Hydrogel bacteria agent for synergistically removing plasticizers and heavy metals in water and preparation method thereof

Abstract

The application discloses a hydrogel bacterial agent for synergistically removing plasticizers and heavy metals in water and a preparation method thereof. 2+ The preparation method comprises the following steps: inducing and culturing functional strains by a concentration gradient, compounding the functional strains to obtain a composite bacterial agent, synchronously synthesizing a lignin hydrogel and loading ferrous sulfide nanoparticles to obtain a composite carrier, and finally coupling the composite bacterial agent and the composite carrier and drying to obtain the hydrogel bacterial agent. The hydrogel bacterial agent breaks through the limitation of the prior art that bacterial agents and carriers are prepared separately and have poor synergy, realizes efficient and synchronous removal of plasticizers and heavy metal Cd, and solves the technical problems of low activity, easy loss and poor treatment effect of the prior art bacterial agents in a composite pollution environment.

Description

Technical Field

[0001] This invention relates to the field of hydrogel bacterial agent preparation technology, specifically to a hydrogel bacterial agent and its preparation method for synergistically removing plasticizers and heavy metals from water. Background Technology

[0002] Water resources are a primary resource for life on Earth, and access to clean water is crucial for humanity and ecosystems. However, in recent decades, continuous population growth, rapid industrialization, urbanization, and the excessive use of natural resources have negatively impacted water quality, leading to the generation and discharge of increasing amounts of toxic pollutants into water bodies. Among these, the combined pollution from phthalate (PAE) plasticizers and the heavy metal cd is particularly prominent, and the lack of effective control and remediation strategies seriously threatens water security. Therefore, there is an urgent need to develop a water purification strategy that can efficiently improve water bodies with combined pollution.

[0003] Bioimmobilization technology has become a research hotspot in the field of water pollution control because it can effectively prevent the loss of biological phases. Hydrogels are widely used as carriers for microbial immobilization due to their advantages of low cost and high biocompatibility. Moreover, their excellent mechanical strength properties give immobilized microbial agents significant advantages over free microorganisms, such as high biomass, reusability, strong resistance to toxicity, good genetic stability, and no cell erosion.

[0004] However, existing technologies still have many problems that need to be solved: First, for the combined pollution of PAEs and Cd, there is a lack of synergistic treatment technologies that can simultaneously achieve the degradation of organic pollutants and the passivation of heavy metals, and individual treatment methods are difficult to achieve ideal purification effects; Second, free functional microorganisms are easily lost in water bodies and have weak anti-pollution capabilities, which limits their degradation efficiency for PAEs; Third, single ferrous sulfide nanoparticles are prone to agglomeration, which reduces their adsorption activity for Cd and poses potential environmental risks, while single hydrogels have limited removal capacity for combined pollutants. Summary of the Invention

[0005] The problems existing in the prior art are: the performance of existing hydrogel bacterial agents in the remediation of complex pollution is insufficient, free functional microorganisms are easily lost in water and have weak anti-pollution ability. In order to address the above technical problems, the present invention provides a hydrogel bacterial agent for the synergistic removal of plasticizers and heavy metals in water and its preparation method.

[0006] The technical solution of this invention is: a hydrogel microbial agent for synergistic removal of plasticizers and heavy metals from water, wherein the hydrogel microbial agent is composed of a composite carrier and a composite microbial agent loaded on the composite carrier; the composite carrier is a lignin hydrogel loaded with ferrous sulfide nanoparticles; the composite microbial agent is formulated from functional strains that are cadmium-resistant and have phthalic acid ester (PAE) degradation function; wherein the plasticizer is one or more of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DnOP).

[0007] Note: Lignin hydrogels loaded with ferrous sulfide nanoparticles possess dual advantages. The high porosity and abundant hydrophilic functional groups of lignin hydrogels, such as carboxyl and hydroxyl groups, can enhance the resistance to Cd. 2+ Through complexation and adsorption, ferrous sulfide nanoparticles form sulfide precipitates through physical adsorption and chemical reactions, further enhancing the passivation efficiency of heavy metals. Furthermore, the stable structure of the hydrogel effectively prevents nanoparticle aggregation and loss, reducing environmental risks. At the microbial level, the composite functional strains acclimated to cadmium tolerance not only tolerate Cd... 2+ Under stress, it can also efficiently degrade various plasticizers, including short / long side-chain PAEs such as DMP, DEP, and DBP, solving the problem of inhibited activity of single strains in complex pollution environments. When the two are combined at an optimized solid-liquid ratio of 1g:20~100ml, the carrier provides a stable microenvironment for the growth of the strain, reducing the loss of bacterial cells through erosion. While degrading plasticizers, the strain reduces the combined toxicity of pollutants, forming a synergistic effect of passivating heavy metals and degrading organic matter, which greatly improves the purification efficiency of complex pollutant wastewater. Moreover, the entire system has widely available and low-cost raw materials, no secondary pollution, and is suitable for engineering application scenarios.

[0008] Furthermore, the functional strains include *Patulibacter brassicae*, *Glutamicibater* sp., *Rhodococcus* sp., and *Acinetobacter pittii*.

[0009] Note: Targeted screening was conducted to identify heterogeneous functional strains exhibiting cadmium tolerance, PAE degradation ability, and colonization ability in complex environments. These strains showed no antagonistic effects and formed functional complementarities. *Pseudomonas* and *Glutamicinus belladonna* strains were used to enhance Cd resistance. 2+Stress tolerance was improved; *Rhodococcus* species enhanced the degradation efficiency of broad-spectrum PAEs, while *Acinetobacter* species optimized the adhesion and colonization of bacteria on the surface of hydrogel carriers, solving the technical challenge of single strains being unable to simultaneously adapt to complex polluted environments. This combination of strains enabled the compound bacterial agent to withstand Cd... 2+ In the coexistence system with PAEs, it maintains high activity and achieves a synergistic effect of heavy metal passivation and organic matter degradation, which greatly improves the adaptability of the bacterial agent to the treatment of complex polluted wastewater.

[0010] Furthermore, the functional strains include *Glutamicibater* sp., *Bacillus* sp., *Patulibacter brassicae*, and *Acinetobacter pittii*.

[0011] Description: This invention employs a targeted, stress-resistant, and carrier-adaptive three-dimensional bacterial strain combination system, distinct from existing technologies that use strain mixtures without clear functional guidance. Bacillus species enhance the environmental stress resistance and stability of the bacterial agent in complex aquatic environments; Corynebacterium brassicae fills the technological gap in the efficient degradation of long-side-chain PAEs; and Acinetobacter pylori further improves the binding strength between the bacterial cells and the lignin hydrogel carrier. This strain combination not only tolerates Cd... 2+ Under stress, it can effectively degrade both short and long side-chain PAEs, and the bacteria are not easily lost on the carrier, which significantly extends the actual application time of the bacterial agent.

[0012] A method for preparing a hydrogel bacterial agent for the synergistic removal of plasticizers and heavy metals from water includes the following steps: S1. Preparation of compound microbial inoculants: Take equal volumes of each functional bacteria and expose them to 0-10 mg / L Cd. 2+ After concentration gradient induction culture, the culture was activated in liquid LB medium at 25-35℃ and 120-180rpm for 22-30 h. The culture was then resuspended in inorganic salt medium 2-4 times and the OD600 was adjusted to 1.0 to obtain bacterial suspensions of each functional strain. The bacterial suspensions of each functional strain were mixed in an equal volume ratio of 1:1:1 to prepare a compound microbial agent. S2. Preparation of composite carrier: Acrylamide, maleic anhydride, N,N'-methylenebisacrylamide, and sodium lignosulfonate were dissolved in pure water to obtain a mixed system. In this mixed system, the mass percentages of acrylamide, maleic anhydride, N,N'-methylenebisacrylamide, and sodium lignosulfonate were 10-14%, 4-6%, 0.3-0.5%, and 1-1.5%, respectively. The mixed system was stirred at room temperature for 2 hours until completely dissolved. The pH of the solution was adjusted to 5.5-6.5 with NaOH. Then, 0.8-1.2% potassium persulfate and 0.6-1.0% N,N,N',N'-tetramethylenediamine were added, and the mixture was stirred evenly at room temperature before being poured into a 1 cm thick container. 3 The lignin hydrogel was obtained by gelling in a mold for 0.8-1.2 h, washing with pure water 2-4 times, and freeze-drying. The lignin hydrogel was then immersed in ferrous sulfate solution and sodium sulfide solution in sequence to load ferrous sulfide nanoparticles. After washing and freeze-drying, a composite carrier was obtained. S3. Preparation of hydrogel bacterial agent: The composite carrier described in S2 and the composite microbial agent described in S1 are mixed at a solid-liquid ratio of 1g:20~100ml, and cultured under shaking conditions at a speed of 100~200 rpm, a temperature of 25~35℃ and in the dark for 24~30 h. After sterile filtration and rinsing, the mixture is dried at a temperature of 22~28℃ to obtain the hydrogel microbial agent.

[0013] Description: This invention constructs an integrated preparation process encompassing targeted acclimatization of microbial agents, functional modification of carriers, and coupled immobilization of microorganisms. It overcomes the limitations of existing technologies where carrier preparation and microbial cultivation are separated and lack synergy. The process precisely matches the parameters for cadmium tolerance acclimatization of the strains with the processes of carrier synthesis and loading. Furthermore, it quantitatively optimizes the raw material ratios and reaction conditions for each step, forming a standardized process that can be replicated on a large scale. This preparation method features a closed-loop process logic and controllable parameters, ensuring both the structural stability and heavy metal passivation capability of the composite carrier while maximizing the preservation of the degradation activity of the composite microbial agent. The microbial carrier is firmly bound, and the resulting immobilized microbial agent possesses both highly efficient multi-contamination treatment capabilities and process operability, making it suitable for engineering applications.

[0014] Furthermore, the induction culture method described in S1 is as follows: a. Preparation of composite inducer: Biochar powder, humic acid, monobutyl phthalate, and betaine were mixed in a mass ratio of 1:0.3~0.5:0.2:0.1 to obtain a premix. Sterile deionized water at a mass ratio of 8~12 times that of the premix was added, and the mixture was ultrasonically dispersed for 30~50 min. The mixture was then centrifuged at 3~5℃ and 8000 rpm for 10 min. The supernatant was filtered through a 0.22μm filter membrane to obtain the composite inducer. The particle size of the biochar powder was 50~100 nm. b. Pre-acclimatization culture: Add Cd to liquid LB medium at a final concentration of 0.3 mg / L. 2+ The compound inducer with 5% volume fraction was added, the pH was adjusted to 7.0, and after sterilization, the functional strain was inoculated. The strain was cultured with shaking at 30-35℃ and 150-180 rpm for 72 hours. 0.5-1.5% volume fraction of the compound inducer was added every 24 hours. After the culture was completed, the strain was centrifuged and washed to obtain the pre-adapted strain. c. Two-way gradient acclimatization: Prepare 5 groups of culture media, including Cd 2+ The concentrations were 0.8, 2.0, 4.0, 7.0, and 10.0 mg / L, corresponding to PAEs (DBP) concentrations of 2, 5, 8, 12, and 15 mg / L, respectively. All five culture media contained 3% (v / v) of the compound inducer. The pre-adapted strains were inoculated into each gradient culture medium sequentially. The pre-adapted strains were first inoculated into the first group of culture medium and cultured with shaking at 25-35℃ and 120-180 rpm for 50-70 h. After centrifugation and washing, they were transferred to the next group. Under the same shaking culture conditions as the first group, they were centrifuged, washed, and transferred to the next group. This process was repeated until the fifth gradient culture medium was completed. After all gradient cultures were completed, the bidirectional acclimatized strains were obtained. d. Enhanced co-culture: The bidirectional acclimatized strain was inoculated into a solution containing 4-6 mg / L Cd. 2+ The mixture of PAEs at a concentration of 10-18 mg / L and a composite inducer at a volume fraction of 1.5-2.5% was cultured in a medium at a temperature of 25-35℃ and a rotation speed of 120-180 rpm for 44-52 h. After centrifugation and washing, the mixture was ready for use. The mixed PAEs consisted of DMP, DEP and DBP in equal mass ratios.

[0015] Note: Design of composite inducers to assist Cd 2+ - PAEs bidirectional gradient acclimatization - This stepwise induction process, enhanced by combined culture with mixed PAEs, differs from existing technologies that rely on single heavy metal gradient acclimatization, lack inducer assistance, or use only single PAEs. The multi-component synergistic design of the composite inducer achieves a triple effect of toxicity buffering, degradative enzyme induction, and cell protection. This induction culture method can significantly enhance the Cd content of the strain. 2+ The strain's tolerance threshold and broad-spectrum PAE degradation ability allow it to withstand Cd224 degradation. 2+ It can maintain high biological activity in a complex pollution environment where it coexists with PAEs, which solves the problem of suppressed activity and low degradation efficiency of existing strains under complex stress.

[0016] Furthermore, the method for loading ferrous sulfide nanoparticles in S2 is as follows: immersing the lignin hydrogel in an immersion solution with a concentration of 0.4~0.6 mol·L⁻¹-1 The sample was immersed in a ferrous sulfate solution under nitrogen atmosphere for 10–14 h, then washed 2–4 times with pure water and freeze-dried; then immersed in a solution with a concentration of 0.4–0.6 mol·L⁻¹. -1 The lignin hydrogel loaded with ferrous sulfide nanoparticles was soaked in sodium sulfide solution under nitrogen atmosphere for 10-14 h, then washed with pure water 2-4 times and freeze-dried to obtain the lignin hydrogel.

[0017] Description: This invention employs a two-step, stepwise soaking and freeze-drying process under nitrogen protection to load ferrous sulfide nanoparticles. This overcomes the limitations of existing technologies where direct mixing and loading easily leads to particle oxidation, agglomeration, or uneven loading. Nitrogen protection prevents the oxidation of ferrous ions, and the two-step soaking process achieves uniform in-situ generation and loading of nanoparticles within the hydrogel pores. This loading method ensures the dispersion and binding strength of ferrous sulfide nanoparticles on the lignin hydrogel, preventing particle loss, while preserving the pore structure and functional group activity of the hydrogel, significantly improving the composite carrier's ability to support Cd. 2+ It has high adsorption and passivation efficiency, and the loading process is gentle and does not damage the structural stability of the hydrogel.

[0018] Furthermore, the average particle size of the ferrous sulfide nanoparticles in the ferrous sulfate solution is 400 nm.

[0019] Note: The average particle size of the ferrous sulfide nanoparticles is precisely defined as 400 nm. Unlike existing technologies that do not explicitly define the particle size or blindly pursue small particle sizes, this particle size balances the specific surface area and structural stability of the nanoparticles, making it the optimal particle size for the pore structure of lignin hydrogels. The 400 nm ferrous sulfide nanoparticles possess sufficient surface active sites, enabling efficient adsorption and precipitation of Cd. 2+ It avoids particle agglomeration and clogging of hydrogel pores due to excessively small particle size, while maintaining stable loading within the hydrogel pores to prevent particle loss in practical applications and ensure the carrier's resistance to Cd. 2+ Durability of the passivation effect.

[0020] Furthermore, the Cd described in S1 2+ The culture medium for concentration gradient induction was liquid LB medium, Cd 2+ The concentration gradients included 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, and 7.5 mg / L.

[0021] Note: Based on the growth characteristics of functional strains, a low-concentration starting solution with stepwise increases of Cd was designed. 2+This method employs a concentration gradient and uses nutrient-balanced liquid LB medium as the acclimatization substrate. Unlike existing technologies that involve large concentration gradients that can lead to acute toxicity and bacterial death, this approach achieves a gentle and gradual induction of cadmium tolerance in the strains. The beneficial effect is that this combination of concentration gradient and culture medium design improves the success rate of strain acclimatization, allowing the strains to gradually adapt to Cd. 2+ Stress is avoided to prevent high concentrations of heavy metals from directly causing strain inactivation. At the same time, liquid LB medium can provide sufficient nutrients for strain growth, ensuring the biomass and activity of the strain during the acclimatization process.

[0022] Furthermore, the drying conditions described in S3 are as follows: after filtering and rinsing under sterile conditions, the product is transferred to an oven and dried at 25°C until the moisture content is ≤8%.

[0023] Description: The drying temperature is precisely limited to 25℃ and the moisture content is dried to ≤8%, overcoming the process defects of existing technologies such as high-temperature drying or lack of clear limits on moisture content. Low-temperature drying avoids bacterial cell inactivation, and precise moisture content control achieves a balance between the storage stability and structural integrity of the bacterial agent. This drying condition can remove excess moisture from the bacterial agent and prevent mold growth during storage, while maximizing the preservation of the biological activity of the bacterial cells and the porous structure of the hydrogel carrier. The dried immobilized bacterial agent is easy to store and transport, and can be quickly rehydrated and activated in practical applications to ensure the pollution treatment effect.

[0024] The beneficial effects of this invention are: (1) This invention provides a complete process for loading ferrous sulfide nanoparticles onto lignin hydrogels, including hydrogel preparation, mixing, and preparation of ferrous sulfide nanoparticles and composite materials. This process ensures that the resulting hydrogel has uniform color, stable structure, controllable shape, and strong mechanical strength and environmental stability. Preferably, the ratio of acrylamide to sodium lignin sulfonate monomers is 6.7:1, which allows the lignin hydrogel to effectively fix and disperse the ferrous sulfide nanoparticles using molecular chains, while preventing the aggregation and shedding of the ferrous sulfide nanoparticles, thus reducing environmental risks during application.

[0025] (2) This invention innovatively employs lignin hydrogel loaded with ferrous sulfide nanoparticles as an immobilization carrier. Ferrous sulfide nanoparticles, with their small particle size, large specific surface area, and abundant surface active sites, are effective Cd adsorbents. The hydrogel possesses a large specific surface area and high porosity, containing numerous hydrophilic groups such as carboxyl and hydroxyl groups, which can effectively adsorb heavy metals. Furthermore, incorporating ferrous sulfide into the hydrogel network structure can physically inhibit ferrous sulfide aggregation, maintain its high activity, and avoid potential toxicity and environmental risks. Moreover, the lignin hydrogel exhibits high mechanical strength and resistance to degradation, can adsorb heavy metals through its hydrophilic functional groups, namely carboxyl and hydroxyl groups, and possesses biocompatibility. It can also serve as an immobilization carrier for functional microbial communities, significantly improving the remediation rate of water bodies contaminated with plasticizers and heavy metal Cd. Attached Figure Description

[0026] Figure 1 This refers to the degradation efficiency of the immobilized bacterial agent on different plasticizers after 1, 3, 5, and 7 days (d) of cultivation in Example 2. Figure 2 This refers to the degradation efficiency of the immobilized bacterial agent on different plasticizers after 1, 3, 5, and 7 days (d) of cultivation in Example 3. Figure 3 This refers to the degradation efficiency of the immobilized bacterial agent on different plasticizers after 1, 3, 5, and 7 days (d) of cultivation in Example 4. Figure 4 This is Example 2, comparing the effects of microbial inoculant carriers on Cd in Comparative Examples 1 and 2. 2+ Comparison of saturated adsorption capacity; Figure 5 The lignin hydrogel-supported FeS nanoparticle (FeS@LH) composite material prepared in Example 2 adsorbs Cd. 2+ XPS spectra before and after, where: (a) Cd adsorption by FeS@LH 2+ XPS spectra before and after; (b) shows the Fe 2p orbital in the adsorption of Cd. 2+ The fine spectrum before and after; (c) represents the S 2p orbital in the adsorption of Cd. 2+ The fine spectrum before and after; (d) represents the O 1s orbital in the adsorption of Cd 2+ The fine spectrum before and after; (e) represents the C 1s orbital in the adsorption of Cd 2+ The fine spectrum before and after; (f) shows the adsorption of Cd by FeS@LH. 2+ The newly emerging fine spectrum of Cd 3d orbitals; Figure 6 The FeS@LH composite material in Example 2 adsorbs Cd 2+ Isothermal adsorption model curves before and after; Figure 7 The FeS@LH composite material in Example 2 adsorbs Cd 2+ Adsorption kinetics model curves before and after. Detailed Implementation

[0027] The reagents and bacterial strains used in this invention were sourced as follows: Plasticizers (PAEs) included dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DnOP), all analytically pure and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; anhydrous CdCl2, acrylamide, maleic anhydride, sodium lignosulfonate, N,N'-methylenebisacrylamide, N,N,N',N'-tetramethylenediamine, sodium hydroxide, potassium persulfate, ferrous sulfate heptahydrate, and sodium sulfide, all analytically pure and purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; *Patulibacter brassicae* was also present. The following bacteria were purchased from the China Industrial Microbial Culture Collection Center (CICC24108): * *Glutamicibaters* sp. (2022- ...

[0028] Example 1: This example is a hydrogel microbial agent for the synergistic removal of plasticizers and heavy metal Cd from wastewater. The hydrogel microbial agent consists of a composite carrier and a composite microbial agent loaded on the composite carrier. The composite carrier is a lignin hydrogel loaded with ferrous sulfide nanoparticles. The composite microbial agent 9 is composed of functional strains that are cadmium-resistant and have the function of degrading phthalic acid esters (PAEs). The functional strains include Patulibacter brassicae, Glutamicibater sp., Rhodococcus sp., and Acinetobacter pittii.

[0029] Example 2: This example describes the preparation method of the hydrogel bacterial agent from Example 1, including the following steps: S1. Preparation of compound microbial inoculants: Functional bacterial strains were collected: *Patulibacter brassicae*, *Glutamicibater* sp., *Rhodococcus* sp., and *Acinetobacter pittii*, which were subjected to Cd... 2+ After concentration gradient induction culture, the culture was activated in liquid LB medium at 30℃ and 150rpm for 24 h. The culture was then resuspended three times in inorganic salt medium and the OD600 was adjusted to 1.0 to obtain bacterial suspensions of each functional strain. The bacterial suspensions of each functional strain were mixed in an equal volume ratio of 1:1:1 to prepare a compound microbial agent. The Cd 2+ The culture medium for concentration gradient induction was liquid LB medium, Cd 2+ The concentration gradients included 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, and 7.5 mg / L; the induction culture method was as follows: each functional strain was inoculated into Cd... 2+ The culture was carried out in 0.5 mg / L liquid LB medium at 30°C and 150 rpm with shaking for 48 h. After the culture was completed, the bacterial culture was transferred to Cd... 2+ The culture was carried out in 1.0 mg / L liquid LB medium under the same conditions for 48 h. This process was repeated sequentially to achieve concentration gradients of 2.0 mg / L, 5.0 mg / L, and 7.5 mg / L. After each gradient was completed, bacterial culture was used for the next gradient. After completing all concentration gradients, the Cd-treated culture was obtained. 2+ Functional strains induced by concentration gradient culture; S2. Preparation of composite carrier: Dissolve 15.00 g of acrylamide, 6.65 g of maleic anhydride, 0.50 g of N,N'-methylenebisacrylamide, and 1.60 g of sodium lignosulfonate in 100 mL of pure water. Stir at room temperature for 2 h until completely dissolved. Adjust the pH of the solution to 6.0 with NaOH. Then add 0.25 g of potassium persulfate and 250 μL of N,N,N',N'-tetramethylenediamine. Stir at room temperature for 5 min and pour into a 1 cm solution. 3 After gelling in a mold for 1 hour, washing three times with pure water, and freeze-drying, a sample with a size of 111 cm was obtained. 3 Lignin hydrogel; the lignin hydrogel was sequentially immersed in ferrous sulfate solution and sodium sulfide solution to load ferrous sulfide nanoparticles, and then washed and freeze-dried to obtain a composite carrier; The loading method for ferrous sulfide nanoparticles is as follows: First, weigh 6.95 g of ferrous sulfate heptahydrate and add it to 50 ml of deionized water. Stir for 15 min to obtain 50 mL of 0.5 mol·L⁻¹ solution. −1 Ferrous sulfate solution; Weigh out 1.95 g of sodium sulfide and dissolve it in 50 mL of deionized water to obtain 50 mL of 0.5 mol·L⁻¹ solution. −1 Sodium sulfide solution; Then, 5 g of the lignin hydrogel sample described in S1 was soaked in 50 mL of 0.5 mol·L⁻¹ solution. −1 The lignin hydrogel was soaked in ferrous sulfate solution under nitrogen atmosphere for 12 h, then the solution was discarded. The soaked lignin hydrogel was taken out, washed three times with oxygen-free pure water, and then freeze-dried. The dried gel was then soaked in 50 mL of 0.5 mol·L⁻¹ solution. −1 The lignin hydrogel loaded with ferrous sulfide nanoparticles (FeS@LH) was prepared by soaking the lignin in sodium sulfide solution under nitrogen atmosphere for 12 h, washing it three times with pure water to remove dissolved oxygen, and then freeze-drying it. The average particle size of the ferrous sulfide nanoparticles in the ferrous sulfate solution was 400 nm. S3. Preparation of hydrogel bacterial agent: Weigh 5 g of the composite carrier (FeS@LH) described in S2, add it to 250 ml of the composite microbial agent described in S1 and mix evenly. Shake and culture for 27 h under conditions of 150 rpm, 30°C and protection from light. After sterile filtration and rinsing, dry at 25°C to obtain 5 g of hydrogel agent. The drying conditions are as follows: after filtration and rinsing under sterile conditions, transfer to an oven and dry at 25°C until the moisture content is ≤8%, which is 6.5% in this example.

[0030] Example 3: This example describes a method for preparing a hydrogel-immobilized bacterial agent for the synergistic removal of plasticizers and heavy metal Cd from wastewater. This example is based on Example 2 with changes to the conditions; all other contents are the same except for the following: In S1: the bacterial suspensions of each functional strain were activated in liquid LB medium at 25°C and 120 rpm for 22 h, resuspended twice in inorganic salt medium, and adjusted to OD600=1.0. In S2: Stir at room temperature for 3 minutes, then pour in 1cm... 3 After gelling in a mold for 0.8 h, washing twice with pure water, and freeze-drying, a sample with a size of 111 cm was obtained. 3 Lignin hydrogel; The loading method for ferrous sulfide nanoparticles is as follows: the lignin hydrogel is immersed in an immersion solution with a concentration of 0.4 mol·L⁻¹. -1The sample was immersed in a ferrous sulfate solution under nitrogen atmosphere for 10 hours, then washed twice with pure water and freeze-dried; subsequently, it was immersed in a 0.4 mol·L⁻¹ solution. -1 The lignin hydrogel loaded with ferrous sulfide nanoparticles was soaked in sodium sulfide solution under nitrogen atmosphere for 10 h, then washed twice with pure water and freeze-dried to obtain lignin hydrogel. In S3: Weigh 5 g of the composite carrier (FeS@LH) described in S2, add it to 100 ml of the composite microbial agent described in step S1, mix evenly, and culture by shaking at 100 rpm and 25°C in the dark for 24 h. After sterile filtration and rinsing, dry at 22°C.

[0031] Example 4: This example describes a method for preparing a hydrogel-immobilized bacterial agent for the synergistic removal of plasticizers and heavy metal Cd from wastewater. This example is based on Example 2 with changes to the conditions; all other contents are the same except for the following: In S1: the bacterial suspensions of each functional strain were obtained by activating the strains in liquid LB medium at 35°C and 180 rpm for 30 h, resuspending them four times in inorganic salt medium, and adjusting OD600 to 1.0. In S2: Stir at room temperature for 6 minutes, then pour in 1cm... 3 After gelling in a mold for 1.2 h, washing four times with pure water, and freeze-drying, a sample with a size of 111 cm was obtained. 3 Lignin hydrogel; The loading method for ferrous sulfide nanoparticles is as follows: the lignin hydrogel is immersed in a solution with a concentration of 0.6 mol·L⁻¹. -1 The sample was immersed in a ferrous sulfate solution under nitrogen atmosphere for 14 hours, then washed four times with pure water and freeze-dried; then it was immersed in a 0.6 mol·L⁻¹ solution. -1 The lignin hydrogel loaded with ferrous sulfide nanoparticles was soaked in sodium sulfide solution under nitrogen atmosphere for 14 h, then washed 4 times with pure water and freeze-dried. In S3: Weigh 5 g of the composite carrier (FeS@LH) described in S2, add it to 500 ml of the composite microbial agent described in step S1, mix evenly, and culture by shaking at 200 rpm and 35°C in the dark for 30 h. After sterile filtration and rinsing, dry at 28°C.

[0032] Example 5: This example describes a method for preparing a hydrogel-immobilized bacterial agent for the synergistic removal of plasticizers and heavy metal Cd from wastewater. This example is based on Example 2 with changes to the conditions. Except for the following, all other contents are the same. The difference between this example and Example 2 is: The induction culture method described in S1 is as follows: a. Preparation of the composite inducer: Biochar powder, humic acid, monobutyl phthalate, and betaine were mixed in a mass ratio of 1:0.4:0.2:0.1 to obtain a premix. Sterile deionized water at a mass ratio of 10 times that of the premix was added, and the mixture was ultrasonically dispersed for 40 min. The mixture was then centrifuged at 4℃ and 8000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain the composite inducer. The particle size of the biochar powder was 60~80 nm. b. Pre-acclimatization culture: Add Cd to liquid LB medium at a final concentration of 0.3 mg / L. 2+ The pH was adjusted to 7.0 with 5% (v / v) of the composite inducer, sterilized, and then inoculated with functional strains. The strains were cultured at 32°C and 170 rpm for 72 h with shaking. 1% (v / v) of the composite inducer was added every 24 h. After the culture was completed, the strains were centrifuged and washed to obtain the pre-adapted strains. c. Two-way gradient acclimatization: Prepare 5 groups of culture media, including Cd 2+ The concentrations were 0.8, 2.0, 4.0, 7.0, and 10.0 mg / L, corresponding to PAEs (DBP) concentrations of 2, 5, 8, 12, and 15 mg / L, respectively. All five culture media contained 3% (v / v) of the compound inducer. The pre-adapted strains were inoculated into each gradient culture medium sequentially. The pre-adapted strains were first inoculated into the first group of culture medium and cultured with shaking at 30℃ and 150 rpm for 60 h. After centrifugation and washing, they were transferred to the next group. Under the same shaking culture conditions as the first group, they were centrifuged, washed, and transferred to the next group. This process was repeated until the fifth gradient culture medium was completed. After all gradient cultures were completed, the bidirectional acclimatized strains were obtained. d. Enhanced co-culture: The bidirectional acclimatized strain was inoculated into a solution containing 5 mg / L Cd. 2+ The mixture of PAEs at a concentration of 15 mg / L and a composite inducer at a volume fraction of 2% was cultured in a medium at a temperature of 30°C and a rotation speed of 150 rpm for 48 h. After centrifugation and washing, the mixture was ready for use. The mixed PAEs consisted of DMP, DEP and DBP in equal mass ratios.

[0033] Example 6: This example describes a method for preparing a hydrogel-immobilized bacterial agent for the synergistic removal of plasticizers and heavy metal Cd from wastewater. This example is based on Example 2 with changes to the conditions. Except for the following, all other contents are the same. The difference between this example and Example 2 is: The induction culture method described in S1 is as follows: a. Preparation of composite inducer: Biochar powder, humic acid, monobutyl phthalate, and betaine were mixed in a mass ratio of 1:0.3:0.2:0.1 to obtain a premix. Sterile deionized water at a mass ratio of 8 times that of the premix was added, and the mixture was ultrasonically dispersed for 30 min. The mixture was then centrifuged at 3℃ and 8000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain the composite inducer. The particle size of the biochar powder was 50~70 nm. b. Pre-acclimatization culture: Add Cd to liquid LB medium at a final concentration of 0.3 mg / L. 2+ The pH was adjusted to 7.0 with 5% (v / v) of the composite inducer, sterilized, and then inoculated with functional strains. The strains were cultured with shaking at 30°C and 150 rpm for 72 h. 0.5% (v / v) of the composite inducer was added every 24 h. After the culture was completed, the strains were centrifuged and washed to obtain the pre-adapted strains. c. Two-way gradient acclimatization: Prepare 5 groups of culture media, including Cd 2+ The concentrations were 0.8, 2.0, 4.0, 7.0, and 10.0 mg / L, corresponding to PAEs (DBP) concentrations of 2, 5, 8, 12, and 15 mg / L, respectively. All five culture media contained 3% (v / v) of the compound inducer. The pre-adapted strains were inoculated into each gradient culture medium sequentially. The pre-adapted strains were first inoculated into the first group of culture medium and cultured with shaking at 25℃ and 120 rpm for 50 h. After centrifugation and washing, they were transferred to the next group. Under the same shaking culture conditions as the first group, they were centrifuged, washed, and transferred to the next group. This process was repeated until the fifth gradient culture medium was completed. After all gradient cultures were completed, the bidirectional acclimatized strains were obtained. d. Enhanced co-culture: The bidirectional acclimatized strain was inoculated into a solution containing 4 mg / L Cd. 2+ The mixture of PAEs at a concentration of 10 mg / L and a composite inducer at a volume fraction of 1.5% was cultured in a medium at a temperature of 25°C and a rotation speed of 120 rpm for 44 h. After centrifugation and washing, the mixture was ready for use. The mixed PAEs consisted of DMP, DEP and DBP in equal mass ratios.

[0034] Example 7: This example describes a method for preparing a hydrogel-immobilized bacterial agent for the synergistic removal of plasticizers and heavy metal Cd from wastewater. This example is based on Example 2 with changes to the conditions. Except for the following, all other contents are the same. The difference between this example and Example 2 is: The induction culture method described in S1 is as follows: a. Preparation of composite inducer: Biochar powder, humic acid, monobutyl phthalate, and betaine were mixed in a mass ratio of 1:0.5:0.2:0.1 to obtain a premix. Sterile deionized water at a mass ratio of 12 times that of the premix was added, and the mixture was ultrasonically dispersed for 50 min. The mixture was then centrifuged at 5℃ and 8000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain the composite inducer. The particle size of the biochar powder was 80~100 nm. b. Pre-acclimatization culture: Add Cd to liquid LB medium at a final concentration of 0.3 mg / L. 2+ The pH was adjusted to 7.0 with 5% (v / v) of the composite inducer, sterilized, and then inoculated with functional strains. The strains were cultured with shaking at 35°C and 180 rpm for 72 h. 1.5% (v / v) of the composite inducer was added every 24 h. After the culture was completed, the strains were centrifuged and washed to obtain the pre-adapted strains. c. Two-way gradient acclimatization: Prepare 5 groups of culture media, including Cd 2+ The concentrations were 0.8, 2.0, 4.0, 7.0, and 10.0 mg / L, corresponding to PAEs (DBP) concentrations of 2, 5, 8, 12, and 15 mg / L, respectively. All five culture media contained 3% (v / v) of the compound inducer. The pre-adapted strains were inoculated into each gradient culture medium sequentially. The pre-adapted strains were first inoculated into the first group of culture medium and cultured with shaking at 35℃ and 180 rpm for 70 h. After centrifugation and washing, they were transferred to the next group. Under the same shaking culture conditions as the first group, they were centrifuged, washed, and transferred to the next group. This process was repeated until the fifth gradient culture medium was completed. After all gradient cultures were completed, the bidirectional acclimatized strains were obtained. d. Enhanced co-culture: The bidirectional acclimatized strain was inoculated into a solution containing 6 mg / L Cd. 2+ The mixture of PAEs at a concentration of 18 mg / L and a composite inducer at a volume fraction of 2.5% was cultured in a medium at a temperature of 35°C and a rotation speed of 180 rpm for 52 h. After centrifugation and washing, the mixture was ready for use. The mixed PAEs consisted of DMP, DEP and DBP in equal mass ratios.

[0035] Comparative Example 1: This example describes a method for preparing a carbon-based composite material for the efficient adsorption and removal of organic-inorganic composite pollutants. This example is based on Example 2 with changes to the conditions. Except for the following, all other contents are the same: After obtaining the lignin hydrogel according to step S1, steps S2 and S3 are not performed again.

[0036] Comparative Example 2: This example describes a method for preparing a carbon-based composite material for the efficient adsorption and removal of organic-inorganic composite pollutants. This example is based on Example 2 with changes to the conditions. Except for the following, all other contents are the same: After the composite carrier is prepared according to the steps described in S2, the steps described in S2-3 and S3 are not repeated.

[0037] Test Example 1: This test example is used to determine the removal rate of PAEs by the hydrogel bacterial agent prepared in Examples 1 to 3.

[0038] The specific steps were as follows: 0.2 g of hydrogel bacterial agent was added to a 100 mL sterilized Erlenmeyer flask, with three replicates for each treatment. Then, 20 mL of inorganic salt culture medium and six stock solutions of PAEs were added to each Erlenmeyer flask to achieve a total PAE concentration of 30 mg / L (5 mg / L for each individual PAE). Finally, the Erlenmeyer flasks were incubated at 30℃ and 150 rpm in the dark. Samples were taken on days 1, 3, 5, and 7 to determine the residual PAE levels in the culture medium. The results are shown in the table below. Figure 1 .

[0039] The results show that the hydrogel bacterial agent in Example 1 had the highest removal rate for the six PAEs, reaching 99.72%, 99.57%, 98.84%, 96.61%, 57.69%, and 26.46%, respectively. From the removal rates of PAEs by the hydrogel bacterial agent in Examples 2 and 3, it can be seen that the removal performance of the hydrogel bacterial agent is optimal when the preparation conditions are FeS@LH: bacterial count = 1 g: 50 mL.

[0040] Experimental Example 2: This experimental example was used to determine the effect of FeS@LH from Example 1, lignin hydrogel from Comparative Example 1, and ferrous sulfide nanoparticles from Comparative Example 2 on Cd. 2+ The adsorption rate.

[0041] The specific steps are as follows: Take 0.040 g of the above material and add it to 20 ml of solution containing 100 mg / L of Cd. 2+ The solution was prepared by maintaining a solid-liquid ratio of 2 mg / ml, and incubated at 25°C with shaking for 24 hours. After centrifugation for 10 minutes, the supernatant was collected for Cd determination. 2+ Concentration, test results are shown in Figure 2 .

[0042] The results show that FeS@LH in Example 1 is effective for Cd. 2+ The saturated adsorption efficiency of FeS@LH in Example 1 was significantly greater than that of Comparative Examples 1 and 2, indicating that FeS@LH significantly improved the adsorption efficiency for Cd compared to lignin hydrogel and ferrous sulfide nanoparticles. 2+ The adsorption efficiency.

[0043] Experimental Example 3: This experiment tested the adsorption of Cd by the hydrogel bacterial agent from Example 1. 2+ Characterization was performed using X-ray photoelectron spectroscopy before and after the experiment, and the results are shown in [Figure number missing]. Figure 3 .

[0044] The results show that, as can be seen from the figure, lignin hydrogel successfully loaded ferrous sulfide nanoparticles, and precipitation reaction is the main mechanism for the adsorption of heavy metal Cd by FeS@LH composite material.

[0045] Experimental Example 4: Take 0.040 g of the hydrogel bacterial agent from Example 1 and add it to 20 ml of solution containing 100 mg / L Cd. 2+ The solution was prepared by maintaining a solid-liquid ratio of 2 mg / ml, and incubated at 25°C with shaking for 24 h. After centrifugation for 10 min, the supernatant was collected for Cd determination. 2+ Concentration, test results are shown in Figure 4 .

[0046] The results show that: From Figure 4 It can be seen that the FeS@LH composite material has a better effect on Cd. 2+ The adsorption can reach equilibrium in about 1 hour. The adsorption conforms to the pseudo-second-order adsorption kinetics (R2=0.931) and the Langmuir adsorption thermodynamic model (R2=0.945). The maximum adsorption capacity of the fitted result can reach 636.8 mg / g.

[0047] Investigating the effect of changes in acclimatization methods on the PAEs reduction efficiency of hydrogel bacterial agents: To determine the reduction efficacy of hydrogel bacterial agents against PAEs, the following experiment was conducted: DEHP and DnOP, both initially at 20 mg / L, and Cd, at 5 mg / L were added. 2+ In an inorganic salt culture medium, the hydrogel bacterial agent prepared in Examples 2 and 5-7 was added at a dosage of 10% (v / v) and cultured with shaking at 30℃ and 150 r / min for 7 days. After the culture was completed, the concentration of the remaining PAEs in the culture medium was determined by organic solvent extraction combined with high performance liquid chromatography, and the removal rate was calculated. Three replicates were set up for each experimental group, and the average value of the results was taken, as shown in Table 1.

[0048] Table 1. Reduction efficacy of hydrogel bacterial agents against PAEs (ΣPAEs)

[0049] The results show that, according to the comparison results in Table 1, the removal rates of PAEs in Examples 5, 6, and 7, which used a compound inducer for bidirectional gradient acclimation, were significantly higher than those using Cd alone. 2+ Example 2, induced by concentration gradient, demonstrates that the composite inducer strategy can more effectively enhance the degradation efficiency of functional microbial communities. Example 5, with a removal rate of 87.67%, exhibits the best performance. Its induction conditions—the specific ratio of biochar and humic acid, and the setting of the acclimatization concentration gradient—are all optimal, fully demonstrating the significant advantages of this composite induction and bidirectional acclimatization method in synergistically enhancing the degradation ability of microbial agents on PAEs.

Claims

1. A hydrogel bacterial agent for synergistic removal of plasticizers and heavy metals from water, characterized in that, The hydrogel microbial agent consists of a composite carrier and a composite microbial agent loaded on the composite carrier; the composite carrier is a lignin hydrogel loaded with ferrous sulfide nanoparticles; the composite microbial agent is a compound of functional strains that are cadmium tolerant and have phthalate degradation function.

2. The hydrogel bacterial agent for synergistic removal of plasticizers and heavy metals from water according to claim 1, characterized in that, The functional strains include Corynebacterium brassicae, Bacillus glutamate, Rhodococcus, and Acinetobacter picelli.

3. The hydrogel bacterial agent for synergistic removal of plasticizers and heavy metals from water according to claim 1, characterized in that, The functional strains include *Glutamica belladonna*, *Bacillus*, *Corynebacterium brassicae*, and *Acinetobacter pilosa*.

4. The method for preparing the hydrogel bacterial agent for synergistic removal of plasticizers and heavy metals from water as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Preparation of compound microbial inoculants: Take equal volumes of each functional bacteria and expose them to 0-10 mg / L Cd. 2+ After concentration gradient induction culture, the culture was activated in liquid LB medium at 25-35℃ and 120-180rpm for 22-30 h. The culture was then resuspended in inorganic salt medium 2-4 times and the OD600 was adjusted to 1.0 to obtain bacterial suspensions of each functional strain. The bacterial suspensions of each functional strain were mixed in equal volume ratio to prepare a compound microbial agent. S2. Preparation of composite carrier: Acrylamide, maleic anhydride, N,N'-methylenebisacrylamide, and sodium lignosulfonate were dissolved in pure water to obtain a mixed system. In this mixed system, the mass percentages of acrylamide, maleic anhydride, N,N'-methylenebisacrylamide, and sodium lignosulfonate were 10-14%, 4-6%, 0.3-0.5%, and 1-1.5%, respectively. The mixed system was stirred at room temperature for 2 hours until completely dissolved. The pH of the solution was adjusted to 5.5-6.5 with NaOH. Then, 0.8-1.2% potassium persulfate and 0.6-1.0% N,N,N',N'-tetramethylenediamine were added. After stirring evenly at room temperature, the mixture was poured into a mold and gelled for 0.8-1.2 hours. h, after washing with pure water 2-4 times and freeze-drying, lignin hydrogel is obtained; the lignin hydrogel is then immersed in ferrous sulfate solution and sodium sulfide solution in sequence to load ferrous sulfide nanoparticles, and after washing and freeze-drying, a composite carrier is obtained. S3. Preparation of hydrogel bacterial agent: The composite carrier described in S2 and the composite microbial agent described in S1 are mixed at a solid-liquid ratio of 1g:20~100ml, and cultured under shaking conditions at a speed of 100~200 rpm, a temperature of 25~35℃ and in the dark for 24~30 h. After sterile filtration and rinsing, the mixture is dried at a temperature of 22~28℃ to obtain the hydrogel microbial agent.

5. The method for preparing the hydrogel bacterial agent for synergistic removal of plasticizers and heavy metals from water according to claim 4, characterized in that, The induction culture method described in S1 is as follows: a. Preparation of composite inducer: Biochar powder, humic acid, monobutyl phthalate, and betaine were mixed in a mass ratio of 1:0.3~0.5:0.2:0.1 to obtain a premix. Sterile deionized water at a mass ratio of 8~12 times that of the premix was added, and the mixture was ultrasonically dispersed for 30~50 min. The mixture was then centrifuged at 3~5℃ and 8000 rpm for 10 min. The supernatant was filtered through a 0.22μm filter membrane to obtain the composite inducer. The particle size of the biochar powder was 50~100 nm. b. Pre-acclimatization culture: Add Cd to liquid LB medium at a final concentration of 0.3 mg / L. 2+ The compound inducer with 5% volume fraction was added, the pH was adjusted to 7.0, and after sterilization, the functional strain was inoculated. The strain was cultured with shaking at 30-35℃ and 150-180 rpm for 72 hours. 0.5-1.5% volume fraction of the compound inducer was added every 24 hours. After the culture was completed, the strain was centrifuged and washed to obtain the pre-adapted strain. c. Two-way gradient acclimatization: Prepare 5 groups of culture media, including Cd 2+ The concentrations were 0.8, 2.0, 4.0, 7.0, and 10.0 mg / L, corresponding to PAEs (DBP) concentrations of 2, 5, 8, 12, and 15 mg / L, respectively. All five culture media contained 3% (v / v) of the compound inducer. The pre-adapted strains were inoculated into each gradient culture medium sequentially. The pre-adapted strains were first inoculated into the first group of culture medium and cultured with shaking at 25-35℃ and 120-180 rpm for 50-70 h. After centrifugation and washing, they were transferred to the next group. Under the same shaking culture conditions as the first group, they were centrifuged, washed, and transferred to the next group. This process was repeated until the fifth gradient culture medium was completed. After all gradient cultures were completed, the bidirectional acclimatized strains were obtained. d. Enhanced co-culture: The bidirectional acclimatized strain was inoculated into a solution containing 4-6 mg / L Cd. 2+ The mixture of PAEs at a concentration of 10-18 mg / L and a composite inducer at a volume fraction of 1.5-2.5% was cultured in a medium at a temperature of 25-35℃ and a rotation speed of 120-180 rpm for 44-52 h. After centrifugation and washing, the mixture was ready for use. The mixed PAEs consisted of DMP, DEP and DBP in equal mass ratios.

6. The method for preparing the hydrogel bacterial agent for synergistic removal of plasticizers and heavy metals from water according to claim 4, characterized in that, The loading method for ferrous sulfide nanoparticles in S2 is as follows: the lignin hydrogel is immersed in an immersion solution with a concentration of 0.4~0.6 mol·L⁻¹. -1 The sample was immersed in a ferrous sulfate solution under nitrogen atmosphere for 10–14 h, then washed 2–4 times with pure water and freeze-dried; then immersed in a solution with a concentration of 0.4–0.6 mol·L⁻¹. -1 The lignin hydrogel loaded with ferrous sulfide nanoparticles was soaked in sodium sulfide solution under nitrogen atmosphere for 10-14 h, then washed with pure water 2-4 times and freeze-dried to obtain the lignin hydrogel.

7. The method for preparing the hydrogel bacterial agent for synergistic removal of plasticizers and heavy metals from water according to claim 6, characterized in that, The average particle size of the ferrous sulfide nanoparticles in the ferrous sulfate solution is 400 nm.

8. The method for preparing the hydrogel bacterial agent for synergistic removal of plasticizers and heavy metals from water according to claim 4, characterized in that, Cd as described in S1 2+ The culture medium for concentration gradient induction was liquid LB medium, Cd 2+ The concentration gradients included 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, and 7.5 mg / L.

9. The method for preparing the hydrogel bacterial agent for synergistic removal of plasticizers and heavy metals from water according to claim 4, characterized in that, The drying conditions described in S3 are as follows: after filtering and rinsing under sterile conditions, the product is transferred to an oven and dried at 25°C until the moisture content is ≤8%.

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