A highly adaptable imidacloprid-degrading bacterial community, its enrichment and domestication methods, and its applications.

Abstract

This invention belongs to the field of microbial remediation technology, specifically relating to a highly adaptable imidacloprid-degrading bacterial community and its enrichment and domestication method and application. The enrichment and domestication method of this invention includes the following steps: (1) placing imidacloprid-contaminated soil in an inorganic salt liquid culture medium containing imidacloprid for cultivation to obtain a primary suspension; (2) centrifuging the primary suspension to separate the solid and liquid, allowing the bacterial community and non-dissolved soil components in the primary suspension to enter the precipitate; adding fresh inorganic salt liquid culture medium containing imidacloprid to the precipitate for cultivation to obtain a secondary suspension; (3) performing subculture according to the culture conditions of step (2), taking the last generation suspension of the last subculture cycle, allowing it to stand, and obtaining a bacterial supernatant; (4) centrifuging the bacterial supernatant obtained in step (3) to obtain a highly adaptable imidacloprid-degrading bacterial community. The imidacloprid-degrading bacterial community obtained by enrichment and domestication in this invention has better environmental adaptability to complex soil conditions.

Description

Technical Field

[0001] This invention belongs to the field of microbial remediation technology, specifically relating to a highly adaptable imidacloprid-degrading bacterial community, its enrichment and domestication method, and its application. Background Technology

[0002] Imidacloprid is a neonicotinoid insecticide that is highly effective and broad-spectrum, and has been widely used for the control of crop diseases and pests since its introduction. Due to its strong systemic properties, long-lasting effect, and relatively stable chemical properties, it has been used extensively in agricultural production for a long time. However, imidacloprid degrades slowly in the environment, easily remaining and accumulating in soil, thus adversely affecting the soil ecosystem. Studies have shown that imidacloprid residues not only inhibit the growth and metabolism of beneficial microorganisms in the soil, disrupting the structure and functional stability of the soil microbial community, but may also accumulate through the food chain, exerting toxic effects on non-target organisms (such as earthworms, pollinating insects, and aquatic organisms), thereby threatening agricultural ecological security and human health. Therefore, exploring efficient and environmentally friendly imidacloprid-contaminated soil remediation technologies is of great significance.

[0003] Among existing soil remediation technologies, microbial remediation has attracted widespread attention due to its advantages such as low cost, low risk of secondary pollution, and wide applicability. By utilizing the metabolic activity of microorganisms to transform organic pollutants into low-toxicity or non-toxic substances, microbial remediation technology can achieve in-situ or ex-situ remediation of contaminated soil without damaging its physical and chemical properties, showing promising application prospects. In particular, the biodegradation of imidacloprid by microorganisms with degradation capabilities is considered a sustainable remediation approach.

[0004] Currently, most related research focuses on isolating and screening single bacterial strains capable of degrading imidacloprid from soil, sludge, or pesticide-contaminated environments, and verifying their degradation performance under pure laboratory culture conditions. However, the single degrading strains screened by such methods are usually obtained in ideal, stable laboratory environments with relatively simple growth and metabolic conditions. When these strains are directly applied to actual soil environments, they often face multiple environmental stresses such as temperature, pH, moisture, nutrient levels, and competing microorganisms, resulting in low survival rates, poor stress resistance, and difficulty in maintaining stable degradation efficiency. The remediation effects differ significantly from laboratory results, severely limiting their engineering applications.

[0005] Furthermore, microorganisms in nature do not exist independently as single species, but rather form stable microbial communities through complex relationships such as symbiosis, mutualism, and competition. In the biodegradation of organic pollutants such as imidacloprid, the synergistic participation of multiple functional bacteria is often required. Some degrading bacteria rely on other strains to provide growth factors, intermediate metabolites, or nutrients to maintain normal growth and reproduction. However, existing studies commonly use fixed culture media with the target pollutant as the sole carbon or nitrogen source for enrichment and screening, such as inorganic salt media and M9 media. These media have simple nutrient compositions and cannot accurately reflect the complex and diverse organic matter and nutrient structure in soil. They fail to simulate the interactions between microorganisms in real soil environments, ultimately resulting in screening microbial communities with fragile structures, limited functions, and poor environmental adaptability and resilience in actual soils.

[0006] Therefore, there is a need to provide an improved technical solution that addresses the shortcomings of the existing technology. Summary of the Invention

[0007] The purpose of this invention is to provide a highly adaptable imidacloprid-degrading microbial community, its enrichment and domestication method, and its application, so as to help solve or improve the problem of poor adaptability or degradation effect of microorganisms screened for the remediation of imidacloprid-contaminated soil under complex soil conditions in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for enriching and acclimatizing highly adaptable imidacloprid-degrading bacteria, comprising the following steps: (1) placing imidacloprid-contaminated soil in an inorganic salt liquid culture medium containing imidacloprid and culturing it under constant temperature and shaking to obtain a primary suspension; (2) centrifuging the primary suspension to separate the solid and liquid, so that the bacteria and non-dissolved soil components in the primary suspension enter the precipitate; adding fresh inorganic salt liquid culture medium containing imidacloprid to the obtained precipitate and continuing to culture it under constant temperature and shaking to obtain a secondary suspension; (3) performing subculture according to the culture conditions of step (2), taking the last generation suspension of the last subculture cycle, letting it stand, and obtaining a supernatant containing bacteria; (4) centrifuging the supernatant containing bacteria obtained in step (3), washing the obtained precipitate and resuspending it to obtain the highly adaptable imidacloprid-degrading bacteria.

[0009] Preferably, in step (2), the centrifugal force of the centrifugation is 8000-10000g.

[0010] Preferably, in step (3), the concentration of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid is 100-500 mg / L; in step (3), the concentration of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid increases in a gradient with the increase of the number of generations.

[0011] Preferably, during the subculture process in step (3), the concentration gradient of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid increases by 100 mg / L.

[0012] Preferably, in step (1), the mass ratio of the imidacloprid-contaminated soil to the imidacloprid-containing inorganic salt liquid culture medium is 1:(5-10); the concentration of imidacloprid in the imidacloprid-containing inorganic salt liquid culture medium is 100 mg / L; in step (2), the concentration of imidacloprid in the imidacloprid-containing inorganic salt liquid culture medium is 200 mg / L; in steps (1)-(3), the components of the inorganic salt liquid culture medium include 1 g of K2HPO4, 1 g of KH2PO4, 0.5 g of (NH4)2SO4, 1 g of KNO3, 0.5 g of MgSO4·7H2O, 0.02 g of CaCl2, 2 drops of 100 g / L FeCl2 solution and 1 L of distilled water; the pH of the inorganic salt liquid culture medium is 7.

[0013] Preferably, in step (1) and / or step (2), the temperature of the isothermal shaking culture is 25-35℃; in step (1) and / or step (2), the time of the isothermal shaking culture is 7-14 days; in step (3), the time of standing is 12-24h.

[0014] The present invention also provides a highly adaptable imidacloprid-degrading bacterial community, which adopts the following technical solution: a highly adaptable imidacloprid-degrading bacterial community, wherein the highly adaptable imidacloprid-degrading bacterial community is obtained by enrichment and domestication using the method described above.

[0015] The present invention also provides the application of the highly adaptable imidacloprid-degrading microbial community as described above, which adopts the following technical solution: the application of the highly adaptable imidacloprid-degrading microbial community as described above in the remediation of imidacloprid-contaminated soil.

[0016] Preferably, the method includes the following steps: mixing a bacterial solution containing the highly adaptable imidacloprid-degrading bacteria with imidacloprid-contaminated soil.

[0017] Preferably, the method further includes a step of adding water to mix with the bacterial solution and the imidacloprid-contaminated soil; the mass ratio of the imidacloprid-contaminated soil to water is 1:(0.6-3); the volume ratio of the bacterial solution to the volume of the mixture of imidacloprid-contaminated soil and water is (0.01-0.10):1; and the concentration of the bacterial solution is 1×10⁻⁶. 6 -1×10 8 CFU / L.

[0018] Beneficial effects: The present invention provides a method for enriching and acclimating highly adaptable imidacloprid-degrading microbial communities. This method fully integrates soil nutrients and employs a gradient pollution stress approach to acclimate and enrich functional microbial communities with high imidacloprid degradation performance and high environmental adaptability. The imidacloprid-degrading microbial communities acclimatized and enriched using this method largely retain the original community structure and diversity of imidacloprid-degrading microorganisms, improving their environmental adaptability under complex soil conditions. This provides a new technical pathway and theoretical basis for the efficient bioremediation of imidacloprid-contaminated soils.

[0019] The enrichment and domestication method for highly adaptable imidacloprid-degrading microorganisms of this invention, through multiple rounds of subculture combined with centrifugation, effectively preserves the degrading microorganisms and soil microstructure while removing water-soluble toxic metabolic byproducts accumulated in the culture system. This maintains the physiological activity and metabolic stability of the microorganisms, facilitating the screening and enrichment of dominant microorganisms with high tolerance and sustained degradation ability to imidacloprid, thereby significantly improving the stress resistance and functional stability of the obtained microorganisms in complex soil environments. Furthermore, the enrichment and domestication method for highly adaptable imidacloprid-degrading microorganisms of this invention is simple in process, highly controllable, and suitable for practical application. Attached Figure Description

[0020] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. Wherein: Figure 1 This is the community composition at the genus level of the highly adaptable imidacloprid-degrading bacteria community of Example 1.

[0021] Figure 2 This is a graph showing the test results of the degradation of imidacloprid in soil by the highly adaptable imidacloprid-degrading bacterial community in Example 1; where the horizontal axis represents degradation time and the vertical axis represents the degradation efficiency of the bacterial community on imidacloprid. CK is the control group, without bacterial treatment; LY, ZZ, and PDS are the experimental groups, with OD added at a dosage ratio (volume ratio) of 10%. 600 Bacterial suspension with a value of 0.3.

[0022] Figure 3 The comparison shows the degradation of imidacloprid in the soil by the conventional imidacloprid-degrading bacteria in Comparative Example 1 on day 14, and the degradation performance of the highly adaptable imidacloprid-degrading bacteria in Example 2; where the horizontal axis represents different soil sources, and the vertical axis represents the degradation efficiency of the bacteria on imidacloprid. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0024] The present invention will now be described in detail with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0025] This invention addresses the problem that existing microorganisms screened for the remediation of imidacloprid-contaminated soil have poor adaptability or degradation efficiency under complex soil conditions, and provides a method for enriching and acclimatizing a highly adaptable imidacloprid-degrading bacterial community.

[0026] The enrichment and domestication method of highly adaptable imidacloprid-degrading microbial community of the present invention constructs a highly adaptable degrading microbial community with diversity and synergy through a cultivation and domestication strategy that is closer to the real soil environment, so as to improve its survival ability and degradation stability under complex soil conditions, thereby providing a new technical approach and theoretical basis for the efficient bioremediation of imidacloprid-contaminated soil.

[0027] The enrichment and domestication method of highly adaptable imidacloprid-degrading bacteria in this invention includes the following steps: (1) placing imidacloprid-contaminated soil in an inorganic salt liquid culture medium containing imidacloprid and culturing it at a constant temperature with shaking to obtain a primary suspension; (2) centrifuging the primary suspension to separate the solid and liquid, so that the bacteria and non-dissolved soil components in the primary suspension enter the precipitate; adding fresh inorganic salt liquid culture medium containing imidacloprid to the obtained precipitate and continuing to culture it at a constant temperature with shaking to obtain a secondary suspension; (3) performing subculture according to the culture conditions in step (2), taking the last generation suspension of the last subculture cycle, letting it stand, and obtaining a supernatant containing bacteria; (4) centrifuging the supernatant containing bacteria obtained in step (3), washing the obtained precipitate and resuspending it to obtain a highly adaptable imidacloprid-degrading bacteria.

[0028] The enrichment and acclimatization method for highly adaptable imidacloprid-degrading microorganisms of the present invention fully integrates the soil's own nutrients (in step (2) of the present invention, the precipitate obtained by centrifuging the primary suspension contains non-dissolved soil components, allowing the soil components to enter the subsequent culture process, thereby making full use of the organic matter and micronutrients contained in the soil itself in the enrichment and screening of microorganisms, and constructing a culture system close to the natural soil environment. Unlike the present invention, in the prior art, after obtaining the primary suspension, only the degrading microorganisms contained therein are usually considered, and it is assumed that the microorganisms are uniformly dispersed in the suspension. A portion of the suspension is directly transferred to a fresh acclimatization culture medium for subsequent acclimatization; therefore, the step of centrifuging the primary suspension is not performed to allow sufficient non-dissolved soil components to enter the subsequent treatment), through gradient pollution stress, to acclimatize and enrich functional microorganisms with high imidacloprid degradation performance and high environmental adaptability. The imidacloprid-degrading microbial community obtained by domestication and enrichment using this method can largely retain the original community structure and diversity of the imidacloprid-degrading microbial community, improve the environmental adaptability of the enriched and domesticated imidacloprid-degrading microbial community under complex soil conditions, and avoid the problems of simplified microbial community structure, single function and poor environmental adaptability caused by the limited nutrient components of single inorganic culture genes. This provides a new technical path and theoretical basis for the efficient bioremediation of imidacloprid-contaminated soil.

[0029] In a preferred embodiment of the enrichment and domestication method for highly adaptable imidacloprid-degrading bacteria of the present invention, in step (2), the centrifugal force is 8000-10000g (e.g., 8000g, 8500g, 9000g, 9500g, or 10000g). If the centrifugal force is too small, the bacterial separation efficiency will be low; if the centrifugal force is too large, the bacterial biofilm may rupture and die.

[0030] Preferably, in step (2), the centrifugation time is 10-30 minutes. The centrifugation time is maintained at more than 10 minutes to help ensure separation efficiency; the centrifugation time generally does not exceed 30 minutes to save energy.

[0031] In a preferred embodiment of the enrichment and domestication method for highly adaptable imidacloprid-degrading bacteria of the present invention, in step (3), the concentration of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid is 100-500 mg / L (e.g., 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L or 500 mg / L); in step (3), the concentration of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid increases in a gradient with the increase of the number of generations.

[0032] In a preferred embodiment of the enrichment and domestication method for highly adaptable imidacloprid-degrading bacteria of the present invention, during the subculturing process in step (3), the concentration gradient of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid increases by 100 mg / L.

[0033] In a preferred embodiment of the enrichment and domestication method for highly adaptable imidacloprid-degrading bacteria of the present invention, in step (1), the mass ratio of imidacloprid-contaminated soil to inorganic salt liquid culture medium containing a first concentration of imidacloprid is 1:(5-10) (e.g., 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10); the concentration of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid is 100 mg / L; in step (2), the concentration of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid is 200 mg / L; in steps (1)-(3), the components of the inorganic salt liquid culture medium include 1 g of K2HPO4, 1 g of KH2PO4, 0.5 g of (NH4)2SO4, 1 g of KNO3, 0.5 g of MgSO4·7H2O, 0.02 g of CaCl2, 2 drops of 100 g / L FeCl2 solution and 1 L of distilled water; the pH of the inorganic salt liquid culture medium is 7.

[0034] In a preferred embodiment of the enrichment and domestication method for highly adaptable imidacloprid-degrading bacteria of the present invention, in step (1) and / or step (3), the temperature of the isothermal shaking culture is 25-35℃ (e.g., 25℃, 28℃, 30℃, 32℃ or 35℃); in step (1) and / or step (2), the isothermal shaking culture time is 7-14 days; in step (3), the static time is 12-24h (e.g., 12h, 16h, 20h or 24h).

[0035] The present invention also proposes a highly adaptable imidacloprid-degrading bacterial community. The highly adaptable imidacloprid-degrading bacterial community of the present invention is obtained by enrichment and domestication using the method described above.

[0036] This invention also proposes the application of a highly adaptable imidacloprid-degrading bacterial community, and the application of the highly adaptable imidacloprid-degrading bacterial community in the remediation of imidacloprid-contaminated soil according to embodiments of this invention.

[0037] In a preferred embodiment of the application of the present invention, the following steps are included: mixing a bacterial solution containing the highly adaptable imidacloprid-degrading bacteria with imidacloprid-contaminated soil.

[0038] Preferably, the bacterial solution containing highly adaptable imidacloprid-degrading bacteria, imidacloprid-contaminated soil, and water are mixed evenly and then cultured by shaking to achieve the remediation of imidacloprid-contaminated soil.

[0039] More preferably, the mass ratio of imidacloprid-contaminated soil to water is 1:(0.6-3) (e.g., 1:0.6, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3), and the volume ratio of the bacterial solution to the volume of the mixture of imidacloprid-contaminated soil and water is (0.01-0.10):1 (e.g., 0.01:1, 0.03:1, 0.05:1, 0.08:1, or 0.1:1); the concentration of the bacterial solution is 1×10⁻⁶. 6 -1×10 8 CFU / L (e.g., 1×10⁻⁶) 6 CFU / L, 5×10 6 CFU / L, 1×10 7 CFU / L, 5×10 7 CFU / L or 1×10 8 CFU / L).

[0040] Preferably, the bacterial solution containing highly adaptable imidacloprid-degrading bacteria is obtained by expanding the culture of highly adaptable imidacloprid-degrading bacteria; including the following steps: Step S1: The highly adaptable imidacloprid-degrading bacteria as described above are transferred to an enrichment medium containing imidacloprid and cultured at 30°C and 180 rpm with constant temperature shaking to obtain an enriched bacterial solution; Step S2: The enriched bacterial solution obtained in Step S1 is centrifuged at high speed, and the precipitate is washed twice with an appropriate amount of sterile phosphate buffer and resuspended to obtain the expanded culture of highly adaptable imidacloprid-degrading bacteria.

[0041] Preferably, the enrichment medium containing imidacloprid is prepared as follows: 0.1-0.5g imidacloprid, 10g peptone, 5g beef extract, 5g NaCl, 1L distilled water, pH adjusted to 7.0, sterilized at 121℃ for 30min.

[0042] The highly adaptable imidacloprid-degrading bacterial community, its enrichment and domestication method, and its application are described in detail below through specific embodiments.

[0043] The main reagents used in the following examples: Inorganic salt liquid culture medium: Imidacloprid 0.1-0.5g, K2HPO4 1g, KH2PO4 1g, (NH4)2SO4 0.5g, KNO3 1g, 0.5g of CaCl2, 0.02g of CaCl2, 2 drops of 100g / L FeCl2 solution, and 1L of distilled water were added. The pH was adjusted to 7.0, and the mixture was sterilized at 121℃ for 30 minutes.

[0044] LB medium: 0.1g imidacloprid, 10g peptone, 5g beef extract, 5g NaCl, 800mL distilled water, adjust pH to 7.0, bring volume to 1L with distilled water, sterilize at 121℃ for 30min.

[0045] Phosphate buffer: 0.2g KCl, 0.24g KH2PO4, 8g NaCl, 1.44g Na2HPO4, 800mL distilled water, adjust pH to 7.0, bring volume to 1L with distilled water, sterilize at 121℃ for 30min.

[0046] Example 1 The enrichment and domestication method for highly adaptable imidacloprid-degrading bacterial communities in this embodiment includes the following steps: (1) Soil contaminated with imidacloprid was collected from a farmland in Luoyang, Henan Province, where imidacloprid had been applied for a long time. 5g of soil was weighed into 50mL of inorganic salt liquid culture medium containing 100mg / L imidacloprid and cultured at 30℃ and 180rpm for 7 days to obtain a first-level suspension. (2) Centrifuge the first-stage suspension at a centrifugal force of 8000g, discard the supernatant, add 50mL of inorganic salt liquid culture medium containing 200mg / L imidacloprid to the precipitate, shake the centrifuge tube vigorously to make the precipitate evenly distributed in the inorganic salt liquid culture medium, and continue to culture at a constant temperature of 30℃ and 180rpm for 7 days. (3) Perform subculture according to the method in step (2). Each time, the content of imidacloprid in the fresh inorganic salt liquid culture medium containing imidacloprid is increased by 100 mg / L compared with the previous one, up to 500 mg / L. When the imidacloprid concentration is ≥300 mg / L, the culture time is extended to 14 days. Take the last generation suspension of the last subculture cycle, let it stand for 12 hours, and obtain the supernatant containing bacteria. (4) Take the bacterial supernatant obtained in step (3), centrifuge at high speed with a centrifugal force of 8000g, add 50mL of phosphate buffer to wash twice and resuspend to obtain the highly adaptable imidacloprid degrading bacteria of this embodiment.

[0047] The highly adaptable imidacloprid-degrading bacterial community of this embodiment was sequenced and analyzed using 16S amplicon sequencing with Illumina sequencing technology. The community composition of the bacterial community is as follows: Figure 1 As shown.

[0048] from Figure 1The relative abundance of each genera in the bacterial community can be seen as follows: Azohydromonas sp. (40.06%), Azospirillum sp. (31.91%), unclassified_f__Rhizobiaceae sp. (9.99%), Bordetella sp. (5.28%), Aminobacter sp. (3.41%), Azoarcus sp. (3.04%), Methylophilus sp. (2.41%), Ensifer sp. (1.24%), Terrimonas sp. (0.53%), Acidovorax sp. (0.43%), Flavobacterium sp. (0.41%), Fuscovulum sp. (0.22%), Pusillimonas sp. (0.22%), Mesorhizobium sp. (0.17%), Sphingopyxis sp. (0.16%), Ralstonia sp. (0.13%), Rhizobium sp. (0.09%), Castellaniella sp. (0.04%), Rhizorhabdus sp. (0.03%), Mycobacterium sp. (0.03%), Rhodopseudomonas sp. (0.03%), Pseudaminobactersp. (0.02%), Brucella sp. (0.02%), Afipia sp. (0.02%), Novosphingobium sp. (0.02%).

[0049] Example 2 The highly adaptable imidacloprid-degrading microbial community obtained in Example 1 was used for the remediation of imidacloprid-contaminated soil: Test soils: Farmland soils were collected from Luoyang (LY), Zhengzhou (ZZ), and Pingdingshan (PDS) in Henan Province. An appropriate amount of imidacloprid solution was added to contaminate the soils so that the imidacloprid content in the soils reached 100 mg / kg. The soils were then aged (by standing at around 25°C) for one week before use.

[0050] Preparation of bacterial suspension: The bacterial population screened in Example 1 was inoculated into 50 mL of LB medium (LB medium with an imidacloprid concentration of 100 mg / L) and enriched at 30 °C and 180 rpm for 18 h. The resulting culture was centrifuged at 4 °C and 8000 g for 10 min, the bacterial cells were collected, washed three times with sterile phosphate buffer, resuspended, and OD was adjusted. 600 The value is 0.3, and a bacterial suspension is obtained.

[0051] Imidacloprid degradation experiment: 100g of imidacloprid-contaminated soil was placed in a 500mL Erlenmeyer flask, and deionized water was added to make the mass ratio of imidacloprid-contaminated soil to deionized water in the flask reach 1:3. Bacterial suspension was added at a 10% addition ratio (volume ratio) (the volume of bacterial suspension is 10% of the volume of the mixture of imidacloprid-contaminated soil and deionized water). After stirring evenly, the flask was covered with a sterile breathable membrane and placed in a constant temperature shaking incubator at 30℃ and 180rpm for 14 days. Samples were taken on days 1, 3, 5, 7, 10 and 14 to detect the concentration of imidacloprid in the experimental system. Each treatment was repeated three times.

[0052] Control group (CK): Compared with the experimental group, the bacterial suspension was replaced with an equal amount of deionized water; all other aspects were the same as the experimental group.

[0053] Imidacloprid detection method: High performance liquid chromatography (HPLC) was used to detect the concentration of imidacloprid in soil. After soil sample collection, moisture was removed by freeze drying. 2g of dry soil was weighed and 10mL of acetonitrile (containing 0.1% acetic acid) was added for ultrasonic extraction for 5min. The extract was centrifuged at 3000g for 5min. 2mL of the supernatant was transferred to a 5mL centrifuge tube containing 100mg PSA (N-propylethylenediamine-bonded solid-phase adsorbent) and 50mg C18 (C18 silica gel crosslinked adsorbent). The tube was vortexed for 0.5min, centrifuged for 1min, and the supernatant was filtered through a 0.2μm organic phase filter membrane into a sample vial for analysis. The high performance liquid chromatography parameters are as follows: column model ACQUITY UPLC BEH C18 (2.1mm×150mm, 1.7μm), mobile phase V (acetonitrile):V (water) = 35:65, flow rate 0.9mL / min, column temperature 35℃, injection volume 10μL, and UV detection wavelength 270nm.

[0054] Example 1: The degradation effect of highly adaptable imidacloprid-degrading bacteria on imidacloprid in soils of three locations is as follows: Figure 2 As shown in the figure, for LY soil from which the microbial community originated, the degradation efficiency of imidacloprid reached as high as 86.34% within 14 days; for ZZ and PDS soils from other locations, the degradation efficiencies within 14 days were also 68.85% and 59.81%, respectively. This result indicates that the imidacloprid-degrading microbial community obtained through the enrichment and domestication method based on the highly adaptable imidacloprid-degrading microbial community provided by this invention can efficiently and rapidly degrade imidacloprid in contaminated soils from multiple locations. This provides a new and efficient method for domesticating imidacloprid-contaminated soils.

[0055] Comparative Example 1 The only difference between this comparative example and Example 1 is that conventional methods were used to enrich and acclimate the imidacloprid-degrading bacteria in the soil; all other aspects are consistent with Example 1.

[0056] Specifically, the conventional method for enriching and acclimating imidacloprid-degrading bacteria is as follows: (1) Obtain a primary suspension as in step (1) of Example 1; (2) Take 1 mL of the primary suspension and add it to 50 mL of fresh inorganic salt liquid culture medium containing 200 mg / L imidacloprid, and culture it at a constant temperature of 30°C and 180 rpm for 7 days; (3) According to the method in step (2), take the suspension for subculture each time, and increase the imidacloprid content in the inorganic salt liquid culture medium by 100 mg / L each time until it reaches 500 mg / L; and when the imidacloprid concentration is ≥300 mg / L, the culture time is extended to 14 days, and the bacteria obtained in the last subculture cycle are the imidacloprid-degrading bacteria obtained by the conventional acclimation method.

[0057] The conventional imidacloprid-degrading bacteria obtained in this comparative example were used in an experiment to degrade imidacloprid in soil contaminated with imidacloprid; the experimental method was the same as in Example 2.

[0058] The degradation effect of conventional imidacloprid-degrading bacteria in Example 1 on imidacloprid in soil and its degradation performance compared with that of highly adaptable imidacloprid-degrading bacteria in Example 2. Figure 3 As shown in the figure, the conventional imidacloprid-degrading microbial community (Comparative Example 1 community) achieved a 67.26% degradation efficiency of imidacloprid in the source soil (LY) after 14 days, while the degradation efficiencies for ZZ and PDS soils from other locations were 31.5% and 17.37%, respectively. Compared with the highly adaptable imidacloprid-degrading microbial community in Example 2, the degradation efficiencies decreased by 16.08%, 37.35%, and 42.44%, respectively. This comparative result indicates that the imidacloprid-degrading microbial community obtained by the enrichment and domestication method of the highly adaptable imidacloprid-degrading microbial community provided by this invention has a higher degradation efficiency and stronger adaptability in actual contaminated soils compared with the imidacloprid-degrading microbial community obtained by the conventional enrichment and domestication method.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for enriching and acclimating a highly adaptable imidacloprid-degrading bacterial community, characterized in that, Includes the following steps: (1) The soil contaminated with imidacloprid was placed in an inorganic salt liquid culture medium containing imidacloprid and cultured under constant temperature and shaking to obtain a primary suspension; (2) Centrifuge the primary suspension to separate the solid and liquid components, so that the bacterial community and non-dissolved soil components in the primary suspension enter the precipitate; add fresh inorganic salt liquid culture medium containing imidacloprid to the obtained precipitate and continue to culture at a constant temperature with shaking to obtain the secondary suspension; (3) Perform subculture according to the culture conditions in step (2), take the last generation suspension of the last subculture cycle, let it stand, and obtain the supernatant containing bacteria. (4) Centrifuge the bacterial supernatant obtained in step (3), wash the precipitate and resuspend it to obtain the highly adaptable imidacloprid-degrading bacterial community.

2. The method for enriching and acclimatizing highly adaptable imidacloprid-degrading bacterial communities as described in claim 1, characterized in that, In step (2), the centrifugal force of the centrifugation is 8000-10000g.

3. The method for enriching and acclimatizing highly adaptable imidacloprid-degrading bacterial communities as described in claim 1, characterized in that, In step (3), the concentration of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid is 100-500 mg / L; In step (3), the concentration of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid increases in a gradient with the number of generations.

4. The method for enriching and acclimatizing highly adaptable imidacloprid-degrading bacterial communities as described in claim 3, characterized in that, During the subculture process in step (3), the concentration gradient of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid increases by 100 mg / L.

5. The method for enriching and acclimatizing highly adaptable imidacloprid-degrading bacterial communities as described in claim 1, characterized in that, In step (1), the mass ratio of imidacloprid-contaminated soil to imidacloprid-containing inorganic salt liquid culture medium is 1:(5-10); the concentration of imidacloprid in the imidacloprid-containing inorganic salt liquid culture medium is 100 mg / L. In step (2), the concentration of imidacloprid in the inorganic salt liquid culture medium containing imidacloprid is 200 mg / L; In steps (1)-(3), the components of the inorganic salt liquid culture medium include 1g of K2HPO4, 1g of KH2PO4, 0.5g of (NH4)2SO4, 1g of KNO3, 0.5g of MgSO4·7H2O, 0.02g of CaCl2, 2 drops of 100g / L FeCl2 solution and 1L of distilled water; the pH of the inorganic salt liquid culture medium is 7.

6. The method for enriching and acclimatizing highly adaptable imidacloprid-degrading bacterial communities as described in claim 1, characterized in that, In step (1) and / or step (2), the temperature for isothermal shaking culture is 25-35℃; In step (1) and / or step (2), the isothermal shaking culture time is 7-14 days; In step (3), the settling time is 12-24 hours.

7. A highly adaptable imidacloprid-degrading bacterial community, characterized in that, The highly adaptable imidacloprid-degrading bacterial community was obtained by enrichment and domestication using the method described in any one of claims 1-6.

8. The application of the highly adaptable imidacloprid-degrading microbial community as described in claim 7 in the remediation of imidacloprid-contaminated soil.

9. The application as described in claim 8, characterized in that, The process includes the following steps: mixing a bacterial solution containing the highly adaptable imidacloprid-degrading bacteria with imidacloprid-contaminated soil.

10. The application as described in claim 9, characterized in that, It also includes the step of adding water and mixing it with the bacterial solution and the imidacloprid-contaminated soil; The mass ratio of the soil and water contaminated with imidacloprid was 1:(0.6-3); The ratio of the volume of the bacterial solution to the volume of the mixture of imidacloprid-contaminated soil and water is (0.01-0.10):1; The concentration of the bacterial solution is 1×10⁻⁶. 6 -1×10 8 CFU / L.

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