CN103898001A - Heterotrophic acidophile and method for inhibiting generation of acid mine wastewater by using same - Google Patents
Heterotrophic acidophile and method for inhibiting generation of acid mine wastewater by using same Download PDFInfo
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- CN103898001A CN103898001A CN201210584735.4A CN201210584735A CN103898001A CN 103898001 A CN103898001 A CN 103898001A CN 201210584735 A CN201210584735 A CN 201210584735A CN 103898001 A CN103898001 A CN 103898001A
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- heterotrophism
- bacterial classification
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Abstract
The invention provides a heterotrophic acidophile the name of which is Alicyclobacillus sp., which is collected in a China General Microbiological Culture Collection Center (CGMCC) which is located in institute of microbiology Chinese Academy of Sciences, NO.3 NO.1 Beichen West Road, Chaoyang District, Beijing, China, with a collection date of 26th, December, 2012, and a collection register number of CGMCC No.7038. The heterotrophic acidophile is capable of growing in an acidic environment, a ferric ion in an environment system can be reduced and an environment potential is lowered by using the heterotrophic acidophile and an external carbon source, the sulphide ore dissolution of a system is reduced, and thus acid mine wastewater is inhibited from generating.
Description
Technical field
The present invention relates to microbial technology field.Particularly relating to a kind of Alicyclobacillus belongs to bacterium, its cultural method and utilizes this bacterium to suppress the method that acid wastewater in mine produces.
Background technology
, contact with ore as long as pyrite is exposed in air or containing the underground water of dissolved oxygen even in alkaline environment in neutrality, ore will slow oxidation generation ferrous ion.Now, because pH is higher, ferrous ion can be oxidized into rapidly ferric ion, and the existence of ferric ion can be accelerated pyritous oxidising process, and pH is declined rapidly, when pH further drops to below 3.5, under the katalysis of microorganism, can further produce continuously ferric ion, and the rate of oxidation of the sulphide oress such as pyrite is further improved.Therefore, ferric ion is the key factor of the raw acid wastewater in mine of oxidation of sulfureted mineral products, suppresses the generation of ferric ion, can reduce environment current potential and reduce the generation of acid wastewater in mine.
Summary of the invention
First object of the present invention is to provide the efficient heterotrophism acidophilic bacteria of strain bacterial classification, and this bacterial classification can significantly reduce pyritous dissolution rate.
Second object of the present invention be to provide a kind of can enrichment, separation, cultivate the substratum of this bacterium.
The 3rd object of the present invention is to provide a kind of this bacterium and carbon source utilized and reduces the method that sulphide ores dissolves.
The object of the invention is to be achieved through the following technical solutions:
A kind of heterotrophism acidophilic bacteria, it is characterized in that, strain name is acidocaldarius Alicyclobacillus sp., depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation day is: on December 26th, 2012, preservation registration number is: CGMCC No.7038.
Heterotrophism acidophilic bacteria as above is pure bacterial strain, utilizes this bacterial strain of 16S rDNA clone library technical evaluation to show, this bacterial strain belongs to Alicyclobacillus and belongs to.
It is ferrous iron that heterotrophism acidophilic bacteria as above can utilize carbon source reduction ferric iron, reduces solution potential and reduce sulphide ores to dissolve.
For the substratum of enrichment, separation and Culture heterotrophism acidophilic bacteria described above, it is characterized in that, get 0.25g yeast powder, 0.5g peptone, 0.25g NaCl and 1000mL distilled water, mix rear interpolation gelling gum 30g/L, adjust pH to 3.0.
Enrichment, the isolation cultivation method of heterotrophism acidophilic bacteria, is characterized in that as mentioned above, and described heterotrophism acidophilic bacteria bacterial classification is inoculated in substratum as above, and under the culture temperature of 40-50 ℃, shaking table is cultivated.
Utilize heterotrophism acidophilic bacteria described above to suppress the method that acid wastewater in mine produces, it is characterized in that, by the bacterial classification of described heterotrophism acidophilic bacteria, adopt method as above to carry out, after enrichment culture, being seeded to discarded Sulphur mine.
Method as above, preferably, the inoculum size that described bacterial classification is seeded to discarded Sulphur mine is every m
3area 2 ~ 4L.
Method as above, more preferably, the inoculum size that described bacterial classification is seeded to discarded Sulphur mine is every m
3the about 3L of area.
Method as above, preferably, after the described bacterial classification of inoculation, if the environment pH of described discarded Sulphur mine reduces by 0.5, continues supplemented medium, and additional amount is every m
3area 2 ~ 4L.
Method as above, more preferably, the additional amount of supplemented medium is every m
3the about 3L of area.
The preparation method of heterotrophism acidophilic bacteria is as mentioned above:
(1) bacterial classification concentration and separation culture medium prescription preparation substratum as described above, adjusting pH is sterilizing 25 minutes at 3.0,121 ℃, every 100mL substratum falls one flat plate afterwards.
(2) 100mL is added to 0.3g yeast powder, light shaking enrichment culture 1 week at 45 ℃ containing the water sample of described heterotrophism acidophilic bacteria.Judge bacterial growth situation by measuring inoculum at the light absorption value at 600nm place, in the time that the numerical value of the OD600 recording is between 0.6-0.8, can carry out next step operation.
(3) the heterotrophism acidophilic bacteria of enrichment is coated to above-mentioned culture medium flat plate, and choose single bacterium colony and carry out further plate streaking.Below isolated single bacterium colony adopts, the described method in (4) (5) is further identified.
(4) bacterium after purifying utilizes scanning electron microscope to carry out bacterial classification morphologic observation.
(5) adopt 16S rDNA clone library technology to identify separating pure bacterial strain, qualification result shows that this bacterial strain belongs to Alicyclobacillus and belongs to.
Beneficial effect of the present invention is:
The invention provides a strain strains A licyclobacillus sp., this bacterium can grow under sour environment, utilizes this bacterium and additional carbon, and the ferric ion in reducible environmental system also reduces environment current potential, the stripping of minimizing system sulphide ores, is produced thereby suppress acid wastewater in mine.
Accompanying drawing explanation
Fig. 1 is the stereoscan photograph of bacterial classification of the present invention.
Embodiment
The invention will be further described by the following examples.
Heterotrophism acidophilic bacteria involved in the present invention has carried out preservation, strain name is acidocaldarius Alicyclobacillus sp., depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation day is: on December 26th, 2012, preservation registration number is: CGMCC No.7038.
The cultural method of heterotrophism acidophilic bacteria of the present invention is as follows:
First prepare the substratum of enrichment culture heterotrophism acidophilic bacteria bacterium: take 0.25g yeast powder, 0.5g peptone, 0.25g NaCl and 1000mL distilled water, mix rear interpolation gelling gum 30g/L.Adjusting pH is sterilizing 25 minutes at 3.0,121 ℃, and every 100mL substratum falls one flat plate afterwards.
The water sample containing peculiar acidophilic bacteria that the present invention obtains is to take from Zijinshan copper mine copper-containing acid waste water and Jiangxi Dexing Copper Mine copper-containing acid waste water compound sample.100mL water sample is added to 0.3g yeast powder, cultivate 1 week 45 ℃ of light shaking.Judge bacterial growth situation by measuring inoculum at the light absorption value at 600nm place, in the time that the numerical value of the OD600 recording is between 0.6-0.8, carry out next step operation.Adopt membrane filtration that the bacterium in above-mentioned nutrient solution is filtered, and bacterium is used under the aseptic washing of 20mL.Inoculum size with 5% is inoculated in respectively in multiple 100mL substratum, and 45 ℃ of degree are cultivated, and contrast (CK) is not inoculated in setting.100rpm cultivates observation bacterial growth situation after two weeks.Nutrient solution gradient dilution is become to substratum 1,2,3,4,5(10
-1, 10
-2, 10
-3, 10
-4, 10
-5), get respectively 100 μ L diluents and be coated with dull and stereotypedly, cultivate three days at 45 ℃.Obtain single bacterium colony.
Use scanning electron microscopic observation thalli morphology, thalli morphology as shown in Figure 1.
With 16S rDNA clone library technical Analysis evaluation bacterial classification.By the centrifugal 1mL bacterium liquid bacterium mud that obtains, extract total DNA, utilize round pcr with prokaryotic organism universal primer 530f and 1490r amplification 16S rDNA fragment.After PCR product purification, be connected with the T-easy carrier of Promega, transform bacillus coli DH 5 alpha.The white colony of picking is determined positive bacterium colony by bacterium colony PCR, through enzyme cutting type, to 4 cloning and sequencings.Institute's calling sequence relatively shows through Blast, and this bacterial strain is that Alicyclobacillus belongs to bacterium.
Be respectively charged into 150mL acid wastewater in mine and 15g pyrite with two 300mL flasks, access heterotrophism acidophilic bacteria Alicyclobacillus sp. in one of them flask, another is blank.Shaking table is cultivated 30 days test pyrite solubility rates, found that the flask of inoculation Alicyclobacillus sp.A bacterium is compared with blank flask, and pyrite solubility rate reduces by 20%, and current potential reduces by 10%, and ferric ion concentration is low by 14%.Prove that bacterial classification of the present invention really has ferric ion in reducing environment system, reduces environment current potential, reduces the function of system sulphide ores stripping.
Claims (8)
1. a heterotrophism acidophilic bacteria, it is characterized in that, strain name is acidocaldarius Alicyclobacillus sp., depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation day is: on December 26th, 2012, preservation registration number is: CGMCC No.7038.
2. for enrichment, the separation and Culture substratum of heterotrophism acidophilic bacteria as claimed in claim 1, it is characterized in that, get 0.25g yeast powder, 0.5g peptone, 0.25g NaCl and 1000mL distilled water, mix rear interpolation gelling gum 30g/L, adjust pH to 3.0.
3. the enrichment of heterotrophism acidophilic bacteria, isolation cultivation method described in claim 1, is characterized in that, described heterotrophism acidophilic bacteria bacterial classification is inoculated in substratum as claimed in claim 2, and under the culture temperature of 40-50 ℃, shaking table is cultivated.
4. one kind is utilized heterotrophism acidophilic bacteria described in claim 1 to suppress the method that acid wastewater in mine produces, it is characterized in that, by the bacterial classification of heterotrophism acidophilic bacteria described in claim 1, adopt method as claimed in claim 3 to carry out, after enrichment culture, being seeded to discarded Sulphur mine.
5. method as claimed in claim 4, is characterized in that, the inoculum size that described bacterial classification is seeded to discarded Sulphur mine is every m
3area 2 ~ 4L.
6. method as claimed in claim 4, is characterized in that, the inoculum size that described bacterial classification is seeded to discarded Sulphur mine is every m
3area 3L.
7. method as claimed in claim 4, is characterized in that, after the described bacterial classification of inoculation, if the environment pH of described discarded Sulphur mine reduces by 0.5, continues supplemented medium, and additional amount is every m
3area 2 ~ 4L.
8. method as claimed in claim 4, is characterized in that, after the described bacterial classification of inoculation, if the environment pH of described discarded Sulphur mine reduces by 0.5, continues supplemented medium, and additional amount is every m
3area 3L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779325A (en) * | 2014-12-25 | 2016-07-20 | 北京有色金属研究总院 | Fatty acid bacillus and method for treatment of heavy metal pollution of mine by in situ mineralization of the same |
| CN109967519A (en) * | 2017-12-28 | 2019-07-05 | 北京有色金属研究总院 | A kind of repairing method of microorganism of Tailings Dam heavy metal pollution |
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| CN1384890A (en) * | 1999-09-07 | 2002-12-11 | 比利顿知识产权有限公司 | Recovery of copper from copper bearing sulphide minerals by bioleaching with controlled oxygen feed |
| CN101173230A (en) * | 2007-10-10 | 2008-05-07 | 邢苗 | Basophilic bacillus cereus, produced interior contact dextranase and application of the same |
| JP2009011270A (en) * | 2007-07-06 | 2009-01-22 | Microbio Corp | Culture medium for microbiological examination and microbiological examination device |
| WO2010012030A1 (en) * | 2008-07-29 | 2010-02-04 | Bhp Billiton Ssm Development Pty Ltd | Process for controlled homogeneous acid leaching |
| CN102161977A (en) * | 2011-01-21 | 2011-08-24 | 天津工业生物技术研究所 | New alicyclobacillus strain and application thereof in bioleaching |
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2012
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Patent Citations (5)
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| CN1384890A (en) * | 1999-09-07 | 2002-12-11 | 比利顿知识产权有限公司 | Recovery of copper from copper bearing sulphide minerals by bioleaching with controlled oxygen feed |
| JP2009011270A (en) * | 2007-07-06 | 2009-01-22 | Microbio Corp | Culture medium for microbiological examination and microbiological examination device |
| CN101173230A (en) * | 2007-10-10 | 2008-05-07 | 邢苗 | Basophilic bacillus cereus, produced interior contact dextranase and application of the same |
| WO2010012030A1 (en) * | 2008-07-29 | 2010-02-04 | Bhp Billiton Ssm Development Pty Ltd | Process for controlled homogeneous acid leaching |
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Non-Patent Citations (2)
| Title |
|---|
| 金一荻 等: "基于结冷胶培养基的乌梁素海水体中可培养细菌的多样性研究", 《内蒙古农业大学学报》, vol. 32, no. 2, 30 April 2011 (2011-04-30), pages 160 - 165 * |
| 陈志伟 等: "云南和广东部分热泉Alicyclobacillus分布及系统发育", 《微生物学通报》, vol. 31, no. 3, 31 December 2004 (2004-12-31), pages 50 - 54 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779325A (en) * | 2014-12-25 | 2016-07-20 | 北京有色金属研究总院 | Fatty acid bacillus and method for treatment of heavy metal pollution of mine by in situ mineralization of the same |
| CN109967519A (en) * | 2017-12-28 | 2019-07-05 | 北京有色金属研究总院 | A kind of repairing method of microorganism of Tailings Dam heavy metal pollution |
| CN109967519B (en) * | 2017-12-28 | 2021-06-08 | 有研资源环境技术研究院(北京)有限公司 | Microbial remediation method for heavy metal pollution of tailing pond |
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Effective date of registration: 20190626 Address after: 101407 No. 11 Xingke East Street, Yanqi Economic Development Zone, Huairou District, Beijing Patentee after: Research Institute of engineering and Technology Co., Ltd. Address before: No. 2, Xinjie street, Xicheng District, Beijing, Beijing Patentee before: General Research Institute for Nonferrous Metals |
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Effective date of registration: 20210412 Address after: 101407 No. 11 Xingke East Street, Yanqi Economic Development Zone, Huairou District, Beijing Patentee after: Youyan resources and Environment Technology Research Institute (Beijing) Co.,Ltd. Address before: 101407 No. 11 Xingke East Street, Yanqi Economic Development Zone, Huairou District, Beijing Patentee before: YOUYAN ENGINEERING TECHNOLOGY RESEARCH INSTITUTE Co.,Ltd. |
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