CN104928205A - Bacillus strain capable of efficiently degrading DMP (dimethyl phthalate), culture method and application thereof to remediation of soil PAEs (phthalic acid esters) pollution - Google Patents
Bacillus strain capable of efficiently degrading DMP (dimethyl phthalate), culture method and application thereof to remediation of soil PAEs (phthalic acid esters) pollution Download PDFInfo
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Abstract
The invention discloses a bacillus strain capable of efficiently degrading DMP (dimethyl phthalate), a culture method and application thereof to remediation of soil PAEs (phthalic acid esters) pollution. A strain QD-9-10 which grows by mainly taking DMP as a carbon source is separated and identified from black soil covered with mulching films for a long term by adopting inorganic salt culture medium. According to morphological characteristic observation of a colony and 16SrDNA base sequence determination and homological analysis, the identification result of the bacterium is that QD-9-10 belongs to Bacillus subtilis. According to results of orthogonal experiments, the optimum culture method of the strain QD-9-10 is to perform culture by adopt inorganic salt culture medium under culture conditions that pH is 8.0, temperature is 35DEG C and shaking speed is 150rpm and DMP initial concentration is 100mg L<-1>, and biomass reaches the maximum at 32h. In addition, according to substrate utilization tests, the strain QD-9-10 can also utilize other common PAEs. Accordingly, the Bacillus subtilis QD-9-10 strain has a capability of degrading DMP and other common PAEs, and has a certain application prospect in aspects of degrading PAEs pollutants and remedying soil PAEs pollution.
Description
Technical field
The present invention relates to strain PAEs degradation bacteria and uses thereof, particularly a strain can efficient degradation DMP Bacillus strain and degraded PAEs and rehabilitating soil PAEs pollute in purposes, the invention belongs to microbial technology field.
Background technology
Phthalic ester (PAEs) is widely used in plastic working and other manufactured consumer goods many as a kind of organic compound.Widely using of phthalic ester result in the ecosystem that increasing phthalate enters into us.PAEs pollutes the common concern that the environment that causes and human health problems have caused people.Because PAEs has carcinogenic, teratogenesis, mutagenesis (Song Jiaoyan, Liu main forces, Wan Yang, Deng. humic acid dibutyl phthalate absorption kinetics and diffuse reflectance infrared spectroscopy research [J]. Southwestern University's journal (natural science edition), 2014, 3:022.), 6 kinds of PAEs are classified as priority pollutants China National Environmental Monitoring Center also by DEP by USEPA, these 3 kinds of PAEs of DMP and DnOP are defined as Environment Priority control polluted articles (Jin DC, Kong X, Cui BJ, et al.Biodegradation of di-n-butyl phthalate by a newly isolated Halotolerant Sphingobium sp.International Journal of Molecular Sciences, 2013, 14:24046-24054.Jin D, Bai Z, Chang D, et al.Biodegradation of di-n-butyl phthalate by an isolated Gordonia sp.strain QH-11:Genetic identification and degradation kinetics [J] .Journal of hazardous materials, 2012, 221:80-85.).
In China, the PAEs in soil is usually from farmland plastics film, plastic waste material, rubbish and sewage irrigation, and wherein PAEs enters the use that the main path in farmland is plastic sheeting for farm use.And black soil region northeastward, due to reasons such as China's black soil region temperature are low, plastic sheeting for farm use usage quantity large and organic matter in black soil content is high, to cause black earth PAEs to pollute comparatively serious.The hydrolysis of PAEs and water splitting are all slowly; biological degradation is main path (the Yang C F that this kind of material decomposes in the environment; Wang C C; Chen C H.Di-n-butyl phthalate removal by strain Deinococcus sp.R5in batch reactors [J] .International Biodeterioration & Biodegradation, 2014.).So obtaining PAEs efficient degrading bacteria is the important step improving its biological degradation efficiency.
Dimethyl phthalate (Dimethyl phthalate, DMP) is one of PAEs pollutant, the main forming agent being used as plastic prod.The whole world has a large amount of DMP to be used every year, and because the combination of itself and plastics is non-covalent bonds, so DMP easily enters in ecotope, therefore, DMP has become transworld synthesis organic pollution materials.DMP pollutes the metabolic characteristics that can change Soil respiration and the enzyme activity to have result of study to show, have impact on the ecosystem function of black earth, and then threatens black soil fertility and grain-production.
Therefore, the PAEs degradation bacteria that the present invention is directed in the soil of Northeast black earth area is studied, and is intended to provide fundamental basis for repairing black soil region PAEs pollution and set up microorganism resource platform.
Summary of the invention
An object of the present invention is to provide a strain can the bacterial strain of efficient degradation DMP, this bacterium has the ability of higher degraded DMP, also can utilize other common PAEs simultaneously, have certain application prospect at degraded PAEs pollutent, particularly DMP and rehabilitating soil PAEs pollution aspect.
Two of object of the present invention is to provide a kind of method of cultivating described Bacillus strain.
Three of object of the present invention is to provide the application of described Bacillus strain in degraded PAEs and rehabilitating soil PAEs pollutes.
In order to achieve the above object, present invention employs following technique means:
A strain of the present invention can the Bacillus strain of efficient degradation DMP, called after subtilis QD-9-10, Classification And Nomenclature is Bacillus subtilis QD-9-10, preservation China typical culture collection center, address is in Wuhan University, deposit number is: CCTCC NO:M 2015199, and preservation date is on April 6th, 2015.
The present invention covers from long-term the bacterial strain that one strain of isolation identification the black soil of plastic sheeting for farm use take DMP as sole carbon source growth: Bacillus subtilis QD-9-10.Orthogonal experiment results shows that the optimal growth condition that bacterial strain QD-9-10 carries out growing in minimal medium is: pH 8.0, temperature 35 DEG C, shaking speed 150rpm, DMP starting point concentration 100mgL
-1.The starting point concentration of DMP is comparatively large on the impact of bacteria growing, and biomass reaches maximum when 32h and can by 100mgL
-1dMP degradable.
Wherein, described minimal medium is prepared in accordance with the following methods: K
2hPO
412H
2o 1.0g, KH
2pO
41.0g, NH
4cl 0.8g, NaCl 1.0g, MgSO
47H
2o 0.5g, CaCl
20.006g, FeSO
47H
2o 0.02g, micro-storing solution 2mL, distilled water complement to 1000ml, pH 7.0;
Wherein, described micro-storing solution is prepared in accordance with the following methods: MnSO
4h
2o 0.1g, ZnSO
47H
2o 0.12g, H
3bO
30.07g, Na
2moO
4h
2o 0.04g, CuSO
45H
2o 0.02g, CoCl
20.04g, distilled water complements to 1000ml.
Meanwhile, substrate utilization test shows that bacterial classification QD-9-10 also can utilize other common PAEs.
Therefore, further, the invention allows for the application of described Bacillus strain in degraded PAEs and rehabilitating soil PAEs pollutes.
Wherein, described PAEs comprises dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dinoctyl phthalate (DOP), dimixo-octyl phthalate (DEHP) and dimixo-octyl phthalate (DIOP).
Preferably, described PAEs is dimethyl phthalate (DMP).
To sum up, the present invention is separated the ability that the strain DMP degradation bacteria Bacillus subtilis QD-9-10 obtained has degraded DMP and other common PAEs, in degraded PAEs pollutent and rehabilitating soil PAEs pollution, have certain application prospect.
Accompanying drawing explanation
Fig. 1 is the phylogenetic analysis of bacterial strain QD-9-10 of the present invention;
Fig. 2 is growth curve and the DMP concentration curve of bacterial strain QD-9-10.
Wherein, vertical bar represents standard deviation (N=3).
Embodiment
Further describe the present invention below in conjunction with specific embodiments and the drawings, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The separation of embodiment 1 bacterial strain QD-9-10 and the application in degraded PAEs thereof
1 materials and methods
1.1 for examination material and substratum
Dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dinoctyl phthalate (DOP), dimixo-octyl phthalate (DEHP), and dimixo-octyl phthalate (DIOP), methyl alcohol, ethyl acetate and hexane, all chemical and solvent are analytical reagent, are purchased from Tianjin recovery fine chemistry industry institute.
Basic inorganic salt (MSM) substratum: K
2hPO
412H
2o 1.0g, KH
2pO
41.0g, NH
4cl 0.8g, NaCl 1.0g, MgSO
47H
2o 0.5g, CaCl
20.006g, FeSO
47H
2o 0.02g, micro-storing solution (MnSO
4h
2o 0.1g, ZnSO
47H
2o 0.12g, H
3bO
30.07g, Na
2moO
4h
2o 0.04g, CuSO
45H
2o 0.02g, CoCl
20.04g, distilled water complements to 1000ml) 2mL, distilled water complements to 1000ml, pH 7.0.
LB substratum: peptone 10.0g, yeast powder 5.0g, sodium-chlor 10.0g, distilled water complement to 1000ml, pH 7.0.
The source of 1.2 mixed cultures and domestication
Gather the pedotheque that black soil region covers plastic sheeting for farm use for a long time, from sample, claim the soil sample of 10mg in containing 200mgL
-1(DMP, DEP, DBP, DOP are respectively 50mgL to phthalic ester
-1) 100mL inorganic salt nutrient solution in, adopt the domestication of gradient pressure method, 30 DEG C of shaking culture 7d, are progressively forwarded to containing 250mgL
-1, 300mgL
-1, 350mgL
-1, 400mgL
-1, 450mgL
-1, 500mgL
-1after the inorganic salt nutrient solution of phthalic ester (DMP, DEP, DBP, DOP equivalent), dip a small amount of bacterium liquid with inoculating needle, again receive enrichment culture in MSM substratum.
The separation and purification of 1.3 degradation bacteria strains and qualification
Mixed culture is after gradient dilution, minimal medium is adopted to be separated DMP degradation bacteria, select the bacterial strain that grows fine carry out morphology and 16S rDNA sequential analysis to bacterial strain identify (reference: Lin Xiangui. soil microorganisms research principle and method [M]. Beijing: Higher Education Publishing House, 2010:123-186; Tang Liang, Zhang Jinzhong, Yu Pingping, Sun Ping. the separation of silicate bacteria, purifying, qualification and biological characteristic research [J]. Shandong agricultural sciences, 2008 (1): 71-73; Clarridge J E.Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases [J] .Clinical microbiology reviews, 2004,17 (4): 840-862).The recombinant clone of bacterial strain is checked order by Shanghai Major Biological Medical Technology Co., Ltd..Comprise the extracting of bacterial genomes DNA, electrophoresis detection, pcr amplification, the electrophoresis detection of PCR primer, order-checking cut glue purification order-checking, analytical results and splicing sequence.PCR reagent: EX Taq enzyme (TaKaRa), dNTP (TaKaRa), primer (Invitrogen synthesis), Marker:DL2000.PCR reaction system: (10 × Ex Taq buffer 2.0 μ l, 2.5mM dNTP Mix 1.6 μ l, 5p Primer10.8 μ l, 5p Primer20.8 μ l, Template 0.5 μ l, 5u Ex Taq 0.2 μ l, ddH
2o 14.1 μ l) Total volume 20 μ l.PCR reaction conditions: 95 DEG C of denaturation 5min, then carry out 24 circulations (95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min), extends 10min after 72 DEG C.Sequencing result carries out Blast comparison in the Gen Bank database of NCBI, then adopts MEGA5.2 software to carry out multisequencing homology analysis, and phylogenetic tree construction (Bootstrap=1000).The inoculation finally separation and purification obtained saves backup in LB substratum.
1.4 degradation condition optimizations
What affect the growth of DMP degradation bacteria is respectively pH, DMP starting point concentration, temperature, shaking speed because have 4, and this experiment is provided with 3 levels respectively to each factor, refers to table 1.Utilize the orthogonal design in spss19.0 software, the 9 groups of experiments devising 4 factor 3 levels refer to table 2.Then get bacterial strain and support shaking culture 12h-24h in base in the inorganic salt of 100mL, centrifugal (10000r/min) 5min, collect thalline, use Na
2hPO
4-NaH
2pO
4(0.02molL
-1pH 7.0) buffer solution 3 times, use 0.02molL
-1thalline is made into pH 7.0, OD by phosphoric acid buffer
600the bacteria suspension of=0.2.Be inoculated in MSM minimal medium.Carry out respectively testing to determine best degradation condition according to the 9 groups of experiment conditions provided in orthogonal design.Test process is 3 repetitions.
Table 1 orthogonal test level of factor table
The best degradation condition development schedule of table 2 DMP
1.5 biological degradation experiments
By inoculation to containing 100mgL
-1the minimal medium conditional of DMP is that (PH:8, temperature: 35 DEG C, shaking speed: 150rpm) cultivates.Measure wavelength with ultraviolet spectrophotometer be at regular intervals the OD value of the bacterium liquid of 600nm and sample centrifuging and taking supernatant, the extraction hexane of phthalic ester and microwave assisted extraction methods.Extraction solvent and dried over sodium sulfate, concentrated by rotary evaporation.Detect the content of remaining DMP in concentrated solution.The mensuration of DMP content adopts high performance liquid chromatography (HPLC) (reference: Yan Jiabao, Wang Qiaofeng, the .Elizabethkingia sp.DBP-WUST such as Wei Xin study [J] the degradation characteristic of dibutyl phthalate. Xuzhou Engineering Institute journal (natural science edition), 2012,27 (3): 16-21).The mensuration of the growth curve of bacterial strain adopts turbidimetry.
.
1.6 degraded substrate diversity experiments
For probing into the diversity of bacterial strain to different substrate utilization, bacteria suspension is added to respectively containing 100mgL
-1following substrate: in cultivating in the minimal medium of DMP, DEHP, DBP, observe cultivate the substratum opacity after 36h assess microbial biomass number.There is no the liquid nutrient medium of inoculating strain (CK) in contrast.
2 results and analysis
The form of 2.1DMP degradation bacteria and colony characteristics and 16S rDNA homology analysis
After more than 10 times switchings are cultivated, the mixed culture in shaking flask can at 500mgL
-1well grow in the inorganic salt nutrient solution of phthalic ester, illustrate that degradation bacteria strains obtains enrichment in the process.After multiple sieve, purifying, to obtain a strain can take DMP as the Strain Designation of carbon source for growth is QD-9-10 in screening.QD-9-10 bacterial strain is on inorganic salt flat board after 30 DEG C of constant temperature culture 1d, and bacterium colony is faint yellow, dimpling, moistening, transparent.Gram-positive.16S rDNA strain identification the results are shown in Figure 1, the 16SrDNA sequence homology of QD-9-10 bacterial strain and Bacillus subtilis reaches 99%, in conjunction with its thalli morphology and colony characteristics, be Bacillus subtilis by QD-9-10 identification of strains, preservation China typical culture collection center, address is in Wuhan University, and deposit number is: CCTCC NO:M 2015199, and preservation date is on April 6th, 2015.
The determination of 2.2 orthogonal experiments analyses and best degradation condition
The intuitive analysis of orthogonal experiments is from table 3, and the impact of orthogonal experiment factor is sequentially strong and weak: DMP starting point concentration >pH> temperature > shaking speed.Shaking speed impact is minimum, and factor DMP starting point concentration and pH basic non-interaction action is each other described, shaking speed can be used as error term.Optimum combination condition: A3B3C2D1, namely tests 9, and its condition is PH:8, temperature: 35 DEG C, shaking speed: 120rpm, DMP starting point concentration: 100mgL
-1.
Table 3 DMP best degradation condition research intuitive analysis table
Note: K
1for the mean value of each horizontal factor l, K
2for the mean value of each horizontal factor 2, K
3for the mean value of each horizontal factor 3, R is extreme difference.
Factor C is as error term, and Orthogonal experiment results, through orthogonal assistant's software processes, obtains table 4.From table 4, the impact of factor DMP starting point concentration is more remarkable.3 strong and weak orders of factor impact that variance analysis obtains are: DMP starting point concentration >PH> temperature, result is consistent with intuitive analysis.
Table 4 orthogonal test variance result
Orthogonal experiment results carries out multiple regression analysis through DPS7.05 statistical software, obtains following multiple regression journey: Y=-0.841+0.1272X
1+ 0.0124X
2+ 0.0004X
3-0.0004X
4(X
1represent A; X
2represent B; X
3represent C; X
4represent D; Y represents OD
600value) coefficient R=0.827957, Durbin-Watson d=1.5158.Multiple regression equation relation conefficient is higher as can be seen from the above results, and close to 2, the value of Durbin-Watson d illustrates that equation model degree is high, do not have obvious dependency between residual error.Work as X
1=8, X
2=35, X
3=150, X
4y=0.623 when=100.Namely best of breed is PH=8, and temperature is 35 DEG C, and shaking speed is 150rpm, DMP starting point concentration is 100mgL
-1.This is substantially identical with the result of intuitive analysis and variance analysis.
Comprehensive above 3 kinds of analytical procedures, finally determining best degradation condition is PH:8, temperature: 35 DEG C, shaking speed: 150rpm, DMP starting point concentration: 100mgL
-1.
The growth curve of 2.3 bacterial strains
As seen from Figure 2 by inoculation to containing 100mgL
-1the minimal medium conditional of DMP is (PH:8, temperature: 35 DEG C, shaking speed: 150rpm) biomass that carries out cultivating bacterial strain in lower 0-4h almost do not have change be in slow its, in 4-32h, the biomass increase of bacterial strain is in logarithmic phase, and the increase of 32-40h biomass eases up and enters stationary phase; The minimizing of 40h-48h biomass enters decline phase.And DMP concentration obviously reduces in 0-18h in substratum, concentration stabilize in 18-32h also tends to be 0.Show along with the DMP in the increase substratum of the growth biomass of bacterium is degraded gradually.
2.4 strains for degrading functional diversities are analyzed
The substrate broad spectrum experimental result of DMP degradation bacteria strains is in table 5.As can be seen from the table: QD-9-10 can utilize different PAEs as DEP, DEHP, DBP for substrate grows.Bacterial strain is containing 100mgL
-1grow fine after cultivating 72h in the minimal medium of DEP, substratum is obviously more muddy than the substratum not connecing bacterium a lot.And containing 100mgL
-172h wild Oryza species turbidity is cultivated obviously not as containing 100mgL in the minimal medium of DBP
-1the minimal medium of DEP, but still comparatively more muddy than the substratum not connecing bacterium.
The substrate broad spectrum experiment of table 5 DMP degradation bacteria strains QD-9-10
+++ turbidity is high; ++ turbidity is higher; + muddy;-not muddy.
3 conclusions
To sum up, the present invention is separated the ability that the strain DMP degradation bacteria Bacillus subtilis QD-9-10 obtained has degraded DMP and other common PAEs, (pH 8.0, temperature 35 DEG C under the degraded conditions of the best, shaking speed 150rpm, DMP starting point concentration 100mgL
-1) degradation rate is 100%.Show that this bacterial strain has certain application prospect in degraded PAEs pollutent and the pollution of rehabilitating soil phthalate.
Claims (6)
1. a strain can the Bacillus strain of efficient degradation DMP, it is characterized in that described Strain Designation is subtilis QD-9-10, preservation China typical culture collection center, address is in Wuhan University, deposit number is: CCTCC NO:M 2015199, and preservation date is on April 6th, 2015.
2. cultivate according to claim 1 can the method for Bacillus strain of efficient degradation DMP, it is characterized in that adopting minimal medium to cultivate, culture condition is: pH 8.0, temperature 35 DEG C, shaking speed 150rpm, DMP starting point concentration 100mgL
-1, biomass reaches maximum when 32h.
3. method as claimed in claim 2, is characterized in that described minimal medium is prepared in accordance with the following methods: K
2hPO
412H
2o 1.0g, KH
2pO
41.0g, NH
4cl 0.8g, NaCl 1.0g, MgSO
47H
2o 0.5g, CaCl
20.006g, FeSO
47H
2o 0.02g, micro-storing solution 2mL, distilled water complement to 1000ml, pH 7.0;
Wherein, described micro-storing solution is prepared in accordance with the following methods: MnSO
4h
2o 0.1g, ZnSO
47H
2o 0.12g, H
3bO
30.07g, Na
2moO
4h
2o 0.04g, CuSO
45H
2o 0.02g, CoCl
20.04g, distilled water complements to 1000ml.
4. the application of Bacillus strain according to claim 1 in degraded PAEs and rehabilitating soil PAEs pollutes.
5. apply as claimed in claim 4, it is characterized in that described PAEs comprises dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dinoctyl phthalate (DOP), dimixo-octyl phthalate (DEHP) and dimixo-octyl phthalate (DIOP).
6. apply as claimed in claim 5, it is characterized in that described PAEs is dimethyl phthalate (DMP).
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| CN106119167A (en) * | 2016-07-07 | 2016-11-16 | 浙江省农业科学院 | A kind of utilize the method for Triadimenol in novel microorganism bacterial strain degraded soil |
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| CN106399179A (en) * | 2016-10-09 | 2017-02-15 | 北京工商大学 | Application of Bacillus mojavensis and method for degrading bis(2-ethylhexyl) phthalate by adopting Bacillus mojavensis |
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| CN114686238A (en) * | 2022-02-24 | 2022-07-01 | 湖北省烟草科学研究院 | Preparation method and application of multifunctional soil microorganism conditioning microbial inoculum |
| CN114686238B (en) * | 2022-02-24 | 2024-03-22 | 湖北省烟草科学研究院 | Preparation method and application of multifunctional soil microorganism conditioning microbial inoculum |
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