CN106834269B - A kind of immobilized microspheres and its preparation method and application of PAEs degradation bacteria - Google Patents

A kind of immobilized microspheres and its preparation method and application of PAEs degradation bacteria Download PDF

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CN106834269B
CN106834269B CN201710167589.8A CN201710167589A CN106834269B CN 106834269 B CN106834269 B CN 106834269B CN 201710167589 A CN201710167589 A CN 201710167589A CN 106834269 B CN106834269 B CN 106834269B
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bacteria
immobilized microspheres
mixed
paes
calcium chloride
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CN106834269A (en
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周震峰
饶潇潇
王亚娟
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Qingdao Agricultural University
Ningxia University
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Qingdao Agricultural University
Ningxia University
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14Enzymes or microbial cells immobilised on or in an inorganic carrier
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B09DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09CRECLAMATION OF CONTAMINATED SOIL
    • B09C1/00Reclamation of contaminated soil
    • B09C1/10Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B09DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09CRECLAMATION OF CONTAMINATED SOIL
    • B09C2101/00In situ

Abstract

The present invention relates to the degradation bacteria immobilized microspheres and preparation method thereof of PAEs in removal agricultural land soil a kind of: (1) preparation of bacteria suspension: by pseudomonas putida RXX-01 (deposit number is CGMCC No.13224) culture to logarithmic growth phase, after thallus is cleaned with physiological saline, thallus is deployed into bacteria suspension with physiological saline again, makes the OD of bacteria suspension600=0.8-1.2;(2) outfit of mixed bacteria liquid: sodium alginate soln is prepared with sterile water, active carbon is added in sodium alginate soln and mixes to obtain mixed liquor, the bacteria suspension that step (1) is added into mixed liquor mixes to obtain mixed bacteria liquid;Weight ratio shared by each component is respectively as follows: bacteria suspension 15-25%, sodium alginate 2-3%, active carbon 2-4%;(3) preparation of immobilized microspheres: the calcium chloride solution of 20-30g/L is prepared with sterile water, and mixed bacteria liquid is instilled in calcium chloride solution, immobilized microspheres are prepared after being crosslinked 20-30h;The volume ratio of mixed bacteria liquid and calcium chloride solution is 1:1.5~1:2.Preparation method of the present invention is simple, high to the degradation efficiency of Phthalate Esters In Soil.

Description

A kind of immobilized microspheres and its preparation method and application of PAEs degradation bacteria
Technical field
The invention belongs to field of agricultural biotechnology, in particular to the degradation bacteria of phthalic acid ester is solid in a kind of removal agricultural land soil Surely change microballoon and its preparation method and application.
Background technique
Phthalic acid ester (Phthalic Acid Esters, PAEs) is a kind of typical incretion interferent, adjacent benzene Dicarboxylic acid dimethyl ester (Dimethyl Phthalate, DMP), dibutyl phthalate (Dibutyl Phthalate, DBP) With phthalic acid two (2- ethylhexyl) ester (6 kinds of phthalic acids such as Di- (2-ethylhexyl Phthalate, DEHP) Ester compounds are classified as priority pollutants by U.S.EPA and environmental monitoring master station, China.As important chemical products, Phthalic acid ester is largely used in production process of plastic products as additive.The fast development of China's industrialized agriculture makes to mould Material products are widely used, with plastic ageing phthalic acid ester will be slow discharge and eventually enter into soil, cause its at For one of the organic pollutant being most often detected in agricultural land soil, the adjacent benzene especially in northern facility vegetable plot, southern vegetable soil Dicarboxylic acid esters content even has reached tens mgkg-1.Wherein, DBP and DEHP be in soil recall rate highest and content compared with 2 kinds of high phthalate compounds, due to the molecule molal weight (278.4gmol of DBP-1) and logKow (4.45, Kow is octanol-water partition coefficient) it is respectively less than DEHP (respectively 390.6gmol-1, 7.50), and water solubility (11.2mg L-1) it is higher than DEHP (0.003mgL-1), cause the microorganism utilization rate of DBP in soil relatively high, degradation rate is higher than Therefore DEHP removes remaining DEHP difficulty in soil and is greater than DBP.A large number of studies show that soil phthalic acid ester pollutes Not only will affect growth and development and the yield and quality of crops, and will cause the accumulation of phthalic acid ester in agricultural product to Human health is caused by food chain potentially hazardous, therefore, repairs the pollution of agricultural land soil phthalic acid ester to protecting me State's agricultural land soil quality ensures that agricultural product security, ecological safety and human health are of great practical significance.
Currently, the research of agricultural land soil phthalic acid ester pollution remediation technology is still at an early stage.Existing soil has Machine pollution amelioration mainly uses peripheral doses, chemical remediation and biological prosthetic method.Wherein, biological prosthetic especially microorganism Recovery technique is safe and efficient, without secondary pollution, good in economic efficiency due to having many advantages, such as, quilt is it is believed that be to carry out organic dirt Contaminate that soil remediation is most effective, a kind of most promising pollutant abatement technology.Domestic and foreign scholars to phthalic acid ester degradation bacteria into It has gone screening, there is certain degradation effect.In practical applications, since the ingredient of pollutant in degradation process is more complicated, one The toxicity of a little secondary metabolites and intermediate catabolite is bigger, free bacterium show effectively to degrade in unit volume bacteria concentration it is low, With the features such as competition of indigenous bacterium is in weak tendency, antitoxin sexual abuse ability is poor.Degradation bacteria is carried out using immobilized microorganism technology It is fixed, microorganism in unit volume medium can be improved to avoid the activity of artificial destruction biological enzyme and the stability of biochemical reaction Cell density.In addition, the microenvironment of degradation bacteria immobilized microspheres is conducive to shield indigenous bacterium, bacteriophage and toxicant to micro- life Harmful competition, phagocytosis and the murder by poisoning of object play consistently it can also in complicated soil environment high-effect.Therefore, The phthalic acid ester efficient degrading bacteria filtered out in soil is immobilized into rear shape by process for fixation such as absorption embeddings The degradation rate of phthalic acid ester in agricultural land soil will greatly be improved by being applied to contaminated soil at microballoon, preferably be played outer The effect of source degradation bacteria.
Patent 201510418972.7 discloses the immobilized microspheres and its system of a kind of dibutyl phthalate degradation bacterium Preparation Method and application, patent prepare microballoon by immobilization material of sodium alginate, bentonite, chitosan and calcium chloride, repair effect Fruit is DBP degradation rate about 80% in 28 days soil.However it is only for soil DBP pollution problem and to DBP in soil Degradation efficiency is lower, can not efficient application in DBP and DEHP combined contamination soil.
Summary of the invention
The purpose of the present invention is to provide a kind of degradation bacteria immobilized microspheres of phthalic acid ester in removal agricultural land soil And preparation method thereof, combined contamination soil is coped with, DBP can not only be efficiently removed, moreover it is possible to be effectively removed more difficult to degrade DEHP。
A kind of preparation method of the immobilized microspheres of PAEs degradation bacteria, the specific steps of which are as follows:
(1) preparation of bacteria suspension: by PAEs degradation bacteria culture to logarithmic growth phase, after thallus is cleaned with physiological saline, Thallus is deployed into bacteria suspension with physiological saline again, makes the OD of bacteria suspension600=0.8-1.2;The PAEs degradation bacteria is that stench is false Monad RXX-01, classify entitled Pseudomonas putida, has been preserved in China Committee for Culture Collection of Microorganisms Common micro-organisms center (CGMCC), address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the deposit date is in November, 2016 1, deposit number was CGMCC No.13224;The physiological saline is sterilized 0.9%NaCl solution;
(2) outfit of mixed bacteria liquid: sodium alginate soln is prepared with sterile water, active carbon is added to sodium alginate soln In mix to obtain mixed liquor, into mixed liquor be added step (1) bacteria suspension mix to obtain mixed bacteria liquid;Weight score shared by each component Not are as follows: bacteria suspension 15-25%, sodium alginate 2-3%, active carbon 2-4%;
(3) preparation of immobilized microspheres: the calcium chloride solution of 20-30g/L is prepared with sterile water, and dropwise by mixed bacteria liquid It instills in calcium chloride solution, immobilized microspheres is prepared after being crosslinked 20-30h;The volume ratio of mixed bacteria liquid and calcium chloride solution For 1:1.5~1:2.
Sodium alginate be electrolysed in aqueous solution generate seaweed acid anion, calcium chloride generate divalent calcium ions, calcium ion with Seaweed acid anion occurs electrostatic interaction and attracts, and is cross-linked to form calcium alginate not soluble in water.
Further, weight ratio shared by mixed bacteria liquid each component is respectively as follows: bacteria suspension 20%, sodium alginate 3%, active carbon 2%;The concentration for being used to prepare the calcium chloride solution of immobilized microspheres is 25g/L.
Further, the sodium alginate, active carbon, calcium chloride are using preceding sterilizing.
Further, the culture medium of pseudomonas putida RXX-01 is beef-protein medium in the step (1), Component are as follows: beef extract 3g, peptone 10g, sodium chloride 5g add water to be settled to 1000mL.
Further, in the step (1) pseudomonas putida RXX-01 condition of culture are as follows: 25-30 DEG C, pH6.5- 7.5,150-200r/min isothermal vibration culture 24-72h.It is preferred that 28 DEG C, the culture of pH7.0,170r/min isothermal vibration for 24 hours.
Further, in the step (1) cleaning thallus method are as follows: by the culture solution of pseudomonas putida RXX-01 in 3500-4500rpm is centrifuged 5-15min, abandons supernatant, and after precipitating is resuspended with the physiological saline of sterilizing, repeated centrifugation and resuspension are heavy The step in shallow lake 1-3 times.
Further, mixed bacteria liquid is instilled in calcium chloride solution using peristaltic pump in the step (3), flow control exists 6mL/min。
Further, the immobilized microspheres that the step (3) prepares are rinsed well with sterile distilled water, 4 DEG C of preservations.
The present invention also provides the immobilized microspheres for the PAEs degradation bacteria being in the above way prepared.
In addition, the present invention also provides the immobilized microspheres of the PAEs degradation bacteria in degradation agricultural land soil phthalic acid Application in ester.
Further, use of the immobilized microspheres of the PAEs degradation bacteria in agricultural land soil of degrading when phthalic acid ester Amount is 0.1-1kg/kg dry ground.
The present invention has the advantages that
(1) present invention uses active carbon, sodium alginate and calcium chloride to prepare immobilized microspheres for raw material, i.e., with active carbon For carrier material, carry out embedding fixed phthalic acid ester efficient degrading bacteria that microballoon, preparation side is prepared by sodium alginate Method is easy to operate.
(2) immobilization effect of degradation bacteria is good, high to the degradation efficiency of Phthalate Esters In Soil, is suitable for large area Agricultural land soil phthalic acid ester pollute in-situ immobilization.To the drop of DBP in soil and DEHP after immobilized microspheres culture 10 days Solution rate respectively reaches 94.0% and 34.5%;In contrast, the degradation rate of blank control group DBP and DEHP is respectively 27.9% He 5.8%, for free bacterium processing group between two groups, the degradation rate of DBP and DEHP respectively reach 91.2% and 25.4%.
Detailed description of the invention
The scribing line cultivation results of Fig. 1 pseudomonas putida RXX-01;
Degradation rate of the 24 kinds of immobilized microspheres prepared under Fig. 2 different condition to DBP in soil and DEHP;
The aspect graph of immobilized microspheres prepared by Fig. 3 embodiment 4;
The degradation rate of immobilized microspheres prepared by Fig. 4 embodiment 4 to DBP in soil;
The degradation rate of immobilized microspheres prepared by Fig. 5 embodiment 4 to DEHP in soil.
Biological material specimens preservation information:
Pseudomonas putida (Pseudomonas putida) RXX-01 is preserved in Chinese microorganism strain preservation management committee Member's meeting common micro-organisms center (CGMCC), address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date: 2016 November 1, deposit number are CGMCC No.13224.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, right below in conjunction with drawings and examples The present invention is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, not For limiting the present invention.
Used culture medium is as follows in the embodiment of the present invention:
Beef-protein medium: beef extract 3g, peptone 10g, sodium chloride 5g add water to be settled to 1000mL.
Minimal medium: K2HPO4·3H2O:1g, NaCl:1g, NH4NO3: 0.5g, MgSO4·7H2O:0.4g, CaCl2: 0.1g, FeCl3·6H2O:0.01g adds water to be settled to 1000mL.
The screening and identification of 1 phthalic acid ester degradation bacteria of embodiment
(1) screening of phthalic acid ester degradation bacteria
A certain amount of soil sample is acquired in the green house of vegetables of Qingdao Suburban Areas, accurately weighs 10g fresh soil sample, and addition fills In the triangular flask of 90mL distilled water and 10mL bead, 30 DEG C, 175rpm oscillation mixing, set aside for use are placed in shaking table.
1mL is taken from the above mixed liquor by 1% inoculum concentration, being added to 100mL initial concentration is 100mg/L O-phthalic In the inorganic salts culture solution of acid esters (DBP, DEHP are 50mg/L), 30 DEG C are placed in shaking table, 175rpm carries out under the conditions of being protected from light Shaken cultivation 7d.Every time switching gradually increase phthalic ester concentration (including 200mg/L, 500mg/L, 800mg/L, 1000mg/L, 1500mg/L and 2000mg/L), every seven days are primary as an acclimation period switching, pass through 6 acclimation periods altogether Persistently cultivated.
100 μ L final period domestication liquids are taken, 2000mg/L phthalic acid ester (DBP be 1000mg/L with DEHP) is coated on On inorganic salts solid plate, it is subsequently placed in stationary culture in 30 DEG C of biochemical cultivation cases.After growing naked eyes visible colonies, it is observed Growth characteristics, filter out the best bacterial strain of growing way, picking single colonie by its repeatedly streak inoculation in new inorganic salts solid culture On base, until microscopy purifying.
(2) identification of phthalic acid ester degradation bacteria
1. carrying out morphologic observation to the single colonie RXX-01 screened, cultural characteristic includes the following contents: being trained in solid It supports on base, observation bacterium colony size, projecting shape, transparency, color, migration, quality, form, edge feature and glossiness etc.. On beef-protein medium plate after cultivating a couple of days, bacterium colony is rounded for RXX-01 bacterial strain, and Chinese red, clayey is opaque, Neat in edge, surface elevation moistens smooth, and diameter is generally at 0.5~1.0mm (see Fig. 1).
2. RXX-01 strain, which is sent to Shanghai Sheng Gong Biological Co., Ltd., carries out molecular biology identification, 16SrDNA sequence is as shown in SEQ ID No.1 in sequence table, through sequence alignment, with pseudomonas putida (Pseudomonas Putida) homology of (GenBank accession number AY647158) reaches 100%.The morphological feature and 16SrDNA sequence of comprehensive bacterial strain Column analysis is as a result, Preliminary Identification bacterial strain RXX-01 is pseudomonas putida (Pseudomonas putida).
2 pseudomonas putida RXX-01 of embodiment detects phthalic acid ester degradation capability
By the culture solution of RXX-01 bacterial strain according to 1% inoculum concentration, it is inoculated in the nothing that DBP and DEHP concentration is 50mg/L In machine salt culture medium (50mL), shaken cultivation under conditions of 25 DEG C, 150r/min.Culture 3 days after measure culture solution in DBP with DEHP concentration, and calculate the degradation rate of DBP and DEHP.The degradation rate of DBP is that the degradation rate of 90.8%, DEHP is as the result is shown 54.7%, show RXX-01 bacterial strain to phthalic acid ester degradation capability with higher.
Degradation rate calculation formula: degradation rate (%)=((phthalic acid ester initial concentration-phthalic acid ester residual is dense Degree)/phthalic acid ester initial concentration) × 100%.
The preparation method of 3 pseudomonas putida RXX-01 immobilized microspheres of embodiment
The present embodiment has selected different bacterium solution contents, sodium alginate dense to determine the optimal proportion of immobilization material Degree, activated carbon content, calcium chloride concentration be factor, using immobilized microspheres to the degradation rate of phthalic acid ester as target, optimization Fixing condition.
4 factors are arranged in experiment of single factor altogether, and (bacteria suspension content, sodium alginate concentration, activated carbon content, calcium chloride are dense Degree), 6 concentration levels are arranged in each factor.Percentage involved in it is mass percent.What the bacteria suspension referred to It is with physiological saline by OD made of thallus600=1.0 suspensions.
Under conditions of sodium alginate concentration 2%, activated carbon content 3%, calcium chloride concentration 25g/L, setting bacteria suspension contains Amount is respectively 0%, 5%, 10%, 15%, 20%, 25%, prepares different types of immobilized microspheres.
Under conditions of bacteria suspension content 15%, activated carbon content 3%, calcium chloride concentration 25g/L, setting sodium alginate is dense Degree is respectively 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, prepares different types of immobilized microspheres.
Under conditions of bacteria suspension content 15%, sodium alginate concentration 2%, calcium chloride concentration 25g/L, setting active carbon contains Amount is respectively 0%, 1%, 2%, 3%, 4%, 5%, prepares different types of immobilized microspheres.
Under conditions of bacteria suspension content 15%, sodium alginate concentration 2%, activated carbon content 3%, calcium chloride concentration is set For 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, different types of immobilized microspheres are prepared.
After designing above-mentioned single factor experiment, immobilized microspheres are prepared according to the following steps:
(1) preparation of bacteria suspension: RXX-01 strain is inoculated in beef extract-peptone fluid nutrient medium, in 28 DEG C, It is cultivated for 24 hours under conditions of 170r/min.After taking out fluid nutrient medium, by liquid subpackage in the sterile centrifugation tube of 50mL, in 10min is centrifuged under 4000rpm.After discarding supernatant liquid, it is centrifuged again after being resuspended with the physiological saline after sterilizing, after cleaning twice, Thallus is deployed into bacteria suspension with physiological saline, makes its OD600=1.0.
(2) outfit of mixed bacteria liquid: sodium alginate soln is prepared with sterile water, active carbon is added to sodium alginate soln In mix to obtain mixed liquor, into mixed liquor be added step (1) bacteria suspension mix to obtain mixed bacteria liquid;
(3) preparation of immobilized microspheres: calcium chloride solution is prepared with sterile water, and dropwise with peristaltic pump pumps mixed bacteria liquid It instills in calcium chloride solution, the volume ratio of mixed bacteria liquid and calcium chloride solution is 1:1.5, and fixation is prepared after being crosslinked 20-30h Change microballoon, is rinsed well with sterile distilled water, 4 DEG C save backup.
Prepare under different condition 24 kinds of immobilized microspheres are under equal conditions applied separately to phthalic acid ester dirt It contaminates in soil (content of DBP and DEHP are respectively 50mg/kg and 50mg/kg), the applied amount of immobilized microspheres is 0.5kg/kg Dry ground after equal conditions culture 5 days, measures the residual quantity of the Phthalate Esters In Soil of each immobilized microspheres processing.It is different Under the conditions of the immobilized microspheres that prepare it is as shown in Figure 2 to the degradation rate of DBP in soil and DEHP.
According to immobilized microspheres to the degradation effect of Phthalate Esters In Soil, the optimum range of bacteria suspension content is determined It is 15-25%, the optimum range of sodium alginate concentration is 2-3%, and the optimum range of activated carbon content is 2-4%, and calcium chloride is dense The optimum range of degree is 20-30g/L.
The optimum condition of degradation bacteria immobilization is determined using single factor experiment result further progress orthogonal test.Immobilization Microballoon Orthogonal Experiment and Design and its result are shown in Tables 1 and 2 respectively.
1. immobilized microspheres orthogonal test designs table of table
2. immobilized microspheres orthogonal experiments of table
By the value of very poor R in table 2 it is found that each factor to the influence degree of experimental result be followed successively by from big to small factor B > because Plain C > factor A > factor D determines that the optimum condition for preparing immobilized microspheres is A2B3C1D2, i.e. bacteria suspension content is 20%, seaweed Sour na concn is 3%, activated carbon content 2%, calcium chloride concentration 25g/L, the immobilized microspheres pair prepared under this condition The total degradation rate of PAEs is 63.1%.
Degradation effect of 4 immobilized microspheres of embodiment to Phthalate Esters In Soil
The optimal proportion and its preparation process of immobilization material are obtained according to the result of orthogonal test, steps are as follows:
(1) preparation of bacteria suspension: strain RXX-01 is inoculated into beef extract-peptone fluid nutrient medium, in 28 DEG C, It is cultivated for 24 hours under conditions of 170r/min.After taking out fluid nutrient medium, by liquid subpackage in the sterile centrifugation tube of 50mL, in 10min is centrifuged under 4000rpm.After discarding supernatant liquid, it is centrifuged again after being resuspended with the physiological saline after sterilizing, after cleaning twice, Thallus is deployed into bacteria suspension with physiological saline, makes its OD600=1.0.
(2) it the outfit of mixed bacteria liquid: weighs the sterilized sodium alginate of 3g and is dissolved in 75mL sterile water, stir evenly, obtain Sodium alginate soln.The sterilized active carbon of 2g is weighed in sodium alginate soln, stirs evenly, obtains mixed liquor.By step (1) prepared bacteria suspension pipettes 20mL in above-mentioned mixed liquor in, stirs evenly, obtains mixed bacteria liquid.
(3) it the preparation of immobilized microspheres: weighs the sterilized calcium chloride of 3.75g and is dissolved in 150mL sterile water, be prepared into The calcium chloride solution of 25g/L.The mixed bacteria liquid obtained in peristaltic pump pumps step (2), it is molten to be slowly dropped into calcium chloride In liquid, immobilized microspheres are prepared in crosslinking afterwards for 24 hours, clean with distilled water flushing, are put it into spare in 4 DEG C of refrigerator.
Immobilized microspheres balling-up effect is good, and form is uniform, and photo is as shown in figure 3, be completely used for following degradation experiment.
The immobilized microspheres prepared in free bacteria suspension and the present embodiment are tested respectively to Phthalate Esters In Soil Degradation effect, if blank control.
Every part weigh 200g contaminated soil sample (phthalic acid ester be DBP and DEHP, DBP and DEHP content distinguish It is 100mg/kg and 100mg/kg) in brown sample bottle.Free bacterium group: the bacterium that OD600=1.0 is added into pedotheque is outstanding Liquid 20mL (bacterial suspension inoculation amount is 10%);Immobilized microspheres group: be added into pedotheque it is above-mentioned be prepared it is all Immobilized microspheres;Blank control group: free bacterium and immobilized microspheres are not added.Sterile deionized water is added into each group again, makes Soil moisture content is the 60% of maxmun field capacity, and dark culture in 25 DEG C of biochemical cultivation cases is put into after sealing, is being cultivated respectively Sampling is analyzed when by the 3rd, 5,10 day.
By indoor culture in 10 days, in applying immobilized microspheres processing group, the degradation rate of DBP and DEHP were respectively reached 94.0% and 34.5%;In contrast, the degradation rate of blank control group DBP and DEHP is respectively 27.9% and 5.8%, free bacterium For processing group between two groups, the degradation rate of DBP and DEHP respectively reach 91.2% and 25.4% (Fig. 4~Fig. 5).Illustrate this hair Bright immobilized microspheres can significantly improve the degradation rate to PAEs, especially to the degradation rate of DEHP.
It should be understood that for those of ordinary skills, it can be modified or changed according to the above description, And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
SEQUENCE LISTING
<110>Qingdao Agricultural University, Ningxia University
<120>a kind of immobilized microspheres and its preparation method and application of PAEs degradation bacteria
<130> 2017
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1447
<212> DNA
<213> Pseudomonas putida
<400> 1
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ggtctgagag gatgatcagt cacactggaa ctgagacacg gtccagactc ctacgggagg 300
cagcagtggg gaatattgga caatgggcga aagcctgatc cagccatgcc gcgtgtgtga 360
agaaggtctt cggattgtaa agcactttaa gttgggagga agggcagtaa gctaatacct 420
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tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg tcaactagcc 780
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acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg 900
tggtttaatt cgaagcaacg cgaagaacct taccaggcct tgacatgcag agaactttcc 960
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gaagtcg 1447

Claims (10)

1. a kind of preparation method of the immobilized microspheres of PAEs degradation bacteria, which is characterized in that specific step is as follows:
(1) preparation of bacteria suspension: PAEs degradation bacteria culture to logarithmic growth phase after being cleaned thallus with physiological saline, then is used Thallus is deployed into bacteria suspension by physiological saline, makes the OD of bacteria suspension600=0.8-1.2;The PAEs degradation bacteria is Pseudomonas putida Bacterium (Pseudomonas putida) RXX-01, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms The heart, the deposit date is on November 1st, 2016, deposit number was CGMCC No.13224;The physiological saline is the 0.9% of sterilizing NaCl solution;
(2) outfit of mixed bacteria liquid: sodium alginate soln is prepared with sterile water, active carbon is added in sodium alginate soln and is mixed It is even that mixed liquor, the bacteria suspension that step (1) is added into mixed liquor mix to obtain mixed bacteria liquid;The difference of weight ratio shared by each component Are as follows: bacteria suspension 15-25%, sodium alginate 2-3%, active carbon 2-4%;
(3) preparation of immobilized microspheres: the calcium chloride solution of 20-30g/L is prepared with sterile water, and mixed bacteria liquid is instilled dropwise In calcium chloride solution, immobilized microspheres are prepared after being crosslinked 20-30h;The volume ratio of mixed bacteria liquid and calcium chloride solution is 1: 1.5~1:2.
2. the preparation method of the immobilized microspheres of PAEs degradation bacteria according to claim 1, which is characterized in that mixed bacteria liquid Weight ratio shared by each component is respectively as follows: bacteria suspension 20%, sodium alginate 3%, active carbon 2%;It is used to prepare immobilized microspheres The concentration of calcium chloride solution is 25g/L.
3. the preparation method of the immobilized microspheres of PAEs degradation bacteria according to claim 1, which is characterized in that the step (1) culture medium of pseudomonas putida RXX-01 is beef-protein medium, component are as follows: beef extract 3g, peptone in 10g, sodium chloride 5g, adds water to be settled to 1000mL.
4. the preparation method of the immobilized microspheres of PAEs degradation bacteria according to claim 1, which is characterized in that the step (1) condition of culture of pseudomonas putida RXX-01 in are as follows: 25-30 DEG C, the training of pH6.5-7.5,150-200r/min isothermal vibration Support 24-72h.
5. the preparation method of the immobilized microspheres of PAEs degradation bacteria according to claim 1, which is characterized in that the step (1) method of cleaning thallus in are as follows: the culture solution of pseudomonas putida RXX-01 is centrifuged 5-15min in 3500-4500rpm, Supernatant is abandoned, after precipitating is resuspended with the physiological saline of sterilizing, step 1-3 times of repeated centrifugation and resuspension precipitating.
6. the preparation method of the immobilized microspheres of PAEs degradation bacteria according to claim 1, which is characterized in that the step (3) mixed bacteria liquid is instilled in calcium chloride solution using peristaltic pump in, flow control is in 6mL/min.
7. the preparation method of the immobilized microspheres of PAEs degradation bacteria according to claim 1, which is characterized in that the step (3) immobilized microspheres prepared are rinsed well with sterile distilled water, 4 DEG C of preservations.
8. the immobilized microspheres for the PAEs degradation bacteria that any the method for claim 1-7 is prepared.
9. application of the immobilized microspheres of PAEs degradation bacteria described in claim 8 in degradation agricultural land soil phthalic acid ester.
10. the application of the immobilized microspheres of PAEs degradation bacteria according to claim 9, which is characterized in that the PAEs drop Solving dosage of the immobilized microspheres of bacterium in agricultural land soil of degrading when phthalic acid ester is 0.1-1kg/kg dry ground.
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Publication number Priority date Publication date Assignee Title
CN108085312A (en) * 2017-12-08 2018-05-29 沈阳化工大学 A kind of preparation method of environment-friendly type organic wastewater biological cleanser
CN108408920A (en) * 2018-03-15 2018-08-17 湖南大学 A method of utilizing Penicillium notatum composite degradation malachite green wastewater
CN108410771A (en) * 2018-04-08 2018-08-17 宁夏大学 The application of Rhodococcus sp YC915 and its absorption microbial inoculum in soil phthalic acid ester of degrading
CN109112082A (en) * 2018-07-31 2019-01-01 宁夏大学 The application of arthrobacterium NX917 and its absorption microbial inoculum in phthalic acid ester contaminated soil remediation
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CN109942098A (en) * 2019-03-26 2019-06-28 华南理工大学 A kind of phthalic acid ester and/or antibiotic contaminating microorganisms preparation for repairing and its preparation and application
CN110252786A (en) * 2019-05-20 2019-09-20 上海市农业科学院 Application of the earthworm in degradation DEHP

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104531576A (en) * 2014-12-19 2015-04-22 东北农业大学 Strain for degrading dibutyl phthalate
CN105063006A (en) * 2015-07-16 2015-11-18 东北农业大学 Dibutyl phthalate (DBP) degrading bacteria immobilizing microsphere, and preparation method and application thereof
CN105861365A (en) * 2016-04-19 2016-08-17 黑龙江八农垦大学 Pseudomonas sp. LD23 and preparation of immobilized microspheres of Pseudomonas sp. LD23

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104531576A (en) * 2014-12-19 2015-04-22 东北农业大学 Strain for degrading dibutyl phthalate
CN105063006A (en) * 2015-07-16 2015-11-18 东北农业大学 Dibutyl phthalate (DBP) degrading bacteria immobilizing microsphere, and preparation method and application thereof
CN105861365A (en) * 2016-04-19 2016-08-17 黑龙江八农垦大学 Pseudomonas sp. LD23 and preparation of immobilized microspheres of Pseudomonas sp. LD23

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Genes in PHT plasmid encoding the initial degradation pathway of phthalate in Pseudomonas putida;YasutoshiNomura等;《Journal of Fermentation and Bioengineering》;19921231;第74卷(第6期);全文 *
Phthalate Degradation in Pseudomonas putida;YASUTOSHI NOMURA等;《Annals of the New York Academy of Sciences banner》;19901231;全文 *
一株邻苯二甲酸二( 2-乙基己基) 酯( DEHP)高效降解菌的筛选及其降解特性;高静静等;《环境化学》;20161115;第35卷(第11期);全文 *
固定化微生物处理邻苯二甲酸酯类废水的研究;颜婷婷;《中国优秀硕士学位论文全文数据库》;20080915;全文 *

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