A microbial-based wastewater treatment process

Abstract

This invention relates to a microbial-based wastewater treatment process, belonging to the field of wastewater biological treatment technology. The invention aims to solve the problems of poor tolerance of activated sludge to heavy metals and water quality fluctuations, low degradation efficiency of polycyclic aromatic hydrocarbons, and large amounts of hazardous waste generated in existing processes. Key technical points include: introducing wastewater containing recalcitrant organic matter and heavy metals into a reaction system, and treating it using a supported composite microbial preparation. This preparation includes a composite carrier made of iron-based biochar, chitosan-modified bentonite, etc., on which a gradient functional microbial membrane is attached: an outer layer is a biological barrier formed by heavy metal-resistant pioneer bacteria, and an inner layer is a core bacteria group for organic matter degradation. This invention is mainly used to treat industrial wastewater containing heavy metals and recalcitrant organic matter, and has the advantages of strong shock resistance, high pollutant removal rate, and significant sludge reduction.

Description

Technical Field

[0001] This invention relates to the field of biological wastewater treatment technology, and in particular to a wastewater treatment process based on microorganisms. Background Technology

[0002] With the intensive development of industrial parks, mixed wastewater typically exhibits the coexistence of recalcitrant organic matter (such as phenols and polycyclic aromatic hydrocarbons) and low to medium concentrations of heavy metal ions (such as copper, chromium, and lead). In current wastewater treatment projects, biological treatment technology is the primary means of degrading organic matter. However, existing biological treatment systems face a significant mechanistic contradiction when treating such mixed wastewater: heavy metal ions possess strong nucleophilic and sulfophilic properties, readily binding to the active sites of microbial enzymes and proteins, thus significantly inhibiting microbial metabolic activity and organic matter degradation efficiency. To address this issue, current engineering practices typically employ a two-stage process, adding a chemical precipitation pretreatment unit before the biological treatment stage to remove heavy metals by adding chemical agents. While this process can reduce the concentration of heavy metals entering the biological stage, it suffers from high operating costs and the generation of hazardous sludge containing heavy metals. Furthermore, when the concentration of heavy metals in the influent fluctuates, the dosage of chemical agents is difficult to control precisely. Overdosing can lead to a high pH in the effluent, while underdosing fails to effectively protect the subsequent microbial system. Existing technologies also employ immobilized microorganisms to improve system tolerance, such as using substrates like sodium alginate gel or polyurethane foam to physically encapsulate microorganisms. However, conventional carrier materials typically lack the specific chelating ability for heavy metals, allowing heavy metal ions to diffuse into the carrier through concentration gradients. Moreover, homogeneous carriers struggle to achieve spatial partitioning of different functional bacterial communities, leading to competition for nutrients between tolerant and highly efficient degrading strains within the same area, thus limiting overall degradation efficiency. Therefore, developing a wastewater treatment process that can effectively intercept heavy metals to reduce local toxicity and achieve synergistic degradation of organic matter through the spatial distribution of functional bacterial communities within a single biological treatment process has significant engineering practical value. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a microbial-based wastewater treatment process. This process introduces wastewater containing recalcitrant organic matter and heavy metals into a reaction system comprising a loaded composite microbial agent for biological treatment. The loaded composite microbial agent includes a composite carrier and a gradient-functional microbial membrane attached to the composite carrier. The composite carrier comprises iron-based biochar, chitosan-modified bentonite, and sodium alginate. The gradient-functional microbial membrane comprises heavy metal-tolerant pioneer bacteria colonizing the outer layer of the composite carrier and organic matter-degrading core bacteria colonizing the inner layer of the composite carrier. The reason for adopting this technical solution is that by spatially combining a composite material with adsorption and barrier functions with microbial communities possessing different metabolic functions, a decay gradient of heavy metal concentration can be formed radially along the carrier, thereby ensuring the resilience of the outer layer microorganisms while maintaining the highly efficient degradation activity of the inner layer microorganisms.

[0004] According to a preferred embodiment of the present invention, the raw materials for preparing the composite carrier, by weight, include: 30 to 40 parts of iron-based biochar, 20 to 30 parts of chitosan-modified bentonite, and 5 to 10 parts of sodium alginate. This ratio range is chosen because: if the iron-based biochar content is less than 30 parts, the complexation sites on the carrier surface are insufficient to rapidly intercept heavy metals in the aqueous phase; if it is greater than 40 parts, it will affect the continuity of the sodium alginate gel network, leading to a decrease in the mechanical strength of the carrier. If the chitosan-modified bentonite content is less than 20 parts, the cation exchange capacity of the system is insufficient; if it is greater than 30 parts, the excessive hydration and swelling of the bentonite will compress and close the gel pores, hindering substrate mass transfer. The sodium alginate content is controlled at 5 to 10 parts, which can maintain a suitable mass transfer porosity while ensuring the carrier's shape and strength. More preferably, the raw materials also include at least one selected from diatomaceous earth and corn starch. Diatomaceous earth plays a role in increasing pore size and enhancing mechanical strength; corn starch is a water-insoluble polysaccharide that is physically embedded in the sodium alginate gel network in the form of microparticles. It can be slowly hydrolyzed into glucose by the action of amylase secreted by microorganisms, serving as a slow-release carbon source embedded in the carrier to provide carbon nutrition support for colonizing microorganisms in the early stage of system startup.

[0005] Furthermore, the composite carrier consists of spherical particles with an average pore size of 20 μm to 80 μm; FeOOH nanoparticles are loaded onto the surface of the iron-based biochar; and the cation exchange capacity of the chitosan-modified bentonite is 120 cmol / kg to 150 cmol / kg. The reason for limiting the average pore size to 20 μm to 80 μm is that the size of microbial cells is typically 1-5 μm, and a pore size larger than 20 μm allows the inner layer of bacteria to enter and colonize smoothly; while a pore size smaller than 80 μm effectively restricts the inflow of external water into the carrier to generate macroscopic convection, ensuring that heavy metal ions mainly enter through diffusion and are subject to mass transfer resistance from the gel framework. The FeOOH nanoparticles can utilize their surface hydroxyl groups to form an inner spherical complex with heavy metals; the cation exchange capacity of 120-150 cmol / kg ensures the deep exchange and chelation capacity of the chitosan-modified bentonite for heavy metal cations.

[0006] In the process of this invention, the heavy metal-tolerant pioneer bacteria include at least one of Bacillus subtilis, Bacillus megaterium, and Pseudomonas putida; the organic matter-degrading core bacteria include at least one of Rhodococcus rubrum, Sphingosine monocytogenes, Paracoccus denitrifyingis, and Xanthomonas xanthogenes. These pioneer bacteria colonize the outer layer of the composite carrier up to a depth of 500 μm, forming an extracellular polymer matrix layer as a biological barrier. The organic matter-degrading core bacteria colonize the inner layer of the composite carrier at a depth of 500 μm to 1500 μm. The reason for using 500 μm as the spatial boundary depth is that, according to diffusion kinetics, the concentration of heavy metals in the external aqueous phase, after penetrating the outer chelating material and the extracellular polymer layer, has decreased to a safe range tolerable by the core bacteria at a depth of 500 μm. If the outer layer is less than 500 μm deep, the heavy metal concentration attenuation is insufficient, which can easily harm the inner bacterial community. If the outer layer is too thick (greater than 500 μm), it will increase the resistance to the transfer of dissolved oxygen and organic substrates to the inner layer, affecting the degradation efficiency of the inner bacterial community. It should be noted that the aforementioned 500 μm boundary depth is not a rigid boundary, but a transitional region naturally formed under the concentration gradient driven by the two-step inoculation method. The actual bacterial community distribution exhibits a continuous gradient characteristic in the radial direction, with an alternating coexistence zone of pioneer and core bacterial communities within a range of approximately ±100 μm centered at 500 μm.

[0007] To achieve the aforementioned gradient distribution, the gradient functional microbial membrane is prepared using a two-step inoculation method, "inside first, then outside," which includes: First, immersing the composite carrier in a first suspension containing organic matter-degrading core bacteria for slow-speed shaking and soaking, allowing the core bacteria to penetrate into the inner pores of the carrier and colonize; after intermediate rinsing, second, immersing the carrier in a second suspension containing heavy metal-tolerant pioneer bacteria for micro-aeration and soaking, allowing the pioneer bacteria to colonize the outer layer of the carrier and secrete extracellular polymers to form a biological barrier layer; and the cell density of the second suspension is greater than that of the first suspension. The reason for adopting the "inner-to-outer" sequence is as follows: In the first inoculation step, the outer pores of the carrier are not yet blocked by EPS and are in an open state. The micron-sized cells (1-5μm) of the core bacterial community can smoothly pass through the 20-80μm pores and enter the inner layer (500-1500μm depth) for colonization under the drive of slow oscillation. If the outer pioneer bacterial community is inoculated first, the EPS secreted by it will occupy and block the outer pores in large quantities, forming a dense biological barrier. The micron-sized cells of the core bacterial community will not be able to penetrate this barrier to enter the interior by slow oscillation alone. In the second inoculation step, the high-density pioneer bacterial community suspension allows the pioneer bacterial community to quickly occupy the outer surface of the carrier and the outer macropores, and vigorously secrete EPS under micro-aeration aerobic conditions, forming a continuous biological barrier on the outer layer. At this time, the inner core bacterial community has completed colonization and established an initial biofilm, and will not be disturbed by the outer inoculation process.

[0008] In terms of system-level process flow, the reaction system includes a bio-adsorption section, an anaerobic hydrolysis acidification section, an anoxic denitrification section, and an aerobic biological contact oxidation section connected in series. The supported composite microbial preparation is placed in the bio-adsorption section, the anaerobic hydrolysis acidification section, and the aerobic biological contact oxidation section. The process sequence follows a standard pre-denitrification (A / O) layout: the effluent from the anaerobic hydrolysis acidification section first enters the anoxic denitrification section for denitrification, and then enters the aerobic biological contact oxidation section for organic matter mineralization and ammonia nitrification; the nitrate-containing effluent from the aerobic section is returned to the inlet of the anoxic section through an internal reflux pipeline, achieving circulating denitrification of the nitrified liquid. In the bio-adsorption section, dissolved oxygen is controlled at 0.5-1.5 mg / L, and the hydraulic retention time is 3-4 hours; this stage mainly utilizes the material properties of the carrier to rapidly remove most of the free heavy metals in the aqueous phase. The anaerobic hydrolysis and acidification section employs an upflow anaerobic biofilm reactor. The volumetric filling rate of the supported composite microbial preparation is 60%-70%. The reactor operates in expanded bed mode, with the bed expansion rate controlled at 10%-20% to reduce the static pressure load between carriers and improve mass transfer conditions. Dissolved oxygen is controlled to be less than 0.2 mg / L, and the hydraulic retention time is 10-14 hours. The reason for choosing a hydraulic retention time of 10-14 hours is that the hydrolysis and chain breaking of recalcitrant macromolecular organic matter and the ring opening of polycyclic compounds require a longer reaction time. Simultaneously, this time window ensures the accumulation of acid produced during fermentation and sufficient sulfur ions generated by sulfate-reducing bacteria (SRB). In the anoxic denitrification section, dissolved oxygen is controlled to be less than 0.5 mg / L, the hydraulic retention time is 4-6 hours, and sodium acetate is supplemented as a carbon source according to a preset carbon-to-nitrogen ratio. Denitrifying bacteria remove NO3 from the nitrified liquor returned from the aerobic section. - -N is reduced to N2, completing the removal of total nitrogen. In the aerobic biological contact oxidation section, dissolved oxygen is controlled at 2.5-4.0 mg / L, and the hydraulic retention time is 8-12 hours to ensure complete mineralization of small molecule organic matter and nitrification of ammonia nitrogen. The effluent from the aerobic biological contact oxidation section is connected to the inlet of the anoxic denitrification section via an internal reflux pipeline, with an internal reflux ratio of 200%-300% (based on the system influent flow rate), to remove NO3 produced by nitrification in the aerobic section. - -N is recirculated to the anoxic zone for denitrification. In a pre-denitrification system, the anoxic zone precedes the aerobic zone, and the NO3 in the effluent from the aerobic zone is... - -N must return to the anoxic zone through the internal reflux pipeline; therefore, the internal reflux ratio directly determines the upper limit of total nitrogen removal efficiency. Under an internal reflux ratio of 200%-300%, the theoretical upper limit of total nitrogen removal rate is approximately 67%-75%. Combined with the contribution of ammoniation in the anaerobic zone of the system and the partial simultaneous nitrification and denitrification, an overall TN removal rate of 75%-80% can be achieved.

[0009] Furthermore, the process of this invention also includes an in-situ self-regeneration step of the carrier. The mechanism is as follows: In the anaerobic hydrolysis acidification stage, the fermentation metabolism of the pioneer bacteria produces organic acids (acetic acid, propionic acid, butyric acid, etc.). Due to the restriction of convective exchange by the gel channels, the organic acids accumulate locally in a micro-region of several hundred micrometers on the outer layer of the carrier, causing the pH of this micro-region to decrease to 5.5-6.5. Under this weakly acidic microenvironment, protonation occurs on the FeOOH surface (≡FeOM). + +H + →≡FeOH2 + +M 2+ The stability of chitosan amino chelate complexes decreases (-NH2+H). + →-NH3 + This process causes acidic desorption of heavy metal ions complexed on the carrier surface, releasing them into a free state. According to mass transfer kinetics, the local concentration of these released free heavy metal ions on the carrier's outer surface is higher than that in the bulk aqueous phase. Therefore, they diffuse outwards along the concentration gradient into the bulk aqueous phase outside the carrier—a diffusion direction that conforms to Fick's law. Simultaneously, in the anaerobic sludge layer at the bottom of the reactor, sulfate-reducing bacteria (SRB) reduce the sulfate added to the influent to sulfur dioxide (S). 2- (SO4) 2- +8H + +8e - →S 2- +4H2O), producing S 2- It diffuses into the liquid phase of the reactor. Due to S 2- With Cu 2+ Pb 2+ The reaction of heavy metal ions has an extremely low solubility product constant (CuS, Ksp≈6×10). -37 PbS, Ksp≈3×10 -28 ), S 2- Upon encountering free heavy metal ions desorbed from the carrier surface into the liquid phase in the reactor liquid phase, a precipitation reaction immediately occurs, forming metal sulfide microcrystals. These microcrystals are then carried by the slow upward flow within the reactor and settle to the bottom sludge layer where they are retained. This process involves: acidic desorption from the carrier surface → diffusion of free heavy metals into the liquid phase → sulfur in the liquid phase. 2- The process follows a path of "precipitation capture → microcrystal settling to the bottom sludge layer," allowing the adsorption sites freed up on the outer layer of the carrier due to heavy metal desorption to be restored in situ, thus achieving self-regeneration of the carrier. It should be noted that the mass transfer pathway of heavy metals during this self-regeneration process completely follows the diffusion law from high concentration to low concentration: desorbed heavy metals diffuse outward (carrier surface → aqueous phase), S 2- The sludge diffuses upwards (from the bottom sludge layer to the aqueous phase), where the two react and combine in the liquid phase. Sodium sulfate is added to the influent to bring the SO4 level to [a certain level]. 2- The concentration is approximately 200-500 mg / L to maintain the supply of SRB metabolic substrates.

[0010] Regarding the feasibility of this pH reduction, the following points need to be clarified: Although the amino groups of chitosan (pKa approximately 6.3-6.5) and the carboxyl groups of sodium alginate (pKa approximately 3.2-3.5) in the carrier constitute a certain pH buffer system, under anaerobic fermentation conditions, the acid production rate of VFAs (total concentration 800-1500 mg / L) continuously produced by the pioneer bacteria is much higher than the neutralization rate of the buffer system. More importantly, the gel pore structure creates mass transfer resistance to the exchange of substances inside and outside the carrier, limiting the dilution and scouring of organic acids on the outer layer of the carrier by the main water flow, resulting in the local accumulation of organic acids in micro-regions of several hundred micrometers on the outer layer of the carrier. Measurements showed that the pH of the rinsing solution on the outer surface of the carrier during stable operation was 5.8 ± 0.3, consistent with the 5.5-6.5 range described in this invention. Furthermore, this weakly acidic range (pH 5.5-6.5) is much higher than the critical pH value (approximately 4.0) for sodium alginate gel shrinkage; therefore, the gel skeleton and pore structure of the carrier will not change significantly under these conditions.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention presents a microbial-based wastewater treatment process that effectively solves the technical challenge of microbial toxicity in wastewater treatment where heavy metals and recalcitrant organic matter coexist. This process utilizes the surface complexation of iron-based biochar, the cation exchange and chelation of chitosan-modified bentonite, and the diffusion resistance of a sodium alginate gel network to construct a heavy metal concentration attenuation gradient from the outside to the inside of the carrier. Simultaneously, extracellular polymers secreted by pioneer bacteria colonizing the outer layer further provide biological-level heavy metal binding sites. The synergistic effect of these physical, chemical, and biological mechanisms creates a stable microenvironment with low heavy metal concentrations within the carrier's inner layer, ensuring the efficient degradation activity of the core microbial community for recalcitrant organic matter such as polycyclic aromatic hydrocarbons. This achieves simultaneous interception of heavy metals and removal of organic matter in a single biological reaction system, avoiding the high reagent costs and hazardous sludge generation associated with traditional chemical precipitation pretreatment. Furthermore, this invention utilizes the organic acids produced by microbial metabolism in the anaerobic stage to promote the desorption of heavy metals from the carrier surface. The desorbed heavy metals diffuse down the concentration gradient into the liquid phase, where they are precipitated and captured as stable metal sulfides by sulfide ions produced by sulfate-reducing bacteria at the bottom of the reactor. This achieves in-situ self-regeneration of the carrier's adsorption sites. This mechanism significantly extends the service life of the composite carrier, reduces the frequency of material replacement, and improves the overall process stability and economic efficiency in response to fluctuations in influent load. Detailed Implementation

[0012] The technical solution of the present invention will be described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0013] This invention provides a microbial-based wastewater treatment process, the core of which lies in constructing a supported composite microbial agent and applying this agent to the comprehensive wastewater treatment of industrial parks containing both recalcitrant organic matter and heavy metals. The technical principle of this process is based on the following synergistic mechanism: Regarding the heavy metal interception mechanism at the material level, the composite carrier is composed of three functional materials: iron-based biochar, chitosan-modified bentonite, and sodium alginate. Among them, the FeOOH nanoparticles grown in situ on the surface of the iron-based biochar provide abundant surface hydroxyl groups (≡FeOH), which can bind with Cu through an internal spheroid complexation mechanism. 2+ Pb 2+ Cr 3+ Equal heavy metal ions form stable monodentate or bidentate coordination bonds (≡FeO-M) + The reaction equation is: ≡FeOH + M 2+ →≡FeOM + +H + The presence of the FeOOH phase on the surface of iron-based biochar was confirmed by X-ray diffraction (XRD) analysis, which detected characteristic diffraction peaks of α-FeOOH (goethite) at 2θ = 21.2°, 33.2°, and 36.6°. Fourier transform infrared spectroscopy (FTIR) at 890 cm⁻¹ further confirmed the presence of the phase. -1 and 795cm -1 The Fe-OH bending vibration absorption peak further corroborates the loading of FeOOH. In chitosan-modified bentonite, the amino (-NH2) and hydroxyl (-OH) groups on the chitosan molecular chain work synergistically with the cation exchange sites between the bentonite layers, not only capturing heavy metal cations in the aqueous phase through electrostatic attraction and ion exchange, but also forming stable five-membered ring complexes through chelation. Sodium alginate, after being cross-linked with CaCl2, forms a three-dimensional gel network. The carboxyl and hydroxyl groups on its polymer chain can further complex heavy metals. At the same time, the microporous structure of the gel generates mass transfer resistance to the diffusion of heavy metal ions, forming a concentration decay gradient from the outer surface of the carrier to the interior.

[0014] Regarding the spatial stratification and synergistic degradation mechanism at the microbial level, a two-step inoculation method ("inner-outer") was employed. Organic matter-degrading core bacteria (Rhodococcus rhodochrous, Sphingomonas aspacimobilis, Paracoccus denitrificans, and Cellulomonas flavipenata) were first colonized in the inner layer region (approximately 500–1500 μm). Subsequently, heavy metal-tolerant pioneer bacteria (Bacillus subtilis, Bacillus megaterium, and Pseudomonas putida) were colonized on the surface of the composite carrier in the outer layer region (approximately 500 μm depth). These pioneer bacteria exhibit strong heavy metal tolerance; their secreted extracellular polymeric substances (EPS) contain numerous carboxyl, phosphate, and thiol functional groups, forming a biological barrier layer that further adsorbs and immobilizes residual heavy metal ions that penetrate the material layer. After being blocked by both the material layer and the EPS biological barrier layer, the heavy metal concentration at a depth of approximately 500 μm in the inner layer of the carrier has decreased to below the safe threshold that the core microbial community can tolerate (typically Cu). 2+ <0.5mg / L, Pb 2+ <0.2mg / L), enabling the inner organic matter-degrading core bacterial community to maintain high metabolic activity, and to achieve ring-opening, chain breaking and final mineralization of recalcitrant organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and phenols through key enzyme systems such as dioxygenases and dehalogenases.

[0015] Regarding the in-situ self-regeneration mechanism of the carrier, in the anaerobic hydrolysis and acidification stage, the pioneer bacteria colonizing the outer layer of the carrier ferment and metabolize organic acids (acetic acid, propionic acid, butyric acid, etc.) under anaerobic conditions. Due to the mass transfer resistance formed by the gel channel structure on the exchange of substances inside and outside the carrier, organic acids accumulate locally in a micro-region of several hundred micrometers on the outer layer of the carrier, causing the pH of the micro-region on the outer layer of the carrier to drop to 5.5~6.5. In this weakly acidic microenvironment, protonation occurs on the FeOOH surface, and the coordination bond breaks (≡FeOM + +H + →≡FeOH2 + +M 2+ ), chitosan amino protonation (-NH2+H) + →-NH3 +This process leads to the dissociation of chelated complexes, causing heavy metal ions to desorb from the carrier surface into a free state. The local concentration of these free heavy metal ions on the carrier's outer surface is higher than the concentration in the bulk aqueous phase. Therefore, according to Fick's diffusion law, they diffuse outwards along the concentration gradient into the bulk liquid phase of the reactor. Simultaneously, in the anaerobic sludge layer at the bottom of the reactor, sulfate-reducing bacteria (SRBs, such as *Desulfovibrio*) reduce the sulfate added to the influent to sulfur dioxide (S). 2- (SO4) 2- +8H + +8e - →S 2- +4H2O), producing S 2- It diffuses upwards into the liquid phase of the reactor. 2- When free heavy metal ions desorbed from the surface of each carrier particle into the liquid phase meet in the reactor liquid phase, the solubility of metal sulfides is extremely low (e.g., CuS, Ksp≈6×10⁻⁶). -37 PbS, Ksp≈3×10 -28 The two react immediately to form a metal sulfide microcrystalline precipitate. The density of the formed microcrystalline particles is greater than that of water (e.g., CuS density is approximately 4.6 g / cm³). 3 The sludge naturally settles at the bottom of the expanded bed in an area with a lower upflow velocity and is trapped in the sludge layer at the bottom of the reactor. As a result, the adsorption sites freed up by acidic desorption on the outer layer of the carrier are restored in situ. In this self-regeneration process, the mass transfer direction of all substances follows a diffusion law from high concentration to low concentration: desorbed heavy metals diffuse from the carrier surface (high concentration) to the aqueous phase (low concentration), S... 2- Diffusion occurs from the bottom sludge layer (high concentration) to the upper liquid phase (low concentration), where the two react and combine. Additionally, a small amount of untreated sludge... 2- When the free heavy metal ions that are precipitated in time migrate upward with the rising water flow, they are recaptured by the fresh outer adsorption sites of the carrier particles in the upper layers, forming a secondary interception barrier and further ensuring the quality of the effluent.

[0016] The following describes the specifications and sources of the main raw materials involved in this invention.

[0017] Iron-based biochar was prepared by pyrolysis at 600°C for 2 hours under a nitrogen atmosphere, using rice husks as the carbon source and FeCl3·6H2O as the iron precursor, with a carbon / iron mass ratio of 3:1. The resulting iron-based biochar had a BET specific surface area of ​​280–350 m² / g. 2 / g, with FeOOH nanoparticles having a particle size of 5~15nm. The pyrolysis product was repeatedly washed five times with deionized water to remove residual soluble chlorides. The washing solution was tested with AgNO3 until no white precipitate was formed, ensuring that the residual chloride ion content during the carrier preparation process did not affect the subsequent adsorption performance of FeOOH for heavy metals. Rice husks were purchased from Jiangsu Nantong Tianhua Agricultural Development Co., Ltd.; FeCl3·6H2O (analytical grade, ≥99.0%) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0018] Chitosan-modified bentonite was prepared by reacting sodium bentonite (montmorillonite content ≥90%, Hebei Lingshou Huayuan Mineral Products Processing Plant) as the matrix and chitosan (degree of deacetylation ≥85%, viscosity-average molecular weight 300,000-500,000, Shanghai Aladdin Biochemical Technology Co., Ltd.) as the modifier in an acetic acid solution at pH 4.5 at a mass ratio of 8:2, stirring at 50°C for 4 hours, followed by filtration and drying. The cation exchange capacity (CEC) of the modified chitosan-modified bentonite was 120-150 cmol / kg.

[0019] Sodium alginate is food grade, with a viscosity (1% w / v aqueous solution, 25°C) of 200~400 mPa·s and an M / G ratio of approximately 1.2~1.6. It was purchased from Qingdao Mingyue Algae Group Co., Ltd.

[0020] Other auxiliary materials include: diatomaceous earth (200 mesh, Jiangsu Yancheng Diatomaceous Earth Mining Co., Ltd.); corn starch (food grade, Shandong Zhucheng Xingmao Corn Development Co., Ltd.). It should be noted that the original design proposed adding water-soluble nutrients such as potassium dihydrogen phosphate, peptone, glucose, and a mixture of trace elements during the carrier preparation stage. However, considering the highly hydrophilic and porous nature of sodium alginate gel, these highly water-soluble small molecules would be largely lost during cross-linking bath soaking (12-24 hours) and subsequent washing due to concentration-driven dialysis, and thus could not be effectively retained in the finished carrier. Therefore, this invention ultimately retains only water-insoluble corn starch and diatomaceous earth as embedded components in the carrier—corn starch particles (average particle size approximately 15 μm) are physically embedded in the gel network as solid microparticles. Their water insolubility ensures a high retention rate during cross-linking and washing (measured retention rate >85%). Subsequently, under the action of α-amylase secreted by microorganisms, glucose can be slowly hydrolyzed and released, thus acting as a slow-release carbon source. Other soluble nutrients (phosphorus source, nitrogen source, trace elements) were instead supplied through external culture medium during the acclimatization and stabilization period of the two-step inoculation.

[0021] The crosslinking agent was CaCl2 (analytical grade, ≥96.0%, Sinopharm Chemical Reagent Co., Ltd.), prepared as a 3% w / v CaCl2 aqueous solution as the crosslinking bath for sodium alginate gel.

[0022] The following bacterial strains used in this invention have been deposited at the China Center for Type Culture Collection (CCTCC). The preservation information for each strain is as follows: Bacillus subtilis, accession number CCTCFB2024306; Bacillus megaterium, accession number CCTCAB209224; Pseudomonas putida, accession number CCTCS2024175; Cellulomonas flavigena, accession number CCTCAB92020.

[0023] The following strains have been deposited at the China General Microbiological Culture Collection Center (CGMCC): *Rhodococcus rhodochrous*, accession number CGMCC1.2362; *Sphingomonaspaucimobilis*, accession number CGMCCNo.7783; and *Paracoccus denitrificans*, accession number CGMCCNo.12633.

[0024] The sulfate-reducing bacteria (SRB) used during the start-up of the anaerobic hydrolysis acidification reactor was Desulfovibriodesulfuricans, preservation number CGMCC1.5197. The inoculum was prepared after anaerobic culture at 30°C for 48-72 hours in Postgate C culture to the logarithmic growth phase.

[0025] The preparation method of the supported compound microbial preparation is described below.

[0026] The preparation process of the composite carrier is as follows. Step 1: Iron-based biochar is ground through a 100-mesh sieve and dry-mixed with chitosan-modified bentonite according to the formula ratio. Diatomaceous earth is added as a pore-forming agent if necessary to obtain a solid-phase mixture. Step 2: Sodium alginate is dissolved in deionized water to prepare a 2-4% w / v sodium alginate solution. The solution is stirred continuously in a 50°C water bath for 30 minutes until fully swollen. The solid-phase mixture obtained in Step 1 is added in batches to the sodium alginate solution, and stirred evenly at a solid-liquid ratio of 1:3-1:5 (g / mL) to form a slurry. Simultaneously, corn starch is added to the slurry according to the formula and mixed evenly (corn starch is dispersed in the slurry as solid particles, which are retained during subsequent cross-linking and washing processes because they are insoluble in water). Step 3: Using a dropper method, the above slurry is dripped into a 3% w / v CaCl2 crosslinking bath at a flow rate of 2-5 mL / min via a peristaltic pump. The inner diameter of the dropper is controlled at 3-4 mm, the liquid level difference at 10-15 cm, and free fall is allowed to form spheres. The crosslinking and curing time is 12-24 h, and the crosslinking temperature is 4-10°C. Step 4: The crosslinked and cured spherical particles are removed and washed three times with sterile deionized water to remove residual CaCl2. They are then placed in a forced-air drying oven and dried at 30°C to constant weight (moisture content <10%), yielding spherical composite carriers with an average particle size of 4-6 mm. Scanning electron microscopy (SEM) characterization shows that the average pore size of the obtained carrier is 20-80 μm, exhibiting a connected hierarchical pore structure.

[0027] The construction of gradient functional microbial membranes employed a two-step inoculation method, proceeding from the inside out. The first step involved inoculating the inner layer of the core microbial community: *Rhodococcus rubrum*, *Sphingosomalidone*, *Paradiazepam*, and *Fibromosynostae* were cultured separately in nutrient broth at 30°C and 150 rpm aerobic for 36–48 h until the logarithmic growth phase. The cells were collected, washed with sterile physiological saline, resuspended, and mixed in a volume ratio of 1:1:1:1 to prepare the first suspension. The total cell density was adjusted to OD0.05. 600 ≈1.0~2.0 (approximately 1×10) 9 ~2×10 9 CFU / mL); The dried composite carrier was immersed in the first suspension at a carrier-to-bacterial volume ratio of 1:4, and subjected to slow shaking (60-80 rpm) at 25-28°C for 48-72 hours. During this stage, the outer pores of the carrier were not yet blocked by EPS and remained completely open. The micron-sized cells (1-5 μm) of the core bacterial community could smoothly penetrate the 20-80 μm pores and colonize the inner pores of the carrier at a depth of approximately 500-1500 μm, driven by the gentle liquid flow generated by the slow shaking. A lower cell density (OD) was used. 600 The concentration of ≈1.0~2.0 is to prevent the core microbial community from excessively attaching to the outer layer, so that it preferentially penetrates deeper into the pores under the drive of the concentration gradient.

[0028] After the first step is completed, the carrier is removed from the first suspension and gently rinsed with sterile physiological saline (rinsing flow rate 1-2 mL / min / carrier, lasting 5-10 min) to remove the loosely attached core bacterial cells and the residual first suspension in the pores on the outer surface of the carrier, but retains the core bacterial population that has penetrated into the inner pores and begun to attach.

[0029] The second step is the outer layer inoculation of the pioneer bacteria: Bacillus subtilis, Bacillus megaterium, and Pseudomonas putida are cultured separately in LB medium at 30°C and 160 rpm aerobic for 24 hours until the late logarithmic growth phase (OD). 600 (≈1.5~2.0), collect bacterial cells, wash with sterile physiological saline and resuspend, mix at a volume ratio of 1:1:1 to prepare a second suspension, and adjust the total cell density to OD. 600 ≈5.0 (approximately 5 × 10) 9 The cell density in the second suspension was higher than that in the first suspension (CFU / mL). The composite carrier, after rinsing and completing the first inoculation step, was immersed in the second suspension at a carrier-to-bacterial volume ratio of 1:3. Micro-aeration (0.1-0.3 vvm) was applied at 25-30°C for 24-36 hours. The high-density pioneer bacteria rapidly occupied the outer surface and outer pores (0-500 μm depth) of the carrier. Micro-aeration maintained aerobic conditions, promoting vigorous secretion of EPS by the pioneer bacteria, forming a continuous biological barrier layer on the outer surface. Since the core bacteria had already established an initial biofilm anchorage in the inner pores during the first step, the second outer layer inoculation process did not disturb the already colonized core bacteria in the inner layer.

[0030] After completing the two-step inoculation, the preparation was transferred to sterile physiological saline containing a small amount of glucose (0.5 g / L), potassium dihydrogen phosphate (0.1 g / L), and a trace element mixture (1 mL / L). It was then statically cultured at 25°C for 3–5 days as an acclimatization and stabilization period. During this stage, soluble nutrients such as phosphorus, nitrogen, and trace elements were provided to the microorganisms through the external culture medium, allowing the two layers of microorganisms to form a stable biofilm structure, thus obtaining the loaded composite microbial preparation. CLSM-FISH characterization showed that the pioneer bacteria (Cy3-labeled, red fluorescence) were mainly distributed on the carrier surface at a depth of approximately 500 μm, while the core bacteria (FITC-labeled, green fluorescence) were mainly distributed at a depth of approximately 500–1500 μm. The two types coexisted interleaved at a depth of approximately 400–600 μm, confirming the effective formation of the gradient stratified structure.

[0031] The following describes the system-level process flow. The reaction system of this invention includes four treatment sections connected in series. The biosorption section uses a fixed-bed reactor, with the supported composite microbial preparation randomly packed at a filling rate of approximately 50%–60%. Dissolved oxygen (DO) is controlled at 0.5–1.5 mg / L (micro-aerobic), and the hydraulic retention time (HRT) is 3–4 h. This stage mainly utilizes the adsorption and complexation functions of the carrier material to rapidly retain most of the free heavy metal ions in the aqueous phase. At the same time, the EPS of the pioneer bacteria also participates in the biosorption of heavy metals. After treatment in this stage, the concentration of free heavy metals in the aqueous phase can be reduced by 60%–80%. The anaerobic hydrolysis acidification section employs an upflow anaerobic biofilm reactor (UAFBR). The volumetric filling rate of the supported composite microbial preparation is 60%–70%. The reactor operates in expanded bed mode, maintaining the bed expansion rate at 10%–20% by controlling the upflow velocity. This avoids excessive mechanical stress caused by static pressure accumulation between carrier particles and enhances liquid-solid mass transfer efficiency. DO is controlled to <0.2 mg / L (strict anaerobic), and the HRT is 10–14 h. This section has a dual function: firstly, anaerobic conditions… Facultative and obligate anaerobic bacteria hydrolyze and break down chain-linking and ring-opening reactions of recalcitrant macromolecular organics, converting macromolecules such as polycyclic aromatic hydrocarbons and long-chain alkanes into small-molecule organic acids (VFAs) and alcohols as intermediate products. On the other hand, the organic acids produced by the pioneer bacteria during fermentation lower the pH of the outer microregion of the carrier to 5.5–6.5, triggering the acidic desorption of complexed heavy metals on the carrier surface. The released free heavy metal ions diffuse down their concentration gradient into the bulk liquid phase of the reactor, where they are metabolized by SRB at the bottom of the reactor. 2- Metal sulfide microcrystals (such as CuS, PbS, Cr2S3) are precipitated and captured in the liquid phase. These microcrystals settle into the bottom sludge layer, achieving in-situ self-regeneration of the carrier adsorption sites. Sodium sulfate is added to the influent to bring the SO4 level to a minimum. 2- The concentration is approximately 200–500 mg / L to maintain the supply of SRB metabolic substrates. During reactor startup in the anaerobic hydrolysis and acidification section, a desulfurization Vibrio enrichment culture is inoculated into the bottom of the reactor (inoculation density approximately 10). 8 With an inoculum size of CFU / mL, the inoculum accounts for 10%-15% of the effective volume of the sludge layer at the bottom of the reactor. SRB independently colonize and metabolize in the anaerobic sludge layer at the bottom of the reactor (pH 6.5-7.5, an acidic micro-region far from the outer layer of the carrier), without needing to enter the carrier interior to compete for space with the core bacterial community. The anoxic denitrification section uses an anoxic stirred tank, with suspended packing material added as the carrier for denitrifying bacteria. DO is controlled to <0.5 mg / L, and HRT is 4-6 h, based on the C / N ratio (in terms of COD / NO3). - Sodium acetate is added as an external carbon source at a ratio of 5-7:1 (based on nitrogen content), while VFAs produced in the anaerobic hydrolysis acidification stage are used as a partial carbon source. Denitrifying bacteria are used to reflux NO3- from the nitrification liquor in the aerobic stage. --N is reduced to N2. The aerobic biological contact oxidation section uses a contact oxidation reactor, loaded with a supported composite microbial agent and commercial combined packing material. DO is controlled at 2.5~4.0 mg / L, and HRT is 8~12 h. This section mainly relies on aerobic heterotrophic bacteria to completely oxidize and mineralize the small molecule organic matter produced in the upstream sections into CO2 and H2O. Simultaneously, autotrophic nitrifying bacteria reduce NH4+ to N2. + -N is converted to NO3 through nitrosation and nitration. - -N, the organic matter-degrading core bacterial community in the inner layer of the loaded compound microbial preparation plays a crucial role in this section, especially *Rhodococcus rubrum* and *Sphingomonas oligosporus*, which deeply degrade residual PAHs and phenols. The effluent from the aerobic biological contact oxidation section is connected to the inlet of the anoxic denitrification section via an internal reflux pipeline, with an internal reflux ratio of 200%-300% (based on the system influent flow rate). In the pre-denitrification (A / O) layout of this invention, the anoxic section is located before the aerobic section, and the NO3-containing components produced in the aerobic section... - -N nitrified liquor cannot automatically reach the anoxic zone via the forward flow; it must be pumped back to the anoxic zone inlet via an internal reflux pipeline to achieve denitrification. This is precisely the necessity of internal reflux and a core feature distinguishing pre-denitrification from post-denitrification processes. Under an internal reflux ratio of 200%-300%, the theoretical upper limit of total nitrogen removal is approximately 67%-75%. Combined with the contribution of ammoniation in the anaerobic zone and partial simultaneous nitrification and denitrification, an overall TN removal rate of 75%-80% can be achieved.

[0032] The performance testing methods are described below. COD determination was performed using the potassium dichromate method, in accordance with GB / T11914-1989. Total copper, total lead, and total chromium concentrations were determined using inductively coupled plasma optical emission spectrometry (ICP-OES), in accordance with HJ776-2015, using a PerkinElmer Optima 8300 instrument. Polycyclic aromatic hydrocarbon (PAHs, represented by naphthalene) concentrations were determined using liquid-liquid extraction-high performance liquid chromatography (HPLC), in accordance with HJ478-2009. Volatile phenol concentrations were determined using the 4-aminoantipyrine spectrophotometric method, in accordance with HJ503-2009. Ammonia nitrogen was determined using Nessler's reagent spectrophotometry, in accordance with HJ535-2009. Total nitrogen was determined using alkaline potassium persulfate digestion ultraviolet spectrophotometry, in accordance with HJ636-2012. pH values ​​were measured using the glass electrode method, in accordance with GB / T6920-1986. The mechanical strength of the carrier was determined using the single-sphere crushing method. A WDW-5 microcomputer-controlled electronic universal testing machine was used to compress spherical carriers at a speed of 1 mm / min until they broke, and the maximum load was recorded. Twenty carriers were tested in each group, and the average value was taken. The specific surface area and pore size of the carrier were analyzed using the N2 adsorption-desorption method. The instrument was a Micromeritics ASAP2460. The specific surface area was calculated using the BET method, and the pore size distribution was calculated using the BJH method. Regarding the evaluation of the carrier's regeneration performance, under continuous operation conditions, samples were taken every 30 days to measure the carrier's effect on Cu. 2+ The adsorption capacity (mg / g) was calculated, and the relative adsorption capacity retention rate was calculated based on the initial adsorption capacity. Microbial activity was evaluated using the dehydrogenase activity (DHA) method, with triphenyltetrazolium chloride (TTC) as the electron acceptor, according to GB / T36866-2018. Spatial distribution verification of the bacterial community was performed using a laser confocal microscope (CLSM, Zeiss LSM880) combined with fluorescence in situ hybridization (FISH). After radially freezing sections of the carrier, specific 16S rRNA probes targeting pioneer and core bacterial groups were used for fluorescent labeling, and the bacterial distribution density at different depths was observed and recorded. In situ pH determination of the outer microregion of the carrier was performed using a microelectrode method (Unisense pH-100 microelectrode, 100 μm tip diameter). During stable operation in the anaerobic hydrolysis acidification section, the microelectrode was inserted radially along the carrier in 100 μm increments, and the pH distribution at different depths was recorded.

[0033] S in the liquid phase of the reactor 2- Concentration determination was performed using the methylene blue spectrophotometric method, in accordance with HJ / T 60-2000. X-ray diffraction (XRD) analysis was used to identify metal sulfides in the sludge layer at the bottom of the reactor to confirm the formation of mineral phases such as CuS and PbS.

[0034] The simulated wastewater was prepared using tap water as a base, with the addition of phenol (100 mg / L), naphthalene (20 mg / L), glucose (appropriate amount to adjust total COD), and NH4Cl (40 mg / L as NH4). + (calculated as -N), KNO3 (30 mg / L as NO3) - The simulated wastewater contains (calculated as -N), CuSO4·5H2O, Pb(NO3)2, and CrCl3·6H2O (to adjust the concentration of each heavy metal), and the pH is adjusted to 6.5~7.0 with NaOH / HCl. Unless otherwise specified, the basic indicators of the simulated wastewater are: COD 800~1000 mg / L, Cu 2+ 10 mg / L, Pb 2+ 5 mg / L, Cr 3+ 3 mg / L, NH4 + -N 40 mg / L, TN 70 mg / L, phenol 100 mg / L, naphthalene 20 mg / L.

[0035] Regarding experimental conditions, unless otherwise specified, all examples and comparative examples were conducted in a laboratory-scale continuous flow reaction system (effective volume 50 L). The system was allowed a 30-day start-up and acclimatization period, followed by continuous monitoring for 60 days after reaching stable operation. The average value during the stable operation period was taken as the test result. The water temperature was controlled at 25±2°C.

[0036] Example 1 The composite carrier formulation, by weight parts, consists of: 35 parts iron-based biochar, 25 parts chitosan-modified bentonite, 8 parts sodium alginate, 5 parts diatomaceous earth, and 3 parts corn starch. Water-soluble substances such as potassium dihydrogen phosphate, peptone, glucose, and a mixture of trace elements are not added during the carrier preparation stage. The preparation method follows the above procedure, with a sodium alginate solution concentration of 3% w / v, an dropper inner diameter of 3.5 mm, a cross-linking time of 18 h, and a cross-linking temperature of 6°C. Parameters for the "inner-outer" two-step inoculation method: OD of the first suspension (core bacterial group)... 600 =1.5, shake at 70 rpm, soak for 60 h; intermediate rinse: rinse with sterile physiological saline at a flow rate of 1.5 mL / min / particle for 8 min; second suspension (Pioneer bacteria) OD 600 =5.0, micro-aeration 0.2vvm, soaking for 30h; acclimatization stabilization period 4d (culture medium contains glucose 0.5g / L, KH2PO4 0.1g / L, trace elements 1mL / L). Process parameters: biosorption section HRT=3.5h, DO=1.0mg / L; anaerobic hydrolysis acidification section HRT=12h, DO<0.2mg / L, filling rate 65%, bed expansion rate 15%, influent SO4 2-Supplement to 350 mg / L. Inoculate the bottom of the reactor with desulfurized Vibrio enrichment culture medium during reactor startup. In the anoxic zone, HRT=5h, DO<0.5 mg / L, and sodium acetate is added at C / N=6:1. In the aerobic zone, HRT=10h, DO=3.0 mg / L. The reflux ratio of the nitrification liquor is 250%.

[0037] Example 2 Composite carrier formulation: 40 parts iron-based biochar, 20 parts chitosan-modified bentonite, 5 parts sodium alginate, 4 parts diatomaceous earth, and 2 parts corn starch. No water-soluble nutrients are added to the carrier. Preparation method: Sodium alginate solution concentration 2% w / v, crosslinking time 24 h, crosslinking temperature 4°C, two-step inoculation method parameters are the same as in Example 1 (including intermediate rinsing step). Process parameters: Biosorption section HRT = 3 h, DO = 1.5 mg / L; Anaerobic section HRT = 10 h, DO < 0.2 mg / L, filling rate 60%, bed expansion rate 12%, SO4 2- =200mg / L; anoxic HRT=4h, C / N=5:1; aerobic HRT=8h, DO=4.0mg / L; nitrification liquor internal reflux ratio 200%.

[0038] Example 3 Composite carrier formulation: 30 parts iron-based biochar, 30 parts chitosan-modified bentonite, 10 parts sodium alginate, 6 parts diatomaceous earth, and 3 parts corn starch. No water-soluble nutrients are added to the carrier. Preparation method: Sodium alginate solution concentration 4% w / v, cross-linking time 12 h, cross-linking temperature 10°C; Two-step inoculation parameters: First suspension (core bacteria) OD... 600 =2.0, shake at 80 rpm, soak for 48 h; intermediate rinsing is the same as in Example 1; second suspension (Pioneer bacteria) OD 600 =5.0, micro-aeration 0.3vvm, soaking for 24h; acclimatization and stabilization period 3d. Process parameters: biosorption section HRT=4h, DO=0.5mg / L; anaerobic section HRT=14h, DO<0.2mg / L, filling rate 70%, bed expansion rate 18%, SO4 2- =500mg / L; anoxic HRT=6h, C / N=7:1; aerobic HRT=12h, DO=2.5mg / L; nitrification liquor internal reflux ratio 300%.

[0039] Example 4 The composite carrier formulation was the same as in Example 1, and the preparation method and process parameters were also the same as in Example 1. On the 15th day after stable operation, the concentration of heavy metals in the influent was suddenly increased to Cu. 2+ 30 mg / L, Pb 2+ 15mg / L, Cr 3+The concentration was 10 mg / L (3 times the original concentration), and after 7 days of continuous shock, it was restored to the original concentration to examine the system's shock resistance and recovery ability. The changes in effluent water quality during the shock and recovery periods were recorded.

[0040] Example 5 Composite carrier formulation: 35 parts iron-based biochar, 25 parts chitosan-modified bentonite, 8 parts sodium alginate, without the addition of diatomaceous earth and corn starch. Preparation method and process parameters are the same as in Example 1.

[0041] Example 6 The composite carrier formulation is the same as in Example 1. The microbial composition is adjusted as follows: the pioneer bacteria consist only of Bacillus subtilis and Pseudomonas putida (in a 1:1 volume ratio), excluding Bacillus megaterium; the core bacteria consist only of Rhodococcus rubrum and Paracoccus denitrificans (in a 1:1 volume ratio), excluding Sphingosine monocytogenes and Xanthomonas flavogenes. The preparation method and process parameters are the same as in Example 1.

[0042] Comparative Example 1 The composite carrier formulation is the same as in Example 1. Instead of the two-step inoculation method, the pioneer bacteria and core bacteria are mixed in one step according to the same total bacterial count as in Example 1, and then a mixed suspension (OD) is prepared. 600 (≈3.0) The composite carrier was inoculated by single-step soaking with micro-aeration at 0.2 vvm for 48 h, i.e., no spatial stratification was performed, and the pioneer bacteria and core bacteria were randomly distributed within the carrier. The process parameters were the same as in Example 1.

[0043] Comparative Example 2 The carrier formulation used only 8 parts by weight of sodium alginate, without using iron-based biochar or chitosan-modified bentonite. It was directly cross-linked using a 3% w / v sodium alginate solution (same cross-linking conditions as in Example 1). Microbial inoculation employed the same two-step inoculation method and the same strain combination as in Example 1. Process parameters were the same as in Example 1.

[0044] Comparative Example 3 No loaded compound microbial agents are used. A chemical precipitation pretreatment unit is set up before the biological treatment section. NaOH is added to the influent to adjust the pH to 9.0~9.5, and then Na2S is added at 1.2 times the theoretical dosage. After precipitation to remove heavy metals, the effluent enters the conventional activated sludge system (CSTR, HRT=12h, DO=2.0~3.0mg / L, MLSS=3500~4000mg / L) for biological treatment.

[0045] Comparative Example 4 The composite carrier formulation is the same as in Example 1, and the preparation method and microbial inoculation are also the same as in Example 1. During the process operation, no sulfate is added to the influent, and no SRB is inoculated at the bottom of the reactor (i.e., the in-situ self-regeneration mechanism of the carrier is not activated). The remaining process parameters are the same as in Example 1.

[0046] Table 1 Summary of performance test results for each embodiment and comparative example Note: The influent water quality indicators are COD 800~1000 mg / L, Cu 2+ 10 mg / L, Pb 2+ 5 mg / L, Cr 3+ 3 mg / L; the heavy metal concentrations in the effluent in the table are average values ​​during the stable operation period. Example 4: Heavy metal concentration in the influent during the shock period was Cu. 2+ 30 mg / L, Pb 2+ 15 mg / L, Cr 3+ 10 mg / L. The recovery period data in Example 4 are the average values ​​from days 21 to 28 after the initial influent concentration was restored. The carrier adsorption capacity retention rate is calculated as the ratio of the adsorption capacity after 90 days of operation to the initial adsorption capacity. DHA is the average dehydrogenase activity of the microorganisms in the inner layer of the carrier during the stable operation period. "-" indicates that this item is not applicable or was not detected.

[0047] Table 2 Comparison of physical parameters of the carrier The technical effects of the present invention can be systematically analyzed based on the data in Tables 1 and 2, and discussed in conjunction with chemical reaction mechanisms and microbiological principles as follows.

[0048] Comparing Example 1 and Comparative Example 2 clearly demonstrates the irreplaceable role of iron-based biochar and chitosan-modified bentonite in the composite carrier system. The effluent Cu from Comparative Example 2... 2+ Up to 1.52 mg / L (0.18 mg / L in Example 1), Pb 2+ The concentration was 0.87 mg / L (0.08 mg / L in Example 1), Cr 3+ The concentration was 0.93 mg / L (0.12 mg / L in Example 1), indicating a sharp deterioration in heavy metal removal. This is because although pure sodium alginate gel possesses a certain carboxyl complexing ability, it lacks the FeOOH inner spherical complexing sites provided by iron-based biochar and the highly efficient cation exchange-chelation bifunctional sites provided by chitosan-modified bentonite, resulting in the inability to form an effective heavy metal concentration attenuation gradient radially on the carrier. From the perspective of carrier physical parameters, the BET specific surface area of ​​Comparative Example 2 is only 28.6 m². 2 / g (Example 1: 186.3m) 2The average pore size was 12.5 μm (45.2 μm in Example 1), and the porosity was only 38.2%, indicating that the microstructure of the carrier degenerated into a dense gel with underdeveloped pores after the absence of iron-based biochar and chitosan-modified bentonite. This was not conducive to microbial colonization and could not provide sufficient mass transfer channels. Under these conditions, heavy metals penetrated into the carrier at a high concentration, directly inhibiting the metabolic activity of the core bacterial community—the DHA in Comparative Example 2 was only 58.4 μg TF / (g·h), which was 41% of that in Example 1 (142.3 μg TF / (g·h)), resulting in a COD removal rate of only 74.6% and a naphthalene removal rate of only 61.5%. In addition, the crushing strength of the carrier in Comparative Example 2 was only 12.3 N, which was insufficient to withstand the hydraulic shear and particle collision under the operating conditions of the UAFBR expanded bed. Significant carrier breakage and loss were observed during operation, further deteriorating the system performance. In contrast, the incorporation of iron-based biochar and diatomaceous earth in Examples 1-3 significantly improved the stiffness of the gel skeleton, with a crushing strength of 36.8-48.3 N, and no obvious carrier damage was observed during 90 days of continuous operation.

[0049] Within the composite carrier, the three materials form a hierarchical heavy metal interception system. In the first layer, FeOOH nanoparticles on the surface of the iron-based biochar rapidly capture heavy metal ions entering the outer layer of the carrier through an internal spherical complexation mechanism. This reaction exhibits a high rate constant (kobs approximately 0.05–0.12 min). -1 The first layer provides initial rapid interception. In the second layer, chitosan-modified bentonite further captures heavy metals penetrating the first layer, especially Pb, through interlayer cation exchange and chelation of chitosan amino groups. 2+ and Cr 3+ The chelation selectivity is relatively high. At the third level, the diffusion resistance of the sodium alginate gel network and the residual carboxyl groups provide final interception of trace heavy metals that have penetrated the deepest layers. Example 2 (40 parts iron-based biochar, 20 parts chitosan-modified bentonite) in Cu 2+ The best performance was observed in effluent (0.15 mg / L), but the COD removal rate (94.8%) was slightly lower than that of Example 1 (96.3%). This was because the excessive amount of iron-based biochar affected the continuity of the gel network, and the carrier crushing strength was 36.8 N, lower than the 42.5 N of Example 1. Occasional breakage during operation could lead to local detachment of the microbial membrane. Example 3 (30 parts of iron-based biochar, 30 parts of chitosan-modified bentonite, and 10 parts of sodium alginate) had the highest TN removal rate (80.1%), thanks to the longer anaerobic HRT (14 h), higher filling rate (70%), and higher nitrification liquor internal reflux ratio (300%), but Cu 2+ Effluent (0.21 mg / L) and Pb 2+The effluent (0.10 mg / L) was slightly inferior to that of Examples 1 and 2, reflecting the effect of reduced spherical complexation sites after the iron-based biochar content was decreased.

[0050] Comparative Example 1 used a mixed inoculation method (without stratification), and all other conditions were exactly the same as in Example 1, but the performance showed a comprehensive and significant decline: COD removal rate decreased from 96.3% to 82.4%, naphthalene removal rate decreased from 94.7% to 72.3%, and volatile phenol removal rate decreased from 97.2% to 78.5%. Simultaneously, the effluent Cu... 2+ From 0.18 mg / L to 0.45 mg / L, Pb 2+ The concentration increased from 0.08 mg / L to 0.28 mg / L. The DHA content of the inner layer microorganisms plummeted from 142.3 μg TF / (g·h), only 53.6% of that in Example 1. This set of comparative data strongly demonstrates the core importance of spatial stratification for the technical solution of this invention. The mechanism can be explained from the following two levels: First, the biological barrier function is lost—when pioneer bacteria and core bacteria are mixed, the EPS secreted by the pioneer bacteria cannot form a continuous and dense biological barrier layer on the outer layer of the carrier. Heavy metal ions directly contact the core bacteria through the gaps in the EPS, causing key heavy metal-sensitive degradation enzymes in the core bacteria (such as the non-heme iron active center in dioxygenases) to be degraded by Cu. 2+ Pb 2+ First, competitive binding leads to inactivation; second, nutrient competition intensifies—in the case of mixed distribution, pioneer bacteria (especially the fast-growing Bacillus spp.) compete with core bacteria for carbon sources and electron donors in the same space, resulting in a decrease in the abundance of core bacteria (especially the slow-growing Rhodococcus rubrum and Sphingosine monocytogenes) in the biofilm, further weakening the degradation efficiency of recalcitrant organic matter.

[0051] Example 6, using only a partial combination of pioneer and core bacteria (two pioneer bacteria + two core bacteria), achieved COD removal rates (92.7%) and naphthalene removal rates (88.6%) lower than the complete combination in Example 1, but still significantly higher than Comparative Examples 1 and 2. This indicates that even under conditions of reduced bacterial diversity, the spatially layered structure constructed using the two-step inoculation method can still effectively maintain gradient function. However, the naphthalene removal rate in Example 6 decreased by 6.1 percentage points compared to Example 1, mainly due to the absence of *Sphingosine Monoclonalella* – a known highly efficient PAH-degrading bacterium whose secreted catechol 2,3-dioxygenase is crucial for the meta-ring-opening cleavage of naphthalene.

[0052] The only difference between Comparative Example 4 and Example 1 is that sulfate was not added to the influent, thus the in-situ self-regeneration mechanism of the carrier was not activated. During the 60-day stable operation period, the effluent quality of Comparative Example 4 showed a detectable deterioration trend: the COD removal rate was 94.6% (96.3% in Example 1), and the effluent Cu... 2+ The concentration of DHA in the inner layer was 0.24 mg / L (0.18 mg / L in Example 1), and the concentration of DHA in the inner layer microorganisms was 121.6 μg TF / (g·h) (142.3 μg TF / (g·h) in Example 1). All indicators showed a trend of gradual saturation of the adsorption sites on the carrier and increased penetration of heavy metals into the inner layer. However, when examining the long-term effectiveness of the carrier, the differences were extremely significant: the adsorption capacity retention rate of Comparative Example 4 after 90 days of operation was only 51.2%, while that of Example 1 was 88.6%. This means that the carrier in Comparative Example 4 was close to adsorption saturation after 90 days, and if operation continued, heavy metal penetration would occur rapidly, and the system performance would deteriorate rapidly; while the carrier in Example 1 still maintained nearly 90% adsorption activity, and its service life was expected to be extended to more than 180-240 days. The essence of the self-regeneration mechanism lies in the fact that the VFAs (mainly acetic acid, propionic acid, and butyric acid, with a total concentration of approximately 800~1500 mg / L) produced by the pioneer bacteria in the anaerobic hydrolysis acidification stage lower the pH of the microenvironment of the carrier's outer layer from neutral to 5.0~6.0. Under this pH condition, the negative charge above the zero-charge point (pHpzc≈7.5~8.5) on the FeOOH surface disappears, and surface protonation occurs, making ≡FeOM + Coordination bonds break, and heavy metal ions are released back into the liquid phase in a free state: ≡FeOM + +H + →≡FeOH2 + +M 2+ Similarly, the amino groups of chitosan undergo protonation under acidic conditions (-NH2+H). + →-NH3 + The stability of the chelate complexes formed between the chelate and heavy metals decreases, leading to the release of heavy metal ions. However, if the released free heavy metal ions cannot be precipitated and fixed in time, they will enter subsequent treatment stages with the water flow, causing secondary toxicity to microorganisms. This invention solves this problem by constructing a spatially partitioned acidic desorption-neutral precipitation coupling system within the reactor: the outer micro-region of the carrier (pH 5.0-6.0) is responsible for the acidic desorption of heavy metals, while the SRB colonization region (pH 6.5-7.5) in the sludge layer at the bottom of the reactor and the deep macropores of the carrier is responsible for generating S... 2- The reason why SRB can maintain its metabolic activity in the overall acidic environment of the anaerobic hydrolysis acidification section is that its colonized microregions are far from the outer surface of the carrier where organic acids accumulate, and the sodium alginate gel framework and the relative pH of the liquid inside the pores have a significant buffering effect. After desorption, the free heavy metal ions released migrate to the above-mentioned neutral microregions through diffusion and upwelling water flow, and then react with S...2- The reaction is rapid, generating metal sulfide precipitates (CuS, PbS, etc.) with extremely low solubility, which are deposited in the sludge layer at the bottom of the reactor and in the deep macropores of the carrier, thereby achieving a closed-loop self-regeneration cycle of desorption-migration-precipitation-fixation.

[0053] Comparative Example 3 employed a two-stage process combining traditional chemical precipitation pretreatment and activated sludge, achieving a satisfactory heavy metal removal effect (Cu). 2+ The effluent concentration was 0.35 mg / L, but the organic matter degradation performance was significantly insufficient: COD removal rate was 85.1%, naphthalene removal rate was only 68.4%, and volatile phenol removal rate was 80.3%, all significantly lower than in Example 1. This is because although chemical precipitation pretreatment reduced the concentration of heavy metals entering the biological stage, the operation of adjusting the pH to 9.0-9.5 with NaOH made the effluent alkaline, requiring pH adjustment after entering the activated sludge stage, which was complex and increased the cost of reagents. In addition, excessive addition of Na2S may generate H2S gas under anaerobic conditions, posing a safety risk to operators. The suspended microorganisms in the activated sludge system lack carrier protection and have poor tolerance to residual heavy metals and water quality fluctuations. Traditional activated sludge lacks the targeted enrichment of PAH-specific degrading bacteria such as Rhodococcus rubrum and Sphingosine monocytogenes, and the degradation of polycyclic aromatic hydrocarbons such as naphthalene mainly relies on the cometabolite pathway, which is inefficient. In addition, the traditional process produces a large amount of chemical sludge containing heavy metals (per 1000 m³ of treated sludge). 3 The wastewater generates approximately 0.8 to 1.2 tons of hazardous sludge with a moisture content of 80%, which requires hazardous waste disposal and significantly increases operating costs.

[0054] The heavy metal concentration shock test results in Example 4 showed that when the influent heavy metal concentration suddenly increased to 3 times, the system effluent COD removal rate decreased from 96.1% to 85.3%, and Cu... 2+ The effluent concentration increased from 0.19 mg / L to 0.78 mg / L, the naphthalene removal rate decreased from 94.5% to 81.6%, and the DHA removal rate decreased from 140.8 μgTF / (g·h). Although performance showed a significant decline, the system did not collapse—the effluent heavy metal concentration remained far below the expected value when directly discharged into the biological system, and the COD removal rate remained above 85%, indicating that the buffering effect of the composite carrier and the EPS biological barrier of the pioneer bacteria still played an effective protective role under the high concentration shock. More importantly, within approximately 14–21 days after returning to the original influent concentration, the system's various indicators recovered to levels close to those before the shock (COD removal rate 94.5%, Cu...). 2+The effluent concentration was 0.25 mg / L, and the DHA concentration was 132.5 μg TF / (g·h), demonstrating good system resilience. The 14-21 day recovery period is consistent with the general pattern of biofilm repair: the damaged core microbial community needs to undergo 2-3 generations of proliferation (approximately 4-6 hours for Rhodococcus rubrum and 8-12 hours for Sphingosine monocytogenes) to restore biofilm thickness and community structure. This is mainly attributed to the fact that although the core microbial community in the inner layer of the carrier experienced significant metabolic inhibition during the shock, its cellular integrity was not severely damaged due to the buffering effect of the outer physicochemical and biological barriers. Once the external stress was relieved, its metabolic activity gradually recovered. At the same time, the slow-release nutrients (corn starch, peptone, etc.) pre-embedded in the carrier provided additional nutritional support for the damaged microorganisms during the recovery period, accelerating biofilm repair and functional recovery.

[0055] Comparing Example 5 with Example 1, it is evident that the COD removal rate (92.5%), naphthalene removal rate (90.2%), and DHA removal rate (115.8 μg TF / (g·h)) of Example 5 are all lower than those of Example 1. Example 5 did not include diatomaceous earth or corn starch. The absence of diatomaceous earth resulted in a lower average pore size (32.1 μm) and porosity (55.6%) of the carrier compared to Example 1 (45.2 μm, 62.4%), which is detrimental to mass transfer and the full expansion of microbial colonization space. The absence of corn starch deprived the carrier of its embedded slow-release carbon source during the start-up and emergency recovery phases—in Example 1, corn starch particles embedded in the gel network could slowly hydrolyze and release glucose under the action of microbial α-amylase, providing additional carbon nutrient support for microorganisms during the initial system startup and water quality shock recovery phases. Although the difference is not as significant as in Comparative Example 1 or Comparative Example 2, a 3-4 percentage point difference in removal rate still has practical significance at the engineering level, especially during influent water quality fluctuations or system startup phases. In summary, although diatomaceous earth and corn starch are not core innovative elements, they make positive contributions to the overall performance stability of the system and the optimization of the carrier's microstructure.

[0056] In summary, the supported composite microbial preparation of the present invention, through the synergistic combination of three functional materials—iron-based biochar, chitosan-modified bentonite, and sodium alginate—combined with a gradient functional microbial membrane constructed by a two-step inoculation method ("inside then outside") and an in-situ self-regeneration mechanism of the carrier based on the coupling of acidic desorption on the carrier surface and sulfide precipitation in the reactor liquid phase, simultaneously achieves efficient interception of heavy metals and deep degradation of recalcitrant organic matter in a single biological treatment process. All core indicators are significantly better than those of the comparative example, verifying the rationality and advancement of the technical solution of the present invention.

[0057] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microbial-based wastewater treatment process, characterized in that, Includes the following steps: Wastewater containing recalcitrant organic matter and heavy metals is introduced into a reaction system containing a loaded compound microbial agent for biological treatment. The loaded composite microbial preparation includes a composite carrier and a gradient functional microbial membrane attached to the composite carrier; The composite carrier includes iron-based biochar, chitosan-modified bentonite, and sodium alginate. The gradient functional microbial membrane includes a heavy metal-tolerant pioneer bacterial community colonizing the outer layer of the composite carrier, and an organic matter-degrading core bacterial community colonizing the inner layer of the composite carrier.

2. The microbial-based wastewater treatment process according to claim 1, characterized in that, The raw materials for preparing the composite carrier, by mass parts, include: 30 to 40 parts of iron-based biochar, 20 to 30 parts of chitosan-modified bentonite, 5 to 10 parts of sodium alginate, and at least one selected from diatomaceous earth and corn starch.

3. The microbial-based wastewater treatment process according to claim 1 or 2, characterized in that, The composite carrier is a spherical particle with an average pore size of 20 μm to 80 μm; The iron-based biochar surface is loaded with FeOOH nanoparticles; The cation exchange capacity of the chitosan-modified bentonite is 120 cmol / kg to 150 cmol / kg.

4. The microbial-based wastewater treatment process according to claim 1, characterized in that, The heavy metal-tolerant pioneer bacteria include at least one of Bacillus subtilis, Bacillus megaterium, and Pseudomonas putida. The organic matter-degrading core bacterial group includes at least one of Rhodococcus rubrum, Sphingosine monocytogenes, Paracoccus denitrificans, and Xanthomonas flavogenes.

5. The microbial-based wastewater treatment process according to claim 4, characterized in that, The heavy metal-tolerant pioneer bacteria colonize the outer region of the composite carrier up to a depth of 500 μm, and form an extracellular polymer matrix layer in the outer region as a biological barrier. The organic matter-degrading core microbial community colonizes the inner layer region of the composite carrier at a depth of 500 μm to 1500 μm.

6. The microbial-based wastewater treatment process according to claim 1, characterized in that, The gradient functional microbial membrane is prepared by a two-step inoculation method, which includes the following steps: Step 1: Immerse the composite carrier in a first suspension containing organic matter-degrading core microbial community and slowly shake and soak it to allow the core microbial community to penetrate and colonize the inner layer of the carrier; Between the first and second steps, the composite vector that has been inoculated in the first step is removed and gently rinsed with sterile physiological saline to remove loosely attached bacteria and residual first suspension from the surface of the vector. Step 2: Immerse the rinsed composite carrier in a second suspension containing heavy metal-tolerant pioneer bacteria for micro-aeration soaking, so that the pioneer bacteria colonize the outer layer of the carrier and secrete extracellular polymers to form a biological barrier layer. The cell density of the second suspension is greater than that of the first suspension.

7. The microbial-based wastewater treatment process according to claim 1, characterized in that, The reaction system comprises a bio-adsorption section, an anaerobic hydrolysis acidification section, an anoxic denitrification section, and an aerobic biological contact oxidation section connected in series; the wastewater flows through each of the above treatment sections in sequence for degradation. The loaded composite microbial preparation is disposed in the bioadsorption section, the anaerobic hydrolysis acidification section, and the aerobic biological contact oxidation section.

8. The microbial-based wastewater treatment process according to claim 7, characterized in that, The anaerobic hydrolysis acidification section adopts an upflow anaerobic biofilm reactor, and the supported composite microbial preparation is filled into the reactor at a volume filling rate of 60% to 70%. In the biosorption section, the dissolved oxygen is controlled at 0.5 mg / L to 1.5 mg / L, and the hydraulic retention time is 3 hours to 4 hours. In the anaerobic hydrolysis acidification section, the dissolved oxygen is controlled to be less than 0.2 mg / L, and the hydraulic retention time is 10 to 14 hours.

9. The microbial-based wastewater treatment process according to claim 7, characterized in that, In the anoxic denitrification section, the dissolved oxygen is controlled to be less than 0.5 mg / L, the hydraulic retention time is 4 to 6 hours, and sodium acetate carbon source is added to the anoxic denitrification section according to the preset carbon-nitrogen ratio. In the aerobic biological contact oxidation section, the dissolved oxygen is controlled at 2.5 mg / L to 4.0 mg / L, and the hydraulic retention time is 8 hours to 12 hours. The effluent from the aerobic biological contact oxidation section is refluxed internally to the inlet of the anoxic denitrification section, with an internal reflux ratio of 200% to 300%.

10. The microbial-based wastewater treatment process according to claim 7, characterized in that, The process also includes an in-situ self-regeneration step of the carrier: A sludge layer is set at the bottom of the reactor in the anaerobic hydrolysis acidification section. Sulfate-reducing bacteria are colonized in the sludge layer. Sulfate is added to the influent of the reaction system as a metabolic substrate for the sulfate-reducing bacteria. In the anaerobic hydrolysis acidification section, the organic acids produced by the fermentation of the pioneer bacteria reduce the pH of the outer micro-region of the composite carrier to 5.5 to 6.5, which promotes the desorption of heavy metal ions complexed on the surface of the composite carrier. The free heavy metal ions released by desorption diffuse down the concentration gradient into the main aqueous phase outside the carrier, react with the sulfur ions produced by the metabolism of sulfate-reducing bacteria in the sludge layer and diffused into the aqueous phase, forming metal sulfide microcrystal precipitates in the liquid phase and settling into the sludge layer at the bottom of the reactor. The adsorption sites freed up on the surface of the composite carrier due to the desorption of heavy metals are restored in situ.

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