Method for denitrification of sewage and generation of substrate based on temperature regulation

Abstract

The application provides a temperature regulation-based wastewater denitrification and substrate generation method, wherein the denitrification substrate generation method is characterized in that the substrate is NO2 ‑ , and comprises the following steps: under anaerobic conditions with dissolved oxygen ≤0.5 mg / L, wastewater containing nitrate nitrogen and a carbon source are introduced into a denitrification reactor, the temperature of the reactor is controlled to be 60-75 DEG C, the pH is controlled to be 7.0-9.0, and the hydraulic retention time is ≥48 hours; through temperature selection, a microbial community with Thermus as dominant bacteria is formed in the reactor, the directional conversion of nitrate nitrogen to nitrite nitrogen is driven, and the nitrite nitrogen accumulation rate is ≥90%. Through temperature directional regulation of the denitrification pathway, the application breaks through the core bottleneck of unstable NO2 ‑ substrate supply in the anaerobic ammonia oxidation technology, forms an efficient, low-carbon and low-cost denitrification process chain, and provides an innovative solution for the engineering treatment of high-ammonia-nitrogen wastewater.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment, and more specifically to a method for wastewater denitrification and substrate generation based on temperature control. Background Technology

[0002] Low-carbon emission reduction and achieving energy self-sufficiency are the future trends in wastewater treatment technology development. Anaerobic ammonia oxidation and its coupled processes, as a novel autotrophic denitrification technology, can provide a low-consumption and high-efficiency denitrification pathway for high-nitrogen wastewater such as kitchen waste leachate and sludge digestion liquid, and have very broad research value and application prospects. Under anaerobic conditions, anaerobic ammonia oxidation functional bacteria utilize nitrite nitrogen (NO2) to produce nitrogen. - NO2 acts as an electron acceptor, converting ammonia nitrogen into N2 to remove nitrogen-containing pollutants. - A stable supply of the nitrogen-containing reaction substrate is crucial for achieving anaerobic ammonium oxidation. Nitrogen in high-nitrogen wastewater typically exists in the form of ammonia nitrogen, NO2. - Nitrite (NO2) is unstable and easily converted by various microorganisms, resulting in low concentrations in wastewater. Furthermore, nitrite itself is toxic; artificial addition would increase treatment costs and even cause secondary pollution to water bodies. Therefore, how to achieve NO2 control is crucial. - The stable and continuous accumulation of -N has a profound impact on the promotion and engineering application of anaerobic ammonia oxidation technology.

[0003] Studies have shown that ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) have different degrees of adaptability to the external environment. AOB activity can be enhanced and regulated by optimizing operating parameters such as hydraulic retention time, sludge age, dissolved oxygen, pH, and free ammonia, creating a suitable environment for AOB survival. This inhibits NOB activity and eliminates it from the reaction environment, improving AOB purity and competitive advantage, and ultimately achieving NO2 reduction. - Stable accumulation. However, even so, NO2 still exists in practical engineering applications. - Numerous problems, such as the difficulty in ensuring the concentration of NO2 matrix, surround NO2. - The reaction conditions and regulation of -N generation require the development of new technologies to support them. Summary of the Invention

[0004] The present invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, the present invention provides a method for wastewater denitrification and substrate generation based on temperature control.

[0005] According to one aspect of the present invention, a method for generating a wastewater denitrification substrate based on temperature control is provided, wherein the substrate is NO2. -The process includes the following steps: (1) Under anaerobic conditions with dissolved oxygen ≤0.5mg / L, wastewater containing nitrate nitrogen and carbon source are introduced into a denitrification reactor, and the reactor temperature is controlled at 60-75℃, pH at 7.0-9.0, and hydraulic retention time ≥48 hours; (2) Through temperature selection, a microbial community with Thermus as the dominant bacteria is formed in the reactor, driving the directional conversion of nitrate nitrogen to nitrite nitrogen, and achieving a nitrite nitrogen accumulation rate ≥90%.

[0006] Preferably, the carbon source in step (1) is acetic acid or lactic acid.

[0007] Preferably, the carbon source is acetic acid, and the molar ratio of acetic acid to nitrate nitrogen is 1:3.5-4.5.

[0008] Preferably, in step (1), the dissolved Fe in the reactor 2+ Concentration ≤5mg / L, S 2- Concentration ≤ 0.3 mg / L.

[0009] According to another aspect of the present invention, a wastewater denitrification method based on temperature-controlled denitrification is provided, comprising the following steps: (1) under anaerobic conditions with dissolved oxygen ≤0.5mg / L, wastewater containing nitrate nitrogen and a carbon source are introduced into a denitrification reactor, and the reactor temperature is controlled at 60-75℃, pH at 7.0-9.0, and hydraulic retention time ≥48 hours; (2) through temperature selection, a microbial community with Thermus as the dominant bacteria is formed in the reactor, driving the directional conversion of nitrate nitrogen to nitrite nitrogen, and achieving a nitrite nitrogen accumulation rate ≥90%; (3) the effluent containing high concentration of nitrite nitrogen generated in step (2) is introduced into an anaerobic ammonia oxidation reactor, and anaerobic ammonia oxidation bacteria are used to convert nitrite nitrogen and ammonia nitrogen into nitrogen gas.

[0010] Preferably, the carbon source in step (1) is acetic acid or lactic acid.

[0011] Preferably, the carbon source is acetic acid, and the molar ratio of acetic acid to nitrate nitrogen is 1:3.5-4.5.

[0012] Preferably, in step (1), the dissolved Fe in the reactor 2+ Concentration ≤5mg / L, S 2- Concentration ≤ 0.3 mg / L.

[0013] This invention overcomes the NO2 problem in anaerobic ammonia oxidation technology by using a temperature-directed denitrification pathway. - Overcoming the core bottleneck of unstable substrate supply, a highly efficient, low-carbon, and low-cost denitrification process chain has been developed, providing an innovative solution for the engineering treatment of high-ammonia nitrogen wastewater. By controlling the reaction temperature (60–75℃) and anaerobic conditions, the denitrification pathway is driven to reduce nitrate nitrogen (NO3).- → Nitrite (NO2) - The phase stalled, and experiments verified NO2. - The accumulation rate can reach over 90%. In high-temperature environments, communities dominated by *Thermus* species exhibit genome deletions of downstream genes (norBC, nosZ) and restricted expression of accessory genes, mechanistically blocking NO2. - Further reduction. NO2 in traditional short-path nitration processes. - The concentration fluctuates greatly and is complex to control. This invention achieves continuous, high-concentration NO2 through high-temperature denitrification. - The output directly meets the requirements of the anaerobic ammonium oxidation reaction (NH4). + +1.32NO2 - →N2). Under high temperature conditions (>60℃), the final product of denitrification is NO2. - It is the main component (accounting for >90%), with extremely low N2O generation (not detected or <0.002mmol), avoiding the carbon footprint problem of large-scale N2O release during traditional denitrification. Attached Figure Description

[0014] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0015] Figure 1 The experiment demonstrated the use of hot spring water as a culture medium with the addition of 1 mM NO3. - NO3 in denitrification cultures - Reduction and NO2 - The timeline of formation. Each subplot corresponds to a different hot spring, and NO3 was monitored under controlled conditions. - Reduction and NO2 - The accumulated situation.

[0016] Figure 2 The following are examples of acetate / NO3 at different temperatures. - Denitrification kinetics (A) and final nitrogen species produced (B) in denitrification enrichment cultured under 1:4 conditions. A: NO3- during denitrification. - NO2 - The time-varying changes in acetate concentration are shown. Each panel represents the results of denitrification enrichment at different temperatures. Each data point is the average of three replicates. Red arrows indicate sampling points for metagenomic and metatranscriptomic analyses; B: Each denitrification enrichment is represented by four bars, corresponding to NO3-. - Consumption and NO2 - The generation of N2O and N2.

[0017] Figure 3 It shows the effect of acetate / NO3 - NO3 in representative denitrification concentrates cultured at different temperatures under 1:4 conditions - and NO2 - Temporal variations in concentration and microbial community composition. Panels A, B, C, and D represent enrichments JZ, JXH, DT, and JXL, respectively. Dashed lines separate the data for each enrichment at different temperatures, and a red background indicates the initial conditions for that enrichment. The temperature conditions in the legend are consistent with those in the "Methods" section. The bar chart in the right panel shows the phylogenetic analysis of the 16S rRNA gene sequences, and the line chart in the left panel shows the corresponding data. Error bars represent the standard deviation (SD) of n=3 biological replicates.

[0018] Figure 4 It shows the effect of acetate / NO3 - Presence / deletion of key metabolic genes in eight denitrification concentrates under a 1:4 ratio. Important metabolic genes are represented by binary presence-deletion counts for each taxonomic unit. Each box contains the species classification and relative abundance of all MAGs in the concentrate.

[0019] Figure 5 It shows the effect of acetate / NO3 - Expression of denitrification genes in eight denitrification enrichments under a 1:4 ratio. Heatmaps show total normalized gene expression (TPM) of the denitrification module (A and B), and pie charts show the relative taxonomic contribution of expression (C and D). Panels A and C represent major species, and panels B and D represent minor species. Genes not annotated in the MAG are indicated by gray boxes.

[0020] Figure 6 The edgeR detection in acetate / NO3 was shown. - Results of differentially expressed denitrification cofactor genes in 1:4 ratio cultures. The bar chart shows genes involved in NO2... - The total read counts of key cofactor genes for denitrification (nirNCJ), NO denitrification (norDQ), and N2O denitrification (nosRD) were categorized into two MAGs: major species (A) and minor species (B). For each gene, three bars represent enrichments grouped according to their major denitrification products: NO2 - (H1, JZ, ZY, JXH), N2O (GY, DT), or N2 (JXL, JZL). Colored portions indicate the contribution of different enrichment cultures. Significance of differences between groups is indicated by *: * indicates p < 0.05, ** indicates p < 0.01.

[0021] Figure 7This study demonstrates the role of a temperature-dependent mechanism in the accumulation of denitrification intermediates. The main products of denitrification differ under different temperature conditions, reflecting changes in the microbial community and related gene expression profiles. As the temperature decreases, the products gradually shift from NO2... - The process transforms into N2O, and then back into N2. Green arrows indicate pathways driven by highly expressed genes, black arrows indicate steps encoded by genes that are present but may be poorly expressed, and red crosses mark missing or unexpressed genes. Detailed Implementation

[0022] While the invention can be embodied in many different forms, what is disclosed herein are specific illustrative embodiments that demonstrate the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter.

[0023] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that the following examples are not intended to limit the scope of protection claimed by the present invention, but are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

[0024] The research of this invention has revealed that high-temperature denitrification can be used for the directional production of NO2. - This invention provides a stable substrate for anammox, solving the problem of nitrogen removal from high-ammonia nitrogen wastewater (such as sludge digestate). The invention also discovers that NO3... - It is reduced to different main products at different temperatures, such as NO2. - Or gaseous N. Besides the influence of temperature, differences in microorganisms also lead to differences in denitrification behavior. But NO3... - Under specific temperatures and the action of specific microorganisms, NO2 can be achieved. - The steady and continuous accumulation of [something].

[0025] The above findings are based on the conclusions drawn from the following embodiments.

[0026] Example 1: Mechanism Study Experiment

[0027] In September 2022 and 2024, a total of twelve hot springs with temperature gradients were selected in Tengchong, Yunnan Province, China for on-site measurements and sampling. The hot spring temperatures ranged from 32°C to 76°C, including H1, JZ, and YL at approximately 75°C, ZY and H2 at approximately 65°C, JXH at approximately 60°C, GY, DT, and H5 at approximately 55°C, JXL and WGQ at approximately 45°C, and JZL at approximately 37°C.

[0028] To gain a comprehensive understanding of the denitrification behavior in the sampled hot springs, a denitrification culture was established. The method involved using the hot spring water as the culture medium and adding only approximately 1 mM NO3. - This method preserves the original geochemical conditions of each hot spring to the greatest extent possible, thus more accurately reflecting the in-situ denitrification behavior within the hot springs. To this end, in the field, using a sterile nitrogen-purged syringe, 20 mL of a mixture of hot spring water and sediment (approximately 2:1) from each hot spring is transferred to a pre-deoxygenated, sterilized anaerobic bottle. Then, pre-prepared anaerobic NaNO3 is added. - The solution reached approximately 1 mM NO3 - The final concentration was determined. The bottles were incubated in the hot spring for 2 days, then transferred to a laboratory incubator set to the temperature of the corresponding hot spring. NO3 was monitored. - NO2 - and NH4 + The concentration is used to check the denitrification process.

[0029] After obtaining the overall denitrification behavior of hot springs at different temperatures, the denitrifying community was further enriched using culture media. Acetic acid, a carbon source commonly found in hot springs, was selected as the electron donor for denitrification, and lactic acid was chosen for comparison. Different acetic acid / NO3 ratios were tested. - The ratios include: 1) 2.5 mM acetic acid and 10 mM NO3 - 2) 5mM acetic acid and 10mM NO3 - 3) 10mM acetic acid and 5mM NO3 - And 4) 5 mM lactic acid and 10 mM NO3 - Inoculate 3 mL of the culture obtained in the above steps using hot spring water as the medium into the prepared anaerobic medium (50 mL) with a sterile syringe purged with nitrogen gas, and incubate at the appropriate temperature. Monitor NO3. - NO2 - and NH4 + The concentration was adjusted to monitor the denitrification process. Once denitrification ceased, the enriched material was transferred to fresh medium (10 / 100, v:v) at least three times to obtain a stable community. Different carbon sources (acetic acid or lactic acid) and acetic acid / NO3 ratios were used in the medium. - The enrichment ratios showed a temperature response pattern consistent with the denitrification revealed by the hot spring water.

[0030] The culture medium can contain (18.2 MΩcm water per liter): 0.38 g KH₂PO₄, 0.39 g K₂HPO₄, 0.85 g NaNO₃, 0.1 g MgCl₂·6H₂O, 0.01 g CaCl₂, 0.12 g NaCl, and 5 ml of trace element solution. The prepared basal medium is adjusted to pH 7.5 with 1 M NaOH and then dispensed into 120 ml anaerobic culture flasks (50 ml per flask). Each flask is purged with O₂-free N₂ gas to remove O₂, then sealed with a butyl rubber stopper and aluminum cap, and autoclaved. Before inoculation, the autoclaved anaerobic medium is supplemented (to a final concentration) with 1 ml / L vitamin solution, 2.5 mM sodium acetate, and 2 mg / L FeCl₂·4H₂O, all filtered through a 0.2 μm filter membrane. The trace element solution (per liter) contains the following components: 0.084g MnSO4·H2O, 0.029g ZnSO4·7H2O, 0.029g Co(NO3)2·6H2O, 0.017g CuCl2·2H2O, 0.024g Na2MoO4·2H2O, 0.024g NiCl·6H2O, 0.031g H3BO3, and 4.53g trisodium ethylenediaminetetraacetate. The vitamin solution (per 100mL) contains: vitamin B3 (100mg), vitamin B1 (100mg), vitamin B7 (5mg), vitamin BX (50mg), vitamin B12 (1mg), vitamin B5 (50mg), vitamin B6 (50mg), and folic acid (0.5mg). However, the formulation of the culture medium is not limited to this component. Consistent results were still observed when experiments were conducted using field water with different formulations. The important factors are the carbon source and the ratio.

[0031] Of the eight stable enrichments using culture medium, using 15 NO3 - The denitrification products were carefully quantified. The experiments mainly focused on acetic acid / NO3. - The experiment was conducted at a ratio of 1:4. Additionally, the reaction was carried out using acetic acid / NO3. - Gaseous products were quantitatively identified in fractional samples with ratios of 1:2 or 2:1 to assess the impact of substrate concentration on the results. During denitrification, NO3 was analyzed in the filtered liquid products. - NO2 -- NH4 + The concentrations of acetic acid and N2 were determined using gaseous samples. Calculations involving both the aqueous and gaseous phases were based on the dimensionless Henry's law constant (HCC) of 0.594 for N2 and 0.01541 for N2.

[0032] NO3 was discovered through cultivation using hot spring water and culture medium. -It is reduced to different main products at different temperatures, such as NO2. - Or gaseous N. Besides the influence of temperature, different denitrifying communities in these hot springs caused by other factors may also lead to differences in denitrification behavior. To further distinguish the effects of microbial variation and temperature changes on denitrification behavior, four representative acetic acid / NO3 cultures in the culture medium were selected. - Concentrates at a ratio of 1:4 (JZ at 75℃, JXH at 60℃, DT at 55℃, and JXL at 45℃) were used to test their denitrification capacity under temperature control. For example, the DT concentrate at 55℃ was cultured at 75℃, 65℃, 55℃, and 37℃, and the corresponding cultures were named DT-75℃, DT-65℃, DT-55℃, and DT-37℃, respectively. In each temperature experiment, three parallel groups were established to monitor NO3. - NO2 - and NH4 + The dynamic processes were analyzed, and community composition was assessed using 16S rRNA gene phylogenetic analysis.

[0033] At the end of the denitrification process, the acetic acid / NO3 in the culture medium was extracted using the FastDNA SPIN Kit and HiPure Universal RNA MiniKit42, respectively. - DNA and RNA were extracted from enriched samples at a ratio of 1:4 and from cultures cultured in hot spring water. Microbial community composition was analyzed by high-throughput 16S rRNA gene sequencing under previous conditions using the universal primer pair 515F / 806R.

[0034] acetic acid / NO3 - Metagenomic and metatranscriptomic analyses were performed on enriched samples at a ratio of 1:4 to investigate the taxonomic composition, functional potential, and gene expression patterns of the microbial community. Selected metagenomically assembled genomes (MAGs) and unbinded contigs were reconstructed and used as references for transcriptome matching. The proportion of metatranscriptomic reads mapped to MAGs ranged from 35% to 68%, with an average of 54%. RNA expression was normalized using the TPM metric, allowing for inter-sample comparisons of gene expression. Differentially expressed genes were identified using edgeR based on the total read count of MAG gene transcriptions, with a significance threshold of q < 0.05 and a log2 fold change of 2. The contributions of community structure variation and individual environmental variables were further assessed using R v4.4.2 (www.r-project.org).

[0035] Overall, the hot spring samples exhibited a broad temperature gradient, a relatively narrow pH range, and generally low concentrations of dissolved chemicals with moderate variation. The twelve hot springs ranged in temperature from 32℃ to 76℃, with an average pH of 7.8 ± 1.2. The average dissolved oxygen concentration was 2.4 ± 1.3 mg / L, and the Fe... 2+ The concentration was 1.4 ± 4 mg / L, S 2- The concentration was 0.1 ± 0.2 mg / L. Most samples contained NH4+. + The concentration was below the detection threshold of 0.01 mg / L, while the average NO2 concentration was... - The concentration was 14±7 mg / L. In contrast, the NO3 concentration in the sample was... - The average level was 3±3 mg / L. It is worth noting that when the temperature exceeds 50℃, NO2 produced in the hot spring through ammonium oxidation or nitrate reduction increases. - The average concentration was 15.6±4.8 mg / L, while the average concentration of spring water below 50℃ was 1.42±0.5 mg / L.

[0036] Using hot spring water as the culture medium and adding 1mM NO3 - In the cultures, denitrification products changed with temperature. In cultures above 60°C, NO2 in the six hot springs... - Accumulated to 0.79±0.04 mM (accounting for NO3 reduction) - (97.6±5%). At 55℃, NO2 in the three cultures... - The concentration decreased to 0.28 ± 0.13 mM (accounting for reduced NO3). - (40.7±5%), and further decreased to 0.18±0.18 mM (of the reduced NO3) in three cultures below 45 °C. - 22±19% Figure 1 At higher temperatures (>55℃), NO3... - Reduce the main accumulated NO2 - This is consistent with the higher NO2 levels observed in high-temperature hot springs. - The concentrations were consistent. In some cultures (e.g., YL, H2, and H5), NO2... - The level decreased after reaching its peak, possibly due to the presence of additional electron donors. Nevertheless, NO2 levels in YL (75°C) and H2 (65°C) were lower than those in H5 (55°C). - Accumulation remained higher. Meanwhile, N2O production was also detected in some cultures, with the highest concentration in H5 (55℃), followed by YL (75℃), H2 (65℃), and WGQ (45℃).

[0037] Microbial composition based on 16S rRNA gene phylogenetic analysis was successfully performed on representative cultures at four different temperatures, revealing significant differences between samples. At the order level, Thermales spp. were dominant in the YL sample (75℃), while microorganisms belonging to the Thermales order were also abundant in H2 (65℃). In contrast, Burkholderiales spp. were dominant in H5 (55℃) and WGQ (45℃), suggesting that different dominant denitrifying bacteria are a potential cause of different denitrification products.

[0038] Using culture media tailored to specific temperature conditions, a total of eight denitrification concentrates were successfully recovered from twelve hot springs. These concentrates covered the following culture temperature ranges: 75℃ (H1 and JZ), 65℃ (ZY), 60℃ (JXH), 55℃ (GY ​​and DT), 45℃ (JXL), and 37℃ (JZL). [The text abruptly shifts to a seemingly unrelated topic:] ...in acetic acid and NO3... - NO3 was monitored for nearly 200 hours for all eight enrichments at a ratio of 1:4. - Reduction kinetics. Acetic acid was completely consumed throughout the experiment, and the total denitrification products (NO2) produced at the end of the culture were... - +N2O+N2) accounts for NO3 - Consumption ranges from 83.16% to 96.84%. This assumes the absence of NH4. + According to stoichiometric calculations, the H+ consumed during the oxidation of acetic acid... + Ions account for approximately 62 ± 14% of the total electron equivalent used, consistent with previous findings.

[0039] Overall, the enrichments revealed similar effects of temperature on denitrification products as observed in hot spring water cultures. Generally, in the enrichments at temperatures ranging from 75°C to 60°C (H1, JZ, ZY, and JXH), NO2... - It is NO3 - The main reduction product is NO2 at 55℃ (GY ​​and DT). - N₂O is the dominant product, while below 45℃ (JXL and JZL), N₂ becomes the main product. Figure 2 Specifically, in concentrates above 60°C, NO2... - The average concentration reached 0.32 ± 0.05 mmol, accounting for a significant portion of the reduced NO3. - The concentration of NO3 was 94±3.1%, while no N2O or N2 was detected at the end of the culture. In the enriched sample at 55℃, NO3... - To NO2 - The conversion rate decreased (63% and 54%), producing 0.14 ± 0.02 mmol NO2.- However, these cultures exhibited the highest N2O yield among all enrichments, producing 0.006 and 0.012 mmol N2O, and 0.018 and 0.022 mmol N2, respectively. In contrast, enrichments cultured below 45°C produced higher levels of N2 (0.08 mmol and 0.079 mmol), while NO2... - Accumulation was low (0.03 ± 0.02 mmol, corresponding to 4% and 20%), and N2O levels were extremely low (0.0005 mmol and 0.002 mmol, respectively).

[0040] NO2 at high temperatures (especially above 60°C) - Accumulation occurs at different carbon sources (acetic acid or lactic acid) and between acetic acid / NO3. - The ratio is consistent under (1:2 or 2:1) conditions, although excess carbon source sometimes promotes the accumulation of NO2 at high temperatures (>55°C). - Further reduction to downstream products. The highest N2O concentration was observed in the GY concentrate at 55°C. In contrast, at 37°C, NO2... - There was no accumulation; N2 was the main gaseous product. These findings suggest that substrate limitation is not the cause of gradual denitrification at high temperatures.

[0041] Use culture medium (acetic acid / NO3) - The taxonomic composition of the 1:4) denitrifying culture was determined by 16S rRNA gene sequencing. Temperature was the dominant environmental factor shaping the microbial community structure, explaining 9.4% of the observed variance, followed by nitrite (NO2). - (5.7%) and nitrates (NO3) - The concentration was 4.5%. Throughout the sampling period, the microbial community composition remained stable, with the denitrifying bacteria community in the culture medium closely correlated with that in natural hot spring water. For example, members of the Thermaceae family dominated at temperatures above 55°C, while members of the Burkholderiaceae family dominated below this threshold. This consistency supports the accuracy of the laboratory cultures of this invention in reflecting real-world environmental conditions.

[0042] By artificially adjusting the culture temperature of the denitrification enrichment, a very interesting change occurred in the denitrification behavior: when the two enrichments (JZ and JXH), which were originally cultured above 60℃, were cultured at a lower temperature (45-55℃), NO2... - It remains the main product; conversely, when the enriched products (DT and JXL) that were originally below 45°C were cultured at higher temperatures (60-75°C), there was little or no NO2 at low temperatures. -It begins to accumulate in large quantities. For example, when in acetic acid / NO3 - When DT enrichment is cultured at 55°C under a ratio of 1:4 at 65 or 75°C, NO2... - The concentration increased from 2–3 mM to 7–8 mM. The JXL concentrate did not produce NO2 at 45 °C. - However, when cultured at 75°C, NO2... - Become the main product ( Figure 3 C and 3D).

[0043] The microbial communities in the enrichment also exhibited different responses to temperature regulation. Microorganisms belonging to the genus *Thermus* remained dominant in the JZ enrichment at temperatures ranging from 45°C to 75°C. Figure 3 A). In the JXH enrichment, *Thermus* spp. became the dominant species in the temperature range of 60–75°C, while the abundance of unclassified microorganisms belonging to the genus *Anoxybacillus* gradually increased in the temperature range of 45–60°C. Figure 3 B). When the temperature of the two enrichments (DT and JXL) is raised to 60–75°C, NO2 - Accumulation begins, and the abundance of the genus *Meiothermus* increases. Figure 3 (C and 3D). These temperature-dependent microbial changes indicate that higher temperatures exert more stringent selection pressures on incomplete denitrifying bacteria, leading to the persistence of NO2 even at lower temperatures. - Accumulation. Conversely, communities at low temperatures change upon warming, tending towards incomplete pathways and altering denitrification outcomes. Overall, temperature strongly influences the composition and function of denitrifying communities.

[0044] Metagenomic analysis revealed the presence / deletion of denitrification functional genes. Culture results highlighted the dual impact of temperature on the composition of denitrifying bacteria and the formation of denitrification intermediates. Therefore, eight groups were tested using a medium (acetic acid / NO3). - Multi-omics analysis was performed on denitrification enrichment at a ratio of 1:4 to investigate the underlying molecular mechanisms. First, metagenomic analysis was applied to examine the composition of the denitrification community and to examine the integrity of key denitrification genes at different temperatures.

[0045] MAGs revealed systematic changes in denitrifying bacterial communities at different temperatures. Figure 4The 16S rRNA gene profiles were consistent with those from hot spring water cultures and culture media. GenomeSPOT predictions also showed that the optimal growth temperature for these MAGs closely matched the in situ hot spring conditions. Various thermophilic bacteria (Thermus spp.), such as T. brockianus and T. antranikianii, were dominant in H1 (85.3%) and JZ (75.3%) concentrates at 75°C, ZY concentrates at 65°C (63.2%), and JXH concentrates at 60°C (41.3%), but were absent in concentrates below 55°C. These thermophilic bacteria (Thermus spp.) lacked some core denitrification genes (such as norBC, nosZ) and most accessory genes, but encoded eNOR, which has recently been reported to have NO reduction capabilities. *Anoxybacillus voinovskiensis* also became an important species at 60–65°C (accounting for 11% and 46% of the ZY and JXH enrichments, respectively), encoding the complete denitrification pathway and several accessory genes. At 55°C, *Tepidimonas taiwanensis* replaced *Thermus spp.* as the dominant species, accounting for 91% and 75% of the GY and DT enrichments, respectively. Although encoding the complete upstream denitrification pathway, *T. taiwanensis* lacked the nosZ gene. Interestingly, in all six enrichments above 55°C, the nosZ gene was encoded by low-abundance microorganisms belonging to the phyla Armatimonadetes and Gemmatimonadetes. In contrast, at lower temperatures, *Thauera hydrothermalis* and *Uliginosibacterium gangwonense* dominated the JXL (68%, 45°C) and JZL (35%, 37°C) enrichments, respectively, and both encoded complete core and auxiliary denitrification gene sets. The low-abundance taxa at these temperatures were more diverse, belonging to genera such as *Azospirillum* and *Azoarcus*.

[0046] Transcriptional activity of core denitrification genes at different temperatures. Metatranscriptome analysis was performed to assess the expression and activity of denitrification genes. At the end of the exponential phase (just in time for NO2...) - (before the peak is generated) Figure 2 A) From denitrifying culture (acetic acid: NO3) -RNA was extracted at a ratio of 1:4 to capture actively transcribed RNA. To account for the differences in the potential contribution of species with different abundances to the denitrification process, MAGs in each enrichment group were divided into dominant species (>30%). Figure 4 ) and minor species (<30%), in order to conduct more direct comparative analysis.

[0047] The core denitrification genes of major species (i.e., those used for NO3) - Reduced narGHI, used for NO2 - The expression integrity of reduced nirK / nirS, norBC for NO reduction, and nosZ for N2O reduction generally increases with decreasing temperature. Figure 5 A). Specifically, *Thermus* spp. expresses narGHI and nirK / nirS, but at lower levels in the H1 and JZ enrichments at 75°C, while expression levels are quite high in the ZY enrichment at 65°C. This is consistent with its genomic composition. Figure 4 NonorBC or nosZ expression was detected in *Thermus* spp. Figure 5 (A and C). In the JXH enrichment at 60℃, *Anoxybacillus voinovskiensis* was the dominant species, and all core denitrification genes (narGHI, nirK / nirS, norBC, nosZ) were expressed, with narGHI expression significantly higher than downstream genes. In the GY and DT enrichments at 55℃, *Tepidimonas taiwanensis* was the dominant species, with narGHI, nirS, and norBC genes highly expressed, while nosZ was absent. Finally, in the JXL and JZL enrichments below 45℃, the dominant species belonging to the genera *Thauera* and *Uliginosibacterium* expressed all denitrification genes relatively evenly, consistent with complete denitrification leading to N2 production.

[0048] Secondary species exhibit more complex denitrification gene expression patterns, which vary with microbial diversity at different temperatures. Figure 5(B and D). Notably, genes typically missing in the major species of thermophilic enrichments (>55°C), such as norBC and nosZ, are transcribed by minor species belonging to the genera *Thermus*, *Armatimonadetes*, *Anoxybacillus*, *Thermonicrobium*, and *Gemmatimonadetes*. In enrichments below 45°C, the diversity of minor species increases, with many belonging to α- and γ-proteobacteria. These diverse minor species express denitrification genes for all steps, indicating a broader contribution to the denitrification process at lower temperatures.

[0049] Besides denitrification genes, the nrfA and nirB genes involved in DNRA are also expressed in the major species of the enrichment. Above 60°C, microorganisms belonging to the genera *Thermus* and *Anoxybacillus* express nrfA, while below 55°C, nirB expression is more prevalent. Figure 5 Despite this expression, NH4 was not detected in the culture. + The production of NH4. Interestingly, the genes encoding glutamine synthase (glna) and glutamate synthase (gltb), which are crucial for ammonium assimilation, were expressed in all enrichments, suggesting that NH4 may be produced via DNRA. + It may be rapidly assimilated into the biomass, preventing its accumulation.

[0050] Expression of denitrification accessory genes at different temperatures. While analyzing the transcriptional activity of core denitrification genes provides a framework for understanding the accumulation of metabolic intermediates observed at different temperatures, some differences remain. For example, the dominant species encoding and weakly expressing the nirS gene above 60°C showed no detectable NO2. - Therefore, this invention further quantifies the transcriptional abundance of accessory genes involved in cofactor biosynthesis and protein assembly, which are crucial for the function of core denitrification genes. Figure 6 Specifically, this invention targets nirNCJ (synthesizing heme d1 and integrating it into NirS), norDQ (promoting non-heme iron insertion to support NorBC function), and nosRD (regulating nosZ transcription and possibly mediating electron transport to NosZ).

[0051] In the enriched samples (H1, JZ, ZY, and JXH) above 60℃, most denitrification accessory genes ( Figure 6 The transcription level of B) was uniformly low / absent in all detected MAGs. Figure 6A). In particular, the lack of nirNCJ may limit the reduction of NO2 by NirS. - The activity, even though nirS ( is present and expressed in these major species) Figure 5 A). In contrast, in the 55°C enrichments (GY and DT), the major species highly expressed nirNCJ and norDQ ( Figure 6 A), which will support the activity of nirS and norB, which are highly expressed in these species. Figure 5 A). Meanwhile, nosRD was not detected because nosZ was not expressed in the major species GY and DT. In the JXL and JZL enrichments below 45°C, most accessory genes were expressed, with nosRD transcription levels significantly higher than in other enrichments, indicating that the activity of core denitrification genes expressed in these cultures is not limited by accessory genes. Overall, the observed nosDQ and nosRD transcriptional abundance patterns correlated with the respective N2O and N2 production levels in different enrichments, highlighting their potential roles in regulating the denitrification process and its gaseous end products.

[0052] Temperature-driven stoichiometry of denitrification products. Along the temperature gradient, NO3... - The final product of denitrification undergoes a systematic transformation, shifting from N2 at low temperatures (<45℃) to NO2 at medium to high temperatures (45-55℃). - The mixture with N2O eventually reacts at high temperatures (>60°C) to form NO2. - Primarily, this has been revealed in denitrifying bacteria, and this invention finds that it is regulated by a combined effect of the interrelationship between the expression and activity of these genes driven by denitrifying microorganisms, changes in the integrity of denitrifying gene clusters, and temperature variations. Figure 7 Consistently, the phenomenon of increased denitrification and N2O production stimulated by rising temperatures from ambient to medium-high levels has been widely observed in various habitats. However, a significant difference emerges when the results of this invention are compared with earlier studies focusing on thermophilic denitrification isolates. Previous culture-based experiments using thermophilic strains at high temperatures (>60°C) did not observe NO2. - A clear preference for NO2 over N2O emerges. This inconsistency likely stems at least in part from differences in carbon substrate availability and electron donor characteristics. Previous studies have frequently used complex organic substrates (e.g., yeast extracts), which may provide excess electrons to promote NO2 production. - Further reduction or even complete denitrification. In contrast, this study used acetic acid, the main fermentation product and primary electron donor in the hot spring, which better simulated the in-situ conditions but may have limited NO2 production. - The restoration.

[0053] In addition, a low C / N ratio has previously been reported to be beneficial for NO3. - NO2 is produced by reduction - However, in this study, at high temperatures, regardless of whether acetic acid was limited or sufficient ( Figure 2 NO2 - The continued accumulation of NO2 suggests that temperature, rather than simply carbon limitation, plays a dominant role in shaping this metabolic outcome. - Accumulation may reflect an adaptive strategy by which denitrifying microorganisms optimize energy efficiency under thermophilic conditions. Under carbon-limited conditions in temperate environments, denitrifying bacteria have been found to preferentially suppress NO2 production. - Thermophilic bacteria conserve energy for carbon storage to ensure long-term survival. This energy-saving strategy appears to be further enhanced at high temperatures, even when carbon is abundant. Under high-temperature stress, thermophilic bacteria face higher metabolic demands to maintain core metabolism and synthesize thermoadaptive molecules, such as maintaining RNA and protein homeostasis, synthesizing heat shock proteins, and maintaining elevated energy production rates (as shown in the transcriptomic analysis of this invention). Therefore, thermophilic denitrifying bacteria may still prioritize core survival functions over complete NO3 production. - Reduction first leads to NO2 - accumulation.

[0054] Metabolic division of labor within microbial communities is another factor contributing to the accumulation of intermediate products such as N2O, particularly when N2O reduction is carried out by minor species at high temperatures. Previous studies have attributed enhanced N2O production at higher temperatures to the incomplete denitrification capacity of thermophilic denitrifying bacteria or the higher activity of NOR enzymes relative to NOS enzymes, while the understanding of subsequent N2O reduction remains limited. The findings of this invention provide supplementary insights into this process. Gemmatimonadetes spp. are potential N2O-reducing bacteria found in soil environments, while N2O reduction by Armatimonadetes spp. at higher temperatures (>60°C) has not been reported. However, this invention provides only transcriptomic evidence supporting this function above 60°C, but N2 production was detected under acetic acid-rich conditions. Interestingly, this metabolic division of labor was exclusively observed in the higher-temperature samples of this invention, suggesting that it may represent a potential cooperative strategy for denitrifying communities to adapt to thermal environments. Since thermophilic major denitrifying bacteria typically have smaller genomes, they may rely more heavily on population-level cooperation to compensate for functional deficiencies.

[0055] Regulation of denitrification-related gene expression under temperature variations. Despite the influence of various environmental factors, denitrification products are fundamentally regulated by the presence and expression of denitrification-related genes. In this study, major denitrifying bacteria at high temperatures generally encode incomplete denitrification pathways. Surprisingly, NO2... - The reducing genes nirS / nirK are widely present in cultures above 60°C, but NO2... - The accumulation of these genes remains substantial, which may be attributed to differential expression and / or activity of denitrifying genes under temperature stress. Transcriptome data indicate that the expression patterns of major denitrifying bacteria at different temperatures are consistent with phenotypic data (e.g., expression of the upstream gene narG is higher than nirS at high temperatures, while denitrifying gene expression is similar at different steps at low temperatures). This may stem from inherent differences in the heat tolerance of denitrifying enzymes or represent adaptive self-regulatory strategies of microorganisms (e.g., the heat adaptation strategies discussed above).

[0056] The expression of essential denitrification accessory genes is another factor in the function of core denitrification genes and the formation of their products. Although some natural denitrifying enzymes have been reported to be accessory protein independent, their function is often impaired without proper cofactor assembly. Indeed, physiological and biochemical studies have demonstrated that accessory proteins are crucial for optimal denitrification pathway activity. For example, studies on hydrocarbon-degrading marine bacteria (Marinobacter spp.) have highlighted delays in the reduction of downstream denitrification products, attributed to the need for cofactor-dependent enzyme maturation. Furthermore, nirS expression, relative to nirK, depends on a broader suite of accessory proteins. In summary, the findings of this invention provide a mechanistic explanation for temperature-driven changes in denitrification products, enhancing our understanding of the temperature-dependent regulatory mechanisms in the denitrification pathway.

[0057] Example 2: Pure bacteria verification of directed NO2 accumulation during high-temperature denitrification -

[0058] Thermus sp. AA / 04. Basic inorganic salt solution (pH 7.8); nitrate nitrogen (NO3) - ) Concentration 2mM; Carbon source acetic acid (concentration 0.5mM, satisfying acetic acid:NO3) - =1:4); Dissolved oxygen ≤0.5mg / L (continuous N2 purging). Constant temperature anaerobic culture flask (75℃), hydraulic retention time (HRT) = 20 days.

[0059] Test Results

[0060]

[0061]

[0062] Example 3: Temperature gradient verification of product differentiation patterns

[0063] Using the same Thermus sp. AA / 04, other conditions were kept constant (acetic acid: NO3). - =1:4, pH 7.8), the effect of temperature on the product was tested, and the other conditions were the same as in Example 2.

[0064] 75℃ 92％ 45℃ 7％ 37℃ 3％

[0065] Conclusion: Temperatures above 60℃ are essential for maintaining Thermus dominance and high NO2 levels. - An irreplaceable temperature window for accumulation rate.

[0066] Example 4: The effect of carbon source ratio on NO2 - The cumulative effect

[0067] Using the same Thermus sp. AA / 04, at a constant temperature of 75℃ and pH 7.8, the acetic acid / NO3 ratio was varied. - The proportions and other conditions are the same as in Example 2.

[0068] 1:4 92％ 2:1 78％

[0069] Conclusion: Excessive carbon source leads to excess reducing power, reducing NO2. - Targeted conversion efficiency.

[0070] Example 5: Subsequent Processing

[0071] The chemical equation for the anaerobic ammonia oxidation reaction is: NH4 + +1.32NO2 - →1.16N2+2H2O.

[0072] Anaerobic ammonia oxidation (Anammox), as an emerging biological treatment technology, has gradually gained attention in the field of wastewater treatment in recent years, demonstrating its potential for efficient and sustainable nitrogen removal. Anaerobic ammonia-oxidizing bacteria (AnAOB) can remove nitrogen as nitrite (NO2) under anaerobic conditions. - As an electron acceptor, ammonia nitrogen (NH4) + -N) is directly converted into nitrogen (N2) without relying on dissolved oxygen (DO) or an external organic carbon source. However, in practical applications, stable formation of NO2 is required. - -N is a key challenge. Based on the findings of this invention, continuous accumulation of NO2 can be achieved. — The effect of N is to stably provide NO2. —While reducing nitrogen (N2O), the formation of the greenhouse gas nitrous oxide (N2O) can be minimized. As a process improvement, wastewater containing nitrate nitrogen can be fed into a high-temperature denitrification reactor along with a carbon source under anaerobic conditions with dissolved oxygen ≤0.5 mg / L. The reactor temperature can be controlled at 60-75℃, pH at 7.0-9.0, and the hydraulic retention time at ≥48 hours. Through temperature selection, a microbial community dominated by thermophilic bacteria can form within the reactor, driving the directional conversion of nitrate nitrogen to nitrite nitrogen, thus achieving NO2 reduction. - The accumulation rate is ≥90%, and furthermore, it will produce high concentrations of NO2. - The effluent is introduced into an anaerobic ammonia oxidation reactor, where anaerobic ammonia oxidizing bacteria convert NO2 into nitrogen. - With NH4 + Convert to N2.

[0073] For those skilled in the art, the resulting high concentration of NO2 - The effluent is introduced into an anaerobic ammonia oxidation reactor, where anaerobic ammonia oxidizing bacteria convert NO2 into nitrogen. - With NH4 + The conversion to N2 is a mature experience in this field. As pointed out in the article "Research Progress on the Supply Process and Enhancement Strategy of Anaerobic Ammonia Oxidation of Nitrite", the biological denitrification process based on anaerobic ammonia oxidation is considered a feasible technology for energy-neutral urban wastewater treatment. It is generally achieved through two processes: short-cut nitrification coupled with anaerobic ammonia oxidation (PNA) or short-cut denitrification coupled with anaerobic ammonia oxidation (PDA).

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for generating a wastewater denitrification substrate based on temperature control, characterized in that, The matrix is This includes the following steps: (1) Under anaerobic conditions with dissolved oxygen ≤0.5 mg / L, wastewater containing nitrate nitrogen and a carbon source are introduced into a denitrification reactor, and the reactor temperature is controlled at 60-75℃, pH at 7.0-9.0, and hydraulic retention time at ≥48 hours. The carbon source is acetic acid, and the molar concentration ratio of acetic acid to nitrate nitrogen is 1:3.5-4.

5. (2) Through temperature selection, a microbial community with Thermus as the dominant bacteria is formed in the reactor, which drives the directional conversion of nitrate nitrogen to nitrite nitrogen, and achieves an accumulation rate of ≥90% of nitrite nitrogen.

2. The method according to claim 1, characterized in that: Solubility in the reactor during step (1) Concentration ≤5mg / L Concentration ≤ 0.3 mg / L.

3. A wastewater denitrification method based on temperature-controlled denitrification, characterized in that, Includes the following steps: (1) Under anaerobic conditions with dissolved oxygen ≤0.5 mg / L, wastewater containing nitrate nitrogen and a carbon source are introduced into a denitrification reactor, and the reactor temperature is controlled at 60-75℃, pH at 7.0-9.0, and hydraulic retention time at ≥48 hours. The carbon source is acetic acid, and the molar concentration ratio of acetic acid to nitrate nitrogen is 1:3.5-4.

5. (2) Through temperature selection, a microbial community dominated by Thermus is formed in the reactor, driving the directional conversion of nitrate nitrogen to nitrite nitrogen, and achieving a nitrite nitrogen accumulation rate of ≥90%; (3) The effluent containing high concentration of nitrite nitrogen generated in step (2) is introduced into the anaerobic ammonia oxidation reactor, and the anaerobic ammonia oxidation bacteria are used to convert nitrite nitrogen and ammonia nitrogen into nitrogen gas.

4. The method according to claim 3, characterized in that: Solubility in the reactor during step (1) Concentration ≤5mg / L Concentration ≤ 0.3 mg / L.

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