CN101067115A - Process of screening microbe strain for repairing As polluted soil - Google Patents

Abstract

The process of screening microbe strain for repairing As contaminated farm soil and vegetation includes screening As tolerant microbe strains; successively extraction via analyzing the capacity of these microbe strains in changing the form of As inside the gamma-ray sterilized As contaminated soil and in generating indolacetic acid (IAA) to promote plant growth, so as to screen out microbe strains capable of changing the content of bioavailable As in the soil; and pot culture experiment for verifying the efficiency of these microbe strains in favoring plant growth and increasing As absorbing amount of plant. The process of the present invention is simple, environment friendly, without secondary pollution and low in cost.

Description

A kind of screening method that is used for the microorganism strains of As contaminated soil reparation

One, technical field

The invention belongs to microbe to screen field in the biotechnology, particularly a kind of screening method that is used for the microorganism strains of As contaminated soil biological restoration.

Two, background technology

Prior art: the reparation research of heavy-metal contaminated soil just is being subjected to extensive concern both domestic and external, and the many especially focuses of being paid close attention to of biological restoration heavy-metal contaminated soil are no exception to the As contaminated soil.Arsenic is more valued pollutent in the environment, and after Bangladesh caused a large amount of crowd's arseniasiss owing to Arsenic Contamination in Groundwater, the research of arsenic contamination obtained the extensive concern of international academic community.Many researchs think that the food chain transmission is that human body is exposed to one of main path of arsenic.For south east asia is the country of staple food with rice, and paddy rice is an important environmental problem that is related to HUMAN HEALTH to the absorption and accumulation of arsenic., because mining activities and natural cause (groundwater pollution) also cause large-area arsenic in soil to pollute local and even regional food safety and HUMAN HEALTH are constituted a serious threat in China.

Many microorganisms are composed heavy metal element (Francis and Dodge, 1988 that are stored in the unavailability form in the mineral by the metabolism dissolving in the soil; Cantafio et al., 1996; Kalinowski et al., 2000), thereby important effect (Gadd, 1990 are being brought into play in the migration in soil to heavy metal; Idris etal., 2004).As mainly exists with arsenate [As (V)] and arsenite [As (III)] form in physical environment usually, and in well-oxygenated environment, As is based on As (V); In reducing environment, As is based on As (III) (Keon et al., 2001).Some microorganism strains can use As electron acceptor(EA) or donor, thereby oxidation or reduction As, change the valence state of As in the As pollution medium, as oxidation As (III) (Thermusaquuaticus, T.thermophilus) (Gihring et al., 2001), (Thiobacillus sp.S1, Ancylobacter sp.OL1) (Rhine et al., 2005), promptly As (III) is changed into As (V), the toxicity of As (V) is low than As (III).The arsenic compound kind that exists in the soil is more, be stored in the mineral but mainly compose in the mode of anionic group, and main the tax is stored in the mineral such as Fe, Al (Wenzel et al., 2001).And the As content of bioavailability is not high usually in the As contaminated soil, when using super enrichment As plant original position to repair the As contaminated soil, has limited the efficient (Meharg andHartley-Whitaker, 2002) of As in the super enriching plant absorption soil.Thereby, be necessary to seek appropriate, the assimilated efficiency when eco-friendly technique means changes phytoremediation As contaminated soil.

Report that the short bacterial strain of giving birth to of plant-growth can help plant to obtain key element (Egamberdiyeva and Hflich, 2004 under the unbalanced condition of rhizosphere nutrition; Belimov et al., 2005), and can significantly promote plant in heavy-metal contaminated soils such as Ni, Pb, Zn, Cd, Cu, grow (Burd et al., 1998,2000; Grichko et al., 2000; Nies et al., 2002; Vivas et al., 2006), but the bacterial strains that use in these researchs normally screen from the concentration that can tolerate heavy metal, are difficult to take into account the bacterial strain that filters out and whether one promote the more heavy metal of plant absorbing surely.In addition, the research that relates to As in the work of this respect does not also almost have.

Existing studies show that, microorganism can influence phytomorph, physiology and nutrition (Rovira, 1965) in the soil, and thinks and produce indolylacetic acid (indole acetic acid, 1AA) waiting growth hormone is that microorganism is beneficial to one of major reason of plant-growth (Brows, 1972).Therefore, under As coerced environment, the bacterial strain that screening can be produced IAA may be beneficial to plant-growth.

From above data as can be seen: at present, when screening is applied to the microorganism strains of heavy-metal contaminated soil phytoremediation, usually be to screen from the angle of bacterial strain tolerance heavy metal concentration, this method is difficult to take into account bacterial strain and whether can helps characteristic that plant grows in Heavy-metal Polluted Environment, whether can promote more high-load heavy metal of plant absorbing and remediation efficiency more do not have a kind of microorganism strains screening method that is suitable for phytoremediation As contaminated soil.

Three, summary of the invention

The present invention is directed to the problem that exists in the present bacterial strain screening, a kind of screening method that is used for the microorganism strains of As contaminated soil biological restoration is proposed, this method can filter out and help plant-growth, also can promote plant efficient ground to absorb more As, phytoremediation As is provided the efficient of contaminated soil.

Technical solution of the present invention is: a kind of screening method that is used for the microorganism strains of As contaminated soil biological restoration, the screening step is: the plant rhizosphere soil of mixing directly inserts in bacterium sterilization, that contain As and the fungi liquid substratum, substratum shaking table under 28 ℃, 120r/min condition is cultivated a week, switching once weekly, 1% inoculum size with the fresh liquid culture volume inserts in the fresh culture, so repeats five times; Then, with gradient dilution method screening bacterial strain, wherein on solid medium, bacterium adopts the line partition method to separate, and fungi adopts the point-like method to separate, and obtains the single bacterium colony of pure bacterium and fungi; Above-mentioned bacterial liquid substratum is a beef-protein medium, its pH value is adjusted to the scope of 7.0-7.2 with NaOH solution; The fungi liquid substratum is the Ma Dingshi substratum, regulates the pH value to about 6.5 with NaOH solution; Also be added with Na in above-mentioned two kinds of liquid nutrient mediums ₂HAsO ₄, make that the As concentration range of two kinds of substratum is 50-1000mg/kg, filter out the different bacterial strain of anti-As degree; In liquid nutrient medium, add 2.0% agar and can obtain corresponding solid medium; This step will filter out multiple, anti-As degree different bacterium and fungal bacterial strain.To go up step gained list bacterium colony is inoculated in respectively and does not contain Na ₂HAsO ₄The liquid beef-protein medium and liquid Ma Dingshi substratum in cultivate, again will above-mentioned two kinds of nutrient solutions respectively 4 ℃ down with whizzers with 8000r/min centrifugal after, the supernatant liquor that inclines, the sterile distilled water washing precipitate is all with absorbance value OD ₆₀₀=0.5 is benchmark, makes the bacteria suspension of bacterium and fungi with sterilized water; The inoculation bacteria suspension is in beef-protein medium (not adding As), what add that gamma-radiation sterilized contains As soil 5g again, after cultivating 10d, solution is poured in the beaker, measure nutrient solution pH, use in the distilled water wash triangular flask residue in beaker, to dry to weight for 60 ℃ again, take by weighing the 1g residue and carry out the analysis of As form, filter out the bacterium and the fungal bacterial strain that can change As form in the soil by the stage-by-stage extraction method; This step from the bacterial strain that filters out, further filter out simplely can remarkably influenced the bacterium and the fungal bacterial strain of As and mineral combination in the soil, and can understand bacterial strain and act on which kind of As structure in the soil.Bacterium, fungal bacterial strain that the last step was filtered out are inoculated in respectively and do not contain Na ₂HAsO ₄7H ₂Cultivate in the liquid beef-protein medium of O and the liquid Ma Dingshi substratum, use whizzer centrifugal under 8000r/min respectively above-mentioned two kinds of nutrient solutions, directly add in the 3mL pH=7.5 phosphate buffered saline buffer (glucose that contains mass concentration 1%) in the gained throw out of centrifugal back, after adding the L-tryptophane of 2mL mass concentration 1% again, cultivate 24h down at 37 ℃, after cultivating end, add 2mL mass concentration 5% trichoroacetic acid(TCA) and 1mL 0.5M CaCl ₂Filter, shift out 3mL filtrate in measuring pipe, Xiang Guanzhong cultivates 30min after adding 2mL Salper solution under 25 ℃ dark condition, cultivate and finish the back with measuring indoleacetic acid content under the 722 type spectrophotometer wavelength 535nm, typical curve uses the scope of indolylacetic acid 0-20mg/L, measures the amount that bacterial strain produces IAA, thereby filters out bacterial strain, the fungal bacterial strain that is beneficial to plant-growth; From the bacterial strain of this screening, can pick out to take into account in step and change the biological bacterial strain that can utilize attitude As content in the soil, can take into account As again and coerce under the environment and can be beneficial to plant strain growth.Pot experiment is carried out in bacterial strain, each strain of fungi that the last step filters out, the influence of plant absorbing As content and efficient when verifying each bacterial strain to phytoremediation nature As contaminated soil.The bacterial strain that this step filters out promptly can be used in the biological restoration of actual As contaminated soil.

The present invention compared with prior art, the bacterial strain that filters out can change As and mineral combination in the soil, makes that the transport property of As strengthens in the soil, and promotes plant-growth by producing IAA, strengthens the absorption of plant to As.When the bacterial strain that the objective of the invention is to filter out is used for phytoremediation As contaminated soil, can help plant-growth, more can promote plant efficient ground to absorb more As, improve the efficient of phytoremediation As contaminated soil.

Four, description of drawings

Fig. 1 is the technological line figure of screening bacterial strain;

Fig. 2,3 is the change of inoculating strain to As form in the soil, and the As morphological differences reaches 5% conspicuous level in the different letter representation different vaccinations processing soil; The As of non-obligate ADSORPTION STATE is representing can be by biological utilisation in the soil, the As form of tool environmental risk, and also representing the As that contains in the soil solution, lower usually (the Wenzel et al. of the As content of this form in soil, 2001,2002), as shown in Figure 2, inoculated bacteria B1, B2, B3 and the true F1 of inoculation, F2, F3 all significantly promotes the rising (p＜0.05) of non-obligate ADSORPTION STATE As content in the soil, B1, B2, it is respectively 3.2 of CK that B3 handles, 4.5,3.4 doubly, F1, F2, it is respectively 4.4 of CK that F3 handles, 4.7,3.7 doubly, promptly inoculating strain has improved bioavailability As content in the soil.Obligate ADSORPTION STATE As content is consistent with the variation tendency of non-obligate ADSORPTION STATE As, inoculation is handled all the significantly rising (p＜0.05) of the non-obligate ADSORPTION STATE As content of promotion (Fig. 3), compare with CK, B1, B2, B3 improve the As content 47%, 48%, 31% of this form in the soil respectively, and F1, F2, F3 then are respectively 26%, 36%, 18%.

The IAA content that Fig. 4 produces for the different strains metabolism; Different letter representation different strains produce the IAA content difference and reach 5% conspicuous level; The IAA content that B1, B2, F1, F2 produce is seen Fig. 6, and it is significantly the highest that B2 handles the IAA content that produces, and is 3.6 times that B1 handles; And the IAA content that the F1 processing produces is significantly higher than the F2 processing equally.As seen, induce by the L-tryptophane, the two strain bacteriums, two fungal strains that filter out can both produce IAA.Brows (1972) research thinks that rhizospheric microorganism can produce the material that promotes plant-growth, as IAA, gibberic acid etc., and thinks that plant has different roots and the overground part form may be relevant with the metabolic IAA of plant rhizosphere microbe.As these inoculation are arrived the plant rhizosphere, will help plant and grow, so these bacterial strains should belong to the short microorganism strains of giving birth to of rhizosphere in this research this coercing in the environment of high As contaminated soil.

As shown in Figure 5, different letter representation different vaccinations processing Herba pteridis vittatae biomass differences reach 5% conspicuous level; The inoculation microbial inoculum all promotes the increase of Herba pteridis vittatae overground part and underground part biomass to some extent.Compared with the control, Herba pteridis vittatae underground part and the upperground part biomass that inoculation F2 handles are significantly higher than contrast, and bacterial strain B1 all has promoter action to Herba pteridis vittatae overground part and underground part biomass, but not significantly (p＜0.05).Help plant strain growth when as seen, inoculation B1 and F2 repair the As contaminated soil to Herba pteridis vittatae.

Fig. 6 is inoculation B1, the F2 influence to Herba pteridis vittatae absorption As concentration, and different letter representation different vaccinations are handled Herba pteridis vittatae As concentration difference and reached 5% conspicuous level; Compared with the control, Herba pteridis vittatae overground part and underground part As concentration that inoculation is handled all are higher than contrast, and especially to the promoter action more remarkable (p＜0.05) of Herba pteridis vittatae overground part As concentration, the overground part As concentration that B1, F2 handle is respectively 2.3,1.9 times of contrast.

Fig. 7 is inoculation B1, the F2 influence to Herba pteridis vittatae absorption As content, and different letter representation different vaccinations are handled Herba pteridis vittatae As content differences and reached 5% conspicuous level; Compared with the control, inoculation B1, F2 have all significantly promoted the As content (p＜0.05) that overground part and underground part absorb, and especially overground part As content B1, F2 processing is respectively 2.5,2.7 times that contrast.Herba pteridis vittatae is super enrichment As plant, the As that excess absorbs mainly is transported to overground part, and during to As or other heavy-metal contaminated soil phytoremediation, that wishes most is transported to the plant overground part to the pollutent in the soil by plant exactly, gathers in overground part then so that remove Pollutant levels in the soil.Bacterial strain B1, the F2 of screening all promote the increase of the As content that the Herba pteridis vittatae overground part absorbs in this research, and especially in high density As contaminated soil, inoculating strain is more remarkable to the promoter action that the Herba pteridis vittatae overground part absorbs As.

By above seven width of cloth accompanying drawings as can be seen:

(1) singly meets B1, B2, B3, F1, F2, F3 in containing As soil, all significantly promote the rising of non-obligate and obligate ADSORPTION STATE As content in the soil, improved the bioavailability of As in the soil.

(2) the three strain bacteriums, three fungal strains that filter out can both produce IAA.

(3) Shai Xuan bacterial strain B1, F2 all significantly promotes Herba pteridis vittatae underground part and overground part to absorb the increase of As content, and inoculating strain is more remarkable to the promoter action that the Herba pteridis vittatae overground part absorbs As, and it is respectively 2.5,2.7 times that contrast that B1, F2 handle.

Five, embodiment

Embodiment 1:

1.1 for examination soil

The soil of isolated strains is taken near the vegetable plot, As mining area, Shimen County, Hunan Province, and soil type is a yellow earth, and soil physico-chemical property is: pH 4.20 (soil ratio, 2.5: 1), and total As 412mg/kg, total P 0.45%, Al 7.8%, and Fe 4.65%.

Soil behind the natural air drying ground 100 order nylon mesh, adopted ⁶⁰The Co sterilization, the disinfectant dose rate is 1.138kGy/h, tyndallization twice, total dose is 25kGy.Soil after the sterilization is kept in-80 ℃ of refrigerators.

1.2 strains tested

Screening and the sepn process of bacterium and fungi are as follows: the 100mL that the rhizosphere of 5g mixing soil directly inserts 121 ℃ of moist heat sterilizations contains in the bacterium and fungi culture medium of 500mg/kg As, and 28 ℃, 120r/min shaking table are cultivated a week.Switching once inserts in the fresh culture with 1% inoculum size of fresh liquid culture volume weekly later on, so repeat five times, and gradient dilution method screening bacterial strain, bacterium adopts the line partition method to separate, and fungi adopts the point-like method to separate, and obtains pure single bacterium colony.Beef-protein medium (bacterium): extractum carnis 3g/L, peptone 5g/L, regulate the scope (Nanjing Soil Inst., Chinese Academy of Sciences microbial room write, 1985) of pH value to 7.0-7.2 with NaOH solution.Ma Dingshi (Martin) substratum (fungi): MgSO ₄7H ₂O0.5g/L, KH ₂PO ₄1.0g/L, glucose 10.0g/L, peptone 5.0g/L, regulate pH value to 6.5 (microbial room of Nanjing Soil Inst., Chinese Academy of Sciences writes, 1985) with NaOH solution.Add Na2HAsO respectively in above-mentioned two kinds of substratum ₄, make that containing As concentration in the substratum is 500mg/kg.In liquid nutrient medium, add 2.0% agar get final product corresponding solid medium.

1.3 test design

This test set handling is as follows: B1, F1 and 1 contrast of not inoculating (CK), amount to 3 processing, and each is handled and repeats 3 times, amounts to 9 bottles.Inoculate the bacteria suspension 2mL that configures respectively and (do not add Na in a 50mL extractum carnis albumen substratum is housed ₂HAsO ₄) triangular flask in, the same 2mL sterilized water that adds in the control treatment of not inoculating (CK) is to guarantee culture solution volume unanimity.After inoculation finishes, take by weighing 5g ⁶⁰Soil behind the Co-γShe Xianmiejun is cultivated after 10 days in above-mentioned nutrient solution, and solution is poured in the beaker, measures pH, and residue is dried to weight for 60 ℃ in beaker in the distilled water wash triangular flask, takes by weighing the analysis that the 1g pedotheque carries out the As form.

1.4 test event

The As form adopts improved stage-by-stage extraction method (Wenzel et al., 2001) in the soil: 1. Ass, non-obligate ADSORPTION STATE, 0.05mol/L (NH ₄) ₂SO ₄, 20 ℃/4h; 2. Asp, obligate ADSORPTION STATE, 0.05mol/LNH ₄H ₂PO ₄, 20 ℃/16h; 3. Aso, amorphous hydration Fe, Al oxidation states of matter, 0.2mol/L (NH ₄) ₂C ₂O ₄, 3.25,20 ℃/4h of pH (under the dark condition); 4. Asc, the hydration Fe of crystalline state, Al oxidation states of matter, 0.2mol/L (NH ₄) ₂C ₂O ₄+ xitix, 3.25,96 ℃/0.5h of pH (with illumination).

The mensuration of indolylacetic acid (IAA) (Vivas et al., 2006): the bacterium, the fungi bacterium liquid centrifugal 8min under 8000r/min that cultivate 24h.Directly in the phosphate buffered saline buffer (containing 1% glucose) of adding 3mL pH=7.5, the thalline of 2mL1%L-tryptophane solution after centrifugal, cultivate 24h down at 37 ℃.After cultivating end, add 2mL5% trichoroacetic acid(TCA) and 1mL0.5MCaCl ₂, filter, shift out 3mL filtrate in measuring pipe, add 2mLSalper solution (prescription: 2mL0.5MFeCl ₃Mix with 98mL35% perchloric acid).This solution is cultivated 30min under 25 ℃ dark condition, cultivate to finish the back under 535nm with 722 type spectrophotometric determinations.

The physico-chemical property of pedotheque: pH is with soil: water (no CO ₂)=1: 2.5 method is measured (Lu Rukun, 2002).Use HNO ₃-HF-HClO ₄After the digestion, ICP-MS measures total P.Adopt 1: 1 postdigestive solution example of chloroazotic acid and As morphological analysis sample all to adopt hydride Generation-Atomic Fluorescence Spectrometry (HG-AFS) to measure As.Select for use Chinese Academy of Geological Sciences's physical chemistry of the earth explore the accuracy of soil standard model (GBW07406) control analysis, the rate of recovery of P, As all is controlled in the limit of error.

1.5 data statistics

Adopt SPSS10.0 software processes testing data, the significance of difference between each processing of Duncan multiple comparisons.

Embodiment 2:

2.1 for examination soil

The soil of isolated strains is taken near the mound in As mining area, Cili County, Hunan Province, and soil type is lime (rock) soil, and soil physico-chemical property is: pH 5.97 (soil ratio, 2.5: 1), and total As 514mg/kg, total P618mg/kg, Al 3.18%, and Fe 2.77%.

2.2 test strain

The isolating method of strains tested for earlier from soil screening can produce the bacterial strain of IAA, screening can change the bacterial strain of As form in the soil from these bacterial strains again, concrete implementation step is with in 1.2.

2.3 experimental design

With 1.3.

The screening bacterial strain is in the soil As form influence experiment, and the soil of selecting for use is with among the embodiment 1 ⁶⁰The Co sterile soil is for supplying examination soil.

2.4 test event

With 1.4.

2.5 data statistics

With 1.5.

Embodiment 3:

3.1 for examination soil

The soil of isolated strains is taken near the mound of Hunan Province Guiyang County local method refining As plant area, and soil type is a red earth, and soil physico-chemical property is: pH 3.91 (soil ratio, 2.5: 1); Total As 373mg/kg; Total P225mg/kg, Al 2.64%, and Fe 1.76%.

3.2 strains tested

The isolating method of strains tested can produce the bacterial strain of IAA, and can change the bacterial strain of As form in the soil for screening from soil, and concrete implementation step selects the bacterial strain of best results with 1.2.

Above-mentioned isolated strains is through preliminary evaluation, and the bacterium that the bacterium of example 1,2 screenings is bacillus brevis genus (Brevibacillus), example 3 screenings is bacillus (Bacillus) (eastern elegant pearl etc., 2001), is designated hereinafter simply as B1, B2, B3; The fungi of example 1,2,3 screenings is respectively (Kang Shi) Trichoderma (Trichoderma), Fusarium (Fusarium), single capsule Erysiphe (Sphaerotheca) (Wei Jingchao, 1979), is designated hereinafter simply as F1, F2, F3.

3.3 test design

With 1.3.

The screening bacterial strain is in the soil As form influence experiment, and the soil of selecting for use is also with among the embodiment 1 ⁶⁰The Co sterile soil is for supplying examination soil.

3.4 test event

With 1.4.

3.5 data statistics

With 1.5.

Embodiment 4:

The B1 of screening, F2 bacterial strain absorb the effect of As content to Herba pteridis vittatae

4.1 materials and methods

4.1.1 test plant

Before Herba pteridis vittatae (Pteris vittata L) experimentizes, earlier spore is seeded in the moistening soil behind the moist heat sterilization, is transplanted to when waiting to grow about 2cm in the moistening soil after the same sterilization.The Herba pteridis vittatae seedling is long can to experimentize when high to about 5cm.

4.1.2 for examination soil

Soil for examination picks up from place, the foot of the hill, As minery, Shimen County, Hunan Province respectively.Reject top layer plant litter when gathering soil, get top layer 0～20cm soil, multiple spot is gathered mixing.Natural air drying is crossed the 2mm nylon mesh.

Table 1 is for examination soil essential property

The mensuration project	pH	C/N	As(mg·kg -1)	P(mg·kg -1)	Effective P (mgkg -1)
						Measured value	5.59±0.01	9.75±0.12	185.26±4.29	512±10	5.05±0.59

The basic physical and chemical of soil sees Table 1: it is more serious that place, the foot of the hill, mining area agricultural land soil is polluted by As, is national edatope 3 grade standard (40mg/kg ^-1) 4.63 times, and these soil are now being cultivated, as seen, the agricultural-food of planting in the soil in this district may constitute health threat to resident in distinguishing.

4.1.3 for the examination microbial inoculum

Two kinds of bacterial strains of B1, F2 are only selected in this test for use.The inoculation bacterial preparation process: inoculation B1, F1 (do not add Na respectively at an above-mentioned extractum carnis albumen training substratum and Ma Dingshi substratum ₂HAsO ₄7H ₂O), behind the cultivation 3d, at 4 ℃ of centrifugal bacterial strains of following 8000r/min, the supernatant of inclining is behind sterile distilled water washing bacterial strain three times, all with absorbance value OD ₆₀₀=0.5 is benchmark, makes bacteria suspension with sterilized water.

4.1.4 test design

Test is carried out in potted plant mode, and soil is unsterilised, and setting is treated to: inoculation B1, F2 and 1 contrast of not inoculating, and 3 repetitions are established in each processing, and totally 3 processing amount to 9 basins.The 1.5L plastic tub is adopted in test, and every basin is adorned native 1kg, inoculation bacteria suspension 20mL, and CK handles and waters 20mL distilled water, with the soil mixing.Every basin sowing moves 3 of Herba pteridis vittatae seedlings.All basin random alignment are watered distilled water in Nanjing Soil Inst., Chinese Academy of Sciences's heliogreenhouse, keep 80% of soil water retaining capacity.

14 week of Herba pteridis vittatae growth, the back gathered in the crops, and overground part and underground part are dried to constant weight in 60 ℃ respectively and taken by weighing dry weight respectively.After rhizosphere soil mixed, every basin was got the 100g soil sample, grinds 100 order nylon mesh behind the natural air drying, and sample retention is for measuring every index.

4.1.5 plant sample is measured

Plant sample HNO ₃: HCl=3: 1 carries out postdigestive sample, adopts hydride generator-atomic fluorescence spectroscopy (HG-AFS) to measure As.Plant sample adopts dense HNO ₃And HClO ₄After the digestion, inductively coupled plasma emission spectrometer (ICP-AES) is measured P.Select for use Chinese Academy of Geological Sciences's physical chemistry of the earth explore the accuracy of plant standard model (GBW07603) control analysis, each element measured value is all in accurate scope.

4.1.6 data statistics

Adopt SPSS10.0 software processes testing data, the significance of difference between each processing of Duncan multiple comparisons.

Claims (1)

1. screening method that is used for the microorganism strains that the As contaminated soil repairs is characterized in that screening step and is:

A. the fresh soil of plant rhizosphere with mixing directly inserts in the bacterium that contains As and fungi liquid substratum of sterilization, wherein, the bacterial liquid substratum is a beef-protein medium, its pH value is adjusted to the scope of 7.0-7.2 with NaOH solution, the fungi liquid substratum is the Ma Dingshi substratum, regulate the pH value with NaOH solution and to about 6.5, two kinds of liquid nutrient mediums, be added with Na ₂HAsO ₄Make that the As concentration of two kinds of substratum is 50-1000mg/kg, two kinds of substratum shaking table under 28 ℃, 120r/min condition is cultivated a week, and switching once weekly, 1～5% inoculum size with the fresh liquid culture volume during each switching inserts in the fresh culture, so repeats three～five times; Then, obtain pure bacterium and the single bacterium colony of fungi with gradient dilution method isolated strains;

B. will go up step gained list bacterium colony is inoculated in respectively and does not contain Na ₂HAsO ₄The liquid beef-protein medium and liquid Ma Dingshi substratum in cultivate, with two kinds of nutrient solutions respectively 4 ℃ centrifugal down after, the supernatant liquor that inclines is used the sterile distilled water washing precipitate, makes the bacteria suspension of bacterium and fungi again with sterilized water;

C. inoculate above-mentioned bacteria suspension in not containing Na ₂HAsO ₄Beef-protein medium in, add gamma-radiation sterilization As contaminated soil again, after cultivating end, solution is poured in the beaker, wash in the triangular flask residue with water in beaker, dry to weight, residue is carried out the analysis of As form, thereby filter out the bacterium and the fungal bacterial strain that can change As form in the soil by the stage-by-stage extraction method;

D. will go up the bacterium, the fungal bacterial strain that filter out of step is inoculated in respectively and does not contain Na ₂HAsO ₄The beef extract-peptone liquid nutrient medium and the Ma Dingshi liquid nutrient medium in cultivate, above-mentioned two kinds of nutrient solutions are centrifugal respectively, directly adding 3mL pH=7.5 in the gained throw out of centrifugal back contains in the phosphate buffered saline buffer of glucose of mass concentration 1%, after adding the L-tryptophane of 2mL mass concentration 1% again, cultivate 24h down at 37 ℃, after cultivating end, add 2mL mass concentration 5% trichoroacetic acid(TCA) and 1mL 0.5mol/LCaCl ₂Filter, shift out 3mL filtrate in measuring pipe, Xiang Guanzhong cultivates 30min after adding 2mL Salper solution under 25 ℃ dark condition, cultivate the end back and under with 722 type spectrophotometer wavelength 535nm, measure indoleacetic acid content, typical curve uses the scope of indolylacetic acid 0-20mg/L, measures the ability that bacterial strain produces IAA, thereby filters out bacterial isolates, the fungal bacterial strain that is beneficial to plant-growth.

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