CN105543153A - Method for screening rhizosphere bacteria promoting heavy metal phytoremediation - Google Patents
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Abstract
The invention discloses a method for screening rhizosphere bacteria promoting heavy metal phytoremediation. The method comprises a sample collecting operation and pretreatment operation process, an enrichment treatment process, a primary screening operation process, a separation treatment process and a secondary screening operation process. In the enrichment bottle manufacturing process in enrichment treatment, an adsorption carrier, a heavy metal compound, nutrient broth and nystatin need to be added in a reagent bottle. By adding the adsorption carrier, most strains easy to adsorb can be adsorbed to the adsorption carrier, and thus treatment on solid substances and extraction and separation of the strains in a later stage can be facilitated; by adding the heavy metal compound, a food preference environment for the bacteria is formed in the enrichment bottle, and presentation of the feature that the bacteria adsorb the heavy metal compound is promoted. The method is a novel method for directly and effectively screening the rhizosphere bacteria promoting heavy metal phytoremediation; by means of the method, the screening efficiency is improved, and thus the screened-out rhizosphere beneficial bacteria with the adsorption feature have higher treatment capability on heavy metal pollution after being inoculated to plants.
Description
Technical field
The present invention relates to Bio-remediation of Heavy Metals Contaminated Soils field, be specifically related to a kind of method of screening promotion heavy metal phytoremediation rhizosphere bacteria.
Background technology
Along with industrial expansion, water pollutions, soil pollution, topsoil are on the rise, wherein, the heavy metal contamination caused due to human factors such as mining, exhaust gas emission, sewage irrigation and use heavy metals exceeding standard goods causes comparatively serious harm because it is difficult to process to HUMAN HEALTH.
Heavy metal is different from Organic pollutants, is difficult to be degraded by microorganisms, and one of approach of restoration of soil polluted by heavy metal is absorbed by super enriching plant absorption.But owing to there is complicated relation between the microorganism in root system of plant environment, root system, soil enzyme, metal ion, so useful rhizospheric microorganism need be filtered out from root system of plant and surrounding enviroment thereof.On the one hand, useful rhizospheric microorganism can promote the growth of plant, contribute to improving the remediation efficiency of plant.As scientist is inoculated in super enriching plant Herba pteridis vittatae the VA Mycorrhizal Fungi be separated from contaminated soil, found that the inoculation of VA Mycorrhizal Fungi significantly increases plant to measuring containing As in the absorption of As and plant.On the other hand, useful rhizospheric microorganism also has the resistance of heavy metal toxicity, can adapt to Heavy-metal Polluted Environment better, and can not cause secondary pollution to the ecosystem.Therefore, screening promotes that the rhizosphere bacteria of heavy metal phytoremediation is a key areas of remediation of heavy metal-contaminated soil.At present, traditional screening method adopts the screening of liquid submerged culture method, and the main drawback that this method exists is the microorganism that only can screen growth fast in suspension liquid, and the beneficial rhizosphere bacterium with characterization of adsorption is difficult to screened arriving.
Summary of the invention
The object of the present invention is to provide a kind of method of screening promotion heavy metal phytoremediation rhizosphere bacteria, for solving the microorganism adopting existing liquid submerged culture method only can screen growth fast in suspension liquid, and be difficult to screen the problem of the beneficial rhizosphere bacterium with characterization of adsorption.
To achieve these goals, the invention provides following technical scheme: a kind of method of screening promotion heavy metal phytoremediation rhizosphere bacteria, comprises the steps:
(1) acquisition operations and pretreatment operation are carried out to plants and soil sample;
(2) sample after step (1) gathers carries out enrichment process;
(3) culture after enrichment process carries out primary dcreening operation operation;
(4) separating treatment is carried out to the culture after primary dcreening operation;
(5) be separated the bacterial classification obtained and carry out the operation of multiple sieve;
The enrichment process of described step (2) comprises enrichment bottle making processes, in enrichment bottle, adds sample and spawn culture process, needs in reagent bottle, add absorption carrier, heavy metal compound, nutrient broth and nystatin in described enrichment bottle making processes.
Compared to prior art, screening of the present invention promotes that the method for heavy metal phytoremediation rhizosphere bacteria has following advantage: first the present invention gathers and pre-treatment plants and soil sample, afterwards enrichment process is carried out to it, and absorption carrier, heavy metal compound, nutrient broth and nystatin is added in the making processes of enrichment bottle, wherein, nutrient broth and nystatin are to supply to bacteriotrophy and play antimycotic effect in enrichment process.The present invention is also added with absorption carrier and heavy metal compound relative to traditional method in enrichment process, the interpolation of absorption carrier can make the bacterial classification major part of easily absorption all be adsorbed on absorption carrier, facilitate the process of later stage for solid matter and the extraction and isolation for bacterial classification, also the extraction to the strong bacterial classification of adsorptivity is easier to, being added in enrichment bottle of heavy metal compound makes bacterium define the environment of a partial eclipse, promotes manifesting of bacterial adsorption heavy metal compound characteristic.After enrichment process, the solid matter obtained is carried out primary dcreening operation and is separated.Finally, by enriching plant cultivation directly multiple sieve and checking, the content of observation vegetation growth state and detection plant heavy metal of body, screens the probiotics promoting plant heavy metal absorption absorptive function.
The invention provides a kind of directly, Effective selection promotes the novel method of heavy metal phytoremediation rhizosphere bacteria, the method can screen the probiotics promoting plant heavy metal absorption absorptive function faster and more accurately, improves screening efficiency.After making the beneficial rhizosphere bacterium with characterization of adsorption filtered out be inoculated into plant, heavy metal is polluted and is had stronger Governance Ability.
Accompanying drawing explanation
By reading hereafter detailed description of the preferred embodiment, various other advantage and benefit will become cheer and bright for those of ordinary skill in the art.Accompanying drawing only for illustrating the object of preferred implementation, and does not think limitation of the present invention.In the accompanying drawings:
Fig. 1 shows the flow chart of steps of the method promoting heavy metal phytoremediation rhizosphere bacteria according to screening of the present invention.
Embodiment
The invention provides many applicable creative concepts, this creative concept can be reflected in a large number of in concrete context.The specific embodiment described in following embodiments of the present invention only as the exemplary illustration of the specific embodiment of the present invention, and does not form limitation of the scope of the invention.
Below in conjunction with accompanying drawing and concrete embodiment, the invention will be further described.
The invention provides a kind of method of screening promotion heavy metal phytoremediation rhizosphere bacteria, first, acquisition operations and pretreatment operation are carried out to plants and soil sample, wherein acquisition operations is gather the good plant sample of growth on the serious place of heavy metal contamination, comprise the complete root system of plant sample and the Soil Surrounding of root system thereof, the general soil that need gather in root system periphery 2mm, if locality distance is studied distant, then the sample collected is preserved under need being in the environment of 2 ~ 5 DEG C.Before collected specimens being started to process, first pre-treatment step is carried out to it, namely adopt sterilized water to carry out preliminary flushing to collected specimens, to rinse out impurity and the poor bacterial classification of adsorptivity.
Then, enrichment process is carried out to pretreated sample.First enrichment bottle is made: using one or more in material stronger for the adsorptivities such as gac, rice husk, perlite or maize straw as absorption carrier in enrichment process, being crushed to after longest edge is less than 1cm drops in reagent bottle, input amount is 15 ~ 20g, in order to make the enrichment space in enrichment larger, and avoid the material in enrichment bottle to volatilize too quickly, select the triangular flask of 150ml as reagent bottle, the amount that now absorption carrier adds just in time can cover in the bottom of triangular flask.The interpolation of absorption carrier can make the bacterial classification major part of easily absorption all be adsorbed on absorption carrier, later stage carries out processing for solid absorption carrier and just can extract the stronger bacterial classification of adsorptivity, thus facilitate the extraction and isolation of later stage for bacterial classification, improve the efficiency of screening, and make to have filtered out the beneficial rhizosphere bacterium with characterization of adsorption be inoculated into plant after heavy metal pollute there is stronger Governance Ability.Add appropriate heavy metal compound again, 8 ~ 12 times of the add-on of heavy metal compound to be the concentration making heavy metal compound in nutrient broth be locality Heavy Metals in Contaminated Soils concentration, be preferably 10 times, being added in enrichment bottle of heavy metal compound makes bacterium define the environment of a partial eclipse, promotes manifesting of bacterial adsorption heavy metal compound characteristic.In order to avoid the material aggregation condensation in enrichment bottle, in enrichment bottle, also add a small amount of granulated glass sphere, be advisable with 2 ~ 3.After the nutrient broth 50ml adding the nutrition of supply thalline in enrichment bottle, carry out sterilising treatment to it, sterilising temp is 121 DEG C, and sterilization time is 20min; Sterilizing terminates, and adds the nystatin of 2.0 ~ 3.0mg after enrichment bottle Temperature fall to room temperature again in enrichment bottle, and be preferably 2.5mg, the making of this enrichment bottle just completes.
Wherein, the formula of above-mentioned nutrient broth is: peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, distilled water 1000ml, and modulation pH is 7.2 ± 0.2.
Take the Soil Surrounding of the root system of plant after pretreatment operation and root system, root system is cut off rear transfer to rapidly above-mentioned making formed enrichment bottle in, carry out shaking table cultivation, the envrionment temperature controlling enrichment bottle in shaking table is cultivated is 30 DEG C, and per minute rotates 150 ~ 180 turns.The amount of above-mentioned root system of plant and root system Soil Surrounding controls to be advisable can be immersed in completely in the nutrient broth in enrichment bottle.After within every 5 days, transferring to 50% of the solid matter in last enrichment bottle in an enrichment bottle, namely first enrichment bottle is after the shaking table cultivation of 5 days, transfer in second new enrichment bottle by 50% of the solid matter in first enrichment bottle, at second enrichment bottle after the shaking table cultivation of 5 days, transfer in the 3rd new enrichment bottle by 50% of the solid matter in second enrichment bottle, shift successively according to said sequence, Dual culture 30 ~ 60 days.
Take out the solid absorption carrier in last the enrichment bottle obtained through enrichment culture, primary dcreening operation and lock out operation are carried out to it, aseptic water washing is adopted to fall soil particle and the non-adsorbent thalline of attachment, be placed on solid medium flat board, smear up and down, be placed in incubator to cultivate, incubator temperature is set as 30 DEG C, cultivates 5 days.Solid medium flat board takes out from incubator, is scraped by the lawn around carrier, and isolates single bacterium colony at the flat lining out of above-mentioned solid medium, adopts slant medium to preserve with inclined-plane.
Wherein, the formula of solid medium is: peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, agar 20g, distilled water 1000ml, the add-on of heavy metal compound is make the concentration of described heavy metal compound in distilled water be 8 ~ 12 times of locality Heavy Metals in Contaminated Soils concentration, and modulation solid medium pH is 7.2 ± 0.2;
The formula of slant medium is: peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, agar 20g, distilled water 1000ml, and makes the pH of slant medium be 7.2 ± 0.2.
Further, the operation of multiple sieve need be carried out to being separated the various thalline obtained, in order to avoid evaporating in reagent bottle is too fast, reagent bottle in multiple sieve operation also selects the triangular flask of 150ml, first take a triangular flask, in triangular flask, the enriching plant of commute plantation is cultivated, and as epipremnum aureum, flame nettle etc., it is for subsequent use that the water generally adding 100ml in triangular flask carries out cultivation.
Be separated through primary dcreening operation the various bacterial classifications obtained to be inoculated into respectively in nutrient broth and to carry out shaking table cultivation further, every portion of selected nutrient broth is 10ml, and during shaking table is cultivated, envrionment temperature is 30 DEG C, rotates with 150r/min, cultivates 48h.It is OD that the bacterial classification that cultivation obtains adopts stroke-physiological saline solution to be diluted to concentration
660=0.3, for subsequent use.
Heavy metal compound is added by the triangular flask of each culturing plants, wherein, above-mentioned heavy metal compound can be zinc chloride, zinc sulfate, cupric chloride etc., diluted bacterium liquid is added respectively in each triangular flask, shaking table cultivation is carried out after fixing plant, the temperature that shaking table is cultivated controls to be 30 DEG C, 60r/min, and cultivate 30 ~ 60 days, in culturing process, every day supplements sterilized water and ensures that root system of plant is immersed in below liquid level in triangular flask, after cultivating observation plant upgrowth situation and filter out the root system probiotics that can promote heavy metal phytoremediation by the content of NITRATE BY FLAME ATOMIC spectrophotometry plant heavy metal of body.Often kind of bacterial classification can do and repeatedly sieve experiment again, thus calculates the mean value of the heavy metal content of plant materials internal adsorption.
Example
Gather the good plant of Daye, hubei Province heavy metal contamination serious field aerial to test according to method of the present invention as sample, experimentation is recorded in in hypothallus experiment and contrast experiment, and the experimental result of thalline experiment and contrast experiment is compared, record is in the following.
Thalline is tested
One, the collection of sample: gather the better herbaceous plant sample of growth on the serious place of Daye, hubei Province heavy metal contamination, comprise the soil in its complete root system and periphery 2mm, and preserve under the environment of 4 DEG C and be transported to laboratory.
Two, the pre-treatment of sample: adopt sterilized water to rinse gently in gained sample.
Three, the enrichment of sample: carry out according to following three steps.
(1) enrichment bottle is made.Adopt 16g maize straw to be cut into the particle of soya bean size, be placed in the triangular flask of 150ml.Adopting atomic absorption spectrophotometer to measure heavy metal concentration in samples-soil is 0.04mg/g, therefore in triangular flask, be added into the zinc sulfate of 20mg, the nutrient broth 50ml simultaneously adding 2 granulated glass spherees and configure, overall triangular flask is carried out sterilising treatment 20min at 121 DEG C, after sterilizing, when temperature in triangular flask reduces to room temperature, in triangular flask, add the nystatin of 2.5mg thus make enrichment bottle.
Wherein, consisting of of above-mentioned nutrient broth: peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, distilled water 1000ml, modulation pH is 7.2.
(2) in enrichment bottle, sample is added.Get soil in the plant sample root system of collection and root system 2mm, cut off by root system, be transferred to rapidly in the enrichment bottle that above-mentioned making formed and carry out shaking table cultivation, in the process that shaking table is cultivated, the temperature controlled in enrichment bottle is 30 DEG C, and rotating speed is 150r/min.
(3) spawn culture.After within every 5 days, transferring to 50% of the solid matter in last enrichment bottle in a new enrichment bottle, Dual culture 30 days.
Four, the primary dcreening operation of culture: adopt aseptic technique to take out the solid absorption carrier in last enrichment bottle of enrichment culture, be placed on solid medium flat board for 2 times, smear up and down with aseptic water washing, cultivates after 5 days and takes out in the incubator of 30 DEG C.Lawn around carrier on solid medium flat board after cultivation is scraped.
Wherein, consisting of of solid medium: peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, agar 20.0g, zinc sulfate 0.4g, distilled water 1000ml, pH are 7.2.
Five, the separation of culture: be separated single bacterium colony at the flat lining out of above-mentioned solid medium, and preserve with inclined-plane, in this sepn process, be divided into out 16 strain bacteriums.
Wherein, the formula of slant medium is: peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, agar 20.0g, distilled water 1000ml, pH are 7.2.
Six, the multiple sieve of bacterial classification: carry out according to following three steps.
(1) cultivation of plant.The water adding 100ml in multiple 150ml triangular flask, for cultivating flame nettle, treats flame nettle well-grown in every bottle, for subsequent use.
(2) bacterial classification process.The bacterial classification obtained by primary dcreening operation is inoculated into respectively in the test tube of 10ml nutrient broth and carries out shaking table cultivation 48h, and the envrionment temperature in shaking table cultivation is 30 DEG C, rotating speed is 150r/min, and after cultivating, taking-up bacterial classification stroke-physiological saline solution dilutes bacterial concentration is OD
660=0.3, for subsequent use.
(3) test.0.04g zinc sulfate is added with in the triangular flask of each cultivation flame nettle, 2 granulated glass spherees, respectively to the bacterium liquid adding the different thalline formation of 10ml after second step process dilution in each triangular flask, experiment is marked and fixes flame nettle shaking table and cultivate 30 days, shaking table cultivate in control temperature be 30 DEG C, rotating speed is 60r/min.And every day all adds sterilized water to 100ml to each triangular flask in culturing process, ensure that flame nettle root system is immersed in below liquid level.Cultivate and terminate, flame nettle absorbs the amount of zinc to adopt atomic absorption spectrophotometry to detect.Two deuterzooid are carried out to often kind of thalline and sieves experimentation again, obtain same time flame nettle under often kind of thalline effect and absorb the mean value of the amount of zinc.
Contrast experiment
Step is tested with thalline, difference is that in the multiple sieved journey of sample in the present embodiment, the 3rd step experimental implementation is: cultivate in the triangular flask of flame nettle at two and be added with 0.04g zinc sulfate, 2 granulated glass spherees, the stroke-physiological saline solution of 10ml is added in each triangular flask, solid is reserved flame nettle and is carried out shaking table and cultivates 30 days, the process that shaking table is cultivated is tested with thalline, and adopt NITRATE BY FLAME ATOMIC spectrophotometry to go out flame nettle in two triangular flasks and absorb the amount of zinc, calculating is averaged.
Conclusion
Be tested with three strain thalline in thalline experiment and there is promotion flame nettle to the absorption absorptive function of zinc, in the experiment that this three strains thalline adds, flame nettle to the mean value of the amount of the absorption of zinc be respectively in control experiment flame nettle to 13.2 of the mean value of the absorbed dose of zinc times, 10.1 times and 8.7 times.
By above-mentioned experimental verification, the invention provides a kind of directly, Effective selection promotes the novel method of heavy metal phytoremediation rhizosphere bacteria, the method can screen the probiotics promoting plant heavy metal absorption absorptive function faster and more accurately, improves screening efficiency.Make plant form super enriching plant absorption after making the beneficial rhizosphere bacterium with characterization of adsorption filtered out be inoculated into plant to absorb, heavy metal is polluted and is had stronger Governance Ability.
It should be noted, the present invention will be described instead of limit the invention for above-described embodiment, and those skilled in the art can design alternative embodiment when not departing from the scope of claims.In the claims, any reference symbol between bracket should be configured to limitations on claims.Word " comprises " not to be got rid of existence and does not arrange element in the claims or step.
Claims (7)
1. screen the method promoting heavy metal phytoremediation rhizosphere bacteria, it is characterized in that, comprise the steps:
(1) acquisition operations and pretreatment operation are carried out to plants and soil sample;
(2) sample after step (1) gathers carries out enrichment process;
(3) culture after enrichment process carries out primary dcreening operation operation;
(4) separating treatment is carried out to the culture after primary dcreening operation;
(5) be separated the bacterial classification obtained and carry out the operation of multiple sieve;
The enrichment process of described step (2) comprises enrichment bottle making processes, in enrichment bottle, adds sample and spawn culture process, needs in reagent bottle, add absorption carrier, heavy metal compound, nutrient broth and nystatin in described enrichment bottle making processes.
2. screening according to claim 1 promotes the method for heavy metal phytoremediation rhizosphere bacteria, it is characterized in that, in described step (1), acquisition operations is for gathering the good plant sample of heavy metal contamination serious field aerial, comprises complete root system and the root system Soil Surrounding thereof of plant sample; In described step (1), pretreatment operation is adopt sterilized water to carry out preliminary flushing to the sample gathered.
3. screening according to claim 1 promotes the method for heavy metal phytoremediation rhizosphere bacteria, it is characterized in that, in the making processes of the enrichment bottle of described step (2) enrichment process, described absorption carrier select in gac, rice husk, perlite or maize straw one or more, absorption carrier quality is 15 ~ 20g and is crushed to the length of side to be less than 1cm;
The add-on of described heavy metal compound is 8 ~ 12 times of interpolations of locality Heavy Metals in Contaminated Soils concentration in step (1) according to the concentration making described heavy metal compound in described nutrient broth;
The add-on of described nutrient broth is 45 ~ 55ml, and the formula of described nutrient broth is: peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, distilled water 1000ml, and modulation pH is 7.2 ± 0.2;
Add and terminate, also need reagent bottle to be carried out sterilising treatment and cooling process, finally add the nystatin of 2.0 ~ 3.0mg.
4. screening according to claim 1 promotes the method for heavy metal phytoremediation rhizosphere bacteria, it is characterized in that, in reagent bottle, add sample in described step (2) enrichment process is be transferred in enrichment bottle after being cut off by the root system of plant of sample to adopt shaking table training method to cultivate 30 ~ 60 days, in culturing process, culture temperature is 30 DEG C, and rotating speed is 150 ~ 180r/min;
Spawn culture process is transferred in a rear new enrichment bottle by the solid matter of 50% in last enrichment bottle for every 5 days, while progressively increasing enrichment bottle quantity, progressively extract solid matter.
5. screening according to claim 4 promotes the method for heavy metal phytoremediation rhizosphere bacteria, it is characterized in that, primary dcreening operation in described step (3) be treated to by last the enrichment bottle obtained through enrichment culture solid carrier adopt aseptic water washing fall attachment soil particle and non-adsorbent thalline be placed on solid medium flat board cultivate formed bacterium colony
Wherein, the formula of described solid medium is: peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, agar 20.0g, distilled water 1000ml, the add-on of heavy metal compound is make the concentration of described heavy metal compound in distilled water be 8 ~ 12 times of locality Heavy Metals in Contaminated Soils concentration, and modulation solid medium pH is 7.2 ± 0.2.
6. screening according to claim 5 promotes the method for heavy metal phytoremediation rhizosphere bacteria, it is characterized in that, lock out operation in described step (4) is for scraping the lawn around the carrier being attached with on the solid medium flat board of carrier after step (3) primary dcreening operation process, and be separated single bacterium colony at the flat lining out of above-mentioned solid medium, preserve with inclined-plane;
Wherein, the formula of described slant medium is: peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, agar 20g, distilled water 1000ml, and makes the pH of slant medium be 7.2 ± 0.2.
7. the screening according to claim 1 ~ 6 any one promotes the method for heavy metal phytoremediation rhizosphere bacteria, it is characterized in that, multiple sieve in described step (5) is operating as takes the various bacterial classifications to separating in step (4) to test respectively, experimentation is cultivated for being inoculated into respectively in the test tube of nutrient broth by the bacterial classification after step (4) separation, bacterium liquid after cultivating joins in the reagent bottle containing enriching plant and heavy metal compound and carries out entirety cultivation, and after entirety is cultivated the upgrowth situation of making plant and the heavy metal content that detects in plant materials and obtain promoting the probiotics of plant heavy metal absorption absorptive function.
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Application publication date: 20160504 |