CN101955256B - Method for preparing ibuprofen-degrading bacterial agent - Google Patents
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- CN101955256B CN101955256B CN2010102862885A CN201010286288A CN101955256B CN 101955256 B CN101955256 B CN 101955256B CN 2010102862885 A CN2010102862885 A CN 2010102862885A CN 201010286288 A CN201010286288 A CN 201010286288A CN 101955256 B CN101955256 B CN 101955256B
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- ibuprofen
- bacterial agent
- degrading bacterial
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- degrading
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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
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- 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
Technical field
The invention belongs to the preparation field of ibuprofen-degrading bacterial agent, particularly relate to a kind of preparation method of ibuprofen-degrading bacterial agent.
Background technology
Medicine and personal care articles (Pharmaceutical and Personal Care Products is called for short PPCPs) receive people's concern day by day as a kind of emerging pollutent.Even the Pharmaceuticals and personal care products trace exists, still have the biological activity of certain degree, cause uncertain impact 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, adjusting 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, different because of the region, the degradation efficiency of Ibuprofen BP/EP is differed.The product of photo-Fenton method degraded Ibuprofen BP/EP, toxicity strengthens on the contrary.Studies show that 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-degrading bacterial agent, and the method is simple, and cost is low; The gained ibuprofen-degrading 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 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 is G
-, thalline is rod-short, without the 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-degrading 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, then cultivates 48h-60h at 30 ℃-35 ℃, purifying, and in beef-protein medium, shaking culture is to logarithmic phase with isolated inoculation;
(2) above-mentioned cultured bacterial classification is pressed the inoculum size access culture medium culturing of 10%-20%, and be get final product;
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, nutrient solution directly was distributed into liquid dosage form or adopts adsorption by peat to be distributed into the solid fungicide formulation with packing bag with plastic barrel or packing bottle after fermentation was finished.
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 is so that the having a extensive future of this bacterium.
Beneficial effect
(1) preparation method of the present invention is simple, and cost is low; The gained ibuprofen-degrading 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's is healthy 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 for explanation the present invention 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 limited range 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 always domestication time 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, separate in the flat board line behind the gradient dilution.Then in 30 ℃ of incubators, cultivate 48h.The picking form is single bacterium colony clearly, and numbering, and purifying repeatedly to slant medium, are stored in isolated inoculation under 4 ℃ and treat the follow-up Degrading experiment that carries out 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.Measure the residual concentration of Ibuprofen BP/EP in the solution after cultivating, 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 initially identified as serratia marcescens (Serratia marcescens).Main Biological is G, and thalline is rod-short, without the 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 Degrading experiment
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-degrading 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, then cultivates 48h-60h at 30 ℃-35 ℃, purifying, and in beef-protein medium, shaking culture is to logarithmic phase with isolated inoculation;
(2) above-mentioned cultured bacterial classification is pressed the inoculum size access culture medium culturing of 10%-20%, and be get final product;
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-degrading bacterial agent according to claim 1, it 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-degrading bacterial agent according to claim 1, it is characterized in that: the domestication condition in the described step (1) is stirring velocity 160-240 rev/min, temperature 30-35 ℃, time 1-2 month.
4. the preparation method of a kind of ibuprofen-degrading 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-degrading bacterial agent according to claim 1, it 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-degrading bacterial agent according to claim 1, it 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.
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|---|---|---|---|
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| 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 |
| CN109574399B (en) * | 2018-12-27 | 2021-07-30 | 南京大学 | Method for removing diclofenac in sewage in enhanced manner based on enrichment of nitrobacteria |
Citations (4)
| 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 |
-
2010
- 2010-09-19 CN CN2010102862885A patent/CN101955256B/en not_active Expired - Fee Related
Patent Citations (4)
| 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 |
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Inventor after: Yang Xing Inventor after: Luo Laisheng Inventor after: Ruan Junjie Inventor after: Gao Pin Inventor after: Zhang Dandan Inventor after: Mao Feifei Inventor after: Mei Shufang Inventor after: Xing Lingjun Inventor after: Wang Beiling Inventor after: Xu Bingjie Inventor after: He Mengqi Inventor after: Xue Gang Inventor after: Liu Chang Inventor after: Li Panshan Inventor after: Shi Kuan Inventor after: Zhao Xiaoxiang Inventor after: Liu Yanan Inventor after: Shi Jin Inventor after: Cheng Qiyue Inventor after: Zhou Ningjuan Inventor after: Sun Chao Inventor after: Tian Shihui Inventor before: Yang Xing Inventor before: Luo Laisheng Inventor before: Ruan Junjie Inventor before: Xue Gang Inventor before: Zhao Xiaoxiang Inventor before: Liu Yanan Inventor before: Shi Jin Inventor before: Cheng Qiyue Inventor before: Zhou Ningjuan Inventor before: Sun Chao Inventor before: Tian Shihui |
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Free format text: CORRECT: INVENTOR; FROM: YANG XING XUE GANG ZHAO XIAOXIANG LIU YANAN SHI JIN CHENG QIYUE ZHOU NINGJUAN SUN CHAO TIAN SHI LUO LAISHENG RUAN JUNJIE TO: YANG XING XUE GANG ZHAO XIAOXIANG LIU YANAN SHI JIN CHENG QIYUE ZHOU NINGJUAN SUN CHAO TIAN SHI LUO LAISHENG RUAN JUNJIE GAO PIN ZHANG DANDAN MAO FEIFEI MEI SHUFANG XING LINGJUN WANG BEILING XU BINGJIE HE MENGQI LIU CHANG LI SHANSHAN SHI KUAN |
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