CN103571771B - The Screening and Identification of one strain phthalic ester efficient degradation genus bacillus and application thereof - Google Patents
The Screening and Identification of one strain phthalic ester efficient degradation genus bacillus and application thereof Download PDFInfo
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Abstract
The invention provides a strain from using bacterial strain JF phthalic ester being had to efficient degradation ability be separated the soil of mulch film in a large number, belonging to biological technical field.This strain growth speed is fast, produces gemma, and environmental resistance is strong to external world, at wide temperature (30 DEG C-45 DEG C) and wide pH(pH5.0-8.0) in scope to the degradation rate of phthalic ester more than 90%.This bacterial strain is through being accredited as subtilis (Bacillus subtilis), accession number is KF364634, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation place is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on 07 22nd, 2013, deposit number: CGMCC No. 7950.This bacterial strain can be used for, by the biological restoration of phthalic ester institute contaminated soil, sewage or biopurification, preserving the ecological environment, and then ensures agricultural product security.
Description
Technical field
The present invention relates to biological technical field, be specifically related to Screening and Identification and the application thereof of a high-efficiency degradation phthalic ester genus bacillus.
Background technology
Phthalic ester (Phthalates, PAEs, phenolphthalein ester) be one of synthetic organic compound of large, the wide application of turnout in the world, belong to aromatic dicarboxylic acid ester, mostly be the liquid of high boiling point, low-vapor pressure, colorless and odorless, they are widely used in softening agent and the tenderizer of additives for plastics, paint solvent, synthetic rubber and coating etc., to increase plasticity-and the toughness of plastics, improve the intensity of plastics.PAEs compounds is the general name of about 30 kinds of compounds, by phthalic acid and corresponding alcohol compound, the kind that China commonly uses has: dibutyl phthalate (DBP), phthalic acid two (2-ethyl hexyl) ester (DEHP), dioctyl phthalate (DOP) (DOP), butyl benzyl phthalate (BBP), Dinonylphthalate (DNP) etc., wherein DBP and DEHP consumption is maximum, DBP and DEHP is classified as priority pollutants by EPA, European Union is also using management and control material that DBP assesses as excessive risk, priority pollutant Black List has also been listed in by China.
In recent years, along with phthalic ester (Phthalates, PAEs) a large amount of uses in the industry, PAEs has been prevalent in the environment such as soil, bed mud, water body, air and atmospheric fallout, becomes one of pollutent important in environment.Generally believe that PAEs is a kind of environment incretion interferent at present, and some kind wherein has teratogenesis, carcinogenic, mutagenesis to human body and animal, phthalate enters human body by approach such as Digestive tract, respiratory system and skin contact, have genotoxicity and development toxicity, this harm is hidden and very long.
Phthalic ester is long half time in the environment, and slowly, belong to hard-degraded substance, the microbiological deterioration of PAEs is the main path in physical environment to the speed of its hydrolysis and photodissociation, and current rarely seen remaining will is bright to be waited and report
gordoniathe patent of sp.QH-9 degradable phthalic ester.Zeng Qiaoyun etc. have studied PAEs and enter farm crop by soil and the volatilization of self, and have enriched character in farm crop, enter human body, be directly detrimental to health through edible.Because genus bacillus can form gemma; resistibility in physical environment is better than other microorganisms; therefore; acquisition PAEs efficient degrading bacteria especially efficient degradation genus bacillus has important practice significance, can be and eliminates environment PAEs pollution, preserve the ecological environment and ensure that agricultural product security provides microorganism resource and technical support.
This patent uses from Sichuan Province's YAAN RAIN CITY DISTRICT the bacterial strain of a highly effective degrading dibutyl phthalate be separated to the soil in the vegetables field of mulch film, to check order qualification through 16S rDNA, this strain gene sequence with
bacillus subtilisstrain sequence very high homology.Dibutyl phthalate can be utilized as sole carbon source and energy growth and breeding through overtesting this bacterial strain known, can by degradable for 200mg/L DBP in LB substratum in 14h, and at wide temperature (30 DEG C-45 DEG C) and wide pH(pH5.0-8.0) all there is good degradation capability to dibutyl phthalate in scope; Meanwhile, this bacterium dimethyl terephthalate, phthalic acid two (2-ethyl hexyl) ester, dioctyl phthalate (DOP), butyl benzyl phthalate, Dinonylphthalate etc. all have stronger degradation capability.Absolutely prove that this bacterial strain contains the application potential in the biological restoration of phthalate compound pollution with uniqueness in process.
Summary of the invention
The object of this invention is to provide bacterial strain and the application thereof of a highly effective degrading phthalate compound.
Degradation bacteria strains provided by the present invention is through being accredited as subtilis (Bacillus subtilis), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation place is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is CGMCC No.7950, and preservation date is on 07 22nd, 2013.
Degradation bacteria strains provided by the present invention
bacillus subtilisthe biological characteristics of strain JF: amphimicrobian, the genus bacillus of Gram-positive, is all positive to glucose, pectinose, wood sugar and mannose ferment; Energy gelatin hydrolysate and starch; Phenylalanine desaturase and yolk lecithin enzyme are negative.
The isolation and screening of degradation bacteria strains provided by the present invention:
Get the pedotheque 4 parts that Sichuan Province's YAAN RAIN CITY DISTRICT uses the vegetables field of mulch film, mix thoroughly respectively, respectively get 5g, be placed in the 250 mL Erlenmeyer flasks that 30mL LB substratum is housed, 10min is heated at 80 DEG C, be chilled to room temperature to add dibutyl phthalate again and make its mass concentration be 50 mg/L, in 35 DEG C, 180 r/min shaking culture 168h.Carry out 3 enrichment culture successively again by 5% inoculum size after cultivation terminates, each cultivation 168 h, and progressively improve dibutyl phthalate concentration in substratum, make it to reach 100 mg/L, 200 mg/L, 300 mg/L respectively.By last step gradient tame after nutrient solution by 5%(v/v) inoculum size transfer in 30 mL contain 100 mg/L dibutyl phthalates LB substratum in, and make blank by stroke-physiological saline solution, in 35 DEG C, 180 r/min shaking culture 120 h, measure dibutyl phthalate residual quantity in each nutrient solution.
The nutrient solution defining dibutyl phthalate minimizing is carried out strains separation, gets nutrient solution dilution spread in containing in the LB flat board of 100 mg/L dibutyl phthalates, after 35 DEG C of cultivation 72 h, separation and purification is carried out to the bacterium colony grown.By the bacterial strain after purifying in containing streak culture on the LB inclined-plane of 100 mg/L dibutyl phthalates, wash lower culture by stroke-physiological saline solution and make seed liquor (adjustment cell concn about 10
8cfu/mL).Seed liquor is inoculated in 30 mL containing in the LB substratum of 100 mg/L dibutyl phthalates by 5% inoculum size, and blank is set by stroke-physiological saline solution.35 DEG C, after 180 r/min shakes cultivate 120 h, measure the residual quantity of dibutyl phthalate in each nutrient solution, determine the degradation capability of isolated strains to dibutyl phthalate.
Strain morphology is identified: Gram-positive, genus bacillus, bacterium colony subcircular, flat, opaque, micro-yellow, not reflective, surface ruffle, edge is irregular.
Bacterial strain Physiology and biochemistry is identified
Table 1 bacterial strain JF physiological and biochemical test result.
Bacterial strain JF molecular genetics is identified.
Bacterial strain JF, through 16S rDNA sequencing, obtains following sequence:
gagtttgatcctggctcaggacgaacgctggcggcgtgcctaatacatgcaagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatgcttgtttgaaccgcatggttcaaacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcgatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtaccgttcgaatagggcggtaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtgatacgtaggtggcaagcgttgtccggaattattgggcgtaaggggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgccgaaggtgggacagatgattggggtgaagtcgtaacaaggtagccgta。
In the 16S rDNA gene sequencing result of this bacterial strain and GenBank database, sequence carries out Blast homology alignment, this strain sequence with
bacillus subtilisstrain gene sequence very high homology, obtaining accession number is KF364634.
The bacterial strain JF of efficient degradation phthalate compound provided by the present invention uses the pedotheque enrichment in the vegetables field of mulch film to obtain from Sichuan Province's YAAN RAIN CITY DISTRICT, this bacterial strain all has very high degradation capability to dibutyl phthalate within the scope of wide temperature and wide pH, its fast growth, produce gemma, environmental resistance is strong to external world, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation place is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is CGMCC No.7950, preservation date is on 07 22nd, 2013.
Used medium of the present invention is: Luria-Bertani substratum (LB): yeast extract (Yeast extract) 5.0g; Tryptones (Casein tryptone) 10g; Sodium-chlor (Sodium chloride) 10g; Distilled water (H
2o) 1000mL; Adjust pH to 7.0; Basic medium (MM): ammonium sulfate ((NH
4)
2sO
4) 1.5g, potassium primary phosphate (KH
2pO
4) 0.5g, dipotassium hydrogen phosphate (K
2hPO
4) 1.5g, magnesium sulfate (MgSO
4) 0.2g, sodium-chlor (NaCl) 0.5g, deionized water (H
2o) 1000mL, adjusts pH to 7.0.These two kinds of solid mediums add 20g/L agar (Agar) in liquid medium within again to make.Cultivate based on 121 DEG C, sterilizing 20min is for subsequent use.
Accompanying drawing explanation
Fig. 1 is the bacterium colony cultural characteristic that degradation bacteria strains JF cultivates 48h.
Fig. 2 is the cellular form figure of degradation bacteria strains JF.
Fig. 3 is the curve of the growth of degradation bacteria strains JF in embodiment example 1 and the degraded relation of dibutyl phthalate.
Fig. 4 is the impact of temperature on bacterial strain JF degraded dibutyl phthalate speed.
Fig. 5 is the impact of pH value on bacterial strain JF degraded dibutyl phthalate speed.
Fig. 6 is the impact of substrate mass concentration on bacterial strain JF degraded dibutyl phthalate speed.
Embodiment
Example 1: determine the growing state of phthalic ester degradation bacteria strains JF and the relation of dibutyl phthalate degradation
Actication of culture: in bacterial strain JF to the LB liquid nutrient medium of picking degraded phthalic ester, 35 DEG C, 180r/min shaking culture 24h; Then line continuously, picking list bacterium colony cultivate twice (35 DEG C), and it is for subsequent use that picking list colony inoculation 35 DEG C to LB inclined-plane cultivates 2d.
Washing thalline on lower inclined plane by stroke-physiological saline solution and adjusting cell concn is 10
8cfu/mL, makes seed liquor.
The mensuration of growth curve: the seed liquor of degradation bacteria strains JF is inoculated in the LB liquid nutrient medium containing 200mg/L dibutyl phthalate (DBP) by 5% inoculum size, packing 30 mL/250mL Erlenmeyer flask, 35 DEG C, 180r/min cultivates 48h, during being 0h-36h sample time, every 2h samples 1 bottle, during 36h-48h, every 4h samples 1 bottle, sample is deposited in 4 DEG C of refrigerators, surveys OD
600.
The mensuration of degradation curve: the seed liquor of degradation bacteria strains JF is inoculated in the LB liquid nutrient medium containing 200mg/L dibutyl phthalate (DBP) by 5% inoculum size, packing 30 mL/250mL Erlenmeyer flask, if inoculation 5% sterilized water is blank, 35 DEG C, 180r/min shaking culture, during being 0h-36h sample time, every 2h samples 1 bottle, during 36h-48h, every 4h samples 1 bottle, even nutrient solution 1mL is got again from Erlenmeyer flask, 10mL is settled to acetonitrile, in the centrifugal 10min of 10000r/min after mixing, get supernatant liquor and analyze for liquid chromatography (HPLC).Condition determination: chromatographic column is Gemini 100 C18 post (150mm × 4.60mm, 5.0 μm); Moving phase is acetonitrile: water (83:17, V/V), flow velocity 1.0mL/min; UV-detector, its wavelength is 210nm; Column temperature is 25 DEG C; Sample size 10 μ L.
Cultivated through 48h by the known degradation bacteria strains of result, the dibutyl phthalate of control group is almost without degraded, in experimental group, the about 100% dibutyl phthalate bacterial strain JF that is degraded degrades, in 14h, dibutyl phthalate and degradation bacteria strains JF grow and are proportionate, bacterial strain is very fast to dibutyl phthalate degradation speed, and 24h Fungal biodiversity reaches maximum, and after this thalli growth tends towards stability.
Example 2: the degradation characteristic of bacterial strain JF degraded dibutyl phthalate
Concentration of substrate is degraded on bacterial strain JF the impact of dibutyl phthalate speed: by 5.0%(v/v) inoculum size, by seed liquor (1.0 × 10
8individual/mL) be inoculated in the different LB liquid nutrient medium of dibutyl phthalate concentration (pH7.0,100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L) in, packing 30 mL/250 mL Erlenmeyer flask, 35 DEG C, 180 r/min shaking culture, interval 4h samples.Dibutyl phthalate degradation rate is measured by [0024] described method.
Culture temperature is degraded on bacterial strain JF the impact of dibutyl phthalate speed: by 5.0%(v/v) inoculum size, by seed liquor (1.0 × 10
8individual/mL) to be inoculated in dibutyl phthalate mass concentration be in the LB liquid nutrient medium (pH7.0) of 200 mg/L, packing 30 mL/250 mL Erlenmeyer flask, under differing temps (30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C) 180 r/min shaking culture, interval 4h samples.Dibutyl phthalate degradation rate is measured by [0024] described method.
PH degrades on bacterial strain JF the impact of dibutyl phthalate speed: by 5.0%(v/v) inoculum size, by seed liquor (1.0 × 10
8individual/mL) be inoculated in different pH(5.0,6.0,7.0,8.0) dibutyl phthalate mass concentration be in the LB liquid nutrient medium of 200 mg/L, packing 30 mL/250 mL Erlenmeyer flask, 35 DEG C, 180 r/min shaking culture, interval 4h samples.Dibutyl phthalate degradation rate is measured by [0024] described method.
The degradation characteristic result of bacterial strain JF degraded dibutyl phthalate: in substrate (dibutyl phthalate) mass concentration of testing, temperature, pH value range, the dibutyl phthalate transformation period is 3.1 h ~ 7.0h, well below its natural degradation transformation period (4.4 d ~ 15.1d), can degradable dibutyl phthalate in 12h when substrate mass concentration is 100mg/L, further demonstrating bacterial strain JF can efficient degradation dibutyl phthalate, and the growth velocity of bacterial strain is very fast.Temperature is within the scope of 30 ~ 45 DEG C, and bacterial strain to the change of dibutyl phthalate degradation rate greatly, and presents the trend raised gradually; PH value is little on the impact of dibutyl phthalate degradation rate; Concentration of substrate is within the scope of 100 ~ 400mg/L, and dibutyl phthalate degradation rate changes greatly, and when substrate mass concentration reaches 400mg/L, dibutyl phthalate degradation rate is lower, and obvious dibutyl phthalate inhibits the growth of bacterial strain.This result of study can be bacterial strain JF and is applied to sewage disposal and soil remediation provides certain data refer.
Example 3: phthalic ester degradation bacteria strains JF in MM substratum to the degradation effect of dibutyl phthalate
The seed liquor of degradation bacteria strains is inoculated in by 5% inoculum size in the MM liquid nutrient medium of 200mg/L dibutyl phthalate (DBP), packing 30 mL/250mL Erlenmeyer flask, if inoculation 5% sterilized water is blank, 35 DEG C, 180r/min shaking culture.
Sample after cultivating 60h, the residual quantity of dibutyl phthalate (DBP) in nutrient solution is measured by [0024] described method, control group dibutyl phthalate (DBP) is not almost degraded, and in experimental group, about 92.66% dibutyl phthalate (DBP) is degraded by bacterial strain JF.
Example 4: phthalic ester degradation bacteria strains JF is to the degradation effect of phthalic acid two (2-ethyl hexyl) ester (DEHP)
The seed liquor of degradation bacteria strains is inoculated in by 5% inoculum size in the LB liquid nutrient medium of 200mg/L phthalic acid two (2-ethyl hexyl) ester (DEHP), packing 30 mL/250mL Erlenmeyer flask, if inoculation 5% sterilized water is blank, 35 DEG C, 180r/min shaking culture.
Sample after cultivating 20h, the residual quantity of phthalic acid two (2-ethyl hexyl) ester in nutrient solution is measured by [0024] described method, control group phthalic acid two (2-ethyl hexyl) ester is almost without degraded, and in experimental group, about 97.01% phthalic acid two (2-ethyl hexyl) ester is degraded by bacterial strain JF.
Example 5: phthalic ester degradation bacteria strains JF is to the degradation effect of the phthalic ester in contaminated soil
Use the soil in the vegetables field of mulch film from Sichuan Province's YAAN RAIN CITY DISTRICT and get a certain amount of pedotheque, adding dibutyl phthalate (DBP) makes its mass concentration be about 200mg/L, by the seed liquor of degradation bacteria strains by 5% inoculum size inoculation (experimental group), if inoculation 5% sterilized water is blank, 35 DEG C of cultivations, every 24h sampling, measures the residual quantity of DBP in pedotheque by [0024] described method.
Detected the residual quantity of dibutyl phthalate in sample by HPLC, in blank pedotheque, dibutyl phthalate content is less, and the pedotheque of experimental group almost can't detect dibutyl phthalate when 5d.
Example 6: phthalic ester degradation bacteria strains JF is to the degradation effect of phthalic ester in plastic cement factory sewage
A certain amount of sewage sample is got from the sewage of Chengdu, Sichuan Province plastic cement factory, adding dibutyl phthalate (DBP) makes its concentration be about 200mg/L, by the seed liquor of degradation bacteria strains by 5% inoculum size inoculation (experimental group), if inoculation 5% sterilized water is blank, 35 DEG C of cultivations, every 24h sampling, measures the residual quantity of DBP in sewage sample by [0024] described method.
Detected the residual quantity of dibutyl phthalate in sample by HPLC, in blank sewage sample, dibutyl phthalate content is less, and the sewage sample of experimental group almost can't detect dibutyl phthalate when 4d.
Above example is only be described embodiments of the present invention; and not scope of the present invention is limited; for a person skilled in the art; can improve above-mentioned explanation or be out of shape, but all these improve or are out of shape the protection domain that all should fall into claim of the present invention and determine.
Claims (5)
1. a bacillus subtilis JF (
bacillus subtilisstrainJF), it is characterized in that being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation place is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCC No.7950, and preservation date is on 07 22nd, 2013; Subtilis JF is characterized as: (1) Gram-positive, genus bacillus, bacterium colony subcircular, and flat, opaque, edge is irregular; (2) amphimicrobian, energy gelatin hydrolysate and starch, Phenylalanine desaturase and yolk lecithin enzyme are negative; (3) this bacterial strain JF with
bacillus subtilisbacterial strain 16S rDNA sequence very high homology, submitting to NCBI to obtain accession number is KF364634.
2. subtilis JF described in claim 1 (
bacillus subtilisstrainJF) in the application of degraded dibutyl phthalate, phthalic acid two (2-ethyl hexyl) ester.
3. subtilis JF according to claim 2 is in the application of degraded dibutyl phthalate, phthalic acid two (2-ethyl hexyl) ester, it is characterized in that, degraded combination condition is: temperature 30-45 DEG C, pH=5.0-8.0.
4. subtilis JF according to claim 2 is in the application of degraded dibutyl phthalate, phthalic acid two (2-ethyl hexyl) ester, it is characterized in that, degraded combination condition is: temperature 35 DEG C, pH=7.0, inoculum size 5%, liquid amount 30mL/250mL Erlenmeyer flask, shaking speed 180r/min.
5. subtilis JF according to claim 2 is in the application of degraded dibutyl phthalate, phthalic acid two (2-ethyl hexyl) ester; this bacterial strain can be used for the biological degradation of dibutyl phthalate, phthalic acid two (2-ethyl hexyl) ester; and the biological restoration of these material institute contaminated soils or biopurification, preserve the ecological environment.
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