CN106591169B - A kind of N-Methyl pyrrolidone degradation bacillus NMP-2 and its application - Google Patents
A kind of N-Methyl pyrrolidone degradation bacillus NMP-2 and its application Download PDFInfo
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- CN106591169B CN106591169B CN201610071081.3A CN201610071081A CN106591169B CN 106591169 B CN106591169 B CN 106591169B CN 201610071081 A CN201610071081 A CN 201610071081A CN 106591169 B CN106591169 B CN 106591169B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Abstract
The present invention provides a kind of degradation bacteria strain NMP-2 of elimination N-Methyl pyrrolidone (abbreviation NMP) pollution and its applications, belong to biological high-tech technical field.Bacterial strain uses therefor be identified as bacillus (Bacillus).Main Biological is G+, thallus be it is rod-shaped, have endospore, aerobic growth.It can be grown by sole carbon source, nitrogen source of NMP, and be thoroughly mineralized to generate ammonia nitrogen.The bacterial strain solves the problems, such as NMP difficult for biological degradation in wastewater treatment to the degradation rate of 500mg/L NMP up to 100% under the conditions of laboratory shake flask.
Description
Technical field
The present invention provides the microbial inoculum of a kind of N-Methyl pyrrolidone (abbreviation NMP) degradation bacteria and its production, belongs to biological height
Science and technology field is the pollutant in the method degrading waste water using microorganism, is suitable for modern waste water treatment process.
Background technique
N-Methyl pyrrolidone is that one kind is important industrial chemicals, is that the polarity that a kind of selectivity is strong and stability is good is molten
Agent is widely used in the purifying of aromatic hydrocarbons extraction, acetylene, alkene, alkadienes, the solvent of polyvinylidene fluoride, the electricity of lithium ion battery
Pole auxiliary material, when synthetic gas desulfurization, lube oil finishing, lubricating oil antifreeze, olefinic extracting agent, indissoluble engineering plastics polymerize
Solvent, agricultural herbicide, insulating materials, production of integrated circuits, clean, the PVC tail gas of semicon industry precision instrument, wiring board
Recycling, cleaning agent, color additive, dispersing agent etc..Be also used for the solvent of polymer and the medium of polymerization reaction, such as engineering plastics and
Aramid fiber.
N-Methyl pyrrolidone is widely present in the production waste water such as organic synthesis, pesticide, dyestuff, at present for first containing N-
The processing of base pyrrolidones waste water mostly uses physico-chemical process, higher cost, and easily causes secondary pollution；And it is biological prosthetic
Technology has huge potentiality due to microorganism itself can be degraded and be converted to all kinds of different organic pollutants.Therefore,
The efficient bacterium that screening obtains to remove N-Methyl pyrrolidone from natural environment is of great significance.
Summary of the invention
It is an object of the invention to develop a kind of N- crassitude for the practical problem and demand in production practices
Ketone degradation bacteria strains and its microbial inoculum can make wastewater to reach standard using this bacterial strain or microbial inoculum with the N-Methyl pyrrolidone in degrading waste water
Discharge, while reducing production and application cost.
The present invention provides a kind of degradation bacteria of elimination N-Methyl pyrrolidone pollution, and bacterial strain is that one plant of Gram's staining is anti-
Positive bacteria NMP-2 is answered, was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 2 9th, 2015
(address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), deposit number CGMCC
NO.10544 is identified as bacillus (Bacillus sp.).Main Biological is G+, logarithmic growth phase thallus bar
Shape has endospore, aerobic growth.Bacterium colony is round, opaque, neat in edge, in faint yellow；It can be with N-Methyl pyrrolidone
It is grown for sole carbon source, nitrogen source, and is thoroughly mineralized to generate ammonia nitrogen.36h is to 500mg/L under the conditions of laboratory shake flask
The degradation rate of N-Methyl pyrrolidone is 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 NMP-2.
The present invention provides the production method for containing the bacillus NMP-2 microbial inoculum, comprising: inclined-plane kind-shaking flask induction-
Seeding 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, specifically:
1) test tube species of bacillus NMP-2 are inoculated in the LB culture medium containing 500mg/L N-Methyl pyrrolidone
In, Fiber differentiation is to logarithmic phase；
2) the cultured strain of step 1) is inoculated with by 5% inoculum concentration and contains 500mg/L N- methylpyrrole into 500 liters
The seeding tank of alkanone, induced growth to logarithmic phase, i.e. seed liquor；
3) the step 2) seed liquor is subjected to fermented and cultured, the training used of production tank by 5% inoculum concentration access production tank
It supports base and seed tank culture base phase is same, culture solution after fermentation is microbial inoculum.
LB culture medium prescription described in step 1) are as follows: 500mg/L N-Methyl pyrrolidone, NaCl10.00g/L, albumen
Peptone 5.00g/L, yeast powder 5.00g/L, pH7.0.
The formula of culture medium mass percent used in seeding tank described in step 2) are as follows: 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.
In the incubation for producing tank described in the step 2) seeding tank and step 3), the ventilatory capacity of filtrated air is 1:
0.6-1.2, mixing speed are 220 revs/min, and cultivation temperature is 3 DEG C, and step 2) is with entire process flow incubation time 3)
48-60 hours.Thalline quantity reaches 1,000,000,000/ml or more after fermentation, and culture solution goes out tank and directly uses plastics after fermentation
Pail pack or Packaging Bottle are distributed into liquid agent.
The present invention also provides application of the microbial inoculum containing bacillus NMP-2 in N-Methyl pyrrolidone degradation.
The degradation bacteria NMP-2 provided by the present invention for eliminating N-Methyl pyrrolidone pollution, laboratory biological degradation experiment
The result shows that reaching 100% to the degradation rate of 500mg/L NMP.
The degradation bacterial agent of invention production has production cost low, and easy to use, the good advantage of removal effect is suitble to containing
Have in the wastewater treatment of NMP and promotes the use of.The present invention protects the health of the mankind, reduces waste water for preserving the ecological environment
Processing cost has great importance.
Detailed description of the invention
Fig. 1 is the thallus violet staining photo (1000x) of bacterial strain NMP-2；
Fig. 2 is the growth degradation curve that bacterial strain NMP-2 utilizes N-Methyl pyrrolidone；
Fig. 3 is influence of the inoculum concentration to N-Methyl pyrrolidone degradation efficiency；
Fig. 4 is influence of the temperature to N-Methyl pyrrolidone degradation efficiency.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention
In the case where essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The separation and identification of embodiment 1, bacterial strain
N-Methyl pyrrolidone is added in the activated sludge 3.0ml obtained in the purification tank for liquid waste containing N-Methyl pyrrolidone
Concentration is formula are as follows: every liter of K containing 1.50g in the 100ml inorganic salt liquid culture of 500mg/L2HPO4、0.50g KH2PO4、
0.20g MgSO4·7H2O, 1.00g NaCl, 7.0 pH, the shaken cultivation under the conditions of 160r/min, 37 DEG C press 3% every 36h
Inoculum concentration be transferred in fresh minimal medium, continuous switching 5 times.
Enrichment bacterium solution 1.0ml obtained above is taken, is added in 9.0ml sterile water, is made into 10-1Pregnant solution, then draw
1.0ml prepare 10-1Pregnant solution be added 9.0ml sterile water in, mix well and be made into 10-2Pregnant solution, and so on, it is right
Pregnant solution carries out gradient dilution.And so on, gradient dilution is carried out to pregnant solution.Draw the dilution 0.1ml coating of each gradient
It is that 500mg/L inorganic salts solid medium (is formulated are as follows: every liter of K containing 1.50g in the concentration of the methyl pyrrolidone containing N2HPO4、
0.50g KH2PO4、0.20g MgSO4·7H2O, 1.00g NaCl, 12.00g agar, pH 7.0) on, 37 DEG C are cultivated 2 days.2 days
Afterwards from picking single colonie on above inorganic salts solid medium, after being cultivated 24 hours in the LB liquid medium of 3.0ml, LB
Liquid Culture based formulas are as follows: NaCl 10.00g/L, peptone 5.00g/L, yeast powder 5.00g/L, pH7.0 is in 12000r/min
Under conditions of be centrifuged 2min, remove supernatant, the sterile water that 3.0ml is added shakes up, and is still centrifuged under conditions of 12000r/min
2min is added 3.0ml sterile water and the bacterium is resuspended according to said method with after twice of sterile washing.The 1.0ml bacterium solution is drawn to be added
100ml concentration containing N-Methyl pyrrolidone is in the inorganic salt liquid culture medium of 500mg/L, under the conditions of 220r/min, 37 DEG C
After shaken cultivation 72h, its degradation effect is surveyed with high performance liquid chromatography.The higher one plant of bacterial strain of degradation efficiency is saved, after progress
Continuous experiment.
Degradation bacteria strains are inoculated on LB solid medium, are formulated are as follows: NaCl 10.00g/L, peptone 10.00g/L,
Colonial morphology is observed after yeast powder 5.00g/L, 20.00g/L, pH7.0,37 DEG C of constant temperature incubation 48h of agar, is seen with optical microscopy
Thalli morphology (1000X) is examined, as shown in Figure 1.The Physiology and biochemistry identification of bacterial strain is carried out according to " common bacteria system identification handbook ".
It is accredited as bacillus (Bacillus sp.)；Name are as follows: NMP-2.Main Biological is G+, logarithmic growth phase thallus
Be it is rod-shaped, have endospore, aerobic growth.Can be grown by sole carbon source, nitrogen source of N methyl pyrrolidone, and by its
Thorough mineralising generates ammonia nitrogen, as shown in Figure 2.The bacterium can be produced with the general Zymolysis Equipment of fermentation industry.
2 laboratory biological degradation experiment of embodiment
Influence of 2.1 inoculum concentrations to N-Methyl pyrrolidone degradation efficiency
NMP-2 was cultivated in LB culture medium to the logarithmic phase later period, was distinguished by 0.1,0.5,1,2,3,4%, 5% inoculum concentration
Access minimal medium (the minimal medium formula are as follows: every liter contains 1.5g of the methyl pyrrolidone containing 500mg/LN-
K2HPO4、0.5g KH2PO4、0.2g MgSO4·7H2O, 1.0g NaCl, pH=7.0 are hereafter same) in, 30 DEG C, 160r/min shakes
Bed culture, the content of N-Methyl pyrrolidone is sampled after 72h, as a result as shown in figure 3, measurement result shows by inoculum concentration from low
It is arranged to height, the degradation rate of N-Methyl pyrrolidone is followed successively by 30%, 58%, 75%, 85%, 95%, 96%, 98%.Thus
As it can be seen that the size of inoculum concentration has direct relationship to the degradation efficiency of N-Methyl pyrrolidone, when inoculum concentration is less than 2%, mention
The degradation efficiency of high inoculum concentration, N-Methyl pyrrolidone is significantly increased, but when inoculum concentration reaches more than 3%, degradation rate only has
Micro raising, may enzyme reaction at this time have been approached saturation.
Influence of 2.2 temperature to N-Methyl pyrrolidone
NMP-2 is cultivated in LB culture medium to late log phase, is connected to the methylpyrrole of N containing 500mg/L by 5% inoculum concentration
It in the minimal medium of alkanone, is respectively placed at a temperature of 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 40 DEG C, the training of 160r/min shaking table
It supports, the content of wherein N-Methyl pyrrolidone is measured after 72h, as a result as shown in Figure 4.At 15 DEG C, NMP-2 is to N- methylpyrrole
The degradation rate of alkanone is 14%；In the range of 20 DEG C~30 DEG C, as the temperature rises degradation bacteria degrade N methyl pyrrolidone
Performance obviously rises, and at 30 DEG C, degradation rate is up to 98%.And when temperature reaches 37 DEG C, NMP-2 is to N-Methyl pyrrolidone
Degradation rate falls to 49%.Above data explanation, the most suitable degradation temperature of NMP-2 is 30 DEG C, and too low or excessively high environment temperature
Degree can inhibit the degradation 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 invention is activated on culture dish, and is measured
Degradation property is inoculated in spare on test tube slant.Test tube species are 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 flask in, constant temperature vibration
Culture is swung to logarithmic phase, prepares inoculation seeding tank.500 liters of seeding tank, 400 liters of inventory, (the formula of culture medium mass percent
Are as follows: N-Methyl pyrrolidone 0.5%, glucose 0.8%, K2HPO40.2%, MgSO40.05%, NaCl 0.01%, CaCO3
0.3%, yeast extract 0.02%, pH value 7.2-7.5.) feed intake after 115 DEG C of high pressure moist heat sterilizations will be upper after being cooled to 30 DEG C
It states cultured shaking flask strain and accesses 500 liters of seeding tanks, culture to logarithmic growth phase, i.e. seed liquor, stirring by 5% inoculum concentration
Speed is 220 revs/min, and filtrated air intake is 1: 0.8.5 tons of tankage size, 4.5 tons of inventory of production.Tank is produced after feeding intake
1.1kg/cm2Pressure under, 115 DEG C of high pressure moist heat sterilizations are cooled to 30 DEG C hereinafter, logical filtrated air keeps sterile shape after sterilizing
State is spare, is up to the seed liquor of logarithmic growth phase by 5% inoculum concentration access production tank culture, production tank used medium at
Divide same with seed tank culture base phase.Production tank temperature degree after inoculation is controlled at 30 DEG C, produces filtrated air in the incubation of tank
Ventilatory capacity be 1: 0.8-1.0, mixing speed be 240 revs/min, entire process flow incubation time be 48-60 hours.Fermentation knot
Thalline quantity reaches 1,000,000,000/ml or more after beam.Culture solution goes out tank and directly uses plastic barrel or Packaging Bottle point after fermentation
Dress up liquid dosage form.
Claims (8)
1. a kind of bacillus (Bacillus sp) NMP-2, deposit number CGMCC for N-Methyl pyrrolidone of degrading
NO .10544 。
2. application of the bacillus NMP-2 described in claim 1 in N-Methyl pyrrolidone degradation.
3. the microbial inoculum containing bacillus NMP-2 described in claim 1.
4. the preparation method of microbial inoculum described in claim 3 characterized by comprising inclined-plane kind-shaking flask induction-seeding tank lures
Tank-product is led-produces, the packaging dosage form of the product is liquid bacterial agent or solid absorption microbial inoculum.
5. according to the method described in claim 4, it is characterized in that, specifically:
1) test tube species of Bacillus strain NMP-2 are inoculated in the LB culture medium containing 500mg/L N-Methyl pyrrolidone
In, Fiber differentiation to logarithmic phase；
2) the cultured strain of step 1) is inoculated with by 5% inoculum concentration and contains 500mg/L N-Methyl pyrrolidone into 500 liters
Seeding tank, induced growth to logarithmic phase, i.e. seed liquor；
3) the step 2) seed liquor is subjected to fermented and cultured by 5% inoculum concentration access production tank, produces tank used medium
Same with seed tank culture base phase, culture solution after fermentation is microbial inoculum.
6. according to the method described in claim 5, it is characterized in that, LB culture medium prescription described in step 1) are as follows: 500mg/L
N-Methyl pyrrolidone, NaCl 10.00g/L, peptone 5.00g/L, yeast powder 5.00g/L, pH7.0.
7. according to the method described in claim 5, it is characterized in that, culture medium prescription used in seeding tank described in step 2)
Are as follows: N-Methyl pyrrolidone 0.5%, glucose 0.8%, K2HPO40.2%, MgSO40.05%, NaCl 0.01%, CaCO3
0.3%, yeast extract 0.02%, pH value 7.2-7.5.
8. application of the microbial inoculum of any one of claim 5-7 the method preparation in N-Methyl pyrrolidone degradation.
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| 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 |
| 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 |
| 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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| CN103232965A (en) * | 2013-05-28 | 2013-08-07 | 江苏长青农化股份有限公司 | Bacillus subtilis FC12 for degrading fluoroglycofen-ethyl and application thereof |
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| CN103232965A (en) * | 2013-05-28 | 2013-08-07 | 江苏长青农化股份有限公司 | Bacillus subtilis FC12 for degrading fluoroglycofen-ethyl and application thereof |
Non-Patent Citations (1)
| Title |
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| N-methyl-2-pyrrolidone-degrading bacteria from activated sludge;Karel Krížek et al.;《Water Science & Technology 》;20151231;第71卷(第5期);摘要 * |
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