CN1683518A - High anti-mercury offensive smell pseudomonas strain CHY-7 and use in treating mercury pollution - Google Patents
High anti-mercury offensive smell pseudomonas strain CHY-7 and use in treating mercury pollution Download PDFInfo
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Abstract
The present invention belongs to the field of environment tackling with microbes. The strain is pseudomonasputida CHY-7 separated from mercury contaminated soil and preserved in Chinese typical culture preserving center. Pseudomonasputida CHY-7 is suitable for culture in neutral, weak acid or weak alkaline culture medium at normal temperature, and may grow in water or soil. Pseudomonasputida CHY-7 is aerobic Gram-negativre bacterium and has size of (2.0-4.0)x(0.7-1.1) micron and no spore generated. It forms yellowish colony with smooth raised edge and can generate fluorescence in chromogenesis culture medium. It has high effect of reducing ionic mercury into volatile mercury element and may be applied widely in tackling mercury contaminated environment.
Description
Technical field
The present invention relates to utilize microorganism to implement the project of environment protection treating, a kind of specifically high anti-mercury offensive pseudomonas CHY-7 bacterial strain and the application in administering mercury pollution thereof.
Background technology
United Nations Environment Programme (UNEP) in 2003 has delivered a report and has shown, since the Industrial Revolution, the content of mercury in atmosphere, water, soil has increased about 3 times, and the content of mercury is higher near the manufacturing district, and the continuous aggravation of mercury pollution has caused great harm to human health and environment.
Mercury is as a kind of natural component in the physical environment, and is not high at natural background level, and for example surface water content is less than 0.1 μ g/m
3, content is 0.001 ~ 50 μ g/m in the atmosphere
3, content average out to 0.1 μ g/g in the soil.Yet the production of the exploitation of non-ferrous metal and processing, chlor-alkali, papermaking, industrial mercury consumption such as electric increase year by year, and mercurous agricultural chemicals for some time be extensive use of, cause mercurous material constantly to enter external environment, cause serious mercury.In addition, the natural conversion rate of mercurous material is very slow, even after removing source of pollution, mercury still can retain in local environment for a long time.
Mercury pollution is mainly by the food chain effect, a large amount of enrichments in aquatic organism, fish of human edible mercury pollution etc. and bringing in the body.Have report to show, the content of mercury is higher than safety standards in the U.S. 1/12 or near 5,000,000 human female.Any type of mercury all can be converted into the methyl mercury of severe toxicity under certain condition in the environment, and methyl mercury is mainly encroached on neural system after entering human body, causes the cranial nerve pathology.Nineteen fifty-three Japan over one hundred routine water Wu disease in nine continents be exactly a typical example.Methyl mercury can be encroached on fetus by placental barrier, makes the newborn infant that congenital disorders take place.Mercury pollution also can cause diseases such as cardiovascular systems.
Pollution at China's mercury is serious equally, and some local water quality, soil, rice field, fish duck etc. all are subjected to pollution in various degree.Find in the investigation of Shandong Nansi Lake fishery products that the pond that the has mercury of breeding fish in the duck body exceeds standard and reaches as high as 2.7 times.Guizhou Province's investigation is found, irritates the field owing to quote the mercury pollution river for a long time, and the result causes mercury significantly accumulation in paddy soil.The investigation in Hebei is also found, the residual overrun of mercury in some soil, vegetables, fish, the soil water.Jiangsu found to a pollution-free vegetable base soil poisonous metal pollution survey that mercury pollution was the most outstanding in 1998, and this may grow with the history that planted vegetables at that time and use mercurous agricultural chemicals relevant.In addition, the research result shows that the settling of river, reservoir area of Three Gorges section also generally is subjected to mercury pollution, and the enrichment factor EF value of mercury is up to 1.4-9.2.
The processing of environment mercury pollution at present all is the removal at mercury in the trade effluent, mainly adopts physicochemical method, and commonly used have active carbon adsorption, Coagulation Method, reduction method, sulphide precipitation and an ion exchange method.These measure cost costlinesses also may stay dangerous by product.And pollute except cutting off source of pollution for nature water body and mercury in soils, these methods are all inapplicable.
Nineteen sixty, reported first was separated to the bacterial strain of anti-mercury from occurring in nature, the microorganism of discovering subsequently has different anti-mercury molecular mechanisms, and wherein complicated, the most general anti-mercury mechanism relates to Mer protein family and relevant mer gene thereof: A, B, C, D, P, R and T.These genes are positioned at the manipulator of anti-mercury (mer operon), have identified to have 11 mer manipulators at least, and merA genes encoding inorganic mercury reductase enzyme wherein is in the reaction that NADPH-relies on, with divalent ionic mercury (Hg
2+) be reduced into nontoxic relatively volatile element mercury (Hg
0).MerB genes encoding organic mercury lytic enzyme is hydrolyzed into the weak relatively inorganic mercury (Hg of toxicity with the organic mercury of strong poison
2+), other mer genes mainly are regulatory gene.At present, think that the anti-mercury microorganism with mer manipulator can be used as water body and the most effective instrument of soil mercury pollution repaired.In addition, by conjugation, the mer manipulator can level transmission between microorganism, makes it have application prospect widely aspect biological degradation mercury pollution.
People such as Brunke in 1993 be separated to nature with the merA genetic engineering bacterium be experiment material, prove that these bacteriums can remove the HgCl in the sewage
2After 1999 German researcher from Spittelwasser river mud by containing 50 μ g/L Hg
2+The LB selective medium separate and to obtain Pseudomonas Putida Spi3 bacterial strain, this bacterial strain removes Hg in chlor-alkali factory electrolysis waste solution
2+Elimination efficiency reach 97%, concrete experiment condition is: first day, with bacterial cultures with the flow velocity of 20ml/h by bio-reactor (column volume is 20ml); Then, substratum was flow through bio-reactor 2 days, 5 days or 10 days, allow bacterium growth and breeding on the microbial film of bio-reactor; At last, with the substratum of flow velocity 2ml/h and the electrolysis waste solution (Hg of 18ml/h
2+Concentration is 1.5mg/L) by the microbial film of bio-reactor, detect the content of the Hg of flowing liquid.In addition, the Hg in the removing electrolysis waste solution of this bacterial strain
2+The maximum concentration scope be 7-9mg/L.They were amplified to 1000L with scale in 20002.Because adopt the microbiological deterioration mercury pollution simple and easy, light to the detrimentally affect of environment, cost is low, be expected to become one of important means of removing mercury pollution.
Show from the pertinent data retrieval that comprises Chinese patent, from the soil of mercury pollution, isolate the bacterial strain of anti-mercury, and utilize anti-mercury microorganism to remove mercury pollution and do not see its relevant report as yet.
Summary of the invention
The objective of the invention is to from occurring in nature soil, directly separate, screen high anti-mercury, growing nutrient requirement low, adapt to natural ecological environment and viability is strong, have efficient reduction ion state mercury (Hg
2+) be volatile element mercury (Hg
0) bacterial isolates and administering the mercury pollution environmental applications.
The technical solution adopted for the present invention to solve the technical problems is:
High anti-mercury offensive pseudomonas CHY-7 bacterial strain provided by the invention, it is characterized in that: this bacterial strain is pseudomonas putida CHY-7, be preserved in " Chinese typical culture collection " center ", its preserving number: CCTCC NO.M 204093. on December 7th, 2004
This pseudomonas putida CHY-7 bacterial strain is to separate to obtain from the soil of plant area of a certain discarded fluorescent lamp factory mercury pollution; The bacterial strain normal temperature that suits in neutrality and slightly acidic or weakly alkaline developing medium is cultivated, and also can grow in tap water and soil; Its bacteriology morphological specificity: be gram negative bacillus, its length is 2.0-4.0 * 0.7-1.1um, does not produce gemma, and with many utmost point hair motions, obligate is aerobic; Belong to pseudomonasputida, bacterium colony is faint yellow, the smooth protuberance in edge, generates in the substratum at pigment and can produce fluorescence.
Its concrete screening step is as follows:
Gather the soil of plant area of fluorescent lamp factory mercury pollution, add and contain HgCl
2LB liquid nutrient medium (Hg
2+Concentration is 25mg/L), to smash evenly, after the natural sedimentation, the centrifuging and taking supernatant is coated and is contained 25mg/L Hg
2+The LB flat board, 28 ℃ of incubation 24h.Select the anti-mercury bacterium of 7 strains and carry out anti-mercury property of medicine level detection.
Use HgCl
2Preparation Hg
2+Concentration is the LB liquid nutrient medium of 1.25-100mg/L doubling dilution, the bacterium liquid to be measured of inoculation logarithmic phase, and 28 ℃ leave standstill cultivation 48h; According to the bacterial growth situation, judge the anti-Hg of bacterial strain
2+Level, see the following form-1.
Table-1: the anti-mercury level detection of the bacterial strain of anti-mercury
Bacterial strain is compiled anti-Hg
2+Level
Number (mg/L)
CHY-1??100
CHY-2??25
CHY-3??25
CHY-4??25
CHY-5??25
CHY-6??25
CHY-7??100
According to reference: detection (Bao Youdi, Yu Shugao, the Dai Gengsun etc. of the research I478 strain dysentery bacterium transitivity R plasmid of dysentery bacterium resistance variation.China's microbiology and Journal of Immunology, 1982,2 (5): method 304-307), the selection wherein 4 strain bacterial strain of anti-mercury CHY-1, CHY-3, CHY-6 and CHY-7 are carried out the detection of the bacterium antibiotics resistant property of medicine, the results are shown in following table-2.
Table-2: the bacterial strain antibiotics resistant property of medicine detects
Annotate: Streptomycin sulphate (SM), paraxin (CM), tsiklomitsin (TC), peace Bian penicillin (AP), kantlex (KM), gentamicin (GM), erythromycin (Ery) and cephalo draw stings (Cep)
Because the anti-Hg of CHY-7 bacterial strain
2+Up to 100mg/L, to antibiotic resistance less (table-2), so the biological characteristics of CHY-7 bacterial strain is done further research.
The physiological and biochemical property of API 20NE test kit (BioMerieux company) and other biochemical identification high anti-mercury offensive pseudomonas CHY-7 bacterial strain sees the following form-3.
Content and the explanation of API 20NE test kit with reference to " Bergy ' s Manual of systemic Bacteriology " Vol.VIII, surely the principle that belongs to according to morphological specificity and cell walls chemical composition, proof CHY-7 bacterial strain is a gram negative bacillus, its length is 2.0-4.0X0.7-1.1 μ m, do not produce gemma, with many utmost point hair motions, obligate is aerobic; Biochemical identification belongs to pseudomonasputida (pseudomonas putida), and its bacterium colony is faint yellow, the smooth protuberance in edge, generates in the substratum at pigment and can produce fluorescence.
Table-3: the physiological and biochemical property of high anti-mercury offensive pseudomonas CHY-7 bacterial strain
| The experiment title | The result | The experiment title | The result |
| Nitrate reduction | ??- | Pectinose | ??- |
| Indole reaction | ??- | N.F,USP MANNITOL | ??- |
| Gelatine liquefication | ??- | N-ethanoyl glucose | ??- |
| Urase | ??- | Maltose | ??- |
| Dihydroorotase | ??- | Gluconate | ??+ |
| Glucose turns sour | ??- | Caprate | ??+ |
| The esculin hydrolysis | ??- | Hexanodioic acid | ??- |
| The beta galactose hydrolysis | ??- | Oxalic acid acetate | ??+ |
| The glucose assimilation | ??+ | Citrate trianion | ??+ |
| The pectinose assimilation | ??- | Toluylic acid | ??+ |
| Fluorescence | ??+ | Terminal oxidase | ??+ |
| N-acetyl Portugal (grape) osamine | ??- | Seminose mannose | ??+ |
| Phenylacetate | ??+ | Malate | ??- |
| 42℃ | ??- | Gill fungus sugar | ??- |
| 4℃ | ??+ | Hydrogen sulfide | ??- |
Further utilize the classification of 16S rRNA gene conservative dna sequence dna to identify the homology of the 16SrRNA gene order 100% of CHY-7 bacterial strain and pseudomonasputida ATTC17484 and ATTC17522, promptly the CHY-7 bacterial strain belongs to pseudomonasputida (Pseudomonas putida).This sequence has been submitted GenBank database (GenBank Accession No.AY834215).
High anti-mercury offensive pseudomonas CHY-7 bacterial strain of the present invention both can contain Hg
2+Cultivate in the nutritional mediums such as LB (100mg/ml), also can in sterilization tap water and sterile soil, grow.This bacterial strain adapts to pH value scope wide (pH5.0-10.0), can between 4 ℃-39 ℃, grow, but can not be 42 ℃ of growths.
The anti-ionic mercury level of this high anti-mercury offensive pseudomonas CHY-7 bacterial strain has catalysis ionic mercury (Hg up to 100mg/L
2+) be converted into element mercury (Hg
0) ability, at Hg
2+Concentration is in the scope of 5-50mg/L, and 48 hours transformation efficiency is more than the 94.3%-82.8%, Hg
2+Concentration low-conversion more is high more.And the Hg of the Pseudomonas Putida Spi3 bacterial strain that German Industrialization is used in removing electrolysis waste solution
2+The maximum concentration scope be 7-9mg/L.
The genomic dna of high anti-mercury offensive pseudomonas CHY-7 bacterial strain provided by the invention contains the merA gene, coding inorganic mercury reductase enzyme (merA), catalysis ionic mercury (Hg
2+) be converted into nontoxic relatively volatile element mercury (Hg
0).The part merA gene fragment of this bacterial strain (about 700bp) is respectively 93.1% (GenBank gi:21322678), 91.2% (gi:24411176), 91.9% (gi:150631), 92.1% (gi:23821228) and 92.9% (gi:21322688) with the merA corresponding DNA sequence homology of GenBank database; This dna sequence dna is submitted GenBank database (GenBank accessionNo.AY834216).
High anti-mercury offensive pseudomonas CHY-7 bacterial strain provided by the invention has high anti-Hg
2+The ability of level and very strong reduction ionic mercury is applicable to the improvement of mercury pollution; Because this bacterial strain nutritional requirement is not high, the pH wide ranges (pH5.0-10.0) of growth, in sterilization nature water, can keep growth more than at least 1 month, and keep the bacterium number to reach 18 days in the normal soil of sterilization, also has 80% viable bacteria body number in the time of 30 days, still can be separated to this bacterial strain after 6 months, show that the CHY-7 bacterial strain has very strong nature viability, is particularly useful for the processing of industrial Mercury sewage, the reparation of pollution-free vegetable base mercury contaminated soil and the processing of aquafarm mercury pollution water quality such as fish, shrimp.
The invention has the beneficial effects as follows: anti-ionic mercury level is up to 100mg/L in liquid owing to high anti-mercury offensive pseudomonas CHY-7 bacterial strain of the present invention, and the anti-ionic mercury level of PseudomonasPutidaSpi3 bacterial strain (being up to 10mg/L) that the Bhide national expenditures is removed electrolysis waste solution in industry is high 10 times; High anti-mercury offensive pseudomonas CHY-7 bacterial strain of the present invention was removed ionic mercury in 48 hours in ionic mercury concentration is the 5-50mg/L scope efficient is 94.3%-82.8%, and Germany is used for the Hg that the Pseudomonas PutidaSpi3 bacterial strain removing electrolysis waste solution of electrolysis waste solution is removed in industry
2+The maximum concentration scope be 7-9mg/L; So high anti-mercury offensive pseudomonas CHY-7 bacterial strain of the present invention has the efficient that the ionic mercury of handling greater concn pollutes and more high definition is removed mercury pollution, is applicable to the processing of industrial Mercury sewage; In addition, because this bacterial strain is to the effect of people, animal no pathogenicity, also be specially adapted to the processing of aquafarm mercury pollution water quality such as the reparation of pollution-free vegetable base mercury contaminated soil and fish, shrimp.
Description of drawings
Accompanying drawing 1 is the growth curve of CHY-7 bacterium of the present invention in water body and edatope, among the figure X-coordinate be the time (my god), ordinate zou is a bacterial concentration (* 10
7/ ml or * 10
7/ g).
Accompanying drawing 2 is CHY-7 bacterial strain reduction Hg of the present invention
2+Ability detect, X-coordinate is Hg among the figure
2+Initial concentration (mg/L), ordinate zou are residual mercury concentration (mg/L).
Accompanying drawing 3 is identified for the PCR method of the merA gene of CHY-7 bacterial strain of the present invention.
Embodiment
Embodiment 1: the screening step of high anti-mercury offensive pseudomonas CHY-7 bacterial strain of the present invention is as follows:
Gather pedotheque from the mercury contaminated soil of a certain depleted fluorescent bulb in Fujian Province factory, sample thief 10g smashes evenly, adds 10ml and contains HgCl
2LB liquid nutrient medium (Hg
2+Concentration is 25mg/L) suspend, after the natural sedimentation, 100g centrifuging and taking supernatant is coated and is contained 25mg/L Hg
2+The LB flat board, 28 ℃ of incubation 24h.
From Hg
2+Selective medium (Hg
2+The LB flat board) go up in single bacterium colony of growth, select the 7 strain bacterial strains of anti-mercury (being numbered CHY-1, CHY-2, CHY-3, CHY-4, CHY-5, CHY-6 and CHY-7) and carry out anti-mercury property of medicine level detection.With colony inoculation to be measured in containing 25mg/L Hg
2+The LB nutrient solution in, bacterial growth is inoculated in HgCl to logarithmic phase with 1: 100 ratio
2The Hg of preparation
2+Concentration is in the LB liquid medium of 1.25-100mg/L doubling dilution, and 28 ℃ leave standstill cultivation 48h.According to the bacterial growth situation, judge the anti-Hg of this bacterial strain
2+Level; Then, utilize the biochemical classification of API20NE System (BioMerieux Inc.) bacterium identification kit to the bacterial strain evaluation of classifying.Biochemical identification of the anti-mercury bacterium of 7 strains and anti-Hg thereof
2+Level sees the following form-1.
Table-1: biochemical identification of anti-mercury bacterium and anti-Hg thereof
2+Level determination
Anti-Hg is identified in the biochemical classification of strain number
2+Level (mg/L)
CHY-1 pseudomonas 100
CHY-2 pseudomonas 25
CHY-3 pseudomonas 25
CHY-4 Bacillus subtilus 25
CHY-5 Bacillus subtilus 25
CHY-6 pseudomonas 25
CHY-7 pseudomonas 100
Adopt liquid culture method that the anti-mercury pseudomonas of 4 strains is carried out the antibiotic resistance analysis, the microbiotic of selecting for use is that Streptomycin sulphate (SM), paraxin (CM), tsiklomitsin (TC), peace Bian penicillin (AP), kantlex (KM), gentamicin (GM), erythromycin (Ery) and cephalo draw and sting (Cep); Concrete grammar reference: detection (Bao Youdi, Yu Shugao, the Dai Gengsun etc. of the research I 478 strain dysentery bacterium transitivity R plasmids of dysentery bacterium resistance variation.China's microbiology and Journal of Immunology, 1982,2 (5): 304-307).The results are shown in Table-2.
Table-2: the bacterial strain antibiotics resistant property of medicine detects
Annotate: "+" sensitivity, "-" resistance.
Detect by the biochemical classification evaluation of screening, the bacterium of ionic mercury selective medium, anti-mercury level and antibiotic resistance, obtain the anti-Hg of a plant height
2+, the high anti-mercury offensive pseudomonas CHY-7 bacterial strain less to antibiotic resistance; And this bacterial strain is preserved in " Chinese typical culture collection " center ", its preserving number CCTCC No.M 204093 on December 7th, 2004.
This high anti-mercury offensive pseudomonas CHY-7 bacterial strain suits to grow in the developing medium of pH5.0-10.0 and tap water and normal soil, and optimum pH is 6.0-8.0; Can between 4 ℃-39 ℃, grow, but can not be 42 ℃ of growths, optimum temperuture is 28-30 ℃; Its bacteriology morphological specificity: be gram negative bacillus, length is 2.0-4.0X0.7-1.1um, does not produce gemma, and with many utmost point hair motions, obligate is aerobic; Its bacterium colony is faint yellow, the smooth protuberance in edge to belong to pseudomonasputida (pseudomonas putida), generates in the substratum at pigment and can produce fluorescence.
Embodiment 2: utilize the classification of 16S rRNA gene to identify high anti-mercury offensive pseudomonas CHY-7 bacterial strain
Adopt Wizard Genomic DNA purification Kit to extract CHY-7 bacterial genomes DNA, according to sequences Design and synthetic primer conservative in the bacterial 16 S rRNA gene.PCR primer: upstream primer 16S-F:5 '-TgCCAgCAgCCgCggTAA-3 '; Downstream primer 16S-R:5 '-AggCCCgggAACgTATTCAC-3 '.The PCR reaction conditions is 94 ℃/3mim; 94 ℃/45s, 56 ℃/30s, 72 ℃/1min, 30 circulations; 72 ℃/7min.The PCR product is checked order by the precious biotech firm in Dalian.The dna sequencing result is as follows:
ACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAATCCC
CGGGCTCAACCTGGGAACTGCATTCAAACTGACAAGCTAGAGTATGGTA
GAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGG
AACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGC
GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTA
AACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCAGCTA
ACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAA
ATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGA
AGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGA
GATGGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGT
CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTT
GTCCTTAGTTACCAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTG
ACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGG
CCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCG
CGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGC
AACTCGACTGCGTGAAGTCGGAATC
Above dna sequence dna is analyzed by BLASTN in the NCBI gene database, found that and the homology of the 16SrRNA gene order 100% of pseudomonasputida ATTC17484 and ATTC17522 that promptly the CHY-7 bacterial strain belongs to pseudomonasputida (Pseudomnonas putida); This sequence has been submitted GenBank database (GenBank Accession No.AY834215).
Embodiment 3: nest-type PRC is identified the mer4 gene of high anti-mercury offensive pseudomonas CHY-7 bacterial strain
According to merA gene order and the homology comparative analysis that the GenBank database provides, utilize biosoftware such as Generunner merA gene high conservative dna sequence dna district design PCR primer (merA-F45:5 '-ggCgCACgTCAAggAAgC-3 '; MerA-F352:5 '-gTCgAgCAAggCgCgCAg-3 '; MerA-R541:5 '-gACACgggCCTgCTgCTg-3 '; MerA-R756:5 '-gTCCAgTAgggTgACTCTTTC-3 '; Forming 4 pairs of primers altogether is merA-F45/merA-R541, merA-F45/merA-R756, merA-F352/merA-R541 and merA-F352/merA-R756), be template with CHY-7 bacterial genomes DNA, pcr amplification corresponding D NA segment.The PCR reaction conditions is 94 ℃/3min:94 ℃/45s, 54-58 ℃/30s, and 72 ℃/1min, totally 30 circulations; 72 ℃/7min.
The PCR product is analyzed through agarose gel electrophoresis, ethidium bromide staining and ultraviolet imagery system imaging, and the result shows the dna segment size and expection consistent (seeing accompanying drawing 3) of PCR product.
With the PCR product of MerA-F45/MerA-756 primer amplification (being that the dna segment that increases in 4 pairs of primers is the longest, about 700bp) directly order-checking (the precious biotech firm in Dalian); The dna sequencing result is as follows:
CAGTCTGCCGTTGTGTCCTACGCCAAGGGCGCGGCCCAGCTCGACCTTGA
CCCCGGTACCGCATCAGACGCACTGACTGCCGTCGTGGCCGGACTCGGCT
ACAGGGCGACGCTCGCTGATACCTCATCGACGGACAACCGCACCGGACTG
CACGACAAGGTACGCGGCTGGATGGGAACCGCCGATAAGGGCAGCGACG
GCGAGCGCCAGTTGCATATCGCCGTCATCGGCAGCGGCGGCGCTGCAATG
GCGGCGGCGCTGAAGGCCGTCGAGCAAGGCGCAAAGGTCACGCTGATCG
AGCGCGGCACCATCGGCGGCACCTGCGTCAACGTCGGTTGTGTGCCGTCC
AAGATCATGATCCGCGCCGCCCACATCGCCCATCTGCGCCGGGAAAGCCCA
TTCGACGGCGGCATGCCACCCACACCGCCGACGATCTTGCGCGAGCGGCT
GCTGGCCCAGCAGCAGGCCCGTGTCGAAGAACTCCGTCATGCCAAGTACG
AAGGCATCCTGGACGGCAATTCAGCCATCACCGTTCTGCACGGTGAAGCG
CGTTTCAAGGACGACCAGAGCCTTATCGTTAGTTTGAACGAGGGTGGTGA
GCGCGTCGTGATGTTCGACCGCTGCCTGGTCGCCACGGGTGCCAGTCCGG
CCATGCCGCCGATTCCGGGCCTGAAAGAGTCACCCTACTGGACA
The protein sequence of prediction is as follows:
QSAVVSYAKGAAQLDLDPGTASDALTAVVAGLGYRATLADTSSTDNRTGLHD
KVRGWMGTADKGSDGERQLHIAVIGSGGAAMAAALKAVEQGAKVTLIERGT
IGGTCVNVGCVPSKIMIRAAHIAHLRRESPFDGGMPPTPPTILRERLLAQQQAR
VEELRHAKYEGILDGNSAITVLHGEARFKDDQSLIVSLNEGGERVVMFDRCL
VATGASPAMPPIPGLKESPYWT
Above dna sequence dna is analyzed by BLASTN in the NCBI gene database, the merA gene fragment (693bp) that found that CHY-7 strain gene group DNA amplification is respectively 93.1% (GenBank gi:21322678), 91.2% (gi:24411176), 91.9% (gi:150631), 92.1% (gi:23821228) and 92.9% (gi:21322688) with the merA corresponding DNA sequence homology of GenBank database, thereby proves that the CHY-7 bacterial strain has anti-mercury and with Hg
2+Be converted into Hg
0Ability be since this strain gene group contain the merA gene; This dna sequence dna is submitted GenBank database (GenBankaccession No.AY834216)
Embodiment 4: the growth characteristics of high anti-mercury offensive pseudomonas CHY-7 bacterial strain in water body and soil
The high anti-mercury offensive pseudomonas CHY-7 bacterium liquid of logarithmic phase is centrifugal, initial inoculation about 10 * 10
6Bacterium number/ml is in 3ml sterilization tap water, and room temperature (25 ℃) leaves standstill cultivation, regularly gathers water sample, dilution coating LB flat board, enumeration.Equally, bacterium liquid to be measured is evenly mixed in the soil of sterilization, every part of 10g soil, the initial inoculation amount is about 10 * 10
6Bacterium number/g, soil are farmland soil; Regularly gather soil sample, add the aqua sterilisa mixing and suspend, natural sedimentation is got supernatant dilution coating LB flat board, enumeration.Three repeated experiments were observed 30 days continuously.
The result shows that this bacterial strain can keep growth more than at least 1 month in sterilization nature water, and keeps the bacterium number to reach 18 days in the normal soil of sterilization, also has 80% viable bacteria body number (seeing accompanying drawing 1) in the time of 30 days.
Embodiment 5: high anti-mercury offensive pseudomonas CHY-7 bacterial strain catalysis ionic mercury (Hg
2+) be converted into element mercury (Hg
0) capability analysis
Use HgCl
2Preparation Hg
2+Concentration is 5.0,10.0,35.0 and the LB liquid nutrient medium of 50.0mg/L, is sub-packed in the 15ml sterilization test tube high anti-mercury offensive pseudomonas CHY-7 bacterial strain of inoculation logarithmic phase, inoculum size about 10 * 10
6Bacterium number/ml, 30 ℃ leave standstill cultivation 48h.Centrifuging and taking nutrient solution supernatant is measured mercury content with the two pass atomic fluorescence spectrophotometer; Establish the parallel control group simultaneously, three repeated experiments.Bacterium transforms Hg
2+Be Hg
0Formulae of efficiency as follows: transformation efficiency (%)=(control group mercury concentration-test group mercury concentration)/control group mercury concentration * 100%.
By accompanying drawing 2 results as can be seen, high anti-mercury offensive pseudomonas CHY-7 bacterial strain of the present invention has the ability of very strong conversion ionic mercury, and the efficient of removing ionic mercury in ionic mercury concentration is the 5-50mg/L scope in 48 hours reaches 94.3%-82.8%.
Claims (8)
1. a plant height anti-mercury offensive pseudomonas CHY-7 bacterial strain, it is characterized in that: this bacterial strain is pseudomonas putida (pseudomonas putida) CHY-7, be preserved in " Chinese typical culture collection " center ", its preserving number CCTCC NO.M 204093 on December 7th, 2004.
2. a plant height anti-mercury offensive pseudomonas CHY-7 bacterial strain is characterized in that: separate to obtain from the soil of mercury pollution.
3, high anti-mercury offensive pseudomonas CHY-7 bacterial strain according to claim 2 is characterized in that: this bacterial strain normal temperature that suits in neutrality and slightly acidic or weakly alkaline developing medium is cultivated, and also can grow in tap water and soil.
4, a plant height anti-mercury offensive pseudomonas CHY-7 bacterial strain, it is characterized in that: its bacteriology morphological specificity: be gram negative bacillus, its length is 2.0-4.0 * 0.7-1.1 μ m, does not produce gemma, with many utmost point hair motions, obligate is aerobic; Belong to pseudomonasputida, bacterium colony is faint yellow, the smooth protuberance in edge, generates in the substratum at pigment and can produce fluorescence.
5, a plant height anti-mercury offensive pseudomonas CHY-7 bacterial strain, it is characterized in that: this bacterial strain nutritional requirement is not high, and the pH wide ranges pH5.0-10.0 of growth has very strong nature viability.
6, a plant height anti-mercury offensive pseudomonas CHY-7 bacterial strain is characterized in that: the anti-Hg of this bacterial strain
2+Level is 100mg/L; Its genomic dna contains the merA gene, coding inorganic mercury reductase enzyme (merA), but catalysis ionic mercury (Hg
2+) be converted into nontoxic relatively volatile element mercury (Hg
0); The merA Gene Partial dna sequence dna of this bacterial strain is submitted the database in NCBI GenBank, and sequence number is AY834216.
7, by the described high anti-mercury offensive pseudomonas of claim 6 CHY-7 bacterial strain, it is characterized in that: this bacterial strain has high anti-mercury characteristic and extremely strong catalysis ionic mercury (Hg
2+) be converted into element mercury (Hg
0) ability.
8, a plant height anti-mercury offensive pseudomonas CHY-7 bacterial strain is in the application of administering mercury pollution, and it is characterized in that: this bacterial strain is applicable to the processing of industrial Mercury sewage, the reparation of pollution-free vegetable base mercury contaminated soil, the processing of place mercury pollution water quality such as aquaculture.
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| DE4227769C2 (en) * | 1992-08-23 | 1997-03-06 | Biotechnolog Forschung Gmbh | Pseudomonas putida strains for mercury reduction and process for their preparation |
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