A long-term stable operation of SRB repair system for heavy metal wastewater treatment and application

Abstract

The application discloses a long-term stable operation SRB repair system for heavy metal wastewater treatment and application, and belongs to the technical field of heavy metal wastewater treatment, and solves the technical problems that the existing mining heavy metal wastewater usually contains high-concentration soluble heavy metals, high-sulfate content and low pH value, causes serious damage to the ecological environment, and the traditional physical and chemical treatment method has the technical problems of large reagent consumption, large sludge quantity, easy secondary pollution and the like. The long-term stable operation SRB repair system for heavy metal wastewater treatment is based on the biological sulfidation of the SRB composite bacterial community in the UASB biological reactor, combines the synergistic effect of the microbial community and the protection mechanism of the extracellular polymeric substance, realizes efficient removal and fixation of heavy metals, constructs the composite SRB bacterial community system, accurately controls operation parameters, and utilizes the synergistic protection mechanism of the EPS to realize efficient and stable removal and fixation of various heavy metals in the AMD in the UASB reactor.

Description

Technical Field

[0001] This invention relates to the field of heavy metal wastewater treatment technology, specifically to an SRB remediation system for treating heavy metal wastewater that can operate stably for a long period of time and its application. Background Technology

[0002] Heavy metal pollution, particularly heavy metal wastewater pollution from mining sources, is a global environmental problem. Among these, cadmium is listed as a priority heavy metal pollutant due to its low toxicity threshold, strong bioaccumulation capacity, and high environmental mobility.

[0003] Mining wastewater containing heavy metals, such as acid mine wastewater (AMD), typically contains high concentrations of dissolved heavy metals like cadmium, lead, and zinc, along with high sulfate content and low pH values. This causes severe damage to the ecological environment. These complex water conditions significantly promote the dissolution and migration of heavy metals, not only severely damaging the ecosystem but also greatly increasing the technical difficulty and cost of wastewater treatment, becoming a technological bottleneck in the field of mining environmental protection. While traditional physicochemical treatment methods are technically mature, they suffer from problems such as high reagent consumption, large sludge production, and the potential for secondary pollution.

[0004] Although sulfate-reducing bacteria (SRB)-mediated anaerobic bioremediation technology has potential advantages in treating heavy metal wastewater from mining, there are still many challenges in transforming it into a stable and reliable engineered treatment process. Summary of the Invention

[0005] This invention addresses the shortcomings and deficiencies of existing technologies and aims to solve the aforementioned technical problems in the treatment of heavy metal wastewater in the mining industry. This invention provides an SRB remediation system for heavy metal wastewater treatment that can be started up quickly, operate stably for a long time, and is resilient to multiple metal stresses, as well as its application.

[0006] To achieve the above objectives, this invention provides an SRB remediation system for treating heavy metal wastewater that can operate stably for a long period. Based on the biosulfurization of SRB complex microbial communities in an upflow anaerobic sludge blanket (UASB) reactor, combined with the synergistic effect of the microbial community and the protective mechanism of extracellular polymeric substances (EPS), this system achieves efficient removal and fixation of heavy metals. Specifically:

[0007] In the UASB bioreactor, the composite SRB microbial community used anaerobic activated sludge and heavy metal-contaminated soil as inoculum sources. The composite SRB microbial community utilized organic carbon sources in the wastewater as electron donors to convert sulfate (SO4) into electrons. 2- ) is reduced to sulfide (S 2- ), sulfides (S 2-It reacts chemically with dissolved heavy metal ions in wastewater to form metal sulfide precipitates, achieving efficient fixation and removal of heavy metals; both anaerobic activated sludge and soil contaminated with heavy metals contain microbial communities including Thermodesulfobacteriota (SRB core), Bacillota (fermentation / tolerance supplementation), Bacteroidota (anaerobic hydrolysis / fermentation framework), Actinomycetota (tolerance / remediation related), Chloroflexota (anaerobic hydrolysis / fermentation framework), Pseudomonadota (tolerance / heterotrophic / opportunistic), and Euryarchaeota (electron sink / anabolism / potential DIET); soil contaminated with heavy metals includes Actinomycetes and Proteobacteria;

[0008] By adjusting the start-up and operation parameters of the UASB bioreactor to maintain them within a range favorable to anaerobic metabolism and SRB activity, the functional stability of sulfate reduction and metal sulfide precipitation can be ensured, while the long-term stability and cell protection of the SRB complex microbial system can be achieved.

[0009] A stress system was constructed by gradient addition of different types and concentrations of heavy metals. The selected composite microbial community was then adapted and domesticated to obtain a composite microbial agent with heavy metal tolerance and fixation ability, thereby achieving efficient fixation and removal of heavy metals in the UASB bioreactor.

[0010] Preferably, the anaerobic activated sludge includes 10%-25% Thermodesulfobacteriota (SRB core), 5%-15% Bacillota (fermentation / tolerance supplement), 5%-15% Bacteroidota (anaerobic hydrolysis / fermentation framework), 0.5-5% Actinomycetota, 5%-15% Chloroflexota (anaerobic hydrolysis / fermentation framework), 2%-15% Pseudomonadota (tolerance / heterotrophic / opportunistic), and 5-15% Euryarchaeota.

[0011] The microbial community in soil contaminated with heavy metals includes: Thermodesulfobacteriota (SRB core) 0.5-10%, Bacillota (fermentation / tolerance supplement) 20%-60%, Bacteroidota (anaerobic hydrolysis / fermentation skeleton) 5%-15%, Actinomycetota 5-25%, Chloroflexota (anaerobic hydrolysis / fermentation skeleton) 0.5%-5%, Pseudomonadota (tolerance / heterotrophic / opportunistic) 5%-25%, and Euryarchaeota 0.5-5%.

[0012] Preferably, the specific method for constructing a heavy metal stress system is as follows: different types and concentrations of the heavy metal Cd are added in a gradient manner. 2+ Pb 2+ Zn 2+ Constructing a coercive system, introducing Cd into the water 2+ The concentration was increased from an initial 0 mg / L to 20 mg / L, and then maintained at Cd. 2+ Concentration 20 mg / L, Pb 2+ The concentration was gradually increased from 0 mg / L to 5 mg / L, 10 mg / L and 15 mg / L; in Cd 2+ Concentration 20 mg / L, Pb 2+ Under the condition of a concentration of 15 mg / L, continue to add Zn 2+ The concentration of the bacteria was gradually increased from 0 mg / L to 5 mg / L, 10 mg / L and 20 mg / L. The selected compound bacterial groups were adapted and domesticated to obtain compound bacterial agents with heavy metal tolerance and fixation ability, so as to achieve efficient fixation and removal of heavy metals in UASB bioreactor.

[0013] Preferably, it includes the following steps:

[0014] S1 Constructing a Composite SRB Microbial Community System – UASB Bioreactor: The composite SRB microbial community system uses anaerobic digested sludge and heavy metal contaminated soil as inoculum sources. The two are mixed in a certain proportion to construct a composite SRB microbial community system with stronger shock resistance and metabolic resilience, so as to improve the functional redundancy and environmental adaptability of the microbial network.

[0015] S2 regulates the start-up and operating parameters of the UASB bioreactor, precisely controlling key operating parameters (stability indicators) within the UASB bioreactor, including pH, alkalinity, and carbon-sulfur ratio (COD / SO4). 2-This keeps it within the optimal range that is conducive to anaerobic metabolism and SRB activity, so as to ensure the functional stability of sulfate reduction and metal sulfide precipitation. Metal sulfides preferentially nucleate and deposit on the extracellular or granular sludge surface, thereby transferring heavy metals from the dissolved phase to the solid phase, achieving rapid and efficient removal and fixation.

[0016] S3 constructs multiple heavy metal Cd 2+ / Pb 2+ / Zn 2+ The gradient dosing acclimatization system employs a mechanism of gradually adding different concentrations and types of heavy metals to acclimatize the SRB complex microbial community, enabling it to gradually adapt to the high-concentration heavy metal stress environment. Even under multiple heavy metal stress scenarios, the UASB bioreactor can still achieve adaptation and stable maintenance of multi-metal stress, maintaining long-term stable operation and efficient removal capacity.

[0017] Preferably, the operation of the UASB bioreactor is divided into three stages: the start-up stage, the heavy metal shock stage (stage I), and the combined stress stage (stage II). In step S2, COD / SO4 2- The specific control methods include:

[0018] Start-up phase: Influent COD / SO4 2- The ratio was adjusted to 1:1 to achieve rapid enrichment of SRB and establishment of a sulfate reduction functional network;

[0019] In Phase I: COD / SO4 2- The ratio is 1.0:1.0 and remains unchanged;

[0020] In stage II: Keep the influent sulfate concentration constant, and reduce the influent COD / SO4 ratio by decreasing the glucose dosage. 2 The ratio was adjusted to 0.8:1.0.

[0021] Preferably, the dynamic control parameters and operating conditions for each stage of the UASB bioreactor operation are as follows:

[0022] Start-up phase (days 0-72): This involves inoculating the sludge with the SRB complex microbial community. The UASB bioreactor uses glucose as the sole carbon source, and the influent is artificially prepared high-sulfate simulated wastewater (SO4). 2- The concentration was 1000 mg / L; the influent COD concentration was controlled at 1.0 g / L during this stage, and the carbon-to-sulfur ratio (COD / SO4) was [not specified]. 2- The ratio was adjusted to 1:1 to achieve rapid enrichment of SRB and establishment of the sulfate reduction functional network. When the sulfate removal rate and sulfide production in the effluent of the UASB bioreactor are continuously stable, or when the index levels exceed the peak value in the early stage of acclimatization, the reactor is judged to have completed the start-up and microbial acclimatization and enter the subsequent stress experiment stage.

[0023] Phase I (Days 73-114, Heavy Metal Impact Phase): A single addition of Cd is made to the UASB bioreactor influent system. 2+ Using CdCl2·2.5 H2O as a simulated heavy metal pollution source, the influent Cd... 2+ With a concentration of 20 mg / L, the pollution conditions of sudden heavy metal impact in actual mining wastewater were simulated to explore the response mechanism of microbial community and reactor performance under single cadmium stress.

[0024] Phase II (Days 115-154, Complex Stress Phase): Maintaining constant influent sulfate concentration, the COD / SO4 ratio of the influent was reduced by decreasing glucose dosage. 2- The ratio was adjusted to 0.8:1.0 to further reduce the amount of organic carbon source added to better match the actual operating conditions, and to study the metabolic regulation and functional maintenance mechanism of microbial communities under combined stress.

[0025] Preferably, in the inoculation of contaminated soil and anaerobic sludge, the contaminated soil accounts for 25%;

[0026] The overall pH is controlled between 5.0 and 7.0; total alkalinity: 1300-1600 mg CaCO3 / L, bicarbonate alkalinity: 1100-1250 mg CaCO3 / L; the reactor operating temperature is controlled by a constant temperature water bath circulation system and maintained at 35-40℃ throughout the process; each reactor group uses a peristaltic pump for independent water intake, and the HRT is set to 4-12h and kept stable.

[0027] Preferably, the UASB bioreactor is an upflow anaerobic sludge bed bioreactor, whose internal structure includes a bottom fluidization zone, a middle membrane module zone, and a top three-phase separator.

[0028] The aforementioned SRB remediation system for treating heavy metal wastewater, which can operate stably for a long period, employs a combined process of "physicochemical conditioning - primary precipitation - UASB bioreactor - secondary precipitation" to achieve acidity neutralization and stable removal of heavy metals. The specific process flow is as follows:

[0029] (1) The influent first enters the equalization tank, where an alkaline agent is added to raise the pH to the suitable range of 5.0-7.0 for microorganisms, and the water quality and quantity are homogenized by stirring;

[0030] (2) Subsequently, the wastewater flows into the primary sedimentation tank, while the final process effluent rich in sulfides is returned to the primary sedimentation tank, where sulfides and some of the heavy metal cadmium in the influent are effectively removed.

[0031] (3) Then, organic matter in the influent is added through the carbon source addition tank to provide a stable and balanced carbon source for the UASB bioreactor. The mixing tank is used to ensure that the influent and carbon source are fully mixed.

[0032] (4) Subsequently, the wastewater enters the core process unit—UASB bioreactor, and the hydraulic retention time is controlled at 4-12h. The sulfur-cycle type composite functional bacteria reduce sulfate to sulfide in an anaerobic environment, and combine with heavy metal ions to generate metal sulfides for further removal, while degrading residual organic matter.

[0033] Preferred options also include:

[0034] (5) The treated effluent enters the secondary sedimentation tank to complete the sludge-water separation, and the supernatant is discharged as the final effluent; part of the settled sludge is returned to the primary sedimentation tank to enhance the front-end sedimentation effect, and the other part is returned to the UASB bioreactor as the remaining sludge to further enrich and remove the residual heavy metals in the sludge, so as to achieve deep purification of heavy metals and sludge stabilization in the system.

[0035] This invention provides an SRB remediation system for treating heavy metal wastewater that can operate stably for a long period and its application. It has the following beneficial effects:

[0036] (1) This invention constructs a composite SRB microbial community system, precisely controls the operating parameters, and utilizes the synergistic protection mechanism of EPS to achieve efficient and stable removal and fixation of various heavy metals in AMD in a UASB bioreactor, and converts them into recyclable metal sulfides, thereby overcoming the technical bottlenecks and high costs faced by existing technologies in treating complex mining wastewater.

[0037] Through the synergistic mechanism of physical adsorption and chemical complexation, the system achieves graded adsorption and buffered fixation of heavy metals, effectively reducing the direct toxicity of heavy metals to microbial cells.

[0038] (2) The SRB remediation system for treating heavy metal wastewater that can operate stably for a long time and its application, and the synergistic protection and fixation of EPS: EPS secreted by SRB plays multiple key roles in the process of heavy metal removal. EPS not only provides structural protection and functional regulation for the microbial community, but also forms the first line of defense by physically adsorbing and chemically complexing heavy metal ions through functional groups such as carboxyl, hydroxyl and amide groups on its surface. The synergistic effect of EPS at different levels (the outer EPS enriches heavy metals and the inner EPS participates in cell structure binding) realizes the hierarchical adsorption and buffer fixation of heavy metals, effectively reducing the direct toxicity of heavy metals to microbial cells, thereby maintaining the metabolic activity of SRB and the stability of the system.

[0039] (3) The SRB remediation system of this invention can be started up quickly, operate stably for a long time, and is resilient to multiple metal stresses. The resilience of the contaminated soil is enhanced by the introduction of indigenous microorganisms from the contaminated soil: the introduction of indigenous microorganisms from the contaminated soil increases the variety of microorganisms in the system that utilize organic matter, transfer electrons, and resist heavy metals, thereby improving the functional redundancy and environmental adaptability of the microbial network. This makes the system superior in terms of pH fluctuation, VFA accumulation mitigation, and long-term operational stability, and enhances the system's shock resistance and metabolic resilience.

[0040] (4) Constructing multiple heavy metal Cd 2+ / Pb 2+ / Zn 2+ A long-term acclimatization system with gradient dosing was used to realize the differences in cell activity and spatial correlation characteristics between heavy metals and microorganisms / sludge particles in different inoculation systems under multi-metal stress. Attached Figure Description

[0041] Figure 1 This is a schematic diagram illustrating the working principle of the UASB bioreactor of the present invention;

[0042] Figure 2 This is a schematic diagram of the process flow for the application of the UASB bioreactor of the present invention in Example 4;

[0043] Figure 3 This is a schematic diagram of the UASB reactor structure of the present invention, wherein... Figure 3 (a) is a schematic diagram of the start-up and operation conditions of the three UASB reactors in the experimental examples of this invention; Figure 3 (b) are photographs of the actual apparatus of the three UASB reactors in the experimental examples of this invention;

[0044] Figure 4 This is a graph showing the heavy metal concentration data of the influent / effluent water of three groups of UASB reactors in the experimental examples of this invention;

[0045] Figure 5 This refers to the phylum-level composition of the microbial community in the activated sludge of the three groups of UASB reactors in Experimental Example 2 of this invention;

[0046] Figure 6 This refers to the genus-level composition of the microbial community in the activated sludge of the three groups of UASB reactors in Experimental Example 2 of this invention;

[0047] Figure 7 This is a photograph of the UASB reactor used in Experimental Example 2 of this invention. Figure 7 (a) An image of the sediments at the bottom of the UASB reactor; Figure 7 (b) for Figure 7 (a) X-ray diffraction (XRD) pattern of the corresponding part;

[0048] Figure 8 for Figure 7 The corresponding SEM images of the bottom sediments of the reactor (the yellow box indicates the elemental mapping area) and SEM-EDS analysis data are shown below. Figure 8 (a) is a SEM image of the sediments; Figure 8 (b) is a Cd distribution; Figure 8 (c) is an S-distribution; Figure 8 (d) is the SEM-EDS energy spectrum. Detailed Implementation

[0049] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0050] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Example 1

[0051] The present invention provides an SRB remediation system for treating heavy metal wastewater that can operate stably for a long time. Based on the biosulfurization effect of SRB complex microbial community in UASB bioreactor, combined with the synergistic effect of microbial community and the protection mechanism of EPS, it achieves efficient removal, fixation and resource recovery of heavy metals.

[0052] like Figure 1 As shown, the SRB remediation system of this invention uses a UASB bioreactor as its core platform. In the UASB bioreactor, the composite SRB bacterial community utilizes the organic carbon source in the wastewater as an electron donor to remove sulfate (SO42-). 2- ) is reduced to sulfide (S 2- The reduction of sulfate and the formation of sulfides are achieved by regulating the start-up and operation parameters of the UASB bioreactor to maintain them within the optimal range for anaerobic metabolism and SRB activity. This ensures the functional stability of sulfate reduction and the precipitation of metal sulfides with extremely low solubility, i.e., the sulfides (S) produced by the SRB. 2- It reacts chemically with dissolved heavy metal ions in wastewater to form metal sulfide precipitates with extremely low solubility, achieving efficient fixation and removal of heavy metals, while also enabling the SRB composite microbial system to maintain long-term stability and cell protection.

[0053] The relative abundance of anaerobic activated sludge includes: Thermodesulfobacteriota (SRB core) 10%-25%, Bacillota (fermentation / tolerance supplement) 5%-15%, Bacteroidota (anaerobic hydrolysis / fermentation skeleton) 5%-15%, Actinomycetota 0.5-5%, Chloroflexota (anaerobic hydrolysis / fermentation skeleton) 5%-15%, Pseudomonadota (tolerance / heterotrophic / opportunistic) 2%-15%, and Euryarchaeota 5-15%.

[0054] The bacterial communities present in soils contaminated with heavy metals, based on relative abundance, include: Thermodesulfobacteriota (SRB core) 0.5-10%, Bacillota (fermentation / tolerance supplement) 20%-60%, Bacteroidota (anaerobic hydrolysis / fermentation skeleton) 5%-15%, Actinomycetota 5-25%, Chloroflexota (anaerobic hydrolysis / fermentation skeleton) 0.5%-5%, Pseudomonadota (tolerance / heterotrophic / opportunistic) 5%-25%, and Euryarchaeota 0.5-5%.

[0055] Meanwhile, a stress system was constructed by adding different concentrations of heavy metals in a gradient manner to achieve efficient fixation, removal and resource recovery of heavy metals in the UASB bioreactor, as well as the adaptation and response mechanisms under single and multiple heavy metal stresses.

[0056] Specifically, the following steps are included:

[0057] S1. Constructing a composite SRB microbial community system – UASB bioreactor

[0058] The composite SRB microbial community system uses anaerobic digested sludge and heavy metal contaminated soil as inoculum sources. The two are mixed at a mass ratio of 25% based on the amount of contaminated soil to construct a composite SRB microbial community system with stronger shock resistance and metabolic resilience, thereby enhancing the functional redundancy and environmental adaptability of the microbial network.

[0059] S2. Adjusting the start-up and operation parameters of the UASB bioreactor

[0060] Precise control of key operating parameters within the UASB bioreactor, including pH, alkalinity, volatile fatty acid (VFA) accumulation, and COD / SO4. 2-The overall pH is controlled between 5.0 and 7.0; total alkalinity: 1300-1600 mg CaCO3 / L, bicarbonate alkalinity: 1100-1250 mg CaCO3 / L. The reactor operating temperature is controlled by a constant temperature water bath circulation system and maintained at 35-40℃ throughout the process. Each UASB bioreactor uses a peristaltic pump for independent water intake, and the HRT is set to 4-12h and kept stable to maintain it within the optimal range for anaerobic metabolism and SRB activity. This ensures the functional stability of sulfate reduction and the precipitation of metal sulfides with extremely low solubility. Metal sulfides preferentially nucleate and deposit on the extracellular or granular sludge surface, thereby transferring heavy metals from the dissolved phase to the solid phase, achieving rapid and efficient removal and fixation.

[0061] The operation of the UASB bioreactor of this invention is divided into three stages: the start-up stage, the heavy metal shock stage (Stage I), and the combined stress stage (Stage II). The carbon-to-sulfur ratio (COD / SO4) is... 2- The specific dynamic control method is as follows:

[0062] S21. During the start-up phase: To inoculate the sludge with the SRB complex microbial community, the UASB bioreactor uses glucose as the sole carbon source, and the influent is artificially prepared high-sulfate simulated wastewater, containing SO42-. 2- The concentration was 1000 mg / L; the influent COD concentration was controlled at this stage in relation to SO4. 2- mass ratio (COD / SO4) 2- The ratio was adjusted to 1.0:1.0 to achieve rapid enrichment of SRB and establishment of the sulfate reduction functional network.

[0063] S22. In Stage I: Cd is added once to the influent system of the UASB bioreactor. 2+ To simulate heavy metal pollution sources, the influent Cd 2+ When the concentration reaches 20 mg / L, metal sulfide precipitate CdS is generated in the UASB bioreactor.

[0064] S23. In Stage II: Maintain the influent sulfate concentration constant, and reduce the influent COD / SO4 ratio by decreasing the glucose dosage. 2 The ratio was adjusted to 0.8:1.0 to further reduce the amount of organic carbon source added, making it more suitable for actual operating conditions. Under high toxicity stress, the enriched composite SRB microbial community system continuously completed sulfate reduction and drove Cd fixation in the form of sulfides. The UASB bioreactor effectively controlled dissolved Cd. 2+ Maintaining high removal efficiency: the metabolic regulation and functional maintenance mechanism of microbial communities under combined stress.

[0065] S3. Constructing a domestication system with multiple heavy metal gradient additions.

[0066] Different concentrations and types of heavy metal Cd were added in a gradient manner. 2+ / Pb 2+ / Zn 2+ The mechanism, specifically the method, involves the gradient addition of different types and concentrations of the heavy metal Cd. 2+ Pb 2+ Zn 2+ Constructing a coercive system, introducing Cd into the water 2+ The concentration was increased from an initial 0 mg / L to 20 mg / L, and then maintained at Cd. 2+ Concentration 20 mg / L, Pb 2+ The concentration was gradually increased from 0 mg / L to 5 mg / L, 10 mg / L and 15 mg / L; in Cd 2+ Concentration 20 mg / L, Pb 2+ Continue adding Zn at a concentration of 15 mg / L 2+ The concentration of the bacteria was gradually increased from 0 mg / L to 5 mg / L, 10 mg / L and 20 mg / L. The selected complex bacterial community was then adapted and domesticated. The SRB complex bacterial community was gradually domesticated to obtain a complex bacterial agent with heavy metal tolerance and fixation ability. This allowed the SRB complex bacterial community to gradually adapt to the high concentration of heavy metal stress environment. Even under multiple heavy metal stress scenarios, the UASB bioreactor can still adapt to and maintain stability under multiple metal stress, maintain long-term stable operation, and achieve efficient fixation and removal of heavy metals by the UASB bioreactor.

[0067] The working principle of this invention is based on the biosulfation of SRB complex bacteria in a UASB bioreactor, combined with the synergistic effect of the microbial community and the protective mechanism of extracellular polymeric substances (EPS), to achieve efficient removal and fixation of heavy metals. (1) Sulfate reduction and sulfide formation: In the UASB bioreactor, the complex SRB bacteria use the organic carbon source in the wastewater as an electron donor to reduce sulfate (SO4) into sulfides. 2- ) is reduced to sulfide (S 2- This process maintains the optimal activity of SRB by precisely controlling key operating parameters such as pH, alkalinity, VFA, and carbon-sulfur ratio, ensuring the continuous and stable generation of sulfides. (2) Heavy metal precipitation and fixation: The sulfides (S) generated by SRB 2- ) and dissolved heavy metal ions in wastewater, such as Cd 2+ Pb 2+ Zn 2+Chemical reactions occur, forming metal sulfide precipitates with extremely low solubility, such as CdS. These metal sulfides preferentially nucleate and deposit on the extracellular or granular sludge surface, thereby transferring heavy metals from the dissolved phase to the solid phase, achieving rapid removal and fixation. (3) Synergistic protection and fixation of EPS: EPS secreted by SRB plays multiple key roles in the removal of heavy metals. EPS not only provides structural protection and functional regulation for the microbial community, but also physically adsorbs and chemically complexes heavy metal ions through functional groups such as carboxyl, hydroxyl, and amide groups on its surface, forming the first line of defense. The synergistic effect of EPS at different levels, with the outer layer EPS enriching heavy metals and the inner layer EPS participating in cell structure binding, achieves hierarchical adsorption and buffer fixation of heavy metals, effectively reducing the direct toxicity of heavy metals to microbial cells, thereby maintaining the metabolic activity of SRB and the stability of the system. (4) Enhanced resilience introduced from contaminated soil: The introduction of indigenous microorganisms from contaminated soil increases the variety of microorganisms in the system that utilize organic matter, transfer electrons, and resist heavy metals, thereby enhancing the functional redundancy and environmental adaptability of the microbial network. This gives the system an advantage in mitigating pH fluctuations, VFA accumulation, and long-term operational stability, enhancing its shock resistance and metabolic resilience.

[0068] (5) Gradual acclimatization and adaptation mechanism: By gradually adding multiple heavy metals, the SRB complex microbial community gradually adapts to the high-concentration heavy metal stress environment. In this process, the microbial community achieves adaptation and stable maintenance to multi-metal stress through multi-dimensional evolutionary mechanisms such as EPS spatial structure reconstruction, enhanced electron transfer capacity, and redistribution of sulfur metabolism pathways, ensuring the long-term efficient operation of the system. Example 2

[0069] The operation process of the UASB bioreactor of this invention consists of three stages: the start-up stage, the heavy metal shock stage (Stage I), and the combined stress stage (Stage II). The dynamic control parameters and operating conditions for each stage are as follows:

[0070] S21. Start-up Phase (Days 0-72): This involves inoculating the sludge with the SRB complex microbial community. The UASB bioreactor uses glucose as the sole carbon source, and the influent is artificially prepared high-sulfate simulated wastewater (SO4). 2- The concentration was 1000 mg / L; during this stage, the influent COD concentration was controlled at 1.0 g / L, and the COD / SO4 ratio was [not specified]. 2- The ratio was adjusted to 1.0:1.0 to achieve rapid enrichment of SRB and establishment of the sulfate reduction functional network. When the sulfate removal rate in the effluent of the UASB bioreactor reaches >50% and the sulfide production is continuously stable at 20~100 mg / L, or when the index level exceeds the peak value in the early stage of acclimatization, the reactor is judged to have completed the start-up and microbial acclimatization and enter the subsequent stress experiment stage.

[0071] S22, Phase I (Days 73-114, Heavy Metal Impact Phase): Cd is added once to the UASB bioreactor influent system. 2+ Using CdCl2·2.5 H2O as a simulated heavy metal pollution source, the influent Cd... 2+ With a concentration of 20 mg / L, the pollution conditions of sudden heavy metal impact in actual mining wastewater were simulated to explore the response mechanism of the microbial community and the reactor operation performance under single cadmium stress.

[0072] Stage S23, II (Days 115-154, Complex Stress Stage): Maintain constant influent sulfate concentration, reduce influent COD / SO4 by decreasing glucose dosage. 2 The ratio was adjusted to 0.8:1.0 to further reduce the amount of organic carbon source added to better match the actual operating conditions, and to study the metabolic regulation and functional maintenance mechanism of microbial communities under combined stress. Example 3

[0073] like Figure 2 As shown, the SRB remediation system for treating heavy metal wastewater that can operate stably for a long time in this invention, based on Example 2, uses an upflow anaerobic sludge bed reactor in the UASB bioreactor. Its internal structure includes a bottom fluidized zone, a middle membrane module zone, and a top three-phase separator. This optimized internal structure design can: (1) improve the effluent quality of sludge retention and achieve sludge recirculation, maintaining high biomass in the reactor. (3) potentially integrate membrane separation technology: the middle membrane module zone provides space for future integration of membrane separation technology, which can further improve the effluent quality and achieve higher standards of discharge or reuse. Example 4

[0074] like Figure 2 As shown, the application of the SRB remediation system for treating heavy metal wastewater, which can operate stably for a long time, is specifically demonstrated by applying the UASB bioreactor from Example 3 to a combined process of "physicochemical adjustment - primary precipitation - UASB bioreactor - secondary precipitation" to achieve acidity neutralization and stable removal of heavy metals. The specific process flow is as follows:

[0075] (1) The influent first enters the equalization tank, where an alkaline agent is added to raise the pH to the suitable range of 5.0-7.0 for microorganisms, and the water quality and quantity are homogenized by stirring;

[0076] (2) Subsequently, the wastewater flows into the primary sedimentation tank, while the final process effluent rich in sulfides is returned to the primary sedimentation tank, where sulfides and some of the heavy metal cadmium in the influent are effectively removed.

[0077] (3) Then, organic matter in the influent is added through the carbon source addition tank to provide a stable and balanced carbon source for the UASB bioreactor. The mixing tank is used to ensure that the influent and carbon source are fully mixed.

[0078] (4) Subsequently, the wastewater enters the core process unit—the UASB bioreactor of Example 3 of this invention, and the hydraulic retention time is controlled at 8h. The sulfur-cycle type composite functional bacteria are used in an anaerobic environment to reduce sulfate to sulfide and combine with heavy metal ions to generate metal sulfides for further removal, while degrading residual organic matter.

[0079] (5) The treated effluent enters the secondary sedimentation tank to complete the mud-water separation, and the supernatant is discharged as the final effluent.

[0080] Part of the settled sludge is returned to the primary sedimentation tank to enhance the front-end sedimentation effect; the other part is returned to the UASB bioreactor as excess sludge to further enrich and remove residual heavy metals in the sludge, thereby achieving deep purification of heavy metals and sludge stabilization within the system.

[0081] This invention S 2- The sulfide-contaminated effluent is returned to the primary sedimentation tank, meaning that the residual sulfur in the treated effluent is fully utilized by recirculating it. 2- Sulfides are introduced into the primary sedimentation tank for initial precipitation, aiming to reduce the deposition of heavy metals in the main reaction zone—the UASB bioreactor. Pre-precipitation before the main reactor reduces the accumulation of heavy metals inside the reactor, helping to maintain long-term stable operation and reduce maintenance costs. Simultaneously, it facilitates the recovery of the photosensitive material cadmium sulfide, improving the economic efficiency and sustainability of wastewater treatment.

[0082] The UASB bioreactor in Example 3 of this invention was specifically tested and applied in the following experiments.

[0083] Experimental Example 1

[0084] Experimental setup: Three sets of UASB bioreactors made of plexiglass, as described in Example 3 of this invention, were constructed. Schematic diagrams of the three reactor sets are shown below. Figures 2-4 As shown, the reactor has a total height of 1455 mm, a radius of 70 mm, and an effective working volume of 9.5 L. The actual structure and schematic diagram of the dual-chamber upflow fluidized anaerobic bioreactor are shown below. Figure 2 As shown, the UASB bioreactor has a fluidized bed at the bottom, a membrane module zone in the middle, and a three-phase separator at the top. The entire anaerobic biological treatment system includes a wastewater substrate tank, a UASB bioreactor, a wet gas flow meter, a thermostatic water bath, a peristaltic pump, a small vacuum pump, and a timer. Both the small vacuum pump and the peristaltic pump are controlled by the timer to operate simultaneously. TMP is recorded by a digital pressure gauge (SIN-Y290, Sino Measure, China). The thermostatic water bath (YCX-6, Moer, China) maintains the operating temperature at 37 ± 0.1 ℃.

[0085] Inoculation with a composite SRB microbial community system: Contaminated soil and anaerobic sludge were used as inoculation sources. The sludge was anaerobically digested and taken from the effluent of an anaerobic bioreactor that had been stably removing sulfate wastewater in the laboratory for a long time. The sludge was highly enriched with SRB, and the original parameters were pH 7.0-7.3, MLSS 18.74 ± 0.03 g / L, and MLVSS 5.42 ± 0.01 g / L. Desulfomonile Abundance 0.22%, Syntrophobacter Abundance 0.61%, Desulfomicrobium Abundance 1.32%, Acetobacterium 11.41%, methylo-trophic Methanomethylovorans 32.0%, acetotrophic Methanosaeta 5.7%.

[0086] Soil samples contaminated with heavy metals were taken from an open-pit mine in Fujian Province. Specific content indicators of relevant substances in the soil are shown in Table 1-1. After inoculation with indigenous microorganisms, the samples were allowed to stand for 24 hours. The supernatant was removed, and the remaining sludge was used as inoculum sludge and mixed with reactor sludge to serve as the activated sludge inoculum for this experiment.

[0087]

[0088] To ensure consistency in microbial biomass across all reactors and to systematically assess the contribution of soil-derived microorganisms, the inoculation process was standardized based on volatile solids (VS) content rather than volume, with a total VS inoculation amount of 94.20 g per reactor.

[0089] The anaerobic activated sludge, by relative abundance, included Thermodesulfobacteriota (16.41%), Bacillota (8.74%), Bacteroidota (10.02%), Chloroflexota (9.33%), Euryarchaeota (7.34%), Pseudomonadota (6.64%), and Actinomycetota (3.03%).

[0090] The bacterial communities contained in the heavy metal contaminated soil, based on relative abundance, include Thermodesulfobacteriota (4.77%), Bacillota (35.67%), Bacteroidota (3.05%), Chloroflexota (2.83%), Euryarchaeota (1.67%), Pseudomonadota (11.98%), and Actinomycetota (15.56%).

[0091] Reactor A is inoculated only with mixed sludge (UASB). 100%sludge ) Pure anaerobic sludge inoculation, reactor B (UASB) 75%sludge+25% soil 25% of the sludge was replaced with contaminated soil of equal volatile solids (VS), and 75% of the anaerobic sludge was mixed with 25% of the contaminated soil for inoculation.

[0092] Reactor C (UASB) 50% sludge+50% soil 50% of the sludge was replaced with contaminated soil of equal VS amount, and 50% of the anaerobic sludge was mixed with 50% of the contaminated soil for inoculation.

[0093] The experiment lasted for 166 days, with the temperature maintained at 35°C throughout. Each reactor group used a peristaltic pump for independent water intake, and the HRT was set to 8 h and kept stable.

[0094] The enrichment and expansion substrate for the composite functional bacteria is (80-100 L): carbon source (such as methanol, glucose, sodium lactate, agricultural waste, etc., COD) 80, KH2PO4 8.00, Na2SO4 118.34, MgCl2·6H2O 12.50, NaHCO3 120.00, FeCl2·4H2O 4.20, KCl 60.00, CoCl2·6H2O 0.42, NH4Cl 68.00, CaCl2·2H2O 1.50, K2HPO4 20.00, NiCl2·6H2O 0.42. When the sulfate removal rate and sulfide yield in the reactor effluent are continuously stable, or when the index levels exceed the peak values ​​of the early acclimatization period, the reactor is considered to have completed start-up and microbial acclimatization.

[0095] The analysis results based on the above three sets of reactor operation test data are as follows:

[0096] Table 2 Results of the three reactor operation test data

[0097] <![CDATA[Dissolved Cd 2+ Continuous removal efficiency]]> Lead (Pb) removal efficiency Zinc (Zn) removal efficiency Firmicutes Thermodesulfobacteriota Bacillota Pseudomonadota Actinomycetota Reactor A ≥97.5% 99.99% 99.99% 8.4% 16.7% 8.4% 8.9% 4.6% Reactor B (25%) ≥97.5% 99.99% 99.99% 19.6% 12.3% 4.2% 4.8% 8.7% Reactor C (50%) ≥97.5% 99.99% 99.99% 36.8% 11.4% 1.7% 6.7% 2.0%

[0098] Table 3 Results of the three reactor operation test data

[0099] Methanothrix Desulfobacca Mesotoga Anaerolinea Clostridium Desulforhabdus Intestinibacillus Oscillibacter Sphaerochaeta Treponema Inert bacteria genus Ignavibacterium Reactor A 6.4% 1.9% 5.5% 1.9% 0.6% 1.2% 1.1% 0.3% 0.8% 1.3% 1.5% Reactor B (25%) 2.2% 2.1% 4.9% 3.2% 1.8% 0.8% 2.7% 0.7% 0.4% 0.9% 0.6% Reactor C (50%) 0.9% 0.7% 1.1% 1.5% 3.0% 1.3% 12.4% 3.0% 2.3% 2.2% 2.0%

[0100] The results of the reactor operation test data in Tables 2 and 3, and Figures 4-8 The data representation results, and the specific analysis results are as follows:

[0101] (1) Cadmium (Cd) removal effect: In the initial stage of the experiment, the carbon-sulfur ratio (COD / SO4) was adjusted. 2- The carbon-to-sulfur ratio was set to 1:1 to achieve rapid enrichment of SRB and establish the sulfate reduction functional network. Subsequently, in stage I / II, the carbon-to-sulfur ratio was adjusted to 0.8:1.0, and Cd at a concentration of 20 mg / L was added. 2+ .

[0102] Experimental results show that in the influent Cd 2+ At a concentration of 20 mg / L, reactors A, B, and C all exhibited almost complete cadmium removal. (Dissolved Cd) 2+ The continuous removal efficiency reaches ≥97.5%, and the effluent Cd 2+ The concentration was below the instrument's detection limit. This indicates that the composite microbial system of the present invention is effective against Cd. 2+ Even under continuous input, it still possesses extremely strong shock resistance and high removal potential. This excellent performance mainly relies on sulfides and Cd produced by SRB metabolism. 2+ The chelation precipitation, supplemented by the adsorption and complexation of the EPS matrix and the physical retention of granular sludge, forms a synergistic fixation system of "chemical precipitation-matrix adsorption-structural retention". At the spatial distribution level, the fixation of Cd within the reactor exhibits a clear vertical differentiation characteristic: UASB 50% sludge+50% soil Cd preferentially accumulates in the upper influent contact zone, easily causing local inhibition of microbial activity; while UASB 100% sludge With UASB 75% sludge+25% soil The Cd migrates more towards the bottom layer and is trapped there, with the lower sludge becoming the main area for stationary Cd. This effectively reduces the heavy metal stress in the upper active reaction zone and lays the structural foundation for the long-term stable operation of the reactor.

[0103] (2) Verification of heavy metal fixation mechanisms: such as Figure 7 and Figure 8 As shown, X-ray diffraction (XRD) analysis of the bottom sediments of the reactor confirmed that during operation, a large amount of Cd was converted into an insoluble sulfide mineral phase (CdS), thus achieving stabilization. Scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) further demonstrated the simultaneous presence of cadmium (Cd) and sulfur (S) in the bottom sediments, indicating the presence of S generated by sulfate reduction metabolism. 2- Able to interact with Cd extracellularly 2+Rapid combination occurs, primarily forming metal sulfide precipitates. This verifies the presence of Cd in the system. 2+ The fixation and biomineralization processes are mediated by SRB.

[0104] Based on multi-scale evidence from ICP-MS, FTIR, and Zeta potentials, this study further demonstrates the different roles of different levels of EPS in heavy metal adsorption and complexation, and their impact on system stability. Quantitative analysis (ICP-MS) of heavy metal content in different levels of extracellular polymeric materials (S-EPS, LB-EPS, and TB-EPS) revealed significant differences in heavy metal distribution across the EPS levels, indicating that EPS is not a homogeneous barrier but rather a protective system with clearly defined functional divisions. Specifically, the outer layer EPS (e.g., S-EPS / LB-EPS) showed higher levels of heavy metal enrichment, suggesting its crucial role as the first line of defense against heavy metal invasion of microbial cells; while the inner layer EPS (TB-EPS) is more likely involved in the stable binding process closely related to cell structures. FTIR analysis further revealed that under heavy metal stress, the carboxyl groups (–COO) in EPS... - Significant shifts and intensities of characteristic functional groups such as hydroxyl (–OH) and amide (–CONH–) groups were observed, indicating that heavy metals primarily bind to EPS through complexation and coordination. Simultaneously, Zeta potential results showed significant changes in the surface charge of EPS with heavy metal adsorption, reflecting the crucial roles of charge neutralization and surface charge reconstruction in the metal binding process. In summary, different levels of EPS, through a synergistic mechanism of physical adsorption and chemical complexation, achieved hierarchical adsorption and buffering of heavy metals, effectively reducing their direct toxicity to microbial cells. This provides a key structural basis for maintaining metabolic activity and system stability of the composite SRB system under heavy metal stress.

[0105] Based on the evolution of EPS fluorescent components and the reconstruction of metagenomic functional modules in long-term experiments, this study demonstrates the characteristics of EPS humification process and the synergistic regulation of ASR / DSR switching and key functional pathways. Combining the 3D-EEM and PARAFAC results from both experiments, the EPS fluorescent components in the system undergo directional succession (changes in the proportion of protein-like / humic components) during stress and recovery, showing a consistent response with reactor stability indicators (pH, VFA, sulfides). Metagenomic data further reveals the differential enrichment and synergistic regulation of key ASR / DSR genes and sulfur metabolism-stress resistance-related pathways under different stress scenarios, indicating that the microbial system achieves adaptation and stable maintenance to multimetallic stress through multidimensional evolution involving EPS spatial structure reconstruction, enhanced electron transport capacity, and redistribution of sulfur metabolism pathways.

[0106] (3) Removal effect of multiple heavy metals: In addition to cadmium, the technical solution of the present invention also showed extremely strong removal performance of heavy metals lead and zinc in mine water. The above-mentioned heavy metal ions were not detected in the effluent, and the removal rate reached 99.99%. Under the gradient addition condition of gradually increasing Pb and Zn concentration (from 5 mg / L to 20 mg / L), the concentrations of Cd, Pb and Zn in the reactor effluent could be maintained at extremely low levels for a long time, which further proved the resilience of the system of the present invention to multiple heavy metal stress.

[0107] Conclusion: This experimental example fully demonstrates that the technical solution of this invention, through the construction of a composite SRB microbial community system, optimization of operating parameters, gradient acclimatization, and the synergistic effect of EPS, can efficiently and stably remove multiple heavy metals from mining wastewater and immobilize them as recyclable metal sulfides, providing an effective solution for heavy metal pollution control. The physical encapsulation and chemical complexation of the EPS matrix, and the local SRB metabolism-mediated S... 2- Precise release, in-situ formation and anchoring of CdS precipitation, and the three elements work synergistically to construct a multi-layered heavy metal defense and fixation mechanism involving physical isolation and chemical adsorption. This mechanism not only achieves efficient and stable fixation of Cd in anaerobic sludge but also provides core support for the microbial community to resist heavy metal stress and maintain long-term stable reactor operation from both structural and functional perspectives. Furthermore, by gradually increasing the concentration of multiple metals, the reactor achieves long-term stable operation under combined stress scenarios.

[0108] Experimental Example 2

[0109] Determination and optimization of the optimal inoculation ratio for contaminated soil

[0110] In Experimental Example 1 of this invention, reactor B, inoculated with 25% contaminated soil, achieves a better balance between enhanced functional redundancy and shock resistance and retention of the sulfate reduction core, while reactor C, inoculated with 50% contaminated soil, carries the risk of excessive contaminated soil inoculation, leading to dilution of the functional architecture and weakening of key processes.

[0111] The results of the functional bacteria enrichment characterization data are analyzed as follows:

[0112] like Figure 5 and Figure 6 As shown, phylum-level analysis of the microbial composition in the three reactors revealed that Reactor A was more predominantly SRB-based, Reactor C was more enriched with soil metal-tolerant bacteria, and Reactor B was intermediate, exhibiting both heavy metal-specific resistance and sulfate-reducing ability.

[0113] Thermodesulfobacteriota is a typical SRB phylum, accounting for a high proportion in all three reactors, with the highest proportion in the 100% sludge group, and is the main force in sulfate reduction.

[0114] Bacillota (Bacillus) was significantly enriched in the 50% soil group and contains a variety of soil bacteria that can enhance the system's stress resistance.

[0115] Chloroflexota participates in organic matter degradation and part of the sulfur cycle, provides substrates for SRB, and maintains the cometabolous network.

[0116] Pseudomonadota (Pseudomonas) contains some metal-resistant, denitrifying, and multifunctional heterotrophic bacteria that participate in electron acceptor diversification and co-metabolism.

[0117] Phyllum-level community composition analysis showed that the dominant groups in the UASB bioreactor of this invention were mainly concentrated in the phyla Bacillota / Firmicutes, Thermodesulfobacteriota, and Euryarchaeota. This indicates that the dominant ecological niche of the system of this invention includes: (1) electron-donating microorganisms that hydrolyze and ferment organic matter; (2) sulfur-reducing microorganisms that utilize electrons to reduce sulfate and produce sulfides; and (3) electron consumers such as methanogenic archaea that maintain metabolic thermodynamics. In general, the basic functional framework of the functional microbial communities in the reactors of experimental groups A, B, and C is consistent. However, different start-up strategies will significantly affect the relative weight and coupling strength of each functional module in the community, thereby determining the system's ability to maintain and recover from heavy metal stress.

[0118] Reactor A (UASB) 100%sludgeThe system exhibited high homeostasis maintenance and recovery capabilities under Cd stress. Among its microbial communities, the thermophilic dethiobacterium phylum (typical SRB) had the highest relative abundance (16.70%), and the relative abundance of the methanogenic archaea phylum was also significantly higher than other groups (8.40%), especially the acetic acid-producing methanogen *Methanothrix*, which had the highest abundance (6.39%). This indicates that the system can effectively maintain sulfide supply and acetic acid consumption, ensuring the sulfide precipitation and fixation of heavy metal Cd and the stable operation of the anaerobic food web. The system better preserves key taxa related to symbiotic energy conservation (acetic acid absorption) and the sulfur cycle, maintaining stronger electron flow continuity and stable functional coupling. Metagenomic analysis showed that it had the highest abundance of DSR (dissimilar sulfate reduction) functional genes (such as the Apr-Dsr module) and high expression levels of antioxidant-related genes (sod, kat, gshA, gshB), indicating that it has stronger ROS scavenging and reducing power and can effectively cope with Cd-induced oxidative stress.

[0119] Reactor B (UASB) 75%sludge+25% soil The system exhibited a more balanced structural and functional microbial community under Cd stress. The relative abundance of *Dethiobacillus thermophilus* (SRB) remained high (12.30%), while *Archaeopterygii* also maintained a certain abundance (4.2%). Simultaneously, the system introduced appropriate amounts of soil microorganisms, such as *Actinomycetes* and *Proteobacteria*, which enhanced the system's functional redundancy and shock resistance. Metagenomic analysis showed that the system excelled in intracellular thiol synthesis (with the highest abundance of *CysE* / *CysK* genes), contributing to Cd adaptation. While enhancing upstream organic matter degradation and utilization and broad-spectrum resistance redundancy, it still relatively preserved sulfur cycling and commensal modules, achieving a good balance between enhanced resistance and maintenance of core functions.

[0120] Reactor C (UASB) 50% sludge+50% soilThe system exhibited weak metabolic resilience and recovery potential under Cd stress. Its microbial community showed a significantly increased proportion of Firmicutes (containing abundant anaerobic or facultative anaerobic heterotrophic bacteria) (36.80%), indicating over-dominance. However, the relative abundance of thermophilic dethiobacteria (SRB) and methanogenic archaea (such as Methanothrix) significantly decreased (11.40% and 1.70%, respectively), indicating dilution of core functional modules. Excessive soil inoculation led to the dominance of more tolerant, broad-spectrum, general heterotrophic bacteria, diluting key functional modules centered on sulfate reduction. This resulted in decreased anaerobic food web coupling, dilution of the functional core, reduced heavy metal fixation efficiency, and weak overall system recovery capacity. Metagenomic analysis also showed persistently low abundance in various functional modules (such as DSR, antioxidant, and electron transport), further confirming its limited metabolic resilience and recovery potential.

[0121] Furthermore, in the soil-introduced experimental group, the relative abundance of soil indicator phyla such as Actinomycetota and Pseudomonadota increased. Actinomycetes typically possess strong capabilities in organic matter degradation and environmental adaptation, while Pseudomonadota comprises diverse groups with varied metabolic pathways, metal tolerance characteristics (such as efflux and regulatory elements), and biofilm-related abilities. These groups are found in the UASB... 75% sludge+25% soil With UASB 50% sludge+50% soil The addition of [amount of pollutants] provides the system with higher functional redundancy and resilience under heavy metal stress, thereby enhancing community resilience and recovery potential. However, when excessive amounts of contaminated soil (50%) were added, SRB and methanogenic-related categories decreased simultaneously, indicating a contradiction between the community building dominated by resistance and broad-spectrum metabolism and the treatment functional architecture centered on sulfate reduction: overemphasizing resistance and diversity may weaken the core functions and key interaction chains required to maintain stable and efficient treatment.

[0122] ETSA and key enzyme activity (ATPS, CYTc, NADPH) results showed that electron transport activity and energy metabolism capacity were significantly enhanced in the contaminated soil system. Further metagenomic annotation revealed upregulation of sulfur metabolism and electron transport-related modules / genes, demonstrating a closer electronic coupling between the SRB sulfur reduction process and other functional microorganisms, thus forming a "sulfur cycle-direct interspecific electron transport synergistic response framework" (SR-DIET), realizing the recovery process and resilience maintenance mechanism of the system under heavy metal stress.

[0123] In summary, the SRB remediation system for treating heavy metal wastewater, which can operate stably for a long time and its application, demonstrates the remediation and removal effect of a composite functional microbial community with SRB as the core on wastewater containing multiple heavy metal ions in a UASB reactor. The SR-DIET framework and functional redundancy enhancement mechanism introduced into contaminated soil, by introducing indigenous microbial communities from heavy metal-contaminated soil, increases the variety of organic matter utilization, electron transport, and heavy metal resistance functional microorganisms in the system. This not only enhances the functional redundancy and environmental adaptability of the microbial network but also forms a "sulfur cycle-direct interspecific electron transport synergistic response framework" (SR-DIET). This enables the remediation system of this invention to exhibit strong rapid recovery and resilience under heavy metal stress.

[0124] The above are merely embodiments of the present invention. For example, anaerobic activated sludge may contain, by relative abundance, 10%-20% of Thermodesulfobacteriota, 5%-15% of Bacillota, 5%-15% of Bacteroidota, 0.5-5% of Actinobacteria, 5%-15% of Chloroflexota, 2%-15% of Pseudomonadota, and 5-15% of Euryarchaeota; soil contaminated with heavy metals may contain, by relative abundance, Thermodesulfobacteriota, 0.5-10%, Bacillota, 20%-60%, Bacteroidota, 5%-15% of Actinobacteria, and Chloroflexota. 0.5%-5%, Pseudomonadota 5%-25%, and Euryarchaeota 0.5-5% can all achieve the long-term stable operation of the SRB remediation system for heavy metal wastewater treatment of the present invention.

[0125] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for the long-term stable remediation of heavy metal wastewater treated with SRB, characterized in that, Based on the biosulfurization effect of SRB composite microbial communities in upflow anaerobic sludge blanket (UASB) reactors, combined with the synergistic effect of microbial communities and the protective mechanism of extracellular polymeric substances (EPS), efficient removal and fixation of heavy metals are achieved. Specifically: In the UASB bioreactor, the composite SRB microbial community used anaerobic activated sludge and heavy metal-contaminated soil as inoculum sources. The composite SRB microbial community utilized organic carbon sources in the wastewater as electron donors to convert sulfate (SO4) into electrons. 2- ) is reduced to sulfide (S 2- ), sulfides (S 2- The anaerobic activated sludge reacts chemically with dissolved heavy metal ions in the wastewater to form metal sulfide precipitates, achieving efficient fixation and removal of heavy metals. Both the anaerobic activated sludge and the heavy metal-contaminated soil contain microbial communities including Thermodesulfobacteriota, Bacillota, Bacteroidota, Actinomycetota, Chloroflexota, Pseudomonadota, and Euryarchaeota. By adjusting the start-up and operation parameters of the UASB bioreactor to maintain them within a range favorable to anaerobic metabolism and SRB activity, the functional stability of sulfate reduction and metal sulfide precipitation can be ensured, while the long-term stability and cell protection of the SRB complex microbial system can be achieved. A stress system was constructed by adding different types and concentrations of heavy metals in a gradient manner. The selected composite microbial community was then adapted and domesticated to obtain a composite microbial agent with heavy metal tolerance and fixation ability, thereby achieving efficient fixation and removal of heavy metals in the UASB bioreactor.

2. The SRB remediation method for treating heavy metal wastewater that can operate stably for a long period of time, as described in claim 1, is characterized in that... The anaerobic activated sludge contains, by relative abundance, 10%-20% of Thermodesulfobacteriota, 5%-15% of Bacillota, 5%-15% of Bacteroidota, 0.5-5% of Actinomycetota, 5%-15% of Chloroflexota, 2%-15% of Pseudomonadota, and 5-15% of Euryarchaeota. The bacterial communities contained in the heavy metal contaminated soil, based on relative abundance, include: Thermodesulfobacteriota (0.5%-10%), Bacillota (20%-60%), Bacteroidota (0.5%-5%), Actinomycetota (5-25%), Chloroflexota (0.5%-5%), Pseudomonadota (5%-25%), and Euryarchaeota (0.5-5%).

3. The SRB remediation method for treating heavy metal wastewater that can operate stably for a long time according to claim 1, characterized in that, The specific method for constructing a heavy metal stress system is as follows: different types and concentrations of heavy metal Cd are added in a gradient. 2+ Pb 2+ Zn 2+ Constructing a coercive system, introducing Cd into the water 2+ The concentration was increased from an initial 0 mg / L to 20 mg / L, and then maintained at Cd. 2+ Concentration 20 mg / L, Pb 2+ The concentration was gradually increased from 0 mg / L to 5 mg / L, 10 mg / L and 15 mg / L; in Cd 2+ Concentration 20 mg / L, Pb 2+ Under the condition of a concentration of 15 mg / L, continue to add Zn 2+ The concentration of the bacteria was gradually increased from 0 mg / L to 5 mg / L, 10 mg / L and 20 mg / L. The selected compound bacterial groups were adapted and domesticated to obtain compound bacterial agents with heavy metal tolerance and fixation ability, so as to achieve efficient fixation and removal of heavy metals in UASB bioreactor.

4. The SRB remediation method for treating heavy metal wastewater that can operate stably for a long time according to claim 1, characterized in that, Specifically, the following steps are included: S1 Constructing a Composite SRB Microbial Community System – UASB Bioreactor: The composite SRB microbial community system uses anaerobic digested sludge and heavy metal contaminated soil as inoculum sources. The two are mixed to construct a composite SRB microbial community system with stronger shock resistance and metabolic resilience, so as to improve the functional redundancy and environmental adaptability of the microbial network. S2 regulates the start-up and operation parameters of the UASB bioreactor, precisely controlling key operating parameters such as pH, alkalinity, and COD / SO4 within the UASB bioreactor. 2- This ensures that the sulfate reduction and metal sulfide precipitation are maintained within the optimal range that is conducive to anaerobic metabolism and SRB activity, thereby ensuring the functional stability of sulfate reduction and metal sulfide precipitation. Metal sulfides preferentially nucleate and deposit on the extracellular or granular sludge surface, thereby transferring heavy metals from the dissolved phase to the solid phase, achieving rapid and efficient removal and fixation. S3 constructs multiple heavy metal Cd 2+ / Pb 2+ / Zn 2+ The gradient dosing acclimatization system employs a mechanism of gradually adding different concentrations and types of heavy metals to acclimatize the SRB complex microbial community, enabling it to gradually adapt to the high-concentration heavy metal stress environment. Even under multiple heavy metal stress scenarios, the UASB bioreactor can still achieve adaptation and stable maintenance of multi-metal stress, maintaining long-term stable operation and efficient removal capacity.

5. The SRB remediation method for treating heavy metal wastewater that can operate stably for a long period of time, as described in claim 4, is characterized in that... The operation of the UASB bioreactor is divided into three stages: the start-up stage, the heavy metal shock stage (Stage I), and the combined stress stage (Stage II). In step S2, COD / SO4 2- The specific control methods include: Start-up phase: Influent COD / SO4 2- The ratio was adjusted to 1.0:1.0 to achieve rapid enrichment of SRB and establishment of the sulfate reduction functional network; In Phase I: COD / SO4 2- The ratio is 1.0:1.0 and remains unchanged; In stage II: Keep the influent sulfate concentration constant, and reduce the influent COD / SO4 ratio by decreasing the glucose dosage. 2 The ratio was adjusted to 0.8:1.

0.

6. The SRB remediation method for treating heavy metal wastewater that can operate stably for a long period of time, as described in claim 5, is characterized in that... The specific dynamic control parameters and operating conditions for each stage of the UASB bioreactor operation are as follows: Start-up phase: The UASB bioreactor is inoculated with SRB-derived microbial communities from the sludge. Glucose is the sole carbon source, and the influent is artificially prepared high-sulfate simulated wastewater (SO4). 2- The concentration was 1000 mg / L; during this stage, the influent COD concentration was controlled at 1.0 g / L, and the COD / SO4 ratio was [not specified]. 2- The ratio was adjusted to 1.0:1.0 to achieve rapid enrichment of SRB and establishment of the sulfate reduction functional network. When the sulfate removal rate and sulfide production in the effluent of the UASB bioreactor are continuously stable, or when the index levels exceed the peak value in the early stage of acclimatization, the reactor is judged to have completed the start-up and microbial acclimatization and enter the subsequent stress experiment stage. Phase I: Cd is added once to the UASB bioreactor influent system. 2+ Using CdCl2·2.5H2O as a simulated heavy metal pollution source, the influent Cd... 2+ With a concentration of 20 mg / L, the pollution conditions of sudden heavy metal impact in actual mining wastewater were simulated to explore the response mechanism of microbial community and reactor performance under single cadmium stress. Phase II: Maintaining the influent sulfate concentration constant, the COD / SO4 ratio in the influent is reduced by decreasing the glucose dosage. 2- The ratio was adjusted to 0.8:1.0 to further reduce the amount of organic carbon source added to better match the actual operating conditions, and to study the metabolic regulation and functional maintenance mechanism of microbial communities under combined stress.

7. The SRB remediation method for treating heavy metal wastewater that can operate stably for a long period of time according to claim 1, characterized in that, In the inoculation of contaminated soil mixed with anaerobic sludge, the contaminated soil accounts for 25%; The overall pH is controlled between 5.0 and 7.0; total alkalinity: 1300-1600 mg CaCO3 / L, bicarbonate alkalinity: 1100-1250 mg CaCO3 / L; the reactor operating temperature is controlled by a constant temperature water bath circulation system and maintained at 35-40℃ throughout the process; each reactor group uses a peristaltic pump for independent water intake.

8. The SRB remediation method for treating heavy metal wastewater that can operate stably for a long period of time according to claim 1, characterized in that, The UASB bioreactor uses a UASB bioreactor, whose internal structure includes a bottom fluidization zone, a middle membrane module zone, and a top three-phase separator.

9. The application of the SRB remediation method for heavy metal wastewater treatment that can operate stably for a long period of time, as described in claim 8, is characterized in that... A combined process of "physicochemical conditioning - primary precipitation - UASB bioreactor - secondary precipitation" is adopted to achieve acidity neutralization and stable removal of heavy metals. The specific process flow is as follows: (1) The influent first enters the equalization tank, where an alkaline agent is added to raise the pH to the suitable range of 5.0-7.0 for microorganisms, and the water quality and quantity are homogenized by stirring; (2) Subsequently, the wastewater flows into the primary sedimentation tank, while the final process effluent rich in sulfides is returned to the primary sedimentation tank, and the sulfides and some of the heavy metal cadmium in the influent are effectively removed. (3) Then, organic matter in the influent is added through the carbon source addition tank to provide a stable and balanced carbon source for the UASB bioreactor. The mixing tank is used to ensure that the influent and carbon source are fully mixed. (4) Subsequently, the wastewater enters the core process unit—UASB bioreactor, and the hydraulic retention time is controlled at 4-12h. The sulfur-cycle type composite functional bacteria reduce sulfate to sulfide in an anaerobic environment, and combine with heavy metal ions to generate metal sulfides for further removal, while degrading residual organic matter.

10. The application of the SRB remediation method for heavy metal wastewater treatment that can operate stably for a long period of time, as described in claim 9, is characterized in that... Also includes: (5) The treated effluent enters the secondary sedimentation tank to complete the mud-water separation, and the supernatant is discharged as the final effluent; Part of the settled sludge is returned to the primary sedimentation tank to enhance the front-end sedimentation effect, while the other part is returned to the UASB bioreactor as excess sludge to further enrich and remove residual heavy metals in the sludge, thereby achieving deep purification of heavy metals and sludge stabilization within the system.

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