CN102952767A - Pseudomonas psychrophila HA-4 low-temperature sulfamethoxazole degrading bacteria as well as screening method and application thereof - Google Patents

Pseudomonas psychrophila HA-4 low-temperature sulfamethoxazole degrading bacteria as well as screening method and application thereof Download PDF

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CN102952767A
CN102952767A CN2012101535527A CN201210153552A CN102952767A CN 102952767 A CN102952767 A CN 102952767A CN 2012101535527 A CN2012101535527 A CN 2012101535527A CN 201210153552 A CN201210153552 A CN 201210153552A CN 102952767 A CN102952767 A CN 102952767A
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sulfamethoxazole
temperature
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pseudomonas
low temperature
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CN102952767B (en
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马放
蒋本超
李昂
蔡蕊
崔迪
邱天
张斯�
庞长泷
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Yixing Environmental Protection Industry Co.,Ltd.
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HIT YIXING ACADEMY OF ENVIRONMENTAL PROTECTION
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Abstract

The invention discloses a pseudomonas psychrophila HA-4 low-temperature sulfamethoxazole degrading bacteria, and belongs to the technical fields of bioengineering and environment engineering technology. The pseudomonas psychrophila HA-4 low-temperature sulfamethoxazole degrading bacteria is a low-temperature pseudomonas strain, which is preserved by the microbial strains preservation management committee center of general microbiology in March 27th, 2012, with the preservation number of CGMCC No.5944. The pseudomonas psychrophila HA-4 low-temperature sulfamethoxazole degrading bacteria is obtained by separating sludge in some sewage treatment plant aeration tank in Haerbin city; the strain can be grown by serving sulfamethoxazole as unique carbon, nitrogen and energy sources; the sulfamethoxazole can be degraded during culture of cell liquid; the cell liquid can be used for biodegradation of the sulfamethoxazole in city sewage; and by the strain, the removal rate of the sulfamethoxazole with the initial concentration of 100mg/L under a low-temperature condition is up to 35.34 percent.

Description

One strain Pseudomonas psychrophila HA-4 low temperature sulfamethoxazole degradation bacteria and screening method and application
Technical field
The invention belongs to biotechnology, field of environment engineering technology, relate to bacterial strain Pseudomonas psychrophilaHA-4 and the application in the degraded sulfamethoxazole thereof.
Background technology
Medicine and personal-care supplies (Pharmaceuticals and personal care products) mainly comprise various prescribed and non prescribed medicines, spices, hair dye, makeup, opalizer, hair jelly, shampoo, perfumed soap etc.Wherein most of medicine just directly is drained in the environment without metabolism in vivo, in addition in the production process of medicine and process in the expired process with not using medicine and also can cause it to enter in the water surrounding; Personal-care supplies also can directly enter water surrounding when washing one's face and rinsing one's mouth and swim.
Although the concentration that most PPCPs exist in environment is very low, because bioconcentration, along with the increase of time, PPCPs will be finally to the ecosystem, especially water surrounding is produced far-reaching and expendable impact.The treatment process that PPCPs is commonly used in the water body has Physical, chemical method and biological process etc., and biological treatment is compared and is considered to the most environmental friendliness and method with low cost.The concentration of the biological treatment of PPCPs and the structure of its compound, starting point concentration, elementary substrate, source and the incubation time of microbe inoculation are relevant.
Sulfamethoxazole is widely used in human body as a kind of antibacterials, and it belongs to a kind of among the PPCPs.At present, in China's effluent of municipal sewage plant, the mean concns level of this medicine is the highest, even has the negative phenomenon of removing to exist, and will be higher than spring in the winter time in its mean concns of northern area.
Foreign study shows, Bacillus firmus, Bacillus cereus, Rhodococcus rhodochrous, Pseudomonas, Flavobacterium, AeromonasDeng the ability with degraded sulfamethoxazole.At present, both at home and abroad the researcher mainly study sulfamethoxazole under normal temperature condition biological degradation and report under cold condition is less.
Summary of the invention
Purpose of the present invention is exactly to remove the usefulness of the PPCPs pollutant take sulfamethoxazole as representative for strengthening the low temp biochemical system, isolates bacterial strain from occurring in nature Pseudomonas psychrophilaHA-4 can promote the degraded of sulfamethoxazole, to the removal based theoretical of the sulfamethoxazole in the low temperature municipal effluent in low temperature environment.It is a strain low temperature pseudomonas strain.
Technical solution of the present invention is: a strain Pseudomonas psychrophilaHA-4 low temperature sulfamethoxazole degradation bacteria it is characterized in that a strain low temperature pseudomonas strain, was preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC No.5944 on March 27th, 2012.
Bacterial strain Pseudomonas psychrophilaHA-4 separates to obtain from active sludge, and design temperature (10 ℃) screening is specifically screened step as follows:
(1) getting the 20mL Harbin fresh active sludge of peaceful municipal wastewater treatment plant Aerobic Pond adds 100mL and contains 20mg/L take sulfamethoxazole as sole carbon source, in the minimal medium triangular flask of the energy, placing temperature is 10 ℃, rotating speed is to cultivate a week in the shaking table of 150r/min, obtains enrichment bacterium liquid;
(2) get 10mL enrichment bacterium liquid and add in the 100mL sulfamethoxazole minimal medium triangular flask, the culture condition of repeating step (1) 3 ~ 4 times, the concentration of each sulfamethoxazole improves step by step, finally to 100mg/L, obtains domestication bacterium liquid;
(3) get 100 μ L domestication bacterium liquid and add and contain in the 100mg/L sulfamethoxazole solid inorganic salt culture medium flat plate, coating method smoothens, and placing temperature is 10 ℃ incubator, cultivates a week, obtains single bacterium colony;
(4) repetitive operation step (3) 3 ~ 4 times is namely finished the acquisition of purebred low temperature sulfamethoxazole degradation bacteria;
The purebred low temperature sulfamethoxazole degradation bacteria that (5) will filter out is inoculated into the inclined-plane enrichment medium, in 4 o Preserve inoculum size 10% under the C;
Wherein the composition of minimal medium is: 3.5g K 2HPO 4, 0.5gNaCl, 1.5g KH 2PO 4, 0.15g MgSO 47H 2O, 0.5g (NH 4) 2SO 4, the 1mL trace element solution is dissolved in the 1000mL distilled water, regulates pH7.0; The composition of inorganic salt solid medium is to add in addition agar 2g/L in the composition of minimal medium;
The enrichment culture based component is: 1.5 g KH 2PO 4, 0.15 g MgSO 47H 2O, 0.5 g (NH 4) 2SO 4, 0.5 g NaCl, 3.5 g K 2HPO 4, 2g peptone, 18g agar, 1.0 mL trace element solutions be dissolved in the distilled water of 1000mL, regulates pH to 7.0;
The composition of trace element solution is: 0.1 g CuSO 45H 2O, 0.3 g MnSO 44H 2O, 0.2 g ZnSO 47H 2O, 2.0 g NaHCO 310H 2O, 0.5 g CoCl 26H 2O, 0.05g CaCl 22H 2O, 0.5g FeSO 47H 2O, 0.02g (NH 4) 6Mo 7O 24H 2O is dissolved in the distilled water of 1000mL.
The OD of the domestication bacterium liquid that step (2) obtains 660=1.192; Substratum all is at 121 ℃, and autoclaving used after 20 minutes.
This bacterial strain Pseudomonas psychrophilaHA-4 suits to grow in the developing medium of (pH 6.0), and growth temperature is 10 ℃, its bacteriology morphological specificity: be gram negative bacillus, atrichia, aerobic, the degradable sulfamethoxazole can be take sulfamethoxazole as sole carbon source and energy growth.
Through strain identification, having obtained length is the 16S rDNA sequence of 1609bp.
The PCR primer adopts universal primer F8 (5 '-AGA GTT TGA TCA TGG CTC AG-3) and the R1522 (5 '-AAG GAC GTC ATC CAG CCG CA-3 ') of bacterial 16 S rDNA.(GeneAmp, PCR System 9700) carries out amplified reaction with the PCR instrument.The reaction system cumulative volume is 25 μ l: ultrapure water 16.2 μ l, and 10 х Buffer, 2.5 μ l, dNTP 2 μ l, F8 primer 1 μ l, R22 primer 1 μ l, ExTaq enzyme 0.3 μ l, total DNA do to get 2 μ l in reaction system after certain dilution.94 ℃ of denaturation 5 min, through 30 circulations, wherein, and 98 ℃ of sex change 20 s, 55 ℃ of annealing 40 s, 72 ℃ are extended 1.5 min, extend 7 min, 1% agarose gel electrophoresis, ultraviolet detection at 72 ℃ again after 30 circulations.Carry out homology relatively by the Blast program, show that the homology of this bacterial strain and Pseudomonas psychrophila is up to 99%.
Bacterial strain of the present invention Pseudomonas psychrophilaHA-4 is applied to the biological treatment of sulfamethoxazole in the sewage under the low temperature.
Bacterial strain of the present invention Pseudomonas psychrophilaHA-4 needs to tame in the minimal medium with sulfamethoxazole before application, culture condition be bacterial strain at 10 ℃, pH 7.0, low-temperature aerobic is cultivated.This bacterial strain has higher degradation capability to sulfamethoxazole in the low temperature habitat, and has widely substrate broad spectrum, sees Table 1:
The substrate broad spectrum of table 1 bacterial strain HA-4
Substrate The result Substrate The result
Carbamzepine + Carbazole +
17 α-lynoral + Oestrone +
Trihydroxy-oestrin + Pentanoic +
Oil of mirbane - P-diaminodiphenyl +
Resorcinol + 1-amino-2-an aromatic plant metioned in ancient books phenol-4-sulfonic acid +
Ibuprofen BP/EP + Dimethylbenzene -
Two different sour toluene esters + The 4-amino-2,2,6,6-tetramethylpiperidine +
Toluene - Benzene -
Annotate :+: well-grown;-: do not grow
The beneficial effect that the present invention reaches is: strain Pseudomonas psychrophila HA-4 provided by the invention has the ability of higher degraded sulfamethoxazole, under the bacterial strain optimal growth condition, sulfamethoxazole to 100 mg/L, at short notice, degradation rate reaches more than 35.34%.
Description of drawings
Fig. 1 is bacterial strain of the present invention Pseudomonas psychrophilaThe stereoscan photograph of HA-4.
Fig. 2 is bacterial strain of the present invention Pseudomonas psychrophilaHA-4 grows in take sulfamethoxazole as unique carbon and nitrogen sources substratum and sulfamethoxazole degradation curve figure.
Fig. 3 is that bacteria suspension of the present invention is to the degradation curve figure of different starting point concentration sulfamethoxazoles.
Fig. 4 determining at the best pH growth conditions of the sulfamethoxazole take starting point concentration as 100mg/L in unique carbon and nitrogen sources substratum that be pseudomonas strain of the present invention.
Fig. 5 determining at the optimum temps growth conditions of the sulfamethoxazole take starting point concentration as 100mg/L in unique carbon and nitrogen sources substratum that be pseudomonas strain of the present invention.
Fig. 6 determining at the optimum revolution growth conditions of the sulfamethoxazole take starting point concentration as 100mg/L in unique carbon and nitrogen sources substratum that be pseudomonas strain of the present invention.
Fig. 7 determining at the optimum inoculation amount growth conditions of the sulfamethoxazole take starting point concentration as 100mg/L in unique carbon and nitrogen sources substratum that be pseudomonas strain of the present invention.
Embodiment
The invention will be further described below in conjunction with accompanying drawing.
Embodiment 1
Strain Pseudomonas psychrophila provided by the invention separates from active sludge and obtains, and design temperature (10 ℃) screening is specifically screened step as follows:
(1) getting the 20mL Harbin fresh active sludge of peaceful municipal wastewater treatment plant Aerobic Pond adds 100mL and contains 20mg/L take sulfamethoxazole as sole carbon source, in the minimal medium triangular flask of the energy, placing temperature is 10 ℃, rotating speed is to cultivate a week in the shaking table of 150r/min, obtains enrichment bacterium liquid;
(2) getting 10mL enrichment bacterium liquid adds in the 100mL sulfamethoxazole minimal medium triangular flask, the culture condition of repeating step (1) 3 ~ 4 times, the concentration of each sulfamethoxazole improves step by step, finally to 100mg/L, obtain domestication bacterium liquid, the OD of the domestication bacterium liquid of acquisition 660=1.192;
(3) get 100 μ L domestication bacterium liquid and add and contain in the 100mg/L sulfamethoxazole solid inorganic salt culture medium flat plate, coating method smoothens, and placing temperature is 10 ℃ incubator, cultivates a week, obtains single bacterium colony;
(4) repetitive operation step (3) 3 ~ 4 times is namely finished the acquisition of purebred low temperature sulfamethoxazole degradation bacteria;
The purebred low temperature sulfamethoxazole degradation bacteria that (5) will filter out is inoculated into the inclined-plane enrichment medium, in 4 o Preserve inoculum size 10% under the C;
Wherein the composition of minimal medium is: 3.5g K 2HPO 4, 0.5gNaCl, 1.5g KH 2PO 4, 0.15g MgSO 47H 2O, 0.5g (NH 4) 2SO 4, the 1mL trace element solution is dissolved in the 1000mL distilled water, regulates pH7.0; The composition of inorganic salt solid medium is to add in addition agar 2g/L in the composition of minimal medium;
The enrichment culture based component is: 1.5 g KH 2PO 4, 0.15 g MgSO 47H 2O, 0.5 g (NH 4) 2SO 4, 0.5 g NaCl, 3.5 g K 2HPO 4, 2g peptone, 18g agar, 1.0 mL trace element solutions be dissolved in the distilled water of 1000mL, regulates pH to 7.0;
The composition of trace element solution is: 0.1 g CuSO 45H 2O, 0.3 g MnSO 44H 2O, 0.2 g ZnSO 47H 2O, 2.0 g NaHCO 310H 2O, 0.5 g CoCl 26H 2O, 0.05g CaCl 22H 2O, 0.5g FeSO 47H 2O, 0.02g (NH 4) 6Mo 7O 24H 2O is dissolved in the distilled water of 1000mL.
Substratum all is at 121 ℃, and autoclaving used after 20 minutes.
This bacterial strain Pseudomonas psychrophilaHA-4 suits to grow in the developing medium of (pH 6.0), and growth temperature is 10 ℃, its bacteriology morphological specificity: be gram negative bacillus, atrichia, aerobic, the degradable sulfamethoxazole can be take sulfamethoxazole as sole carbon source and energy growth.
This bacterial strain Pseudomonas psychrophilaThe HA-4 cultural characteristic:
This bacterium on the solid medium take sulfamethoxazole as sole carbon source 10 ℃ cultivated 6-7 days, bacterium colony is little, and is rounded, neat in edge, projection slightly, bacterium colony is creamy white, and very easily provokes.Scanning electron microscopic observation is the tyrothricin (see figure 1).
Bacterial strain provided by the invention Pseudomonas psychrophilaThe 16S rDNA sequence of HA-4.
The PCR primer adopts universal primer F8 (5 '-AGA GTT TGA TCA TGG CTC AG-3) and the R1522 (5 '-AAG GAC GTC ATC CAG CCG CA-3 ') of bacterial 16 S rDNA.(GeneAmp, PCR System 9700) carries out amplified reaction with the PCR instrument.The reaction system cumulative volume is 25 μ l: ultrapure water 16.2 μ l, and 10 х Buffer, 2.5 μ l, dNTP 2 μ l, F8 primer 1 μ l, R22 primer 1 μ l, ExTaq enzyme 0.3 μ l, total DNA do to get 2 μ l in reaction system after certain dilution.94 ℃ of denaturation 5 min, through 30 circulations, wherein, and 98 ℃ of sex change 20 s, 55 ℃ of annealing 40 s, 72 ℃ are extended 1.5 min, extend 7 min, 1% agarose gel electrophoresis, ultraviolet detection at 72 ℃ again after 30 circulations.Carry out homology relatively by the Blast program, show this bacterial strain with Pseudomonas psychrophilaHomology up to 99%, sequence is as follows:
TTGCATGCCTGCAGGTCGACGATTAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGGTGCTTGCACCTCTTGAGAGCGGCGGACGGGTGAGTAATACCTAGGAATCTGCCTGATAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCTACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACGTTAGTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGAATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGTCTTGAACTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCTAGAGATAGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTTACCACGGTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTAATCTCTAGAGGATCCCCGGGTACCGAGCT
Embodiment 2
The present invention in take sulfamethoxazole as unique carbon and nitrogen sources substratum to its degraded and bacterial strain Pseudomonas psychrophilaThe application of HA-4 upgrowth situation research, its step is as follows:
Bacterial strain Pseudomonas psychrophilaHA-4 is at 10 ℃ of temperature, initial pH value 6.0, shaking speed 150r/min, under the condition of 50mL Erlenmeyer flask liquid amount 20mL, the inoculum size of HA-4 bacteria suspension is 15%, the starting point concentration 100mg/L of sulfamethoxazole degraded system, cultured continuously 192h, every 24h detects the concentration of sulfamethoxazole.Adopt high performance liquid chromatography, chromatographic condition is to adopt Waters e2695 Alliance HPLC, and chromatographic column is Symmetry
Figure 941708DEST_PATH_IMAGE001
-C18 post (4.6 * 250 mm, 5 μ m); Moving phase is acetonitrile: formic acid water=40:60(V/V); Detect wavelength 265nm; 30 ℃ of column temperatures; Flow velocity 1.0mL/min; Sample size 10 μ L; Retention time 8min.The degradation rate that records sulfamethoxazole in the 192h is 35.34%.
Preparation HA-4 bacteria suspension:
To divide with the HA-4 bacterium liquid that enrichment medium is cultivated and install in the centrifuge tube, centrifugal 15 min under 8000 r/min conditions, abandoning supernatant, then in centrifuge tube, add the phosphate buffer solution that bacterium is crossed in death of monks or nuns, stir evenly the thalline of centrifuge tube bottom settlings, recentrifuge so washs thalline 3 times, to remove the substratum of thalline surface adsorption.The thalline that obtains is configured with minimal medium, namely obtain bacteria suspension.Fig. 2 is bacterial strain Pseudomonas psychrophilaThe growth of HA-4 in enrichment medium and the degraded situation of sulfamethoxazole; As seen from the figure, the degraded of sulfamethoxazole occurs in the logarithmic phase of strain growth.
Embodiment 3
The present invention in take sulfamethoxazole as unique carbon and nitrogen sources substratum to the application of its Study on degradation:
With the starting point concentration of sulfamethoxazole degraded system in the alternate embodiment 2, other step is with embodiment 2.Fig. 3 is that the HA-4 bacteria suspension is to the degraded situation of different starting point concentration sulfamethoxazoles; As seen from Figure 3, bacterial strain Pseudomonas psychrophilaHA-4 has efficient degradation capability to sulfamethoxazole, can tolerate sulfamethoxazole concentration up to 200mg/L.
Embodiment 4
The application of the optimal growth condition research of pseudomonas strain of the present invention:
To be changed to enrichment medium take sulfamethoxazole as unique carbon and nitrogen sources substratum among the embodiment 2, change pH, temperature, shaking speed and inoculum size, other step is with embodiment 2.By Fig. 4, Fig. 5, Fig. 6 and Fig. 7 as seen, in the sulfamethoxazole degraded system of starting point concentration 100mg/L, cultured continuously 192h, bacterial strain Pseudomonas psychrophilaThe optimal growth condition of HA-4, namely 10 ℃ of temperature, pH=6.0, shaking speed 150r/min, inoculum size are 10%.
TTGCATGCCTGCAGGTCGACGATTAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGGTGCTTGCACCTCTTGAGAGCGGCGGACGGGTGAGTAATACCTAGGAATCTGCCTGATAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCTACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACGTTAGTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGAATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGTCTTGAACTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCTAGAGATAGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTTACCACGGTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTAATCTCTAGAGGATCCCCGGGTACCGAGCT

Claims (6)

1. a strain Pseudomonas psychrophilaHA-4 low temperature sulfamethoxazole degradation bacteria it is characterized in that a strain low temperature pseudomonas strain, was preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC No.5944 on March 27th, 2012.
2. a strain according to claim 1 Pseudomonas psychrophilaHA-4 low temperature sulfamethoxazole degradation bacteria, it is characterized in that bacterial strain suits to grow in the developing medium of pH 6.0, growth temperature is 10 ℃, its bacteriology morphological specificity: be gram negative bacillus, atrichia, aerobic, the degradable sulfamethoxazole can be take sulfamethoxazole as sole carbon source and energy growth.
3. a strain according to claim 1 and 2 Pseudomonas psychrophilaThe screening method of HA-4 low temperature sulfamethoxazole degradation bacteria is characterized in that strain Pseudomonas psychrophila HA-4 separates to obtain from active sludge, 10 ℃ of screenings of design temperature are specifically screened step as follows:
(1) getting the 20mL Harbin fresh active sludge of peaceful municipal wastewater treatment plant Aerobic Pond adds 100mL and contains 20mg/L take sulfamethoxazole as sole carbon source, in the minimal medium triangular flask of the energy, placing temperature is 10 ℃, rotating speed is to cultivate a week in the shaking table of 150r/min, obtains enrichment bacterium liquid;
(2) get 10mL enrichment bacterium liquid and add in the 100mL sulfamethoxazole minimal medium triangular flask, the culture condition of repeating step (1) 3 ~ 4 times, the concentration of each sulfamethoxazole improves step by step, finally to 100mg/L, obtains domestication bacterium liquid;
(3) get 100 μ L domestication bacterium liquid and add and contain in the 100mg/L sulfamethoxazole inorganic salt solid medium flat board, coating method smoothens, and placing temperature is 10 ℃ incubator, cultivates a week, obtains single bacterium colony;
(4) repetitive operation step (3) 3 ~ 4 times is namely finished the acquisition of purebred low temperature sulfamethoxazole degradation bacteria;
The purebred low temperature sulfamethoxazole degradation bacteria that (5) will filter out is scoring to the inclined-plane enrichment medium, in 4 oPreserve inoculum size 10% under the C;
Wherein the composition of minimal medium is: 3.5g K 2HPO 4, 0.5gNaCl, 1.5g KH 2PO 4, 0.15g MgSO 47H 2O, 0.5g (NH 4) 2SO 4, the 1mL trace element solution is dissolved in the 1000mL distilled water, regulates pH7.0; The composition of inorganic salt solid medium is to add in addition agar 2g/L in the composition of minimal medium;
The enrichment culture based component is: 1.5 g KH 2PO 4, 0.15 g MgSO 47H 2O, 0.5 g (NH 4) 2SO 4, 0.5 g NaCl, 3.5 g K 2HPO 4, 2g peptone, 18g agar, 1.0 mL trace element solutions be dissolved in the distilled water of 1000mL, regulates pH to 7.0;
The composition of trace element solution is: 0.1 g CuSO 45H 2O, 0.3 g MnSO 44H 2O, 0.2 g ZnSO 47H 2O, 2.0 g NaHCO 310H 2O, 0.5 g CoCl 26H 2O, 0.05g CaCl 22H 2O, 0.5g FeSO 47H 2O, 0.02g (NH 4) 6Mo 7O 24H 2O is dissolved in the distilled water of 1000mL.
4. screening method according to claim 3 is characterized in that the OD of the domestication bacterium liquid that step (2) obtains 660=1.192.
5. screening method according to claim 3 is characterized in that substratum all is at 121 ℃, and autoclaving used after 20 minutes.
6. low temperature sulfamethoxazole degradation bacteria according to claim 1 is characterized in that being applied to the biological treatment of sulfamethoxazole in the sewage under the low temperature.
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