CN109628337B - Efficient phenol degrading bacterium and application thereof - Google Patents
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- 238000006731 degradation reaction Methods 0.000 claims description 20
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- 238000011081 inoculation Methods 0.000 description 4
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- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- 108010080698 Peptones Proteins 0.000 description 2
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
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- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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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
-
- 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
-
- 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
- C02F2101/34—Organic compounds containing oxygen
- C02F2101/345—Phenols
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- Bioinformatics & Cheminformatics (AREA)
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- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
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Abstract
The invention discloses a high-efficiency phenol degrading bacterium and application thereof, wherein the preservation number of the strain is CGMCC NO. 16470. The strain can effectively degrade high-concentration wastewater containing phenol.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to a high-efficiency phenol degrading bacterium and application thereof.
Background
Phenol is a main pollutant in industrial wastewater of oil refining, plastics, textiles and the like, is high in toxicity and difficult to degrade, and is a pollutant which is preferably controlled by environmental protection organizations of a plurality of countries. The treatment method of the phenol-containing wastewater comprises a physical chemical method and a biological method, and the physical and chemical treatment methods have the defects of high cost, complex operation, easy secondary pollution and the like.
In recent years, with the development of new biological technologies, the research on the treatment of phenol-containing wastewater by microbial degradation has been receiving more and more attention. Wherein, most of the separated strains are suitable for treating the waste water containing phenol with low concentration, and less strains can treat the waste water containing phenol with high concentration. The efficient degradation of phenol in sewage is a hot problem in the current water pollution treatment field.
In order to obtain a stable and efficient degradation effect, the phenol can be degraded into non-toxic or low-toxic substances by adopting a microbial remediation technology. Among them, the most important point is to screen the functional strains which can degrade phenol with high efficiency. Therefore, it is necessary to provide a strain which can effectively degrade phenol and can be used in the field of water pollution treatment.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a high-efficiency phenol degrading bacterium and application thereof. The strain can efficiently degrade phenol, and particularly has high degradation effect on high-concentration phenol-containing polluted water.
The purpose of the invention is realized by the following technical scheme:
the strain for degrading phenol has a preservation number of CGMCC NO. 16470.
The application of the strain for degrading phenol in repairing phenol-containing water pollution.
Preferably, the concentration of the strain used for degrading phenol is 108MPN/ml-1010MPN/ml。
Preferably, the applicable pH value of the strain is 5-9.
Preferably, the phenol concentration used for degradation is from 1000mg/L to 2500 mg/L.
Microbial preservation for patent procedure:
the preservation date is as follows: 9/13/2018
The preservation unit: china general microbiological culture Collection center
The preservation number is: CGMCC NO.16470
And (4) storage address: xilu No.1 of Beijing, Chaoyang, Beijing, and the institute for microbiology, Chinese academy of sciences, which are assigned by classification (strains): acinetobacter (Acinetobacter sp.QC-01).
Compared with the prior art, the high-efficiency phenol degrading bacterium and the application thereof provided by the invention have the following technical effects: 1) the strain Acinetobacter sp.QC-01 for degrading phenol can take phenol as a unique carbon nitrogen source, so that the economic benefit is improved for biologically degrading phenol; 2) acinetobacter sp.QC-01, which is a strain that degrades phenol, can degrade phenol at high concentrations, e.g., completely degrade phenol at an initial concentration of 200mg/L within 10 h; 3) the phenol degrading strain Acinetobacter sp.QC-01 can quickly repair phenol-containing polluted water.
Drawings
FIG. 1 is a graph showing the growth of strains according to example 2 of the present invention;
FIG. 2 is a graph of the degradation rate of phenol by the beads shown in example 3 according to the present invention at different initial phenol concentrations;
FIG. 3 is a graph showing the degradation rate of phenol under different pH conditions by the strain shown in example 4 according to the present invention.
FIG. 4 is a graph showing the phenol degradation rate of wastewater remediated with the strain shown in example 5 according to the present invention.
The following detailed description of the embodiments of the present invention is provided in connection with the examples to facilitate understanding and understanding of the technical solutions.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available without specific reference, and the following examples are not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention are intended to be included in the scope of the present invention.
Example 1
Isolation and characterization of strains
Adding polluted soil of a pesticide factory into an inorganic salt culture medium with phenol (200mg/L) as a unique carbon source, and performing shaking table domestication culture at the constant temperature of 30 ℃ and the constant temperature of 150 r/min; inoculating the domesticated culture solution into a fresh phenol inorganic salt culture medium containing 200mg/L every 5 days, wherein the inoculation amount is 10% (V/V); after continuous acclimation for 30 days, diluting the enrichment culture, coating the enrichment culture in a 200mg/L phenol solid culture medium, and culturing in a constant temperature incubator at 30 ℃ for 2-3 days to form single colonies. Selecting colony, purifying and culturing to obtain single colony with same size and shape, streaking to slant solid culture medium, culturing for 2-3 days to obtain target colony, and storing at about 4 deg.C.
In some embodiments, the mineral salts medium can include K2HPO4、KH2PO4、NaCl、MgSO4.H2O、MnS04.H20、Fe2(S04)3.H 20、(NH4)2S04Or phenol or a combination thereof. In some embodiments, K can be included in 1000ml of mineral salts medium2HPO4 0.4g、KH2PO4 0.1g、NaCl 0.1g、MgSO4.H2O 0.2g、MnS04.H20 0.01g、Fe2(S04)3.H 20 0.01g、(NH4)2S04Any one or combination of 0.4g, or 0.5g phenol.
In some embodiments, the solid medium may comprise KH2PO4、Na2HPO4.H2O, sucrose, peptone, yeast powder, NaCl, MgSO4.7H2O、FeSO4.7H2O, or agar, or a combination thereof; in some embodiments, 1000ml of solid medium may comprise KH2PO4 0.9g、Na2HPO4.H26.5g of O, 3g of sucrose, 1g of peptone, 0.5g of yeast powder, 1g of NaCl and MgSO4.7H2O 0.2g、FeSO4.7H20.01g of O, or 15-20g of agar, or a combination thereof.
In some embodiments, the inorganic salt medium initial pH can be 7.0-8.0 and the solid medium initial pH can be 7.0-8.0.
In some embodiments, the mineral salts medium can be sterilized at high pressure, 121-; the solid medium can be sterilized at high pressure, 121-.
To facilitate understanding of the isolation of the strains, the isolation process of the strains is exemplified below:
(1) taking 10g of polluted soil of the pesticide factory, adding the polluted soil into a 100ml of triangular conical flask containing 200mg/L of phenol inorganic salt culture medium, and performing acclimatization culture on the soil at the constant temperature of 30 ℃ for 150 r/min; inoculating the domesticated culture solution into a fresh phenol inorganic salt culture medium containing 200mg/L every 5 days, wherein the inoculation amount is 10% (V/V);
(2) after 30 days of continuous acclimation, the enrichment culture was diluted 10 times respectively-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9Spreading 0.1ml of each of the above solutions on 200mg/L phenol solid plate, and culturing in 30 deg.C constant temperature incubator for 2-3 days;
(3) selecting single bacterial colony to perform strain purification culture, repeating for more than 3 times until single bacterial colony with the same shape and size appears on the plate;
(4) and (4) selecting the single colony in the step (3), streaking the single colony to a slant solid culture medium, culturing for 2-3 days to obtain a target strain, and storing at 4 ℃.
Identification of strain morphology and physiological and biochemical levels
The bacterial strain is gram negative, a single bacterium is rod-shaped and has no spore, the bacterial strain grows on an inorganic salt fixed culture medium, the bacterial colony is milky white, raised and slightly moist, and is dotted, and the diameter of the bacterial colony is about 1.5 mm. The strain can grow by taking phenol as the only carbon-nitrogen source, and can completely degrade phenol with the initial concentration of 200mg/L within 12 h. The optimal growth temperature of the strain is 30-35 ℃, the optimal pH value is 7.0-8.0, and the optimal rotation speed of a production shaker is 100-.
Molecular biology level identification:
bacterial DNA was extracted using a DNA extraction kit (Sangon) following conventional extraction procedures and then subjected to PCR amplification of the desired fragment.
A pair of universal primers designed to amplify a 16s rDNA sequence was:
27F 5'-AGTTTGATCMTGGCTCAG-3'(SEQ ID NO.:1)；
1492R 5'-GGTTACCTTGTTACGACTT-3(SEQ ID NO.:2)；
using genome DNA as a template, adding Premix TapTM, performing PCR amplification, detecting PCR products by using 1% agarose gel electrophoresis, purifying by using a DNA purification recovery kit, connecting to a pGM-T vector, transforming into Escherichia coli DH5 alpha competent cells, coating into an LB solid culture medium containing ampicillin, culturing at 37 ℃ for 12h, picking colonies into a liquid LB culture medium, performing shaking culture at 37 ℃ and 180rpm overnight, and extracting plasmids by using a plasmid extraction kit for determination.
Wherein, the PCR reaction condition is pre-denaturation at 94 ℃ for 5 min; then 30 cycles of denaturation at 94 ℃ for 1 min; annealing at 55 deg.C for 1 min; extension was then carried out at 72 ℃ for 5 min. The PCR amplification product is sent to Shanghai Biotech company for sequencing. The sequence is shown as SEQ ID NO. 3:
the strain is identified by 16S rDNA, and the results are subjected to homology comparison by a Genebank Blast program to obtain that the homology of the strain and Acinetobacter sp is 99 percent, and further obtain that the strain is Arthrobacter. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 9 and 30 months, and the preservation number is as follows: CGMCC NO. 16470.
EXAMPLE 2 growth curves of the strains
Selecting solid culture medium at 4 deg.C for preserving Acinetobacter sp.QC-01, culturing in phenol culture medium at 100mg/L to 108MPN/ml, inoculating the bacterial suspension into a fresh inorganic salt liquid culture medium according to the inoculation amount of 5% (V/V), and culturing in a shaking table at 30 ℃ and 150 r/min. Wherein the initial pH of the liquid medium was 7.0, and the growth curve was plotted with time as abscissa and absorbance OD600 as ordinate. The OD600 values were measured by sampling every 1 hour. The growth curve of the strain is shown in figure 1, the strain is in the regulation phase within 0-2h and enters the logarithmic growth phase from the 2 h; the OD600 value was 0.8 at 12h, and the OD600 value was 1.0 at 20h when the bacteria continued to grow.
Example 3 degradation rate of phenol by beads at different initial phenol concentrations
Selecting solid culture medium at 4 deg.C for preserving Acinetobacter sp.QC-01, culturing in phenol solid culture medium at 100mg/L to 108MPN/ml, inoculating the bacterial suspension into fresh inorganic salt culture medium of 50mg/L, 100mg/L, 200mg/L, 500mg/L and 1000mg/L according to the inoculation amount of 5% (V/V), and culturing in a shaking table of 30 ℃ and 150 r/min. Wherein, the substrate concentration time of the inorganic salt culture medium is an abscissa, the phenol degradation rate is an ordinate, and a curve is drawn to investigate the phenol degradation rate of the bacteria beads under different initial phenol concentrations. As shown in FIG. 2, the strain can degrade phenol at a rate of 100% in the range of 0mg/L to 200mg/L of the initial phenol concentration.
Example 4 degradation rate of phenol by the strains under different pH conditions
Selecting solid culture medium at 4 deg.C for preserving Acinetobacter sp.QC-01, culturing in phenol solid culture medium at 100mg/L to 108MPN/ml, the bacterial suspension was inoculated into fresh inorganic salt medium of pH 5, pH 6, pH 7, pH 8, pH 9 in an inoculum size of 5% (V/V), and cultured in a shaker at 30 ℃ and 150 r/min. Wherein, the initial pH of the inorganic salt liquid culture medium is an abscissa, the phenol degradation rate is an ordinate, and a curve is drawn. And (3) investigating the degradation rate of the strain on phenol under different pH conditions.
The phenol degradation rate of the strain under different initial phenol concentration conditions is shown in fig. 3, and the phenol degradation rate of the strain is highest under the condition that the initial pH value is 7.
EXAMPLE 5 Strain remediation of phenol-containing Water contamination
As shown in FIG. 4, wastewater containing phenol with a concentration of 1500mg/L is taken, a carbon source, a nitrogen source and a phosphorus source are added according to a ratio of 200:5:1 to prepare wastewater with a COD concentration of 3500mg/L, phenol degrading bacteria seed liquid Acinetobacter sp.QC-01 is added, and the strain density is 108MPN/ml, after 48 hours of action, the concentration of phenol in the wastewater is detected by 4-aminoantipyrine spectrophotometry, the concentration of phenol in the wastewater is 0.6mg/L and the COD effluent is 36mg/L, and therefore, the degradation rate of phenol degradation by the strain in the embodiment reaches 99%.
The foregoing description shows and describes several preferred embodiments of the invention, but as aforementioned, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> environmental protection technology Limited, Jiangsu Yiyu
<120> high-efficiency phenol degrading bacterium and application thereof
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tgggcgtaaa gcgtgcgtag gcggctgatt aagtcggatg tgaaatccct gagcttaact 600
taggaattgc attcgatact ggtcagctag agtatgggag aggatggtag aattccaggt 660
gtagcggtga aatgcgtaga gatctggagg aataccgatg gcgaaggcag ccatctggcc 720
taatactgac gctgaggtac gaaagcatgg ggagcaaaca ggattagata ccctggtagt 780
ccatgccgta aacgatgtct actagccgtt ggggcctttg aggctttagt ggcgcagcta 840
acgcgataag tagaccgcct ggggagtacg gtcgcaagac taaaactcaa atgaattgac 900
gggggcccgc acaagcggtg gagcatgtgg tttaattcga tgcaacgcga agaaccttac 960
ctggtcttga catagtaaga actttccaga gatggattgg tgccttcggg aacttacata 1020
caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080
agcgcaaccc ttttccttat ttgccagcac ttcgggtggg aactttaagg atactgccag 1140
tgacaaactg gaggaaggcg gggacgacgt caagtcatca tggcccttac gaccagggct 1200
acacacgtgc tacaatggtc ggtacaaagg gttgctacct agcgatagga tgctaatctc 1260
aaaaagccga tcgtagtccg gattggagtc tgcaactcga ctccatgaag tcggaatcgc 1320
tagtaatcgc ggatcagaat gccgcggtga atacgttccc gggccttgta cacaccgccc 1380
gtcacaccat gggagtttgt tgcaccagaa gtagctagcc taactgcaaa gagggcggtt 1440
accacggtgt ggccgatgac tggggtgaag tcgtaaca 1478
Claims (5)
1. The efficient phenol degrading bacterium is characterized in that the preservation number of the phenol degrading bacterium is CGMCC NO.16470, and the phenol degrading bacterium is classified and named as Acinetobacter sp.
2. The use of the high efficiency phenol degrading bacteria of claim 1 in remediating phenol-containing water pollution.
3. Use according to claim 2, characterized in that the concentration of the strain used for the efficient degradation of phenol is 108MPN/ml-1010MPN/ml。
4. The use according to claim 2, wherein the phenol-degrading bacteria are suitable for use at a pH of 5 to 9.
5. Use according to claim 2, characterized in that the phenol concentration used for the degradation is 1500 mg/L.
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| KR20010097469A (en) * | 2000-04-24 | 2001-11-08 | 손 경 식 | Pseudomonas putida CJ-PEN a) and Acinetobacteria lwoffii CJ-PEN b) having an improved property for degrading phenol and/or phenol chloride |
| CN102851251A (en) * | 2012-04-24 | 2013-01-02 | 黑龙江省科学院大庆分院 | Genetically engineered bacteria for degrading phenol in petrochemical wastewater |
| CN103695351A (en) * | 2013-12-18 | 2014-04-02 | 浙江工商大学 | Acinetobacter baumannii and application thereof |
| CN104528951A (en) * | 2014-12-29 | 2015-04-22 | 天津凯英科技发展有限公司 | Microbial agent for treating coking wastewater and application thereof |
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| KR20010097469A (en) * | 2000-04-24 | 2001-11-08 | 손 경 식 | Pseudomonas putida CJ-PEN a) and Acinetobacteria lwoffii CJ-PEN b) having an improved property for degrading phenol and/or phenol chloride |
| CN102851251A (en) * | 2012-04-24 | 2013-01-02 | 黑龙江省科学院大庆分院 | Genetically engineered bacteria for degrading phenol in petrochemical wastewater |
| CN103695351A (en) * | 2013-12-18 | 2014-04-02 | 浙江工商大学 | Acinetobacter baumannii and application thereof |
| CN104528951A (en) * | 2014-12-29 | 2015-04-22 | 天津凯英科技发展有限公司 | Microbial agent for treating coking wastewater and application thereof |
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