Complex microbial inoculant, reinforced sludge containing complex microbial inoculant and application of complex microbial inoculant in DMF (Dimethyl Formamide) sewage treatment

Abstract

The invention provides a complex microbial inoculant for treating DMF (dimethylformamide) wastewater, a preparation method of the complex microbial inoculant, an enhanced sludge system, application of the complex microbial inoculant in treatment of wastewater containing DMF and a biological treatment method of the DMF wastewater. The complex microbial inoculant comprises at least two of paracoccus denitrificans, pseudomonas stutzeri and salt-tolerant bacillus, the active quantity ratio of the paracoccus denitrificans to the pseudomonas stutzeri to the salt-tolerant bacillus is (0-3): (0-3): (0-3), and the condition is that the active quantity of at least two of the paracoccus denitrificans, the pseudomonas stutzeri and the salt-tolerant bacillus is not 0. Through synergistic combination of at least two of paracoccus denitrificans, pseudomonas stutzeri and salt-tolerant bacillus, the degradation rate of DMF in wastewater is remarkably improved, and the activity and stability of activated sludge added with compound bacteria are improved.

Description

Technical Field

[0001] This invention belongs to the field of biological wastewater treatment technology, specifically relating to a compound microbial agent composition for treating wastewater containing dimethylformamide (DMF), a pretreatment method for the compound microbial agent, an enhanced sludge system for the compound microbial agent, the application of the compound microbial agent composition for treating wastewater containing dimethylformamide, and a biological treatment method for wastewater containing dimethylformamide by applying the compound microbial agent. Background Technology

[0002] Dimethylformamide (DMF, CAS 68-12-2) is an important chemical solvent widely used in synthetic leather, pharmaceuticals, electronic cleaning, and dye synthesis. my country's synthetic leather industry accounts for over 65% of global DMF consumption, with individual production lines discharging 200-500 tons of wastewater per day (containing 500-2000 mg / L of DMF). Furthermore, in the polyurethane industry, as a washing and curing agent, dimethylformamide (DMF) is a good solvent for various polymers such as polyethylene, polyvinyl chloride, polyacrylonitrile, and polyamide, and can be used in the wet spinning of synthetic fibers such as polyacrylonitrile fibers and the synthesis of polyurethane. In the pharmaceutical industry, as a synthetic drug intermediate, DMF is widely used in the production of doxycycline, cortisone, and sulfonamide drugs. In the acrylic fiber industry, as a solvent, DMF is mainly used in the dry spinning production of acrylic fibers. In the pesticide industry, DMF is used to synthesize highly effective and low-toxicity pesticides and insecticides. In the dye industry, DMF is used as a dye solvent. In the petrochemical industry, DMF is used as a gas absorbent for the separation and purification of gases. In the leather industry, DMF is mainly used in the wet-process synthetic leather production. In most industrial production processes, DMF exists only as a solvent and is discharged with the production wastewater with virtually no consumption.

[0003] In recent years, with the increasing discharge of DMF industrial wastewater, DMF pollution has become increasingly serious. Many studies have concluded that DMF is carcinogenic and poses environmental risks. Table 2 of China's "Emission Standard of Pollutants for Synthetic Leather and Artificial Leather Industry" (GB 21902-2008) stipulates a DMF emission limit of 2 mg / L, but the actual concentration of DMF in industrial wastewater often exceeds this limit by 20-100 times. Furthermore, DMF is chemically stable, with a B / C ratio of 0.065, is difficult to biodegrade, and possesses a certain degree of toxicity, which inhibits the biological treatment process of wastewater and affects treatment efficiency. In addition, DMF wastewater is characterized by high pollutant concentrations, particularly high total nitrogen concentrations, especially high organic nitrogen concentrations, but low ammonia nitrogen concentrations. Therefore, DMF wastewater has long been considered a highly toxic and difficult-to-biodegrade organic industrial wastewater.

[0004] Currently, the main methods for treating DMF wastewater are physicochemical and chemical methods, including distillation, extraction, adsorption, advanced oxidation, and alkaline hydrolysis. However, these methods have drawbacks such as high cost and susceptibility to secondary pollution. In contrast, biological treatment methods have become a research hotspot in DMF wastewater treatment technology due to their advantages such as large treatment capacity, low cost, mild conditions, and no secondary pollution. Patent document CN116814483B isolated a highly efficient anoxic denitrifying strain, Paracoccus denitrificans, from activated sludge of an environmental protection company and used it to treat actual DMF wastewater. Shao Ruying et al. (Screening and Application Research of N-Dimethylformamide Wastewater Degrading Bacteria, Fine Chemical Intermediates, Vol. 53, No. 5, October 2023) prepared a bacterial agent by mixing Pseudomonas aeruginosa and Paracoccus in a certain proportion and adding it to wastewater, which showed good degradation effect on dimethylformamide polluted water.

[0005] However, there are few reports in China on the use of pure microorganisms to enhance the degradation of DMF by activated sludge, and even fewer related applications of highly efficient and stable DMF degradation. Therefore, it is important to develop a new, highly efficient composite microbial agent for treating DMF wastewater and to study its application. Summary of the Invention

[0006] To achieve the above objectives, this invention utilizes a synergistic combination of denitrifying paracocci, Pseudomonas schrenckii, and halophilic Bacillus for the treatment of DMF-containing wastewater, which significantly improves the degradation rate of DMF in the wastewater and enhances the activity and stability of the activated sludge containing the combined bacteria.

[0007] Specifically, the present invention adopts the following technical solution:

[0008] 1. A compound bacterial agent for treating wastewater containing dimethylformamide, comprising at least two of *Paracococcus denitrificans*, *Pseudomonas schrenckii*, and halophilic Bacillus, wherein the ratio of the number of active *Paracococcus denitrificans*, *Pseudomonas schrenckii*, and halophilic Bacillus is (0-3):(0-3):(0-3), provided that the number of active *Paracococcus denitrificans*, *Pseudomonas schrenckii*, and halophilic Bacillus is not 0.

[0009] Optionally, the compound microbial agent contains *Paracoccus denitrificans*, *Pseudomonas schrenckii*, and halophilic Bacillus, wherein the ratio of the number of active *Paracoccus denitrificans*, *Pseudomonas schrenckii*, and halophilic Bacillus is (1-3):(1-3):(1-3).

[0010] Optionally, the *Paragonimococcus denitrificans* is *Paragonimococcus denitrificans* GDMCC 1.335, which was purchased from the Guangdong Provincial Center for Microbial Culture Collection, with the purchase code GDMCC 1.335; the *Pseudomonas schlegelii* is *Pseudomonas schlegelii* GDMCC NO.:1.446, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with the accession number GDMCC NO.:1.446; and the *Bacillus halophilus* is *Bacillus halophilus* CICC 24727, which was purchased from the China Center for Type Culture Collection (CCTCC), with the strain number CICC 24727.

[0011] 2. A method for preparing the above-mentioned compound microbial agent, comprising:

[0012] The bacterial strain activation step involves inoculating at least two of the following strains into a culture medium and culturing them to the logarithmic phase: denitrifying paracoccus, Pseudomonas schrenckii, and halophilic Bacillus; activating the strains in LB liquid medium; transferring the activated bacterial agent to LB liquid medium for further culture to obtain a bacterial suspension; and then adding the bacterial suspension to the DMF biological treatment tank.

[0013] Liquid seed preparation steps: Inoculate the activated bacterial strains separately into sterile liquid culture medium, culture until the logarithmic growth phase, and prepare a bacterial suspension; and

[0014] Mixing step - Mix the prepared bacterial turbid solution according to the ratio, and add the mixed bacterial solution to the liquid sterile culture medium for expansion culture to obtain the final compound bacterial agent.

[0015] 3. An enhanced sludge system comprising activated sludge and a composite microbial agent as described above, added to the activated sludge.

[0016] Optionally, the final number of active bacteria per unit mass of enhanced sludge is 2.5 × 10⁻⁶. 6 cfu / g -2.25×10 7 The activated sludge is the return sludge from the secondary sedimentation tank of a wastewater treatment plant, with an initial mixed liquor suspended solids concentration (MLSS) of 2800-3200 mg / L and a sludge volume index (SVI) of 90-120 mL / g.

[0017] Optionally, the activated sludge is a three-stage progressive gradient acclimation sludge. The gradient acclimation conditions include: using DMF as the sole carbon source in a sequencing batch reactor, with the concentration of DMF in the added wastewater increasing in a gradient, the DMF concentration of the wastewater added each time being 200 mg / L → 500 mg / L → 1000 mg / L, and the operating mode of each acclimation being: influent (0.25 h) → anoxic stirring (2 h) → aerobic treatment (8 h) → sedimentation treatment (1.5 h) → effluent (0.25 h), with an HRT of 12 h, wherein the dissolved oxygen concentration (DO) in the aerobic treatment is 3.0-5.0 mg / L, the pH is 7.0-7.5 and remains constant throughout the gradient acclimation process.

[0018] 4. The application of the compound microbial agent described in any one of the above-mentioned items or the enhanced sludge system described in any one of the above-mentioned items in the treatment of wastewater containing dimethylformamide.

[0019] 5. A biological treatment method for wastewater containing dimethylformamide, the biological treatment method comprising the following steps:

[0020] S1 - Provide the compound microbial agent according to the method described above;

[0021] S2 - The composite microbial agent is added to the activated sludge contained in the DMF biological treatment tank to obtain microbially enhanced sludge; then...

[0022] S3 - The dimethylformamide (DMF) wastewater is fed into a DMF biological treatment tank containing the strain-enhanced sludge to treat the DMF wastewater.

[0023] Optionally, the activated sludge is graded-acclimatized before adding the compound microbial agent, and the compound microbial agent is added 1-5 days after graded-acclimatization, so that the final number of active bacteria in a unit mass of enhanced sludge is 2.5 × 10⁻⁶. 6 cfu / g -2.25×10 7 .

[0024] Optionally, the operating conditions of step S3 include: a hydraulic retention time (HRT) of 12 hours, a dissolved oxygen concentration (DO) of 3.0-5.0 mg / L during the aerobic phase, a temperature of 30°C to 35°C, a pH of 7.0-7.5, and continuous operation for at least 20 days.

[0025] Beneficial effects

[0026] 1. Overcoming the bottleneck of DMF degradation efficiency and improving DMF degradation efficiency. This invention achieves a DMF degradation rate of ≥89.7% in wastewater (compared to only 60.8% in the control group) through the synergistic metabolism of denitrifying paracocci, Pseudomonas schistosomiasis and halophilic Bacillus (preferably in an activity ratio of 3:2:1) combined with a gradient acclimatization process of sludge for wastewater treatment.

[0027] 2. Achieve complete mineralization of DMF. A single bacterial species cannot cover the entire metabolic pathway of DMF, resulting in intermediate products inhibiting microbial activity. The composite bacterial agent-enhanced sludge system of this invention can achieve complete mineralization of DMF (COD removal rate ≥80.4%), that is, DMF is basically converted into inorganic nitrogen such as ammonia nitrogen, which is then degraded by nitrifying and denitrifying bacteria, ultimately achieving complete denitrification of pollutants.

[0028] 3. Solves the problem of poor sludge stability in wastewater treatment: Traditional sludge systems for wastewater treatment are prone to sludge bulking (sludge index SVI > 150 mL / g) when DMF concentration > 500 mg / L. The compound microbial agent or compound microbial agent-enhanced sludge system of this invention fully utilizes the synergistic effect between various microbial species to efficiently degrade DMF, and the sludge SVI is stabilized at 85-105 mL / g. Attached Figure Description

[0029] Figure 1 The bar chart shows the effects of control groups 2-4 and different combinations of bacteria (experimental groups 2-1 to 2-3) on DMF degradation rate and synergistic index.

[0030] Figure 2 The bar chart shows the effects of control group 3-10 and different combinations of three bacteria (experimental groups 3-1 to 3-9) on DMF degradation rate and synergistic index. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0033] Conversely, this invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of the invention as defined in the claims. Furthermore, to provide a better understanding of the invention, certain specific details are described in detail below. However, those skilled in the art will fully understand the invention even without these detailed descriptions.

[0034] definition

[0035] As used in this article, DMF refers to dimethylformamide (CAS No.: 68-12-2), with the molecular formula C3H7NO.

[0036] As used in this article, SVI (Sludge Volume Index) refers to the sludge volume index, which reflects the sludge settling performance (unit: mL / g). This is specifically described in Song Hongqiang et al.'s "Application of Sludge Settling Ratio in Activated Sludge Process for Wastewater Treatment" (Song Hongqiang et al., Application of Sludge Settling Ratio in Activated Sludge Process for Wastewater Treatment [J]. Oil and Gas Field Environmental Protection, 2010, 20(04): 18-19+23+57-58).

[0037] As used in this article, MLSS (Mixed Liquor Suspended Solids) refers to the concentration of suspended solids in a mixed liquor (unit: mg / L). The test method for MLSS is the gravimetric method, as detailed in Section 5.2 of the Standard Test Method for Municipal Sludge, CJ / T221-2023.

[0038] As used in this article, synergistic enhancement refers to the improved efficiency achieved through metabolic complementarity among denitrifying paracocci (DMF → formic acid), Pseudomonas schistosomiasis (formic acid → CO2 / H2O), and halophilic Bacillus (maintaining osmotic pressure) in the treatment of DMF-containing wastewater.

[0039] As used in this article, DMF mineralization refers to the complete decomposition of dimethylformamide (DMF, C3H7NO) into inorganic small molecules (such as CO2, H2O, NH3 / NO3) through biological or chemical processes. - The process of (etc.).

[0040] Although existing technologies utilize various bacteria that degrade DMF, there is still a lack of applications for pure-culture microorganism-enhanced activated sludge DMF degradation. Unexpectedly, the inventors discovered that combining at least two of *Paracoccus denitrifyingans* (DMF → formic acid), *Pseudomonas schistosomiasis* (formic acid → CO2 / H2O), and halophilic Bacillus (maintaining osmotic pressure) can synergistically enhance the degradation of DMF in wastewater, achieving a synergistic effect greater than the sum of its parts.

[0041] Therefore, in one aspect, the present invention provides a compound bacterial agent for treating dimethylformamide (DMF) wastewater, comprising at least two of *Paracococcus denitrificans*, *Pseudomonas schrengeri*, and halophilic Bacillus, wherein the ratio of the number of active *Paracococcus denitrificans*, *Pseudomonas schrengeri*, and halophilic Bacillus is (0-3):(0-3):(0-3), provided that the number of active *Paracococcus denitrificans*, *Pseudomonas schrengeri*, and halophilic Bacillus is not zero.

[0042] In a preferred embodiment, the compound microbial agent comprises *Paragonimus denitrificans*, *Pseudomonas schrenckii*, and halophilic Bacillus. The ratio of the active quantities of *Paragonimus denitrificans*, *Pseudomonas schrenckii*, and halophilic Bacillus is (1-3):(1-3):(1-3). Specifically, the ratio of the active quantities of *Paragonimus denitrificans*, *Pseudomonas schrenckii*, and halophilic Bacillus can be 1:1:1, 1:2:2, 1:3:3, 2:1:1, 2:2:3, 2:3:1, 3:1:3, 3:2:1, or 3:3:2.

[0043] Optionally, *Paragonimococcus denitrificans* can be *Paragonimococcus denitrificans* GDMCC 1.335, which is deposited at the Guangdong Provincial Microbiological Culture Collection Center with accession number GDMCC 1.335. *Pseudomonas schrenckii* is *Pseudomonas schrenckii* GDMCC NO.: 1.446, which is also deposited at the Guangdong Provincial Microbiological Culture Collection Center with accession number GDMCC NO.: 1.446. GDMCC NO.: 1.446 can be purchased from the GDMCC official website, the website address is: https: / / www.gdmcc.net / # / index The halophilic Bacillus is CICC 24727, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CICC 24727. Halophilic Bacillus (C) primarily plays a tolerance role in the combination; its individual degradation ability is weak, but it significantly improves system stability when combined with A / B bacteria (especially in high-salt environments).

[0044] The above-described aspects of the present invention can solve the problems of poor degradation efficiency and poor salt tolerance of existing single-strain bacteria for DMF. In particular, even under conditions of 3% salinity, the composite bacterial agent of the present invention can improve the synergistic degradation efficiency of DMF in wastewater to over 89.7%.

[0045] In a second aspect, the present invention provides an enhanced sludge system comprising activated sludge and any one of the aforementioned compound microbial agents added to the activated sludge.

[0046] Optionally, the final number of active bacteria per unit mass of enhanced sludge is 2.5 × 10⁻⁶. 6 cfu / g -2.25×10 7

[0047] Optionally, the activated sludge can be return sludge from the secondary sedimentation tank of a wastewater treatment plant, with an initial mixed liquor suspended solids concentration (MLSS) of 2800-3200 mg / L and a sludge volume index (SVI) of 90-120 mL / g.

[0048] In a preferred embodiment, the activated sludge can be subjected to a three-stage progressive gradient acclimation. The gradient acclimation conditions include: using DMF as the sole carbon source in a sequencing batch reactor (SBR), with the concentration of DMF in the added wastewater increasing in a gradient, the DMF concentration of the wastewater added each time being 200 mg / L → 500 mg / L → 1000 mg / L, and the operating mode of each acclimation being: influent (0.25 h) → anoxic stirring (2 h) → aerobic treatment (8 h) → sedimentation treatment (1.5 h) → effluent (0.25 h), with a HRT of 12 h, wherein the dissolved oxygen concentration (DO) in the aerobic treatment is 3.0-5.0 mg / L, the pH is 7.0-7.5, and it remains constant throughout the gradient acclimation process.

[0049] The three-stage progressive gradient acclimatization described above solves the problems of insufficient adaptability and stability in existing sludge systems, ensuring that the sludge SVI remains stable at 80-100 mL / g without bulking. Data from the examples show that the F / M of the sludge system of this invention can be maintained at 0.25-0.35 kg / (kg·d), within the highly efficient degradation range, with an OUR of 15.2 mg O2 / (g MLSS·h) (salinity 3%), demonstrating that the microbial community maintains high metabolic activity.

[0050] Preferably, the compound microbial agent is added to the activated sludge during the first 1-5 days of sludge gradient acclimation, so that the final number of active bacteria in a unit mass of enhanced sludge is 2.5 × 10⁻⁶. 6 cfu / g -2.25×10 7 .

[0051] A third aspect of the present invention provides the application of the above-described compound microbial agent in the treatment of wastewater containing dimethylformamide (DMF).

[0052] In a fourth aspect, a method for preparing a compound microbial agent is provided, comprising:

[0053] Strain activation steps - Inoculate at least two of the following strains into the culture medium: Paracoccus denitrificans, Pseudomonas schrenckii, and halophilic Bacillus, and culture until the logarithmic phase;

[0054] Liquid seed preparation steps: Inoculate the activated bacterial strains separately into sterile liquid culture medium, culture until the logarithmic growth phase, and prepare a bacterial suspension; and

[0055] Mixing step - The prepared bacterial suspensions are mixed according to the formula, and the mixed bacterial solution is added to a liquid sterile culture medium for expansion culture to obtain the final compound bacterial agent.

[0056] More specifically, the activation culture of the strain includes the following steps:

[0057] Strain activation: Take 5-15 μL of each frozen target strain and inoculate it into 100 mL of LB liquid sterile medium. Incubate at 30-35℃ with shaking at 160-180 rpm until the logarithmic phase.

[0058] Liquid seed preparation: 100 mL of the above-mentioned activated bacterial strains (single strain and mixed strains added in proportion) were transferred to 1000 mL of LB liquid sterile medium and cultured at 30-35℃ and 160-180 rpm until the logarithmic phase, and finally a bacterial suspension was prepared.

[0059] In a fifth aspect, a biological treatment method for dimethylformamide (DMF) wastewater is provided, the biological treatment method comprising the following steps:

[0060] S1 - Prepare the compound microbial agent according to the above method;

[0061] S2 - The composite microbial agent is added to the activated sludge contained in the DMF biological treatment tank to obtain microbially enhanced sludge; then...

[0062] S3 - The dimethylformamide (DMF) wastewater is fed into a DMF biological treatment tank containing the strain-enhanced sludge to treat the DMF wastewater.

[0063] Preferably, the dimethylformamide (DMF) wastewater can be derived from at least one of synthetic leather wastewater or electronic cleaning wastewater. Electronic cleaning wastewater refers to wastewater containing high concentrations of photoresist residue and organic solvents generated during the development / stripping process after photolithography, when organic solvents (such as DMF, NMP, etc.) are used to remove excess photoresist from silicon wafers.

[0064] Optionally, in the influent of dimethylformamide (DMF) wastewater, the DMF concentration is 800-2000 mg / L, the COD is 2000-5000 mg / L, the salinity is 2%-5%, and the ammonia nitrogen concentration in the effluent is less than 10 mg / L.

[0065] Preferably, the biological treatment method achieves a DMF degradation rate of 89.7-97.2%, a COD removal rate of 60.3-75.6%, and a TN removal rate of 60.3%-75.6%.

[0066] Optionally, in step S2, the activated sludge is gradient-acclimated before adding the compound microbial agent, and the compound microbial agent is added 1-5 days after gradient acclimation, so that the final amount of compound microbial agent in a unit mass of enhanced sludge is 2.5 × 10⁻⁶. 6 cfu / g -2.25×10 7 cfu / g.

[0067] Optionally, the operating conditions of step S3 include: a hydraulic retention time (HRT) of 12 hours, a dissolved oxygen concentration (DO) of 3.0-5.0 mg / L during the aerobic phase, a temperature of 30°C to 35°C, a pH of 7.0-7.5, and continuous operation for at least 20 days.

[0068] In conclusion, subsequent tests proved that:

[0069] Degradation rate: Under low salinity, the composite microbial agent of the present invention + activated sludge achieved a DMF degradation rate of 97.2% (12 hours), while the control group (conventional sludge) only achieved 69.3%.

[0070] Salt tolerance: At a salinity of 3%, the degradation rate of the compound microbial agent + activated sludge was still >89.7%, while the control group decreased to 60.8%.

[0071] Sludge activity: The MLSS of the compound microbial agent + activated sludge remained stable at 4000-4500 mg / L, the SVI remained stable at 80-100 mL / g after gradient acclimation, and the F / M ratio remained at 0.25-0.35 kg / (kg·d), which is within the high-efficiency degradation range. The OUR was 15.2 mg O2 / (gMLSS·h) (salinity 3%), proving that the microbial community maintained high metabolic activity.

[0072] Furthermore, for the compound microbial agent, the three-strain combination of 3:2:1 (active quantity ratio) exhibited the best degradation performance (degradation rate of 99.3%). Example 9 (dosage ratio 3:2:1, quantity of compound microbial agent 2.5 × 10⁻⁶) 6 The optimal scheme (CFU / g, addition time: day 1) showed a 28.9% increase in DMF degradation rate compared to the control group, and the best sludge stability (SVI = 85 ± 5 mL / g).

[0073] The invention will be further described in detail below with examples.

[0074] Example 1: Activated sludge acclimatization

[0075] 1. Construction of the domestication system

[0076] Reactor configuration: Sequencing batch reactor (SBR) with an effective volume of 10L, built-in aeration device and mechanical agitator (50-100rpm).

[0077] Sludge Source: The inoculum sludge was taken from the secondary sedimentation tank return sludge of a wastewater treatment plant of an environmental protection company (initial MLSS concentration 3000±200mg / L, SVI 90-120mL / g). The volume range of activated sludge used was 8.5L-9.75L. The sludge was subjected to gradient acclimation in the SBR reactor using DMF as the sole carbon source. The gradient acclimation process was as follows: First, DMF wastewater with a concentration of 200mg / L was added to the activated sludge in the SBR reactor, and then the reactor was run as follows: influent (0.25h) → anoxic stirring (2h) → aerobic treatment (8h) → sedimentation treatment (1.5h) → effluent (0.25h), HRT was 12h; then, DMF wastewater with a concentration increased to 500mg / L was added to the activated sludge in the SBR reactor, and then the process was as follows... The process was as follows: influent (0.25h) → anoxic stirring (2h) → aerobic treatment (8h) → sedimentation (1.5h) → effluent (0.25h), with a heat treatment time (HRT) of 12h. Then, DMF wastewater with a concentration of 1000mg / L was added to the activated sludge in the SBR reactor, and the process was repeated: influent (0.25h) → anoxic stirring (2h) → aerobic treatment (8h) → sedimentation (1.5h) → effluent (0.25h), with an HRT of 12h. Therefore, the concentration of DMF increased in each gradient from 200mg / L to 500mg / L to 1000mg / L. The aerobic treatment conditions during each acclimation process were: pH 7.0-7.5, DO 3-5mg / L.

[0078] 2. Gradient acclimatization process

[0079] Stage Division: The acclimatization process is divided into concentration gradient acclimatization, using a three-stage gradual regulation method (Table 1):

[0080] Table 1

[0081]

[0082] Domestication termination condition: The fluctuation rate of the data detected for 3 consecutive days is ≤5% (calculation formula: |current day value - previous day value| / previous day value × 100%). When the condition is met, the domestication is considered to be completed.

[0083] Table 2 shows the changes in sludge characteristics during the acclimation process.

[0084] Table 2

[0085] Domestication time (days) SVI (mL / g) MLSS (g / L) Acclimation of microbial community status in sludge 0 (Initial) 120±5 2.5±0.2 The flocculent material is loose, and the bacterial flora has not adapted. 7 95±4 3.8±0.3 The flocs are dense, dominated by A / B bacteria. 14 85±3 4.5±0.2 Biofilm formation, tri-microbe symbiosis 21 80±2 5.2±0.3 Steady-state symbiosis, peak mineralization capacity

[0086] Example 2: Verification of the degradation effect of target bacteria

[0087] Purchased bacterial strains (Denitrifying Paracoccus GDMCC 1.335, Pseudomonas schlegelii DSM 5190, and halophilic Bacillus CICC 24727) were added to the sludge system in combination, and the DMF degradation rate was determined. The degradation rate was determined by HPLC, under the following conditions:

[0088] Chromatographic conditions for DMF detection: C18 column, mobile phase methanol-water (70:30).

[0089] Column: Agilent ZORBAX SB-C18 (4.6×150mm, 5μm)

[0090] Flow rate: 1.0 mL / min

[0091] Column temperature: 35℃

[0092] Detection wavelength: 210nm

[0093] Injection volume: 15 μL

[0094] The bacterial culture method is as follows:

[0095] Strain activation: Take 5-15 μL of each frozen target strain and inoculate it into 100 mL of LB liquid sterile medium. Incubate at 30-35℃ with shaking at 160-180 rpm until the logarithmic phase.

[0096] Liquid seed preparation: 100 mL of the above-mentioned activated bacterial strains (single strain and mixed strains added in proportion) were transferred to 1000 mL of LB liquid sterile medium and cultured at 30-35℃ and 160-180 rpm until the logarithmic phase, and finally a bacterial suspension was prepared.

[0097] Specifically: If it is a single bacterial strain, transfer 100 mL of the activated single bacterial strain to 1000 mL of LB liquid sterile medium for culture.

[0098] If it is a mixed strain, the activated corresponding single strain is prepared according to the corresponding ratio of the mixed strain, and 100 mL of the mixture is cultured in 1000 mL of LB liquid sterile medium.

[0099] The bacterial suspension obtained above was added to a DMF-containing inorganic salt medium at a volume ratio of 5%. The components of the LB medium and the inorganic salt medium in this medium are as follows:

[0100] LB medium is prepared by adding 10.0g peptone, 5.0g yeast extract, and 10.0g sodium chloride to water to a final volume of 1.0L, adjusting the pH to 7.0±0.2, and sterilizing at 121℃ for 20 minutes before use.

[0101] Inorganic salt culture medium: KH2PO4, 0.75g, Na2HPO4·12H2O, 0.75g, MgSO4·7H2O, 0.1g, trace element solution*, 1ml, add water to 1.0L.

[0102] Trace element solution: FeSO4·7H2O, 0.1g, CaCl2, 0.02g, ZnSO4, 0.01g, MnSO4·4H2O, 0.01g, prepared by adding water to 1.0L.

[0103] The preparation procedure for DMF-containing inorganic salt culture medium is as follows (taking 500 mg / L DMF as an example):

[0104] 1. Measure 800 mL of deionized water, add 0.5 g of DMF (purity ≥ 99%), and stir until completely dissolved;

[0105] 2. Add the LB medium and inorganic medium in sequence, and stir for 5 minutes;

[0106] 3. Add 1 mL of trace element solution, bring the volume to 1 L, and adjust the pH to 7.0 ± 0.1 (calibrated with a pH meter); the trace element solution is prepared as above.

[0107] 4. Use after sterilization at 121℃ for 20 minutes.

[0108] Experiments on the dosage ratios of single, double, and triple strains:

[0109] As shown in Table 3, the single-bacterial suspensions obtained above were inoculated at a volume ratio of 5% (corresponding to a live bacterial count of 2.5 × 10⁻⁶). 6 CFU / g was added to DMF-containing inorganic salt medium. Four experimental groups were set up for the single-cell suspension: three experimental groups and one control group; each group had three replicates, as shown in Table 3 below.

[0110] Table 3

[0111]

[0112] Table 4 below shows the DMF degradation performance data of each bacterial strain after individual addition (initial DMF concentration 1000 mg / L, salinity 3%, pH = 7.0-7.5, operation time 12 hours, during which the dissolved oxygen concentration (DO) in the aerobic phase was 3.0-5.0 mg / L, temperature was 30℃ to 35℃, and pH was 7.0-7.5):

[0113] Table 4

[0114] Serial Number DMF degradation rate (%) COD removal rate (%) 1-1 85.2％±2.3 81.0±3.1 1-2 75.1±1.9 71.3±2.8 1-3 55.3±2.1 52.5±1.8 1-4 0 0

[0115] As shown in Table 4 above, the degradation rate of DMF by halophilic Bacillus is low, and the increase in dosage has a limited contribution to the degradation rate of DMF.

[0116] Under the same conditions as above, the resulting dual-bacterial suspension was inoculated at a volume ratio of 5% (corresponding to a live bacterial count of 2.5 × 10⁻⁶). 6 The cfu / g) was added to the DMF-containing inorganic salt medium. A total of 3 experimental groups and 1 control group were set up for the two bacterial suspensions. Each group was set up in 3 replicates. The specific settings are shown in Table 5 below.

[0117] Table 5

[0118]

[0119] Table 6 below and Figure 1 The data on DMF degradation performance after dual-strain addition are shown (initial DMF concentration 1000 mg / L, salinity 3%, pH 7.0-7.5, operation for 12 hours, during which the dissolved oxygen concentration (DO) in the aerobic phase was 3.0-5.0 mg / L, temperature was 30℃ to 35℃, and pH was 7.0-7.5).

[0120] Table 6

[0121]

[0122] Synergy Index (SI) = Theoretical Degradation Rate / Actual Degradation Rate

[0123] Theoretical degradation rate of DMF = Σ(expected degradation rate of a single bacterium × weight of the bacterium in the combination)

[0124] For example, when the dosage ratio is 1:1:0, with *Paragonimus denitrifyingus* and *Pseudomonas schwanniferus* each accounting for 0.5%, the theoretical degradation rate = expected degradation rate of *Paragonimus denitrifyingus* (A) * 0.5 + expected degradation rate of *Pseudomonas schwanniferus* (B) * 0.5 = 85.2% * 0.5 + 75.1% * 0.5 = 42.6% + 37.55% = 80.15%.

[0125] Three-strain addition ratio experiment: Under the same conditions as above, the three-strain addition ratio experiment adopted the L9(34) orthogonal experimental design, with denitrifying paracoccus (A), Pseudomonas schrenckii (B), and halophilic Bacillus (C) as the three factors. The different addition ratios of the three bacteria were three levels (1-3), with a total of 9 groups of experiments. Each group was set up with 3 parallels, and 1 control group was set up. The specific settings are shown in Table 7 below.

[0126] Table 7

[0127]

[0128] Table 8 below and Figure 2The DMF degradation performance data of each of the three bacterial strains after addition are shown (initial DMF concentration 1000 mg / L, salinity 3%, pH = 7.0-7.5, operation for 12 hours, during which the dissolved oxygen concentration (DO) in the aerobic phase was 3.0-5.0 mg / L, temperature was 30℃ to 35℃, and pH was 7.0-7.5).

[0129] Table 8

[0130]

[0131]

[0132] The definitions of synergistic index and theoretical degradation rate are as above.

[0133] in conclusion:

[0134] 1. The three-strain combination of 3:2:1 exhibits the best degradation performance (DMF degradation rate of 99.3%).

[0135] 2. Salt-tolerant Bacillus (C) primarily plays a tolerance role in the combination, but it significantly affects the synergistic effect. Data shows that, for example, in group 2-1, no salt-tolerant Bacillus (C) was added, but the synergistic index was the highest, indicating the weakest synergistic effect. In groups 2-2 and 2-3, both included salt-tolerant Bacillus (C), resulting in a lower synergistic index and a stronger synergistic effect. Furthermore, in the three-strain experiment, increasing the proportion of salt-tolerant Bacillus (C) did not significantly change the actual DMF degradation rate. This clearly demonstrates the strong synergistic effect of salt-tolerant Bacillus (C). Its individual degradation ability is weak, but when combined with A / B bacteria, it significantly improves system stability (especially in high-salt environments).

[0136] Example 3: Microbial enhancement of sludge degradation of DMF

[0137] 1. Experimental Design

[0138] Experimental and control group setup:

[0139] Experimental group: The three optimal bacterial strain ratios selected in Example 2 (3:1:3, 3:2:1, 3:3:2) were added to the activated sludge acclimated in Example 1 at different times (acclimation days 1, 3, and 5) and at different amounts (inoculation volume ratio: 5%, 10%, and 15%).

[0140] Control group: containing only sludge acclimated for 21 days (without the addition of exogenous microbial agents).

[0141] Operating conditions:

[0142] Reactor: Erlenmeyer flask, operating parameters: DMF 1000mg / L, salinity 3.0-5.0mg / L, temperature 30℃ to 35℃, pH 7.0-7.5.

[0143] Cycle: Continuous operation for 20 days, with each cycle lasting 12 hours (2 hours of anaerobic stirring → 6 hours of aerobic stirring → 1 hour of sedimentation → 3 hours of drainage).

[0144] 2. Effect monitoring and data collection

[0145] Degradation performance indicators:

[0146] DMF concentration: DMF concentration was determined by HPLC during daily drainage sampling (same as Example 2);

[0147] COD / TN / ammonia nitrogen: Test 3 times a week, on the first, third and seventh day of each week, using the methods of HJ828-2017 (COD) and HJ 636-2012 (TN / ammonia nitrogen);

[0148] Sludge activity: MLSS (GB / T 11901-1989), SVI (CJ / T 221-2005).

[0149] Stability assessment: DMF degradation rate fluctuation ≤3% for 5 consecutive days, and SVI stable at 80-120mL / g.

[0150] 3. Result verification and determination of the optimal dosing method

[0151] The degradation efficiencies are compared in Table 9.

[0152] Table 9

[0153]

[0154] Optimal strategy:

[0155] Experimental group 1 (dosage ratio 3:2:1, dosage 5%, dosing time on day 5) was the optimal scheme, with a DMF degradation rate 23.2% higher than the control group and the best sludge stability (SVI = 95 ± 5 mL / g).

[0156] Example 4: Verification of actual wastewater treatment effects

[0157] 1. Actual wastewater sources and water quality analysis

[0158] Wastewater type:

[0159] Wastewater from synthetic leather factories: DMF concentration 800-1200 mg / L, COD 2000-3000 mg / L, salinity 2%-3%, containing a small amount of tanning agents (formaldehyde compounds);

[0160] Electronics factory cleaning wastewater (wastewater containing high concentrations (requiring replenishment of photoresist within the required concentration range) of photoresist residue and organic solvents generated during the post-lithography development / stripping process using DMF to remove excess photoresist from silicon wafers): DMF concentration 1500-2000 mg / L, COD 3000-5000 mg / L, salinity 3%-5%, containing trace amounts of heavy metals (Cu). 2+ <1mg / L, Ni 2+ <0.5mg / L).

[0161] Pretreatment: The above-mentioned synthetic leather wastewater and electronics factory cleaning wastewater are passed through a screen, an oil separator, and a neutralization tank (pH adjusted to 7.0-7.5) to remove large particulate suspended solids and grease.

[0162] 2. Experimental group setup and operating parameters

[0163] Microbial agent addition:

[0164] The optimal combination determined in Example 3 (Denitrifying Paracoccus: Pseudomonas schrenckii: Salt-tolerant Bacillus = 3:2:1, total dosage 5%) was added to the actual wastewater treatment system on the 5th day.

[0165] Experimental Group A: Synthetic leather wastewater + sludge acclimated with the optimal ratio of microbial strains;

[0166] Experimental Group B: Electronic cleaning wastewater + sludge acclimated with the optimal ratio of bacterial strains;

[0167] Control groups A / B: Same wastewater type + sludge without bacterial acclimation.

[0168] Process parameters:

[0169] Reactor: Continuous flow activated sludge system (aeration tank volume 10m³) 3 HRT = 14h; divided into 4h of anoxic stirring and 8h of aerobic aeration; the parameters of the aerobic stage are DO 3.0-5.0mg / L, temperature 30-35℃, pH 7.0-7.5;

[0170] Monitoring cycle: 60 consecutive days of operation, with sampling and testing every 3 days.

[0171] 3. Detection Indicators and Methods

[0172] DMF / COD / ammonia nitrogen / TN: Same detection methods as in Examples 2 and 3;

[0173] Heavy metal residues: ICP-MS method (HJ 700-2014);

[0174] Sludge activation: SVI (measuring cylinder method, CJ / T 221-2005);

[0175] 4. Experimental Results and Analysis

[0176] (1) The DMF degradation effect is shown in Table 10.

[0177] Table 10

[0178]

[0179] (2) Comprehensive water quality indicators (Table 11)

[0180] The comprehensive indicators of the effluent from Example 4 are shown in Table 11 below.

[0181] Table 11

[0182]

[0183] (3) Sludge activity and stability

[0184] SVI: The experimental group remained stable at 85-105 mL / g, while the control group fluctuated frequently (120-200 mL / g);

[0185] The above results indicate that

[0186] Degradation efficiency: The experimental group (combined bacteria + activated sludge) achieved a DMF degradation rate of approximately 97.2% (12 hours), while the control group (conventional sludge) achieved only 69.3%.

[0187] Salt tolerance: At a salinity of 3% (corresponding to cleaning wastewater from an electronics factory), the DMF degradation rate in the experimental group was still >89.7%, while that in the control group decreased to 60.8%.

[0188] Sludge activity (electronics factory cleaning wastewater and synthetic leather wastewater): In the experimental group, the MLSS of the sludge remained stable at 4000-4500 mg / L, and the SVI remained stable at 80-100 mL / g after gradient acclimation. The F / M ratio was maintained at 0.25-0.35 kg / (kg·d), which is within the efficient degradation range. The OUR was 15.2 mg O2 / (g MLSS·h) (salinity 3%), demonstrating that the microbial community maintained high metabolic activity.

[0189] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the embodiments of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the protection scope of the present invention.

Claims

1. A composite microbial agent for treating wastewater containing dimethylformamide, characterized by The complex microbial agent comprises at least two of Paracoccus denitrificans, Pseudomonas stutzeri and Bacillus halodurans, wherein the active quantity ratio of Paracoccus denitrificans, Pseudomonas stutzeri and Bacillus halodurans is (0-3):(0-3):(0-3), provided that the active quantity of at least two of Paracoccus denitrificans, Pseudomonas stutzeri and Bacillus halodurans is not 0.

2. The complex bacterial agent according to claim 1, characterized by The complex microbial agent comprises Paracoccus denitrificans, Pseudomonas stutzeri and Bacillus halodurans, wherein the active quantity ratio of Paracoccus denitrificans, Pseudomonas stutzeri and Bacillus halodurans is (1-3):(1-3):(1-3).

3. The complex bacterial agent according to claim 1 or 2, characterized in that The Paracoccus denitrificans is Paracoccus denitrificans GDMCC 1.335, which is purchased from Guangdong Microbial Culture Collection Center with the purchase number GDMCC 1.335; the Pseudomonas stutzeri is Pseudomonas stutzeri GDMCC NO.: 1.446, which is preserved in Guangdong Microbial Culture Collection Center with the preservation number GDMCC NO.: 1.446; and the Bacillus halodurans is Bacillus halodurans CICC 24727, which is purchased from China Center for Type Culture Collection (CCTCC) with the strain number CICC 24727.

4. A method for preparing the complex microbial agent according to any one of claims 1 to 3, characterized by It comprises: a strain activation step: inoculating at least two of Paracoccus denitrificans, Pseudomonas stutzeri and Bacillus halodurans in culture medium respectively and culturing to logarithmic phase; a liquid seed preparation step: inoculating the activated strains in liquid sterile culture medium respectively, culturing to logarithmic phase and preparing bacterial suspension; and a mixing step: mixing the prepared bacterial suspensions according to the ratio and adding the mixed bacterial liquid into liquid sterile culture medium for large-scale culture to obtain the final complex microbial agent.

5. A system for enhancing sludge, characterized by The active sludge is a three-stage progressive gradient acclimated sludge, and the gradient acclimation conditions comprise: using DMF as the only carbon source in a sequencing batch reactor, increasing the DMF concentration in the added wastewater in a gradient, the DMF concentration in the wastewater added each time being 200 mg / L→500 mg / L→1000 mg / L, and the operation mode of each acclimation being influent (0.25 h)→anoxic stirring (2 h)→oxygen treatment (8 h)→sedimentation treatment (1.5 h)→effluent (0.25 h), and the HRT being 12 h, wherein the dissolved oxygen concentration DO in the oxygen treatment is 3.0-5.0 mg / L, the pH is 7.0-7.5 and is kept constant throughout the gradient acclimation process.

6. The enhanced sludge system of claim 5, wherein, The number of final active bacteria in the unit mass of the enhanced sludge is 2.5 x 10 6 cfu / g-2.25 x 10 7 cfu / g, the active sludge is the return sludge of the secondary sedimentation tank of a sewage treatment plant, the initial concentration of mixed liquor suspended solids MLSS is 2800-3200 mg / L, and the sludge volume index SVI is 90-120 mL / g.

7. The enhanced sludge system of claim 5, wherein 8. The complex microbial agent according to any one of claims 1 to 3 or the sludge system according to any one of claims 5 to 7 for use in treating wastewater containing dimethylformamide. The biological treatment method comprises the following steps:

9. A method for the biological treatment of wastewater containing dimethylformamide, characterized in that S1-providing the complex microbial agent according to the method of claim 4; S2-adding the complex microbial agent to the active sludge contained in the DMF biological treatment tank to obtain strain-strengthened sludge; and then ​ S3 - passing dimethylformamide DMF wastewater into a DMF biological treatment tank containing the strain-strengthened sludge to treat the DMF wastewater.

10. The biological treatment method according to claim 9, characterized in that In step S2, the activated sludge is gradient acclimated before the addition of the composite microbial agent, and the composite microbial agent is added 1-5 days after the gradient acclimation, so that the number of final active bacteria in the unit mass of the reinforced sludge is 2.5×10 6 cfu / g-2.25×10 7 ; the operation conditions of the step S3 include a hydraulic retention time HRT of 12 hours, a dissolved oxygen concentration DO of 3.0-5.0 mg / L in the aerobic stage, a temperature of 30-35°C, a pH value of 7.0-7.5, and continuous operation for at least 20 days.

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