CN106754496B - Siderophore high-yield bacterium preparation and application thereof in contaminated soil heavy metal remediation - Google Patents
Siderophore high-yield bacterium preparation and application thereof in contaminated soil heavy metal remediation Download PDFInfo
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- CN106754496B CN106754496B CN201611136114.4A CN201611136114A CN106754496B CN 106754496 B CN106754496 B CN 106754496B CN 201611136114 A CN201611136114 A CN 201611136114A CN 106754496 B CN106754496 B CN 106754496B
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- bacillus
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
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- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Soil Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Processing Of Solid Wastes (AREA)
Abstract
The invention belongs to the field of bioremediation of environmental pollution, and particularly relates to a siderophore high-yield bacterium preparation and application thereof in remediation of heavy metals in polluted soil. A siderophore high-yield bacterium preparation, which contains Bacillus bacteria (Bacillus bacterium)Bacillussp.) S86 or Pseudomonas bacteria (M.) (Pseudomonassp.) one or a combination of S17. The siderophore high-yield bacterium preparation can be used for restoring heavy metal in farmland polluted soil, and particularly can be used for reinforcing the restoration of heavy metal polluted soil by sweet sorghum.
Description
Technical Field
The invention belongs to the field of bioremediation of environmental pollution, and particularly relates to a siderophore high-yield bacterium preparation and application thereof in remediation of heavy metals in polluted soil.
Background
Heavy metal pollution of soil is now a global environmental problem, is concerned by society and people, and needs to be solved urgently. In many areas of China (especially in east China), the soil is polluted by heavy metals in different degrees, and the contents of the heavy metals such as cadmium, arsenic, copper, lead, zinc and the like in grains, vegetables and fruits in many areas exceed the standard or are close to the critical value, so that the health of people is seriously harmed.
The heavy metal pollution treatment of soil is urgently difficult, and the main reasons are that heavy metal can not be biodegraded like organic pollutants, and the heavy metal has poor mobility in soil. The traditional physical and chemical methods such as soil dressing, leaching, ion exchange and the like are only suitable for treating small-scale heavily polluted soil, and are suffered from problems of high cost, damage to soil structures, easiness in causing secondary pollution and the like. Compared with the prior art, bioremediation is favored because of the advantages of low cost, good effect, simplicity, easy implementation, no secondary pollution and the like. The microorganism-plant combined remediation is one of bioremediation, is popular because of the advantages of effectively reducing the toxicity of heavy metals in soil, adsorbing the heavy metals and changing the microenvironment of the rhizosphere, further improving the absorption or fixation efficiency of plants on the heavy metals, having relatively low implementation cost, being environment-friendly, being capable of large-scale in-situ remediation and the like, and is a new technology for treating the heavy metal pollution of large-area soil, which is vigorously developed and promoted in recent years. However, the efficiency of the above-described combined remediation techniques depends on the biomass of the remediating plant, as well as the bioavailability of heavy metals in the soil. In other words, the larger the plant biomass and the ability to absorb as much heavy metals from the soil as possible, the higher the remediation efficiency and the better the effect. How to improve the phytoremediation efficiency becomes a hot spot for research and attention. Hyperaccumulating plants (such as brassica, heliopsis and pennisetum) are generally low in biomass and difficult to grow on a large scale, and thus have very limited application in practical remediation. And some plants which grow rapidly and have large biomass and heavy metal enrichment capacity are increasingly applied to the remediation of heavy metal contaminated sites. As an energy crop for producing bioethanol, sweet sorghum has the advantages of strong pressure resistance, rapid growth, large biomass and the like, and becomes the most competitive heavy metal restoration plant at present.
Siderophore (Siderophore) is a microbially produced organic compound with superior complexing power for iron, and functions to provide microbial cells with iron nutrients, especially in low iron environments. The combination of siderophores with heavy metals is mainly reflected in the biological function of siderophores, i.e. while concentrating iron in the environment and promoting its transport into cells, siderophores can also affect the bioavailability of other metals by complexation, thereby reducing their toxicity. In addition, the microbial siderophore can also improve the disease resistance of the plant root system by inhibiting the growth of pathogenic microorganisms. The siderophore producing strain is a popular candidate for repairing microorganism parts in a heavy metal pollution system by combining microorganism and plants due to the advantages and the characteristics of the siderophore producing strain.
At present, many siderophore producing bacteria are separated from the environment by artificial enrichment, screening and other techniques, and pure culture is realized. However, among these strains, siderophore high-producing strains have been reported to be limited; the strain which has multiple advantages and characteristics (such as multiple heavy metal resistance and pathogenic fungus knot resistance) is deficient; there are few strains that actually enhance the ability of plants to absorb heavy metals in soil. Therefore, finding and obtaining a siderophore high-producing strain with excellent properties has been a hot spot and focus in this research field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a siderophore high-yield bacterium preparation.
The invention also aims to provide the application of the siderophore high-yield bacterium preparation in the heavy metal remediation of farmland polluted soil, and the application is mainly used for enhancing the remediation of the typical heavy metal polluted soil by sweet sorghum.
The technical problem of the invention is implemented by the following technical scheme:
the high-yield siderophore bacteria Pseudomonas sp.S17 and Bacillus sp.S86 related by the invention are originated from the soil of a test field in Zhejiang science and technology institute in Hangzhou, Zhejiang, and are obtained by artificial enrichment culture, pressure screening, separation and purification. The S17 strain is pseudomonas bacteria, gram stain is negative, catalase is positive, oxidase is negative, V.P. test is negative, indole test is positive, the shape of the bacteria is short rod-shaped, the bacterial colony is light yellow and round, the edge of the bacterial colony is slightly burr and smooth and moist, other physiological and biochemical characteristics are shown in table 1, the strain is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) at 2016, 10, 12 and is numbered CGMCC No. 13104.
A siderophore high-yield strain is a strain S86 of Bacillus bacteria (Bacillus sp.) which is preserved in the China general microbiological culture Collection center in 2016, 10, 12 and 12 days with the number of CGMCC No.13105 and the preservation unit address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing. The S86 strain is bacillus, gram stain positive, catalase positive, oxidase negative, V.P. test positive, indole test negative, the thallus morphology is rod-shaped, the colony is white, round and waxy, and other physiological and biochemical characteristics are shown in Table 2.
TABLE 1 physiological and biochemical characteristics of Strain S17
Note: "+" indicates growth or a positive reaction; "-" indicates no growth or negative reaction (same below).
TABLE 2 physiological and biochemical characteristics of Strain S86
The optimum growth temperature of the S17 strain was 29 ℃. With one fifth LB (Luria-Bertani) or industrial fermentation medium (corn flour 10g/L, soybean meal 8g/L, (NH)4)2SO46g/L, 6g/L corn steep liquor and MgSO4·7H2O 1g/L，K2HPO4·3H2O 2g/L，MnSO40.05g/L, pH 7.0) for 24 hours, the bacterial concentration of the bacterial liquid can reach 6.5-9.2 x 109CFU/mL (CFU, colony forming unit). The bacterium is sensitive to ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline (all of which are common antibiotics). It can be considered that: when the thallus is released into the natural environment, super bacteria can not be generated due to the problem of drug resistance (drug resistance). In a pathogenic bacteria antagonism test, the strain is found to inhibit the growth of three pathogenic fungi, namely penicillium, aspergillus niger and sclerotinia rot of colza. The strain is used for testing 12 types of metal ions (Li)+、Ag+、Cd2+、Cu2+、Hg2+、Ba2+、Pb2+、Zn2+、Mn2+、Ni2+、Fe3+And Co4 +) Exhibiting varying degrees of tolerance (or resistance). In view of the current situation of heavy metal contaminated soil in China, that multiple heavy metal pollutes, the strain is considered to have less influence on the repairing effect by the inhibition of the heavy metal in the implementation of the reinforced repairing process compared with other strains without tolerance or resistance of multiple heavy metals. The strain has 99.2 percent of Siderophore Unit (SU) and reaches the highest level of Siderophore bacteria, and is the strain with the strongest Siderophore production capability in domestic Siderophore bacteria. In addition, S17 has certain phosphorus dissolving and 3-indoleacetic acid producing capacity. 1.5 percent of glucose is used as a carbon source, 0.2 percent of ammonium chloride is used as a nitrogen source, the temperature is 30 ℃, the pH value is 7.2, the culture time is 66 hours (the initial inoculation amount is 2 percent), and the phosphorus content of the bacterial solution is 120.35 mg/L. The strain was inoculated (inoculum size: 1%) into a general LB liquid medium containing L-tryptophan (200mg/L), cultured at 28 ℃ at 160r/min for 3 days, and then the 3-indoleacetic acid content in the fermentation broth was measured, and found to be 72.36 mg/L.
The optimum growth temperature of the S86 strain was 28 ℃. After one fifth of LB or industrial fermentation culture medium is used for 24 hours, the bacterial concentration of the bacterial liquid can reach 6.1-8.6 multiplied by 109CFU/mL. The bacterium is sensitive or highly sensitive to ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline. It can be considered that: when the thallus is released into the natural environment, super bacteria can not be generated due to the problem of drug resistance (drug resistance). In a pathogenic bacterium antagonism test, the strain is found to be capable of inhibiting the growth of pathogenic fungi of tomato gray mold, tomato early blight, eggplant wilt, cotton wilt, rape sclerotinia rot and melon gummy stem blight. The strain is used for testing 12 types of metal ions (Li)+、Ag+、Cd2+、Cu2+、Hg2+、Ba2+、Pb2+、Zn2+、Mn2+、Ni2+、Fe3+And Co4+) Exhibiting varying degrees of tolerance (or resistance). It is considered that, compared with other strains without tolerance or resistance of various heavy metals, the strain has small influence on the repair effect by the inhibition of the heavy metals in the implementation of the strengthening repair process. The strain has an SU value of 76.4%, and belongs to a strong siderophore producing strain. This is achieved byIn addition, when the S86 strain and the S17 strain were co-cultured in one fifth of LB or industrial fermentation medium at 29 ℃ and pH7.3 at 150r/min for 24 hours (the initial inoculum size of S86 and S17 was half of that of each individual inoculum size), the cell density of the bacterial suspension was found (7.4-10.6X 10)9CFU/mL) is significantly higher than the cell density of the bacterial suspension when each is cultured alone.
An application of the siderophore high-yield strain in the field of remediation of heavy metals in farmland polluted soil.
Preferably, the application is to strengthen the remediation of heavy metal contaminated soil by sweet sorghum, and the method is to spray a bacterial solution containing the siderophore high-yield bacteria at the root of the growing sweet sorghum, wherein the concentration of the bacterial solution is 1-5 multiplied by 108CFU/mL。
Preferably, the bacterial liquid is a logarithmic phase siderophore high-yield bacterial liquid.
Preferably, the bacterial liquid is obtained by culturing one fifth of LB (Luria-Bertani) or an industrial fermentation medium for 22-24 hours, and the formula of the industrial fermentation medium is as follows: 10g/L of corn flour, 8g/L of soybean meal, (NH)4)2SO46g/L, 6g/L corn steep liquor and MgSO4·7H2O 1g/L，K2HPO4·3H2O 2g/L，MnSO40.05g/L。
Preferably, the liquid culture temperature is 25-30 ℃, and the pH of the basic inorganic salt liquid culture medium is 6.8-7.2.
Preferably, the liquid culture temperature is 28 ℃, and the pH of the basic inorganic salt liquid culture medium is 7.0.
A siderophore high-yield bacterium preparation, which contains one or the combination of Bacillus bacteria (Bacillus sp.) S86 or Pseudomonas sp.S 17.
Preferably, the preparation is obtained by inoculating a strain to one fifth LB (Luria-Bertani) or an industrial fermentation medium for culturing for 22-24 hours, and the formula of the industrial fermentation medium is as follows: 10g/L of corn flour, 8g/L of soybean meal, (NH)4)2SO46g/L, 6g/L corn steep liquor and MgSO4·7H2O 1g/L，K2HPO4·3H2O 2g/L，MnSO40.05 g/L. Furthermore, the temperature of the preparation fermentation culture is 29 plus or minus 1 ℃, and the pH value is 7.2-7.4.
The application of the siderophore high-yield bacterium preparation in the field of remediation of heavy metals in farmland polluted soil.
Preferably, the application is to strengthen the remediation of heavy metal contaminated soil by sweet sorghum, the method is to spray the high-yield bacterial preparation containing the iron carrier at the root of the sweet sorghum in the growth period, and the total concentration of thalli in the high-yield bacterial preparation containing the iron carrier is 1-5 multiplied by 108CFU/mL。
In summary, compared with the prior art, the invention has the following advantages:
the Pseudomonas sp.S17 and Bacillus sp.S86 strains are strong siderophore producing strains. Wherein, the Pseudomonas sp.S17 strain reaches the highest siderophore production capability, and the siderophore production capability is the strongest among strains reported in China. The S17 and S86 strains are sensitive to common antibiotics, show different degrees of tolerance or resistance to 12 heavy metals, and have pathogenic fungus knot resistance. When the two are cultured together, the density of the thallus is obviously higher than that of the thallus cultured under the same condition, and the complementation proliferation effect exists. In addition, the S17 strain also has the phosphorus dissolving capacity and the 3-indoleacetic acid producing capacity. The advantages and the characteristics are significant for the combined microorganism-plant remediation of the heavy metal polluted land! The remediation system can be applied to remediation of the polluted site more practically and more efficiently.
Detailed Description
The present invention is further illustrated by the following examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1: separation of Pseudomonas sp.S17 and Bacillus sp.S86 and test of siderophore production Capacity
100g of test field soil in Zhejiang science and technology college, Hangzhou, Zhejiang province is collected on site by a universal five-point sampling method, contained in a sterile envelope and quickly brought back to a laboratory.
General CAS detection solutions and MSA liquid media were prepared according to Chengxing et al reports (Chenshaoxing, Zhao Xiang, Shen, etc.. plate detection method for highly sensitive Pseudomonas siderophore. microbiological bulletin 2006, 33: 122-. The solid detection plate is formed by adding 5% of CAS detection liquid and 2% of agar powder into MSA liquid culture medium and solidifying.
Soil samples were diluted 10-fold in increments and plated onto one-fifth LB solid plates (i.e., 1/5 with all nutrients as universal LB medium). After the bacterial colonies appear after the culture at 25 ℃, picking out the bacterial colonies with different forms one by using sterile toothpicks, transferring the bacterial colonies onto a new fifth LB solid plate until the bacterial colonies are purified into a single bacterial colony. All pure strains are numbered in sequence, and are preserved by conventional strains and stored in a refrigerator at 4 ℃ in a slant way.
The purified strains are transferred to a solid detection plate one by one, inverted culture is carried out for 2 days at the temperature of 28 ℃, whether a color-changing ring is generated around a bacterial colony is observed and recorded in detail, the diameter of the color-changing ring is used as the primary judgment of the strength of the siderophore capability of siderophore bacteria, and in addition, an Escherichia coli DH5 α strain is required to be arranged as a negative control.
The quantitative determination of the siderophore is carried out according to the reports of Chenshaoxing et al (Chenshaoxing, Zhao Xiang, Shen, et al. plate detection method of high-sensitivity pseudomonad siderophore. microbiological report, 2006, 33: 122-. The OD of each sample was measured after the sample was fully left at room temperature680The (As) value was zeroed against double distilled water. The absorbance of each liquid medium containing the non-cultured bacteria as the supernatant was determined in the same manner, and this was used as a reference value (Ar).The concentration of siderophores is expressed in siderophor activity units and is repeated 4 times per treatment. Subsequently, the siderophore productivity of each strain was ranked according to the report of Persmark et al (Persmark M, Expert D, Neilands JB. isolation, chromatography, and synthesis of microorganisms, a compound with siderophoreactivity from Erwinia chrysophyte Chemistry, the Journal of Biological Chemistry,1989,264: 3187-.
As a result, 126 strains of culturable strains were obtained in total on one fifth of the LB solid plates, 9 of which had a siderophore productivity (see Table 3). Among the strains having the ability to produce siderophores, the S17 strain had the strongest ability, and the S86 strain was the second best.
TABLE 3 siderophore production ability record table for siderophore producing bacteria
Example 2: antibiotic susceptibility test and pathogenic fungus antagonistic capability test of Pseudomonas sp.S17 and Bacillus sp.S86
The antibiotic sensitivity test adopts a filter paper sheet method, five common antibiotics of ampicillin, tetracycline, chloramphenicol, kanamycin and streptomycin are selected, and the diameter of a bacteriostatic zone of each antibiotic filter paper sheet on plates for culturing siderophore producing bacteria S17 and S86 is used as a judgment standard of sensitivity or drug resistance. The results of the tests given in tables 4 and 5 show that strains S17 and S86 both respond more or less sensitively to the above antibiotics tested.
TABLE 4 antibiotic susceptibility test results for strain S17
Note: the diameter of the inhibition zone is greater than the upper limit value of the medium sensitivity range, the high sensitivity is determined, and the diameter of the inhibition zone is less than the lower limit value of the sensitivity range, the resistance is determined (the same below).
TABLE 5 antibiotic susceptibility test results of Strain S86
This example illustrates that when the Pseudomonas sp.s17 and Bacillus sp.s86 are released into the natural environment, they will not become super-bacteria due to the problem of drug resistance, which provides a safety guarantee for the subsequent practical application.
The antagonistic capacity of pathogenic bacteria and fungi of S17 and S86 is determined by a plate confronting method by taking 10 pathogenic bacteria, namely tomato gray mold pathogen, tomato early blight pathogen, eggplant fusarium wilt pathogen, melon gummy stem blight pathogen, sclerotinia sclerotiorum, penicillium, cotton fusarium wilt pathogen, aspergillus niger, wheat sharp eyespot pathogen and wheat leaf blight pathogen, as indicator bacteria. Indicator bacteria are inoculated in the center of a universal PDA plate, S17 and S86 are respectively inoculated at the periphery of the universal PDA plate, each group of experiments are repeated for 5 times, and the antagonistic capacity of the strains S17 and S86 is determined through the colony radius and the inhibition zone width.
As shown in Table 6, Pseudomonas sp.S17 showed significant antagonistic activity against the tested Penicillium, Aspergillus niger and sclerotinia rot of rape, while Bacillus sp.S86 showed resistance to gray mold of tomato, early blight of tomato, eggplant blight, cotton blight, sclerotinia rot of rape and melon vine blight of melon to various degrees.
TABLE 6 antagonistic capability test of Pseudomonas sp.S17 and Bacillus sp.S86
Note: negatives are "-"; marking the diameter of the inhibition zone of 0-5mm as plus "; the diameter of the inhibition zone is 5-10mm and is marked as "+"; the diameter of the inhibition zone is 10-15mm and is marked as "+ +"; the diameter of the inhibition zone is larger than 15mm and is marked as ++++.
Example 3: heavy metal Minimum Inhibitory Concentration (MIC) test for Pseudomonas sp.S17 and Bacillus sp.S86
MIC assay design was performed according to the study of Filali et al (Filali BK, Taoufik J, Zeroual Y, Dzairi FZ, Talbi M, Blaghen M. Water bacteria resistant to heavymetals and antibiotics. Current Microbiology, 2000, 41: 151. times.156), by picking equal size, activated S17 and S86 single colonies, respectively, in culture tubes, and shaking at 30 ℃ and 160r/min for 32 hours. And determining the MIC value according to the growth vigor of the strains, setting 3 repeats for each group, and respectively testing the tolerance conditions of the strains to 12 metal ions. As shown in Table 7, the test strains showed multiple heavy metal tolerance (resistance) and excellent properties.
TABLE 7 MIC test results
Note: the data in the table are the average of 3 measurements.
Example 4: phosphorus dissolving and 3-indoleacetic acid producing capability test of Pseudomonas sp.S17 and Bacillus sp.S86
Inoculating loops of Pseudomonas sp.S17 and Bacillus sp.S86 from the slant of the test tube, respectively, into a triangular flask containing 50mL of liquid LB medium, shaking at 160r/min for 24h, and collecting 1mL (about 1X 10) of each loop8CFU) cultures, inoculated with Ca respectively3(PO4)2100mL of inorganic phosphorus fermentation medium [ glucose 10g, (NH) as the sole phosphorus source4)2SO41g，NaCl 0.3g，KCl 0.3g，MgSO4·7H2O 0.3g，FeSO4·7H2O 0.002g，MnSO4·H2O 0.002g，Ca3(PO4)25g, adding distilled water to 1000mL, adjusting pH to 7.5 ], performing shaking culture (160r/min) at 30 ℃ for 48h, and repeating the steps for 3 times. The phosphorus content is determined by applying a universal molybdenum-antimony colorimetric method, and 1mL of culture solution is respectively taken to be in corresponding numbersCentrifuging the centrifugal tube at 5000r/min for 10 min. 1mL of the supernatant is accurately taken and placed in a volumetric flask with 25mL of the corresponding number, 2.5mL of the molybdenum-antimony anti-revealing agent is added, and then the volume is determined by using distilled water. After reacting at room temperature for 30min, measuring the absorbance at 700nm with spectrophotometer, and measuring with mg P2O5Is expressed by/L. The treatment without inoculation was used as a blank control and the experiment was repeated 3 times. As a result, it was found that Pseudomonas sp.S17 had a phosphorus-solubilizing ability of 88.6. + -. 2.26mg/L, and Bacillus sp.S86 had no phosphorus-solubilizing ability.
Pseudomonas sp.S17 and Bacillus sp.S86 were inoculated into LB liquid medium containing L-tryptophan (200mg/L), respectively, and cultured at 28 ℃ and 160r/min for 3 days. The OD of the respective bacterial suspensions is subsequently determined spectrophotometrically600The bacterial suspension was then centrifuged at 10000r/min for 10min, and the supernatant was added to an equal volume of Salkowski colorimetric solution (Libbert Ead Risch H. interactions between plants and epithelial bacteria regarding the same amount of auxin metabolism [ J]Physiologia Plantarum, 1969, 22: 51-58), standing in dark for 30min for color development, and determining OD530Calculating the content of 3-indoleacetic acid in unit volume of bacterial liquid, setting 3 times of repetition, and taking E.coli DH5 α which does not produce 3-indoleacetic acid as a negative control, and measuring that the yield of the S17 strain 3-indoleacetic acid is 62.8 +/-0.86 mg/L, and the S86 strain and the negative control do not detect the 3-indoleacetic acid.
People find that many repairing plants can not play a repairing and absorbing role to the maximum extent due to the reasons of nutrition deficiency (particularly lack of phosphorus elements), limited absorption, growth inhibition and the like in the traditional heavy metal polluted site repairing process. This example shows that Pseudomonas sp.S17 has the ability to solubilize phosphorus and produce 3-indoleacetic acid, which is very practical and important for enhancing plant survival, growth and repair. Example 5: application of Pseudomonas sp.S17 and Bacillus sp.S86 together for strengthening absorption of heavy metals by sweet sorghum
A siderophore high-yield bacterium preparation is prepared by compounding bacterium liquid A and bacterium liquid B.
Bacterial liquid A: s17 as initial inoculum, 29 ℃, industrial fermentation medium (10 g/L corn flour, 8g/L soybean meal, (NH)4)2SO46g/L, 6g/L corn steep liquor and MgSO4·7H2O 1g/L，K2HPO4·3H2O 2g/L，MnSO40.05g/L, pH 7.2) for 24 hours.
B, bacterial liquid: s86 Bacillus sp.as initial inoculum, 28 ℃, industrial fermentation medium (corn flour 10g/L, soybean meal 8g/L, (NH)4)2SO46g/L, 6g/L corn steep liquor and MgSO4·7H2O 1g/L，K2HPO4·3H2O 2g/L，MnSO40.05g/L, pH 7.5) for 24 hours.
Before compounding, the bacterial liquid A and B are separately regulated to 1.0 × 10 with sterile water or tap water8CFU/mL. Then, according to VA:VBCompounding according to the volume ratio of 1:1 (namely the equal volume ratio) to obtain the siderophore high-yield bacterium preparation.
The soil used in the test is collected from a vegetable garden which is not polluted by heavy metal, and the main physicochemical properties are as follows: 4.84 percent of organic matter, 832.6mg/kg of total nitrogen, 292.1mg/kg of total phosphorus, 220.3mg/kg of total potassium, 68.2mg/kg of quick-acting nitrogen, 18.1mg/kg of quick-acting phosphorus, 58.3mg/kg of quick-acting potassium and pH 6.2. The heavy metal content is 0.21mg/kg cadmium, 58mg/kg copper and 101mg/kg zinc. Air drying, sieving, adding solution cadmium chloride, zinc chloride and copper sulfate to obtain Cd in soil2+In a concentration of 25mg/kg, Zn2+Has a concentration of 800mg/kg, Cu2+The concentration of (2) is 800 mg/kg. The contaminated soil was thoroughly mixed and tested after 2 months of stability at 60% humidity.
The seeds of the repairing plant sweet sorghum are purchased from the bridge city of the river of the Linan city. The seeds germinate first, when the seedlings grow to about 10cm, the seedlings with consistent growth vigor are selected and transplanted into 1.5kg of plastic pots (13 cm in height and 17cm in diameter) of the previously stabilized heavy metal contaminated soil, and 1 seedling is planted in each pot. After transplanting for 7 days, when the plants grow normally, 1mL of fresh preparation with uniformly shaken siderophore high-yield bacterium preparation is inoculated to the roots of the respective plants.
In order to avoid the influence of the nutrient components in the culture medium on the test result, the inoculated bacterial liquid is washed for 3 times by sterile water and then resuspended by the sterile water. Test forThe experiment set 4 treatments: blank control (C) without plants and without added bacteria, no inoculated bacteria (P) with only plants, no inoculated bacteria (M) with no plants, and both plants and bacteria (PM), 5 replicates were set for each treatment. Watering regularly, keeping illumination for 8h every day, growing sweet sorghum in a greenhouse for 82 days, harvesting, cleaning root, soaking in 10mmol of EDTA solution for 30min, and washing with deionized water for 2 times. The plants are dried to constant weight at 80 ℃ after being enzyme-deactivated at 105 ℃, and the dry weight of the roots and the overground parts is weighed. Digestion of plant tissue (HClO)4∶HNO31: 4), the heavy metal content is determined and the whole procedure is blanked to eliminate interference.
As shown in Table 8, the biomass and heavy metal absorption of sweet sorghum can be significantly improved by inoculating Pseudomonas sp.S17 and Bacillus sp.S86. Wherein the dry weight of the overground part and the root part was increased by 31.8% and 36.0%, respectively, compared with the control without inoculation (PM treatment compared with P treatment), while Cd in the overground part of sweet sorghum2+、Zn2+And Cu2+The total content of the extract is respectively increased by 87.5 percent, 87.3 percent and 88.4 percent. The S17 and S86 compound microbial inoculum is proved to have the functions of promoting the growth of the sweet sorghum and strengthening the absorption capacity of the sweet sorghum to test heavy metals.
TABLE 8 absorption of heavy metals from the aerial parts and roots of sweet sorghum
Note: the content of heavy metal is biomass multiplied by the concentration of heavy metal; the extraction efficiency is the ratio of the total heavy metal content (the sum of the root part and the overground part) extracted by the plant to the total heavy metal content in the initial soil; indicates that the values were significantly different from the corresponding control values (P < 0.05).
Compared with the closest prior art (xylonite, siderophore bacteria for strengthening sweet sorghum to restore soil heavy metal pollution, environmental science and technology, 2014, 37: 74-79), the invention adopts dry overground part weight and Cd aboveground part treated by' plant and microorganism2+、Zn2+And Cu2+The total content and the respective extraction efficiency are respectively improved by 9.4%, 25.8%, 22.2% and 31.9%, and 24.5%, 22.2% and 28.6%, and the effect is betterGood and remarkable advantages. In the aspect of extraction rate improvement (PM treatment extraction rate/P treatment extraction rate), the extraction rate improvement effects of the three heavy metals are respectively improved by 56.5%, 123.5% and 44.0%, and the advantages are obvious. It should be noted that Pseudomonas sp.S17 is not the same strain as Pseudomonas sp.T07 in the above study on xylonite. Because, it has obvious differences in physiological and biochemical characteristics (see table 1), which are shown in the four test items of catalase, oxidase, glucose utilization ability and methyl red test.
Claims (7)
1. A siderophore high-yield bacterium which is a bacillus bacterium (A)Bacillussp.) in 2016, 10, 12 days, and has the serial number of CGMCCNo No. 13105.
2. An application of the siderophore high-producing strain as described in claim 1 in the field of remediation of heavy metals in farmland polluted soil.
3. A siderophore high-yield bacterium preparation is characterized in that: the siderophore high-productivity bacterial preparation comprising the Bacillus bacterium (B) of claim 1Bacillussp.) S86 or Pseudomonas bacteria (M.) (Pseudomonassp.) S17, the Pseudomonas bacteria S17 was deposited in 2016, 10, 12 days, in the China general microbiological culture Collection center (CGMCC), with the number of CGMCC 13104.
4. The siderophore high productivity bacterial preparation as claimed in claim 3, wherein: the preparation is obtained by inoculating one fifth of LB (Luria-Bertani) or an industrial fermentation culture medium with strains for culturing for 22-24 hours, and the formula of the industrial fermentation culture medium is as follows: 10g/L of corn flour, 8g/L of soybean meal, (NH)4)2SO46g/L, 6g/L corn steep liquor and MgSO4·7H2O 1 g/L，K2HPO4·3H2O 2 g/L，MnSO40.05 g/L。
5. The siderophore high productivity bacterial preparation as claimed in claim 3, wherein: the temperature of the preparation fermentation culture is 29 plus or minus 1 ℃, and the pH value is 7.2-7.4.
6. The application of the siderophore high-yield bacterium preparation as described in claim 3 in the field of remediation of heavy metals in farmland polluted soil.
7. The use of claim 6, wherein: the application is to strengthen the remediation of heavy metal contaminated soil by sweet sorghum, the method is to spray the high-yield bacterial preparation containing the iron carrier at the root of the sweet sorghum in the growing period, and the total concentration of thalli in the high-yield bacterial preparation containing the iron carrier is 1-5 multiplied by 108CFU/mL。
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| CN102643760A (en) * | 2011-02-18 | 2012-08-22 | 中国热带农业科学院环境与植物保护研究所 | Antagonistic bacterium capable of generating siderophore for controlling plant diseases |
| CN104928212A (en) * | 2015-06-03 | 2015-09-23 | 华南农业大学 | Bacillus megaterium strain X3 and preparation method and application thereof |
| CN105985922A (en) * | 2016-01-20 | 2016-10-05 | 华南农业大学 | Bacillus aryabhattai J5 and application thereof |
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2016
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643760A (en) * | 2011-02-18 | 2012-08-22 | 中国热带农业科学院环境与植物保护研究所 | Antagonistic bacterium capable of generating siderophore for controlling plant diseases |
| CN104928212A (en) * | 2015-06-03 | 2015-09-23 | 华南农业大学 | Bacillus megaterium strain X3 and preparation method and application thereof |
| CN105985922A (en) * | 2016-01-20 | 2016-10-05 | 华南农业大学 | Bacillus aryabhattai J5 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Bio prospecting of Pseudomonas aeruginosa For Their Potential to Produce Siderophore, Process Optimization and Evaluation of Its Bioactivity;Marathe RJ等;《International Journal of Bioassays》;20151231;第4卷(第2期);第3667-3675页 * |
| 产铁载体细菌强化甜高粱修复土壤重金属污染;张璐;《环境科学与技术》;20140430;第37卷(第4期);第74-79页 * |
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