Treatment method for secondary sedimentation tank float sludge initiated by DCM control agent in sulfate process pulping

Abstract

The invention relates to the field of sewage treatment, in particular to a treatment method for secondary sedimentation tank float sludge initiated by a DCM control agent in sulfate pulping. The method comprises a step of adding a fusion protein as shown in SEQ ID NO.3 into a secondary sedimentation tank, and further comprises the following steps: regulating the concentration of dissolved oxygen to 2.0-4.0 mg / L, the temperature to 35-40 DEG C and the pH value to 6.8-7.2, hydrolyzing extracellular proteins or extracellular polysaccharides generated by various zoogloea bacteria in the secondary sedimentation tank, destroying a foam skeleton and reducing adhesion of microhyphae, thereby realizing the purpose of removing the microhyphae in the secondary sedimentation tank. And the problem of'combined action of a DCM control agent and filamentous bacteria 'is solved, and the problem of float sludge in a secondary sedimentation tank is solved.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment, specifically to a method for treating floating sludge in the secondary sedimentation tank induced by DCM control agent in sulfate pulping. Background Technology

[0002] Sulfate pulping is one of the main processes in current chemical pulping. To produce low-extractant pulp that meets the stringent requirements of high-quality pulps (such as specialty paper and food packaging paper), the industry commonly uses the addition of a special dichloromethane (DCM) control agent to effectively reduce the DCM content in the pulp. However, while this process addresses product quality issues, it also introduces new wastewater treatment challenges.

[0003] DCM control agents are typically highly foaming surfactants. When these substances enter aerobic biological treatment systems along with industrial wastewater, they trigger a series of chain reactions, resulting in severe environmental pollution.

[0004] Existing technologies for addressing this issue mainly include: chemical defoaming / sterilization, which involves spraying defoamers, sterilizers, or modified flocculants onto the surface of aeration tanks or secondary sedimentation tanks to quickly eliminate foam or inhibit filamentous bacteria activity. However, the addition of chemical agents introduces new chemical residues, causing secondary pollution. Single-parameter control methods offer only short-term effects, and foam and sludge problems are prone to recurrence, making long-term stable control difficult. Physical interception / separation methods are only end-of-pipe treatments and are passive responses that only address the symptoms, not the root cause. Physical interception cannot prevent the continuous proliferation of filamentous bacteria within the biological system, nor can it stop the continuous generation of sludge. With the continuous impact of DCM control agents, the rate of sludge generation often exceeds the physical removal capacity, leaving the system in an unstable state. Therefore, there is currently no effective method for handling sludge generated in secondary sedimentation tanks by DCM control agents in sulfate pulping. Summary of the Invention

[0005] When wastewater containing large amounts of DCM control agents enters an aerobic biological treatment system, it triggers a series of chain reactions: 1. Provides a foam framework: As an exogenous surfactant, DCM control agent significantly reduces the surface tension of the liquid, providing a stable "framework" for the large number of microbubbles generated during aeration.

[0006] 2. Induction of filamentous bacteria proliferation: The components of this control agent synergistically interact with specific filamentous bacteria in activated sludge. The filamentous bacteria, with their hydrophobic cell walls, adhere to the bubbles stabilized by the DCM control agent, and rapidly proliferate using these bubbles as a carrier.

[0007] 3. Leading to system deterioration: The combined effect of this "DCM control agent-filamentous bacteria" rapidly exacerbates the biological foam problem in the aeration tank. After these lightweight foams containing a large number of filamentous bacteria flow into the secondary settling tank, they cannot settle effectively, forming a large area of ​​stubborn floating sludge layer.

[0008] 4. Comprehensive Impact: The floating sludge in the secondary sedimentation tank directly leads to a significant loss of activated sludge, resulting in a substantial decrease in the suspended solids concentration of the activated sludge mixed liquor and a reduction in biological treatment efficiency. To ensure that the final effluent meets standards, it is necessary to significantly increase the chemical dosage in subsequent advanced treatment units (such as advanced oxidation and coagulation sedimentation), causing a surge in operating costs and posing secondary environmental risks.

[0009] To address the shortcomings of existing technologies, this invention provides a control method for a sulfate pulping wastewater treatment system. This method is the first to identify the synergistic pathogenic mechanism between DCM control agents and filamentous bacteria. Through the synergistic effects of precise dissolved oxygen elevation, high-temperature control, and narrow-range pH regulation, coupled with targeted fusion protein supplementation, the combined pathogenic chain of "DCM control agent-filamentous bacteria" is severed at its source, achieving directional succession of the microbial community and fundamentally solving the problem of floating sludge in the secondary sedimentation tank. This method offers significant advantages in terms of long-term stability, economy, and environmental friendliness.

[0010] In a first aspect, the present invention provides a method for treating floating sludge in a secondary settling tank caused by a DCM control agent in sulfate pulping, the method comprising the step of adding the fusion protein shown in SEQ ID NO.3 to the secondary settling tank.

[0011] Furthermore, the method also includes setting the dissolved oxygen concentration (DO) at the inlet of the aerobic tank to 2.0-4.0 mg / L.

[0012] Optionally, the dissolved oxygen concentration at the inlet of the aerobic tank is significantly increased from a conventionally considered sufficiently low level (approximately 1.0 mg / L) and stabilized at 2.0-4.0 mg / L, preferably 2.5-3.5 mg / L. This increase aims to completely eliminate localized microaerobic zones within the aeration tank, depriving microfilamentous bacteria of their optimal growth conditions, while ensuring that floc-forming bacteria receive sufficient oxygen for metabolism and floc formation.

[0013] Furthermore, the method also includes setting the temperature to 38°C-40°C. Preferably, it is 38.5°C-39.5°C. This high-temperature strategy aims to leverage the temperature sensitivity differences between specific filamentous bacteria (such as microfilamentous bacteria) and flocculent bacteria, which are better adapted to high temperatures, to promote flocculent microorganisms to become the dominant microbial population in the system by creating an optimal "heat stress" environment for non-filamentous bacteria.

[0014] Furthermore, the method also includes setting the pH value to 6.8-7.2. This pH range is designed to fine-tune the competitive landscape of microorganisms, slightly deviating them from the optimal growth points of certain foaming filamentous bacteria, while ensuring the efficient conduct of overall biochemical reactions (especially nitrification).

[0015] The above-mentioned regulation of dissolved oxygen, temperature and pH should be implemented as a whole and continuously for no less than 3 months to ensure that the microbial community completes the complete replacement from filamentous bacteria dominance to floc bacteria dominance and forms a new and stable ecosystem balance.

[0016] Further, the method includes the step of inoculating a secondary sedimentation tank with a recombinant engineered bacterium comprising the fusion protein shown in SEQ ID NO. 3. In one specific embodiment of the present invention, the recombinant engineered bacterium is Pichia pastoris.

[0017] In a second aspect, the present invention provides a fusion protein for treating secondary sedimentation tank sludge caused by DCM control agents in sulfate pulping, wherein the fusion protein is the fusion protein shown in SEQ ID NO.3.

[0018] Optionally, the fusion protein further includes an amino acid sequence that has more than 95% identity with the amino acid sequence of the fusion protein shown in SEQ ID NO.3, obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of the fusion protein shown in SEQ ID NO.3; Optionally, the fusion protein further includes the amino acid sequence of a fusion protein with the same function obtained by attaching a tag protein to the N-terminus and / or C-terminus of the amino acid sequence of the fusion protein shown in SEQ ID NO.3.

[0019] Those skilled in the art will understand that reasonable sequence alterations can be made to the sequence shown in SEQ ID NO:3 without affecting its protease activity, and these altered sequences should also fall within the scope of protection of this application. Such alterations include, but are not limited to: conserved amino acid substitution, partial amino acid deletion, addition, and N-terminal or C-terminal truncation; the altered protein should still retain protease activity equivalent to the sequence in SEQ ID NO:2. Furthermore, the binding peptide can be chemically modified in accordance with conventional techniques, including but not limited to: cyclization, acetylation, PAS conversion, PEGylation, and fatty acid modification; modifications can occur at the N-terminus, C-terminus, main chain, side chain, or specific amino acid residues of the peptide.

[0020] In a third aspect, the present invention provides a biomaterial selected from at least one of the following: A1) The nucleic acid molecule encoding the fusion protein described above; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2); A4) Recombinant microorganisms containing nucleic acid molecules described in A1), or recombinant microorganisms containing the expression cassette described in A2), or recombinant microorganisms containing the recombinant vector described in A3); A5) A whole-cell catalyst containing the nucleic acid molecule described in A1), or a whole-cell catalyst containing the expression cassette described in A2), or a whole-cell catalyst containing the recombinant vector described in A3).

[0021] Further, the nucleic acid molecule described in A1) includes a publicly disclosed nucleic acid molecule encoding the fusion protein and / or a nucleic acid molecule optimized as needed. Optionally, the nucleic acid molecule described in A1) includes at least one of the nucleotide sequences shown in SEQ ID NO.4.

[0022] It should be understood that those skilled in the art can optimize the nucleic acid molecule encoding SEQ ID NO.3 according to different expression systems (such as other engineered bacteria), and such variants are all within the scope of protection of this application.

[0023] Further, the recombinant vector described in A3) includes at least one of the following: pET series vectors, pBAD vectors, pGEX series vectors, pCAl-n / pCAl-pelB vectors, pPOW3.0 vectors, pPIC series vectors, and pYES2 vectors.

[0024] Furthermore, the recombinant microorganisms described in A4) include at least one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0025] Optionally, the recombinant microorganism is Pichia pastoris.

[0026] In a fourth aspect, the present invention provides the use of the fusion protein or the biomaterial in the preparation of hydrolyzed protein or hydrolyzed polysaccharide products.

[0027] In a fifth aspect, the present invention provides the application of the method, the fusion protein, or the biomaterial described herein in the preparation of wastewater treatment products.

[0028] Optionally, the wastewater treatment product uses the fusion protein shown in SEQ ID NO.3 to hydrolyze the extracellular proteins or extracellular polysaccharides produced by various bacterial flocs in the secondary sedimentation tank, thereby disrupting the foam skeleton, reducing the attachment of microhyphae, and thus reducing the floating sludge in the secondary sedimentation tank.

[0029] In a sixth aspect, the present invention provides a wastewater treatment product, the product comprising the fusion protein or the biomaterial described above.

[0030] The beneficial effects of the present invention include, but are not limited to: Targeted framework: By hydrolyzing the extracellular proteins or extracellular polysaccharides produced by various bacterial flocs in the secondary sedimentation tank, the foam framework is destroyed, reducing the attachment of microfibrils, thereby reducing the floating sludge in the secondary sedimentation tank.

[0031] Targeting the root cause: For the first time, it clearly points out and solves the fundamental cause of the disease as the "combined effect of DCM control agent and filamentous bacteria", rather than the symptoms of treating foam or floating mud.

[0032] Ecological regulation: Abandoning the traditional approach of relying on chemical agents, it reshapes the microbial community structure of activated sludge through the ecological principle of "supporting the superior and suppressing the inferior," resulting in long-lasting effects and no side effects.

[0033] System resilience: It can not only effectively eliminate floating sludge in the secondary sedimentation tank, but also restore MLSS concentration and biochemical system efficiency, thereby significantly reducing the amount of downstream chemicals added and achieving cost savings throughout the entire process.

[0034] Industrial validation: This solution has been validated by continuous and stable operation on an industrial scale for three months, proving its high reliability and effectiveness in solving complex biochemical problems caused by process additives. Attached Figure Description

[0035] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is the OD in the embodiment of the present invention. 540 - Standard curve of reducing sugar concentration. Detailed Implementation

[0036] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0037] Example 1: Screening of protein glycosidases Studies have shown that surfactants in DCM control agents combine with proteins or polysaccharides secreted by microorganisms in the secondary sedimentation tank. Alginate, a polysaccharide derivative composed of β-D-mannuronic acid and α-L-guluronic acid, is mainly produced by Pseudomonas (such as P. aeruginosa) and Azotobacter. It is the core substance for forming stable biofilms and gel-like flocs, but excessive amounts can lead to sludge bulking and a sharp increase in viscosity. Alginate is a major component of the foam skeleton. To break down the foam skeleton, an enzyme that can simultaneously hydrolyze proteins and lyse alginate is provided.

[0038] Alginate Lyase: Accession number B2FSW8 in the Uniport database (https: / / www.uniprot.org / uniprotkb / B2FSW8 / entry); Subtilisin: Accession number P04189 in the Uniport database (https: / / www.uniprot.org / uniprotkb / P04189 / entry), derived from Bacillus subtilis and other Bacillus subtilis bacteria. It is one of the most classic and broad-spectrum proteases. Belonging to the serine protease family, it can cleave peptide bonds on the carboxyl side of many amino acids (especially aromatic and aliphatic residues). It is alkali-resistant (optimal pH 8-11), heat-resistant, and widely used in detergents, leather, and wastewater treatment.

[0039] The functional regions of the two proteins were analyzed using bioinformatics methods, and the alginate lyase peptide AL1 (SEQ ID NO.1) and the subtilisin protease peptide ST1 (SEQ ID NO.2) were obtained. AL1 and ST1 were spliced ​​together to form a fusion protein, which obtained the fusion protein FP1 (SEQ ID NO.3) with both broad-spectrum protease activity and alginate lyase activity.

[0040] SEQ ID NO.1: LAAGTLYRLTGDRAYVDYARDMLLQYAQLYPTLGPHPEGRGQIPGRVFWQVLNDSVWLVNAIQGYDAIRDALSAEDRNTIESKVFRPMAEFLVSEPKNYDQIHNHATWAVAATGMTGYVLRDQELVEKSLRGSQKDDKFGFLRQIDLLFSPDGYYEEGPYYQRYALAPFLLFAN AIERNEPQRKIFARRDGVLLKAVDVLVQSSYGGLFFPINDAILDKGIDTEELVAGIGIAYARTGDDRLLSVAEQQKRLLLSPEGLQVAQALAANKAKPFDYHPMLLRDGPDGDRGGLAILRMNGERGQALVQKDTMQGMGHGHFDKLNWLFYDNGNPVVTDYGAARFLNVEAK; SEQ ID NO.2: EKGGKVQKQFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGS; SEQ ID NO.3: MLAAGTLYRLTGDRAYVDYARDMLLQYAQLYPTLGPHPEGRGQIPGRVFWQVLNDSVWLVNAIQGYDAIRDALSAEDRNTIESKVFRPMAEFLVSEPKNYDQIHNHATWAVAATGMTGYVLRDQELVEKSLRGSQKDDKFGFLRQIDLLFSPDGYYEEGPYYQRYALAPFLLFANAIERNEPQRKIFARRDGVLLKAVDVLVQSSYGGLFFPINDAILDKGIDTEELVAGIGIAYARTGDDRLLSVAEQQKRLLLSPEGLQVAQALAANKAKPFDYHPMLLRDGPDGDRGGLAILRMNGERGQALVQKDTMQGMGHGHFDKLNWLFYDNGNPVVTDYGAARFLNVEAKEKGGKVQKQFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGS; Example 2: Expression of the fusion protein Culture medium E. coli liquid medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0.

[0041] Escherichia coli solid medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1.5% (w / v) agar, 1% (w / v) NaCl, pH 7.0.

[0042] Yeast solid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, 2% (w / v) agar.

[0043] Yeast liquid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, PBS buffer, pH=7.0.

[0044] BMGY yeast culture medium: 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) biotin, PBS solution.

[0045] Yeast induction medium BMMY: 1% (w / v) yeast extract, 2% (w / v) peptone, 0.5% methanol (v / v), 1.34% (w / v) YNB, 0.00004% (w / v) biotin, PBS solution.

[0046] This invention uses Pichia pastoris for expression, and the specific steps are as follows: 1. Construction of expression carriers The fusion protein was optimized according to the Pichia pastoris codon to obtain the nucleotide sequence encoding the fusion protein FP1. The nucleotide sequence encoding FP1 is shown in SEQ ID NO. 4. The optimized nucleotide sequence was synthesized by Thermo Fisher Scientific and introduced into a His-tagged pGAPZα A vector (which carries a GAP constitutive promoter; carries an α-factor signal peptide for efficient secretion; and allows for efficient screening using Zeocin™ antibiotics, directly screening for high-copy transformants) to obtain the recombinant vector pGAPZα A-FP1. A small amount of the recombinant vector pGAPZα A-FP1 was transformed into competent BL21(DE3) E. coli and plated on LB plates containing Zeocin (25 µg / mL) to amplify the plasmid.

[0047] SEQ ID NO.4:

[0048] The expression vector was linearized using the DNA restriction endonuclease BglII, and Pichia pastoris GS115 was transformed by electroporation. The cells were then plated on YPDZ selective plates containing different high concentrations of zeocin antibiotic (100, 500, and 1000 µg / mL, respectively) and incubated at 30°C for 2-4 days until single colonies appeared. Positive transformants were identified by colony PCR and stored in glycerol tubes.

[0049] Positive transformants were selected and cultured in BMGY medium to the late logarithmic phase. The cells were collected by centrifugation, resuspended in BMGY medium, and induced for 48 h at 30°C and 250 rpm.

[0050] The sample was centrifuged at 4°C and 12,000 rpm for 2 minutes, and the supernatant was collected to obtain the crude enzyme solution of the fusion protein FP1.

[0051] Example 3: Functional Verification of Fusion Protein 1. Protease activity assay: Enzyme sample: Test group: crude enzyme solution of Pichia pastoris fermentation supernatant containing the target fusion protein (diluted 10 times).

[0052] Positive control: 1 mg / mL commercial subtilisin (Sigma, catalog number: P8038) solution.

[0053] Negative control: The same batch of crude enzyme solution inactivated by heating at 95°C for 15 minutes.

[0054] Substrate: 2% (w / v) azocasein dissolved in 50 mM Tris-HCl buffer (pH 7.5).

[0055] Reaction system: 100 μL enzyme solution + 400 μL substrate solution. React precisely in a 37°C water bath for 30 minutes.

[0056] Termination and Detection: The reaction was terminated by adding 500 μL of 10% (w / v) trichloroacetic acid (TCA). The mixture was placed on ice for 10 minutes to precipitate unhydrolyzed proteins, followed by centrifugation at 13,000 rpm for 10 minutes. 600 μL of the supernatant was collected and 700 μL of 0.5 M NaOH was added. After mixing, the absorbance (OD) was measured at 440 nm. 440 ).

[0057] Activity definition: Under the above conditions, the rate at which OD increases per minute 440 The amount of enzyme required to increase the value by 0.01 is defined as 1 activity unit (U).

[0058] The results are shown in Table 1. The results indicate that the protease activity of the crude enzyme solution of the fusion protein was 10.73 U / mL (mean ± standard deviation). The activity of commercial subtilisin (1 mg / mL) was 16.07 U / mL. The relative activity of the fusion protein was approximately 66.8% of that of the commercial standard enzyme. Considering that this was an unpurified crude enzyme solution and that the protease in the fusion protein represents only one domain, this activity level is very ideal, demonstrating the correct folding and functional realization of this domain.

[0059] Table 1 Raw absorbance data and activity calculation

[0060] *Note: Net increase in OD value (ΔOD) = Sample average OD 440 - Negative control mean OD 440 Activity (U / mL) = (ΔOD / 0.01) / (reaction time 30 min) / enzyme solution added volume 0.1 mL. 2. Assay for alginate lyase activity: Enzyme sample: Test group: crude enzyme solution of Pichia pastoris fermentation supernatant containing the target fusion protein.

[0061] Positive control: Commercial alginate lyase (Sigma-Aldrich, catalog number: A1603).

[0062] Negative control: The same batch of crude enzyme solution that has been heat-inactivated (95°C, 15 minutes).

[0063] Blank control: An equal volume of buffer solution.

[0064] Substrate: 0.2% (w / v) sodium alginate (sodium salt, low viscosity) dissolved in 20 mM Tris-HCl buffer (containing 2 mM CaCl2, pH 7.0).

[0065] Reaction system (using the reduction end-addition method): Take 400 μL of substrate solution preheated to 37°C, add 100 μL of appropriately diluted enzyme solution (100-fold dilution), and quickly vortex to mix. React precisely in a 37°C water bath for 30 minutes. Immediately place in a boiling water bath for 10 minutes to terminate the reaction.

[0066] Detection method: DNS method Take 500 μL of the above-terminated reaction solution and add 750 μL of DNS reagent.

[0067] Heat in a boiling water bath for 10 minutes to allow the reducing sugar and DNS to react fully and produce a brownish-red substance.

[0068] After cooling, dilute with 2.75 mL of deionized water.

[0069] Absorbance (OD) was measured at a wavelength of 540 nm. 540 ).

[0070] Preparation of standard curve: D-mannuronic acid (the monosaccharide component of alginate) was used as a standard, with concentration gradients of 0, 0.1, 0.2, 0.4, 0.6, and 0.8 mM.

[0071] The DNS method was used to determine the OD values, just like with the sample, and the OD plot was generated. 540 - Standard curve of reducing sugar concentration.

[0072] Activity definition: Under the above reaction conditions, the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar (calculated as mannouronic acid) per minute is defined as 1 enzyme activity unit (U).

[0073] Calculation formula: Enzyme activity (U / mL) = [(C × Vt) ​​ / (t × Vs)] × D C: Reducing sugar concentration (μmol / mL) calculated from the standard curve Vt: Total volume of the reaction system (0.5 mL) t: Reaction time (30 min) Vs: Volume of enzyme solution added (0.1 mL) D: Enzyme solution dilution factor (100) The equation of the standard curve is: y = 0.8358x + 0.0187, R² = 0.996 ( Figure 1 This indicates good linearity within the measurement range and that it can be used for accurate calculations.

[0074] The results are shown in Table 2. The results indicate that the alginate lyase activity of the crude enzyme solution of the fusion protein was 5.28 U / mL, while the activity of the commercial enzyme was 7.85 U / mL. The relative activity of the fusion protein was approximately 67.1% of that of the commercial enzyme. Considering that this was a crude enzyme solution and part of a fusion protein, this result is very positive, indicating that the domain was successfully expressed and possesses efficient catalytic function.

[0075] Table 2 Absorbance and Activity Calculation

[0076] Note: The reducing sugar concentration and enzyme activity in the table have been adjusted after deducting the blank control values.

[0077] Example 4: Simulated sludge floating experiment in a secondary sedimentation tank 1. Experimental materials: Simulated wastewater and sludge: Fresh mixed liquor (MLSS = 3,200 mg / L, pH = 7.2) taken from the end of the aeration tank of a sulfate pulping wastewater treatment plant.

[0078] Sludge inducer: Sodium dodecylbenzenesulfonate (LAS), as a mimic of the high foaming surfactant in DCM control agent, was prepared into a 10 g / L stock solution.

[0079] Test reagent: Fusion protein crude enzyme solution: The crude enzyme solution of protein prepared in Example 2 has a protease activity of 10.73 U / mL and an alginate lyase activity of 5.50 U / mL.

[0080] Positive control 1: 10% FeCl3 solution (industrial grade).

[0081] Negative control: Equal volume of buffer (50 mM Tris-HCl, pH 7.5).

[0082] Experimental setup: 6 1 L graduated transparent sedimentation columns, with matching stirring paddles.

[0083] 2. The experimental design is shown in Table 3.

[0084] Table 3 Experimental procedures for each group

[0085] 3. Experimental steps: Step 1: Problem Induction. Add 1L of mixed solution to the sedimentation columns of groups G2-G5, add LAS mother liquor as designed, and stir slowly (60 rpm) for 15 minutes to mix evenly, simulating the impact of incoming water.

[0086] Step 2: Initial Settling Observation. Stop stirring and start timing. Record the mud-water interface height every 5 minutes for 30 minutes. At the end of this stage (T=30min), measure and record the thickness of the floating mud layer and the turbidity (OD) of the supernatant. 650 ), and calculate SVI for each group. 30 .

[0087] Step 3: Add treatment agents. Slowly stir the settled sludge at the bottom of each column until it is in a uniform suspension. According to the design plan, add the corresponding treatment agents to groups G3-G5 respectively, and stir slowly for 5 minutes to mix the agents evenly. Add an equal amount of buffer solution to groups G1 and G2.

[0088] Step 4: Post-treatment settling observation. Stop stirring and begin the second timing (T'). Record the mud-water interface height every 2 minutes for the first 10 minutes, and then every 5 minutes thereafter, for a total observation time of 30 minutes. At T'=30min, record the thickness of the floating mud layer and the turbidity of the supernatant again, and calculate the SVI after treatment.30 .

[0089] Step 5: Endpoint Sampling and Analysis. After the experiment, surface sludge from each column was collected for EPS extraction, and the supernatant from the middle layer was collected to determine the residual LAS concentration.

[0090] 4. Analytical Indicators and Methods: Settling performance: Sludge volume index (SVI) 30 ), interface settling rate.

[0091] Mud control: Mud layer thickness reduction rate.

[0092] Effluent water quality: Turbidity of supernatant (OD) 650 LAS residue (methylene blue process).

[0093] Sludge properties: Protein / polysaccharide content in EPS (BCA / phenol-sulfuric acid process).

[0094] 5. Experimental Results and Analysis (1) Improved settling performance SVI 30 Changes: The results before and after treatment are shown in Table 4. The results indicate that the SVI in group G2 was as high as 217, confirming that LAS successfully induced deterioration of sedimentation. The fusion protein treatment was significantly effective: the high-dose group (G5) showed the best effect, with an SVI reduction of 45%, superior to the FeCl3 group (37%) and the commercial protease group (23%). Its interfacial sedimentation rate was the fastest (0.35 cm / min), indicating dense flocs and rapid sedimentation. The low-dose group (G4) was comparable to FeCl3, demonstrating its high efficiency.

[0095] Table 4 SVI after treatment with different reagents 30 Results of the change

[0096] Group G2 had an SVI of 217, confirming that LAS successfully induced a deterioration in sedimentation.

[0097] The fusion protein treatment showed significant effects: the high-dose group (G5) showed the best results, with a 45% reduction in SVI, which was superior to the FeCl3 group (37%). Its interface settling rate was the fastest (0.35 cm / min), indicating that the flocs were dense and settled rapidly.

[0098] Significant dose-response effect: The effect of the low-dose group (G4) was comparable to that of FeCl3, demonstrating its high efficiency.

[0099] (2) Effect of controlling the floating mud layer After 30 minutes of treatment, the reduction rate of the mud layer thickness was: Group G5 >95% (almost completely eliminated), Group G3 ~85%, and Group G4 ~60%.

[0100] Visual observation: The floating sludge in Group G5 quickly disintegrated from a stable foam layer into fine flocs and sank within 10 minutes after dosing, and the supernatant quickly became clear. <00>

[0101] (3)Improvement of supernatant water quality Turbidity removal rate (OD 650 ): Group G5 (86%) > Group G3 (78%) > Group G4 (70%).

[0102] LAS residue rate: Group G5 (42%) < Group G3 (71%). The fusion protein group showed a certain ability to directly degrade surfactants, while FeCl3 mainly removed them by coagulation and encapsulation.

[0103] (4)Analysis of sludge properties EPS components: The contents of protein and polysaccharide in the loose EPS (LB-EPS) of the floating sludge in Group G5 decreased by 58% and 49% respectively compared with Group G2, and the decrease was significantly greater than that of other treatment groups. It was proved that the fusion protein could effectively hydrolyze two key components of EPS.

[0104] Example 5: Sewage treatment in the secondary sedimentation tank The first stage: System preparation and baseline survey (weeks 1-2) 1.1 Equipment installation and calibration Parameter regulation system: Calibrate the on-line DO meter, pH meter and temperature sensor in the aerobic tank to ensure the measurement accuracy: DO error ≤ ±0.1 mg / L, pH error ≤ ±0.05, temperature error ≤ ±0.3℃, Check the uniformity of the aeration system, repair the blocked or damaged aeration heads, and ensure that there is no dead zone for aeration. ]Debug the heating / heat exchange system (steam valve, heat exchanger, cooling tower), and verify the heating capacity (up to 42℃ at most). Debug the automatic acid-base dosing system to ensure that the dosing response time ≤ 30 seconds and the pH control accuracy ≤ ±0.1.

[0105] Drug dosing system: Install a fusion protein dosing system (storage tank, metering pump, static mixer) at the sludge return pump well. The storage tank is equipped with a temperature control device to maintain storage in the dark at 4±1℃ to ensure the stability of enzyme activity. Conduct a water linkage test run, calibrate the metering pump, and the dosing accuracy error ≤ ±2%. Confirm the installation position of the dosing point and the static mixer to ensure that the fusion protein is fully mixed with the returned sludge.

[0106] 1.2 Baseline data collection Without changing the existing operating parameters or adding any reagents, conduct a complete monitoring of the aerobic biochemical unit and the secondary sedimentation tank for at least 7 days to establish a "background value". Various monitoring indicators are shown in Table 5.

[0107] Table 5 Monitoring Indicators

[0108] 1.3 Parameter Status Confirmation and Impact Mode Identification During the baseline period, the dissolved oxygen (DO) was approximately 1.0 mg / L, the water temperature was at a normal level (record the actual value), the pH fluctuation range was confirmed, the dominance of filamentous bacteria was confirmed (SVI > 180 mL / g, microscopic examination showed filamentous bacteria abundance ≥ 4), the sludge coverage in the secondary sedimentation tank was > 30%, and the dosing pattern of the DCM control agent was coordinated with the production department to record the dosing time, dosage, frequency, and duration. The peak shock period was identified and marked as a critical control point.

[0109] Phase Two: Dual-track parallel coordinated regulation (Weeks 3-14) 2.1 Stage Division and Operational Parameters The "step-by-step" approach was adopted, with each stage running stably for 2-3 weeks. Parameter control and drug administration were carried out simultaneously. The parameters for each stage are shown in Table 6.

[0110] Table 6 Parameters for each stage

[0111] Phase 3: Effectiveness Evaluation and Post-Month Monitoring (Weeks 15-16) 3.1 Stop drug administration and maintain parameter control. Completely stop the addition of fusion protein Continue to maintain DO, temperature, and pH within the target range (2.5-3.5 mg / L, 38.5-39.5℃, 6.8-7.2). Observe whether the floating sludge problem rebounds and assess the independent maintenance capability of parameter control. 3.2 Post-effect monitoring Continue intensive monitoring for 14 days, with monitoring indicators and frequencies the same as at baseline. The monitoring standards are shown in Table 7.

[0112] Table 7 Monitoring Indicators and Judgment Criteria

[0113] Phase 4: Data Compilation and Effectiveness Evaluation 4.1 Comparison of Core Performance Indicators Table 8 Core Effects of Each Stage

[0114] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for treating floating sludge in the secondary sedimentation tank caused by DCM control agent in sulfate pulping, characterized in that, The method includes the step of adding the fusion protein shown in SEQ ID NO.3 to a secondary sedimentation tank.

2. The method according to claim 1, characterized in that, The method also includes setting the dissolved oxygen concentration (DO) at the inlet of the aerobic tank to 2.0-4.0 mg / L.

3. The method according to claim 1, characterized in that, The method also includes setting the temperature to 35℃-40℃.

4. The method according to claim 1, characterized in that, The method also includes setting the pH value to 6.8-7.

2.

5. The method according to claim 1, characterized in that, The method includes the step of inoculating a secondary sedimentation tank with recombinant engineered bacteria comprising the fusion protein described in SEQ ID NO.

3.

6. A fusion protein for treating secondary sedimentation tank sludge induced by DCM control agents in sulfate pulping, characterized in that, The fusion protein is the fusion protein shown in SEQ ID NO.

3.

7. A biomaterial, characterized in that, The biomaterial is selected from at least one of the following: A1) A nucleic acid molecule encoding the fusion protein as described in claim 6; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2); A4) Recombinant microorganisms containing nucleic acid molecules described in A1), or recombinant microorganisms containing the expression cassette described in A2), or recombinant microorganisms containing the recombinant vector described in A3); A5) A whole-cell catalyst containing the nucleic acid molecule described in A1), or a whole-cell catalyst containing the expression cassette described in A2), or a whole-cell catalyst containing the recombinant vector described in A3).

8. The use of the fusion protein of claim 6 or the biomaterial of claim 7 in the preparation of products containing hydrolyzed proteins and / or hydrolyzed polysaccharides.

9. The application of the method according to any one of claims 1-5, the fusion protein according to claim 6, or the biomaterial according to claim 7 in the preparation of wastewater treatment products.

10. A wastewater treatment product, characterized in that, The product includes the fusion protein of claim 6 or the biomaterial of claim 7.

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