Screening method of low-temperature nitrifying flora

Abstract

The screening method specifically comprises the following steps: carrying out gradient low-temperature domestication culture on activated sludge containing the nitrifying flora by using a liquid screening culture medium to obtain the low-temperature nitrifying flora; the liquid screening culture medium comprises the following components: a core nitrogen source, an inorganic carbon source, a basic inorganic salt, a synergistic component and a trace element mixed solution, the synergistic component is prepared from spermidine, betaine, L-proline, humic acid and a vitamin B group compound. By improving components of the culture medium and combining gradient low-temperature domestication, the nitrifying flora with high low-temperature stability is obtained, the enrichment period of the low-temperature nitrifying bacteria is shortened, and the constructed flora has extremely high adaptability and resilience to temperature fluctuation and can keep stable denitrification efficiency in a complex and changeable actual sewage treatment environment.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, and in particular to a method for screening low-temperature nitrifying bacteria. Background Technology

[0002] With increasingly stringent water environmental protection standards in my country, wastewater discharge standards, especially restrictions on ammonia nitrogen emissions, are becoming increasingly stringent. Biological denitrification processes, due to their economic efficiency and environmental friendliness, have become the mainstream technology for wastewater treatment. Nitrification, as a key step in biological denitrification, relies on ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) to convert ammonia nitrogen into nitrate. However, in winter or in northern my country, wastewater treatment plants generally face the challenge of water temperatures dropping to 8-15℃ or even lower. Under these low-temperature conditions, the metabolic enzyme activity of traditional nitrifying bacteria is significantly inhibited, cell membrane fluidity decreases, and the proliferation rate slows down, leading to a sharp decline in the efficiency of the entire nitrification process. In existing technologies, the ammonia oxidation load of nitrification systems at low temperatures is typically below 30 mg / (L·d), far from meeting the requirements for efficient denitrification. To maintain effluent quality standards, conventional operations often rely on external heating to raise the water temperature or compensating for insufficient activity by extending the hydraulic retention time or increasing the sludge concentration. These methods not only significantly increase the system's energy consumption and operating costs, but also lead to a larger reactor footprint. Furthermore, they still cannot guarantee stable operation under frequent temperature fluctuations, which has become a common technical bottleneck restricting the stable operation of wastewater treatment plants in cold regions.

[0003] To address the aforementioned bottlenecks, existing technologies primarily seek breakthroughs on two levels: first, by screening and acclimatizing low-temperature resistant microbial strains; and second, by optimizing reactor processes and operating conditions. Regarding strain screening, existing methods largely focus on long-term acclimatization at constant, low temperatures. However, conventional low-temperature acclimatization methods have significant drawbacks: firstly, the acclimatization cycle is lengthy, typically requiring over 90 days, resulting in low efficiency; secondly, the acclimatization process is often conducted in batch shake flasks, leading to poor adaptability of the resulting microbial communities to common engineering continuous flow operation modes and biofilm attachment growth patterns, making it difficult to directly translate laboratory performance into engineering efficiency; furthermore, the acclimatization conditions are singular, resulting in weak adaptability and recovery ability of the microbial communities to temperature fluctuations. When temperature fluctuations exceed 5°C, their activity is prone to a significant decrease of over 30%, indicating insufficient system resistance to shock loads. At the process level, although some studies have attempted to combine carrier biofilms and pH / dissolved oxygen control, they often lack an integrated design that synergizes with specific low-temperature bacterial strain screening methods. This leads to a disconnect between the bacterial strain, carrier, process, and control links, making it impossible to systematically improve nitrification load and operational stability at low temperatures. Therefore, developing a technical solution that can rapidly screen out highly active, stable, and engineered low-temperature nitrifying bacterial communities, and form a matching, efficient application process, is of urgent practical significance and important application value for solving the challenges of low-temperature wastewater treatment. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for screening low-temperature nitrifying bacteria. By improving the culture medium composition and combining it with gradient low-temperature acclimatization, a nitrifying bacteria community with strong low-temperature stability is obtained, shortening the enrichment cycle of low-temperature nitrifying bacteria. The constructed community has extremely strong adaptability and resilience to temperature fluctuations and can maintain stable denitrification efficiency in complex and variable actual wastewater treatment environments.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for screening low-temperature nitrifying bacteria is specifically as follows: activated sludge containing nitrifying bacteria is subjected to gradient low-temperature acclimatization culture using a liquid screening medium to obtain low-temperature nitrifying bacteria; the liquid screening medium contains the following components: core nitrogen source, inorganic carbon source, basic inorganic salts, synergistic components and trace element mixture; the synergistic components include spermidine, betaine, L-proline, humic acid and vitamin B complex.

[0007] Preferably, the mass ratio of the aforementioned spermidine, betaine, L-proline, humic acid and vitamin B complex is (1-3):(10-20):(5-10):(8-12):(0.5-1).

[0008] Preferably, the aforementioned liquid screening culture medium comprises the following components per 1000 mL of distilled water:

[0009] Core nitrogen source: ammonium chloride 0.5-1.5 g, ammonium sulfate 0.2-0.6 g; Inorganic carbon source: sodium bicarbonate 1.5-2.1 g, sodium carbonate 0.2-0.3 g; Basic inorganic salts: KH2PO4 0.1-0.2 g, K2HPO4 0.3-0.4 g, MgSO4·7H2O 0.2-0.3 g, CaCl2 0.05-0.1 g, NaCl 1.5-2.5 g; Synergistic components: spermidine 0.01-0.03 g, betaine 0.1-0.2 g, L-proline 0.05-0.1 g, humic acid 0.08-0.12 g, vitamin B complex 0.005-0.01 g; Trace element mixture: 0.5-1.5 mL, containing ferrous sulfate, manganese sulfate, sodium molybdate and zinc sulfate.

[0010] Preferably, the aforementioned gradient low-temperature acclimatization culture specifically includes the following steps:

[0011] (1) First round of acclimatization: The enrichment solution was transferred to fresh liquid culture medium at an inoculation rate of 10% and aerobic culture was carried out at a temperature of 15-16℃ for 6-8 days;

[0012] (2) Second round of acclimatization: The qualified bacterial solution from the first round of acclimatization was transferred at an inoculation rate of 10% and cultured aerobically at a temperature of 10-12℃ for 7-9 days;

[0013] (3) Third round of acclimatization: The temperature is reduced to 5-8℃, the ammonia nitrogen concentration in the culture medium is increased to 1.5-2.0 g / L, and aerobic culture is carried out for 8-10 days. Low temperature acclimatization is completed.

[0014] Preferably, the aforementioned gradient low-temperature acclimatization culture step further includes an initial enrichment culture step:

[0015] The activated sludge was inoculated into a liquid screening medium and cultured aerobically at 20-22℃ for 5-7 days to complete the first round of enrichment and obtain the enriched solution.

[0016] Preferably, the aforementioned gradient low-temperature acclimatization culture step is followed by a purification and enrichment culture step:

[0017] The acclimatized bacterial culture was transferred to a liquid screening medium containing 0.5-1 g / L bentonite and cultured stably at 5-8℃. After 2-3 rounds of transfer, a pure culture of highly efficient low-temperature nitrifying bacteria was obtained.

[0018] Preferably, the aforementioned method operates entirely using a continuous flow reactor and implements threshold-triggered flow ramp control based on ammonia nitrogen removal rate.

[0019] During the gradient low-temperature acclimatization and cultivation process, the ammonia nitrogen removal rate of the effluent is monitored in real time. When the ammonia nitrogen removal rate is ≥90% and remains stable for 24-72 hours, the influent flow rate is increased by 10%-30%. When the ammonia nitrogen removal rate is <70%, the flow rate is directly reduced to the flow rate when the ammonia nitrogen removal rate was stable above 80% last time, and the stabilization time is extended until the ammonia nitrogen removal rate recovers and stabilizes above 85%, and is maintained at this level for more than 24 hours before attempting to increase the flow rate again.

[0020] Preferably, the dissolved oxygen concentration during the aforementioned gradient low-temperature acclimatization culture process is 2-3 mg / L, the stirring speed is 120-150 r / min, and the pH is 7.5-8.2.

[0021] The application of the low-temperature nitrifying bacteria obtained by the above screening method in the biological denitrification system for wastewater treatment at 8-15℃.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) This invention improves the culture medium composition and combines gradient low temperature acclimatization to obtain a nitrifying bacterial community with strong low temperature stability, shortens the enrichment period of low temperature nitrifying bacteria, and enables them to maintain high ammonia oxidation activity in a wide temperature range of 9-33℃, with a small decrease in activity when the temperature fluctuates; and constructs an integrated technology of gradient low temperature acclimatization + continuous flow + carrier biofilm + threshold trigger flow enhancement + pH / DO closed loop control to achieve efficient and stable nitrification at low temperatures of 8-15℃; at the same time, it provides a wide temperature range verification path to prove that the bacterial community has excellent adaptability and recovery ability to temperature fluctuations;

[0024] (2) The present invention uses spermidine as the core synergistic component, which plays a dual regulatory role in the low-temperature nitration system. It can be embedded in the cell membrane phospholipid bilayer and inhibit the low-temperature induced membrane lipid phase transition through electrostatic interaction and molecular wedge effect, thereby maintaining the fluidity and integrity of the cell membrane, thus ensuring the conformational activity of membrane-bound ammonia monooxygenase and the smooth flow of substrate transmembrane transport channels. Moreover, as a specific inducing factor, spermidine can bind to nucleic acids to stabilize the DNA / RNA secondary structure, relieve the inhibition of transcription and translation by low temperature, and significantly upregulate the expression levels of key functional genes of ammonia oxidation and nitrite oxidation, thereby increasing the amount of functional enzymes synthesized from the source.

[0025] (3) The spermidine of the present invention can synergistically enhance the effects of betaine and L-proline. Speridine maintains the permeability of the cell membrane, ensuring that betaine and L-proline compatible solutes can be efficiently transported into the cell. The betaine and L-proline enriched in the cell prevent intracellular enzyme protein denaturation and ice crystal damage by regulating osmotic pressure and forming a hydration layer, which greatly improves the survival rate of the bacterial community under low temperature and temperature fluctuation. Moreover, spermidine can induce the synthesis of enzyme precursor proteins, and B vitamins provide the necessary coenzyme factors. The combination of the two can ensure that the newly synthesized protein is rapidly converted into a metabolically active holoenzyme, which significantly shortens the lag period of low temperature acclimatization. Detailed Implementation

[0026] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to explain the solution.

[0027] The nitrifying bacteria of this invention were directly screened from activated sludge in an aerobic biological treatment tank of a wastewater treatment plant. The specific formulation, culture conditions, and screening steps of the liquid screening medium are as follows:

[0028] (1) Based on 1000 mL of distilled water, the distribution of each group of liquid screening medium is as follows:

[0029] Core nitrogen sources: ammonium chloride 0.5-1.5 g, ammonium sulfate 0.2-0.6 g. The compound ammonium salt is suitable for AOB / NOB synergistic metabolism, avoiding the limitation of a single nitrogen source.

[0030] Inorganic carbon source: 1.5-2.1 g sodium bicarbonate and 0.2-0.3 g sodium carbonate. A buffered carbon source is used to meet the carbon requirements of autotrophic nitrifying bacteria while stabilizing the pH.

[0031] Basic inorganic salts: KH2PO4 0.1-0.2 g, K2HPO4 0.3-0.4 g, MgSO4·7H2O 0.2-0.3 g, CaCl2 0.05-0.1 g, NaCl 1.5-2.5 g, to supplement essential elements such as phosphorus, magnesium, and calcium, and maintain cell membrane integrity at low temperatures;

[0032] Synergistic components: spermidine 0.01-0.03 g, betaine 0.1-0.2 g, L-proline 0.05-0.1 g, humic acid 0.08-0.12 g, B vitamins complex 0.005-0.01 g; spermidine can stabilize membrane structure, reduce damage at low temperatures by 50%, accelerate amoA / nxrA gene expression, and improve ammonia nitrogen removal rate; betaine and L-proline are low-temperature stress protectants, improving the low-temperature survival rate of bacterial communities; B vitamins complex is a nitrification function inducer, which can accelerate the expression of amoA / nxrA functional genes;

[0033] Trace element mixture: 0.5-1.5 mL, containing ferrous sulfate, manganese sulfate, sodium molybdate and zinc sulfate.

[0034] Key parameters: pH adjusted to 7.5-8.2, sterilized at 121℃ for 20 min, and cooled to the target temperature before use.

[0035] (2) The screening and culture conditions are as follows:

[0036] Temperature control: The initial screening stage is 20-22℃. The transition temperature will reduce the impact on the bacterial community's adaptation. The temperature will be gradually reduced to the target low temperature range in the subsequent stage.

[0037] Dissolved oxygen conditions: Aerobic culture, maintain dissolved oxygen concentration of 2-3 mg / L, which can meet the respiratory needs of autotrophic nitrifying bacteria and avoid excessive dissolved oxygen inhibition at low temperature;

[0038] Stirring conditions: 120-150 r / min to ensure sufficient contact between nutrients and microorganisms and avoid local substrate deficiency.

[0039] (3) Specific screening steps

[0040] a. Initial enrichment culture

[0041] Take 5 g / L of activated sludge from the aerobic biological treatment tank of a wastewater treatment plant, inoculate it into the above-mentioned liquid culture medium, and incubate it aerobically in a shaker at 20-22℃ for 5-7 days. Monitor ammonia nitrogen (NH4+) every 24 hours during the incubation process. + -N), nitrite nitrogen (NO2) - -N), nitrate nitrogen (NO3) - The first round of enrichment is completed when the ammonia nitrogen removal rate reaches 60% or more, based on the ammonia nitrogen (N-N) and pH index.

[0042] b. Gradient low-temperature acclimatization

[0043] First round of acclimatization: Transfer the first round of enrichment solution to fresh liquid culture medium at an inoculation rate of 10%, lower the temperature to 15-16℃, continue aerobic culture for 6-8 days, monitor nitrogen indicators, and when the ammonia nitrogen removal rate is stable at more than 70% and there is no obvious accumulation of nitrite nitrogen, the acclimatization is qualified.

[0044] Second round of acclimatization: Transfer qualified bacterial solution at an inoculation rate of 10%, lower the temperature to 10-12℃, and incubate for 7-9 days. During this period, ammonium chloride is added to adjust and maintain a stable ammonia nitrogen concentration to ensure that nitrifying bacteria are in continuous contact with the substrate. When the ammonia nitrogen removal rate is maintained above 75%, proceed to the next round.

[0045] The third round of acclimatization: the temperature is lowered to 5-8℃, the ammonia nitrogen concentration in the culture medium is gradually increased to 1.5-2.0 g / L, and cultured for 8-10 days. When the monitoring indicators meet the requirements of ammonia nitrogen removal rate ≥80% and nitrification rate ≥0.8 mg / (L·h), the low temperature acclimatization is completed.

[0046] c. High-efficiency bacterial culture purification and enrichment

[0047] The successfully acclimatized bacterial culture was transferred at a 1% inoculation rate to a fresh, optimized culture medium containing auxiliary components (modified bentonite 0.8 g / L) that help the target microorganisms quickly and stably attach to the carrier. The culture was then incubated at 5-8°C, with one-third of the medium replaced every 3-4 days to prevent the accumulation of metabolic products. After 2-3 rounds of inoculation, when the ammonia nitrogen removal rate stabilized above 85% and the nitrite nitrogen accumulation was ≤0.1 g / L at 5-8°C, a pure culture of highly efficient low-temperature nitrifying bacteria was obtained. The nitrifying bacteria, acclimatized and enriched using the above method, can be directly applied to the supporting system of this invention. Their excellent low-temperature nitrification performance and stress resistance are key to ensuring the stable and efficient operation of the system.

[0048] (4) System device composition

[0049] Reactor: Acrylic glass reactor, with an effective volume that can be switched between 1-4L for pilot testing and 2-3L for validation; internally equipped with a customized packing support.

[0050] Carrier filler: Plastic carrier suitable for biofilm attachment, with adjustable filling amount (50%-100%).

[0051] Inlet and outlet water and flow control: Peristaltic pump accurately measures inlet and outlet water; continuous flow operation.

[0052] Aeration system: microporous aeration, DO controlled at 2.0-4.0 mg / L, preferably 2.5-3.5 mg / L.

[0053] pH closed-loop control: pH sensor + acid-base dosing unit to maintain pH at 7.5-8.0.

[0054] Temperature control: During the low-temperature phase, ice packs / heat exchange units are used to maintain the temperature at 9-14℃; during the wide-temperature range verification phase, heating rods are used to raise the temperature to 33℃ and then gradually lower it to 11℃.

[0055] Online / offline monitoring: ammonia nitrogen, nitrite nitrogen, temperature, pH; MLSS and ammonia oxidation load should be monitored when necessary.

[0056] (5) Influent substrate and operating conditions

[0057] Simulated wastewater: using ammonium chloride as the nitrogen source (NH4) +-N is 40-100 mg / L), sodium bicarbonate is an inorganic carbon source (0.4-0.7 g / L), and potassium dihydrogen phosphate is a phosphorus source (0.03-0.04 g / L).

[0058] Inoculation source: Nitrifying system microbial community, initial inoculation of approximately 150 mL of mud-water mixture.

[0059] Operating mode: continuous flow, stable at 9-14℃ during the low-temperature stage, pH 7.5-8.0, DO 2-4 mg / L.

[0060] When the device operates in continuous flow mode, a peristaltic pump is used to precisely control the influent and effluent flow rates, ensuring stable hydraulic conditions of the system. The aeration system employs microporous aeration, and the reactor integrates an automated pH control system. A pH sensor monitors the acidity and alkalinity of the reaction system in real time and links with the acid-base dosing device to precisely control the pH value to the appropriate range.

[0061] The core control logic of this invention is as follows:

[0062] (1) Low-temperature intensive acclimatization (4L system)

[0063] Start-up: Temperature 9-14℃, initial influent NH4 + -N is 100 mg / L.

[0064] Threshold-triggered flow increase: When the effluent ammonia nitrogen removal rate is ≥90% and remains stable, the influent flow rate is increased by 10%-30% to gradually enhance the hydraulic and substrate load.

[0065] Low temperature maintenance: When the ambient temperature rises, place ice packs on the reactor and the inlet water tank regularly to ensure that the temperature is below 14°C.

[0066] Results: After about 40 days, the low-temperature ammonia oxidation load increased from the initial 23 mg / L·d to 474 mg / L·d (9-14℃).

[0067] (2) Load enhancement and loading optimization (2L system)

[0068] The initial packing material was 100% filled. After stabilizing at 4L, the packing material was reduced to 50%, and the effective volume was adjusted to 2L. (Influent NH4...) + -N was adjusted to 40 mg / L, influent flow rate was adjusted to 64 L / d, and HRT was adjusted to 0.75 h.

[0069] Results: At 12-15℃, the ammonia oxidation load reached 598 mg / L·d.

[0070] (3) Unit mass activity assessment (1L shaker system)

[0071] Take a carrier with an attached bacterial film, configure a 1L aeration bottle, and adopt two modes: full water replacement and dynamic feeding.

[0072] Calculation formula: Ammonia nitrogen degradation rate per unit mass v = ammonia oxidation rate / sludge concentration, unit mg / (g·h).

[0073] Representative results (low temperatures of 12.8℃ and 10.8℃): v≈7.53-21.2 mg / (g·h) (MLSS of the vector system is approximately 0.25 g / L).

[0074] (4) Wide temperature range adaptability verification (3L continuous flow)

[0075] Temperature program: Start at 14-19℃ → rise to 30-33℃ → then drop to 11-21℃. After each stage stabilizes, increase the inlet flow rate by 20%.

[0076] Results: The ammonia oxidation load increased from 27 mg / L·d to 286 mg / L·d, indicating that the bacterial community has good adaptability and recovery ability over a wide temperature range.

[0077] The MLSS of the new packing material biofilm in the 3L system is about 0.065 g / L, corresponding to a low-temperature shaker v≈7.05-24.8 mg / (g·h) (10.7-14.0℃).

[0078] (5) Core control logic

[0079] Temperature (low temperature conditions): 9-14℃ (target 8-15℃);

[0080] Wide temperature range verification: 33℃ then dropped to 11℃.

[0081] pH: 7.5-8.0; DO: 2.5-3.5 mg / L; HRT: 0.75-4 h (adjust according to scale and load).

[0082] Threshold triggering conditions: After the removal rate is ≥90% for 24-72 hours, increase the flow rate by 20%; if the removal rate is <70% or there is abnormal accumulation of nitrite, reduce the flow rate by one level and extend the stabilization time.

[0083] Carrier filling: 30%-60% of the reactor volume, which can be adjusted according to the age of the bacteria.

[0084] Inlet water formula: NH4 + -N 40-100 mg / L; Sodium bicarbonate 0.4-0.7 g / L; KH2PO4 0.03-0.04 g / L.

[0085] Example 1

[0086] The low-temperature nitrification experimental system employed a 4L plexiglass reactor equipped with a customized packing support to provide a carrier for microbial attachment and growth. The system operated in a continuous flow mode. The microbial strains used in the experiment were derived from a short-cut nitrification system using biogas slurry, with 150 mL of sludge-water mixture as inoculum to achieve rapid enrichment of the dominant nitrifying bacteria and system start-up. During the experiment, when the ammonia nitrogen removal rate was consistently maintained above 90%, a step-by-step control strategy was adopted to increase the influent flow rate, with each increase set at 20%, gradually enhancing the system's hydraulic load. The nitrification process was assessed by continuously monitoring the dynamic changes in ammonia nitrogen and nitrite nitrogen concentrations within the reactor.

[0087] In the initial stage of the experiment, the basic formulation of the simulated wastewater was: ammonia nitrogen concentration 100 mg / L, sodium bicarbonate concentration 0.7 g / L, and potassium dihydrogen phosphate concentration 0.04 mg / L, to establish stable influent substrate conditions. After the system reached a stable state through adaptive operation, the influent formulation was optimized and adjusted by doubling the concentration to explore the operational efficiency and microbial response mechanism of the low-temperature nitrification system. The experimental data are shown in Table 1.

[0088] Table 1 Experimental Data

[0089]

[0090] Analysis of the data in Table 1 shows that, after 40 days of targeted acclimatization, the enrichment of nitrifying bacteria suitable for low-temperature environments of 9-14℃ was successfully achieved. The experimental results indicate a significant increase in the ammonia oxidation load of the low-temperature nitrification system, from the initial 23 mg / L·d to 474 mg / L·d, providing crucial data support for optimizing the efficiency of wastewater treatment processes and controlling operating parameters under low-temperature conditions.

[0091] Following a random sampling principle, three packing cells were selected from the central area of ​​the packing rack in the 4L reactor. The biofilm and sludge-water mixture adhering to the surface of the selected packing cells were completely washed away and collected. The samples were processed using the standard gravimetric method, and the sludge concentration was accurately determined through drying and constant weight procedures to obtain quantitative data on the biofilm microbial biomass in that area. As shown in Table 2 below, the sludge concentration in the 4L system low-temperature nitrification test reactor was 0.25 g / L.

[0092] Table 2 Sludge Concentration

[0093]

[0094] Example 2

[0095] After the experiment in Example 1, the process parameters were optimized and adjusted: the packing material loading on the packing rack in the 4L plexiglass reactor was reduced to 50% of its initial state, and the effective reactor volume was simultaneously adjusted to 2L, while maintaining the original continuous flow feed mode. The influent ammonia nitrogen concentration was set to 40 mg / L, and the influent flow rate was adjusted to 64 L / d (hydraulic retention time HRT = 0.75 h) using a peristaltic pump. The low-temperature nitrification efficiency was studied under these conditions. This adjustment mainly investigated the effects of changes in packing density and reactor volume on the metabolic activity of nitrifying bacteria and the ammonia nitrogen removal efficiency under low-temperature conditions. The experimental data are shown in Table 3.

[0096] Table 3 Experimental Data

[0097]

[0098] Analysis of the data in Table 3 shows that, with an influent flow rate of 64 L / d, the ammonia oxidation load can reach 598 mg / L·d at an environment of 12-15℃.

[0099] Example 3

[0100] The experiment was conducted in a low-temperature shaker. The reactor used a glass bottle with an effective volume of 1L and was equipped with an aeration system to meet the dissolved oxygen requirements of microbial metabolism, providing a suitable aerobic environment for the enrichment and maintenance of nitrifying bacteria. Five packing materials with surface-attached bacterial sludge were selected from the packing rack of the experimental device in Example 1, and five unused blank packing materials of the same specification were added simultaneously and placed into the reactor. Artificially prepared simulated wastewater was used as the influent in this experiment, and a total of four comparative experiments were conducted.

[0101] In the first and second experiments, the influent water composition was: ammonia nitrogen concentration 100 mg / L, sodium bicarbonate concentration 0.7 g / L, and potassium dihydrogen phosphate concentration 0.04 mg / L. During operation, water samples were collected from the reactor to measure the ammonia nitrogen concentration. When the ammonia nitrogen concentration in the reactor remained consistently below 2 mg / L, a full water replacement operation was performed, replacing the wastewater with freshly prepared simulated wastewater to maintain consistent system operating conditions.

[0102] The third and fourth experiments employed a dynamic substrate replenishment mode, eliminating the need for water exchange during operation. When the ammonia nitrogen concentration in the reactor fell below 2 mg / L, ammonia nitrogen (10 mg / L) and sodium bicarbonate (concentration range 0.4-0.7 g / L) were added to maintain a stable substrate concentration gradient. Water samples were taken during the experiments to measure the ammonia nitrogen concentration. When the ammonia nitrogen concentration dropped below 2 mg / L, the complete reaction time and measurement data were recorded. Based on the sludge concentration measurement results, combined with the ammonia nitrogen degradation rate and reaction time, the ammonia nitrogen degradation rate per unit mass of this microbial community was calculated in mg / (g·h). The experimental data are shown in Table 4, and the calculation formula is as follows:

[0103] The ammonia nitrogen degradation rate per unit mass of microbial community = ammonia oxidation rate / sludge concentration;

[0104] The ammonia oxidation rate was mg / L·h; the sludge concentration was 0.25 g / L (see Table 2 for details); and the ammonia nitrogen degradation rate per unit mass of this microbial community was mg / (g·h).

[0105] Table 4 Experimental Data

[0106]

[0107] The purpose of the first experiment was to start up the nitrification system and acclimate the microorganisms to their adaptation under low-temperature conditions. The operation primarily aimed to activate the microbial community and achieve initial system stability. After a significant decline in ammonia nitrogen removal efficiency and the microbial metabolic activity reached a stage of saturation, water exchange was performed to adjust the system substrate conditions. Therefore, the experimental data from this stage are not considered for reference.

[0108] According to the data analysis in Table 4, in the second experiment, when the ambient temperature was maintained at 12.8℃, after 27 hours of operation, the ammonia nitrogen concentration in the system successfully dropped to below 2 mg / L. The low-temperature nitrifying bacteria showed significant ammonia nitrogen degradation activity. The ammonia nitrogen concentration change data during the 6-20 h period of the reaction process were used for calculation, and the ammonia nitrogen degradation rate per unit mass of this microbial community reached 21.2 mg / (g·h).

[0109] In the third and fourth experiments, the system exhibited more efficient ammonia nitrogen removal performance, with the ammonia nitrogen concentration decreasing to below 2 mg / L within 5-6 hours. The low-temperature nitrifying bacteria demonstrated a certain ammonia nitrogen conversion capacity under different temperature conditions. Since the ammonia nitrogen concentration in this stage of the experiment decreased to below 2 mg / L within 5-6 hours, the actual reaction time was selected for calculation based on this rapid degradation characteristic to ensure the accuracy and reliability of the data. Specifically, when the ambient temperature was stable at 12.8℃, the ammonia oxidation rate of the low-temperature nitrifying bacteria was 8.32 mg / (g·h); while when the ambient temperature decreased to 10.8℃, the ammonia nitrogen degradation rate per unit time of this bacterial group was correspondingly adjusted to 7.53 mg / (g·h).

[0110] Example 4

[0111] Eight biofilm-attached packing materials were selected from the packing rack of the experimental apparatus in Example 1, and eight blank packing materials of the same specification were added simultaneously. All eight were placed together in an acrylic reactor with an effective volume of 3L and a total volume of 5L. The reactor was operated in a continuous flow mode.

[0112] This experiment aimed to investigate the nitrogen removal performance of nitrifying bacteria under dynamic temperature changes. A temperature gradient system was constructed to simulate a temperature fluctuation from 14℃ to 33℃, followed by a stepwise decrease to 11℃. The system monitored the effects of different temperature stages on ammonia oxidation load and sludge activity of the nitrifying bacteria, providing theoretical basis and data support for the optimized operation and control of biological nitrification processes under low-temperature conditions.

[0113] In the initial stage of the experiment, the influent composition was: ammonia nitrogen concentration 100 mg / L, sodium bicarbonate concentration 0.7 g / L, and potassium dihydrogen phosphate concentration 0.04 mg / L, thus establishing stable initial influent substrate conditions. After the reactor start-up and operation phase was completed, the concentration changes of ammonia nitrogen and nitrite nitrogen in the reactor were monitored. When the ammonia nitrogen removal rate stabilized above 90%, the influent flow rate was precisely adjusted using a peristaltic pump, with each increase set at 20%. The experimental data are shown in Table 5.

[0114] Table 5 Experimental Data

[0115]

[0116] As can be seen from the data analysis in Table 5, after 20 days of experimentation, the ammonia oxidation load of the low-temperature nitrifying bacteria adapted to 9-14℃ increased from 27 mg / L·d to 286 mg / L·d after the ambient temperature was increased, indicating that the low-temperature nitrifying bacteria still have good biological nitrification performance over a wide temperature gradient range.

[0117] Three new packing materials were selected from the 3L reactor following a random sampling principle. The biofilm and sludge-water mixture adhering to the surface of the selected packing materials were completely eluted and collected. The samples were processed using the standard gravimetric method, and the sludge concentration was accurately determined through drying and constant weight procedures to obtain quantitative data on the biofilm microbial biomass in this area. As shown in Table 6 below, the sludge concentration in the 3L insulated nitrification test reactor was 0.065 g / L.

[0118] Table 6 Sludge Concentration

[0119]

[0120] Example 5

[0121] The experiment was conducted in a low-temperature shaker. The reactor used a glass bottle with an effective volume of 1L and was equipped with an aeration system to meet the dissolved oxygen requirements of microbial metabolism and provide a suitable aerobic environment for the enrichment and maintenance of nitrifying bacteria. Five packing materials that had been newly added to the 3L reaction system and had already completed biofilm attachment were selected from the experimental apparatus of Example 4 and transferred to the reactor.

[0122] This experiment used artificially prepared simulated sewage as the influent source and conducted three sets of control experiments.

[0123] In the first two experiments, the influent water quality was strictly controlled as follows: ammonia nitrogen concentration 70 mg / L, sodium bicarbonate concentration 0.5 g / L, and potassium dihydrogen phosphate concentration 0.03 mg / L. During operation, water samples were collected from the reactor to measure the ammonia nitrogen concentration. When the ammonia nitrogen concentration in the reactor remained consistently below 2 mg / L, a complete water replacement was performed, replacing the wastewater with freshly prepared simulated wastewater to maintain consistent system operating conditions.

[0124] The third experiment employed a dynamic substrate replenishment mode, eliminating the need for water exchange during operation. When the ammonia nitrogen concentration in the reactor fell below 2 mg / L, 10 mg / L ammonia nitrogen and 0.4-0.7 g / L sodium bicarbonate were added to maintain a stable substrate concentration gradient. Water samples were taken during the experiment to measure the ammonia nitrogen concentration. When the ammonia nitrogen concentration dropped below 2 mg / L, the complete reaction time and measurement data were recorded. Based on the sludge concentration measurement results, combined with the ammonia nitrogen degradation rate and reaction time, the ammonia nitrogen degradation rate per unit mass of this microbial community was calculated (mg / g·h). The sludge concentration was 0.065 g / L, and the experimental data are shown in Table 7.

[0125] Table 7 Experimental Data

[0126]

[0127] In the first experiment, the nitrifying bacteria underwent a significant environmental adaptation transition due to the abrupt shift from room temperature to a low-temperature environment. During this period, the metabolic activity of the nitrifying bacteria was inhibited, nitrification was difficult to proceed normally, and the ammonia nitrogen removal function was temporarily disabled. Therefore, the data from the first experiment did not effectively reflect the true treatment efficiency of nitrifying bacteria under low-temperature conditions and was not included in the scope of valid data analysis.

[0128] Table 7 shows that in the second experiment, when the ambient temperature was maintained at a stable 10.7℃, the ammonia nitrogen concentration in the system was effectively reduced to below 2 mg / L after 44 hours of continuous operation. Based on the actual reaction time of 44 hours, the ammonia nitrogen degradation rate per unit mass of microbial community reached 24.8 mg / (g·h). In the third experiment, the system achieved the treatment target of reducing the ammonia nitrogen concentration to below 2 mg / L within 24 hours under a stable ambient temperature of 10.7℃. Based on the actual reaction time of 24 hours, the ammonia nitrogen degradation rate per unit mass of microbial community was 7.05 mg / (g·h).

[0129] Example 5

[0130] This study verifies the core role and synergistic effect of the synergistic components (spermidine, betaine, L-proline, humic acid, and vitamin B complex) in the liquid screening medium of this invention, clarifies the influence of each component on the ammonia oxidation activity of low-temperature nitrifying bacteria, and demonstrates the superiority of the full-component medium.

[0131] The screening method in this embodiment is consistent with that in Example 2, ensuring that the single variable is the composition of the culture medium synergistic components:

[0132] Reactor: Acrylic glass reactor, effective volume 2L, internally filled with 50% carrier packing;

[0133] Operating mode: continuous flow, influent flow rate 64 L / d, hydraulic retention time (HRT) = 0.75 h;

[0134] Environmental parameters: temperature 12±1℃, dissolved oxygen (DO) 2.5-3.5 mg / L, pH 7.5-8.0;

[0135] Influent water quality: NH4 + -N=40 mg / L, sodium bicarbonate 0.6 g / L, potassium dihydrogen phosphate 0.035 g / L, trace element mixture 1.0 mL / L;

[0136] Inoculum: Nitrifying bacteria that had been successfully acclimatized in the 4 L reactor in Example 1, with an inoculum amount of 10% (200 mL of mud-water mixture).

[0137] Experimental period: 15 days, with the first 5 days being the adaptation period and the last 10 days being the stabilization period. The average value of the data during the stabilization period was taken as the final result. Three parallel samples were set up for each group. The experimental data are shown in Table 8.

[0138] Experimental group: The liquid culture medium formula for screening (based on 1000mL of distilled water) is as follows: ammonium chloride 1.0 g, ammonium sulfate 0.4 g, sodium bicarbonate 1.8 g, sodium carbonate 0.25 g, KH2PO4 0.15 g, K2HPO4 0.35 g, MgSO4・7H2O 0.25 g, CaCl2 0.08 g, NaCl 2 g, spermidine 0.02 g, betaine 0.15 g, L-proline 0.08 g, humic acid 0.1 g, vitamin B complex 0.008 g, and 1mL of trace element mixture including: ferrous sulfate 0.003 g, manganese sulfate 0.002 g, sodium molybdate 0.0008 g, and zinc sulfate 0.0004 g.

[0139] Control group 1: The difference between the control group and the experimental group is that spermidine was not added to the culture medium, while the other components and amounts were the same.

[0140] Control group 2: The difference between the control group and the experimental group was that betaine was not added to the culture medium, while the other components and amounts were the same.

[0141] Control group 3: The difference between the control group and the experimental group was that no vitamin B complex was added to the culture medium, while the other components and amounts were the same.

[0142] Control group 4: The difference between the control group and the experimental group is that no synergistic components were added to the culture medium. It only contained the core nitrogen source, inorganic carbon source, basic inorganic salts, and trace elements. The other components and their amounts were the same.

[0143] Table 8 Experimental Data

[0144]

[0145] As shown in Table 8, the experimental group had the best performance: the ammonia oxidation load reached 605 mg / L·d, which was significantly higher than all control groups, and the nitrite nitrogen accumulation was only 0.08 mg / L. This indicates that the whole-component synergistic system can maximize the activation of the metabolic activity of low-temperature nitrifying bacteria, while ensuring the synergistic effect of AOB and NOB and avoiding the accumulation of intermediate products, thus verifying the rationality of the culture medium formula of this invention.

[0146] The activity retention rate of control group 1 was only 51.6%, with an ammonia oxidation load less than 52% of that of the experimental group, and a significant increase in nitrite nitrogen accumulation. This is because spermidine is the core synergistic component; its absence leads to the inability to inhibit cell membrane lipid phase transition, impaired membrane permeability, and decreased activity of membrane-bound ammonia monooxygenase. Upregulation of amoA / nxrA functional gene expression is also inhibited, resulting in insufficient synthesis of functional enzymes. Even with the presence of betaine and humic acid, the core issues of membrane structure stability and gene expression activation cannot be resolved, directly demonstrating the core initiating role of spermidine. The activity of control group 2 was significantly decreased, higher than control group 1 but lower than the experimental group. Although spermidine can maintain cell membrane integrity, the lack of betaine prevents the effective accumulation of intracellular compatible solutes, increases the risk of low-temperature denaturation of enzyme proteins, and reduces the resistance of the bacterial community, confirming the synergistic mechanism of spermidine and betaine. The activity retention rate of control group 3 was 68.6%, lower than that of the experimental group. As essential coenzymes for nitrification enzymes, the deficiency of B vitamins prevents the rapid conversion of spermidine-induced enzyme precursor proteins into active holoenzymes, leading to decreased enzymatic reaction efficiency and prolonged metabolic lag phase in the gut microbiota. This highlights the crucial role of B vitamins in activating functional enzymes. In contrast, control group 4 exhibited only a 36.0% activity retention rate, a mere 218 mg / L·d ammonia oxidation load, and the highest nitrite nitrogen accumulation, further demonstrating that the synergistic component system of this invention can significantly overcome the low-temperature nitrification bottleneck and is key to enhancing low-temperature nitrification activity.

[0147] In summary, the culture medium of this invention forms a highly efficient and synergistic system through the core regulation of spermidine, the synergistic protection of betaine and L-proline, and the enzyme activation of B vitamins. Its performance is significantly better than that of culture media lacking a single component or without synergistic components, providing direct experimental support for the innovation and superiority of the screening method.

[0148] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for screening low-temperature nitrifying bacteria, characterized in that, The specific method is as follows: Activated sludge containing nitrifying bacteria was subjected to gradient low-temperature acclimatization culture using liquid screening medium to obtain low-temperature nitrifying bacteria. The liquid screening medium contains the following components: a core nitrogen source, an inorganic carbon source, basic inorganic salts, synergistic components, and a mixture of trace elements; The synergistic components include spermidine, betaine, L-proline, humic acid, and B vitamins.

2. The method for screening low-temperature nitrifying bacteria according to claim 1, characterized in that, The mass ratio of spermidine, betaine, L-proline, humic acid and vitamin B complex is (1-3): (10-20): (5-10): (8-12): (0.5-1).

3. The method for screening low-temperature nitrifying bacteria according to claim 1, characterized in that, The liquid screening medium contains the following components per 1000 mL of distilled water: Core nitrogen sources: ammonium chloride 0.5-1.5 g, ammonium sulfate 0.2-0.6 g; Inorganic carbon source: 1.5-2.1 g sodium bicarbonate, 0.2-0.3 g sodium carbonate; Basic inorganic salts: KH₂PO₄ 0.1-0.2 g, K₂HPO₄ 0.3-0.4 g, MgSO₄·7H₂O 0.2-0.3 g, CaCl₂ 0.05-0.1 g, NaCl 1.5-2.5 g; Synergistic components: spermidine 0.01-0.03 g, betaine 0.1-0.2 g, L-proline 0.05-0.1 g, humic acid 0.08-0.12 g, vitamin B complex 0.005-0.01 g; Trace element mixture: 0.5-1.5 mL, containing ferrous sulfate, manganese sulfate, sodium molybdate and zinc sulfate.

4. The method for screening low-temperature nitrifying bacteria according to claim 1, characterized in that, The gradient low-temperature acclimatization culture specifically includes the following steps: (1) First round of acclimatization: The enriched solution was transferred to fresh liquid culture medium at an inoculation rate of 10% and aerobic culture was carried out at a temperature of 15-16℃ for 6-8 days; (2) Second round of acclimatization: The qualified bacterial solution from the first round of acclimatization was transferred at an inoculation rate of 10% and cultured aerobically at a temperature of 10-12℃ for 7-9 days; (3) Third round of acclimatization: The temperature is reduced to 5-8℃, the ammonia nitrogen concentration in the culture medium is increased to 1.5-2.0 g / L, and aerobic culture is carried out for 8-10 days. Low temperature acclimatization is completed.

5. The screening method according to claim 4, characterized in that, The gradient low-temperature acclimatization culture step is preceded by an initial enrichment culture step: The activated sludge was inoculated into a liquid screening medium and cultured aerobically at 20-22℃ for 5-7 days to complete the first round of enrichment and obtain the enriched solution.

6. The screening method according to claim 1, characterized in that, The gradient low-temperature acclimatization culture step is followed by a purification and enrichment culture step: The acclimatized bacterial culture was transferred to liquid screening medium and cultured stably at 5-8℃. After 2-3 rounds of transfer, a pure culture of highly efficient low-temperature nitrifying bacteria was obtained.

7. The screening method according to claim 6, characterized in that, The method operates entirely using a continuous flow reactor and implements threshold-triggered flow boosting control based on ammonia nitrogen removal rate. During the gradient low-temperature acclimatization and cultivation process, the ammonia nitrogen removal rate of the effluent was monitored in real time. When the ammonia nitrogen removal rate was ≥90% and remained stable for 24-72 hours, the influent flow rate was increased by 10%-30%. When the ammonia nitrogen removal rate was <70%, the flow rate was reduced and the stabilization time was extended.

8. The screening method according to claim 1, characterized in that, The dissolved oxygen concentration during the gradient low-temperature acclimatization culture process was 2-3 mg / L, the stirring speed was 120-150 r / min, and the pH was 7.5-8.

2.

9. The application of the low-temperature nitrifying bacteria population obtained by the screening method according to any one of claims 1-8 in a biological denitrification system for wastewater treatment at 8-15℃.

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