Microbial remediation method for soil tetracycline antibiotic pollution

Abstract

The application provides a microbial remediation method for soil tetracycline antibiotic pollution and relates to the technical field of contaminated soil remediation. The microbial remediation method for soil tetracycline antibiotic pollution mainly uses a microbial remediation agent to remediate soil. The microbial remediation agent is composed of an adsorption carrier material and a microbial inoculum in a mass ratio of 6-8:1-3. The adsorption carrier material is composed of calcined diatomite, modified lignin, modified vermiculite, modified sugar cane residue, modified starch, multi-stage particle size biochar, chitin, nano silicon dioxide and sodium dodecyl sulfonate. The microbial inoculum is composed of tobacco arthrobacter, bacillus subtilis, xylose-oxidizing achromobacter and stenotrophomonas maltophilia. The application overcomes the shortcomings of the prior art. The microbial remediation agent composed of the adsorption carrier material and the microbial inoculum is used to remediate soil, which effectively improves the remediation efficiency, guarantees the long-term remediation effect of soil, and improves the convenience of actual use.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of contaminated soil remediation, in particular to a microbial remediation method for soil tetracycline antibiotic pollution. BACKGROUND

[0002] Antibiotics are widely used to treat various diseases of humans and animals, and to promote the growth of animals, etc. However, about 59% of the antibiotics taken by humans and animals cannot be absorbed and utilized, and are then discharged into the environment with urine and feces. The antibiotics entering the environment have a negative impact on environmental microorganisms, and the antibiotics in the soil can harm plants, soil animals and microorganisms, seriously affecting environmental safety.

[0003] Since the antibiotics used for animal feeding in China are mostly tetracycline antibiotics, and tetracycline antibiotics are a class of broad-spectrum antibiotics produced by actinomycetes, including chlortetracycline, oxytetracycline, tetracycline, and semi-synthetic derivatives methacycline, doxycycline, minocycline, etc., long-term use will lead to enrichment of a large amount of tetracycline antibiotics in the soil, seriously polluting the environment and ecology.

[0004] At present, the main degradation pathways of tetracycline antibiotics in soil are biological degradation, hydrolysis, photodegradation and micro-electrolysis. Biological degradation has great advantages in both cost and efficiency, but traditional biological degradation mostly selects bacteria that can degrade tetracycline antibiotics, and adds them to the soil at a certain concentration to remediate the soil. However, the use of single strain has some shortcomings in overall efficiency and effect, and cannot continue to remediate the soil for a long time, which has certain limitations for actual use. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a microbial remediation method for soil tetracycline antibiotic pollution, which uses a microbial remediation agent composed of an adsorption carrier material and a microbial inoculant to remediate the soil, effectively improving the remediation efficiency while ensuring long-term remediation effect, and improving the convenience of actual use.

[0006] To achieve the above purposes, the technical scheme of the present application is implemented as follows:

[0007] The application discloses a method for repairing soil tetracycline antibiotic pollution by using microorganisms, and relates to the field of soil remediation.

[0008] Preferably, the preparation methods of the modified wood lignin and the modified sugarcane residue are both acid-base modification after air-blast treatment of raw materials.

[0009] Preferably, the modified vermiculite is obtained by microwave expansion treatment after acid washing of vermiculite.

[0010] The preparation method of the modified starch comprises the following steps: dissolving starch in water, semi-pasting after temperature rising, freeze-drying and crushing, rapid treatment by high-temperature steam at 220-250 DEG C for 60-80 s, and finally drying.

[0011] The multi-level particle size biochar is composed of biochar particles with particle sizes of (0.1-2 mm), (10-20 mm) and (30-50 mm) at a mass ratio of 4:5:1.

[0012] Preferably, each strain in the microbial agent is obtained by continuous passage domestication of original strains in a tetracycline-containing basic solid culture medium.

[0013] Preferably, the continuous passage domestication process comprises the following steps: inoculating each original strain into a basic solid culture medium containing 100 mg / L of tetracycline, culturing at 25-37 DEG C for 2-4 days, screening out resistant live bacteria, transferring into a basic solid culture medium containing 200 mg / L of tetracycline for passage culture, repeating for 5 times, increasing the concentration of tetracycline by 100 mg / L each time, so that the concentration of tetracycline reaches 500 mg / L in the last culture, and completing the passage domestication.

[0014] Preferably, the preparation method of the microbial remediation agent comprises the following steps:

[0015] (1) mixing the microbial agent with water to prepare a precursor solution for standby use;

[0016] (2) stirring the calcined diatomite into the precursor solution for 15-20 min, then adding the modified starch, chitin, nano-silicon dioxide and sodium dodecyl sulfonate, and continuing to stir for 10-15 min to obtain a pretreatment liquid;

[0017] (3) after the pretreatment liquid is left to stand for 2-3 h, adding the modified lignin, modified vermiculite, modified bagasse and multi-level particle size biochar into the inner part and stirring uniformly under pressure, and then drying and solidifying to obtain the microbial remediation agent.

[0018] Preferably, the stirring speed in the step (2) is 800-1000 r / min.

[0019] Preferably, the pressure stirring mode in the step (3) is to increase the pressure to 2-4 MPa, and to stir at a speed of 400-600 r / min for 10-15 min.

[0020] The present application provides a microbial remediation method for soil tetracycline antibiotic pollution, and has the following advantages compared with the prior art:

[0021] (1) The present application uses calcined diatomite, modified lignin, modified vermiculite, modified bagasse, modified starch and multi-level particle size biochar as the adsorption carrier material, which can effectively load the microbial raw materials, ensure the survival rate of the microorganisms during use, and further improve the degradation efficiency of the microorganisms on tetracycline, and ensure the long-term degradation of tetracycline in the soil.

[0022] (2) The present application obtains strains with good degradation effect on tetracycline by passing tetracycline through the tobacco arthrobacter, bacillus subtilis, xanthobacter, and stenotrophomonas maltophilia, which can effectively and comprehensively degrade tetracycline in the soil, and is loaded by the adsorption carrier material to ensure the remediation efficiency in the soil. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0024] Embodiment 1:

[0025] Subculture of each strain:

[0026] 1. Arthrobacter nicotianae OTC-16 is inoculated on the basic solid medium containing 100 mg / L tetracycline antibiotics, and cultured at 32°C for 3 days. Then, the resistant live bacteria are selected and inoculated on the basic solid medium containing 200 mg / L tetracycline antibiotics for subculture, and cultured at 30°C for 3 days. The resistant live bacteria are continuously selected and repeated in the above manner. The content of tetracycline antibiotics in the basic solid medium is increased by 100 mg / L each time until the content of tetracycline antibiotics in the basic solid medium reaches 500 mg / L, and the subculture domestication is completed.

[0027] 2. Bacillus subtilis is inoculated on the basic solid medium containing 100 mg / L tetracycline antibiotics, and cultured at 32°C for 3 days. Then, the resistant live bacteria are selected and inoculated on the basic solid medium containing 200 mg / L tetracycline antibiotics for subculture, and cultured at 30°C for 3 days. The resistant live bacteria are continuously selected and repeated in the above manner. The content of tetracycline antibiotics in the basic solid medium is increased by 100 mg / L each time until the content of tetracycline antibiotics in the basic solid medium reaches 500 mg / L, and the subculture domestication is completed.

[0028] 3. Achromobacter xylosoxidans is inoculated on the basic solid medium containing 100 mg / L tetracycline antibiotics, and cultured at 32°C for 3 days. Then, the resistant live bacteria are selected and inoculated on the basic solid medium containing 200 mg / L tetracycline antibiotics for subculture, and cultured at 30°C for 3 days. The resistant live bacteria are continuously selected and repeated in the above manner. The content of tetracycline antibiotics in the basic solid medium is increased by 100 mg / L each time until the content of tetracycline antibiotics in the basic solid medium reaches 500 mg / L, and the subculture domestication is completed.

[0029] 4. Stenotrophomonas maltophilia strain DT1 is inoculated on the basic solid medium containing 100 mg / L tetracycline antibiotics, and cultured at 32°C for 3 days. Then, the resistant live bacteria are selected and inoculated on the basic solid medium containing 200 mg / L tetracycline antibiotics for subculture, and cultured at 30°C for 3 days. The resistant live bacteria are continuously selected and repeated in the above manner. The content of tetracycline antibiotics in the basic solid medium is increased by 100 mg / L each time until the content of tetracycline antibiotics in the basic solid medium reaches 500 mg / L, and the subculture domestication is completed.

[0030] Example 2:

[0031] Preparation of the microbial repair agent:

[0032] (1)The lignin and bagasse are respectively subjected to steam explosion treatment at a high temperature of 160℃ for 1 min under a pressure of 2Mpa, and then are sequentially immersed in solutions with pH values of 4 and 7.5 for 15 min, and after the end of the immersion, the pH is adjusted to neutral and dried, to obtain modified lignin and modified bagasse;

[0033] (2)The vermiculite is pickled in an acid solution with a pH of 6 for 20 min, and then the pH is adjusted to neutral and dried, and then is subjected to expansion treatment at a microwave power of 600W for 70s, and then is crushed through a 20-mesh sieve, to obtain modified vermiculite;

[0034] (3)The sweet potato starch is dissolved in water, heated to 62℃, and subjected to half-gelatinization treatment for 15 min while stirring, and then is freeze-dried and crushed, and is subjected to rapid treatment with high-temperature hot steam at 240℃ for 70s, and then is dried, to obtain modified starch;

[0035] (4)The biochar particles with particle sizes of (0.1-2mm), (10-20mm), and (30-50mm) are mixed according to a mass ratio of 4:5:1, to obtain multi-level particle size biochar;

[0036] (5)The tobacco arthrobacter, bacillus subtilis, acidovorax facilis, and stenotrophomonas maltophilia in the domestication of Example 1 are collected and mixed to prepare a microbial inoculant, and the viable bacterial count in the microbial inoculant is ≥200 billion viable bacterial count / g;

[0037] (6)10g of the above microbial inoculant is added to 1000ml of water, mixed uniformly to prepare a precursor solution, 14.5g of calcined diatomite is added to the precursor solution, stirred at 800r / min for 15 min, and then 2.9g of modified starch, 1.74g of chitin, 1.16g of nanometer silicon dioxide, and 0.58g of sodium dodecyl sulfonate are added, and continue to stir for 15 min to obtain a pretreatment liquid;

[0038] (7)After the pretreatment liquid is allowed to stand for 3h, 2.32g of modified lignin, 2.9g of modified vermiculite, 6.38g of modified bagasse, and 7.52g of multi-level particle size biochar are added, and then the pressure is increased to 2Mpa, and the stirring speed is 500r / min, and the stirring time is 15 min, and then the mixture is dried and solidified, to obtain a microbial remediation agent.

[0039] Example 3:

[0040] For the preparation method of the microbial remediation agent in Example 2, unmodified lignin, bagasse, starch, and biochar with a particle size range of 30-50mm are selectively used instead of modified lignin, modified vermiculite, modified bagasse, modified starch, and multi-level particle size biochar, respectively, and the remaining preparation steps are the same as those of Example 2, and are shown in Table 1 as follows:

[0041] Table 1

[0042]

[0043] The "√" in the above Table 1 means adding, and "-" means not adding.

[0044] Detection:

[0045] Taking terramycin as an example, the terramycin is added to the subculture of each strain in Example 1 as a tetracycline antibiotic, and the soil containing 100 g / Kg of terramycin prepared in the laboratory is used as the experimental soil,

[0046] The microbial repair agents prepared in Experimental Groups 1-5 in Example 2 are mixed with the experimental soil in a mass ratio of 1:2000, and the microbial agent prepared in Example 2 is mixed with the experimental soil in a mass ratio of 1:4000 to form a control group, and the content of terramycin in each group of soil is detected after 24h, 48h, 72h and 168h under the simulation of outdoor light time at a temperature of 25±2℃, and the degradation rate is calculated, and the specific results are shown in Table 2 below:

[0047] Table 2

[0048]

[0049]

[0050] As can be seen from the above Table 2, the microbial repair agent prepared in Experimental Group 1 has a lower degradation of terramycin within 24h compared with the control group, but it can effectively improve the degradation efficiency after 48h, and the degradation efficiency is greatly enhanced compared with the control group, that is, the colonization effect of the microbial agent using the adsorption carrier material can be improved, and in the subsequent long-term observation (168h), the degradation efficiency of the experimental group is still increasing stably, while the overall increase of the control group is not large, that is, the microbial repair agent of the experimental group 1 of the present application can stably and long-acting repair the pollution of tetracycline antibiotics in the soil.

[0051] It should be noted that, in this document, relational terms such as first and second and the like can be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element preceded by "comprises... " does not, without more constraints, foreclose the existence of additional identical elements in the process, method, article, or apparatus that comprises the stated elements.

[0052] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those ordinarily skilled in the art should understand: the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for the microbial remediation of soil contaminated with tetracycline antibiotics, characterized in that, The microbial remediation method for soil tetracycline antibiotic pollution mainly uses a microbial remediation agent to remediate the soil, wherein the microbial remediation agent is composed of an adsorption carrier material and a microbial inoculant in a mass ratio of 6-8:1-3; The adsorption carrier material is made of the following raw materials in parts by weight: calcined diatomite 20-30 parts, modified lignin 3-5 parts, modified vermiculite 4-6 parts, modified sugar cane residue 10-12 parts, modified starch 4-6 parts, multi-level particle size biochar 10-15 parts, chitin 2-4 parts, nano silicon dioxide 1-3 parts, and sodium dodecyl sulfonate 0.6-1.2 parts; The microbial inoculant is made of a combination of Arthrobacter nicotinovorans, Bacillus subtilis, Acidovorax xylosoxidans, and Stenotrophomonas maltophilia. The preparation method of the modified lignin and the modified sugar cane residue is as follows: the lignin and the sugar cane residue are respectively subjected to steam explosion treatment at a pressure of 2 MPa and a high temperature of 160°C for 1 min, then are sequentially immersed in solutions with pH values of 4 and 7.5 for 15 min, and after the immersion, the pH values are adjusted to neutral and the modified lignin and the modified sugar cane residue are dried. The modified starch is prepared by dissolving starch in water, heating for semi-pasting, freeze-drying and crushing, then rapidly treating with high-temperature steam at 220-250°C for 60-80 s, and finally drying. The multi-level particle size biochar is composed of biochar particles with particle sizes of (0.1-2 mm), (10-20 mm), and (30-50 mm) in a mass ratio of 4:5:

1.

2. The method of microbial remediation of soil tetracycline antibiotic pollution according to claim 1, characterized in that: The modified vermiculite is obtained by acid washing and microwave expansion treatment of vermiculite.

3. The method of microbial remediation of soil tetracycline antibiotic pollution according to claim 1, characterized in that: Each strain in the microbial inoculant is obtained by continuous passage domestication of the original strain in a tetracycline-containing basic solid culture medium.

4. The method of microbial remediation of soil tetracycline antibiotic pollution according to claim 3, characterized in that, The continuous passage domestication process is as follows: each original strain is inoculated into a basic solid culture medium containing 100 mg / L of tetracycline, and is cultured at 25-37°C for 2-4 days, then the resistant live bacteria are selected and transferred into a basic solid culture medium containing 200 mg / L of tetracycline for passage culture, and the process is repeated for 5 times, with the concentration of tetracycline being increased by 100 mg / L each time, so that the concentration of tetracycline reaches 500 mg / L in the last culture, and the passage domestication is completed.

5. The method of microbial remediation of soil tetracycline antibiotic pollution according to claim 1, characterized in that, The preparation method of the microbial remediation agent comprises the following steps: (1) mixing the microbial inoculant with water to prepare a precursor solution; (2) adding calcined diatomite into the precursor solution and stirring for 15-20 min, then adding modified starch, chitin, nano silicon dioxide, and sodium dodecyl sulfonate, and continuing to stir for 10-15 min to obtain a pretreatment liquid; (3) after the pretreatment liquid is statically placed for 2-3 h, adding modified lignin, modified vermiculite, modified sugar cane residue, and multi-level particle size biochar into the pretreatment liquid, stirring uniformly, and then drying and solidifying to obtain the microbial remediation agent.

6. The method of microbial remediation of soil tetracycline antibiotic pollution according to claim 5, characterized in that, The stirring speed in step (2) is 800-1000 r / min.

7. The method of microbial remediation of soil tetracycline antibiotic pollution according to claim 5, characterized in that, In step (3), the pressure is increased to 2-4 MPa, and the stirring speed is 400-600 r / min for 10-15 min.

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