CN106591169A - NMP (N-Methyl Pyrrolidone) degradation bacillus NMP-2 and application thereof - Google Patents
NMP (N-Methyl Pyrrolidone) degradation bacillus NMP-2 and application thereof Download PDFInfo
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Abstract
The invention relates to an NMP (N-Methyl Pyrrolidone) degradation bacillus NMP-2 and application thereof. The invention provides a degradation bacterium strain NMP-2 for NMP pollution elimination and application thereof, and belongs to the technical field of biological high technology. Through identification, the bacterium strain is the bacillus, has the major biological characteristic of G<+>, the coryneform thalli and endogenous spores, and realizes the aerobic growth. The growth can be performed by using the NMP as a unique carbon source and nitrogen source; and the NMP can be thoroughly mineralized to generate ammonia nitrogen. Under the laboratory flask shaking condition, the degradation rate of the bacterium strain on 500mg/L of NMP reaches 100 percent; and the problem of difficult NMP biodegradation in wastewater treatment is solved.
Description
Technical field
The present invention provides a kind of N-Methyl pyrrolidone（Abbreviation NMP）Degradation bacteria and its microbial inoculum of production, belong to biological high-tech technical field, are the pollutant in the method degrading waste water using microorganism, it is adaptable to modern waste water treatment process.
Background technology
N-Methyl pyrrolidone be it is a kind of be important industrial chemicals, it is a kind of strong and good stability the polar solvent of selectivity, it is widely used in the purification of aromatic hydrocarbons extraction, acetylene, alkene, alkadienes, the solvent of polyvinylidene fluoride, the electrode auxiliary material of lithium ion battery, solvent when synthetic gas desulfurization, lube oil finishing, lubricating oil antifreeze, olefinic extracting agent, the polymerization of indissoluble engineering plastics, agricultural herbicide, insulant, production of integrated circuits, semicon industry precision instrument, wiring board clean, PVC tail gas recycles, abluent, color additive, dispersant etc..It is also used for the solvent of polymer and the medium of polyreaction, such as engineering plastics and aramid fiber.
N-Methyl pyrrolidone is widely present in the production waste water such as organic synthesiss, pesticide, dyestuff, adopts physico-chemical process for the process containing N-Methyl pyrrolidone waste water at present more, and its is relatively costly, and easily causes secondary pollution；And bioremediation technology has huge potentiality because microorganism itself all kinds of different organic pollutions can be degraded and be converted.Therefore, screening obtains removing the efficient bacterium of N-Methyl pyrrolidone and is significant from natural environment.
The content of the invention
Present invention aims to the practical problem and demand in production practices, develop a kind of N-Methyl pyrrolidone degradation bacteria strains and its microbial inoculum, discharged wastewater met the national standard can be made with the N-Methyl pyrrolidone in degrading waste water using this bacterial strain or microbial inoculum, while reducing production and use cost.
The present invention provides a kind of degradation bacteria for eliminating N-Methyl pyrrolidone pollution, and its bacterial strain is one plant of gram positive bacterium NMP-2, and on 2 9th, 2015 China Committee for Culture Collection of Microorganisms's common micro-organisms center (address was preserved in：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), deposit number CGMCC NO.10544 are identified as bacillus cereuss (Bacillus sp.).Main Biological is G+, exponential phase thalline is shaft-like, with endospore, aerobic growth.Bacterium colony is circular, opaque, neat in edge, in faint yellow；Can with N-Methyl pyrrolidone as sole carbon source, nitrogen source grown, and be thoroughly mineralized to produce ammonia nitrogen.Under the conditions of laboratory shake flask 36h to the degradation rate of 500mg/L N-Methyl pyrrolidone up to 100%.The bacterium can be produced with the general Zymolysis Equipment of fermentation industry.
The present invention provides the microbial inoculum containing bacillus cereuss NMP-2.
The present invention provides the production method containing the bacillus cereuss NMP-2 microbial inoculums, including：Inclined-plane kind-shaking flask induction-seed tank induces-produces tank-product, and the packaging dosage form of the product is liquid bacterial agent or solid absorption microbial inoculum.
The production method of the microbial inoculum, specially：
1）The test tube kind of bacillus cereuss NMP-2 is inoculated in the LB culture medium containing 500mg/L N-Methyl pyrrolidone, inducing culture is to logarithmic (log) phase；
2）By step 1）Cultured strain is inoculated with into 500 liters of seed tanks containing 500mg/L N-Methyl pyrrolidone, induced growth to logarithmic (log) phase, i.e. seed liquor by 5% inoculum concentration；
3）By step 2）The seed liquor accesses production tank and carries out fermentation culture by 5% inoculum concentration, and production tank used medium is identical with seed tank culture base, and the culture fluid after the completion of fermentation is microbial inoculum.
Step 1）Described in LB culture medium prescriptions be：500mg/L N-Methyl pyrrolidone, NaCl 10.00g/L, peptone 5.00g/L, yeast powder 5.00g/L, pH7.0.
Step 2）The formula of the culture medium mass percent used by described seed tank is：N-Methyl pyrrolidone 0.5%, glucose 0.8%, K2HPO40.2%, MgSO40.05%, NaCl 0.01%, CaCO30.3%, yeast extract 0.02%, pH value 7.2-7.5.
Step 2）The seed tank and step 3）In the incubation of described production tank, the ventilation of filtrated air is 1：0.6-1.2, mixing speed is 220 revs/min, and cultivation temperature is 3 DEG C, step 2）With 3）Whole technological process incubation time be 48-60 hours.Thalline quantity reaches 1,000,000,000/more than ml after fermentation ends, and culture fluid goes out tank and is directly distributed into liquid agent with plastic barrel or Packaging Bottle after the completion of fermentation.
The present invention also provides application of the microbial inoculum containing bacillus cereuss NMP-2 in N-Methyl pyrrolidone degraded.
Provided by the present invention to eliminate degradation bacteria NMP-2 that N-Methyl pyrrolidone pollutes, laboratory biological degradation experiment result shows, to the degradation rate of 500mg/L NMP 100% is reached.
The degradation bacterial agent of the invention production has low production cost, and easy to use, the good advantage of removal effect is adapted to be promoted the use of in the wastewater treatment containing NMP.The present invention is for preserving the ecological environment, and the protection mankind's is healthy, reduces cost for wastewater treatment and has great importance.
Description of the drawings
Fig. 1 is the thalline violet staining photo of bacterial strain NMP-2（1000x）；
Fig. 2 is growth degradation curves of the bacterial strain NMP-2 using N-Methyl pyrrolidone；
Fig. 3 is impact of the inoculum concentration to N-Methyl pyrrolidone degradation efficiency；
Fig. 4 is impact of the temperature to N-Methyl pyrrolidone degradation efficiency.
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 are well known to those skilled in the art in embodiment.
The separation and identification of embodiment 1, bacterial strain
During the activated sludge 3.0ml obtained from the purification tank for liquid waste containing N-Methyl pyrrolidone adds N-Methyl pyrrolidone concentration for the 100ml inorganic salt liquid cultures of 500mg/L, its formula is：Per liter contains 1.50 g K2HPO4、0.50 g KH2PO4、0.20 g MgSO4×7H2O, 1.00 g NaCl, pH 7.0, in 160r/min, shaken cultivation under the conditions of 37 DEG C is transferred in fresh minimal medium every 36h by 3% inoculum concentration, continuous switching 5 times.
Enrichment bacterium solution 1.0ml obtained above is taken, in adding 9.0ml sterilized water, 10 is made into-1Pregnant solution, then draw 1.0ml is prepared 10-1Pregnant solution add in 9.0ml sterilized water, fully mix and be made into 10-2Pregnant solution, by that analogy, gradient dilution is carried out to pregnant solution.By that analogy, gradient dilution is carried out to pregnant solution.The diluent 0.1ml for drawing each gradient coats the concentration containing N methyl pyrrolidones (formula is for 500mg/L inorganic salt solid mediums：Per liter of K containing 1.50g2HPO4、0.50g KH2PO4、0.20g MgSO4×7H2O, 1.00g NaCl, 12.00g agar, pH 7.0) on, 37 DEG C are cultivated 2 days.Picking single bacterium colony on inorganic salt solid medium after 2 days more than, after cultivating 24 hours in the LB fluid mediums of 3.0ml, LB liquid culture based formulas are：NaCl 10.00g/L, peptone 5.00g/L, yeast powder 5.00g/L, pH7.0 is centrifuged 2min under conditions of 12000r/min, supernatant is removed, the sterilized water for adding 3.0ml shakes up, and 2min is still centrifuged under conditions of 12000r/min, according to said method with twice of aseptic washing after, add the resuspended bacterium of 3.0ml sterilized water.It is in the inorganic salt liquid culture medium of 500mg/L, in 220r/min, under the conditions of 37 DEG C after shaken cultivation 72h, with high performance liquid chromatography its degradation effect to be surveyed to draw 1.0ml bacterium solution addition 100ml concentration containing N-Methyl pyrrolidone.One plant of higher bacterial strain of degradation efficiency is preserved, subsequent experimental is carried out.
Degradation bacteria strains are inoculated on LB solid mediums, its formula is：Colonial morphology is observed after NaCl 10.00g/L, peptone 10.00g/L, yeast powder 5.00g/L, agar 20.00g/L, pH7.0,37 DEG C constant temperature culture 48h, observation by light microscope thalli morphology is used（1000X）, as shown in Figure 1.Bacterial strain Physiology and biochemistry identification according to《Common bacteria system identification handbook》Carry out.It is accredited as bacillus（Bacillus sp.）；It is named as：NMP-2.Main Biological is G+, exponential phase thalline is shaft-like, with endospore, aerobic growth.Can with N methyl pyrrolidones as sole carbon source, nitrogen source grown, and be thoroughly mineralized to produce ammonia nitrogen, as shown in Figure 2.The bacterium can be produced with the general Zymolysis Equipment of fermentation industry.
The laboratory biological degradation experiment of embodiment 2
Impact of 2.1 inoculum concentrations to N-Methyl pyrrolidone degradation efficiency
NMP-2 was cultivated to the logarithmic (log) phase later stage in LB culture medium, and by 0.1,0.5,1,2,3,4%, 5% inoculum concentration the minimal medium containing 500mg/LN- methyl pyrrolidones is respectively connected to（Minimal medium formula is：Per liter of K containing 1.5g2HPO4、0.5g KH2PO4、0.2g MgSO4·7H2O, 1.0g NaCl, pH=7.0 are hereafter same）In, 30 DEG C, 160r/min shaking table cultures sample the content of N-Methyl pyrrolidone after 72h, as a result it is as shown in Figure 3, measurement result shows, arranges from low to high by inoculum concentration that the degradation rate of N-Methyl pyrrolidone is followed successively by 30%, 58%, 75%, 85%, 95%, 96%, 98%.As can be seen here, the size of inoculum concentration has direct relation to the degradation efficiency of N-Methyl pyrrolidone, when inoculum concentration is less than 2%, improve inoculum concentration, the degradation efficiency of N-Methyl pyrrolidone is significantly increased, but when inoculum concentration reaches more than 3%, degradation rate only has micro raising, may now enzyme reaction close saturation.
2. impact of 2 temperature to N-Methyl pyrrolidone
NMP-2 is cultivated to late log phase in LB culture medium, it is connected in the minimal medium of the methyl pyrrolidones of N containing 500mg/L by 5% inoculum concentration, be respectively placed in 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, at a temperature of 40 DEG C, 160r/min shaking table cultures, the content of wherein N-Methyl pyrrolidone is determined after 72h, as a result as shown in Figure 4.At 15 DEG C, NMP-2 is 14% to the degradation rate of N-Methyl pyrrolidone；In the range of 20 DEG C～30 DEG C, as the rising degradation bacteria degraded N methyl pyrrolidone performances of temperature substantially rise, at 30 DEG C, degradation rate is up to 98%.And when temperature reaches 37 DEG C, NMP-2 drops to 49% to the degradation rate of N-Methyl pyrrolidone.Data above illustrates that the most suitable degradation temperature of NMP-2 is 30 DEG C, and too low or too high ambient temperature can suppress the degraded of N-Methyl pyrrolidone.
The production of embodiment 3 is implemented
The original seed of microorganism N-Methyl pyrrolidone degradation bacteria NMP-2 of the present invention is activated on culture dish, and determines degradation property, be inoculated in standby on test tube slant.Test tube kind is inoculated in the culture medium of LB containing 200ml (LB culture medium prescription g/L：N-Methyl pyrrolidone 0.5, yeast powder 5.0, peptone 10.0, NaCl 10.00, pH=7. 0) 1000ml shaking flasks in, constant-temperature shaking culture to logarithmic (log) phase, prepare inoculation seed tank.500 liters of seed tank, 400 liters of inventory,（The formula of culture medium mass percent is：N-Methyl pyrrolidone 0.5%, glucose 0.8%, K2HPO4
0.2%, MgSO40.05%, NaCl 0.01%, CaCO30.3%, yeast extract 0.02%, pH value 7.2-7.5.）Feed intake 115 DEG C of high pressure moist heat sterilizations after finishing, and after being cooled to 30 DEG C, above-mentioned cultured shaking flask strain is accessed into 500 liters of seed tanks by 5% inoculum concentration, cultivates to exponential phase, i.e. seed liquor, and mixing speed is 220 revs/min, and filtrated air intake is 1: 0.8.5 tons of tankage size of production, 4.5 tons of inventory.The 1kg/cm of tank 1. is produced after feeding intake2Pressure under, 115 DEG C of high pressure moist heat sterilizations, are cooled to less than 30 DEG C after sterilizing, lead to filtrated air and keep aseptic condition standby, the seed liquor for being up to exponential phase accesses production tank culture by 5% inoculum concentration, and production tank used medium composition is identical with seed tank culture base.At 30 DEG C, the ventilation for producing filtrated air in the incubation of tank is 1: 0.8-1.0 to postvaccinal production tank temperature control, and mixing speed is 240 revs/min, and whole technological process incubation time is 48-60 hours.Thalline quantity reaches 1,000,000,000/more than ml after fermentation ends.Culture fluid goes out tank and is directly distributed into liquid dosage form with plastic barrel or Packaging Bottle after the completion of fermentation.
Claims (9)
1. a kind of N-Methyl pyrrolidone is degraded bacillus cereuss NMP-2, and its deposit number is CGMCC NO.10544.
2. applications of the Bacillus strain NMP-2 described in claim 1 in N-Methyl pyrrolidone degraded.
3. containing the microbial inoculum of Bacillus strain NMP-2 described in claim 1.
4. the preparation method of microbial inoculum described in claim 3, it is characterised in that include：Inclined-plane kind-shaking flask induction-seed tank induces-produces tank-product, and the packaging dosage form of the product is liquid bacterial agent or solid absorption microbial inoculum.
5. method according to claim 4, it is characterised in that specially：
1）The test tube kind of Bacillus strain NMP-2 is inoculated in the LB culture medium containing 500mg/L N-Methyl pyrrolidone, inducing culture is to logarithmic (log) phase；
2）By step 1）Cultured strain is inoculated with into 500 liters of seed tanks containing 500mg/L N-Methyl pyrrolidone, induced growth to logarithmic (log) phase, i.e. seed liquor by 5% inoculum concentration；
3）By step 2）The seed liquor accesses production tank and carries out fermentation culture by 5% inoculum concentration, and production tank used medium is identical with seed tank culture base, and the culture fluid after the completion of fermentation is microbial inoculum.
6. method according to claim 5, it is characterised in that step 1）Described in LB culture medium prescriptions be：500mg/L N-Methyl pyrrolidone, NaCl 10.00g/L, peptone 5.00g/L, yeast powder 5.00g/L, pH7.0.
7. method according to claim 5, it is characterised in that step 2）Culture medium prescription used by described seed tank is：N-Methyl pyrrolidone 0.5%, glucose 0.8%, K2HPO4
0.2%, MgSO4
0.05%, NaCl 0.01%, CaCO30.3%, yeast extract 0.02%, pH value 7.2-7.5.
8. method according to claim 5, it is characterised in that step 2）The seed tank and step 3）In the incubation of described production tank, the ventilation of filtrated air is 1：0.6-1.2, mixing speed is 220 revs/min, and cultivation temperature is 3 DEG C, step 2）With 3）Whole technological process incubation time be 48-60 hours.
9. application of the microbial inoculum that prepared by any one of claim 5-8 methods described in N-Methyl pyrrolidone degraded.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109913387A (en) * | 2019-03-19 | 2019-06-21 | 南京理工大学 | Degrade N-Methyl pyrrolidone enterobacteria and application in the treatment of waste water |
| CN112574916A (en) * | 2020-12-16 | 2021-03-30 | 南京理工大学 | N-methylpyrrolidone degrading bacterium and application thereof in wastewater treatment |
| CN114854620A (en) * | 2022-03-08 | 2022-08-05 | 黄河三角洲京博化工研究院有限公司 | N-methylpyrrolidone degrading strain and application thereof |
| CN117106662A (en) * | 2023-09-14 | 2023-11-24 | 武汉格林环源净化工程有限公司 | Bacillus bailii and culture method and application thereof |
| CN114854620B (en) * | 2022-03-08 | 2024-06-04 | 黄河三角洲京博化工研究院有限公司 | Degradation strain of N-methylpyrrolidone and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913387A (en) * | 2019-03-19 | 2019-06-21 | 南京理工大学 | Degrade N-Methyl pyrrolidone enterobacteria and application in the treatment of waste water |
| CN109913387B (en) * | 2019-03-19 | 2022-01-11 | 南京理工大学 | Enterobacter for degrading N-methylpyrrolidone and application of enterobacter in wastewater treatment |
| CN112574916A (en) * | 2020-12-16 | 2021-03-30 | 南京理工大学 | N-methylpyrrolidone degrading bacterium and application thereof in wastewater treatment |
| CN112574916B (en) * | 2020-12-16 | 2022-04-29 | 南京理工大学 | N-methylpyrrolidone degrading bacterium and application thereof in wastewater treatment |
| CN114854620A (en) * | 2022-03-08 | 2022-08-05 | 黄河三角洲京博化工研究院有限公司 | N-methylpyrrolidone degrading strain and application thereof |
| CN114854620B (en) * | 2022-03-08 | 2024-06-04 | 黄河三角洲京博化工研究院有限公司 | Degradation strain of N-methylpyrrolidone and application thereof |
| CN117106662A (en) * | 2023-09-14 | 2023-11-24 | 武汉格林环源净化工程有限公司 | Bacillus bailii and culture method and application thereof |
| CN117106662B (en) * | 2023-09-14 | 2024-03-15 | 武汉格林环源净化工程有限公司 | Bacillus bailii and culture method and application thereof |
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Address after: 211132 No. 16, ancient spring road, Tangshan street, Jiangning District, Nanjing, Jiangsu Applicant after: JIANGSU NANZI ENVIRONMENTAL PROTECTION SCIENCE & TECHNOLOGY Co.,Ltd. Address before: 211132 No. 16, ancient spring road, Tangshan street, Jiangning District, Nanjing, Jiangsu Applicant before: JIANGSU NANZI ENVIRONMENTAL PROTECTION CO.,LTD. |
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Address after: No. 16 Guquan Road, Tangshan Street, Jiangning District, Nanjing City, Jiangsu Province, 211100 Patentee after: Zhongzhi (Jiangsu) Environmental Construction Co.,Ltd. Address before: 211132 No. 16, ancient spring road, Tangshan street, Jiangning District, Nanjing, Jiangsu Patentee before: JIANGSU NANZI ENVIRONMENTAL PROTECTION SCIENCE & TECHNOLOGY Co.,Ltd. |
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