CN1306026C - Bacillus alcaigenes and its application for degrading methamidophos - Google Patents
Bacillus alcaigenes and its application for degrading methamidophos Download PDFInfo
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- CN1306026C CN1306026C CNB2005100745246A CN200510074524A CN1306026C CN 1306026 C CN1306026 C CN 1306026C CN B2005100745246 A CNB2005100745246 A CN B2005100745246A CN 200510074524 A CN200510074524 A CN 200510074524A CN 1306026 C CN1306026 C CN 1306026C
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- acephatemet
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Abstract
The present invention relates to a bacterium and provides bacillus alcaigenes and an application thereof in degrading methamidophos. The bacillus alcaigenes is Alcaligenes sp. LY-5 and has the preserving number of CCTCC NO: M205027. Methamidophos is used as a unique nutrient source and degradated to form phosphate radicals, and phosphate precipitation is formed from the phosphate radicals and a heavy metal source in the environment, so the action of attenuation and dephosphorization is realized once.
Description
Technical field
The present invention relates to a kind of bacterium, especially a kind of Alcaligenes and the application in degrading methamidophos thereof.
Background technology
Biotechnology is existing historical for many years in the application of environmental area, and Wastewater Treated by Activated Sludge Process trade effluent early and the bioremediation technology that grows up over nearly 20 years are wherein arranged.The degradable pollutant kind of microorganism is broadly divided into polycyclic aromatic hydrocarbons (PAHs), organic dye and pigment, tensio-active agent, agricultural chemicals, phenols and halohydrocarbon etc.At present, the applying biological degradation principles is administered the potential advantages that contaminated environment has and has been obtained extensive approval.It is economy, safety not only, and the treatable pollutent threshold value of institute is low, residual few, and its application prospect is good.
Aspect the efficient degrading bacteria of organic pollutant, domestic have many units obtained different organic pollutants in requisition for degradation bacteria strains, some is Application and Development successfully.For example, people (2000) such as Chinese Academy of Agricultural Sciences's atom the subject of knowledge and the object of knowledge Chen Ming from the water body of industrial wastewater pollution and mud separation screening to 6 strains to chlorobenzene and phenol organic pollutant high density tolerance and the bacterial isolates of degradation capability in various degree; People's (calendar year 2001) such as the Ceng Feng of Zhongshan University isolate a strain phthalic ester degradation bacteria FS1 (Pseudomonas fluorescens FS1) from the active sludge of handling petrochemical plant waste water; People's (calendar year 2001) such as the Li Qiufen of Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science screen organic pollutant degradation bacterium 10 strains of shrimp pond, cultured prawn is not had pathogenic effects, and wherein 6 strains can improve the surviving rate of young shrimp; (2000) isolation and selections from municipal effluent and trade effluent such as the Jia Sheng of Institute of Microorganism, Academia Sinica sweet smell go out a kind of high-efficiency multi-function mixed bacterial, comprise genus bacillus 7 strains, other bacterium 6 strains and yeast 7 strains etc.; (1999) such as the Hou Yi of chemical engineering institute of South China Science ﹠ Engineering University are separated to the effectively subtilis of degrading nitrobenzene of 1 strain, directly use the aerobic technology just can waste water is innoxious fully; Zhang Zhijie etc. (2000) people isolates 2 strain naphthalene degradation bacteria and 1 pyridine degradation bacterium strain from the active sludge of handling waste water, through the microorganism degraded of metabolizable energy promotion hardly degraded organic substance altogether, effect is best when the three acts synergistically; (1998) such as the Cai Baoli of Nankai University are separated to 5 energy from industrial sewage be the bacterial isolates of the sole carbon source and the energy with the phenylformic acid; People (1997) such as the Zhang Jian of Agricultural University Of Anhui screen the 2 strains bacterium very capable to degrading aniline from be subjected to the aniline Contaminated soil for a long time; Li Shubin etc. (1999) report is separated to the pseudomonas of strain energy efficiently degrading organophosphorus pesticides acephatemet; Aspergillus M-2 and the degraded Rogor active higher aspergillus ornatus Z58 of Liu Yuhuan etc. (2000) from being separated to the energy degrading methamidophos the organophosphorus Contaminated soil; Village iron really waits the result of study of (2000) to show, endures the predominant bacteria population that exists degrading methamidophos in the mangrove forest soil that industrial or agricultural and sanitary sewage pollute to the fullest extent; Huang Zhiyongs etc. (1999) are separated to a strain Rhodopseudomonas palustris H3 from printing and dyeing mill's mud clock, and this bacterium has very strong anaerobic decolorization and Degradation to Acid Red B 2GL dyestuff; The strain white-rot fungi OGC101 that Li Huirong etc. are separated to all has wide spectrum and effective degradation capability to big classes of dyestuff such as azoic dyestuff, thiazine dyes, phthalocyanine pigments; Cao Mengde etc. are separated to the gas sporangium RD2 of a strain to the azo dye activity bright red X-3B colour of camel's hair from the dyeing waste water contaminate environment.
Acephatemet is the organic pesticide of a kind of water miscible wide spectrum, severe toxicity, and chemical name is O, S-dimethyl amido thio-phosphamide.This agricultural chemicals has stomach toxicity, tags and systemic action, is used to prevent and treat various pests, widely uses on agricultural.1964, developed acephatemet in succession by the West Germany and the U.S..In recent years, along with the forbidding of organochlorine pesticide, the consumption of acephatemet rises rapidly, has become one of pesticide species of the present consumption maximum of China.1996 annual consumptions have reached 60,000 tons, account for 21.4% of sterilant total amount, occupy (Hua Xiaomei, 1996) first of many sterilants.People cause the pesticide residue of grain, vegetables etc. to exceed standard in a large amount of its pest controls of use, and methamidophos pesticide poisoning and cooupational intoxication case increase (Bellis, 1994 in succession; Sundaranc, 1993).And, the factory effluent of the methamidophos pesticides of a large amount of dischargings, its organophosphorus concentration height, component complexity, biological degradability is poor, the biochemical treatment effect is low, causes soil, water body environment pollution, and the eubiosis is destroyed.It has now become one of the preferential ten big pesticide species of monitoring of China (Jiang Xiliu, 1993).Therefore, should strengthen its Research on degradation.In the ecosystem, the metabolic type of microorganism is many, has very strong biochemical adaptive faculty, and can produce corresponding enzyme quickly is to utilize the agricultural chemicals of all kinds of synthetic as carbon, nitrogenous source growth, and often can be degraded into inorganics fully, these all are that other approach is incomparable.Microorganism to the decomposition of agricultural chemicals as a kind of effective environmental control measures, in have important practical significance aspect decontamination, the environment purification (Ruan Shaojiang, 2000).
Summary of the invention
The object of the present invention is to provide a strain Alcaligenes (Alcaligenes sp.), and the people for green fluorescent protein (GFP) plasmid that changes Ka Na (Kn) resistance over to as biomarker, can in the open environment degradation process, detect the abundance of this bacterial strain at any time.
Another object of the present invention is to provide the application of Alcaligenes in degrading methamidophos.
The invention provides a strain Alcaligenes, be Alcaligenes sp.LY-5, be deposited in Chinese typical culture collection center on April 1st, 2005, deposit number is CCTCC NO:M205027.
Bacterium Alcaligenes sp.LY-5 provided by the present invention is an Alcaligenes through the 16srDNA sequential analysis, and green fluorescent protein (GFP) plasmid that is changed over to Ka Na (Kn) resistance is as biomarker, has Kn resistance and inductor sec.-propyl-Beta-D-thiogalactoside (ITPG) inductive GFP ability to express, its luminescence phenomenon can be under fluorescent microscope, detected, the abundance of this bacterium can be in the open environment degradation process, detected at any time.
Alcaligenes Alcaligenes sp.LY-5 can be unique nutrition source with acephatemet, and acephatemet is degraded to the phosphate radical form and forms calcium phosphate precipitation with heavy metal source in the environment, once reaches the effect of attenuation and dephosphorization.During with Alcaligenes Alcaligenes sp.LY-5 degrading methamidophos, liquid nutrient medium consist of NaCl 0.2g, KCl 0.2g, anhydrous MgSO
40.2g, MnCl
20.2g, NH
4NO
31.0g, pure water 1000mL, pH=7.0.Acephatemet 1g.
Acephatemet is degraded to nontoxic phosphate radical form can passes through high performance liquid chromatograph (HPLC) and NMR detection.
The selection of Alcaligenes Alcaligenes sp.LY-5 is as follows:
1. the collection of degradation bacteria: pollute the sections such as sewage draining exit, biochemistry pool of the insecticide factory around the section along the husky small stream of Sanming City, totally 6 sampling points: the 1. total sewage draining exit in chemical plant, Sanming City, Fujian Province (be called for short three and change total mouthful) mud; 2. the NaCl of slag factory piles up pond mud; 3. biochemistry pool one-level and secondary pond combined sewage; 4. Fujian Province's Sanming City phosphorus sewage draining exit (be called for short yellow phosphorus mouth) sewage; 5. biochemistry pool mud; 6. slag factory percolate.
The domestication of acephatemet degradation bacteria with separate
Soil sampling 5g or water sample 10ml, inoculating 80ml acephatemet concentration respectively is in the basic medium of 100mg/L, in 37 ℃, after the 120r/min shaking table is cultivated a week, getting 10ml access acephatemet concentration is in the basic medium of 200mg/L, and inoculation once progressively improves acephatemet concentration weekly later on, be followed successively by 400mg/L, 800mg/L, and under the concentration conditions of 800mg/L, cultivate again a week.
Cultivate in .37 ℃ of incubator of coating bacterium liquid on containing the LB solid medium flat board of 800mg/L acephatemet, it is streak culture again to choose single bacterium colony, occurs until not assorted bacterium.
3. the detection of acephatemet degraded:
High performance liquid chromatograph (HPLC) condition: Varian Prostar210 high performance liquid chromatograph, (150 * 4.6mm), moving phase is 20% methyl alcohol to the C18 reversed-phase column, 80% water; Sensing range 210-300nm, the monitoring wavelength is 215nm.
Phosphorus spectrum: Varian UNITY+500 nuclear magnetic resonance analyser, single colony inoculation of picking is gone into to be equipped with the screw-cap test tube of 5ml enrichment medium (methylamine phosphate content is 500mg/L), place 37 ℃, 120r/min shaking table overnight incubation, every strain is according to 10% inoculum size, insert methylamine phosphate content and be in the basic medium of 1000mg/L (20ml), place 37 ℃, the 120r/min shaking table, take out bacterium liquid 0.5ml behind the 10min, centrifugal (3000rpm, 10min) after. get supernatant and be used for HPLC and detect, later on every 3 days, get sample one time, carry out HPLC and detect, simultaneously above centrifugal bacterium liquid supernatant is got 0.5mL and be used for 500,000,000 nuclear-magnetisms and carry out the phosphorus spectrum analysis.
4, result
The HPLC detected result shows, filter out 14 strain acephatemet bacterium for degrading for the degradation efficiency of acephatemet all than higher, after cultivating 4,7,14 days, the average degradation rate of acephatemet is respectively 67.6%, 81.1% and 85.9%.Sample is carried out synchronous phosphorus spectrum analysis, and discovery is along with acephatemet peak area in the chromatogram weakens, and the peak at acephatemet 42.6ppm place also weakens gradually in the phosphorus spectrum, and to low drift, at 10ppm, 24ppm and the new peak of appearance, 1ppm place.The phosphorus of different strains for degrading acephatemet after products spectrum peak is incomplete same, 14 strain bacterial strains can be divided into 3 types (A, B, C).The nuclear-magnetism of degrading after a week the results are shown in Figure 1.
Choose and to be degraded to the highest strain of degradability in the three strain bacteriums of A group of phosphate radical to acephatemet, carry out the 16sRNA sequencing analysis, find that it is the Alkaligenes bacterium.This bacterium is changed over to the GFPxm18 plasmid that cell tumour key lab of Xiamen University makes up, makes it produce the Kn resistance, and induce, cultivate down for 17 ℃ at low temperature by inductor (ITPG),
Can under fluorescent microscope, detect its luminescence phenomenon.
Shaking table cultivation stage in this strains for degrading process (14 days) leaves standstill about a week after finishing, and finds to occur in the nutrient solution tiny crystal.It is carried out X-ray diffraction analysis, learn that it is MnHPO
4Crystal.This crystal is a rhombic system, spatial mode P2 (1), and brilliant bag parameter is: a=11.234 (5) nm, b=6.145 (3) nm, c=6.948 (3) nm; α=90deg; β=90deg, γ=90deg.Z=4.As seen the Mn ion in its substratum can be used for phosphate anion that degrading methamidophos produces in conjunction with precipitation, reaches the effect of phosphorus just.
Description of drawings
Fig. 1 is the phosphorus spectrum result of the degrading methamidophos in 3 groups of one weeks of bacterium.
Embodiment
Get the Kn resistance Alcaligenes that we make up, in open environment, carry out mimic acephatemet degradation experiment.Get the basic medium of 3 groups of 2L, wherein first group adds acephatemet 2g, does not artificially inoculate any bacterium, in contrast group, second group of Kn resistance Alcaligenes nutrient solution that adds 2g acephatemet and our structure of 20ml, the 3rd group of basis at second group adds the leach liquor of the 20g soil of 20ml again.Aerated culture, sampling in per 2 days detects its acephatemet degradation rate and bacterial growth amount (colony number in the colony number in the non-resistant flat board/Kn resistant panel) by HPLC and coated plate respectively.Detect 8 days, the result is as shown in the table.And get second day work bacterium liquid, add the IPTG low temperature induction and cultivate, under fluorescent microscope, detect luminescence phenomenon.Can confirm by above experiment, our bacterium keeps its acephatemet degrading activity in open environment, and is the absolute predominance bacterial classification in the water surrounding that contains higher acephatemet concentration, after acephatemet pressure is removed, its advantage disappears, and bacterial strain distributes and is tending towards normal in the environment.
Acephatemet degradation rate (%)
| | 0 day | 2 days | 4 days | 8 days |
| | 0 | 0 | 0 | 0 |
| | 1 | 70 | 75 | 85 |
| | 1 | 68 | 70 | 82 |
Increment (bacterium number/105)
| | 0 day | 2 days | 4 days | 8 days |
| | 0 | 0 | 0 | 2 |
| Second group | 4/4 | 46/44 | 78/70 | 54/20 |
| Second group | 7/4 | 52/47 | 72/70 | 129/28 |
Claims (1)
1. the application of Alcaligenes in degrading methamidophos, described Alcaligenes are Alcaligenes sp.LY-5, are deposited in Chinese typical culture collection center on April 1st, 2005, and deposit number is CCTCC NO:M205027.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101134943B (en) * | 2007-05-11 | 2010-05-19 | 华东理工大学 | Bacillus alcaligenes and method for preparing single enantiomer amygdalic acid |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100429304C (en) * | 2006-12-05 | 2008-10-29 | 中国科学院武汉病毒研究所 | Bacillus alcaligenes strain for degrading ortho-nitrophenol and preparing method |
| CN101144063B (en) * | 2007-09-03 | 2011-08-17 | 哈尔滨工业大学 | Alkalibacillus huanghai |
| CN102517230B (en) * | 2011-12-12 | 2013-06-19 | 温州医学院 | Bifidobacterium breve and detection method of methamidopho pesticide residue in foodstuff |
| CN102989760A (en) * | 2012-11-21 | 2013-03-27 | 苏州科技学院 | Restoring method for soil polluted by acephate pesticide |
| CN107446855B (en) * | 2017-08-29 | 2020-09-04 | 侨康生物科技(广东)有限公司 | Alcaligenes strain, enzyme preparation and application of enzyme preparation in degrading pesticide residue |
| CN108410751B (en) * | 2018-02-05 | 2021-01-26 | 温州大学 | Alcaligenes faecalis and application thereof in degradation and decoloration of azo dyes |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1141381A (en) * | 1995-07-07 | 1997-01-29 | 泷源制造株式会社 | Covering assembly for insertion port of detachable key-handle |
| CN1055119C (en) * | 1996-06-05 | 2000-08-02 | 南京农业大学 | Chemical residual degradation vaccine and its prodn. method |
| CN1502688A (en) * | 2002-11-22 | 2004-06-09 | 中国农业科学院生物技术研究所 | Bacterial strain with broad-spectrum degradation ability to organic phosphorus pesticide and screening method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1141381A (en) * | 1995-07-07 | 1997-01-29 | 泷源制造株式会社 | Covering assembly for insertion port of detachable key-handle |
| CN1055119C (en) * | 1996-06-05 | 2000-08-02 | 南京农业大学 | Chemical residual degradation vaccine and its prodn. method |
| CN1502688A (en) * | 2002-11-22 | 2004-06-09 | 中国农业科学院生物技术研究所 | Bacterial strain with broad-spectrum degradation ability to organic phosphorus pesticide and screening method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101134943B (en) * | 2007-05-11 | 2010-05-19 | 华东理工大学 | Bacillus alcaligenes and method for preparing single enantiomer amygdalic acid |
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