CN106591171A - Variovorax paradoxus DEA-3 and immobilization and application thereof - Google Patents
Variovorax paradoxus DEA-3 and immobilization and application thereof Download PDFInfo
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Abstract
The invention discloses variovorax paradoxus DEA-3 which is classified and named as variovorax paradoxus DEA-3, the preservation date is February 9, 2015, the full name of the preservation institution is China General Microbiological Culture Collection Center (CGMCC), and the preservation accession number is CGMCCNO.10543. The variovorax paradoxus DEA-3 can grow by use of N, N-dimethyl ethanolamine as the sole carbon source and nitrogen source, the 200mg /L N, N-dimethyl ethanolamine degradation rate of the variovorax paradoxus DEA-3 can reach 94.5%; the 200mg /L N, N-dimethyl ethanolamine degradation rate of an immobilization small ball prepared by the variovorax paradoxus DEA-3 can reach more than 90%, the immobilization small ball prepared by the variovorax paradoxus has a very good degradation effect, and in addition, the immobilization small ball prepared by the variovorax paradoxus is low in production cost, convenient to use, and suitable for promotion and use in the treatment of wastewater containing the N, N-dimethyl ethanolamine.
Description
Technical field
The present invention relates to technical field of environmental microorganism, more particularly toOne kind arguement variovorax DEA-3 And its immobilization and application。
Background technology
N, N- dimethylethanolamines are a kind of broad-spectrum organic intermediates, it is a kind of colourless or flaxen liquid, it can be used for synthesizing cationic exchanger resin, can be used as the stabilizer of coating, the additive of fuel oil, boiler scale preventative, with the development of polyurethane industrial, the important auxiliary agent of polyurethane processing is also used as.Because dimethylethanolamine is miscible with water, a large amount of dimethylethanolamines are had during use into waste water hence into environment, cause water pollution.
Microbial degradation has the advantages that low cost, efficiency high, non-secondary pollution, ecological recovery are good, is applied at many aspects, so filtering out N, N- dimethylethanolamines degradation bacteria is significant for process N, the waste water of N- dimethylethanolamines.
The content of the invention
The invention provides a kind of arguement variovorax DEA-3, it can be with N, and N- dimethylethanolamines are that sole carbon source and nitrogen source are grown, and to containing 200mg/L N in waste water, the degradation rate of N- dimethylethanolamines is up to 94.5%.
Such, one kind arguement variovorax DEA-3 is the technical scheme is that, its Classification And Nomenclature is Variovorax paradoxus DEA-3,PreservationDate is on 2 9th, 2015,PreservationUnit full name is Chinese microorganism strain preservation managementThe committeeCommon micro-organisms center, itsPreservationRegistration number is CGMCC NO.10543.
Present invention also offers immobilized spherule prepared by above-mentioned arguement variovorax DEA-3, its preparation is, using CaCl2 and being fixed of bamboo charcoal, concrete preparation process, to comprise the following steps：
1) with oese, a small amount of bacterial classification of picking is inoculated in LB fluid nutrient mediums in 30-37 DEG C on the slant medium, and shaken cultivation is to logarithmic phase under the conditions of 160-200r/min；
2) above-mentioned cultured bacterium solution is centrifuged into 2-3min under conditions of 10000-12000r/min, outwells supernatant, add the sterilized water of same volume, shake up rear 10000-12000r/min centrifugations 2-3min, so wash twice, with the aseptic aqueous suspension of same volume, make thalline liquid；
3) 0.8g sodium alginates are weighed to be dissolved in 20ml distilled water, adds 1000mg/L bamboo charcoals, ultrasonic disperse；
4) solution for obtaining step 3 syringe instills the 3%CaCl2 solution under normal temperature, is crosslinked in 4 DEG C after calcification 6h and obtains calcium alginate gel immobilized spherule, aseptic washing be stored in for twice 4 DEG C it is standby.
Step 1) described in LB culture mediums be：NaCl 10.0g/L, peptone 10.0g/L, dusty yeast 5.0g/L, pH7.0.
Present invention also offers application of the immobilized spherule in N, N- dimethyl ethanol amine degradations.
Application of the immobilized spherule in N, N- dimethyl ethanol amine degradations, specially：1) by immobilized spherule, aeration 15-24h is activated in waste water, and water temperature 26-30 DEG C is kept in activation process, and aeration rate is 1.2-1.5L/min；2) by the N in waste water, N- dimethylethanolamines concentration adjustment≤200mg/L, temperature is adjusted to 25-35 DEG C, pH value is adjusted to after 6.8-7.2, immobilized spherule after activation is added into waste water, aeration is carried out with 1.2-1.5L/min aeration rates, hydraulic detention time is more than 36h, wherein, volume ratio 18-20 of the addition of immobilized spherule and waste water：100.
Preferably, wastewater temperature of intaking is adjusted to 26-30 DEG C.
Preferably, hydraulic detention time is more than 72h.
Arguement variovorax DEA-3 of the present invention can be with N, and N- dimethylethanolamines are that sole carbon source and nitrogen source are grown, and to 200mg/L N, the degradation rate of N- dimethylethanolamines is up to 94.5%；Immobilized spherule prepared by arguement variovorax DEA-3 of the present invention, to 200mg/L N, the degradation rate of N- dimethylethanolamines reaches more than 90%, with good degradation effect, in addition, immobilized spherule has low production cost, it is easy to use, it is adapted to containing N, promotes the use of in the wastewater treatment of N- dimethylethanolamines.
Description of the drawings
Figure 1It is arguement variovorax DEA-3 thalline violet staining photo (1000x)；
Figure 2It is that arguement variovorax DEA-3 utilizes N, the growth degradation curve of N- dimethylethanolamines；
Figure 3Be under condition of different temperatures immobilized spherule to N, the impact of N- dimethyl ethanol amine degradations.
Figure 4It is immobilized spherule to N, the time dependent curve of N- dimethyl ethanol amine degradation clearancesFigure；
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification made to the inventive method, step or condition or replacement, belong to the scope of the present invention.
If not specializing, the conventional meanses that technological means used is well known to those skilled in the art in embodiment.
The separation and identification of arguement variovorax DEA-3 of embodiment 1
Arguement variovorax DEA-3 is obtained from Fine Chemical Works Sewage Disposal activated sludge tank separation screening.
The detailed process of screening is：
1) Fine Chemical Works Sewage Disposal activated sludge 5ml is taken, N containing 200mg/L is seeded to, in the minimal medium of N- dimethylethanolamines, its formula is：200mg/LN, N- dimethylethanolamines, 1.5g/LK2HPO4,0.5g/L KH2PO4,0.2g/L MgSO47H2O, 1.0g/L NaCl, 30 DEG C -37 DEG C of shaking table culture case was cultivated to after 7 days, mixed liquor 5m is gone to be seeded to containing 400mg/LN, in the minimal medium of N- dimethylethanolamines, filling a prescription is：400mg/L N, N- dimethylethanolamine, 1.5g/L K2HPO4,0.5g/L KH2PO4,0.2g/L MgSO47H2O, 1.0g/L NaCl, culture obtains pregnant solution after 7 days；
2) N is taken, N- dimethylethanolamines enrichment bacterium solution 1.0ml in adding 9.0ml sterilized waters, is fully mixed and is made into 10-1Pregnant solution, then draw 1.0ml is prepared 10-1Pregnant solution add in 9.0ml sterilized waters, fully mix and be made into 10-2Pregnant solution, by that analogy, gradient dilution is carried out to pregnant solution.The dilution 0.1ml for drawing each gradient is coated containing 200mg/LN, and on the nitrogen-free inorganic salts solid medium of N- dimethylethanolamines, its formula is：Per liter of K containing 1.5g2HPO4、0.5g KH2PO4、0.2g MgSO4·7H2O, 1.0g NaCl, 20g agar, 7.0,30 DEG C of culture 72h of pH.Picking single bacterium colony on inorganic salts solid medium after 72h more than, after cultivating 24 hours in the LB fluid nutrient mediums of 5ml, 3min is centrifuged under conditions of 10000r/min, remove supernatant, the sterilized water for adding 5ml shakes up, 3min is still centrifuged under conditions of 10000r/min, according to said method with twice of aseptic washing after, add the aseptic aqueous suspension bacterium of 5ml.1ml bacterium solution addition 100mlN are drawn, during N- dimethylethanolamines concentration is for the nitrogen-free inorganic salt liquid culture medium of 200mg/l, its formula is：Per liter of K containing 1.5g2HPO4、0.5g KH2PO4、0.2g MgSO4·7H2O, 1.0g NaCl, pH 7.0, in 160r/min, under the conditions of 30 DEG C after shaken cultivation 36h, with high performance liquid chromatography its degradation effect is surveyed.
One plant of bacterial strain of degradation efficiency highest is preserved, its thalline Gram's staining photo (1000x)As schemed 1Shown, Jing 16s rDNA identifications belong to arguement variovorax category (Variovorax paradoxus)；The 16S rDNA gene orders such as SEQ ID NO of the bacterial strain：Shown in 1, its Classification And Nomenclature of the bacterial strain was Variovorax paradoxus DEA-3, on 2 9th, 2015PreservationIn Chinese microorganism strainPreservationManagementThe committeeCommon micro-organisms center,PreservationAddress:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica,PreservationRegistration number is CGMCC NO.10543.
The arguement variovorax DEA-3 Main Biological is Gram-negative, with polar flagella motion, does not form gemma, chemoorganotrophy, strict aerobic, respiratory metabolism, non-zymocyte.
Free arguement variovorax DEA-3 of embodiment 2 can be with N, and N- dimethylethanolamines are the situation that sole carbon source and carbon source are grown
There is arguement to covet the single bacterium colony of picking one on the flat board of thermophilic bacterium DEA-3 from growth, in being inoculated in LB fluid nutrient mediums, in 30 DEG C, 12h (OD600 ≈ 1.0) is cultivated in 160rpm shaking tables, after 8000-12000rpm centrifugations, abandoning supernatant, with aseptic water washing three times, is resuspended in isopyknic sterilized water as seed liquor.
Load containing 200mg/L to N in 250mL triangular flasks, the minimal medium of N- dimethylethanolamines, its formula is：200mg/LN, N- dimethylethanolamines, 1.5g/L K2HPO4,0.5g/L KH2PO4,0.2g/L MgSO47H2O, 1.0g/L NaCl, PTA-2 seed liquors are accessed by the inoculum concentration of 1% (volume fraction), is cultivated in 30 DEG C, 160rpm shaking tables, every 2h 4mL nutrient solutions are taken out, sample abandoning supernatant Jing after 8000rpm centrifuges, it is resuspended in isopyknic sterilized water and determines OD600, growth curve of arguement variovorax DEA-3 using p-TSA is drawn according to experimental dataAs schemed 2Shown, it shows that Rhodococcus ruber PTA-2 can be with N, and N- dimethylethanolamines are that sole carbon source and carbon source are grown, and Jing is determined, and to 500mg/LN, the degradation rate of N- dimethylethanolamines is up to 94.5%.
The method that the arguement of embodiment 3 variovorax DEA-3 is prepared into immobilized spherule
1) with oese, a small amount of bacterial classification of picking is inoculated in LB fluid nutrient mediums in 30 DEG C on the slant medium, and shaken cultivation is to logarithmic phase under the conditions of 160r/min；
2) above-mentioned cultured bacterium solution is centrifuged into 3min under conditions of 10000r/min, outwells supernatant, add the sterilized water of same volume, shake up rear 10000r/min centrifugations 3min, so washed twice, with the aseptic aqueous suspension of same volume, make thalline liquid.
3) 0.8g sodium alginates are weighed to be dissolved in 20ml distilled water, adds 1000mg/L bamboo charcoals, ultrasonic disperse；
4) solution for obtaining step 3 syringe instills the 3%CaCl under normal temperature2Solution, is crosslinked after calcification 6h in 4 DEG C and obtains calcium alginate gel immobilized spherule, aseptic washing be stored in for twice 4 DEG C it is standby.
Wherein, step 1) described in LB culture mediums be：NaCl 10.0g/L, peptone 10.0g/L, dusty yeast 5.0g/L, pH7.0.
Application process of the immobilized spherule of embodiment 4 in N, N- dimethyl ethanol amine degradations
1) by the immobilized spherule of embodiment 3, aeration 24h is activated in waste water, water temperature 26-30 DEG C in activation process, and aeration rate is in 1.2L/min；
2) wastewater temperature is adjusted to respectively 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, pH value is adjusted in 6.8-7.2, N in water inlet waste water, N- dimethylethanolamine concentration is adjusted to and be adjusted to respectively after 500mg/L, and 185mL immobilized spherules are added in every liter of waste water, aeration is carried out with 1.5L/min aeration rates, hydraulic detention time is 72h, the degradation rate of N, N- dimethylethanolamineAs schemed 3It is shown.
When wastewater temperature is 30 DEG C, with the increase of hydraulic detention time, the degradation rate of N, N- dimethylethanolamineAs schemed 4It is shown.
FromFigure 3Can be seen that, too high or too low for temperature to process N to arguing variovorax DEA-3, the effect of N- dimethylethanolamines has suppression, and at 25 DEG C -35 DEG C, degradation rate can maintain more than 80% to optimal temperature scope.
FromFigure 4Can be seen that, when wastewater temperature of intaking is 30 DEG C, with the increase of hydraulic detention time, the degradation rate of N, N- dimethylethanolamine is elevated, when hydraulic detention time is 72h, the clearance of N, N- dimethylethanolamine, up to maximum, is more than 90%.
Waste water is configured and forms in embodiment 4, and its formula is：500mg/L N, N- dimethylethanolamine, 1.5g/L K2HPO4、0.5g/L KH2PO4、0.2g/L MgSO4·7H2O、1.0g/L NaCl。
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all any modification, equivalent substitution and improvements within the spirit and principles in the present invention, made etc., be should be included within the scope of the present invention.
Claims (3)
1. a kind of to argue variovorax DEA-3, its Classification And Nomenclature isVariovorax paradoxusDEA-3, preservation date is on 2 9th, 2015, and depositary institution's full name is Chinese microorganism strain preservation management committee common micro-organisms center, and its preservation registration number is CGMCC NO.10543.
2. the immobilized spherule that prepared by red arguement variovorax DEA-3 described in claim 1.
3. application of the immobilized spherule described in claim 2 in N, N- dimethyl ethanol amine degradations.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122480A (en) * | 2021-05-07 | 2021-07-16 | 河北萌帮生物科技有限公司 | Variovorax controversial, application and method for producing humic acid by converting lignite |
| CN113249263A (en) * | 2021-06-01 | 2021-08-13 | 江苏大学 | Bacillus subtilis UJSQL 01 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1571833A (en) * | 2001-10-17 | 2005-01-26 | 克莱沃尔有限公司 | Variovarax strains capable of degrading methyl tert-butyl either and their use |
| CN102787129A (en) * | 2012-07-20 | 2012-11-21 | 南京师范大学 | Amidase gene of Variovorax boronicumulans CGMCC 4969, and its application in biological degradation of acrylamide |
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- 2016-04-01 CN CN201610204789.1A patent/CN106591171A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1571833A (en) * | 2001-10-17 | 2005-01-26 | 克莱沃尔有限公司 | Variovarax strains capable of degrading methyl tert-butyl either and their use |
| CN102787129A (en) * | 2012-07-20 | 2012-11-21 | 南京师范大学 | Amidase gene of Variovorax boronicumulans CGMCC 4969, and its application in biological degradation of acrylamide |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122480A (en) * | 2021-05-07 | 2021-07-16 | 河北萌帮生物科技有限公司 | Variovorax controversial, application and method for producing humic acid by converting lignite |
| CN113122480B (en) * | 2021-05-07 | 2022-04-15 | 河北萌帮生物科技有限公司 | Variovorax controversial, application and method for producing humic acid by converting lignite |
| CN113249263A (en) * | 2021-06-01 | 2021-08-13 | 江苏大学 | Bacillus subtilis UJSQL 01 and application thereof |
| CN113249263B (en) * | 2021-06-01 | 2022-04-29 | 江苏大学 | Bacillus subtilis UJSQL 01 and application thereof |
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