CN103571771A - Screening and identification and application of bacillus for efficiently degrading phthalate - Google Patents
Screening and identification and application of bacillus for efficiently degrading phthalate Download PDFInfo
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Abstract
The invention provides a bacterial strain JF which is separated out from a large number of soils with mulching films used and can efficiently degrade phthalate, and belongs to the technical field of biology. The bacterial strain grows fast, can produce spore, is high in resistance to external environment, and reaches more than 90% of degradation rate regarding to phthalate within a broad range that the temperature is 30 to 45 DEG C and the pH (Potential of Hydrogen) is 5.0 to 8.0. The identification result shows that the bacterial strain is bacillus subtilis (Bacillus Subtilis), and the accession number is KF364634; and the bacterial strain is stored in China General Microbiological Culture Collection Center (CGMCC) located at No.3, Courtyard 1, West Beichen Road, Chaoyang District, Beijing, on July 22, 2013, with the storage number of CGMCC No.7950. The bacterial strain can be applied to bioremediation or biological purification of soil and sewage polluted by phthalate, and protects the ecological environment, and thus the safety of agricultural products is ensured.
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 the 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, formed, the conventional kind of China 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 are maximum, DBP and DEHP are classified as priority pollutants by EPA, European Union is the management and control material of assessment using DBP as excessive risk also, priority pollutant Black List has also been listed in by China.
In recent years, along with a large amount of uses of phthalic ester (Phthalates, PAEs) in 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 at present PAEs is a kind of environment incretion interferent, and some kind wherein has teratogenesis, carcinogenic, mutagenesis to human body and animal, phthalate can enter 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 environment, and its hydrolysis and the speed of photodissociation are very slow, belong to hard-degraded substance, and the microbiological deterioration of PAEs is the main path in physical environment, and at present 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 can enter farm crop by soil and the volatilization of self, and has enriched character in farm crop, through edible, enters human body, is directly detrimental to health.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.
The bacterial strain of the highly effective degrading dibutyl phthalate being separated in the soil in vegetables field of mulch film is used in Yucheng District, Ya'an, this patent Shi Cong Sichuan Province, through 16S rDNA order-checking, identify, this strain gene sequence with
bacillus subtilisbacterial strain sequence height homology.Through known this bacterial strain of overtesting, can utilize dibutyl phthalate as sole carbon source and energy growth and breeding, can 200mg/L DBP in LB substratum is degradable in 14h, and at wide temperature (30 ℃-45 ℃) and wide pH(pH5.0-8.0) all dibutyl phthalate is all had to good degradation capability 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 aspect the biological restoration that phthalate compound pollutes and has unique application potential in processing.
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 4 parts of the pedotheques that the vegetables field of mulch film is used in Yucheng District, Ya'an, Sichuan Province, mix thoroughly respectively, respectively get 5g, be placed in the 250 mL Erlenmeyer flasks that 30mL LB substratum is housed, at 80 ℃, heat 10min, being chilled to room temperature, to add dibutyl phthalate to make its mass concentration be 50 mg/L again, in 35 ℃, 180 r/min shaking culture 168h.After cultivation finishes, by 5% inoculum size, carry out successively 3 times again enrichment culture, cultivate 168 h at every turn, and progressively improve dibutyl phthalate concentration in substratum, make it to reach respectively 100 mg/L, 200 mg/L, 300 mg/L.By the nutrient solution after last step gradient domestication by 5%(v/v) inoculum size transfers and contains in the LB substratum of 100 mg/L dibutyl phthalates in 30 mL, and make blank by stroke-physiological saline solution, in 35 ℃, 180 r/min shaking culture 120 h, measure dibutyl phthalate residual quantity in each nutrient solution.
The nutrient solution that defines dibutyl phthalate minimizing is carried out to strains separation, get nutrient solution dilution spread in containing in the LB flat board of 100 mg/L dibutyl phthalates, cultivate after 72 h, the bacterium colony growing is carried out to separation and purification for 35 ℃.Bacterial strain after purifying, in containing streak culture on the LB inclined-plane of 100 mg/L dibutyl phthalates, is washed to lower culture by stroke-physiological saline solution and made seed liquor (adjustment cell concn approximately 10
8cfu/mL).Seed liquor is inoculated in to 30 mL containing in the LB substratum of 100 mg/L dibutyl phthalates by 5% inoculum size, and by stroke-physiological saline solution, blank is set.35 ℃, 180 r/min shakes are cultivated after 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, and 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 Physiology and biochemistry measurement 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 is carried out the contrast of Blast homology, this bacterial strain sequence with
bacillus subtilisstrain gene sequence height homology, obtaining accession number is KF364634.
The Yucheng District, Ya'an, bacterial strain JFShi Cong Sichuan Province of efficient degradation phthalate compound provided by the present invention is used the pedotheque enrichment in the vegetables field of mulch film to obtain, 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, environment resistibility 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, sal epsom (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 are in liquid medium within, to add 20g/L agar (Agar) to make again.Cultivation is based on 121 ℃, and sterilizing 20min is standby.
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 ℃, 180r/min shaking culture 24h; Then line continuously, picking list bacterium colony are cultivated twice (35 ℃), and 35 ℃ to LB inclined-plane cultivation 2d are standby for picking list colony inoculation.
By stroke-physiological saline solution, washing on lower inclined plane thalline and adjust 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 that contains 200mg/L dibutyl phthalate (DBP) by 5% inoculum size, packing 30 mL/250mL Erlenmeyer flasks, 35 ℃, 180r/min are cultivated 48h, be 1 bottle of every 2h sampling during 0h-36h sample time, during 36h-48h, every 4h sampling is 1 bottle, sample is deposited in 4 ℃ 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 that contains 200mg/L dibutyl phthalate (DBP) by 5% inoculum size, packing 30 mL/250mL Erlenmeyer flasks, if inoculate 5% sterilized water, it is blank, 35 ℃, 180r/min shaking culture, be 1 bottle of every 2h sampling during 0h-36h sample time, during 36h-48h, every 4h sampling is 1 bottle, from Erlenmeyer flask, get again even nutrient solution 1mL, with acetonitrile, be settled to 10mL, after mixing, in the centrifugal 10min of 10000r/min, get supernatant liquor and analyze for liquid chromatography (HPLC).Condition determination: chromatographic column is Gemini 100 C18 posts (150mm * 4.60mm, 5.0 μ m); Moving phase is acetonitrile: water (83:17, V/V), flow velocity 1.0mL/min; UV-detector, its wavelength is 210nm; Column temperature is 25 ℃; Sample size 10 μ L.
By the known degradation bacteria strains of result, through 48h, cultivated, the dibutyl phthalate of control group is almost without degraded, be degraded bacterial strain JF degraded of approximately 100% dibutyl phthalate in experimental group, in 14h, dibutyl phthalate and degradation bacteria strains JF growth are proportionate, bacterial strain is very fast to dibutyl phthalate degradation speed, and it is maximum that 24h thalline biomass reaches, and after this thalli growth tends towards stability.
Example 2: the degradation characteristic of bacterial strain JF degraded dibutyl phthalate
The impact of concentration of substrate on bacterial strain JF degraded dibutyl phthalate speed: press 5.0%(v/v) inoculum size, by seed liquor (1.0 * 10
8individual/mL) be inoculated in the LB liquid nutrient medium that dibutyl phthalate concentration is different (pH7.0,100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L) in, packing 30 mL/250 mL Erlenmeyer flasks, 35 ℃, 180 r/min shaking culture, interval 4h sampling.By [0024] described method, measure dibutyl phthalate degradation rate.
The impact of culture temperature on bacterial strain JF degraded dibutyl phthalate speed: press 5.0%(v/v) inoculum size, by seed liquor (1.0 * 10
8individual/mL) be inoculated in the LB liquid nutrient medium (pH7.0) that dibutyl phthalate mass concentration is 200 mg/L packing 30 mL/250 mL Erlenmeyer flasks, under differing temps (30 ℃, 35 ℃, 40 ℃, 45 ℃) 180 r/min shaking culture, interval 4h sampling.By [0024] described method, measure dibutyl phthalate degradation rate.
The impact of pH on bacterial strain JF degraded dibutyl phthalate speed: press 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 flasks, 35 ℃, 180 r/min shaking culture, interval 4h sampling.By [0024] described method, measure dibutyl phthalate degradation rate.
The degradation characteristic result of bacterial strain JF degraded dibutyl phthalate: within the scope of substrate (dibutyl phthalate) mass concentration of testing, temperature, pH value, 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 proved that 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 ℃, and bacterial strain changes greatly dibutyl phthalate degradation rate, and presents the trend raising 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 obviously dibutyl phthalate has suppressed the growth of bacterial strain.This result of study can be that bacterial strain JF is applied to sewage disposal and soil remediation provides certain data refer.
Example 3: phthalic ester degradation bacteria strains JF degradation effect to dibutyl phthalate in MM substratum
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 flasks, establishing inoculation 5% sterilized water is blank, 35 ℃, 180r/min shaking culture.
After cultivating 60h, sample, by [0024] described method, measure the residual quantity of dibutyl phthalate (DBP) in nutrient solution, control group dibutyl phthalate (DBP) is not degraded almost, and in experimental group, approximately 92.66% dibutyl phthalate (DBP) is degraded by bacterial strain JF.
Example 4: the degradation effect of phthalic ester degradation bacteria strains JF to phthalic acid two (2-ethyl hexyl) ester (DEHP)
The seed liquor of degradation bacteria strains is inoculated in the LB liquid nutrient medium of 200mg/L phthalic acid two (2-ethyl hexyl) ester (DEHP) by 5% inoculum size, packing 30 mL/250mL Erlenmeyer flasks, if inoculate 5% sterilized water, be blank, 35 ℃, 180r/min shaking culture.
After cultivating 20h, sample, by [0024] described method, measure the residual quantity of phthalic acid two (2-ethyl hexyl) ester in nutrient solution, control group phthalic acid two (2-ethyl hexyl) ester is almost without degraded, and in experimental group, approximately 97.01% phthalic acid two (2-ethyl hexyl) ester is degraded by bacterial strain JF.
Example 5: the degradation effect of phthalic ester degradation bacteria strains JF to the phthalic ester in contaminated soil
From being used the soil in vegetables field of mulch film, Yucheng District, Ya'an, Sichuan Province gets a certain amount of pedotheque, adding dibutyl phthalate (DBP) makes its mass concentration be about 200mg/L, the seed liquor of degradation bacteria strains is inoculated to (experimental group) by 5% inoculum size, if inoculate 5% sterilized water, it is blank, 35 ℃ of cultivations, every 24h sampling, by the residual quantity of DBP in [0024] described method mensuration pedotheque.
By HPLC, detect the residual quantity of dibutyl phthalate in sample, in blank pedotheque, dibutyl phthalate content is less, and the pedotheque of experimental group almost can't detect dibutyl phthalate when 5d.
Example 6: the degradation effect of phthalic ester in phthalic ester degradation bacteria strains JFDui plastic cement factory sewage
From the sewage of Chengdu, Sichuan Province plastic cement factory, get a certain amount of sewage sample, adding dibutyl phthalate (DBP) makes its concentration be about 200mg/L, the seed liquor of degradation bacteria strains is inoculated to (experimental group) by 5% inoculum size, if inoculate 5% sterilized water, it is blank, 35 ℃ of cultivations, every 24h sampling, by the residual quantity of DBP in [0024] described method mensuration sewage sample.
By HPLC, detect the residual quantity of dibutyl phthalate in sample, 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 that embodiments of the present invention are described; and not scope of the present invention is limited; for a person skilled in the art; can above-mentioned explanation be improved or is out of shape, but these all improvement or distortion all should fall into the definite protection domain of claim of the present invention.
Claims (6)
1. a bacillus subtilis JF(Bacillus subtilis strainJF), 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, preserving number is CGMCC No.7950, and preservation date is on 07 22nd, 2013.
2. subtilis JF(described in claim 1
bacillus subtilisstrainJF), it is characterized in that: (1) individual morphology feature: Gram-positive, genus bacillus; (2) colony characteristics: bacterium colony subcircular, flat, opaque, micro-yellow, not reflective, surface ruffle, edge is irregular; (3) biochemical indicator feature: amphimicrobian, is all positive to glucose, pectinose, wood sugar and mannose ferment; Energy gelatin hydrolysate and starch; Phenylalanine desaturase and yolk lecithin enzyme are negative; (4) 16S rDNA sequence signature: in the 16S rDNA gene sequencing result of this bacterial strain and GenBank database, sequence is carried out the contrast of Blast homology, this bacterial strain sequence with
bacillus subtilisstrain gene sequence height homology, obtaining accession number is KF364634.
Subtilis JF described in claim 1 (
bacillus subtilisstrainJF) application in degraded phthalate compound.
4. the application of subtilis JF according to claim 3, is characterized in that, degraded combination condition is: temperature 30-45 ℃, pH=5.0-8.0.
5. the application of subtilis JF according to claim 3, is characterized in that, degraded combination condition is: 35 ℃ of temperature, pH=7.0, inoculum size 5%, liquid amount 30mL/250mL Erlenmeyer flask, shaking speed 180r/min.
6. the application of subtilis JF according to claim 3; this bacterial strain can be used for the biological degradation of the phthalic esters such as dibutyl phthalate (DBP), phthalic acid two (2-ethyl hexyl) ester (DEHP), dimethyl phthalate (DMP), dioctyl phthalate (DOP) (DOP), butyl benzyl phthalate (BBP), diisononyl phthalate (DINP); and biological restoration or the biopurification of these material contaminated soils, water body; preserve the ecological environment, and then ensure agricultural product security.
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