | 重庆工商大学 | Bacillus tropicalis and application thereof in reduction of Cr (VI) |
| CN114289497B (en) * | 2021-12-29 | 2023-07-25 | 有研资源环境技术研究院(北京)有限公司 | Chemical-microorganism step-by-step repair process for treating Cr (VI) in chromium slag storage field |
| CN115094005B (en) * | 2022-07-06 | 2023-03-31 | 四川大学 | Bacillus subtilis, biological material and application in hexavalent chromium pollution treatment |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008146958A1 (en) * | 2007-05-04 | 2008-12-04 | Pyung Kang Special Vehicle Co., Ltd. | Bacillus megaterium bc1-1 kccm 10856p reducing nasty odor and heavy metal and clarification methods for resource recovery from food waste or livestock waste water by using it |
| CN102174543A (en) * | 2011-02-28 | 2011-09-07 | 中国科学院南京土壤研究所 | Dioxygenase gene, clone expression thereof and conversion of pyrene and benzo [a] pyrene |
| CN105274025A (en) * | 2015-10-29 | 2016-01-27 | 深圳市中南环保科技控股有限公司 | Compound bacterium and treatment process for heavy-metal sewage treatment |
| CN106734159A (en) * | 2016-12-02 | 2017-05-31 | 青岛蔚蓝生物集团有限公司 | A kind of restorative procedure of heavy-metal contaminated soil |
| CN110317764A (en) * | 2019-08-09 | 2019-10-11 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of complex micro organism fungicide and its application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017757B (en) * | 2014-06-03 | 2016-05-18 | 江南大学 | The one plant height effect end, changed bacillus and the composite base made changes microbial inoculum and application |
-
2019
- 2019-12-31 CN CN201911407441.2A patent/CN113122463B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008146958A1 (en) * | 2007-05-04 | 2008-12-04 | Pyung Kang Special Vehicle Co., Ltd. | Bacillus megaterium bc1-1 kccm 10856p reducing nasty odor and heavy metal and clarification methods for resource recovery from food waste or livestock waste water by using it |
| CN102174543A (en) * | 2011-02-28 | 2011-09-07 | 中国科学院南京土壤研究所 | Dioxygenase gene, clone expression thereof and conversion of pyrene and benzo [a] pyrene |
| CN105274025A (en) * | 2015-10-29 | 2016-01-27 | 深圳市中南环保科技控股有限公司 | Compound bacterium and treatment process for heavy-metal sewage treatment |
| CN106734159A (en) * | 2016-12-02 | 2017-05-31 | 青岛蔚蓝生物集团有限公司 | A kind of restorative procedure of heavy-metal contaminated soil |
| CN110317764A (en) * | 2019-08-09 | 2019-10-11 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of complex micro organism fungicide and its application |
Non-Patent Citations (2)
| Title |
|---|
| 一株抗铬芽孢杆菌的分离鉴定及16S rDNA序列分析;龚汉雨等;《湖北农业科学》;20090605(第06期);摘要 * |
| 耐Cr(Ⅵ)菌株的筛选及条件优化;杨文玲等;《河南科学》;20130912(第08期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113122463A (en) | 2021-07-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113122463B (en) | Bacillus megaterium and method for restoring chromium-contaminated soil by using same | |
| KR20210124232A (en) | Bacillus tianshenni strain, its application in the field of microbial preparation and heavy metal recovery | |
| CN106867921B (en) | Acidithiobacillus and method for treating acid mine wastewater and recycling iron resources by using same | |
| JP7062322B2 (en) | Use in the treatment of membrane concentrates of Pseudomonas balealica and its debris leachate | |
| CN112592855B (en) | Bacillus subtilis and method for treating uranium and cadmium polluted water body by using same | |
| CN112322550B (en) | Stenotrophomonas maltophilia and application thereof in heavy metal treatment | |
| CN110669706A (en) | Leersia hexandra endophytic bacterium capable of reducing hexavalent chromium as well as preparation method and application thereof | |
| CN112007947A (en) | Application of pseudomonas rhodesiae in repairing heavy metal polluted environment and heavy metal tolerance | |
| CN111375631B (en) | Pseudomonas and microorganism in-situ solidification method for restoring chromium-contaminated soil by using same | |
| CN113118206A (en) | Microbial remediation method for heavy metal contaminated soil of ex-service slag field of smelting plant | |
| CN108977370B (en) | Yeast for degrading phenol compounds and application thereof | |
| CN108866105B (en) | Method for producing nano cadmium sulfide by using enterobacter LY6 | |
| CN111378592B (en) | Bacillus licheniformis and method for treating malodorous organic wastewater by using same to purify water | |
| CN114657089B (en) | Chromium-reducing bacillus and method for repairing chromium-polluted soil by using same | |
| CN113249276B (en) | Bacillus cereus and application thereof | |
| Zhao et al. | Isolation of a sulfate reducing bacterium and its application in sulfate removal from tannery wastewater | |
| CN105779325A (en) | Fatty acid bacillus and method for treatment of heavy metal pollution of mine by in situ mineralization of the same | |
| CN114644992B (en) | Coxsackie strain and method for in-situ remediation of high-concentration heavy metal lead and uranium contaminated soil by same | |
| CN105670965B (en) | Strain with iron reduction capacity and application thereof | |
| CN109957523B (en) | Oligotrophic sulfate reducing bacteria and process for restoring heavy metal pollution of bottom mud of river channel by using oligotrophic sulfate reducing bacteria | |
| CN113583896A (en) | Enterobacter huoshanense and application thereof | |
| CN106967636A (en) | A kind of bacterial strain GX 3 and the method that nanogold is reclaimed from waste water containing gold ion | |
| CN103898001B (en) | A kind of heterotrophism acidophilic bacteria and the method utilizing the suppression acid wastewater in mine generation of this bacterium | |
| CN113122464B (en) | Halophytic nitrogen-fixing bacteria and method for treating chromium-polluted soil in high-saline-alkali environment by using same | |
| CN106085923B (en) | A kind of preparation method and application of bacillus amyloliquefaciens and its biological flocculant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |