Method for improving nitrification efficiency under alkaline conditions by regulating extracellular polymeric substances and ion transport

Abstract

The application discloses a method for improving the nitrification efficiency under alkaline conditions by regulating extracellular polymeric substances and ion transport. The method induces the secretion of more acidic polysaccharides and acidic organic substances in the extracellular polymeric substances of nitrifying bacteria and regulates the activities of anti-transport proteins and F0-F1 ATPase by gradually increasing the pH value of the system, the operation days and other comprehensive strategies, thereby promoting the intracellular H + input to maintain the intracellular pH stability. At the same time, the biological mineralization effect of carbonic anhydrase is utilized to accelerate the consumption of extracellular OH ‑ , improve the alkaline environmental adaptability and nitrification performance of the nitrification sludge, and significantly enrich Nitrosomonas and Nitrospira and other alkali-tolerant nitrifying bacteria, and the average ammonia nitrogen removal rate of the system under the condition of pH 10.0 can be as high as 96.6%.

Description

Technical Field

[0001] The present invention belongs to the field of sewage biological treatment technology, and relates to a method for improving nitrification efficiency under alkaline conditions by regulating extracellular polymers and ion transport. Background Art

[0002] The effect of nitrification in biological denitrification systems is affected by various operating parameters such as temperature, hydraulic retention time (HRT), solid retention time (SRT) and pH. It is worth noting that due to wastewater quality shock or system characteristics (such as the pH value of the bacterial-algal symbiotic system fluctuates between 6.0 and 10.0 during photosynthesis), the alkaline environment becomes one of the main challenges faced by nitrifying bacteria. Normally, when the pH is greater than 9.6, the biological activity of nitrifying bacteria will be inhibited or even stopped. The high pH value has a significant adverse effect on nitrifying bacteria, which poses a challenge to the actual stable operation of the activated sludge system. Therefore, it is necessary to develop a method to improve the adaptability and nitrification efficiency of nitrifying bacteria in an alkaline environment.

[0003] pH value is an important environmental factor affecting the performance of nitrifying sludge and the structure of microbial communities. Nitrifying bacteria are very sensitive to its changes. Too high pH will not only inhibit ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB), but also destroy NH4 + -N and NH3-N chemical balance, resulting in NH3 gas escape, free ammonia (FA) concentration increased. FA, as an inhibitor of nitrifying bacteria, can not only passively diffuse into the cytoplasm through the cell membrane, combined with the protons in the cell to be converted into ammonium, resulting in cell alkalization, but also inhibit the synthesis of intracellular ATP, affecting cell energy supply. Therefore, most nitrifying bacteria (such as AOB Nitrosomonas) are difficult to survive in a highly alkaline environment, and NOB Nitrospira has a slightly stronger adaptability, but in the current study, only two strains of this genus can grow in an environment with a pH value higher than 8.0 (Daebeler A, Kitzinger K, Koch H, et al. Exploring the upper pH limits of nitrite oxidation: diversity, ecophysiology, and adaptive traits of haloalkalitolerant Nitrospira [J]. ISME J, 2020, 14 (12): 2967-2979.).

[0004] At present, some studies have explored the acclimation method of alkaline-resistant activated sludge. For example, Chinese patent CN201710835023.8 dehydrates and reactivates the activated sludge, and then adds a certain amount of ammonia solution every day to achieve alkali-resistant acclimation. In addition, Chinese patent CN201210340270.8 adds organic alkaline wastewater to the system to gradually increase the pH value to acclimate the alkaline-resistant activated sludge. However, these acclimation methods cannot ensure that the activated sludge is always kept in a highly alkaline environment, and none of them systematically evaluates the denitrification performance of the activated sludge after acclimation, especially the research on nitrification activity is relatively lacking. Therefore, exploring how to improve the nitrification efficiency under alkaline conditions through adaptive regulation of nitrifying bacteria has important research and application value for ensuring the stable operation of the activated sludge system in a high pH environment. Summary of the Invention

[0005] To address the problems of suppressed microbial activity, increased toxicity, sludge bulking, and poor sedimentation in activated sludge systems under high pH conditions, the present invention provides a method for improving nitrification efficiency under alkaline conditions by regulating extracellular polymeric substances (EPPs) and ion transport. This method significantly improves the adaptability of nitrifying bacteria through a series of pathways, including the secretion of acidic organic matter in EPPs and the maintenance of intracellular pH stability by cation / proton antiporters, through a comprehensive strategy that gradually increases the system pH and controls the number of days of operation. This method, which results in the enrichment of highly alkaline-resistant nitrifying sludge, achieves high ammonia nitrogen removal rates and nitrification activity at a constant pH of 10.0.

[0006] The technical solutions of the present invention are as follows:

[0007] The method for improving nitrification efficiency under alkaline conditions by regulating extracellular polymers and ion transport is as follows:

[0008] A nitrifying membrane bioreactor was constructed, which adopted a continuous flow operation mode, with continuous aeration at the bottom of the reactor and the effluent was filtered using a submerged microfiltration membrane module to separate biomass and wastewater;

[0009] During the operation of the nitrifying membrane bioreactor, the environmental pH value in the reactor is gradually increased through three periods, including the stabilization period, the alkaline stimulation period, and the alkaline adaptation period. The number of operating days is controlled by monitoring the level of reactive oxygen species in the system. Using these comprehensive control strategies, the OH production is accelerated by promoting the secretion of acidic substances in the extracellular polymers of nitrifying bacteria, regulating antiporters and F0-F1 ATPase, and utilizing the biomineralization process involving carbonic anhydrase. - Consumption and H + The input of nitrifying sludge can further improve the alkaline adaptability and nitrification performance of nitrifying sludge;

[0010] After entering the alkaline adaptation period, the nitrifying membrane bioreactor continues to operate in a constant high pH environment. When the activity of nitrifying bacteria recovers to the same level as that in the stable period and the content of extracellular polymers and the activity of related functional proteins change significantly, an alkali-resistant and efficient nitrifying microbial community is obtained.

[0011] Preferably, the stable period is a neutral environment; the alkaline stimulation period is a slightly alkaline environment; and the alkaline adaptation period is a highly alkaline environment.

[0012] Preferably, the method for controlling the number of operations is to gradually increase the pH value of the environment and enter the next stage when the active oxygen level in the system drops to less than 50% of the mid-stage.

[0013] Preferably, an automated pH control system is used to control the pH value in the reactor in real time. When the pH value is lower than the set value, the dosing pump is turned on to deliver NaOH solution to the reactor to increase the pH value; when the pH value is higher than the set value, the dosing pump is turned off. Due to the ammonia oxidation effect of microorganisms, the pH value will further decrease. The automated pH control system consists of a pH probe, a pH controller, a dosing pump, and a NaOH solution storage tank.

[0014] Preferably, when the active oxygen level drops to less than 50% of that in the middle period during each pH gradient, sludge microbial samples are collected to measure the content of extracellular polymers and the activity of related functional proteins.

[0015] Preferably, the initial sludge concentration in the nitrifying membrane bioreactor is 2000-3000 mg / L, the dissolved oxygen concentration in the system is maintained at 3.0-4.0 mg / L, and the hydraulic retention time is 6-9 h.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) The present invention utilizes comprehensive control strategies such as pH gradient enhancement and operation days control. At pH 10.0, the alkali-resistant nitrifying bacteria are controlled to secrete acidic polysaccharides in the extracellular polymer (EPS), while glutamate dehydrogenase (GDH) is used to increase acidic organic matter to neutralize the external alkaline environment. In addition, the antiporter (such as NhaA, TrkA and KefB) and F0-F1 ATPase are regulated to promote H + The input of OH is used to maintain the intracellular pH stability, and a series of biomineralization processes involving carbonic anhydrase (CA) are used to accelerate the OH - The consumption of nitrifying bacteria increased the abundance of Nitrosomonas and Nitrospira to 1.3% and 5.4%, respectively, significantly improving the adaptability of nitrifying bacteria in high alkaline environments.

[0018] (2) The present invention successfully obtained alkali-resistant nitrifying sludge that maintained efficient nitrification performance under highly alkaline conditions using a nitrifying bioreactor. The average ammonia removal rate at pH 10.0 was as high as 96.6±2.2%. At the same time, the alkali-resistant nitrifying bacteria were applicable to a wide pH range and showed high activity at pH 7.5-10.0, with NOB activity reaching 14.6 mg N·g -1 MLVSS·h -1 , which is equivalent to the activity level in a neutral environment, showing high stability and strong adaptability, and has potential application prospects in the field of sewage biological treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the nitrifying membrane bioreactor.

[0020] Figure 2 (a) Protein, (b) Polysaccharide and (c) Total EPS content changes in EPS, (df) Three-dimensional fluorescence parallel factor analysis of the three components in EPS and their (gi)F max Changes in value.

[0021] Figure 3 The changes in the content of relevant functional proteins were obtained by PICRUSt2 based on the KEGG database.

[0022] Figure 4 This shows the daily changes in inlet and outlet water quality during the operation of the nitrifying membrane bioreactor.

[0023] Figure 5 (a) AOB and NOB activities of nitrifying sludge at the end of each stage, (b) AOB and NOB activities of alkaline-tolerant nitrifying sludge at pH values ​​of 7.5, 8.0, 9.0, and 10.0 after acclimation, and (c) changes in reactive oxygen species (ROS) levels in nitrifying sludge at the middle and end of each stage.

[0024] Figure 6 The abundance changes of some functional bacterial genera in the reactor at different stages.

[0025] Figure 7 (a) Changes in activated sludge concentration, (b) changes in SVI during sludge acclimation, (c) comparison of XRD spectra of seed sludge and alkali-resistant nitrification sludge, (d) comparison of photos after calcination, (e) comparison of infrared spectra, (f) scanning electron microscope images of seed sludge and (g) alkali-resistant nitrification sludge. DETAILED DESCRIPTION

[0026] The present invention will be described in further detail below with reference to specific embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0027] The reagents used in the following examples can all be obtained from commercial channels.

[0028] Example 1

[0029] Construction of nitrifying membrane bioreactor:

[0030] A continuous flow nitrification membrane bioreactor with an effective volume of about 2 L was established. Figure 1 As shown, the reactor is constructed of plexiglass. The effluent is filtered using a submerged microfiltration membrane assembly (PVDF membrane, 1.6 mm inner diameter, 2.8 mm outer diameter, 0.1 μm pore size) to separate biomass from wastewater, ensuring that all biomass remains within the bioreactor. The initial sludge concentration is 2560 mg / L. Microporous aeration is provided at the bottom of the reactor, with continuous aeration provided by an air pump. pH and dissolved oxygen (DO) probes are installed within the reactor to monitor pH and DO in real time, maintaining DO concentrations between 3.0 and 4.0 mg / L. pH is controlled by an automated pH control system. The reactor inlet is synthetic domestic sewage without a carbon source, and both inlet and outlet flow rates are controlled by peristaltic pumps. The HRT is 8.94 h, which was shortened to 6.19 h after day 75 to increase the nitrification rate.

[0031] The nitrifying membrane bioreactor operates through three phases, gradually increasing the pH within the reactor: first, a stabilization phase (Phase I, 1-28 days), with an ambient pH of 7.5. This is followed by an alkaline stimulation phase, during which the pH is gradually increased to 8.0±0.1 (Phase II, 29-58 days), 8.5±0.1 (Phase III, 59-90 days), 9.0±0.1 (Phase IV, 91-120 days), and finally to 9.5±0.1 (Phase V, 121-150 days). Finally, an alkaline acclimation phase occurs, with the pH rising to 10.0±0.1 (Phase VI, 151-320 days). Furthermore, a control strategy for the number of days of operation is used to further acclimate the nitrifying sludge. Whenever the reactive oxygen species level in the system drops below 50% of the mid-phase level, the pH is gradually increased, marking the start of the next phase.

[0032] The composition of synthetic domestic sewage was as follows: NH4Cl (229.28 mg / L), K2HPO4 (21.95 mg / L), MgSO4·7H2O (91.2 mg / L), CaCl2 (11.1 mg / L), and a trace element solution (1 mL / L). The trace element solution consisted of 1.5 g / L FeCl3·6H2O, 0.18 g / L KI, 0.15 g / L H3BO3, 0.12 g / L MnCl2·4H2O, 0.12 g / L ZnSO4·7H2O, 0.06 g / L NaMoO4·2H2O, 0.03 g / L CuSO4·5H2O, and 10 g / L EDTA-2Na.

[0033] Example 2

[0034] Methods for improving the alkali resistance of nitrifying bacteria and the effect of improving nitrification efficiency:

[0035] By regulating the various physiological characteristics of nitrifying bacteria, the alkali resistance of nitrifying bacteria can be improved. Figure 2 As shown in the figure, in a high alkaline environment, by promoting bacteria to secrete a large amount of acidic organic matter (such as acidic polysaccharides, humic acid, etc.) in EPS, these substances can act as buffers to neutralize the external alkalinity, thereby inhibiting the excessive OH - Damage to cells. Figure 3 As shown, it catalyzes Na + / K + The output and H + The input of , can maintain the intracellular pH at around 7-8.5. This pathway is the core of bacteria to maintain intracellular pH homeostasis. Generally, a high pH environment can provide a higher transmembrane potential under high aeration conditions. Therefore, a continuous high dissolved oxygen environment in the reactor can increase the transcription of F0-F1 ATPase, which is a key factor for H + Provides sufficient energy for the pumping into the cells. At the same time, by increasing the GDH content, the production of organic acids (i.e. glutamate) is increased to ensure the normal energy metabolism of the cells. In addition, by increasing the content of carbonic anhydrase, CO2 is accelerated to HCO3 - The conversion of HCO3 - Converted to CO3 2- To further consume OH - , and then with Ca in the water 2+ The reaction forms calcite crystals CaCO3, and the formation of this biomineralization accelerates the formation of OH - consumption, preventing cell damage.

[0036] Figure 4 The NH4 in the influent and effluent during the operation of the nitrification membrane bioreactor is shown.+ -N, NO2 - -N and NO3 - -N concentration. Obviously, when the pH is below 9.0, the ammonia nitrogen removal rate is always 100%, so the HRT is shortened to 6.19h after 75 days to improve the nitrification efficiency. Although the stability of the effluent decreases slightly when the pH increases to 9.5 and 10.0, the average ammonia nitrogen removal rate remains at 96.6±2.2%, which significantly improves the nitrification efficiency in high pH environment. Figure 5 As shown in (a), the activity of AOB decreased significantly by 78.5% at high pH, ​​while NOB was inhibited at pH 8.5 and 9.0, but its activity gradually recovered to 14.6 mg N·g with the improvement of adaptability. -1 MLVSS·h -1 , has been significantly improved, achieving efficient and stable nitrification. At the same time, the applicable pH range of the alkaline nitrification sludge is wide, and it maintains high activity in the pH range of 7.5 to 10.0 ( Figure 5 (b)) and reached the highest activity at pH 10.0, indicating the effective enrichment of alkali-tolerant nitrifying bacteria. In addition, the reduction of ROS levels at the end of each stage ( Figure 5 (c)) The nitrifying sludge gradually adapted to the stress stimulation of the external alkaline environment and improved its adaptability. In addition, the bacteria significantly improved their alkaline adaptability through the above-mentioned multiple pathways, so nitrifying bacteria such as AOB Nitrosomonas (1.3%), norank_f__JG30-KF-CM45 (2.0%) and NOB Nitrospira (5.4%) were significantly enriched ( Figure 6 ), which means the successful domestication of alkali-resistant and efficient nitrifying sludge.

[0037] according to Figure 7 (f) and (g) show that there are large pieces of flaky sediments and some granular agglomerates on the surface of the alkali-resistant nitrification sludge, mainly CaCO3 ( Figure 7 (c)-(e)), but its structure is relatively loose, while the seed sludge has stronger agglomeration. Despite this, the alkali-resistant nitrification sludge still has good sedimentation performance, with an SVI value of only 121.4±3.6mL / g, and no sludge bulking phenomenon occurs ( Figure 7 (a)-(b)), which is attributed to the increase of carbonic anhydrase resulting in a large amount of CaCO3 attached to the sludge surface, which improves the sedimentation of sludge flocs.

[0038] In summary, the method of the present invention accelerates OH by promoting the secretion of acidic polysaccharides, regulating cation / proton antiporters and F0-F1 ATPase, and increasing the activity of carbonic anhydrase without causing sludge bulking and maintaining good sedimentation performance. -The results show that the adaptability of nitrifying bacteria to highly alkaline environments has been significantly improved through various channels such as the consumption of water and wastewater. At the same time, the nitrification efficiency has been dynamically improved, and the alkali-resistant nitrifying bacteria have been successfully enriched. This has shown significant environmental benefits and has good practical value, which is of great significance for promoting the development of sewage treatment technology.

[0039] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not specifically limited by the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0040] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0041] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for improving nitrification efficiency under alkaline conditions by regulating extracellular polymers and ion transport, characterized in that: The details are as follows: Construct a nitrification membrane bioreactor, adopt a continuous flow operation mode, continuously aerate the bottom of the nitrification membrane bioreactor, and use a submerged microfiltration membrane module to separate biomass and wastewater from the effluent; During the operation of the nitrifying membrane bioreactor, the environmental pH value in the nitrifying membrane bioreactor is gradually increased through three periods, including the stabilization period, the alkaline stimulation period and the alkaline adaptation period. The operation days are controlled by monitoring the level of reactive oxygen species in the system. These comprehensive control strategies are used to promote the secretion of acidic substances in the extracellular polymers of nitrifying bacteria, regulate antiporters and F0-F1 ATPase, and accelerate the biomineralization process involving carbonic anhydrase. - Consumption and H + The input of nitrifying sludge can further improve the alkaline adaptability and nitrification performance of nitrifying sludge; After entering the alkaline adaptation period, the nitrifying membrane bioreactor continues to operate in a constant high pH environment. When the activity of nitrifying bacteria recovers to the same level as that in the stable period and the content of extracellular polymers and the activity of related functional proteins change significantly, an alkaline-resistant and efficient nitrifying microbial flora is obtained. The stable period is a neutral environment, the alkaline stimulation period is a slightly alkaline environment, and the alkaline adaptation period is a highly alkaline environment. The method for controlling the number of operating days is to gradually increase the environmental pH value when the active oxygen level in the system drops to less than 50% of the mid-stage to enter the next stage.

2. The method according to claim 1, characterized in that An automated pH control system is used to control the pH value in the nitrification membrane bioreactor in real time. When the pH value is lower than the set value, the dosing pump is turned on to transport NaOH solution into the nitrification membrane bioreactor to increase the pH value; when the pH value is higher than the set value, the dosing pump is turned off, and the pH value will be further reduced due to the ammonia oxidation effect of microorganisms.

3. The method according to claim 2, characterized in that The automatic pH control system consists of a pH probe, a pH controller, a dosing pump, and a NaOH solution storage tank.

4. The method according to claim 1, wherein When the reactive oxygen species level dropped below 50% of that at the middle stage during each pH gradient, sludge microbial samples were collected and the content of extracellular polymers and the activity of related functional proteins were determined.

5. The method according to claim 1, wherein The initial sludge concentration in the nitrifying membrane bioreactor was 2000~3000 mg / L, the dissolved oxygen concentration in the system was maintained at 3.0~4.0 mg / L, and the hydraulic retention time was 6~9 h.

Images