CN103160491B - Immobilized cell of mutagenized bacterium M1 of Rhodococcus ruber SD3 and application thereof in degradation of phenol pollutants - Google Patents
Immobilized cell of mutagenized bacterium M1 of Rhodococcus ruber SD3 and application thereof in degradation of phenol pollutants Download PDFInfo
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- CN103160491B CN103160491B CN201310129238.XA CN201310129238A CN103160491B CN 103160491 B CN103160491 B CN 103160491B CN 201310129238 A CN201310129238 A CN 201310129238A CN 103160491 B CN103160491 B CN 103160491B
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Abstract
The invention relates to an immobilized cell of a mutagenized bacterium M1 of Rhodococcus ruber SD3 and application thereof in degradation of phenol pollutants. For the mutagenized bacterium M1 prepared by mutagenizing Rhodococcus ruber SD3 with 0.3% lithium chloride and screening, the degradation rate on 1.5 g/L phenol within 72 hours is 99.77%. The immobilized cell having a diameter of 6 mm is prepared by embedding the mutagenized bacterium M1 with 1% sodium alginate and 1% polyvinyl alcohol. When the immobilized cell is repeatedly used 5 times, the degradation rate on 2 g/L phenol within 72 hours is more than 98%. Thus, the mutagenized bacterium M1 of Rhodococcus ruber SD3 and the immobilized cell thereof can efficiently degrade phenol pollutants, and can be used for biological treatment of phenol-containing industrial wastewater.
Description
Technical field
The invention belongs to Environmental Biotechnology field, the application of the immobilized cell of mutagenesis bacterium M1 that relates in particular to a kind of Rhodococcus ruber SD3 in degradation of phenol pollutent.
Background technology
Phenol is the principal pollutant in trade effluent.China lists phenol among priority pollutant Black List, and the discharge of phenolic wastewater is had to strict regulation: under general condition, the concentration containing Volatile Phenols of tap water is 0.001 mg/L, in source water, containing phenol maximum permissible concentration, is 0.002 mg/L.Biological process is method and most widely used treatment technology comparatively advanced in current field of waste water treatment, the main method of Ye Shi China phenolic wastewater harmless treatment.But due to the toxicity of phenolic wastewater and the characteristic of difficult degradation, so the bacterial classification of introducing must have stronger adaptive faculty and fall phenol ability.From physical environment, separation has obtained some Phenol-degrading Bacteria Strains at present, wherein
pseudomonas putida, Bacillus brevis, Candida maltosa, Candida tropicaliswith
alcaligenes faecaliscan tolerate the L up to 1000mg
-1above phenol.Yet relevant
rhodococcus ruberpyrogentisinic Acid's degraded is not but reported.
In addition, for degradation of phenol, immobilized cell is a kind of effective means, and it can improve cell Pyrogentisinic Acid's tolerance, and can reuse.Up to now, there are multiple immobilization technology and immobilization material for wastewater treatment.Entrapping method is most widely used method.Alginate calcium is because price is low, fixing step simple, do not have toxicity to become a kind of common embedding medium to cell.But the bead that alginate calcium is made is frangible, and easily by microbial destruction.If add polyvinyl alcohol to form mixture, that will improve mechanical property and the thermostability of bead.
Summary of the invention
The object of the present invention is to provide immobilized cell and the application in degradation of phenol pollutent thereof of the mutagenesis bacterium M1 of a kind of Rhodococcus ruber SD3, can effectively improve phenol degrading performance.
The present invention realizes like this, the immobilized cell of the mutagenesis bacterium M1 of a kind of Rhodococcus ruber SD3, massfraction 1 % sodium alginate and massfraction 1 % polyvinyl alcohol embedding preparation for immobilized cell, getting 10 g wet thallus evenly mixes at normal temperatures with 10 ml 1 % sodium alginate-1 % polyvinyl alcohol solutions, above mixed solution is added in aseptic syringe, splash into slowly in the ice-cold aseptic saturated boric acid-calcium chloride solution of 0. 1 mol/L, saturated boric acid-the calcium chloride solution that contains gel beads is put into 4 ℃ of refrigerators and fix 24 h, discard saturated boric acid-calcium chloride solution, add sterile distilled water washing once, add again sterile distilled water, 4 ℃ save backup.
The diameter of described immobilized cell is 6mm.
Described immobilized cell is reused five times, in 72h to the phenol degrading rate of 2g/L all more than 98%.
Described thalline is the mutagenesis bacterium M1 of Rhodococcus ruber SD3, now be kept at depositary institution's preservation of State Intellectual Property Office's appointment, depositary institution is Chinese Typical Representative culture collection center, preservation date is on March 28th, 2013, deposit number is CCTCC NO:M 2013112, and Latin name is Rohodococcus ruber M1.
Rhodococcus ruber SD3 of the present invention is preservation disclosed bacterial classification, now be kept at depositary institution's preservation of State Intellectual Property Office's appointment, depositary institution is Chinese Typical Representative culture collection center, preservation date is on February 26th, 2012, deposit number is CCTCC NO:M 2012035, and Latin name is Rhodococcus ruber SD3.
Rhodococcus ruber SD3 by 0.3 % lithium chloride mutagenesis after the mutagenesis bacterium M1 that obtains of screening at phenol minimal medium (NaCl 1 g, NH
4cl 1 g, MgSO
47H
2o 3 g, K
2hPO
41.5 g, KH
2pO
40.5 g, phenol 1.5g, is settled to 1000 mL without phenol distilled water) in, 35 ℃, the concussion of 200r/min shaking table are cultivated after 72h, and phenol degrading rate is 99.77%.Therefore the mutagenesis bacterium M1 of Rhodococcus ruber SD3 has high efficiency at degradation of phenol pollutent, can be used for biological process and processes containing phenol trade effluent.
Technique effect of the present invention is: immobilized cell is reused five times, in 72h to the phenol degrading rate of 2g/L all more than 98%, therefore the immobilized cell of the mutagenesis bacterium M1 of Rhodococcus ruber SD3 has high efficiency at degradation of phenol pollutent, can be used for biological process and processes containing phenol trade effluent.
Accompanying drawing explanation
Fig. 1 is the degradation of phenol performance map of the mutagenesis bacterium M1 of Rhodococcus ruber SD3.
Fig. 2 is the immobilized cell figure of the mutagenesis bacterium M1 of Rhodococcus ruber SD3.
Embodiment
Embodiment 1: the degradation of phenol performance of the mutagenesis bacterium M1 of Rhodococcus ruber SD3
Mutagenesis bacterium M1 with transfering loop picking Rhodococcus ruber SD3, brings back to life in LB substratum, treats that growth is to logarithmic phase.By the amount of 2 %, be inoculated in phenol minimal medium (NaCl 1 g, NH
4cl 1 g, MgSO
47H
2o 3 g, K
2hPO
41.5 g, KH
2pO
41.5 g, phenol 1.5g, is settled to 1000 mL without phenol distilled water), 35 ℃, 200 r/min shaking table concussion cultivation 72 h.The degradation rate that adopts 4-aminoantipyrene spectrophotometry to record phenol is 99.77%(Fig. 1).
Embodiment 2: the process for fixation of the mutagenesis bacterium M1 of Rhodococcus ruber SD3
1, the mutagenesis bacterium M1 of picking Rhodococcus ruber SD3 from the inclined-plane of preserving, is inoculated in LB medium liquid, 35 ℃, 200 rpm shaking table vibration activation culture 28 h.The bacterium liquid of getting activation according to the inoculum size inoculation of 2 % after 35 ℃, 200 rpm shaking table shaking culture.
2, by the bacterium liquid of cultivating respectively at centrifugal 10 min of 4000 rpm, collect thalline in the aseptic centrifuge tube of 50 ml, add centrifugal 10 min of 4000 rpm after 50 ml sterilized waters vibration washings, repeat above-mentioned steps and wash once again.
3, getting 10 g wet thallus evenly mixes at normal temperatures with 10 ml 1 % sodium alginate (SA)-1 % polyvinyl alcohol (PVA) solution.
4, above mixed solution is added in aseptic syringe, splash into slowly in the ice-cold aseptic saturated boric acid-calcium chloride solution of 0. 1 mol/L.
5, the saturated boric acid-calcium chloride solution that contains gel beads is put into 4 ℃ of refrigerators and fix 24 h.
6, discard saturated boric acid-calcium chloride solution, add sterile distilled water washing once, then add sterile distilled water, 4 ℃ save backup (Fig. 2).
Embodiment 3: the reusability of the immobilized cell of the mutagenesis bacterium M1 of Rhodococcus ruber SD3
1, immobilized cell is added to phenol minimal medium (NaCl 1 g, NH
4cl 1 g, MgSO
47H
2o 3 g, K
2hPO
41.5 g, KH
2pO
40.5 g, phenol 2.0g, is settled to 1000 mL without phenol distilled water) in, 35 ℃, 200 rpm shaking table shaking culture 72 h.
2, in the substratum from cultivating, take out immobilized cell, use sterilized water washed twice.Immobilized cell after washing is added to new phenol minimal medium (NaCl 1 g, NH
4cl 1 g, MgSO
47H
2o 3 g, K
2hPO
41.5 g, KH
2pO
40.5 g, phenol 2.0g, is settled to 1000 mL without phenol distilled water) in, 35 ℃, 200 rpm shaking table shaking culture 72 h, reuse five times.Five phenol degrading rates of for the first time to the are respectively 99.99%, 99.99%, 98.98%, 99.78%, 98.53%.
Claims (2)
1. the immobilized cell of the mutagenesis bacterium M1 of a Rhodococcus ruber SD3, it is characterized in that massfraction 1 % sodium alginate and massfraction 1 % polyvinyl alcohol embedding preparation for immobilized cell, getting 10 g wet thallus evenly mixes at normal temperatures with 10 ml 1 % sodium alginate-1 % polyvinyl alcohol solutions, above mixed solution is added in aseptic syringe, splash into slowly in the ice-cold aseptic saturated boric acid-calcium chloride solution of 0. 1 mol/L, saturated boric acid-the calcium chloride solution that contains gel beads is put into 4 ℃ of refrigerators and fix 24 h, discard saturated boric acid-calcium chloride solution, add sterile distilled water washing once, add again sterile distilled water, 4 ℃ save backup, described thalline is the mutagenesis bacterium M1 of Rhodococcus ruber (Rohodococcus ruber) SD3, deposit number is CCTCC NO:M 2013112.
2. the immobilized cell of the mutagenesis bacterium M1 of Rhodococcus ruber SD3 according to claim 1, is characterized in that, the diameter of described immobilized cell is 6mm.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103627653B (en) * | 2013-10-17 | 2016-01-20 | 浙江省环境保护科学设计研究院 | A kind of Rhodococcus ruber bacterial strain and the application in the wastewater treatment containing organic pollutant thereof |
| CN104673710B (en) * | 2014-12-31 | 2017-09-01 | 浙江至美环境科技有限公司 | Rhodococcus strain and its application |
| CN104894012A (en) * | 2015-05-15 | 2015-09-09 | 南京农业大学 | 17 beta-estradiol degrading strain and application thereof |
| CN105800775B (en) * | 2016-05-20 | 2019-02-15 | 浙江新三印印染有限公司 | A kind of discoloration method of wastewater in textile printing and dyeing industry |
| KR20210114436A (en) | 2019-01-15 | 2021-09-23 | 랴오닝 그레이티스트 바이오-파마슈티컬 컴퍼니 리미티드 | Products derived from Rhodococcus louver and pharmaceutical uses thereof |
| CN112218647B (en) | 2019-04-24 | 2023-07-04 | 辽宁天安生物制药股份有限公司 | Use of rhodococcus ruber products in the treatment of thermal injury |
| CN110564716B (en) * | 2019-08-05 | 2021-07-20 | 武汉大学 | Bacterium-carrying composite microsphere for synchronously removing phenol and aniline, and preparation method and application thereof |
| US20230069441A1 (en) | 2020-01-21 | 2023-03-02 | Liaoning Greatest Bio-Pharmaceutical Co., Ltd. | Use of rhodococcus ruber cell wall skeleton in regenerative medicine |
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| CN101857846A (en) * | 2010-04-20 | 2010-10-13 | 深圳清华大学研究院 | Rhodococcus erythropolis and microbial inoculum and application thereof |
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| CN101857846A (en) * | 2010-04-20 | 2010-10-13 | 深圳清华大学研究院 | Rhodococcus erythropolis and microbial inoculum and application thereof |
| CN102604875A (en) * | 2012-03-26 | 2012-07-25 | 江西师范大学 | Rhodococcus ruber and application thereof in degradation of phenol pollutants |
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