CN109810930A - It is a kind of can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application - Google Patents
It is a kind of can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application Download PDFInfo
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Abstract
The present invention provides it is a kind of can restoration of soil polluted by heavy metal microbial bacterial agent, its active constituent includes Rhizobium of Milk Vetch, Pseudomonas fluorescens, saccharomyces cerevisiae, bacillusmusilaginosiengineering, Bacillus cercus, its dosage form is microcapsule formulation, and total viable count is up to 5 × 1010~6×1010cfu/g.The preparation method of the microbial bacterial agent includes activation culture, tame culture step by step, mixing fermentation culture, the preparation of bacteria suspension, magnetic field-intensification microcapsules prepare several steps.The present invention is high using the microbial bacterial agent thallus survival rate of spray drying microcapsules technology preparation, embedding efficiency is high, storage-stable is good, lasting period is long, the magnetic fields that carrier generates can further increase microorganism to the removal rate of available heavy metal, realize efficient reparation to heavy-metal contaminated soil, and it is nontoxic, pollution-free, effect on environment is small, there is high practical value.
Description
Technical field
The present invention relates to technical field of biological remediation, more particularly to it is a kind of can restoration of soil polluted by heavy metal microbial bacteria
Agent and its preparation method and application.
Background technique
Soil is the most basic means of production of the mankind, is material base for the survival of mankind, and current soil pollutes
Become three overall situation pollution problem of China together with atmosphere pollution, water pollution.In agricultural production process, soil pollution can be led
Soil fertility decline is caused, crops quality and yield reduce, the serious sustainable development for hindering agricultural.Meanwhile harmful substance
It gathers, can be eaten by food chain by the mankind, and then endanger the health of the mankind in crops body.
Heavy metal refers to that atomic density is greater than 5g/cm3Metallic element, about 40 kinds, mainly include cadmium, mercury, lead,
Chromium, copper, zinc, manganese, nickel etc..But generally arsenic, aluminium etc. are also included within from toxicity angle interior.From early in the twentieth century with industry
The fast development of technology, the mankind are more and more wider using the range of heavy metal, are discharged into the heavy metal contaminants in environment and also get over
Come it is more, heavy metal due to the features such as its residence time is long, toxicity is big, difficult to degrade as soil pollution major pollutants it
One.The approach of heavy-metal contaminated soil mainly includes the following: 1) the excessive application of chemical fertilizer, pesticide.The volume increase of agricultural needs
Chemical fertilizer and pesticide are applied, and long-term a large amount of application chemical fertilizer and pesticide can not only destroy soil texture, wherein the heavy metal contained
It can be directly entered soil, cause soil pollution.2) industrial pollution.In industrial area, by plant produced waste discharge, waste residue, give up
Gas, dust etc. can be by soil, waters and the atmosphere around surface water body runoff, atmosphere floating dust pollution, and constantly accumulation causes
Serious pollution.3) improper use of Technique for Agricultural Films.The development of Technique for Agricultural Films improves the production of crop, but agriculture to a certain extent
The unreasonable utilization of membrane technology can generate a large amount of residual agricultural film, and these agricultural films usually contain heavy metal, and not degradable,
Residual causes soil pollution in the soil.4) traffic pollution.It is highway containing heavy metals such as lead, cadmiums in the tail gas of motor vehicle emission
The main source of two sides heavy metal pollution of soil.Studies have shown that the pollution from other organic compounds is different, heavy metal pollution is very
Difficult natural degradation, and there is enriching, the self-purification capacity of soil is accumulated and destroyed for a long time, and soil is made to become " the storage of pollutant
Library ".Crops are planted on this kind of soil, heavy metal can be absorbed by root system of plant, cause crop production reduction or output containing a huge sum of money
" malicious grain ", " the malicious vegetables " belonged to.According to statistics, the arable land in China about 20% is different degrees of by various heavy metal-polluted at present
Dye, therefore, solves the problems, such as that heavy metal pollution of soil is urgent and quite important.
In-situ passivation reparation refers to applies passivator in contaminated soil, change/reduction heavy metal migration and biology
Validity, to reduce the content of beary metal entered in plant.It play an important role in Heavy Metal Pollution Control.In-situ passivation
Recovery technique mainly include engineering control reparation, peripheral doses, chemical remediation and biological prosthetic four kinds of methods.And first three side
Method is the traditional administering method of comparison, although achieving certain effect in practical applications, there are manpower and financial resources expend it is big,
Implement the defects of complicated, control expense is high, Yi Yinqi soil fertility reduces, is unfavorable for promoting and applying on a large scale, therefore, take
It is biological prosthetic to administer heavy metal pollution as the heat subject in current global range.Biological prosthetic includes phytoremediation, animal
Reparation and microorganism remediation, wherein for phytoremediation using technologies such as plant absorption, volatilization, extractions, repairing effect is obvious, but due to
The survival of plant is larger by Environmental Factors, in addition, the general biomass of hyperaccumulative plant is smaller etc. to limit pushing away for the technology
Wide application.
Heavy-metal Polluted Environment reparation is carried out using microorganism, effect is good, not will cause secondary pollution, has and preferably answers
Use prospect.Researchers have discovered that it is many have patience to heavy metal, and can be changed by modes such as enrichment, sorption and conversions
Become the environmental chemical behavio4 of heavy metal, and then to controlling, administer the heavy metal-polluted microorganism for being infected with positive effect.At present research compared with
More bacteriums with repairing heavy metal pollution function mainly have bacillus, pseudomonas, rhizobium etc.;Fungi master
There are aspergillus niger, aspergillus flavus, Candida, saccharomyces cerevisiae etc..Microorganism mainly includes biological suction to the repair mechanisms of heavy metal
Echo bioconversion.A large number of studies show that there are hydroxyl, carboxyl, sulfydryl, amino, aldehyde radicals etc. to live on bacterium and fungal cell wall
Property group, heavy metal ion thus can be adsorbed or be complexed.On the other hand, microorganism passes through redox, complex coordination, first
The effects of base, demethylation, converts heavy metal ion, to reduce the environmental toxicity of heavy metal.On the whole, micro- life
Object be by reducing the migration and validity of heavy metal to the absorption of heavy metal in environment, precipitating, fixation, covalently conversion etc.,
To drop the toxicity of heavy metal.In recent years, carried out both at home and abroad and a large amount of eliminated grinding for heavy metal pollution of soil using microorganism
Study carefully, also makes great progress.
But there is also many problems to need to solve for microorganism remediation technology at present.First suitable for the micro- of soil remediation
Most of bacteria agent does not have commercialization condition also also in the laboratory research stage.Secondly, existing microorganism in the market
There is, available heavy metal removal rate not strong to heavy metal tolerance be not high and reparation to heavy-metal contaminated soil for microbial inoculum
The undesirable problem of ability.And the dosage form of microbial bacterial agent is mostly freeze dried powder and wettable powder, this kind of dosage form product
Unstable quality, storage life is short, and long-term preservation will lead to the reduction of living bacteria count content, and in practical application, microorganism
Vulnerable to external environment influence and inactivate, or by indigenous microorganism interference and lose competitive advantage, so as to cause micro- life
The lasting period of object microbial inoculum is not grown, and repairing effect is influenced.
Summary of the invention
In view of the above drawbacks of the prior art and problem, the object of the present invention is to provide one kind can repairing heavy metal pollution soil
The microcapsule-type microbial bacterial agent of earth, it is a further object of the present invention to provide the preparation methods of this kind of microbial bacterial agent, and by its
Applied to the heavy metal pollution reparation of agricultural land soil, the removal rate of available heavy metal is made to reach 90% or more, realized to heavy metal
The efficient reparation of polluted agricultural land.
The present invention is achieved through the following technical solutions above-mentioned technical effect:
It is a kind of can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application, which is characterized in that it is described micro-
The active constituent of bacteria agent includes Rhizobium of Milk Vetch, Pseudomonas fluorescens, saccharomyces cerevisiae, bacillusmusilaginosiengineering, wax-like
Bacillus, dosage form are microcapsule formulation, and total viable count is 5 × 1010~6×1010cfu/g。
The preparation method of the microbial bacterial agent includes the following steps:
1) activation culture: in gnotobasis, respectively by Rhizobium of Milk Vetch, Pseudomonas fluorescens, saccharomyces cerevisiae, gel-shaped bud
Spore bacillus, Bacillus cercus strain be restored to room temperature state from preservation state, then strain is conventionally shaken
Bed activation culture, is prepared into seed liquor;
2) domestication culture step by step: above-mentioned seed liquor is inoculated into respectively containing heavy metal ion Cd2+、Hg2+、Pb2+、Cu2+、Zn2+
Domestication culture medium in cultivated, period sampling measuring bacterium solution absorbance indicates to have tamed after absorbance reaches maximum value
At bacterium solution at this time is transferred in the domestication culture medium containing higher concentration heavy metal ion and is cultivated, is so carried out continuously 5
The other domestication culture of concentration level;
3) mixing fermentation culture: selecting liquid fermentation tank, and fermentation medium liquid amount is 60 ~ 70%, the Chinese milk vetch after inoculation domestication
Rhizobium, Pseudomonas fluorescens, saccharomyces cerevisiae, bacillusmusilaginosiengineering, Bacillus cercus seed liquor, total inoculum concentration be 6 ~
10%, the inoculative proportion of each microorganism is 2:1:1:3:3, controls 30 ~ 35 DEG C of fermentation temperature, 250 ~ 300rpm of revolving speed, ventilatory capacity
1.0 ~ 1.4VVM, 36 ~ 48h of fermented and cultured under this fermentation condition;
4) preparation of bacteria suspension: being centrifuged 8 ~ 10min for fermentation liquid under the conditions of 6000 ~ 7000 rpm, abandon supernatant collection bacterium mud,
With brine 2-3 times, bacterium mud is suspended in protective agent (5% antierythrite solution) and is prepared into bacteria suspension, keeps viable bacteria close
Degree reaches 1 × 1011Cfu/ml or more;
5) preparation of magnetic field-intensification microcapsules: Fe of the selection degree of compression 20 ~ 30%3O4Particle is carrier, outstanding according to carrier and bacterium
The ratio of liquid is 1:10, carrier is added into bacteria suspension, then stirring evenly makes carrier adsorption thallus, is dried to obtain core at 37 DEG C
Material;It is 1:5 according to the ratio of core material and wall material, core material is added in wall material, obtains mixed liquor after mixing well;By mixed liquor
Through the uniform sample introduction of peristaltic pump to spray dryer, 115 ~ 125 DEG C of inlet air temperature, 0.18 ~ 0.22MPa of atomisation pressure are controlled, sample introduction
13 ~ 15ml/min of flow, collects product up to the microcapsule formulation by 45 ~ 55 DEG C of leaving air temp after spray drying.
Conventional method described in step 1) refers to Rhizobium of Milk Vetch, Pseudomonas fluorescens, bacillusmusilaginosiengineering, wax
The strain of shape bacillus is inoculated in bacterium basal liquid medium, 30 ~ 35 DEG C, 160 ~ 200r/min shaking table culture 18 ~
24h;The strain of saccharomyces cerevisiae is inoculated in yeast fluid nutrient medium, 28 ~ 32 DEG C, 140 ~ 180r/min shaking table culture 30 ~
36h。
Cd described in step 22+、Hg2+、Pb2+、Cu2+、Zn2+, 5 concentration ranks be set as 60mg/L, 120mg/L,
180mg/L、240mg/L、300mg/L。
The basic recipe of culture medium is tamed described in step 2 are as follows: 16 ~ 20g/L of glucose, 5 ~ 10g/L of peptone, yeast
Cream 4 ~ 6g/L, CaCO3 2.4 ~ 3.0g/L, KH2PO40.5 ~ 0.9g/L, K2HPO40.5 ~ 0.8g/L of 1.8 ~ 2.2g/L, NaCl,
MgSO4·7H20.3 ~ 0.5g/L of O, 0.12 ~ 0.14mg/L of methyl jasmonate, additionally adds CdCl2、Pb(NO3)2、HgCl2,
CuSO4, ZnSO4, surplus is water, pH5.5 ~ 6.5.
The formula of fermentation medium described in step 3) are as follows: 20 ~ 28g/L of wheatfeed, 27 ~ 33g/L of beancake powder, mushroom bran powder 7 ~
11g/L, CaCO34 ~ 5g/L, KH2PO40.5 ~ 1.0g/L, K2HPO40.9 ~ 1.5g/L of 1.8 ~ 2.2g/L, NaCl, fulvic acid 1.5
~ 2.5g/L, Tween 80 0.8 ~ 1.2g/L, 6-BA 1.3 ~ 1.5mg/L of 0.6 ~ 1.0mg/L, IAA, surplus are water, pH5.5 ~
6.5。
Fe described in step 5)3O4Particle, needs before use by magnetization treatment, make its surface induction intensity reach 0.3 ~
0.6mT。
Wall material described in step 5) the preparation method comprises the following steps: maltodextrin, microcrystalline cellulose and water are mixed by the mass ratio of 3:1:6
It closes, heats while stirring in 60 DEG C of water-baths uniformly, again through the high-pressure homogeneous processing 10min of 20 ~ 30MPa after cooling, obtain compound
Wall material.
The application of the microbial bacterial agent is that microbial bacterial agent is directly applied to heavy metal pollution farmland soil to be repaired
In earth, amount of application is 20 ~ 50g/m2, handled under field conditions (factors) 30 days or so after turning over mixing.
Technical solution provided by the invention has following significant advantage compared with prior art:
1) present invention chooses a variety of microorganisms for having effects that heavy metal pollution is repaired and composite bacteria agent is made, these microorganism counterweights
The repair mechanisms multiplicity of metal, can not only accumulate heavy metal adsorption, additionally it is possible to heavy metal be converted into stable state, not to ring
Border causes to administer heavy metal pollution to greatest extent on the basis of secondary destruction, improves comprehensive repair ability.
2) present invention carries out the domestication culture step by step of contents of many kinds of heavy metal ion to the microorganism of selection respectively, after taming
For every kind of microorganism to these heavy metals tolerance all with higher, compounding can adapt to weight in various degree after composite bacteria agent is made
The soil of metallic pollution is answered even if being still able to maintain activity in polluting more serious soil and carrying out efficient repairing.
3) present invention is with Fe3O4Particle is as carrier adsorption microbial cells, Fe3O4Particle is not only in the mistake of spray drying
There is protective effect, and Fe to thallus in journey3O4Particle can generate low-intensity magnetic field after magnetization treatment, can promote microorganism
Growth metabolism improves the stability of microorganism growth, to improve its absorption and conversion capability to heavy metal, reaching raising has
Imitate the purpose of state heavy metal removing rate.
4) using spray drying microcapsules technology microcapsule formulations are made in microbial bacterial agent by the present invention, are protected by optimization
Agent, carrier and spray drying condition etc. improve thallus survival rate and embedding rate.Compared with the dosage form of other microbial bacterial agents,
Microcapsule formulation of the invention can it is intact save strain activity, solve the problems, such as microorganism long-term preservation activity decline,
Microorganism is affected by the external environment smaller when in use, and good stress resistance, the lasting period is long, can play microorganism pair to the maximum extent
The removal effect of heavy metal, available heavy metal removal rate can reach 90% or more.
5) raw material of the invention and production process are nontoxic, it can be achieved that non-pollutant discharge, and raw material sources are wide
General, cheap, microcapsule preparation process is simple, is suitble to industrialized mass production, and the commercialization for biological remediation microbial inoculum is established
Basis.
Detailed description of the invention
Fig. 1 is influence of the different protective agents to thallus survival rate and embedding rate;
Fig. 2 is the influence of different carriers and bacteria suspension ratio to thallus survival rate and embedding rate;
Fig. 3 is the influence of different core materials and wall material ratio to thallus survival rate and embedding rate;
Fig. 4 is influence of the different inlet air temperature to thallus survival rate and embedding rate;
Fig. 5 is influence of the different atomisation pressures to thallus survival rate and embedding rate.
Specific embodiment
In order to more fully understand technology contents of the invention, technical solution of the present invention is made combined with specific embodiments below
It is further described and illustrates.
Raw materials used or reagent in the embodiment of the present invention can be obtained unless otherwise specified by conventional commercial approach.
Strain used:
Rhizobium of Milk Vetch, number 58262, saccharomyces cerevisiae, number 098896 are bought limited in hundred Ou Bowei biotechnology of Beijing
Company;
Pseudomonas fluorescens, number 21093 are bought in Chinese industrial Microbiological Culture Collection administrative center;
Bacillusmusilaginosiengineering, number 180688, Bacillus cercus, number 132551, purchase create connection biotechnology in Bei Na
Co., Ltd.
Used medium:
Bacterium basal liquid medium: nutrient broth medium is formulated as peptone 10g/L, powdered beef 3g/L, NaCl 5g/L,
Surplus is water, pH7.0;
Yeast fluid nutrient medium: YPD culture medium is formulated as yeast extract 10g/L, peptone 20g/L, glucose 20g/L, and surplus is
Water, pH5.5;
Tame culture medium: basic recipe is glucose 18g/L, peptone 7.5g/L, yeast extract 5g/L, CaCO3 2.7g/L
KH2PO40.7g/L, K2HPO42.0g/L, NaCl 0.65g/L, MgSO4·7H2O 0.4g/L, methyl jasmonate 0.13mg/L,
Additional addition CdCl2、Pb(NO3)2、HgCl2, CuSO4, ZnSO4, surplus is water, pH6.0;
Fermentation medium: it is formulated as wheatfeed 24g/L, beancake powder 30g/L, mushroom bran powder 9g/L, CaCO34.5g/L, KH2PO4
0.75g/L, K2HPO42.0g/L, NaCl 1.2g/L, fulvic acid 2.0g/L, Tween 80 1.0g/L, 6-BA 0.8g/L, IAA
1.4mg/L, surplus are water, pH6.0.
Embodiment 1
It is a kind of can restoration of soil polluted by heavy metal microbial bacterial agent, active constituent includes that Rhizobium of Milk Vetch, fluorescence are false single
Born of the same parents bacterium, saccharomyces cerevisiae, bacillusmusilaginosiengineering, Bacillus cercus, total viable count are 5 × 1010~6×1010Cfu/g,
Dosage form is microcapsule formulation, and the preparation method of the microbial bacterial agent includes the following steps:
1) activation culture: in gnotobasis, respectively by Rhizobium of Milk Vetch, Pseudomonas fluorescens, saccharomyces cerevisiae, gel-shaped bud
Spore bacillus, Bacillus cercus strain be restored to room temperature state from preservation state, by Rhizobium of Milk Vetch, Pseudomonas
The strain of bacterium, bacillusmusilaginosiengineering and Bacillus cercus is inoculated in nutrient broth medium respectively, in 33 DEG C, 180r/
Min shaking table culture 18 ~ be prepared into seed liquor for 24 hours;The strain of saccharomyces cerevisiae is inoculated in YPD culture medium, in 30 DEG C, 160r/
30 ~ 36h of min shaking table culture is prepared into seed liquor.
2) domestication culture step by step: above-mentioned seed liquor is inoculated into respectively containing heavy metal ion Cd2+、Hg2+、Pb2+、Cu2+、
Zn2+Domestication culture medium in cultivated, period sampling measuring bacterium solution absorbance, after absorbance reaches maximum value indicate domestication
It completes, bacterium solution at this time is transferred in the domestication culture medium containing higher concentration heavy metal ion and is cultivated, is so carried out continuously 5
A other domestication culture of concentration level;Cd2+、Hg2+、Pb2+、Cu2+、Zn2+5 concentration ranks be set as 60mg/L, 120mg/L,
180mg/L、240mg/L、300mg/L。
3) mixing fermentation culture: selecting liquid fermentation tank, and fermentation medium liquid amount is 65%, the purple cloud after inoculation domestication
English rhizobium, Pseudomonas fluorescens, saccharomyces cerevisiae, bacillusmusilaginosiengineering, Bacillus cercus seed liquor, total inoculum concentration are
6 ~ 10%, the inoculative proportion of each microorganism is 2:1:1:3:3, controls 33 DEG C of fermentation temperature, revolving speed 280rpm, ventilatory capacity 1.2VVM,
The fermented and cultured 48h under this fermentation condition.
4) preparation of bacteria suspension: fermentation liquid is centrifuged 8 ~ 10min under the conditions of 6000 ~ 7000rpm, abandons supernatant collection bacterium
Bacterium mud is suspended in protective agent (5% antierythrite solution) with brine 2-3 times and is prepared into bacteria suspension, made to live by mud
Strain density reaches 1 × 1011Cfu/ml or more.
5) preparation of magnetic field-intensification microcapsules: Fe of the selection degree of compression 20 ~ 30%3O4Particle is carrier, needed before use through
Magnetization treatment is crossed, its surface induction intensity is made to reach 0.3 ~ 0.6mT;It is 1:10 according to the ratio of carrier and bacteria suspension, to bacterium
Carrier is added in suspension, then stirring evenly makes carrier adsorption thallus, is dried to obtain core material at 37 DEG C;According to the ratio of core material and wall material
Example is 1:5, and core material is added to wall material (the preparation method comprises the following steps: by maltodextrin, microcrystalline cellulose and water by the mass ratio of 3:1:6
Mixing heats while stirring uniformly in 60 DEG C of water-baths, again through the high-pressure homogeneous processing 10min of 20 ~ 30MPa after cooling) in, sufficiently
Mixed liquor is obtained after mixing;By mixed liquor through the uniform sample introduction of peristaltic pump to spray dryer, 120 DEG C of inlet air temperature are controlled, by spraying
Pressure 0.20MPa, sample introduction flow 14ml/min, collect product up to microcapsule formulation by 50 DEG C of leaving air temp after spray drying.
Embodiment 2, it is protectant preferably
It selects trehalose, starch, antierythrite, gelatin, whey powder as protective agent, bacterium mud is suspended in respectively in each protective agent
It is prepared into bacteria suspension, microcapsule formulation is prepared into according still further to the method in 1 step 5) of embodiment, calculates different protective agent conditions
Under thallus survival rate.
Interpretation of result: in spray-drying process, due to high temperature, hyperosmosis, thallus is be easy to cause to damage, leads to microorganism
It is dead.In general, can effectively reduce the damage in spray-drying process to phage structure by the way that protective agent is added, spray drying is protected
Protecting agent mainly includes protide and carbohydrate.Fig. 1 is the thallus survival rate under the conditions of different protective agents, as seen from Figure 1, erythrose
Alcohol, trehalose, the thallus survival rate of whey powder are higher, illustrate that protecting effect is preferable.And the thallus survival rate highest of antierythrite,
Protecting effect is best, therefore selects antierythrite as most suitable protection agent.
Embodiment 3, carrier and bacteria suspension ratio it is preferred
The ratio that carrier and bacteria suspension is arranged is variable: ratio setting 1:16,1:14,1:12,1:10,1:8, remaining parameter item
Part remains unchanged, and is prepared into microcapsule formulation according to the method in 1 step 5) of embodiment, calculates different carriers and bacteria suspension ratio
Thallus survival rate and embedding rate under the conditions of example.
Interpretation of result: in spray-drying process, carrier has protective effect, however the mistake of carrier adsorption thallus to thallus
Journey is sufficiently complex, the too high or too low stability that can all influence carrier Yu thallus compound of carrier concn, to influence thallus
Survival rate and embedding rate.Fig. 2 is the thallus survival rate and embedding rate under the conditions of different carriers and bacteria suspension ratio.From Figure 2 it can be seen that
When the ratio of carrier and bacteria suspension is 1:10, thallus survival rate and embedding rate highest, therefore select 1:10 as carrier and
The optimal proportions of bacteria suspension.
Embodiment 4, core material and wall material ratio it is preferred
The ratio that core material and wall material is arranged is variable: ratio setting 1:1,1:3,1:5,1:7,1:9, remaining Parameter Conditions is kept
It is constant, it is prepared into microcapsule formulation according to the method in 1 step 5) of embodiment, under the conditions of calculating different core materials and wall material ratio
Thallus survival rate and embedding rate.
Interpretation of result: wall material plays decisive role in microencapsulation, and it is shadow that wall material, which has protective effect to thallus,
Ring the principal element of thallus survival rate.Fig. 3 is the thallus survival rate and embedding rate under the conditions of different core materials and wall material ratio.By scheming
3 as it can be seen that with wall material concentration increase, thallus survival rate also gradually rises;And embedding rate is as the increase of wall material concentration is in first
The trend reduced after raising, when wall material concentration is excessively high, the viscosity of material is larger, is unfavorable for being spray-dried, influences embedding efficiency,
Therefore comprehensively consider the optimal proportions for selecting 1:5 as core material and wall material.
The optimization of embodiment 5, spray drying condition
1) optimization of inlet air temperature
It is variable that inlet air temperature core when spray drying, which is arranged: 80,100,120,140,160 DEG C of temperature setting, remaining parameter
Condition remains unchanged, and is prepared into microcapsule formulation according to the method in 1 step 5) of embodiment, calculates under different inlet air temperature
Thallus survival rate and embedding rate.
Interpretation of result: in spray-drying process, inlet air temperature is one of major control factors, when inlet air temperature is too low
When, the water content of product increases, and mobility reduces and be easy agglomeration, influences embedding efficiency;When inlet air temperature is excessively high, it is easy to make
It is damaged at thallus, can also reduce protective effect of the wall material to core material.Fig. 4 be different leaving air temps under the conditions of thallus survival rate and
Embedding rate.From fig. 4, it can be seen that when inlet air temperature is 120 DEG C, thallus survival rate and embedding rate highest, therefore select 120 DEG C of works
For most suitable inlet air temperature.
2) optimization of atomisation pressure
The atomisation pressure being arranged when spray drying is variable: pressure is set as 0.10,0.15,0.20,0.25,0.30MPa, remaining
Parameter Conditions remain unchanged, and are prepared into microcapsule formulation according to the method in 1 step 5) of embodiment, calculate different atomisation pressures
Under thallus survival rate and embedding rate.
Interpretation of result: in spray-drying process, atomisation pressure is also one of major control factors, when atomisation pressure is too low
When, the partial size of product is excessive, the rapid evaporation of moisture is influenced, to influence embedding efficiency;When atomisation pressure is excessively high, it is easy broken
Bad bacterial cell structure, to influence thallus survival rate.Fig. 5 is the thallus survival rate and embedding under the conditions of different atomisation pressures
Rate.As seen from Figure 5, when atomisation pressure is 0.20MPa, thallus survival rate and embedding rate highest, therefore 0.20MPa is selected to make
For most suitable atomisation pressure.
Comparative example 1
It is a kind of can restoration of soil polluted by heavy metal microbial bacterial agent, active constituent includes that Rhizobium of Milk Vetch, fluorescence are false single
Born of the same parents bacterium, saccharomyces cerevisiae, bacillusmusilaginosiengineering, Bacillus cercus, dosage form are freeze dried powder, the preparation of the microbial bacterial agent
Method is to prepare bacteria suspension according to the method in 1 step 4) of embodiment, in ultra low temperature freezer after -20 DEG C of 5 ~ 6 h of pre-freeze
Vacuum freeze drying is carried out, -30 DEG C of primary drying phase, -40 DEG C of whole drying stage is lower than the water content of final freeze dried powder
5%。
Comparative example 2
It is a kind of can restoration of soil polluted by heavy metal microbial bacterial agent, active constituent includes that Rhizobium of Milk Vetch, fluorescence are false single
Born of the same parents bacterium, saccharomyces cerevisiae, bacillusmusilaginosiengineering, Bacillus cercus, dosage form are wettable powder, the system of the microbial bacterial agent
Preparation Method is to prepare bacteria suspension according to the method in 1 step 4) of embodiment, by bacteria suspension and white carbon black according to the ratio of 10:1
Wettable powder is made in mixing, 37 DEG C of forced air dryings.
Comparative example 3
It is a kind of can restoration of soil polluted by heavy metal microbial bacterial agent, active constituent includes that Rhizobium of Milk Vetch, fluorescence are false single
Born of the same parents bacterium, saccharomyces cerevisiae, bacillusmusilaginosiengineering, Bacillus cercus, dosage form are microcapsule formulation, the system of the microbial bacterial agent
Preparation Method difference from Example 1 is cancellation step 2) domestication culture, other steps are same as Example 1 step by step.
Comparative example 4
It is a kind of can restoration of soil polluted by heavy metal microbial bacterial agent, active constituent includes that Rhizobium of Milk Vetch, fluorescence are false single
Born of the same parents bacterium, saccharomyces cerevisiae, bacillusmusilaginosiengineering, Bacillus cercus, dosage form are microcapsule formulation, the system of the microbial bacterial agent
Preparation Method difference from Example 1 is, directly using bacteria suspension as core material in step 5), according to the ratio of core material and wall material
For 1:1, core material is added in wall material, obtains mixed liquor after mixing well;Other spray drying conditions are same as Example 1.
Test example 1
1) the thermodynamic stability test of preparation
The microbial bacterial agent of embodiment 1, comparative example 1, comparative example 2 is placed in insulating box, stores 14d under the conditions of 54 ± 2 DEG C,
It is counted once every 2d, measures the variation of its thallus survival rate.
2) the normal temperature storage stability test of preparation
The microbial bacterial agent of embodiment 1, comparative example 1, comparative example 2 is placed in insulating box, is stored under the conditions of 25 ± 2 DEG C
360d counts once every 60d, measures the variation of its thallus survival rate.
Interpretation of result: from the thermodynamic stability test result of preparation it is found that 1 microcapsule formulation of embodiment when storage 14d
Thallus survival rate is 92.20%, is significantly higher than the thallus survival of the freeze dried powder of comparative example 1 and the wettable powder of comparative example 2
Rate;From the normal temperature storage stability test result of preparation it is found that storage 360d when 1 microcapsule formulation of embodiment thallus survival rate
It is the thallus survival rate of 91.08%, the also significantly greater than freeze dried powder of comparative example 1 and the wettable powder of comparative example 2, shows micro-
Capsule preparations have good storage stability.
Test example 2
In June, 2017, the dirty farmland for filling area outside Jiangsu Province, Guannan County chemical industrial park, Lianyungang that has drawn from is tested, test is preceding right
0 ~ the 20cm of agricultural land soil surface layer sampling, carries out available heavy metal content detection, and testing result is cadmium 1.02mg/kg, mercury
2.76mg/kg, lead 136.7mg/kg, copper 80.9mg/kg, zinc 215.1mg/kg, soil pH value is 5.5 ~ 6.0, then by the agriculture
Field is divided into 5 regions, and micro- life of embodiment 1, comparative example 1, comparative example 2, comparative example 3, comparative example 4 is respectively adopted in each region
Object microbial inoculum carries out repair process, and processing method is to be directly applied to microbial bacterial agent in soil to be repaired, and amount of application is
30g/m2, handled under field conditions (factors) 30 days or so after turning over mixing.After treatment again has the soil in each region
Effect state content of beary metal is detected, and is calculated the removal rate of several heavy metal species, be the results are shown in Table 3.
Test example 3
In September, 2017 tests the farmland on Jiangsu Province, city stone Dangshan, the Jurong copper mine field periphery that has drawn from, to the agricultural land soil before test
0 ~ 20cm of surface layer sampling, carries out available heavy metal content detection, and testing result is cadmium 5.95mg/kg, mercury 4.18mg/kg, lead
58.3mg/kg, copper 247.4mg/kg, zinc 328.6mg/kg, soil pH value are 6.5 ~ 7.0, and the farmland is then divided into 5 areas
Domain, each region be respectively adopted embodiment 1, comparative example 1, comparative example 2, comparative example 3, comparative example 4 microbial bacterial agent repaired
Multiple processing, processing method are to be directly applied to microbial bacterial agent in soil to be repaired, amount of application 40g/m2, turn over mixing
It handles 30 days or so under field conditions (factors) afterwards.After treatment again to the soil in each region carry out available heavy metal content into
Row detection, calculates the removal rate of several heavy metal species, the results are shown in Table 4.
Interpretation of result: from the result of test example 2 and test example 3 it is found that being repaired using the microbial bacterial agent of embodiment 1
Processing, has reached 90% or more to the removal rate of the heavy metals available state such as cadmium, mercury, lead, copper, zinc, has been significantly higher than comparative example 1-
4 removal rate shows that microbial bacterial agent of the invention is significant to the repairing effect of heavy-metal contaminated soil.
The available heavy metal removal rate of embodiment 1, comparative example 3 and comparative example 4 is above comparative example 1 and comparative example 2, this
Be since the microbial bacterial agent of embodiment 1, comparative example 3 and comparative example 4 is microcapsule formulations, can be preferably than common pulvis
The activity of microorganism is saved, microorganism is affected by the external environment smaller when in use, and good stress resistance, the lasting period is long, can be maximum
Microorganism is played to limit to the removal effect of heavy metal.
Comparing embodiment 1 and comparative example 3, the microbial bacterial agent of embodiment 1 is during the preparation process to the microorganism of selection point
Not carry out contents of many kinds of heavy metal ion domestication culture step by step, every kind of microorganism after taming all has these heavy metals higher
Tolerance, the soil that can adapt to different degrees of heavy metal pollution after composite bacteria agent is made in compounding, even if more tight polluting
Activity, which is still able to maintain, in the soil of weight and carries out efficient repairing answers.
Comparing embodiment 1 and comparative example 4, the microbial bacterial agent of embodiment 1 is during the preparation process with Fe3O4Particle is as load
Body adsorbs thallus, Fe3O4Particle not only has protective effect, but also Fe to thallus during spray drying3O4Particle is through magnetic
Low-intensity magnetic field can be generated after change processing, the growth metabolism of microorganism can be promoted, the stability of microorganism growth is improved, to improve
Its absorption and conversion capability to heavy metal achievees the purpose that improve available heavy metal removal rate.
In conclusion although the present invention by specific embodiment to the present invention have been described in detail, this field one
As technical staff it is to be understood that above-described embodiment is only description to the preferred embodiment of the present invention, rather than to this hair
The limitation of bright protection scope, persons skilled in the art within the technical scope disclosed by the invention, the change that can be readily occurred in
Change, it is within the scope of the present invention.
Claims (9)
1. one kind can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application, which is characterized in that it is described
The active constituent of microbial bacterial agent includes Rhizobium of Milk Vetch, Pseudomonas fluorescens, saccharomyces cerevisiae, bacillusmusilaginosiengineering, wax
Shape bacillus, dosage form are microcapsule formulation, and total viable count is 5 × 1010~6×1010cfu/g。
2. it is according to claim 1 can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application,
It is characterized in that, the preparation method of the microbial bacterial agent includes the following steps:
1) activation culture: in gnotobasis, respectively by Rhizobium of Milk Vetch, Pseudomonas fluorescens, saccharomyces cerevisiae, gel-shaped bud
Spore bacillus, Bacillus cercus strain be restored to room temperature state from preservation state, then strain is conventionally shaken
Bed activation culture, is prepared into seed liquor;
2) domestication culture step by step: above-mentioned seed liquor is inoculated into respectively containing heavy metal ion Cd2+、Hg2+、Pb2+、Cu2+、Zn2+'s
It is cultivated in domestication culture medium, period sampling measuring bacterium solution absorbance, indicates that domestication is completed after absorbance reaches maximum value,
Bacterium solution at this time is transferred in the domestication culture medium containing higher concentration heavy metal ion and is cultivated, be so carried out continuously 5 it is dense
Spend the domestication culture of rank;
3) mixing fermentation culture: selecting liquid fermentation tank, and fermentation medium liquid amount is 60 ~ 70%, the Chinese milk vetch after inoculation domestication
Rhizobium, Pseudomonas fluorescens, saccharomyces cerevisiae, bacillusmusilaginosiengineering, Bacillus cercus seed liquor, total inoculum concentration be 6 ~
10%, the inoculative proportion of each microorganism is 2:1:1:3:3, controls 30 ~ 35 DEG C of fermentation temperature, 250 ~ 300rpm of revolving speed, ventilatory capacity
1.0 ~ 1.4VVM, 36 ~ 48h of fermented and cultured under this fermentation condition;
4) preparation of bacteria suspension: being centrifuged 8 ~ 10min for fermentation liquid under the conditions of 6000 ~ 7000 rpm, abandon supernatant collection bacterium mud,
With brine 2-3 times, bacterium mud is suspended in protective agent (5% antierythrite solution) and is prepared into bacteria suspension, keeps viable bacteria close
Degree reaches 1 × 1011Cfu/ml or more;
5) preparation of magnetic field-intensification microcapsules: Fe of the selection degree of compression 20 ~ 30%3O4Particle is carrier, outstanding according to carrier and bacterium
The ratio of liquid is 1:10, carrier is added into bacteria suspension, then stirring evenly makes carrier adsorption thallus, is dried to obtain core at 37 DEG C
Material;It is 1:5 according to the ratio of core material and wall material, core material is added in wall material, obtains mixed liquor after mixing well;By mixed liquor
Through the uniform sample introduction of peristaltic pump to spray dryer, 115 ~ 125 DEG C of inlet air temperature, 0.18 ~ 0.22MPa of atomisation pressure are controlled, sample introduction
13 ~ 15ml/min of flow, collects product up to the microcapsule formulation by 45 ~ 55 DEG C of leaving air temp after spray drying.
3. it is according to claim 2 can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application,
It is characterized in that, conventional method described in step 1) refers to Rhizobium of Milk Vetch, Pseudomonas fluorescens, gel-shaped gemma bar
Bacterium, Bacillus cercus strain be inoculated in bacterium basal liquid medium, 30 ~ 35 DEG C, 160 ~ 200r/min shaking table train
Feeding 18 ~ for 24 hours;The strain of saccharomyces cerevisiae is inoculated in yeast fluid nutrient medium, is trained in 28 ~ 32 DEG C, 140 ~ 180r/min shaking table
Support 30 ~ 36h.
4. it is according to claim 2 can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application,
It is characterized in that, Cd described in step 22+、Hg2+、Pb2+、Cu2+、Zn2+, 5 concentration ranks are set as 60mg/L, 120mg/
L、180mg/L、240mg/L、300mg/L。
5. it is according to claim 2 can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application,
It is characterized in that, taming the basic recipe of culture medium described in step 2 are as follows: 16 ~ 20g/L of glucose, 5 ~ 10g/L of peptone, ferment
Female 4 ~ 6g/L of cream, CaCO3 2.4 ~ 3.0g/L, KH2PO40.5 ~ 0.9g/L, K2HPO40.5 ~ 0.8g/ of 1.8 ~ 2.2g/L, NaCl
L, MgSO4·7H20.3 ~ 0.5g/L of O, 0.12 ~ 0.14mg/L of methyl jasmonate, additionally adds CdCl2、Pb(NO3)2、HgCl2,
CuSO4, ZnSO4, surplus is water, pH5.5 ~ 6.5.
6. it is according to claim 2 can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application,
It is characterized in that, the formula of fermentation medium described in step 3) are as follows: 20 ~ 28g/L of wheatfeed, 27 ~ 33g/L of beancake powder, mushroom bran
Powder 7 ~ 11g/L, CaCO34 ~ 5g/L, KH2PO40.5 ~ 1.0g/L, K2HPO40.9 ~ 1.5g/L of 1.8 ~ 2.2g/L, NaCl, it is yellow rotten
1.5 ~ 2.5g/L of acid, Tween 80 0.8 ~ 1.2g/L, 6-BA 1.3 ~ 1.5mg/L of 0.6 ~ 1.0mg/L, IAA, surplus is water,
pH5.5~6.5。
7. it is according to claim 2 can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application,
It is characterized in that, Fe described in step 5)3O4Particle needs to reach its surface induction intensity by magnetization treatment before use
0.3~0.6mT。
8. it is according to claim 2 can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application,
It is characterized in that, wall material described in step 5) the preparation method comprises the following steps: by maltodextrin, microcrystalline cellulose and water press 3:1:6 quality
Than mixing, heats while stirring in 60 DEG C of water-baths uniformly, again through the high-pressure homogeneous processing 10min of 20 ~ 30MPa after cooling, obtain
Compound wall materials.
9. it is according to claim 1 can restoration of soil polluted by heavy metal microbial bacterial agent and its preparation method and application,
It is characterized in that, the application of the microbial bacterial agent is that microbial bacterial agent is directly applied to heavy metal pollution farmland to be repaired
In soil, amount of application is 20 ~ 50g/m2, handled under field conditions (factors) 30 days or so after turning over mixing.
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