AT-BC rotating disk artificial microbial cell denitrification technology and its application

Abstract

The invention discloses an AT-BC turntable artificial microbial cell denitrification technology and an application thereof. Shewanella sp. ZJB22168, with a deposit number of CCTCC NO: M20221609, can be applied to the denitrification treatment of nitrogen-containing sewage. Shewanella sp. has a good removal effect on high-concentration ammonia nitrogen (≥500 mg / L), and the ammonia nitrogen degradation rate reaches more than 90% within 24 hours, and has a good removal effect on nitrate nitrogen, nitrite nitrogen and total nitrogen. It also has a good denitrification effect in actual landfill leachate, with an ammonia nitrogen removal rate of 97.59% within 48 hours, and can be used to effectively remove high-concentration ammonia nitrogen in landfill leachate; in actual domestic sewage, the ammonia nitrogen removal rate is 95.17% within 24 hours. When Shewanella is inoculated into the AT-BC biological rotary disc system, the ammonia nitrogen removal rate is increased to 92.17% compared with no addition of Shewanella, and the gaseous nitrogen conversion rate is increased to 37.54%. This provides a new idea for finding efficient denitrification microorganisms and reducing the cost of biological treatment of nitrogen-containing sewage such as landfill leachate and domestic sewage.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and in particular relates to an AT-BC rotating disk artificial microbial cell denitrification technology and an application thereof. Background Art

[0002] The nitrogen in water comes from a wide range of sources, mainly from domestic and agricultural sources, followed by industrial sources and centralized sewage discharge. Excessive discharge of nitrogen will lead to eutrophication of water bodies, deteriorate water quality, and affect the growth and reproduction of aquatic organisms. Therefore, denitrification of nitrogen-containing sewage is particularly important.

[0003] Among the existing sewage treatment methods, the physical method mainly uses gravity or mechanical force to remove floating and suspended matter in sewage to improve water quality, but this method usually cannot treat the water completely and needs to be coordinated with other methods; the chemical method adds flocculants, oxidants and other chemical reagents to the sewage to achieve the purpose of further treating the sewage through neutralization, redox, coagulation and other reactions, but this method usually produces secondary waste, increasing the difficulty of subsequent treatment; and the biological method uses the metabolic function of microorganisms to further treat the difficult-to-degrade organic matter, nitrogen, phosphorus and other substances in the sewage to purify the sewage.

[0004] Among the biological treatment processes for nitrogen-containing wastewater, the activated sludge process is the mainstream process. However, this method requires aeration, is energy-intensive, and has high costs. In recent years, biological nitrogen removal (BNR) has been widely and deeply studied, such as simultaneous nitrification and denitrification (SND), nitrification-denitrification (NDN), short-range nitrification-denitrification (PND), partial nitrification-denitrification, denitrification and dissimilatory nitrate reduction to ammonium (DNRA), simultaneous nitrification-anaerobic ammonium oxidation-denitrification (SNAD), and complete autotrophic denitrification of nitrite (CANON). Among them, the microbial aerobic nitrification and heterotrophic denitrification system for nitrogen-containing wastewater treatment based on the AT-BC biological rotary disc treatment system is a simple operation, stable effect, and highly practical urban sewage treatment process. In urban sewage treatment, efficient denitrification of landfill leachate and domestic sewage has gradually become a key issue. Among them, landfill leachate is a typical high-ammonia nitrogen wastewater. Due to its complex composition, low C / N ratio, high organic matter content, and frequent fluctuations in water quality indicators, it has become a major problem in the field of water treatment. The domestic sewage discharge is large and contains a variety of organic pollutants, and there is an urgent need to find an efficient treatment method. Compared with the traditional activated sludge method, the AT-BC biological rotary disc treatment system has the advantages of high total nitrogen removal rate, good sludge sedimentation, high effluent clarity, and no sludge swelling and floating. It overcomes the problems of sludge swelling, difficulty in mud-water separation, water temperature fluctuations, carbon-nitrogen ratio imbalance affecting treatment efficiency, and low temperature, high ammonia nitrogen and high concentration in sewage treatment.

[0005] The technical core of the microbial aerobic nitrification and heterotrophic denitrification treatment system lies in the AT-BC biological turntable system. The AT-BC biological turntable system is composed of a rotary mesh biological contactor. By adjusting the dissolved oxygen concentration in the system and adding bacterial nutrient solution, the dominant bacterial species can be multiplied in large quantities, and the relationship between the bacterial growth of the membrane disk, the sludge thickness of the membrane disk, the rotation speed of the turntable and the influent water quality can be adjusted to control the reflux ratio, so that the entire system is in the optimal state. Alienation refers to the process in which aerobic denitrifying bacteria degrade complex nitrogen sources into simple gaseous nitrogen (N2O, N2, etc.) through step-by-step reactions, that is, the heterotrophic nitrification-aerobic denitrification metabolic pathway. Studies have shown that there are mainly two nitrogen alienation pathways in aerobic denitrifying bacteria, namely the incomplete nitrification-denitrification pathway and the complete nitrification-denitrification pathway. Among them, the incomplete nitrification-denitrification pathway: NH4 + →NH2OH→N2O→N2; Complete nitrification-denitrification pathway: NH4 + →NH2OH→NO2 - / NO3 - →NO2 - →N2O→N2. Synchronous heterotrophic nitrification-aerobic denitrification (HNAD) bacteria attached to the AT-BC rotating disk use the complete nitrification-denitrification pathway: NH4 + →NH2OH→NO2 - / NO3 - →NO2 - →N2O→N2, it can utilize a variety of nitrogen sources in wastewater, carry out nitrification and denitrification simultaneously under aerobic conditions, reduce the accumulation of nitrate and nitrite, and finally remove them efficiently in the form of gaseous nitrogen. It has a wide range of biological resources and environmental adaptability, and is the key to efficient nitrogen removal by AT-BC biological rotary disc. However, the current structure of natural denitrification microbial flora is easily disturbed by factors such as raw water quality and ambient temperature changes, and the proportion of functional denitrification microorganisms is unstable, which ultimately affects the denitrification effect of simultaneous nitrification and denitrification. In addition, due to the high assimilation ratio of the flora in the activated sludge denitrification process, nitrogen components are easily accumulated in situ in the form of cells, etc., and timely sludge discharge is required after long-term treatment. The treatment process is relatively complicated, and it is urgent to increase the nitrogen conversion ratio in the microbial denitrification process. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention obtains a strain of Shewanella with high dissimilatory effect through artificial screening and applies it to AT-BC rotary disc for efficient denitrification for wastewater treatment.

[0007] The present invention specifically adopts the following technical solutions:

[0008] On the one hand, the present invention provides a strain of Shewanella sp. ZJB22168, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20221609 and a deposit date of October 20, 2022.

[0009] Another aspect of the present invention provides the use of a strain of Shewanella in the denitrification treatment of nitrogen-containing wastewater.

[0010] Further, the application includes the following steps: directly inoculating the Shewanella into nitrogen-containing sewage, adding a carbon source to a C / N ratio of 5-20, culturing at pH=6-8, 30-40°C, and 160-220rpm to remove ammonia nitrogen from the sewage. Further, the carbon source is sodium succinate (C4H4Na2O4).

[0011] The present invention also provides a related application of inoculating Shewanella into an AT-BC biological rotary disc system for denitrification.

[0012] Compared with the prior art, the advantages of the present invention are:

[0013] The Shewanella sp. ZJB22168 of the present invention has a good removal effect on ammonia nitrogen, and has a good removal effect on nitrate nitrogen, nitrite nitrogen and total nitrogen. When sodium succinate is used as a carbon source and ammonium sulfate is used as the only nitrogen source, ammonia nitrogen can be gradually converted into nitrate nitrogen within 72 hours of aerobic culture under the conditions of C / N of 10, pH of 7, temperature of 30°C and rotation speed of 160 rpm, and the degradation efficiency of ammonia nitrogen in 24 hours is 90.69%, and the degradation efficiency of ammonia nitrogen in 72 hours is 97.96%, and there is no accumulation of nitrite nitrogen. Nitrate nitrogen is completely removed within 96 hours, and total nitrogen is degraded by 62.19% within 96 hours. In the actual denitrification of landfill leachate, the ammonia nitrogen removal rate of Shewanella within 48 hours is 97.59%; in the actual denitrification process of domestic sewage, the ammonia nitrogen removal rate of Shewanella within 24 hours is 95.17%, which has good practical application value. When Shewanella was inoculated into the AT-BC bio-rotating disc system, the ammonia nitrogen removal rate increased to 92.17%, and the gaseous nitrogen conversion rate increased to 37.54%. The accumulation of sediment was reduced, which has important practical significance for the development of efficient denitrification biological agents and the reduction of the treatment cost of microbial denitrification. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the Gram staining picture of Shewanella ZJB22168 of the present invention.

[0015] Figure 2 This is the phylogenetic tree of Shewanella ZJB2216816S rDNA of the present invention.

[0016] Figure 3Schematic diagram of the ammonia nitrogen degradation ability of Shewanella ZJB22168 of the present invention.

[0017] Figure 4 It is a schematic diagram of the denitrification efficiency of Shewanella ZJB22168 in actual landfill leachate of the present invention.

[0018] Figure 5 This is a schematic diagram of the denitrification treatment efficiency of Shewanella ZJB22168 of the present invention in actual domestic sewage. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby. In the embodiments, if there is no special explanation, all are conventional methods, and the reagents used are conventional reagents or reagents prepared according to conventional methods if there is no special explanation.

[0020] Example 1: Screening and identification of a strain of Shewanella

[0021] Take 5 mL of leachate from a wastewater treatment company in Hangzhou and add it to the sterilized enrichment culture medium, and culture it at 30°C and 160 rpm for 5 days.

[0022] The components of the above-mentioned enrichment medium are: 2.36 g of ammonium sulfate ((NH4)2SO4), 16.88 g of sodium succinate (C4H4Na2O4), 6.5 g of dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O), 1.5 g of potassium dihydrogen phosphate (KH2PO4), 20 mL of trace elements, distilled water to 1000 mL, natural pH.

[0023] The ingredients of the above trace elements are: zinc sulfate (ZnSO4) 4.4 mg, manganese chloride tetrahydrate (MnCl2·4H2O) 10.2 mg, calcium chloride (CaCl2) 11 mg, ferrous sulfate heptahydrate (FeSO4·7H2O) 10 mg, cupric sulfate pentahydrate (CuSO4·5H2O) 3.2 mg, ammonium molybdate tetrahydrate ((NH4)6Mo7O2·4H2O) 2.2 mg, cobalt chloride hexahydrate (CoCl2·6H2O) 3.2 mg, EDTA-Na 144 mg, distilled water 1000 mL, natural pH.

[0024] The enriched culture solution was inoculated into a test tube filled with sterile water and diluted in proportion to obtain a bacterial concentration of 10 -4 , 10 -5Gradient bacterial suspension; then use the plate spreading method to take 100 μL from each gradient bacterial suspension and spread it on LB solid culture medium, culture at 30℃ for 2 days, pick colonies with different morphology for streak separation, separate and purify for more than 3 times until the colony characteristics are basically consistent, and then inoculate on slant culture medium and store in a 4℃ refrigerator.

[0025] The ingredients of the LB medium are: 10 g of tryptone, 5 g of yeast powder, 10 g of sodium chloride, distilled water to 1000 mL, and natural pH.

[0026] A bacterial strain screened from LB solid culture medium was amplified by PCR using bacterial universal primers 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′, 1492R: 5′-TACGGYTACCTTGTTACGACTT-3′, and verified by agarose gel electrophoresis (1%); electrophoresis detection, gel excision, purification and sequencing were performed, and the sequence was determined by Qingke Biotechnology.

[0027] PCR reaction system (50 μL): 25 μL of the high-fidelity enzyme used was Phusion High-Fidelity PCRMasterMix with HF Buffer; 3 μL (10 μM) of each front and rear F / R primer; 10 μL DNA template 6 μL ddH2O; the configured PCR system was used for PCR amplification according to the following reaction conditions: pre-denaturation at 98°C for 30 s, followed by 25 cycles: denaturation at 98°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 15 s; final extension at 72°C for 1 min. PCR products were purified using AMPure XP Beads (Beckman Coulter, Indianapolis, IN) and quantified using PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA).

[0028] The 16S rDNA sequence of strain ZJB22168 is 1399 bp in length, and its gene sequence is shown in SEQ ID NO.1.

[0029] The strain sequence results were uploaded to the NCBI database and compared with the existing bacterial 16S rDNA gene sequences in the database. The results showed that the bacteria was Shewanella sp. The phylogenetic tree was constructed by the proximity joining method of Mega7.0 software to analyze the genetic characteristics of the strain, determine its species and genetic evolutionary status, and the results are shown in Figure 2 .

[0030] The strain ZJB22168 was named Shewanella sp., strain number ZJB22168, deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M20221609, and the deposit date was October 20, 2022.

[0031] Gram staining of strain ZJB22168 Figure 1 Its biological characteristics are: Gram-negative bacteria, yellow-white round small colonies, smooth edges, moist, sticky, easy to pick, suitable pH is 7.0-9.0, suitable growth temperature is 30-40℃.

[0032] Example 2: Verification of the removal effect of high concentration ammonia nitrogen from Shewanella

[0033] The formula of high concentration ammonia nitrogen culture medium is: ammonium sulfate ((NH4)2SO4) 2.36g, sodium succinate (C4 H4Na2O4) 16.88g, potassium dihydrogen phosphate (KH2 PO4) 1.5g, potassium dihydrogen phosphate trihydrate (K2HPO4·3H2O) 6.5g, trace element solution 20mL, distilled water to 1000mL, pH value 7.0-7.5; the trace element solution includes: zinc sulfate (ZnSO4) 4.4mg, manganese chloride tetrahydrate (MnCl2·4H2O) 10.2mg, calcium chloride (CaCl2) 11mg, ferrous sulfate heptahydrate (FeSO4·7H2O) 10mg, cupric sulfate pentahydrate (CuSO4·5H2O) 3.2mg, ammonium molybdate tetrahydrate ((NH4)6Mo7O2·4H2O) 2.2mg, cobalt chloride hexahydrate (CoCl2·6H2O) 3.2mg, EDTA-Na 144mg, distilled water 1000mL, natural pH.

[0034] The treatment method is: inoculate 2% seed liquid (seed liquid is a bacterial suspension cultured in LB medium at 30°C and 160rpm for 12h), culture at 30°C and 160rpm. Regular sampling is performed to determine the ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen and OD in the supernatant. 600 Value, see the result Figure 3 .

[0035] Conclusion: It can be seen that the bacterial growth reached the maximum bacterial concentration in 72 hours, and the bacterial concentration began to decline after 72 hours; the degradation rate of ammonia nitrogen reached 90.69% in 24 hours and 97.96% in 72 hours, and then as the bacterial concentration decreased, ammonia nitrogen was released and accumulated again. Nitrate nitrogen accumulated in the first 48 hours, and the nitrate nitrogen concentration decreased after 48 hours, and was completely degraded in 96 hours; total nitrogen decreased by 62.19% in 96 hours; no accumulation of nitrite nitrogen was monitored during the experiment.

[0036] Example 3: Effect of Shewanella in denitrification of actual landfill leachate

[0037] The medium was replaced with 2.36 g of ammonium sulfate ((NH4)2SO4) in the high-concentration ammonia nitrogen medium in Example 2 by diluted landfill leachate raw water collected from a sewage treatment plant in Hangzhou (the ammonia nitrogen concentration in the raw water was about 2000 mg·L -1 , diluted four times, the ammonia nitrogen concentration after dilution is about 500mg·L -1 ), the rest of the recipe remains unchanged.

[0038] The treatment method is: inoculate 2% seed liquid (seed liquid is a bacterial suspension cultured in LB medium at 30°C and 160rpm for 12h), culture at 30°C and 160rpm. Regularly sample and measure the ammonia nitrogen, nitrite nitrogen, nitric nitrogen and OD in the supernatant. 600 Value, see the result Figure 4 .

[0039] Conclusion: In the actual process of landfill leachate denitrification, the bacteria continued to grow within 48 hours, and the ammonia nitrogen removal efficiency reached 97.59%. During the experiment, no accumulation of nitrite nitrogen was monitored. The nitrate nitrogen concentration reached the highest within 24 hours and then decreased. This strain can tolerate high concentrations of ammonia nitrogen and effectively treat wastewater containing high concentrations of ammonia nitrogen, providing a new solution for the biological treatment of landfill leachate.

[0040] Example 4: Effect of Shewanella in denitrification of actual domestic sewage

[0041] The medium was replaced with 2.36 g of ammonium sulfate ((NH4)2SO4) in the high-concentration ammonia nitrogen medium in Example 2 and raw domestic sewage (ammonia nitrogen concentration of about 150 mg·L) collected from a sewage treatment plant in Hangzhou. -1 , basically free of nitrate and nitrite), add exogenous carbon source sodium succinate to a C / N ratio of 10, and keep the rest of the formula unchanged.

[0042] The treatment method is: inoculate 2% seed liquid (seed liquid is a bacterial suspension cultured in LB medium at 30°C and 160rpm for 12h), culture at 30°C and 160rpm. Take samples regularly to measure the ammonia nitrogen and OD in the supernatant. 600 Value, see the result Figure 5 .

[0043] Conclusion: In the actual domestic sewage denitrification process, the bacteria continued to grow within 24 hours, the ammonia nitrogen removal efficiency reached 95.17%, and the accumulation of nitrite nitrogen and nitrate nitrogen was not monitored during the experiment. This strain has a wide range of applications. It can not only effectively treat landfill leachate containing high concentrations of ammonia nitrogen, but also effectively treat domestic sewage, enrich the strain library of environmental microorganisms, provide environmental microorganisms with efficient denitrification, and provide solutions for polluted water bodies of varying degrees.

[0044] Example 5: Nitrogen balance analysis of Shewanella denitrification in AT-BC bio-rotating disk system

[0045] The treatment method is: the seed liquid (the seed liquid is a bacterial suspension cultured in LB medium at 30°C and 160rpm for 12h) is connected to the AT-BC biological turntable system at an inoculation rate of 2%, and the initial ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and total nitrogen concentrations in the landfill leachate and the ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and intracellular nitrogen concentrations in the effluent supernatant after 96h are determined, and the concentration of lost gaseous nitrogen is calculated and a table is drawn up.

[0046] As shown in Table 1, when Shewanella was not added, the sewage contained denitrifying microorganisms, and the ammonia nitrogen degradation rate was 73.96% in 96 hours. The accumulation of nitrite nitrogen was never detected. The initial total nitrogen contained 273.33 mg·L -1 The conversion rate was 53.85% and 99.93 mg·L -1 The conversion rate was 19.71%. After adding Shewanella, the ammonia nitrogen degradation rate increased to 92.17% in 96 hours. No accumulation of nitrite nitrogen was detected during the experiment, and the accumulated nitrate nitrogen was completely degraded. In the initial total nitrogen, there were 141.15 mg·L -1 The conversion rate was 24.5%, and 215.88 mg·L -1 The conversion rate into gaseous nitrogen was 37.54%, which was significantly higher than that without adding Shewanella.

[0047] Table 1 Nitrogen balance analysis of landfill leachate in AT-BC rotary disc system

[0048]

[0049] -: not detected;

[0050] Intracellular nitrogen = total nitrogen after bacterial crushing - total dissolved nitrogen before crushing; gaseous nitrogen = nitrogen removal rate × initial total nitrogen.

Claims

1. A strain of Shewanella ( Shewanella sp.) ZJB22168, the deposit number is CCTCC NO: M20221609.

2. Use of Shewanella as claimed in claim 1 in denitrification of nitrogen-containing wastewater, characterized in that: The application is to directly inoculate Shewanella ZJB22168 into sewage, add a carbon source to a C / N ratio of 5-20, and culture at pH=6-8, 30-40°C, and 160-220rpm to remove ammonia nitrogen in the sewage, wherein the carbon source is sodium succinate.

3. Use of the Shewanella according to claim 1 in denitrification of nitrogen-containing wastewater, characterized in that The application is to inoculate Shewanella ZJB22168 into an AT-BC biological rotary disc system for denitrification.

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