Pandoravirus agent and low-temperature filamentous fungus sludge bulking inhibition method based on the pandoravirus agent

Abstract

This invention discloses a pineapple-based *Ulva prolifera* inoculant and a low-temperature filamentous sludge bulking inhibition method based on the pineapple-based *Ulva prolifera* inoculant. Addressing the problem of deteriorated sludge settling performance caused by excessive proliferation of filamentous bacteria at low temperatures, the method involves adding *Ulva prolifera* in its logarithmic growth phase to the bulking activated sludge system. Through nutrient competition and interference with the filamentous bacteria's quorum sensing system, the growth of filamentous bacteria is specifically inhibited. This method can significantly reduce SVI by more than 50% within 24 hours of operation at 10℃ without damaging the system's denitrification function and leaving no chemical residues. This invention has advantages such as fast response and simple operation, and is suitable for the stable operation of wastewater treatment plants in winter.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment, specifically relating to a pineapple pantothecin agent, a method for inhibiting low-temperature filamentous sludge bulking based on the pineapple pantothecin agent, and their applications. Background Technology

[0002] In wastewater treatment process optimization, biological denitrification and sludge bulking control are two independent and even mutually restrictive objectives. The significant differences between them in terms of micro-ecological regulation are as follows: Biological denitrification and sludge bulking control have significant conflicting objectives in terms of micro-ecological regulation. Biological denitrification aims to convert ammonia nitrogen into nitrogen gas using nitrifying and denitrifying bacteria, and its core process is to enrich functional bacterial communities through alternating anoxic / aerobic environments. On the other hand, the key to sludge bulking control lies in maintaining the ecological balance between floc-forming bacteria and filamentous bacteria, and preventing the excessive proliferation of filamentous bacteria from damaging sludge settling performance (i.e., increasing SVI).

[0003] To achieve efficient nitrogen removal, long sludge retention time (SRT) and low dissolved oxygen (DO) strategies are often employed to enrich slow-growing nitrifying bacteria. However, long SRT and low DO are precisely the conditions conducive to the overgrowth of filamentous bacteria (especially microfilamentous bacteria). This means that simply optimizing operating parameters for nitrogen removal often inadvertently creates an ecological environment conducive to the explosive growth of filamentous bacteria. Existing nitrogen removal enhancement technologies (such as adding carbon sources and adjusting the reflux ratio) mainly focus on nitrogen conversion pathways, lacking effective intervention in the quorum sensing (QS) system of filamentous bacteria and its competitive relationship with flocs. Especially under low-temperature conditions, filamentous bacteria, with their huge specific surface area advantage, far surpass flocs in the uptake of long-chain fatty acids. This nutrient competition mechanism is completely different from the electron acceptor competition mechanism in the nitrogen removal process, further exacerbating the difficulty of control.

[0004] Sludge bulking is mainly divided into filamentous bulking and non-filamentous bulking (high-viscosity bulking). Among them, filamentous bulking accounts for a very high proportion. Its characteristic is that filamentous microorganisms proliferate excessively in the flocs to form a skeletal structure, which hinders sludge compaction, leading to a sharp increase in SVI and difficulty in solid-liquid separation in the secondary sedimentation tank.

[0005] Temperature is a key factor influencing the competitive relationship between filamentous bacteria and floc-forming bacteria. Based on kinetic selection theory, at low temperatures (especially around 10°C), the metabolic activity and maximum specific growth rate of floc-forming bacteria are significantly inhibited. Meanwhile, some filamentous bacteria, due to their larger specific surface area and stronger affinity for substrates and dissolved oxygen (i.e., lower half-saturation constant), can gain a competitive advantage in low-temperature, low-substrate-concentration, or low-dissolved-oxygen environments, leading to excessive proliferation and bulking. When the water temperature rises above 22°C, the growth advantage of floc-forming bacteria is restored, and this bulking phenomenon is usually suppressed. This mechanism has been widely verified in winter operations in northern my country and some industrial wastewater treatment scenarios.

[0006] Existing technologies for addressing filamentous sludge bulking mainly include the following categories, but all have significant drawbacks: Operational parameter regulation: Based on kinetic selection theory, methods such as increasing dissolved oxygen (DO), adjusting the load, optimizing the nutrient ratio, or setting selectors are used to attempt to create selection pressure favorable to microbial flocs. However, under low-temperature conditions, the overall metabolic rate of microorganisms decreases, and relying solely on parameter regulation often results in slow response, limited effectiveness, and difficulty in reversing the already formed community structure.

[0007] Chemical agent addition: As an emergency measure, adding oxidants (such as liquid chlorine, hydrogen peroxide, ozone) or coagulants can have a rapid effect, but there are problems such as high cost, easy poisoning of beneficial microorganisms, secondary pollution, and inability to establish a long-term biological inhibition mechanism.

[0008] Physical methods, such as adding inert substances or using ultrasonic or magnetic separation, are often difficult to promote on a large scale due to their complexity or high cost.

[0009] In recent years, biocontrol using antagonistic interactions between microorganisms has gradually become a research hotspot due to its environmental friendliness, high specificity, and good sustainability. *Panthera pineapple* (… Pantoea ananatisAs a Gram-negative bacterium widely distributed in the rhizosphere, phyllosphere, and soil environment of plants, *Ubiquitinella pineapple* has shown application potential in multiple fields. In environmental remediation and resource utilization, *Ubiquitinella pineapple* has demonstrated unique application value. Rasheed, N., et al. (2023) reviewed the application mechanism of microalgal symbiosis systems in wastewater treatment and resource utilization in *Frontiers in Bioengineering and Biotechnology*, pointing out that this technology can significantly improve microalgal biomass and lipid accumulation efficiency, providing new ideas for advanced wastewater treatment and biomass energy production. Furthermore, some *Ubiquitinella* strains have been shown to possess the ability to degrade recalcitrant organic pollutants such as petroleum hydrocarbons, and have shown significant potential in biocatalysis fields such as biosurfactant production and chiral compound resolution.

[0010] In activated sludge wastewater treatment systems, sludge bulking is primarily caused by the overgrowth of filamentous microorganisms. However, current technologies often treat filamentous bacteria as a single category, ignoring the fundamental differences between filamentous fungi and filamentous bacteria in their biological characteristics, growth environment, and pathogenic mechanisms, resulting in a serious lack of targeted treatment methods. Filamentous fungi are eukaryotes with a well-defined nucleus and complex organelles, and their cell walls are mainly composed of chitin; while the main culprits causing low-temperature bulking (such as microfilamentous bacteria) are... Microthrix parviceL La Zoothecoleum Haliscomenobacter hydrossis Filamentous fungi (e.g., *Phyllostachys*) belong to prokaryotes, lack a formed nucleus, and have cell walls composed of peptidoglycan. This fundamental taxonomic difference determines their drastically different sensitivities to inhibitors. Filamentous fungi typically dominate in industrial wastewater with low pH (<6.0), high C / N ratio, and high levels of recalcitrant organic matter; while filamentous bacteria (especially microfilamentous fungi) prefer neutral pH, long sludge retention time (SRT), low dissolved oxygen, and domestic wastewater rich in long-chain fatty acids (LCFAs). Especially under low-temperature conditions (<15°C), the growth rate of filamentous bacteria is significantly higher than that of flocculent bacteria, becoming the dominant factor in winter sludge bulking. Currently, mainstream sludge bulking control methods (such as chlorination, hydrogen peroxide, ozone, or specific fungicides) are mostly based on empirical development of methods to inhibit filamentous fungi, or work through non-specific oxidation that disrupts cell walls / membranes. These methods are often ineffective against filamentous bacteria with different cell wall structures and metabolic mechanisms, and may even exacerbate bulking by accidentally killing flocculent bacteria. Furthermore, regulatory strategies for filamentous fungi (such as adjusting the pH to acidic) are completely inapplicable to the expansion of filamentous bacteria, which mainly occur in neutral environments.

[0011] Therefore, there is an urgent need for a precise regulation technology specifically targeting the physiological characteristics of filamentous bacteria. Summary of the Invention

[0012] This invention aims to provide a *Panthera pineapple* inoculant to inhibit the excessive proliferation of bulking filamentous bacteria such as *Thiopyrea*. One of the metabolites of *Panthera pineapple*, acyl homoserine lactones (AHLs), acts as an interfering agent, disrupting the quorum sensing system (QS) of filamentous bacteria or utilizing interspecies competition. This addresses the problems of poor control, low specificity, and sludge runoff in secondary sedimentation tanks of existing wastewater treatment technologies under low-temperature conditions. Specifically, the objective of this invention is to provide a *Panthera pineapple* inoculant that maintains high activity and metabolic function at low temperatures (5℃-15℃), overcoming the shortcomings of existing conventional microbial agents that exhibit reduced activity and poor colonization at low temperatures. Utilizing specific metabolites produced by *Panthera pineapple* (such as quorum sensing signal molecules or their degrading enzymes), it specifically interferes with the quorum sensing system of filamentous bacteria (such as microfilamentous bacteria, zoosporidioidomycetes, etc.) or competes for their survival resources, thereby inhibiting the excessive proliferation of filamentous bacteria. The active ingredient of the bacterial agent is *Panthera pineapple* strain; the *Panthera pineapple* strain exhibits low-temperature growth tolerance, can grow and reproduce normally at temperatures between 5℃ and 15℃, and the bacterial concentration at the stable stage when cultured at 10℃ is not less than 1×10⁻⁶. 8 CFU / mL.

[0013] Furthermore, the filamentous bacteria inhibited by the *Panthera pineapple* strain are selected from *Thiobacillus* (…). Thiothrix ), Microfilamentous fungi ( Microthrix parviceL La Zoothecoleum ( Haliscomenobacter hydrossis ) or Nocardia ( Nocardia spp. One or more of the following.

[0014] Furthermore, the *Panthera pineapple* strain can secrete AHLs signaling molecules, which have the activity of interfering with the quorum sensing system of filamentous bacteria or competing for ecological niches.

[0015] Furthermore, the formulation of the bacterial agent is a lyophilized powder.

[0016] Another object of the present invention is to provide a method for inhibiting low-temperature filamentous bacterial sludge bulking with the aforementioned pineapple pancreatic bacteria agent, comprising the following steps: (1) Preparation of bacterial agent; Pure culture of Pantotheca pineapple was carried out, and the bacterial solution in the logarithmic growth phase was collected. After centrifugation to remove the culture medium, the bacterial precipitate was resuspended to obtain a concentrated bacterial solution. (2) System addition; The bacterial concentrate obtained in step (1) is added to the activated sludge system in the state of filamentous bacterial sludge expansion; (3) Substrate replenishment; replenishing the activated sludge system with influent substrate; (4) Process operation; The Sequencing Batch Reactor (SBR) process system with a pre-anoxic denitrification section and a subsequent aerobic nitrification section is adopted. In particular, under low-temperature conditions, the growth of filamentous bacteria is inhibited by the competitive ecological niche or secretion of signaling molecules by the pineapple pancreatic bacteria, thereby inhibiting sludge bulking.

[0017] Furthermore, the specific process for preparing the microbial agent in step (1) is as follows: The lyophilized Pantotheca pineapple powder was inoculated into LB liquid medium for activation culture, and then transferred to fresh LB liquid medium for further culture for 24-48 hours to obtain a high concentration of bacterial solution. The high-concentration bacterial solution is then transferred to LB broth medium at a certain inoculation amount and cultured for 24-48 hours until the bacterial solution is in the logarithmic growth phase, for subsequent centrifugation.

[0018] Furthermore, the specific operation of adding the substance in step (2) is as follows: The bacterial culture in the logarithmic growth phase was centrifuged, the supernatant was discarded to remove residual culture medium, and the bacterial precipitate was collected. After resuspending and mixing the bacterial precipitate with sterile water or buffer solution, it is directly added to the expanded activated sludge mixture.

[0019] Furthermore, the composition and concentration of the influent substrate mentioned in step (3) are: ammonia nitrogen (NH4+) + The concentration of nitrogen (N) is 30 mg / L; the concentration of chemical oxygen demand (COD) is 150 mg / L; and the pH value is adjusted to 7.5-8.5.

[0020] Furthermore, the operating control conditions for the aerobic nitrification section in step (4) are as follows: the dissolved oxygen (DO) concentration in the reactor is maintained above 2.0 mg / L.

[0021] Furthermore, the mixed liquor suspended solids (MLSS) concentration in the activated sludge system described in step (4) is controlled within the range of 2500-2600 mg / L.

[0022] Furthermore, the low-temperature condition mentioned in step (4) specifically refers to controlling the operating temperature of the activated sludge system within the range of 5-15℃.

[0023] Furthermore, the operating cycle and time allocation of the SBR process described in step (4) are as follows: Each operating cycle consists of an anaerobic denitrification process for 8 hours, followed by an aerobic nitrification process for 13 hours. The operating cycle also includes subsequent sedimentation, drainage, and idle periods.

[0024] This method maintains a stable total nitrogen (TN) removal rate while inhibiting filamentous bacterial sludge bulking. Compared with the control group without bacterial agent, the reduction in nitrification and denitrification rates after adding the pineapple pantothecin agent does not exceed 15%, and the effluent ammonia nitrogen and total nitrogen concentrations are not higher than those of the control group. Beneficial effects

[0025] This invention provides an environmentally friendly sludge bulking control method: by replacing or reducing the addition of chemical agents (such as chlorine, hydrogen peroxide, etc.) through biological regulation, the risk of secondary pollution is reduced, providing a sludge bulking control technology solution that is simple to operate, has low operating costs, and high ecological safety.

[0026] AHLs signaling molecules exhibit two opposing effects across different bacterial species. For example, patent CN202311447274.0 discloses the positive promoting effect of AHLs on nitrifying bacteria; however, the inventors unexpectedly discovered that specific amounts of AHLs (especially a mixed spectrum secreted in situ by *Ubiquitinella pineapple*) have a significant quorum sensing interference or growth inhibition effect on filamentous bacteria (such as microfilaments). This species-specific response difference allows the present invention to precisely inhibit filamentous swelling bacteria without harming denitrifying bacteria, a level of ecological balance regulation that is difficult to achieve with simple exogenous chemical AHLs. The present invention utilizes the low-temperature resistant *Ubiquitinella pineapple* to secrete appropriate concentrations of AHLs in situ, continuously, and stably within the sludge system. This bioregulation method not only maintains a long-term anti-swelling effect but also avoids secondary pollution or uncontrollable impacts on non-target bacterial communities that may result from exogenous chemicals. Attached Figure Description

[0027] Figure 1 The sludge settling index is shown for the control group and the experimental group with added Pantotheca pineapple.

[0028] Figure 2 The sludge settling ratio of the control group and the experimental group with added Pantotheca pineapple after 30 minutes of static sedimentation.

[0029] Figure 3 Microscopic images of the control group and the experimental group with added Pantotheca pineapple in phase contrast mode. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments, but it is not intended to limit the present invention.

[0031] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to 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 commercially available conventional products. The pineapple pantothecin lyophilized powder was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number BMZ137657.

[0032] In this embodiment of the invention, a 250mL high borosilicate glass screw-top bottle was used as the reactor, with an effective operating volume of 100mL. A uniformly sized magnetic stir bar was placed inside the bottle, and the stirring speed was controlled at 600rpm to ensure uniform mixing of the sludge and water and aerobic conditions in the system (DO concentration in the water greater than 2mg / L). Artificially synthesized wastewater was used as the test water. Operating conditions were: dissolved oxygen concentration ≥2.00mg / L, influent substrate containing NH4⁺-N=30mg / L, COD=150mg / L, pH 7.5-8.5, and sludge concentration 2505mg / L. The reaction system operated for 24 hours, including 0.5 hours of influent, 8 hours of anoxic denitrification, 13 hours of aerobic nitrification, 2 hours of sedimentation, and 0.5 hours of effluent discharge.

[0033] Example 1: Preparation and Activation of Microbial Agent Take lyophilized Pantotheca pineapple and inoculate it into 5 mL of LB liquid medium. Activate and culture at 28℃ and 150 rpm for 12 h. Then, transfer it to 50 mL of fresh LB medium at a 5% (v / v) inoculation rate and continue to culture for 36 h to obtain a highly active concentrated bacterial solution.

[0034] The above concentrated bacterial suspension was inoculated into 100 mL of LB broth at a 10% (v / v) inoculation rate and cultured at 28°C and 150 rpm for 36 h. The culture was centrifuged at 4000 rpm for 10 min, and the supernatant was discarded to remove residual culture medium. The bacterial pellet was collected, resuspended in sterile physiological saline, mixed well, and the bacterial concentration was adjusted to obtain the final OD. 600 For experiments with a bacterial culture of ≥2.0, bacterial culture solution was added.

[0035] Example 2: Constructing a Simulated Wastewater Treatment System A 250mL high borosilicate glass screw-top bottle was used as the SBR, with an effective working volume of 100mL. The reactor has a built-in magnetic stirrer and is connected to a micro air pump that continuously supplies air through the aeration head. The stirring speed was set to 600rpm to ensure uniform mixing of the mud and water and aerobic conditions (DO≥2.0mg / L).

[0036] The influent was synthetic wastewater with an NH4⁺-N concentration of 30 mg / L and a COD concentration of 150 mg / L (carbon source: anhydrous sodium acetate), and the pH was adjusted to 8. The inoculum sludge was taken from expanded sludge in the secondary sedimentation tank of a wastewater treatment plant, with an initial MLSS controlled at 2505±50 mg / L and an initial SVI value of 285±10 mL / g. The entire reaction system was placed in a constant-temperature shaking incubator (or constant-temperature water bath), with the operating temperature controlled at 10±1℃. The operating cycle was 24 hours per cycle, specifically: 0.5 hours of influent, 8 hours of anoxic denitrification (closed stirring, aeration stopped), 13 hours of aerobic nitrification (open or continuous aeration, maintaining DO ≥ 2.0 mg / L), 2 hours of sedimentation (stirring and aeration stopped), and 0.5 hours of effluent (effluent ratio 50%).

[0037] Experimental setup: At the start of the first running cycle, the *Ulva pineapple* bacterial suspension prepared in Example 1 was added to the reaction system. The amount added was controlled to ensure that the initial concentration of *Ulva pineapple* in the system reached 1 × 10⁻⁶. 7 CFU / mL. No further bacterial agent is added in subsequent cycles; the system is maintained solely through periodic water changes.

[0038] Control group setup: Except for not adding Pantothecin pineapple bacterial solution (instead adding an equal volume of sterile physiological saline), the operating conditions were exactly the same as those of the experimental group.

[0039] Both groups ran continuously for 6 cycles (a total of 144 hours).

[0040] The SVI values ​​were monitored at 0h, 6h, 12h, 18h, and 24h, and the results are analyzed below: (1) At the beginning of the experiment (0 h), the sludge volume index (SVI) of both the experimental group and the control group was around 285 mL / g, and the sludge was in a state of expansion. After 12 h, the SVI of the experimental group decreased to 215 mL / g (a decrease of 24.6%), while the control group remained at 290 mL / g. By the end of the first cycle (24 h), the SVI of the experimental group had significantly decreased to 145 mL / g, successfully recovering to the normal activated sludge range (SVI < 150 mL / g), with a total decrease of 49.1%; in contrast, the SVI of the control group further increased to 295 mL / g, and the expansion intensified. The results show that the method of the present invention has significant rapid response characteristics and can significantly improve sludge settling performance within 24 h.

[0041] (2) After six consecutive cycles (144 h in total), the SVI of the experimental group remained stable between 135-150 mL / g without any rebound. Microscopic observation showed that the abundance rating of filamentous bacteria in the sludge of the experimental group decreased from the initial level 4 to level 0-1, and the floc structure was dense and compact. In contrast, the SVI of the control group remained above 280 mL / g, with a large number of filamentous bacteria proliferating and the sludge being loose and flocculent. Combined with the statistical distribution in Figure 1, it can be seen that the SVI data of the control group had a very large dispersion (large tank span, wide distribution range of scattered points), indicating that its settling performance was extremely unstable; while the SVI data points of the experimental group were highly concentrated (flat tank, dense clustering of scattered points), indicating excellent treatment effect and good reproducibility. There was a highly significant difference in the SVI values ​​between the two groups (P<0.01, marked as ** in the figure), further confirming the high efficiency and reliability of this method in controlling sludge bulking.

[0042] (3) After six cycles of operation, the effluent quality was tested. The average NH4⁺-N concentration in the experimental group was 2.0 mg / L, with a removal rate of 93.3%; the average NH4⁺-N concentration in the control group was 1.8 mg / L, with a removal rate of 94.0%. Statistical analysis showed no significant difference in ammonia nitrogen concentration between the two groups (P>0.05). This indicates that the addition of pineapple biomass inoculant did not inhibit the activity of nitrifying bacteria in the system, and the denitrification function of the wastewater treatment system remained intact.

[0043] Experimental results showed that the addition of the microbial agent exhibited a rapid response characteristic. At the end of the first operating cycle (24 hours) after the addition of the agent, the sludge was in a severely bulky state. However, after treatment with *Pantodactylus lactis*, the SVI in the experimental group decreased to approximately 145 mL / g, stabilizing within the non-bulking range of normal activated sludge. The SVI reduction reached 50%. The data points in the experimental group were highly concentrated, and the box plot showed low dispersion, indicating that the method produced consistent and reproducible results in different repeated experiments, demonstrating excellent stability and reliability.

[0044] The inhibition method provided by this invention can usually be observed to improve sludge settling performance within 24 hours after addition, and can maintain a stable bulking control effect for a long time, with the advantages of rapid response and continuous stability.

[0045] The static settling experiment visually confirmed the above quantitative results, such as Figure 2As shown, the sludge layer at the bottom of the measuring cylinder in the control group was large in volume and loosely structured, with a turbid supernatant and a small proportion of the total volume. In contrast, the sludge in the experimental group settled rapidly and compacted tightly, with a clear and transparent supernatant and a large volume proportion, fully demonstrating a significant improvement in settling performance. This invention not only achieves a rapid and significant decrease in SVI values ​​but also maintains their long-term stability within a safe range, effectively solving the problem of sludge bulking caused by excessive growth of filamentous bacteria under low-temperature conditions, and possesses promising engineering application prospects.

[0046] Prior to this embodiment, observation and identification of the initial bulking sludge taken from the wastewater treatment plant revealed that the dominant filamentous bacteria causing sludge bulking were mainly filoths (approximately 60%) and zoosporidioides (approximately 25%), with a small amount of Nocardia. The filoths appeared as short, rod-shaped, hydrophobic, spherical aggregates; the zoosporidioides exhibited slender, sheath-like structures; and the Nocardia exhibited branching structures. After 24 hours of operation with *Pantoinella pineapple*, re-sampling revealed significant breakage, protoplasmic shrinkage, and dissolution of the hyphae of the filoths and zoosporidioides, with their abundance rating rapidly decreasing from level 4 to level 1. The branching structure of Nocardia was significantly reduced, indicating that the method of this invention has an inhibitory effect on the aforementioned filamentous bacteria. *Pantoinella pineapple* rapidly restores sludge settling performance by secreting specific extracellular enzymes or through competitive inhibition mechanisms, thereby disrupting the cell wall integrity of the aforementioned filamentous bacteria or inhibiting their elongation growth.

[0047] Morphological identification of initial sludge, such as Figure 3 As shown, the main swelling filamentous bacteria were identified as microfilamentous bacteria and zoosheathed bacteria. In the control group, a large number of these bacteria extended outwards from the surface of the activated sludge flocs, forming a stable network framework that hindered sludge compaction. In the experimental group with added *Pantotheca pineapple*, sampling and observation after 24 hours revealed that: the characteristic spherical aggregates of microfilamentous bacteria disintegrated, and the hyphae broke into short rods; the long sheath structure of zoosheathed bacteria disintegrated; and the branching of *Nocardia* was significantly reduced. The sludge flocs became dense and compacted, and no complete extended hyphae were observed. The flocs are mainly composed of floc-forming bacteria such as *Flocetomyces*, which secrete extracellular polymers, causing themselves to aggregate into high-density, tightly structured particles. High-density flocs can settle rapidly and compact under gravity. The results clearly demonstrate that Pantotheca pineapple can not only broadly inhibit filamentous bacteria, but also specifically disrupt the growth structure of specific dominant bacterial species such as microfilamentous bacteria, zoosporous bacteria, Nocardia bacteria, and actinomycetes, fundamentally solving the problem of sludge bulking caused by the excessive proliferation of these specific filamentous bacteria.

[0048] This invention provides an inhibition method that introduces a specific strain of *Ubiquitin bromide* as a bioregulator to suppress filamentous sludge bulking under low-temperature conditions. This method achieves targeted regulation through natural competition and antagonism among microorganisms: *Ubiquitin bromide* specifically inhibits filamentous sludge growth through nutrient competition and interference with quorum sensing, while maintaining stable floc function. Compared to traditional methods relying on chemical agents or complex process adjustments, this invention can still take effect rapidly at low temperatures of 10-15℃, significantly improving sludge settling performance (SVI recovers to below 150 mL / g) within 24 hours of addition, with no risk of chemical residues, and is simple to operate and low in cost. This method provides an efficient, green, and easy-to-implement biological solution for the stable operation of wastewater treatment plants in winter. This invention utilizes the metabolic activities of *Ubiquitin bromide*, employing biological pathways such as nutrient competition and interference with quorum sensing to achieve specific inhibition of filamentous bacteria, precisely regulating the microbial community structure without harming other beneficial microorganisms.

[0049] This invention employs biological control methods, eliminating the risk of chemical residues and secondary pollution. Experiments show that this method, while specifically inhibiting the excessive growth of filamentous bacteria, does not suppress the system's organic matter degradation or nitrification capabilities, ensuring the stable operation of the core process performance of the wastewater treatment system. Compared to traditional methods, this invention significantly improves response speed and treatment stability under low-temperature conditions, providing a reliable technical solution for filamentous sludge bulking.

[0050] It should be noted that the sludge concentration mentioned in this article refers to the suspended solids concentration of the mixed liquor after the wastewater and activated sludge are mixed.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Any equivalent changes and modifications made within the scope of protection of this invention, such as simple substitutions, combinations, or simplifications of the technical solutions of this invention, as long as they do not depart from the technical principles of this invention, should be covered within the scope of protection of this invention.

Claims

1. A pineapple pancreatic fungicide, characterized in that: The active ingredient of the microbial agent is Pantotheca pineapple strain, and the microbial agent inhibits the bulking of filamentous bacterial sludge at a temperature of 5-15℃.

2. The pineapple pantothecin inoculant according to claim 1, characterized in that: The filamentous bacteria inhibited by the *Panthera pineapple* strain are selected from one or more of *Thiobacillus*, *Microfilariae*, *Zoothecae*, or *Nocardia*.

3. The pineapple pantothecin inoculant according to claim 1, characterized in that: The bacterial agent is in the form of a lyophilized powder.

4. A method for inhibiting low-temperature filamentous sludge bulking using the pineapple pantothecin inoculant as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preparation of bacterial agent: Pure culture of Pantotheca pineapple was carried out, and the bacterial solution in the logarithmic growth phase was collected. After centrifugation to remove the culture medium, the bacterial precipitate was resuspended to obtain the bacterial concentrate. (2) System addition: The bacterial concentrate obtained in step (1) is added to the activated sludge system in the state of filamentous sludge expansion; (3) Substrate replenishment: Replenish the influent substrate to the activated sludge system; (4) Process operation: The system adopts a sequencing batch activated sludge process with a pre-anoxic denitrification section and a subsequent aerobic nitrification section. In particular, under low-temperature conditions, the growth of filamentous bacteria is inhibited by the competitive ecological niche or secretion of signaling molecules by the pineapple pancreatic bacteria, thereby inhibiting sludge bulking.

5. The method according to claim 4, characterized in that, The specific process for preparing the microbial agent in step (1) is as follows: The lyophilized Pantotheca pineapple powder was inoculated into LB liquid medium for activation culture, and then transferred to fresh LB liquid medium for further culture for 24-48 hours to obtain a high concentration of bacterial solution. The high-concentration bacterial solution is then transferred to LB broth medium at a certain inoculation amount and cultured for 24-48 hours until the bacterial solution is in the logarithmic growth phase, for subsequent centrifugation.

6. The method according to claim 4, characterized in that, The specific operation of adding the substance in step (2) is as follows: The bacterial culture in the logarithmic growth phase was centrifuged, the supernatant was discarded to remove residual culture medium, and the bacterial precipitate was collected. After resuspending and mixing the bacterial precipitate with sterile water or buffer solution, it is directly added to the expanded activated sludge mixture.

7. The method according to claim 4, characterized in that, The components and concentrations of the influent substrate mentioned in step (3) are as follows: ammonia nitrogen concentration is 30 mg / L; chemical oxygen demand concentration is 150 mg / L; and pH value is adjusted to 7.5-8.

5.

8. The method according to claim 4, characterized in that, In step (4), the concentration of suspended solids in the mixed liquor of the activated sludge system is controlled within the range of 2500-2600 mg / L; the concentration of dissolved oxygen in the aerobic nitrification reactor is maintained above 2.0 mg / L.

9. The method according to claim 4, characterized in that, The low-temperature conditions mentioned in step (4) are specifically as follows: The operating temperature of the activated sludge system is controlled within the range of 5-15℃.

10. The method according to claim 4, characterized in that, The operating cycle and time allocation of the SBR process in step (4) are as follows: in each operating cycle, the anoxic denitrification operation is carried out for 8 hours first, followed by the aerobic nitrification reaction for 13 hours; the operating cycle also includes subsequent sedimentation, drainage and idle stages.

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