Fermentation method of nitrobacteria consortia based on microelement regulation and control

Abstract

The invention relates to the technical field of environmental microorganisms and fermentation engineering, in particular to a nitrifying bacteria consortium fermentation method based on microelement regulation and control. Culturing consortia formed by ammonia oxidizing bacteria AOB and nitrite oxidizing bacteria NOB into a fermentation seed solution; in the fermentation process, microelement supplementing liquid is added according to the oxygen consumption rate value, stage supplementing is conducted, a mixed fermentation system with AOB and NOB synchronously and stably increased is formed, the problems that nitrifying bacteria are unstable in fermentation and have no nitrite nitrogen degradation capacity are solved, the single-tank fermentation time is shortened, the production cost is reduced, and the production efficiency is improved. The ammoxidation rate of a finished product is improved.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology and fermentation engineering technology, and in particular to a fermentation method based on trace element regulation of nitrifying bacterial aggregates. Background Technology

[0002] Autotrophic nitrifying bacteria are a mature product in the wastewater treatment industry. They are typically a class of bacteria capable of oxidizing ammonia nitrogen or ammonium salts to nitrite or oxidizing nitrite to nitrate. These include ammonia-oxidizing bacteria (AOB) (e.g., *Nitrosomonas*, *Nitrococcus*) and nitrite-oxidizing bacteria (NOB) (e.g., *Nitrobacterium*, *Nitrococcus*). Under aerobic conditions, ammonia-oxidizing bacteria first oxidize ammonia to nitrite, and nitrite-oxidizing bacteria then oxidize nitrite to nitrate; the two work together to complete the entire nitrification process. Unlike traditional heterotrophic nitrifying microorganisms, autotrophic nitrifying bacteria have a nitrification capacity 10³-10⁶ times greater and are a core component of nitrification in nature. For nitrifying bacteria, the ammonia oxidation rate is the core standard for evaluating their nitrification effect, reflecting to some extent the strength of their nitrification activity in practical applications. In 2020, HG / T5748-2020 "Performance Evaluation Method of Nitrifying Bacteria Agents for Water Treatment", which was under the jurisdiction of the Water Treatment Subcommittee of the National Technical Committee on Standardization of Chemicals, further confirmed the use of ammonia oxidation performance: under aerobic conditions, the mass of ammonia nitrogen oxidized per unit volume of nitrifying bacteria agent per unit time is used to evaluate the nitrification capacity of nitrifying bacteria.

[0003] The ammonia oxidation performance of nitrifying bacteria is closely related to the fermentation process; however, nitrifying bacteria are strictly inorganic chemoautotrophic microorganisms. Relevant studies at home and abroad have used inorganic nutrients as substrates. Therefore, the fermentation process of nitrifying bacteria is different from that of heterotrophic bacteria in the following main ways: 1. It is a pure inorganic fermentation, and the requirements for the fermentation environment are relatively low; 2. Recent reports often use semi-batch fermentation for improvement, with an average generation time of more than 10 hours for nitrifying bacteria and a single-tank fermentation time of about 10 days; 3. Although nitrifying bacteria include ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB), single-strain fermentation of ammonia oxidizing bacteria (AOB) is often performed.

[0004] Due to the long fermentation time and special fermentation method, nitrifying bacteria fermentation is often accompanied by many problems: for example, the single-strain fermentation of ammonia-oxidizing bacteria (AOB) has the ability to degrade nitrite nitrogen, but the single-strain fermentation technology of nitrite-oxidizing bacteria (NOB) is still relatively scarce; the fermentation products of ammonia-oxidizing bacteria (AOB) are severely inhibited, the fermentation concentration is low and the rate is low, and a large amount of nitrite nitrogen is produced in the tailwater, which increases the difficulty of tailwater treatment; the fermentation stability is poor, the cycle is long, miscellaneous bacteria proliferate, and the data fluctuates frequently, which further increases the fermentation time of a single tank.

[0005] Therefore, there is an urgent need for a mixed fermentation process of autotrophic nitrifying bacteria to address the problems of instability and lack of nitrite degradation capacity in current nitrifying bacteria fermentation processes on the market, thereby reducing the fermentation time per tank and increasing the ammonia oxidation rate of the finished product. Summary of the Invention

[0006] In view of this, this invention proposes a fermentation method based on trace element regulation of nitrifying bacterial aggregates. In the seed production stage, AOB and NOB are combined to form stable nitrifying bacterial aggregates as the seed liquid for the upper fermentation tank. The aggregates are continuously passaged to increase their abundance and improve the ammonia oxidation rate of the upper fermentation tank seed, which is controlled at 50-60 mgNH3-N / L / hr. In the early stage of mixed fermentation, trace element regulation is used to promote NOB proliferation while balancing the AOB / NOB biomass, maintaining a dynamic growth balance. This ensures that the amount of nitrite nitrogen produced during fermentation is less than or equal to the amount required for NOB proliferation, thereby achieving a balanced mixed fermentation of nitrifying bacteria. This results in a mixed fermentation system where AOB and NOB grow synchronously and stably, solving the problems of unstable nitrifying bacterial fermentation and lack of nitrite nitrogen degradation ability, reducing the fermentation time per tank, and improving the ammonia oxidation rate of the finished product.

[0007] The technical solution of this invention is implemented as follows: The first invention provides a fermentation method for nitrifying bacterial aggregates, comprising the following steps: S1, culturing nitrifying bacteria aggregates into a fermentation seed liquid; the nitrifying bacteria aggregates are aggregates formed by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB); S2, the seed culture is inoculated into the fermentation medium for fermentation. During fermentation, feed solution is added according to the oxygen consumption rate, and feeding is carried out in stages. Phase 1: When the oxygen consumption rate is <40 mg O2 / (gVSS·h), batch feeding is carried out; Phase Two: When the oxygen consumption rate reaches 40 mg O2 / (gVSS·h), automatic feeding is activated. Phase 3: When the oxygen consumption rate is >40 mg O2 / (gVSS·h), automatic feeding with increased speed will be activated. In stage four, when the fermentation rate reaches <40 mg O2 / (gVSS·h), feeding is stopped and fermentation is terminated.

[0008] Based on the above technical solutions, preferably, in step S1, the ammonia oxidation rate of the polymer in the fermentation seed liquid is 50~60 mgNH3-N / L\hr.

[0009] By continuously subculturing to increase the relative abundance of aggregates in the seed culture, and according to HG / T 5748-2020 "Performance Evaluation Method of Nitrifying Bacteria Agents for Water Treatment", the ammonia oxidation rate at the time of sample collection of each generation of seed culture was tested and controlled within an appropriate range to ensure the stable fermentation of nitrifying bacteria aggregates, achieve the mixed fermentation balance of nitrifying bacteria, and form a mixed fermentation system in which AOB and NOB grow synchronously and stably.

[0010] Based on the above technical solutions, preferably, in step S2, the feed liquid contains Fe, Cu, Ca and Se elements.

[0011] Fe participates in the electron transport chain (cytochrome) during biomolecular fermentation and is a core component of ammonia monooxygenase and ferrous oxidase. It promotes the conversion of NH3 to NH2OH catalyzed by ammonia monooxygenase (the first step of nitration) and the electron transport and energy production processes of cytochrome. Cu is a key component of nitrite oxidoreductase, mainly promoting the catalysis of NO2 by nitrite oxidoreductase. - To NO3 - The conversion process (second step of nitrification); Ca mainly plays a role in maintaining cell wall stability and signal transduction, improving membrane permeability, promoting the proliferation of NOB in nitrite-oxidizing bacteria, and acting as an enzyme activator to promote metabolic activity; Se can act as an antioxidant, protecting cells from oxidative damage and helping bacteria resist damage from reactive oxygen species.

[0012] Based on the above technical solutions, preferably, the molar concentration of Fe is 144~216 μmol / L, the molar concentration of Cu is 8~20 μmol / L, the molar concentration of Ca is 13.5~20.5 μmol / L, and the molar concentration of Se is 0.75~2 μmol / L.

[0013] Based on the above technical solution, further preferredly, the Fe element is selected from FeSO4·7H2O at a concentration of 0.04~0.06 g / L, the Cu element is selected from CuSO4·5H2O at a concentration of 2~5 g / L, the Ca element is selected from CaCl2·2H2O at a concentration of 2~3 g / L, and the Se element is selected from Na2SeO3·5H2O at a concentration of 0.2~0.5 mg / L.

[0014] Based on the above technical solution, and further preferably, the Fe element is 0.05 g / L FeSO4·7H2O, the Cu element is 3.5 g / L CuSO4·5H2O, the Ca element is 2.5 g / L CaCl2·2H2O, and the Se element is selected from 0.35 mg / L Na2SeO3·5H2O.

[0015] Based on the above technical solutions, preferably, the feed solution also includes Mn, Co and Zn elements, as well as inorganic salts.

[0016] Based on the above technical solutions, preferably, the feed solution further includes 0.2~0.5 mg / L of MnCl2·4H2O, 0.2~0.5 mg / L of CoSO4·7H2O, and 60~90 μg / L of ZnSO4·7H2O; the inorganic salts include 100 g / L of NH4Cl, 0.3~0.5 g / L of NaCl, 0.03~0.05 g / L of MgSO4·7H2O, 0.3~0.5 g / L of K2HPO4·3H2O, and 0.01~0.03 g / L of KH2PO4.

[0017] Based on the above technical solutions, preferably, in step S2, the flow rate of the automatic feeding in stage two is 38~42 mL / h; more preferably, the initial flow rate of the automatic feeding is 40 mL / h.

[0018] Based on the above technical solutions, preferably, in step S2, the speed-up of the automatic feeding in stage three is 40~80mL / h, and the feeding interval is 23~25h.

[0019] Based on the above technical solutions, a further optimized method is to use a material replenishment interval of 24 hours.

[0020] Based on the above technical solutions, preferably, the ammonia nitrogen and nitrite nitrogen in the accelerated automatic feeding process are both <5 mg / L.

[0021] Based on the above technical solutions, preferably, in step S2, the indicator for stopping feeding in stage four also includes feeding flow rate reaching 600 mL / h.

[0022] Based on the above technical solutions, preferably, in step S2, the pH during the fermentation process is 7.0~8.0.

[0023] Based on the above technical solutions, the following further preferred options are available: Based on the pre-controlled seed culture in the Erlenmeyer flask, the AOB proliferation rate was still much better than NOB in the early stage of fermentation. This invention maintains the above-mentioned micronutrient ratio in the feed solution throughout the fermentation process, aiming to promote the rapid proliferation of NOB, so that the nitrite nitrogen generated by AOB consuming ammonia nitrogen is always less than or equal to the proliferation amount required by NOB, maintaining the high-activity proliferation state of NOB, and dynamically controlling the changes in pH and OUR (oxygen consumption rate).

[0024] Online monitoring parameters: Real-time monitoring of pH, DO (dissolved oxygen), OUR (oxygen consumption rate), temperature, and rotation speed. A sharp increase in OUR and a rapid decrease in pH indicate that the bacteria are in a vigorous metabolic phase with sufficient substrate. If OUR begins to stabilize or decrease, and pH changes gradually, it indicates that the substrate is about to be depleted or product inhibition has begun, and bacterial metabolism is slowing down. The system automatically compensates for pH based on the monitoring values ​​from the pH probe. If the pH decreases rapidly, the alkali addition rate increases continuously per hour; conversely, if the pH changes gradually, the alkali addition rate decreases per hour. However, if there is no alkali addition per unit time and no pH control is performed, the growth of nitrifying bacteria in the fermenter will be inhibited or the bacteria will die, leading to fermentation failure.

[0025] Dynamic control: Phase 1: Fermentation start-up. After inoculation, batch feeding is prioritized. At this time, the oxygen consumption rate is <40mg O2 / (gVSS·h), and the bacterial count is accumulated. The batch feed is 100mL / time. When the levels of ammonia nitrogen and nitrite nitrogen are 0mg / L, the next batch feed is continued. Phase 2, Feeding Start-up Phase: When the OUR reaches the set threshold of 40 mg O2 / (gVSS·h), it indicates that the cells have entered a high-activity state. The automatic feeding system is then started, with the initial flow rate set at 40 mL / h and the feeding interval at 24 h. During this period, both ammonia nitrogen and nitrite nitrogen need to be controlled to be <5 mg / L. Phase 3: Accelerated Feeding Phase. When the oxygen consumption rate is >40mg O2 / (gVSS·h), automatic accelerated feeding is activated, with an acceleration rate of 40~80mL / h and a feeding interval of 24h. During the acceleration, it is necessary to follow the parameter judgment standard, that is, control both ammonia nitrogen and nitrite nitrogen to be <5mg / L. If the parameters are abnormal, the acceleration time should be delayed or the rate should be reduced as a buffer. Phase Four: Stop Feeding or Fermentation Termination. When fermentation reaches an oxygen consumption rate < 40 mg O2 / (gVSS·h) or a feed flow rate reaches 600 mL / h, feeding should be stopped and fermentation terminated. The overall feed pump's on / off state and speed are controlled by the real-time value of OUR. The higher the OUR, the greater the feed rate should be to meet the demands of high-speed metabolism.

[0026] The fermentation method for nitrifying bacterial aggregates of the present invention has the following advantages over the prior art: 1. By regulating the balance of AOB and NOB in the mixed fermentation of aggregates, the nitrite concentration in the fermentation broth can be kept below 5 mg / L. Through strategic regulation, the nitrite conversion rate can be increased to 95.97%, which is conducive to the continuous fermentation application of nitrifying bacteria. The working time of a single tank can be reduced from 10 days to 7 days.

[0027] 2. This invention stabilizes the microbial community structure by precisely controlling the addition of trace elements during fermentation, effectively solving the inhibition phenomenon that may be caused by the accumulation of metabolites, reducing fermentation over-fermentation, significantly improving fermentation stability, and greatly increasing the operational error tolerance.

[0028] 3. The mixed fermentation method can enhance the ability of nitrifying bacteria to degrade nitrite nitrogen. Under optimal conditions, the maximum removal rate of nitrite nitrogen can reach 40.38 mg / (L·h), and 120 mg / L of nitrite nitrogen can be completely converted within 3 hours, thus expanding the application of nitrifying bacteria in aquariums and aquaculture. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a streak plate diagram of the AOB strain of the present invention; Figure 2 This is a streak plate diagram of the NOB strain of the present invention; Figure 3 This is a diagram of the seed culture of nitrifying bacteria aggregates of the present invention; Figure 4 This is a graph showing the changing trends of ammonia nitrogen and nitrite nitrogen during fermentation by nitrifying bacterial aggregates according to the present invention. Figure 5 The ammonia oxidation rate of nitrifying bacteria aggregates according to the present invention is shown in the ammonia nitrogen fitting curve. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] The fermentation basal culture medium formula used in this invention is as follows: The inorganic salt composition of the fermentation basal medium is as follows: NaCl 0.3~0.5 g / L, MgSO4·7H2O 0.03-0.05 g / L, K2HPO4·3H2O 0.3-0.5 g / L, KH2PO4 0.01-0.03 g / L; The trace element composition is as follows: FeSO4·7H2O 0.04-0.06 g / L, MnCl2·4H2O 0.2-0.5 mg / L, CuSO4·5H2O 2-5 mg / L, ZnSO4·7H2O 60-90 μg / L, CoSO4·7H2O 0.2-0.5 mg / L, NiSO4·6H2O 0.2-0.5 mg / L.

[0033] The pH adjusting agent in this invention is a 10% H2SO4 solution; the pH adjusting agent is a 100 g / L sodium carbonate solution.

[0034] The accession numbers of the ammonia-oxidizing bacteria AOB and the nitrite-oxidizing bacteria NOB in this invention are CCTCC NO: M20232716 and CCTCC NO: M2022808, respectively.

[0035] Example 1: Preparation of nitrifying bacteria aggregates.

[0036] AOB and NOB were purified using traditional microbial isolation and purification methods: 1. Enrichment of AOB and NOB The enriched sludge samples were obtained from pharmaceutical, petrochemical, chemical, resin, coal wastewater, electroplating / battery, food, and municipal wastewater. An appropriate amount of glass beads and 1 mL of ammonium chloride were added, and the pH was adjusted to 8 daily with sodium carbonate. The samples were then incubated at 28°C with shaking at 200 rpm in the dark for at least one week.

[0037] 2. Screening, purification, and assembly Both AOB and NOB were screened and purified using traditional solid culture media through plating, streak plating, liquid transfer, and pure culture. The streak plots of AOB and NOB are shown below. Figure 1 and Figure 2 As shown, purified AOB and NOB were cultured and spliced ​​into nitrifying bacteria aggregates at an inoculum ratio of 1:3.

[0038] Example 2: Preparation of seed culture for nitrifying bacteria aggregates for fermentation.

[0039] The nitrifying bacteria seed culture was continuously subcultured. After each subculture, the decision to continue subculturing was made based on the actual ammonia oxidation rate. During the subculture process, qualitative observations of ammonia nitrogen and nitrite nitrogen were performed every morning and evening (system: 10 μl chromogenic reagent + 0.5 ml sample). Specific adjustments are shown in Table 1.

[0040] Subculture method for nitrifying bacteria aggregate seeds: At the time of each generation, the ammonia oxidation rate was measured, and the inoculum size for subculture was 10% (v / v).

[0041] Table 1. Feeding schedule for nitrifying bacteria aggregates during passage.

[0042] The above procedures were followed for continuous subculturing until the ammonia oxidation rate was adjusted to 50-60 mg NH3-N / L / hr, at which point the seeds were stored for the next stage.

[0043] The ammonia oxidation rate was adjusted to 40, 50, 55, 60, 70, 80, 90 and 100 mgNH3-N / L\hr, respectively, and fermentation was carried out under the same conditions. The ammonia oxidation rate was measured, and the results are shown in Table 2.

[0044] Table 2 Comparison of seed fermentation of nitrifying bacteria at different rates

[0045] Through a series of fermentation comparisons, it was found that excessively high or low ammonia oxidation rates (AOB) in nitrifying bacteria directly affect fermentation stability. Furthermore, the concentration of AOB directly influences the determination of the ammonia oxidation rate. Therefore, when the ammonia oxidation rate is ≤40 mgNH3-N / L / hr, the AOB concentration is too low. After inoculation, substrate accumulation is severe, the rate of nitrite production (first-order product) is low, and NOB cannot proliferate rapidly. When AOB proliferates to the optimal concentration, the low relative abundance of NOB leads to slow proliferation, and the large accumulation of nitrite severely inhibits product growth, preventing normal fermentation. When the ammonia oxidation rate is ≥60 mgNH3-N / L / hr, NOB still cannot keep up with the AOB proliferation rate in the early stages of fermentation. During the fermentation process, nitrite levels remain >300 mg / L, preventing normal acceleration or causing repeated fluctuations in nitrite levels, requiring more time to adjust the bacterial ratio balance. When the ammonia oxidation rate is 50~60 mgNH3-N / L / hr, substrate consumption and first-order product consumption are relatively balanced, allowing for normal fermentation. Therefore, before loading the fermentation tank, the ammonia oxidation rate should be 50~60 mgNH3-N / L / hr. mgNH3-N / L\hr is the standard for loading seed liquid into the tank.

[0046] Example 3: Screening and optimization of trace elements.

[0047] Basic formula for feed solution: Inorganic salt composition of the fed culture medium: NH4Cl 100 g / L, NaCl 0.4 g / L, MgSO4·7H2O 0.04 g / L, K2HPO4·3H2O 0.4 g / L, KH2PO4 0.02 g / L; The typical trace element composition is as follows: MnCl2·4H2O 0.35 mg / L, CoSO4·7H2O 0.35 mg / L, ZnSO4·7H2O 75 μg / L.

[0048] Based on the above feed solution formulation, a single-factor experiment was conducted using nitrite-oxidizing bacteria NOB as the target strain to regulate the microelements (Fe, Cu, Ca, and Se).

[0049] 1. Single-factor experiment on Fe element.

[0050] Based on the above feed solution formulation, FeSO4·7H2O was added at concentrations of 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 200 mg / L, and 500 mg / L, respectively, to subculture the NOB strain and determine the NOB rate. The results are shown in Table 3.

[0051] Table 3. Record Sheet for Single-Factor Experiment (NOB) on Iron (Fe)

[0052] 2. Single-factor experiment on Cu element.

[0053] Based on the above feed solution formulation, 0.01 mg / L, 0.25 mg / L, 0.5 mg / L, 1.5 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 30 mg / L, 100 mg / L and 500 mg / L of CuSO4·5H2O were added respectively to subculture the NOB strain, and the NOB growth rate was measured. The results are shown in Table 4.

[0054] Table 4. Record Sheet for Single-Factor Experiment (NOB) on Copper (Cu)

[0055] 3. Single-factor experiment on Ca element.

[0056] Based on the above feed solution formulation, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 10 mg / L, 20 mg / L and 50 mg / L of CaCl2·2H2O were added respectively to subculture the NOB strain, and the NOB rate was measured. The results are shown in Table 5.

[0057] Table 5. Record Sheet of Single-Factor Experiment (NOB) for Calcium (Ca)

[0058] 4. Single-factor experiment on Se element.

[0059] Based on the above feed solution formulation, 0.01 mg / L, 0.05 mg / L, 0.15 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 3 mg / L, 10 mg / L and 50 mg / L of Na2SeO3·5H2O were added respectively to subculture the NOB strain, and the NOB growth rate was measured. The results are shown in Table 6.

[0060] Table 6. Record Sheet of Selenium (Se) Single-Factor Experiment (NOB)

[0061] As shown in Tables 3-6, the regulation of trace elements needs to be matched with the proliferation and metabolism of the target bacteria. If the concentration is lower than the target concentration, the trace elements required for NOB proliferation cannot be met, and the promotion effect cannot be achieved, resulting in a low NOB growth rate. If the concentration is higher than the target concentration, it will directly lead to the inhibition of NOB growth or the production of toxicity, resulting in passage failure.

[0062] 5. Experiment on the main regulation of single component deficiency of trace elements.

[0063] Based on the above feed solution formula, add the following respectively: Group 1: Fe, Cu, Ca and Se elements, i.e., adding 40 mg / L FeSO4·7H2O, 2.5 mg / L CuSO4·5H2O, 4 mg / L CaCl2·2H2O and 0.5 mg / L Na2SeO3·5H2O; Group 2: Cu, Ca, and Se elements, with the same concentrations as in Group 1; Group 3: Fe, Ca, and Se elements, with the same concentrations as in Group 1; Group 4: Fe, Cu and Se elements, with the same concentrations as in Group 1; Group 5: Fe, Cu and Ca elements, with the same concentration as Group 1; NOB strains were passaged and the NOB rate was measured. The results are shown in Table 7.

[0064] Table 7 Experimental Record Table of Single Component Deficiency of Major Regulatory Trace Elements

[0065] It is evident that the four elements Fe, Cu, Ca, and Se do not act in isolation, but rather form a multi-layered, multi-target synergistic support network, jointly ensuring the efficiency of nitrification and the stability of the system. Among them, Fe and Cu are the core elements, related to the activity of key enzymes in nitrification (AMO, HAO, NXR), responsible for energy acquisition and conversion. Fe and Cu both participate in the ammonia oxidation process, and neither is dispensable. The nitrite oxidation process mainly relies on Cu catalysis, thus ensuring the integrity and efficiency of nitrification. Although Ca does not directly participate in catalysis, it plays an important role in maintaining the structural integrity of cells and fungi. Se, as a key component of glutathione peroxidase, helps to remove toxic byproducts generated during nitrification.

[0066] Example 4: Fermentation of nitrifying bacteria aggregates.

[0067] Fermentation culture conditions: Temperature: 30℃; pH: 7.0-8.0; relative dissolved oxygen ≥20%.

[0068] Dynamic control of the fermentation process: Based on Example 2, the ammonia oxidation rate of nitrifying bacteria aggregates was adjusted to 50~60 mg NH3-N / L\hr, and inoculated into the basic fermentation medium at an inoculation rate of 10% (v / v) for fermentation.

[0069] Real-time monitoring of pH, DO (dissolved oxygen), OUR (oxygen consumption rate), temperature, and rotation speed; adjust to temperature 30℃, pH 7.0~8.0, and relative dissolved oxygen ≥20%.

[0070] Dynamic control: Phase 1: Fermentation start-up. After inoculation, batch feeding is prioritized. At this time, the oxygen consumption rate is <40mg O2 / (gVSS·h), and the bacterial count is accumulated. The batch feed is 100mL / time. When the levels of ammonia nitrogen and nitrite nitrogen are 0mg / L, the next batch feed is continued. Phase 2, Feeding Start-up Phase: When the OUR reaches the set threshold of 40 mg O2 / (gVSS·h), it indicates that the cells have entered a high-activity state. The automatic feeding system is then started, with the initial flow rate set at 40 mL / h and the feeding interval at 24 h. During this period, both ammonia nitrogen and nitrite nitrogen need to be controlled to be <5 mg / L. Phase 3: Accelerated feeding phase. When the oxygen consumption rate is >40mg O2 / (gVSS·h), automatic accelerated feeding is activated, with an acceleration of 40~80mL / h and a feeding interval of 24h. During the acceleration, it is necessary to use the parameter judgment standard, that is, control both ammonia nitrogen and nitrite nitrogen to be <5mg / L. Phase 4: Stop feeding or terminate fermentation. When the fermentation reaches an oxygen consumption rate of <40 mg O2 / (gVSS·h) or the feeding flow rate reaches 600 mL / h, feeding is stopped and fermentation is terminated.

[0071] Data recording and changes during fermentation, such as Figure 4 As shown. By Figure 4 The changing trends of ammonia nitrogen and nitrite nitrogen during continuous fermentation show that the overall fermentation process is stably controlled, and the concentration range of nitrite nitrogen in the intermediate product is <5 mg / L, which enables stable fermentation of nitrifying bacteria polymers.

[0072] Simultaneously, the fermentation rate of the obtained product was measured. Ammonia oxidation rate was tested according to HG / T 5748-2020 "Performance Evaluation Method for Nitrifying Bacteria Agents for Water Treatment" (for rates exceeding 500, the original solution was diluted 1-fold). The fitted curve is shown below. Figure 5 As shown, based on the above standard, the nitrifying bacteria rate in the lower tank is 678 mgNH3-N / L\hr, which is far higher than the Class I standard specified in the standard.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fermentation method for nitrifying bacterial aggregates, characterized in that, Includes the following steps: S1, culturing nitrifying bacteria aggregates into a fermentation seed liquid; the nitrifying bacteria aggregates are aggregates formed by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB); S2, the seed culture is inoculated into the fermentation medium for fermentation. During fermentation, feed solution is added according to the oxygen consumption rate, and feeding is carried out in stages. Phase 1: When the oxygen consumption rate is <40 mg O2 / (gVSS·h), batch feeding is carried out; Phase Two: When the oxygen consumption rate reaches 40 mg O2 / (gVSS·h), automatic feeding is activated. Phase 3: When the oxygen consumption rate is >40 mg O2 / (gVSS·h), automatic feeding with increased speed will be activated. In stage four, when the fermentation rate reaches <40 mg O2 / (gVSS·h), feeding is stopped and fermentation is terminated.

2. The fermentation method for nitrifying bacterial aggregates as described in claim 1, characterized in that: In step S1, the ammonia oxidation rate of the polymers in the fermentation seed liquid is 50~60 mgNH3-N / L\hr.

3. The fermentation method for nitrifying bacterial aggregates as described in claim 1, characterized in that: In step S2, the feed solution contains Fe, Cu, Ca and Se elements.

4. The fermentation method for nitrifying bacterial aggregates as described in claim 3, characterized in that: The molar concentration of Fe is 144~216 μmol / L, the molar concentration of Cu is 8~20 μmol / L, the molar concentration of Ca is 13.5~20.5 μmol / L, and the molar concentration of Se is 0.75~2 μmol / L.

5. The fermentation method for nitrifying bacterial aggregates as described in claim 3, characterized in that: The feed solution also includes Mn, Co and Zn elements, as well as inorganic salts.

6. The fermentation method for nitrifying bacterial aggregates as described in claim 1, characterized in that: In step S2, the flow rate of the automatic feeding described in stage two is 38~42 mL / h.

7. The fermentation method for nitrifying bacterial aggregates as described in claim 1, characterized in that: In step S2, the speed-up of the automatic feeding described in stage three is 40~80mL / h, and the feeding interval is 23~25h.

8. The fermentation method for nitrifying bacterial aggregates as described in claim 7, characterized in that: The ammonia nitrogen and nitrite nitrogen in the accelerated automatic feeding process are both <5 mg / L.

9. The fermentation method for nitrifying bacterial aggregates as described in claim 1, characterized in that: In step S2, the indicator for stopping feeding as described in stage four also includes feeding flow rate reaching 600 mL / h.

10. The fermentation method for nitrifying bacterial aggregates as described in claim 1, characterized in that: In step S2, the pH during the fermentation process is 7.0 to 8.0.

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