Anaerobic ammonia oxidation granular sludge cultivation device and method under low temperature conditions

Abstract

This invention discloses a cultivation device and method for anaerobic ammonia oxidation granular sludge under low-temperature conditions. The device includes a cultivation reactor, an inlet tank, an outlet tank, and a water tank. The method includes the following steps: S1, constant temperature cultivation; S2, low-temperature shock; S3, index detection and correction. This invention cultivates anaerobic ammonia oxidation granular sludge suitable for use under low-temperature conditions using a dedicated device. The low-temperature shock induces excessive secretion of extracellular polymeric substances (EPS), which, combined with the decrease in AnAOB metabolic activity caused by low temperature, forms a "low-temperature-structural compensation" effect. Simultaneously, the decrease in fluid shear force and EPS gelation jointly promote microparticle aggregation, ultimately forming a stable compensating granular structure, which is of great significance for the operation of anaerobic ammonia oxidation processes under low-temperature conditions.

Description

Technical Field

[0001] This invention relates to the field of environmental engineering wastewater treatment technology, specifically to a low-temperature anaerobic ammonia oxidation granular sludge cultivation device and cultivation method. Background Technology

[0002] In the field of wastewater treatment, traditional biological nitrogen removal processes are energy-intensive, complex, require large amounts of organic carbon sources, and generate significant amounts of residual sludge and greenhouse gas emissions, making it difficult to meet the demands of low-carbon, high-efficiency, and economical wastewater treatment under the "dual carbon" objective. To overcome the many drawbacks of traditional biological nitrogen removal processes, anammox (anammox) technology emerged. Since the 1990s, Professor Kuenen Gijs's team first discovered and proved the existence of anammox, opening up a completely new avenue for nitrogen removal in wastewater treatment.

[0003] As a novel and efficient biological nitrogen removal technology, anaerobic ammonia oxidation (ANAO) offers numerous significant advantages over traditional biological nitrogen removal processes. It eliminates the need for external organic carbon sources, directly converting ammonia nitrogen and nitrite nitrogen into nitrogen gas, greatly simplifying the nitrogen removal process, reducing operating costs and energy consumption, and also decreasing sludge production and greenhouse gas emissions. However, despite its immense theoretical and practical potential, ANAO still faces significant challenges in practical applications, especially in low-temperature environments (7-15℃), severely hindering its large-scale adoption.

[0004] Currently, over 150 commercial biological nitrogen removal wastewater treatment plants based on anaerobic ammonia oxidation (ANAO) have been established globally, but reports of their operation in low-temperature environments are scarce. Under low-temperature conditions, the nitrogen removal efficiency of ANAO processes often deteriorates significantly, primarily manifested as low sludge granulation, easy expansion, and easy floating, ultimately potentially leading to system failure. The main reasons for this are as follows:

[0005] First, low temperatures inhibit the metabolic activity and growth rate of anaerobic ammonia-oxidizing bacteria. As an autotrophic chemosynthetic bacterium, anaerobic ammonia-oxidizing bacteria grow slowly, with a doubling time of up to 14 days under suitable temperature conditions. At low temperatures, their metabolic activity and growth rate are further reduced, which hinders the sludge granulation process and makes it difficult to form stable granular sludge.

[0006] Secondly, low temperatures affect the secretion of extracellular polymeric substances (EPS). EPS is a key binding material in the sludge granulation process, helping microbial cells aggregate and form granular structures. However, in low-temperature environments, the ability of microorganisms to secrete EPS decreases, thereby weakening the formation and stability of sludge particles. This makes granular sludge prone to disintegration and floating, leading to sludge loss and affecting denitrification efficiency and the stable operation of the system.

[0007] Third, low temperatures may cause frostbite to microbial cells. Under low-temperature conditions, the water inside microbial cells may freeze and expand in volume, thereby causing mechanical damage to the cell membrane and internal structure, affecting the normal physiological functions and metabolic activities of microorganisms, and further aggravating the deterioration of sludge properties.

[0008] Furthermore, existing technologies for anammox sludge granulation and recycling under low-temperature conditions are relatively limited, lacking systematic investigation and exploration. Effective solutions have not yet been developed for how to stably, rapidly, and sustainably form anammox granular sludge in low-temperature environments, or for overcoming the aggregation and floating of granular sludge under low-temperature shocks. This limits the current application of anammox processes primarily to industrial wastewater denitrification in suitable temperature environments (30-37℃), while its application in low-temperature environments is severely restricted. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a device and method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions.

[0010] The technical solution of this invention is:

[0011] A low-temperature anaerobic ammonia oxidation granular sludge cultivation device includes: a cultivation reactor, a plurality of inlet tanks connected to the bottom of the cultivation reactor, an outlet tank connected to one side of the top of the cultivation reactor, and a water tank located outside the cultivation reactor.

[0012] A three-phase separator is provided at the top of the culture reactor, and a stirrer is provided above the outside of the culture reactor. The stirring rod at the bottom of the stirrer extends to the bottom of the culture reactor. An exhaust pipe is provided on the side wall of the culture reactor above the three-phase separator.

[0013] The inlet pipe at the top of the water tank and the outlet pipe at the bottom of the water tank are both connected to the external cooler and heater to form a circulating water circuit.

[0014] Furthermore, there are 2 to 4 inlet tanks.

[0015] Note: By setting up multiple inlet tanks, wastewater, glucose culture substrate, amino acid culture substrate, and sludge can be injected and replenished separately.

[0016] Furthermore, the outlet pipe is connected to the cooler and the heater via a first branch pipe and a second branch pipe, respectively. The outlets of the cooler and the heater are both connected to a water storage tank. The inlet pipe is connected to the water storage tank. Valves are provided on the outlet pipe, the first branch pipe, and the second branch pipe. A thermometer is provided on the outer wall of the water tank.

[0017] Note: The cooler and heater can supply cold and hot water to the water tank respectively, thereby regulating the temperature of the culture reactor.

[0018] Furthermore, the inlet tank is connected to the culture reactor via an inlet pipe, and the outlet tank is connected to the culture reactor via an outlet pipe. A guide pump is installed on the inlet pipe, the inlet pipe, and the outlet pipe.

[0019] Note: Water inlet and outlet are achieved through a diversion pump.

[0020] The present invention also provides a method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions, based on the anaerobic ammonia oxidation granular sludge cultivation device under low-temperature conditions described in any one of the above-mentioned methods, comprising the following steps:

[0021] S1. Constant temperature culture: The anaerobic ammonia oxidation microparticle sludge is enriched in the culture reactor, and glucose culture substrate or amino acid culture substrate is added. The mass ratio of the anaerobic ammonia oxidation microparticle sludge to the glucose culture substrate or amino acid culture substrate is 1 to 1.2:3. The mixture is cultured at a constant temperature of 18 to 23°C for 7 days.

[0022] S2. Low-temperature shock: The anaerobic ammonia oxidation micro-particle sludge after constant temperature cultivation is subjected to low-temperature shock. Specifically, the temperature is lowered to 7-7.5℃ at a rate of 0.5-2℃ / h and maintained for 2-3 days. Then, the temperature is raised to 13-15℃ at a rate of 0.5-2℃ / min and maintained for 2-3 days. The temperature is then lowered again. Each cooling-heating cycle is one cycle, and a total of 4-5 cycles are performed.

[0023] S3. Index Detection and Correction: Detect the extracellular polymer secretion of anammox microgranular sludge after low-temperature shock and compare it with the extracellular polymer secretion of anammox microgranular sludge before isothermal incubation. If the increase in extracellular polymer secretion of anammox microgranular sludge exceeds 33-35%, the incubation is completed. If the increase in extracellular polymer secretion of anammox microgranular sludge is less than 33-35%, repeat the cycle in S2 1-2 times until the increase in extracellular polymer secretion of anammox microgranular sludge exceeds 33-35%, and the incubation is completed.

[0024] Furthermore, the anaerobic ammonia oxidation microparticle sludge in S1 needs to be screened to obtain anaerobic ammonia oxidation microparticle sludge with a particle size of less than 0.25 mm.

[0025] Note: By optimizing the size of anaerobic ammonia oxidation microparticle sludge, it is more conducive to the generation of extracellular polymers and sludge cultivation.

[0026] Furthermore, the glucose concentration in the glucose culture substrate in S1 is 900–1000 mg / L, and the amino acid culture substrate is composed of L-leucine, L-lysine, L-glutamic acid, L-alanine, and L-cysteine, each with a concentration of 180–200 mg / L.

[0027] Note: The addition of glucose or amino acid culture substrates can promote the formation of extracellular polymers.

[0028] Furthermore, in step S3, if the increase in extracellular polymer secretion of anammox microparticle sludge is less than 33-35% for the first time, and the cycle in step S2 is repeated 1-2 times, and if the increase in extracellular polymer secretion of anammox microparticle sludge is still less than 33-35% after another test, then 20-25% of the glucose culture substrate or amino acid culture substrate in step S1 is added to the culture reactor, and the cycle in step S2 is repeated 1-2 times until the increase in extracellular polymer secretion of anammox microparticle sludge exceeds 33-35%, thus completing the culture.

[0029] Note: By testing and correcting, we ensure that the cultured anaerobic ammonia oxidation microparticle sludge can maintain good activity at low temperatures.

[0030] The present invention also provides an application of a method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions, which is applied to denitrification of industrial wastewater under low-temperature conditions, wherein the low-temperature temperature is 0-10℃.

[0031] Preferably, the specific application method is as follows: two anammox granular sludge cultivation devices are used as a treatment group. One anammox granular sludge cultivation device is used for anammox granular sludge cultivation, and the other anammox granular sludge cultivation device is used for wastewater treatment. The treated wastewater is separated by a three-phase separator and then discharged into an outlet tank through an outlet pipe. The generated exhaust gas is separated by a three-phase separator and then discharged through an exhaust pipe. After each cultivation cycle, the process is repeated. The anammox granular sludge cultivation device after anammox granular sludge cultivation is used for wastewater treatment, and the anammox granular sludge cultivation device after wastewater treatment is used for anammox granular sludge cultivation. This process is repeated in a cycle.

[0032] The beneficial effects of this invention are:

[0033] The present invention discloses a low-temperature anaerobic ammonia oxidation granular sludge cultivation device and method. The device is used to cultivate anaerobic ammonia oxidation granular sludge suitable for use under low-temperature conditions. In this method, the excessive secretion of extracellular polymeric substances (EPS) is induced by low-temperature shock, which, together with the decrease in AnAOB metabolic activity caused by low temperature, forms a "low-temperature-structure compensation" effect. At the same time, the decrease in fluid shear force and EPS gelation jointly promote the aggregation of microparticles, ultimately forming a stable compensating granular structure. This is of great significance for the operation of anaerobic ammonia oxidation process under low-temperature conditions. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the overall structure of an anaerobic ammonia oxidation granular sludge cultivation device under low-temperature conditions according to the present invention.

[0035] Figure 2 This is a schematic diagram of the overall structure of the device in Embodiment 14 of the present invention;

[0036] Figure 3 This is a schematic diagram of the growth mode and aggregation characteristics of anammox microparticle sludge in the experimental examples of this invention;

[0037] Figure 4 This is a schematic diagram of the particle size variation structure of anammox micro-particle sludge in the experimental example of this invention;

[0038] Figure 5 This is a diagram of the full life cycle cycle model of anammox microparticle sludge in the experimental example of this invention.

[0039] Among them, 1-culture reactor, 11-three-phase separator, 12-exhaust pipe, 2-inlet tank, 21-inlet pipe, 3-outlet tank, 31-outlet pipe, 4-water tank, 41-inlet pipe, 42-outlet pipe, 421-first branch pipe, 422-second branch pipe, 43-valve, 44-thermometer, 5-stirrer, 51-stirring rod, 6-water storage tank, 61-cooler, 62-heater, 7-diversion pump. Detailed Implementation

[0040] Example 1

[0041] like Figure 1 As shown, an anaerobic ammonia oxidation granular sludge cultivation device under low temperature conditions includes: a cultivation reactor 1, two inlet tanks 2 connected to the bottom of the cultivation reactor 1, an outlet tank 3 connected to one side of the top of the cultivation reactor 1, and a water tank 4 located outside the cultivation reactor 1.

[0042] A three-phase separator 11 is provided at the top inside the culture reactor 1, and a stirrer 5 is provided on the outside of the culture reactor 1. The stirrer 5 is a commercially available stirring motor. The stirring rod 51 at the bottom of the stirrer 5 extends to the bottom inside the culture reactor 1. An exhaust pipe 12 is provided on the side wall of the culture reactor 1 above the three-phase separator 11.

[0043] The water inlet pipe 41 at the top of the water tank 4 and the water outlet pipe 42 at the bottom of the water tank 4 are both connected to the externally installed cooler 61 and heater 62. The cooler 61 and heater 62 are commercially available products, forming a circulating water circuit. The end of the water outlet pipe 42 is connected to the cooler 61 and heater 62 through the first branch pipe 421 and the second branch pipe 422 respectively. The water outlets of the cooler 61 and heater 62 are both connected to a water storage tank 6. The water inlet pipe 41 is connected to the water storage tank 6. The water outlet pipe 42, the first branch pipe 421, and the second branch pipe 422 are all equipped with valves 43. The valves 43 are commercially available solenoid valves. The water tank 4 is equipped with a thermometer 44 on its outer wall. The thermometer 44 is a commercially available water temperature thermometer.

[0044] The liquid inlet tank 2 is connected to the culture reactor 1 through the liquid inlet pipe 21, and the liquid outlet tank 3 is connected to the culture reactor 1 through the liquid outlet pipe 31. The water inlet pipe 41, the liquid inlet pipe 21, and the liquid outlet pipe 31 are all equipped with a flow guide pump 7, which is a commercially available high-power flow guide pump.

[0045] Example 2

[0046] The difference between this embodiment and Embodiment 1 is that:

[0047] There are 3 liquid inlet tanks.

[0048] Example 3

[0049] The difference between this embodiment and Embodiment 1 is that:

[0050] There are 4 liquid inlet tanks.

[0051] Example 4

[0052] This embodiment is a method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions, based on a low-temperature anaerobic ammonia oxidation granular sludge cultivation device according to Embodiment 1, including the following steps:

[0053] S1. Constant temperature incubation: Anaerobic ammonia oxidation microparticle sludge is enriched in incubation reactor 1. The anaerobic ammonia oxidation microparticle sludge needs to be screened to obtain anaerobic ammonia oxidation microparticle sludge with a particle size of less than 0.25 mm. Glucose culture substrate is added, and the glucose concentration in the glucose culture substrate is 950 mg / L. The mass ratio of anaerobic ammonia oxidation microparticle sludge to glucose culture substrate is 1.1:3. The mixture is incubated at a constant temperature of 20℃ for 7 days.

[0054] S2. Low-temperature shock: The anaerobic ammonia oxidation micro-particle sludge after constant temperature cultivation is subjected to low-temperature shock. Specifically, the temperature is lowered to 7.2℃ at a rate of 1℃ / h and maintained for 2.5 days. Then, the temperature is raised to 14℃ at a rate of 1℃ / min and maintained for 2.5 days. The temperature is lowered again. Each cooling-heating cycle is one cycle, and a total of 4 cycles are performed.

[0055] S3. Index Detection and Correction: The extracellular polymer secretion of anammox microgranular sludge after low-temperature shock was detected and compared with that before constant temperature culture. The results showed that the extracellular polymer secretion of anammox microgranular sludge increased by 39%, exceeding 34%, and the culture was completed.

[0056] Example 5

[0057] The difference between this embodiment and embodiment 4 is that:

[0058] S1. Constant temperature culture: Anaerobic ammonia oxidation microparticle sludge is enriched in the culture reactor, and amino acid culture substrate is added. The amino acid culture substrate consists of L-leucine, L-lysine, L-glutamic acid, L-alanine and L-cysteine, each with a concentration of 180 mg / L.

[0059] Example 6

[0060] The difference between this embodiment and embodiment 4 is that:

[0061] S1. Constant Temperature Cultivation: Anaerobic ammonia oxidation microparticle sludge is enriched in the cultivation reactor, and amino acid culture substrate is added. The amino acid culture substrate consists of L-leucine, L-lysine, L-glutamic acid, L-alanine and L-cysteine, each with a concentration of 190 mg / L. The mass ratio of anaerobic ammonia oxidation microparticle sludge to amino acid culture substrate is 1:3. The mixture is incubated at 18°C ​​for 7 days.

[0062] Example 7

[0063] The difference between this embodiment and embodiment 4 is that:

[0064] S1. Constant Temperature Cultivation: Anaerobic ammonia oxidation microparticle sludge is enriched in the cultivation reactor, and amino acid culture substrate is added. The amino acid culture substrate consists of L-leucine, L-lysine, L-glutamic acid, L-alanine and L-cysteine, each with a concentration of 200 mg / L. The mass ratio of anaerobic ammonia oxidation microparticle sludge to amino acid culture substrate is 1.2:3. The mixture is incubated at 23°C for 7 days.

[0065] Example 8

[0066] The difference between this embodiment and embodiment 4 is that:

[0067] S1. Constant temperature culture: The glucose concentration in the glucose culture substrate is 900 mg / L, and the mass ratio of anaerobic ammonia oxidation microparticle sludge to glucose culture substrate is 1:3. The culture is carried out at 18℃ for 7 days.

[0068] Example 9

[0069] The difference between this embodiment and embodiment 4 is that:

[0070] S1. Constant temperature culture: The glucose concentration in the glucose culture substrate is 1000 mg / L, and the mass ratio of anaerobic ammonia oxidation microparticle sludge to glucose culture substrate is 1.2:3. The culture is carried out at 23℃ for 7 days.

[0071] Example 10

[0072] The difference between this embodiment and embodiment 4 is that:

[0073] S2. Low-temperature shock: The anaerobic ammonia oxidation micro-particle sludge after constant temperature cultivation is subjected to low-temperature shock. Specifically, the temperature is lowered to 7℃ at a rate of 0.5℃ / h and maintained for 2 days. Then, the temperature is raised to 13℃ at a rate of 0.5℃ / min and maintained for 2 days. The temperature is lowered again. Each cooling-heating cycle is one cycle, and a total of 5 cycles are performed.

[0074] Example 11

[0075] The difference between this embodiment and embodiment 4 is that:

[0076] S2. Low-temperature shock: The anaerobic ammonia oxidation micro-particle sludge after constant temperature cultivation is subjected to low-temperature shock. Specifically, the temperature is lowered to 7.5℃ at a rate of 2℃ / h and maintained for 3 days. Then, the temperature is raised to 15℃ at a rate of 2℃ / min and maintained for 3 days. The temperature is lowered again. Each cooling-heating cycle is one cycle, and a total of 4 cycles are performed.

[0077] Example 12

[0078] The difference between this embodiment and embodiment 4 is that:

[0079] S3. Index Detection and Correction: The extracellular polymer secretion of anammox microgranular sludge after low-temperature shock was detected and compared with that before constant temperature culture. The results showed that the extracellular polymer secretion of anammox microgranular sludge increased by 37.6%, exceeding 33%, and the culture was completed.

[0080] Example 13

[0081] The difference between this embodiment and embodiment 4 is that:

[0082] S3. Index Detection and Correction: The extracellular polymer secretion of anammox microgranular sludge after low-temperature shock was detected and compared with that before constant temperature culture. The results showed that the extracellular polymer secretion of anammox microgranular sludge increased by 35.5%, which exceeded 35%, and the culture was completed.

[0083] Example 14

[0084] The difference between this embodiment and embodiment 4 is that:

[0085] S3. Index Detection and Correction: The extracellular polymer secretion of anammox microgranular sludge after low-temperature shock was detected and compared with that before isothermal culture. The results showed that the increase in extracellular polymer secretion of anammox microgranular sludge was 31.7%, which was less than 33%. Therefore, the cycle in S2 was repeated once until the increase in extracellular polymer secretion of anammox microgranular sludge exceeded 33%, and the culture was completed.

[0086] In the first test, the extracellular polymer secretion of the anammox microparticle sludge increased by less than 33%. After one cycle in S2, the extracellular polymer secretion of the anammox microparticle sludge increased by 33.2%, exceeding 33%, and the culture was completed.

[0087] Example 15

[0088] The difference between this embodiment and embodiment 4 is that:

[0089] S3. Index Detection and Correction: The extracellular polymer secretion of anammox microgranular sludge after low-temperature shock is detected and compared with that before isothermal culture. The test results show that the increase in extracellular polymer secretion of anammox microgranular sludge is 30.5%, which is less than 34%. Therefore, the cycle in S2 is repeated once until the increase in extracellular polymer secretion of anammox microgranular sludge exceeds 34%, and the culture is completed.

[0090] In the first test, the extracellular polymer secretion of the anammox microgranular sludge increased by less than 33%. After one cycle in S2, the extracellular polymer secretion of the anammox microgranular sludge increased by 32.6%, still less than 34%. Therefore, 20% of the glucose culture substrate in S1 was added to culture reactor 1, and the cycle in S2 was repeated. The result showed that the extracellular polymer secretion of the anammox microgranular sludge increased by 35.5%, exceeding 34%, and the culture was completed.

[0091] Example 16

[0092] The difference between this embodiment and embodiment 4 is that:

[0093] S3. Index Detection and Correction: The extracellular polymer secretion of anammox microgranular sludge after low-temperature shock was detected and compared with that before isothermal culture. The results showed that the increase in extracellular polymer secretion of anammox microgranular sludge was 28.8%, which was less than 35%. Therefore, the cycle in S2 was repeated once until the increase in extracellular polymer secretion of anammox microgranular sludge exceeded 34%, and the culture was completed.

[0094] In the first test, the increase in extracellular polymer secretion of the anammox microgranular sludge was less than 35%. After one cycle in S2, the increase was 30.1%, still less than 35%. Therefore, 22% of the mass of the amino acid culture substrate in S1 was added to culture reactor 1, and the cycle in S2 was repeated. This time, the increase in extracellular polymer secretion reached 33.4%, still less than 35%, and the culture was completed. Then, 25% of the mass of the amino acid culture substrate in S1 was added to culture reactor 1, and the cycle in S2 was repeated. This time, the increase in extracellular polymer secretion reached 37.6%, exceeding 35%, and the culture was completed.

[0095] Example 17

[0096] This embodiment is an application of a low-temperature anaerobic ammonia oxidation granular sludge cultivation method based on Example 4, applied to industrial wastewater denitrification under low-temperature conditions (7°C). The specific application method is as follows:

[0097] Two anaerobic ammonia oxidation granular sludge cultivation devices are used as a treatment group, such as Figure 2 As shown, the two devices share a water storage tank 6. One anaerobic ammonia oxidation granular sludge cultivation device is used for anaerobic ammonia oxidation granular sludge cultivation, while the other anaerobic ammonia oxidation granular sludge cultivation device is used for wastewater treatment. The treated wastewater is separated by a three-phase separator 11 and then discharged into an outlet tank 3 through an outlet pipe 31. The generated exhaust gas is separated by a three-phase separator 11 and then discharged through an exhaust pipe 12. After each cultivation cycle, the process is repeated: the anaerobic ammonia oxidation granular sludge cultivation device is used for wastewater treatment, and the anaerobic ammonia oxidation granular sludge cultivation device after wastewater treatment is used for anaerobic ammonia oxidation granular sludge cultivation. This process is repeated in a cycle.

[0098] Example 18

[0099] The difference between this embodiment and embodiment 17 is that:

[0100] An application of a method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions was described, which was then applied to denitrification of industrial wastewater at a low-temperature environment of 0°C.

[0101] Example 19

[0102] The difference between this embodiment and embodiment 17 is that:

[0103] An application of a method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions was described, which was then applied to denitrification of industrial wastewater at a low-temperature environment of 10℃.

[0104] Experimental Example

[0105] To verify the feasibility of the method of the present invention and to show that the prepared sludge can adapt to low-temperature conditions, we conducted the following experiments as a theoretical basis.

[0106] First, under the same nitrogen volumetric loading conditions, nitrogen-containing industrial wastewater at different temperatures (35℃, 25℃, 15℃, and 7℃) was treated. Anammox microparticles with a particle size less than 0.25 mm were enriched in four 5L anammox culture reactors using a sieve for long-term stability experiments. Using computational fluid dynamics, rapid in-situ absorption spectroscopy, aberration-corrected scanning transmission electron microscopy, stable isotope tracing, and high-throughput bioinformatics sequencing, the changes in water quality and flow regime parameters, microparticle sludge properties and morphology, and microbial community and gene parameters in the reactors were monitored and evaluated. This revealed the anammox microparticle growth pattern and aggregation characteristics, establishing a theory of anammox growth-type granulation under low-temperature conditions.

[0107] Secondly, to simulate the floating phenomenon of granular sludge caused by temperature fluctuations and shocks, two experimental groups were established: a cooling type (35℃→25℃→15℃→7℃) and a heating type (7℃→15℃→25℃→35℃). Anammox microgranular sludge with a particle size greater than 1 mm was enriched in two 5L anammox culture reactors using a sieve for long-term stability experiments. X-ray diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, atomic force microscopy, high-resolution transmission electron microscopy-energy dispersive spectroscopy, and laser scanning confocal microscopy were used to monitor and evaluate the changes in parameters such as the properties and morphology of large granular sludge, the physicochemical and aggregation indicators of EPS, and the activity and community indicators of expanded functional filaments in the reactors. This revealed the biological and microbiological mechanisms underlying the anaerobic granular sludge enlargement and easy disintegration, and established a theory for the regeneration of new anammox particles under temperature shock conditions.

[0108] Based on the above research findings, further research was conducted on the construction and evaluation of a set of water quality parameters, granular property parameters, and process operation parameters for granular sludge recycling. Actual operational data was used to investigate the condition of granular sludge, involving reactor parameters (design parameters), physical indicators (water temperature, etc.), and chemical indicators (NH4+, etc.). 4+ and NO 2– The goal is to establish an information set for enriching granular sludge, based on five dimensions: parameters (such as HRT, etc.), particle characteristics (such as sludge concentration, etc.). Ensemble learning algorithms such as decision trees and random forests are employed. Based on the results of parameter identification and evaluation, a granular sludge recycling model is constructed using numerical fitting. Machine learning is used to simulate the changing patterns and trends of particles, achieving sustainable multiplication of Anammox granular sludge.

[0109] 1. Water quality and denitrification performance parameters

[0110]

[0111]

[0112] Key findings:

[0113] When the temperature dropped from 35℃ to 7℃, the total nitrogen removal rate of the sludge decreased by 32.5%, but the EPS secretion increased by 35.1%, indicating that low temperature induces EPS as a structural compensation mechanism.

[0114] Particle growth rate is linearly positively correlated with temperature (Rg). 2 =0.92), growth almost stopped at 7℃, but still maintained basic denitrification function.

[0115] 2. Computational Fluid Dynamics (CFD) Simulation Results

[0116]

[0117] Flow characteristics:

[0118] Low temperatures lead to increased fluid viscosity, decreased flow velocity and turbulence intensity, and a 18.8% reduction in particle surface shear force (35℃→7℃), thus weakening the particle breakage effect.

[0119] The mass transfer coefficient is positively correlated with temperature; the substrate diffusion rate decreases by 33.7% at 7°C, which may limit the metabolic activity of microorganisms.

[0120] 3. Characterization by aberration-corrected scanning transmission electron microscopy (AC-STEM)

[0121] Microstructure parameters:

[0122] 35℃: The particle core has an ordered crystal structure (lattice spacing 0.28nm), the biofilm layer thickness is 200-300nm, and the EPS is in the form of a fibrous network.

[0123] 7℃: Defects appear in the core crystal (lattice spacing fluctuates by ±0.05nm), the biofilm layer thickens to 400-500nm, EPS forms a dense gel-like matrix, encapsulating more extracellular polymer particles (50-100nm in diameter).

[0124] 4. Stable isotope tracing (15N-labeled ammonia nitrogen)

[0125] Nitrogen conversion pathway parameters:

[0126] At 35℃, the rate at which 15N-labeled ammonia nitrogen is converted to nitrogen gas is 0.15 μmol / (gVSS·h), and the isotopic fractionation factor α = 1.023.

[0127] At 7℃, the conversion rate dropped to 0.06 μmol / (gVSS·h) and α=1.018, indicating that the metabolic preference of anaerobic ammonia oxidizing bacteria (AnAOB) changed at low temperature and the nitrogen isotope fractionation effect weakened.

[0128] Conclusion 1: The dual regulatory mechanism of low temperature on Anammox microparticle formation:

[0129] Lowering the temperature directly inhibits the metabolic activity of AnAOB (nitrogen removal rate decreases by 32.5% at 7℃), but compensates for the stability of particle structure by inducing excessive secretion of EPS (increases by 35.1%), forming a "low temperature-structure compensation" effect.

[0130] The decrease in fluid shear force and EPS gelation jointly promote the aggregation of microparticles. At 7°C, the average particle size can still increase from 0.25 mm to 0.42 mm, confirming that growth-type particle formation can still occur at low temperatures.

[0131] Temperature dependence of Anammox microparticle enlargement mode:

[0132] At 35℃, the main growth pattern is "synchronous growth of core crystallization and biofilm layer", and the particles are regular spherical. Below 15℃, the growth pattern changes to "EPS encapsulation and disordered stacking", and the particle surface becomes rough and the porosity increases by 12.3%.

[0133] Low temperature induced a 105.6% increase in the abundance of Candidatus Anammoxoglobus, suggesting that this genus may promote particle aggregation by secreting highly viscous EPS.

[0134] Establishment of a theoretical model for low-temperature granulation:

[0135] The low-temperature particle formation kinetic equation was obtained by fitting experimental data:

[0136]

[0137] Where D is the particle diameter (mm), T is the temperature (°C), and [EPS] is the extracellular polymeric substance concentration (mg / gVSS). This model reveals that EPS contributes 62.7% to the compensation of particle growth at low temperatures (7°C).

[0138] In addition, the following parameters were obtained through real-time monitoring with in-situ probes, coupling spectral data with chemometric models, and inverting the dynamics of substance concentration:

[0139] UV-Vis absorption peaks (e.g., organic absorption at 254 nm, pigment absorption at 420 nm), absorbance change rate; substrate concentration change (e.g., ammonia nitrogen, nitrate), electron transfer rate.

[0140] Particles were ultrathin sections (50-100 nm) loaded onto a copper mesh. Accelerating voltage (e.g., 200 kV) and spherical aberration corrector settings were applied (to eliminate chromatic aberration and achieve a resolution below 0.1 nm). High-angle annular dark-field (HAADF) imaging, combined with energy-dispersive X-ray spectroscopy (EDX) for elemental analysis, yielded the following structural parameters:

[0141] Granular crystal structure (such as lattice spacing and defect distribution) and elemental distribution (such as surface scan spectra of elements such as C, N, O, and Fe).

[0142] Interfacial features between the biomembrane layer and the core, and the nanoscale structure of extracellular polymeric substances (EPS).

[0143] Isotope-labeled substrates are added to the system, and isotope-labeled substances are obtained through periodic sampling: such as 15N-labeled ammonia nitrogen and 13C-labeled organic compounds. Isotope enrichment (e.g., abundance of 15N in nitrogen products) and turnover rate (e.g., substrate consumption rate) are used; metabolic pathway parameters are also included: isotope enrichment (e.g., abundance of 15N in nitrogen products) and turnover rate (e.g., substrate consumption rate). The distribution of labeled isotopes in the samples is detected using mass spectrometry (e.g., MAT-253), and the substance transformation pathways are analyzed using metabolic models.

[0144] like Figure 3 As shown, this study reveals the growth pattern and aggregation characteristics of anammox microparticles, the mechanism of action and functional aggregation microbiology of the biofilm EPS in the reactor operation system, elucidates the biofilm layered growth mechanism in microparticles over time, the granulation law and dynamic transformation of granular sludge in multiple particle sizes (<0.25mm, 0.25-0.5mm, 0.5-1mm and >1mm), and explores the multi-biofilm layer-multi-particle-transformation behavior of microparticles under long-term operation conditions, revealing the particle size-specific mechanism of granular sludge.

[0145] like Figure 4 As shown, we can understand the morphological changes during the floating and hollowing process of granular sludge, the particle size growth pattern of granular sludge, and the spatial distribution and aggregation mechanism of bulking filamentous bacteria in the granules.

[0146] like Figure 5 As shown, ensemble learning algorithms such as decision trees and random forests were employed. Based on the results of parameter identification and evaluation, a granular sludge recycling model was constructed using numerical fitting, and machine learning was used to simulate the changing patterns and trends of granules. Through small-scale granular sludge parameter surveys, commercial granular sludge parameter surveys, and Anammox granular sludge bibliometrics, the elements in the granular sludge recycling mechanism were further identified and verified, establishing a full life-cycle granular sludge recycling model.

[0147] Ultimately, the sludge prepared by the method of this invention can operate stably for a long time (>240 days) at 7°C, with a total nitrogen removal rate maintained above 60%, which is 8°C better than the cold resistance of the traditional Anammox process (minimum tolerance 15°C).

[0148] When the temperature is below 7°C (e.g., 5°C), the removal rate drops sharply to below 40%, and the particles begin to disintegrate, indicating that 7°C is the critical cold resistance temperature in practical applications. Therefore, in the preparation process, we prefer 7°C in Example 10 as the temperature for low-temperature impact.

[0149] Low temperature recovery ability

[0150] Sludge that has been cultured at 7°C for a long time can recover to 75% of total nitrogen removal rate within 5 days after being reheated to 35°C, but it takes 10 days to fully recover to the initial level, indicating that low temperature does not cause irreversible damage to microorganisms.

[0151] Engineering application value

[0152] In cold regions (such as northern regions where winter water temperatures are ≤10℃), the sludge can be optimized for application in the following ways:

[0153] Control the reactor temperature to ≥7℃, and add EPS precursors (such as glucose) to enhance particle stability;

[0154] Using an intermittent warming strategy (such as raising the temperature to 15°C and maintaining it for 2 hours each day) can increase the expression level of cold-resistance genes by 1.8 times.

Claims

1. A method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions, characterized in that, The cultivation method is based on a cultivation device, which includes: a cultivation reactor (1), a plurality of inlet tanks (2) connected to the bottom of the cultivation reactor (1), an outlet tank (3) connected to one side of the top of the cultivation reactor (1), and a water tank (4) located outside the cultivation reactor (1). The top of the culture reactor (1) is provided with a three-phase separator (11), and the top of the outside of the culture reactor (1) is provided with a stirrer (5). The stirring rod (51) at the bottom of the stirrer (5) extends to the bottom of the culture reactor (1). The side wall of the culture reactor (1) above the three-phase separator (11) is provided with an exhaust pipe (12). The inlet pipe (41) at the top of the water tank (4) and the outlet pipe (42) at the bottom of the water tank (4) are connected to the external cooler (61) and heater (62) to form a circulating water circuit. The cultivation method includes the following steps: S1. Constant temperature culture: The anaerobic ammonia oxidation micro-particle sludge is enriched in the culture reactor (1), and glucose culture substrate or amino acid culture substrate is added. The mass ratio of the anaerobic ammonia oxidation micro-particle sludge to the glucose culture substrate or amino acid culture substrate is 1~1.2:

3. The mixture is cultured at a constant temperature of 18~23℃ for 7 days. S2. Low-temperature shock: The anaerobic ammonia oxidation micro-particle sludge after constant temperature cultivation is subjected to low-temperature shock. Specifically, the temperature is lowered to 7-7.5℃ at a rate of 0.5-2℃ / h and maintained for 2-3 days. Then, the temperature is raised to 13-15℃ at a rate of 0.5-2℃ / min and maintained for 2-3 days. The temperature is then lowered again. Each cooling-heating cycle is one cycle, and a total of 4-5 cycles are performed. S3. Index Detection and Correction: Detect the extracellular polymer secretion of anammox microgranular sludge after low-temperature shock and compare it with the extracellular polymer secretion of anammox microgranular sludge before isothermal incubation. If the increase in extracellular polymer secretion of anammox microgranular sludge exceeds 33-35%, the incubation is completed. If the increase in extracellular polymer secretion of anammox microgranular sludge is less than 33-35%, repeat the cycle in S2 1-2 times until the increase in extracellular polymer secretion of anammox microgranular sludge exceeds 33-35%, then the incubation is completed.

2. The method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions according to claim 1, characterized in that, There are 2 to 4 liquid inlet tanks (2).

3. The method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions according to claim 1, characterized in that, The outlet pipe (42) is connected to the cooler (61) and the heater (62) respectively through the first branch pipe (421) and the second branch pipe (422). The outlet ends of the cooler (61) and the heater (62) are both connected to a water storage tank (6). The inlet pipe (41) is connected to the water storage tank (6). Valves (43) are provided on the outlet pipe (42), the first branch pipe (421) and the second branch pipe (422). A thermometer (44) is provided on the outer wall of the water tank (4).

4. The method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions according to claim 1, characterized in that, The inlet tank (2) is connected to the culture reactor (1) through the inlet pipe (21), and the outlet tank (3) is connected to the culture reactor (1) through the outlet pipe (31). A guide pump (7) is provided on the water inlet pipe (41), the inlet pipe (21), and the outlet pipe (31).

5. The method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions according to claim 1, characterized in that, The anaerobic ammonia oxidation microparticle sludge in S1 needs to be screened to obtain anaerobic ammonia oxidation microparticle sludge with a particle size of less than 0.25 mm.

6. The method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions according to claim 1, characterized in that, The glucose concentration in the glucose culture substrate in S1 is 900~1000 mg / L, and the amino acid culture substrate is composed of L-leucine, L-lysine, L-glutamic acid, L-alanine and L-cysteine, each with a concentration of 180~200 mg / L.

7. The method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions according to claim 1, characterized in that, In S3, if the increase in extracellular polymer secretion of anammox microparticle sludge is less than 33-35% for the first time, and the cycle in S2 is repeated 1-2 times, and if the increase in extracellular polymer secretion of anammox microparticle sludge is still less than 33-35% after another test, then 20-25% of the mass of glucose culture substrate or amino acid culture substrate in S1 is added to the culture reactor (1), and the cycle in S2 is repeated 1-2 times until the increase in extracellular polymer secretion of anammox microparticle sludge exceeds 33-35%, and the culture is completed.

8. The application of the method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions according to claim 1, characterized in that, It is applied to denitrification of industrial wastewater in low-temperature environments, where the low temperature is 0~10℃.

9. The application of the method for cultivating anaerobic ammonia oxidation granular sludge under low-temperature conditions according to claim 8, characterized in that, The specific application method is as follows: two anaerobic ammonia oxidation granular sludge cultivation devices are used as a treatment group. One anaerobic ammonia oxidation granular sludge cultivation device is used for anaerobic ammonia oxidation granular sludge cultivation, and the other anaerobic ammonia oxidation granular sludge cultivation device is used for wastewater treatment. The treated wastewater is separated by a three-phase separator (11) and then discharged into the outlet tank (3) through the outlet pipe (31). The generated waste gas is separated by a three-phase separator (11) and then discharged through the exhaust pipe (12). After each cultivation cycle, the process is repeated. The anaerobic ammonia oxidation granular sludge cultivation device after anaerobic ammonia oxidation granular sludge cultivation is used for wastewater treatment, and the anaerobic ammonia oxidation granular sludge cultivation device after wastewater treatment is used for anaerobic ammonia oxidation granular sludge cultivation. This process is repeated in a cycle.

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