CN105950502A - Complex endophytic bacterial inoculant and application thereof in phytoremediation of heavy metal contaminated soil - Google Patents
Complex endophytic bacterial inoculant and application thereof in phytoremediation of heavy metal contaminated soil Download PDFInfo
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Abstract
The invention discloses a complex endophytic bacterial inoculant and an application thereof in phytoremediation of heavy metal contaminated soil. The complex inoculant is composed of acinetobactercalcoaceticus, pseudomonas fluorescens, bacillusmegaterium, stenotrophomonasmaltophilia, bacillus pumilus and pseudomonas putida. The complex inoculant disclosed by the invention, when applied to heavy metal pollution remediation, integrates advantages of various endophytic bacteria and overcomes disadvantages of phytoremediation; and the complex inoculant can promote the growth of sedum alfredii hance, enhance the absorbing capacity of the sedum alfredii hance to heavy metals and improve the efficiency of phytoremediation.
Description
Technical field
The present invention relates to phytoremediation technology field, be specifically related to a kind of compound endogenetic bacteria microbial inoculum and
Application in phytoremediation.
Background technology
Along with quickly propelling of industrial high speed development, the modernization of agricultural production and urbanization, soil
Earth Heavy Metal Pollution is the most serious.
Heavy metal cannot be degraded by microorganisms, and has poor mobility, holdup time length, disguise, no
The features such as reversibility, soil once contaminated by heavy metals with regard to extremely difficult reduction.Heavy metal one in soil
Aspect can affect the multiple metabolic activity such as plant growing, cell division, the growth of suppression crop thus
Reduce crop yield.On the other hand, the heavy metal in soil can be entered by Removed In Soil-crop System
Enter food chain, bring great risk to human health.In the face of tens huge sum of moneys occurred in the past few years
Belong to the Mass disturbance caused, reduce Mobility of Heavy Metals In Soil Environment content, reduce its dirt to agricultural product
Dye, ensures that ecosystem and human health are extremely the most urgent.
In face of limited land resource, the reparation of heavy-metal contaminated soil becomes agricultural sustainable development
A vital task.Owing to heavy metal can not can directly be passed through planting as organic pollutant
Degraded in thing rhizosphere or plant eliminates pollutant, the difficulty of the phytoremediation of heavy metal in soil
Much greater.
Phytoremediation (phytoremediation) is to utilize green plants transfer, receiving or conversion environment
The based technique for in-situ remediation of middle pollutant, with conventional art such as soil moved in improve the original method, soil fixing, Soil leaching etc.
Physics is compared with chemical method, has efficiently, environmental friendliness, low cost, does not cause secondary pollution etc.
Advantage.Sedum alfredii Hance initially finds on location, ancient Pb/Zn ore deposit, Quzhou City of Zhejiang Province, is in first
The Zn/Cd hyperaccumulative plant that state is primary, it is possible to efficiently extract Zn, Cd, Pb element in soil,
It is not only research plant absorption, transhipment and ultraproduct and tires out the good model material of Zn/Cd mechanism, also carry
Rising the good material of the heavy metal-polluted dyeing technique of phytoremediation, in its plant, heavy metal concentration is in soil
Several times even tens times.Although Sedum alfredii Hance compares most heavy metal in growth rate with Biomass
Hyperaccumulative plant wants some advantage, but the efficiency polluted of its repairing heavy metal in soil also suffer from growth slow,
Biomass is little, adaptability is weak and the restriction of giving property of heavy metal-polluted soil, therefore, how to promote southeast scape
It growth, degeneration-resistant, improve Biomass and to the absorption of heavy metal-polluted soil, transport, accumulate heavy metal energy
Power, this has highly important for the actual application of application Sedum alfredii Hance restoration of soil polluted by heavy metal
Meaning.
Summary of the invention
The invention provides a kind of endogenetic bacteria microbial inoculum, this microbial inoculum can gather the excellent of several endogenetic bacteria
Gesture, overcomes the weakness of phytoremediation, effectively facilitates the growth of Sedum alfredii Hance, strengthens Sedum alfredii Hance counterweight
The absorbability of metal, improves phytoremediation efficiency.
A kind of compound endogenetic bacteria microbial inoculum, by acinetobacter calcoaceticus
(Acinetobactercalcoaceticus), pseudomonas fluorescens (Pseudomonas fluorescens),
Bacillus megaterium (Bacillusmegaterium), germ oligotrophy unit cell
(Stenotrophomonasmaltophilia), bacillus pumilus (Bacillus pumilus) and stench
Pseudomonas (Pseudomonas putida) forms.
Above six strain endogenetic bacterias are all saved in and are positioned in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
In China Committee for Culture Collection of Microorganisms's common micro-organisms of institute of microbiology of academy of science of state
The heart, preservation information is as follows:
Acinetobacter calcoaceticus (Acinetobactercalcoaceticus) was protected on March 19th, 2013
Hiding, Classification And Nomenclature is acinetobacter calcoaceticus (Acinetobactercalcoaceticus), and bacterial strain number is
Sasm3, deposit number is CGMCC No.7334.This bacterial strain is at Publication No. CN 103266073A
Application for a patent for invention document disclosed in.
Pseudomonas fluorescens (Pseudomonas fluorescens) in preservation on March 3 in 2016,
Classification And Nomenclature is pseudomonas fluorescens (Pseudomonas fluorescens), and bacterial strain number is SM05,
Deposit number is CGMCC No.12173.
Bacillus megaterium (Bacillusmegaterium) is in preservation on March 11 in 2013, classification
Named bacillus megaterium (Bacillusmegaterium), bacterial strain number is SAN1, deposit number
For CGMCC No.7295.This bacterial strain is in the application for a patent for invention of Publication No. CN 103275891A
Disclosed in document.Germ oligotrophy unit cell (Stenotrophomonasmaltophilia) is in 2016
Preservation on March 3, Classification And Nomenclature is germ oligotrophy unit cell
(Stenotrophomonasmaltophilia), bacterial strain number is SMN1, and deposit number is CGMCC
No.12171。
Bacillus pumilus (Bacillus pumilus) is in JIUYUE in 2014 preservation on the 19th, classification life
Entitled bacillus pumilus (Bacillus pumilus), bacterial strain number is SrN7, and deposit number is
CGMCC No.9685.This bacterial strain is at the application for a patent for invention literary composition of Publication No. CN104862255A
Disclosed in offering.
Pseudomonas putida (Pseudomonas putida) is in preservation on March 3 in 2016, classification
Named pseudomonas putida (Pseudomonas putida), bacterial strain number is SMN6, deposit number
For CGMCC No.12172.
The compound endogenetic bacteria microbial inoculum named plant growth-promoting composite bacteria agent capable PLSa01 of the present invention.
6 strain endophytic bacterial controlled effects involved by this microbial inoculum all screen the hyperaccumulative plant southeast scape from surface sterilizing
It stem and blade.Using conventional endophyte separation method, whole strain Sedum alfredii Hance is removed stale
Blade, tap water is rinsed well, then by every some respectively with 70% ethanol leaching 3min, aseptic
Wash 3 times, 0.5%HgCl2Soak 1min, then use aseptic water washing more than 5 times, and will be
The sterilized water of rear once washing is coated with LB flat board to confirm the aseptic condition on plant surface.
Prepare LB solid medium.In clean superclean bench, with aseptic operation cutter, (flame burns
Cool down after burning sterilizing) cutting plant sample thin slice, and thin slice is laid in the culture medium of culture dish,
Each culture medium about 9 (as shown in Figure 1), seals culture dish with sealed membrane and is inverted in 30 DEG C of cultivations
Case is cultivated one week.Lower Sedum alfredii Hance section bacterial plaque around, line three times points repeatedly is chosen with inoculating loop
Separate out single bacterium colony, be inoculated in LB test tube slant, 4 DEG C of preservations (can preserve 2 months) respectively.
Identify the physicochemical property being screened the endogenetic bacteria obtained, find that wherein 6 strain endogenetic bacterias can
Resistance to higher concentration heavy metal, ability produce IAA and acc deaminase, the most do not grow short of money
Anti-factor, manufactures simple, and above-mentioned six strain bacterial strains are mixed to get multiple by composite bacteria agent capable mixed method routinely
Closing microbial inoculum, in composite bacteria agent capable, each strain quantity is stable, it is possible to form respond well endophyte compound bacteria
Agent.
6 strain endogenetic bacterias of composition composite bacteria agent capable are each other without antagonism, and the speed of growth is consistent,
Normal inoculation and cultivation can obtain, such as: every strain antibacterial takes bacterium solution 20 μ L, joins 100mL
Liquid LB (every 1000mL culture medium contains: sodium chloride 10g, yeast powder 5g, peptone 10g,
PH is 7.2) in culture medium, 150rpm, 35 DEG C, 48h, i.e. can get microbial inoculum bacterium solution, measure bacterium
Liquid obtains each strain bacterial concentration all not less than 108CFU。
The physicochemical property of 6 strain antibacterials of the compound endogenetic bacteria microbial inoculum of composition is as shown in table 1:
Table 1 is combined the composition of endogenetic bacteria microbial inoculum and the physicochemical property relevant to growth-promoting
"+" expression possesses respective capabilities；"-" represents do not possess respective capabilities.
The present invention also provides for a kind of described compound endogenetic bacteria microbial inoculum in phytoremediation heavy metal pollution
Application.
Preferably, at least one during described heavy metal is Pb, Zn and Cd.
Preferably, described plant is Sedum alfredii Hance.
When described plant is Sedum alfredii Hance, a kind of application mode is: use water culture to Sedum alfredii Hance
Carry out Fast-propagation, in the 4th week, in culture fluid, add described compound endogenetic bacteria microbial inoculum.
Further, described nutritional solution is: K2SO4, 0.7mmol/L；KCl, 0.1mmol/L；
Ca(NO3)2·4H2O, 2mmol/L；MgSO4·7H2O, 0.5mmol/L；KH2PO4, 0.1mmol/L；
H3BO3, 10 μm ol/L；MnSO4·H2O, 0.5 μm ol/L；ZnSO4·7H2O, 1 μm ol/L；
(NH4)4MoO244H2O, 0.01 μm ol/L；FeSO4·7H2O is dissolved in 20 μm ol/L boiled
Na2In EDTA solution, 20 μm ol/L, adjust nutrition pH at night 5.8 with NaOH or HCl.
Described method for quickly breeding is: the branch that clip Sedum alfredii Hance is newborn, every treaty 10cm length,
Leaving behind 5～8 blades of shoot apex, insert it in 0.1 times of mill water culture nutrient solution, temperature is
23～25 DEG C, illumination condition is 200～300 μ E/m2S, after 3 weeks, shoot growth goes out fraction of,
Again with 1 times of above-mentioned Solution culture method, and ventilate in nutritional solution.Within every 3～4 days, change one time of nutrition liquid.
The inoculum concentration of composite bacteria agent capable described in nutritional solution is 104～108CFU, inoculation January after to the southeast
Herba hylotelephii erythrosticti root system scanning survey, finds 104CFU system general length and tip of a root quantity are substantially less than at other
Reason, 105～108Between CFU, difference is not notable, according to economy principle, selects minimum 105CFU conduct
Inoculum density.
Process Sedum alfredii Hance by this kind of method, after grow 1 month and do not inoculate PLSa01 to photograph
Ratio, the Sedum alfredii Hance growth of inoculation microbial inoculum PLSa01 is more vigorous, and root system is more flourishing.Collect east
The root system of south Herba hylotelephii erythrosticti measures its weight and length, and after inoculation PLSa01, the gross weight of root system increases by 200%
Above, system general length increases nearly 300%.Owing to Sedum alfredii Hance trophophase is shorter, the life of overground part
Thing amount does not embodies significant difference.
When plant is Sedum alfredii Hance, another kind of application mode is: will be well-grown in nutritional solution
Sedum alfredii Hance root system immerse described compound endophyte microbial inoculum in 3～5h, continue water planting transplant after one week
To mine soil potted plant in.
Preferably, described Sedum alfredii Hance is 4～June at the mine potted plant middle growth time of soil.
Soil in potted plant is the mine soil of heavy metal pollution, it is further preferred that described mine is native
Physicochemical property as shown in table 3.
According to said method processing Sedum alfredii Hance, after inoculation PLSa01, Sedum alfredii Hance the upperground part biomass is bright
Aobvious increase.After Sedum alfredii Hance is gathered in the crops, the Biomass of overground part and underground part (i.e. root) is carried out
Weigh and analyze discovery, compared with control treatment, after inoculation PLSa01, the overground part of Sedum alfredii Hance
Dramatically increasing in various degree, wherein, the increase of the upperground part biomass is all had with the Biomass of underground part
59%, underground part Biomass increases by 61%, illustrates that endogenetic bacteria microbial inoculum PLSa01 can remarkably promote
Sedum alfredii Hance overground part and the growth of underground part.
The compound endophyte agent of the present invention can also be used with for continuous repair heavy-metal contaminated soil, it is preferable that
Described application includes: take Sedum alfredii Hance top branch root culture in nutritional solution, by described compound interior
Bacteria-promoting agent is seeded in the root system of Sedum alfredii Hance, by the Sedum alfredii Hance plantation after compound for inoculation endophyte agent
In heavy-metal composite pollution mine soil.
Need not be again in the Growing season that Sedum alfredii Hance after the compound endophyte agent of inoculation is planted for the first time
Secondary inoculation endophyte, the inoculation of the most each season of growth once, cradles second after plantation for the first time
Individual Growing season starts, and gathers in the crops overground part, and during harvesting, overground part retains stem 4～about 8cm, the most often
Individual Growing season is gathered in the crops once.
When postvaccinal Sedum alfredii Hance is just planted, growth is relatively slow, and 9 annual～December and 3～June are it
Growing season.In i.e. 1 year 2 seasons of results, plant the most continuously, it is achieved gather in continuously, high-efficiency and continuous reparation.
Described Sedum alfredii Hance seedling density in soil is every square metre of plantation 70～100 strains.
The method not having individual Growing season to inoculate Sedum alfredii Hance afterwards is: during inoculation, will wash away cultivation
The endogenetic bacteria composite bacteria agent capable PLSa01 of base mixes as substrate with fertilizer or Nutrition Soil, keeps bacterium
Bulk concentration is 108CFU substrate, humidity 60～70%, ditching deep placement (also can spill and execute), dig the rainy day
Ditch depth amount of application is 0.12～0.15kg/m2。
By this kind of application process, after inoculation endophyte, the survival rate of Sedum alfredii Hance is close to 100%, logical
Often not inoculating endophyte plant survival rate is 71.0～90.1%, and the Sedum alfredii Hance inoculation grown in the wild
Being caused Seedling strain mortality rate to be substantially reduced more than 50% by harvesting afterwards, Seedling strain regrows more vigorous.?
The Sedum alfredii Hance inoculation endophyte of field grown is after 3 months, and same Biomass overground part absorbs a huge sum of money
Zn, Cd amount that belongs to has been respectively increased 18.7% and 47.48%.
Accompanying drawing explanation
Figure 1A and Figure 1B is the result figure that line determines the antagonism relationship between each bacterial strain of antibacterial.
Fig. 2 is that inoculation compares figure with the growing state of Sedum alfredii Hance root system under non-inoculation condition.
Fig. 3 is that earth culture experiment lower inoculation endophyte microbial inoculum affects figure to Sedum alfredii Hance growing way.
Fig. 4 inoculates microbial inoculum PLSa01 to Sedum alfredii Hance root top and the impact of root biomass.
Fig. 5 inoculates microbial inoculum PLSa01 to Sedum alfredii Hance root top and the impact of root system Zn concentration.
Fig. 6 inoculates microbial inoculum PLSa01 to Sedum alfredii Hance root top and the impact of root system Cd concentration.
Fig. 7 inoculates microbial inoculum PLSa01 to Sedum alfredii Hance root top and the impact of root system Zn accumulation.
Fig. 8 inoculates microbial inoculum PLSa01 to Sedum alfredii Hance root top and the impact of root system Cd accumulation.
Detailed description of the invention
Embodiment 1 promotees Sedum alfredii Hance biological restoration and is combined the overall characteristic of endogenetic bacteria microbial inoculum
The microbial inoculum made takes 200 μ L be seeded in 100mL culture medium, cultivate 36h for 35 DEG C,
The microbial inoculum part arrived, in order to preservation, adds 300 μ L sterile glycerols in 1.5mL sterile centrifugation tube
With 700 μ L logarithmic (log) phase bacterium solution ,-80 DEG C of preservations (can preserve 2 years), remaining liquid microbial inoculum closes
The qualification of key physiological and biochemical index.
1, pathogenic qualification
Above-mentioned bacterium solution is inoculated into fresh damaged blade face (Sedum alfredii Hance, Nicotiana tabacum L., Brassica campestris L), with nothing
Bacterium water compares, and does not finds blade Disease Characters；100 μ L bacterium solution injections are extracted with asepsis injector
In the Bulbus Allii Cepae stem of fresh and healthy, compare with sterilized water, cultivate one week, do not find that Bulbus Allii Cepae has for 30 DEG C
Disease Characters.
2, antagonistic experiment between plant-growth promoting rhizobacteria
By whether there is obvious the antagonism relationship between line measuring each growth-promoting antibacterial.Such as Figure 1A
Shown in the black wire frame in Figure 1B, we, when the selection of growth promoting bacteria agent, find between some antibacterial
There is obvious the antagonism relationship, at this moment need the quality according to growth-promoting effect to remove growth-promoting effect poor
Strain.
3, resistance to Zn/Cd feature
To containing Zn/Cd LB culture medium (contain respectively Concentraton gradient: Zn (1,2,4,5,8,
10,12,15,20,25,30,35 or 40mM) or Cd (0.05,0.1,0.2,0.3,
0.4,0.5,0.6,0.7,0.8,1.0,1.2,1.5,2.0,2.5,3.0,3.5,4.0 or 5.0mM))
Middle inoculation 10 μ L logarithmic (log) phase bacterium solution, cultivates 36 hours for 37 DEG C, observes endogenetic bacteria at different Zn/Cd
Growing state under concentration.Bacterium solution cultivation obtained dilutes 10 respectively5～109Times, according to bacterial community
Form judges each strain quantitative proportion in bacterium solution；For the antibacterial that cannot be differentiated by colonial morphology,
Then tried by different characteristics such as the two heavy metal patience, product IAA and product acc deaminases
Pipe is cultivated and judges the two ratio in bacterial community.
4, acc deaminase ability is produced
When by growth-promoting rhizosphere bacterium field planting to seed coat or root system of plant, can synthesize and secrete growth hormone
IAA, newly synthesized IAA will be absorbed by plant and form Endogenous auxin, thus stimulates plant thin
The division of born of the same parents and prolongation.Meanwhile, IAA can excite the activity of ACC synzyme, by S-adenosine
Methionine changes into 1-amino-cyclopropane-1-carboxylic acid.Acc deaminase is to measure plant growth-promoting rhizobacteria to promote
Enter one of index of plant growing ability.
With reference to Honma M and Shimomura T (Honma M&Shimomura T.Metabolism
Of 1-aminocyclopropane-1-carboxylic acid.AgricBiolChem, 1978,42:
That 1825-1831) sets up produces the activity of acc deaminase with mensuration α-one butanoic acid to detect endogenetic bacteria
Method.
5, IAA ability is produced
Heteroauxing (IAA) be a kind of can coordinate plant growth grow auxin.
Use Salkowski colorimetry (Sarwar&Kremer.Determination of bacterially
Derived auxins using a microplate method.Letters in Applied Microbiology,
2008,20:282-285) measure separator and produce the ability of auxin class material IAA.
6, secretion is addicted to the mensuration of ferrum element ability
It is small molecule amount, the special chelated iron of energy synthesized under the conditions of low ferrum by microorganism addicted to ferrum element
The chelate of ion, secretes the antibacterial addicted to ferrum element and can be used for preventing and treating fungal disease.
Utilize CAS flat band method (Schwyn&Neilands.Universal chemical assay for the
Detection and determination of siderophores.Analytical Biochemistry, 1987,
160:47-56) detection bacterial secretory is addicted to the ability of ferrum element.
By detection, this microbial inoculum has resistance to Zn/Cd ability, produces acc deaminase ability, product IAA
Ability, secretion are addicted to ferrum element ability, and the minimum patience of microbial inoculum heavy metal to be significantly greater than single bacterial strain
The minimum patience of heavy metal, the ability of producing IAA and the ability of ACC are also remarkably reinforced.This bacterium
Every physiological and biochemical index of agent sees table 2 below.
The physiological and biochemical index that table 2 endogenetic bacteria microbial inoculum is relevant to promoting reparation
6, endogenetic bacteria composite bacteria agent capable PLSa01 determines, in plant, the situation of growing
The Sedum alfredii Hance root system taken root is soaked 4h in bacterium solution, is then transferred to normal water planting nutrition
Liquid is cultivated 1 week, takes the root of plant, stem, each 2g of leaf respectively, grind with phosphate buffer and plant
Fabric texture, draws and dilutes 10 times, 100 times and 1000 times, takes 200 μ L and is coated with solid LB cultivation
Flat board, lucifuge is inverted incubated overnight 24h, is added up the number of each endogenetic bacteria according to endophyte COMMUNITY CHARACTERISTICS
Amount, for the antibacterial that COMMUNITY CHARACTERISTICS is similar, can be by the ability of its resistance to heavy metal, product IAA, ACC
The different difference checkings of the ability of deaminase.
Embodiment 2 endogenetic bacteria composite bacteria agent capable is on Sedum alfredii Hance growth and the impact of heavy metal soil remediation
Nutritional solution consists of: K2SO4, 0.7mmol/L；KCl, 0.1mmol/L；Ca(NO3)2·4H2O,
2mmol/L；MgSO4·7H2O, 0.5mmol/L；KH2PO4, 0.1mmol/L；H3BO3, 10 μm ol/
L；MnSO4·H2O, 0.5 μm ol/L；ZnSO4·7H2O, 1 μm ol/L；(NH4)4MoO244H2O,
0.01μmol/L；FeSO4·7H2O is dissolved in the 20 μm ol/L Na boiled2In EDTA solution,
20μmol/L.Nutrition pH at night 5.8 is adjusted) with NaOH or HCl
(1) water planting environment
Use water culture that Sedum alfredii Hance is carried out Fast-propagation.The branch that clip Sedum alfredii Hance is newborn, often
Treaty 10cm length, leaves behind 5～8 blades of shoot apex, inserts it into 0.1 times of water planting battalion
In nutrient solution, temperature is 23～25 DEG C, and illumination condition is 200～300 μ E/m2S, after 3 weeks, branch is raw
Grow fraction of, then with 1 times of above-mentioned Solution culture method, and ventilate in nutritional solution.Every 3～4 days
Change one time of nutrition liquid.Thalline (the centrifugal receipts of endogenetic bacteria microbial inoculum are added in the 4th circumference nutritional solution
Collection logarithmic (log) phase thalline, sterilized water washs 3 times), inoculum concentration is that the thalline in every milliliter of nutritional solution is
105CFU, changes nutritional solution afterwards and does not inoculates.
Observe the growing state of Sedum alfredii Hance after growing 1 month, as in figure 2 it is shown, with do not inoculate
The comparison of PLSa01 is compared, and the Sedum alfredii Hance growth of inoculation microbial inoculum PLSa01 is more vigorous, and root system is more
Add prosperity.The root system collecting Sedum alfredii Hance measures its weight and length, root system after inoculation PLSa01
Gross weight increases by more than 200%, and system general length increases nearly 300%.Due to Sedum alfredii Hance trophophase relatively
Short, the Biomass of overground part does not embodies significant difference.
(2) earth culture environment
Use earth culture method plantation Sedum alfredii Hance.The best Sedum alfredii Hance root will be grown in nutritional solution
About 4h in microbial inoculum is immersed in system, and continuation water planting, after one week, is transplanted to mine soil potted plant central, mine soil
Physicochemical property be shown in Table 3, keep the humidity of soil about 55%, growth time is 4～June, totally 2
Individual month.
As seen from Figure 3, after inoculation PLSa01, Sedum alfredii Hance the upperground part biomass substantially increases.Will
After Sedum alfredii Hance results, the Biomass of overground part and underground part (i.e. root) is weighed and is analyzed,
Find, compared with control treatment, after inoculation PLSa01, the overground part of Sedum alfredii Hance and underground part
Biomass all has dramatically increasing in various degree, wherein, the increase by 59% of the upperground part biomass, underground
Portion's Biomass increases by 61% (as shown in Figure 4), illustrates that microbial inoculum PLSa01 can remarkably promote southeast scape
It overground part and the growth of underground part.
The basic physical and chemical of mine used soil tested by table 3
Heavy metal quantitative analysis and accumulation to Sedum alfredii Hance root and overground part are surveyed further
Determining and analyze, the detection of Zn and Cd uses conventional method.As shown in figures 5-8, inoculation PLSa01
After, Sedum alfredii Hance overground part and the heavy metal concentration of underground part and accumulation all dramatically increase, wherein,
Overground part and root Zn content concn increase by 30% and 41% respectively, the content concn of overground part Cd
Increasing by 54%, root Cd concentration increase is inconspicuous；Matched group Sedum alfredii Hance overground part Zn's is long-pending
Tired amount is 304.3mg, and the accumulation of root Zn is 5.6mg, after inoculation endophyte PLSa01,
The accumulation of Sedum alfredii Hance overground part Zn can reach 627.7mg, improves 106%, root Zn's
Accumulation also reaches 12.7mg, improves 126%.Meanwhile, after inoculation PLSa01, Sedum alfredii Hance
The accumulation of overground part Cd has reached 41.2mg, improves 144%, root accumulation raising 73%.
Compared with not inoculating PLSa01 process, plant-microorganism recovery technique system significantly improves east
South Herba hylotelephii erythrosticti Biomass (P < 0.01) and overground part heavy metal Cd concentration (P < 0.01).
It addition, we find also by test, under laboratory condition, do not inoculate the survival of endophyte plant
Rate is about 71.0～90.1%, and after inoculating endophyte, makes Sedum alfredii Hance seedlings plugging survival rate 96.8
～99.5%.Owing to mine soil soil property is barren, if increasing by about 20% Nutrition Soil microbial inoculum in mine soil
(pasture and water of rotting, concentration is 107～108The microbial inoculum PLSa01 of/g) after, Biomass more improve to
94.7～136.2%.
Embodiment 4 promotees the application of Sedum alfredii Hance biological restoration endogenetic bacteria
Sedum alfredii Hance Seedling strain Fast-propagation (with reference to the method for embodiment 3): take Sedum alfredii Hance top branch
Bar is taken root about 45 days in nutritional solution.LB fluid medium incubated overnight PLSa01 microbial inoculum, nothing
Bacterium washing goes culture medium, by 105CFU is inoculated into takes root in Seedling basket, aerobic culture 1 week.Now east
South Herba hylotelephii erythrosticti endophyte will be grown inside root, with root growth symbiosis surely.
Cottage propagation: Sedum alfredii Hance inoculation endophyte is after 1 week, and endophyte will be grown inside its root surely,
Planting in heavy-metal composite pollution mine soil by Sedum alfredii Hance Seedling strain, seedling density is every square metre
Plantation 70～100 strains.Just during plantation, growth is relatively slow, and 9 annual～December and 3～June are its Growing season.
Endophyte, inoculation of later each season of growth one need not be again inoculated in one Growing season of first plantation
Secondary, during inoculation, the Sasm3 bacterium solution washing away culture medium is mixed as substrate with fertilizer or Nutrition Soil,
Keeping cell concentration is 108CFU substrate, humidity 60～70%, ditching deep placement (also can spill and execute) rainy day,
Ditching deep placement consumption is 0.12～0.15kg/m2, after two season of growth, more planting can be grown
Strain.
Cradle results: cradle from the beginning of postvaccinal second Growing season for the first time, treat that Biomass is maximum
Time cradle, gather in the crops overground part, during harvesting overground part retain stem 4～about 8cm, later each growth
Season gathers in the crops once, in i.e. 1 year 2 seasons of results, plants the most continuously, it is achieved gather in continuously, high-efficiency and continuous
Repair.
Statistics finds, after inoculation endophyte, the survival rate of Sedum alfredii Hance, close to 100%, does not connects
Planting endophyte plant survival rate is 71.0～90.1%, and is received after the Sedum alfredii Hance inoculation grown in the wild
Cutting and cause Seedling strain mortality rate to be substantially reduced more than 50%, Seedling strain regrows more vigorous.Out of office external
Long Sedum alfredii Hance inoculation endophyte is after 3 months, same Biomass overground part absorb heavy metal Zn,
Cd amount has been respectively increased 18.7% and 47.48%.
Claims (10)
1. a compound endogenetic bacteria microbial inoculum, it is characterised in that by acinetobacter calcoaceticus
(Acinetobactercalcoaceticus), pseudomonas fluorescens (Pseudomonas fluorescens),
Bacillus megaterium (Bacillusmegaterium), germ oligotrophy unit cell
(Stenotrophomonasmaltophilia), bacillus pumilus (Bacillus pumilus) and evil
Smelly pseudomonas (Pseudomonas putida) forms.
2. the most compound endogenetic bacteria microbial inoculum is in heavy metal pollution phytoremediation
Application.
Apply the most according to claim 2, it is characterised in that described heavy metal is Pb, Zn,
With at least one in Cd.
Apply the most according to claim 2, it is characterised in that described plant is Sedum alfredii Hance.
Apply the most according to claim 4, it is characterised in that described application includes: use water
Training method carries out Fast-propagation to Sedum alfredii Hance, raw in adding described being combined in the 4th week in culture fluid
Antibacterial microbial inoculum.
Apply the most according to claim 5, it is characterised in that described compound endogenetic bacteria microbial inoculum
Inoculum concentration be 105～108CFU。
Apply the most according to claim 4, it is characterised in that described application includes: will be in battalion
In nutrient solution, well-grown Sedum alfredii Hance root system immerses in described compound endogenetic bacteria microbial inoculum 3～5h, continues
Continuous water planting be transplanted to after one week mine soil potted plant in.
Apply the most according to claim 7, it is characterised in that described Sedum alfredii Hance is at mine soil
Potted plant middle growth time is 4～June.
Apply the most according to claim 4, it is characterised in that described application includes: take the southeast
Herba hylotelephii erythrosticti top branch root culture in nutritional solution, is seeded to the southeast by described compound endogenetic bacteria microbial inoculum
In the root system of Herba hylotelephii erythrosticti, the Sedum alfredii Hance after compound for inoculation endogenetic bacteria microbial inoculum is planted and is combined in heavy metal
Pollute in mine soil.
Apply the most according to claim 9, it is characterised in that the compound endogenetic bacteria bacterium of inoculation
This microbial inoculum need not be again inoculated in the Growing season that Sedum alfredii Hance after agent is planted for the first time, the most each
The season of growth is inoculated once, cradles for the first time from the beginning of second Growing season after plantation, and results are on the ground
Portion, during harvesting, overground part retains stem 4～about 8cm, and later each Growing season is gathered in the crops once.
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