CN115404191A - Soil remediation composite microbial agent containing Delftia D39 and Junpian M1031 and preparation method thereof - Google Patents
Soil remediation composite microbial agent containing Delftia D39 and Junpian M1031 and preparation method thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention relates to the technical field of soil remediation microbial inoculum, in particular to a soil remediation compound microbial inoculum of Delftia D39 and Jun-bacterium M1031 and a preparation method thereof, wherein the raw materials comprise 20 to 30 parts by weight of Delftia (Blastoud), namelyDelftia sp.) D39 fermentation liquor, 5 to 10 parts by weight of Junpian bacteria (a mixture of fermented liquor and a fermentation liquor)Lampropedia sp.) M1031 fermentation liquid, 8 to 10 parts by weight of wetting agent, 60 to 70 parts by weight of dispersing agent and 1 to 5 parts by weight of ultraviolet protective agent. The soil remediation complex microbial inoculum has the advantages of degradation rate of phoxim in soil of more than 98%, long activity period, wider application range and great significance for remediation of soil polluted by phoxim pesticide residues.
Description
Technical Field
The invention relates to the technical field of soil remediation microbial inoculum, in particular to a soil remediation compound microbial inoculum of Delftia D39 and Junpian M1031 and a preparation method thereof.
Background
Phoxim is one of the most widely used organophosphorus insecticides in China, and the dosage is ranked first in 2015 in various main crops. In the spraying process, only a small part of pesticide plays a role, and 50-60% of phoxim can be remained in the soil. The phoxim is slowly degraded in the soil without direct sunlight, and belongs to a pesticide difficult to digest.
Two strains having a function of degrading phoxim have been isolated from the genus delford so far. Shenshijia et al reported in 2007 that a delford XSP-1 strain with phoxim as a unique carbon source can completely degrade 100mg/L of phoxim within 7h, and the strain has good degradation capability on methyl parathion, chlorpyrifos and fenitrothion. In 2020, the inventor also isolated a Delftia D39 strain, and in patent CN109679875A, an enzyme preparation for degrading phoxim is disclosed to be prepared by utilizing Delftia D39, and the enzyme preparation can degrade more than 90-99% of phoxim residue in grain storage of granaries and byproducts of agricultural product processing. The enzyme preparation is prepared by ultrasonically crushing D39 bacteria of Delftia sp, and subsequent experiments prove that D39 intracellular enzyme mainly plays a role in degrading phoxim, and D39 extracellular enzyme does not play a role in degrading phoxim. The enzyme preparation is directly applied to soil, intracellular enzymes are easy to inactivate, and the soil needs to be repaired by using a viable bacteria agent. Tests show that when the concentration of phoxim in the soil is more than 150mg kg -1 In time, D39 soil remediation microbial inoculum for 48h of phoximThe degradation rate of the soil is only about 81 percent, and the capability of repairing highly polluted soil is poor.
Disclosure of Invention
In order to improve the degradation effect of the delford D39 microbial inoculum on phoxim in soil, the invention provides a soil remediation composite microbial inoculum of the delford D39 and Junpian M1031 and a preparation method thereof, so as to realize efficient remediation of phoxim-polluted soil.
The first aspect provides a soil remediation complex microbial inoculum of Delftia D39 and Jun-myco M1031, which comprises 20 to 30 parts by weight of Delftia (Delftia sp.) D39 fermentation liquor, 5 to 10 parts by weight of Jun-myco (Lampurpedia sp.) M1031 fermentation liquor, 8 to 10 parts by weight of a wetting agent, 60 to 70 parts by weight of a dispersing agent and 1 to 5 parts by weight of an ultraviolet protective agent;
the Daerfurt D39 is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of West Lu No.1 of North Chen in the south of the morning of Beijing, the preservation number is CGMCC No.16844, the preservation date is 11 months and 30 days in 2018, and the Daerfurt D39 is disclosed in the invention patent CN109679875A of China.
The Junpian bacteria M1031 is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Beijing's republic of Chaoyang district, xilu No.1, the preservation number is CGMCC No.20988, the preservation date is 2020, 11 and 2 days, and the Junpian bacteria M1031 is disclosed in the Chinese patent CN 113249274A.
Further, the wetting agent includes calcium lignosulfonate and naphthalene sulfonate polycondensate.
Further, the mass ratio of the calcium lignosulfonate to the naphthalenesulfonate polycondensate is 3.
Further, the dispersing agent is diatomite, and the ultraviolet protective agent is carboxymethyl cellulose.
Further, the inoculation amount of the soil remediation complex microbial inoculum in soil is 2-10g/8729kg -1 。
In a second aspect, the invention also provides a preparation method of the soil remediation complex microbial inoculum, which comprises the following steps:
(1) Mixing the Daerfurt D39 fermentation liquor, the Junpian M1031 fermentation liquor, the wetting agent, the dispersing agent and the ultraviolet protective agent, and uniformly stirring;
(2) And (3) putting the mixed raw materials into a jet mill for crushing, and sieving after crushing to prepare the soil remediation composite microbial inoculum.
Further, the fermentation liquor of the delford D39 is prepared by the following method:
s1, drawing a preserved Delftia D39 strain on an LB solid culture medium, and performing inverted activation culture at 25-30 ℃ for 1-2d to prepare an activated strain;
s2, filling 100-150mL of LB liquid culture medium into a 500mL triangular flask, wherein the pH of the LB liquid culture medium is 7.0-7.5, inoculating the activated strain into the LB liquid culture medium, wherein the inoculation amount is 4-8%, and performing shaking culture at 30-32 ℃ for 2-3d to obtain a bacterial liquid;
s3, inoculating a bacterial liquid into a 500L fermentation tank containing a fermentation medium A, wherein the initial inoculation amount is 4%, and the rotating speed is 350r 8729min -1 Dissolved oxygen 5mmol 8729L -1 And adding ammonia water in a flowing manner, controlling the pH to be 7.0 and the temperature to be 32 ℃, and fermenting for 40 to 52h to prepare the Delftia D39 fermentation liquor.
Further, the fermentation medium A comprises, by mass, 2 parts of corn flour, 2 parts of bean flour, 1 part of glucose, 0.1 part of ammonium sulfate, 0.2 part of dipotassium hydrogen phosphate, 0.2 part of potassium dihydrogen phosphate, 0.03 part of magnesium sulfate heptahydrate and deionized water which are supplemented to 100 parts.
The invention has the beneficial effects that:
the invention provides a soil remediation composite microbial inoculum containing deloft D39 and jun-pai M1031, experiments show that the jun-pai M1031 has certain vitalizing and synergistic effects on the degradation of phoxim by the deloft D39, and the jun-pai M1031 is beneficial to maintaining the good activity of the deloft D39 and promoting the degradation of the phoxim in the long-term degradation process. The soil remediation complex microbial inoculum has the advantages of high degradation efficiency, long active period and wide application range.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a line graph of the soil remediation effect of two soil remediation microbial inoculum under different dosage.
FIG. 2 is a line graph of the remediation effect of two soil remediation microbial inoculum on soils with different phoxim initial concentrations.
FIG. 3 shows that the contents of two soil remediation microbial inoculum, namely, the phoxim, are 300mg -1 Is a line graph of the soil remediation rate of (1).
Detailed Description
In order to make those skilled in the art better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of D39 soil remediation microbial inoculum
1. The strain source is as follows: the Delftia dellofoenum D39 is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of No.1 Xilu Beijing North Chen of the Chaoyang district, the preservation number is CGMCC No.16844, and the strain is classified and named as Delftia dellofoenum (A), (B), (C) and (C)Delftia sp.) The preservation date is 11 months and 30 days in 2018.
2. Culture medium:
(1) LB solid medium: 5g of yeast extract, 10g of peptone, 10g of NaCl10g agar, 15g of deionized water to 1000mL, pH7.0, and sterilizing at 121 ℃ for 20min.
(2) LB liquid medium: 5g of yeast extract, 10g of peptone, 10g of NaCl10g, deionized water to 1000mL, pH7.0, and sterilizing at 121 ℃ for 20min.
(3) Fermentation medium a: 20g of corn flour, 20g of bean flour, 10g of grapes, 1g of ammonium sulfate, 2g of dipotassium phosphate, 2g of monopotassium phosphate, 0.3g of magnesium sulfate heptahydrate and deionized water, wherein the deionized water is supplemented to 1kg, the pH value is 7.0, and the sterilization is carried out for 20min at the temperature of 115 ℃.
3. The preparation method comprises the following steps:
s1, drawing a line of the preserved Delftia D39 on an LB solid culture medium, and performing inverted activation culture at 30 ℃ for 2D to obtain an activated Delftia D39 strain;
s2, filling 150mL of LB liquid culture medium into a 500mL triangular flask, inoculating the activated strain into the LB liquid culture medium, wherein the inoculation amount is 4%, and performing shaking culture for 2D at 30-32 ℃ in a shaking table to obtain a D39 bacterial liquid;
s3, inoculating the D39 bacterial liquid into a 500L fermentation tank containing the fermentation medium A, wherein the initial inoculation amount is 4%, and the rotating speed is 350r 8729min -1 Dissolved oxygen 5mmol 8729L -1 Ammonia water is fed in a flowing manner to control the pH to be about =7.0, the temperature is controlled to be 32 ℃, and fermentation is carried out for 48 hours, so as to prepare the Delftia D39 fermentation liquor;
s4, taking 30 parts by weight of Delftia D39 fermentation liquor, adding 70 parts by weight of diatomite, 6 parts by weight of calcium lignosulfonate, 4 parts by weight of naphthalenesulfonate polycondensate Morwet D425 and 2.5 parts by weight of carboxymethyl cellulose, and uniformly mixing;
and S5, putting the mixed raw materials into a jet mill for grinding, and sieving to obtain the D39 soil remediation microbial inoculum.
Example 2 preparation of D39-M1031 Complex microbial inoculum for soil remediation
1. The strain source is as follows:
daerfurt D39, which is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, with the preservation address of No. 3 of Xilu No.1 of the North Chen in the south of the morning of Beijing, the preservation number of CGMCC No.16844, and the classified name of the strain being Daerfurt (a)Delftia sp.) The preservation date is 2018, 11 and 30 months, and related information is disclosed in the chinese patent CN 109679875A.
Junpian bacteria M1031, preserved by China general microbiological culture Collection center with preservation number CGMCC No.1, no. 3 of Beijing's republic of Chaoyang, preservation number CGMCC No.20988, and classified name of Junpian bacteria (Zhanpian bacteria) (C)Lampropedia sp.) The preservation date is 11 months and 2 days in 2020, and related information is disclosed in the invention special for ChinaAccording to CN 113249274A.
2. Culture medium:
LB solid medium, LB liquid medium and fermentation medium A were prepared as in example 1.
Fermentation medium B: 20g of corn flour, 20g of bean flour, 0.5g of glucose, 1g of ammonium sulfate, 0.5g of dipotassium phosphate, 0.5g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate and deionized water, wherein the balance is 1kg, the pH value is 7.0, and the mixture is sterilized for 20min at the temperature of 115 ℃.
3. The preparation steps are as follows:
(1) the preparation method of the Daerford bacterium D39 fermentation liquor is the same as that of the example 1;
(2) preparing a jun pano bacterium M1031 fermentation broth:
s1, taking a preserved Junpian M1031 strain;
s2, 150mL of LB liquid medium is filled into a 500mL triangular flask, the activated Juniperus M1031 strain is inoculated into the LB liquid medium, the inoculum size is 4%, and shaking culture is carried out on a shaker at 30 to 32 ℃ for 2d to obtain Juniperus M1031 bacterial liquid;
s3, inoculating Junpian bacteria M1031 bacterial liquid into a 500L fermentation tank containing fermentation medium B, wherein the initial inoculation amount is 4%, and the rotation speed is 200r 8729min -1 Dissolved oxygen 5mmol 8729L -1 And (3) continuously adding ammonia water, controlling the pH to be about =7.0, and fermenting for 48 hours at the temperature of 30 ℃ to obtain the zymotic fluid of the Juniperus chinensis M1031.
(3) Taking 20 parts by weight of Daerfurt D39 fermentation liquor and 10 parts by weight of Juniperus M1031 fermentation liquor, adding 70 parts by weight of diatomite, 6 parts by weight of calcium lignosulfonate, 4 parts by weight of naphthalene sulfonate polycondensate Morwet D425 and 2.5 parts by weight of carboxymethyl cellulose, and uniformly mixing;
and S5, putting the mixed raw materials into a jet mill for crushing, and sieving to obtain the D39-M1031 soil remediation composite microbial inoculum.
Example 3 determination of the number of two soil remediation microbial inoculum and related indexes
The number of the soil remediation microbial inoculum prepared in examples 1 and 2 was determined by a flat viable count method. Determining each technical index of the soil remediation microbial inoculum according to national standard of the people's republic of China: the wetting time is measured by GB5451-2001, the suspension rate is measured by GB/T14825-2006, the fineness is measured by GB/T16150-1995, the water content is measured by GB/T1600-2001, and the pH value is measured by GB/T1601-1993. The detection results are shown in table 1 and all accord with the national standard.
TABLE 1 technical indices of two remediation agents
Example 4 shelf life of two soil remediation Agents
The water contents of the D39 soil remediation microbial inoculum and the D39-M1031 soil remediation composite microbial inoculum are both 4%, and the pH value is 7.0. The results of the survival rate test of the storage period are shown in Table 2.
Table 2 number of viable bacteria (x 10) with different storage periods of two soil remediation microbial inoculum 8 cfu∙g -1 ) And survival rate
As can be seen from the table 2, the two microbial inoculums can maintain high survival rate, are suitable for processing and storing of the microbial inoculums, have long shelf life, and have relatively high survival rate of the D39-M1031 soil remediation composite microbial inoculums.
Example 5 determination of remediation effects of two soil remediation microbial inoculum on phoxim-contaminated soil
(1) Method for measuring phoxim content
Adopting high performance liquid chromatography (HPLC method), taking 1mL of liquid to be detected, placing the liquid in a graduated test tube with a plug, adding 5mL of dichloromethane, violently oscillating, standing for layering, removing an upper aqueous phase, dehydrating an organic phase through anhydrous sodium sulfate, taking 1mL of the liquid in a microcentrifuge tube, blowing nitrogen to dry, and adding 1mL of methanol for dissolving (pure chromatogram). The measurement conditions were as follows, with the mobile phase being methanol: water =80:20, the flow rate is 1mL/min, the sample injection amount is 10 mu L, the chromatographic column is a TC-C18 chromatographic column, the thickness is 5 mu m, the thickness is 250mm multiplied by 4.6mm, the working wavelength of the variable wavelength detector is 280 nm, and the quantification is carried out by adopting an external standard method.
(2) Determination of optimal dosage of microbial inoculum in soil
Weighing 200g of soil, and air-dryingSieving with 20 mesh sieve, adding a certain amount of phoxim solution to make its concentration be 50 mg/kg -1 And (4) uniformly mixing. 200g of soil is divided into 10 parts on average and randomly divided into two groups A and B, and each group comprises 5 parts. D39 soil remediation microbial inoculum is doped into the group A, and D39-M1031 soil remediation complex microbial inoculum is doped into the group B, so that the concentration of each group of microbial inoculum is 0.5 g/kg -1 、1g·kg -1 、2g·kg -1 、4g·kg -1 、10g·kg -1 And (5) culturing for 2d under natural laboratory conditions.
Taking 2g of soil sample by a five-point sampling method, measuring the content of the phoxim by an HPLC method, calculating the degradation rate of the phoxim, and repeating the treatment for three times by taking soil without the fungicide as a control. The water holding capacity of the treated soil samples is kept at 50 percent, and the distilled water is not sufficient for supplying. The results were statistically calculated and line-plotted as shown in fig. 1.
As can be seen from FIG. 1, the concentration of the microbial inoculum in the soil was varied from 0.5 g/kg -1 Increased to 4 g.kg -1 The degradation rate of the phoxim is in an increasing trend, which shows that in the soil containing the phoxim with a certain concentration, the larger the using amount of the microbial inoculum, the faster the degradation rate of the phoxim, and the faster the soil remediation speed.
The dosage of the microbial inoculum is increased to 10 g/kg -1 In the process, the degradation rate of the phoxim of the D39 soil remediation microbial inoculum group is reduced, while the degradation rate of the phoxim of the D39-M1031 soil remediation composite microbial inoculum is still increased, but the increase amplitude is not obvious. Therefore, when the dosage of the D39 soil remediation microbial inoculum is too high, the degradation rate of the phoxim is reduced on the contrary, and the D39-M1031 soil remediation composite microbial inoculum with the same dosage is added, the degradation rate of the phoxim is slightly increased, and the junipecaceae M1031 can be seen to have a certain vitamin stability and synergism effect on the degradation of the phoxim by the Daerfurtia sp D39.
Therefore, the optimal dosage of the D39-M1031 soil remediation complex microbial inoculum is 4 g/kg -1 And the degradation of phoxim is not obviously promoted by continuously increasing the dosage of the microbial inoculum.
(3) Repairing effect of microbial inoculum in phoxim soil with different concentrations
Weighing 200g of soil, air-drying and sieving by a 20-mesh sieve, evenly dividing the 200g of soil into 10 parts, randomly dividing the soil into two groups A and B, and 5 parts of each group. Each group is added with each partAdding phoxim pesticide to make the concentration of phoxim in soil sample be 10mg kg -1 、50mg·kg -1 、75mg·kg -1 、100mg·kg -1 、150mg·kg -1 . Adding D39 soil remediation microbial inoculum into each concentration soil sample of group A, and adding D39-M1031 soil remediation complex microbial inoculum into each concentration soil sample of group B to make the microbial inoculum concentration be 4 g.kg -1 And culturing for 48h under natural laboratory conditions.
Taking 2g of soil sample by a five-point sampling method, measuring the content of the phoxim by an HPLC method, calculating the degradation rate of the phoxim, and repeating the treatment for three times by taking soil without the fungicide as a control. The water holding capacity of each treated soil sample is kept at 50 percent, and the distilled water is not sufficient for supplying. The results were counted and line-plotted as shown in fig. 2.
As can be seen from fig. 2, the degradation rate of phoxim decreased with increasing initial concentration of phoxim in the soil. When the content of phoxim in the soil is less than or equal to 50mg kg -1 In the process, the remediation microbial inoculum can be completely degraded within 48 hours, and the degradation rate is 100%. When the concentration of phoxim in the soil is between 50 and 100mg-kg -1 In the meantime, the degradation rate of phoxim of the D39 soil remediation bacterium agent 48h is more than or equal to 92.08%, and the degradation rate of phoxim of the D39-M1031 soil remediation compound bacterium agent 48h is more than or equal to 95.11%. When the concentration of phoxim in the soil is 150mg kg -1 In the process, the degradation rate of the D39 soil remediation complex microbial inoculum for 48h on the phoxim is reduced to 81.05 percent, while the degradation rate of the D39-M1031 soil remediation complex microbial inoculum for 48h on the phoxim can reach more than 90 percent, so that the soil remediation effect is better.
(4) Remediation rate of microbial inoculum on soil seriously polluted by phoxim
The collected soil sample is mixed with phoxim, so that the content of phoxim in the soil is 300mg kg -1 The soil sample is divided into three groups A, B and C, wherein the group A soil without the remediation microbial inoculum is used as a control group, the group B is added with the D39 soil remediation microbial inoculum, the group C is added with the D39-M1031 soil remediation compound microbial inoculum, and the soil treatment method by adding the remediation microbial inoculum comprises the following steps: the remediation microbial inoculum is mixed into the soil to ensure that the concentration of the microbial inoculum is 4 g.kg -1 . Each treatment was set up in triplicate and incubated for 7 days in laboratory conditions. Storing in 1d, 2d, 3d, 5d, and 7d refrigerator at-20 deg.C to be testedAnd (4) the degradation rate of the repaired phoxim. The water holding capacity of each treated soil sample is kept at 50 percent, and the distilled water is not sufficient for supplying. The content of phoxim was measured by HPLC method and the degradation rate of phoxim was calculated, the results were counted and plotted as a line graph, as shown in fig. 3.
As can be seen from FIG. 3, when the content of phoxim in the soil is 300mg kg -1 When the D39 soil remediation microbial inoculum is applied, the degradation rate of the phoxim is increased and then decreased along with the increase of the degradation time. Within the first 5d, the degradation rate of phoxim increased to 92.67% with time, and by the time of 7d, the degradation rate of phoxim decreased to 81.30%.
When the content of phoxim in the soil is 300mg kg -1 In the process, a D39-M1031 soil remediation complex microbial inoculum is applied, within 7D, the degradation rate of phoxim always shows a rising trend along with the increase of degradation time, the growth speed is gradually slowed down, and the degradation rate of phoxim reaches 98.01% at the 7 th D. Therefore, in the long-term degradation process of high-concentration phoxim, the jun-tablet bacterium M1031 helps to maintain the activity of the delford D39 and promotes the delford D39 to degrade the phoxim, so that the ability of the D39-M1031 soil remediation compound microbial inoculum to remediate soil is more durable.
Example 6 colonization by two soil remediation Agents
Sterilizing collected soil, and adding phoxim to make the content of phoxim in soil be 150 mg-kg -1 The inoculation amount of the remediation microbial inoculum is 4 g/kg -1 And taking soil samples at 8h, 12h, 24h, 36h and 48h under natural conditions of a laboratory for viable bacteria counting. The water holding capacity of each treated soil sample is kept at 50 percent, and the distilled water is not sufficient for supplying. The colonization results of the repairing microbial agents are shown in Table 3.
TABLE 3 colonization results of two soil remediation Agents
From table 3, it can be seen that delftia D39 can colonize phoxim-contaminated soil, and the number of D39 colonizers tends to increase and decrease with time.
After the D39 soil remediation microbial inoculum is applied for 24 hours, the maximum number of the colonizers is 6.32 multiplied by 10 8 cfu·g -1 (ii) a After the D39 soil remediation microbial inoculum is applied for 48 hours, the number of the colonizers is reduced to 5.39 multiplied by 10 7 cfu·g -1 This is probably due to the fact that the decrease in phoxim content in the soil at a later stage leads to a decrease in the amount of carbon source available to the strain.
When the D39-M1031 soil remediation microbial inoculum is applied for 1h, the number of the colonization bacteria is slightly lower than that of the D39 soil remediation microbial inoculum, and after 24h, the maximum number of the colonization bacteria is 7.51 multiplied by 10 8 cfu·g -1 The soil remediation microbial inoculum is higher than D39; after the D39-M1031 soil remediation complex microbial inoculum is applied for 48 hours, the number of the colonizers is reduced to 6.12 multiplied by 10 8 cfu·g -1 But still higher than the D39 soil remediation microbial inoculum. Therefore, the colonization rate of the D39-M1031 soil remediation microbial inoculum is faster, the maximum value of the number of the colonization bacteria is larger, and the reduction rate of the number of the colonization bacteria is slow.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention.
Claims (8)
1. A soil remediation compound microbial inoculum of Delftia D39 and Jun-bacterium M1031 is characterized in that the raw materials comprise 20 to 30 parts by weight of Delftia (D) (Delftia sp.) D39 fermentation liquor, 5 to 10 parts by weight of Junpian bacteria (a mixture of fermented liquor and a fermentation liquor)Lampropedia sp.) M1031 fermentation liquor, 8 to 10 parts by weight of wetting agent, 60 to 70 parts by weight of dispersing agent and 1 to 5 parts by weight of ultraviolet protective agent;
the Delftia sp D39 is preserved by the common microorganism center of China general microbiological culture Collection center, the preservation address is No. 3 of Xilu No.1 of Beijing, chaoyang, the preservation number is CGMCC No.16844, and the preservation date is 11 months and 30 days in 2018;
the Jun Pacific bacteria M1031 is preserved by the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of West Lu No.1 of Chacheng of the Chaoyang district in Beijing, the preservation number is CGMCC No.20988, and the preservation date is 2020 years, 11 months and 2 days.
2. The soil remediation complex formulation of claim 1 wherein the wetting agent comprises calcium lignosulfonate and a naphthalene sulfonate polycondensate.
3. The soil remediation complex microbial inoculant of claim 2, wherein the mass ratio of the calcium lignosulfonate to the naphthalene sulfonate polycondensate is 3.
4. The soil remediation complex bacterial agent of claim 1, wherein the dispersant is diatomaceous earth and the ultraviolet protectant is carboxymethylcellulose.
5. The soil remediation complex microbial inoculum of claim 1, wherein the inoculation amount of the soil remediation complex microbial inoculum in soil is 2-10g 8729kg -1 。
6. The preparation method of the soil remediation complex microbial inoculum according to claim 1, which comprises the following steps:
(1) Mixing the Daerfurt D39 fermentation liquor, the Junpian M1031 fermentation liquor, the wetting agent, the dispersing agent and the ultraviolet protective agent, and uniformly stirring;
(2) And (3) putting the mixed raw materials into a jet mill for crushing, and sieving after crushing to prepare the soil remediation composite microbial inoculum.
7. The method of claim 6, wherein the deloford bacterium D39 fermentation broth is prepared by:
s1, drawing a preserved Delftia D39 strain on an LB solid culture medium, and performing inverted activation culture at 25 to 30 ℃ for 1 to 2d to prepare an activated strain;
s2, filling 100 to 150mL of LB liquid culture medium into a 500mL triangular flask, wherein the pH of the LB liquid culture medium is 7.0 to 7.5, inoculating the activated strain into the LB liquid culture medium, wherein the inoculation amount is 4 to 8 percent, and performing shaking table shaking culture at 30 to 32 ℃ for 2 to 3d to prepare a bacterial liquid;
s3, inoculating a bacterial liquid into a 500L fermentation tank containing a fermentation medium A, wherein the initial inoculation amount is 4%, and the rotating speed is 350r 8729min -1 Dissolved oxygen 5mmol 8729L -1 And (3) feeding ammonia water, controlling the pH to be =7.0, and fermenting at the temperature of 32 ℃ for 40-52h to prepare the Delftia D39 fermentation liquor.
8. The preparation method according to claim 7, wherein the fermentation medium A comprises 2 parts by mass of corn flour, 2 parts by mass of bean flour, 1 part by mass of glucose, 0.1 part by mass of ammonium sulfate, 0.2 part by mass of dipotassium hydrogen phosphate, 0.2 part by mass of potassium dihydrogen phosphate, 0.03 part by mass of magnesium sulfate heptahydrate, and the balance of deionized water to 100 parts.
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| CN104531585A (en) * | 2014-12-31 | 2015-04-22 | 中国水稻研究所 | Delftia tsuruhatensis and application thereof |
| CN107475155A (en) * | 2017-09-15 | 2017-12-15 | 黑龙江宝基农业科技股份有限公司 | The soil remediation composite bacteria agent and its soil remediation method for soil fertilizer medicine residual of degrading |
| CN109679875A (en) * | 2019-01-17 | 2019-04-26 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | A kind of Dell's Ford bacterium and application |
| CN110484473A (en) * | 2019-08-31 | 2019-11-22 | 暨南大学 | A method of promote the field He Yu Dell Ford bacterium to colonize in plant rhizosphere |
| CN113249274A (en) * | 2021-05-24 | 2021-08-13 | 齐鲁工业大学 | Junpian bacteria and junpian bacteria cell suspension and application thereof in preventing and treating pepper wilt |
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| CN104531585A (en) * | 2014-12-31 | 2015-04-22 | 中国水稻研究所 | Delftia tsuruhatensis and application thereof |
| CN107475155A (en) * | 2017-09-15 | 2017-12-15 | 黑龙江宝基农业科技股份有限公司 | The soil remediation composite bacteria agent and its soil remediation method for soil fertilizer medicine residual of degrading |
| CN109679875A (en) * | 2019-01-17 | 2019-04-26 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | A kind of Dell's Ford bacterium and application |
| CN110484473A (en) * | 2019-08-31 | 2019-11-22 | 暨南大学 | A method of promote the field He Yu Dell Ford bacterium to colonize in plant rhizosphere |
| CN113249274A (en) * | 2021-05-24 | 2021-08-13 | 齐鲁工业大学 | Junpian bacteria and junpian bacteria cell suspension and application thereof in preventing and treating pepper wilt |
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