CN107755424B - Method for improving plant extraction efficiency by utilizing interaction of carbon dioxide enrichment and endophyte - Google Patents
Method for improving plant extraction efficiency by utilizing interaction of carbon dioxide enrichment and endophyte Download PDFInfo
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- CN107755424B CN107755424B CN201710961149.XA CN201710961149A CN107755424B CN 107755424 B CN107755424 B CN 107755424B CN 201710961149 A CN201710961149 A CN 201710961149A CN 107755424 B CN107755424 B CN 107755424B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention relates to the field of heavy metal polluted plant extraction, and aims to provide a method for improving plant extraction efficiency by utilizing carbon dioxide enrichment and endophyte interaction. The method comprises the following steps: pre-culturing Sedum alfredii Hance with tap water, selecting seedling with root length of 1-2cm, inoculating with endophyte compound bacterial liquid, and transplanting to black plastic barrel, wherein the nutrient solution contains cadmium; adding endophyte compound bacteria liquid, culturing in a plant growth box, keeping the enrichment of carbon dioxide in the plant growth box, replacing the nutrient solution every 3 days, and maintaining the concentration of thalli; after 1 month of carbon dioxide enrichment culture, the plants were harvested. The invention utilizes the carbon dioxide enrichment technology to improve photosynthesis and biomass, and simultaneously applies endophyte to promote absorption and accumulation of the hyperaccumulator plant on heavy metal cadmium, thereby overcoming the defects of slow plant growth and small biomass in the plant extraction technology, enhancing the environmental adaptability of the hyperaccumulator plant sedum alfredii hance and obviously improving the plant extraction efficiency.
Description
Technical Field
The invention relates to the field of heavy metal polluted plant extraction, in particular to a method for improving plant extraction efficiency by utilizing the interaction of a carbon dioxide enrichment technology and endophyte.
Background
The plant extraction and restoration means that one or more heavy metals are absorbed from the environment by heavy metal enrichment or hyper-accumulation plants, transferred and stored to the overground part. Compared with the traditional restoration engineering measures such as physics, chemistry and the like, the plant extraction restoration has the advantages of low cost, no damage to ecological environment, no secondary pollution and the like, and has wide application prospect. The application of the plant extraction and restoration technology has two prerequisites, one is that the plant must be a heavy metal enrichment or super-accumulation plant, can accumulate heavy metal elements with high concentration, and the other is that the biomass of the plant is large and the growth rate is high. However, most of the super-accumulated plants discovered at present have small biomass and slow growth rate, and limit the large-area popularization and application of plant extraction and repair technology, so that the establishment of a novel efficient plant extraction and repair enhancement technology system is very urgent.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for improving the plant extraction efficiency by utilizing the interaction of carbon dioxide enrichment and composite endophyte.
In order to solve the technical problem, the solution of the invention is as follows:
the method for improving the plant extraction efficiency by utilizing the interaction of carbon dioxide enrichment and endophyte comprises the following steps:
(1) pre-culturing hyperaccumulative plant Sedum alfredii Hance with tap water, selecting seedling with root length of 1-2cm, and inoculating the root system of the seedling into endophyte compound bacterial liquid for 2 h;
the endophyte compound bacterial liquid comprises four endophytes: rhodococcus (Rhodococcus), Buthus (Buttiauxella), Stenotrophomonas (Stenotrophomonas) and Herbaspirillum (Herbaspirillum), wherein the endophytes of Rhodococcus and Buthus are derived from the root system of the hyperaccumulating plant Sedum alfredii, and the endophytes of Stenotrophomonas and Spirospirillum are derived from the stem; the endophyte composite bacterial liquid is formed by mixing bacterial liquids of four endophytes according to the volume ratio of 1: 1, and the content of thalli in each bacterial liquid is 109CFU mL-1；
(2) Transplanting the inoculated Sedum alfredii Hance seedling into a black plastic barrel filled with a nutrient solution, wherein the concentration of cadmium in the nutrient solution is 50 mu M, and adding endophyte compound bacteria liquid into the nutrient solution to enable the thallusAt a concentration of 106CFU/ml; putting the black plastic barrel into a plant growth box for culturing, and keeping the enrichment concentration of carbon dioxide in the plant growth box at 800 muL.L during the culturing process-1Changing the nutrient solution every 3 days, and maintaining the thallus concentration in the nutrient solution at 106CFU/ml；
(3) After culturing for 1 month in carbon dioxide enrichment, harvesting the hyperaccumulator plant Sedum alfredii Hance, and analyzing biomass and cadmium extraction amount.
In the present invention, the preculture in step (1) means: placing the super-accumulator plant Sedum alfredii Hance in an artificial climate chamber, and pre-culturing with tap water for 1 week; the culture is carried out under the conditions of 14 hr illumination, 25/20 deg.C day and night, and relative humidity of artificial climate chamber maintained at 70% in day and 85% in night.
In the present invention, the cells of the four endophytes are obtained by: grinding stems and roots of the super-accumulator plant Sedum alfredii Hance respectively, and then culturing the grinding liquid by using a DF-ACC-Cd solid culture medium respectively; selecting bacterial colonies, streaking, and respectively purifying to obtain the four endophytes;
activating the four endophytes by using an LB liquid culture medium, and culturing each endophyte for 1-2 days at the temperature of 25-35 ℃; centrifuging the obtained bacterial liquid at 10000rpm for 5min, and removing supernatant; washing the precipitate with sterile phosphate buffer solution, centrifuging, removing supernatant for 3 times to obtain precipitate as thallus, adding appropriate amount of sterile water to obtain thallus content of 109CFU mL-1(ii) a Mixing the four bacterial liquids according to the volume ratio of 1: 1, and adjusting the OD of the bacterial suspension by using 0.85% sterile NaCl solution6001.0-1.2, obtaining the endophyte composite bacterial liquid, wherein the concentration of the bacterial liquid is 109CFU mL-1。
In the invention, the formula of the nutrient solution in the step (2) is as follows: k2SO4，0.7mmol/L；KCl，0.1mmol/L；Ca(NO3)2·4H2O，2mmol/L；MgSO4·7H2O，0.5mmol/L；KH2PO4，0.1mmol/L；H3BO3，10μmol/L；MnSO4·H2O，0.5μmol/L；ZnSO4·7H2O，0.1μmol/L；(NH4)4MoO24·4H2O, 0.01 mu mol/L; 20 mu mol/LFe EDTA solution; the pH of the nutrient solution was adjusted to 5.8 with NaOH or HCl.
In the invention, the control conditions of the plant growth box in the step (2) are as follows: humidity 70%, day and night temperature 25 deg.C and 20 deg.C, and day light illumination 100 μmol/m-2·s-1The light was applied for 14 hours per day.
Description of the inventive principles:
the super-accumulation plant Sedum alfredii Hance is a perennial herb with fleshy stems and leaves; the rootstock moves transversely, leaves grow alternately, and the lower leaves often fall off. Different from common plants, the sedum alfredii hance is a cadmium hyper-accumulation plant and has strong tolerance, absorption and accumulation capacities on cadmium. Under the condition of water culture, the sedum alfredii hance can normally grow under the cadmium concentration of 50 mu M and has strong enrichment capacity on cadmium. Therefore, the hyperaccumulator sedum alfredii hance has important application value in cadmium pollution extraction and remediation.
The increase of the concentration of the carbon dioxide can enhance the photosynthesis of the plant, promote the growth of the root system of the plant and improve the utilization efficiency of nutrients and water, thereby improving the biomass of the plant. Carbon dioxide is therefore often used as a fertilizer to increase the yield of crops. Meanwhile, the microorganism-enhanced plant extraction and restoration is considered to be an economic, environment-friendly, safe, efficient and reliable method, endophytes of plants can be colonized in plants without harming the growth of the plants, and meanwhile, the growth of the plants can be promoted through the processes of secreting plant growth hormone and siderophores, relieving the generation of ethylene and fixing nitrogen, dissolving mineral state nutrient elements and the like, so that the accumulation of heavy metals in the hyperaccumulation plants is improved. Therefore, the endophyte has wide application prospect in the aspect of improving plant extraction and repair as a biological enhancer.
Compared with the prior art, the invention has the beneficial effects that:
the invention combines the traditional agricultural measures of applying high-concentration carbon dioxide with the idea of endophyte biological strengthening technology, invents a novel agricultural measure, namely, the photosynthesis and biomass of the super-accumulation plant are improved by utilizing the carbon dioxide enrichment technology, and meanwhile, the endophyte is applied to promote the absorption and accumulation of the super-accumulation plant on heavy metal cadmium, solves the defects of slow plant growth and small biomass in the plant extraction technology, enhances the environmental adaptability of the super-accumulation plant of sedum alfredii hance, avoids secondary pollution caused by adding a chemical soil modifier in the regulation and control of other agricultural chemical measures, obviously improves the plant extraction efficiency, and is a novel agricultural measure with high efficiency, safety, economy and environmental protection.
Drawings
FIG. 1 shows the effect of carbon dioxide enrichment technology and composite endophyte interaction on the biomass of super-accumulated Sedum alfredii Hance;
FIG. 2 shows the effect of carbon dioxide enrichment technology and composite endophyte interaction on the concentration of cadmium in the super-accumulated plant Sedum alfredii Hance.
FIG. 3 shows the effect of carbon dioxide enrichment technology and composite endophyte interaction on the extraction amount of cadmium from the super-accumulated plant Sedum alfredii Hance.
In the figure: CO 22+ M represents inoculating composite endophyte under carbon dioxide enrichment condition; CO 22Represents carbon dioxide enrichment only; m represents inoculation of only composite endophytes; CK represents the control.
Detailed Description
The feasibility and the application effect of the method of the invention will be further illustrated by the following specific examples.
(1) Placing the super-accumulator plant Sedum alfredii Hance in a climatic chamber, pre-culturing with tap water for 1 week to allow it to root, wherein the illumination period is 14 hr and the day and night temperature is 25/20 deg.C. The relative humidity of the climatic chamber was maintained at 70% day and 85% night.
(2) Taking fresh stems and roots of a hyperaccumulative plant sedum alfredii hance which normally grows in an old lead-zinc mine, respectively carrying out surface sterilization and grinding, and then respectively culturing the grinding liquid by using a DF-ACC-Cd solid culture medium; selecting bacterial colonies, streaking, and respectively purifying to obtain thalli of four endophytes: rhodococcus SaSR1(Rhodococcus), Buthus SaSR9(Buttiauxella), Stenotrophomonas SaSS5 (Stenotrophoromonas) and Phlebacillus SaSS13 (Herbaspirillum). Wherein Rhodococcus SaSR1 and SacR 9 of genus Buthus are derived from root system, and stenotrophomonas SaSS5 and Salicornia SaSS13 are derived from stem.
The following are specifically mentioned: the culture, screening and identification methods of the four endophytes are all the prior known technologies, and the technicians in the field can completely and repeatedly reproduce the culture, screening and purification results of the four endophytes according to the test conditions of the invention. The naming systems of the four endophytes are determined according to the universal naming rules in the industry, the numbers can be determined according to the needs, and the numbers after the name of the endophyte are the numbers used by the applicant in the fungus screening process.
Endophytes SaSR1, SaSR9, SaSS5 and SaSS13 are activated by LB liquid culture medium, the obtained bacterial liquid is centrifuged for 5min at 10000rpm, and the supernatant is discarded. Washing the precipitate with sterile phosphoric acid buffer solution, centrifuging, removing supernatant, continuously operating for 3 times to obtain precipitate as thallus, adding appropriate amount of sterile water to obtain bacteria solution with thallus content of 109CFU mL-1. Mixing the four bacterial liquids according to the volume ratio of 1: 1, and adjusting the OD of the bacterial suspension by using 0.85% sterile NaCl solution6001.0-1.2, obtaining the endophyte compound bacterial liquid, wherein the bacterial concentration is about 109CFU mL-1。
Selecting a strong and consistent hyperaccumulation plant Sedum alfredii Hance with the root length of about 1-2cm, inoculating the compound bacterial liquid, immersing the root system in the prepared endophyte compound bacterial liquid, and inoculating for 2 h.
(3) According to CO2The difference of different concentrations and whether add compound fungus liquid sets up four and handles, is respectively: 350 μ L. L-1CO2，350μL·L-1CO2+ Compound bacterial liquid, 800. mu.L.L-1CO2，800μL·L-1CO2+ a compound bacterial liquid. Transplanting the inoculated Sedum alfredii Hance seedlings into black plastic barrels, planting 4 plants in each plastic barrel, adding endophyte composite bacteria liquid into the nutrient solution to ensure that the concentration of the bacteria is 106CFU/ml。
The nutrient solution formula is as follows: k2SO4，0.7mmol/L；KCl，0.1mmol/L；Ca(NO3)2·4H2O，2mmol/L；MgSO4·7H2O，0.5mmol/L；KH2PO4，0.1mmol/L；H3BO3，10μmol/L；MnSO4·H2O，0.5μmol/L；ZnSO4·7H2O，0.1μmol/L；(NH4)4MoO24·4H2O, 0.01 mu mol/L; 20 mu mol/LFe EDTA solution; the pH of the nutrient solution was adjusted to 5.8 with NaOH or HCl.
Transferring the black plastic barrel into a plant growth box for culturing, wherein the concentration of carbon dioxide in the plant growth box is maintained at 800 muL.L during the culturing process-1The nutrient solution is replaced every 3 days, and for the nutrient solution added with the compound bacterial solution, the compound bacterial solution needs to be supplemented every time the nutrient solution is replaced, so that the concentration of the thalli in the nutrient solution is maintained at 106CFU/ml. Other control conditions of the plant growth chamber are as follows: humidity 70%, day and night temperature 25 deg.C and 20 deg.C, and day light illumination 100 μmol/m-2·s-1The light was applied for 14 hours per day. After the plants grow for one month, the rhodiola rosea super-accumulated in each plant growth box is harvested, and the biomass and the cadmium absorption accumulation amount of the rhodiola rosea super-accumulated in each plant growth box are detected.
As shown in fig. 1-3, when the composite endophyte is inoculated under the condition of carbon dioxide enrichment, the plant biomass is obviously increased (fig. 1), the biomass of the root system and the overground part are respectively increased by 242.9 percent and 121.6 percent, the cadmium concentration and the cadmium extraction amount are also obviously increased (fig. 2 and 3), the cadmium concentration of the overground part and the root system are respectively increased by 57.4 percent and 61.6 percent, and the cadmium extraction amount of the overground part and the root system are respectively increased by 287.8 percent and 233.1 percent. The contrast experiment shows that the carbon dioxide enrichment technology and the composite endophyte have good promotion effect on plant growth and extraction and accumulation of heavy metal cadmium, and can remarkably improve the plant extraction efficiency.
Claims (5)
1. A method for improving plant extraction efficiency by utilizing interaction of carbon dioxide enrichment and endophyte is characterized by comprising the following steps:
(1) pre-culturing hyperaccumulative plant Sedum alfredii Hance with tap water, selecting seedling with root length of 1-2cm, and inoculating the root system of the seedling into endophyte compound bacterial liquid for 2 h;
the endophyte composite bacterial liquid comprises four endophytes of Rhodococcus, Buthus, stenotrophomonas and Oenospira, wherein the endophytes of Rhodococcus and Buthus are derived from the root system of the super-accumulation plant Sedum alfredii, and the endophytes of stenotrophomonas and Oenospira are derived from stems; the endophyte composite bacterial liquid is formed by mixing bacterial liquids of four endophytes according to the volume ratio of 1: 1, and the content of thalli in each bacterial liquid is 109CFU mL-1；
(2) Transplanting the inoculated Sedum alfredii Hance seedling into a black plastic barrel filled with a nutrient solution, wherein the cadmium concentration in the nutrient solution is 50 mu M, and adding endophyte compound bacteria liquid into the nutrient solution to ensure that the thallus concentration is 106CFU/ml; putting the black plastic barrel into a plant growth box for culturing, and keeping the concentration of carbon dioxide in the growth box at 800 muL.L during the culturing process-1Changing the nutrient solution every 3 days, and maintaining the thallus concentration in the nutrient solution at 106CFU/ml；
(3) After culturing for 1 month in carbon dioxide enrichment, harvesting the hyperaccumulator plant Sedum alfredii Hance, and analyzing biomass and cadmium extraction amount.
2. The method according to claim 1, wherein the pre-culturing in step (1) is: placing the super-accumulator plant Sedum alfredii Hance in an artificial climate chamber, and pre-culturing with tap water for 1 week; the culture is carried out under the conditions of 14 hr illumination, 25/20 deg.C day and night, and relative humidity of artificial climate chamber maintained at 70% in day and 85% in night.
3. The method of claim 1, wherein the thallus of four endophytes is obtained by: grinding stems and roots of the super-accumulator plant Sedum alfredii Hance respectively, and then culturing the grinding liquid by using a DF-ACC-Cd solid culture medium respectively; selecting bacterial colonies, streaking, and respectively purifying to obtain the four endophytes;
activating the four endophytes by using an LB liquid culture medium, and culturing each endophyte for 1-2 days at the temperature of 25-35 ℃; centrifuging the obtained bacterial liquid at 10000rpm for 5min, and removing supernatant;washing the precipitate with sterile phosphate buffer solution, centrifuging, removing supernatant, continuously operating for 3 times to obtain precipitate as thallus, adding appropriate amount of sterile water to make thallus content in the bacteria liquid be 109CFU mL-1(ii) a Mixing the four bacterial liquids at a volume ratio of 1: 1, and adjusting the bacterial suspension OD with 0.85% sterile NaCl solution6001.0-1.2, obtaining the endophyte composite bacterial liquid, wherein the concentration of the bacterial liquid is 109CFU mL-1。
4. The method of claim 1, wherein the nutrient solution of step (2) is formulated as: k2SO4，0.7mmol/L；KCl，0.1mmol/L；Ca(NO3)2·4H2O，2mmol/L；MgSO4·7H2O，0.5mmol/L；KH2PO4，0.1mmol/L；H3BO3，10μmol/L；MnSO4·H2O，0.5μmol/L；ZnSO4·7H2O，0.1μmol/L；(NH4)4MoO24·4H2O, 0.01 mu mol/L; 20 mu mol/LFe EDTA solution; the pH of the nutrient solution was adjusted to 5.8 with NaOH or HCl.
5. The method according to claim 1, wherein the control conditions of the plant growth chamber in step (2) are: humidity 70%, day and night temperature 25 deg.C and 20 deg.C, and day light illumination 100 μmol/m-2·s-1The light was applied for 14 hours per day.
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