CN101955256A - Method for preparing ibuprofen-degrading bacterial agent - Google Patents

Method for preparing ibuprofen-degrading bacterial agent Download PDF

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CN101955256A
CN101955256A CN2010102862885A CN201010286288A CN101955256A CN 101955256 A CN101955256 A CN 101955256A CN 2010102862885 A CN2010102862885 A CN 2010102862885A CN 201010286288 A CN201010286288 A CN 201010286288A CN 101955256 A CN101955256 A CN 101955256A
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ibuprofen
bacterial agent
preparation
culture medium
described step
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CN101955256B (en
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杨兴
薛罡
赵晓祥
刘亚男
史锦
程起跃
周宁娟
孙超
田世烜
罗来盛
阮俊杰
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Donghua University
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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    • Y02W10/10—Biological treatment of water, waste water, or sewage

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Abstract

The invention relates to a method for preparing an ibuprofen-degrading bacterial agent, which comprises the following steps of: (1) inoculating activated sludge in a secondary sedimentation tank of a sewage treatment plant into a culture medium, domesticating by using a gradient pressure domestication method, standing, taking an upper-layer water sample, gradually diluting, streaking for separation, culturing, purifying, inoculating the separated strain into a beef extract peptone culture medium, and performing shake culture until a logarithmic phase; and (2) inoculating the cultured strain into the culture medium to prepare the ibuprofen-degrading bacterial agent. The method has the advantages of simpleness and low cost; and the prepared ibuprofen-degrading bacterial agent has the advantages of convenient use, high efficiency of degrading ibuprofen, easy inactivation of the strain, and application to ibuprofen removal in water treatment.

Description

A kind of preparation method of Ibuprofen BP/EP degradation bacterial agent
Technical field
The invention belongs to the preparation field of Ibuprofen BP/EP degradation bacterial agent, particularly relate to a kind of preparation method of Ibuprofen BP/EP degradation bacterial agent.
Background technology
Medicine and personal care articles (Pharmaceutical and Personal Care Products is called for short PPCPs) are subjected to people's attention day by day as a kind of emerging pollutent.Even medicine and personal-care supplies trace exist, still have the biological activity of certain degree, cause uncertain influence to environment, and may produce potential hazard to HUMAN HEALTH by approach such as tap water and foods.
In common PPCPs compound, microbiotic, antiphlogistic drug, antiepileptic drug, regulating blood fat medicine in all kinds of surrounding mediums to detect frequency higher.Wherein non-steroidal antipyretic-antalgic and anti-inflammatory drug Ibuprofen BP/EP (Ibuprofen, molecular structure such as Fig. 1) as nonprescription drugs, are used in a large number, are one of PPCPs pollutants at present of greatest concern.
Traditional activated sludge process because of the region difference, differs to the degradation efficiency of Ibuprofen BP/EP.The product of photo-Fenton method degraded Ibuprofen BP/EP, toxicity strengthens on the contrary.Studies show that the clearance and the microbial strains of Ibuprofen BP/EP are closely related.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of Ibuprofen BP/EP degradation bacterial agent, and this method is simple, and cost is low; Gained Ibuprofen BP/EP degradation bacterial agent is easy to use, the efficient degradation Ibuprofen BP/EP, and bacterial strain is easy to deactivation.
A kind of efficient degradation Ibuprofen BP/EP bacterial strain that is used for strengthening the ability of removing the sewage Ibuprofen BP/EP among the present invention is a kind of gramstaining reaction negative bacterial strain BL-1, through being accredited as serratia marcescens (Serratia marcescens).Main biological characteristics is G -, thalline is rod-short, no brood cell, and about 1.54 * 1.08 μ m of size, the tool peritrichous, facultative aerobic; V-P reaction, catalase, nitrate reaction, the gelatin liquification test positive, methyl red test, oxydase, phenylalanine reaction, hydrogen sulfide production test feminine gender; Can utilize sucrose, glucose, wood sugar, N.F,USP MANNITOL, pectinose to produce acid, can not utilize lactose to produce acid; This bacterial strain all loses activity behind uviolizing 2h.The Genbank accession number of this bacterial strain 16S rRNA is HM136577.
The preparation method of a kind of Ibuprofen BP/EP degradation bacterial agent of the present invention comprises:
(1) get in the inoculum size access substratum of active sludge by 5%-10% of sewage work's second pond, with the domestication of gradient pressure type method for domesticating, ibuprofen concentration is 10-35mg/L; After the domestication, leave standstill, get the upper strata water sample, line separates behind the gradient dilution, cultivates 48h-60h at 30 ℃-35 ℃ then, purifying, and in beef-protein medium, shaking culture is to logarithmic phase with isolated inoculation;
(2) inoculum size of above-mentioned cultured bacterial classification being pressed 10%-20% inserts culture medium culturing, promptly;
The air flow of sterile air turns to 1 in the described culturing process: 0.6-1.2, and stirring velocity is 180-240 rev/min, culture temperature is 30-35 ℃; Incubation time is 48-60 hour, after the fermentation ends thalline quantity reach 1,000,000,000/more than the mL, fermentation is finished the back nutrient solution and directly is distributed into liquid dosage form with plastic barrel or packing bottle or adopts peat to adsorb and be distributed into the solid fungicide formulation with packing bag.
Culture medium prescription in the described step (1) is: K 2HPO 44.35g, KH 2PO 41.7g, NH 4Cl 2.1g, MgSO 40.2g, MnSO 40.05g, FeSO 47H 2O 0.01g, CaCl 22H 2O 0.03g, distilled water 1000mL, pH=6.8-7.0.
Domestication condition in the described step (1) is stirring velocity 160-240 rev/min, temperature 30-35 ℃, and time 1-2 month.
Beef-protein medium in the described step (1) is: extractum carnis 3.0g, peptone 10.0g, NaCl 5.0g, distilled water 1000mL, pH=7.0～7.2.
The concentration of the Ibuprofen BP/EP in the described step (1) is 10mg/L, 15mg/L, 20mg/L, 25mg/L, 30mg/L, 35mg/L.
Culture medium prescription in the described step (2) is: sucrose 10g/L, extractum carnis 3g/L, KH 2PO 41.7g/L, NH 4Cl2.1g/L, MgSO 40.2g/L, CaCO 30.3g/L, pH=7.0-7.5, Ibuprofen BP/EP 10-35mg/L.
Obtained strains is applied to the removal of the Ibuprofen BP/EP in the water treatment.
The present invention behind domestication, concentration and separation, purifying, obtains a strain has the efficient degradation effect to Ibuprofen BP/EP bacterium by the thought of biological reinforcing technology from the active sludge of municipal sewage plant's second pond.This bacterium source is in Waste Water Treatment, so this bacterial strain can adapt to complicated physical condition, and this makes having a extensive future of this bacterium.
Beneficial effect
(1) preparation method of the present invention is simple, and cost is low; Gained Ibuprofen BP/EP degradation bacterial agent is easy to use, the efficient degradation Ibuprofen BP/EP, and bacterial strain is easy to deactivation, guaranteed the ecological security that uses; The present invention is for improving and preserving the ecological environment, and the protection people health is significant;
(2) small-scale sewage treatment facility (BAF) result shows, can obviously improve the removal efficient of Ibuprofen BP/EP with the microbial inoculum of the present invention's production.
Description of drawings
The chemical structure of Fig. 1 Ibuprofen BP/EP;
Fig. 2 thalline electromicroscopic photograph;
Fig. 3 do not degrade (a) and degrade GC-MS total ion chromatogram of (b) sample;
Fig. 4 aeration biological filter device figure.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
1. the separation of bacterial strain and evaluation
(1) active sludge of taking from municipal sewage plant's second pond is inoculated in the minimal medium by 5% access amount, tame with gradient pressure type method for domesticating, ibuprofen concentration is 10,15,20,25,30,35mg/L, at 30 ℃, 160r/min, tame cultivation under the condition of loadings 100mL in the 250mL Erlenmeyer flask, the time of domestication always is 2 months.The minimal medium prescription is K 2HPO 44.35g, KH 2PO 41.7g, NH 4Cl 2.1g, MgSO 40.2g, MnSO 40.05g, FeSO 47H 2O0.01g, CaCl 22H 2O 0.03g, distilled water 1000mL, pH=6.8-7.0.
(2) get the sample of domestication after 2 months, left standstill 30 minutes, get upper strata water sample 1mL, line separation on flat board behind the gradient dilution.In 30 ℃ of incubators, cultivate 48h then.The single clearly bacterium colony of picking form, numbering, and purifying repeatedly to slant medium, are stored in isolated inoculation under 4 ℃ and treat the follow-up test of degrading in the refrigerator.Wherein enrichment medium is extractum carnis 3.0g, peptone 10.0g, NaCl 5.0g, distilled water 1000mL, pH=7.0～7.2.The agar powder that adds 17g/L in the solid medium.
(3) a certain amount of single bacterium colony after separation and purification of difference picking is in minimal medium separately, the Ibuprofen BP/EP that all contains 15mg/L in each minimal medium, at 30 ℃, 160r/min, to cultivate under the condition of loadings 100mL in the 250mL Erlenmeyer flask, incubation time is 48h.Cultivate the back and measure the residual concentration of Ibuprofen BP/EP in the solution, thereby determine the bacterial strain BL-1 that Ibuprofen BP/EP is had the efficient degradation effect;
Described inorganic medium is sucrose 10g/L, extractum carnis 3g/L, KH 2PO 41.7g/L, NH 4Cl 2.1g/L, MgSO 40.2g/L, CaCO 30.3g/L, pH=7.0-7.5, Ibuprofen BP/EP 15mg/L.
(4) identify and 16S rRNA sequential analysis and homology comparison that through Physiology and biochemistry bacterial strain BL-1 is serratia marcescens (Serratia marcescens) by preliminary evaluation.Main biological characteristics is G, and thalline is rod-short, no brood cell, and about 1.54 * 1.08 μ m of size, the tool peritrichous, facultative aerobic; V-P reaction, catalase, nitrate reaction, the gelatin liquification test positive, methyl red test, oxydase, phenylalanine reaction, hydrogen sulfide production test feminine gender; Can utilize sucrose, glucose, wood sugar, N.F,USP MANNITOL, pectinose to produce acid, can not utilize lactose to produce acid; This bacterial strain all loses activity behind uviolizing 2h.The Genbank accession number of this bacterial strain 16S rRNA is HM136577.
2. laboratory Ibuprofen BP/EP degraded test
A strain Ibuprofen BP/EP efficient degrading bacteria provided by the present invention is about 7. in initial pH value, temperature is 30 ℃, oscillation rate is 160r/min, the liquid amount of 250mL Erlenmeyer flask is under the condition of 100mL, the inoculation amount is 0.6%, the Ibuprofen BP/EP starting point concentration is 15mg/L, incubation time 48h, and the degradation rate of Ibuprofen BP/EP is up to 90%.
3. the strenuous test of small-scale sewage treatment facility (BAF)
The bacterium liquid of fermentation by 2% ratio, is being intake 15.6 ℃～24.5 ℃ of water temperatures, and under the condition of pH 6.88～7.27, influent load is 0.2m 3/ m 2H, gas-water ratio is 1.5: 1 o'clock, the clearance that has added Ibuprofen BP/EP in the BAF of microbial inoculum is 86%～91%.And the clearance that does not add Ibuprofen BP/EP in the BAF of microbial inoculum is 48%～53%.

Claims (6)

1. the preparation method of an Ibuprofen BP/EP degradation bacterial agent comprises:
(1) get in the inoculum size access substratum of active sludge by 5%-10% of sewage work's second pond, with the domestication of gradient pressure type method for domesticating, ibuprofen concentration is 10-35mg/L; After the domestication, leave standstill, get the upper strata water sample, line separates behind the gradient dilution, cultivates 48h-60h at 30 ℃-35 ℃ then, purifying, and in beef-protein medium, shaking culture is to logarithmic phase with isolated inoculation;
(2) inoculum size of above-mentioned cultured bacterial classification being pressed 10%-20% inserts culture medium culturing, promptly;
The air flow of sterile air turns to 1 in the described culturing process: 0.6-1.2, and stirring velocity is 180-240 rev/min, culture temperature is 30-35 ℃; Incubation time is 48-60 hour, after the fermentation ends thalline quantity reach 1,000,000,000/more than the mL.
2. the preparation method of a kind of Ibuprofen BP/EP degradation bacterial agent according to claim 1 is characterized in that: the culture medium prescription in the described step (1) is: K 2HPO 44.35g, KH 2PO 41.7g, NH 4Cl 2.1g, MgSO 40.2g, MnSO 40.05g, FeSO 47H 2O 0.01g, CaCl 22H 2O 0.03g, distilled water 1000mL, pH=6.8-7.0.
3. the preparation method of a kind of Ibuprofen BP/EP degradation bacterial agent according to claim 1 is characterized in that: the domestication condition in the described step (1) is stirring velocity 160-240 rev/min, temperature 30-35 ℃, and time 1-2 month.
4. the preparation method of a kind of Ibuprofen BP/EP degradation bacterial agent according to claim 1, it is characterized in that: the beef-protein medium in the described step (1) is: extractum carnis 3.0g, peptone 10.0g, NaCl 5.0g, distilled water 1000mL, pH=7.0～7.2.
5. the preparation method of a kind of Ibuprofen BP/EP degradation bacterial agent according to claim 1 is characterized in that: the concentration of the Ibuprofen BP/EP in the described step (1) is 10mg/L, 15mg/L, 20mg/L, 25mg/L, 30mg/L, 35mg/L.
6. the preparation method of a kind of Ibuprofen BP/EP degradation bacterial agent according to claim 1 is characterized in that: the culture medium prescription in the described step (2) is: sucrose 10g/L, extractum carnis 3g/L, KH 2PO 41.7g/L, NH 4Cl 2.1g/L, MgSO 40.2g/L, CaCO 30.3g/L, pH=7.0-7.5, Ibuprofen BP/EP 10-35mg/L.
CN2010102862885A 2010-09-19 2010-09-19 Method for preparing ibuprofen-degrading bacterial agent Expired - Fee Related CN101955256B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103255161A (en) * 2013-05-15 2013-08-21 西北农林科技大学 Method for knocking out ibuprofen fosmid catechol dioxygenase gene
CN109574399A (en) * 2018-12-27 2019-04-05 南京大学 A method of Diclofenac in removal sewage is strengthened based on enrichment nitrobacteria

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0567102A2 (en) * 1992-04-22 1993-10-27 Canon Kabushiki Kaisha Method of biologically decomposing phenol or furan compounds by microorganisms
CN1837356A (en) * 2006-04-21 2006-09-27 哈尔滨工业大学 Method for acclimating, separating and screening highly effective phosphate-accumulating bacteria from intermittent circulating active sludge-membrane biological reaction system
CN101031637A (en) * 2005-04-21 2007-09-05 揖斐电株式会社 Method of treating wastewater containing organic compound
CN101260373A (en) * 2007-10-24 2008-09-10 大连理工大学 Micrococcus luteus with novel metabolic characteristic and application thereof in biological degradation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0567102A2 (en) * 1992-04-22 1993-10-27 Canon Kabushiki Kaisha Method of biologically decomposing phenol or furan compounds by microorganisms
CN101031637A (en) * 2005-04-21 2007-09-05 揖斐电株式会社 Method of treating wastewater containing organic compound
CN1837356A (en) * 2006-04-21 2006-09-27 哈尔滨工业大学 Method for acclimating, separating and screening highly effective phosphate-accumulating bacteria from intermittent circulating active sludge-membrane biological reaction system
CN101260373A (en) * 2007-10-24 2008-09-10 大连理工大学 Micrococcus luteus with novel metabolic characteristic and application thereof in biological degradation

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103255161A (en) * 2013-05-15 2013-08-21 西北农林科技大学 Method for knocking out ibuprofen fosmid catechol dioxygenase gene
CN103255161B (en) * 2013-05-15 2015-07-29 西北农林科技大学 A kind of method knocking out Ibuprofen BP/EP FOX plasmid catechol dioxygenase gene
CN109574399A (en) * 2018-12-27 2019-04-05 南京大学 A method of Diclofenac in removal sewage is strengthened based on enrichment nitrobacteria

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