CN100560713C - The Achromobacter xylosoxidans SY8 and the purposes of purifying arsenic contamination - Google Patents
The Achromobacter xylosoxidans SY8 and the purposes of purifying arsenic contamination Download PDFInfo
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- CN100560713C CN100560713C CNB2007100520646A CN200710052064A CN100560713C CN 100560713 C CN100560713 C CN 100560713C CN B2007100520646 A CNB2007100520646 A CN B2007100520646A CN 200710052064 A CN200710052064 A CN 200710052064A CN 100560713 C CN100560713 C CN 100560713C
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Abstract
The invention belongs to the environmental microorganism technical field, be specifically related to that a strain has the Achromobacter xylosoxidans SY8 of oxygenizement to inorganic trivalent arsenic and in the application aspect the purifying arsenic contamination.Feature of the present invention is to separate to obtain a strain has oxygenizement to inorganic trivalent arsenic Achromobacter xylosoxidans SY8 from the soil of arsenic contamination.This bacterial strain can be oxidized to inorganic pentavalent arsenic with the inorganic trivalent arsenic in the arsenic contamination environment, greatly reduced the toxicity of arsenic in the environment and strengthened the adsorbable removal of arsenic.Bacterial strain of the present invention is named as Achromobacter xylosoxidans SY8 (Achromobacter xylosoxidans), is a kind of newfound arsenic oxidizing bacteria, and its preserving number is CCTCC NO:M207048.Preliminary study shows that bacterial strain of the present invention has application promise in clinical practice aspect the improvement environment As pollution.
Description
Technical field
The invention belongs to the environmental microorganism technical field, be specifically related to a strain inorganic trivalent arsenic is had oxygenizement Achromobacter xylosoxidans SY8 screening and aspect purifying arsenic contamination in purposes.
Background technology
Heavy metal contamination has at present become global problem, especially an arsenic contamination.The official of the World Health Organization announces that the whole world has at least more than 5,000 ten thousand populations being faced with the threat of endemic arsenic poisoning, and wherein, great majority are Asian countries, and China is subjected to one of the most serious country of arseniasis harm.Though at Chinese arsenic contamination geologic agent is arranged, human factor is more obvious, severe contaminations such as the processing of for example exploitation in ore deposit and smelting, arsenic product, burning of coal soil, underground water and river.
At arsenic contamination, present international and domestic occurred some improvement methods (pay and to pass the civil service examinations. brief talk the improvement technology of arsenic-containing waste water. Jiangxi energy .2006,4:75-76), mainly contain following several: the one, chemical process, such as chemical precipitation, waste residue is difficult to handle though this method effect is obvious, has been subjected to certain restriction in developed country at present; The 2nd, physical method, such as physical adsorption, this method expense height is unfavorable for large-scale purification; The 3rd, plant restoration method, certain plants (as Herba pteridis vittatae) can absorb the arsenic of high density and enrichment in vivo, and a large amount of plantations collect that the back is appropriate handles, and this method is not suitable for containing in a large number the improvement of arsenic sewage; The 4th, emerging microorganism active mud method, it mainly is the culturing micro-organisms flora, add the aeration tank mixed solution to reach metabolism and adsorption to heavy metal ion, this method is as the most promising a kind of arsenic contamination improvement method, has economy, advantage such as efficient and harmless, all very in vogue at home and abroad, but this method maximum limitation on technology is the clearance height of active sludge to pentavalent arsenic, and it is low to the stronger arsenious clearance of toxicity, mainly be because pentavalent arsenic is electronegative, easily by sludge absorption and trivalent arsenic is not charged, so be necessary trivalent arsenic is oxidized to pentavalent arsenic when handling arsenic-containing waste water, carry out oxidation if in active sludge, add chemical substance, will influence the activity of mud, press in the practice and solve this technical bottleneck, and arsenic oxidation bacterium be exactly a well selection.
Arsenic in the environment mainly exists with the inorganic form of trivalent and pentavalent, toxicity is also embodied on these two kinds of valence states, wherein arsenious toxicity exceeds about 100 times than pentavalent arsenic, and trivalent arsenic does not have electric charge, movability is strong, easier harm environment, Achromobacter xylosoxidans SY8 of the present invention can be oxidized to the trivalent arsenic of strong toxicity the pentavalent arsenic a little less than the toxicity, not only greatly reduce the toxicity of arsenic in the environment, but also can cooperate emerging microorganism active mud method, reach effective purification to the arsenic contamination environment.
Summary of the invention
The objective of the invention is to overcome the defective of existing environment As pollution purification techniques, separate obtaining the novel arsenic oxidation of strain bacterium, this bacterial strain can be oxidized to the trivalent arsenic of high poison in the environment pentavalent arsenic of low toxicity, by using the arsenic contamination in the environment purification.The invention still further relates to its purposes.
The present invention is achieved through the following technical solutions:
The inventor separates, screens the novel arsenic oxidation of strain bacterium, this bacterial strain is named as SY8, belong to a kind of Achromobacter xylosoxidans (Achromobacter xylosoxidans), this bacterial strain is deposited in Chinese typical culture collection center (CCTCC) on April 16th, 2007, and its preserving number is CCTCC NO:M207048.
The screening scheme of Achromobacter xylosoxidans SY8 is referring to accompanying drawing 1.By accompanying drawing 1, take the topsoil sample of somewhere, Shayang County, Chinese Hubei Province arsenic contamination earlier, add finite concentration (detailed description of seeing below, down together) NaAsO
2Carry out enrichment culture, again the soil sample of enrichment culture is diluted and be coated with and contain finite concentration NaAsO
2CDM solid medium flat board, cultivate and to grow arsenic resistance bacterium, the bacterium colony of picking different shape rule mono-clonal, use KMnO again
4Method (T.M.Salmassi et al.Oxidation of Arsenite by Agrobacterium albertimagni, AOLl5, sp.Nov., Isolated from Hot Creek, California.Geomicrobiology Journal.2002,19:53-66.) detect arsenic oxidation bacterium, more detected arsenic oxidation bacterium is cooked relevant evaluation work such as 16S ribosomal RNA gene (16S rDNA), morphology and functional gene, finally obtain Achromobacter xylosoxidans SY8.
More detailed technological step is as follows:
(1) sample is taked; Take the topsoil of somewhere, Shayang County, Hubei Province arsenic contamination in mid-May, 2006.
(2) example enrichment: accurately take by weighing soil sample 100g in 250ml sterilization triangular flask, add the NaAsO of 5ml 80mM
2, stir evenly gently to put in 28 ℃ of incubators and cultivate a week, note adding sterilized water, guarantee that sample is dried.
(3) arsenic resistance bacterium separates: accurately take by weighing NaAsO
2Enrichment soil sample 10g puts in 28 ℃ of shaking tables and vibrates half an hour in the triangular flask that the 90ml stroke-physiological saline solution is housed, and gets 1ml more successively and progressively be diluted to 10 in the 9ml stroke-physiological saline solution
-3, l0
-4, 10
-5, get the 0.1ml coating respectively and contain 800uM NaAsO
2CDM solid medium flat board, 3 flat boards of each extent of dilution coating are put in 28 ℃ of incubators and are cultivated a week, the bacterial strain that grows is an arsenic resistance bacterium, puts in 4 ℃ of refrigerators flat board stand-by.CDM solid culture based formulas is as follows, the 100ml solution A, and the 2.5ml solution B, the 10ml solution C is added in the agar of 1.5% sterilization and fusion, and making its final volume is 1L, and final pH is 7.2, wherein solution A (81.2mMMgSO
47H
2O, 187mM NH
4Cl, 70mM Na
2SO
4, 0.574mM K
2HPO
4, 4.57mM CaCl
22H
2O, 446mMC
3H
5NaO
3), solution B (4.8mM FeSO
47H
2O), solution C (950mM NaHCO
3), all solution are prepared with deionized water, 121 ℃ of sterilizations of solution A 20 minutes, solution B and solution C filtration sterilization.
(4) line separates: the bacterium colony line that the arsenic resistance bacterium picking that obtains in the step (3) is different, guarantee to obtain mono-clonal, and the TSA culture medium flat plate is used in line, treats to put after bacterium grows in 4 ℃ of refrigerators stand-by and a in-80 ℃ of refrigerators with the preservation of glycerine freeze pipe.The TSA culture medium prescription is as follows, takes by weighing 40g Soybean-Casein Digest (Bacto Tryptic Soy Agar) and adds deionized water to 1L, sterilizes 15 minutes for 121 ℃.
(5) arsenic oxidation bacterium screening: the arsenic resistance bacterium mono-clonal that obtains in the step (4) is transferred in the CDM liquid nutrient medium, adds the NaAsO of 1ml 80mM
2, making its final concentration is 800uM, puts shaking culture in 28 ℃ of shaking tables, treats that bacterium is long to use KMnO after dense
4Detect its oxidisability.Method is as follows, gets 20ul 0.01M KMnO
4In the 1.5ml centrifuge tube, add the CDM bacterium liquid of the above-mentioned cultivation of 1ml again, if KMnO
4Becoming orange explanation by pink does not have oxidation, if KMnO
4Still be that oxidation (KMnO takes place in the pink explanation
4Can be with NaAsO
2Be oxidized to Na
3AsO
4, in the bacterium liquid as have a NaAsO
2, KMnO then
4Variable color reacts).CDM liquid culture based formulas does not just contain agar with the CDM solid medium.
(6) classification of arsenic oxidation bacterium is identified: the one, and utilize 16S rDNA to identify, (concrete PCR method is 200510120584.7 referring to the application number that the applicant proposed December 30 in 2005 promptly to adopt prokaryotic organism 16S rDNA universal primer 27F (5 ' AGAGTTTGATCMTGGCTCAG3 ') and 1492R (5 ' GGYTACCTTGTTACGACTT3 ') to be PCR, denomination of invention is the patent application specification of " a kind of small quality fast extraction method for soil total DNA "), increase its 16S rDNA and the order-checking, compare with international NCBI GenBank (www.ncbi.nlm.nih.gov) Nucleotide database again, nucleotide homology is 99%, is accredited as Achromobacter xylosoxidans Achromobacter xylosoxidans; The 2nd, utilize scanning electron microscope identification of morphology (seeing accompanying drawing 3), gramstaining analysis and growth characteristics to identify.The mycology feature is as follows: the thalline direct rod shape, and long 2-3um, wide 0.6-0.8um, Gram-negative bacteria, suitable growth temperature 26-32 ℃, appropriate pH 7.0-7.5, facultative aerobic, on LB, CDM and TSA solid medium, be the bacterium colony of white, circle, projection; The 3rd, the arsenic oxidase gene is identified, earlier with degenerate pcr (W.P.Inskeep et al.Detection, diversity and expression of aerobic bacterial arsenite oxidase genes.Environmental Microbiology.2007,9 (4): 934-943) cutting out partial sequence, again as probe, method by genomic library is (available from the test kit CopyControl Fosmid Library Production Kit of EPICENTRE Technologies company, working method is according to the specification sheets of this test kit) clone full gene of arsenic oxydase and downstream correlated series (about 5kb), this sequence has been delivered to NCBI GenBank database, and its number of registration is EF523515.
The preservation of Achromobacter xylosoxidans SY8:
Achromobacter xylosoxidans SY8 can make short term storage after the cultivation 28 ℃ of cultivations on LB, TSA or CDM liquid or solid substratum under 4 ℃.If long-term preservation, can use glycerine freeze pipe or lyophilize pipe (referring to Zhao Bin, He Shaojiang. the microbiology experiment. first version. the .2002:202-205 of Science Press) preservation strain is proper.
Positively effect of the present invention is:
Have data to show, inorganic trivalent arsenic is compared with inorganic pentavalent arsenic, and the former toxicity is not only strong 100 times than the latter, and the former neutral, is difficult for removing, having very strong movability, easier harm environment.Separation screening of the present invention to Achromobacter xylosoxidans SY8 inorganic trivalent arsenic can be oxidized to inorganic pentavalent arsenic, greatly reduced the harm of arsenic in the environment, can also cooperate emerging microorganism active mud ruling by law reason arsenic contamination.Bacterial strain of the present invention from the arsenic contamination environment separation screening to, the arsenic contamination environment is had stronger resistance and adaptability, be expected to aspect purifying arsenic contamination, play a significant role.
Description of drawings
Fig. 1: be technological line figure of the present invention.
Fig. 2: be the arsenic oxidation graphic representation of Achromobacter xylosoxidans SY8 of the present invention, X-axis represent the time (hour), cell density (OD is represented on the Y-axis left side
600), pentavalent arsenic concentration (uM) is represented on Y-axis the right.
Fig. 3: be the stereoscan photograph of Achromobacter xylosoxidans SY8 of the present invention, magnification and scale are indicated in the drawings.
Embodiment
Embodiment 1: separate Achromobacter xylosoxidans SY8 from As polluted soil
Experiment soil is taken from the topsoil of somewhere, Shayang County, Chinese Hubei Province arsenic contamination, and the concrete operations step is as follows:
(1) example enrichment: accurately take by weighing soil sample 100g in 250ml sterilization triangular flask, add the NaAsO of 5ml 80mM
2, stir evenly gently to put in 28 ℃ of incubators and cultivate a week, note adding sterilized water, guarantee that sample is dried.
(2) arsenic resistance bacterium separates: accurately take by weighing NaAsO
2Enrichment soil sample 10g puts in 28 ℃ of shaking tables and vibrates half an hour in the triangular flask that the 90ml stroke-physiological saline solution is housed, and gets 1ml more successively and progressively be diluted to 10 in the 9ml stroke-physiological saline solution
-3, 10
-4, 10
-5, get the 0.1ml coating respectively and contain 800uM NaAsO
2CDM solid medium flat board, 3 flat boards of each extent of dilution coating are put in 28 ℃ of incubators and are cultivated a week, the bacterial strain that grows is an arsenic resistance bacterium, puts in 4 ℃ of refrigerators flat board stand-by.CDM solid culture based formulas is as follows, the 100ml solution A, and the 2.5ml solution B, the 10ml solution C is added in the agar of 1.5% sterilization and fusion, and making its final volume is 1L, and final pH is 7.2, wherein solution A (81.2mMMgSO
47H
2O, 187mM NH
4Cl, 70mM Na
2SO
4, 0.574mM K
2HPO
4, 4.57mM CaCl
22H
2O, 446mMC
3H
5NaO
3), solution B (4.8mM FeSO
47H
2O), solution C (950mM NaHCO
3), all solution are prepared with deionized water, 121 ℃ of sterilizations of solution A 20 minutes, solution B and solution C filtration sterilization.
(3) line separates: the bacterium colony line that the arsenic resistance bacterium picking that obtains in the step (2) is different, guarantee to obtain mono-clonal, and the TSA culture medium flat plate is used in line, treats to put after bacterium grows in 4 ℃ of refrigerators stand-by and a in-80 ℃ of refrigerators with the preservation of glycerine freeze pipe.The TSA culture medium prescription is as follows, takes by weighing 40g Soybean-Casein Digest (Bacto Tryptic Soy Agar) and adds deionized water to 1L, sterilizes 15 minutes for 121 ℃.
(4) arsenic oxidation bacterium screening: the arsenic resistance bacterium mono-clonal that obtains in the step (3) is transferred in the CDM liquid nutrient medium, adds the NaAsO of 1ml 80mM
2, making its final concentration is 800uM, puts shaking culture in 28 ℃ of shaking tables, treats that bacterium is long to use KMnO after dense
4Detect its oxidisability.Method is as follows, gets 20ul 0.01M KMnO
4In the 1.5ml centrifuge tube, add the CDM bacterium liquid of the above-mentioned cultivation of 1ml again, if KMnO
4Becoming orange explanation by pink does not have oxidation, if KMnO
4Still be that oxidation (KMnO takes place in the pink explanation
4Can be with NaAsO
2Be oxidized to Na
3AsO
4, in the bacterium liquid as have a NaAsO
2, KMnO then
4Variable color reacts).CDM liquid culture based formulas does not just contain agar with the CDM solid medium.
(5) classification of arsenic oxidation bacterium is identified: the one, and utilize 16S rDNA to identify, (concrete PCR method is 200510120584.7 referring to the application number that the applicant proposed December 30 in 2005 promptly to adopt prokaryotic organism 16S rDNA universal primer 27F (5 ' AGAGTTTGATCMTGGCTCAG3 ') and 1492R (5 ' GGYTACCTTGTTACGACTT3 ') to be PCR, denomination of invention is the patent application specification of " a kind of small quality fast extraction method for soil total DNA "), increase its 16S rDNA and the order-checking, compare with international NCBI GenBank (www.ncbi.nlm.nih.gov) Nucleotide database again, nucleotide homology is 99%, is accredited as Achromobacter xylosoxidans Achromobacter xylosoxidans; The 2nd, utilize scanning electron microscope identification of morphology (seeing accompanying drawing 3), gramstaining analysis and growth characteristics to identify.The mycology feature is as follows: the thalline direct rod shape, and long 2-3um, wide 0.6-0.8um, Gram-negative bacteria, suitable growth temperature 26-32 ℃, appropriate pH 7.0-7.5, facultative aerobic, on LB, CDM and TSA solid medium, be the bacterium colony of white, circle, projection; The 3rd, the arsenic oxidase gene is identified, earlier with degenerate pcr (W.P.Inskeep et al.Detection, diversity and expression of aerobic bacterial arsenite oxidase genes.Environmental Microbiology.2007,9 (4): 934-943) cutting out partial sequence, again as probe, method by genomic library is (available from the test kit CopyControl Fosmid Library Production Kit of EPICENTRE Technologies company, working method is according to the specification sheets of this test kit) clone full gene of arsenic oxydase and downstream correlated series (about 5kb), this sequence has been delivered to NCBI GenBank database, and its number of registration is EF523515.
Embodiment 2: the arsenic oxidation curve of Achromobacter xylosoxidans SY8
Picking Achromobacter xylosoxidans SY8 mono-clonal is inoculated in the 100ml CDM liquid nutrient medium, puts in 28 ℃ of shaking tables shaking culture 48 hours, this moment cell density OD
600Be about 0.15, be kept at 4 ℃ of refrigerators, be used for inoculation as kind of daughter bacteria liquid.Draw 1ml in the fresh CDM liquid nutrient medium of 100ml with 1% inoculum size, put in 28 ℃ of shaking tables shaking culture and add NaAsO after 12 hours
2Mother liquor, making its final concentration is 800uM, continues to cultivate to begin sampling after 9 hours, gets sample one time every 1 hour, until NaAsO
2Till the complete oxidation, get 1.2ml and 0.6ml respectively at every turn, survey cell density (OD respectively with spectrophotometry again
600) and pentavalent arsenic concentration (OD
838), all enchashment is now done.Specific practice is as follows: the one, survey cell density (OD
600), be reference with the deionized water, directly measure its light absorption value at wavelength 600nm place with the sample of 1.2ml.The 2nd, pentavalent arsenic concentration (OD
838); 0.6ml sample 12000rpm after centrifugal 2 minutes; get the 0.3ml supernatant in the 10ml volumetric flask; with the arsenic molybdenum blue method (Zhou Yuewen. the arsenic molybdenum blue method is measured trivalent arsenic and pentavalent arsenic. environmental protection science .1990; 16 (4): 45-47) measure its light absorption value, calculate the concentration of pentavalent arsenic again according to typical curve or formula at wavelength 838nm place.The Achromobacter xylosoxidans SY8 arsenic oxidation curve of drawing is seen accompanying drawing 2.
Embodiment 3: Achromobacter xylosoxidans SY8 the simulation arsenic polluted water body in to arsenious oxidation effectiveness
Adopt the lake water (this lake water is fresh water, is three class water quality of China national Classification of water Qualities, and pH is 7.79) of the other South Lake of deionized water and Chinese Wuhan City, Hubei Province Hua Zhong Agriculture University to add NaAsO
2Be used for simulating arsenic polluted water body, investigate Achromobacter xylosoxidans SY8 arsenious oxidation effectiveness.Specific practice is as follows: prepare three 250ml triangular flasks, adorn (121 ℃ of 100ml deionized waters for one, sterilized 30 minutes), adorn 100ml lake water respectively (one of them triangular flask is in 121 ℃ for all the other two, sterilized 30 minutes, another triangular flask is unsterilised), every bottle of cultivation thalline of adding Achromobacter xylosoxidans SY8 makes the whole density (OD of its cell then
600) be 0.1-0.2, in 3 triangular flasks, add NaAsO respectively again
2Mother liquor, making its final concentration is 800uM, puts in 28 ℃ of shaking tables shaking culture 24 hours, during use KMnO
4The method detected result sees Table 1.
Table 1 KMnO
4Method detects the result of 3 kinds of simulation arsenic polluted water bodies respectively
| Remove centrifugal water | Sterilization lake water | Unsterilised lake water | |
| Add NaAsO 2Detect before | Nondiscoloration | Nondiscoloration | Nondiscoloration |
| Add NaAsO 2Do not cultivate detection afterwards | Variable color | Variable color | Variable color |
| Add NaAsO 2Cultivate detection in 24 hours afterwards | Nondiscoloration | Nondiscoloration | Nondiscoloration |
Find out that by table 13 kinds of water are adding NaAsO
2Detect KMnO before
4The no reduced form material of nondiscoloration explanation exists, and does not influence experiment and carries out; Adding NaAsO
2Do not cultivate detection, KMnO afterwards
4Variable color explanation NaAsO
2Have reductibility, cause KMnO
4Variable color; Adding NaAsO
2Cultivate detection in 24 hours afterwards, KMnO
4Nondiscoloration explanation NaAsO
2Be oxidized to Na
3AsO
4, no reducing substances exists, so KMnO
4Can variable color, simultaneously by control experiment, promptly 3 kinds of water only add NaAsO
2Do not add Achromobacter xylosoxidans SY8, as a result NaAsO
2Oxidation can not take place, this has proved absolutely that Achromobacter xylosoxidans SY8 makes NaAsO
2Oxidation takes place.In sum, 3 kinds of water add Achromobacter xylosoxidans SY8 and all can make NaAsO
2Oxidation takes place, illustrate Achromobacter xylosoxidans SY8 low nutrition, single existence, with the situation of other microorganism coexistence under all trivalent arsenic is had good oxidation effectiveness, demonstration the present invention has application promise in clinical practice.
Claims (2)
1, a strain can be oxidized to tervalent arsenic in the inorganic arsenic chemicals Achromobacter xylosoxidans (Achromobacter xylosoxidans) of the arsenic of pentavalent in the inorganic arsenic chemicals, it is characterized in that: be deposited in Chinese typical culture collection center, its preserving number is CCTCC NO:M207048.
2, the application of the described Achromobacter xylosoxidans of claim 1 aspect purifying arsenic contamination.
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| CN102146353B (en) * | 2009-12-18 | 2012-12-12 | 南京工业大学 | Gene engineering bacterium capable of both tolerating high-concentration As (Arsenic) (III) and oxidizing As (III) and application thereof |
| CN103834589B (en) * | 2014-01-13 | 2016-10-05 | 福建农林大学 | One Achromobacter xylosoxidans and application thereof |
| CN103937704B (en) * | 2014-03-10 | 2016-04-13 | 赵晗 | One Achromobacter xylosoxidans and the application in heavy-metal ion removal thereof |
| CN107794051B (en) * | 2017-10-27 | 2019-02-12 | 迦百农生态农业发展股份有限公司 | A kind of biological agent of purifying arsenic contamination soil |
| CN107841476B (en) * | 2017-12-08 | 2021-03-30 | 福建农林大学 | Application of arsenic oxidizing bacteria in colonization of soil in trivalent arsenic polluted paddy field |
| CN107841477B (en) * | 2017-12-08 | 2020-09-11 | 福建农林大学 | Application of arsenic oxidizing bacteria in reducing rice trivalent arsenic pollution |
| CN107974415B (en) * | 2017-12-08 | 2020-09-11 | 福建农林大学 | Application of arsenic oxidizing bacteria in repairing paddy field trivalent arsenic pollution |
| CN115011529B (en) * | 2022-07-19 | 2022-12-30 | 广东省科学院动物研究所 | Heavy metal resistant achromobacter xylosoxidans and application thereof |
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