A nitrification-denitrification composite bacterial agent, its preparation method and application

Abstract

This invention discloses a nitrification-denitrification composite bacterial agent, its preparation method, and its application, belonging to the field of wastewater treatment technology. The composite bacterial agent uses activated sludge from a microaerobic reactor selected by a triple pressure screening process ("low DO-FA-FNA") as the initial bacterial strain. It is enriched through composite nitrogen source supply, multi-factor synergistic selection pressure regulation, and multi-generation cyclic sequence transfer culture. The composite bacterial agent includes core functional bacteria and nitrite-oxidizing bacteria (NOB), with the core bacteria accounting for no less than 80% of the total bacterial count, and the batch-to-batch Bray-Curtis dissimilarity index not exceeding 0.08. Designed for wastewater with high ammonia nitrogen and low carbon-to-nitrogen ratio, the bacterial agent has advantages such as high denitrification efficiency, strong stability, low energy consumption, and low carbon source requirement. It can be directly applied to the enhancement or rapid start-up of wastewater biological treatment systems, solving the technical problems of low purity, poor batch consistency, incomplete inhibition of nitrite-oxidizing bacteria, and high application cost of existing bacterial agents.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, and in particular to a highly stable short-cut nitrification and denitrification composite bacterial agent for wastewater with high ammonia nitrogen and low carbon-to-nitrogen ratio, its preparation method and application. Background Technology

[0002] Wastewater such as sludge filtrate generated during the treatment of urban sewage and industrial wastewater is characterized by "three highs and one low"—high ammonia nitrogen, high salinity, high suspended solids, and low carbon-to-nitrogen ratio. The ammonia nitrogen concentration often reaches 500-2000 mg / L. Traditional full-process nitrification and denitrification processes face technical bottlenecks such as carbon source scarcity, high aeration energy consumption, and poor system stability, making it difficult to achieve wastewater discharge standards.

[0003] Short-cut nitrification-denitrification technology, by controlling the nitrification process at the nitrite stage, can save 25% of aeration and 40% of carbon source, making it an ideal solution for treating this type of wastewater. However, in existing technologies, the short-cut nitrification-denitrification function largely relies on in-situ acclimation, and the functional microbial community is easily affected by influent fluctuations, resulting in poor stability. Furthermore, many publicly available compound microbial agents suffer from low purity of core microbial communities, poor batch-to-batch consistency, and incomplete inhibition of nitrite-oxidizing bacteria (NOB), leading to unstable nitrogen removal efficiency and difficulty in large-scale promotion in practical applications. In addition, existing microbial agent preparation often employs single selective pressure or single nitrogen source acclimation, making it difficult to achieve synergistic enrichment of ammonia-oxidizing bacteria (AOB) and denitrifying bacteria, thus limiting the overall performance of the agents. Ammonia-oxidizing bacteria (AOB) mainly include genera such as *Nitrosomonas* and *Nitrosospira*, while nitrite-oxidizing bacteria (NOB) mainly include genera such as *Nitrobacter* and *Nitrospira*. Existing technologies struggle to simultaneously achieve efficient enrichment of AOB and precise inhibition of NOB.

[0004] For microbial inoculants, reproducibility is crucial. If the preparation method relies solely on random acclimatization without clear directional control measures, it lacks practicality. Therefore, developing a short-cut nitrification-denitrification composite inoculant with a clearly defined core microbial community, high purity, strong stability, synergistic function, and a reproducible and controllable preparation process, along with its directional enrichment preparation method, is of great significance for solving the problem of treating wastewater with high ammonia nitrogen and low carbon-to-nitrogen ratio. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing short-cut nitrifying and denitrifying bacterial agents, such as low purity, poor batch consistency, incomplete NOB inhibition, insufficient functional synergy, poor reproducibility of preparation processes, and difficulty in large-scale production. It provides a composite bacterial agent with a clearly defined core bacterial community, strong stability, and high denitrification efficiency. At the same time, it provides a method for its targeted preparation and its application in the treatment of wastewater with high ammonia nitrogen and low carbon-to-nitrogen ratio, solving the technical problems of high carbon source consumption, high energy consumption, and system instability in the treatment of wastewater with high ammonia nitrogen and low carbon-to-nitrogen ratio.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A nitrification-denitrification compound microbial agent includes a core functional microbial community and nitrite-oxidizing bacteria (NOB). Its core functional microbial community includes ammonia-oxidizing bacteria (AOB) and denitrifying bacteria, with the total proportion of core bacteria not less than 80%.

[0007] The relative abundance of the nitrite-oxidizing bacteria (NOB) is no higher than 0.5%. The nitrite-oxidizing bacteria (NOB) can block the oxygen-consuming pathway of "nitrite-nitrate", avoid energy waste, and ensure the stability of the short-cut nitrification process.

[0008] The compound microbial agent has a Shannon index of 4.2-4.5, a Simpson index of 0.03-0.05, and a batch-to-batch Bray-Curtis dissimilarity index of no more than 0.08.

[0009] The ammonia-oxidizing bacteria include the genus *Nitrosomonas*, with a relative abundance of 20%-40%. Ammonia-oxidizing bacteria can efficiently oxidize ammonia nitrogen to nitrite and are key functional bacteria for short-cut nitrification.

[0010] The denitrifying bacteria include core denitrifying bacteria and auxiliary denitrifying bacteria.

[0011] The core denitrifying bacteria include *Dokdonella* and *Zoogloea*. *Dokdonella* is mentioned in conjunction with the accompanying drawings of the embodiments of this invention. The technical solutions in the embodiments of this invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort belong to the category of *Dokdonella* with a relative abundance of 15%-30% and *Zoogloea* with a relative abundance of 10%-25%. *Zoogloea* possesses both floc formation and denitrification functions, which can enhance the colonization stability of the bacterial agent in the wastewater treatment system and prevent bacterial loss.

[0012] The relative abundance of auxiliary denitrification bacteria is 5%-15%, including Thaurea and Dechloromonas, with Thaurea accounting for 3%-8% and Dechloromonas for 2%-7%. These auxiliary denitrification bacteria can form a functionally redundant denitrification network, ensuring efficient reduction of nitrite to nitrogen and improving the integrity of denitrification.

[0013] The present invention also provides a method for preparing the above-mentioned nitrification-denitrification composite bacterial agent, comprising the following steps: (1) Initial strain screening: Activated sludge that has been acclimatized under the triple pressure of "low DO-FA-FNA" in the micro-aerobic reactor for a long time is selected as the initial strain. The relative abundance of ammonia oxidizing bacteria (AOB) in the activated sludge is not less than 10%, which lays the foundation for efficient enrichment.

[0014] (2) Preparation of targeted enrichment medium: An inorganic salt basal medium was prepared using ammonium chloride and sodium nitrite as a combined nitrogen source, with ammonium chloride content of 500-800 mg / L and sodium nitrite content of 100-200 mg / L; the carbon-to-nitrogen ratio of the medium was not higher than 2. This provides substrates for AOB to maintain ammonia oxidation activity and provides electron acceptors for denitrifying bacteria. At the same time, the FNA formed by the accumulation of sodium nitrite synergistically inhibits NOB at low pH. The inorganic salt basal medium removes complex organic matter, creates a highly selective environment, and eliminates non-target bacterial groups.

[0015] (3) Synergistic selection pressure regulation: The above initial strains were inoculated into the above culture medium, and the culture conditions were controlled as follows: dissolved oxygen 0.3-0.8 mg / L, pH 7.5-8.0, and temperature 25-30℃. Utilizing the difference in oxygen affinity between AOB and NOB, NOB was inhibited while meeting the metabolic needs of AOB. The weakly alkaline environment promoted the conversion of ammonia nitrogen to FA, further enhancing NOB inhibition and forming a multi-layered inhibition barrier.

[0016] (4) Multigenerational cyclical transfer culture: When the nitrite accumulation rate is not less than 80% and the ammonia nitrogen removal rate is not less than 90%, the culture is transferred to the above culture medium at an inoculation ratio of 10%-12%, and the culture is repeated for 3-5 generations, with each generation having a culture cycle of 48-72 hours, to obtain a compound bacterial solution; (5) Preparation of bacterial agent: The enriched compound bacterial solution is centrifuged and concentrated, and a protective agent is added to prepare a liquid bacterial agent; or it is freeze-dried to prepare a solid bacterial agent.

[0017] Furthermore, the DO content in step (1) is 0.3-0.8 mg / L, the mass ratio of ammonium chloride to sodium nitrite in the composite nitrogen source in step (2) is 4-8:1, and the effective bacterial count of the liquid bacterial agent in step (5) is not less than The effective bacterial count of solid bacterial agents shall not be less than

[0018] Furthermore, the protective agent in step (5) includes glycerol and trehalose.

[0019] The present invention also provides an application of the above-mentioned nitrification-denitrification composite bacterial agent in wastewater treatment, wherein the application method is to add 1-5 / L of solid bacterial agent or 5-10mL of liquid bacterial agent to the wastewater biochemical treatment system.

[0020] Furthermore, the wastewater includes sludge dewatering liquid, coal chemical wastewater, and aquaculture wastewater, wherein the ammonia nitrogen concentration is 500-2000 mg / L and the carbon-to-nitrogen ratio is less than 3; the domestic wastewater treatment system includes AO process, MBR process and SBR process.

[0021] Compared with the prior art, the present invention, employing the above technical solution, has the following beneficial effects: (1) High purity and clear function of microbial community: The total proportion of core functional bacteria is not less than 80%, NOB is completely inhibited, and the relative abundance is not higher than 0.5%. It has been verified by metagenomics and has a complete "short-range nitrification-denitrification" metabolic pathway, without the need for additional functional matching.

[0022] (2) Strong batch consistency: Through multi-generation cyclic sequence transfer culture and synergistic selection pressure regulation, the Bray-Curtis dissimilarity index between batches of bacterial agents is no higher than 0.08, which meets the requirements of standardized production.

[0023] (3) The preparation process is efficient and controllable: the initial strain is pre-screened under triple pressure, the enrichment cycle is short, the culture conditions are precise and controllable, and it can be produced on a large scale.

[0024] (4) Low application cost and wide adaptability: This bacterial agent is designed for wastewater with high ammonia nitrogen and low carbon-nitrogen ratio. After being added, it can quickly colonize and form a functional dominant bacterial community. The total nitrogen removal rate of the system is ≥85%, which is more than 25% higher than that of traditional processes. The aeration volume is reduced by 25%-30%, and the amount of organic carbon source added is reduced by 40%-45%, which significantly reduces the operating cost. The system start-up cycle is shortened by 30%-50%, the ability to resist shock load is enhanced, and it is compatible with a variety of mainstream biochemical treatment processes. It has good treatment effect on different types of wastewater with high ammonia nitrogen and low carbon-nitrogen ratio. Attached Figure Description

[0025] Figure 1 This is a comparison chart of the Shannon index between the compound microbial agent of Example 1 and the original process sludge; Figure 2 This is a comparison chart of the Chao1 index of the compound microbial agent in Example 1 and the original process sludge; Figure 3 This is a batch-to-batch PCA analysis chromatogram of the compound microbial agent in Example 1; Figure 4 This is a stacked diagram of the horizontal community structure of the compound microbial agent in Example 1; Figure 5 The composite microbial agent of Example 1 is shown in the horizontal heat map. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Preparation of Compound Microbial Agent 1. Initial microbial strains: Activated sludge acclimated in a microaerobic reactor for 6 months was selected. The relative abundance of AOB was 12% and the relative abundance of NOB was 3.5%. 2. Culture medium preparation: Ammonium chloride 600 mg / L, sodium nitrite 100 mg / L, add...

[0028] 3. Enrichment culture: Inoculum size 10%, dissolved oxygen controlled at 0.5 mg / L, temperature 28℃, cultured for 72 h, ammonia nitrogen removal rate 92%, nitrite accumulation rate 83%; 4. Repeated subculturing: Transfer to fresh culture medium at a 15% inoculum ratio, repeat culture for 4 generations, with ammonia nitrogen removal rate ≥90% and nitrite accumulation rate ≥80% during the stationary phase of each generation; 5. Preparation of bacterial agent: Centrifuge and concentrate (8000 r / min, 10 min), add 10% glycerol as a preservative to prepare liquid bacterial agent with an effective viable count of 1.2 × 10⁻⁶ cells / min. 8 CFU / mL; Analysis revealed that Nitrosomonas comprised 32%, Dokdonella 25%, Zoogloea 18%, Thaurea 6%, and Dechloromonas 4%, with the core genera accounting for 85% of the total. NOB accounted for 0.3%, the Shannon index was 4.35, the Simpson index was 0.036, and the batch-to-batch Bray-Curtis dissimilarity index was 0.06. A comparison of alpha diversity with the original process sludge is shown in Table 1.

[0029] Table 1. Statistical table of microbial Alpha diversity index Original aerobic sludge 1506.69 0.99702 0.00707 1431 Sludge domestication 1133.88 0.99698 0.02444 1042 raw anaerobic sludge 1510.54 0.99812 0.00608 1475 Table 1 (in conjunction with) Figure 1 , Figure 2It can be seen that the Observed OTUs and Ace indices of the original aerobic sludge and the original anaerobic sludge are significantly higher than those of the acclimated sludge, while the Shannon index is lower than that of the acclimated sludge. This indicates that after the targeted enrichment in Example 1, the diversity of the microbial community is reduced and the functional microbial community is effectively enriched. The Simpson index is higher than that of the original sludge, which further verifies the dominant position of the core functional microbial community.

[0030] Figure 3 The results showed that the samples were clustered tightly, indicating that the community structure of different batches of bacterial agents was not significantly different and that the batch consistency was good, which meets the requirement of Bray-Curtis dissimilarity index ≤ 0.08 between batches in this invention.

[0031] Figure 4 The study demonstrated the dominance of Pseudomonadota and the low abundance of Nitrospirota (the main phylum to which NOB belongs), thus validating the effective suppression of NOB.

[0032] Figure 5 This visually demonstrates the dominant position and consistent distribution of core functional bacterial groups such as Nitrosomonas, Dokdonella, and Zoogloea, further proving the rationality and stability of the bacterial composition of this invention.

[0033] Example 2: Application of microbial agents in sludge filtrate treatment 1. Wastewater quality: ammonia nitrogen concentration 850 mg / L, COD concentration 1200 mg / L, C / N = 1.4, salinity 3%; 2. Processing system: MBR process reactor, effective volume 10L; 3. Dosing of microbial agent: Add the liquid microbial agent prepared in Example 1 at a dosage of 8 mL / L; 4. Operating parameters: Dissolved oxygen 0.6 mg / L, pH 7.7, temperature 28℃, HRT=24h; 5. Treatment effect: The system stabilized 7 days after addition, with ammonia nitrogen removal rate of 95% and total nitrogen removal rate of 88%; compared with the control group without bacterial agent addition, the aeration volume decreased by 28%, the sodium acetate dosage decreased by 42%, and the start-up cycle was shortened by 40%.

[0034] Example 3: Application of bacterial agents in coal chemical wastewater treatment 1. Wastewater quality: ammonia nitrogen concentration 1500 mg / L, COD concentration 2200 mg / L, C / N = 1.5, salinity 5%; 2. Processing system: AO process reactor, effective volume 20L; 3. Addition of microbial agent: Add the freeze-dried solid microbial agent prepared in Example 1 (effective viable count 1.5 × 10⁻⁶). Dosage: 3g / L; 4. Operating parameters: Dissolved oxygen 0.4 mg / L, pH 7.6, temperature 27℃, HRT=36h; 5. Treatment effect: The system stabilized 10 days after addition, with an ammonia nitrogen removal rate of 93% and a total nitrogen removal rate of 86%. Compared with the traditional process, the operating energy consumption was reduced by 26%, the carbon source cost was reduced by 43%, the system's resistance to shock loads was significantly improved, and the ammonia nitrogen concentration could still stably meet the standard when it fluctuated by ±200mg / L.

[0035] Example 4: Application of microbial agents in the treatment of pig farm wastewater Wastewater quality: ammonia nitrogen concentration 1000 mg / L, COD concentration 2500 mg / L, C / N = 1.3, salinity 2%; Processing system: Process reactor, effective volume 15L; Inoculum dosing: Add the liquid inoculum prepared in Example 1 at a dosage of 10 mL / L; Operating parameters: dissolved oxygen 0.5 mg / L, pH 7.8, temperature 28℃, HRT=30h; Treatment results: The system stabilized 8 days after addition, with an ammonia nitrogen removal rate of 94% and a total nitrogen removal rate of 85%. Compared with the control group without bacterial agent addition, the aeration volume was reduced by 30%, the sodium acetate dosage was reduced by 45%, the start-up period was shortened by 42%, the system's resistance to shock loads was significantly improved, and the ammonia nitrogen concentration could still stably meet the standard when it fluctuated by ±250mg / L. At the same time, the sludge production rate was reduced by 21%, effectively reducing the cost of sludge disposal.

[0036] This invention achieves high purity, high stability, and functional integrity of short-cut nitrification and denitrification bacteria agents through strain screening, culture condition optimization, and innovative preparation process. It provides a new technical solution for the efficient treatment of wastewater with high ammonia nitrogen and low carbon-to-nitrogen ratio, and has significant engineering application value and environmental benefits.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention; equivalent substitutions or modifications made by those skilled in the art without departing from the spirit and substance of the present invention should all fall within the scope of protection of the present invention.

Claims

1. A nitrification-denitrification composite microbial agent, characterized in that, The compound microbial agent includes a core functional microbial community and nitrite-oxidizing bacteria (NOB); the core functional microbial community includes ammonia-oxidizing bacteria (AOB) and denitrifying bacteria, with the total proportion of core bacteria not less than 80%, and the relative abundance of nitrite-oxidizing bacteria (NOB) not higher than 0.5%; the Shannon index of the compound microbial agent is 4.2-4.5, the Simpson index is 0.03-0.05, and the batch-to-batch Bray-Curtis dissimilarity index is not higher than 0.

08.

2. The compound microbial agent according to claim 1, characterized in that, The ammonia-oxidizing bacteria (AOB) include *Nitrosomonas*, with a relative abundance of 20%-40%; the denitrifying bacteria include *Dokdonella*, *Zoogloea*, and auxiliary denitrifying bacteria, with *Dokdonella* having a relative abundance of 15%-30%, *Zoogloea* having a relative abundance of 10%-25%, and auxiliary denitrifying bacteria having a relative abundance of 5%-15%.

3. The compound microbial agent according to claim 2, characterized in that, The auxiliary denitrifying bacteria include Thaurea and Dechloromonas, with Thaurea having a relative abundance of 3%-8% and Dechloromonas having a relative abundance of 2%-7%.

4. The method for preparing the compound microbial agent according to claim 1, 2, or 3, characterized in that, Includes the following steps: (1) Initial strain screening: Activated sludge that has been acclimatized under the triple pressure of "low DO-FA-FNA" in the micro-aerobic reactor for a long time was selected as the initial strain, wherein the relative abundance of ammonia oxidizing bacteria (AOB) in the activated sludge was not less than 10%; (2) Preparation of directional enrichment culture medium: An inorganic salt basal culture medium is prepared using ammonium chloride and sodium nitrite as a composite nitrogen source, wherein the ammonium chloride content is 500-800 mg / L and the sodium nitrite content is 100-200 mg / L; the carbon-nitrogen ratio of the culture medium is not less than that of a nitrification-denitrification composite bacterial agent, characterized in that the composite bacterial agent includes a core functional bacterial group and nitrite oxidizing bacteria (NOB); the core functional bacterial group includes ammonia oxidizing bacteria (AOB) and denitrifying bacteria, the total proportion of core bacterial genera is not less than 80%, and the relative abundance of nitrite oxidizing bacteria (NOB) is not higher than 0.5%; the Shannon index of the composite bacterial agent is 4.2-4.5, the Simpson index is 0.03-0.05, and the batch-to-batch Bray-Curtis dissimilarity index is not higher than 0.08 and not higher than 2; (3) Synergistic selection pressure regulation: The above initial strains were inoculated into the above culture medium, and the culture conditions were controlled as follows: dissolved oxygen 0.3-0.8 mg / L, pH 7.5-8.0, and temperature 25-30℃; (4) Multigenerational cyclical transfer culture: When the nitrite accumulation rate is not less than 80% and the ammonia nitrogen removal rate is not less than 90%, the culture is transferred to the above culture medium at an inoculation ratio of 10%-12%, and the culture is repeated for 3-5 generations, with each generation having a culture cycle of 48-72 hours, to obtain a compound bacterial solution; (5) Preparation of bacterial agent: The enriched compound bacterial solution is centrifuged and concentrated, and a protective agent is added to prepare a liquid bacterial agent; or it is freeze-dried to prepare a solid bacterial agent.

5. The method for preparing the compound microbial agent according to claim 4, characterized in that, The DO content in step (1) is 0.3-0.8 mg / L.

6. The method for preparing the compound microbial agent according to claim 4, characterized in that, In step (2), the mass ratio of ammonium chloride to sodium nitrite in the composite nitrogen source is 4-8:

1.

7. The method for preparing the compound microbial agent according to claim 4, characterized in that, In step (5), the effective bacterial count of the liquid bacterial agent is not less than The effective bacterial count of solid bacterial agents shall not be less than .

8. The application of the compound microbial agent according to claim 1, 2 or 3 in wastewater treatment.

9. The application according to claim 8, characterized in that, The application method is to add 1-5 / L of solid bacterial agent or 5-10mL of liquid bacterial agent to the wastewater biological treatment system.

10. The application according to claim 9, characterized in that, The wastewater includes sludge dewatering liquid, coal chemical wastewater, and aquaculture wastewater, with an ammonia nitrogen concentration of 500-2000 mg / L and a carbon-to-nitrogen ratio of less than 3; the domestic wastewater treatment system includes AO process, MBR process, and SBR process.

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