CN108048371A - A kind of multifunctional agriculture soil remediation microbial inoculum and its preparation method and application - Google Patents
A kind of multifunctional agriculture soil remediation microbial inoculum and its preparation method and application Download PDFInfo
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- CN108048371A CN108048371A CN201810083683.XA CN201810083683A CN108048371A CN 108048371 A CN108048371 A CN 108048371A CN 201810083683 A CN201810083683 A CN 201810083683A CN 108048371 A CN108048371 A CN 108048371A
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Abstract
A kind of multifunctional agriculture soil remediation microbial inoculum and its preparation method and application, is related to a kind of microbial inoculum and its preparation method and application.Be easily competed to solve microorganism in the microbial degradation method of existing DBP by indigenous microorganism, the Soil Factors such as the soil moisture are influenced, it is difficult to the problem of keeping higher bioactivity and more single function.The microbial inoculum includes microbial bacterial agent A and DBP degradation fermented liquid, and the microbial bacterial agent A includes sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid.Method：Prepare microbial bacterial agent A；Prepare DBP degradation fermented liquids；DBP degradation fermented liquids and microbial bacterial agent A are mixed, obtain multifunctional agriculture soil remediation microbial inoculum.The microbial inoculum can keep higher bioremediation activity for a long time in soil environment, effectively improve the remediation efficiency of DBP pollutions, while DBP contaminated soils are repaired, and contaminated soil fertility can be improved.The present invention is used for soil remediation field.
Description
Technical field
The present invention relates to a kind of microbial inoculums and its preparation method and application.
Background technology
The fast development of agricultural results in a large amount of uses of agricultural mulching, usage amount the first in the world of Chinese agricultural mulching,
Agricultural film causes white pollution, and the situation is tense.Dibutyl phthalate (DBP) is a kind of important phthalate ester (PAEs) compound,
As a kind of plasticizer, it is widely used in plastic film.A large amount of uses of agricultural film, cause DBP in China's agricultural soil
Seriously polluted, DBP pollutions not only result in soil soil property variation, influence agricultural production, DBP can also be accumulated by crop, passed through
Food chain is entered in human body, and the generations such as human endocrine, nervous system are endangered.Chinese environmental detection master station and the U.S. at present
DBP is classified as priority pollutants by Environmental Protection Administration (EPA), therefore the prevention that DBP pollutes in soil is very urgent.
DBP not facile hydrolysis and photodissociation in soil environment, microbial degradation are still that the main of agricultural non-point source pollution is controlled at present
Reason method.Agricultural non-point source pollution refers to as caused by the scattered pollution sources such as deposit, pesticide, waste material, pathogenic bacteria to water layer, lake
The pollution of the ecosystems such as pool, riverbank, shore bank, air.Compared with point-source pollution, the space-time unique of pollution of area source is wider, not really
Qualitative bigger, ingredient, process are more complicated, it is more difficult to control.Currently, in China's rural activity, non-science through management read and
The backward mode of production is that the use, excessive chemical fertilizer such as highly toxic pesticide are applied an important factor for causing agricultural environment pollution of area source
It spreads, non-degradable agricultural film is abandoned every year in field, open incineration stalk, that large-scale plant that raises fowl and animal excrement does not do harmless treatment is random
Stack etc..Pollution of these pollution sources to environment, it is especially maximum to the pollution effect of water environment, according to statistics, agricultural non-point source pollution
Account for the 60%~80% of river and lake eutrophication problem.
In the existing soil using microbiological treatment in the method for DBP, microbial function is more single, and easily by the micro- life of original inhabitants
The influence of the Soil Factors such as object competition, the soil moisture, it is difficult to keep higher bioactivity.Therefore in the agriculture of natural environment
The ineffective of DBP is removed in industry soil.
The content of the invention
The present invention is easily to be competed to solve microorganism in the microbial degradation method of existing DBP by indigenous microorganism, is native
The influence of the Soil Factors such as earth temperature, it is difficult to which the problem of keeping higher bioactivity and more single function provides one
Kind multifunctional agriculture soil remediation microbial inoculum and its preparation method and application.
Multifunctional agriculture soil remediation microbial inoculum of the present invention includes microbial bacterial agent A and DBP degradation fermented liquid, micro- life
Object microbial inoculum A includes sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid.
The mass ratio of wherein microbial bacterial agent A and DBP degradation fermented liquids is (5-10)：(2-4).
Sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium fermentation in the microbial bacterial agent A
Liquid mass ratio is (1-2)：(1-2)：(5-10).
The concentration of DBP degradation fermented liquids is 108More than cfu/mL.
The concentration of sphingolipid fermented liquid is 108More than cfu/mL.
The concentration of colloid bacillus cereus zymotic fluid is 108More than cfu/mL.
The concentration of acid-producing Klebsiella bacterium zymotic fluid is 108More than cfu/mL.
The DBP degradation bacterias are pseudomonad (Pseudomonas sp.) DNB-S1, are deposited in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center (CGMCC) is hidden, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation
Date is on November 6th, 2014, and deposit number is CGMCC No.9526.Disclosed in patent 201410798113.0.
The sphingolipid bacterium purchase is protected from China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
It is CGMCC No.4400 to hide number.
The colloid bacillus cereus is bought from China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), deposit number is CGMCC No.7240.
The acid-producing Klebsiella bacterium is bought from China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), deposit number is CGMCC No.4155.
By above-mentioned multifunctional agriculture soil remediation microbial inoculum and carrier according to mass ratio 1:1 mixing can obtain solid and repair bacterium
Agent.The carrier is according to mass ratio 2 by attapulgite, kaolin and humic acid:1:1 composition.
The preparation method of above-mentioned multifunctional agriculture soil remediation microbial inoculum, is realized according to the following steps：
First, microbial bacterial agent A is prepared：
Sphingolipid bacterium is inoculated in Pikovskava solution Phos solid mediums, in 27~29 DEG C, 120~130r/min items
24~26h of activation culture is carried out under part；Then the strain of activation is inoculated in Pikovskava solution Phos fluid nutrient mediums again
After expanding 4~6d of culture, sphingolipid fermented liquid is obtained；
Colloid bacillus cereus is inoculated in silicate bacteria solid medium in 27~29 DEG C, 120~130r/min conditions
24~26h of lower progress activation culture；Then again by the strain of activation be inoculated in silicate bacteria fluid nutrient medium expand culture 4~
After 6d, colloid bacillus cereus zymotic fluid is obtained；
Acid-producing Klebsiella bacterium is inoculated in Ah 's bayesian fixed nitrogen solid medium in 27~29 DEG C, 120~130r/min
Under the conditions of carry out activation culture 24~26h；Then the strain of activation is inoculated in Ah 's bayesian fixed nitrogen fluid nutrient medium again to expand
After cultivating 4~6d, sphingolipid fermented liquid is obtained；
Sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid are mixed, obtained compound
Microbial bacterial agent A；
2nd, DBP degradation bacterias are inoculated in inorganic salts fixed nitrogen solid medium in 27~29 DEG C, 120~130r/min conditions
24~26h of lower progress activation culture；Then the strain of activation is inoculated in inorganic salt liquid culture medium again and expands 24~26h of culture
Afterwards, the DBP degradation germ liquids that will be enlarged by culture are inoculated by 10%~15% inoculum concentration in inexpensive fermentation culture medium, and 27~29
DEG C, 72~74h of activation culture is carried out under the conditions of 120~130r/min, obtain DBP degradation fermented liquids；
3rd, DBP degradation fermented liquids and microbial bacterial agent A are mixed, obtains multifunctional agriculture soil remediation microbial inoculum.
Sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid mass ratio are in step 1
(1-2)：(1-2)：(5-10).
The mass ratio of microbial bacterial agent A and DBP degradation fermented liquid is (5-10) in step 3：(2-4).
The solution Phos solid culture based components of Pikovskava described in step 1 include：10g calcium phosphate, 0.5g sulfuric acid
Ammonium, 10g glucose, 0.3g magnesium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.03g ferric sulfate, 0.03g manganese sulfates, 1000mL
Distilled water, 15g agar, pH=7.0；
The solution Phos Liquid Culture based components of Pikovskava described in step 1 include：10g calcium phosphate, 0.5g sulfuric acid
Ammonium, 10g glucose, 0.3g magnesium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.03g ferric sulfate, 0.03g manganese sulfates, 1000mL
Distilled water, pH=7.0.
The based component of silicate bacteria solid culture described in step 1 includes：3g disodium hydrogen phosphates, 1.0g ferric sulfate, 10g
Glucose, 1.0g magnesium sulfate, 0.2g sodium chloride, 1.5g potassium feldspars, 1000mL distilled water, 15g agar, pH=7.0；
The based component of silicate bacteria Liquid Culture described in step 1 includes：3g disodium hydrogen phosphates, 1.0g ferric sulfate, 10g
Glucose, 1.0g magnesium sulfate, 0.2g sodium chloride, 1.5g potassium feldspars, 1000mL distilled water, pH=7.0；
The fixed nitrogen solid culture based component of Ah 's bayesian described in step 1 includes：0.3g potassium dihydrogen phosphates, 0.2g magnesium sulfate,
0.12g sodium chloride, 1.0g calcium carbonate, 10g sucrose, 1000mL distilled water, 15g agar, pH=7.0；
The fixed nitrogen Liquid Culture based component of Ah 's bayesian described in step 1 includes：0.3g potassium dihydrogen phosphates, 0.2g magnesium sulfate,
0.12g sodium chloride, 1.0g calcium carbonate, 10g sucrose, 1000mL distilled water, pH=7.0.
The based component of inorganic salts solid culture described in step 2 includes, 1.0g potassium dihydrogen phosphates, 0.4g magnesium sulfate, 1.0g chlorine
Change sodium, 0.01g iron chloride, 0.075g calcium chloride, 0.5g ammonium nitrate, 500mg/ml DBP, 1000mL distilled water, 15g agar, pH
=7.0；
Inorganic salt liquid medium component described in step 2 includes, 1.0g potassium dihydrogen phosphates, 0.4g magnesium sulfate, 1.0g chlorine
Change sodium, 0.01g iron chloride, 0.075g calcium chloride, 0.5g ammonium nitrate, 500mg/ml DBP, 1000mL distilled water, pH=7.0.
Inexpensive fermentation medium component described in step 2 includes, 23.9g corn flour, 37.5g analysis for soybean powder, 0.8g di(2-ethylhexyl)phosphates
Hydrogen potassium, 0.4g magnesium sulfate, 0.4g sodium chloride, 0.04g iron chloride, 0.1g calcium chloride, 1000mL distilled water, pH=7.0.
Above-mentioned multifunctional agriculture soil remediation microbial inoculum is repairing the application in polluting agricultural soil.
Specific method is：Multifunctional agriculture soil remediation microbial inoculum is added in soil to be repaired, multifunctional agriculture soil
The dosage of remediation microbial inoculum is the 1 ‰ -1.5 ‰ of soil volume to be repaired.
Beneficial effects of the present invention：
Multifunctional agriculture soil remediation microbial inoculum can effectively solve the problem that solve in existing PAEs dye repair process microorganism easily by
Indigenous microorganism competition, the influence of Soil Factors, it is difficult to the problems such as keeping higher bioactivity and more single function；
Agricultural soil can be effectively improved simultaneously, improved or replied soil quality, improve crop quality and yield.
Multi-functional microbial inoculum prepared by the present invention is a kind of compounding microbial bacterial agent, does not have antagonism between compounding strain, each
There is different, the biological function holding stabilization of strain；It is synergistic between each strain, it mutually promotes, it can be notable
The ability of DBP in DBP degradation bacterias degradation soil is improved, DBP pollution removal rates reach more than 94% in soil, false with simple application
Monad (Pseudomonas sp.) DNB-S1 compares, and the removal rate of DBP improves 8%-12% in agricultural soil.Simultaneously
The content of effective N P and K in soil can substantially be increased, available nitrogen, rapid available phosphorus and quick-acting potassium content are respectively increased in soil
11%~12%, 11%~13%, 9%~10%.Soil quality can be improved, soil fertility is improved, make biological activity of soil
Increase, significantly improve urase and sucrase active in soil, promote soil renewal, so as to promote the growth of crops.
The preparation method of the multi-functional microbial inoculum is easy to operate, not high to environmental requirement.And the immobilized microorganism used
Quantity that technology effectively overcomes traditional microbial bacterial agent is few, activity is low, stability is poor and the shortcomings that intolerant to environmental impact,
The pollution of DBP in soil can be effectively eliminated.
The present invention realizes soil remediation and the complex function of improvement, suitable for agricultural extension and application, to realizing that agricultural can
Lasting target is of great significance.
Description of the drawings
Fig. 1 is sphingolipid bacterium in embodiment 2, colloid bacillus cereus, the tablet face-off result of acid-producing Klebsiella bacterium；
Fig. 2 is the tablet face-off of DBP degradation bacterias in embodiment 2, sphingolipid bacterium, colloid bacillus cereus, acid-producing Klebsiella bacterium
As a result.
Specific embodiment
Technical solution of the present invention is not limited to act specific embodiment set forth below, further includes between each specific embodiment
Any combination.
Specific embodiment one：Present embodiment multifunctional agriculture soil remediation microbial inoculum is dropped including microbial bacterial agent A and DBP
Fermented liquid is solved, the microbial bacterial agent A includes sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium
Zymotic fluid.
The DBP degradation bacterias are pseudomonad (Pseudomonas sp.) DNB-S1, are deposited in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center (CGMCC) is hidden, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation
Date is on November 6th, 2014, and deposit number is CGMCC No.9526.Disclosed in patent 201410798113.0.
The microbial inoculum of present embodiment can improve contaminated soil fertility while DBP contaminated soils are repaired,
Have great importance for clean manufacturing, physical geography and the food security of agricultural.
Specific embodiment two：The present embodiment is different from the first embodiment in that：Microbial bacterial agent A and DBP drop
The mass ratio for solving fermented liquid is (5-10)：(2-4).It is other same as the specific embodiment one.
Specific embodiment three：The present embodiment is different from the first embodiment in that：The microbial bacterial agent A mesothecas
Fat fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid mass ratio are (1-2)：(1-2)：(5-
10).It is other same as the specific embodiment one.
Specific embodiment four：The present embodiment is different from the first embodiment in that：DBP degradation fermented liquid it is dense
It spends for 108More than cfu/mL.It is other same as the specific embodiment one.
Specific embodiment five：The present embodiment is different from the first embodiment in that：The concentration of sphingolipid fermented liquid
For 108More than cfu/mL.It is other same as the specific embodiment one.
The sphingolipid bacterium purchase is protected from China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
It is CGMCC No.4400 to hide number.
Specific embodiment six：The present embodiment is different from the first embodiment in that：Colloid bacillus cereus zymotic fluid
Concentration be 108More than cfu/mL.It is other same as the specific embodiment one.
The colloid bacillus cereus is bought from China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), deposit number is CGMCC No.7240.
Specific embodiment seven：The present embodiment is different from the first embodiment in that：Acid-producing Klebsiella bacterium ferments
The concentration of liquid is 108More than cfu/mL.It is other same as the specific embodiment one.
The acid-producing Klebsiella bacterium is bought from China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), deposit number is CGMCC No.4155.
Specific embodiment eight：The present embodiment is different from the first embodiment in that：It is repaiied in multifunctional agriculture soil
It answers and carrier is additionally added in microbial inoculum, the mass ratio of multifunctional agriculture soil remediation microbial inoculum and carrier is 1:1, the carrier is by bumps
Stick soil, kaolin and humic acid are according to mass ratio 2:1:1 composition.It is other same as the specific embodiment one.
Specific embodiment nine：The preparation method of present embodiment multifunctional agriculture soil remediation microbial inoculum, according to the following steps
It realizes：
First, microbial bacterial agent A is prepared：
Sphingolipid bacterium is inoculated in Pikovskava solution Phos solid mediums, in 27~29 DEG C, 120~130r/min items
24~26h of activation culture is carried out under part；Then the strain of activation is inoculated in Pikovskava solution Phos fluid nutrient mediums again
After expanding 4~6d of culture, sphingolipid fermented liquid is obtained；
Colloid bacillus cereus is inoculated in silicate bacteria solid medium in 27~29 DEG C, 120~130r/min conditions
24~26h of lower progress activation culture；Then again by the strain of activation be inoculated in silicate bacteria fluid nutrient medium expand culture 4~
After 6d, colloid bacillus cereus zymotic fluid is obtained；
Acid-producing Klebsiella bacterium is inoculated in Ah 's bayesian fixed nitrogen solid medium in 27~29 DEG C, 120~130r/min
Under the conditions of carry out activation culture 24~26h；Then the strain of activation is inoculated in Ah 's bayesian fixed nitrogen fluid nutrient medium again to expand
After cultivating 4~6d, sphingolipid fermented liquid is obtained；
Sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid are mixed, obtained compound
Microbial bacterial agent A；
2nd, DBP degradation bacterias are inoculated in inorganic salts fixed nitrogen solid medium in 27~29 DEG C, 120~130r/min conditions
24~26h of lower progress activation culture；Then the strain of activation is inoculated in inorganic salt liquid culture medium again and expands 24~26h of culture
Afterwards, the DBP degradation germ liquids that will be enlarged by culture are inoculated by 10%~15% inoculum concentration in inexpensive fermentation culture medium, and 27~29
DEG C, 72~74h of activation culture is carried out under the conditions of 120~130r/min, obtain DBP degradation fermented liquids；
3rd, DBP degradation fermented liquids and microbial bacterial agent A are mixed, obtains multifunctional agriculture soil remediation microbial inoculum.
Specific embodiment ten：Present embodiment is unlike specific embodiment nine：Sphingolipid bacterium is fermented in step 1
Liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid mass ratio are (1-2)：(1-2)：(5-10).It is other with
Specific embodiment nine is identical.
Specific embodiment 11：Present embodiment is unlike specific embodiment nine：Microbial bacteria in step 3
The mass ratio of agent A and DBP degradation fermented liquid is (5-10)：(2-4).It is other identical with specific embodiment nine.
Specific embodiment 12：Present embodiment is unlike specific embodiment nine：Described in step 1
Pikovskava solution Phos solid culture based components include：10g calcium phosphate, 0.5g ammonium sulfate, 10g glucose, 0.3g sulfuric acid
Magnesium, 0.3g sodium chloride, 0.3g potassium chloride, 0.03g ferric sulfate, 0.03g manganese sulfates, 1000mL distilled water, 15g agar, pH=
7.0.It is other identical with specific embodiment nine.
Specific embodiment 13：Present embodiment is unlike specific embodiment nine：Described in step 1
Pikovskava solution Phos Liquid Culture based components include：10g calcium phosphate, 0.5g ammonium sulfate, 10g glucose, 0.3g sulfuric acid
Magnesium, 0.3g sodium chloride, 0.3g potassium chloride, 0.03g ferric sulfate, 0.03g manganese sulfates, 1000mL distilled water, pH=7.0.It is other with
Specific embodiment nine is identical.
Specific embodiment 14：Present embodiment is unlike specific embodiment nine：Silicic acid described in step 1
Salt bacterium solid culture medium ingredient includes：3g disodium hydrogen phosphates, 1.0g ferric sulfate, 10g glucose, 1.0g magnesium sulfate, 0.2g chlorine
Change sodium, 1.5g potassium feldspars, 1000mL distilled water, 15g agar, pH=7.0.It is other identical with specific embodiment nine.
Specific embodiment 15：Present embodiment is unlike specific embodiment nine：Silicic acid described in step 1
Salt bacterial liquid medium component includes：3g disodium hydrogen phosphates, 1.0g ferric sulfate, 10g glucose, 1.0g magnesium sulfate, 0.2g chlorine
Change sodium, 1.5g potassium feldspars, 1000mL distilled water, pH=7.0.It is other identical with specific embodiment nine.
Specific embodiment 16：Present embodiment is unlike specific embodiment nine：Ah palpus described in step 1
Bayesian fixed nitrogen solid culture based component includes：0.3g potassium dihydrogen phosphates, 0.2g magnesium sulfate, 0.12g sodium chloride, 1.0g calcium carbonate,
10g sucrose, 1000mL distilled water, 15g agar, pH=7.0.It is other identical with specific embodiment nine.
Specific embodiment 17：Present embodiment is unlike specific embodiment nine：Ah palpus described in step 1
Bayesian fixed nitrogen Liquid Culture based component includes：0.3g potassium dihydrogen phosphates, 0.2g magnesium sulfate, 0.12g sodium chloride, 1.0g calcium carbonate,
10g sucrose, 1000mL distilled water, pH=7.0.It is other identical with specific embodiment nine.
Specific embodiment 18：Present embodiment is unlike specific embodiment nine：It is inorganic described in step 2
Salt solid culture based component includes, 1.0g potassium dihydrogen phosphates, 0.4g magnesium sulfate, 1.0g sodium chloride, 0.01g iron chloride, 0.075g
Calcium chloride, 0.5g ammonium nitrate, 500mg/ml DBP, 1000mL distilled water, 15g agar, pH=7.0.Other and specific embodiment party
Formula nine is identical.
Specific embodiment 19：Present embodiment is unlike specific embodiment nine：It is inorganic described in step 2
Salt liquid medium component includes, 1.0g potassium dihydrogen phosphates, 0.4g magnesium sulfate, 1.0g sodium chloride, 0.01g iron chloride, 0.075g
Calcium chloride, 0.5g ammonium nitrate, 500mg/ml DBP, 1000mL distilled water, pH=7.0.It is other identical with specific embodiment nine.
Specific embodiment 20：Present embodiment is unlike specific embodiment nine：It is cheap described in step 2
Fermentation medium components include, 23.9g corn flour, 37.5g analysis for soybean powder, 0.8g potassium dihydrogen phosphates, 0.4g magnesium sulfate, 0.4g chlorinations
Sodium, 0.04g iron chloride, 0.1g calcium chloride, 1000mL distilled water, pH=7.0.It is other identical with specific embodiment nine.
Specific embodiment 21：Present embodiment multifunctional agriculture soil remediation microbial inoculum is repairing pollution agricultural soil
In application.
Specific embodiment 22：Present embodiment is unlike specific embodiment 21：Repair pollution agriculture
The specific method of industry soil is：Multifunctional agriculture soil remediation microbial inoculum is added in soil to be repaired, multifunctional agriculture soil
The dosage of remediation microbial inoculum is the 1 ‰ -1.5 ‰ of soil volume to be repaired.It is other identical with specific embodiment 21.
Elaborate below to the embodiment of the present invention, following embodiment under based on the technical solution of the present invention into
Row is implemented, and gives detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following realities
Apply example.
Embodiment 1：
The preparation of the present embodiment multifunctional agriculture soil remediation microbial inoculum, is realized according to the following steps：
First, complex micro organism fungicide A is prepared：
Sphingolipid bacterium (CGMCC No.4400) seed is taken, Pikovskava solution Phos solid mediums are inoculated in, 28
DEG C, carry out activation culture for 24 hours under the conditions of 125r/min；Then the strain of activation is inoculated in Pikovskava solution Phos liquid again
After body culture medium expands culture 5d, sphingolipid bacterium (CGMCC No.4400) cell concentration reaches 4.0 × 109cfu/mL。
The Pikovskava solutions Phos solid culture based component includes：10g calcium phosphate, 0.5g ammonium sulfate, 10g grapes
Sugar, 0.3g magnesium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.03g ferric sulfate, 0.03g manganese sulfates, 1000mL distilled water, 15g
Agar, pH=7.0；
The Pikovskava solutions Phos Liquid Culture based component includes：10g calcium phosphate, 0.5g ammonium sulfate, 10g grapes
Sugar, 0.3g magnesium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.03g ferric sulfate, 0.03g manganese sulfates, 1000mL distilled water, pH=
7.0。
Take colloid bacillus cereus (CGMCC No.7240) seed, be inoculated in silicate bacteria solid medium 28 DEG C,
Activation culture is carried out under the conditions of 125r/min for 24 hours；Then the strain of activation is inoculated in silicate bacteria fluid nutrient medium again to expand
After big culture 5d, colloid bacillus cereus (CGMCC No.7240) cell concentration reaches 4.0 × 109cfu/mL。
The silicate bacteria solid culture based component includes：3g disodium hydrogen phosphates, 1.0g ferric sulfate, 10g glucose,
1.0g magnesium sulfate, 0.2g sodium chloride, 1.5g potassium feldspars, 1000mL distilled water, 15g agar, pH=7.0；
The silicate bacteria Liquid Culture based component includes：3g disodium hydrogen phosphates, 1.0g ferric sulfate, 10g glucose,
1.0g magnesium sulfate, 0.2g sodium chloride, 1.5g potassium feldspars, 1000mL distilled water, pH=7.0；
Acid-producing Klebsiella bacterium (CGMCC No.4155) seed is taken, is inoculated in Ah 's bayesian fixed nitrogen solid medium 28
DEG C, carry out activation culture for 24 hours under the conditions of 125r/min；Then the strain of activation is inoculated in Ah 's bayesian fixed nitrogen Liquid Culture again
After base expands culture 5d, sphingolipid bacterium (CGMCC No.4400) cell concentration reaches 8.0 × 108cfu/mL。
Ah 's bayesian fixed nitrogen solid culture based component includes：0.3g potassium dihydrogen phosphates, 0.2g magnesium sulfate, 0.12g chlorine
Change sodium, 1.0g calcium carbonate, 10g sucrose, 1000mL distilled water, 15g agar, pH=7.0；
Ah 's bayesian fixed nitrogen Liquid Culture based component includes：0.3g potassium dihydrogen phosphates, 0.2g magnesium sulfate, 0.12g chlorine
Change sodium, 1.0g calcium carbonate, 10g sucrose, 1000mL distilled water, pH=7.0.
Sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid mass ratio are 1：1：5.
2nd, take DBP degradation bacterias (CGMCC No.9526) seed, be inoculated in inorganic salts fixed nitrogen solid medium 28 DEG C,
Activation culture is carried out under the conditions of 125r/min for 24 hours；Then the strain of activation is inoculated in inorganic salt liquid culture medium again and expands training
After supporting for 24 hours, the DBP degradation bacterias (CGMCC No.9526) that will be enlarged by culture are inoculated into inexpensive fermentation by the 10% of fermentating liquid volume
In culture medium, 28 DEG C, activation culture 72h, DBP degradation bacteria (CGMCC No.9526) cell concentration is carried out under the conditions of 125r/min
Reach 1.0 × 1010cfu/mL。
The inorganic salts solid culture based component includes, 1.0g potassium dihydrogen phosphates, 0.4g magnesium sulfate, 1.0g sodium chloride,
0.01g iron chloride, 0.075g calcium chloride, 0.5g ammonium nitrate, 500mg/ml DBP, 1000mL distilled water, 15g agar, pH=
7.0；
The inorganic salt liquid medium component includes, 1.0g potassium dihydrogen phosphates, 0.4g magnesium sulfate, 1.0g sodium chloride,
0.01g iron chloride, 0.075g calcium chloride, 0.5g ammonium nitrate, 500mg/ml DBP, 1000mL distilled water, pH=7.0.
The inexpensive fermentation medium component includes, 23.9g corn flour, 37.5g analysis for soybean powder, 0.8g potassium dihydrogen phosphates,
0.4g magnesium sulfate, 0.4g sodium chloride, 0.04g iron chloride, 0.1g calcium chloride, 1000mL distilled water, pH=7.0.
3rd, it is 3 according to mass ratio by DBP degradation fermented liquids and microbial bacterial agent A：After 5 mixing, then press with solid carrier
According to mass ratio 1:1 mixing, you can obtain multifunctional agriculture soil remediation microbial inoculum.The carrier be by attapulgite, kaolin and
Humic acid is according to mass ratio 2:1:1 composition.
Embodiment 2：
The present embodiment whether there is antagonism between further analyzing four kinds of strains.
The antagonism of microorganism, refer between microorganism it is mutual resist, be mutually exclusive, even killing mutually show
As.Substantially have and change the diversified forms such as microenvironment, occupy-place, contention nutrition, secretion antibacterial material, immunization.
By four kinds of bacteriums, DBP degradation bacterias (CGMCC No.9526), sphingolipid bacterium (CGMCC No.4400), colloid gemma bar
Bacterium (CGMCC No.7240) and acid-producing Klebsiella bacterium (CGMCC No.4155) carry out tablet dual test respectively.By preservation
Strain activated after, on solid medium respectively two-by-two intersected line culture, in 28 DEG C of constant incubators, training
Support 2d~5d, 3 repetitions.The bacterial growth situation of bacterium colony formation and line intersection on observation tablet daily.If in bacterium
Two kinds of bacterium colonies of intersection of strain have growth, then illustrate between two bacterium there is no the antagonism relationship or have facilitation each other；
If two kinds of bacterium colonies of intersection are all without growth phenomenon, the situation of growth is poor or only a kind of colony growth, and another bacterium
It does not grow, just illustrates that there are the antagonism relationships between bacterial strain.
Fig. 1 is sphingolipid bacterium (CGMCC No.4400), colloid bacillus cereus (CGMCC No.7240) and the sour citric acid of production
Bacterium (CGMCC No.4155) tablet stands facing each other situation, and the bacterium colony bacterium of 3 kinds of bacterium is formed at 3d rear plate line, and line intersection is not
Colony growth with strain is normal, and there is no antagonisms.Fig. 2 is DBP degradation bacterias (CGMCC No.9526), sphingolipid bacterium (CGMCC
No.4400), colloid bacillus cereus (CGMCC No.7240) and acid-producing Klebsiella bacterium (CGMCC No.4155) tablet face-off feelings
Condition, the bacterium colony bacterium of 4 kinds of bacterium is formed at 3d rear plate line, and the colony growth for intersection different strain of ruling is normal, is not present
Antagonism.
Embodiment 3：
(1) carry out in greenhouse and apply cell of the multifunctional agriculture soil remediation microbial inoculum reparation by DBP contaminated soils
Experiment, validator environment remediation and reclamation result, science is provided for application of the composite bacteria agent in agricultural production practice
Foundation.
(2) test material and method
(2.1) for studying object：It is pakchoi for examination vegetables, kind is just prosperous up to 88.
(2.2) for examination soil：Experimental field belong to the continental monsoon climate average temperature of the whole year in temperate zone mostly between -5-5 DEG C, soil
Earth type belongs to black earth, and fertile soil, the content of organic matter are high.Practice ground is without preceding crop, the basic physics and chemistry of pilot soil
Matter is shown in Table 1.
Table 1 tests the basic physical and chemical of soil
Note：4 repetitions are tested, lowercase (a, b, c) represents the otherness between same time different disposal in form
(p<0.05)。
(2.3) experimental design
Pakchoi seed germinates in unpolluted soil, while is carried out with the Huo Gelan nutrient solutions without DBP
Culture.Until pakchoi three pieces true leaf is fully deployed, the close and healthy seedling of selection growing way is transplanted to equipped with dirt
In the pottery pot for contaminating soil (20mg/kg DBP).Multifunctional agriculture soil remediation microbial inoculum or degradation bacteria are added in into soil simultaneously
DNB-S1.Contaminated soil 300g is contained in each small basin, a pakchoi is colonized per basin, pours a water at regular intervals, is protected
Soil is held in small basin in appropriate humidity.It is sampled respectively in the 7th, 14,21,28,35d, the growth of harvest time pakchoi is referred to
Protein content and Vitamin C content content are detected in mark, blade, while detect DBP residual quantities, Soil Available in soil
Nutrient, soil urease and sucrase active.Untainted soil is set simultaneously and adds in the DBP of 20mg/kg and is not added with multi-functional
The soil of agricultural soil remediation microbial inoculum compares.Each processing experiment sets 4 repetitions.
The multifunctional agriculture soil remediation microbial inoculum that this experiment uses is multifunctional agriculture soil remediation prepared by embodiment 1
Microbial inoculum.
(3) result and analysis
(3.1) multifunctional agriculture soil remediation microbial inoculum analyzes soil fertility and soil DBP residual conditions
Research finds that DBP pollutions will not have apparent influence to the content of quick-acting carbon nitrogen phosphorus in soil, not using multi-functional
DBP contents are 14.37mg/kg after 35d in the DBP contaminated soils of agricultural soil remediation microbial inoculum, still in higher concentration；Only
It is little to add available nitrogen in the soil of DBP degradation bacterias DNB-S1, rapid available phosphorus and quick-acting potassium content variation, after 35d, DBP in soil
Content is 3.53mg/kg；After adding in multifunctional agriculture soil remediation microbial inoculum 35d, DBP contents are only in by DBP contaminated soils
0.28mg/kg, repairing effect clearly, and available nitrogen in soil after application multifunctional agriculture soil remediation microbial inoculum 35d
Content adds 11.52%, and available phosphorus contents add 11.01%, and quick-acting potassium content adds 9.19%, adds in multi-functional agriculture
Industry soil remediation microbial inoculum effectively improves soil fertility compared with DBP pollutions and untainted control soil.
2 soil physico-chemical property of table and soil DBP residual conditions
Note：4 repetitions are tested, lowercase (a, b, c) represents the otherness between same time different disposal in form
(p<0.05)。
(3.2) multifunctional agriculture soil remediation microbial inoculum is to Soil Enzyme Activities
Compared with untainted control soil, DBP pollutions can significantly inhibit urase and sucrase active in soil, and apply
It is significantly improved with urase and sucrase active in soil after multifunctional agriculture soil remediation microbial inoculum 35d, compared to DBP contaminated soils
24.51 and 17.18% have been respectively increased in urase and sucrase active, have reached the level of not comtaminated control soil, have illustrated bacterium
The addition of agent effectively alleviates toxic actions of the DBP to soil, promotes the recovery of soil enzyme activities.
Influence of the 3 function microbial inoculum of table to urease activity
Note：4 repetitions are tested, lowercase (a, b, c) represents the otherness between same time different disposal in form
(p<0.05)。
(3.3) multifunctional agriculture soil remediation microbial inoculum analyzes Growth of Cabbage Index Influence
When pakchoi is in DBP pollutions, DBP substantially inhibits the growth of pakchoi root and the accumulation of dry matter,
Soluble protein and ascorbic content in plant are reduced simultaneously, reduces the yield and quality of pakchoi；And apply more work(
After energy agricultural soil remediation microbial inoculum 35d, the growth of pakchoi root, the accumulation of dry matter, plant soluble protein, vitamin C are equal
It shows significantly to recover, does not apply multifunctional agriculture soil remediation microbial inoculum contaminated soil, pakchoi root long rises
5.52%, root dry weight rises 6.45%, and plant weights rise 11.97%, and soluble protein improves 24.93%, dimension life
Plain C improves 13.67%, substantially returns to no DBP levels of pollution, illustrates that multifunctional agriculture soil remediation microbial inoculum is relieved
The growth that DBP pollutes to pakchoi is coerced.
4 Growth of Cabbage index situation of change of table
Note：4 repetitions are tested, lowercase (a, b, c) represents the otherness between same time different disposal in form
(p<0.05)。
To sum up, phosphate solubilizing bacteria, potassium solubilizing bacteria, nitrogen-fixing bacteria and DBP degradation bacterias are configured as multifunctional agriculture according to a certain percentage
The active ingredient of soil remediation microbial inoculum, microbial inoculum bacterial action is good, and the antagonism relationship is not present between four kinds of strains.Microbial inoculum is pressed with carrier
Solid fungicide is prepared into after ratio mixing, solid-state carrier includes attapulgite, kaolin and humic acid, can normal temperature condition preservation effect
Fruit is good.Multifunctional agriculture soil remediation microbial inoculum is notable to DBP pollutions removal effect in soil, reaches more than 94%；Can have
Effect improves Soil Available nitrogen, rapid available phosphorus, quick-acting potassium content；Function microbial inoculum generates different journeys to soil enzyme activities in repair process
The influence of degree, the activation played to soil urease, sucrase active；At the same time, DBP pollutions can effectively be alleviated to generation
The inhibition of table industrial crops Growth of Cabbage effectively facilitates the development of pakchoi root and the accumulation of internal dry matter, improves
Soluble protein content and Vitamin C content in plant improve the product and quality of pakchoi；Multifunctional agriculture soil is repaiied
Multiple microbial inoculum plays soil fertility certain improving effect, and can promote available nitrogen, rapid available phosphorus in the soil for growth of crop
And quick-acting potassium content has been respectively increased 11.52%, 11.01%, 9.19% compared with control.
Claims (10)
1. a kind of multifunctional agriculture soil remediation microbial inoculum, it is characterised in that the microbial inoculum is sent out including microbial bacterial agent A and DBP degradation bacteria
Zymotic fluid, the microbial bacterial agent A include sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium fermentation
Liquid.
2. a kind of multifunctional agriculture soil remediation microbial inoculum according to claim 1, it is characterised in that microbial bacterial agent A and
The mass ratio of DBP degradation fermented liquids is (5-10)：(2-4).
A kind of 3. multifunctional agriculture soil remediation microbial inoculum according to claim 1 or 2, it is characterised in that the microbial bacteria
Sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid mass ratio are (1-2) in agent A：(1-
2)：(5-10).
4. a kind of multifunctional agriculture soil remediation microbial inoculum according to claim 3, it is characterised in that DBP degradation bacterias are fermented
The concentration of liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid is 108More than cfu/mL.
5. a kind of multifunctional agriculture soil remediation microbial inoculum according to claim 4, it is characterised in that in multifunctional agriculture soil
It is additionally added carrier in earth remediation microbial inoculum, the mass ratio of multifunctional agriculture soil remediation microbial inoculum and carrier is 1:1, the carrier be by
Attapulgite, kaolin and humic acid are according to mass ratio 2:1:1 composition.
6. the preparation method of multifunctional agriculture soil remediation microbial inoculum as described in claim 1, it is characterised in that this method includes
Following steps：
First, microbial bacterial agent A is prepared：
Sphingolipid bacterium is inoculated in Pikovskava solution Phos solid mediums, under the conditions of 27~29 DEG C, 120~130r/min
Carry out 24~26h of activation culture；Then the strain of activation is inoculated in Pikovskava solution Phos fluid nutrient mediums again to expand
After cultivating 4~6d, sphingolipid fermented liquid is obtained；
By colloid bacillus cereus be inoculated in silicate bacteria solid medium under the conditions of 27~29 DEG C, 120~130r/min into
24~26h of row activation culture；Then the strain of activation is inoculated in silicate bacteria fluid nutrient medium again and expands 4~6d of culture
Afterwards, colloid bacillus cereus zymotic fluid is obtained；
Acid-producing Klebsiella bacterium is inoculated in Ah 's bayesian fixed nitrogen solid medium in 27~29 DEG C, 120~130r/min conditions
24~26h of lower progress activation culture；Then the strain of activation is inoculated in Ah 's bayesian fixed nitrogen fluid nutrient medium again and expands culture 4
After~6d, sphingolipid fermented liquid is obtained；
Sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid are mixed, obtain composite microbial
Object microbial inoculum A；
2nd, by DBP degradation bacterias be inoculated in inorganic salts fixed nitrogen solid medium under the conditions of 27~29 DEG C, 120~130r/min into
24~26h of row activation culture；Then after the strain of activation being inoculated in 24~26h of inorganic salt liquid culture medium expansion culture again,
The DBP degradation germ liquids that will be enlarged by culture are inoculated by 10%~15% inoculum concentration in inexpensive fermentation culture medium, 27~29 DEG C,
72~74h of activation culture is carried out under the conditions of 120~130r/min, obtains DBP degradation fermented liquids；
3rd, DBP degradation fermented liquids and microbial bacterial agent A are mixed, obtains multifunctional agriculture soil remediation microbial inoculum.
7. the preparation method of multifunctional agriculture soil remediation microbial inoculum according to claim 6, it is characterised in that：In step 1
Sphingolipid fermented liquid, colloid bacillus cereus zymotic fluid and acid-producing Klebsiella bacterium zymotic fluid mass ratio are (1-2)：(1-2)：(5-
10)。
8. the preparation method of multifunctional agriculture soil remediation microbial inoculum according to claim 6, it is characterised in that：In step 3
The mass ratio of microbial bacterial agent A and DBP degradation fermented liquid is (5-10)：(2-4).
9. multifunctional agriculture soil remediation microbial inoculum described in claim 1 is repairing the application in polluting agricultural soil.
10. repairing the agrological specific method of pollution is：Multifunctional agriculture soil remediation microbial inoculum is added to soil to be repaired
In, the dosage of multifunctional agriculture soil remediation microbial inoculum is the 1 ‰ -1.5 ‰ of soil volume to be repaired.
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