An estrogen-polluted soil plant-microorganism combined remediation and evaluation method based on rhizosphere microenvironment construction

Abstract

The application discloses a kind of based on rhizosphere microenvironment construction estrogen contaminated soil plant-microorganism combined repair and evaluation method, belong to environmental pollution bioremediation technical field.The method is by standardizing pretreatment to soil, control soil water content, and adopt solution dispersion mode to construct estrogen contaminated soil model;Quantitative inoculation is carried out to contaminated soil by the functional microorganism with estrogen degradation ability after washing treatment, and plants are planted to construct rhizosphere system of plant-microorganism combined repair;Through cultivation under controllable temperature and humidity and illumination conditions, in combination with dead bacteria control, plant-free control and other experimental systems, rhizosphere soil sample is collected, and estrogen residue and microbial community structure are synchronously detected and evaluated.The method has the advantages of operation specification, good repeatability, controllable environmental conditions, etc., and is suitable for experimental simulation, mechanism research and repair effect evaluation of plant-microorganism combined repair technology for estrogen contaminated soil.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollution bioremediation technology, and discloses a plant-microbe co-remediation and evaluation method for estrogen-polluted soil based on rhizosphere microenvironment construction. Technical Background With the development of industry and agriculture and the intensification of human activities, the accumulation of steroid estrogens such as estradiol (E2) in the soil environment is becoming increasingly serious, posing a potential threat to ecosystems and human health. Currently, remediation technologies for estrogen pollution in soil mainly include physical, chemical, and biological methods. Physical and chemical methods suffer from high costs and are prone to causing secondary pollution, while bioremediation technologies have attracted much attention due to their environmental friendliness and low cost.

[0002] Existing research primarily employs microbial degradation or phytoremediation methods for estrogen pollution remediation. Current technologies, such as CN105039301A, utilize immobilized microbial carriers to remediate estradiol-contaminated soil. The core of this method is to immobilize microbial communities using carrier materials (such as pyrophyllite, cottonseed cake, etc.) to improve their colonization rate and degradation efficiency in the soil. However, this method suffers from limitations due to the carrier dependence of microorganisms, and the degradation capacity of immobilized microorganisms is unstable in soil, easily affected by environmental conditions. Furthermore, its remediation system mainly relies on the single action of microorganisms, without involving the construction of the rhizosphere environment, or systematically studying the synergistic effects of plants and microorganisms, and it is difficult to reflect the impact of the rhizosphere microenvironment on the estrogen degradation process in actual soil. In addition, immobilized microorganisms are costly and may perform poorly in certain soil types. On the other hand, some technologies propose constructing plant-related functional microbial communities to improve plant growth or enhance soil health. However, these technologies are mostly aimed at increasing agricultural production or improving ecology. They do not establish standardized pollution simulation and evaluation processes for the pollution characteristics of estrogen-like endocrine disruptors, nor do they involve the simultaneous analysis of the dynamic residual changes of estrogen pollutants in the rhizosphere and the succession of microbial communities.

[0003] Therefore, single-microbial remediation is easily limited by the soil environment, and strains are difficult to colonize stably; single-phytoremediation has limited degradation efficiency and makes it difficult to distinguish between rhizosphere action and contributions from abiotic processes. For example, some bacterial strains have been shown to degrade E2 in liquid culture media, but their activity is inhibited in actual soil systems; some plants, such as alfalfa and ryegrass, have been shown to absorb or convert estrogen, but the remediation cycle is long and the efficiency is limited. Many co-remediation experiments are conducted in petri dishes or hydroponically, failing to realistically simulate the soil rhizosphere environment, and the operation process lacks fine-tuning of soil moisture, microbial inoculum size, and rhizosphere microenvironment control, resulting in poor reproducibility and low practical application value. In addition, existing methods for monitoring pollutant residues and microbial community dynamics during remediation rely on crude sampling methods that easily damage the rhizosphere microzone, affecting data accuracy and making it difficult to achieve simultaneous analysis of pollutant residues and microbial community changes. Plant-microbe co-remediation, on the other hand, can improve the microbial habitat and promote pollutant degradation through plant root exudates, and has significant advantages.

[0004] Therefore, there is an urgent need to develop a plant-microbe co-remediation and evaluation method for estrogen-contaminated soil based on rhizosphere microenvironment construction, for the standardization research of plant-microbe co-remediation systems. Summary of the Invention

[0005] This invention aims to provide a plant-microbe co-remediation and evaluation method for estrogen-contaminated soil based on rhizosphere microenvironment construction. This method enables the stable construction of estrogen-contaminated soil, quantitative inoculation of functional microorganisms, controllable cultivation of plant rhizosphere systems, and dynamic detection of pollutant residues and microbial community succession, thereby improving the reproducibility and comparability of experiments.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Step 1: Soil sample pretreatment Topsoil was collected at a depth of 0–10 cm. The collected soil was dried at a temperature of 40–80℃, then ground and sieved with a sieve aperture size ≤2 mm to obtain homogeneous soil.

[0007] Step 2: Soil Moisture Control Ultrapure water is added to the homogeneous soil to adjust the soil moisture content to 20%–30% of the maximum water holding capacity, so as to form a soil microenvironment suitable for rhizosphere microbial activation and plant germination.

[0008] Step 3: Construction of Estrogen Pollution Simulation An estrogen-based contaminant solution is added to moistened soil, and the contaminant is fully dispersed through continuous turning or mechanical stirring to construct estrogen-contaminated soil. The final concentration of the estrogen-based contaminant is 1–20 mg / kg, preferably 10 mg / kg. This concentration can simulate the conditions of moderately to highly contaminated sites and effectively assess remediation efficiency. The estrogen-based contaminant is one or more of estradiol (E2), estrone (E1), and ethinylestradiol (EE2).

[0009] Step 4: Preparation and quantitative inoculation of functional microorganisms Functional microorganisms capable of degrading estrogen were cultured in a suitable medium until the logarithmic growth phase. The bacterial cells were collected by centrifugation and washed 2–5 times with PBS buffer to remove residual culture medium. The bacterial cells were then resuspended in an inorganic salt medium or a carbon-free solution to prepare a bacterial suspension. This suspension was inoculated into estrogen-contaminated soil, achieving an inoculum concentration of OD0.05. 600 An inoculum concentration of 0.05 g / g soil is used to ensure effective colonization of the bacteria in the rhizosphere. An inactivated bacterial treatment was used as a control to assess abiotic effects.

[0010] Step 5: Rhizosphere System Construction and Plant Cultivation The inoculated contaminated soil is divided into seedling containers or potted containers, preferably seedling trays, with each container containing 10g of soil. Revived plant seeds are sown on the soil surface or at a depth of 0.5–2cm, with 3 seeds sown per container to form a dense root system, enhance the rhizosphere effect, and construct a plant-microbe co-remediation rhizosphere system. The plants are preferably leguminous plants, grasses, or pollution-tolerant plants, with alfalfa and ryegrass being more preferred.

[0011] Step 6: Control of Rhizosphere Culture Conditions The constructed rhizosphere system was cultured in a constant temperature and humidity environment. The culture conditions included: temperature 30℃, relative humidity 75%, a 12-hour light / dark cycle, and a culture period of 30 days. During the culture period, soil moisture content was maintained by quantitative watering, with watering frequency either daily or every other day.

[0012] Step 7: Compare system settings Set up at least one control system: (1) Dead bacteria control group: Inactivated bacteria were added to contaminated soil; (2) Aseptic control group: Contaminated soil was not inoculated with functional microorganisms; (3) Control group without plants: soil contaminated with functional microorganisms was inoculated but no plants were planted; The control system was used to differentiate between plant rhizosphere effects, microbial degradation effects, and abiotic loss effects.

[0013] Step 8: Dynamic Sampling and Detection Evaluation Soil samples were collected in a time series during the cultivation process, with sampling points including days 0, 3, 6, 10, 15, and 30 after the start of cultivation. Samples were collected for: (1) Determine the residual concentration of estrogen-like pollutants in the soil; (2) Extract soil microbial DNA and perform community structure analysis; (3) Assess the colonization of functional bacteria in the rhizosphere and the pattern of community succession.

[0014] Each treatment should have ≥3 independent replicates, preferably 5 independent replicates, and each sampling time point should have ≥2 parallel samples, preferably 3 parallel samples.

[0015] Compared with the prior art, the present invention has the following advantages: 1) Improve the stability and consistency of estrogen-contaminated soil construction through uniform pollutant dispersion and quantitative inoculation strategies; 2) Achieve controllable cultivation of the rhizosphere microenvironment and improve experimental reproducibility through rhizosphere moisture regulation, temperature and humidity control, and photoperiod control; 3) Effectively distinguish between plant effects, microbial effects, and abiotic losses by setting up systems such as dead bacteria controls and plant-free controls; 4) Achieve simultaneous analysis of pollutant residue changes and microbial community succession through time-series dynamic sampling; 5) Applicable to the evaluation of remediation effects of different estrogen pollutants, different plant species, and different functional microbial agents, and can be promoted as a standardized rhizosphere experimental technology system. Attached Figure Description

[0016] Figure 1 This invention relates to the degradation curves of estradiol in soil by the alfalfa-strain C1 combined system (Plant+C1), alfalfa (Plant), and C1 in the experiment of remediating estradiol-contaminated soil by alfalfa-strain C1.

[0017] Figure 2 This invention relates to the experiment of using alfalfa-strain C1 to remediate estradiol-contaminated soil, and to the degradation curves of alfalfa-strain C1 combined system, alfalfa, and C1 on estrone, a metabolite of estradiol in soil.

[0018] Figure 3 The top ten bacterial taxa with the highest relative abundance at the phylum level in soil samples during the culture period were the alfalfa assorted strain C1 (C1 group) and the uninoculated strain C1 (CK group) in Example 1.

[0019] Figure 4 The two groups, alfalfa assorted strain C1 (C1 group) and uninoculated strain C1 (CK group), belonged to the top ten bacterial taxa in terms of relative abundance in soil samples during the culture period in Example 1. Detailed Implementation

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] The embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the invention. For those skilled in the art, other equivalent implementation methods can be obtained based on the content of this application without creative effort, and all such methods should be considered to fall within the protection scope of the present invention.

[0022] Unless otherwise specified, the raw materials used in the following embodiments are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0023] Example 1 An experimental method for the joint remediation of estrogen-contaminated soil using a plant-microorganism approach, the specific steps of which are as follows: 1. Fresh topsoil (0-10 cm) was collected from the green area of ​​Shanghai Jiao Tong University. After air drying, it was ground and passed through a 2 mm sieve to remove impurities. The sieved soil was then mixed with deionized water to adjust the moisture content to 25% (approximately 28% field capacity). An appropriate amount of soil was weighed and E2 stock solution was added. The mixture was stirred thoroughly to ensure uniform contamination, resulting in contaminated soil with an E2 concentration of 10 mg / kg.

[0024] 2. Inoculate E2-degrading strain C1 into LB liquid medium and incubate overnight at 30°C with shaking at 180 rpm. Collect the bacterial cells by centrifugation at 8000 rpm for 5 min, resuspend in PBS buffer and wash three times, and finally resuspend in ISM basal salt medium to adjust OD. 600 To an appropriate value.

[0025] 3. Place 10 g of contaminated soil in a seedling tray and inoculate with C1 bacterial suspension to achieve a bacterial cell density of 0.05 OD per gram of soil. 600 Mix lightly. Use the treatment with inactivated bacteria as a blank control.

[0026] 4. Sow 3 surface-sterilized and rehydrated alfalfa seeds evenly in the top 0.5–2 cm of soil in each seedling tray. Place the seedling trays in an artificial climate chamber, set at 30℃, 75% humidity, and a 12h / 12h light cycle, and cultivate for 30 days. Replenish the water lost through evaporation daily to maintain a constant weight.

[0027] 5. Samples were collected on days 0, 3, 6, 10, 15, and 30, with three parallel samples taken at each time point for each treatment. The plants were carefully removed, and the rhizosphere soil was collected for E2 residue detection.

[0028] The results showed that in estradiol-contaminated soil, the plant-microbe co-remediation system constructed from functional strain C1 and alfalfa exhibited higher removal efficiency for both estradiol and its metabolite estrone (E1). Figure 1 As shown, for estradiol, the removal rate of the combined remediation system reached 94.5% on day 3 of cultivation, significantly higher than that of the alfalfa-only treatment group (62.2%) and the functional strain-only treatment group (92.5%). Within 15 days of cultivation, both the combined remediation system and the functional strain-only treatment group achieved complete removal of estradiol. However, the removal half-life of estradiol in the combined remediation system was shortened to 2.48 days, lower than the 4.62 days of the phytoremediation system alone and the 2.77 days of the microbial remediation system alone, indicating that the synergistic effect of plants and microorganisms can accelerate the removal rate of estradiol.

[0029] In the removal of estrone, an intermediate product of estradiol degradation, plant-microbe co-remediation systems show a more significant advantage. For example... Figure 2 As shown, after 30 days of cultivation, the removal rate of estrone by the co-remediation system was approximately 60%, significantly higher than the approximately 9% in the alfalfa-only treatment group and the approximately 43% in the functional bacterial strain-only treatment group. These results indicate that the plant-microbe co-remediation system can effectively remove estradiol and its metabolite estrone simultaneously, which is beneficial in reducing the accumulation and migration risk of estrogenic pollutants in the soil environment.

[0030] Example 2 To verify the effect of the introduced functional strains on the original strains in the alfalfa rhizosphere soil while degrading estrogen pollutants, total soil microbial DNA was extracted from parallel samples collected in Example 1 at days 0, 6, 15, and 30, along with the uninoculated strain C1 (CK group), and 16S rRNA gene sequencing was performed to analyze changes in community composition.

[0031] Microbial diversity analysis showed that, at the phylum level, such as Figure 3 The results showed that the rhizosphere soil of alfalfa was consistently dominated by Proteobacteria ( ). Proteobacteria ) and acidobacteria ( Acidobacteria Actinobacteria (C1) were the dominant phylum, indicating that the overall bacterial community structure of the rhizosphere soil did not change drastically after the introduction of functional strain C1, and the original dominant phyla remained stable, thus avoiding the rhizosphere microecological imbalance caused by the introduction of exogenous strains. Compared with the control group without functional strain inoculation, the treatment group inoculated with functional strain C1 had a higher proportion of Actinobacteria (C1). ActinobacteriaThe presence of a phased enrichment during the mid-stage of cultivation, followed by a gradual decline and stabilization of its relative abundance in the later stages of cultivation, indicates that the functional strains introduced in this invention can participate in the construction of the rhizosphere microbial community for a certain period of time, but will not cause a long-term or irreversible monopoly of dominance, which is conducive to maintaining the dynamic balance of the rhizosphere soil microbial community.

[0032] like Figure 4 Furthermore, at the genus level, it is demonstrated that the method of this invention does not lead to the complete disappearance of the original dominant bacterial genera, but rather optimizes the community structure by adjusting their relative abundance. For example, Altererythrobacter genus and Pseudomonas The genera showed a gradual decline in both treatment groups, indicating that estrogen contamination conditions exerted a certain selective pressure on this type of bacteria, rather than the introduction of functional strains directly causing their extinction. Pseudoxanthomonas The abundance change was more pronounced after inoculation with the functional strain, reflecting that the functional strain SJTC1 participated in the microbial competition process in the rhizosphere soil, but did not disrupt the overall community structure. Furthermore, the results of Example 2 showed that in the treatment group inoculated with the functional strain C1, Brevundimonas The genera showed significant enrichment during the mid-stage of cultivation, and this enrichment was a phased change, indicating that the rhizosphere system constructed by the method of this invention can induce the synergistic participation of potential functional bacterial genera, thereby facilitating the continuous transformation of estrogen pollutants and their intermediate products.

[0033] In summary, the results of Example 2 further demonstrate that the plant-microbe co-remediation method of the present invention can effectively introduce exogenous estrogen-degrading strains while gently regulating the original microbial community of alfalfa rhizosphere soil. It not only does not damage the rhizosphere microecological structure, but also promotes the phased enrichment and synergistic succession of functionally related microbial communities, providing good rhizosphere microbial environment support for the safe, stable and sustainable remediation of estrogen-contaminated soil.

[0034] Example 3 The plant-microbe combined remediation rhizosphere experimental evaluation method described in this invention was used to analyze and evaluate the effect of functional strain C1 combined with alfalfa in remediating estradiol (E2) contaminated soil.

[0035] The results show that the plant-microbe co-remediation rhizosphere system constructed using the method of this invention can form a stable rhizosphere microenvironment under estrogen pollution conditions, allowing for simultaneous monitoring and evaluation of both the pollutant removal process and changes in the rhizosphere microbial community. In this rhizosphere system, the removal efficiency of estradiol and its metabolite estrone was significantly higher than that of plant-based or microbial-based remediation systems alone, indicating that the rhizosphere microenvironment constructed using the method of this invention can effectively enhance the synergistic remediation effect between plants and microorganisms.

[0036] Further analysis of rhizosphere soil using the method of this invention, including zoned sampling and microbial community structure analysis, revealed that the overall structure of the rhizosphere soil bacterial community remained relatively stable after the introduction of functional microorganisms, with no drastic changes in the original dominant bacterial groups. This indicates that the rhizosphere microenvironment constructed by this invention possesses good ecological stability. Simultaneously, some bacteria related to estrogen conversion showed phased enrichment during the remediation process and gradually stabilized in the later stages of cultivation, reflecting that this method can achieve dynamic regulation and process evaluation of the rhizosphere microbial community.

[0037] In summary, the rhizosphere microenvironment constructed by the method of this invention can simultaneously achieve stable operation and multi-index evaluation of the plant-microbe co-remediation process in estrogen-contaminated soil. This not only verifies the feasibility and reproducibility of the rhizosphere microenvironment construction method, but also proves its applicability in co-remediation research and effect evaluation of estrogen-contaminated soil.

[0038] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for plant-microbe co-remediation and evaluation of estrogen-contaminated soil based on rhizosphere microenvironment construction, characterized in that, Includes the following steps: A) Collect topsoil and dry, grind, and sieve it to obtain homogeneous soil; B) Add water to the homogeneous soil to adjust the moisture content to 20%–30% of the maximum water holding capacity; C) Add estrogen-based pollutant solution to the conditioned soil and mix well to construct estrogen-contaminated soil with a pollutant concentration of 10 mg / kg; D) Cultivate functional microorganisms with estrogen-degrading capabilities, collect bacterial cells, wash with buffer solution, resuspend, and inoculate into the estrogen-contaminated soil to an inoculum size of OD. 600 0.05 g / gram of soil; E) Place the inoculated contaminated soil into seedling containers or potted containers and plant revived plant seeds to construct a plant-microbe joint remediation rhizosphere system; F) Cultivate under constant temperature and humidity conditions for 30 days and replenish water to maintain stable moisture content; G) Collect rhizosphere soil samples according to time series, determine estrogen residual concentration and analyze changes in microbial community structure to evaluate the effect of plant-microbe joint remediation.

2. The method according to claim 1, characterized in that, In step A, the surface soil sampling depth is 0–10 cm.

3. The method according to claim 1, characterized in that, The sieving process described in step A uses a sieve aperture size ≤ 2 mm.

4. The method according to claim 1, characterized in that, The estrogen-like pollutant mentioned in step C is one or more of estrone, estradiol, and ethinylestradiol.

5. The method according to claim 1, characterized in that, The plant mentioned in step E is a legume or a grass.

6. The method according to claim 1, characterized in that, In step F, the culture temperature is 30℃, the relative humidity is 75%, the light cycle is 12 hours, and the light / dark cycle is alternating for 30 days.

7. The method according to claim 1, characterized in that, A dead bacteria control group and / or a plant-free control group were set up to distinguish between the microbial degradation effect and the plant rhizosphere effect.

8. The method according to claim 1, characterized in that, The sampling time points for collecting rhizosphere soil samples according to the time series in step G include days 0, 3, 6, 10, 15, and 30 after the start of cultivation.

9. The method according to claim 1, characterized in that, The method for analyzing changes in microbial community structure in step G is as follows: extract total microbial DNA from the soil sample and perform high-throughput sequencing to analyze changes in microbial community structure.

10. The application of the method according to any one of claims 1 to 9 in the screening of remediation technologies for estrogen-contaminated soil, the study of rhizosphere microbial community succession, or the analysis of plant-microbe joint remediation mechanisms.

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