CN103160491B - Immobilized cell of mutagenic bacterium M1 of Rhodococcus ruber SD3 and application of immobilized cell in degradation of phenol pollutants - Google Patents
Immobilized cell of mutagenic bacterium M1 of Rhodococcus ruber SD3 and application of immobilized cell 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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- phenol
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- rhodococcus ruber
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- 210000001822 immobilized cell Anatomy 0.000 title claims abstract description 26
- 241000187563 Rhodococcus ruber Species 0.000 title claims abstract description 24
- 241000822976 bacterium M-1 Species 0.000 title claims abstract description 24
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 title abstract description 42
- 230000015556 catabolic process Effects 0.000 title abstract description 13
- 238000006731 degradation reaction Methods 0.000 title abstract description 13
- 239000003344 environmental pollutant Substances 0.000 title abstract description 5
- 231100000719 pollutant Toxicity 0.000 title abstract description 5
- 231100000219 mutagenic Toxicity 0.000 title abstract 6
- 230000003505 mutagenic effect Effects 0.000 title abstract 6
- 231100000350 mutagenesis Toxicity 0.000 claims abstract description 20
- 238000002703 mutagenesis Methods 0.000 claims abstract description 20
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 8
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 8
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 5
- 239000000661 sodium alginate Substances 0.000 claims abstract description 5
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 5
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- UKVHQGHRJIEIML-UHFFFAOYSA-L calcium boric acid dichloride Chemical class [Cl-].[Cl-].[Ca+2].OB(O)O UKVHQGHRJIEIML-UHFFFAOYSA-L 0.000 claims description 7
- 239000011324 bead Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 229920006395 saturated elastomer Polymers 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 230000000593 degrading effect Effects 0.000 abstract description 7
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 2
- 238000010170 biological method Methods 0.000 abstract 1
- 239000010842 industrial wastewater Substances 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 230000031018 biological processes and functions Effects 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002351 wastewater Substances 0.000 description 3
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 229940072056 alginate Drugs 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000004065 wastewater treatment Methods 0.000 description 2
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- 241000193764 Brevibacillus brevis Species 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000222128 Candida maltosa Species 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
An immobilized cell of a mutagenic bacterium M1 of Rhodococcus ruber SD3 and application thereof in degradation of phenol pollutants, wherein the degrading rate of the mutagenic bacterium M1 obtained by screening the mutagenic bacterium M1 of the Rhodococcus ruber SD3 after 0.3% lithium chloride mutagenesis to 1.5g/L phenol in 72 hours is 99.77%. The mutagenic bacterium M1 was embedded with 1% sodium alginate and 1% polyvinyl alcohol to prepare immobilized cells with a diameter of 6 mm. The immobilized cells are repeatedly used for 5 times, and the degradation rate of the immobilized cells to 2g/L phenol within 72 hours is over 98 percent. Therefore, the mutagenic bacterium M1 of the rhodococcus ruber SD3 and the immobilized cells thereof have high efficiency in degrading phenol pollutants and can be used for treating phenol-containing industrial wastewater by a biological method.
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 |
|---|---|---|---|---|
| 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 |
| CN111727235B (en) | 2019-01-15 | 2024-03-15 | 辽宁天安生物制药股份有限公司 | Rhodococcus ruber product and pharmaceutical application thereof |
| KR20220004091A (en) | 2019-04-24 | 2022-01-11 | 랴오닝 그레이티스트 바이오-파마슈티컬 컴퍼니 리미티드 | Use of Rhodococcus louver 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 |
| KR20220130162A (en) | 2020-01-21 | 2022-09-26 | 랴오닝 그레이티스트 바이오-파마슈티컬 컴퍼니 리미티드 | Use of Rhodococcus louver cell wall scaffold in regenerative medicine |
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