A method for mitigating carbon dioxide inhibition of denitrification and promoting denitrification

Abstract

The application discloses a method for relieving the inhibition of carbon dioxide on denitrification and promoting denitrification. The method comprises the following steps: inputting 0.3%-20% of CO2 into a closed reactor, inoculating Proteiniphilum aceticum into a denitrification system of P.denitrificans, and realizing the effect of relieving the inhibition of CO2 on denitrification and promoting the denitrification efficiency through the interaction of the two bacteria, wherein the inoculation ratio of P.denitrificans and Proteiniphilum is 1:2-1:15, the carbon-nitrogen ratio of the denitrification system is 1-5, the pH of the denitrification system is 6.0-9.0, and the temperature of the denitrification culture is 30-37 DEG C. The application promotes anaerobic denitrification by using the interaction of Proteiniphilum and P.denitrificans, has low cost, has no secondary pollution, can obviously relieve the inhibition of CO2, improves the nitrate removal effect, and reduces the accumulation of nitrite and nitrous oxide.

Description

Technical Field

[0001] This invention relates to the field of biological denitrification technology, and in particular to a method for mitigating the inhibition of denitrification by carbon dioxide and promoting denitrification, especially a method using Proteiniphilum to mitigate the inhibition of denitrification by CO2 and promote anaerobic denitrification efficiency. Background Technology

[0002] With industrial and economic development, the burning of large quantities of fossil fuels and deforestation have led to a gradual increase in atmospheric CO2 concentration, and the rate of increase is accelerating. This rise in atmospheric CO2 concentration will result in a series of major environmental problems, including ocean acidification and global warming. Carbon dioxide sequestration is considered an effective means of addressing rising CO2 concentrations, but due to CO2 leakage issues at sequestration sites, the CO2 concentration around underground sequestration sites can rise dramatically, reaching up to 100 times the atmospheric concentration, posing a severe challenge to various biological and environmental cycles on Earth. On the other hand, the extensive use of nitrogen-containing substances such as fertilizers has led to increasingly serious nitrogen pollution in the environment, causing a series of environmental problems such as eutrophication of water bodies and groundwater pollution. The use of biological denitrification to remove nitrate pollution from water is receiving increasing attention. However, CO2 inhibits the denitrification process, slowing it down, and also leads to the accumulation of toxic and harmful intermediate products of nitrate reduction (such as nitrite and nitrous oxide). Therefore, it is essential to find a method to alleviate the inhibitory effect of CO2 on denitrification, promote the high-speed denitrification process, and prevent the accumulation of intermediate products. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention aims to provide a method for mitigating the inhibitory effect of carbon dioxide while simultaneously promoting denitrification. This method involves adding cultured *Paracococcus denitrifyingans* and *Acetobacter acetophilus* in a specific ratio to a reactor filled with CO2. This mitigates the inhibitory effect of CO2 on denitrification while simultaneously increasing the denitrification rate and reducing the accumulation and release of the denitrification intermediate product N2O. In this invention, the mixed microbial composition mitigates the adverse effects of CO2 on denitrification because of the synergistic effect between *Paracococcus denitrifyingans* and *Acetobacter acetophilus*. They counteract the adverse effects of CO2 by producing microbial secondary metabolites or increasing the content of extracellular polymers in the system, thereby restoring and promoting the denitrification process. This method relies solely on microbial regulation, is simple to implement, and produces no secondary pollution.

[0004] The purpose of this invention is to provide a method for mitigating the inhibition of denitrification by carbon dioxide and promoting denitrification by adding denitrifying paracocci and acetic acid-producing proteinophilic bacteria to synergistically alleviate the inhibitory effect of carbon dioxide on denitrification and promote microbial denitrification.

[0005] In one embodiment of the present invention, the following steps are included:

[0006] S1. Add culture medium and trace elements to the reactor and perform anaerobic treatment. Then, introduce carbon dioxide and sterilize to obtain an anaerobic denitrification culture medium system.

[0007] S2. Provide suspensions of denitrifying paracocci and acetic acid-producing proteinophilic bacteria;

[0008] S3. The denitrifying paracoccus suspension and the acetic acid-producing proteinophilic bacteria suspension are inoculated into the anaerobic denitrification culture medium system obtained in step S1 for anaerobic denitrification culture.

[0009] In one embodiment of the present invention, in step S1, the molar percentage of carbon dioxide is 0.3% to 20%.

[0010] In one embodiment of the present invention, in step S1, the carbon-to-nitrogen ratio in the culture medium is 1 to 5.

[0011] In one embodiment of the present invention, in step S1, the culture medium includes a carbon source and inorganic salts.

[0012] Furthermore, the carbon source is glucose or sodium acetate.

[0013] Furthermore, the trace elements include 7.30 g / L disodium ethylenediaminetetraacetate, 2.50 g / L ferrous sulfate heptahydrate, 0.02 g / L manganese chloride, 0.242 g / L sodium molybdate, 0.02 g / L calcium chloride, 0.135 g / L copper chloride crystals, and 0.34 g / L zinc chloride.

[0014] Further, the inorganic salts include 2.16 g / L potassium nitrate, 0.5 g / L ammonium chloride, 0.07–0.1 g / L magnesium sulfate, 0.5–4.65 g / L disodium hydrogen phosphate, 1–2.44 g / L potassium dihydrogen phosphate, 0.2 g / L cysteine, and 1 mL / L trace elements.

[0015] In one embodiment of the present invention, the trace elements include 7.30 g / L disodium ethylenediaminetetraacetate, 2.50 g / L ferrous sulfate heptahydrate, 0.02 g / L manganese chloride, 0.242 g / L sodium molybdate, 0.02 g / L calcium chloride, 0.135 g / L copper chloride crystals, and 0.34 g / L zinc chloride.

[0016] In some embodiments of the present invention, in step S1, the anaerobic treatment refers to filling the reactor with inactive material for at least 30 minutes to remove oxygen.

[0017] Furthermore, the inactive gas includes nitrogen.

[0018] In one embodiment of the present invention, in step S2, the culture medium in the denitrifying paracoccus suspension is LB medium.

[0019] Furthermore, the LB medium comprises 10 g / L trypsin, 5 g / L yeast extract, and 10 g / L NaCl.

[0020] Furthermore, the culture medium for the acetic acid-producing proteinophilic bacteria suspension comprises 5 g / L trypsinized casein, 5 g / L peptone, 10 g / L yeast extract, 5 g / L beef extract, 5 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L cysteine, 1 mg / L resazurin, 2 g / L sodium chloride, 5 mg / L heme chloride, 10 mg / L calcium chloride, 20 mg / L magnesium sulfate heptahydrate, and 40 mg / L potassium dihydrogen phosphate.

[0021] In one embodiment of the present invention, in step S2, the initial OD of the denitrifying paracoccus suspension is... 600 It ranges from 0.01 to 0.02.

[0022] In one embodiment of the present invention, in step S2, the initial OD of the acetophilic bacteria suspension is... 600 It ranges from 0.02 to 0.2.

[0023] In one embodiment of the present invention, in step S3, the inoculation ratio of the *Paragonimella denitrificans* suspension to the *Acetobacter acetophilus* suspension is 1:2 to 1:15. Within this range, the synergistic effect of the mixed bacteria is better, with a promoting effect within this range; however, beyond this range, the effect deteriorates.

[0024] In one embodiment of the present invention, in step S3, the carbon-to-nitrogen ratio of the anaerobic denitrification culture medium system is 1 to 5.

[0025] In one embodiment of the present invention, in step S3, the pH of the anaerobic denitrification culture medium system is 6.0 to 9.0.

[0026] In one embodiment of the present invention, in step S3, the temperature of the anaerobic denitrification culture is 30-37°C.

[0027] The technical solution of the present invention has the following advantages compared with the prior art:

[0028] (1) The present invention only adds microorganisms to the bioreactor without adding metals or other organic matter, which is simple to operate and has no secondary pollution.

[0029] (2) In this invention, the cultivation of microorganisms can alleviate the inhibitory effect of CO2 on denitrification while appropriately improving the effect of biological denitrification, increasing the denitrification rate, and reducing the accumulation of intermediate products.

[0030] Instruction manual illustrations

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0032] Figure 1 This is a flowchart of the operation of the present invention. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0034] The bacterial strains used in this invention are: *Proteiniphilum* purchased from China (General Microbiological Culture Collection Center, CGMCC 1.5024, TB 107); and *P. denitrificans* purchased from the American Type Culture Collection (ATCC), strain number ATCC 19367.

[0035] Example 1

[0036] (1) Add culture medium and 1 mL of trace elements to a sterile serum bottle, adjust the pH of the system to 7.2 with NaOH and HCl, purge with nitrogen for 30 minutes to maintain an anaerobic state. Then purge with 0.3% CO2 gas in the headspace, seal with a sterile butyl rubber stopper, and sterilize at 121℃ for 20 minutes to obtain sterilized culture medium system A.

[0037] The culture medium comprises a carbon source and inorganic salts. The carbon source is 1.15 g / L sodium acetate. The inorganic salts include 2.16 g / L potassium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate, 4.65 g / L disodium hydrogen phosphate, 2.44 g / L potassium dihydrogen phosphate, 0.2 g / L cysteine, and 1 mL / L trace elements. The trace elements include 7.30 g / L disodium ethylenediaminetetraacetate, 2.50 g / L ferrous sulfate heptahydrate, 0.02 g / L manganese chloride, 0.242 g / L sodium molybdate, 0.02 g / L calcium chloride, 0.135 g / L copper chloride crystals, and 0.34 g / L zinc chloride. The carbon-to-nitrogen ratio in the culture medium is 3.

[0038] (2) The denitrifying microorganism P. denitrificans was cultured in sterile LB medium under aerobic conditions to OD. 600The OD value was 1.8–2.0. Under anaerobic conditions, *Proteiniphilum* was cultured to OD values ​​using sterile acetogenic proteinophilic bacteria medium. 600 The concentration was 0.5–1.0. The pre-cultured denitrifying microorganisms P. denitrificans and Proteiniphilum were washed three times with PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4 and 0.24 g / L KH2PO4, pH = 7.4) and then concentrated to 30 mL.

[0039] (3) In step (1), 0.05 mL of the pre-cultured denitrifying microorganism P. denitrificans was inoculated into the 100 mL anaerobic denitrification medium after sterilization, and then 5 mL of Proteiniphilum was inoculated, so that the inoculation volume ratio of P. denitrificans and Proteiniphilum was 1:10; at the same time, the denitrifying microorganisms P. denitrificans and Proteiniphilum were inoculated separately into the denitrification medium as controls. The anaerobic bottle was placed on a shaker and anaerobic denitrification culture was carried out at 37°C.

[0040] Experimental Results: The results showed that *Proteiniphilum* itself has a very low nitrate reduction capacity, and denitrification is mainly accomplished by the denitrifying microorganism *P. denitrificans*. Under a CO2 atmosphere, the denitrification efficiency decreased by 21.9%, and the rate decreased by 45.6%. After adding *Proteiniphilum*, the denitrification efficiency of the co-cultured anaerobic denitrification system increased from 43.5% to 99.4%, ultimately resulting in no nitrite accumulation and a 74.7% reduction in nitrous oxide accumulation.

[0041] Example 2

[0042] (1) In a sterile serum bottle, add culture medium and 1 mL of trace elements, adjust the pH of the system to 6.8 with NaOH and HCl, purge with nitrogen for 30 minutes to maintain an anaerobic state. Then purge with 0.3% CO2 gas in the headspace, seal with a sterile butyl rubber stopper, and sterilize at 121℃ for 20 minutes to obtain sterilized culture medium system B.

[0043] The culture medium comprises a carbon source and inorganic salts. The carbon source is glucose, prepared at 250 g / L and sterilized at 115°C for 15 minutes. The inorganic salts include 1.44 g / L potassium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate, 4.65 g / L disodium hydrogen phosphate, 2.44 g / L potassium dihydrogen phosphate, 0.2 g / L cysteine, and 1 mL / L trace elements. The trace elements include 7.30 g / L disodium ethylenediaminetetraacetate, 2.50 g / L ferrous sulfate heptahydrate, 0.02 g / L manganese chloride, 0.242 g / L sodium molybdate, 0.02 g / L calcium chloride, 0.135 g / L copper chloride crystals, and 0.34 g / L zinc chloride. The carbon-to-nitrogen ratio in the culture medium is 3.

[0044] (2) The denitrifying microorganism P. denitrificans was cultured in sterile LB medium under aerobic conditions to OD. 600 The OD value was 1.8–2.0. Under anaerobic conditions, *Proteiniphilum* was cultured to OD values ​​using sterile acetogenic proteinophilic bacteria medium. 600 The concentration was 0.5–1.0. The pre-cultured denitrifying microorganisms P. denitrificans and Proteiniphilum were washed three times with PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4 and 0.24 g / L KH2PO4, pH = 7.4) and then concentrated to 30 mL.

[0045] (3) Add 0.18 ml of sterilized glucose to 100 mL of the sterilized culture medium to make the glucose concentration 0.9 g / L. Then, inoculate 0.05 mL of the pre-cultured denitrifying microorganism P. denitrificans into 100 mL of the above anaerobic denitrification culture medium, and then inoculate 0.5 mL of Proteiniphilum to make the inoculation volume ratio of P. denitrificans to Proteiniphilum 1:10. The denitrifying microorganisms P. denitrificans and Proteiniphilum are inoculated separately into the denitrification culture medium as controls. Place the anaerobic bottle on a shaker and carry out anaerobic denitrification culture at 37°C.

[0046] The results showed that *Proteiniphilum* itself has a very low nitrate reduction capacity, and denitrification is mainly accomplished by the denitrifying microorganism *P. denitrificans*. Under a CO2 atmosphere, the denitrification efficiency decreased by 32.1%, and the rate decreased by 66.9%. After adding *Proteiniphilum*, the denitrification efficiency of the co-cultured anaerobic denitrification system increased from 38.5% to 90.8%, ultimately resulting in no nitrite accumulation and a 52.7% reduction in nitrous oxide accumulation.

[0047] Example 3

[0048] (1) Add culture medium and 1 mL of trace elements to a sterile serum bottle, adjust the pH of the system to 7.2 with NaOH and HCl, purge with nitrogen for 30 minutes to maintain an anaerobic state. Then purge with 20% CO2 gas in the headspace, seal with a sterile butyl rubber stopper, and sterilize at 121℃ for 20 minutes to obtain the sterilized culture medium system C.

[0049] The culture medium comprises a carbon source and inorganic salts. The carbon source is glucose, prepared at 250 g / L and sterilized at 115°C for 15 minutes. The inorganic salts include 2.16 g / L potassium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate, 4.65 g / L disodium hydrogen phosphate, 2.44 g / L potassium dihydrogen phosphate, 0.2 g / L cysteine, and 1 mL / L trace elements. The trace elements include 7.30 g / L disodium ethylenediaminetetraacetate, 2.50 g / L ferrous sulfate heptahydrate, 0.02 g / L manganese chloride, 0.242 g / L sodium molybdate, 0.02 g / L calcium chloride, 0.135 g / L copper chloride crystals, and 0.34 g / L zinc chloride. The carbon-to-nitrogen ratio in the culture medium is 2.

[0050] (2) The denitrifying microorganism P. denitrificans was cultured in sterile LB medium under aerobic conditions to OD. 600 The OD value was 1.8–2.0. Under anaerobic conditions, *Proteiniphilum* was cultured to OD values ​​using sterile acetogenic proteinophilic bacteria medium. 600 The concentration was 0.5–1.0. The pre-cultured denitrifying microorganisms P. denitrificans and Proteiniphilum were washed three times with PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4 and 0.24 g / L KH2PO4, pH = 7.4) and then concentrated to 30 mL.

[0051] (3) Add 0.12 ml of sterilized glucose to 100 mL of the sterilized culture medium to make the glucose concentration 0.6 g / L. Then, inoculate 0.05 mL of the pre-cultured denitrifying microorganism P. denitrificans into 100 mL of the above anaerobic denitrification culture medium, and then inoculate 0.6 mL of Proteiniphilum to make the inoculation volume ratio of P. denitrificans to Proteiniphilum 1:12. The denitrifying microorganisms P. denitrificans and Proteiniphilum are inoculated separately into the denitrification culture medium as controls. Place the anaerobic bottle on a shaker and carry out anaerobic denitrification culture at 37°C.

[0052] The results showed that *Proteiniphilum* itself has a very low nitrate reduction capacity, and denitrification is mainly accomplished by the denitrifying microorganism *P. denitrificans*. Under a CO2 atmosphere, the denitrification efficiency decreased by 46.3%, and the rate decreased by 96.5%. After adding *Proteiniphilum*, the denitrification efficiency of the co-cultured anaerobic denitrification system increased from 23.4% to 87.3%, ultimately resulting in no nitrite accumulation and a 34.7% reduction in nitrous oxide accumulation.

[0053] Example 4

[0054] (1) In a sterile serum bottle, add culture medium and 1 mL of trace elements, adjust the pH of the system to 7.2 with NaOH and HCl, purge with nitrogen for 30 minutes to maintain an anaerobic state. Then purge with 0.3% CO2 gas in the headspace, seal with a sterile butyl rubber stopper, and sterilize at 121℃ for 20 minutes to obtain the sterilized culture medium system D.

[0055] The culture medium comprises a carbon source and inorganic salts. The carbon source is glucose, prepared at 250 g / L and sterilized at 115°C for 15 minutes. The inorganic salts include 2.16 g / L potassium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate, 4.65 g / L disodium hydrogen phosphate, 2.44 g / L potassium dihydrogen phosphate, 0.2 g / L cysteine, and 1 mL / L trace elements. The trace elements include 7.30 g / L disodium ethylenediaminetetraacetate, 2.50 g / L ferrous sulfate heptahydrate, 0.02 g / L manganese chloride, 0.242 g / L sodium molybdate, 0.02 g / L calcium chloride, 0.135 g / L copper chloride crystals, and 0.34 g / L zinc chloride. The carbon-to-nitrogen ratio in the culture medium is 5.

[0056] (2) The denitrifying microorganism P. denitrificans was cultured in sterile LB medium under aerobic conditions to OD. 600The OD value was 1.8–2.0. Under anaerobic conditions, *Proteiniphilum* was cultured to OD values ​​using sterile acetogenic proteinophilic bacteria medium. 600 The concentration was 0.5–1.0. The pre-cultured denitrifying microorganisms P. denitrificans and Proteiniphilum were washed three times with PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4 and 0.24 g / L KH2PO4, pH = 7.4) and then concentrated to 30 mL.

[0057] (3) Add 0.3 ml of sterilized glucose to 100 mL of the sterilized culture medium to make the glucose concentration 1.5 g / L. Then, inoculate 0.05 mL of the pre-cultured denitrifying microorganism P. denitrificans into 100 mL of the above anaerobic denitrification culture medium, and then inoculate 0.4 mL of Proteiniphilum to make the inoculation volume ratio of P. denitrificans to Proteiniphilum 1:8. The denitrifying microorganisms P. denitrificans and Proteiniphilum are inoculated separately into the denitrification culture medium as controls. Place the anaerobic bottle on a shaker and carry out anaerobic denitrification culture at 30 °C.

[0058] The results showed that *Proteiniphilum* itself has a very low nitrate reduction capacity, and denitrification is mainly accomplished by the denitrifying microorganism *P. denitrificans*. Under a CO2 atmosphere, the denitrification efficiency decreased by 21.5%, and the rate decreased by 44.8%. After adding *Proteiniphilum*, the denitrification efficiency of the co-cultured anaerobic denitrification system increased from 73.4% to 99.8%, ultimately resulting in no nitrite accumulation and a 58.7% reduction in nitrous oxide accumulation.

[0059] Example 5

[0060] (1) Add culture medium and 1 mL of trace elements to a sterile serum bottle, adjust the pH of the system to 7.0 with NaOH and HCl, purge with nitrogen for 30 minutes to maintain an anaerobic state. Then purge with 5% CO2 gas in the headspace, seal with a sterile butyl rubber stopper, and sterilize at 121℃ for 20 minutes to obtain sterilized culture medium system E.

[0061] The culture medium comprises a carbon source and inorganic salts. The carbon source is glucose, prepared at 250 g / L and sterilized at 115°C for 15 minutes. The inorganic salts include 2.16 g / L potassium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate, 4.65 g / L disodium hydrogen phosphate, 2.44 g / L potassium dihydrogen phosphate, 0.2 g / L cysteine, and 1 mL / L trace elements. The trace elements include 7.30 g / L disodium ethylenediaminetetraacetate, 2.50 g / L ferrous sulfate heptahydrate, 0.02 g / L manganese chloride, 0.242 g / L sodium molybdate, 0.02 g / L calcium chloride, 0.135 g / L copper chloride crystals, and 0.34 g / L zinc chloride. The carbon-to-nitrogen ratio in the culture medium is 3.

[0062] (2) The denitrifying microorganism P. denitrificans was cultured in sterile LB medium under aerobic conditions to OD. 600 The OD value was 1.8–2.0. Under anaerobic conditions, *Proteiniphilum* was cultured to OD values ​​using sterile acetogenic proteinophilic bacteria medium. 600 The concentration was 0.5–1.0. The pre-cultured denitrifying microorganisms P. denitrificans and Proteiniphilum were washed three times with PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4 and 0.24 g / L KH2PO4, pH = 7.4) and then concentrated to 30 mL.

[0063] (3) Add 0.18 ml of sterilized glucose to 100 mL of the sterilized culture medium to make the glucose concentration 0.9 g / L. Then, inoculate 0.05 mL of the pre-cultured denitrifying microorganism P. denitrificans into 100 mL of the above anaerobic denitrification culture medium, and then inoculate 0.75 mL of Proteiniphilum to make the inoculation volume ratio of P. denitrificans to Proteiniphilum 1:15. The denitrifying microorganisms P. denitrificans and Proteiniphilum are inoculated separately into the denitrification culture medium as controls. Place the anaerobic bottle on a shaker and carry out anaerobic denitrification culture at 30 °C.

[0064] The results showed that *Proteiniphilum* itself has a very low nitrate reduction capacity, and denitrification is mainly accomplished by the denitrifying microorganism *P. denitrificans*. Under a CO2 atmosphere, the denitrification efficiency decreased by 28.3%, and the rate decreased by 58.9%. After adding *Proteiniphilum*, the denitrification efficiency of the co-cultured anaerobic denitrification system increased from 38.5% to 99.9%, ultimately resulting in no nitrite accumulation and a 74.6% reduction in nitrous oxide accumulation.

[0065] Example 6

[0066] (1) Add culture medium and 1 mL of trace elements to a sterile serum bottle, adjust the pH of the system to 7.2 with NaOH and HCl, purge with nitrogen for 30 minutes to maintain an anaerobic state. Then purge with 20% CO2 gas in the headspace, seal with a sterile butyl rubber stopper, and sterilize at 121℃ for 20 minutes to obtain the sterilized culture medium system F.

[0067] The culture medium comprises a carbon source and inorganic salts. The carbon source is glucose, prepared at 250 g / L and sterilized at 115°C for 15 minutes. The inorganic salts include 2.16 g / L potassium nitrate, 0.5 g / L ammonium chloride, 0.07–0.1 g / L magnesium sulfate, 0.5–4.65 g / L disodium hydrogen phosphate, 1–2.44 g / L potassium dihydrogen phosphate, 0.2 g / L cysteine, and 1 mL / L trace elements. The trace elements include 7.30 g / L disodium ethylenediaminetetraacetate, 2.50 g / L ferrous sulfate heptahydrate, 0.02 g / L manganese chloride, 0.242 g / L sodium molybdate, 0.02 g / L calcium chloride, 0.135 g / L copper chloride crystals, and 0.34 g / L zinc chloride. The carbon-to-nitrogen ratio in the culture medium is 4.

[0068] (2) The denitrifying microorganism P. denitrificans was cultured in sterile LB medium under aerobic conditions to OD. 600 The OD value was 1.8–2.0. Under anaerobic conditions, *Proteiniphilum* was cultured to OD values ​​using sterile acetogenic proteinophilic bacteria medium. 600 The concentration was 0.5–1.0. The pre-cultured denitrifying microorganisms P. denitrificans and Proteiniphilum were washed three times with PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4 and 0.24 g / L KH2PO4, pH = 7.4) and then concentrated to 30 mL.

[0069] (3) Add 0.24 ml of sterilized glucose to 100 mL of the sterilized culture medium to make the glucose concentration 1.2 g / L. Then, inoculate 0.05 mL of the pre-cultured denitrifying microorganism P. denitrificans into 100 mL of the above anaerobic denitrification culture medium, and then inoculate 0.4 mL of Proteiniphilum to make the inoculation volume ratio of P. denitrificans to Proteiniphilum 1:8. The denitrifying microorganisms P. denitrificans and Proteiniphilum are inoculated separately into the denitrification culture medium as controls. Place the anaerobic bottle on a shaker and carry out anaerobic denitrification culture at 35 °C.

[0070] The results showed that *Proteiniphilum* itself has a very low nitrate reduction capacity, and denitrification is mainly accomplished by the denitrifying microorganism *P. denitrificans*. Under a CO2 atmosphere, the denitrification efficiency decreased by 25.6%, and the rate decreased by 53.5%. After adding *Proteiniphilum*, the denitrification efficiency of the co-cultured anaerobic denitrification system increased from 48.6% to 96.8%, ultimately resulting in no nitrite accumulation and a 72.6% reduction in nitrous oxide accumulation.

[0071] Example 7

[0072] (1) Add culture medium and 1 mL of trace elements to a sterile serum bottle, adjust the pH of the system to 7.0 with NaOH and HCl, purge with nitrogen for 30 minutes to maintain an anaerobic state. Then purge with 20% CO2 gas in the headspace, seal with a sterile butyl rubber stopper, and sterilize at 121℃ for 20 minutes to obtain sterilized culture medium system G.

[0073] The culture medium comprises a carbon source and inorganic salts. The carbon source is glucose, prepared at 250 g / L and sterilized at 115°C for 15 minutes. The inorganic salts include 1.44 g / L potassium nitrate, 0.5 g / L ammonium chloride, 0.07–0.1 g / L magnesium sulfate, 0.5–4.65 g / L disodium hydrogen phosphate, 1–2.44 g / L potassium dihydrogen phosphate, 0.2 g / L cysteine, and 1 mL / L trace elements. The trace elements include 7.30 g / L disodium ethylenediaminetetraacetate, 2.50 g / L ferrous sulfate heptahydrate, 0.02 g / L manganese chloride, 0.242 g / L sodium molybdate, 0.02 g / L calcium chloride, 0.135 g / L copper chloride crystals, and 0.34 g / L zinc chloride. The carbon-to-nitrogen ratio in the culture medium is 5.

[0074] (2) The denitrifying microorganism P. denitrificans was cultured in sterile LB medium under aerobic conditions to OD. 600The OD value was 1.8–2.0. Under anaerobic conditions, *Proteiniphilum* was cultured to OD values ​​using sterile acetogenic proteinophilic bacteria medium. 600 The concentration was 0.5–1.0. The pre-cultured denitrifying microorganisms P. denitrificans and Proteiniphilum were washed three times with PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4 and 0.24 g / L KH2PO4, pH = 7.4) and then concentrated to 30 mL.

[0075] (3) Add 0.3 mL of sterilized glucose to 100 mL of the sterilized culture medium to make the glucose concentration 1.5 g / L. Then, inoculate 0.05 mL of the pre-cultured denitrifying microorganism P. denitrificans into 100 mL of the above anaerobic denitrification culture medium, and then inoculate 0.5 mL of Proteiniphilum to make the inoculation volume ratio of P. denitrificans to Proteiniphilum 1:10. The denitrifying microorganisms P. denitrificans and Proteiniphilum are inoculated separately into the denitrification culture medium as controls. Place the anaerobic bottle on a shaker and carry out anaerobic denitrification culture at 35 °C.

[0076] The results showed that *Proteiniphilum* itself has a very low nitrate reduction capacity, and denitrification is mainly accomplished by the denitrifying microorganism *P. denitrificans*. Under a CO2 atmosphere, the denitrification efficiency decreased by 23.4%, and the rate decreased by 48.8%. After adding *Proteiniphilum*, the denitrification efficiency of the co-cultured anaerobic denitrification system increased from 59.8% to 93.5%, ultimately resulting in no nitrite accumulation and a 73.5% reduction in nitrous oxide accumulation.

[0077] Table 1

[0078]

[0079]

[0080] Analysis of the data in Table 1 and the examples shows that in a denitrification system with a defined content of *Paracococcus denitrifyingus*, the presence of carbon dioxide inhibits the denitrification process of *Paracococcus denitrifyingus*. Adding a certain concentration of acetophilic bacteria can restore or even enhance the nitrate reduction rate. Specifically, when the nitrate content is 300 mg / L, the carbon-to-nitrogen source ratio is 3, and the inoculum volume ratio of *Paracococcus denitrifyingus* to acetophilic bacteria is 1:8 or 1:10, the denitrification rate can be significantly restored and enhanced, increasing to 99.4%, with low accumulation of intermediate products. The pH of 7.2 and the temperature of 37°C are both within the optimal range for both microorganisms, thus achieving the optimal culture environment for both bacteria. In a carbon dioxide atmosphere with a carbon-to-nitrogen ratio of 2–5, a low carbon-to-nitrogen ratio may lead to insufficient carbon source for nitrate bioreduction or nitrite accumulation. This method can maximize the utilization of carbon source in the system, thereby improving the denitrification reduction rate. Among these, the best effect is achieved when the inoculation volume ratio of denitrifying paracocci to acetophilic bacteria is 1:10 at a carbon-to-nitrogen ratio of around 3. However, a higher ratio of bacteria will not enhance the promoting effect, as the growth of microorganisms requires a certain amount of carbon source.

[0081] In this invention, a certain proportion of mixed bacteria can mitigate the adverse effects of CO2 on denitrification because there is a synergistic effect between *Paracococcus denitrifyingans* and *Acetobacter acetophilus*. This synergistic effect counteracts the adverse effects of CO2 by producing microbial secondary metabolites or increasing the content of extracellular polymers in the system, thereby restoring and promoting the denitrification process. Compared with physical and chemical methods, this approach not only saves costs but also produces fewer harmful intermediate products and generates less secondary pollution.

[0082] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for mitigating the inhibition of denitrification by carbon dioxide and promoting denitrification, characterized in that, By adding denitrifying paracocci and acetic acid-producing proteinophiles, the inhibitory effect of carbon dioxide on denitrification is synergistically alleviated and microbial denitrification is promoted; Includes the following steps: S1. Add culture medium and trace elements to the reactor and perform anaerobic treatment. Then, introduce carbon dioxide and sterilize to obtain an anaerobic denitrification culture medium system. S2. Provide suspensions of denitrifying paracocci and acetic acid-producing proteinophilic bacteria; S3. The denitrifying paracoccus suspension and the acetic acid-producing proteinophilic bacteria suspension are inoculated into the anaerobic denitrification culture medium system obtained in step S1 and anaerobic denitrification culture is carried out. In step S1, the molar percentage of carbon dioxide is 0.3% to 20%, and the carbon-to-nitrogen ratio in the culture medium is 1 to 5. In step S3, the carbon-to-nitrogen ratio of the anaerobic denitrification culture medium system is 1 to 5; The inoculation ratio of the denitrifying paracoccus suspension and the acetic acid-producing proteinophilic bacteria suspension is 1:2 to 1:15; The pH of the anaerobic denitrification culture medium system is 6.0–9.0; The anaerobic denitrification culture temperature is 30–37°C.

2. The method for mitigating carbon dioxide's inhibition of denitrification and promoting denitrification according to claim 1, characterized in that, In step S1, the culture medium includes a carbon source and inorganic salts.

3. The method for mitigating carbon dioxide's inhibition of denitrification and promoting denitrification according to claim 1, characterized in that, In step S2, the initial OD of the denitrifying paracoccus suspension 600 It ranges from 0.01 to 0.

02.

4. The method for mitigating carbon dioxide's inhibition of denitrification and promoting denitrification according to claim 1, characterized in that, In step S2, the initial OD600 of the acetic acid-producing proteinophilic bacteria suspension is 0.02~0.2.

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