Preparation method of space functionalized sulfate reducing bacteria blanket

Abstract

The invention relates to the technical field of sulfate reducing bacteria, in particular to a preparation method of a space-functionalized sulfate reducing bacteria blanket, which comprises the following steps: firstly, preparing a three-layer composite carrier consisting of a carbon felt bottom layer, a nitrogen-doped graphene / Fe3O4 conductive middle layer and a slow-release nutrition top layer; then, utilizing the synergistic effect of an electric field with specific intensity and an alternating / constant magnetic field to directionally colonize an acid-producing and hydrogen-producing flora and an acetic acid oxidation type and hydrogen nutrition type sulfate reducing flora in different space depths of the carrier in sequence to form a vertical metabolism partition; and finally, maturing the fungus blanket through multi-stage gradient domestication containing a fluctuating carbon source and weak current stimulation. According to the improvement, the problems of low treatment efficiency, poor impact resistance and long-term operation performance degradation caused by disordered spatial distribution of functional microorganisms, easy breakage of metabolic chains, unstable flora structure and lack of in-situ sensing regulation capability when the traditional sulfate reducing flora is applied to high-volatility rare earth tail water treatment are solved.

Description

Technical Field

[0001] This invention relates to the field of sulfate-reducing bacteria technology, and specifically to a method for preparing spatially functionalized sulfate-reducing bacterial mats. Background Technology

[0002] Sulfate-reducing bacteria (SRB) biofilm technology has been widely used in the treatment of acidic mine wastewater. It achieves SRB immobilization and functional enhancement by forming a biofilm (bacterial mat) on a carrier surface. Current techniques typically involve directly inoculating SRB bacterial solutions onto the surface of porous or fibrous carriers (such as activated carbon, volcanic rock, or polyurethane fillers), forming a biofilm through natural adsorption and static cultivation. Further improvements include incorporating conductive substances (such as activated carbon or graphene) into the carrier material to promote electron transfer; or applying a weak electric field during cultivation to stimulate bacterial growth.

[0003] However, the existing methods for preparing mycelial mats have technical bottlenecks that are not yet fully recognized by those skilled in the art: Traditional inoculation methods result in the random mixing and distribution of microorganisms with different metabolic functions (such as acid-producing bacteria, acetic acid oxidizing SRBs, and hydrogen-nutritive SRBs) within the carrier. This disordered spatial arrangement leads to lengthy and inefficient metabolite transport pathways within the microbial community, especially when treating wastewater with fluctuating composition, where metabolic synergy between different functional microbial communities is prone to break down.

[0004] In randomly formed biofilms, non-target bacteria with faster growth rates (such as acid-producing bacteria) tend to gradually gain an advantage in competition, while the ecological niche of key SRB strains is difficult to stabilize, leading to irreversible "drift" and performance degradation of the biofilm function during long-term operation.

[0005] Existing conductive carriers serve only as passive electron transfer media or merely provide surface area, lacking the ability to actively and directionally guide specific functional microorganisms to colonize the carrier at their optimal spatial location during the inoculation stage. Microbial attachment relies entirely on random collisions and natural chemotaxis, failing to achieve the orderly construction of functional zones.

[0006] The microbial community structure of the mats formed by conventional static culture and domestication is optimized for stable substrate conditions. When faced with drastic fluctuations in the type, concentration, and pH of carbon sources in actual rare earth tailings, these mats lack inherent metabolic resilience and structural buffering capacity, making them prone to dysfunction.

[0007] Therefore, there is an urgent need for a novel preparation method for sulfate-reducing bacterial mats that can actively construct spatially ordered, functionally partitioned, structurally stable, and inherently resistant to fluctuations. Summary of the Invention

[0008] The technical problem to be solved by this invention is: how to overcome the problems of low treatment efficiency, poor shock resistance and long-term performance degradation caused by the disordered spatial distribution of functional microorganisms, easy breakage of metabolic chains, unstable microbial community structure and lack of in-situ sensing and regulation capabilities when traditional sulfate-reducing bacteria are applied to the treatment of highly fluctuating rare earth tailwater.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: The preparation method of spatially functionalized sulfate-reducing bacterial mat includes the following steps: S1: Prepare a three-layer composite carrier, which includes a carbon felt layer as the bottom biological attachment matrix, a nitrogen-doped graphene / Fe3O4 composite layer as the middle conductive and magnetic response layer, and a polycaprolactone / hydroxyapatite porous layer as the upper slow-release nutrient supply layer, and pre-activate the carrier with microbial secretions. S2: Electric-Magnetic Field Synergistic Spatial Directed Colonization: Under the synergistic effect of a weak electric field and an alternating / constant magnetic field, acid-producing and hydrogen-producing bacteria, acetic acid-oxidizing sulfate-reducing bacteria, and hydrogen-nutritive sulfate-reducing bacteria are sequentially and directionally colonized at different spatial depths of the carrier to form preliminary spatial functional zoning. S3: Multi-substrate coupled gradient acclimatization and mat mat maturation: The colonized carrier is placed in a continuous flow reactor and undergoes three stages of incremental acclimatization with a single organic acid, a mixed complex carbon source, and a fluctuating carbon source as the influent. In-situ weak electrical stimulation is applied during the maturation period to finally obtain a mature spatially functionalized mat mat.

[0010] Furthermore, in the above-mentioned method for preparing spatially functionalized sulfate-reducing bacterial mats, in step S1, the Fe3O4 nanoparticles in the nitrogen-doped graphene / Fe3O4 composite layer have a particle size of 20-50 nm and a loading of 1-3 mg / cm³. 2 The polycaprolactone / hydroxyapatite porous layer contains sustained-release microcapsules encapsulating diammonium hydrogen phosphate and sodium molybdate.

[0011] Furthermore, in the above-mentioned method for preparing spatially functionalized sulfate-reducing bacterial mats, in step S2, the strength of the weak electric field is 0.3-0.8 V / cm, and its direction is towards the carrier when colonizing acid-producing and hydrogen-producing bacteria, and towards the solution when colonizing acetic acid-oxidizing SRB bacteria; the magnetic field adopts an alternating magnetic field with a frequency of 5-15 Hz and an intensity of 30-70 mT in the early stage of colonization, and a constant magnetic field with an intensity of 80-120 mT when colonizing HSRB.

[0012] Furthermore, in the above-mentioned method for preparing spatially functionalized sulfate-reducing bacterial mats, in step S2, the acid-producing and hydrogen-producing bacterial communities mainly colonize the bottom layer of the carbon felt, the acetic acid-oxidizing SRB communities mainly colonize the conductive layer and the upper and middle layers of the carbon felt, and the hydrogen-nutritive SRB communities mainly colonize the deep layer of the conductive layer and the lower layer of the polycaprolactone / hydroxyapatite slow-release layer.

[0013] Furthermore, in the above-mentioned method for preparing spatially functionalized sulfate-reducing bacterial mats, in step S3, during the fluctuating carbon source acclimatization stage, the type of influent carbon source changes randomly or periodically among one or more of acetic acid, propionic acid, lactic acid, and ethanol, and the sulfate concentration changes stepwise within a preset range; the in-situ weak electrical stimulation is the application of a DC constant voltage of 0.1-0.4 V through the conductive layer.

[0014] The beneficial effects of this invention are as follows: By coupling electric and magnetic fields in a coordinated spatial orientation colonization with multi-substrate gradient domestication, precise spatial arrangement and niche locking of functional microorganisms on a three-dimensional carrier are achieved, thereby constructing a biomimetic biofilm system (bacterial mat) with vertical metabolic partitioning, efficient electron transport pathways, and inherent resistance to fluctuations. This method surpasses the limitations of traditional mixed culture or random biofilm formation. Specifically, the ordered spatial arrangement of acid-producing bacteria, acetic acid oxidizing SRBs, and hydrogen-nutritive SRBs shortens the physical distance of the substrate transformation chain, maximizes the transfer efficiency of metabolic intermediates, reduces competition and inhibition, and thus increases the sulfate reduction rate per unit biomass. By using physical (electric and magnetic fields) and chemical (gradient domestication) methods to "anchor" key functional microbial communities to specific functional layers of the carrier (e.g., SRBs enriched in the conductive layer), and supplemented by a slow-release nutrient layer for continuous supply, the "drift" of the microbial community caused by fluctuations in influent or competition is effectively resisted. The abundance fluctuation of core functional bacteria during long-term operation is less than ±5%, solving the problem of functional bacteria being easily squeezed out in traditional processes. The spatial functional zoning of the microbial carpet itself acts as a buffer system. When the influent carbon source type or sulfate concentration changes abruptly, the functional microorganisms in different zones can form metabolic relay and complement each other, preventing the entire chain from collapsing. Combined with the fluctuation acclimatization during the maturation period, the microbial carpet possesses "metabolic resilience," exhibiting a small drop in removal rate when facing shocks and rapidly recovering to a highly efficient state within 24 hours. This overcomes the problems of low treatment efficiency, poor shock resistance, and long-term performance degradation caused by the disordered spatial distribution of functional microorganisms, easily broken metabolic chains, unstable microbial community structure, and lack of in-situ sensing and regulation capabilities when traditional sulfate-reducing bacteria are applied to the treatment of highly volatile rare earth wastewater. Detailed Implementation

[0015] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments.

[0016] This invention relates to a method for preparing a spatially functionalized sulfate-reducing bacterial mat, comprising the following steps: S1: A three-layer composite carrier module was prepared. The bottom layer (in contact with water flow) was a carbon felt layer (5 mm thick, fiber diameter 10-15 μm), providing high specific surface area and initial bioattachment sites. The middle layer was a nitrogen-doped graphene / magnetic Fe3O4 composite conductive layer (2 mm thick), which was formed by coating nitrogen-doped graphene hydrogel with Fe3O4 nanoparticles (20-50 nm in diameter) prepared by co-precipitation onto the carbon felt and vacuum drying at 60 °C. This layer constructs an efficient electron transport network and provides magnetic responsiveness. The top layer (away from water flow) was a polycaprolactone / hydroxyapatite (PCL / HA) porous sustained-release layer (3 mm thick), prepared by electrospinning, with a porosity >80% and a pore size of 50-200 μm. It internally loaded with microcapsules of diammonium hydrogen phosphate ((NH4)2HPO4) and sodium molybdate (Na2MoO4) as a long-term sustained-release N / P source and a cofactor for the key SRB enzyme.

[0017] The composite carrier was immersed in Postgate C medium containing 10% local rare earth tailwater sludge supernatant and pre-cultured at 35°C with low-speed shaking (50 rpm) for 48 hours to allow a layer of natural microbial secretions to be adsorbed on the carrier surface, which is beneficial for subsequent specific colonization.

[0018] S2: Enrich three types of functional bacterial communities: (a) Acid-producing and hydrogen-producing bacteria (AHB): Enriched from effluent sludge using glucose and yeast extract as substrates, mainly including Clostridium; (b) Acetic acid oxidizing SRB (ASRB): Enriched using the previously patented method, with Desulfobacter and Desulfocurvus as the dominant bacteria; (c) Hydrogen-nutritive SRB (HSRB): Enriched from sludge of the same source using H2 / CO2 (80:20) as substrate, with Desulfovibrio as the dominant bacteria.

[0019] The activated composite carrier was placed in a colonization reaction tank. First, a steady, weak electric field (0.5 V / cm, perpendicular to the carrier plane) was applied to the bottom of the tank. Since SRB cells are typically negatively charged, they migrate directionally towards the anode (carrier direction) under the influence of the electric field. Subsequently, an alternating magnetic field (10 Hz, 50 mT) was activated, exciting the Fe3O4 nanoparticles in the intermediate conductive layer to generate heat and micro-perturbations, promoting the microorganisms that had approached the carrier to overcome the energy barrier and firmly attach to the micropores of the conductive layer and carbon felt layer.

[0020] AHB bacterial solution was injected into the reaction tank and colonized for 2 hours under the influence of an electric field (directed towards the carrier). During this stage, AHB mainly adhered to the bottom layer of the carbon felt. Due to its rapid utilization of glucose and acid production characteristics, it easily formed a biofilm at the carrier-water interface. The solution was then replaced with ASRB bacterial solution, and the direction of the electric field was reversed (0.3 V / cm, pointing towards the solution). A trace amount of N2 was introduced to dispel residual O2, and colonization continued for 3 hours. During this stage, ASRB tended to colonize in the conductive layer and the upper layer of the carbon felt, slightly away from the interface, to utilize the acetic acid produced by the bottom AHB. In the third step, HSRB bacterial solution was injected, the electric field was turned off, and a constant weak magnetic field (100 mT) was turned on. Utilizing the possible magnetosomes within the HSRB cells or their attraction to the magnetic field, they further colonized in the deeper layers of the conductive layer and the lower layer of the PCL / HA slow-release layer to utilize the H2 generated within the system.

[0021] S3: The influent adopts a three-stage incremental coupling substrate strategy: Phase I (Start-up period, 7 days): The influent contains sodium acetate (500 mg / L COD), sodium lactate (300 mg / L COD), and a low concentration of sulfate (800 mg / L, from the effluent). This phase encourages the synergistic growth of ASRB and HSRB, and the bacterial mat begins to exhibit reducing capabilities.

[0022] Phase II (Enhanced Period, 14 days): The influent is switched to a complex carbon source (a mixture of glucose and ethanol, with no change in total COD) and a high concentration of sulfate (1500 mg / L, all from the effluent). This forces AHB and SRB to establish a tight interspecific metabolic coupling. The bottom-layer AHB ferments glucose to produce acid and hydrogen, while the middle and upper-layer SRB consumes these products to reduce sulfate.

[0023] Phase III (Maturation and Stabilization Period, 21 days): Fluctuation simulation was introduced, with the type of influent carbon source (acetic acid, propionic acid, lactic acid, ethanol, or a mixture thereof) and sulfate concentration randomly changed daily (with step changes between 1200-2000 mg / L). Simultaneously, in-situ weak electrical stimulation (applying a constant bias voltage of 0.2 V through the carrier's conductive layer) was activated to enhance the direct interspecies electron transport (DIET) pathway within the microbial mat. After this phase, a mature, spatially functionalized microbial mat (S-FSRB Mat) approximately 2-3 mm thick, with a dense structure and a dark gray color, formed on the carrier.

[0024] The above embodiments describe the integrated S-FSRB Mat bioelectrochemical-membrane composite reaction system and its operation: System Composition: The main body of the reactor is an upflow anaerobic biofilm reactor (UBF), internally packed with S-FSRBMat carrier modules in a regular pattern. A micro-bioelectrochemical sensor is embedded in the carrier array: consisting of a pair of microelectrodes (the working electrode is a carbon fiber electrode modified with an SRB-specific receptor, and the counter electrode is an Ag / AgCl electrode), used for in-situ, real-time monitoring of H2S generation and acetic acid consumption rates on the surface of the biofilm. The system integrates a PLC control system to receive sensor signals.

[0025] Operating process: The mature S-FSRB Mat carrier module was loaded into the UBF reactor, and conventional SRB suspended sludge was inoculated at 8% (v / v) as a supplement. The influent was actual rare earth tailwater with pH adjusted to 6.5-7.0, supplemented with necessary nutrients (C / N / P at a ratio of 100:5:1).

[0026] The temperature is maintained at 35±1℃; the hydraulic retention time (HRT) is dynamically adjusted according to the sulfate load of the influent, with a baseline of 24h, and is allowed to be automatically adjusted by PLC between 18-36h; the upflow velocity is controlled at 1.0-1.5 m / h to ensure good mass transfer.

[0027] Carbon source dosing control based on H2S sensor signals: When the in-situ H2S generation rate sensor signal is lower than the set threshold (corresponding to a decrease in sulfate reduction rate), and the sulfate concentration in the effluent does not exceed the standard, the PLC determines that the carbon source is relatively insufficient or the type is mismatched. The system automatically adds a small amount of carbon source (increasing the impact load by 10-20% COD) in a pulsed manner from the backup carbon source storage tanks (which store sodium acetate, ethanol, and sodium lactate respectively) according to a preset algorithm (such as based on the type of the last effective carbon source). After two hours, the sensor signal recovery is evaluated.

[0028] pH-coordinated regulation based on acetic acid sensor signals: When the in-situ acetic acid consumption rate signal remains low while the pH shows a downward trend, it is determined that the acid production process is stronger than the acid consumption process. The PLC not only adjusts the carbon source addition but also starts the NaHCO3 dosing pump to precisely control the pH within a narrow range of 7.0-7.2, avoiding acid accumulation that inhibits SRB.

[0029] HRT dynamic adjustment based on performance feedback: The sulfate removal load per unit carrier volume is used as the main performance indicator. When the influent concentration suddenly increases, the system will automatically extend the HRT to the upper limit (36h). If the removal load continues to decrease, a synergistic remedial procedure of carbon source replenishment and weak electrical stimulation (applying 0.3V to the carrier for 4 hours) will be triggered.

[0030] This section systematically verifies the synergistic effect and necessity of various technical features in the "Preparation Method of Spatially Functionalized Sulfate-Reducing Bacterial Mat (S-FSRB Mat)" of this invention through detailed examples and a series of comparative examples. All experiments used the same actual rare earth wastewater (influent SO4²⁻: 2500±500 mg / L, pH 3.8-4.5) as the treatment object and were evaluated after running for 90 days in the same integrated bioelectrochemical-membrane composite reaction system (UBF reactor, effective volume 10L).

[0031] Example 1 A method for preparing a spatially functionalized sulfate-reducing bacterial mat includes the following steps: S1: Purchase commercial polyacrylonitrile-based carbon felt (thickness 5.0 mm ± 0.2 mm, areal density 0.10 g / cm³). 3 Fiber diameter 12 μm, specific surface area ≥1200 m² 2 / g), cut into 10 cm × 10 cm cubes.

[0032] A modified Hummers method was used to prepare an aqueous solution of graphene oxide (GO) (concentration 5 mg / mL). 100 mL of GO solution was mixed with 2.0 g of trisodium citrate and 1.5 g of urea, and the mixture was stirred in a 95°C water bath for 6 hours to induce nitrogen doping, yielding a nitrogen-doped graphene oxide (N-GO) dispersion. Separately, 4.0 g of FeCl3·6H2O and 1.6 g of FeCl2·4H2O were dissolved in 50 mL of deionized water. Under nitrogen protection, at 60°C, and with vigorous stirring, this solution was added dropwise to the N-GO dispersion. Subsequently, 10% ammonia was added dropwise until the pH reached 10, and the reaction continued for 2 hours. The product was then magnetically separated, washed three times with water and ethanol, and redispersed in 50 mL of water to obtain a nitrogen-doped graphene / Fe3O4 composite conductive paste. The average particle size of the Fe3O4 nanoparticles was determined to be 35 nm ± 8 nm by TEM.

[0033] Polycaprolactone (PCL, Mw = 80,000) was dissolved at a concentration of 12% (w / v) in a mixed solvent of dichloromethane and N,N-dimethylformamide (volume ratio 7:3). Nano-hydroxyapatite (HA, particle size <100 nm) at 20% of the PCL mass and sustained-release microcapsules (microcapsules with gelatin-gum arabic as the wall material and a core material of diammonium hydrogen phosphate and sodium molybdate, mass ratio 10:1, encapsulation efficiency 86% ± 3%) were added, and the mixture was magnetically stirred for 24 hours until homogeneous.

[0034] The carbon felt block was completely immersed in the aforementioned conductive slurry, ensuring thorough penetration. A wet film was formed on both sides of the carbon felt using a dip-coating method (dip speed 5 cm / min). It was then dried in a 60°C vacuum drying oven for 12 hours, followed by annealing in a tube furnace at 450°C for 2 hours under argon protection to complete the reduction and fixation of the conductive layer. The dry weight load of the conductive layer was measured to be approximately 2.2 mg / cm³. 2 .

[0035] Using an electrospinning apparatus, the above PCL / HA / microcapsule spinning solution was loaded into a 10 mL syringe. The needle (21G) was positioned 15 cm away from the receiving roller (coated with aluminum foil), and a voltage of 18 kV was applied at a feed rate of 0.8 mL / h. A carbon felt coated with a conductive layer was fixed onto the receiving roller, and spinning was performed for 30 minutes, forming a porous fiber membrane (slow-release layer) with a thickness of approximately 3.2 mm on one side. Analysis of SEM images using ImageJ software revealed an average fiber diameter of 2.1 μm, an average pore size of 125 μm, and a porosity of approximately 83%.

[0036] The final three-layer composite carrier structure is as follows: carbon felt layer (bottom layer, 5 mm) - nitrogen-doped graphene / Fe3O4 conductive layer (middle layer, <0.2 mm) - PCL / HA porous slow-release layer (top layer, 3.2 mm).

[0037] The prepared composite carrier was immersed in a mixture of 500 mL Postgate C medium (sulfate- and reducing agent-free) and 50 mL of aerobic sludge supernatant from a rare earth wastewater treatment plant (0.22 μm after centrifugation and filtration). It was then placed in a 35°C shaker and cultured at 50 rpm for 48 hours. After removal, the surface was gently rinsed with sterile anaerobic phosphate buffer (pH 7.0) and set aside for later use.

[0038] S2: Acid- and hydrogen-producing bacteria (AHB): Take 200 mL of rare earth tailings sediment and add it to 800 mL of culture medium containing glucose (5 g / L) and yeast extract (2 g / L). Incubate anaerobicly at 35°C (N2 atmosphere) for 5 days, manually shaking twice daily. Collect the late logarithmic growth phase culture by centrifugation (6000 rpm, 10 min), and resuspend in sterile anaerobic buffer to OD. 600 ≈ 1.0. 16S rRNA sequencing showed that the main genera were Clostridium (~35%) and Enterobacter (~22%).

[0039] Acetic acid oxidizing SRB (ASRB): The SRB-MCC group (Desulfobacter and Desulfocurvus as the dominant bacteria) that had been graded and domesticated in the background technique was centrifuged and resuspended to OD.600 ≈ 1.0.

[0040] Hydrogen-nutritive SRB (HSRB) bacterial culture: Take 100 mL of SRB-MCC bacterial suspension and inoculate it into a mineral salt medium with H2 / CO2 (80:20, 1 atm) as the sole carbon and energy source. Incubate anaerobically at 35°C for 2 weeks, and subculture 3 times. Centrifuge and resuspend to OD. 600 ≈0.8. FISH analysis confirmed that *Desulfovibrio* spp. was the dominant bacterium.

[0041] Electrophoresis-Magnetic Field Co-colonization Device and Process: A customized acrylic colonization reaction cell (internal dimensions: 12 cm L × 12 cm W × 8 cm H) was used. Two platinum wire electrodes (1 mm in diameter, 10 cm apart) were embedded parallel to each other at the bottom of the cell and connected to a DC regulated power supply. The cell was placed at the center of a Helmholtz coil that could generate an alternating / constant magnetic field.

[0042] AHB colonization (bottom layer): Place the activated carrier (slow-release side up) horizontally at the bottom of the reaction tank, near the anode. Inject Postgate C basal medium containing AHB bacteria (the volume should be just enough to submerge the carrier by 5 mm). Turn on the power and apply a steady electric field of 0.5 V / cm (anode below the carrier), while simultaneously turning on an alternating magnetic field of 10 Hz, 50 mT. Let it stand under these conditions for 2 hours. The electric field promotes the migration of negatively charged AHB cells towards the carrier (anode), and the perturbations generated by the alternating magnetic field help them penetrate into the deep pores of the carbon felt.

[0043] ASRB colonization (middle layer): Carefully remove the colonization solution and gently rinse the carrier surface with anaerobic buffer. Inject ASRB culture medium. Reverse the electric field direction (cathode below the carrier) and apply a weak electric field of 0.3 V / cm (avoiding strong disturbance to the colonized ASRB), while simultaneously purging high-purity N into the liquid surface for 25 minutes to remove oxygen, maintaining an alternating magnetic field of 10 Hz and 50 mT. Let stand for 3 hours. Use the reverse electric field to guide the ASRB to colonize in a direction away from the anode (i.e., the upper middle layer of the carrier).

[0044] HSRB colonization (upper and middle layers / lower layers of the slow-release layer): Remove the colonization solution and rinse gently. Inject HSRB culture medium. Turn off the electric field and switch the magnetic field to a constant 100 mT, then let it stand for 3 hours. Utilizing the potential attraction of HSRB to the magnetic field and its diffusion effect, it further colonizes into the depths of the conductive layer and the lower layer of the porous slow-release layer.

[0045] Step S3: The colonized carrier modules (10 in total) are vertically suspended and evenly arranged in an upflow anaerobic acclimation reactor with an effective volume of 2L. A constant temperature water jacket is installed outside the reactor to maintain the temperature at 35±0.5℃. The water inlet is controlled by a precision peristaltic pump.

[0046] Three-stage acclimatization process: Phase I (Start-up period, 7 days): The influent is artificially prepared, containing sodium acetate (COD 500 mg / L), sodium lactate (COD 300 mg / L), and sulfate (provided by analytical grade MgSO4 and filtered effluent at a 1:1 ratio) at a concentration of 800 mg / L. The pH is adjusted to 7.0. HRT = 48 h.

[0047] Phase II (Enhanced Phase, 14 days): The influent was switched to a mixed carbon source of glucose (COD 400 mg / L) and ethanol (COD 400 mg / L), and the sulfate concentration was increased to 1500 mg / L (all supplied by the filtered effluent). HRT = 36 h.

[0048] Phase III (Maturation and Stabilization Period, 21 days): Fluctuation simulation was implemented. The carbon source type (one or a mixture of two of sodium acetate, sodium propionate, sodium lactate, and ethanol, maintaining a total COD of 800 mg / L) and sulfate concentration (randomly selected from three levels: 1200, 1600, and 2000 mg / L) were randomly switched daily via an automated sampling system. Simultaneously, in-situ weak electrical stimulation was activated: the conductive layer of the carrier module was connected in parallel with a DC power supply via wires, applying a constant bias voltage of 0.2 V (relative to the Ag / AgCl reference electrode inserted into the reactor) to the entire carrier array. HRT = 24 h.

[0049] Maturity assessment: At the end of Stage III, a uniform, dense, dark gray biofilm formed on the carrier surface, with a thickness of approximately 2.5-3.0 mm (measured by microelectrode puncture). Small samples were taken for activity testing; the specific sulfate reduction rate in standard sodium acetate-sulfate medium reached 455 mg SO4. 2- / g VSS·d indicates that the preparation of mature S-FSRB Mat is complete.

[0050] Comparative Example 1 The scheme described in Example 1 was adopted, except that the application of electric and magnetic fields was omitted in step S2. Equal volumes of AHB, ASRB, and HSRB bacterial solutions were mixed and uniformly inoculated onto a pre-activated composite carrier in one go. After standing for 5 hours for adsorption, the gradient acclimatization in step S3 was carried out in the same manner as in Example 1.

[0051] Comparative Example 2 The scheme described in Example 1 is adopted, except that in step S1, only a single carbon felt material (approximately 10 mm thick) is used as the carrier, without the composite nitrogen-doped graphene / Fe3O4 layer and PCL / HA slow-release layer. Steps S2 and S3 are exactly the same as in Example 1.

[0052] Comparative Example 3 The scheme described in Example 1 is adopted, except that in step S1, the carrier consists only of a carbon felt layer and a nitrogen-doped graphene / Fe3O4 conductive layer, omitting the upper PCL / HA slow-release layer. Steps S2 and S3 are exactly the same as in Example 1, with all nutrients added through the influent.

[0053] Comparative Example 4 The scheme described in Example 1 is adopted, except that in step S3, the "fluctuation simulation acclimatization" and "weak electrical stimulation" in the third stage are cancelled. Only stage I (single organic acid) and stage II (mixed carbon source) acclimatization are performed, and the total acclimatization time is shortened to 21 days.

[0054] Comparative Example 5 The scheme described in Example 1 is adopted, except that in step S2, the colonization order of the functional bacterial community is changed, and electric field-magnetic field directional colonization is carried out in the order of HSRB -> AHB -> ASRB.

[0055] Comparative Example 6 The scheme described in Example 1 is adopted, except that in stages II and III of step S3, sodium acetate is used as the sole carbon source throughout the process, and the sulfate concentration is still increased and fluctuated in a gradient.

[0056] Comparative Example 7 The scheme described in Example 1 is adopted, except that in the directional colonization process in step S2, the electric field strength is increased to 2.0 V / cm (AHB) and 1.5 V / cm (ASRB), while the magnetic field parameters remain unchanged.

[0057] Comparative Example 8 The scheme described in Example 1 is adopted, except that in step S2, only one functional bacterial group, acetic acid oxidized SRB (ASRB), is inoculated, and its colonization on the carrier is guided by an electric field-magnetic field. The carbon source for domestication in S3 is only sodium acetate.

[0058] Comparative Example 9 The scheme described in Example 1 is adopted, except that in the entire directional colonization process in step S2, only a constant magnetic field of 100mT is used, and an alternating magnetic field is not used, while the electric field parameters remain unchanged.

[0059] Water quality related indicator testing methods: 1. Sulfate radical (SO4 2- Determination of removal rate: Test method: Ion chromatography (IC), referring to "Determination of Inorganic Anions in Water by Ion Chromatography" (HJ 84-2016).

[0060] Specific operations: Sample pretreatment: Collect 100 mL samples of reactor influent and effluent at a fixed time each day (e.g., 9:00 AM) and immediately filter them through a 0.45 μm water-based microporous membrane to remove suspended solids and bacteria.

[0061] Storage: The filtrate should be stored at 4°C and the determination should be completed within 24 hours.

[0062] Instrument parameters: Chromatograph: Thermo Scientific™ Dionex™ ICS-600 Analytical column: Dionex™ IonPac™ AS11-HC (4 × 250 mm) Protective pillars: Dionex™ IonPac™ AG11-HC (4×50 mm) Eluent: KOH gradient elution, 30 mM (0-10 min), 30-45 mM (10-20 min). Flow rate: 1.0 mL / min Column temperature: 30℃ Detector: Conductivity detector, Suppression current: 75 mA Standard curve: SO4 concentrations of 1, 5, 10, 50, and 100 mg / L were prepared using Na2SO4. 2- Standard solution series, establish standard curve (R 2 >0.999).

[0063] Calculation: Sulfate removal rate (%) = [(Influent SO42-)] 2- Concentration - Effluent SO4 2- Concentration) / Influent SO4 2- [Concentration] × 100%.

[0064] 2. Determination of impact recovery time: Test method: Artificial impact load test and continuous monitoring method Specific operations: Baseline period: The average SO4 concentration measured over three consecutive days under stable operating conditions. 2- The removal rate is used as the baseline value (R0).

[0065] Impact Implementation: On the 4th day, by increasing the proportion of concentrated effluent to the influent, SO4 in the influent was reduced within 2 hours. 2-Increase the concentration to 150% of the baseline value (e.g., from 2500 mg / L to 3750 mg / L) and maintain this concentration in the influent.

[0066] Monitoring frequency: After the shock begins, SO4 levels in the effluent are measured every 4 hours. 2- Concentration, calculate instantaneous removal rate (R) t ).

[0067] Recovery determination: Recovery time = From the start of the impact to R t The continuous time experienced by ≥ 0.9 × R0.

[0068] Recovery verification: After the recovery standard is met, continue monitoring for 24 hours to confirm that the removal rate is stable at the level after recovery.

[0069] 3. Determination of COD consumption per unit sulfate removal: Test method: Chemical oxygen demand (COD) determination and material balance method. COD determination refers to the dichromate method for the determination of chemical oxygen demand in water quality (HJ 828-2017).

[0070] Specific operations: COD measurement: The total COD of the influent is measured daily (including the COD of the effluent and the COD of the added carbon source).

[0071] A Hach DRB200 digester and a DR3900 spectrophotometer were used. 2.0 mL of filtered water sample was added to a pre-filled COD reagent tube (containing potassium dichromate, mercuric sulfate, and silver sulfate), and digested at 150°C for 2 hours.

[0072] After cooling, the absorbance was measured, and the COD concentration was calculated.

[0073] Carbon source addition record: Accurately record the daily addition amount and concentration of each carbon source (sodium acetate, ethanol, etc.) and convert it into COD contribution value.

[0074] Sulfur removal rate calculation: based on daily influent and effluent SO4 2- Calculate the daily SO4 removal based on concentration gradient and flow rate. 2- Mass, and converted to sulfur element mass (S, molecular weight 32).

[0075] Calculate: Unit SO4 2- COD removal consumption (kg COD / kg S) = (Mass of COD corresponding to total COD in the influent + Mass of COD added from external carbon source) / Mass of sulfur removed.

[0076] 4. Determination of total abundance of core SRB: Testing methods: High-throughput sequencing and bioinformatics analysis.

[0077] Specific operations: Sample collection: On day 90 of operation, three 1cm×1cm samples of the mycelial mat were randomly taken from different positions (upper, middle, and lower) of each reactor carrier. The samples were mixed and used as a biological replicate, with three replicates for each group.

[0078] DNA extraction: The FastDNA® Spin Kit for Soil (MP Biomedicals) was used according to the instructions. DNA concentration and quality were assessed using Nanodrop and agarose gel electrophoresis.

[0079] PCR amplification and library preparation: Primers: Targeting the V4 region of the bacterial 16S rRNA gene. 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3').

[0080] Reaction system: 25 μL, containing 12.5 μL KAPA HiFi HotStart ReadyMix, 0.5 μM each of upstream and downstream primers, and 20-30 ng template DNA.

[0081] Program: 95℃ for 3 min; 30 cycles (95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s); 72℃ for 5 min.

[0082] Sequencing: The purified amplicon was sent to Shanghai Paisenuo Biotechnology Co., Ltd. for PE250 sequencing on the Illumina NovaSeq6000 platform.

[0083] Data Analysis: Use QIIME2 (version 2020.11) for raw data quality control, noise reduction (DADA2), and generation of ASV tables.

[0084] Species annotations were performed using the Silva 138 database.

[0085] Core SRB definition: The relative abundance of known typical sulfate-reducing bacteria genera, including Desulfovibrio, Desulfobulbus, Desulfobacter, Desulfocurvus, Desulfomicrobium, Desulfatibacillum, Desulfococcus, and Desulfosarcina, is added together to obtain the "core SRB total abundance".

[0086] 5. Observation of the structural characteristics of the mycelial mat: Test method: Laser confocal microscopy (CLSM) combined with fluorescence in situ hybridization (FISH).

[0087] Specific procedures: Sample fixation and sectioning: Take the carrier mycelium and fix it with 4% paraformaldehyde (PBS buffer, pH 7.4) at 4°C for 4 hours. After dehydration and clearing, use a cryostat (Leica CM1950) to cut along a direction perpendicular to the carrier surface to obtain longitudinal sections 10-20 μm thick, and mount them on glass slides.

[0088] FISH hybridization: Using SRB-specific oligonucleotide probes: DSV698 (5'-CGTTCGTCGCGGTCCTT-3'), for Desulfovibrio spp., Cy3 marked (red).

[0089] DSS658 (5'-TCCACTTCCCTCTCCCAT-3'), for Desulfobulbus spp., Cy5 notation (cyan).

[0090] EUB338 I-III (mixed), for most bacteria, FITC-marked (green).

[0091] Hybridization buffer contains 0.9 M NaCl and 35% formamide (toughness). Hybridize at 46°C in the dark for 3 hours.

[0092] Wash with washing buffer at 48°C for 15 minutes.

[0093] CLSM observations: Instrument: Leica TCS SP8. Objective: 63× oil immersion lens. Excitation / emission wavelengths: FITC (488 / 500-550 nm), Cy3 (552 / 560-620 nm), Cy5 (638 / 650-720 nm).

[0094] Z-axis scan: Scan the entire slice thickness in 0.5 μm increments to obtain a 3D image stack.

[0095] Image analysis: Three-dimensional reconstruction and fluorescence signal analysis were performed using ImageJ and Imaris software.

[0096] "Clear zoning" criteria: Different fluorescent markers (representing different functional bacterial groups) show a clear stratified distribution along the Z-axis, rather than being uniformly mixed.

[0097] "SRB enriched in the conductive layer" determination: The maximum intensity layer of red (DSV698) and cyan (DSS658) fluorescence signals highly coincides with the physical location of the conductive layer in the carrier structure (determined by the reflected light channel).

[0098] Key data for Example 1 and Comparative Examples 1 to 9 during the 90-day operation period are shown in Table 1: Table 1 The results above show that Comparative Example 1 (mixed inoculation) resulted in chaotic spatial distribution of microorganisms and a disjointed metabolic chain, manifested as the lowest removal rate (78.6%), the slowest recovery, and high COD consumption. This demonstrates that random attachment without physical field guidance cannot self-organize to form efficient zoning.

[0099] Comparative Example 5 (incorrect colonization order) resulted in SRB being incorrectly colonized in the bottom layer near the influent, making them susceptible to direct effects from water flow shear and substrate type changes, leading to decreased stability. This demonstrates that the colonization order must match the metabolic flow direction (influent → products).

[0100] Comparative Example 8 (a single bacterial community), although having high SRB abundance, lacked the pre-metabolic support of acid-producing bacteria, resulting in low efficiency when facing complex carbon sources and a lack of metabolic flexibility. This demonstrates the necessity of spatial cooperation among multiple bacterial communities.

[0101] While the bacterial mat in Comparative Example 2 (without a conductive layer) exhibited some removal capability, it lacked efficient electron transport channels and could not integrate sensing and electrical stimulation, resulting in lower performance and shock resistance (recovery time 48 hours) compared to Example 1. This demonstrates that the conductive layer is the core element for enhancing DIET and achieving intelligent interaction.

[0102] In Comparative Example 3 (without a slow-release layer), the upper layer of SRB in the mycelial mat experienced poor growth due to unstable nutrient supply (especially trace elements such as Mo) during long-term operation, resulting in insufficient overall structural strength and sustainability. This demonstrates that the slow-release layer is crucial for maintaining the activity and structural stability of the deep mycelial community.

[0103] The bacterial mats in Comparative Example 4 (without fluctuation acclimatization / electric stimulation) performed reasonably well during routine operation, but their recovery ability was significantly weaker than that in Example 1 when faced with actual fluctuating water inflow (30h vs <24h). This demonstrates that active fluctuation acclimatization and electrical stimulation can pre-enhance the adaptability of the bacterial community and interspecies electrical connections.

[0104] Comparative Example 6 (single carbon source acclimatization) showed high acetic acid utilization efficiency (high SRB abundance), but high COD consumption, and its adaptability to other carbon sources that may appear in the effluent is questionable. This demonstrates that multi-carbon source fluctuation acclimatization can broaden the substrate spectrum of the bacterial community and enhance the robustness of the system.

[0105] Comparative Example 7 (high-intensity electric field) may adversely affect the cell membrane potential of microorganisms or even cause damage, thus reducing bacterial activity and colonization success rate. This demonstrates that a "weak" electric field and an "alternating-constant" combined magnetic field are the key parameter windows for achieving effective and non-destructive directional guidance in this method.

[0106] Comparative Example 9 (constant magnet only) did not provide sufficient agitation and penetration of microorganisms in the early stage of colonization, resulting in uneven colonization of the bottom layer and affecting the uniformity and stability of the overall fungal carpet.

[0107] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for the preparation of a spatially functionalized sulfate-reducing bacteria carpet, characterized in that, The method comprises the following steps: S1: preparing a three-layer composite carrier, which comprises a carbon felt layer as a bottom bio-attachment matrix, a nitrogen-doped graphene / Fe3O4 composite layer as an intermediate conductive and magnetic response layer, and a polycaprolactone / hydroxyapatite porous layer as an upper slow-release nutrient supply layer, and pre-activating the carrier with microbial secretions; S2: electric field-magnetic field synergistic spatial directional colonization: under the synergistic effect of a weak electric field and an alternating / constant magnetic field, acid-producing and hydrogen-producing bacteria, acetate-oxidizing sulfate-reducing bacteria, and hydrogenotrophic sulfate-reducing bacteria are sequentially colonized in different spatial depths of the carrier to form a preliminary spatial functional division; S3: multi-substrate coupled gradient acclimation and bacterial carpet maturation: the colonized carrier is placed in a continuous flow reactor and sequentially undergoes three-stage increasing acclimation with single organic acid, mixed complex carbon source, and fluctuating carbon source as influent, and in-situ weak electric stimulation is applied in the maturation period to finally obtain a mature spatial functional bacterial carpet.

2. The method of claim 1, wherein the method further comprises, In the S1, the Fe3O4 nanoparticles in the nitrogen-doped graphene / Fe3O4 composite layer have a particle size of 20-50 nm, and a loading amount of 1-3 mg / cm 2 ; the polycaprolactone / hydroxyapatite porous layer is loaded with slow-release microcapsules encapsulating diammonium hydrogen phosphate and sodium molybdate.

3. The method of claim 1, wherein the method further comprises, In S2, the intensity of the weak electric field is 0.3-0.8 V / cm, and the direction is directed to the carrier when colonizing acid-producing and hydrogen-producing bacteria, and is directed to the solution when colonizing acetate-oxidizing SRB; the magnetic field uses an alternating magnetic field with a frequency of 5-15 Hz and an intensity of 30-70 mT in the early colonization stage, and uses a constant magnetic field with an intensity of 80-120 mT when colonizing HSRB.

4. The method of claim 1, wherein the method further comprises, In S2, the acid-producing and hydrogen-producing bacteria mainly colonize in the carbon felt bottom layer, the acetate-oxidizing SRB mainly colonize in the conductive layer and the upper layer of the carbon felt, and the hydrogenotrophic SRB mainly colonize in the deep layer of the conductive layer and the lower layer of the polycaprolactone / hydroxyapatite slow-release layer.

5. The method of claim 1, wherein the method further comprises, In S3, in the fluctuating carbon source acclimation stage, the type of influent carbon source randomly or periodically changes among one or more of acetic acid, propionic acid, lactic acid, and ethanol, and the sulfate concentration changes in steps within a preset range; the in-situ weak electric stimulation is a direct current constant voltage of 0.1-0.4 V applied through the conductive layer.

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