Composite functional bacteria preparation, enrichment method thereof and application thereof in anambr

Abstract

The application discloses a composite functional bacteria preparation, an enrichment method thereof and application of the composite functional bacteria preparation in an AnMBR, and belongs to the technical field of sewage anaerobic membrane biological treatment. The application adopts anaerobic sludge as a substrate, provides gamma-caprolactone (GCL) and glucose as a composite carbon source pulse supply under the condition of electric stimulation assistance, combines two-stage enrichment and gradient screening processes, and obtains composite functional bacteria which has both quorum quenching (QQ) and methanogenesis capacity. 3+ The composite microspheres are used for AnMBR dosing operation. The electric stimulation strengthens degradation of AHLs and proliferation of methanogens in the enrichment stage, accelerates formation of a bacterial community, and improves biological stability and methane production of the system. Compared with existing functional bacteria enrichment methods, the application introduces electric stimulation as a metabolic regulation means in the enrichment stage, significantly improves the functional expression level of the bacterial community, and is a core technical path for realizing membrane pollution mitigation and collaborative improvement of methane production capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of anaerobic membrane bioreactor technology, in particular to a composite functional bacteria preparation, an enrichment method thereof and application of the composite functional bacteria preparation in an AnMBR. BACKGROUND

[0002] Anaerobic Membrane Bioreactor (AnMBR) couples anaerobic biological conversion with membrane separation technology, which has the dual advantages of efficient organic matter removal and methane energy recovery, and is suitable for high-concentration organic wastewater treatment, municipal sludge reuse and other fields. However, in the AnMBR environment, due to the characteristics of high sludge age, low dissolved oxygen, high organic load and metabolic product enrichment in the operating conditions, biofilm formation is particularly significant, so the stable operation of AnMBR still faces the severe challenge of biofouling, which is manifested as continuous increase of transmembrane pressure difference (TMP), attenuation of membrane flux, increase of cleaning frequency and increase of operating energy consumption. Biofilm pollution mainly comes from the following three aspects: (1) soluble microbial products (SMP) and extracellular polymeric substances (EPS) are enriched on the membrane surface to form a dense gel layer; (2) microbial flocs and bacteria adhere and grow on the membrane surface to form biofilm; (3) physical damage caused by membrane pore blockage and irreversible adsorption.

[0003] Studies have shown that the Quorum Sensing (QS) mechanism plays a key regulatory role in bacterial adhesion, EPS synthesis and biofilm homeostasis. With acyl homoserine lactone (AHL) as a typical signal molecule, the LuxI / LuxR system regulates the coordination of group behavior, including extracellular enzyme expression, EPS enhancement and biofilm structure stability. There is a significant correlation between the concentration of AHLs in the AnMBR system and the severity of membrane pollution (TMP increase rate, EPS protein / polysaccharide content). Therefore, to inhibit membrane pollution, Quorum Quenching (QQ) technology has become an important biological control path. Common strategies include: (1) adding AHL-degrading enzymes such as lactonase and acylase; (2) enriching / adding QQ functional bacteria; (3) immobilizing the above enzymes or bacterial flora in the system to improve stability and mass transfer efficiency. However, traditional QQ methods still have multiple bottlenecks: enzyme preparations are expensive and easy to lose activity, free bacteria are not easy to maintain, and the enrichment system with AHLs as an inducer is high in cost and complex in operation. In addition, such methods often do not take into account the promotion of methanogenesis efficiency, making it difficult to achieve the synergy of pollution control and energy recovery.

[0004] To this end, in recent years, there are gradually some researches exploring the introduction of low-intensity electric stimulation (Electric Stimulation, ES) into the anaerobic system as an auxiliary means of biological enrichment. Under the premise of ensuring anaerobicity is not destroyed, moderate electric field can produce positive effects in the following aspects: (1) by regulating the cell membrane potential and proton gradient, activating the electron transport chain and extracellular enzyme expression in specific metabolic pathways; (2) enhancing the extracellular electron transfer capacity, promoting the anaerobic respiration process closely related to methane production; (3) selectively promoting the dominance of functional bacteria with QQ activity and methane production capacity, improving the community structure; (4) accelerating the AHLs degradation kinetics process, accelerating the functional bacteria enrichment cycle and improving the biological stability. At present, the researches on electric stimulation in strengthening anaerobic granular sludge formation and improving the hydrogen / methane production capacity of electroactive bacteria have achieved initial success, but the coupling application of electric stimulation in the "enrichment stage" of functional bacteria is still in the early stage of exploration, and most of the researches focus on the electrochemical reaction or the body of the membrane electrode system. There is still a lack of systematic scheme on how to accurately regulate the functional bacteria population succession and QS quenching process through electric stimulation. In addition, the parameters such as electric stimulation intensity, duration and electrode layout have not formed a mature window, and there are risks of non-target side effects such as pH deviation, electrode passivation and bacterial selection deviation.

[0005] Therefore, it is urgent to construct a new type of complex bacteria enrichment method with strong engineering adaptability, which has the functions of electric stimulation regulation, low-cost induction, quorum sensing quenching, methane metabolism activation and engineering adaptability. SUMMARY

[0006] The purpose of the present application is to provide a complex functional bacteria preparation and its enrichment method and application in AnMBR. Combining the low-cost simulation characteristics of γ-hexalactone (GCL) as a QS-like signal inducer and glucose as an auxiliary carbon source for metabolic support, and through the intermittent action of low-intensity direct current electric field, a ternary synergistic enrichment strategy of "low-intensity electric stimulation + GCL + glucose pulse supply" is constructed as the key path of functional bacteria strengthening. Cooperating with the two-stage anaerobic selection process, the AHLs degradation rate is systematically improved, the functional bacteria co-metabolic process is stabilized, and the membrane fouling control and methane production dual performance of the subsequent immobilized microspheres in AnMBR are enhanced, thereby solving the problems of membrane fouling aggravation, methane production decline, high cost and poor stability of existing functional bacteria enrichment process in AnMBR operation.

[0007] To achieve the above purpose, the present application provides an enrichment method of a complex functional bacteria preparation, which specifically comprises the following steps:

[0008] S1, substrate pre-culture: using anaerobic digestion sludge as inoculation substrate, preparing bicarbonate buffer anaerobic basic medium, and pre-culturing under N2 gas phase and 35±2 ℃ conditions;

[0009] S2, pre-enrichment: adding GCL and glucose double carbon source to the culture system of step S1 for pulse induction, and applying low-intensity direct current stimulation for pre-enrichment to obtain mixed bacteria liquid rich in QQ and methanogens;

[0010] S3, gradient screening: inoculating the mixed bacteria liquid obtained in step S2 into a new culture medium, and increasing the GCL concentration for gradient screening, taking the removal rate of AHLs within 240 min and the methane yield as screening indexes to obtain composite functional bacteria with high QQ activity and methane production capacity;

[0011] S4, immobilized carrier construction: immobilizing the composite functional bacteria obtained in step S3 in sodium alginate-chitosan-Fe 3+ In the composite gel microspheres, the prepared immobilized microspheres are the target product.

[0012] Preferably, in step S1, the anaerobic basal medium contains NaHCO3, NH4Cl, KH2PO4, KCl, Mg 2+ , Ca 2+ , trace element and vitamin solution.

[0013] Preferably, in step S2, the voltage gradient of the low-intensity direct current stimulation is 0.8 V / cm, and the electrode material is a graphite rod.

[0014] Preferably, in step S2, the initial mass concentration of GCL is 50 mg / L, and the initial mass concentration of glucose is 100 mg / L; each 48 hours is a period, the glucose concentration is maintained at 50-100 mg / L, and the GCL concentration is increased to 100-200 mg / L.

[0015] Preferably, in step S3, the inoculation amount of gradient screening is 10% of the volume of the mixed bacteria liquid of step S2; and the screening index is specifically that the removal rate of AHLs within 240 min is greater than or equal to 70%, and the methane yield is increased by greater than or equal to 15% compared with the un-enriched control.

[0016] Preferably, in step S4, the sodium alginate-chitosan-Fe 3+ The enrichment method of the composite gel microspheres is as follows: the mixed solution of sodium alginate and chitosan containing the composite functional bacteria is added dropwise into a 0.1 M FeCl3 solution for crosslinking for 10-20 min, and then transferred into a 0.05 M Na2SO4 solution for secondary crosslinking for 5-15 min, so that the particle size of the obtained gel microspheres is 2.5-3.0 mm, and the water content is 70-75%.

[0017] The application also provides a composite functional bacteria preparation obtained by the above enrichment method.

[0018] The present invention also provides the application of the above-mentioned composite functional bacterial preparation, which is applied in AnMBR to reduce the rate of increase of transmembrane pressure gradient, reduce the accumulation of extracellular polymers and increase methane yield.

[0019] Preferably, the compound functional microbial preparation is added to the AnMBR for operation, wherein the initial addition amount of the compound functional microbial preparation in the AnMBR is 0.05% of the effective volume of the reactor, and 30-50% of the initial addition amount is replenished every 30-45 days.

[0020] Preferably, the AnMBR is a submerged hollow fiber membrane with the following operating conditions: operating temperature of 35±2℃, hydraulic retention time of 24 h, and sludge age of 60-100 days.

[0021] The core innovation of this invention lies in the fact that, based on traditional carbon source induction, it is the first time that low-intensity electrical stimulation and GCL+glucose dual carbon source induction are synergistically coupled in an anaerobic enrichment system. By intervening with an external electric field, the metabolic behavior of the microbial community is precisely regulated, thereby achieving a synergistic enhancement of quorum sensing quenching (QQ) ability and methanogenesis pathway from the source.

[0022] The compound functional bacterial preparation, its enrichment method, and its application in AnMBR provided by this invention, compared with the control group that did not adopt the measures of this invention, show the following effects within the same membrane configuration and operating window (taking statistics from continuous operation ≥30 days as an example):

[0023] 1) Film inhibition effect: TMP growth slope is reduced by 30-60%, and the cleaning cycle is extended by 25-50%;

[0024] 2) EPS control: EPS is reduced by 15-20%, with the protein portion showing a more significant decrease;

[0025] 3) Energy recovery: Cumulative methane production increased by 10–35%;

[0026] 4) Stability: The immobilized support maintained structural integrity for ≥30 days under normal stirring and shock conditions at 35±2 °C, and QQ activity remained well maintained;

[0027] 5) Enrichment efficiency: Applying stable electrical stimulation (0.8 V / cm) during the enrichment stage significantly promoted the synergistic proliferation of QQ bacteria and methanogens, resulting in a stable bacterial community structure and a longer duration of functional maintenance.

[0028] 6) Cost / Feasibility: GCL is significantly cheaper than AHLs as an inducer, and dual carbon source pulses and online micro-maintenance avoid the problems of frequent enzyme replenishment and high cost.

[0029] The above range represents the reproducible range for engineering verification. The specific values ​​vary depending on the influent water quality, membrane material, and operating window, but do not affect the substantial improvements of this invention.

[0030] The composite functional bacterial preparation, its enrichment method, and its application in AnMBR provided by this invention represent substantial differences and advancements compared to existing technologies:

[0031] 1) Different induction strategies: Non-AHLs bulk induction, pulse induction with GCL+ co-carbon source and introduction of electrical stimulation for synergistic induction, reduce cost and nitrogen side effects, and improve enrichment efficiency;

[0032] 2) Different enrichment process: A two-stage process (pre-enrichment + gradient screening) is proposed, and electrical stimulation is applied in the gradient screening stage. The AHLs 240 min removal rate and methane hourly yield are used for joint screening to take into account both "controlling membrane fouling and improving gas production efficiency".

[0033] 3) Different carrier structures: Alginic acid-chitosan-Fe 3+ Composite cross-linked microspheres improve shear resistance and mass transfer stability, outperforming common Ca 2+ alginic acid;

[0034] 4) Improved indicator system: Closed-loop verification from multiple dimensions such as TMP slope, EPS classification, AHLs residue, SMA / gas production to achieve consistency between mechanism and performance;

[0035] 5) Novel enhancement methods: Electrical stimulation is introduced to enhance the bioaccumulation process, forming a stable and efficient QQ+ methanogenic bifunctional bacterial community, achieving a breakthrough improvement over existing pure biological induction methods.

[0036] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is an embodiment of the AnMBR device and schematic diagram of the present invention;

[0039] Figure 2 Sodium alginate-chitosan-Fe obtained in the embodiments of the present invention 3+ Image of composite microspheres;

[0040] Figure 3This is a comparison of the degradation efficiency of C6-HSL by the composite functional bacteria in Example 3 and Comparative Example 1 of the present invention;

[0041] Figure 4 This is a comparison graph of the transmembrane pressure difference (TMP) versus time during the operation of AnMBR in Example 4 and Comparative Example 2 of the present invention.

[0042] Figure 5 This is a comparison chart of the cumulative methane production during the operation of AnMBR between Example 4 and Comparative Example 2 of the present invention;

[0043] Figure 6 This is a comparison chart of the polysaccharide and protein content in EPS during AnMBR operation between Example 4 and Comparative Example 2 of the present invention. Detailed Implementation

[0044] This invention provides a compound functional bacterial preparation, its enrichment method, and its application in AnMBR. The enrichment method includes the following steps:

[0045] S1, Substrate Pre-culture

[0046] Under anaerobic conditions, municipal digested sludge or AnMBR waste sludge was selected as the inoculum (preferably VSS 3–8 g / L). The culture medium contained NaHCO3, NH4Cl, KH2PO4, KCl, and Mg. 2+ Ca 2+ The bicarbonate buffer system was used, and trace elements / vitamins were added. The culture was carried out under gaseous N2, temperature 35±2 °C, and shaking speed 80–120 rpm. Furthermore, in order to inhibit acidification accumulation, VFA could be monitored online and regulated by carbon supplementation strategy.

[0047] S2, combined induction by dual carbon source pulse induction and electrical stimulation (Stage I, pre-enrichment).

[0048] Under anaerobic conditions, γ-caprolactone (GCL, initial concentration 50 mg / L) and glucose (100 mg / L) were added as inducing substrates to initiate the pre-enrichment reaction. Each cycle lasted 48 hours, maintaining a glucose concentration of 50–100 mg / L while gradually increasing the GCL concentration to 200 mg / L, creating a pulsed supply mode. Simultaneously, a low-intensity direct current stimulation device (voltage gradient 0.8 V / cm) was installed in the reaction system, using graphite rods as the anode and cathode materials, positioned at both ends of the reactor.

[0049] Electrical stimulation enhances the screening efficiency of functional bacteria through the following mechanisms:

[0050] It activates extracellular electron transfer reactions in anaerobic metabolic pathways, promoting the rapid proliferation of methanogens.

[0051] It promotes the high expression of AHLs degrading enzyme systems (such as lactones and oxidoreductases) and improves the efficiency of QS signal interference.

[0052] Synergistic GCL induction optimizes the metabolic state of target functional microbiota under hypoxic / low redox potential conditions.

[0053] Shorten the enrichment cycle and improve the biological stability of the microbial community, forming a dominant population with well-defined functions within 7–10 days.

[0054] This synergistic induction process constitutes the most significant difference between this invention and existing functional bacteria enrichment processes. Its "electrostimulation + carbon source coupling induction" method is the core technical path for this invention to achieve simultaneous improvement in membrane fouling control and methane production capacity, and provides key biological support for the subsequent construction of immobilized carriers and the stable operation of the AnMBR system.

[0055] S3, Gradient Filtering (Phase II)

[0056] 10% (v / v) of the Stage I supernatant was inoculated into the new system, and the GCL was gradually increased to the range of 100–200 mg / L, while maintaining glucose at 50–100 mg / L. Using AHLs removal rate at 240 min and methanogenic yield as the core screening indicators, a combined functional bacterium with both QQ activity and enhanced methanogenesis was screened. Furthermore, when the methanogenic yield decreased by more than 20% in a short period, glucose was reduced to 50 mg / L and acetate was supplemented at 100 mg / L to stabilize the state.

[0057] S4, Construction of Immobilized Carrier

[0058] The compound functional bacteria were inoculated at a concentration of 10% (wet volume / gel system) into the sodium alginate-chitosan-Fe group. 3+ Composite system: Sodium alginate 2% (w / v), chitosan 0.5% (w / v, dissolved in 0.5% acetic acid), added dropwise to 0.1 M FeCl3 for crosslinking for 15 min, then transferred to 0.05 M Na2SO4 for secondary crosslinking for 10 min, yielding 2.5–3.0 mm wet beads with a moisture content of 70–75%. The outer chitosan layer provides a cationic network, and the middle Fe... 3+ - The alginate network structure improves shear strength and enhances pore connectivity.

[0059] The product obtained above was added to an AnMBR (Animated Microbiota Blot) system with a submerged hollow fiber membrane and operated at a dosage of 0.05% v / v wet beads, with a replenishment of 30–50% every 30–45 days. The operating conditions of the AnMBR were: temperature 35±2 °C, hydraulic retention time (HRT) 24 h, and sludge retention time (SRT) 60–100 days.

[0060] The following indicators were set: TMP–t, corrected flux, SMP / EPS (S-EPS / L-EPS / T-EPS), methane yield, and AHLs residual concentration. The TMP growth slope and washing cycle were used as the core of the engineering evaluation, while the AHLs removal rate and EPS protein / polysaccharide ratio were used as mechanistic evidence. AHLs were quantified using LC-MS / MS; EPS fractionation was performed using sodium chloride thermal extraction.

[0061] When QQ activity decreases or TMP slope increases, replenish beads.

[0062] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims and are all within the protection scope of the present invention.

[0063] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0064] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0065] Unless otherwise specified, the reagents, instruments, and equipment used in this invention are all commonly used by those skilled in the art, and the testing standards all use national or international standards commonly used in the field, without further explanation.

[0066] Example

[0067] S1, Two-stage anaerobic enrichment and screening of multifunctional bacteria

[0068] S1.1 Inoculation substrate: municipal anaerobic digestion sludge or AnMBR waste sludge, preferably VSS 3 g / L.

[0069] S1.2 Anaerobic basal culture medium (per liter): NaHCO3 2.5 g; NH4Cl 0.3 g; KH2PO4 0.3 g; KCl 0.1 g; MgCl2·6H2O 0.1 g; CaCl2·2H2O 0.05 g; Trace element solution 1 mL, Trace element composition (mg / L): FeSO4∙7H2O (2100), H3BO3 (30), MnCl2∙4H2O (100), CoCl2∙4H2O (190), NiCl2∙4H2O (24), CuCl2∙4H2O (144), Na2MoO4∙2H2O (36); Vitamin solution 1 mL, Vitamin solution composition (mg / L): Folic acid (10); pH 7.3.

[0070] S1.3 Anaerobic environment: In anaerobic serum bottles, the solution is replaced with N2 three times; 35 ℃; shaking speed 120 rpm; liquid-to-volume ratio 60% (v / v).

[0071] S2, Pre-enrichment (Stage I, 1–14 d)

[0072] S2.1 Induction and carbon source: Start with GCL 50 mg / L + glucose 100 mg / L, and feed once after 24 h; then supplement the carbon source every 48 h, maintain glucose at 100 mg / L, and gradually increase the GCL concentration to 200 mg / L.

[0073] S2.2, Electrical Stimulation Configuration

[0074] Electrode materials: The anode and cathode are graphite rods, both led out by stainless steel wires.

[0075] Electrode placement: The anode and cathode are fixed on both sides of the inner wall of the serum bottle.

[0076] Voltage control: A constant voltage DC power supply is used, with a voltage gradient of 0.8 V / cm.

[0077] Oxygen control: All electrode components undergo deoxygenation treatment before installation, and N2 replacement is maintained throughout the operation to ensure anaerobic conditions.

[0078] Monitoring items: Record changes in voltage, current (≤10 mA), pH, VFA, methane yield, and AHLs concentration every 24 h.

[0079] S3, Gradient Screening (Phase II, 15–45 days)

[0080] S3.1, Inoculation and Step-up: Introduce the new system with 10% (v / v) of the Stage I supernatant; increase GCL to 100→150→200 mg / L every 48 h, and maintain glucose at 100 mg / L (adjusted with VFA).

[0081] Phase II maintains the same electrode structure and voltage settings as Phase I; maintain pH at 7.0–7.4 to avoid acid-base shifts caused by electrolytic side reactions.

[0082] S3.2 Screening Indicators:

[0083] a) Removal rate of AHLs (using C6-HSL as an indicator) ≥70% within 240 min;

[0084] b) Methane yield increased by ≥15% compared to the control.

[0085] Furthermore, when short-term methanogenesis decreased by >20%, glucose was reduced to 50 mg / L and acetate was added at 100 mg / L to stabilize the state.

[0086] S3.3 Obtain composite functional bacteria for subsequent immobilization.

[0087] S4, Preparation of immobilized microspheres (sodium alginate-chitosan-Fe) 3+ complex)

[0088] S4.1 Formulation and Solution:

[0089] a) Internal phase solution: 2.0% sodium alginate (w / v) + 10% inoculated compound functional bacteria (wet volume / gel volume), gently mix and degas.

[0090] b) External phase / crosslinking solution: 0.1 M FeCl3 (pre-cooled in an ice bath to improve bead strength); Secondary crosslinking solution: 0.05 M Na2SO4.

[0091] c) Chitosan coating solution: 0.5% chitosan (w / v, dissolved in 0.5% acetic acid), filtered and sterilized for later use.

[0092] S4.2 Bead Formation and Crosslinking

[0093] a) Dropping method: Injection pump + 0.8 mm needle, flow rate 12 mL / min, drop distance 20 cm.

[0094] b) Crosslinking: Immerse in FeCl3 for 15 min; remove and crosslink again with Na2SO4 for 10 min; wash twice with purified water.

[0095] c) Chitosan coating: Place in chitosan solution for 15 minutes with gentle shaking, then remove and rinse once. This produces a product as shown in the image. Figure 2The immobilized microspheres shown have a particle size of 3.0 mm, a water content of 70–75%, and a wet mass of 35 mg of bacteria per bead.

[0096] S4.3, Recovery and Storage

[0097] a) Incubate in basal culture medium for 24 h to recover; 35 ℃.

[0098] b) Short-term storage at 4 ℃ ≤7 days; for long-term storage, a trace amount of GCL (0.5–1 mg / L) can be added weekly in an anaerobic basal matrix.

[0099] The product obtained above was added to an AnMBR with a submerged hollow fiber membrane for application and operation.

[0100] S1 and AnMBR System Setup and Startup

[0101] like Figure 1 As shown, the reactor volume is 1 L (glass material); the membrane module is submerged hollow fiber (PTFE) with a pore size of 0.1 μm; the circulation / stirring method is paddle stirring; online monitoring includes TMP (0–40 kPa), pH / temperature, and ORP; sampling ports are influent / effluent, mixed liquor, and membrane sludge.

[0102] S2, Startup Process

[0103] (1) Inoculation: Inoculate with anaerobic methanogenic sludge (VSS 3 g / L).

[0104] (2) Domestication: HRT 24 h, temperature 35 ℃, SRT 60 d.

[0105] (3) Bead addition: After revival, the immobilized microspheres prepared in the example are added to the reactor at a dosage of 0.05% (v / v) all at once.

[0106] (4) Influent: Synthetic wastewater (chemical oxygen demand is mainly supplied by glucose (COD 400 mg / L), nitrogen is supplied by NH4Cl (30 mg N / L), phosphorus is supplied by KH2PO4 (10 mg P / L), and alkalinity is adjusted by NaHCO3 (800 mg / L). The load is gradually increased; after stabilizing for 5 days, the evaluation stage is entered.

[0107] S3, Continuous Operation and Maintenance Strategy

[0108] (1) Steady-state conditions: HRT 24 h; temperature 35℃; SRT 60 d.

[0109] (2) Replenishment of beads: 50% of the original amount of beads are added every 30 days.

[0110] (3) Cleaning strategy: Physical cleaning (or replacement of membrane module) is triggered when TMP reaches 40 kPa; chemical cleaning is carried out according to the membrane supplier's recommendation.

[0111] Comparative Example 1 (Enrichment without Electrical Stimulation)

[0112] Except for the absence of electrodes and an electrical stimulation module, all other conditions (inoculation sludge, culture medium, temperature, and carbon source addition) were consistent with the previous examples. Under the same carbon source and culture conditions, the functional expression of the microbial community in the enrichment process without electrical stimulation was delayed, and the degradation of AHLs was significantly lower than that in the electrically stimulated group.

[0113] Comparative Example 2 (Enrichment of Non-functional Bacteria)

[0114] No compound functional bacteria were added and the GCL was maintained; other AnMBR operating parameters were the same as in the example. Experimental results showed that the TMP growth slope was significantly higher than in the example; the total amount of EPS protein and polysaccharides increased.

[0115] Comparative Example 3 (AHLs induced + Ca) 2+ Alginic acid mononetwork)

[0116] The bacterial community obtained using AHLs as an inducer was formed into beads by cross-linking an alginate mononetwork with CaCl2; the rest was the same as in the example. Early control of membrane fouling was effective, but the carrier mechanical degradation and activity maintenance were poor, and long-term TMP control and methanogenesis enhancement were not as good as in the example.

[0117] Figure 3 This is a comparison of the degradation efficiency of C6-HSL by the composite functional bacteria in Example 3 and Comparative Example 1. The detection method for the signal molecules was liquid chromatography-mass spectrometry (LC-MS), and the main instruments and equipment were a LC-MS system (Thermo Fisher, TSQ02-10001), electrospray ionization (ESI, Waters, USA), and a CORTECS UPLC C18 column (2.1×100mm, 1.6mm, Waters, USA). The mobile phase consisted of water containing 0.1% formic acid and methanol containing 0.1% formic acid. The specific operating steps were as follows: (1) Turn on the HPLC exhaust switch and exhaust for 25 minutes; (2) Connect the water containing 0.1% formic acid and methanol containing 0.1% formic acid to the mass spectrometer and turn on the pump to rinse; (3) Replace the water containing 0.1% formic acid and methanol containing 0.1% formic acid with water containing 0.1% formic acid and formic acid containing 0.1% acetonitrile, and exhaust for 5 minutes with each of the two solutions; (4) Turn on the nitrogen emitter, connect it to the mass spectrometer, and start the analysis and data processing; (5) After the analysis and processing were completed, repeat step 2 to rinse, cool down and turn off the machine. Figure 3It can be seen that, compared with the example without voltage enrichment, the QQ bacteria enriched by voltage in Comparative Example 1 showed an approximately 12% higher removal rate of C6-HSL, indicating that voltage enrichment helps to improve the activity of QQ bacteria.

[0118] Figure 4 This is a comparison graph showing the change in transmembrane pressure differential (TMP) over time during AnMBR operation between Example 4 and Comparative Example 2 of the present invention. Through previous functional testing and analysis, a QQ bacterium with optimal performance was obtained. The optimal QQ bacterium was encapsulated and introduced into a laboratory-built AnMBR to monitor the actual effect of the strain. During daily operation of the reactor, the influent and effluent were controlled by peristaltic pumps, and a pressure sensor was installed at the effluent end to measure the transmembrane pressure differential (TMP) every 10 minutes. No sludge was discharged except for sampling and analysis. Each reactor was connected to a gas collection bag to monitor the specific methane production in the reactor. Figure 4 It was found that Comparative Example 2, without the addition of QQ microbeads, showed signs of clogging within 6.8 ± 0.50 days. However, with the addition of QQ microbeads, the clogging cycle in this example was extended to approximately 13.2 ± 0.64 days, roughly twice the original cycle. This indicates that the functional microbeads exhibit superior bioclogging control performance due to the QQ effect.

[0119] Figure 5 This is a comparison chart of the cumulative methane production during the operation of AnMBR in Example 4 and Comparative Example 2 of the present invention. The amount of biogas produced by AnMBR was measured using gas chromatography (GC). The main steps are as follows: (1) Sample collection: Use an airtight glass syringe to collect gas samples to avoid gas leakage; (2) Pretreatment: Use a 0.45 hydrophobic filter membrane to filter and remove particulate matter; (3) Instrument preparation: Turn on the gas chromatograph (equip with a flame ionization detector FID), pre-age the HP-PLOT / Q capillary column (30m×0.53mm×40μm), set the column temperature to 80℃, the detector to 200℃, and the injection port to 150℃; (4) Standard curve calibration: Inject methane standard gas (0.1%~10% gradient concentration) and establish a peak area-degree linear equation (R²≥0.999); (5) Sample injection analysis: Inject the sample through the injector; (6) Separation and detection: The methane retention time is about 2.1min, and the FID response value is proportional to the concentration; (7) Data processing: Integrate the peak area on the chromatography workstation, quantify using the external standard method, and the parallel sample deviation is ≤5%; (8) System cleaning: After analysis, purify the system with high-purity nitrogen for 30min to remove residues, cool down and turn off the system. pass Figure 5It was found that during the 28-day operation period, the cumulative methane production of Comparative Example 2 was 1.26 L, while the methane production of the example with added QQ beads reached 1.52 L, an increase of 20.63% compared to Comparative Example 2. The methane conversion efficiency was 13.61% higher than that of Comparative Example 2. It is speculated that the addition of QQ beads can retain methanogenic bacteria that have adapted to the new environment, thereby enhancing their activity.

[0120] Figure 6 This is a comparison chart of polysaccharide and protein content in EPS during AnMBR operation between Example 4 and Comparative Example 2 of the present invention. Soluble microbial metabolites (SMP) and extracellular polymeric substances (EPS) were extracted using a modified NaCl heating technique. The specific steps are as follows: 1) SMP: 30 mL of the mud-water mixture was placed in a centrifuge tube, and the supernatant was filtered through a 0.45 µm filter at 3000 rpm for 15 minutes; 2) LB-EPS: 0.05% NaCl preheated to 60°C was added to a centrifuge tube to a final volume of 30 mL, vortexed for 1 minute, and the supernatant was filtered through a 0.45 µm filter at 4000 rpm for 10 minutes; 3) TB-EPS: 0.05% NaCl was added back to a centrifuge tube to a final volume of 30 mL. The mixture was vortexed for 1 minute, then poured into an Erlenmeyer flask and placed in a 60°C constant temperature water bath shaker for 30 minutes. Finally, the supernatant was filtered through a 0.45µm filter at 11000 rpm for 15 minutes. 5) Polysaccharides and proteins were determined using the sulfuric acid-phenol method and the Lowry method, respectively. Figure 6 It can be seen that the EPS content of Comparative Example 2 in each stage is higher than that of Example 1. The degradation of AHLs by QQ bacteria is the main reason for the decrease in PN and PS secretion.

[0121] Therefore, this invention provides a low-cost, long-term sustainable method for the enrichment and application of composite functional bacteria based on the synergistic induction of GCL, glucose, and electrical stimulation. This method combines quorum sensing quenching with the synergistic enrichment strategy of methanogenic microorganisms, relying on low-intensity electrical stimulation to regulate the functional expression of the bacterial community. It overcomes the limitations of traditional chemical induction and static screening, significantly improving the degradation capacity of AHLs and the efficiency of methane metabolism, and achieving simultaneous optimization of membrane fouling control and energy recovery. It solves the problem of rapid TMP rise and flux decline caused by QS-driven EPS secretion and biofilm homeostasis in AnMBR operation, while overcoming the shortcomings of traditional AHLs-induced enrichment, such as high cost, easy loss of free QQ bacteria, and easy pulverization of immobilized carriers.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for enriching a compound functional bacterial preparation, characterized in that, Specifically, the following steps are included: S1. Substrate pre-culture: Using anaerobic digested sludge as the inoculation substrate, prepare a bicarbonate-buffered anaerobic basic culture medium and pre-culture it under N2 gas phase and 35±2℃ conditions. S2, Pre-enrichment: Add GCL and glucose dual carbon source to the culture system of step S1 for pulse induction, and simultaneously apply low-intensity DC stimulation for pre-enrichment to obtain a mixed bacterial culture rich in QQ and methanogens. S3. Gradient screening: The mixed bacterial solution obtained in step S2 is inoculated into a new culture medium, and the GCL concentration is increased to carry out gradient screening. The removal rate of AHLs within 240 min and the methane production rate are used as screening indicators to obtain a compound functional bacteria with both high-efficiency QQ activity and methanogenic ability. S4. Construction of the immobilization carrier: The composite functional bacteria obtained in step S3 were immobilized on sodium alginate-chitosan-Fe... 3+ In composite gel microspheres, the immobilized microspheres are the target product; In step S2, the initial mass concentration of GCL is 50 mg / L and the initial mass concentration of glucose is 100 mg / L; every 48 hours is a cycle, maintaining the glucose concentration at 50-100 mg / L and increasing the GCL concentration to 200 mg / L; the voltage gradient of the low-intensity DC stimulation is 0.8 V / cm, and the electrode material is a graphite rod. In step S3, the inoculum size for gradient screening is 10% of the mixed bacterial culture volume from step S2, gradually increasing the GCL to the range of 100–200 mg / L while maintaining glucose at 50–100 mg / L. The specific screening criteria are: AHLs removal rate ≥70% within 240 min, and methane yield ≥15% higher than the non-enriched control. In step S4, sodium alginate-chitosan-Fe 3+ The enrichment method for composite gel microspheres is as follows: a mixed solution of sodium alginate and chitosan containing composite functional bacteria is added dropwise to a 0.1M FeCl3 solution for cross-linking for 10-20 min, and then transferred to a 0.05M Na2SO4 solution for secondary cross-linking for 5-15 min. The resulting gel microspheres have a particle size of 2.5-3.0 mm and a water content of 70-75%.

2. The enrichment method for a compound functional bacterial preparation according to claim 1, characterized in that: In step S1, the anaerobic basal medium contains NaHCO3, NH4Cl, KH2PO4, KCl, and Mg. 2+ Ca 2+ Trace elements and vitamin solutions.

3. The application of the compound functional bacterial preparation enriched by the enrichment method according to any one of claims 1-2, characterized in that: The compound functional bacterial preparation is used in AnMBR to reduce the rate of increase in transmembrane pressure gradient, reduce extracellular polymer accumulation, and increase methane yield.

4. The application of the compound functional microbial preparation according to claim 3, characterized in that: The compound functional microbial preparation is added to the AnMBR for operation. The initial dosage of the compound functional microbial preparation in the AnMBR is 0.05% of the effective volume of the reactor, and 30-50% of the initial dosage is replenished every 30-45 days.

5. The application of the compound functional bacterial preparation according to claim 3, characterized in that: AnMBR is a submerged hollow fiber membrane structure, with the following operating conditions: operating temperature of 35±2℃, hydraulic retention time of 24h, and sludge age of 60-100d.

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