Method for chemical-biological treatment of nitrogen contaminants

Abstract

The present disclosure provides a chemical-biological treatment method for removing nitrogen contaminants from nitrogen-containing wastewater. The method comprises pre-treating the nitrogen-containing wastewater with an oxidant in the presence of a catalyst to form an oligomer mixture; precipitating the oligomer mixture by further addition of an oxidizing agent to obtain a solid precipitate and a supernatant wherein the supernatant comprises one or more inorganic nitrogen compounds; separating the supernate from the solid precipitate; and contacting the supernatant with a microbial culture to convert the one or more inorganic nitrogen compounds to nitrogen wherein the catalyst comprises an iron-tetraamido macrocyclic ligand (Fe-TAML) or a peroxidase; and wherein the microbial culture comprises denitrifying bacteria and anaerobic ammonia oxidizing bacteria.

Description

[0001] Cross-reference with related applications

[0002] This application claims priority to Singapore Patent Application No. 10202403153X, filed on October 9, 2024, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] This disclosure generally relates to a chemical-biological treatment method for nitrogen pollutants. More specifically, this disclosure relates to a chemical-biological treatment method for removing nitrogen pollutants from wastewater. Background Technology

[0004] Nitrogen pollution is one of the environmental challenges we face today. Both organic and inorganic nitrogen pollutants are produced by humans as plastics and fertilizers, but these products often end up in the environment as pollutants. For a long time, biological processes have been considered a suitable and economical method for removing nitrogenous pollutants such as ammonia and nitrates from wastewater. For example, in nitrification / denitrification, ammonia nitrogen is oxidized to nitrite, nitrate, and ultimately N2. However, these processes are too slow for high-intensity industrial wastewater. The processes are also hampered by toxic substances such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and other compounds such as surfactants.

[0005] In chemical treatment processes, advanced oxidation processes (AOPs) are commonly used to treat organic nitrogen pollutants such as amines, nitrosamines, and nitriles. In AOPs, oxidants such as ozone are used to oxidize the pollutants into ammonia or nitrates. Although AOPs are an effective method for treating nitrogen pollutants, they are costly to operate due to the expense of reagents and energy. Unwanted byproducts that are more toxic than the original pollutants may also be formed during AOP processes.

[0006] Therefore, it is desirable to provide an alternative treatment method for nitrogen pollutants in wastewater, which seeks to solve at least one of the above-mentioned problems, or at least provide an alternative solution. Summary of the Invention

[0007] In one aspect, a chemical-biological treatment method for removing nitrogen pollutants from nitrogen-containing wastewater is provided. The method includes pretreating the nitrogen-containing wastewater with an oxidant in the presence of a catalyst to form an oligomer mixture; precipitating the oligomer mixture by further adding an oxidant to obtain a solid precipitate and a supernatant, wherein the supernatant contains one or more inorganic nitrogen compounds; separating the supernatant from the solid precipitate; and...

[0008] The supernatant is contacted with a microbial culture to convert one or more inorganic nitrogen compounds into nitrogen gas, wherein the catalyst comprises an iron-tetraamide macrocyclic ligand (Fe-TAML) or a peroxidase; and wherein the microbial culture comprises denitrifying bacteria and anaerobic ammonia-oxidizing bacteria. Attached Figure Description

[0009] In the accompanying drawings, similar reference numerals in different views generally refer to the same parts. The drawings are not necessarily drawn to scale; rather, the emphasis is usually on illustrating the principles of various embodiments. In the following description, various embodiments of the invention are described with reference to the accompanying drawings.

[0010] Figure 1A and 1B The effect of the catalyst Fe-TAML on the oxidation of 1 g / L aniline with 1 g / L hydrogen peroxide (H2O2) at 60 °C was described. Figure 1A The results showed that, in the presence of 3 mg / L Fe-TAML and H2O2, the aniline-containing solution darkened after 1 h. After 24 h, the solution remained dark and a solid precipitate formed at the bottom. Figure 1B This is a control experiment, showing that when a solution containing aniline is oxidized in the absence of Fe-TAML, only a slight color change is observed after 24 h.

[0011] Figure 1C The graph shows the aniline conversion rate as a function of Fe-TAML dosage after 24 h.

[0012] Figures 2A to 2C It is a bar chart, showing the bars in ( ) Figure 2A 20℃, ( Figure 2B 40℃ and ( Figure 2C Precipitates formed at 60℃ using different reaction times (w) s ) and suspended solids (w p The total dry weight of H2O2 was fixed at 1 g / L.

[0013] Figure 3 The effects of H2O2 concentration and temperature on the formation of solid precipitates are shown.

[0014] Figure 4A and 4B A two-step chemical process for the pretreatment of aniline wastewater according to some embodiments of this disclosure is shown.

[0015] Figure 5 This is a diagram illustrating the removal of ammonium and nitrite using highly enriched bacterial cultures according to some embodiments of this disclosure. Detailed Implementation

[0016] The following description illustrates exemplary methods, parameters, etc. To enable those skilled in the art to practice the invention, the embodiments have been described in sufficient detail. Other embodiments may also be used, and structural and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments.

[0017] Features described in the context of one embodiment may be applicable accordingly to the same or similar features in other embodiments. Features described in the context of one embodiment may be applicable accordingly to other embodiments, even if not explicitly described in those other embodiments. Furthermore, additions and / or combinations and / or substitutions described for features in the context of one embodiment may be applicable accordingly to the same or similar features in other embodiments.

[0018] In the context of the various implementations, the use of designations without a specific number for a feature or element includes designations for one or more of the features or elements.

[0019] In the context of the various implementations, the term “about” or “approximately” applied to numerical values ​​covers both precise values ​​and reasonable variations, such as within 10% of the specified value.

[0020] As used herein, the term “and / or” includes any and all combinations of one or more of the items listed herein.

[0021] "Includes" means, but is not limited to, anything that follows the word "includes". Therefore, the use of the term "includes" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present.

[0022] "Composed of" means including but not limited to anything that follows the phrase "composed of". Therefore, the phrase "composed of" indicates that the listed elements are necessary or mandatory, and no other elements may exist.

[0023] In one aspect, this disclosure relates to a chemical-biological treatment method for removing nitrogenous pollutants from nitrogenous wastewater. The method includes pretreating the nitrogenous wastewater with an oxidant in the presence of a catalyst to form an oligomer mixture; precipitating the oligomer mixture by further adding an oxidant to obtain a solid precipitate and a supernatant, wherein the supernatant contains at least one or more inorganic nitrogen compounds; separating the supernatant from the solid precipitate; and contacting the supernatant with a microbial culture to convert the one or more inorganic nitrogen compounds into nitrogen gas, wherein the catalyst comprises an iron-tetraamide macrocyclic ligand (Fe-TAML) or a peroxidase; and wherein the microbial culture comprises denitrifying bacteria and anaerobic ammonia-oxidizing bacteria.

[0024] As used herein, "nitrogenous wastewater" refers to a stream of wastewater that may contain nitrogen pollutants in various chemical forms. This nitrogenous wastewater may contain organic nitrogen pollutants, such as amines (including aniline and nitrosamines), nitriles, and other nitrogen-containing organics, and / or inorganic nitrogen pollutants or compounds, such as ammonium, ammonia, nitrites, and nitrates. The nitrogenous wastewater may be obtained from any source, including industrial, agricultural, aquaculture, or domestic sources. Aniline is one of the most common amines found in industrial wastewater. In one exemplary embodiment, the nitrogenous wastewater may contain aniline at a concentration of 1 g / L.

[0025] The oxidant can be any suitable oxidant used for treating nitrogen-containing wastewater. Such oxidants may include, but are not limited to, hydrogen peroxide, oxygen, ozone, sodium percarbonate, and hypochlorite. In various embodiments, the oxidant includes hydrogen peroxide (H₂O₂). In various embodiments, the oxidant used in the pretreatment step has a concentration of 1 g / L.

[0026] The pretreatment step is carried out in the presence of a catalyst. Iron-tetraamide macrocyclic ligands (Fe-TAML) and peroxidases are considered green catalysts that do not produce or lead to the formation of toxic byproducts during the oxidation of nitrogenous wastewater. For example, the metal center of Fe-TAML can be activated by hydrogen peroxide to form a highly active reagent, which can then react with the amino group of a nitrogenous contaminant (e.g., aniline) to initiate an oligomerization reaction. In some embodiments, the catalyst comprises Fe-TAML. In other embodiments, the catalyst comprises peroxidase. The catalyst plays an important role in the oxidation reaction. For example, in the presence of Fe-TAML, the oxidation of aniline in nitrogenous wastewater can be completed within one hour, while in the absence of Fe-TAML, oxidation does not occur even after 24 hours. For example, the degree of aniline conversion may vary depending on the amount of catalyst used. In various embodiments, the catalyst used in the methods of this disclosure has a concentration of 3 mg / L.

[0027] In various embodiments, the pretreatment step is carried out under neutral or alkaline conditions at ambient temperature.

[0028] In various embodiments where the nitrogenous wastewater contains aniline, the pretreatment step produces oligomers having 3-6 monomer units and some inorganic nitrogen compounds (including ammonium and nitrates). In conventional methods, the oligomers form a suspension and cannot be easily separated without additional membrane filtration or centrifugation. This problem is addressed in the method of this disclosure by a treatment process that forms a solid precipitate at the end of the reaction, thereby allowing for easy separation of contaminants in the form of a solid precipitate.

[0029] In various embodiments, precipitation of the oligomer mixture is achieved by adding an additional oxidant to continue the oxidation reaction (but at a higher temperature). In various embodiments, the precipitation step can be carried out at a temperature in the range of 60°C to 80°C. In some embodiments, the precipitation step can be carried out at a lower temperature (e.g., 20°C to 40°C) for a longer time, while the precipitation yield is reduced. The precipitation duration can be 24 hours or longer.

[0030] In the precipitation step, increasing the temperature or heating to 60°C to 80°C or 80°C may promote the cyclization reaction. Products such as N-phenylphenazine may be formed, and these can be separated from the solution as solid precipitates.

[0031] In some embodiments, the concentration of the oxidant added in the precipitation step is in the range of 1 g / L to 10 g / L. In one exemplary embodiment, 1 g / L H2O2 may be added to continue the oxidation reaction.

[0032] The supernatant obtained from the precipitation step contains one or more inorganic nitrogen compounds. In various embodiments, the inorganic nitrogen compounds are selected from ammonium, nitrates, and nitrites. In some embodiments, nitrates and ammonium, as byproducts of the oxidation reaction, are detected after pretreatment of the nitrogen-containing wastewater. In other embodiments, nitrates and ammonium, including trace amounts of nitrite, are detected as byproducts of the oxidation reaction after pretreatment of the nitrogen-containing wastewater.

[0033] In some embodiments, nitrite is produced by the microbial culture during denitrification. Specifically, the supernatant has a carbon-to-nitrogen ratio of less than 0.5. This allows the denitrifying bacteria and anaerobic ammonia-oxidizing bacteria to convert nitrate in the supernatant into nitrite.

[0034] In some embodiments, the method further includes adding a carbon source before or during the step of contacting the supernatant with the microbial culture to adjust the carbon-to-nitrogen ratio of the supernatant to less than 0.5, allowing the denitrifying bacteria and anaerobic ammonia-oxidizing bacteria to convert nitrates in the supernatant into nitrites. This applies when the supernatant may be carbon-free. In some exemplary embodiments, the carbon source may include sodium acetate, lactate, or glycerol.

[0035] After nitrate is converted to nitrite, the supernatant mainly contains ammonium and nitrite. Under anaerobic conditions, in the absence of a carbon source, both ammonium and nitrite are simultaneously converted to nitrogen gas by the microbial culture, without any byproducts. In various embodiments, under anaerobic conditions, the ammonium and nitrite in the supernatant are classified as... The bacteria convert the gas into nitrogen.

[0036] In various embodiments, the denitrifying bacteria belong to the genus *Taurella* (…). ), and the anaerobic ammonia-oxidizing bacteria belong to Specifically, the denitrifying bacteria are species of the genus *Tauriella*. N101, and the anaerobic ammonia-oxidizing bacteria are G56. In some embodiments, a *Daucus* species N101 containing two bacterial strains and... G56 uses highly enriched microbial cultures to treat inorganic nitrogen compounds in the nitrogen-containing wastewater.

[0037] Some bacteria require very little carbon to sustain their growth. For example, G56 requires very little carbon to sustain its growth and prevents further nitrite reaction. In such embodiments, the carbon-to-nitrogen (C / N) ratio can be less than 0.5, depending on the carbon requirements of the microbial culture. Other suitable C / N ratio ranges can be used for denitrification with other types of denitrifying bacteria and anaerobic ammonia-oxidizing bacteria.

[0038] The solid precipitate can be separated from the supernatant using any suitable separation method. Examples of separation methods include, but are not limited to, filtration, centrifugation, decantation, precipitation, and combinations thereof. The choice of separation method may depend on a variety of factors, such as the particle size of the solid, the concentration of the solid in the liquid phase, and cost considerations. In some embodiments, the separation is performed by decanting the supernatant after the solid particles have deposited to form the solid precipitate.

[0039] Advantageously, this disclosure provides a method for overcoming the challenges of advanced oxidation processes, such as the Fenton reaction. Typically, the Fenton reaction is used to treat high-intensity industrial wastewater that is not biodegradable. Although the Fenton reaction is effective, it is costly to operate due to the nonspecificity of the chemical reaction. Furthermore, the Fenton reaction occurs only within a very narrow pH range (2.8–3.2). Therefore, pH adjustment is necessary before treatment. At the end of the reaction, ferrous ions are oxidized to ferric ions and precipitate along with the remaining contaminants as iron sludge. Incineration is the only solution due to the hazardous nature of this sludge, but incineration is costly and subject to strict government regulation due to air pollution.

[0040] Compared to traditional biological processes for treating inorganic nitrogen, the method disclosed in this disclosure is more versatile and energy-efficient. It utilizes only hydrogen peroxide, bacteria, and a small amount of metal catalyst, making it a green and environmentally friendly process. Hydrogen peroxide is considered green because it decomposes into water and oxygen after use. As for the nitrogen pollutants, the final products are oligomers and nitrogen gas. The former can be collected and used as a supercapacitor. The latter can be released into the air. The method is applicable to the removal of both organic and inorganic pollutants, and the combination of chemical and biological processes results in high treatment efficiency and low cost.

[0041] Furthermore, the method disclosed herein achieves a 96.8% removal efficiency for aniline, demonstrating its effectiveness in treating industrial wastewater containing nitrogenous contaminants, making it a cost-effective solution for industrial applications. The highly enriched microbial culture exhibits high activity towards inorganic nitrogen compounds. It is capable of simultaneously converting ammonium, nitrite, and nitrate into nitrogen gas without any inhibition or formation of any undesirable byproducts. The formation of a solid precipitate after the chemical treatment allows for easy separation of the reaction products from water. This feature simplifies downstream separation processes and reduces the need for complex and expensive separation technologies.

[0042] To facilitate a better understanding of this disclosure, embodiments of specific implementations are provided below. These embodiments should not in any way be construed as limiting or defining the entire scope of this disclosure. Those skilled in the art will recognize that the embodiments described below are not an exhaustive list of implementations of this disclosure.

[0043] Example

[0044] Example 1 : Chemical process for treatment of aniline waste water

[0045] 1.1 Effect of catalyst on aniline oxidation

[0046] The effect of catalysts on the oxidation of aniline was investigated. Because high-strength wastewater containing organic nitrogen pollutants is too toxic for biological treatment, a chemical process was introduced prior to biological treatment to reduce its toxicity. Here, synthetic wastewater containing 1 g / L aniline was used as a model system.

[0047] First, 1 g / L H₂O₂ was added to the solution, with an initial pH of 6.9. The reaction was carried out at 60 °C for 24 h. To investigate the effect of the catalyst, 3 mg / L Fe-TAML was also added to the solution. The conversion rate of aniline was defined as:

[0048]

[0049] Where c0 is the initial aniline concentration. It is the aniline concentration at time t.

[0050] Figure 1A The oxidation of aniline using H₂O₂ in the presence of Fe-TAML (3 mg / L) is shown. After 1 h, the solution turned black. After extending the reaction time to 24 h, the solution color became even darker, and a solid precipitate formed at the bottom. However, in the absence of Fe-TAML, only a slight color change was observed after 24 h. Figure 1B This experiment confirms the important role of the catalyst in this reaction.

[0051] Next, the effect of Fe-TAML dosage on aniline conversion after 24 h was investigated, and the results are shown in Figure 1C In the absence of the catalyst Fe-TAML, the conversion rate of aniline was only 15.9%. This result indicates that the oxidation of aniline using H₂O₂ alone is too slow. In contrast, the conversion rate was 64.6% in the presence of 1 mg / L Fe-TAML. This result suggests that 1 mg / L Fe-TAML is insufficient, and more Fe-TAML is needed to complete the reaction within 1 h. Figure 1C The conversion rate was shown to increase with increasing Fe-TAML dosage. When 3 mg / L Fe-TAML was used, the aniline conversion rate reached 98.0%. Therefore, this Fe-TAML dosage was chosen for subsequent studies.

[0052] 1.2 Formation of solid precipitate

[0053] It has been shown that aniline can be oxidized in the presence of H₂O₂ and Fe-TAML, and the solution darkens after 1 h. However, the reaction products become suspended in the solution and cannot be easily separated. Membrane filtration or centrifugation is required to remove them from the solution. This additional step is costly and poses a practical limitation to the process's application in wastewater treatment.

[0054] Therefore, this disclosure develops a method for treating the solid precipitate formed at the end of the reaction, which allows for easier removal of the solid precipitate. It has been found that when the sample is left to stand for one day without stirring, a layer of solid precipitate is observed, and the supernatant is then decanted to recover the solid precipitate. The suspended solids in the supernatant are also recovered using centrifugation. Both the solid precipitate and the suspended solids are dried and weighed.

[0055] Here, the total solid weight (wt.) is defined as:

[0056]

[0057] Where w p and w s These are the dry weights of the solid precipitate and the suspended solids in the supernatant, respectively.

[0058] Figures 2A to 2C The results showed that the total solid weight increased with reaction time. At 20°C, the total solid weight was only 0.330 ± 0.099 g after 1 h of reaction, and increased to 0.79 g after 24 h of reaction. Figure 2A The same trend was observed at 40℃ and 60℃. Figure 2B and 2C The results indicate that the formation of the solid is much slower than the oxidation of aniline. Further oxidation of the initial reaction products may occur, leading to the formation of the solid precipitate. Furthermore, as... Figure 2C As shown, the total solid weight after 4 hours of reaction reached 0.44 g, slightly higher than the total solid mass at 20℃ and 40℃. This indicates that higher temperatures favor the formation of solids in the reaction, but the effect is negligible.

[0059] To obtain more solids in the reaction, the H2O2 concentration was increased, and the reaction was repeated at 20°C, 40°C, and 60°C. Figure 3 The results showed that the amount of solid precipitate increased with increasing H2O2 concentration. Adding 8 g / L H2O2 at 60℃ had a significant effect, forming a thick layer of solid precipitate at the bottom. Simultaneously, the supernatant turned purple, indicating that the larger amount of H2O2 led to further oxidation of the reaction products in the solution.

[0060] 1.3 Two-step treatment process for aniline waste water

[0061] Because aniline is oxidized very quickly, within 1 hour, but the formation of solid precipitates occurs on a much longer timescale, a two-step treatment process has been developed for the treatment of aniline wastewater.

[0062] In the first step, 3 mg / L Fe-TAML and 1 g / L H2O2 were added to the solution to react with aniline. The solution was kept at ambient temperature for 1 h. It was noted that the solution turned black within 1 h after the addition of the Fe-TAML catalyst, as shown... Figure 4A As shown in the diagram. However, no solid precipitate was found at the bottom. In the second step, an additional dose of H2O2 (1 g / L) was added to continue the oxidation reaction, and the temperature was raised to 80°C, as shown. Figure 4B As shown in the image.

[0063] After the reaction, a solid precipitate forms at the bottom, the solution becomes clear, and some black particles are suspended in the solution. The supernatant is then decanted and the solid precipitate at the bottom is recovered. After drying and weighing, the solid precipitate accounts for 62.5% of the added aniline. Based on mass spectrometry results, it is speculated that the solid particles are aniline oligomers with 4-6 units. These molecules have an N-phenylphenazine or phenazine structure, whose large conjugated macrocyclic structure facilitates precipitation via π-π stacking. Simultaneously, the supernatant is filtered to remove suspended particles from the solution. The solid on the filter paper is dried and weighed. It accounts for 1.3 ± 0.2% of the aniline. The solid precipitate at the bottom and the suspended solid particles are combined, and the total solids yield is 63.8 ± 1.6%. This figure indicates the amount of aniline that can be converted to a solid and recovered by filtration. Overall, the total conversion of aniline can reach 96.8 ± 0.1%.

[0064] In the filtered supernatant, 17.62 mg-N / L of nitrate and 3.28 mg-N / L of ammonium were detected, indicating that a portion of aniline can be converted into inorganic nitrogen substances through a series of oxidation reactions. The presence of nitrate (or other inorganic nitrogen substances) can be treated using biological processes with highly enriched cultures, as described in the next section.

[0065] In the second step, the effect of temperature was further investigated. Table 1 shows that the higher temperature in the second step favored precipitation and total solids yield. When the temperature was 20°C (i.e., no heating in the second step), the precipitate yield was only 19.1 ± 1.1%. However, the total solids yield was 56.5 ± 1.8%, only slightly lower than the yield at 80°C. These results indicate that the reaction products at lower temperatures are suspended solid particles that do not precipitate spontaneously. However, they can still be separated from the solution by filtration. Based on these results, optimized conditions (3 mg / L Fe-TAML, additional 1 g / L H2O2, 80°C) were selected for treating real aniline wastewater.

[0066] Table 1: Effect of heating temperature on precipitation yield, total solids yield, and total aniline conversion in a two-step chemical treatment process used for both synthetic and real aniline wastewater. An additional dose of 1 g / L H₂O₂ was added during the heating step.

[0067]

[0068] "±" represents the standard error (n=3).

[0069] 1.4 Treatment of real aniline waste water

[0070] The optimized procedure was then applied to treat real aniline wastewater obtained from a local wastewater treatment plant. The sample contained approximately 1 g / L aniline (HPLC analysis showed 992.8 mg / L). As shown in Table 1, the aniline conversion and precipitation yield after treatment were 89.4% and 55.4%, respectively, slightly lower than the results for synthetic aniline solutions. These results may be due to interference from other contaminants in the wastewater sample. The results indicate that the process can be applied to the treatment of real industrial wastewater.

[0071] Example 2: Biological process for treatment of inorganic nitrogen species

[0072] 2.1 Simultaneous removal of ammonium and nitrite using a highly enriched culture

[0073] To obtain a highly enriched culture for denitrification, activated sludge (5 mL) was transferred from the anaerobic chamber to a sealed serum bottle containing 45 mL of sterile culture medium and stirred to obtain a homogeneous suspension. Nitrogen was purged into the medium to maintain anaerobic conditions, and then the medium was transferred back into the anaerobic chamber. After several days of incubation, nitrite and ammonium concentrations were measured to determine nitrogen removal activity. The culture with the highest nitrogen removal rate and efficiency was transferred to fresh medium (10% V / V). This transfer was repeated until a precipitate-free culture was obtained. The precipitate-free culture was then subjected to absolute dilution (from 10... -1 Up to 10 -9The highest dilution exhibiting nitrogen removal activity was then transferred to fresh medium (10% v / v). This absolute dilution process was repeated three times until a highly enriched culture containing anaerobic ammonia-oxidizing bacteria with high nitrogen removal rates and short doubling times was obtained. Based on the 16S rRNA gene sequence, this highly enriched culture contained two species: *Taurizoa* species N101 and... G56.

[0074] In the presence of 100 mg-N / L ammonium and nitrite, the highly enriched culture was able to remove both nitrite and ammonium simultaneously without a carbon source. After 24 h, the concentrations of ammonium and nitrite decreased to 18 mg-N / L and 0 mg-N / L, respectively. Simultaneously, the nitrate concentration increased to 62 mg-N / L. Since both ammonium and nitrite were consumed by the bacterial culture, an additional dose of 100 mg-N / L ammonium and nitrite was added. Both ammonium and nitrite were depleted again within one day. This result indicates that the bacterial culture can simultaneously remove ammonium and nitrite, converting them into nitrate or nitrogen gas. The experiment lasted for 6 days, with 100 mg-N / L ammonium and nitrite added daily. At the end of day 6, the bacterial culture had consumed a total of 600 mg-N / L of ammonium and nitrite, and produced 143 mg-N / L of nitrate.

[0075] 2.2 Effect of carbon to nitrogen (C / N) ratio on nitrite and nitrate removal

[0076] The effect of the C / N ratio on nitrogen removal was investigated using highly enriched cultures. In this study, acetate was used as the sole carbon source. The concentrations of ammonium and nitrite were each maintained at 100 mg⁻⁶ N / L. No nitrate was added to the solution.

[0077] When the C / N ratio was 0.2, the nitrite concentration decreased to 6.6 mg-N / L after 24 h, while the nitrate concentration increased to 42.8 mg-N / L. This result indicates that nitrite can be converted to nitrate during the process, and that the microbial culture cannot convert nitrate to nitrogen gas.

[0078] When the C / N ratio increased to 0.5, the nitrite concentration decreased to 2.3 mg-N / L and the nitrate concentration decreased to 21.0 mg-N / L, indicating that a higher C / N ratio is beneficial for the removal of both nitrite and nitrate. The results also show that nitrate can be converted into nitrogen gas in the presence of a carbon source.

[0079] When the C / N ratio is 1.5, neither nitrite nor nitrate can be detected after 24 hours.

[0080] The removal rate of ammonium was relatively slow. When the C / N ratio was 0.2, the residual ammonium concentration after 24 h was 12.5 mg-N / L. In contrast, when the C / N ratio was 1.5, the residual ammonium in the solution after 24 h was 33.5 mg-N / L. This result indicates that a high C / N ratio has an adverse effect on ammonium removal, but it is necessary for nitrate removal.

[0081] Table 2: Effect of C / N ratio on the removal of nitrite and ammonium by highly enriched culture over 24 h. Initial concentrations of nitrite, nitrate, and ammonium were 100 mg-N / L, 0 mg-N / L, and 100 mg-N / L, respectively.

[0082]

[0083] 2.3 Removal of nitrate in waste water after a chemical pre-treatment process

[0084] It has been shown that wastewater containing 1 g / L aniline can be treated with H2O2 and Fe-TAML, producing a solid precipitate. Simultaneously, 17.62 mg-N / L nitrate and 3.28 mg-N / L ammonium, as byproducts of the oxidation reaction, were detected in the solution. To remove nitrate and ammonium from the wastewater, 1 L of supernatant was taken, and 20 mL of enrichment culture and 100 mg of sodium acetate were added to the solution. The solution was kept in a sealed bottle, and samples were taken periodically to analyze the concentrations of nitrate, nitrite, and ammonium. As shown in Table 3, both nitrate and ammonium decreased rapidly over time. After 24 h, only 8.34 mg-N / L nitrate and 1.02 mg-N / L ammonium remained in the solution. Simultaneously, 13.27 mg-N / L nitrite was detected. These results indicate that the bacterial culture can convert nitrate to nitrite in the presence of a carbon source. Finally, all nitrogenous substances were removed within 2 days using the highly enriched culture.

[0085] Table 3: Removal of nitrates and ammonium from aniline wastewater using enriched bacterial cultures and an additional carbon source (1 g / L sodium acetate).

[0086]

[0087] In summary, a chemical-biological pretreatment process for nitrogen pollutants has been developed. This process requires only low levels of hydrogen peroxide and a Fe-TAML catalyst. Following the pretreatment, the nitrogen pollutants are oxidized to form a readily separable solid precipitate. The supernatant contains inorganic nitrogenous substances such as ammonium, nitrite, and nitrate, which can be obtained using bacteria belonging to the genus *Taurizoa* and... Two species of the genus, especially species N101 of the genus *Taugella* and Further treatment with highly enriched microbial cultures of G56, which exhibit aerobic denitrification behavior, is required. Only a very small amount of carbon source is needed to convert all three inorganic nitrogenous substances (ammonium, nitrite, and nitrate) into nitrogen gas.

[0088] The methods disclosed herein are generally applicable to the removal of nitrogenous pollutants from wastewater, including industrial and domestic wastewater. In some embodiments, the biological processes can be used independently to remove inorganic nitrogenous pollutants from agricultural and aquaculture wastewater.

[0089] Although embodiments of the invention have been shown and described, the invention is not limited to the described embodiments. Rather, those skilled in the art will understand that various modifications and variations can be made to the embodiments of the invention without departing from the scope of the invention, which is set forth in the claims.

Claims

1. A chemical-biological treatment method for removing nitrogen pollutants from nitrogen-containing wastewater, the method comprising: The nitrogen-containing wastewater was pretreated with an oxidant in the presence of a catalyst to form an oligomer mixture; The oligomer mixture is precipitated by further adding an oxidant to obtain a solid precipitate and a supernatant, wherein the supernatant contains one or more inorganic nitrogen compounds. Separate the supernatant from the solid precipitate; and The supernatant is contacted with a microbial culture to convert the one or more inorganic nitrogen compounds into nitrogen gas. The catalyst described herein comprises an iron-tetraamide macrocyclic ligand (Fe-TAML) or a peroxidase; and The microbial culture mentioned therein contains denitrifying bacteria and anaerobic ammonia-oxidizing bacteria.

2. The method of claim 1, wherein the denitrifying bacteria belong to the genus Thauera ), and the anaerobic ammonia oxidizing bacteria belong to the genus Brocadia .

3. The method according to claim 1, wherein the precipitation step is carried out at a temperature in the range of 60°C to 80°C.

4. The method according to claim 1, wherein the one or more inorganic nitrogen compounds include ammonium, nitrite and nitrate.

5. The method according to claim 4, wherein the supernatant has a carbon-to-nitrogen ratio of less than 0.5, for converting nitrates in the supernatant into nitrites.

6. The method of claim 4, further comprising: A carbon source is added before or during the step of contacting the supernatant with the microbial culture to adjust the carbon-to-nitrogen ratio of the supernatant to less than 0.5, thereby converting nitrates in the supernatant into nitrites.

7. The method according to claim 5 or 6, wherein under anaerobic conditions, in the absence of a carbon source, the ammonium and nitrite in the supernatant are simultaneously converted into nitrogen gas.

8. The method according to claim 1, wherein the nitrogen-containing wastewater contains aniline at a concentration of 1 g / L.

9. The method of claim 1, wherein the oxidant comprises hydrogen peroxide.

10. The method of claim 1, wherein the pretreatment step is performed using the oxidant at a concentration of 1 g / L.

11. The method of claim 1, wherein the oxidant added during the precipitation step has a concentration in the range of 1 g / L to 10 g / L.

12. The method according to claim 1, wherein the catalyst has a concentration of 3 mg / L.

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