CN106591277A - Immobilized Bacillus having phosphate dissolving and heavy metal passivation functions and preparation method and application thereof - Google Patents
Immobilized Bacillus having phosphate dissolving and heavy metal passivation functions and preparation method and application thereof Download PDFInfo
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- CN106591277A CN106591277A CN201611116120.3A CN201611116120A CN106591277A CN 106591277 A CN106591277 A CN 106591277A CN 201611116120 A CN201611116120 A CN 201611116120A CN 106591277 A CN106591277 A CN 106591277A
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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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
Abstract
The invention discloses an immobilized Bacillus having phosphate dissolving and heavy metal passivation functions and a preparation method and an application thereof, which belong to the technical field of cadmium pollution soil biological restoration. The immobilized Bacillus comprises biological carbon and Bacillus cereus B19, can be used for restoring the soil with severe cadmium pollution, and has better restoration effect. The composite microbe fertilizer contains the immobilized Bacillus, an organic fertilizer and an inorganic nutrient having the phosphate dissolving and heavy metal passivation functions. The composite microbe fertilizer has the advantages that cost is low, soil available nutrient content is increased, yield increase is achieved, the soil with cadmium pollution is effectively repaired, introduction of heavy metal cadmium in human body with crops can be effectively controlled, agricultural product safety is guaranteed; in addition, the composite microbe fertilizer cannot change a farmland utilization mode without secondary pollution, is conveniently used, and is economic and feasible.
Description
Technical field
The invention belongs to cadmium pollution soil technical field of biological remediation, has phosphorus decomposing, a passivation huge sum of money more particularly, to a kind of
Immobilized Bacillus Sp of the function of dominant and preparation method and application.
Background technology
Cadmium (Cd) is the extremely strong Heavy Metallic Elements of bio-toxicity, and chemism in the environment is strong, mobility is big, poison
Property persistently, easily human health is jeopardized by the enrichment of food chain, liver, kidney, skeleton pathological changes, also carcinogenic work can be induced
With.For the restorative procedure of the heavy-metal contaminated soils such as cadmium has peripheral doses, chemical redemption and biological restoration.Peripheral doses are main
Including soil moved in improve the original, Soil leaching and electro reclamation, although repairing effect is good, but rehabilitation cost is high, it is difficult to large-scale to promote.Change
Method mainly adds chemical reagent (such as Calx, ground phosphate rock and organic substance) to hold heavy metal, reduces it biological
Effectiveness, but chronic administration chemical reagent easily causes soil compaction, destroys Soil structure, or even cause secondary pollution.It is biological
Reparation includes plant extraction and microorganism remediation, and plant extraction is enriched with by over-accumulated plant, then by collecting plant
The purpose of removing heavy metals is reached, but this method is taken up an area greatly, and efficiency is low, and during planting super accumulation crop grain can not be all carried out
Food production.Microorganism remediation mainly uses some microorganisms with functions such as absorption, precipitation of heavy metals, reduces weight in soil
The biological effectiveness of metal, reduces the risk that heavy metal enters agricultural product, ensures agricultural product security.
Substantial amounts of research shows that biological restoration can effectively reduce bioavailability of heavy metals, reduces the absorption of crop,
Ensure agricultural product security.For example, Tang Basheng etc. (A of CN 103361072) reports a kind of cadmium pollution bioremediation agent and is applied to
The plantation of Oryza sativa L., as a result shows to apply after bioremediation agents, wattenshlick field soil Cd2+Content reduces 0.096mgkg-1, yellow mud
Field soil Cd2+Content reduces 0.042mgkg-1, and the cadmium content in Brown Rice and Caulis et Folium Oryzae is also significantly lower than compareed, and
Brown rice cadmium content has reached national food sanitary standard (GB2715-2005).Jin Jingxun (A of CN 104531551) report by
A kind of cadmium pollution soil microorganism renovation agent is applied in contaminated soil, it is found that microorganism renovation agent can be greatly lowered soil
Middle cadmium content, and effect can go directly inside soil.But, the research of microorganism remediation Cd pollutions focuses mostly in middle low-pollution water
It is flat, the with serious pollution soil of Cd is received with microorganism remediation, its repairing effect is often undesirable, and for example, He Mengmeng etc. (2013) grinds
Cadmium has been studied carefully in the Aqui-cinnamon soil of Beijing to corn seedling and its poisonous effect of rhizosphere microorganism, as a result shown that cadmium pollution can cause jade
Rice rhizosphere microorganism structure of community changes, and high concentration cadmium can cause part microbial population to reduce or even disappear completely
Lose.Charcoal and Pseudomonas putida are made degraded of the immobilized microorganism to PAH by Chen et al. (2012) researchs,
As a result show that immobilized microorganism can improve degradation effects of the Pseudomonas putida to PAH.But charcoal is used as micro-
It is less that bio-carrier repairs area research in heavy metal.
It is low by cadmium pollution soil nutrient, when repairing redoubling utilizes such soil, it is impossible to which the biology of simple reduction cadmium has
Effect property, will also increase soil fertility, and ensure the growth of crop.But because present microorganism remediation product focuses mostly in a huge sum of money
The removal effect of category, few people's research multi-functional bacterial strain especially has growth promotion and heavy metal-passivated microorganism simultaneously
Preparation, has the function of volume increase concurrently while removing heavy metals are removed, and secure agricultural production tool is of great significance.
The content of the invention
In order to overcome the shortcoming of prior art with it is not enough, the primary and foremost purpose of the present invention is to provide a kind of to have phosphorus decomposing, blunt
Change the Immobilized Bacillus Sp of the huge sum of money function of dominant.
Another object of the present invention is to provide above-mentioned with phosphorus decomposing, the Immobilized Bacillus Sp of heavy metal-passivated function
Preparation method.
Another object of the present invention is to provide above-mentioned with phosphorus decomposing, the Immobilized Bacillus Sp of heavy metal-passivated function
Using.
Another object of the present invention is to provide a kind of for activating soil nutrient and restoring cadmium polluted composite microbial
Thing fertilizer.The composite microbiological fertilizer of the present invention realizes the increasing of agricultural output on the premise of Land-Use is not changed
Plus and contaminated soil biological restoration, effectively prevent heavy metal cadmium from entering human body by crops, ensure agricultural product security.
Another object of the present invention is to provide above-mentioned for activating soil nutrient and restoring cadmium polluted composite microbial
The preparation method of thing fertilizer.
It is still another object of the present invention to provide above-mentioned for activating soil nutrient and restoring cadmium polluted composite microbial
The application of thing fertilizer.
The purpose of the present invention is achieved through the following technical solutions：
A kind of Immobilized Bacillus Sp with phosphorus decomposing, heavy metal-passivated function, including charcoal and bacillus cereuss
(Bacillus cereus)B19。
Described charcoal is with the mass volume ratio of the fermentation liquid of bacillus cereuss (Bacillus cereus) B19
(0.1～0.3) g：30mL；Preferably 0.1g：30mL.
Described bacillus cereuss (Bacillus cereus) B19, depositary institution：Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center, preservation date：On 04 27th, 2016, deposit number：CGMCC NO.12405, preservation ground
Location：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；And in Chinese patent " application number
201610391094.9, title：It is public in bacillus cereuss and application with heavy metal passivation and promotion plant growing function "
Open.
Described charcoal is the charcoal that biological material is fired；
Preferably, described charcoal is the charcoal of 300 DEG C of firings of cattle manure biological material；
The described preparation method with phosphorus decomposing, the Immobilized Bacillus Sp of heavy metal-passivated function, comprises the steps：
By bacillus cereuss (Bacillus cereus) B19 activation, culture, fermentation liquid is obtained, then add charcoal
Enter to fermentation liquid, continue to cultivate, centrifugation, with sterile water wash, collect precipitation, that is, obtain with phosphorus decomposing, heavy metal-passivated function
Immobilized Bacillus Sp.
The condition of described culture is 30 ± 2 DEG C, 150～180rpm, 12～16h of shaking table；
The condition that described continuation is cultivated is 30 ± 2 DEG C, 150～180rpm, 18～30h of shaking table；Preferably 30 ± 2 DEG C,
150～180rpm shaking table 24h.
The condition of described centrifugation is that 5000～8000rpm is centrifuged 5～10min；Preferably 7000rpm is centrifuged 5min.
The number of times of described use sterile water wash is 2～3 times.
The described application with phosphorus decomposing, the Immobilized Bacillus Sp of heavy metal-passivated function, including for decomposing indissoluble
Property phosphorus, for producing auxin, for salt tolerant, for heavy metal tolerance, for promoting plant seed germination, has for increasing soil
Effect phosphorus, Microbial nitrogen, and for reducing soil available heavy metal.
It is a kind of for activating soil nutrient and restoring cadmium polluted composite microbiological fertilizer, comprising with phosphorus decomposing, passivation
The Immobilized Bacillus Sp of the huge sum of money function of dominant, fertilizer and inorganic nutrients.
Effective bacterial content reaches 0.2 hundred million cfug in described composite microbiological fertilizer-1More than；
Water content is kept to be 18～22% in described composite microbiological fertilizer；Preferably 20%.
Described fertilizer is decomposed cow dung；Its physicochemical property is as follows：Full nitrogen 4.00gkg-1, full phosphorus 1.51gkg-1,
Full potassium 2.45gkg-1, pH 8.25, organic matter 202.98gkg-1, full cadmium 0.15mgkg-1；
Described inorganic nutrients are from the pure carbamide of analysis of Test reagents Technology Co., Ltd. Guangzhou, Guangdong, tricalcium phosphate and chlorination
Potassium reagent.
N in described inorganic nutrients:P (in terms of tricalcium phosphate):K is (with K2O is counted)=4%：6%：6%；
It is described for activating soil nutrient and the preparation method of restoring cadmium polluted composite microbiological fertilizer, including as
Lower step：
Inorganic nutrients are added in fertilizer；Immobilized Bacillus Sp is added, contains effective bacterium in composite microbiological fertilizer
Amount reaches 0.2 hundred million cfug-1More than；Keep water content to be 18～22%, obtain for activating soil nutrient and repair cadmium dirt
The composite microbiological fertilizer of dye.
It is described for activating soil nutrient and restoring cadmium polluted composite microbiological fertilizer in Plantula Brassicae chinensis are potted plant
Using.According to 0.1～0.2mgNg-1The dose of soil is applied fertilizer, and 0.2mgNg-1The dose of soil carries out fertilising effect
Fruit is optimal.
The present invention has the following advantages and effect relative to prior art：
(1) charcoal is a kind of excellent carrier, and bacillus cereuss are protected wherein, strengthens bacillus cereuss and resists unfavorable ring
The ability in border.The Immobilized Bacillus Sp of the present invention can be used for restoring cadmium polluted serious soil, and having preferably repair effect
Really.
(2) composite microbiological fertilizer that cadmium pollution soil of the invention is repaired is with low cost, by increasing capacitance it is possible to increase soil is effectively supported
Point content, reaches production-increasing function, at the same can effective cadmium pollution soil repair, effective prevention and control heavy metal cadmium enters people with crops
Body, the composite microbiological fertilizer that in addition cadmium pollution soil of the invention is repaired, does not change the Land use systems in farmland, without secondary dirt
Dye, easy to use, economically feasible.
(3) show through substantial amounts of experiment, the composite microbiological fertilizer has raising crop product, heavy metal-passivated cadmium,
Ensure the effect of agricultural product security.With the increase of composite microbiological fertilizer consumption, plant height, root is long, and fresh weight and dry weight are notable
Higher than CK process.In addition, applying the nutrient content that Plantula Brassicae chinensis and soil are a significant increase after the composite microbiological fertilizer, apply
Low-level composite microbiological fertilizer is used, Nitrogen and phosphorus content increases by 10%, 22%, 21% compared with CK, respectively；Apply Gao Shui
After flat composite microbiological fertilizer, Nitrogen and phosphorus content increases by 14%, 41%, 33% compared with CK, respectively；Apply low-level to answer
Microbial manure is closed, soil alkali-hydrolyzable nitrogen, available phosphoruss, available potassium, microbial biomass carbon and microbial biomass nitrogen content are distinguished compared with CK
Increase by 27%, 29%, 45%, 31%, 18%；After applying high-level composite microbiological fertilizer, soil alkali-hydrolyzable nitrogen, available phosphoruss, speed
Effect potassium, microbial biomass carbon and microbial biomass nitrogen content increase by 34%, 49%, 65%, 48%, 33% compared with CK, respectively.Except this
Outside, the complex microorganism can significantly reduce Plantula Brassicae chinensis and soil cadmium content, apply low-level composite microbiological fertilizer, plant
Strain cadmium content reduces 37% compared with CK；After applying high-level composite microbiological fertilizer, plant cadmium content drops compared with CK
It is low by 57%；Low-level composite microbiological fertilizer is applied, effective cadmium content of soil reduces 8% compared with CK；Apply Gao Shui
After flat composite microbiological fertilizer, effective cadmium content of soil reduces 39% compared with CK.
Description of the drawings
Fig. 1 is charcoal NF300 microscope photographing photos under ordinary light source.
Fig. 2 is charcoal NF300 microscope photographing photos under blue-fluorescence.
Fig. 3 is that charcoal NF300 absorption GFP labellings bacillus cereuss B19 (B19-NF300) is micro- under ordinary light source
Mirror shoots photo.
Fig. 4 is that charcoal NF300 absorption GFP labellings bacillus cereuss B19 (B19-NF300) is micro- under blue-fluorescence
Mirror shoots photo.
Fig. 5 is passivation effect of the Immobilized Bacillus Sp to cadmium in different cadmium concentration fluid mediums；Wherein, numerical value in figure
For meansigma methodss ± SD values (n=3).
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
In following examples unless otherwise indicated, it is normal experiment method and operating procedure in the art.
Embodiment 1 has phosphorus decomposing, the preparation of the Immobilized Bacillus Sp of heavy metal-passivated function
Bacillus cereuss are activated, according to 1% amount 30mL LB culture medium (beef-protein medium) is inoculated into
In, 30 ± 2 DEG C, 150～180rpm, 12～16h of shaking table prepare fermentation liquid, weigh 0.1g charcoals and add fermentation liquid, and 30 ± 2
DEG C, 150～180rpm shaking table 24h, 7000rpm centrifugations 5min after taking-up, with sterile water wash 2～3 times, collect precipitation, obtain final product
To the Immobilized Bacillus Sp with phosphorus decomposing, heavy metal-passivated function.
Embodiment 2 has phosphorus decomposing, the Immobilized Bacillus Sp absorption representation of heavy metal-passivated function
The Biomass adsorbed on charcoal is determined with fat phosphorus method, and with GFP labelling bacillus cereuss, in fluorescence microscopy
Microscopic observation biology carbon surface whether transmitting green fluorescence, characterizes the bacillus cereuss situation adsorbed on charcoal.
1st, fat phosphorus method determines charcoal
(1) with 200 mesh nylon screens the biological high-area carbon that microorganism is fixed is separated and is transferred to from bacteria suspension
100mL conical flasks, chloroform 10mL, methanol 20mL, distilled water 8mL (volume ratios are rapidly joined successively into conical flask with cover
For 1：2：0.8) the extraction mixed liquor of biolipid phosphorus, is constituted, conical flask is put back to into constant temperature oscillator and is firmly shaken 10min, taken out
Stand 12h.
(2) stand in then backward each conical flask and be separately added into chloroform and each 10mL of water so that final chloroform：Methanol：Water
For 1：1：0.9, sealing stands again 12h.
(3) take out lower floor's chloroform phase 10mL containing lipidic component and be transferred to 25mL tool plug scale color comparison tubes, water bath method.
The potassium persulfate solution of 0.8mL 5% is added in test tube, and adds water to 10mL scales, 121 DEG C in high-pressure steam sterilizing pan
30min is cleared up, by the phosphate concn in the Ammonium Molybdate Spectrophotometric Method for Determination digestion solution of total phosphorus determination.As a result with nmol P
g-1Charcoal represents that 1nmol P are approximately equivalent to the cell 10 of escherichia coli size8cfu.1 is the results are shown in Table, charcoal NF300 pair
It is an excellent absorption carrier for B19 bacterial strains.
The Immobilized Bacillus Sp of table 1 adsorbs bacterium amount result
| Numbering | Phosphorus content (nmol Pg-1) | Charcoal adsorbs bacterium amount (cfug-1) |
| B19-NF300 | 450.39 | 4.50×1010 |
2nd, charcoal absorption GFP labelling bacillus cereuss situations
(1) prepared by competent cell
1), B19 strain culturing in 2 × YT culture medium (tryptone 16.0g, yeast extract powder 10.0g, NaCl 5.0g,
The incubated overnight of distilled water 1000mL, pH 7.0～7.2)；
2), be inoculated with 0.5mL overnight cultures in 50mL pre-temperature 2 × YT culture medium, 250mL flasks (1:100 inoculations).37
DEG C, 200rmin-1Culture, makes bacterium solution OD600Reach between 0.2 and 0.4；
3), supernatant is outwelled, is eliminated as much as many liquid；
4), 7.5mL pre-cooling FSB (KCl 7.4g, CaCl are added2·2H2O 7.5g, glycerol 100mL, 1M KCO2CH3
10.0mL, distilled water 1000mL, pH 6.2) gentle re-suspended cell precipitation；
5) 15min, is incubated on ice；
6)、3000r·min-1Centrifugation 10min sedimentation cells pour out FSB；
7) the gentle re-suspended cell precipitations of the cold FSB of 2mL, are added, 0.2mL aliquots is drawn with pipette and is arrived 1.5mL's
In microtest tube, in -80 DEG C cell is preserved.
(2) GFP plasmids conversion
According to following method by plasmid transformed competence colibacillus cell, minimum 3 repetitions of each bacterium, and add negative control
(blank bacterial strain).
1), thaw at room temperature cell, until suspension is all liquid.Centrifuge tube is placed on ice；
2) DNA, is added in 100 μ L competent cells, and (green fluorescent protein plasmid pGFPuv is (by U.S. The Samuel
Doctor Mysore of Roberts Noble Foundation give)), it is gently mixed and shakes up (typically<1 μ L superhelix miniprep dnas
Or 50～100ng connection mixture is advisable)；
3) 30min, is incubated on ice；
4), centrifuge tube is put into into 2min in 42 DEG C of water-baths；
5), ice bath 5min；
6), 1mL 2 × YT culture medium is added；
7), 37 DEG C of incubation 30min,
8), 100 μ L cultures are added in simple centrifugation backward selection culture medium (addition 40mg/mL kalamycins)；
9), 37 DEG C of incubated overnight.
(3) screening of recon is converted
The bacterium colony that can be grown on the selective medium for the addition of antibiotic is selected, negative control does not grow, converted
Can grow afterwards, when can tentatively judge plasmid has been imported.Also need to simultaneously further using fluorescence microscope detection sun
Whether property bacterium colony can send green fluorescence, if can actually observe green fluorescence in the case where blue light is excited, convert successfully,
By positive bacterium colony culture, -80 DEG C are stored in.
By the successful bacterial strain activation of GFP labellings, in adding the LB culture medium containing charcoal, after 30 DEG C of shaking table 24h
7000rpm is centrifuged 5min, takes and is deposited in microscope slide, in fluorescence microscope experimental result.Experimental result is shown in Fig. 1～Fig. 4, from
Fig. 1 can be seen that the B19 bacterial strains of GFP labellings and excite lower display green fluorescence in blue light, and adsorb in charcoal NF300 tables
Face, again demonstrates absorption situations of the charcoal NF300 to B19 bacterial strains.
Embodiment 3 has the Soluble phosphorus energy of phosphorus decomposing, the Immobilized Bacillus Sp of heavy metal-passivated function to Phos culture medium
Power
Weigh the addition Phos liquid of the Immobilized Bacillus Sp with phosphorus decomposing, heavy metal-passivated function that 0.1g is prepared
(calcium phosphate, iron phosphate and aluminum phosphate culture medium replace calcium phosphate with iron phosphate and aluminum phosphate as unique phosphorus source in culture medium.
Phos liquid culture based formulas：Glucose 10g, calcium phosphate 5g, magnesium chloride 5g, magnesium sulfate 0.25g, potassium chloride 0.2g, sulphuric acid
Ammonium 0.1g, distilled water 1 000mL, pH 7.0), at the same do only plus bacillus cereuss B19 processs, only plus charcoal process and
Blank, 30 ± 2 DEG C, 150～180rpm shaking table 72h take out and take after 7000rpm centrifugation 5min supernatant measure amount of phosphorus dissolved,
Study its phosphate solubilization.Experimental result is shown in Table 2, and B19 bacterial strains absorption as can be seen from Table 2 forms fixation in charcoal NF300
Changing bacillus cereuss does not reduce phosphate solubilization of the B19 bacterial strains to calcium phosphate, but significantly improves B19 bacterial strains to iron phosphate and phosphorus
The phosphate solubilization of sour aluminum.
Phosphate solubilization of the Immobilized Bacillus Sp of table 2 to Phos culture medium
Note：Numerical value is meansigma methodss ± SD values (n=3) in table；Indicate poor without significance with same letter with column data
Different (Duncan methods, p<0.05)
Embodiment 4 has the Immobilized Bacillus Sp of phosphorus decomposing, heavy metal-passivated function to containing different cadmium concentration liquid cultures
The passivation effect of cadmium in base
Weigh the Immobilized Bacillus Sp with phosphorus decomposing, heavy metal-passivated function that 0.1g prepares add it is dense containing different cadmiums
In the fluid medium of degree (0,0.2,0.5,1,2,10,25,30,35mgL-1Cd, uses 0.01M NaNO3Do background solution),
Do simultaneously only plus charcoal is processed and blank, 30 ± 2 DEG C, after 150～180rpm shaking table 24h, 7000rpm centrifugation 5min
Supernatant AAS (Atomic Absorption) measure is taken, clearance of the material to cadmium is calculated.Experimental result is shown in Fig. 5, as can be seen from Figure 5, biological
After charcoal NF300 absorption B19, with Cd in solution2+The increase of concentration, its clearance is slightly above charcoal NF300, shows immobilization
Bacillus cereuss are not occupied by the site that charcoal adsorbs cadmium, and compared with charcoal, removal effect also has increased trend.It is raw
After thing charcoal NF300 absorption B19, its clearance improves 2.3 times compared with B19.
It is prepared by the composite microbiological fertilizer of embodiment 5
According to inorganic nutrients N:P (in terms of tricalcium phosphate):K is (with K2O is counted)=4%：6%：6% ratio adds fertilizer
In；Immobilized Bacillus Sp is added, makes effective bacterial content in composite microbiological fertilizer reach 0.2 hundred million cfug-1, while being not added with
The control treatment of bacterium.Each processes and prepares 1kg fertilizer, keeps water content to be 20%, in being fitted into sealed bag, puts at room temperature, and
In using front sampling, nutrient content in fertilizer is determined.Experimental result is shown in Table 3, from table 3 it can be seen that composite microbiological fertilizer is full
Foot effectively bacterial content >=0.2 hundred million cfug-1。
The composite microbiological fertilizer physicochemical property of table 3
Note：A is the organic-inorganic compound fertilizer for being not added with bacterium；B is composite microbiological fertilizer.
Application of the composite microbiological fertilizer of embodiment 6 in Plantula Brassicae chinensis are potted plant
Cadmium pollution rice soil is gathered from Guangdong Shaoguan Qujiang River Ma Bazhen, soil physico-chemical property is as follows：Full nitrogen 1.85gkg-1,
Full phosphorus 0.80gkg-1, full potassium 9.74gkg-1, soil alkali-hydrolyzable nitrogen 180.54mgkg-1, available phosphoruss 38.02mgkg-1, speed
Effect potassium 123.28mgkg-1, organic matter 28.38gkg-1, pH 5.26, full cadmium 2.90mgkg-1, (DTPA soaks available Cd
Put forward state) content be 1.55mgkg-1.5mm sieves are crossed after air-drying, dress 2kg is native per basin, and soil moisture content maintains field capacity
60%.Plantula Brassicae chinensis are planted according to following experimental design：
Soil and Plant samples are gathered after results, for the analysis of physiological and biochemical index and heavy metal index.Experiment knot
Fruit is shown in Table 4, table 5 and table 6.
The plant height of Plantula Brassicae chinensis is increased after composite microbiological fertilizer from table 4 it is known that applying, root is long, fresh weight and dry
Weight, and with the increase of fertilising consumption, plant height, root is long, fresh weight and dry weight are significantly higher than CK process.Likewise, from experiment
As a result from the point of view of, apply composite microbiological fertilizer and contrast with the composite organic-inorganic fertilizer for being not added with bacterium, composite microbiological fertilizer can show
The Biomass of the increase Plantula Brassicae chinensis of work.
From table 5 it is known that applying the N-P-K content that Plantula Brassicae chinensis are a significant increase after composite microbiological fertilizer, apply
Low-level composite microbiological fertilizer, Nitrogen and phosphorus content increases by 10%, 22%, 21% compared with CK, respectively；Apply high level
After composite microbiological fertilizer, Nitrogen and phosphorus content increases by 14%, 41%, 33% compared with CK, respectively.In addition, applying compound
Microbial manure is compared with the composite organic-inorganic fertilizer for being not added with bacterium, and composite microbiological fertilizer significantly increases plant N P and K and contains
Amount.Likewise, from the point of view of experimental result, applying composite microbiological fertilizer can significantly reduce the cadmium content of Plantula Brassicae chinensis enrichment,
Low-level composite microbiological fertilizer is applied, plant cadmium content reduces 37% compared with CK；Apply high-level composite microbe fertilizer
After material, plant cadmium content reduces 57% compared with CK.
Be a significant increase Soil Available state N-P-K content after composite microbiological fertilizer from table 6 it is known that applying, apply
Use low-level composite microbiological fertilizer, soil alkali-hydrolyzable nitrogen, available phosphoruss and quick-acting potassium content compared with CK, respectively increase by 27%,
29%th, 45%；After applying high-level composite microbiological fertilizer, soil alkali-hydrolyzable nitrogen, available phosphoruss and quick-acting potassium content divide compared with CK
Zeng Jia by 34%, 49%, 65%.In addition, apply composite microbiological fertilizer comparing with the composite organic-inorganic fertilizer for being not added with bacterium, it is combined
Microbial manure significantly increases soil alkali-hydrolyzable nitrogen, available phosphoruss and quick-acting potassium content.Additionally, apply composite microbiological fertilizer can
Increase Microbial nitrogen content, and as the increase of fertilizer amount is significantly raised, apply low-level composite microbiological fertilizer, soil
Earth Microbial nitrogen content increases by 31%, 18% compared with CK, respectively；After applying high-level composite microbiological fertilizer, soil is micro-
Biological carbon nitrogen content increases by 48%, 33% compared with CK, respectively.Likewise, from the point of view of experimental result, applying composite microbe fertilizer
Material can significantly reduce effective cadmium content of soil, apply low-level composite microbiological fertilizer, effective cadmium content of soil with
CK is compared, and reduces 8%；After applying high-level composite microbiological fertilizer, effective cadmium content of soil is reduced compared with CK
39%.
Impact of the composite microbiological fertilizer Different Fertilization level of table 4 to Chinese cabbage plant height, root length and Biomass
| Numbering | Plant height (cm strains-1) | (the cm strains of root length-1) | Fresh weight (g strains-1) | Dry weight (g strains-1) |
| CK | 16.30±1.04c | 3.83±0.29b | 5.10±1.70c | 0.59±0.12c |
| T1 | 17.22±1.41bc | 3.82±0.49b | 5.61±0.38c | 0.69±0.02bc |
| T2 | 17.83±1.02bc | 3.87±0.22b | 6.57±0.92bc | 0.80±0.11b |
| T3 | 18.40±0.45ab | 5.41±0.49a | 9.78±1.99b | 0.82±0.07b |
| T4 | 20.00±0.19a | 6.04±0.07a | 14.12±2.84a | 0.98±0.45a |
Note：Numerical value is meansigma methodss ± SD values (n=3) in table, with indicating without significance with same letter person in column data
Difference (Duncan methods, p ＜ 0.05).
Impact of the composite microbiological fertilizer Different Fertilization level of table 5 to Chinese cabbage nutrient and heavy metal cadmium
| Numbering | Full nitrogen (gkg-1) | Full phosphorus (gkg-1) | Full potassium (gkg-1) | Full cadmium (mgkg-1) |
| CK | 12.08±0.33d | 1.51±0.08c | 39.03±3.65d | 2.10±0.04a |
| T1 | 12.63±0.29c | 1.64±0.05c | 44.30±2.27c | 1.94±0.12a |
| T2 | 13.39±0.10b | 1.94±0.28b | 49.26±1.77b | 1.32±0.17b |
| T3 | 13.49±0.11b | 2.15±0.10b | 51.01±0.16b | 1.17±0.06b |
| T4 | 14.02±0.12a | 2.54±0.18a | 57.90±2.28a | 0.90±0.05c |
Note：Numerical value is meansigma methodss ± SD values (n=3) in table, with indicating without significance with same letter person in column data
Difference (Duncan methods, p ＜ 0.05).
Impact of the composite microbiological fertilizer Different Fertilization level of table 6 to soil nutrient and available Cd
Note：Numerical value is meansigma methodss ± SD values (n=3) in table, with indicating without significance with same letter person in column data
Difference (Duncan methods, p ＜ 0.05).
The invention is not limited in aforesaid specific embodiment.The present invention is expanded to and any in this manual disclosed
New feature or any new combination, and the arbitrary new method that discloses or the step of process or any new combination.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any spirit without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of Immobilized Bacillus Sp with phosphorus decomposing, heavy metal-passivated function, it is characterised in that including charcoal and waxy
Bacillus cereuss (Bacillus cereus) B19.
2. the Immobilized Bacillus Sp with phosphorus decomposing, heavy metal-passivated function according to claim 1, it is characterised in that：
Described charcoal is (0.1 with the mass volume ratio of the fermentation liquid of bacillus cereuss (Bacillus cereus) B19
～0.3) g：30mL；
Described charcoal is the charcoal that biological material is fired.
3. the preparation method with phosphorus decomposing, the Immobilized Bacillus Sp of heavy metal-passivated function described in claim 1 or 2, its
It is characterised by comprising the steps：
By bacillus cereuss (Bacillus cereus) B19 activation, culture, obtain fermentation liquid, then by charcoal add to
Fermentation liquid, continues to cultivate, centrifugation, with sterile water wash, collect precipitation, that is, obtain consolidating with phosphorus decomposing, heavy metal-passivated function
Surely bacillus cereuss are changed.
4. the described preparation with phosphorus decomposing, the Immobilized Bacillus Sp of heavy metal-passivated function according to claim 3
Method, it is characterised in that：
The condition of described culture is 30 ± 2 DEG C, 150～180rpm, 12～16h of shaking table；
The condition that described continuation is cultivated is 30 ± 2 DEG C, 150～180rpm, 18～30h of shaking table；
The condition of described centrifugation is that 5000～8000rpm is centrifuged 5～10min.
5. the application with phosphorus decomposing, the Immobilized Bacillus Sp of heavy metal-passivated function described in claim 1 or 2, its feature
It is：Application of the described Immobilized Bacillus Sp in following either side：For decomposing insoluble phosphorus, for producing length
Element, for salt tolerant, for heavy metal tolerance, for promoting plant seed germination, for increasing soil available phosphorus, Microbial
Nitrogen, and for reducing soil available heavy metal.
6. it is a kind of for activating soil nutrient and restoring cadmium polluted composite microbiological fertilizer, it is characterised in that will comprising right
Ask described in 1 or 2 with phosphorus decomposing, the Immobilized Bacillus Sp of heavy metal-passivated function, fertilizer and inorganic nutrients.
7. according to claim 6 for activating soil nutrient and restoring cadmium polluted composite microbiological fertilizer, it is special
Levy and be：
Effective bacterial content reaches 0.2 hundred million cfug in described composite microbiological fertilizer-1More than；
Water content is kept to be 18～22% in described composite microbiological fertilizer.
8. the preparation for activating soil nutrient and restoring cadmium polluted composite microbiological fertilizer described in claim 6 or 7
Method, it is characterised in that comprise the steps：
Inorganic nutrients are added in fertilizer；Immobilized Bacillus Sp is added, effective bacterial content in composite microbiological fertilizer is reached
To 0.2 hundred million cfug-1More than；Keep water content to be 18～22%, obtain for activating soil nutrient and restoring cadmium polluted
Composite microbiological fertilizer.
9. described in claim 6 or 7 for activating soil nutrient and restoring cadmium polluted composite microbiological fertilizer in little Bai
Application in nappy cultivation.
10. application according to claim 9, it is characterised in that：According to 0.1～0.2mgNg-1The dose of soil is applied
Fertilizer.
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