An extracellular respiration type anammox process without nitrite

Abstract

The application discloses an extracellular respiration type ANAMMOX process without nitrite, belongs to the field of water treatment, and overcomes defects such as difficulty in nitrite supply, accumulation of by-products, limitation of physiological denitrification limit and the like. The application comprises the following steps: step 1, constructing a conventional ANAMMOX system; step 2, introducing an extracellular electron acceptor embedded by bacterial extracellular polymers, domesticating and stimulating direct transmission of ammonia nitrogen electrons to the cell surface, and the selected bacterial extracellular polymers are extracted from residual sludge of a sewage treatment plant, and meet the principle of 'waste treatment with waste'. Step 3, using the extracellular respiration type ANAMMOX reactor constructed in step 2 to treat sewage containing only ammonia. The application in detail provides selection of the extracellular electron acceptor and a treatment flow, and a detailed ANAMMOX electron transmission path, and confirms the feasibility of denitrification without nitrite supply, greatly expands the application scene of the ANAMMOX process, and is helpful to direct implementation of single ANAMMOX in mainstream municipal wastewater.

Description

Technical Field

[0001] This invention belongs to the field of water treatment technology, specifically relating to an extracellular respiration-based anaerobic ammonia oxidation process that does not require nitrite. Background Technology

[0002] Given the increasingly serious water pollution problem worldwide, more and more research is dedicated to developing efficient, energy-saving, and low-carbon biological nitrogen removal technologies. Anaerobic ammonia oxidation (ANAMMOX) is a disruptive technology in the field of wastewater nitrogen removal. This novel process effectively overcomes the shortcomings of traditional nitrogen removal technologies (nitrification-denitrification), such as high energy consumption for oxygen supply, large carbon source requirements, and high treatment costs, greatly advancing the energy-saving and emission-reduction process in wastewater nitrogen removal treatment. Under strictly anaerobic conditions, anaerobic ammonia oxidizing bacteria (AnAOB) use HCO3- - Alternatively, CO2 can be used as a carbon source to transfer nitrite (NO2) onto the anaerobic zymocyte membrane structure. - -N) is used as an electron acceptor to directly convert ammonia nitrogen (NH4) + Nitrite (NO2) is converted to N2, thus completing nitrogen removal. According to existing biochemical models of ANAMMOX, the detailed process of nitrogen transformation in ANAMMOX is as follows: Nitrite (NO2) is converted to N2. - Nitrite (NH4+) is reduced to nitric oxide (NO) by nitrite reductase (NIR). Subsequently, hydrazine synthase (HZS) catalyzes the reaction of NO with ammonia nitrogen (NH4+). + -N) condenses to form hydrazine (N2H4; E'0 = -700 mV), the strongest chemical reducing agent in nature. Finally, hydrazine dehydrogenase (HDH) oxidizes N2H4 to N2. Simultaneously, N2H4 releases four low-potential electrons and, through the electrorespiratory complex contained in ANAMMOX, establishes a membrane potential via a series of intracellular electron transfer pathways and returns to the anaerobic zymocyte, becoming NO2. - -N reduction and N2H4 synthesis provide electrons, thus forming a complete intracellular electron cycle pathway. As an inorganic autotrophic growth process, the electrons consumed in CO2 fixation are partially converted into NO2 by nitrite oxidoreductase (NXR). - -N is oxidized to NO3 - -N compensation.

[0003] However, ANAMMOX, which relies on intracellular electron transport, still has many associated problems. Firstly, ANAMMOX relying on intracellular electron transport must utilize NO2 from wastewater. - -N acts as an electron acceptor. Because NO2 - -N is readily oxidized or reduced to other forms, so NO2 is almost non-existent in actual wastewater. - -N. Therefore, NO2 is rarely found in wastewater. - -N has become the biggest obstacle to the widespread adoption of the ANAMMOX process. Secondly, the oxidation of NO2... - -N is NO3- -N, used to compensate for electron vacancies in the intracellular electron transport chain, also contributes to 11% of nitrogenous byproducts (compared to influent NH4). + The accumulation of NO-N content limits the physiological denitrification limit of ANAMMOX to only 89%. Therefore, there is an urgent need to develop a denitrification method different from that based on NO2. - -N is the extracellular respiratory ANAMMOX pathway of the electron acceptor, thus solving NO2. - -N supply restriction and removal of all NO3 - The limitations of -N byproducts. Shi et al. (2016) provided a design concept for extracellular electron transport pathways based on Shewanella and Geobacter: ① Certain microorganisms can utilize metal minerals as terminal electron sinks for respiration, directly overcoming the barrier of intracellular electron acceptors; ② The microbial cell membrane is a physical barrier to electron exchange, which can be overcome by a transfer pathway composed of redox proteins (e.g., C-type cytochromes) and structural proteins (e.g., microbial nanowires and other cellular structures). This pathway is expected to appear in the same or different species and participate in electron crossing across the entire cell domain, enabling the exchange of intracellularly produced electrons with extracellular electron acceptors. However, due to the unique cellular structure of AnAOB, whether this process is compatible with the ANAMMOX system remains largely unknown, which makes its feasibility still face challenges from basic research to industrial application.

[0004] Based on this, this study utilizes native functional bacterial strains and known enzyme libraries of the ANAMMOX system to drive efficient extracellular respiration-type nitrogen metabolism through in-situ activation of a novel non-nitrite-dependent ANAMMOX pathway, and provides a detailed extracellular electron transfer pathway and molecular mechanism. This invention is the first to develop a nitrite-free extracellular respiration-type anaerobic ammonia oxidation process, fundamentally solving bottlenecks such as obstructed ANAMMOX electron acceptor supply and byproduct accumulation. Summary of the Invention

[0005] In summary, NO2 - -N stable supply and NO3 - -N byproduct accumulation has become a fundamental obstacle to the practical application and widespread adoption of the ANAMMOX process. Therefore, this invention provides an extracellular respiration-based anaerobic ammonia oxidation process that does not require nitrite. It innovatively develops a nitrite-independent ANAMMOX process using an extracellular electron acceptor as the terminal electron sink, directly delivering NH4. + -N electrons are released extracellularly, successfully avoiding ANAMMOX's interference with the intracellular electron acceptor NO2. - -N demand and NO3 -The generation of NO-N significantly expands the application scope and governance depth of the ANAMMOX process. The main challenge is how to rapidly transform the "normal" intracellular respiration-dependent ANAMMOX process into an extracellular respiration-dependent system, thereby eliminating the need for NO2 in the ANAMMOX process. - -N can achieve a single NH4 + -N removal and breakthroughs in physiological denitrification limits create possibilities.

[0006] To this end, the present invention provides the following technical solution.

[0007] The aforementioned anaerobic ammonia oxidation process that does not require nitrite respiration includes the following steps:

[0008] Step 1: Construct NO2 - Anaerobic ammonia oxidation system with simultaneous supply of nitrogen and ammonia nitrogen:

[0009] The inoculated sludge is fed into a sequencing batch reactor (SBR) containing NO2. - -N and NH4 + -N artificial water is used as the reactor feed water to meet the nutritional needs of ANAMMOX bacteria. It adopts intermittent operation with a single cycle duration of 6-12 hours, including five stages: water inlet, stirring, sedimentation, water outlet, and idle.

[0010] The inoculated sludge characteristics meet at least one of the following conditions:

[0011] A. The volume of the inoculated sludge accounts for 20-40% of the total reactor volume;

[0012] B. The total amount of suspended solids in the initial mixture formed after stirring is 5000~6000 mg / L;

[0013] C. In the initial mixture formed after stirring, the mass ratio of volatile suspended solids (i.e., organic biomass content) to total suspended solids is 0.4~0.6, ensuring that the sludge has sufficient organic microorganisms.

[0014] (1) The wastewater mentioned in this step contains NO2 - -N, NH4 + -N and media solutions containing trace elements: NO2 - -N concentration is 30-80 mg / L, NH4 + -N is configured to set the concentration to NO2. - The concentration of -N is 12-1.3 times that of deionized water; a solution containing 5-20 mg / L PO4 is prepared using deionized water. 3- - A trace element nutrient solution containing P, 50-80 mg / L CaCl2, and 500-1000 mg / L KHCO3;

[0015] (2) By adjusting the hydraulic retention time of the reactor, the total nitrogen removal rate of the effluent is made higher than 85%, and the start-up of the “normal” anaerobic ammonia oxidation system is finally completed.

[0016] Step 2: Based on the anaerobic ammonia oxidation system obtained in Step 1, an extracellular electron acceptor substance is added, and NO2 in the influent is adjusted every 3-4 days. - -N mass concentration of 5-10 mg / L, thereby reducing NO2 in the influent. - -N / NH4 + The ratio of -N influent mass concentration is used to in-situ stimulate the electron transfer potential of AnAOB, ultimately constructing an extracellular respiration-type anaerobic ammonia oxidation process that does not require nitrite.

[0017] Step 3: Use the extracellular respiration-type anaerobic ammonia oxidation reactor constructed in Step 2 to treat wastewater containing only ammonia;

[0018] Furthermore, the reactor also includes a pH and DO meter, the probe of which is installed in the reactor to continuously monitor and adjust the pH and DO levels during the denitrification process to promote the survival of functional bacteria. Preferably, the probe of the pH and DO meter is located at the reactor's drain outlet.

[0019] Furthermore, it also includes a raw water tank, which is connected to an inlet via an inlet pipe, and the artificial water distribution maintains dissolved oxygen at 0-0.5 mgO2 / L through aeration.

[0020] The pH of the wastewater is controlled at 7.3 to 7.5. The pH of the wastewater is adjusted using HCl or NaOH solution. For example, the concentration of HCl or NaOH solution is 1 M.

[0021] In addition to the ANAMMOX domestication unit, the present invention is also equipped with an extracellular electron acceptor substance modification unit, that is, the extracellular electron acceptor needs to be modified by this unit to more easily become the extracellular electron acceptor of ANAMMOX bacteria.

[0022] Further, the selection of extracellular electron acceptor substances involves adding substances with electron vacancies on their surface, which can then accept electrons transferred from ANAMMOX. Suitable options include ferric oxide (Fe2O3), magnetite (Fe3O4), manganese dioxide (MnO2), ferrihydrite (γFeO(OH)), and humic substances. Ferrihydrite (γFeO(OH)) is preferred, considering that ANAMMOX bacteria are iron-loving bacteria and iron sources participate in the synthesis of various functional proteins and heme, and that ferrihydrite (γFeO(OH)) is widely available and inexpensive.

[0023] Furthermore, to ensure that the extracellular electron acceptor material better adheres to the bacterial surface to accept electrons transferred from inside and outside the bacteria, extracellular polymers are extracted from the surface of surviving microorganisms as encapsulating agents to encapsulate the added extracellular electron acceptor material, thereby increasing the adhesion between the bacterial agent and the material; preferably, the surviving microorganisms used are taken from the residual sludge of the secondary sedimentation tank of the sewage treatment plant, which conforms to the principle of "treating waste with waste".

[0024] Furthermore, the extraction of extracellular polymers was completed in a separate modification unit. The specific process was as follows: 15 ml of the remaining sludge was taken into a centrifuge tube and diluted to 40 ml. The centrifuge tube was then subjected to centrifugation, ultrasonic treatment in an ice-water bath, sodium cation exchange resin, a second centrifugation, and filtration. The supernatant was then collected.

[0025] Furthermore, the centrifugation force required is 1500-2500g (first) and 4500-5500g (second), with a time of 20-30 min; the ultrasonic treatment frequency is 20-26 kHz, with a time of 3 min; filtration uses a 0.22 μm fiber filter membrane; and the dosage of sodium ion exchange resin is 60-90 g / g sludge sample.

[0026] Furthermore, the supernatant was thoroughly mixed with the extracellular electron acceptor, frozen at -20 to -30°C, and then freeze-dried to obtain powdered EPS-embedded extracellular electron acceptor.

[0027] Furthermore, the content of the external electron acceptor substance to be embedded in the EPS supernatant is 20-50 mg / ml of supernatant, and it is added to the ANAMMOX reactor;

[0028] Furthermore, the dosage of EPS-embedded extracellular electron acceptor, by mass, is 50-100 mg / L relative to the volume of sludge in the reactor.

[0029] Finally, based on the functional bacterial strains and related enzyme annotation capabilities, this invention also proposes a coherent and feasible microbial mechanism for ammonia electron transfer, thereby supporting the ANAMMOX system's NO2-free capability. - -N, acting as an electron acceptor, can achieve closed-loop electron transport.

[0030] Preliminary findings on transanaerobic zymosomal electron transport of hydrazine: AnAOB oxidizes N₂H₄ via an unknown oxidoreductase, releasing four electrons which are stored in a methylquinoline pool. This establishes a proton motive force on the anaerobic zymosomal membrane, driving ATP synthesis. Subsequently, the Rieske / cytbbc1 complex of AnAOB oxidizes the dual-electron carrier quinoline, simultaneously coupling with a high redox potential electron acceptor (such as NAD(P)) to synthesize NAD(P)H, releasing free electrons. Simultaneously, the electrons contained in NAD(P)H are released by NADH:quinone oxidoreductase and transferred to the Rnf-type electron transport complex to generate ferric oxidoreductase (Fd).red Ultimately, N2H4 oxidation releases 4 electrons as cytoplasmic free electrons, NAD(P)H, and Fd. red They successfully crossed the anaerobic enzyme membrane in various forms.

[0031] Furthermore, cytochrome-mediated cytoplasmic migration occurs: AnAOB expresses an inner membrane c-type cytochrome similar to CymA (Cyt A: 4 heme atoms) that transfers electrons released from the methylquinoline pool (i.e., the cytoplasmic free electrons mentioned above) to the periplasmic monoheme c-type cytochrome (Cyt A: 1 heme atom). Subsequently, AnAOB secretes an outer membrane protein complex (OmpA family protein) and transfers electrons from the periplasm to the bacterial surface via the electron transport chain.

[0032] Ultimately, electron transition occurs on the bacterial surface: Since AnAOB lacks a carrier for long-distance electron transport, the electrons released by AnAOB can only reach the surface of the extracellular electron acceptor through direct contact. This undoubtedly requires a closer adhesion between the bacteria and the extracellular acceptor. The modified extracellular electron acceptor provided by this invention precisely meets this requirement. The outer extracellular polymeric encapsulating agent of the extracellular electron acceptor facilitates adhesion to the abundant secretions of AnAOB, further shortening the mass transfer distance from intracellular electrons to the extracellular acceptor.

[0033] The extracellular respiration-based anaerobic ammonia oxidation process described in this invention, which does not require nitrite, has the following technical advantages:

[0034] 1. The extracellular respiration-based anaerobic ammonia oxidation process provided by this invention uses an extracellular electron acceptor substance to replace unstable NO2 in water. - -N, completely eliminate NO3 - The challenge of NH4+ byproduct accumulation is overcome, reducing the high cost and complex operation management of coupling processes, greatly expanding the application scenarios of ANAMMOX technology, and facilitating the direct implementation of single ANAMMOX in mainstream municipal wastewater. In the absence of intracellular electron acceptors, high-quality extracellular electron acceptors stimulate the expression of various extracellular electron transport-related enzymes in AnAOB, utilizing extracellular solid-state electron acceptors as terminal electron sinks for high-efficiency NH4+ transport. + -N removal. Specifically, AnAOB transfers NH4+ across anaerobic zymocytes (intrinsic membrane organelles unique to AnAOB) via Rieske / cytb bc1, relying on cytoplasmic migration of polyhemoglobin (Cyt A) and electron transitions on the cell surface. + -N electrons are transported outside the cell. Therefore, the novel ANAMMOX process provided by this invention does not require NO2. - -N can also be used as a necessary electron donor to achieve efficient NH4+ production. +-N removal has brought hope for efficient, energy-saving, and low-carbon treatment of urban wastewater rich in ammonia nitrogen.

[0035] Furthermore, the microbial community-mediated ANAMMOX process is considered a game-changer in wastewater nitrogen removal technology, with numerous successful implementation cases. However, 11% of NO3... - The direct emission of NO3- byproducts not only causes secondary pollution but also limits the physiological limit of nitrogen removal for the ANAMMOX process to 89%. Therefore, conventional ANAMMOX processes require treatment of residual NO3 before effluent discharge. - -N content will be further monitored and removed. The non-nitrosodependent ANAMMOX process proposed in this invention no longer involves the oxidation of NO2. - -N is NO3 - -N enables electron closure within bacteria, fundamentally eliminating NO3-. - -N byproduct emissions.

[0036] 2. The extracellular respiration-based anaerobic ammonia oxidation process provided by this invention significantly enhances the metabolic level of functional bacteria, accelerates cell self-replication, shortens cell aging time, and effectively improves reactor start-up time. Despite these significant advantages, the engineering application of ANAMMOX technology has been very slow. According to incomplete statistics, there are currently only a dozen or so ANAMMOX engineering cases in my country. This predicament is closely related to the extremely low growth rate exhibited by AnAOB. ​​Previous studies have confirmed that the maximum specific growth rate of ANAMMOX bacteria in granular sludge form at 32-33℃ is only 0.05-0.06 days. -1 (Generation time as long as 10-12 days). Therefore, AnAOB has long been considered an extremely slow-growing species, resulting in a very long start-up period for the ANAMMOX system without exogenous inoculation (the first ANAMMOX project in Rotterdam took nearly 3 years to start up), greatly limiting the widespread application of ANAMMOX technology. The extracellular respiration-dependent anaerobic ammonia oxidation process proposed in this invention effectively improves the growth rate of AnAOB. ​​On the one hand, the extracellular respiration-dependent process produces more ferric reductase (Fd) to assist in the transanaerobic transport of ammonia electrons. Compared to intracellular respiration, it eliminates the need for NO2. - The -N extracellular respiration-based ANAMMOX process will accumulate more iron-reducing proteins (Fd) in microorganisms, thereby improving inorganic carbon and NH4+. +Inorganic components such as nitrogen (N) synthesize biomass, promoting the growth and accumulation of AnAOB. ​​The main reason is that in organisms using the acetyl-CoA pathway, electron transfer during the CO2 reduction step is primarily mediated by ferrous d (Fd) with low redox potential. In the novel ANAMMOX process, more Fd acts as an electron shuttle between the anaerobic zymosomal respiratory complex and the central carbon metabolism and nitrogen assimilation of AnAOB, accelerating electron communication efficiency and thus improving the population accumulation efficiency of AnAOB.

[0037] 3. More importantly, the extracellular respiration-based anaerobic ammonia oxidation process provided by this invention overcomes the diffusion flux limitation of ANAMMOX, accelerating the uptake of nutrient substrates by bacteria, thereby providing synthetic elements and energy for cell division. Firstly, it eliminates the need for NO2. - The operating characteristics of -N allow the new system to be no longer limited by NO2. - The diffusion flux of -N; secondly, intracellular electrons are continuously released into the aqueous environment, thereby reducing the overall redox level of the reactor and making it more favorable for positive ions (NH4+). + The migration and transport of NH4+ (-N) will benefit more NH4+. + -N transport to the surface of AnAOB; thirdly, AnAOB has the ability to take up extracellular inorganic electrons. Taking ferric compounds or iron-based minerals as extracellular electron acceptors as an example, after accepting ammonia electrons, they are reduced to free ferrous ions (Fe(II)). Free ferrous ions (Fe(II)) can also participate in metabolism as electrons, accelerating the efficiency of ATP synthesis, thereby providing energy for transmembrane transport of the extracellular matrix; fourthly, excessive NH4+... + -N demand and ample ATP supply lead to the synthesis of large amounts of ammonia transport-specific protein (AMT) on the surface of ANAMMOX, increasing NH4+. + -N transport channels for intracellular transfer. In summary, the establishment of the extracellular respiration-based anaerobic ammonia oxidation process has comprehensively overcome the substrate diffusion flux limitations in anaerobic environments. Rapid nutrient uptake and targeted biomass synthesis improve the metabolic level of AnAOB itself, accelerate cell self-replication, and are expected to break through the physiological limits of ANAMMOX enrichment, completing population expansion in a very short time, thereby cultivating modified ANAMMOX granular sludge with high bacterial density, greatly expanding the application scope and treatment depth of the new process. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 This is a schematic diagram of the extracellular respiration-type anaerobic ammonia oxidation device of the present invention that does not require nitrite;

[0040] Figure 2 Anaerobic ammonia-oxidizing bacteria do not require NO2. - -N extracellular electron transfer pathway;

[0041] Figure label:

[0042] ANAMMOX Directional Acclimation Unit: 1-Raw Water Tank, 2-First Time Controller, 3-Inlet Pump, 4-Inlet Pipe, 5-Second Time Controller, 6-Stirring Device, 7-Stirring Paddle, 8-pH Measurement Probe, 9-DO Measurement Probe, 10-pH and DO Meters, 11-Third Time Controller, 12-Solenoid Valve, 13-Drain Pipe, 14-Outlet Water Tank; Extracellular Electron Acceptor Modification Unit: 15-0℃ Ice Bath Device, 16-High-Speed ​​Centrifuge, 17-Ultrasonic Device, 18-Sodium-Type Cation Exchange Resin, 19-Extracellular Electron Acceptor Substance, 20-Freeze Dryer. Detailed Implementation

[0043] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0044] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0045] Example 1

[0046] This embodiment provides an extracellular respiration-based anaerobic ammonia oxidation process that does not require nitrite, and its device structure is as follows: Figure 1 As shown, it includes an ANAMMOX domestication unit and an extracellular electron acceptor substance modification unit;

[0047] Specifically, the core reactor of the ANAMMOX acclimatization unit adopts a sequencing batch reactor (SBR) operation mode, strictly adhering to five processes: water inlet, stirring, sedimentation, water outlet, and idle period. The reactor structure has an inlet at the top and an outlet at the bottom.

[0048] The stirring device includes a drive motor 6 and a stirring unit 7, the stirring unit being disposed in the reactor;

[0049] To facilitate monitoring of the pH value and DO content of the reaction, a pH and DO meter 10 is also included. The pH measuring probe 8 and DO measuring probe 9 of the pH and DO meter are installed in the reactor, located in the ANAMMOX reaction area.

[0050] like Figure 1 As shown, the device in this embodiment also includes a raw water tank 1, which is connected to an inlet via an inlet pipe 4. An inlet pump 3 is installed on the inlet pipe 4 to pump the wastewater in the raw water tank 1 into the reactor. A first time controller 2 is connected to the inlet pump 3 to control the operating time of the inlet pump 3.

[0051] In this embodiment, the rotation axis of the propeller blades is coaxial with that of the reactor, and the drive motor of the propeller blades is connected to the third time controller 5. The drain outlet of the reactor is connected to the drain pipe 13, and a solenoid valve 12 is installed on the drain pipe 13. The solenoid valve 12 is connected to the fourth time controller 11, and the drain pipe 13 discharges the treated wastewater to the effluent pool 14.

[0052] The extracellular electron acceptor material modification unit includes the following steps: the remaining sludge is extracted in sequence through a first centrifugation treatment 16, an ultrasonic treatment 17 in an ice-water bath environment 15, a sodium-type cation exchange resin 18, a second centrifugation treatment 16, and a filtration stage. After being homogenized with the extracellular electron acceptor material, it is then freeze-dried 20 to complete the encapsulation process.

[0053] This method integrates the first and second centrifugation processes, ultrasonic treatment, and sodium-type particle exchange resin into a single centrifuge tank. The required material parameters for each step are clearly specified, including centrifugal forces of 1500-2500g (first) and 4500-5500g (second), for 20-30 min; ultrasonic treatment frequency of 20-26 kHz, for 3 min; filtration using a 0.22 μm fiber membrane; and sodium-type ion exchange resin dosage of 60-90 g / g sludge sample.

[0054] Example 2

[0055] This embodiment provides an extracellular respiration-type anaerobic ammonia oxidation process that does not require nitrite, using the apparatus in Example 1 and employing a sequencing batch reactor process to treat artificial wastewater;

[0056] The sequential batch reaction process has a single cycle duration of 12 hours, including 10 minutes of water inlet, 680 minutes of stirring, 15 minutes of sedimentation, 10 minutes of water outlet, and 5 minutes of idle time.

[0057] The extracellular respiration-based anaerobic ammonia oxidation process and high-density functional bacteria culture method without nitrite include the following steps:

[0058] Step 1: Construct a stable "normal" anaerobic ammonia oxidation system:

[0059] (1) “Normal” ANAMMOX sludge was used as inoculation sludge. In this embodiment, the ANAMMOX sludge was identified as having Candidatus Brocadia as the dominant functional bacteria.

[0060] (2) Inoculate sludge into the reactor, with the volume of the inoculated sludge accounting for 20% of the total reactor volume. Prepare a solution containing 130 mg / L NO2 using deionized water. - -N, 100 mg / LNH4 + -N, 10 mg / LPO4 3- Artificial wastewater containing -P, 60 mg / L CaCl2, and 1000 mg / L KHCO3 was prepared and its pH was maintained at 7.5. This artificial wastewater was pumped into the reactor and stirred. The initial mixture contained approximately 5000 mg / L of total suspended solids, with a volatile suspended solids / total suspended solids (VSS) / SS ratio of 0.6 and an average sludge particle size of 800.6 μm. Sedimentation and effluent were then carried out. The growth rate of the ANAMMOX sludge was measured to be only 0.07 μg / d. -1 Its corresponding generation time is as high as 12 days.

[0061] The total nitrogen removal rate in the reactor effluent is higher than 85%, indicating that the denitrification effect of the system has reached a stable state.

[0062] Step 2: In the "normal" anaerobic ammonia oxidation system prepared in step (1), 100 mg / L of extracellular acceptor substance - extracellular polymer modified powder is added. In this embodiment, [γFeO(OH)] ferriferrite is preferred as the extracellular acceptor substance to gradually reduce the NO2 in the influent. - -N / NH4 + -N ratio, in situ construction of extracellular respiration-type anaerobic ammonia oxidation process without nitrite.

[0063] Step 3: The artificially synthesized wastewater is treated using the extracellular respiration-type anaerobic ammonia oxidation reactor constructed in Step 2;

[0064] NO2 in the reactor inlet and effluent of Example 2 - -N, NH4 + -N and PO4 3- The content of -P was measured daily, and the growth rate of AnAOB was also determined.

[0065] Table 1. Concentration of pollutants in influent and effluent

[0066]

[0067] Table 2 Pollutant treatment rate in Example 2

[0068]

[0069] The experimental results above show that, compared with the "normal" ANAMMOX system (experimental example 0-13 days), the present invention achieves significantly lower NO3 levels than theoretically required. - -N / NH4 + In the -N case, the introduction of [γFeO(OH)]-extracellular polymer modified powder for single NH4 + The removal efficiency of -N and total nitrogen can be effectively enhanced, significantly higher than previous studies, with the overall denitrification efficiency approaching 100%. The average peak phosphorus removal rate also increased from 45.01% to 80.58%, and the reaction system operated stably without NO3. - -N accumulation. Most importantly, the growth rate of ANAMMOX was significantly optimized in this embodiment ( Figure 2 The excellent living environment and the activation of ferric reductase greatly increased the bacterial growth rate by about 5 times, with the growth rate of ANAMMOX bacteria reaching 0.33 days. -1 (Generation time 2.1 days, 30℃), which is basically the same as that of nitrifying bacteria.

[0070] Simultaneously, metagenomics and metatranscriptomics data were used to compare the genomic data of inoculated sludge and the sludge at the end of Experiment 2 to identify the electron outflow pathway relied upon by ANAMMOX bacteria: Previous studies have confirmed that the Rieske / cytb complex in Anammox bacteria plays a central role in the oxidation of quinoline and the reduction of type C cytochromes. Utilizing this electron bifurcation mechanism, the synthesis of NAD(P)H via the reduction reaction of (methyl)quinoline oxidation coupled with a high redox potential electron acceptor (such as NAD(P)) is thermodynamically feasible, thereby releasing free electrons. Comparative transcriptomics analysis showed that when [γFeO(OH)]-extracellular polymer modified powder was introduced, the expression level of the Rieske / cytbbc1 complex increased by 3.40-fold (Table 3). Clearly, more bc1 complexes may be used to catalyze the oxidation of (methyl)quinoline to NAD(P)H(Reaction A; Figure 2It participates in extracellular electron transport. Correspondingly, in the cytoplasm, the expression of the gene encoding NADH (metF) is increased by 1.35-fold (Table 3). According to existing knowledge of Anammox biochemistry, the [γFeO(OH)]-extracellular polymer modified powder also stimulates a series of metabolic responses in AnAOB to cope with NAD(P)H accumulation. First, NAD(P)H is oxidized by NADH:quinone oxidoreductase, and the released energy (∆G0'=-47 kJ) is preferentially used to upregulate the sodium-dependent Rnf-type electron transport complex (Table 1) to transfer Na ions, thereby generating Na kinetics (Reaction B; Figure 2 Furthermore, Na kinetics promotes the upregulation of NAD-dependent oxidoreductases, quinoline dehydrogenases, or NAD-dependent dehydrogenases to drive the opposite, unfavorable NAD+ reduction (Table 3), further generating NAD(P)H(Reaction C; Figure 2 Ultimately, in the membrane-bound Rnf complex, electrons from NADH oxidation are transferred to the ferrooxidase protein (Fd). red (ReactionD; Figure 2 Due to Fd red The redox potential (e0'Fd = -500 ~ -420 mV) is more negative than that of NAD+ / NADH (e0'NADH = -320 mV), and the excess energy is used for Fd. re d. Transmembrane transport. Therefore, hydrazine dehydrogenase (HDH) releases some energy as cytoplasmic free electrons, NAD(P)H, and Fd. red They cross the anaerobic enzyme membrane in various forms.

[0071] In addition to the complex trans-anaerobic zymosomal electron transport described above, subsequent electron transfer follows a similar electron transfer pathway design to Shewanella and Geobacter. In the extracellular respiration-based anaerobic ammonia oxidation process, AnAOB expresses an inner membrane C-type cytochrome (Cyt A: 4 heme) similar to CymA to transfer methylquinoline oxidation electrons (i.e., the cytoplasmic free electrons mentioned above) to a highly upregulated periplasmic monoheme C-type cytochrome (Cyt A: 1 heme). Figure 2 Table 3). Cyt A (one heme) is also homologous to MtrA in the metal-oxidizing bacterium *Shewanella*, thus serving as an electron shuttle between Cyt A (four heme) and outer membrane cytochromes. Subsequently, the outer membrane pore-cytochrome complex homolog (OmpA family protein) was highly expressed in AnAOB (expression level increased by 1.06-fold). Electrons are transferred from the periplasm to the bacterial surface via the electron transport chain. Figure 2However, transcriptomic data showed no detectable cytochromes (such as riboflavin) for long-range electron transport (Table 3). Due to the lack of long-range electron transport carriers, AnAOB-released electrons can only reach the surface of extracellular electron acceptors through direct contact. Therefore, following the pathway described above, ammonia electrons do not need to be transported by NO2. - With -N receiving, efficient NH4 can be achieved. + -N is removed.

[0072] Table 3. List of energy conservation-related genes with similar expression under two experimental conditions ("normal" ANAMMOX and extracellular respiration-dependent ANAMMOX system).

[0073]

[0074] Example 3

[0075] This embodiment provides an extracellular respiration-based anaerobic ammonia oxidation process that does not require nitrite, and is basically the same as in Embodiment 2, except that this embodiment uses actual wastewater and the extracellular electron acceptor is humic matter encapsulating bacterial extracellular polymers. Since actual wastewater only contains NH4+... + -N, and the concentration was lower than that of the artificially synthesized wastewater in Example 2. Therefore, the single-cycle duration of the sequencing batch reactor was adjusted to 6 hours, including 10 minutes of influent, 320 minutes of stirring, 15 minutes of sedimentation, 10 minutes of effluent, and 5 minutes of idle time. To further verify the feasibility of this invention in practical applications, NO3 in the reactor influent and effluent of Example 2 was tested. - -N, NO2 - -N, NH4 + -N content was measured daily.

[0076] In Example 3, the influent used in the reactor was domestic sewage, taken from the sewer system of the Beijing University of Technology staff quarters, with the following water quality indicators: NO3. - -N is 3-5 mg / L, NH4 + -N concentration was 40-46 mg / L, NO2 - -N concentration was 0.8-1.1 mg / L. Experimental results showed that under the extracellular respiration-type ANAMMOX process, the average NO3 in the effluent was [data missing]. - -N, NO2 - -N, NH4 + The -N concentrations were 1.89 mg / L, 0.32 mg / L, 0 mg / L, and 0.49 mg / L, respectively, meeting the Class A discharge standard for urban wastewater. Meanwhile, the sludge retention time was measured to be only 3-4 days, and the growth rate of AnAOB increased to 0.3 μ·d⁻¹. -1 above.

[0077] Comparative Example 1

[0078] To demonstrate that the present invention effectively improves the NO2 problem encountered in the ANAMMOX process. - -N stable supply and NO3 - The problem of -N byproduct accumulation. Table 2 shows the reactor treatment performance in steps 1 (0-13 days) and 2 (55-60 days) of Example 2. Compared to the normal "ANAMMOX" system, the extracellular respiration-type anaerobic ammonia oxidation process provided by this invention, which does not require nitrite, reduces NO2 in the ANAMMOX system. - -N / NH4 + The -N ratio gradually decreased from 1.3 to 0, and the pollutant removal efficiency increased from 87% to 98%, resulting in reduced NO3 in the effluent. - -N content decreased from 15 mg / L to 1 mg / L. The generation time of AnAOB decreased from 12 days to 3 days, which is conducive to the rapid self-replication of AnAOB.

[0079] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An extracellular respiration-based anaerobic ammonia oxidation process that does not require nitrite, characterized in that, By introducing extracellular electron acceptors based on the anaerobic ammonia oxidation process, an extracellular respiration-type anaerobic ammonia oxidation system without the need for nitrite is constructed to achieve the direct removal of ammonia nitrogen without the generation of nitrate byproducts. Includes the following steps: Step 1: Construct NO2 - Anaerobic ammonia oxidation system with simultaneous supply of nitrogen and ammonia nitrogen: The inoculated sludge is added to the reactor, and then wastewater is added to the reactor; the inoculated sludge meets at least one of the following conditions: A. The volume of the inoculated sludge accounts for 20-40% of the total reactor volume; B. The total amount of suspended solids in the initial mixture formed after stirring is 5000~6000 mg / L; C. In the initial mixture formed after stirring, the mass ratio of volatile suspended solids to total suspended solids is 0.4~0.6, ensuring sufficient organic microorganisms in the sludge; the wastewater mentioned in this step contains NO2. - -N, NH4 + -N and media solutions containing trace elements; NO2 - -N concentration is 30-80 mg / L, NH4 + -N is configured to set the concentration to NO2. - The concentration of -N is 1.2-1.3 times that of PO4; 5-20 mg / L PO4 is prepared using deionized water. 3- The nutrient solution contains trace elements such as P, 50-80 mg / L CaCl2, and 500-1000 mg / L KHCO3; the pH of the wastewater is controlled at 7.0-7.

5. Step 2: Based on the anaerobic ammonia oxidation system obtained in Step 1, add an extracellular electron acceptor substance; the dosage of the extracellular electron acceptor substance, by mass, is 50-100 mg / L of sludge in the reactor; start adjusting the influent NO2 every 3-4 days. - -N mass concentration 5-10 mg / L, until NO2 in the influent - With -N mass of 0 mg / L, an extracellular respiration-based anaerobic ammonia oxidation process that does not require nitrite was finally constructed. Step 3: Treat wastewater containing only ammonia; The extracellular electron acceptor substances include ferric oxide (Fe2O3), iron(II) oxide (Fe3O4), manganese dioxide (MnO2), ferrihydrite (γFeO(OH)) or humic substances; the extracellular electron acceptor substances need to be pretreated by encapsulation with bacterial extracellular polymers (EPS) before addition to ensure a complete and convenient electron transport channel between anaerobic ammonia-oxidizing bacteria and extracellular acceptors.

2. The extracellular respiration-based anaerobic ammonia oxidation process without nitrite as described in claim 1, characterized in that, The mass concentration of the external electron acceptor material to be embedded in the EPS supernatant is 20-50 mg / ml EPS supernatant.

3. The extracellular respiration-type anaerobic ammonia oxidation process according to claim 1, characterized in that: NH4 + -N releases electrons that travel through the bacterial cell's electron transport pathway to the extracellular receptor, rather than NO2. - -N; The detailed electron transfer pathway is as follows: anaerobic ammonia-oxidizing bacteria via Rieske / cytb bc1 Mediating transanaerobic zymosomal transfer, relying on cytoplasmic migration of polyhemoglobin cytochromes and electron transitions on the cell surface, NH4+... + -N oxidation electrons are transported directly outside the cell.

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