Special composite microbial agent for sewage denitrification and preparation method of carbon source thereof
By combining compound microbial agents and modified carbon sources, the problem of low nitrogen removal efficiency in wastewater under low carbon-to-nitrogen ratio conditions is solved, achieving efficient and environmentally friendly wastewater nitrogen removal. It also utilizes waste resources and forms a triple electron transfer mechanism that combines autotrophic and heterotrophic processes.
Patent Information
- Application Number
- CN202610783441.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-02
- Publication Date
- 2026-08-25
AI Technical Summary
Existing wastewater denitrification technologies have high carbon source addition costs, large carbon emissions, and are prone to secondary pollution under low carbon-to-nitrogen ratio conditions. Furthermore, the microbial community has a single function, low denitrification efficiency, and difficulty in achieving synergistic coupling between autotrophic and heterotrophic processes, which affects large-scale application.
A composite microbial agent consisting of iron-oxidizing autotrophic denitrifying bacteria, heterotrophic nitrifying-aerobic denitrifying bacteria, and iron mineral mineralization associated bacteria, combined with self-made nano-zero-valent iron and modified carbon source, forms a triple electron transfer enhancement mechanism to achieve synchronous synergy between autotrophic and heterotrophic processes. Using waste Chinese medicinal residues and corn cobs as raw materials, a multifunctional carbon source is prepared.
Achieving a total nitrogen removal rate of over 85% under low-temperature and low-carbon conditions improves electron transfer efficiency, avoids secondary pollution, and realizes resource-based reuse of carbon sources and efficient nitrogen removal.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of wastewater treatment technology, and in particular to a special composite microbial agent for wastewater denitrification and its carbon source preparation method. Background Technology
[0002] Nitrogen pollution is one of the main causes of eutrophication in water bodies, and wastewater denitrification is a crucial step in maintaining the ecological health of the aquatic environment. Currently, biological denitrification technology is widely used due to its cost-effectiveness and efficiency. However, the denitrification process often requires the addition of organic matter (such as sodium acetate or methanol) as an electron donor. Nevertheless, traditional heterotrophic denitrification processes that rely on organic carbon sources face significant challenges when treating wastewater with low C / N ratios, including high carbon source input costs, large carbon emissions, susceptibility to secondary pollution, and low denitrification efficiency.
[0003] In recent years, iron-based autotrophic denitrification technology has attracted widespread attention as a novel wastewater denitrification method. This technology utilizes zero-valent iron (ZVI) or divalent iron as an electron donor and nitrate as an electron acceptor, ultimately producing trivalent iron and nitrogen gas, achieving autotrophic denitrification. It boasts advantages such as environmental friendliness, economic efficiency, and the absence of organic carbon sources. Studies have shown that iron-based denitrification technology has achieved a nitrogen removal rate of approximately 80%, but its volumetric nitrogen loading and removal rate are still lower than those of traditional heterotrophic denitrification, and Fe²⁺… + The oxidation pathway mechanism remains controversial, which affects the large-scale application of the technology.
[0004] Most of the currently available denitrifying microbial agents are single heterotrophic denitrifying bacterial groups with limited functions. They rely solely on exogenous organic carbon to complete denitrification, have weak resistance to shock loads, and their activity decreases significantly under low temperature and low nutrient water conditions. At the same time, conventional agents lack an electron transfer enhancement system, resulting in a slow denitrification reaction rate, easy loss of bacterial strains, and poor sludge settling performance.
[0005] Furthermore, existing carbon source products only possess a single carbon release function and cannot simultaneously provide electron donors and catalytic reaction sites for denitrifying bacteria. A mature and integrated system for the coupled and synergistic technology of autotrophic and heterotrophic denitrification has not yet been formed, and there are also significant technological gaps in the integrated solution of bacteria-carbon source for iron-based biomineralization synergistic denitrification. Therefore, developing a wastewater treatment method and system that can integrate autotrophic and heterotrophic pathways, enhance electron transfer, achieve low carbon source addition, and eliminate secondary pollution is of great significance. Summary of the Invention
[0006] The purpose of this invention is to provide a special compound microbial agent for wastewater denitrification, which can achieve efficient removal of total nitrogen under low temperature, low carbon, and low energy consumption conditions, and solve the long-standing problem of excessive total nitrogen in wastewater in the industry.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a special compound microbial agent for wastewater denitrification. The compound microbial agent comprises a mixed bacterial solution consisting of iron-oxidizing autotrophic denitrifying bacteria, heterotrophic nitrifying-aerobic denitrifying bacteria, and iron mineral mineralization associated bacteria. The iron-oxidizing autotrophic denitrifying bacteria is *Paracococcus iron-oxidizing*, the heterotrophic nitrifying-aerobic denitrifying bacteria is *Pseudomonas schlegelii*, and the iron mineral mineralization associated bacteria is *Shewanella*.
[0008] Preferably, the *Paragonimococcus ferruginosa*, *Pseudomonas schwanniferus*, and *Shewanella* are compounded in a volume ratio of 2-4:4-6:1-3, and the viable bacterial concentration of each is ≥1.5×10⁻⁶. 9 CFU / mL.
[0009] Preferably, the composite microbial agent further includes sodium alginate, carboxymethyl chitosan, nano-zero valent iron, and trace elements.
[0010] Preferably, the mass ratio of the mixed bacterial solution, sodium alginate, carboxymethyl chitosan, nano-zero valent iron, and trace elements in the composite microbial agent is 40~50:20~25:15~20:8~12:3~7.
[0011] The present invention also provides a method for preparing a carbon source for wastewater denitrification using the aforementioned composite microbial agent combination, comprising the following steps: (1) After crushing the plant biomass raw material, add dilute acid solution to hydrolyze it to obtain a hydrolyzed mixture; (2) Add ferrous salt to the hydrolyzed mixture, and then inoculate with Shewanella bacterial solution to carry out anaerobic mineralization reaction to obtain the mineralized mixture; (3) Add hydroxyapatite, polycaprolactone and crosslinking agent to the mineralized mixture to carry out crosslinking and curing reaction; (4) The reaction product is hot-pressed into shape, cooled and then cut to obtain a solid carbon source.
[0012] Preferably, the plant biomass raw material in step (1) is salvia miltiorrhiza residue and corn cob, with a mass ratio of 4~5:3~4; The dilute acid solution is a dilute acetic acid solution with a mass fraction of 1.0% to 1.5%. The ratio of plant biomass raw material to dilute acid solution is 1 g: 7 to 9 mL. The hydrolysis temperature is 50 to 70°C and the hydrolysis time is 1 to 3 h.
[0013] Preferably, the ferrous salt mentioned in step (2) is ferrous chloride, and the amount added is such that the Fe in the system is reduced. 2+ The concentration is 2000–2500 mg / mL; the amount of Shewanella bacterial solution added is 5%–10% of the volume of the hydrolysis mixture; the temperature of the anaerobic mineralization reaction is 25–35℃, and the time is 40–50 h.
[0014] Preferably, the crosslinking agent in step (3) is a citrate crosslinking agent; the mass ratio of hydroxyapatite, polycaprolactone and crosslinking agent is 50~70:70~90:15~25; the crosslinking curing reaction temperature is 20~30℃ and the reaction time is 80~100min.
[0015] The present invention also provides a wastewater denitrification composition of the aforementioned composite microbial agent and the carbon source prepared by the aforementioned preparation method, wherein the mass ratio of the composite microbial agent to the carbon source is 1~2:3~5.
[0016] The present invention also provides an application of the aforementioned wastewater denitrification composition in wastewater treatment.
[0017] Beneficial effects
[0018] This invention couples three functional microbial communities: autotrophic denitrification through iron oxidation, simultaneous heterotrophic nitrification and denitrification, and iron mineralization. The autotrophic and heterotrophic pathways proceed synchronously and synergistically, complementing each other, achieving a total nitrogen removal rate of over 85%. The resulting carbon source possesses four functions: carbon release, catalysis, electron transfer, and mineralization stabilization, expanding the traditional single-function carbon source into a multi-functional integrated system, significantly improving carbon source utilization. Through the in-situ generation of conductive iron minerals by associated bacteria in iron mineralization, the conductivity of nano-zero-valent iron in the magnetic microsphere agent, and the electron shuttle function of Fe3O4 in the carbon source, a triple electron transfer enhancement mechanism is formed, significantly improving electron transfer efficiency. The carbon source uses waste Chinese medicinal herb residue and corn cobs as raw materials, realizing the resource-based reuse of agricultural and Chinese medicinal herb waste, which is green and environmentally friendly, and the entire process does not produce harmful intermediate products, eliminating the risk of secondary pollution from greenhouse gases and chemical agents. Detailed Implementation
[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0020] Example 1
[0021] 1. Preparation of microbial agents
[0022] *Paragonimococcus ferrooxidans*, *Pseudomonas schwanniferus*, and *Shewanella* (all commercially available strains) were cultured and expanded, with each strain having a viable cell concentration ≥1.5 × 10⁻⁶. 9 CFU / mL. Three bacterial strains were mixed in a volume ratio of 3:5:2 and activated at room temperature in the dark for 30 min to obtain a mixed bacterial solution. Under a 45℃ water bath, the mixed bacterial solution, sodium alginate, carboxymethyl chitosan, nano-zero valent iron, and trace elements (composed of magnesium sulfate, calcium chloride, and potassium dihydrogen phosphate in a mass ratio of 3:2:45) were mixed evenly in a mass ratio of 45:22:18:10:5. 2% by volume of calcium chloride curing solution was added, and the mixture was cured and shaped. It was then air-dried at 25℃ with a moisture content controlled at 10% to obtain a composite granular bacterial agent.
[0023] 2. Preparation of carbon source
[0024] Take 450 g of Salvia miltiorrhiza residue and 350 g of corn cob, wash and crush them, and pass them through a 40-mesh sieve; add dilute acetic acid solution (mass fraction 1.2%) at a material-to-liquid ratio of 1 g: 8 mL and soak at 60℃ for 2 hours for hydrolysis; add ferrous chloride to the hydrolysate mixture to reduce the Fe content in the system. 2+ The concentration was approximately 2300 mg / mL. Activated Shewanella bacterial solution (8% of the total liquid volume) was inoculated and anaerobic mineralized at 30°C for 48 h to generate Fe3O4 minerals. 60 g of hydroxyapatite, 80 g of polycaprolactone, and 20 g of citrate crosslinking agent were added to the mineralized mixture, and the mixture was crosslinked and cured at room temperature for 90 min. The mixture was then hot-pressed at 80°C and 10 MPa, cooled, and cut to obtain columnar slow-release solid carbon sources with a diameter of 5 mm and a length of 10 mm.
[0025] Experimental Example 1
[0026] Six parallel experiments were conducted, including an experimental group, a compound granular bacterial agent group, a single bacterial agent group, a common commercial bacterial agent group, a blank group, and a group without mineralized carbon source. The water quality was uniformly simulated as low carbon-to-nitrogen ratio domestic sewage, with influent TN: 42.36±2 mg / L and NH4+: 2 mg / L. + -N: 26.45±1.5 mg / L, COD: 95±5 mg / L, C / N=2.75. Water temperature was divided into two groups: normal temperature (25℃) and low temperature (9℃). Three parallel samples were set up for each group of experiments. The experimental period was 15 days, and samples were taken every 24 hours to test water quality indicators. The final average value was taken as the experimental result. The detection methods were: TN was detected by alkaline potassium persulfate digestion ultraviolet spectrophotometry, and COD was detected by potassium dichromate digestion.
[0027] (1) Experimental group: 400 mL of sewage was placed in the reactor, 200 mg of the compound granular bacterial agent prepared in Example 1 and 700 mg of solid carbon source were added, the reactor was sealed to create an anaerobic environment, the stirring speed was 80 r / min, and the hydraulic retention time was 10 h; the reactor was placed in constant temperature incubators at 25℃ and 9℃ respectively and kept away from light; the reactor was stirred twice a day for 30 min each time to maintain the uniformity of water quality and prevent the bacterial agent and carbon source from depositing; the experimental period was 15 days, and the upper clear liquid was extracted every 24 h, filtered through a 0.45 μm filter membrane and the water quality indicators were tested. Each group was tested in parallel 3 times and the average value was calculated.
[0028] (2) Ordinary carbon source group: 400 mL of sewage was placed in the reactor, and 200 mg of the compound granular bacterial agent prepared in Example 1 and 700 mg of ordinary unmodified corn cob carbon source (simply crushed and dried) were added. The reactor was sealed to create an anaerobic environment. The stirring speed was 80 r / min and the hydraulic retention time was 10 h. The reactor was placed in constant temperature incubators at 25℃ and 9℃ respectively and kept away from light. The reactor was stirred twice a day for 30 min each time to maintain the uniformity of water quality and prevent bacterial agent deposition. The experimental period was 15 days. The upper clear liquid was extracted every 24 h and filtered through a 0.45 μm filter membrane to detect water quality indicators. Each group was tested in parallel for 3 times and the average value was calculated.
[0029] (3) Single bacterial group: 400 mL of sewage was placed in the reactor and granular bacterial agent prepared solely by Pseudomonas stearothermiae was added (the preparation method is the same as the compound granular bacterial agent in Example 1). The reactor was sealed to create an anaerobic environment. The stirring speed was 80 r / min and the hydraulic retention time was 10 h. The reactor was placed in constant temperature incubators at 25℃ and 9℃ respectively and kept away from light. The reactor was stirred twice a day for 30 min each time to maintain the uniformity of water quality and prevent bacterial agent deposition. The experimental period was 15 days. The upper clear liquid was extracted every 24 h and filtered through a 0.45 μm filter membrane to detect water quality indicators. Each group was tested in parallel for 3 times and the average value was calculated.
[0030] (4) Common commercial bacterial agent group: 400 mL of sewage was placed in the reactor, and commercially available bacterial agent (ammonia nitrogen degrading bacteria, model QWWS-02) was added. The reactor was sealed to create an anaerobic environment. The stirring speed was 80 r / min and the hydraulic retention time was 10 h. The reactor was placed in constant temperature incubators at 25℃ and 9℃ respectively and kept away from light. The reactor was stirred twice a day for 30 min each time to maintain the uniformity of water quality and prevent bacterial agent deposition. The test period was 15 days. The upper clear liquid was extracted every 24 h and filtered through a 0.45 μm filter membrane to test the water quality indicators. Each group was tested in parallel for 3 times and the average value was calculated.
[0031] (5) Non-mineralized bacteria group: 400 mL of sewage was placed in the reactor, and granular bacterial agent (prepared according to the method of Example 1) and 700 mg of solid carbon source were added. The reactor was sealed to create an anaerobic environment. The stirring speed was 80 r / min and the hydraulic retention time was 10 h. The reactor was placed in constant temperature incubators at 25℃ and 9℃ respectively and kept away from light. The reactor was stirred twice a day for 30 min each time to maintain the uniformity of water quality and prevent the bacterial agent and carbon source from depositing. The test period was 15 days. The upper clear liquid was extracted every 24 h and filtered through a 0.45 μm filter membrane to detect water quality indicators. Each group was tested in parallel for 3 times and the average value was calculated.
[0032] (6) Blank group: 400 mL of sewage was placed in the reactor without adding any bacterial agent. The reactor was sealed to create an anaerobic environment. The stirring speed was 80 r / min and the hydraulic retention time was 10 h. The reactor was placed in constant temperature incubators at 25℃ and 9℃ respectively and kept away from light. The reactor was stirred twice a day for 30 min each time to maintain uniform water quality. The test period was 15 days. The upper clear liquid was extracted every 24 h and filtered through a 0.45 μm filter membrane to test water quality indicators. Each group was tested in parallel for 3 times and the average value was calculated.
[0033] The average TN removal rate at room temperature, the TN removal rate at low temperature, and the COD value of the above six groups were statistically analyzed. The results are shown in Table 1.
[0034] Table 1 Water quality test results
[0035] As shown in Table 1, (1) Synergistic analysis of strain combination: The TN removal rate of the single Pseudomonas schlegelii (single strain group) at room temperature was only 55.74%; the removal rate of the dual strain system after removing mineralizing bacteria (non-mineralizing bacteria group) at room temperature increased to 72.33%; while the denitrification rate of the three strains compound system of the present invention reached 86.26% at room temperature. This proves that there is a significant positive synergistic effect among iron-oxidizing autotrophic bacteria, heterotrophic denitrifying bacteria, and iron-mineralizing bacteria. Iron-oxidizing paracoccus undertakes the autotrophic denitrification and replenishment, Pseudomonas schlegelii undertakes the main heterotrophic denitrification, and Shewanella constructs electron transport channels. All three strains are indispensable, and the strain combination is irreplaceable. (2) Enhancement analysis of iron-based mineralized carbon source: Compared with the experimental group and the ordinary carbon source group, the bacterial groups of the two groups are completely consistent, only the carbon source is different; the denitrification rate of ordinary biomass carbon source at room temperature is only 63.41%, while the denitrification rate of the iron-based mineralized modified carbon source of the present invention is increased by 22.85%. The reason lies in the in-situ generation of nano-Fe3O4 inside the mineralized carbon source, which constructs an iron-based electron shuttle system and reduces the activation energy of the denitrification reaction; at the same time, the slow-release structure can controllably release small molecule organic acids, continuously and stably supplying the carbon source and avoiding the instantaneous depletion of the carbon source, proving that the mineralization modification process is the core technology for improving the carbon source.
[0036] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A special compound microbial agent for wastewater denitrification, characterized in that, The compound microbial agent comprises a mixed bacterial solution consisting of iron-oxidizing autotrophic denitrifying bacteria, heterotrophic nitrifying-aerobic denitrifying bacteria, and iron mineral mineralization associated bacteria; the iron-oxidizing autotrophic denitrifying bacteria is *Paracococcus iron-oxidizing*, the heterotrophic nitrifying-aerobic denitrifying bacteria is *Pseudomonas schlegelii*, and the iron mineral mineralization associated bacteria is *Shewanella*.
2. The compound microbial agent as described in claim 1, characterized in that, The P. ferrireducens, P. stutzeri and Shewanella are compounded according to a volume ratio of 2-4:4-6:1-3, and the live bacteria concentration is all ≥1.5×10 9 CFU / mL.
3. The compound microbial agent as described in claim 2, characterized in that, The compound microbial agent also includes sodium alginate, carboxymethyl chitosan, nano-zero valent iron, and trace elements.
4. The compound microbial agent as described in claim 3, characterized in that, The mass ratio of the mixed bacterial solution, sodium alginate, carboxymethyl chitosan, nano-zero valent iron, and trace elements in the composite microbial agent is 40~50:20~25:15~20:8~12:3~7.
5. A method for preparing a carbon source for wastewater denitrification, used in combination with the composite microbial agent according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) After crushing the plant biomass raw material, add dilute acid solution to hydrolyze it to obtain a hydrolyzed mixture; (2) Add ferrous salt to the hydrolyzed mixture, and then inoculate with Shewanella bacterial solution to carry out anaerobic mineralization reaction to obtain the mineralized mixture; (3) Add hydroxyapatite, polycaprolactone and crosslinking agent to the mineralized mixture to carry out crosslinking and curing reaction; (4) The reaction product is hot-pressed into shape, cooled and then cut to obtain a solid carbon source.
6. The preparation method according to claim 5, characterized in that, The plant biomass raw materials mentioned in step (1) are Salvia miltiorrhiza residue and corn cob, with a mass ratio of 4~5:3~4; The dilute acid solution is a dilute acetic acid solution with a mass fraction of 1.0% to 1.5%. The ratio of plant biomass raw material to dilute acid solution is 1 g: 7 to 9 mL. The hydrolysis temperature is 50 to 70°C and the hydrolysis time is 1 to 3 h.
7. The preparation method according to claim 5, characterized in that, The ferrous salt mentioned in step (2) is ferrous chloride, and the amount added is such that the Fe in the system is reduced. 2+ The concentration is 2000~2500 mg / mL; the amount of Shewanella bacterial solution added is 5%~10% of the volume of the hydrolysis mixture; the temperature of the anaerobic mineralization reaction is 25~35℃, and the time is 40~50 h.
8. The preparation method according to claim 5, characterized in that, The crosslinking agent in step (3) is a citrate crosslinking agent; the mass ratio of hydroxyapatite, polycaprolactone and crosslinking agent is 50~70:70~90:15~25; the crosslinking curing reaction temperature is 20~30℃ and the reaction time is 80~100 min.
9. A wastewater denitrification composition comprising the composite microbial agent according to any one of claims 1 to 4 and a carbon source prepared by the preparation method according to claims 5 to 8, characterized in that, The mass ratio of the composite microbial agent to the carbon source is 1~2:3~5.
10. The use of the wastewater denitrification composition of claim 9 in wastewater treatment.