Pseudomonas aeruginosa, denitrification composite filler as well as preparation method and application of denitrification composite filler

Abstract

The invention relates to a pseudomonas aeruginosa strain, a denitrification composite filler as well as a preparation method and application of the pseudomonas aeruginosa strain and the denitrification composite filler. The pseudomonas aeruginosa strain ZZU PseZJJ-01 is preserved in Guangdong Microbial Culture Collection Center on December 7, 2023, and the preservation number is GDMCC No: 1.4478. The invention further discloses a preparation method of the denitrification composite filler. The denitrification composite filler is mainly prepared by cross-linking and curing an embedding material hydrogel, a culture bacterium solution of the pseudomonas aeruginosa, an iron matrix and / or a ferrous sulfide matrix or a mixture of auxiliaries in a cross-linking solution. The invention further discloses application of the pseudomonas aeruginosa and a preparation method and application of the composite filler. The pseudomonas aeruginosa is high in denitrification efficiency and long in biological activity maintaining time, the composite filler is good in denitrification effect and good in microorganism immobilization and activity retentivity, and the pseudomonas aeruginosa and the composite filler can be put underground in situ. The preparation method is simple, low in cost and suitable for industrial production.

Description

Technical Field

[0001] The present invention specifically relates to a strain of Pseudomonas aeruginosa, a denitrification composite filler, and a preparation method and application thereof. Background Art

[0002] Due to agricultural non-point source pollution and urban sewage leakage, the concentration of nitrate in groundwater continues to rise, threatening ecological safety and human health. Biological denitrification is considered to be an economical, efficient and environmentally friendly in-situ denitrification technology. Among them, in recent years, the sulfur-iron co-substrate denitrification system has been widely used because of its ability to simultaneously utilize reduced iron (Fe 2+ ) and reduced sulfur (S 2- ) as an electron donor, it can achieve efficient nitrate reduction under anoxic conditions without the need for an external organic carbon source, and has long-term stability, gradually becoming a hot topic in groundwater denitrification research. In this system, the screening and optimization of functional bacterial communities are the core technology. The microbial population structure in natural water bodies or underground environments is complex. Although functional groups such as sulfur-oxidizing bacteria, iron-oxidizing bacteria, and denitrifying bacteria are common, their activity, adaptability, and reaction efficiency vary, resulting in unstable performance in engineering applications. At the same time, in actual engineering, bacterial communities face problems such as easy loss in the in-situ system, difficulty in maintaining activity, and low colonization efficiency.

[0003] my country has a vast distribution of low-grade pyrite resources (such as pyrite FeS2 and ferrous sulfide FeS). Due to their low sulfur content, high impurities, and poor crystallinity, these ores are difficult to directly use in traditional metallurgy or for the extraction of high-purity materials, resulting in them being left idle or abandoned for a long time. However, they still contain sulfur and iron, which can serve as electron donors. If these ores can be utilized as resources through structural modification and synergistic biological processes, they could not only effectively replace high-cost chemical agents (such as Na2S and FeSO4), but also achieve the ecological transformation of industrial solid waste resources and enhance their environmental value. The recent rise of porous active materials (such as modified minerals, bio-support materials, and functionalized porous particles) provides a new technological path for microbial immobilization and enhanced reaction interfaces.

[0004] CN118702276A discloses a sulfur autotrophic denitrification filler and its preparation method. The filler comprises the following raw materials: ferric chloride, calcium chloride, polyacrylamide, calcium phosphate, humic acid, starch, sulfur powder, pyrite powder, baking soda, and water. However, the filler is not bioactive and does not contain high-efficiency bacterial strains, resulting in limited denitrification effectiveness.

[0005] CN117602735A discloses a MXene-enhanced pyrite-sulfur autotrophic denitrification composite filler, its preparation method, and its application. Sulfur, pyrite, few-layer MXene, clay, and a binder are mixed in a certain proportion, mixed with an aqueous sodium thiosulfate solution, and then extruded and granulated. MXene acts as an electron transfer bridge, improving electron transfer efficiency in biological systems. However, the filler is not bioactive and does not contain efficient bacterial strains, resulting in limited denitrification effectiveness.

[0006] In summary, it is urgent to find a functional bacterium with high denitrification reaction efficiency of natural bacteria and long biological activity maintenance time, good denitrification effect, low cost, high resource utilization, good microbial fixation and activity retention, strong acid and alkali resistance, uniform spatial distribution, good mechanical strength and stability of denitrification composite filler, simple process, low cost, and suitable for industrial production of denitrification composite filler preparation method, as well as the application of Pseudomonas aeruginosa and denitrification composite filler with good denitrification effect and can be placed in situ underground. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a natural bacterial strain of Pseudomonas aeruginosa with high denitrification reaction efficiency and long-term biological activity.

[0008] The technical problem to be further solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a denitrification composite filler with good denitrification effect, low cost, high resource utilization, good microbial fixation and activity retention, strong acid and alkali resistance, uniform spatial distribution, and good mechanical strength and stability.

[0009] A further technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a method for preparing a denitrifying composite filler with a simple process, low cost and suitable for industrial production.

[0010] A further technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide an application of Pseudomonas aeruginosa and a denitrification composite filler which has a good denitrification effect and can be placed in situ underground.

[0011] The technical solution adopted by the present invention to solve the technical problem is as follows: a strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) ZZU PseZJJ-01, the Pseudomonas aeruginosa ZZU PseZJJ-01 was deposited with the Guangdong Provincial Microbial Culture Collection on December 7, 2023, with the deposit number GDMCC No: 1.4478. The Pseudomonas aeruginosa was screened from excess sludge from a sewage treatment plant and has nitrogen conversion capabilities. The strain belongs to the genus Pseudomonas and has autotrophic, heterotrophic, and mixotrophic nitrogen conversion capabilities.

[0012] The Pseudomonas aeruginosa of the present invention is isolated and cultured through a functional bacterial community system, and screening conditions are set according to the oxygen-poor and carbon-poor characteristics of groundwater to obtain an autotrophic denitrification efficient strain suitable for groundwater conditions.

[0013] The screening method for Pseudomonas aeruginosa comprises the following steps: (1) Acclimation and enrichment of excess sludge: add 180 mL of groundwater inorganic salt culture medium (add 1.5-2.5 g KNO3 to 1 L of groundwater actually taken) into a 250 mL conical flask, inoculate 20 mL of bacterial source sludge (taken from the excess sludge of the secondary sedimentation tank of a sewage treatment plant), seal the conical flask with a rubber stopper with two glass tubes A and B, where tube A is used to flush pure N2 and tube B is used to collect the generated N2 gas; blow nitrogen for 8-12 minutes to put the bacterial solution in an anaerobic growth environment, and culture it in a constant temperature shaker at a temperature of 28-32 ° C and a speed of 100-140 r / min. Replace the groundwater inorganic salt culture medium every 4 days, and take 20 mL of the acclimated bacterial solution and add it to the new groundwater inorganic salt culture medium after replacement. Repeat this operation to allow the bacteria to fully reproduce. After acclimation and culture for 32-40 days, a stable bacterial solution for enrichment culture is obtained; (2) Strain separation and purification: add physiological saline to the enriched culture stable bacterial solution obtained in step (1) at a volume ratio of 1:1 to prepare an enriched culture suspension, and perform 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 The concentration was diluted in a gradient manner, and the gradient dilution solution was used as an inoculum to be spread on LB solid medium for inoculation. The LB solid medium was then placed in an anaerobic incubator at 28-32°C for static culture, and the growth of the colonies was observed. When colonies visible to the naked eye appeared on the LB solid medium, a single colony was picked with an inoculating loop for streak separation, and the colony was placed in an anaerobic incubator at 28-32°C for further static culture. The streak separation operation was repeated several times until the colony morphology was consistent, thereby obtaining a purified strain. The LB solid medium (1 L) contained 5 g yeast extract powder, 10 g NaCl, 10 g tryptone, and 20 g agar powder, and the pH value was 7.00±0.2. (3) Identification of bacterial species: The purified strain obtained in step (2) was sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA identification. The sequencing results were submitted to GenBank and compared with the GenBank ribosome database using the blast comparison program on NCBI (http: / / rdp.cme.msu / index.jsp). The identification result was: Pseudomonas aeruginosa. The strain was then sent to the Guangdong Provincial Microbial Culture Collection for preservation and was numbered ZZU PseZJJ-01.

[0014] The present invention further solves the technical problem by adopting the following technical solution: a denitrification composite filler mainly composed of embedding material hydrogel, the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) ZZU PseZJJ-01 culture solution, an iron matrix and / or a ferrosulphide matrix, or a mixture thereof with an additive, are cross-linked and solidified in a cross-linking solution. The denitrifying composite filler of the present invention utilizes an embedding material as a framework to encapsulate nitrogen-converting bacterial strains isolated and purified from excess sludge. This embedding material serves as a material framework and electron donor source to achieve efficient denitrification and functionalize the reuse of minerals, thus possessing significant economic and environmental significance. The embedding material has a high specific surface area, excellent mass transfer properties, and a controllable pore structure, significantly enhancing microbial activity and pollutant transport efficiency, thereby achieving bacteria-material coupling for in-situ groundwater remediation. The cross-linking solution serves to secure the bacteria into spheres.

[0015] Preferably, the embedding material hydrogel and the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The volume ratio of the culture solution of ZZU PseZJJ-01 is 100:40~60.

[0016] Preferably, the embedding material includes polyvinyl alcohol and / or sodium alginate, etc. More preferably, the embedding material includes polyvinyl alcohol and sodium alginate, etc. Polyvinyl alcohol (PVA) is a synthetic polymer produced by the hydrolysis of polyvinyl acetate. Its structure contains hydroxyl groups, which promotes the chemical cross-linking process. Due to its non-toxic properties, good biodegradability and gel properties, PVA is a suitable choice for enhancing the biological properties, durability and chemical stability of SA, and has attracted widespread attention due to its low cost and good biocompatibility. The present invention adds SA to the PVA gel system, which can effectively improve the network structure of the existing matrix material pore structure that is not conducive to material transfer, increase the mass transfer rate, reduce the tendency of agglomeration, improve the mechanical properties, and enhance the mechanical strength of the carrier.

[0017] Preferably, in the embedding material hydrogel, the mass concentration of polyvinyl alcohol is 0-15%, the mass concentration of sodium alginate is 0-4%, and the difference between the two is 0. More preferably, in the embedding material hydrogel, the mass concentration of polyvinyl alcohol is 5-15%, and the mass concentration of sodium alginate is 1-4%.

[0018] Preferably, the embedding material hydrogel is prepared by adding the embedding material into water, heating it in a water bath to 85-95° C. until it is completely dissolved, and then naturally cooling it to room temperature.

[0019] Preferably, the mass volume ratio of the iron matrix and / or ferrosulfide matrix, the auxiliary agent and the embedding material hydrogel is 1-6:0-4:100 (more preferably 2-4:1-3:100) in g / g / mL.

[0020] Preferably, the particle size of the iron matrix or ferrosulfide matrix is ​​75 to 150 μm.

[0021] Preferably, the iron matrix is ​​iron powder with an iron content of ≥95%.

[0022] Preferably, the pyrite matrix includes one or more of pyrite, FeS2, or FeS. The pyrite includes pyrite, ferrous sulfide, etc., wherein pyrite mainly contains FeS2, and the mass content of FeS2 is ≥90%, and the mass content of ferrous sulfide mainly contains FeS, and the mass content of Fe is ≥60%, and the mass content of S is ≥25%.

[0023] Preferably, the additive includes activated carbon and / or calcium carbonate. Because the three-dimensional network structure formed by cross-linking PVA-SA is relatively dense, adding activated carbon (C) with a high specific surface area to the PVA-SA mixture can increase the porosity of the hydrogel and its bacterial adsorption capacity, thereby improving its performance in removing water pollutants. Adding calcium carbonate can also enhance the mechanical properties of the denitrifying composite filler.

[0024] Preferably, the particle size of the auxiliary agent is sieved through 100 mesh.

[0025] Preferably, the cross-linking liquid includes a saturated boric acid cross-linking liquid containing calcium chloride, etc. The cross-linking material has high chemical stability, good biocompatibility, and high cross-linking efficiency. By cross-linking calcium chloride and saturated boric acid, a stable gel structure can be formed. Taking the embedding material as polyvinyl alcohol PVA-sodium alginate SA as an example, PVA is the main chain of a representative polymer because it has a crystalline domain generated by strong hydrogen bonds, which can provide many physical cross-linking sites; calcium chloride is selected as a hydrogen bond acceptor to prepare a metal salt-based DES hydrogel, which can make full use of the conductivity of anions and cations, and at the same time, through the coordination of metal cations and PVA hydroxyl groups, the hydrogel has excellent mechanical properties and conductivity; the formation of hydrogel fibers originates from the formation of the lattice between PVA molecules, in which Ca2+ The ions in the spinning solution combine with the hydroxyl groups of the PVA molecules to form a cross-linked structure, and the coordinated divalent ions between SA and Ca play a role in this process; boric acid (B(OH)3) can form a complex with the hydroxyl groups (-OH) in polyvinyl alcohol in the solution, promoting the cross-linking and gel formation of the polyvinyl alcohol chains, and can also cooperate with the carboxyl groups in sodium alginate to further enhance the network structure.

[0026] Preferably, the concentration of calcium chloride in the cross-linking solution is 15 to 25 g / L. By adjusting the concentration of calcium chloride, the mechanical strength and porosity can be adjusted.

[0027] Preferably, the OD of the culture solution is 600 is 0.6~0.8. 600 The bacterial liquid below is in a rapid growth period.

[0028] Preferably, the preparation method of the culture solution is: taking the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) ZZU PseZJJ-01 was cultured in LB liquid medium with a shaker, centrifuged, and washed with phosphate buffer for ≥2 times, and then resuspended with sterile saline.

[0029] Preferably, the LB liquid culture medium (1 L) contains 5 g yeast extract powder, 10 g NaCl, 10 g tryptone, and has a pH value of 7.00±0.2.

[0030] Preferably, the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The volume ratio of ZZU PseZJJ-01 to LB liquid culture medium is 1:80-120.

[0031] Preferably, the shaking incubation temperature is 28-32°C, the rotation speed is 100-140 r / min, and the time is 10-14 h until the OD 600 The OD of the bacterial solution is 0.6 to 0.8. 600 When it is 0.6-0.8, it is a rapid growth period, and the experimental effect of inoculating the bacterial solution at this stage is better.

[0032] Preferably, the rotation speed of the centrifugal precipitation is 8000-12000 r / min, and the time is 8-12 min.

[0033] Preferably, the resuspending to OD 600 It is 0.6~0.8.

[0034] In summary, the denitrifying composite filler of the present invention combines a pyrite matrix and / or low-grade pyrite (such as pyrite, ferrous sulfide, etc.) with a functional bacterial community through structural modification. Using an embedding material (such as polyvinyl alcohol (PVA)-sodium alginate (SA)) as the matrix and utilizing a saturated boric acid crosslinking solution containing calcium chloride as a dual-crosslinking system, the material's spherical formability and structural density are enhanced, forming a stable porous structure. Ultimately, this improves the material's electron donor capacity, microbial stability, and reaction sustainability during the denitrification process, significantly enhancing bacterial fixation efficiency and long-term bioactivity maintenance. When supplemented with activated carbon as a bio-adjuvant, the resulting three-dimensional network structure not only exhibits excellent porosity, mass transfer performance, mechanical stability, and pH buffering properties, but also provides excellent attachment space and nutrient exchange conditions for the functional bacterial community. Calcium carbonate primarily enhances the mechanical stability of the denitrifying composite filler. Compared with the traditional method of adding bacteria, the denitrifying composite filler of the present invention can realize the three-in-one integrated construction of "functional bacteria + electron donor + active matrix" to construct an enhanced sulfur-iron co-matrix active material, which shows stronger reaction controllability and operational stability in engineering practice, and has broad application prospects and practical significance.

[0035] The present invention further solves the technical problem by adopting the following technical solution: a method for preparing a denitrification composite filler, wherein the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The culture solution of ZZUPseZJJ-01, iron matrix and / or ferrosulphur matrix, or auxiliary agents, are mixed evenly, and then added dropwise into the cross-linking solution for cross-linking and solidification into balls, which are filtered and refrigerated to obtain a denitrifying composite filler.

[0036] Preferably, the cross-linking and curing into balls is carried out at room temperature for 18 to 30 hours.

[0037] Preferably, the particle size of the cross-linked and solidified spheres is 3 to 6 mm.

[0038] The present invention further solves the technical problem by adopting the following technical solution: a Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) Application of ZZU PseZJJ-01, in nitrogenous wastewater, autotrophic, heterotrophic or mixed culture raw materials are added, or inorganic carbon sources are added, and under anaerobic conditions, the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The culture solution of ZZU PseZJJ-01 is sealed after adjusting the pH value, and placed in a shaking incubator for autotrophic, heterotrophic or mixed culture.

[0039] Preferably, in the nitrogen-containing wastewater, NO3 - -N system initial concentration is 10 ~ 80 mg / L. The NO3 - -N system initial concentration refers to NO3- -N based on NO3 - -N is the concentration of the total volume of all materials including groundwater, culture solution, sludge lysate, etc.

[0040] Preferably, in the autotrophic culture, the autotrophic raw material is added to the nitrogen-containing wastewater system at an amount of 15-25 g / L. In the autotrophic environment, the strain uses sulfur, iron and organic matter as electron donors and sodium bicarbonate as an inorganic carbon source for autotrophic denitrification.

[0041] Preferably, in the polyculture, the autotrophic raw material is added to the nitrogen-containing wastewater system in an amount of 5 to 15 g / L.

[0042] Preferably, the carbon-nitrogen ratio of the heterotrophic raw material to the nitrogen-containing wastewater added in the heterotrophic culture is 4 to 8: 1. In the environment where the heterotrophic raw material exists, the strain uses organic matter as an organic carbon source to perform heterotrophic denitrification.

[0043] Preferably, the carbon-nitrogen ratio of the heterotrophic feedstock to the nitrogen-containing wastewater added in the polyculture is 3 to 5:1. In the presence of the polyculture feedstock, the strain uses sulfur, iron, and organic matter as electron donors, sodium bicarbonate as an inorganic carbon source, and organic matter as an organic carbon source for polyculture denitrification.

[0044] Preferably, the autotrophic raw material comprises one or more of an iron matrix, pyrite, FeS2, or FeS. The iron matrix is ​​iron powder having an iron content of ≥95%. The pyrite comprises pyrite, ferrous sulfide, etc., wherein pyrite primarily contains FeS2 with a mass content of ≥90%, and ferrous sulfide primarily contains FeS with a mass content of ≥60% and a mass content of ≥25%.

[0045] Preferably, the heterotrophic raw material includes one or more of glucose, sludge lysate or sodium succinate. When glucose or sodium succinate is used as heterotrophic or mixed culture raw material, carbon-nitrogen ratio = chemical oxygen demand (COD): NO3 in nitrogen-containing wastewater - -N system initial concentration; when using sludge lysate as heterotrophic or mixed culture raw material, carbon-nitrogen ratio = dissolved chemical oxygen demand (SCOD): NO3 in nitrogen-containing wastewater - -The initial concentration of N in the system.

[0046] Preferably, an inorganic carbon source is added to the nitrogen-containing wastewater system at a dosage of 0.5 to 1.5 g / L while adding the autotrophic or polytrophic raw materials. More preferably, the inorganic carbon source is sodium bicarbonate.

[0047] Preferably, the anaerobic condition refers to exposing nitrogen-containing wastewater to helium for 15 to 25 minutes.

[0048] Preferably, the Pseudomonas aeruginosa ( Pseudomonas aeruginosa.The volume ratio of the culture solution of ZZU PseZJJ-01 to the nitrogen-containing wastewater system is 4 to 6:100. The preparation method of the culture solution is as described above.

[0049] Preferably, the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The bacterial protein concentration of the culture solution of ZZU PseZJJ-01 is 40-60 mg / L. The bacterial protein concentration is the bacterial solution OD 600 Measured at 0.6 to 0.8.

[0050] Preferably, the pH value is adjusted to 7.5 to 8.5. When autotrophy or polytrophy is adopted in the present invention, sodium bicarbonate is added as an inorganic carbon source, and the pH value is generally alkaline. Adjusting the pH value to the above value is more conducive to autotrophic, heterotrophic or polytrophic reactions.

[0051] Preferably, the autotrophic, heterotrophic, or mixotrophic culture is carried out at a temperature of 25-35°C and a rotational speed of 100-200 rpm. The autotrophic culture is carried out for 2-30 days, the heterotrophic culture for 10-30 hours, and the mixotrophic culture for 10 hours-10 days (more preferably 5-10 days), until both nitrate and nitrite nitrogen are completely reduced. Under these culture conditions, the bacterial solution is in a rapid growth phase, and the inoculated bacterial solution exhibits good activity.

[0052] In the nitrogen-containing wastewater system of the present invention, the amount of each material is based on the NO3 - -N is the concentration of the total volume of all materials including groundwater, culture solution, sludge lysate, etc.

[0053] The present invention further solves the technical problem by adopting the following technical solution: an application of a denitrification composite filler, wherein the denitrification composite filler is used for denitrification to remove nitrate.

[0054] Preferably, the denitrification composite filler and the inorganic carbon source are added to the nitrogen-containing wastewater, placed in a shaking table, and subjected to autotrophic cultivation.

[0055] Preferably, the solid-liquid ratio of the denitrification composite filler to the nitrogen-containing wastewater is 1 to 3 g / mL: 100. At this dosage, both nitrate nitrogen and nitrite nitrogen can be completely removed while saving filler.

[0056] Preferably, the amount of the inorganic carbon source added is 0.5 to 1.5 g / L nitrogen-containing wastewater. More preferably, the inorganic carbon source is sodium bicarbonate.

[0057] Preferably, in the nitrogen-containing wastewater, NO3 - The concentration of -N is 10 to 80 mg / L. The purpose of the present invention is to treat nitrate nitrogen wastewater using immobilized microorganisms, so the selected nitrate nitrogen concentration is relatively low.

[0058] Preferably, the temperature of the autotrophic culture is 25-35° C., the rotation speed is 100-200 r / min, and the time is 10-20 days, until the nitrate nitrogen and nitrite nitrogen are completely reduced.

[0059] The beneficial effects of the present invention are as follows: (1) The natural bacterial flora of Pseudomonas aeruginosa of the present invention has high denitrification reaction efficiency and maintains biological activity for a long time; (2) The denitrification composite filler of the present invention has a good denitrification effect; it can efficiently utilize low-grade pyrite, solving the problem of high cost of pyrite substrate and difficulty in directly using traditional pyrite in the denitrification process, with low raw material cost and high resource utilization rate; based on the PVA-SA network, a good bacterial carrier environment is constructed, with good microbial fixation and activity retention, strong acid and alkali resistance, and no significant changes in salt solution; the porous structure + activated carbon co-carrier design can improve the spatial distribution uniformity of electron donors and bacterial communities, promote material exchange; and it has good mechanical strength and stability; (3) The preparation method of the denitrifying composite filler of the present invention is simple in process, low in cost, and suitable for industrial production. The material composition can be flexibly adjusted according to parameters such as nitrate nitrogen concentration and temperature in groundwater; (4) The application of the Pseudomonas aeruginosa and denitrifying composite filler of the present invention in water denitrification has a good denitrification effect, has stability adapted to the groundwater environment, and can be placed in situ underground; it provides an efficient and sustainable new material for the treatment of groundwater nitrate pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Picture 1 Pseudomonas aeruginosa of the present invention ( Pseudomonas aeruginosa. ) Application Example 1 and Comparative Example 1 of ZZU PseZJJ-01 at different initial NO3 - After autotrophic denitrification at the concentration of - -N concentration (a) and NO2 - -N concentration (b) comparison chart; Picture 2 Pseudomonas aeruginosa of the present invention ( Pseudomonas aeruginosa. ) Application Example 1 of ZZU PseZJJ-01 After heterotrophic denitrification under different carbon sources, NO3 - -N concentration (a), NO2 - -N concentration (b) comparison chart; Picture 3 Pseudomonas aeruginosa of the present invention ( Pseudomonas aeruginosa. ) Application Example 1 of ZZU PseZJJ-01 After heterotrophic denitrification under different carbon sources, NH4 + -Comparison of changes in N concentration (c) and TN concentration (d); Picture 4 Pseudomonas aeruginosa of the present invention ( Pseudomonas aeruginosa. ) Application Example 1 of ZZU PseZJJ-01 After denitrification under different polyculture conditions, NO3 - -N concentration (a), NO2 - -N concentration (b) comparison chart; Picture 5 Pseudomonas aeruginosa of the present invention ( Pseudomonas aeruginosa. ) Application Example 1 of ZZU PseZJJ-01 After denitrification under different polyculture conditions, NH4 + -Comparison of changes in N concentration (c) and TN concentration (d); Picture 6 The application examples 1 to 6 of the denitrification composite filler of the present invention are NO3 in autotrophic denitrification. - -N concentration (a), NO2 - -N concentration (b) comparison chart; Picture 7 The application examples 1 to 6 of the denitrification composite filler of the present invention are NH4 + -Comparison of changes in N concentration (c) and TN concentration (d); Picture 8 This is a bar graph comparing the total nitrogen removal rates of application examples 1 to 6 of the denitrifying composite filler of the present invention in autotrophic denitrification. DETAILED DESCRIPTION

[0061] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0062] The examples and comparative examples of the present invention used NO3 --N groundwater was obtained by adding KNO3; the iron powder used in the embodiments of the present invention and the comparative example had a particle size of 75 to 150 μm and an iron content of ≥99%, purchased from Tianjin Komeo Chemical Reagent Co., Ltd.; the pyrite (denoted as FeS2) had a particle size of 75 to 150 μm and a FeS2 mass content of ≥90%, purchased from Guangxi Wanbao Mineral Store, produced in Shangbao, Hunan; the ferrous sulfide (denoted as FeS) had a particle size of 75 to 150 μm, a Fe mass content of ≥60%, and a S mass content of ≥25%, purchased from Henan Yuzhong New Materials Co., Ltd.; the particle size of the activated carbon used was passed through a 100-mesh sieve; the LB liquid culture medium (1 L) used in the embodiment of the present invention contained 5 g yeast extract, 10 g NaCl, 10 g tryptone, and a pH value of 7.00; the sludge lysis solution used in the embodiment of the present invention was prepared by a hot alkaline method using excess sludge: the pH value of the excess sludge was adjusted to 11 with a 2 mol / L NaOH solution, and then placed in a pressure cooker at 121°C for 60 sludge lysis. After 30 min, the mixture was centrifuged at 10,000 r / min, and the supernatant was taken. The pH value of the supernatant was adjusted to 7 with 1 mol / L hydrochloric acid and centrifuged for 10 min. The resulting supernatant was the sludge lysate (SCOD was 23063±2236 mg / L). Unless otherwise specified, the chemical reagents used in the Examples and Comparative Examples of the present invention were obtained through conventional commercial channels.

[0063] A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) ZZU PseZJJ-01 Example 1 The Pseudomonas aeruginosa ZZU PseZJJ-01 was deposited in the Guangdong Provincial Microbiological Culture Collection on December 7, 2023, with the deposit number GDMCC No: 1.4478.

[0064] The screening method of Pseudomonas aeruginosa ZZU PseZJJ-01 comprises the following steps: (1) Acclimation and enrichment of excess sludge: 180 mL of groundwater inorganic salt culture medium (2.0 g of KNO3 was added to 1 L of groundwater actually taken) was added to a 250 mL conical flask, and 20 mL of bacterial source sludge (taken from the excess sludge in the secondary sedimentation tank of a sewage treatment plant) was inoculated. The conical flask was sealed with a rubber stopper with two glass tubes A and B, where tube A was used to flush pure N2 and tube B was used to collect the generated N2 gas. Nitrogen was blown off for 10 min to place the bacterial solution in an anaerobic growth environment, and the culture was carried out in a constant temperature shaker at a temperature of 30 °C and a speed of 120 r / min. The groundwater inorganic salt culture medium was replaced every 4 days, and 20 mL of the acclimated bacterial solution was added to the replaced new groundwater inorganic salt culture medium. This operation was repeated to allow the bacteria to fully reproduce. After acclimation and culture for 36 days, a stable bacterial solution was obtained for enrichment culture. (2) Strain separation and purification: add physiological saline to the enriched culture stable bacterial solution obtained in step (1) at a volume ratio of 1:1 to prepare an enriched culture suspension, and perform 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Performing gradient dilution, using the gradient dilution solution as an inoculum, spreading and inoculating the culture medium on LB solid medium, then placing the LB solid medium in a 30°C anaerobic incubator for static culture, and observing the growth of the colonies. When visible colonies appear on the LB solid medium, use an inoculation loop to pick a single colony for streak separation, and continue static culture in a 30°C anaerobic incubator. Repeat the streak separation operation several times until the colonies have consistent morphology, thereby obtaining a purified strain. The LB solid medium (1 L) contains 5 g yeast extract powder, 10 g NaCl, 10 g tryptone, and 20 g agar powder, with a pH of 7.00. (3) Identification of bacterial species: The purified strain obtained in step (2) was sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA identification. The sequencing results were submitted to GenBank and compared with the GenBank ribosome database using the blast comparison program on NCBI (http: / / rdp.cme.msu / index.jsp). The identification result was: Pseudomonas aeruginosa. The strain was then sent to the Guangdong Provincial Microbial Culture Collection for preservation and was numbered ZZU PseZJJ-01.

[0065] Reference Example 1 for Preparation of Embedding Material Hydrogel 100 g of polyvinyl alcohol and 20 g of sodium alginate were added to 880 mL of water, heated to 90°C in a water bath until they were completely dissolved, and naturally cooled to room temperature to obtain an embedding material hydrogel with a polyvinyl alcohol mass concentration of 10% and a sodium alginate mass concentration of 2%.

[0066] Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) Reference Example 2 for Preparation of ZZU PseZJJ-01 Culture Solution Take 1 mL of Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) ZZU PseZJJ-01 was cultured in 100 mL LB liquid medium at 30°C and 120 r / min on a shaking platform for 12 h until OD 600The precipitate was centrifuged at 10000 r / min for 10 min, and the precipitate was washed three times with phosphate buffer and resuspended with sterile saline to OD 600 It is 0.6-0.8 and it is done.

[0067] Denitrification composite filler embodiments 1 to 6 Denitrification composite filler embodiments 1 to 6 of the present invention are composed of the embedding material hydrogel obtained in reference example 1, the Pseudomonas aeruginosa obtained in reference example 2 ( Pseudomonas aeruginosa. ) A mixture of a culture solution of ZZU PseZJJ-01, an iron matrix or a sulfide-iron matrix, and an additive is cross-linked and cured in a saturated boric acid cross-linking solution containing calcium chloride (the concentration of calcium chloride is 20 g / L); the components and amounts are shown in Table 1.

[0068] Table 1 Components and dosage of denitrification composite fillers in Examples 1 to 6 of the present invention

[0069] In the table, “-” means not added.

[0070] Examples 1 to 6 of a method for preparing a denitrification composite filler According to the components and dosages in Examples 1 to 6 of the denitrifying composite filler in Table 1, the Pseudomonas aeruginosa obtained in Reference Example 2 was added to the embedding material hydrogel obtained in Reference Example 1. Pseudomonas aeruginosa. ) The culture solution of ZZU PseZJJ-01, the iron matrix or the ferrous sulfide matrix, and the additives are uniformly mixed and then added dropwise into a saturated boric acid cross-linking solution containing calcium chloride using a 20 mL sterile syringe. The mixture is cross-linked and solidified into spheres at room temperature for 24 hours. The spheres are filtered and refrigerated to obtain denitrifying composite fillers 1 to 6 with particle sizes of 3 to 6 mm, respectively.

[0071] A Pseudomonas aeruginosa ( Pseudomonas aeruginosa. )Application Example 1 of ZZU PseZJJ-01 In group 4 NO3 - The initial concentrations of the -N system were 10, 20, 30, and 50 mg / L of NO3 - -N groundwater, the autotrophic raw material FeS was added at a dosage of 20g / L, and sodium bicarbonate was added at a dosage of 1g / L, and the NO3 - -N groundwater, after being exposed to helium for 20 min, 10 mL of Pseudomonas aeruginosa obtained in Reference Example 2 was inoculated under anaerobic conditions. Pseudomonas aeruginosa.) ZZU PseZJJ-01 culture solution (with a bacterial protein concentration of 50 mg / L) was adjusted to pH 8.0, with a total volume of 200 mL. The culture solutions were sealed and placed in a shaker for autotrophic cultivation at 30°C and 150 r / min for 3, 6, 9, and 28 days, respectively, until both nitrate nitrogen and nitrite nitrogen were completely reduced.

[0072] In order to detect the autotrophic denitrification ability of ferrous sulfide on Pseudomonas aeruginosa, NO3 - -N and NO2 - -N concentration, the sampling process was carried out in a sterile anaerobic box.

[0073] like Picture 1 As shown in (a), with the initial NO3 - -N concentration increased from 10 mg / L to 50 mg / L, NO3 - -N is completely restored from 2 days to 7 days; Picture 1 (b) shows that at different initial NO3 - -N concentration system, NO3 - -N reduction process has NO2 - The accumulation phenomenon, when the initial NO3 - When the -N concentration is lower than or equal to 30 mg / L, NO2 - -N was finally completely reduced in 9 days; when the initial NO3 - When -N is 50 mg / L, NO2 - The accumulation of NO3 reached its maximum on the 7th day and decreased to 1.70 ± 2.40 mg / L on the 26th day. This indicates that the autotrophic denitrification process first reduces nitrate nitrogen to nitrite nitrogen, which is then further reduced to nitrogen gas or other gaseous nitrogen-containing products, ultimately achieving denitrification. The Pseudomonas aeruginosa of the present invention has good autotrophic NO3 - -N conversion ability.

[0074] A Pseudomonas aeruginosa ( Pseudomonas aeruginosa. )Application Example 2 of ZZU PseZJJ-01 In group 3 NO3 - The initial concentration of the system containing NO3 is 35 mg / L - -N groundwater, heterotrophic raw materials glucose, sludge lysate, and sodium succinate (carbon-nitrogen ratio of 6:1) were added at a COD concentration of 210 mg / L, and NO3 - -N groundwater, after being exposed to helium for 20 min, 10 mL of Pseudomonas aeruginosa obtained in Reference Example 2 was inoculated under anaerobic conditions. Pseudomonas aeruginosa.) ZZU PseZJJ-01 culture solution (with a bacterial protein concentration of 50 mg / L) was adjusted to pH 8.0, with a total volume of 200 mL. The culture solutions were sealed and placed in a shaker for heterotrophic culture at 30°C and 150 r / min for 29 h, 26 h, and 24 h, respectively, until both nitrate nitrogen and nitrite nitrogen were completely reduced.

[0075] In order to detect the heterotrophic denitrification ability of Pseudomonas aeruginosa with different carbon sources, samples were taken every 2 h to measure NO3 - -N, NO2 - -N、NH4 + -N and TN concentrations, the sampling process was carried out in a sterile anaerobic box.

[0076] like Picture 2 As shown in (a), during the entire heterotrophic denitrification process, the Pseudomonas aeruginosa strain of the present invention uses glucose, sludge lysate and sodium succinate as carbon sources to generate NO3 - The time for complete reduction of -N was 16h, 14h and 12h respectively. For the strains, the use of different carbon sources as electron donors for NO3 - The different -N reduction rates may be related to the different redox properties of the strains to the carbon source and the preference of the strains to utilize the carbon source; Picture 2 As shown in (b), NO2 - The concentration first increases to a peak value and then decreases to zero. When glucose, sludge lysate, and sodium succinate are used as carbon sources, NO2 - The peak concentrations of -N were 4.90 mg / L, 5.09 mg / L, and 7.07 mg / L, respectively, indicating that there were sufficient denitrifying enzymes and electrons to reduce NO2 in time during the reaction. - -N, nitrogen in the denitrification process from NO3 - -N is converted to nitrite NO2 - -N. During the entire heterotrophic denitrification process, when sodium succinate was used as the carbon source, the strain reduced NO3 - -N rate was the fastest, followed by sludge lysate and finally glucose, indicating that the strain grew best when using sodium succinate as a carbon source. - -N removal rate is close to 100%.

[0077] like Picture 3 (c) shows that during the whole heterotrophic denitrification process, the amount of NH4 + -N concentration is very low, less than 0.5 mg / L; Picture 3 As shown in (d), as the reaction proceeds, the removal rate of TN increases with time, which is consistent with the reduction of NO3 - -N's trend is basically the same.

[0078] In summary, the Pseudomonas aeruginosa strain of the present invention has good heterotrophic NO3 - -N conversion ability.

[0079] A Pseudomonas aeruginosa ( Pseudomonas aeruginosa. )Application Example 3 of ZZU PseZJJ-01 In group 3 NO3 - The initial concentration of the system containing NO3 is 35 mg / L - -N groundwater, heterotrophic sludge lysate (carbon-nitrogen ratio of 4:1) was added at a SCOD concentration of 140 mg / L, and then autotrophic raw materials Fe powder, FeS2, and FeS were added at a dosage of 10 g / L, and sodium bicarbonate was added at a dosage of 1 g / L, and the NO3 - -N groundwater, after being exposed to helium for 20 min, 10 mL of Pseudomonas aeruginosa obtained in Reference Example 2 was inoculated under anaerobic conditions. Pseudomonas aeruginosa. ) The culture solution of ZZUPseZJJ-01 (with a bacterial protein concentration of 50 mg / L) was adjusted to pH 8.0, with a total volume of 200 mL. The solution was sealed and placed in a shaker for 5, 9, and 7 days, respectively, at a temperature of 30°C and a rotation speed of 120 r / min, until the nitrate nitrogen and nitrite nitrogen were completely reduced.

[0080] In order to detect the denitrification ability of the Pseudomonas aeruginosa in the mixed culture of different carbon sources + iron substrates or sulfide iron substrates, samples were taken every 1 day to measure NO3 - -N, NO2 - -N、NH4 + -N and TN concentrations, the sampling process was carried out in a sterile anaerobic box.

[0081] like Picture 4 As shown in (a), during the whole polyculture denitrification process, the Pseudomonas aeruginosa strain of the present invention was cultured under the conditions of Fe powder + sludge lysate, FeS2 + sludge lysate, and FeS + sludge lysate respectively, and NO3 - -N was completely reduced in 5d, 8d, and 6d, respectively. The reduction time of NO3 by the strain was compared under the mixed culture conditions with Fe, FeS2, and FeS as inorganic electron donors and organic carbon sources. - The rate of addition of the inorganic electron donor system was significantly faster than that of the inorganic electron donor system with the same addition amount, indicating that the addition of carbon source promoted nitrate reduction, and the addition of inorganic electron donors Fe, FeS2, and FeS reduced the cost of purchasing carbon source and realized the resource utilization of waste iron matrix or sulfide iron matrix; Picture 4 As shown in (b), NO2 -The -N concentration reached peak values ​​on the 2nd, 5th, and 4th days, and then gradually decreased, with peak concentrations of 9.79 mg / L, 11.95 mg / L, and 9.82 mg / L, respectively.

[0082] like Picture 5 (c) shows that, in addition to the Fe powder + sludge lysis liquid group NH4 + The maximum concentration of -N was 2.10 mg / L (lower than the emission limit), and the concentrations of the other two groups were all lower than 1 mg / L; Picture 5 (d) shows that the change trend of TN is similar to that of NO3 - -N is basically the same. On the 7th day of the reaction, the total nitrogen concentration of the FeS+sludge lysate group was the lowest, only 0.30 mg / L; this shows that under the mixed culture state, the total nitrogen removal effect is better than that in the inorganic environment where Fe, FeS2, and FeS are added alone as inorganic electron donors.

[0083] In summary, Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) ZZU PseZJJ-01 application examples 1 to 3 show that the present invention Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) ZZU PseZJJ-01 can convert 35 mg / L NO3 into 0.5% NO3 with zero-valent iron, pyrite or ferrous sulfide as inorganic electron donors, or glucose, sludge lysate or sodium succinate as organic carbon sources, regardless of autotrophic, heterotrophic or mixed culture conditions. - -N Complete restore.

[0084] A Pseudomonas aeruginosa ( Pseudomonas aeruginosa. )Comparative Example 1 of ZZU PseZJJ-01 The difference between this comparative example and application example 1 is that: - -N system with an initial concentration of 100 mg / L containing NO3 - -N groundwater, and cultured autotrophically for 28 days.

[0085] In order to detect the autotrophic denitrification ability of ferrous sulfide on Pseudomonas aeruginosa, NO3 - -N and NO2 - -N concentration, the sampling process was carried out in a sterile anaerobic box.

[0086] like Picture 1 As shown in (a), when the initial NO3 - When the -N concentration is 100 mg / L, NO3 - Failed to completely reduce, at 16 days, NO3 - The concentration was 17.79 ± 1.83 mg / L, and NO3 - -N concentration did not change significantly; Picture 1 As shown in (b), when the initial NO3 - When -N is 100 mg / L, NO2 - The -N concentration maintained an upward trend and eventually stabilized at 36.50 ± 2.15 mg / L, indicating that the accumulation of nitrite inhibited the reduction of nitrate nitrogen.

[0087] The NO3 - -N system initial concentration and the amount of each material are based on the NO3 - -N is the concentration of the total volume of all materials including groundwater, culture solution, sludge lysate, etc.

[0088] Application Examples 1 to 6 of a Denitrification Composite Filler 4g of denitrification composite filler 1-6 and 0.2g of sodium bicarbonate were added to 200mL of NO3 - -N concentration of 35mg / L containing NO3 - -N groundwater in a 250mL anaerobic bottle, placed in a shaker, at a temperature of 30℃ and a rotation speed of 120r / min, and cultured autotrophically for 16d, 12d, 18d, 16d, 20d, and 10d respectively, until the nitrate nitrogen and nitrite nitrogen were completely reduced.

[0089] In order to detect the autotrophic denitrification ability of the denitrifying composite filler, samples were taken every 1 day, and the water samples were filtered through a 0.45µm filter membrane to determine the NO3 - -N, NO2 - -N、NH4 + The concentrations of -N and TN were measured in a sterile anaerobic chamber. Two parallel samples were set for each group, and the average value of the two groups was taken for calculation.

[0090] like Picture 6 As shown in (a), the denitrifying composite fillers 1 to 6 of the present invention can achieve the removal of NO3 in the entire autotrophic denitrification process. - Among them, the removal speed of denitrification composite filler 2 Fe+C and denitrification composite filler 4 FeS2+C was the fastest, and nitrate could be completely removed on the 10th day of reaction; while denitrification composite filler 1 Fe and denitrification composite filler 3FeS2 removed NO3 on the 14th day and 16th day of reaction respectively. - -N is completely reduced, indicating that the addition of activated carbon to the denitrification composite filler helps to increase the rate of nitrate reduction. Picture 6 As shown in (b), NO2 --N gradually began to accumulate after the reaction started. In the denitrification composite filler 2 Fe+C, denitrification composite filler 4 FeS2+C and denitrification composite filler 6 FeS+C, on the 6th day of the reaction, NO2 - The concentrations of -N reached 8.28 mg / L, 18.05 mg / L, and 4.83 mg / L, respectively, and all reached their peak concentrations first. They were completely reduced on the 12th, 16th, and 10th day of the reaction, respectively. On the other hand, the concentrations of denitrification composite filler 1 Fe, denitrification composite filler 3 FeS2, and denitrification composite filler 5 FeS were completely reduced on the 16th, 18th, and 20th day of the reaction, respectively. - -N is fully restored.

[0091] like Picture 7 As shown in (c), denitrification composite fillers 1 to 6 make the NH4 + -N concentration showed a trend of increasing first and then stabilizing, and the final concentration was below 6.5 mg / L. NH4 + -N may be generated during the growth of the strain in the denitrifying composite filler. Picture 7 As shown in (d), the denitrifying composite fillers 1 to 6 caused the total nitrogen concentration in the entire reaction system to be in a decreasing trend, and the final concentration was basically below 7 mg / L.

[0092] like Picture 8 As shown in the figure, during the entire autotrophic denitrification process, the total nitrogen removal rates of denitrification composite fillers 1 to 6 in the system were all above 82%. Among them, the total nitrogen removal rates of denitrification composite filler 3 FeS2 and denitrification composite filler 4 FeS2+C were the highest, reaching 90.86% and 92.94% respectively, followed by denitrification composite filler 5 FeS and denitrification composite filler 6 FeS+C, reaching 87.33% and 87.31% respectively.

[0093] In summary, the denitrification composite fillers with iron matrix or sulfide iron matrix as autotrophic raw materials can complete the autotrophic denitrification of nitrate. Among them, the comprehensive investigation of NO3 - -N, NO2 - -N、NH4 + The removal rate and effect of -N and TN are higher when pyrite (FeS2) is used as the sulfide-iron matrix than when ferrous sulfide (FeS) is used as the sulfide-iron matrix, and the reaction system runs for a long time. Adding activated carbon to the denitrification composite filler helps to improve its denitrification capacity. The reaction efficiency can be further improved by forming mixed culture conditions by adding carbon sources (such as sludge lysis liquid).

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa. )ZZU PseZJJ-01, characterized by: The Pseudomonas aeruginosa ZZU PseZJJ-01 was deposited in the Guangdong Provincial Microbiological Culture Collection on December 7, 2023, with the deposit number GDMCC No: 1.4478.

2. A denitrification composite filler, characterized in that: Mainly composed of embedding material hydrogel, Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The culture solution of ZZU PseZJJ-01, the iron matrix and / or the ferrosulfur matrix, or a mixture thereof with an auxiliary agent are cross-linked and solidified in a cross-linking solution.

3. A denitrification composite filler as claimed in claim 2, characterized in that: The embedding material hydrogel and the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The volume ratio of the culture solution of ZZU PseZJJ-01 is 100:40-60; the embedding material includes polyvinyl alcohol and / or sodium alginate; in the embedding material hydrogel, the mass concentration of polyvinyl alcohol is 0-15%, the mass concentration of sodium alginate is 0-4%, and the difference between the two is 0; the embedding material hydrogel is prepared by adding the embedding material to water, heating it in a water bath to 85-95°C until it is completely dissolved, and naturally cooling it to room temperature; the iron matrix and / or sulfur The mass volume ratio of the iron matrix, the auxiliary agent and the embedding material hydrogel is 1-6:0-4:100 in g / g / mL; the particle size of the iron matrix or the ferrosulfide matrix is ​​75-150 μm; the ferrosulfide matrix includes one or more of pyrite, FeS2 or FeS; the auxiliary agent includes activated carbon and / or calcium carbonate; the particle size of the auxiliary agent is capable of passing through a 100-mesh sieve; the cross-linking liquid includes a saturated boric acid cross-linking liquid containing calcium chloride; and the concentration of calcium chloride in the cross-linking liquid is 15-25 g / L.

4. The denitrification composite filler according to claim 2 or 3, characterized in that: The OD of the culture solution 600 is 0.6 to 0.8; the preparation method of the culture solution is: taking the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) ZZU PseZJJ-01 was cultured in LB liquid medium on a shaking platform, centrifuged, and washed with phosphate buffer for ≥2 times before being resuspended with sterile physiological saline to obtain the product; the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The volume ratio of ZZU PseZJJ-01 to LB liquid culture medium is 1:80-120; the shaking culture temperature is 28-32°C, the speed is 100-140 r / min, and the time is 10-14 h, until OD 600 The centrifugal precipitation speed is 8000-12000 r / min, and the time is 8-12 min; the resuspending to OD 600 It is 0.6~0.

8.

5. A method for preparing the denitrifying composite filler according to any one of claims 2 to 4, characterized in that: In the embedding material hydrogel, the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The culture solution of ZZU PseZJJ-01, the iron matrix and / or the ferrosulfur matrix, or an auxiliary agent, are mixed evenly, and then added dropwise into the cross-linking solution to cross-link and solidify into balls, which are filtered and refrigerated to obtain a denitrifying composite filler.

6. The method for preparing the denitrifying composite filler according to claim 5, characterized in that: The temperature for cross-linking and curing to form balls is room temperature, and the time is 18 to 30 hours; the particle size of the balls after cross-linking and curing is 3 to 6 mm.

7. A Pseudomonas aeruginosa as claimed in claim 1 ( Pseudomonas aeruginosa. ) Application of ZZU PseZJJ-01, characterized by: In nitrogen-containing wastewater, autotrophic, heterotrophic or mixed culture raw materials are added, or an inorganic carbon source is added, and under anaerobic conditions, the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) After adjusting the pH value of the culture solution of ZZUPseZJJ-01, seal it, place it in a shaking incubator, and carry out autotrophic, heterotrophic or mixed culture.

8. Pseudomonas aeruginosa according to claim 7 ( Pseudomonas aeruginosa. ) Application of ZZU PseZJJ-01, characterized by: In the nitrogen-containing wastewater, NO3 - -N system initial concentration is 10-80 mg / L; in the autotrophic culture, the autotrophic raw material is added to the nitrogen-containing wastewater system in an amount of 15-25 g / L; in the mixed culture, the autotrophic raw material is added to the nitrogen-containing wastewater system in an amount of 5-15 g / L; the carbon-nitrogen ratio of the heterotrophic raw material added to the heterotrophic culture and the nitrogen-containing wastewater is 4-8:1; the carbon-nitrogen ratio of the heterotrophic raw material added to the mixed culture and the nitrogen-containing wastewater is 3-5:1; the autotrophic raw material includes one or more of iron substrate, pyrite, FeS2 or FeS; the heterotrophic raw material includes one or more of glucose, sludge lysate or sodium succinate; while adding the autotrophic or mixed raw material, an inorganic carbon source is added to the nitrogen-containing wastewater system in an amount of 0.5-1.5 g / L; the anaerobic condition refers to: exposing the nitrogen-containing wastewater to helium for 15-25 minutes; the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The volume ratio of the culture solution of ZZU PseZJJ-01 to the nitrogen-containing wastewater system is 4 to 6:100; the Pseudomonas aeruginosa ( Pseudomonas aeruginosa. ) The bacterial protein concentration of the culture solution of ZZU PseZJJ-01 is 40-60 mg / L; the pH value is adjusted to 7.5-8.5; the temperature of the autotrophic, heterotrophic or mixed culture is 25-35° C., and the rotation speed is 100-200 r / min, wherein the autotrophic culture time is 2-30 days, the heterotrophic culture time is 10-30 hours, and the mixed culture time is 10 hours to 10 days, until the nitrate nitrogen and nitrite nitrogen are completely reduced.

9. Use of the denitrification composite filler according to any one of claims 2 to 4, characterized in that: The denitrification composite filler according to any one of claims 2 to 4 is used for denitrification to remove nitrate.

10. The use of the denitrification composite filler according to claim 9, characterized in that: The denitrification composite filler and the inorganic carbon source according to any one of claims 2 to 4 are added to the nitrogen-containing wastewater, placed in a shaking table, and cultured autotrophically to obtain the product; the solid-liquid ratio of the denitrification composite filler to the nitrogen-containing wastewater is 1 to 3:100 g / mL; the amount of the inorganic carbon source added is 0.5 to 1.5 g / L nitrogen-containing wastewater; in the nitrogen-containing wastewater, NO3 - -N concentration is 10-80 mg / L; the temperature of the autotrophic culture is 25-35°C, the rotation speed is 100-200 r / min, and the time is 10-20 days, until nitrate nitrogen and nitrite nitrogen are completely reduced.

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