CN102533586A

CN102533586A - Pandora bacterium with dichloromethane degrading capability and application thereof

Info

Publication number: CN102533586A
Application number: CN2011103700702A
Authority: CN
Inventors: 陈建孟; 成卓韦; 蒋轶锋; 傅凌霄; 於建明; 朱润晔
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2011-11-18
Filing date: 2011-11-18
Publication date: 2012-07-04
Anticipated expiration: 2031-11-18
Also published as: CN102533586B

Abstract

The invention provides a dichloromethane degrading bacterium, namely a pandoraeapnomenusa strain flx<-1>, and an application thereof. The strain is preserved in China Center for Type Culture Collection in Wuhan University (430072), Wuhan, China, the preservation number is CCTCC NO.: M 2011242, and the preservation date is July 8, 2011. The pandoraeapnomenusa strain flx<-1> is taken from a wastewater treatment unit and has good degrading effects on chlorinated hydrocarbon compounds, in particular on DCM (dichloromethane), and the DCM can be completely converted into CO2, H2O, biologic cells and other harmless substances. Meanwhile, benzene, toluene and other common industrial pollutants can be degraded to certain extent through the strain. Therefore, the strain has broad application prospects in biological purification of industrial exhaust gas and wastewater.

Description

Pandora bacterium and application thereof with methylene dichloride degradation capability

(1) technical field

The present invention relates to a strain---Pandora bacterium (Pandoraea pnomenusa strain) flx-1, and the application in microbiological deterioration methylene dichloride, chlorobenzene, benzene, toluene or ethylbenzene.

(2) background technology

Methylene dichloride (CH ₂Cl ₂, Dichloromethane is called for short DCM) and be a kind of use water-insoluble organic solvent very widely, be widely used in metallic substance degreasing, paint removal industry and pharmaceutical industry.Along with industriallization, quickening of urbanization process, various countries also increase the demand of DCM day by day.One of DCM is not only atmospheric principal pollutant, or the important component of municipal wastewater and industrial sewage, the DCM more than 90% derives from industry and uses and waste combustion.Because it is difficult for being degraded, the transformation period of DCM in atmosphere and water surrounding was respectively 70 days and 700 years.DCM has cumulative bad and persistence in environment, human habitat has been produced great negative impact, has been classified as one of toxic pollutant of 129 kinds of preferential controls by EPA (EPA).Therefore the purification techniques of studying DCM just seems very urgent and important.

The biopurification technology has characteristics such as the efficient of removal is high, processing costs is low, secondary pollution is little, is widely used in the degraded and the purification of toxic pollutant gradually.One of key that adopts biotechnology processing DCM is the bacterial strain that acquisition has efficient degradation DCM ability.At present; Chinese scholars has been carried out big quantity research to the biological degradation of alkane derivative; But owing to contain the chlorine atom in extremely low, the structure of DCM solubleness; The DCM degradation bacteria that is separated to so far is also more limited; Mainly comprise pseudomonas (Pseudomonas), methyl bacillus (Methylobacterium), raw silk germ (Hyphomicrobium), genus bacillus (Bacillaceae), mycobacterium (Mycobacterium) and fusobacterium (Fusobacterium) etc., these its degradation efficiencies of bacterial strain that is separated to are still waiting further raising.Big quantity research shows the efficient DCM degradation bacteria of separation screening from environment, remains one of important method of eliminating organo-chlorine pollutant in the environment.

(3) summary of the invention

The object of the invention provides that a plant height is imitated, tolerance is strong has Pandora bacterium of DCM degradation capability and uses thereof.

The technical scheme that the present invention adopts is:

One strain methylene dichloride degradation bacteria---Pandora bacterium (Pandoraea pnomenusa strain) flx-1 is preserved in Chinese typical culture collection center, address: China; Wuhan, Wuhan University, 430072; Deposit number: CCTCC NO:M 2011242, preservation date: on July 8th, 2011.

Said Pandora bacterium flx-1 colony characteristics is following: bacterium colony is white in color, and neat in edge is smooth moistening; The form that transmission electron microscope is observed this thalline down is a tyrothricin, does not have flagellum, Gram-negative, and oxidase positive, starch hydrolysis and indole test are positive, can't utilize lactose.

Can the degrade DCM of starting point concentration 0～1500mg/L of this bacterial strain flx-1, the eventual degradation product is CO ₂, H ₂O and cellular biomass, average mineralization rate 74.15%, average yield coefficient 0.1269mgcells/mg DCM.The discovery of this degradation bacteria is significant to the efficient purification of DCM in the trade effluent waste gas.This bacterial strain Pandora bacterium flx-1 is other industrial common organic pollutant, especially chlorobenzenes of degradable, benzene, toluene, ethylbenzene etc. also.

The invention still further relates to the application of described Pandora bacterium flx-1 in microbiological deterioration methylene dichloride, benzene, toluene, ethylbenzene, o-Xylol, chlorobenzene, THF, methyl alcohol or ethyl propenoate.The starting point concentration of this type material is preferably 0～1500mg/L in the waste water.

Said degraded is at 25～35 ℃, pH 7～9, and salinity is counted with NaCl under the condition of 0～1% (w/w) and carried out.

Preferably, said degraded is at 33 ℃, pH 7.3, and salinity is counted with NaCl under 0.66% the condition and carried out.

With the bacteria suspension of Pandora bacterium flx-1 through the cultivation acquisition; Put into and contain 100mg/L benzene (B), toluene (T), ethylbenzene (E), o-Xylol (X), chlorobenzene (CB), THF (THF), methyl alcohol (MeOH) respectively, in the simulated wastewater of ethyl propenoate (EA), 33 ℃ of temperature; PH7.3; Shaking culture under the condition of salinity (in NaCl) 0.66%, said Pandora bacterium flx-1 these pollutents of in 92h, degrading to some extent, degradation rate is 57～100%.

With the bacteria suspension that Pandora bacterium flx-1 obtains after cultivating, put in the deionized water of the methylene dichloride (DCM) that contains 100mg/L, respectively with O ₂, NO ₂ ^-, NO ₃ ^-, SO ₄ ^2-And Fe ³⁺Be unique electron donor, leave standstill and shaking culture that described Pandora bacterium flx-1 all can utilize NO ₂ ^-, NO ₃ ^-, SO ₄ ^2-And Fe ³⁺As electron donor, under anaerobic realize dechlorination to DCM.With Fe ³⁺During for electron donor, described Pandora bacterium flx-1 can realize the dechlorination to DCM in the short period of time, and average chlorine release rate has surpassed under the same terms with O ₂The release rate of chlorine during for electron donor shows that described Pandora bacterium flx-1 is one type of facultative aerobe.

Pandora bacterium among the present invention is a kind of common tyrothricin, through patent searching and other pertinent literatures, does not find to utilize the report of Pandora bacterium degraded DCM as yet.The discovery of this degradation bacteria is significant for the efficient purification of chlorinated hydrocarbon pollutent such as DCM in the trade effluent waste gas and other hydrocarbons pollutants.

Preferably, the said Pandora bacterium flx-1 starting point concentration that is used to degrade is the DCM waste water of 50～1500mg/L, and described culture condition is that temperature is 33 ℃, pH7.3, and salt concn (in NaCl) is 0.66%.Said Pandora bacterium flx-1 is 0.1269mg cells/mg DCM for average yield coefficient in this concentration range, and the average mineralization rate of DCM is 74.15%, and the release rate of average cl ions is 96.8%.

Pandora bacterium flx-1 of the present invention takes from treatment unit for waste water, and for chlorinated hydrocarbon, especially DCM has degradation effect preferably, can comparatively fully be converted into CO to DCM ₂, H ₂Innoxious substance such as O, cellular biomass; Simultaneously, this bacterial strain common pollutent of industry such as benzene, toluene of also degrading to some extent, thereby in the industrial gaseous waste biological purification of waste water, have broad application prospects.

(4) description of drawings

Fig. 1 is gramstaining photo and the transmission electron microscope photo of Pandora bacterium flx-1 of the present invention;

Fig. 2 is the phylogeny tree graph of Pandora bacterium flx-1 of the present invention;

Fig. 3 for Pandora bacterium flx-1 of the present invention to cellular biomass change curve in the degradation curve of different concns DCM, the degradation process and than degradation rate matched curve;

Fig. 4 is Pandora bacterium flx-1 of the present invention CO in the process of degraded DCM ₂Growing amount matched curve, cl ions release profiles and cellular biomass formation curve;

Fig. 5 is that Pandora bacterium flx-1 of the present invention is to the common organic pollutant degradation performance analysis of industry;

Fig. 6 investigates for the dechlorination effect of hydrocarbons pollutant organochlorine under aerobic and anaerobic condition respectively for Pandora bacterium flx-1 of the present invention.

(5) embodiment

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

Separation, purifying and the evaluation thereof of embodiment 1:Pandoraea pnomenusa flx-1

1.Pandoraea the separation of pnomenusa flx-1 and purifying

Pandora bacterium flx-1 is the strain Gram-negative bacteria that domestication from the active sludge of waste water processing station of Ningbo of Zhejiang refinery factory, separation obtain.Concrete steps are following:

Get the active sludge of waste water processing station of Ningbo of Zhejiang refinery factory.After tap water was eluriated five times, the sky 48h that exposes to the sun removed residual organic as far as possible.Preparation inorganic salt nutrient solutions (pH7) are that sole carbon source carries out the orientation domestication to active sludge with DCM, and 3d changes fresh inorganic salt nutrient solution, measures the pH of nutrient solution every day, and 40d left and right sides pH obviously descends.

Preparation DCM starting point concentration is the 50mg/L minimal medium, and compound method is following: KH ₂PO ₄0.376g, K ₂HPO ₄0.456g, (NH ₄) ₂SO ₄0.48g, NaNO ₃0.68g, Mg (NO ₃) ₂0.25g, CaCl ₂2H ₂O 0.011g, trace element (MnCl ₂H ₂O 0.06g, ZnCl ₂0.088g, KI 0.01g, NaMoO ₄2H ₂O 0.1g, H ₃BO ₃0.05g), zero(ppm) water, 1000mL; PH 7.0-7.2 is sub-packed in the sealing saline bottle (50mL/) of 250mL, 110 ℃ of sterilization 40min.After treating substratum cooling, add DCM as sole carbon source, with the mud of domestication by volume mark be that 5% amount joins in the substratum, in 30 ℃, the shaking table shaking culture of 160rpm.Be transferred in the minimal medium after the fresh sterilization shaking table shaking culture about 3d with 10% amount.Separate in conjunction with agar inorganic salt plate streaking, picking list bacterium colony inserts the minimal medium shaking table and continues shaking culture, up to the single bacterium that obtains the degradable DCM that growth is fast in above-mentioned substratum, bacterium colony is regular and proterties is stable.

2.Pandoraea the evaluation of pnomenusa flx-1

Through 16S rDNA sequential analysis and Physiology and biochemistry experimental identification, confirm that bacterial strain flx-1 is Pandoraea pnomenusa.Concrete steps are following:

Adopt the DNA of the centrifugal environmental sample DNA recovery test kit of 3S post (V2.2, Shanghai Shenergy Biocolor BioScience & Technology Company) extraction and purifying bacterial strain, 4 ℃ of preservations.Universal primer BSF8/20 and the BSR1541/20 that selects bacterium for use carries out pcr amplification to the DNA of purifying, and primer sequence is respectively:

BSF8/20：5′-AGAGT?TTGAT?CCTGG?CTCAG-3′

BSR1541/20：5′-AAGGAGGTGATCCAG?CCGCA-3′

The PCR response procedures is set at: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 1min then, 59 ℃ of annealing 1min, 72 ℃ are extended 1.5min, circulate 35 cycles; 72 ℃ are extended 10min then; Last 4 ℃ keep 10min.The PCR product is checked order (the prompt base in the English Weihe River, Shanghai), sequencing result is seen sequence table.

The 16S rDNA sequence (SEQ ID NO.1) of bacterial strain flx-1 is uploaded to Genbank; Obtain the accession number JN021530 of Genbank; Carry out homology relatively with the gene order among the Genbank simultaneously; Find that it belongs to Pandoraea and belongs to, the highest with Pandoraea pnomenusa (AY268168) homology, reach 100%.Fig. 1 is gramstaining and the transmission electron microscope photo of bacterial strain flx-1, and Fig. 2 is the phylogeny tree graph of bacterial strain flx-1, and table 1 is the part physiological and biochemical test result of bacterial strain flx-1.

Table 1: the Physiology and biochemistry character of bacterial strain flx-1

Annotate :+represent positive ,-represent feminine gender

Based on sequencing result and physiological and biochemical test result, confirm that flx-1 belongs to Pandoraea pnomenusa.Therefore, with this bacterium called after Pandoraea pnomenusa strain flx-1, be preserved in Chinese typical culture collection center, deposit number: CCTCC NO:M 2011242.

Embodiment 2: response surface is optimized the environmental factor of bacterial strain Pandoraeapnomenusa flx-1 (CCTCC NO:M2011242) degraded DCM

1. the acquisition of response surface test design and strains for degrading DCM rate prediction model

Investigated of the influence of 3 factors such as nutrient solution pH, culture temperature and nutrient solution salinity to the DCM degradation effect; Utilize SAS system software that the DCM degradation rate that test obtains is carried out model-fitting, thereby obtain to predict bacterial strain Pandoraea pnomenusa flx-1 degradation rate to DCM under different culture condition.The practical implementation step is following:

Be central value with pH7 (Code=0), temperature 30 ℃ of (Code=0), salt concn 0.5% (Code=0) respectively, utilize SAS system software to carry out three factors, three hydraulic tests designs (table 2).

Table 2: contrived experiment response value and predictor

* be the Code value of investigation factor :-1,0,1 corresponds respectively to pH 7,8,9;

Temperature

20,30,40 ℃; Salt concn 0%, 0.5%, 1.0%.

Press table 2 preparation minimal medium, be sub-packed in the 250mL saline bottle, liquid amount is 50mL/, 110 ℃ of sterilization 40min.After treating the nutrient solution cooling, add the flx-1 bacteria suspension that is in logarithmic phase in each saline bottle, make the initial biological amount reach 14.7mg/L (in wet thallus).With DCM is sole carbon source, and starting point concentration is 100mg/L, and sealing is placed on the shaking table shaking culture of differing temps.Other gets the saline bottle that identical nutrient solution is housed, and the sterilization back adds DCM but do not add the flx-1 bacteria suspension, places under the identical condition and cultivates, as blank.After cultivating 18h, analyze DCM residual concentration in the nutrient solution, measure living weight simultaneously.According to the degradation rate that records, utilize SASsystem software that it is carried out the secondary multiple regression, the predictive model that match obtains is:

Y＝-96.326+14.93646*X ₁+2.876025*X ₂+1.842833*X ₃-0.847083*X ₁*X ₁-0.0735*X ₁*X ₂-0.2655*X ₁*X ₃-0.037343*X ₂*X ₂+0.1693*X ₂*X ₃-4.125333*X ₃*X ₃

In the formula, Y is the degradation rate [mg/ (Lh)] of methylene dichloride, X ₁, X ₂, X ₃Be respectively the Code value of medium pH value, culture temperature and nutrient solution salinity.The coefficient R of multiple regression equation ²=0.9930, explain that degradation rate and actual degradation rate that this predictive model is predicted have good dependency (table 2), can be used for predicting that bacterial strain is to the degradation rate of DCM under the different culture condition.

2. the optimum environment factor of bacterial strain Pandoraea pnomenusa flx-1 degraded DCM

Utilize SAS system software that the predictive model that obtains is analyzed; Make that its single order partial derivative is zero; Environmental factor combination when acquisition bacterial strain Pandoraea pnomenusa flx-1 degraded DCM speed reaches peak; Be 33 ℃ of culture temperature, pH7.3, salt concn (in NaCl) is 0.66%, and the degradation rate that this moment, model was predicted is 5.34mg/ (Lh).Bacterial strain is 5.40mg/ (Lh) to the actual degradation rate of DCM under above-mentioned culture condition, and is comparatively approaching with predictor.The practical implementation step is following:

Get 200mL (pH7.2) inorganic salt nutrient solution, add NaCl and make its concentration reach 0.66%, regulate nutrient solution pH to 7.3, divide to be filled in the saline bottle of 4 250mL, 110 ℃ of sterilization 40min.After treating nutrient solution cooling, getting wherein the flx-1 bacteria suspension that three bottles of addings are in the growth logarithmic phase, make the initial cell amount reach 15.1mg/L, is sole carbon source with DCM, and starting point concentration is 100mg/L.Other gets one bottle and only adds DCM and do not add bacteria suspension as blank.Above-mentioned 4 bottles of saline bottles place 33 ℃ of shaking table shaking culture, and behind the 20h, residual DCM concentration in the analyzing and testing liquid phase is measured the living weight in the nutrient solution simultaneously, obtains the degradation rate of bacterial strain flx-1.

At nutrient solution pH7.3; When 33 ℃ of culture temperature, nutrient solution salinity 0.66%; Bacterial strain Pandoraea pnomenusa flx-1 is 5.37,5.41 and 5.42mg/ (Lh) to the actual degradation rate of DCM; Average degradation rate is 5.40mg/ (Lh), approaches model predication value, shows that this environmental factor is combined as the optimum environment combinations of factors of bacterial strain flx-1 degraded DCM.Embodiment 3:Pandoraeapnomenusa flx-1 detects the degradation property of different concns DCM

Under optimum environment factor condition (nutrient solution pH7.3,33 ℃ of culture temperature, nutrient solution salinity 0.66%), investigated the degradation property of bacterial strain flx-1 to starting point concentration 25～2000mg/L DCM.The result shows, bacterial strain flx-1 25～1500mg/L DCM that can degrade fully, but the starting point concentration of can only partly degrading is 2000mg/L DCM.Under optimal culture condition, bacterial strain high specific degradation rate has reached 0.8561mg DCM/ (mg cellsh).The practical implementation step is following:

Preparation pH is 7.3, salinity (in NaCl) is 0.66% inorganic salt nutrient solution 1000mL, is sub-packed in the saline bottle of 250mL every bottle of 50mL, 110 ℃ of sterilization 40min.After treating the nutrient solution cooling; Get wherein the flx-1 bacteria suspension that 10 bottles of addings are in logarithmic phase; Make the initial biological amount reach 36.5mg/L, add DCM simultaneously, make its concentration reach 25,50,100,200,300,500,800,1000,1500 and 2000mg/L as sole carbon source; All the other 10 bottles only add the DCM of same amount and do not add the flx-1 bacteria suspension as blank.Saline bottle seals back 33 ℃ of shaking culture, regularly measures DCM concentration and living weight residual in the nutrient solution, draws the degradation curve of bacterial strain flx-1 for different starting point concentration DCM, and calculates accordingly than degradation rate.

The result is shown in Fig. 3 (a-d).The bacterial strain flx-1 starting point concentration of degrading is lower than 1500mgL ^-1DCM, and in this scope along with the increase of concentration, the significantly lag-phase occurred comparatively, but its degradation process just becomes comparatively rapid after bacterial strain adapts to gradually.When initial DCM concentration is 2000mgL ^-1The time, bacterial strain flx-1 only has comparatively significantly Degradation to DCM in preceding 80h, obvious variation does not all take place in residual DCM concentration in the liquid phase afterwards, this possibly be since bacterial strain when the DCM of this concentration of degraded, some meta-bolites (CH of generation ₃OH, H ⁺, Cl ^-Deng) accumulation in a large number in nutrient solution, bacterial strain is produced serious toxic action, and then suppressed the degrading activity of cell.Utilize the cell proliferation amount to calculate the ratio degradation rate of bacterial strain, pass through Haldane ' s inhibition growth kinetics model contrast degradation rate simultaneously and carried out match (Fig. 3 e), obtained the high specific degradation rate for 25-1500mg/L DCM.When DCM concentration was 585mg/L, DCM reached peak 0.8561h than degradation rate ^-1

Embodiment 4:Pandoraea pnomenusa flx-1 is to mineralization rate, cl ions release rate and the average yield coefficient analysis of DCM

At optimum environment factor condition (nutrient solution pH7.3; 33 ℃ of culture temperature, nutrient solution salinity 0.66%) under, mineralization rate, cl ions release rate and the average yield coefficient of bacterial strain Pandoraea pnomenusa flx-1 investigated to initial DCM concentration 50-1500mg/L.The result shows that described bacterial strain flx-1 can finally be converted into CO to DCM ₂, H ₂O and cellular biomass, average mineralization rate is 74.15%, and average cl ions release rate is 96.8%, and the average yield coefficient is 0.1269mgcells/mg DCM.The practical implementation step is following:

Preparation pH is 7.3, salinity (in NaCl) is 0.66% inorganic salt nutrient solution 1200mL, is sub-packed in the saline bottle of 250mL every bottle of 50mL, 110 ℃ of sterilization 40min.After treating the nutrient solution cooling; Get wherein the flx-1 bacteria suspension that 16 bottles of addings are in logarithmic phase; Make the initial biological amount reach 36.0mg/L (in wet thallus); Add DCM simultaneously as sole carbon source, make its concentration reach 50,100,200,300,500,800,1000 and 1500mg/L (2 bottles of each concentration); All the other 8 bottles only add the DCM of same amount and do not add the flx-1 bacteria suspension as blank.Above-mentioned saline bottle seals back 33 ℃ of shaking culture, regularly measures DCM concentration in the nutrient solution, cl ions (Cl ^-) DCM concentration, CO in concentration, cellular biomass and the sealing saline bottle top gas phase ₂Concentration.CO in gas phase ₂In the sealing saline bottle, inject the HCl1mL of 30% (w/w) when concentration remains unchanged, make the HCO in the liquid phase ₃ ^-, CO ₃ ^2-Deng being converted into CO fully ₂, measure CO in the gas phase behind the ready to balance once more ₂Concentration.Draw mineralising and the dechlorination curve of bacterial strain flx-1 to different DCM concentration, match obtains average mineralization rate and the average release rate of cl ions, and combines the cell proliferation amount, draws the relation curve between cellular biomass and the DCM degradation amount, calculates average cell yield.

The result is as shown in Figure 4.Cl ^-The amount of removing and DCM concentration linear, its fitting a straight line equation is y=0.8083x (R ²=0.9972), shows the DCM of the every 1mg of utilization of bacterial strain, can produce 0.8353mg Cl ^-, and theoretical Cl ^-The amount of removing be 0.8353mg/mg DCM, so Cl ^-Average release rate be 96.8%.CO ₂Curve y=0.3838x (the R also linear between growing amount and the DCM degradation amount, that match obtains ²=0.9498), promptly bacterial strain flx-1 permineralization 1mg DCM can produce 0.3838mg CO ₂In theory, when the DCM complete oxidation be H ₂O and CO ₂The time, CO ₂Growing amount is 0.5176mg/mg DCM, so the average mineralization rate of bacterial strain flx-1 is 74.15%.Bacterial strain flx-1 can utilize organic carbon to synthesize self cell thing in the process of degraded DCM.Linear relationship between cell proliferation amount and the DCM degradation amount is y=0.1269x (R ²=0.9923), shows that the every degraded of bacterial strain flx-1 1mg DCM can synthesize self cellular biomass of 0.1269mg.

Embodiment 4:Pandoraea pnomenusa flx-1 is to the common organic pollutant degradation performance analysis of industry

Respectively with benzene (B), toluene (T), ethylbenzene (E), o-Xylol (X), chlorobenzene (CB), THF (THF), methyl alcohol (MeOH); Organic pollutant common in the ethyl propenoate industry such as (EA) is investigated the degradation capability of bacterial strain Pandoraea pnomenusa flx-1 for these materials as sole carbon source.The result shows, the bacterial strain flx-1 above-mentioned organic pollutant of degrading to some extent, can comparatively fully degrade in the 24h chlorobenzene, THF, methyl alcohol, can degrade in the 92h benzene, toluene, ethylbenzene, o-Xylol and ethyl propenoate.Specific embodiments is following:

Preparation pH is 7.3, salt concn (in NaCl) is 0.66% inorganic salt nutrient solution 800mL, is sub-packed in the saline bottle of 250mL every bottle of 50mL, 110 ℃ of sterilization 40min.After treating the nutrient solution cooling; Getting wherein, 8 bottles of addings are in logarithmic phase flx-1 bacteria suspension; Make the initial biological amount reach 38.4mg/L (in wet thallus); Respectively with benzene, toluene, ethylbenzene, o-Xylol, chlorobenzene, THF, methyl alcohol and ethyl propenoate as sole carbon source, starting point concentration is 100mg/L; Other gets the above-mentioned organism of 8 bottles of adding same amounts but does not add the flx-1 bacteria suspension as blank.The saline bottle sealing is placed on 33 ℃ shaking table shaking culture, analyzes residual organic concentration in the liquid phase respectively at 24h and 92h, measures corresponding living weight, draws the degradation rate of different organic carbon sources and the relation curve between the bacterial strain flx-1 cell proliferation amount.

The result is as shown in Figure 5.When incubation time is 24h; Bacterial strain flx-1 can comparatively fully degrade chlorobenzene, THF and methyl alcohol; Degradation rate has reached 99.7%, 95.7% and 80.2% respectively; And this moment, bacterial strain had only 19.2%, 56.8% and 45.2% for the degradation rate of benzene, o-Xylol and ethyl propenoate, was 0% to the degradation rate of toluene and ethylbenzene.When incubation time is 92h; Bacterial strain flx-1 has reached 57.2%, 100%, 47.2%, 83.1% and 81.3% respectively for the degradation rate of benzene, toluene, ethylbenzene, o-Xylol and ethyl propenoate; Some are difficult during by biodegradable organic pollutant (like BTEX such as benzene, toluene) in degraded to show bacterial strain flx-1; Usually need certain adaptive phase, treat the above-mentioned organic pollutant of just comparatively fully degrading after bacterial strain adapts to.The explanation of above test-results, the bacterial strain Pandoraea pnomenusa flx-1 multiple common organic pollutant of degrading is used for the actual industrial Pollution abatement for this bacterial strain and has certain directive significance.

Embodiment 5: Pandoraea pnomenusa flx-1 analyzes for the hydrocarbons pollutant dechlorination effect organochlorine under aerobic and the anaerobism culture condition

Respectively with O ₂, NO ₂ ^-, NO ₃ ^-, SO ₄ ^2-And Fe ³⁺Be unique electron donor, investigate the dechlorination ability of bacterial strain Pandoraea pnomenusa flx-1 DCM and CB (chlorobenzene).The result shows that bacterial strain flx-1 can be with O ₂, NO ₃ ^-, SO ₄ ^2-And Fe ³⁺Be electron donor, realize dechlorination DCM, and with Fe ³⁺Be the optimized electronic donor, dechlorination ability and effect are considerably beyond other electron donors; Simultaneously, bacterial strain flx-1 also can be respectively with O ₂And Fe ³⁺Be electron donor, realize dechlorination effect CB.The practical implementation step is following:

Get the 600mL deionized water, be sub-packed in 12 250mL saline bottles, every bottle of 50mL, 2 bottles is one group.Get wherein 4 groups, add NaNO respectively ₃, NaNO ₂, Na ₂SO ₄And FeCl ₃110 ℃ of above-mentioned substratum sterilization 40min, treat the nutrient solution cooling after, add the flx-1 bacteria suspension that is in logarithmic phase, make the initial biological amount reach 37.5mg/L.Getting with the deionized water is 2 bottles of the saline bottles of nutrient solution, adds DCM, makes its concentration reach 100mg/L, and (this culture system is with O in sealing ₂Be unique electron donor), place 33 ℃ of shaking table shaking culture; Remaining 10 bottles are filled the nitrogen processing, drive dissolved oxygen and top air in the saline bottle with high nitrogen gas stream as far as possible, treat to seal after nitrogen replaces bottle interior oxygen fully, with adding DCM in the past bottle of micro-sampling pin, make its concentration reach 100mg/L and (are dissolved with NaNO ₃Deng the culture system of inorganic salt respectively with NO ₂ ^-, NO ₃ ^-, SO ₄ ^2-, Fe ³⁺Be unique electron donor, and only be that the system of nutrient solution does not have electron donor with the deionized water), place 33 ℃ of anaerobism incubators to leave standstill cultivation.Chlorine ion concentration in the timing analysis nutrient solution is drawn the chlorine ion concentration change curve.Other gets the 200mL deionized water, is divided into 2 groups.Wherein one group adds FeCl ₃After be sub-packed in 2 250mL saline bottles, every bottle of 50mL.Another group direct packaging is in 2 saline bottles.110 ℃ of above-mentioned substratum sterilization 40min, treat the nutrient solution cooling after, add the flx-1 bacteria suspension that is in logarithmic phase, make the initial biological amount reach 38.4mg/L.Get and be dissolved with Fe ³⁺2 flask culture bases; Drive dissolved oxygen and top air in the saline bottle with high nitrogen gas stream as far as possible, treat to seal after nitrogen replaces bottle interior oxygen fully, add DCM and CB respectively with microsyringe; Make both concentration all reach 100mg/L, place 33 ℃ of anaerobism incubators to leave standstill cultivation.Remaining 2 bottles directly add DCM and CB, and sealing places 33 ℃ of shaking table shaking culture.Chlorine ion concentration in the timing analysis nutrient solution is drawn the chlorine ion concentration change curve.

The result is as shown in Figure 6.Bacterial strain flx-1 can not only be with O under the competent situation of oxygen ₂For unique electron donor is realized the dechlorination to DCM, also can be under anaerobic with NO ₂ ^-, NO ₃ ^-, SO ₄ ^2-And Fe ³⁺For unique electron donor is realized the dechlorination to DCM.And Fe ³⁺During for electron donor, dechlorination effect will be better than other electron donors far away, and dechlorination efficiency has reached 96.5% in the 20h, and under the same terms, with O ₂During for electron donor, dechlorination efficiency is merely 69.6% in the 20h.Bacterial strain flx-1 can be respectively with Fe ³⁺And O ₂Be electron donor, realize that the 24h dechlorination rate has reached 90.0% and 64.6% respectively to the CB dechlorination.Above experimental result shows that bacterial strain flx-1 is a kind of facultative aerobe, can not only under aerobic condition, realize the dechlorination to DCM, also can under anaerobic can be with NO ₂ ^-, NO ₃ ^-, SO ₄ ^2-And Fe ³⁺Be unique electron donor, realize the dechlorination to DCM, this bacterium also can be realized dechlorination to CB simultaneously, thereby in the improvement of chlorinated hydrocarbon pollutent, has broad application prospects.

SEQUENCE?LISTING

< 110>Zhejiang Polytechnical University

< 120>have the Pandora bacterium and the application thereof of methylene dichloride degradation capability

<130>

<160> 3

<170> PatentIn?version?3.4

<210> 1

<211> 1471

<212> DNA

<213> Pandoraea?sp.

<400> 1

cgccgtggcg?gctgccatta?acatgcagtc?gaacggcagc?acgggtgctt?gcacctggtg 60

gcgagtggcg?aacgggtgag?taatacatcg?gaacgtacct?tgtagtgggg?gatagctcgg 120

cgaaagccgg?attaataccg?catacgctct?gaggaggaaa?gcgggggacc?ttcgggcctc 180

gcgctacaag?agcggccgat?gtcagattag?ctagttggtg?aggtaaaagc?tcaccaaggc 240

gacgatctgt?agctggtctg?agaggacgac?cagccacact?gggactgaga?cacggcccag 300

actcctacgg?gaggcagcag?tggggaattt?tggacaatgg?gcgaaagcct?gatccagcaa 360

tgccgcgtgt?gtgaagaagg?ccttcgggtt?gtaaagcact?tttgtccgga?aagaaatcct 420

ctgggttaat?acctcggggg?gatgacggta?ccggaagaat?aagcaccggc?taactacgtg 480

ccagcagccg?cggtaatacg?tagggtgcaa?gcgttaatcg?gaattactgg?gcgtaaagcg 540

tgcgcaggcg?gttttgtaag?acggatgtga?aatccccggg?cttaacctgg?gaactgcatt 600

cgtgactgca?aggctagagt?atggcagagg?ggggtagaat?tccacgtgta?gcagtgaaat 660

gcgtagagat?gtggaggaat?accgatggcg?aaggcagccc?cctgggccaa?tactgacgct 720

catgcacgaa?agcgtgggga?gcaaacagga?ttagataccc?tggtagtcca?cgccctaaac 780

gatgtcaact?agttgttggg?gattcatttc?cttagtaacg?tagctaacgc?gtgaagttga 840

ccgcctgggg?agtacggtcg?caagattaaa?actcaaagga?attgacgggg?acccgcacaa 900

gcggtggatg?atgtggatta?attcgatgca?acgcgaaaaa?ccttacctac?ccttgacatg 960

tacggaatcc?tgctgagagg?tgggagtgct?cgaaagagaa?ccgtaacaca?ggtgctgcat 1020

ggctgtcgtc?agctcgtgtc?gtgagatgtt?gggttaagtc?ccgcaacgag?cgcaaccctt 1080

gtccttagtt?gctacgcaag?agcactctaa?ggagactgcc?ggtgacaaac?cggaggaagg 1140

tggggatgac?gtcaagtcct?catggccctt?atgggtaggg?cttcacacgt?catacaatgg 1200

tcggtacaga?gggctgccaa?accgcgaggt?ggagctaacc?ccagaaaacc?gatcgtagtc 1260

cggatcgcag?tctgcaactc?gactgcgtga?agctggaatc?gctagtaatc?gcggatcagc 1320

atgtcgcggt?gaatacgttc?ccgggtcttg?tacacaccgc?ccgtcacacc?atgggagtgg 1380

gttttgccag?aagtaggtag?cctaaccgca?aggagggtgc?ttaccacggc?aggattcatg 1440

actgggggaa?gtcgaatcaa?gtgtctgcca?c 1471

<210> 2

<211> 20

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 2

agagtttgat?cctggctcag 20

<210> 3

<211> 20

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 3

aaggaggtga?tccagccgca 20

Claims

1. a strain methylene dichloride degradation bacteria---Pandora bacterium (Pandoraea pnomenusa strain) flx-1; Be preserved in Chinese typical culture collection center, address: China, Wuhan; Wuhan University; 430072, deposit number: CCTCC NO:M 2011242, preservation date: on July 8th, 2011.

2. Pandora bacterium flx-1 as claimed in claim 1, it is characterized in that said Pandora bacterium flx-1 colony characteristics is following: bacterium colony is white in color, and neat in edge is smooth moistening; The form that transmission electron microscope is observed this thalline down is a tyrothricin, does not have flagellum, Gram-negative, and oxidase positive, starch hydrolysis and indole test are positive, can't utilize lactose.

3. the application of Pandora bacterium flx-1 as claimed in claim 1 in microbiological deterioration methylene dichloride, benzene, toluene, ethylbenzene, o-Xylol, chlorobenzene, THF, methyl alcohol or ethyl propenoate.

4. application as claimed in claim 3 is characterized in that said degraded at 25～35 ℃, pH 7～9, and salinity is counted with NaCl under 0～1% the condition and carried out.

5. application as claimed in claim 4 is characterized in that said degraded at 33 ℃, pH 7.3, and salinity is counted with NaCl under 0.66% the condition and carried out.