CN110184225A - One plant of rhizosphere growth-promoting endophytic bacteria PHE-2 and its application with PAHs degradation capability - Google Patents

One plant of rhizosphere growth-promoting endophytic bacteria PHE-2 and its application with PAHs degradation capability Download PDF

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CN110184225A
CN110184225A CN201910561730.1A CN201910561730A CN110184225A CN 110184225 A CN110184225 A CN 110184225A CN 201910561730 A CN201910561730 A CN 201910561730A CN 110184225 A CN110184225 A CN 110184225A
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CN110184225B (en
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刘娟
陈志高
高彦征
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Nanjing Agricultural University
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    • A—HUMAN NECESSITIES
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    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/32—Hydrocarbons, e.g. oil
    • C02F2101/327—Polyaromatic Hydrocarbons [PAH's]
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Abstract

The invention discloses one plant of rhizosphere growth-promoting endophytic bacteria PHE-2 with PAHs degradation capability, classification naming is series bacillus (Paenibacillus sp.), it is preserved on August 26th, 2014 China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.1.12900.The function microbial inoculum of bacterial strain PHE-2 of the present invention preparation in 5d is 100mgL to initial concentration under shake flask culture conditions‑1Luxuriant and rich with fragrance degradation rate up to 95% or more；Meanwhile bacterial strain PHE-2 anthracene also capable of being fast degraded, pyrene and.Bacterial strain PHE-2 has good table film forming ability, and has a variety of growth-promoting characteristics, can produce heteroauxin, siderophore, and have phosphate solubilization.Bacterial strain PHE-2 of the present invention can be directly used for lowering PAHs polluted-water, soil and the intracorporal luxuriant and rich with fragrance pollution risk of plant, and can significantly promote plant growth.

Description

One plant of rhizosphere growth-promoting endophytic bacteria PHE-2 and its application with PAHs degradation capability
Technical field
The invention belongs to environment polycyclic aromatic hydrocarbon (PAHs) to pollute biological prosthetic field, and being related to one plant has PAHs degradation capability Rhizosphere growth-promoting endophytic bacteria (Paenibacillus sp.) PHE-2 and its application.
Background technique
Polycyclic aromatic hydrocarbon (Polycyclic aromatic hydrocarbon, PAHs) is by two and more than two phenyl ring The compound of structure composition, as common persistence organic pollutant (POPs) a kind of in soil, PAHs has chronic toxicity, Mutagenesis, carcinogenic, teratogenesis " three cause " effect and bio-refractory.Due to the poisonous and harmful property of PAHs, China already PAHs is included in the pollutant blacklist of Environment Priority detection.What national environmental protection portions in 2014 and Ministry of Land and Resources's joint were issued " national Soil Pollution Investigation bulletin " is pointed out, the point exceeding standard rate of national PAHs in soil (PAHs) is 1.4%.Ring PAHs in border can be entered in plant by plant absorption, to plant on plant organ, tissue, cell and gene level The normal physiological activity of object has an impact, and causes the intracorporal oxidative stress of plant, even results in cell death.In addition, PAHs can Human health is threatened to transmit enrichment by food chain.Thus, how to lower plant PAHs pollution risk is current agricultural One of the hot spot of environmental area research.
In recent decades, it is gradually risen using the plant combined recovery technique processing environment pollution problem of microorganism-, the technology With without secondary pollution, environmental-friendly, the cheap simultaneous advantage beautified the environment etc. by extensive concern, it is suitble to poisonous and harmful have The reparation of machine pollutant large area pollution of area source, rehabilitation expense are lower.Although extensive using microorganism remediation technology merely Better repairing effect would generally be obtained applied to the removing of environmental organic pollutant, but by microorganism and plant combined use, Such as the root table bacterial biof iotalm etc. that mycorrhizal fungi, endophytic bacterium and rhizosphere bacteria are formed.Plant rhizosphere there are quantity can The bacterium of sight, root system constantly secrete the root exudates such as polysaccharide, amino acid and low molecular organic acids, root to rhizosphere by root table Border bacterium competitively colonizes in plant roots table to obtain these nutriments, and forms miniature clone, aggregation and thin in root table Born of the same parents' cluster.The structure that these more bacteriums cooperatively form has shown the feature of bacterial biof iotalm at many aspects, thus studies It is referred to as root table bacterial biof iotalm together with mature bacterial biof iotalm by person.Root table bacterial biof iotalm has generation plant more Hormone promotes crop yield, improves plant to the physiological ecologicals function such as absorption of minerals, while can be by generating antibiotic etc. Secondary metabolite assists plant to resist invading pathogens.For example, at typical plant growth-promoting rhizobacteria (PGPR) In Paenibacillus sp., more plants of bacteriums all can colonize to form bacterial biof iotalm in plant roots table, and pass through a variety of growth-promoting machines System promotes host plant growth.
In recent years, research shows that the bacterium for having the function of that organic pollutant degradation ability bacterium is formed in plant roots table is raw Object film plays an important role in terms of the metabolism of rhizosphere pollutant and removal.The peculiar structure such as EPS of root table bacterial biof iotalm can To be enriched with the organic pollutant of rhizospheric environment, and in biomembrane the synergistic effect of various bacteria and functional gene horizontal transfer Etc. mechanism can then strengthen metabolism of the bacterial biof iotalm to organic pollutant, and then accelerate the degradation of organic pollutant.It is so far Only, due to a lack of corresponding microorganism resource, using the research of the root telogenesis film bacterial degradation PAHs with growth-promoting ability and using still It has not been reported.So, in complicated rhizospheric environment, if there is the rhizosphere growth-promoting endophytic bacteria with PAHs degradation capability? if In the presence of can this bacterium colonize in plant roots table and form bacterial biof iotalm to guarantee that it is long-term steady in rhizospheric environment It is fixed to exist? if can exist steadily in the long term, which is introduced into PAHs and pollutes environment, it can be effective Lower PAHs content in plant?
Summary of the invention
The purpose of the present invention is in view of the above technical problems, divide from the plant roots table being grown in PAHs long-term pollution soil There is the rhizosphere growth-promoting endophytic bacteria PHE-2 of PAHs degradation capability from obtaining one plant, and provide its application in pollution environment.
The purpose of the present invention can be achieved through the following technical solutions:
One plant of rhizosphere growth-promoting endophytic bacteria PHE-2 with PAHs degradation capability, classification naming is series bacillus (Paenibacillus sp.) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on August 26th, 2014 Center, culture presevation number are CGMCC No.1.12900.
It is a further object of the present invention to provide a kind of function microbial inoculums containing rhizosphere growth-promoting endophytic bacteria PHE-2.
The function microbial inoculum containing rhizosphere growth-promoting endophytic bacteria PHE-2 is obtained by the following method:
(1), rhizosphere growth-promoting endophytic bacteria PHE-2 is inoculated in fermentation medium, shaken cultivation to logarithmic phase；
(2), the strain of culture to logarithmic phase is connect by 5% inoculum concentration (V/V, on the basis of culture volume, similarly hereinafter) Kind enters seeding tank, cultivates to logarithmic phase, obtains seed liquor；Seed liquor is produced into tank culture by 10% inoculum concentration access；
Wherein, seeding tank and production tank used in fermentation medium it is identical as the fermentation medium of step (1), fermented and cultured The formula of base are as follows: peptone 10.0gL-1, yeast 5.0gL-1, NaCl 10.0gL-1, pH 7.0-7.4；
The ventilatory capacity of filtrated air is 1:1~1.2 in the incubation of seeding tank and production tank, and mixing speed is 180~220 revs/min, cultivation temperature is 28~30 DEG C, and whole process incubation time is 55~60 hours, after fermentation thallus number Amount reaches 1,000,000,000/ml or more；
(3), culture solution goes out tank after fermentation, adjusts culture solution concentration to OD600nm=1.0 with fermentation medium, obtains To function microbial inoculum.
It is yet another object of the invention to provide the rhizosphere growth-promoting endophytic bacteria PHE-2 with PAHs degradation capability degradation PAHs's Using the application of PAHs preferably in degradation water body, soil and plant.
It is yet another object of the invention to provide the answering in degradation PAHs of the function microbial inoculum containing rhizosphere growth-promoting endophytic bacteria PHE-2 With the application of PAHs preferably in degradation water body, soil and plant.
The PAHs be selected from phenanthrene, anthracene, pyrene andIn any one or more.
A further object of the present invention is to provide the rhizosphere growth-promoting endophytic bacteria PHE-2 with PAHs degradation capability and is promoting crop The application of growth.
A further object of the present invention is to provide the function microbial inoculum containing rhizosphere growth-promoting endophytic bacteria PHE-2 and is promoting plant growth Application.
The crop is preferably water spinach.
Beneficial effects of the present invention:
Using the function microbial inoculum of series bacillus of the present invention (Paenibacillus sp.) bacterial strain PHE-2 preparation in shaking flask It is 100mgL to initial concentration that 5d is interior under condition of culture-1Luxuriant and rich with fragrance degradation rate up to 95% or more；Bacterial strain PHE-2 of the present invention has Stronger heat-resisting and resistance to alkali ability is inoculated with institute of the present invention according to inoculum concentration 5% when temperature is 30-40 DEG C, pH is 7.0-10.0 The function microbial inoculum stated stablizes 90% or more luxuriant and rich with fragrance degradation rate.Meanwhile bacterial strain PHE-2 anthracene also capable of being fast degraded, pyrene and Bacterial strain PHE-2 has good table film forming ability, and has a variety of growth-promoting characteristics, can produce heteroauxin, siderophore, and Has phosphate solubilization.It is planted after water spinach root table by soaking to colonize bacterial strain PHE-2 in a manner of root in the nutrition by phenanthrene pollution In liquid, discovery bacterial strain PHE-2 can form bacterial biof iotalm in water spinach root table.Bacterial strain PHE-2 colonizing into water spinach root table Film can effectively facilitate the growth of water spinach, and lower its intracorporal luxuriant and rich with fragrance pollution risk.After water planting 15d, inoculation PHE-2 group is hollow The biomass of dish, which is significantly greater than, is inoculated with inactivated bacteria group；And compared to inoculation inactivated bacteria group, it is inoculated with the intracorporal phenanthrene of PHE-2 group water spinach Content and accumulation are all significantly reduced.For example, in 10.0mgL-1Under pollution concentration, after water planting 15d, go out compared to inoculation Viable bacteria group plant, the content for being inoculated with PHE-2 group root system of plant and cauline leaf China and Philippines reduce 40.93% and 53.8% respectively.
Bacterial strain PHE-2 of the present invention can be produced with the general Zymolysis Equipment of fermentation industry, have production cost low, be used Convenient, the good advantage of removal effect can be directly used for lowering PAHs polluted-water, soil and the intracorporal luxuriant and rich with fragrance pollution wind of plant Danger, and can significantly promote plant growth, there is wide industrial prospect and important environment, economic and social benefit.The present invention Bacterial strain PHE-2 is provides microorganism resource and technical support in the agricultural product of the contaminated area PAHs production health, simultaneously for guarantee Agricultural product security under PAHs pollution environment is of great significance.
Detailed description of the invention
Fig. 1 is the bacterium colony photo (A) and transmission electron microscope photo (B) of bacterial strain PHE-2 on LB plate.
Fig. 2 be bacterial strain PHE-2 with luxuriant and rich with fragrance (a), anthracene (b), pyrene (c),(d) be carbon source when growth and degradation curve.
Fig. 3 is that environmental condition (a. temperature, b. initial pH value, c. inoculum concentration, d. concentration of substrate) is with phenanthrene to bacterial strain PHE-2 The influence of carbon source for growth and degradation phenanthrene.
Fig. 4 is the Soluble phosphorus circle that bacterial strain PHE-2 is formed on Phos culture medium (A) and organic phosphorus culture medium (B).
Fig. 5 is bacterial biof iotalm (the A. plant root tip that bacterial strain PHE-2 is formed after water spinach root table colonizes under scanning electron microscope Portion's overview；B. it is gathered in the bacterial strain of plant roots table；C. the bacterial biof iotalm of plant roots table package；D. the space knot of bacterial biof iotalm Structure).
Biomaterial preservation information
Rhizosphere growth-promoting endophytic bacteria PHE-2, classification naming are series bacillus (Paenibacillus sp.), are preserved in China Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), address are city, BeiJing, China, North Star West Road, Chaoyang District 1 Number institute, deposit number are CGMCC NO.1.12900, and the deposit date is on August 26th, 2014.
Specific embodiment
The separation and identification of 1 bacterial strain PHE-2 of embodiment
The amur foxtail sample near Nanjing aromatic hydrocarbons factory's blowdown mouth region is acquired, gently removes plant with sterilizing hairbrush After object root table soil, root is placed in the centrifuge tube equipped with 5mL sterilizing ultrapure water and acutely shakes 30s, to destroy root table bacterium Biofilm structure makes bacterium separate out, and the bacterial suspension of acquisition is spare as the bacterial community in root surface biomembrane.
Bacterial suspension is added in the minimal medium of 100mL with 2% inoculum concentration, and is added luxuriant and rich with fragrance to 100mg L-1As sole carbon source, in 30 DEG C, 180rmin-1Shaking table culture 7d is transferred to identical culture medium with 1% inoculum concentration In, after continuous switching three times, gradient dilution pregnant solution takes 10-4~10-7Each 0.1mL of the pregnant solution of dilution is coated on addition 100mg·L-1On luxuriant and rich with fragrance inorganic salts plate, after 30 DEG C of constant temperature incubation 3d, observes, occur around choosing colony in the UV lamp The bacterial strain of obvious hydrolysis circle, isolates and purifies to obtain single colonie by scribing line.The single colonie that further picking is grown be inoculated in containing 100mg·L-1In luxuriant and rich with fragrance minimal medium, in 30 DEG C, 180rmin-1Shaking table culture 7d verifies it and imitates to luxuriant and rich with fragrance degradation Fruit.Minimal medium formula are as follows: NH4NO31.0g·L-1, KH2PO40.5g·L-1, K2HPO41.5g·L-1, NaCl 1.0g L-1, MgSO4·7H2O 0.2g·L-1, pH 7.0；Solid medium adds 18gL-1Agar.
The verification method of degradation effect: after isometric chromatography methanol is added into culture solution, it is ultrasonically treated 30min, makes bottle Sino-Philippines all dissolutions, 12000rmin-1Supernatant membrane filtration (0.22 μm of aperture) is taken after centrifugation removal precipitating, with efficient Liquid chromatography for measuring luxuriant and rich with fragrance content therein.High performance liquid chromatography (Shimadzu LC-20AT, detector models SPD-20A) setting ginseng Number are as follows: Inertsil ODS-SP-C18 reverse-phase chromatographic column (150mm × 4.6mm, 5 μm), mobile phase is methanol: water=90:10 (v:v), flow velocity 0.90mLmin-1, 40 DEG C of column temperature, ultraviolet detection wavelength 245nm, 10 μ L of sample volume.External standard method is fixed by peak area Amount.
It is separated to one plant of luxuriant and rich with fragrance bacterium for degrading from pregnant solution, is named as bacterial strain PHE-2.The liquid chromatogram inspection of the luxuriant and rich with fragrance front and back of degradation It surveys the result shows that being 100mgL to initial concentration in bacterial strain PHE-25d-1Luxuriant and rich with fragrance degradation rate be greater than 95%.Bacterial strain PHE-2 is in LB After cultured on solid medium 4d, bacterium colony is circle, and surface is smooth, and median rise, edge is irregular, and be translucent shape, there is light Pool, thallus stickiness is big, easily provokes (Figure 1A)；The bacterium is in the shape of a rod under transmission electron microscope, amphitrichous, forms gemma (figure 1B), physio-biochemical characteristics are shown in Table 1.The form and physiological and biochemical property of bacterial strain PHE-2 and " Bergey's manual of Systematic bacteriology " in series bacillus (Paenibacillus sp.) description it is consistent.
Using the genomic DNA of bacterial strain PHE-2 as template, PCR expansion is carried out with bacterial 16 S rRNA gene order universal primer Increase, obtains the 16S rRNA gene order that length is 1400bp.In RDP database (https: //rdp.cme.msu.edu/) Carry out Blast, the results showed that bacterial strain PHE-2 and the homology of series bacillus (Paenibacillus sp.) are nearest, sequence phase Reach 99% or more like property.Determine that the bacterium is bacillus genus (Paenibacillus sp.) thin in conjunction with physiological and biochemical property Bacterium.The bacterium is delivered into China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, deposit number is CGMCCNO.1.12900, the deposit date is on August 26th, 2014.
The physio-biochemical characteristics of 1. bacterial strain PHE-2 of table
Note: "+" indicates reaction for the positive；"-" indicates reaction for feminine gender.
The fermentation of 2 function microbial inoculum of embodiment
Use the technique of bacterial strain PHE-2 production function microbial inoculum are as follows: inclined-plane kind --- shake-flask seed liquid --- seeding tank --- Product (packaging dosage form is liquid bacterial agent).
(1), series bacillus (Paenibacillus sp.) PHE-2 that deposit number is CGMCCNO.1.12900 is tried Pipe kind is inoculated in fermentation medium, shaken cultivation to logarithmic phase；
(2), above-mentioned cultured strain is inoculated with by 5% inoculum concentration into seeding tank, cultivates to logarithmic phase, obtains seed Liquid；Seed liquor is produced into tank culture by 10% inoculum concentration access；
Wherein, the fermentation of fermentation medium and step (1) used in fermentation medium used in seeding tank and production tank is trained It is same to support base phase, formula is equal are as follows: peptone 10.0gL-1, yeast 5.0gL-1, NaCl 10.0gL-1, pH 7.0-7.4；
Seeding tank and production tank incubation in filtrated air ventilatory capacity be 1:1.2, mixing speed be 200 turns/ Point, cultivation temperature is 30 DEG C, and whole process incubation time is 60 hours, and thalline quantity reaches 1,000,000,000/ml or more after fermentation；
(3), culture solution goes out tank after fermentation, adjusts culture solution concentration to OD600nm=1.0, i.e., with fermentation medium For function microbial inoculum.
Bacterial strain PHE-2 tests luxuriant and rich with fragrance and other PAH biodegrade in 3 culture medium of embodiment
It is 100mgL that initial concentration is added in minimal medium (with embodiment 1)-1Phenanthrene (or initial concentration is 100mg·L-1Anthracene or initial concentration be 50mgL-1Pyrene or initial concentration be 50mgL-1's), it is connect with 5% inoculum concentration Enter the function microbial inoculum of embodiment 2, the PHE-2 of setting inoculation inactivation is control, 30 DEG C of constant-temperature table 180rmin-1Shaken cultivation. Timing sampling detects growing state and degradation situation of the bacterial strain PHE-2 using above-mentioned various PAH as carbon source for growth when.
The quantitative measurement of bacterial strain PHE-2 is counted using gradient dilution spread plate in culture medium, and culture medium China and Philippines residual contains The measurement of amount is the same as embodiment 1；The measuring method of anthracene residual content is identical as phenanthrene in culture medium.Pyrene andDetection method by as follows Carry out: after diploid product chromatography methanol is added into culture solution, being ultrasonically treated 30min, make in bottle pyrene orAll dissolutions, 12000r·min-1Supernatant membrane filtration (0.22 μm of aperture) is taken after centrifugation removal precipitating, uses high effective liquid chromatography for measuring Pyrene therein orContent.High performance liquid chromatography (Shimadzu LC-20AT, detector models SPD-20A) setup parameter are as follows: Inertsil ODS-SP-C18 reverse-phase chromatographic column (150mm × 4.6mm, 5 μm), mobile phase is methanol: water=90:10, flow velocity 1.00mL·min-1, 40 DEG C of column temperature, ultraviolet detection wavelength 245nm, 20 μ L of sample volume.External standard method presses peak area quantification.
The result shows that bacterial strain PHE-2 can luxuriant and rich with fragrance be that carbon source for growth is good, it is 100mgL to initial concentration in 5d-1's Luxuriant and rich with fragrance degradation rate reaches 95% or more；And when culture is to 6d, bacterial strain reaches 99.3% (Fig. 2 a) to luxuriant and rich with fragrance degradation rate.Bacterial strain PHE-2 with anthracene, pyrene orFor same well-grown on the minimal medium of carbon source: 30 DEG C of shaking table culture 84h, bacterial strain PHE- The degradation rate of 2 pairs of anthracenes reaches 91.1% (Fig. 2 b)；30 DEG C of shaking table culture 7d reach 62.9% (Fig. 2 c) to the degradation rate of pyrene；30 DEG C shaking table culture 9d is rightDegradation rate reach 92.6% (Fig. 2 d).
The influence luxuriant and rich with fragrance to bacterial strain PHE-2 degradation of 4 environmental condition of embodiment
By measure containing in luxuriant and rich with fragrance inorganic salts culture solution bacterial number and luxuriant and rich with fragrance residual concentration come probe into temperature, initial pH, The influence luxuriant and rich with fragrance to bacterial strain PHE-2 degradation of inoculum concentration, substrate phenanthrene concentration.Specific implementation details are as follows:
To 3mL contain luxuriant and rich with fragrance minimal medium (with embodiment 1, luxuriant and rich with fragrance initial content 100mgL-1) in be added 5% bacterium it is outstanding Liquid (i.e. the function microbial inoculum of embodiment 2, similarly hereinafter), temperature are set as 20,25,30,35,40,45 DEG C, 180rmin-1Shaking table is protected from light training Support 6d.
Adjusting minimal medium (with embodiment 1, luxuriant and rich with fragrance initial content 100mgL-1) initial pH value be 4.0,5.0, 6.0,7.0,8.0,9.0,10.0, to 3mL difference pH value containing the bacteria suspension that 5% is added in luxuriant and rich with fragrance minimal medium, 30 DEG C, 180r·min-1Shaking table is protected from light culture 6d.
To 3mL contain luxuriant and rich with fragrance minimal medium (with embodiment 1, luxuriant and rich with fragrance initial content 100mgL-1) in be added 2%, 4%, 6%, 8%, 10%, 12% bacteria suspension, 30 DEG C, 180rmin-1Shaking table is protected from light culture 6d.
It is 50,100,150,200,250,300,400mgL to initial luxuriant and rich with fragrance concentration-13mL contain in luxuriant and rich with fragrance minimal medium 5% bacteria suspension, 30 DEG C, 180rmin are added-1Shaking table is protected from light culture 6d.
Three groups of repetitions are arranged in each processing, and the PHE-2 of setting inoculation inactivation is control.The number of bacterial strain PHE-2 in culture medium It measures and is counted surely using gradient dilution spread plate, the measurement of culture medium China and Philippines residual content is the same as embodiment 1.
The result shows that environmental condition significantly affects the growth of bacterial strain PHE-2 and to luxuriant and rich with fragrance degradation, but all in all, bacterial strain PHE-2 has stronger heat-resisting and resistance to alkali ability.When temperature is 30-40 DEG C (Fig. 3 a) or pH is 7.0-10.0 (Fig. 3 b), bacterium Strain PHE-2 all well-growns and 90% or more of (30 DEG C of temperature, pH7.0, similarly hereinafter) is up under optimum condition to luxuriant and rich with fragrance degradation rate. It increases inoculum concentration and is remarkably improved bacterial strain PHE-2 to luxuriant and rich with fragrance degradation rate, and after inoculum concentration is greater than 6%, bacterial strain is to phenanthrene Degradation rate stablizes 90% or more (Fig. 3 c) under optimum condition；Concentration of substrate also will affect the growth of bacterial strain PHE-2 and its right Luxuriant and rich with fragrance degradation.When luxuriant and rich with fragrance concentration is in 100mgL-1Within when, bacterial strain PHE-2 is to luxuriant and rich with fragrance degradation rate more than 90% in 6d；And work as Luxuriant and rich with fragrance concentration is greater than 200mgL-1When, the growth of bacterial strain PHE-2 is obviously suppressed, and is also remarkably decreased (figure to luxuriant and rich with fragrance degradation rate 3d)。
The growth-promoting characteristic of 5 bacterial strain PHE-2 of embodiment
The determination step that bacterial strain PHE-2 produces heteroauxin (IAA) ability is as follows: by bacteria suspension (the i.e. function bacterium of embodiment 2 Agent) with what 5% inoculum concentration access 5mL contained L-Trp there is (20%2.5mgmL in nitrogen culture medium-1L-Trp mother liquor, V:v), 30 DEG C, 180rmin-1Shaking table culture for 24 hours after, will be enlarged by culture solution and be placed in 8000rmin-1High speed centrifugation 10min. It takes 1mL supernatant to be added in 5mL centrifuge tube, while 50 μ L 10mmolL is added-1Phosphoric acid solution and 2mL color developing agent (1mL 0.5mol·L-1FeCl3·7H2O and 49mL 35%HClO4Mixed liquor) it mixes well, it is placed in dark place, 25 DEG C of standing 30min, Using ultraviolet specrophotometer in 530nm colorimetric, the PHE-2 of setting inoculation inactivation is control.Wherein, there is nitrogen culture medium prescription (g·L-1): sucrose 10.0, (NH4)2SO41.0, K2HPO42.0, MgSO4·7H2O 0.5, NaCl 0.1, yeast powder 0.5, CaCO30.5, pH 7.0.
The determination step that bacterial strain PHE-2 produces siderophore (Siderophore) ability is as follows: 1mL bacteria suspension is accessed 20mL Have in nitrogen culture medium, 30 DEG C, 180rmin-1Shaken cultivation 48h, 8000rmin-1High speed centrifugation 10min takes 3mL supernatant In 10mL centrifuge tube, be added equivalent CAS detection liquid, mix well, stand 2h after, using ultraviolet specrophotometer in 630nm measures absorbance.The PHE-2's for separately taking 3mL CAS detection liquid and 3mL inoculation to inactivate has the mixing of nitrogen medium supernatant to make For blank control group (CK).According to the ratio of experimental group and CK, judge that bacterial strain PHE-2 produces the size of siderophore ability.
The measurement of bacterial strain PHE-2 phosphate solubilization uses organic phosphorus (egg yolk lecithin EYPC) and Phos (Ca3(PO4)2) solid Body culture medium Soluble phosphorus circle method measures the ratio of the transparent loop diameter of strains tested bacterium colony Soluble phosphorus (D) and colony diameter (d).Culture Measurement and ratio calculated (D/d) after 12d.Ratio is bigger, and phosphate solubilization is stronger, and ratio is smaller, and phosphate solubilization is weaker, ratio 1 When indicate bacterium colony without phosphate solubilization.Organic phosphorus solvability measurement culture medium uses Meng Jinna culture medium, the measurement of Phos Soluble phosphorus Culture medium uses PKO culture medium.
Plant growth-promoting effect test result shows that bacterial strain PHE-2 can generate up to 21.10mgL in having nitrogen culture medium-1 IAA, chromogenic reaction pinkiness, this illustrate bacterial strain PHE-2 produce IAA ability it is stronger；And bacterial strain PHE-2 has certain production Siderophore ability.In addition, the Soluble phosphorus circle that bacterial strain PHE-2 is formed on phosphorous culture medium is as shown in figure 4, show PHE-2 pairs of bacterial strain Phos and it is organic phosphorus have certain solvability, the ability of dissolved metals is greater than the ability of dissolved organic phosphorus.
The application of 6 function bacterium microbial inoculum of embodiment
It is tested by Greenhouse Water Culture, mode is colonized using leaching root, bacterial strain PHE-2 is colonized into water spinach root table, studies it Influence to water spinach absorption and accumulation phenanthrene, specific test procedure are as follows:
(1), bacterial strain label and seed disinfection vernalization
Fresh PHE-2 bacteria suspension (i.e. the function microbial inoculum of embodiment 2, similarly hereinafter) gradient dilution is coated on various concentration Antibiotic LB plate (antibiotic: gentamicin, ampicillin, kanamycins, tetracycline, chloramphenicol, spectinomycin, strepto- Element；Concentration: 10,20,40,60,80,100mgL-1).Using the LB solid plate of not added with antibiotic as control.In 30 DEG C of constant temperature 48h is cultivated, bacterium colony growing state in culture dish is observed.The result shows that bacterial strain PHE-2 to the ampicillin of high concentration, block that Mycin, streptomysin and gentamicin (100mgL-1) resistant.Therefore, select ampicillin and kanamycins as bacterium The dual anti-label of strain, to track distribution situation of the bacterial strain in plant roots table and culture solution.
It takes water spinach seed ultrapure water clean, 10-20min, surface sterilization is impregnated in 1% liquor natrii hypochloritis 8-12h is impregnated in sterile water afterwards, seed is uniformly sprinkling upon in seedlings nursing plate, vernalization in artificial time incubator is placed in, during vernalization Paying attention to being protected from light, incubator temperature is set as 25 DEG C of daytime, and 20 DEG C of night.
(2), pot experiment designs
Experimental design 8 processing.CK1: contain 1.0mgL-1The Hoagland solution of phenanthrene pollution does not plant water spinach；CK2: contain 10.0mg·L-1The Hoagland solution of phenanthrene pollution does not plant water spinach；UP1: Hoagland solution kind water spinach is inoculated with inactivated bacteria；UP2: Hoagland solution kind water spinach inoculating strain PHE-2；CP1: contain 1.0mgL-1The Hoagland solution kind water spinach inoculation of phenanthrene pollution Inactivated bacteria；CP2: contain 10.0mgL-1The Hoagland solution kind water spinach of phenanthrene pollution is inoculated with inactivated bacteria；CPR1: contain 1.0mgL-1 The Hoagland solution kind water spinach inoculating strain PHE-2 of phenanthrene pollution；CPR2: contain 10.0mgL-1The Hoagland solution kind of phenanthrene pollution Water spinach inoculating strain PHE-2.5 repetitions are arranged in each processing.
When water spinach seedling length to three pieces true leaf, it is transplanted in the brown, wide-mouth bottle containing Hoagland solution.For Water spinach seedling is placed in fresh PHE-2 bacteria suspension after impregnating 6h, then transplants in being equipped with by the processing for needing inoculating strain PHE-2 (concentration is respectively 1,10mgL to the pollution of 250mL phenanthrene-1) Hoagland solution brown, wide-mouth bottle in.Every bottle is planted 8 plants, artificial Plant is cultivated in climate box, diurnal temperature is respectively set to 25 and 20 DEG C, and the time is respectively 12h, cultivates 15d.It periodically adds fresh Hoagland solution maintain liquid level, sampled in 15d, measure plant fresh weight, and plant is freeze-dried 72h, planted Object dry weight.
Plant weighing the result shows that, under without luxuriant and rich with fragrance pollutional condition, inoculating strain PHE-2 group (UP2) plant goes out with inoculation Viable bacteria group (UP1) compare, the fresh weight of plant and dry weight have been respectively increased 7.86%, 39.28% after water planting 15d.And in 1.0mg L-1Under luxuriant and rich with fragrance pollution concentration, after water planting 15d, inoculating strain PHE-2 group (CPR1) than inoculation inactivated bacteria group (CP1) plant fresh weight and 48.59% and 62.51% has been respectively increased in dry weight.Illustrate regardless of whether inoculating strain PHE-2 can all promote under luxuriant and rich with fragrance pollutional condition Into the growth of water spinach.
(3), the observation of water spinach root table bacterial biof iotalm
After water spinach water planting 3d, takes its root system (being about 3cm) segment to be placed in 5mL centrifuge tube, 2.5% glutaraldehyde is added Solution submerges root system, and centrifuge tube is placed in -4 DEG C of refrigerators and fixes 2h or more, sample is sent to Agricultural University Of Nanjing's life science Institute's Electron Microscopy Room is further processed, finally with scanning electron microscope (SEM) observation bacterial strain PHE-2 water spinach root table at Film situation.
SEM observed result shows (Fig. 5), is handled by the leaching root of function stem PHE-2, after water planting 3d, water spinach root Surface has a large amount of bacterial accumulation (Fig. 5 B), and the bacterium colonized in water spinach root table forms biofilm structure, part bacterium It is adhered on film, biomembrane is in the space structure (Fig. 5 C and 5D) of accordion.Functions bacterium PHE-2, which can stablize, to be colonized in sky Heart dish root table simultaneously forms bacterial biof iotalm.Counting discovery, water planting phase further are carried out to the bacterial strain PHE-2 colonized in plant roots table Between, it is 1.0mgL in luxuriant and rich with fragrance content-1Culture solution in, colonize the bacterial strain PHE-2 quantity in water spinach root table in 5.37- 6.92LogCFU·g-1Between fresh weight；It and is 10.0mgL in luxuriant and rich with fragrance content-1Culture solution in, colonize the bacterium in water spinach root table Strain PHE-2 quantity is in 5.73-7.01LogCFUg-1Between fresh weight.
(4), the extraction and measurement of plant sample China and Philippines
The root of water spinach and cauline leaf are cut with scissors, is sub-packed in valve bag, is placed in freeze drier and is freeze-dried 2-3 days, by after freeze-drying plant roots and cauline leaf with mortar be fully ground crushing, then weigh a certain amount of plant sample in 30mL In glass centrifuge tube, the solution ultrasonic extraction 30min of 10mL methylene chloride and n-hexane (V:V=1:1) is added, is repeated 3 times. Whole extract liquors are crossed into anhydrous sodium sulfate-silica gel column purification, with methylene chloride and n-hexane (V:V=1:1) mixed liquor of 10mL Elution.Collect eluent dress in a round bottom flask, 40 DEG C of constant temperature are concentrated to dryness, and add 2mL methanol constant volume, cross 0.22 μm of aperture filter Film, with the content of HPLC/UV (with embodiment 1) measurement plant roots and cauline leaf China and Philippines.
The result shows that the luxuriant and rich with fragrance content in root system of plant and cauline leaf is increased, and is inoculated with function with the increase of luxuriant and rich with fragrance concentration The luxuriant and rich with fragrance content of plant root and leaves and stems can be effectively reduced in energy bacterium PHE-2.Specifically, in 1.0mgL-1Under pollution concentration, After water planting 15d, in CP1In group, the content of root system of plant China and Philippines is 2.33mgkg-1, the content of cauline leaf China and Philippines is 0.38mg kg-1；And in CPR1In group, the content of root system of plant China and Philippines is 1.25mgkg-1, the content of cauline leaf China and Philippines is 0.36mgkg-1.With CP1It compares, CPR1The content of root system of plant and cauline leaf China and Philippines reduces 46.35%, 5.26% respectively.And in 10.0mg L-1Under pollution concentration, after water planting 15d, in CP2In group, the content of root system of plant China and Philippines is 20.67mgkg-1；Cauline leaf China and Philippines Content is 1.58mgkg-1；And in CPR2In group, the content of root system of plant China and Philippines is 12.21mgkg-1；Cauline leaf is Sino-Philippines to be contained Amount is 0.73mgkg-1.With CP2It compares, CPR2The content of root system of plant and cauline leaf China and Philippines reduces 40.93% respectively, 53.8%.Correspondingly, the luxuriant and rich with fragrance accumulation in water spinach is also substantially reduced after inoculation function stem PHE-2.Specifically, In 1.0mgL-1Under pollution concentration, after water planting 15d, compared with CP1 group, the accumulation of CPR1 group root system of plant and cauline leaf China and Philippines 73.17% and 39.34% are reduced respectively；And in 10.0mgL-1Under pollution concentration, after water planting 15d, compared with CP2 group, CPR2 group root system of plant and the accumulation of cauline leaf China and Philippines reduce 25.48% and 22.36% respectively.

Claims (10)

1. one plant of rhizosphere growth-promoting endophytic bacteria PHE-2 with PAHs degradation capability, classification naming is series bacillus (Paenibacillus sp.) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on August 26th, 2014 Center, culture presevation number are CGMCC No.1.12900.
2. a kind of function microbial inoculum containing rhizosphere growth-promoting endophytic bacteria PHE-2 described in claim 1.
3. the function microbial inoculum according to claim 2 containing rhizosphere growth-promoting endophytic bacteria PHE-2, it is characterised in that it be by with Made from the method for lower section:
(1), rhizosphere growth-promoting endophytic bacteria PHE-2 is inoculated in fermentation medium, shaken cultivation to logarithmic phase；
(2), the strain of culture to logarithmic phase is inoculated with by 5% inoculum concentration into seeding tank, cultivates to logarithmic phase, obtains seed Liquid；Seed liquor is produced into tank culture by 10% inoculum concentration access；
Wherein, seeding tank and production tank used in fermentation medium it is identical as the fermentation medium of step (1), fermentation medium Formula are as follows: peptone 10.0gL-1, yeast 5.0gL-1, NaCl 10.0gL-1, pH 7.0-7.4；
The ventilatory capacity of filtrated air is 1:1~1.2 in the incubation of seeding tank and production tank, and mixing speed is 180~ 220 revs/min, cultivation temperature is 28~30 DEG C, and whole process incubation time is 55~60 hours, and thalline quantity reaches after fermentation 1000000000/ml or more；
(3), culture solution goes out tank after fermentation, adjusts culture solution concentration to OD600nm=1.0 with fermentation medium, obtains function It can microbial inoculum.
4. the rhizosphere growth-promoting endophytic bacteria PHE-2 described in claim 1 with PAHs degradation capability is in the application of degradation PAHs.
5. application according to claim 4, it is characterised in that the application is in degradation water body, soil and plant The application of interior PAHs.
6. the function microbial inoculum as claimed in claim 2 containing rhizosphere growth-promoting endophytic bacteria PHE-2 is in the application of degradation PAHs.
7. application according to claim 6, it is characterised in that the application is in degradation water body, soil and plant The application of interior PAHs.
8. the application according to claim 4 or 6, it is characterised in that the PAHs be selected from phenanthrene, anthracene, pyrene andIn it is any It is one or more.
9. the rhizosphere growth-promoting endophytic bacteria PHE-2 described in claim 1 with PAHs degradation capability is in the application for promoting plant growth.
10. the function microbial inoculum as claimed in claim 2 containing rhizosphere growth-promoting endophytic bacteria PHE-2 is in the application for promoting plant growth.
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