Remediation fungicide for drug-resistant contaminated soil and preparation method of remediation fungicide

Abstract

The invention provides a drug-resistant contaminated soil remediation fungicide and a preparation method thereof, and belongs to the technical field of soil remediation. The remediation fungicide for the drug-resistant contaminated soil is prepared from 5 to 10 parts of bacillus subtilis, 5 to 10 parts of bacillus megatherium, 5 to 10 parts of bacillus coagulans, 5 to 10 parts of actinomycetes, 4 to 5 parts of modified chitosan, 2 to 3 parts of agarose and 3 to 5 parts of earthworm content, according to the method, the content of the earthworms cultured by livestock and poultry manure containing antibiotics is adopted, so that the soil polluted by drug resistance can be repaired in a targeted manner; the added bacillus subtilis and other bacteria can decompose organic substances in the soil and promote updating and circulation of the organic substances, and the bacteria and the earthworm content have a synergistic effect to jointly repair the drug-resistant contaminated soil; meanwhile, the added bacteria can also generate metabolites beneficial to plant growth, so that the environment is improved after the drug-resistant polluted soil is repaired, and the growth and development of plants are promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of soil remediation, and in particular to a drug-resistant contaminated soil remediation bacterial agent and a preparation method thereof. Background Art

[0002] With the widespread use of antibiotics, the problem of drug resistance has become a hot topic of global concern. As an important part of the ecosystem, soil is increasingly contaminated by drug resistance. The presence of drug-resistant genes in the soil makes the bacteria in the soil insensitive to antibiotics, which increases the difficulty of treating infectious diseases. In addition, drug-resistant genes may enter the human body through the food chain, posing a potential threat to human health. The impact of drug-resistant contaminated soil on the environment and human health cannot be ignored. On the one hand, it destroys the balance of the soil ecosystem and affects the diversity and function of soil microorganisms; on the other hand, it increases the difficulty of treating infectious diseases and poses a potential threat to human health. In addition, drug-resistant genes may enter the human body through the food chain, further exacerbating health risks.

[0003] At present, the methods for soil remediation include physical methods: including soil heat treatment, soil aeration and soil leaching. These methods remove pollutants and drug-resistant genes in the soil by physical means, but may damage the soil structure and affect soil fertility. Chemical methods: such as redox method, acid-base method and electrochemical method. These methods change the material state in the soil by adding chemical reagents, thereby removing pollutants and drug-resistant genes, and secondary pollution problems that may be caused by chemical reagents. Therefore, it is of great practical significance to provide a soil remediation bacterial agent with high remediation efficiency and no secondary pollution to the soil. Summary of the invention

[0004] The purpose of the present invention is to provide a drug-resistant soil remediation bacterial agent and a preparation method thereof. The soil remediation bacterial agent of the present invention uses earthworm contents cultured with livestock and poultry manure containing antibiotics, so that it can carry out targeted remediation of drug-resistant contaminated soil. At the same time, bacteria are added to facilitate the decomposition of organic matter in the soil, promote the renewal and circulation of organic matter, and the earthworm contents and bacteria work synergistically to remediate the drug-resistant contaminated soil.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a drug-resistant contaminated soil remediation bacterial agent, comprising:

[0007] 5-10 parts of Bacillus subtilis, 5-10 parts of Bacillus megaterium, 5-10 parts of Bacillus coagulans, 5-10 parts of actinomycetes, 4-5 parts of modified chitosan, 2-3 parts of agarose and 3-5 parts of earthworm contents.

[0008] The present invention also provides a method for preparing the drug-resistant contaminated soil remediation bacterial agent described in the above technical solution, comprising the following steps:

[0009] Bacillus subtilis, Bacillus megaterium, Bacillus coagulans and actinomycetes are first cultured respectively, bacterial liquids obtained by the culture are first mixed, and a mixed bacterial liquid is obtained and refrigerated for standby use;

[0010] The earthworms are subjected to a second culture and then decomposed to obtain earthworm contents;

[0011] Modifying chitosan to obtain modified chitosan;

[0012] The mixed bacterial solution, earthworm contents, modified chitosan and agarose are mixed to obtain the drug-resistant contaminated soil remediation bacterial agent.

[0013] Furthermore, the first culture conditions are: temperature of 25-30° C. and time of 3-4 days.

[0014] Furthermore, the refrigeration temperature is 0-4°C.

[0015] Furthermore, the ratio of Bacillus subtilis, Bacillus megaterium, Bacillus coagulans and actinomycetes in the first mixture is 1:1:1:1.

[0016] Furthermore, the second culture is to place the earthworms in livestock and poultry manure for breeding, the breeding time is 10 to 15 days, and the livestock and poultry manure includes antibiotics.

[0017] Furthermore, the earthworm contents are earthworm midgut contents.

[0018] Furthermore, the modification is to first mix the chitosan with dilute hydrochloric acid for acidification, then mix the acidified chitosan with lactobionic acid solution and N,N'-carbonyldiimidazole, and finally perform filtration and dialysis.

[0019] Furthermore, the mass fraction of the dilute hydrochloric acid is 3-7%; the mass concentration of the lactobionic acid solution is 50g / L, and the usage ratio of the chitosan, dilute hydrochloric acid, lactobionic acid solution and N,N'-carbonyldiimidazole is 5-10g:1-1.2L:4.5-5L:2.5-3L.

[0020] The present invention also provides the use of the drug-resistant contaminated soil remediation bacterial agent described in the above technical solution in the remediation of drug-resistant contaminated soil. Furthermore, the mass ratio of the drug-resistant contaminated soil remediation bacterial agent to soil is 1-2:500-1000.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: the earthworm contents cultured with livestock and poultry manure containing antibiotics are added to the drug-resistant contaminated soil remediation bacterial agent of the present invention, so that it can carry out targeted remediation of drug-resistant contaminated soil; in addition, the present invention limits the addition of bacteria such as Bacillus subtilis and Bacillus megaterium, and the added bacteria can decompose organic matter in the soil, promote the renewal and circulation of organic matter, and synergize with the earthworm contents to jointly remediate the drug-resistant contaminated soil; at the same time, the added bacteria can also produce metabolites that are beneficial to plant growth, which is convenient for improving the environment after the remediation of the drug-resistant contaminated soil and promoting the growth and development of plants. DETAILED DESCRIPTION

[0022] The present invention provides a drug-resistant contaminated soil remediation bacterial agent, comprising:

[0023] 5-10 parts of Bacillus subtilis, 5-10 parts of Bacillus megaterium, 5-10 parts of Bacillus coagulans, 5-10 parts of actinomycetes, 4-5 parts of modified chitosan, 2-3 parts of agarose and 3-5 parts of earthworm contents.

[0024] Furthermore, the drug-resistant contaminated soil remediation bacterial agent of the present invention preferably includes 5 to 8 parts of Bacillus subtilis, 5 to 8 parts of Bacillus megaterium, 5 to 8 parts of Bacillus coagulans, 5 to 8 parts of actinomycetes, 4 to 4.5 parts of modified chitosan, 2.5 to 3 parts of agarose and 3 to 4 parts of earthworm contents.

[0025] The present invention also provides a method for preparing the drug-resistant contaminated soil remediation bacterial agent described in the above technical solution, comprising the following steps:

[0026] Bacillus subtilis, Bacillus megaterium, Bacillus coagulans and actinomycetes are first cultured respectively, bacterial liquids obtained by the culture are first mixed, and a mixed bacterial liquid is obtained and refrigerated for standby use;

[0027] The earthworms are subjected to a second culture and then decomposed to obtain earthworm contents;

[0028] Modifying chitosan to obtain modified chitosan;

[0029] The mixed bacterial solution, earthworm contents, modified chitosan and agarose are mixed to obtain the drug-resistant contaminated soil remediation bacterial agent.

[0030] Bacillus subtilis, Bacillus megaterium, Bacillus coagulans and actinomycetes are first cultured respectively, bacterial liquids obtained by the culture are first mixed, and a mixed bacterial liquid is obtained and refrigerated for standby use;

[0031] In the present invention, the Bacillus subtilis, Bacillus megaterium, Bacillus coagulans and actinomycetes are inoculated into existing culture media for culture respectively. The culture media selected for the Bacillus subtilis, Bacillus megaterium and Bacillus coagulans are preferably LB culture media; the culture media selected for the actinomycetes are preferably Go's culture media.

[0032] In the present invention, the conditions for the first culture are preferably: a temperature of 25 to 30° C. and a time of 3 to 4 days; more preferably, a temperature of 25 to 28° C. and a time of 3 to 3.5 days.

[0033] In the present invention, the refrigeration temperature is preferably 0-4°C.

[0034] In the present invention, the ratio of Bacillus subtilis, Bacillus megaterium, Bacillus coagulans and actinomycetes is preferably 1:1:1:1.

[0035] The earthworms are subjected to a second culture and then decomposed to obtain earthworm contents.

[0036] The present invention requires that earthworms be specially cultured first and then decomposed to obtain the earthworm contents required for the drug-resistant soil remediation bacterial agent of the present invention. The present invention does not specifically limit the type of earthworms, and any earthworm type familiar to those skilled in the art can be used.

[0037] In the present invention, the second culture is preferably to culture earthworms in livestock and poultry manure; the culture time is preferably 10 to 15 days, more preferably 10 to 12 days; and the livestock and poultry manure preferably includes antibiotics.

[0038] In the present invention, the earthworm contents are preferably earthworm midgut contents.

[0039] The present invention uses earthworms bred with livestock and poultry manure containing antibiotics to further carry out targeted repair of drug-resistant contaminated soil. According to the specific situation that the drug-resistant contaminated soil is contaminated by different types of drugs, the type of antibiotics in the livestock and poultry manure is selected, which can improve the efficiency of soil repair.

[0040] The chitosan is modified to obtain modified chitosan.

[0041] In the present invention, the modification is preferably carried out by first mixing the chitosan with dilute hydrochloric acid for acidification, then mixing the acidified chitosan with lactobionic acid solution and N,N'-carbonyldiimidazole, and finally filtering and dialyzing to obtain the modified chitosan.

[0042] In the present invention, the mass fraction of the dilute hydrochloric acid is preferably 3-7%, and more preferably 3-5%; the mass concentration of the lactobionic acid solution is preferably 50 g / L, and the dosage ratio of the chitosan, dilute hydrochloric acid, lactobionic acid solution and N,N'-carbonyldiimidazole is preferably 5-10 g:1-1.2 L:4.5-5 L:2.5-3 L, and more preferably 5-8 g:1 L:5 L:3 L.

[0043] The invention mixes acidified chitosan with lactobionic acid solution and N,N'-carbonyldiimidazole, and then dialyzes the mixture for 5 days through a dialysis bag with a retention molecular weight of 10000-12000 to obtain a retained solution in the bag, and then vacuum dries the retained solution in the bag at a temperature of 45°C to obtain modified chitosan.

[0044] The present invention modifies chitosan, thereby promoting its effect as a bacterial carrier, making it easier to maintain bacterial activity and facilitating bacteria to repair contaminated soil; in addition, the modified chitosan can further enhance its ability to improve soil structure, such as increasing soil permeability and water retention, promoting plants to better absorb nutrients and water, and facilitating the recovery of the ecological environment after soil repair.

[0045] Finally, the mixed bacterial solution, earthworm contents, modified chitosan and agarose are mixed to obtain the drug-resistant contaminated soil remediation bacterial agent.

[0046] The present invention adopts agarose and modified chitosan as carriers of bacteria to maintain the activity of bacteria in repairing soil, so that the bacteria of the present invention have high activity after being mixed with earthworm contents, and jointly repair drug-resistant contaminated soil.

[0047] The present invention also provides the use of the drug-resistant contaminated soil remediation bacterial agent described in the above technical solution in the remediation of drug-resistant contaminated soil. Furthermore, the mass ratio of the drug-resistant contaminated soil remediation bacterial agent to soil is preferably 1 to 2:500 to 1000.

[0048] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1

[0050] (1) placing 5 parts of Bacillus subtilis, 5 parts of Bacillus megaterium and 5 parts of Bacillus coagulans in three LB culture media respectively, placing 5 parts of actinomycetes in Gould's medium, culturing the above four culture media at a temperature of 25° C. for 3 days, then mixing the bacterial liquids obtained by the above cultures, and refrigerating the mixed bacterial liquid at a temperature of 0 to 4° C. for later use;

[0051] (2) taking an appropriate amount of earthworms and placing them in livestock and poultry manure containing antibiotics for breeding, taking them out after 15 days of breeding, decomposing the earthworms, and obtaining the midgut contents of the earthworms;

[0052] (3) 5 g of chitosan was dissolved in 1 L of 3% dilute hydrochloric acid for acidification, and then the acidified chitosan was mixed with 4.5 L of lactic acid solution and 2.5 L of N, N'-carbonyldiimidazole for modification for 3 days to obtain a mixture, and then the mixture was filtered to obtain a filtrate, and then the filtrate was dialyzed for 5 days through a dialysis bag with a retention molecular weight of 10000 to 12000 to obtain a retained solution in the bag, and then the retained solution in the bag was placed at a temperature of 45°C for vacuum drying to obtain modified chitosan;

[0053] (4) 4 parts of modified chitosan, 2 parts of agarose, 3 parts of earthworm midgut contents and the mixed bacterial solution are mixed to obtain the drug-resistant contaminated soil remediation bacterial agent.

[0054] Example 2

[0055] (1) placing 8 parts of Bacillus subtilis, 8 parts of Bacillus megaterium and 8 parts of Bacillus coagulans in three LB culture media respectively, placing 8 parts of actinomycetes in Gould's medium, culturing the above four culture media at a temperature of 28° C. for 3 days, then mixing the bacterial liquids obtained by the above cultures, and refrigerating the mixed bacterial liquid at a temperature of 0 to 4° C. for later use;

[0056] (2) taking an appropriate amount of earthworms and placing them in livestock and poultry manure containing antibiotics for breeding, taking them out after 12 days of breeding, decomposing the earthworms, and obtaining the midgut contents of the earthworms;

[0057] (3) 8 g of chitosan was dissolved in 1 L of 3% dilute hydrochloric acid for acidification, and then the acidified chitosan was mixed with 5 L of lactic acid solution and 3 L of N,N'-carbonyldiimidazole for modification for 3 days to obtain a mixture, and then the mixture was filtered to obtain a filtrate, and the filtrate was dialyzed for 5 days through a dialysis bag with a molecular weight of 10,000 to 12,000 to obtain a retained solution in the bag, and then the retained solution in the bag was placed at a temperature of 45°C for vacuum drying to obtain modified chitosan;

[0058] (4) 4.5 parts of modified chitosan, 2.5 parts of agarose, 4 parts of earthworm midgut contents and the mixed bacterial solution are mixed to obtain the drug-resistant contaminated soil remediation bacterial agent.

[0059] Example 3

[0060] (1) 10 parts of Bacillus subtilis, 10 parts of Bacillus megaterium and 10 parts of Bacillus coagulans were placed in three LB culture media respectively, and 10 parts of actinomycetes were placed in Gould's medium, and the above four culture media were cultured at a temperature of 30° C. for 3 days, and then the bacterial liquids obtained by the above cultures were mixed, and the mixed bacterial liquid was placed in a refrigerator at a temperature of 0 to 4° C. for standby use;

[0061] (2) taking an appropriate amount of earthworms and placing them in livestock and poultry manure containing antibiotics for breeding, taking them out after 15 days of breeding, decomposing the earthworms, and obtaining the midgut contents of the earthworms;

[0062] (3) 10 g of chitosan was dissolved in 1.2 L of 7% dilute hydrochloric acid for acidification, and then the acidified chitosan was mixed with 5 L of lactic acid solution and 3 L of N, N'-carbonyldiimidazole for modification for 3 days to obtain a mixture, and then the mixture was filtered to obtain a filtrate, and then the filtrate was dialyzed through a dialysis bag with a retention molecular weight of 10000 to 12000 for 5 days to obtain a retained solution in the bag, and then the retained solution in the bag was placed at a temperature of 45°C for vacuum drying to obtain modified chitosan;

[0063] (4) 5 parts of modified chitosan, 3 parts of agarose, 5 parts of earthworm midgut contents and the mixed bacterial solution are mixed to obtain the drug-resistant contaminated soil remediation bacterial agent.

[0064] Application Example 1

[0065] The drug-resistant contaminated soil remediation bacterial agent obtained in Example 1 was mixed with the contaminated soil at a mass ratio of 1:1000, and the contaminated soil was remediated. The soil was tested 30 days after the remediation.

[0066] Comparative application example 1

[0067] Clean water was mixed with the contaminated soil at a mass ratio of 1:1000, and the soil was tested 30 days after mixing.

[0068] According to the test results, the content of genes and harmful organic matter in the contaminated soil in Application Example 1 decreased by more than 85%, and the biological content increased. In contrast, the composition of the contaminated soil in Application Example 1 did not decrease significantly.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A drug-resistant contaminated soil remediation bacterial agent, characterized in that: include: 5-10 parts of Bacillus subtilis, 5-10 parts of Bacillus megaterium, 5-10 parts of Bacillus coagulans, 5-10 parts of actinomycetes, 4-5 parts of modified chitosan, 2-3 parts of agarose and 3-5 parts of earthworm contents.

2. The method for preparing the drug-resistant contaminated soil remediation bacterial agent according to claim 1, characterized in that: The following steps are involved: Bacillus subtilis, Bacillus megaterium, Bacillus coagulans and actinomycetes are first cultured respectively, bacterial liquids obtained by the culture are first mixed, and a mixed bacterial liquid is obtained and refrigerated for standby use; The earthworms are subjected to a second culture and then decomposed to obtain earthworm contents; Modifying chitosan to obtain modified chitosan; The mixed bacterial solution, earthworm contents, modified chitosan and agarose are mixed to obtain the drug-resistant contaminated soil remediation bacterial agent.

3. The method for preparing the drug-resistant contaminated soil remediation bacterial agent according to claim 2, characterized in that: The first culture conditions are: temperature of 25-30° C. and time of 3-4 days.

4. The method for preparing the drug-resistant contaminated soil remediation bacterial agent according to claim 2, characterized in that: The refrigeration temperature is 0-4°C.

5. The method for preparing the drug-resistant contaminated soil remediation bacterial agent according to claim 2, characterized in that: The ratio of Bacillus subtilis, Bacillus megaterium, Bacillus coagulans and actinomycetes is 1:1:1:

1.

6. The method for preparing the drug-resistant contaminated soil remediation bacterial agent according to claim 2, characterized in that: The second culture is to place the earthworms in livestock and poultry manure for breeding, the breeding time is 10 to 15 days, and the livestock and poultry manure includes antibiotics.

7. The method for preparing the drug-resistant contaminated soil remediation bacterial agent according to claim 2, characterized in that: The earthworm contents are earthworm midgut contents.

8. The method for preparing the drug-resistant contaminated soil remediation bacterial agent according to claim 2, characterized in that: The modification is to first mix the chitosan with dilute hydrochloric acid for acidification, then mix the acidified chitosan with lactobionic acid solution and N,N'-carbonyldiimidazole, and finally perform filtration and dialysis.

9. The method for preparing the drug-resistant contaminated soil remediation bacterial agent according to claim 8, characterized in that: The mass fraction of the dilute hydrochloric acid is 3-7%; the mass concentration of the lactobionic acid solution is 50g / L, and the dosage ratio of the chitosan, dilute hydrochloric acid, lactobionic acid solution and N,N'-carbonyldiimidazole is 5-10g:1-1.2L:4.5-5L:2.5-3L.

10. Use of the drug-resistant contaminated soil remediation bacterial agent according to claim 1 or any one of claims 2 to 9 in the remediation of drug-resistant contaminated soil, characterized in that: The mass ratio of the drug-resistant contaminated soil remediation bacterial agent to the soil is 1-2:500-1000.