CN101920262B

CN101920262B - Plasmide carried by Sphingobacterium sp DDT-6

Info

Publication number: CN101920262B
Application number: CN2010102768031A
Authority: CN
Inventors: 虞云龙; 高春明; 方华
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2010-09-09
Filing date: 2010-09-09
Publication date: 2012-06-06
Anticipated expiration: 2030-09-09
Also published as: CN101920262A

Abstract

The invention discloses a bioremediation method for soil contaminated by residual DDT, comprising the following steps: inoculating Sphingobacterium sp. DDT-6 or plasmid carried thereby or transformant containing the plasmid to soil with residual DDT, wherein the collection number of the Sphingobacterium sp. DDT-6 is CCTCC M 208116. The plasmid carried by the Sphingobacterium sp. DDT-6 has genes for degrading DDT, thus having the performance of degrading DDT-6, more importantly, the plasmid is transferable, and can be transformed into soil microorganisms and even possibly is subject to gene recombination with genes in soil microorganisms through inoculating the plasmid or strains containing the plasmid to the soil, thereby solving the problem that exogenous microorganisms are difficult to live in soil on one hand, and ensuring that degrading bacteria can be maximally dispersed to degrade the DDT as much as possible on the other hand.

Description

The plasmid that a kind of Sphingobacterium (Sphingobacterium sp.) DDT-6 carries

Technical field

The present invention relates to environmental protection technical field, relate in particular to a kind of DDT residual contamination soil organisms restorative procedure.

Background technology

Agricultural chemicals is one of primary pollution source of soil; Banned pesticides residual contaminations such as DDT; This residual level in the soil of certain areas is still high, and we detect DDT recall rate 100% in the soil of somewhere, and content is up to 1.19mg/kg; Great majority are at 0.46～0.93mg/kg, and low reaches between 0.14～0.29.Pesticide residual contamination constitutes harm to a certain degree to soil ecosystem in the soil, the more important thing is agricultural-food and Chinese medicinal materials are polluted, and is detrimental to health, and influences the foreign exchange earning of agricultural-food and Chinese medicinal materials.Therefore, research and develop the bioremediation technology and the product of forbidding such as DDT persistent pesticide residual contamination soil simultaneously, for ensureing soil environment quality and agricultural-food, Chinese medicinal materials safety, promote national economy healthy move significant.

Biological reinforced is the important means that promotes organic pollutant degradation, optimizes the geobiology function; With the contaminated soil biological prosthetic is the biological reinforced efficient degrading bacteria that mainly utilizes of purpose, and the efficient degrading bacteria that adopts usually comprises indigenous bacterium, external source bacterium and genetic engineering bacterium.Efficient degrading bacteria gets into contaminated soil and faces two problems: 1) owing to biological and abioticly coerce effect, the mikrobe of introducing is difficult to the adapted soil environment and keeps secular degrading activity; 2) degradation bacteria of survival is difficult to reach good extensively disperseing deeply in soil, has limited raising (Gentry etal., 2004 of whole degradation capability of soil and purification function; Moran et al., 2006).How to promote effectively that soil microorganisms is the key issue and the technical bottleneck of soil pollution biological prosthetic to the formation of contaminant degradation functional network.

Organic pollutant is mainly driven by mikrobe in degradation in soil; Soil bacteria adapts to and the molecular mechanism of degradation of contaminant comprises that gene level shifts (horizontal gene transfer), sudden change and DNA reorganization; Wherein the gene level transfer is important mechanisms (Ochman et al., 2000 that bacterial genomes and degradation function are evolved; Arber, 2000; Gogarten and Townsend, 2005; Lin Xiaoyan etc., 2005), Lawrence and Ochman (2002) point out that through sequential analysis Escherichia coli genome 27% shifts acquisition through gene level.Soil bacteria is positioned at host's IncP-1 plasmid (Nojiri et al., 2004) widely mostly to the degradation function gene of exogenous organic pollutant, like organic phosphorous insecticide degrading genes opd (Bhadbhade et al., 2002 on the plasmids such as pMCP424; Horne et al., 2002); Sterilant carbaryl hydrolase gene (Hashimotoet al., 2006) on the plasmid pRC1; Degrading genes atzABC, atrzD, atzBC (Rousseaux et al., 2002, Trefault et al., 2004 of the herbicide atrazine on the plasmids such as pJP4, pADP-1, pEST4011; Aislabie et al., 2005).The IncP-1 plasmid has the characteristics of transitivity and host's popularity; Can between soil bacteria, shift through approach such as conversion, joint and transductions; Like weedicide 2; 4-D dissimilation plasmid pJP4 can change Burkholderia, many kinds of soil bacterias of Pseudomonas over to from Escherichia coli ATCC 15224, and transformant reaches 10 ⁷-10 ⁸/ gram soil, the latter obtains 2, the 4-D degradation capability (Newby et al., 2000a); G-30027 dissimilation plasmid pADP1::Tn5 can change 4 kinds of Variovorax soil bacterias (Devers etal., 2005) over to from donor bacterium Agrobacterium tumefaciens; Gelder etc. (2005) detect r plasmid pB10 and are transferred to 15 genus from different donor bacterium Pseudomonas putida SM1443, Ralstonia eutropha JMP228, Sinorhizobium meliloti RM1021 (bacterium of α-and γ-Proteobacteria) is separated to 306 conversions altogether in active sludge.

Summary of the invention

The invention provides a kind of DDT residual contamination soil organisms restorative procedure, solved the problem that the traditional method mikrobe is difficult to adapted soil environment and difficulties in dispersion.

A kind of biological renovation method of DDT residual contamination soil comprises: Sphingobacterium (Sphingobacterium sp.) DDT-6 or its plasmid that carries or the transformant that comprises this plasmid are inoculated in the soil of residual DDT.

Sphingobacterium (Sphingobacterium sp.) DDT-6 is preserved in the Chinese typical culture collection center (CCTCC) that is positioned at Lopa Nationality an ancient woman's ornament mountain, Wuhan City Wuhan University; Preservation date is on August 5th, 2008; Preserving number is CCTCC M 208116, and the physiologic character of this bacterial strain and separation purification method are open in patent 200810120741.8.

The inventive method is applicable to all soil, and preferred, described soil pH value is 5～9; Described soil DDT content is at 0.1～2mg/kg; Described soil organic matter content 5～50g/kg.

Preferably, a kind of DDT residual contamination soil organisms restorative procedure specifically comprises: the bacterium liquid of sphingosine bacillus DDT-6 or said transformant is sprayed onto on the soil, and every square metre is sprayed thalline quantity is 5 * 10 ¹⁰～5 * 10 ¹²

Preferably, the host bacterium of said transformant is E.Coli TG1.

The present invention also provides the described Sphingobacterium of a kind of claim 1 (Sphingobacteriumsp.) plasmid that DDT-6 carries and the transformant that comprises this plasmid.

The plasmid that Sphingobacterium DDT-6 of the present invention carries has the gene of degraded DDT; Make it have the performance of degraded DDT; The more important thing is that this plasmid is transferable plasmid, through in soil, inoculating the bacterial strain that this plasmid perhaps contains this plasmid, this plasmid can be transformed in the soil microorganisms; Even possibly recombinate with gene in the soil microorganisms; Solved on the one hand the inoculating microbe difficult problem of in soil, survive, degradation bacteria can be disperseed to greatest extent on the other hand, and DDT as much as possible degrades.

Description of drawings

The Fluirescence observation figure that Fig. 1 shifts between soil microorganisms for DDT dissimilation plasmid in the shop experiment soil;

Fig. 2 respectively handles the degraded situation map (A:CK of DDT in the soil for field test; B:pDOD-gfp; C:Sphingobacterium sp.DDT-6 (pDOD-gfp); D:E.Coli TG1 (pDOD-gfp));

Fig. 3 is the DDT degradation rate of each bacterial strain and plasmid different time;

Fig. 4 is inoculated into the degradation bacteria quantity of different time behind the soil for each bacterial strain and plasmid.

Embodiment

Sphingobacterium (Sphingobacterium sp.) DDT-6 is preserved in Chinese typical culture collection center (CCTCC) on August 5th, 2008; Preserving number is CCTCC M 208116, and the physiologic character of this bacterial strain and separation purification method are open in patent 200810120741.8.

Substratum

Luria-Bertani substratum (LB substratum): yeast extract paste 5.0g; Peptone 10.0g; Sodium-chlor 10.0g; Zero(ppm) water 1000ml; PH7.0, and high pressure steam sterilization (121 ℃, 20min);

M substratum: K ₂HPO ₄3H ₂O 2.96g; KH ₂PO ₄0.87g; (NH ₄) ₂SO ₄1.1g; MgSO ₄0.097g; MnSO ₄H ₂O 0.025g; FeSO ₄7H ₂O 0.005g; CaSO ₄0.0015g; Xitix 0.005g; Zero(ppm) water 1000ml; PH7.0, and high pressure steam sterilization (121 ℃, 20min);

Minimal medium: MgSO ₄7H ₂O 0.4g; FeSO ₄7H ₂O 0.02g; K ₂HPO ₄0.2g; (NH ₄) ₂SO ₄0.2g CaSO ₄0.08g; Zero(ppm) water 1000ml; PH7.0; High pressure steam sterilization (121 ℃, 20min);

The LB solid medium: in the LB substratum, add 20g agar, high pressure steam sterilization (121 ℃, 20min);

The M solid medium: in the M substratum, add 20g agar, high pressure steam sterilization (121 ℃, 20min).

Dissimilation plasmid and function thereof

DNA among the Sphingobacterium DDT-6 uses the Axygen DNA to extract test kit in a small amount and extracts; This test kit adopts improved SDS alkaline lysis; Prepare the optionally method fast purifying DNA of adsorption of DNA of film in conjunction with DNA, the size of the DNA that extracts and quality detect through 1% agarose gel electrophoresis.

Single bacterium colony of picking Sphingobacterium DDT-6 is in 3ml LB substratum, and shaking culture is spent the night in shaking table under 30 ℃ of conditions, gets 200 μ L bacterium liquid then, inserts respectively that to contain SP 15 Lemon Yellow concentration be 150mgL ^-1, 300mgL ^-1, 450mgL ^-15ml LB substratum in, 30 ℃ of shaking table shaking culture 24h are diluted to 10 ^-5, and coat on the LB solid medium, cultivate 24h for 42 ℃, in 30 ℃ of cultivation 24h, get several single bacterium colonies then and be diluted to 10 respectively again ^-5And coat on the LB solid medium, 30 ℃ of overnight cultures, the dibbling of picking list bacterium colony difference (contains p, p '-DDT 20mgL in LB solid medium and M solid medium again ^-1) correspondence position, cultivate 2-3d and observe growing state at two kinds of solid mediums.(contain p, p '-DDT 20mgL at the M solid medium ^-1) on the bacterium colony of not growing can tentatively be regarded as plasmid and eliminate bacterium.

Dissimilation plasmid DNA 20 μ L add (wherein Klebsiella sp.TST is a test materials, and no specificity requires) among competence E.Coli TG1, indigenous bacterium klebsiella (Klebsiella sp.) TST, shake up gently; Ice bath is placed 30min, places ice bath cooling 5min in 42 ℃ of water-baths behind the thermal shock 90s rapidly, adds 1ml LB liquid nutrient medium; Behind the mixing at 37 ℃ of shaking culture 1h; Make the bacterium state that restore normal growth, get respectively that 100 μ L coating M is dull and stereotyped (to contain p, p '-DDT 20mgL ^-1), cultivate 2-3d in 30 ℃ and observe the bacterial growth situation, screening transformant E.Coli TG1 (pDOD) and Klebsiella sp.TST (pDOD).

In the 100ml triangular flask of sterilization, add the 20ml minimal medium, the interpolation glucose concn is 50mgL ^-1, p, p '-DDT concentration is 20mgL ^-1, inoculate Sphingobacterium sp.DDT-6, E.Coli TG1, Klebsiella sp.TST, E.Coli TG1 (pDOD), Klebsiella sp.TST (pDOD) (bacteria concentration OD respectively ₆₀₀=0.7).At PH7.0,30 ℃, 150rmin ^-1Condition under shaking culture, timing sampling behind the 21d.Establish the contrast that does not add bacterium simultaneously, contrast and processing respectively are 3 repetitions.Each is handled DDTs degraded situation and sees table 1.

Table 1 different strains is to p, the degraded of p '-DDT

Table 1 result shows, do not have all to obtain degradation function after E.Coli TG1, the Klebsiella sp.TST of DDTs degradation function changes plasmid pDOD over to, and plasmid pDOD encoding D DTs degradation function is described.The structure of fluorescent mark plasmid pDOD-gfp

The structure of fluorescent mark plasmid pDOD-gfp adopts triparental mating; Concrete operations are following: single bacterium colony of Sphingobacterium sp.DDT-6, E.coli DH5 α (pZP201-gfp), E.coli HB 101 (pRK2013) is seeded in the 4ml LB liquid nutrient medium shaking culture 7h in 30 ℃ of shaking tables respectively; With Sphingobacterium sp.DDT-6:E.coli DH5 α (pZP201-gfp): E.coliHB101 (pRK2013)=1: 1: 1,1: 2: 1,2: 1: 1 three mixed; Take out 50 μ l mixed bacteria liquid to LB liquid nutrient mediums (p, p '-DDT 10mgL behind 150rpm, 30 ℃ of shaking table shaking culture 5h ^-1) in leave standstill and cultivate 24h, nutrient solution is diluted to 10 ^-3Insert sulfur acid kantlex and Spectinomycin HCL (Veterinary) (50mgL ^-1) LB nutrient agar flat board in cultivate, observe the colony growth situation behind the 24h.Utilize crossed resistance (table 2), fluorescent mark and degradation function to filter out Sphingobacterium sp.DDT-6 (pDOD-gfp).Adopt the method in " dissimilation plasmid and function thereof ", preparation pDOD-gfp, E.Coli TG1 (pDOD-gfp), Klebsiella sp.TST (pDOD-gfp).

The crossed resistance of table 2 Sphingobacterium sp.DDT-6, E.coli DH5 α (pZP201-gfp), E.coli HB101 (pRK2013)

Annotate: (+) positive; (-) feminine gender

What DDTs degraded in the soil under the laboratory condition is biological reinforced

Sphingobacterium (Sphingobacterium sp.) DDT-6, E.Coli TG1, Klebsiella sp.TST, Sphingobacterium sp.DDT-6 (pDOD-gfp), E.Coli TG1 (pDOD-gfp), Klebsiella sp.TST (pDOD-gfp) are respectively in the 100mlLB substratum, at 30 ℃, 150rmin ^-1Cultivate 24h under the condition, 8000rmin ^-1Centrifugal 5min abandons supernatant, cleans centrifugal 3 times with minimal medium, and is for use with the colony counting method counting.

Pedotheque picks up from the fresh soil sample of the 0-15cm in experimental plot, Zhejiang University Hua Jiachi school district, and (the soil main physical and chemical is seen table 3, removes impurity such as weeds tree root chad, crosses the 2mm sieve, and is air-dry.Each test takes by weighing dry ground and heavily is 1kg soil, adds p, and p '-DDT to 1ppm also adds plasmid (1mg/kg) or degradation bacteria, and bacterium is measured to 10 ⁵CFU/g; Cross 2mm sieve three times, make p, p '-DDT, bacterium and soil thorough mixing as far as possible; The Culture basin of packing into; Cover Culture basin (opening five aperture ventilations above that) with tinfoil paper, lucifuge cultivation maintenance soil moisture content is 60% of a water retaining capacity in 30 ℃ of incubators, and processing back 0d, 7d, 15d, 30d, 60d, 90d timing sampling are analyzed the DDT residual quantity and adopted laser confession focusing observation degraded in soil bacterium number change situation simultaneously.

Table 3 test soil physico-chemical property

Each handles p in the soil, and the degraded situation of p '-DDT is seen Fig. 3.Control soil, non-degradation bacteria E.ColiTG1 and Klebsiella sp.TST handled 120 days, p in the soil, and p '-DDT is almost constant, and its degradation rate is respectively 8.7%, 9.6% and 9.2%.Plasmid pDOD-gfp handled 120 days, p in the soil, and the degradation rate of p '-DDT reaches 66.7%.Because dissimilation plasmid only just shows degradation function after changing mikrobe acquisition expression over to, therefore, plasmid pDOD handles p in the soil, and the degraded of p '-DDT comes from soil microorganisms and changes the degradation function that obtains because of plasmid pDOD-gfp over to.

As shown in Figure 1; Soil was obviously observed the gfp microorganisms marked in 15 days after plasmid pDOD-gfp handles; Adopt the same period MPN method to record DDT degradation bacteria quantity and show that soil detected the existence of degradation bacteria in 7 days after plasmid pDOD-gfp handles, progressively increase subsequently (like Fig. 4, unit is CFU/g).The result shows after plasmid pDOD-gfp handles soil can change soil microorganisms formation degradation flora over to, thereby improves p in the soil, the degraded of p '-DDT.Degradation bacteria Sphingobacterium sp.DDT-6 (pDOD-gfp), E.Coli TG1 (pDOD-gfp) handle soil and are similar results (Fig. 3, Fig. 4, Fig. 1), handle indifference at degradation rate and degradation bacteria quantitative aspects with direct plasmid.Consider that plasmid extracts time-consuming, time-consuming, it is more economical, convenient in practical application, to adopt plasmid donor (like Sphingobacteriumsp.DDT-6 (pDOD-gfp), E.Coli TG1 (pDOD-gfp)) inoculation to handle.

The DDTs degraded is biological reinforced in the field soil

Cixi City An Dong town carries out in the vegetables base in Zhejiang Province in test, and the experimental plot is divided into 12 sub-districts, and the area of each sub-district is 2.0 * 5.0m ², establish Sphingobacterium sp.DDT-6 (pDOD), Sphingobacterium sp.DDT-6 (pDOD-gfp), E.coli TG1 (pDOD-gfp) respectively and spray processing, 100ml 5.0 * 10 ¹⁰The bacterium liquid of CFU/ml adds 4Kg water to and is sprayed onto Tanaka, sprays bacterium liquid again one time with same bacterium amount and method behind the 15d, spray for the first time before the bacterium with the spray bacterium after DDTs residual quantity (Fig. 2) in 15,30,90,120 days sampling analysis soil.The initial residual amount of DDT is 0.79mg/kg in the control treatment soil, and residual quantity is 0.73mg/kg after 90 days, and degradation rate is merely 7.6%; The degradation rate that Sphingobacterium sp.DDT-6 (pDOD), Sphingobacterium sp.DDT-6 (pDOD-gfp), E.coli TG1 (pDOD-gfp) handle DDT in the soil of back significantly improves, and degradation rate is respectively 33.8%, 42.5% and 40.7%.The field test results explanation; Sphingobacterium sp.DDT-6 (pDOD), Sphingobacterium sp.DDT-6 (pDOD-gfp), E.coli TG1 (pDOD-gfp) microbial inoculum are handled soil and all can effectively significantly be accelerated the residual degraded of DDT in the soil, are a kind of effective biological renovation methods.

Claims

1. the plasmid that carries of a Sphingobacterium (Sphingobacterium sp.) DDT-6, described Sphingobacterium DDT-6 preserving number is CCTCC M 208116.

2. one kind contains the transformant that right requires 1 described plasmid.