CN101914467B - Achromobacter xylosoxidans strain and application thereof - Google Patents
Achromobacter xylosoxidans strain and application thereof Download PDFInfo
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- CN101914467B CN101914467B CN2010102246506A CN201010224650A CN101914467B CN 101914467 B CN101914467 B CN 101914467B CN 2010102246506 A CN2010102246506 A CN 2010102246506A CN 201010224650 A CN201010224650 A CN 201010224650A CN 101914467 B CN101914467 B CN 101914467B
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Abstract
The invention discloses an Achromobacter xylosoxidans strain and application thereof. The strain is Achromobacter xylosoxidans N4 with the collection number of CGMCC No.3632. The Achromobacter xylosoxidans N4 GCMCC No.3632 can quickly and efficiently degrade isatin and o-aminobenzoic acid, and can be used for biologically treating calico printing wastewater.
Description
Technical field
The present invention relates to an Achromobacter xylosoxidans strain and application thereof.
Background technology
The denim dyeing waste water contains indigo in a large number, causes colourity higher, and indigo biodegradability is poor, is difficult to contain indigo dyeing waste water with direct processing of microorganism.Use the laccase indigo treatment of dyeing and printing of decolouring, with the photooxidation method of present employing physics or throw the indigo method of chemical reagent sedimentation and handle and compare, have non-secondary pollution, reusable, advantage that cost is low, and not only contain indigo a kind of dyestuff in the denim printing and dyeing mill waste water at present, also has a large amount of black sulfide dyes, if adopt the laccase decolouring indigo, sulphur black just is easy to precipitation and reclaims, otherwise, only just together, also need further to handle this waste two kinds of dye precipitated with chemical reagent precipitation dyeing waste water.
Use the laccase decolouring and contain indigo dyeing waste water, decolouring back BOD significantly raises, and COD is constant substantially, and is still higher, can not qualified discharge.Contain istain and anthranilic acid in the decolouring product, use the indigo conceptual phase that still is in of laccase decolouring at present, the microbial degradation method report of decolouring after product istain and anthranilic acid is less.
Summary of the invention
The purpose of this invention is to provide an Achromobacter xylosoxidans strain and application thereof.
Bacterial strain provided by the invention is Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4, and its preservation registration number is CGMCC № .3632.
Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 02 08th, 2010, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC № .3632.
Provided by the inventionly be applied as the application of Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 in degraded istain or istain and anthranilic acid.
In the described application, the method for described degraded istain is Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 to be inoculated in the system that contains istain cultivate.
In the described application, the method for described degraded istain and anthranilic acid is Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 to be inoculated in the system that contains istain and anthranilic acid cultivate.
In the described application, described Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is that 5% inoculum size be inoculated in the system that contain istain with cultivating the fermented liquid that obtains with volume percent earlier through 28 ℃ of liquid culture 24-48h.The substratum that described liquid culture is used is by extractum carnis 3g/L, peptone 10g/L, the substratum that NaCl 5g/L and water are formed.
In the described application, above-mentioned Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 the system that contains istain or contain istain and the system of anthranilic acid in the temperature of cultivating be 25 ℃-35 ℃, be preferably 28 ℃.
In the described application, the concentration of istain is below the 500mg/L, to be preferably 100mg/L in the described system that contains istain.In the described system that contains istain and anthranilic acid, in the system of described istain and anthranilic acid, the concentration of istain and anthranilic acid is below the 500mg/L, is preferably 100mg/L.
Experiment showed, Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 can degrade quickly and efficiently istain and anthranilic acid, can be used for biological process and handle indigo dyeing waste water after the laccase decolouring.Achromobacter xylosoxidans (Achromobacter xylosoxidans) the N4 CGMCC № .3632 istain of both can having degraded, also can degrading o-aminobenzoic acid, can be used for containing separately the system of istain or anthranilic acid, the mixed system that also can be used for them, degraded istain or anthranilic acid.
Description of drawings
Fig. 1 is 28 ℃, and Achromobacter xylosoxidans under the 180rpm shaking table condition (Achromobacter xylosoxidans) N4CGMCC № .3632 bacterial strain is to the degradation curve of 100mg/L istain.
Fig. 2 is 28 ℃, and Achromobacter xylosoxidans under the 180rpm shaking table condition (Achromobacter xylosoxidans) N4CGMCC № .3632 bacterial strain is to the degradation curve of 100mg/L istain and 100mg/L o-amino benzoyl acid mixture.
Fig. 3 is 28 ℃, and Achromobacter xylosoxidans under the 180rpm shaking table condition (Achromobacter xylosoxidans) N4CGMCC № .3632 bacterial strain is to the degradation curve of 50-150mg/L anthranilic acid.
Fig. 4 is 28 ℃, and Achromobacter xylosoxidans under the 180rpm shaking table condition (Achromobacter xylosoxidans) N4CGMCC № .3632 bacterial strain is to the degradation curve of 200-500mg/L anthranilic acid.
Embodiment
Experimental technique described in the following embodiment if no special instructions, is ordinary method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels.
Separation, purifying and the evaluation of embodiment 1, Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632.
In May, 2009, the application sterilized water dilutes the active sludge (picking up from Zhejiang Province Haining City denim printing and dyeing mill) in denim treatment of dyeing wastewater pond, (the 1L solid medium contains extractum carnis 3g to be coated with solid medium, peptone 10g, NaCl 5g, agar 15g, all the other are water) flat board, be separated to 18 strain bacterium, adopt the method purifying bacterial strain of plate streaking.18 strain bacterium after the separation and purification are inserted liquid nutrient medium with transfering loop respectively, and (the 1L substratum contains extractum carnis 3g, peptone 10g, NaCl 5g, all the other are water) carry out liquid culture, culture temperature is 28 ℃, and 180rpm joins in istain or the o-amino benzoyl aqueous acid with 5% (volumn concentration) inoculum size after cultivating 24-48h, the liquid chromatogram measuring result shows N4 bacterial strain can degrade quickly and efficiently istain and anthranilic acid, can be used for biological process and handles indigo dyeing waste water.
N4 bacterial strain Physiology and biochemistry experimental result is as shown in table 1; 16S rRNA gene order is shown in sequence in the sequence table 1.According to cell microscopic morphology, Physiology and biochemistry data and 16S rRNA gene order data, bacterial strain N4 is accredited as Achromobacter xylosoxidans (Achromobacter xylosoxidans), and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 02 08th, 2010 and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC № .3632.
Table 1, physical and chemical experiment result
One, to the degradation effect proof test of 100 mg/L istains
The substratum of Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632:
Solid medium (1L): extractum carnis 3g, peptone 10g, NaCl 5g, agar 15g, all the other are water.
Liquid nutrient medium (1L): extractum carnis 3g, peptone 10g, NaCl 5g, all the other are water.
Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is inoculated in the liquid nutrient medium, 28 ℃, cultivates 24-48h under the 180 seal m conditions, to the OD of nutrient solution
600Be 0.6, then in non-sterile environment, is that 5% amount is inoculated in the aqueous solution of 100 mg/L istains with nutrient solution according to volume percent, the control group adding is 5% distilled water according to volume percent, 28 ℃, 180rpm cultivates, centrifugal in the different time sampling, supernatant liquor 0.22um filtering with microporous membrane, using high performance liquid chromatograph (adopts the U.S. Agilem of Agilent company 1200 high performance liquid chromatographs to analyze, chromatographic column is a C18 post (4.6 * 150mm), moving phase: V (methyl alcohol): V (water)=60: 40, flow velocity 1 mL/min, detector are UV-visible detection device, the detection wavelength is 254nm, and sample size is 10 μ L) detect the content of istain.Istain is to be 100% with initial content, and other detection level value is a relative content for the percentage composition that the ratio with initial content converts in the degradation process.Novel substance is to be 100% with high-content in the degradation process, and other detection level value is a relative content for the percentage composition that converts with the most high-load ratio.
The result shows that under the effect of N4 bacterial strain, the yellow of the istain aqueous solution is faded.Liquid chromatography shows that under the effect of N4 bacterial strain, the istain peak diminishes gradually, and until disappearance, and control group istain before and after experiment does not have remarkable change.
As shown in Figure 1,12h is cultivated in the 180rpm concussion in the 100 mg/L istain aqueous solution of N4 bacterial strain, is 94.42% ± 0.29 to the degradation rate of istain, and 18h reaches degraded fully; The N4 bacterial strain produces a kind of novel substance in the degraded istain, this material reaches the highest at the 18h relative content, then reduces gradually, and content during 48h (8.05%) is lower than experiment initial content (24.12%).Control group istain concentration under the same experiment condition remains unchanged before and after experiment substantially.
Two, the compliance test result of different concns istain degraded is tested
Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is to the degraded of different concns istain
Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is inoculated in the liquid nutrient medium, and at 28 ℃, 180rpm cultivates 24-48h, and thalline is collected in centrifugal back, (contains 1.5gK in the 1L water with buffered soln
2HPO
4, 0.5gKH
2PO
4, 0.2gMgSO
4.7H
2O, 1.0gNaCl, 1.0g (NH
4)
2SO
4, all the other are water) and wash bacterium 3 times, obtain OD with the resuspended thalline of buffered soln
600It is 0.6 bacteria suspension, is that 5% amount is inoculated into 50mg/L respectively with bacteria suspension according to volume percent, 100mg/L, 150mg/L, 200mg/L, 300mg/L, in the istain aqueous solution of 400mg/L or 500mg/L, 28 ℃, the 180rpm concussion, centrifugal in the different time sampling, supernatant liquor 0.22um filtering with microporous membrane, using high performance liquid chromatograph (adopts the U.S. Agilent of Agilent company 1200 high performance liquid chromatographs to analyze, chromatographic column is a C18 post (4.6 * 150mm), moving phase: V (methyl alcohol): V (water)=60: 40, flow velocity 1mL/min, detector is a UV-visible detection device, the detection wavelength is 254nm, and sample size is 10 μ L) detect the content of istain.
The result shows, after inserting Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632, the istain of 50mg/L, 100mg/L, 150mg/L, 200mg/L, 300mg/L, 400mg/L, 500mg/L reaches degraded fully respectively in 16h, 24h, 24h, 36h, 36h, 72h, 84h; The novel substance of Sheng Chenging reaches the highest at 16h, 24h, 24h, 24h, 36h, 48h, 60h respectively simultaneously, then is lower than the experiment initial content respectively at 32h, 56h, 56h, 60h, 72h, 96h, 96h.
The substratum of Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632:
Solid medium (1L): extractum carnis 3g, peptone 10g, NaCl 5g, agar 15g, all the other are water.
Liquid nutrient medium (1L): extractum carnis 3g, peptone 10g, NaCl 5g, all the other are water.
Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is inoculated in the liquid nutrient medium, 28 ℃, cultivates 24-48h under the 180rpm condition, to the OD of nutrient solution
600Be 0.6, then in non-sterile environment, is that 5% amount is inoculated in the mixed system that contains 100mg/L istain and 100mg/L anthranilic acid with fermented liquid according to volume percent, control group is 5% distilled water for adding volume percent in mixed system, 28 ℃, 180rpm cultivates, centrifugal in the different time sampling, supernatant liquor 0.22um filtering with microporous membrane, using high performance liquid chromatograph (adopts the U.S. Agilent of Agilent company 1200 high performance liquid chromatographs to analyze, chromatographic column is a C18 post (4.6 * 150mm), moving phase: V (methyl alcohol): V (water)=60: 40, flow velocity 1mL/min, detector are UV-visible detection device, the detection wavelength is 254nm, and sample size is 10 μ L) detect the content of istain and anthranilic acid.With initial content is 100%, and other detection level value is a relative content for the percentage composition that the ratio with initial content converts in the degradation process.
The result shows that under the effect of N4 bacterial strain, the yellow of solution is faded.Liquid chromatography result shows, under the effect of N4 bacterial strain, the istain peak diminishes gradually, until disappearance, the anthranilic acid peak value presents the back downward trend that rises earlier, may be the influence of the novel substance that produces in the istain degradation process, and control group istain and anthranilic acid peak value before and after experiment have remarkable change to the anthranilic acid peak value.
As shown in Figure 2, the 180rpm concussion in the mixed system of 100mg/L istain and 100mg/L anthranilic acid of N4 bacterial strain is cultivated in the 42h, thalline is in the cessation of growth cessation phase, and 42h-72h is a logarithmic phase, and 42h-66h o-amino benzoyl acid content during this period reduces rapidly.Cultivate 60h, the N4 bacterial strain reaches 100% to the degradation rate of istain, and the degradation rate of anthranilic acid is reached 53.26%; Cultivate 66h, the N4 bacterial strain is 100% to the degradation rate of istain, is 93.72% to the degradation rate of anthranilic acid.
The substratum of Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632:
Solid medium (1L): extractum carnis 3g, peptone 10g, NaCl 5g, agar 15g, all the other are water.
Liquid nutrient medium (1L): extractum carnis 3g, peptone 10g, NaCl 5g, all the other are water.
One, Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is to the degraded of 100mg/L o-amino benzoyl acid solution
Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is inoculated in the liquid nutrient medium, 28 ℃, cultivates 24-48h under the 180rpm condition, to the OD of nutrient solution
600Be 0.6, then in non-sterile environment, is that 5% amount is inoculated in the aqueous solution of 100mg/L anthranilic acid with nutrient solution according to volume percent, the control group adding is 5% distilled water according to volume percent, 28 ℃ of 180rpm concussions are cultivated, centrifugal in the different time sampling, supernatant liquor 0.22um filtering with microporous membrane, using high performance liquid chromatograph (adopts the U.S. Agilent of Agilent company 1200 high performance liquid chromatographs to analyze, chromatographic column is a C18 post (4.6 * 150mm), moving phase: V (methyl alcohol): V (water)=60: 40, flow velocity 1mL/min, detector is a UV-visible detection device, and the detection wavelength is 254nm, and sample size is 10 μ L) detect the content of anthranilic acid.
Liquid chromatography result shows that under the effect of N4 bacterial strain, the anthranilic acid peak value diminishes gradually, and control group anthranilic acid peak value before and after experiment does not have remarkable change.
12h is cultivated in the 180rpm concussion in 100mg/L o-amino benzoyl aqueous acid of N4 bacterial strain, and the o-amino benzoyl acid concentration is 68.01 ± 9.66mg/L, and 24h o-amino benzoyl acid concentration is 3.74 ± 0.05mg/L.Control group o-amino benzoyl acid concentration under the same experiment condition remains unchanged before and after experiment substantially.
Two, Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 under condition of different temperatures to the degraded of anthranilic acid
Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is inoculated in the liquid nutrient medium, and at 28 ℃, 180rpm cultivates 24-48h, to the OD of nutrient solution
600Be 0.6, then in non-sterile environment, is that 5% amount is inoculated in the o-amino benzoyl aqueous acid of 100mg/L with nutrient solution according to volume percent, under the room temperature situation (10 ℃-20 ℃) respectively, 25 ℃, 30 ℃, under 35 ℃ or the 40 ℃ of conditions, the 180rpm concussion, centrifugal in the different time sampling, supernatant liquor 0.22um filtering with microporous membrane, using high performance liquid chromatograph (adopts the U.S. Agilent of Agilent company 1200 high performance liquid chromatographs to analyze, chromatographic column is a C18 post (4.6 * 150mm), moving phase: V (methyl alcohol): V (water)=60: 40, flow velocity 1mL/min, detector is a UV-visible detection device, and the detection wavelength is 254nm, and sample size is 10 μ L) detect the content of anthranilic acid.
The result shows Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 at 10 ℃-40 ℃ equal energy degrading o-aminobenzoic acids, and the optimal temperature scope is 25 ℃-35 ℃, and the 24h degradation rate is all more than 85%; Under the bigger room temperature situation of temperature fluctuation amplitude (10 ℃-20 ℃), the 24h degradation rate is 28.43%, and the 72h degradation rate is 89.18%; The 24h degradation rate is 22.61% under 40 ℃ of conditions, and the 48h degradation rate is 92.26%.
Three, Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is to the degraded of different concns anthranilic acid
Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4 CGMCC № .3632 is seeded in the liquid nutrient medium, and at 28 ℃, 180rpm cultivates 24-48h, to the OD of nutrient solution
600Be 0.6, then in non-sterile environment, is that 5% amount is inoculated into 50mg/L respectively with nutrient solution according to volume percent, 75mg/L, 100mg/L, 150mg/L, 200mg/L, 300mg/L, in the o-amino benzoyl aqueous acid of 400mg/L or 500mg/L, 28 ℃, the 180rpm concussion, centrifugal in the different time sampling, supernatant liquor 0.22um filtering with microporous membrane, using high performance liquid chromatograph (adopts the U.S. Agilent of Agilent company 1200 high performance liquid chromatographs to analyze, chromatographic column is a C18 post (4.6 * 150mm), moving phase: V (methyl alcohol): V (water)=60: 40, flow velocity 1mL/min, detector is a UV-visible detection device, and the detection wavelength is 254nm, and sample size is 10 μ L) detect the content of anthranilic acid.
The result as shown in Figure 3 and Figure 4, this bacterial strain is respectively 61.19%, 49.73%, 46.49% and 8.09% to the degradation rate of the o-amino benzoyl aqueous acid 16h of 50mg/L, 75mg/L, 100mg/L and 150mg/L, the degradation rate of 24h is respectively 94.05%, 95.06%, 96.27% and 18.69%, is 93% to the degradation rate of the o-amino benzoyl aqueous acid 32h of 150mg/L; Degradation rate to the o-amino benzoyl aqueous acid 1d of 200mg/L is 4.64%, and the degradation rate of 2d is 97.92%; 3d before the o-amino benzoyl aqueous acid of 300mg/L, 400mg/L is not seen degraded, degraded fully in 4d one day; To the o-amino benzoyl aqueous acid of 500mg/L, then before 4d do not see degraded, and degraded fully in 5d one day.
Claims (9)
1. Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4, its preservation registration number is CGMCC № .3632.
2. Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4CGMCC № .3632 is in degraded istain or the application in degraded istain and anthranilic acid.
3. application according to claim 2 is characterized in that: described degraded istain is Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4CGMCC № .3632 to be inoculated in the system that contains istain cultivate.
4. application according to claim 2, it is characterized in that: described degraded istain and anthranilic acid are Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4CGMCC № .3632 to be inoculated in the system that contains istain and anthranilic acid cultivate.
5. according to claim 3 or 4 described application, it is characterized in that: described culture temperature is 25 ℃-35 ℃.
6. application according to claim 3 is characterized in that: the concentration of istain is below the 500mg/L in the described system that contains istain.
7. application according to claim 4 is characterized in that: the concentration of istain and anthranilic acid is below the 500mg/L in the described system that contains istain and anthranilic acid.
8. application according to claim 5, it is characterized in that: described Achromobacter xylosoxidans (Achromobacter xylosoxidans) N4CGMCC № .3632 is earlier through 28 ℃ of liquid culture 24-48 hours, inoculate the system that contains istain or contain istain and the system of anthranilic acid in.
9. application according to claim 8 is characterized in that: the substratum that described liquid culture is used is by extractum carnis 3g/L, peptone 10g/L, the substratum that NaCl5g/L and water are formed.
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| CN102876612B (en) * | 2012-10-12 | 2013-08-28 | 中国地质大学（北京） | Culture for cometabolic degradation of 1, 1-dichloroethylene (DCE) by using benzene as matrix and usage method of culture |
| CN104232522B (en) * | 2014-08-28 | 2017-12-12 | 广西科学院 | A kind of Achromobacter xylosoxidans bacterial strains for producing carboxymethylcelluloenzyme enzyme |
| CN112694999B (en) * | 2021-01-27 | 2022-09-02 | 石河子大学 | Achromobacter xylosoxidans, microbial inoculum comprising same, and preparation method and application thereof |
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