A high-efficiency denitrification method based on coupling of overall ammonia oxidation and sulfur autotrophic denitrification and a reaction system thereof

Abstract

This invention provides a coupled nitrogen removal system and method based on an SBBR reactor for complete ammonia oxidation and sulfur autotrophic denitrification. The system contains both biofilm sludge and sedimentary activated sludge, both enriched with both ammonia-oxidizing bacteria (Comammox) and sulfur autotrophic denitrifying bacteria. Under microaerobic conditions, the ammonia-oxidizing bacteria convert ammonia nitrogen to nitrate nitrogen. Under anoxic conditions, using sodium thiosulfate as an electron donor, the sulfur autotrophic denitrifying bacteria convert nitrate nitrogen to nitrogen gas. By optimizing the operating cycle and oxygen supply strategy, the system achieves synergistic effects between the two bacterial communities, completing the complete oxidation of ammonia nitrogen and efficiently removing nitrate nitrogen without the need for external organic carbon sources. This method features low energy consumption, low carbon source dependence, and stable nitrogen removal performance, making it suitable for deep nitrogen removal treatment of low C / N wastewater and showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biological nitrogen removal technology for wastewater, specifically to a highly efficient nitrogen removal method and reaction system based on the synergistic effect of ammonia-oxidizing bacteria (Comammox) and sulfur-autotrophic denitrifying bacteria (SAD). Background Technology

[0002] With the continuous development of industrial and agricultural production, human production activities inevitably generate more and more nitrogen. This nitrogen is discharged into the atmosphere, lakes, oceans, and soil, disrupting the original nitrogen balance and causing nitrogen pollution. Eutrophication of water bodies caused by excessive nitrogen levels is the most direct manifestation of nitrogen pollution. Currently, most mainstream biological nitrogen removal technologies adopt the traditional nitrification-denitrification model. The nitrification stage includes two processes: first, ammonia-oxidizing bacteria (AOB) or ammonia-oxidizing archaea (AOA) oxidize ammonia nitrogen to nitrite nitrogen, and then nitrite-oxidizing bacteria (NOB) oxidize nitrite nitrogen to nitrate nitrogen. The denitrification stage refers to the process in which, under anaerobic or hypoxic conditions, denitrifying bacteria use organic matter in wastewater as an electron donor and nitrate as an electron acceptor to reduce it to gaseous nitrogen (N2). However, in the traditional nitrification-denitrification model, to ensure NH4+ during the nitrification stage... + Effective removal of -N requires extensive aeration to maintain the activity of ammonia-oxidizing bacteria, which consumes a huge amount of energy. Therefore, during the denitrification stage, it is often necessary to add an additional carbon source, resulting in high wastewater treatment costs.

[0003] In 2015, Daims' team and VanKessel's team successfully enriched and demonstrated microorganisms capable of complete ammonia oxidation (Comammox) in different environments, a discovery that overturned traditional nitrification concepts. Compared to traditional nitrifying bacteria, complete ammonia oxidizing bacteria can independently complete the conversion of ammonia nitrogen to nitrate under low dissolved oxygen conditions, exhibiting higher affinity for ammonia nitrogen and oxygen, and demonstrating significant growth advantages under low ammonia nitrogen concentrations and microaerobic conditions. The discovery of Comammox bacteria provides new possibilities for constructing more efficient and low-energy-consumption denitrification systems, especially suitable for my country's current wastewater treatment needs where carbon sources are scarce and energy consumption is controlled.

[0004] Sulfur-based autotrophic denitrification (SAD) technology, as a nitrogen removal pathway independent of organic carbon sources, has attracted widespread attention. This technology uses reduced sulfur-containing substances as electron donors, and under anoxic or anaerobic conditions, sulfur-based autotrophic denitrifying bacteria reduce nitrates and nitrites to nitrogen gas, achieving highly efficient nitrogen removal without a carbon source. Sulfur-based autotrophic denitrification technology exhibits significant advantages in several aspects: firstly, it significantly reduces dependence on exogenous organic carbon; secondly, it has high denitrification efficiency, meeting increasingly stringent emission standards; and thirdly, it has low operating costs, demonstrating good economic viability. Therefore, this technology has become a research hotspot in the treatment of low C / N wastewater.

[0005] Although both ammonia-oxidizing bacteria and sulfur-autotrophic denitrifying bacteria have excellent denitrification potential, synergistic denitrification systems based on the two have been rarely studied. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a highly efficient denitrification method and reaction system based on the coupling of full-process ammonia oxidation and sulfur autotrophic denitrification, so as to realize the synergistic effect of full-process ammonia oxidizing bacteria and sulfur autotrophic denitrifying bacteria, and efficiently treat low ammonia nitrogen wastewater. Compared with traditional methods, oxygen consumption is significantly reduced, aeration energy consumption is saved, no additional carbon source is required, dependence on external carbon source is reduced, and sludge production can be reduced to a certain extent.

[0007] To achieve the above objectives, this invention provides a highly efficient nitrogen removal method based on the coupling of full-process ammonia oxidation and sulfur autotrophic denitrification, the specific start-up and control steps of which are as follows:

[0008] 1) Enrichment of key functional bacteria: In SBBR1 and SBBR2 reactors, 3L of sludge was inoculated in an aerobic / anoxic zone ratio of 1:1. Both reactors used polyurethane sponge as packing material at a filling ratio of 30%, which was fixed to the inner wall of the reactor to facilitate the attachment and growth of microorganisms. SBBR1 aimed to enrich Commammox bacteria, using ammonia nitrogen as the sole nitrogen source and maintaining an influent concentration of 20mg / L. The nitrogen load in the reactor was increased by shortening the HRT (reducing it every 40 days) to avoid the adverse effects of increased ammonia nitrogen concentration on the ammonia-oxidizing bacteria throughout the process. The dissolved oxygen in the reactor was controlled at 0.2-0.5mg / L, and the pH was 7.5±0.5. The reactor was operated for 120 days. SBBR2 was designed to enrich high-abundance sulfur-autotrophic denitrifying bacteria. NaS₂O₃·5H₂O was used as the electron donor at an influent concentration of 886.4 mg / L, with nitrate nitrogen as the sole nitrogen source, maintained at a concentration of 50 mg / L. The reactor's nitrogen loading was increased by shortening the heating time (HRT) every 30 days to enrich the bacteria. Both reactors used pH and DO probes to monitor pH and dissolved oxygen levels. pH was adjusted by adding HCl or NaOH, and aeration was controlled by adjusting the rotor flow meter. Both reactors were protected from light. During the enrichment process, the concentration of the substrate and the concentration of products formed after substrate consumption were monitored. At the end of the enrichment process, biofilm sludge samples were removed for 16S rRNA high-throughput sequencing and metagenomic analysis to confirm the enrichment of the two functional bacteria.

[0009] 2) Start-up of the coupled system: Take half of the sludge from SBBR1 and SBBR2 and inoculate it into the SBBR3 reactor. SBBR3 also uses polyurethane sponge as filler, with a filling ratio of 30%, and fixes it to the inner wall of the reactor. The influent of the SBBR3 system consists of two parts: a nitrogen source and a sulfur source. The nitrogen source concentration is 20 mg / L ammonia nitrogen (an additional 10 mg / L nitrate nitrogen is added at the beginning of anaerobic treatment from day 30 to day 111). The sulfur source is provided by sodium thiosulfate (NaS2O3·5H2O). The system operates in aerobic / anoxic mode for the first 70 days. The reactor was subjected to 16 hours of aerobic treatment (including 5 minutes of influent) followed by 8 hours of anoxic treatment (including 30 minutes of sedimentation and drainage). For the next 41 days, the aerobic treatment consisted of 12 hours of aerobic treatment (including 5 minutes of influent) followed by 12 hours of anoxic treatment (including 30 minutes of sedimentation and drainage). Simultaneously, the reactor was maintained at a pH of 7.2 ± 0.2 and a dissolved oxygen level of 0.2-0.5 mg / L using pH and DO probes. The pH was adjusted by adding HCl or NaOH, and the aeration rate was controlled by adjusting the rotor flow meter. The reactor was kept in the dark. During the construction of the coupled system, the concentration of the substrate and the concentration of the products formed after substrate consumption were monitored. At the end of the enrichment process, biofilm sludge samples were removed for 16S rRNA high-throughput sequencing and metagenomic analysis to confirm changes in the two functional bacteria.

[0010] This invention also provides a reaction system based on the coupling of full-process ammonia oxidation and sulfur autotrophic denitrification, comprising: a nitrogen source inlet tank (1), a sulfur source inlet tank (2), a peristaltic pump (3), an inlet valve (4), a stirrer (5), polyurethane sponge packing (6), sedimented activated sludge (7), a dissolved oxygen probe (8), a pH probe (9), a multi-parameter water quality analyzer (10), an aeration pump (11), a rotor flow meter (12), an aeration head (13), an outlet valve (14), and an outlet tank (15). The reaction system based on the coupling of full-process ammonia oxidation and sulfur autotrophic denitrification is constructed by inoculating enriched functional bacteria into this system.

[0011] Therefore, the efficient nitrogen removal method and reaction system based on the coupling of full-process ammonia oxidation and sulfur autotrophic denitrification provided by this invention have the following advantages compared with the prior art:

[0012] (1) The enrichment conditions of the whole process ammonia oxidation are special. The present invention provides it with low dissolved oxygen (0.2-0.5 mg / L), low ammonia nitrogen (20 mg / L), and medium temperature (28℃) conditions. By shortening the hydraulic retention time, the nitrogen load in the system is enhanced, which not only reduces the adverse effects of high ammonia nitrogen on the whole process ammonia oxidation bacteria, but also improves the ammonia nitrogen removal efficiency of the whole process ammonia oxidation bacteria, and enriches highly active whole process ammonia oxidation bacteria.

[0013] (2) Inoculate the whole process ammonia oxidizing bacteria and sulfur autotrophic denitrifying bacteria into the coupling system. At this time, the coupling system uses the whole process ammonia oxidizing bacteria to convert ammonia nitrogen into nitrate nitrogen, and then uses sulfur autotrophic denitrifying bacteria to convert nitrate nitrogen into nitrogen gas. Compared with the traditional nitrification-denitrification mode, this process saves aeration energy consumption and does not require organic carbon source, resulting in lower wastewater treatment costs.

[0014] (3) This coupled system is a "dual-site microbial system," containing both biofilm sludge and sedimentary activated sludge. Compared to the traditional SBBR model, the sedimentary activated sludge does not remain suspended during stirring but settles at the bottom of the reactor, resulting in poor dissolved oxygen mass transfer and providing a growth environment for anoxic or even anaerobic microorganisms. Simultaneously, the low dissolved oxygen conditions within the system also favor the growth of ammonia-oxidizing bacteria throughout the process. This dual-site microbial system provides two growth environments simultaneously for aerobic and anoxic bacteria, which helps reduce the inhibition of anoxic microorganisms by dissolved oxygen and establishes a synergistic effect between aerobic and anoxic bacteria.

[0015] (4) The sludge age of the entire coupling system is relatively long, which can reduce the production of excess sludge. Attached Figure Description

[0016] Figure 1 Community structure composition diagram at the taxonomic level for the genus SBBR1

[0017] Figure 2 Community structure composition diagram at the taxonomic level for the genus SBBR2

[0018] Figure 3 This is a schematic diagram of a coupled system for ammonia oxidation and sulfur autotrophic denitrification. The diagram includes a nitrogen source inlet tank (1), a sulfur source inlet tank (2), a peristaltic pump (3), an inlet valve (4), a stirrer (5), polyurethane sponge packing (6), sedimented activated sludge (7), a dissolved oxygen probe (8), a pH probe (9), a multi-parameter water quality analyzer (10), an aeration pump (11), a rotor flow meter (12), an aeration head (13), an outlet valve (14), and an outlet tank (15).

[0019] Figure 4 Community structure composition diagram at the taxonomic level for genus SBBR3 Detailed Implementation

[0020] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, kits, and instruments used in the following examples are commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0021] Example 1: Enrichment of ammonia-oxidizing bacteria and sulfur-autotrophic denitrifying bacteria throughout the entire process.

[0022] First, sludge from the aerobic and anoxic zones of the Lanzhou Heping Wastewater Treatment Plant was inoculated into SBBR1 and SBBR2 reactors at a 1:1 ratio, with 3L of sludge inoculated into each reactor. Both SBBR1 and SBBR2 reactors were selected with an inner diameter of 20cm, a height of 40cm, and an effective volume of 6.0L. Polyurethane sponge was added to the reactors to provide a carrier for microbial attachment and growth, with a filling rate of 30%. The packing material was fixed to the inner wall of the reactors around the perimeter using fine iron wire. The top was equipped with a sealing cap, and all materials were plexiglass. The side outlet of the reactors was controlled by a solenoid valve, and the pH and dissolved oxygen levels were monitored in real time using a water quality parameter analyzer. A peristaltic pump was used for water intake. Except during the influent and sedimentation / drainage stages, the stirring device ran continuously to ensure sufficient reaction, with a volume exchange ratio of 1 / 2.

[0023] SBBR1 aims to enrich bacteria that oxidize ammonia throughout the entire process. The hydraulic retention time changes from 48h to 36h to 24h, shortening every 40 days. During each cycle, except for 5 minutes of influent, 25 minutes of sedimentation, and 5 minutes of effluent, the system is in aeration mode. DO is 0.2-0.5 mg / L, and the influent NH4+ is... + -N was consistently maintained at 20 mg / L, and the temperature was controlled at 28±1℃. The system's efficiency was assessed through routine monitoring of ammonia nitrogen, nitrite, and nitrate nitrogen. After 30 days, the ammonia nitrogen removal efficiency stabilized at approximately 95% and remained at this level until the enrichment process was complete. No abnormal accumulation of nitrite was detected. On day 120, biofilm sludge was collected, and 16S rRNA and metagenomic analysis revealed a 41-fold increase in the abundance of *Nitrospira*, belonging to the *Comammox* species *CladeA*, to 7.0%. Furthermore, *Nitrospira_sp._UW-LDO-01*, *Candidatus_Nitrospira_nitrosa*, and *Candidatus_Nitrospira_nitrificans*, all belonging to the *Comammox* species *CladeA*, were detected. This demonstrates that the enrichment method for end-stage ammonia oxidizing bacteria in this invention can effectively enrich highly active end-stage ammonia oxidizing bacteria.

[0024] SBBR2 is designed to enrich sulfur-autotrophic denitrifying bacteria. The hydraulic retention time (HURD) is changed from 48h to 24h to 12h to 6h, decreasing every 30 days. The reactor is sealed, and each cycle is maintained in anoxic conditions except for 5 minutes of influent, 25 minutes of sedimentation, and 5 minutes of effluent, ensuring the influent contains sufficient S₂O₃. 2-The concentration was 886.4 mg / L, the temperature was controlled at 25±1℃, and the efficiency of the system was evaluated by routine monitoring of nitrate nitrogen, ammonia nitrogen, and nitrite. After 30 days of operation, nitrate nitrogen was basically completely removed, and the removal rate remained stable until the end of enrichment. No abnormal accumulation of nitrite was detected. On day 120, biofilm sludge was collected, and 16S rRNA detection revealed that after enrichment and acclimatization, typical sulfur autotrophic denitrifying bacteria Sulfurimonas, Thiobacillus, and Sulfuritalea became the main genera in the system. The total abundance of sulfur autotrophic denitrifying bacteria increased from 0.39% to 51.6%, an increase of 132 times. This indicates that under this S / N ratio, the enrichment method of immobilized biofilm is very effective.

[0025] Example 2: Constructing a coupled system of full-process ammonia oxidation and sulfur autotrophic denitrification.

[0026] Half of the enriched ammonia-oxidizing bacteria and half of the sulfur-autotrophic denitrifying bacteria were inoculated into SBBR3 cells to construct a coupled system of ammonia oxidation and sulfur autotrophic denitrification. This coupled system is a "dual-state microbial system" that simultaneously contains both biofilm sludge and sedimentary activated sludge. Figure 3 As shown in the figure, the nitrogen source inlet tank (1), sulfur source inlet tank (2), peristaltic pump (3), inlet valve (4), agitator (5), polyurethane sponge packing (6), sedimented activated sludge (7), dissolved oxygen probe (8), pH probe (9), multi-parameter water quality analyzer (10), aeration pump (11), rotor flow meter (12), aeration head (13), outlet valve (14) and outlet tank (15).

[0027] The coupled system operates in 24-hour cycles. After 70 days of operation with 16 hours of aerobic and 8 hours of anoxic cycles, it transitions to 12 hours of aerobic and 12 hours of anoxic cycles. Each cycle includes 5 minutes of influent, 25 minutes of sedimentation, and 5 minutes of effluent. The influent consists of a nitrogen source and a sulfur source. The nitrogen source is 20 mg / L ammonia nitrogen (with an additional 10 mg / L nitrate nitrogen added at the beginning of the anoxic phase from day 30 to day 111). The sulfur source is provided by sodium thiosulfate (NaS₂O₃·5H₂O) at a concentration of 354.56 mg / L, added at the beginning of the anoxic phase. The control system maintains a temperature of 30℃ and a pH of 7±0.5, keeping dissolved oxygen at 0.2-0.5 mg / L during the aerobic phase and below 0.2 mg / L during the anoxic phase. Since some of the SBBR3 activated sludge will flow out with the effluent at these times, no additional sludge discharge is required from the reactor. After 111 days of system operation, the total nitrogen removal rate reached approximately 90%, with both ammonia nitrogen and nitrate nitrogen in the effluent below 2 mg / L, and no nitrite accumulation was detected at any stage of the system. Figure 4As shown, using 16S rRNA and metagenomic techniques, the abundance of Nitrospira (belonging to Commammox) in the fixed biofilm sludge increased from 7.0% after the SBBR1 acclimatization period to 7.18%. The abundances of sulfur autotrophic denitrifying bacteria Thiobacillus and Sulfuritalea were also 5.7% and 2.2%, respectively. At this point, Nitrospira and sulfur autotrophic denitrifying bacteria coexisted in the biofilm. Metagenomic analysis indicated that the abundance of ammonia-oxidizing bacteria throughout the coupled system was slightly increased (from 1.27% to 1.29%). In the sedimentary activated sludge, the abundances of sulfur autotrophic denitrifying bacteria Thiobacillus were 6.47% and Sulfuritalea was 2.84%, while Nitrospira (belonging to Commammox) was 6.26%. The abundance of sulfur autotrophic denitrifying bacteria remained basically unchanged compared to before inoculation, and the abundance of Nitrospira (belonging to Commammox) was also not significantly affected. The above indicates that the synergistic effect of the two types of functional bacteria is also achieved in the sedimentary activated sludge, and the abundance of sulfur autotrophic denitrifying bacteria is higher. Both types of sludge play an important role in the denitrification process of the system.

Claims

1. A highly efficient nitrogen removal method based on the coupling of full-process ammonia oxidation and sulfur autotrophic denitrification, characterized in that, Includes the following steps: (1) Enrichment of key functional bacteria: Polyurethane sponge packing with a filling ratio of 30% was set in both SBBR1 and SBBR2 reactors and fixed to the inner wall of the reactor for a week to promote microbial attachment. SBBR1 was designed to enrich Commammox bacteria. The influent was ammonia nitrogen (concentration of 20 mg / L). The nitrogen load was increased by gradually shortening the hydraulic retention time (HRT from 48 h → 36 h → 24 h). The reaction conditions were maintained at dissolved oxygen 0.2–0.5 mg / L, pH 7.5 ± 0.5, and temperature 28 ± 1 °C for 120 days. SBBR2 aims to enrich sulfur-autotrophic denitrifying bacteria. Sodium thiosulfate is used as the electron donor (concentration of 886.4 mg / L) and nitrate nitrogen is used as the only nitrogen source (concentration of 50 mg / L) in the influent. The nitrogen load is increased by gradually shortening the HRT (48h→24h→12h→6h). During operation, pH and DO are controlled and light-avoidance measures are taken. The concentrations of substrates and products were monitored during the culture process, and finally, biofilm samples were taken for 16S rRNA sequencing and metagenomic analysis to confirm the enrichment of functional bacteria. (2) Start-up of the coupled system: half of the sludge from SBBR1 and SBBR2 was inoculated into the SBBR3 reactor. At this time, the coupled system contained both biofilm sludge and sedimented activated sludge. The same packing method and light-blocking measures were set up. The influent contained 20 mg / L ammonia nitrogen and the sulfur source provided by sodium thiosulfate (10 mg / L nitrate nitrogen was added at the end of the aerobic period from day 30 to day 111). The system was operated in alternating aerobic / hypoxic cycles: 16 hours of aerobic operation followed by 8 hours of hypoxia for the first 70 days, and 12 hours of aerobic operation followed by 12 hours of hypoxia for the next 41 days. The reaction conditions were controlled at pH 7.2 ± 0.2 and DO 0.2–0.5 mg / L during the aerobic phase. Substrate changes were monitored and 16S rRNA sequencing and metagenomic analysis were performed during operation to confirm the coupling effect.

2. The method according to claim 1, characterized in that: (1) The culture medium per liter contains the following components: 20 mg NH4Cl, 50 mg KH2PO4, 75 mg KCl, 50 mg MgSO4·7H2O, 584 mg NaCl, 4 g CaCO3, 1 mL TES, and 1 mL SWS. The TES components (per 1 L) are: 34.4 mg MnSO4·1H2O, 50 mg H3BO3, 70 mg ZnCl2, 72.6 mg Na2MoO4·2H2O, 20 mg CuCl2·2H2O, 24 mg NiCl2·6H2O, 80 mg CoCl2·6H2O, and 1 g FeSO4·7H2O. The SWS components (per 1 L) are: 0.5 g NaOH, 3 mg Na2SeO3·5H2O, and 4 mg Na2WO4·2H2O. (2) The culture medium per liter contains the following components: 50 mg NaNO3, 886.4 mg NaS2O3·5H2O, 600 mg NaHCO3, 6 mg MgCl2, and 3.3 mg KH2PO4; The components in each liter of culture medium described in (3) are based on those in each liter of culture medium described in (1), with an additional 354.56 mg NaS2O3·5H2O (added at the end of aerobic treatment), and an additional 10 mg NaNO3 (added at the end of aerobic treatment) is added from day 30 of the coupled system until the end of the experiment.

3. The reaction system used to initiate the coupling system according to claim 1, characterized in that, It includes the following components: Nitrogen source inlet tank (1), sulfur source inlet tank (2), peristaltic pump (3), inlet valve (4), agitator (5), polyurethane sponge packing (6), sedimented activated sludge (7), dissolved oxygen probe (8), pH probe (9), multi-parameter water quality analyzer (10), aeration pump (11), rotor flow meter (12), aeration head (13), outlet valve (14) and outlet tank (15).

4. The coupled reaction system according to claim 1, characterized in that... Both biofilm sludge and sedimentary activated sludge exist within the system, and both types of sludge play a role in the denitrification process.

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