Heterotrophic nitrification-aerobic denitrification strain capable of synchronously degrading antibiotics and application of heterotrophic nitrification-aerobic denitrification strain
By screening out Klebsiella oxytoca TY, the problem of simultaneous nitrogen removal and degradation in the treatment of antibiotic-containing wastewater in traditional sewage treatment plants has been solved. This has enabled efficient removal of nitrogen and antibiotics in a single reactor, simplifying the process and expanding the scope of application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-07
AI Technical Summary
When treating antibiotic-containing wastewater, existing wastewater treatment plants employ complex biological denitrification processes, which are prone to the accumulation of intermediate products, result in severe competition for carbon sources, and make it difficult to achieve simultaneous denitrification and degradation due to the inhibition of microbial activity by antibiotics. Furthermore, existing heterotrophic nitrification-aerobic denitrification strains have narrow environmental adaptability and are difficult to apply to antibiotic-containing wastewater.
Klebsiella oxytoca TY was screened and isolated. This strain can simultaneously denitrify and degrade antibiotics under heterotrophic aerobic conditions, exhibits broad-spectrum environmental adaptability, and can achieve nitrogen removal and antibiotic degradation in a single reactor, avoiding the accumulation of intermediate products and competition for carbon sources.
It achieves efficient removal of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen from wastewater in a single reactor, while simultaneously removing antibiotics. This simplifies the process, reduces operating costs, and expands the application areas of HN-AD bacteria.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology and relates to a strain of Klebsiella oxytoca (TY) and its applications. This strain has the function of simultaneous heterotrophic nitrification-aerobic denitrification and antibiotic removal, and can achieve efficient removal of carbon, nitrogen, phosphorus and antibiotics from wastewater containing amoxicillin by utilizing organic carbon sources and phosphates. Background Technology
[0002] With the rapid development of the pharmaceutical industry, the problem of wastewater containing large amounts of ammonia nitrogen and residual antibiotics, such as amoxicillin, being discharged into sewage treatment plants is becoming increasingly prominent. Improper treatment of such complex pollutants in sewage treatment plants can not only lead to eutrophication and ecotoxicity, but may also induce the spread of antibiotic resistance genes (ARGs), posing a potential threat to public health and safety. Traditional biological nitrogen removal processes in sewage treatment plants typically rely on the synergistic effect of autotrophic nitrifying bacteria and heterotrophic denitrifying bacteria, requiring staged treatment through aerobic nitrification and anoxic denitrification. This presents technical bottlenecks such as complex process flows, easy accumulation of intermediate products (nitrate or nitrite nitrogen), carbon source competition, and poor microbial population stability. Furthermore, antibiotic pollutants have a significant inhibitory effect on microbial activity, making it difficult to simultaneously achieve nitrogen removal and antibiotic degradation in conventional biological treatment systems.
[0003] Currently, most treatments for antibiotic-containing wastewater employ a combination of physicochemical pretreatment (such as adsorption and oxidation) and biological processes. However, these methods suffer from high costs, large carbon emissions, and a tendency to generate secondary pollution. Although studies have shown that some heterotrophic nitrification-aerobic denitrification (HN-AD) strains can simultaneously remove nitrogen and organic matter, their application is still limited by the fact that their nitrogen removal performance is easily affected by environmental conditions such as pH (neutral range) and temperature (25-35℃), and few HN-AD strains can simultaneously tolerate and degrade pollutants such as antibiotics, making them difficult to directly apply to scenarios involving antibiotic-containing wastewater.
[0004] Against this backdrop, developing multifunctional strains possessing efficient nitrogen removal, broad-spectrum environmental adaptability, and antibiotic degradation capabilities has become crucial for overcoming existing technological limitations. Coupled nitrogen removal and antibiotic degradation through microbial cometabolism can simplify processes and reduce operating costs in a single reactor, holding significant importance for antibiotic wastewater treatment and water remediation. However, there are currently few reports on HN-AD bacteria that simultaneously denitrify and degrade antibiotics, which limits their development and application. Therefore, discovering and isolating more novel functional HN-AD bacteria is essential for the innovation and development of simultaneous nitrogen removal and antibiotic degradation processes, expanding the application areas of HN-AD bacteria, and advancing our understanding of the roles of microorganisms in nature.
[0005] This invention screens, isolates, and purifies an HN-AD bacterium capable of simultaneously denitrifying and degrading antibiotics from sludge in wastewater treatment plants. Under heterotrophic and aerobic conditions, this strain can efficiently perform simultaneous denitrification and antibiotic degradation within a certain pH and temperature range, providing an innovative solution to this technological need. Summary of the Invention
[0006] This invention provides a multi-substrate co-utilization antibiotic degradation-heterotrophic nitrifying aerobic denitrifying bacterium, identified and named Klebsiella oxytoca TY. This strain was isolated from sludge in a wastewater treatment plant and overcomes the shortcomings of existing microorganisms in terms of their narrow adaptability to pH, temperature, and antibiotics. It can achieve simultaneous denitrification and antibiotic degradation of wastewater in a single reactor, overcoming the technical bottlenecks of existing biological denitrification and antibiotic biodegradation, and has broad application prospects.
[0007] Compared to traditional biological denitrification-antibiotic degradation processes, the application described in this invention is characterized by the fact that the nitrification process of Klebsiella oxytoca TY is under heterotrophic conditions with no intermediate product accumulation, the denitrification process is under aerobic conditions, and it can co-metabolize and degrade antibiotics with organic carbon sources. It does not require complex operating conditions, does not rely on the synergistic participation of different functional microorganisms, and does not have problems such as carbon source competition between microorganisms.
[0008] The Klebsiella oxytoca TY strain provided by this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 33884 and deposit date of April 8, 2025.
[0009] The Klebsiella oxytoca TY strain provided by this invention grows on a basic solid culture medium. The preparation method of the basic solid culture medium is as follows: Weigh 5g of sodium citrate, 0.5g of ammonium sulfate, 0.05g of dipotassium hydrogen phosphate, 0.05g of potassium dihydrogen phosphate, 0.05g of magnesium sulfate, 0.01g of ferrous sulfate, 0.01g of manganese sulfate, 1mL of trace elements, and 20-30g of agar. Dissolve the above-mentioned reagents in 1L of deionized water, sterilize at 121℃ for 120min, and pour into a petri dish irradiated with ultraviolet light to prepare a plate culture medium.
[0010] The Klebsiella oxytoca TY strain provided by this invention, after being inoculated into the basal culture medium and cultured for 24 hours, showed that the colonies in the culture dish were yellow, transparent, round, and had a smooth and moist surface.
[0011] The optimal culture conditions for Klebsiella oxytoca TY to exhibit excellent performance, provided by this invention, are: carbon source = sodium citrate, nitrogen source = ammonia nitrogen, C / N ratio (mass ratio, the same below) = 20-40, P / N ratio (mass ratio, the same below) = 0.1, initial ammonia nitrogen concentration = 80 mg / L, pH = 5.5-9.5, and temperature = 20-37℃. The optimal growth conditions are: carbon source = sodium citrate, C / N ratio (mass ratio, the same below) = 35, P / N ratio (mass ratio, the same below) = 0.1, initial ammonia nitrogen concentration = 80 mg / L, pH = 7.5, and temperature = 30℃.
[0012] The Klebsiella oxytoca TY strain provided by this invention can oxidize ammonia nitrogen to nitrite and nitrate nitrogen through nitrification using organic carbon sources as electron donors under aerobic conditions. It can also assimilate ammonia nitrogen into intracellular organic nitrogen to meet its own growth requirements. It can also dissimilate / assimilate nitrite and nitrate nitrogen to nitrogen-containing gases or intracellular organic nitrogen, thereby achieving the removal of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen. It can also achieve simultaneous nitrification and denitrification denitrification processes under aerobic conditions using ammonia nitrogen and nitrate nitrogen, ammonia nitrogen and nitrite nitrogen, or ammonia nitrogen, nitrate nitrogen and nitrite nitrogen as mixed nitrogen sources.
[0013] The Klebsiella oxytoca TY strain provided by this invention can utilize phosphate for aerobic denitrification, and there is no significant accumulation of nitrite and nitrate nitrogen, which shows good application prospects.
[0014] The Klebsiella oxytoca TY strain provided by this invention can combine heterotrophic nitrification-aerobic denitrification and amoxicillin metabolism, and can simultaneously remove ammonia nitrogen and amoxicillin from wastewater under a single aerobic environment, showing good application prospects. Attached Figure Description
[0015] Figure 1 The growth and denitrification / phosphorus removal performance of Klebsiella oxytoca TY under different carbon sources were investigated.
[0016] Figure 2 The growth and denitrification / phosphorus removal performance of Klebsiella oxytoca TY under different nitrogen sources were investigated.
[0017] Figure 3 The growth and denitrification / phosphorus removal performance of Klebsiella oxytoca TY under different C / N ratios were investigated.
[0018] Figure 4 Klebsiella oxytoca TY was tested under different initial NH4+ conditions.+ Growth and denitrification / phosphorus removal performance under [conditions].
[0019] Figure 5 The growth and denitrification / phosphorus removal performance of Klebsiella oxytoca TY under different P / N ratios were investigated.
[0020] Figure 6 The growth and denitrification / phosphorus removal performance of Klebsiella oxytoca TY at different pH values were investigated.
[0021] Figure 7 The growth and denitrification / phosphorus removal performance of Klebsiella oxytoca TY at different temperatures were investigated.
[0022] Figure 8 Klebsiella oxytoca TY with NH4 + Growth and denitrification / phosphorus removal performance when nitrogen is the sole nitrogen source.
[0023] Figure 9 Klebsiella oxytoca TY with NO3 - Growth and denitrification / phosphorus removal performance when nitrogen is the sole nitrogen source.
[0024] Figure 10 Klebsiella oxytoca TY with NO2 - Growth and denitrification / phosphorus removal performance when nitrogen is the sole nitrogen source.
[0025] Figure 11 Klebsiella oxytoca TY with NH4 + and NO3 - Growth and denitrification / phosphorus removal performance when using mixed nitrogen sources.
[0026] Figure 12 Klebsiella oxytoca TY with NH4 + and NO2 - Growth and denitrification / phosphorus removal performance when using mixed nitrogen sources.
[0027] Figure 13 Klebsiella oxytoca TY with NH4 + NO3 - and NO2 - Growth and denitrification / phosphorus removal performance when using mixed nitrogen sources.
[0028] Figure 14The growth and denitrification performance of Klebsiella oxytoca TY using sodium citrate and amoxicillin as a mixed carbon source were investigated.
[0029] Figure 15 This describes the degradation mechanism of amoxicillin by Klebsiella oxytoca TY using sodium citrate and amoxicillin as a mixed carbon source.
[0030] Figure 16 It is an intermediate product produced by the biodegradation of Klebsiella oxytoca TY using sodium citrate and amoxicillin as a mixed carbon source. Detailed Implementation
[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.
[0032] The culture media used in the examples are as follows:
[0033] Basic culture medium: 5g sodium citrate, 0.5g ammonium sulfate, 0.05g dipotassium hydrogen phosphate, 0.05g potassium dihydrogen phosphate, 0.05g magnesium sulfate, 0.01g ferrous sulfate, 0.01g manganese sulfate, 1mL trace element solution, and 1L deionized water.
[0034] Heterotrophic nitrification medium: 5.418g sodium citrate, 0.377g ammonium sulfate, 0.029g dipotassium hydrogen phosphate, 0.018g potassium dihydrogen phosphate, 0.05g magnesium sulfate, 0.01g ferrous sulfate, 0.01g manganese sulfate, 1mL trace element solution, and 1L deionized water.
[0035] Denitrification medium I: sodium citrate 5.418g, potassium nitrate 0.577g, dipotassium hydrogen phosphate 0.029g, potassium dihydrogen phosphate 0.018g, magnesium sulfate 0.05g, ferrous sulfate 0.01g, manganese sulfate 0.01g, trace element solution 1mL, deionized water 1L.
[0036] Denitrification medium II: sodium citrate 5.418g, sodium nitrite 0.394g, dipotassium hydrogen phosphate 0.029g, potassium dihydrogen phosphate 0.018g, magnesium sulfate 0.05g, ferrous sulfate 0.01g, manganese sulfate 0.01g, trace element solution 1mL, deionized water 1L.
[0037] Simultaneous nitrification and denitrification medium I: sodium citrate 5.418g, ammonium sulfate 0.189g, potassium nitrate 0.289g, dipotassium hydrogen phosphate 0.029g, potassium dihydrogen phosphate 0.018g, magnesium sulfate 0.05g, ferrous sulfate 0.01g, manganese sulfate 0.01g, trace element solution 1mL, deionized water 1L.
[0038] Simultaneous nitrification and denitrification medium II: sodium citrate 5.418g, ammonium sulfate 0.189g, sodium nitrite 0.197g, dipotassium hydrogen phosphate 0.029g, potassium dihydrogen phosphate 0.018g, magnesium sulfate 0.05g, ferrous sulfate 0.01g, manganese sulfate 0.01g, trace element solution 1mL, deionized water 1L.
[0039] Simultaneous nitrification and denitrification medium III: sodium citrate 5.418g, ammonium sulfate 0.096g, potassium nitrate 0.192g, sodium nitrite 0.131g, dipotassium hydrogen phosphate 0.029g, potassium dihydrogen phosphate 0.018g, magnesium sulfate 0.05g, ferrous sulfate 0.01g, manganese sulfate 0.01g, trace element solution 1mL, deionized water 1L.
[0040] Antibiotic removal-denitrification medium: sodium citrate 5.418g, ammonium sulfate 0.377g, dipotassium hydrogen phosphate 0.029g, potassium dihydrogen phosphate 0.018g, magnesium sulfate 0.05g, ferrous sulfate 0.01g, manganese sulfate 0.01g, amoxicillin 0.03g, trace element solution 1mL, deionized water 1L.
[0041] Trace elements: 1g zinc sulfate, 0.3g manganese chloride, 3g boric acid, 2g cobalt chloride, 0.1g copper chloride, 0.2g nickel chloride, 0.3g sodium molybdate, 1L deionized water.
[0042] Example 1
[0043] Optimization of optimal growth and denitrification / phosphorus removal conditions for Klebsiella oxytoca TY.
[0044] The bacterial strain preserved in glycerol at -20℃ (deposited on April 8, 2025, at the China General Microbiological Culture Collection Center, accession number CGMCC No. 33884) was inoculated into 100 mL of basal medium sterilized at 121℃ for 20 min and cultured in an air bath shaker at 30℃ and 120 rpm for 48 h to allow the cells to grow to the logarithmic phase. The bacterial suspension was then harvested for inoculation (the same applies below). 5 g / L sodium citrate, 3.31 g / L sodium acetate, 3.5 g / L sodium succinate, 2.3 g / L sucrose, and 2.4 g / L glucose were added as carbon sources to five Erlenmeyer flasks containing 100 mL of basal medium, respectively. Then, 10 mL of bacterial suspension (OD) was inoculated into each flask. 600The sample was incubated in a 30℃, 120rpm air bath shaker (with a concentration of 0.1%), and OD was directly measured at 0h and 24h. 600 The values were determined, and the COD and NH4+ of the supernatant were measured after centrifugation at 8000 rpm for 10 min. + -N,PO4 3- The concentration of -P. Results are as follows: Figure 1 As shown, when the carbon source is sodium citrate, the strain exhibits the highest growth and removal rates of COD, ammonia nitrogen, and phosphate. Therefore, the optimal carbon source condition for the growth of this bacterium is sodium citrate.
[0045] Similarly, with sodium citrate as the optimal carbon source, ammonium sulfate, potassium nitrate, sodium nitrite, glutamic acid, and L-alanine (total nitrogen content 80 mg / L) were added to five Erlenmeyer flasks containing 100 mL of basal medium as nitrogen sources. The inoculation, culture, and sample testing conditions were the same as above. The results are shown in Figure 2. The strain's removal capacity for both inorganic and organic nitrogen was above 75%, demonstrating good degradation ability for multiple nitrogen sources. The strain exhibited the highest total nitrogen removal capacity when ammonia nitrogen was used as the nitrogen source; therefore, ammonia nitrogen is the optimal nitrogen source for the growth of this bacterium.
[0046] Similarly, under the conditions that the optimal carbon source is sodium citrate and the optimal nitrogen source is ammonia nitrogen, the C / N ratio (mass ratio, the same below) in the basal culture medium was adjusted to 5, 10, 15, 20, 25, 30, 35, and 40, respectively, with the inoculation, culture, and testing conditions remaining the same. The results are as follows... Figure 3 As shown, when the C / N ratio is between 25 and 40, the removal rates of COD and ammonia nitrogen by the strain remain above 75%. When the C / N ratio is 35, the removal rates of COD, ammonia nitrogen, and phosphate by the strain are the highest. Therefore, the optimal C / N ratio for the growth of this bacterium is 35.
[0047] Similarly, under the conditions of optimal carbon source (sodium citrate), optimal nitrogen source (ammonia nitrogen), and C / N = 35, the initial ammonia nitrogen concentration of the basal culture medium was adjusted to 40 mg / L, 80 mg / L, 100 mg / L, 140 mg / L, and 180 mg / L, with the inoculation, culture, and testing conditions remaining the same. The results are as follows... Figure 4 As shown, the strain achieved the highest removal rates of COD, ammonia nitrogen, and phosphate when the initial ammonia nitrogen concentration was 80 mg / L. Therefore, the optimal initial ammonia nitrogen concentration for the growth of this bacterium is 80 mg / L.
[0048] Similarly, under the conditions of optimal carbon source (sodium citrate), optimal nitrogen source (ammonia nitrogen), C / N = 35, and initial ammonia nitrogen concentration of 80 mg / L, the P / N of the basal medium was adjusted to 0.1, 0.2, 0.4, 0.7, and 1, with the inoculation, culture, and testing conditions remaining the same. The results are as follows. Figure 5 As shown, the strain achieved the highest removal rates of COD, ammonia nitrogen, and phosphate when P / N = 0.1, therefore the optimal P / N for the growth of this bacterium is 0.1.
[0049] Similarly, under the conditions of optimal carbon source (sodium citrate), optimal nitrogen source (ammonia nitrogen), C / N = 35, initial ammonia nitrogen concentration (80 mg / L), and P / N = 0.1, the pH of the basal medium was adjusted to 3.5, 5.5, 7.5, 9.5, and 11.5, and the inoculation, culture, and testing conditions were the same as above. The results are as follows. Figure 6 As shown, the OD of the strain was [data missing] when pH = 5.5-7.5. 600 All values were above 1.00, and the strain achieved the highest removal rates of COD, ammonia nitrogen, and phosphate at pH 7.5. Therefore, the optimal pH for the growth of this bacterium is 7.5.
[0050] Similarly, under the conditions of optimal carbon source (sodium citrate), optimal nitrogen source (ammonia nitrogen), C / N = 35, initial ammonia nitrogen concentration (80 mg / L), P / N = 0.1, and pH = 7.5, the temperatures were adjusted to 20℃, 25℃, 30℃, 37℃, and 44℃, and the inoculation, cultivation, and testing conditions were the same as above. The results are as follows... Figure 7 As shown, when the temperature is 20-37℃, the removal rates of COD, ammonia nitrogen, and phosphate of the strain are all above 75%, while the removal rates of COD, ammonia nitrogen, and phosphate are highest when the temperature is 30℃. Therefore, the optimal temperature for the growth of this bacterium is 30℃.
[0051] In summary, the optimal culture conditions are: carbon source = sodium citrate, nitrogen source = ammonia nitrogen, C / N ratio (mass ratio, the same below) = 20-40, P / N ratio (mass ratio, the same below) = 0.1, initial ammonia nitrogen concentration = 80 mg / L, pH = 5.5-9.5, and temperature = 20-37℃. The optimal conditions for the growth and nitrogen and phosphorus removal of Klebsiella oxytoca TY are: carbon source = sodium citrate, C / N = 35, initial ammonia nitrogen concentration = 80 mg / L, P / N = 0.1, pH = 7.5, and temperature = 30℃.
[0052] Example 2
[0053] Growth and denitrification / phosphorus removal characteristics of Klebsiella oxytoca TY when ammonia nitrogen is the sole nitrogen source.
[0054] Inoculate 1 mL of bacterial suspension into an Erlenmeyer flask containing 100 mL of heterotrophic nitrification medium. Incubate at 30 °C and 120 rpm. Measure OD every 6 hours. 600 The NH4+ value was determined after centrifugation at 8000 rpm for 10 min, and the NH4+ of the supernatant was measured. + -N, NO3 - -N, NO2 - -N,PO4 3- -P and COD concentrations.
[0055] The results are as follows Figure 8As shown. The strain entered the logarithmic growth phase 6 hours after inoculation and reached the stationary phase after 24 hours. No significant NO2 was observed during the culture period. - -N and NO3 - -N accumulation, COD, NH4 + -N and PO4 3- -P removal mainly occurred during the rapid growth phase of the strain, and ammonia nitrogen was completely removed by 48 hours. The COD removal rate was 97.153%, and PO4 removal was... 3- -P removal rate was 95.019%. Maximum NH4 + -N and PO4 3- -P removal rates were 7.83 mg N / L / h and 1.60 mg N / L / h, respectively.
[0056] Example 3
[0057] The growth and denitrification / phosphorus removal characteristics of Klebsiella oxytoca TY when nitrate nitrogen is the sole nitrogen source.
[0058] Inoculate 1 mL of bacterial suspension into an Erlenmeyer flask containing 100 mL of denitrification medium I, and incubate at 30 °C and 120 rpm. Measure OD every 6 hours. 600 The NH4+ value was determined after centrifugation at 8000 rpm for 10 min, and the NH4+ of the supernatant was measured. + -N, NO3 - -N, NO2 - -N,PO4 3- -P and COD concentrations.
[0059] The results are as follows Figure 9 The results showed that the strain entered the logarithmic growth phase 12 hours after inoculation and entered the stationary phase after 30 hours. This was accompanied by OD... 600 The increase in COD and NO3 - -N and PO4 3- -P gradually decreases, with a small amount of nitrite nitrogen accumulating, then decreases, COD, NO3... - -N and PO4 3- The removal rates of -P were 79.533%, 90.598%, and 100%, respectively, with the highest removal rates for COD and NO3. - -N and PO4 3- The removal rates of -P were 77.43 mgN / L / h, 3.67 mgN / L / h, and 1.38 mgN / L / h, respectively.
[0060] Example 4
[0061] Growth and denitrification / phosphorus removal characteristics of Klebsiella oxytoca TY when nitrite is the sole nitrogen source.
[0062] Inoculate 1 mL of bacterial suspension into an Erlenmeyer flask containing 100 mL of denitrification medium II, and incubate at 30 °C and 120 rpm. Measure OD every 6 hours. 600 The NH4+ value was determined after centrifugation at 8000 rpm for 10 min, and the NH4+ of the supernatant was measured. + -N, NO3 - -N, NO2 - -N,PO4 3- -P and COD concentrations.
[0063] The results are as follows Figure 10 The results showed that the strain experienced a 6-hour stasis period, entered the logarithmic growth phase after 12 hours, and entered the stationary phase at 30 hours. This was accompanied by an OD... 600 The values gradually increased, COD and NO2 - -N and PO4 3- -P gradually decreases, with a small amount of nitrate nitrogen accumulating, then decreases, COD, NO2 - -N and PO4 3- The removal rates of -P were 73.348%, 73.440%, and 100%, respectively, with the highest removal rates for COD and NO2. - -N and PO4 3- The removal rates of -P were 116.19 mgN / L / h, 1.96 mgN / L / h, and 2.41 mgN / L / h, respectively. During the degradation of nitrite nitrogen, a small amount of ammonia nitrogen accumulated during the logarithmic growth phase and then decreased, indicating that the bacteria may convert nitrite nitrogen into ammonia nitrogen and then assimilate it into biomass.
[0064] Example 5
[0065] The growth and denitrification / phosphorus removal characteristics of Klebsiella oxytoca TY when using ammonia nitrogen and nitrate nitrogen as a mixed nitrogen source.
[0066] Inoculate 1 mL of bacterial suspension into an Erlenmeyer flask containing 100 mL of simultaneous nitrification and denitrification medium I, and incubate at 30 °C and 120 rpm. Measure OD every 6 hours. 600 The NH4+ value was determined after centrifugation at 8000 rpm for 10 min, and the NH4+ of the supernatant was measured. + -N, NO3 - -N, NO2 - -N,PO4 3- -P and COD concentrations.
[0067] The results are as follows Figure 11The results showed that during cultivation using ammonia and nitrate nitrogen as a mixed nitrogen source, the increase in bacterial biomass occurred simultaneously with the removal of both ammonia and nitrate nitrogen. The strain entered the logarithmic growth phase 12 hours after inoculation, preferentially utilizing ammonia nitrogen for growth and development, while nitrate nitrogen utilization occurred at 6 hours. When nitrate nitrogen began to degrade, nitrite accumulated and was then gradually degraded. This phenomenon indicates that the strain can utilize NH4+. + -N and NO3 - When -N is a mixed nitrogen source, simultaneous nitrification and denitrification occur, maximizing COD and NH4+. + -N, NO3 - -N and PO4 3- -P removal rates were 95.648%, 85.799%, 98.421%, and 92.304%.
[0068] Example 6
[0069] The growth and denitrification / phosphorus removal characteristics of Klebsiella oxytoca TY when using ammonia nitrogen and nitrite nitrogen as a mixed nitrogen source.
[0070] Inoculate 1 mL of bacterial suspension into an Erlenmeyer flask containing 100 mL of simultaneous nitrification-denitrification medium II, and incubate at 30 °C and 120 rpm. Measure OD every 6 hours. 600 The NH4+ value was determined after centrifugation at 8000 rpm for 10 min, and the NH4+ of the supernatant was measured. + -N, NO3 - -N, NO2 - -N,PO4 3- -P and COD concentrations.
[0071] The results are as follows Figure 12 The results showed that, using ammonia and nitrite nitrogen as a mixed nitrogen source, the strain entered the logarithmic growth phase 6 hours after inoculation, while nitrite nitrogen utilization occurred at 12 hours. This was accompanied by OD... 600 With the increase of COD, nitrogen and phosphorus, the highest levels of COD and NH4+ are degraded. + -N, NO2 - -N and PO4 3- The phosphorus removal rates were 94.971%, 93.814%, 67.863%, and 51.718%, respectively. During the degradation of ammonia nitrogen, nitrate nitrogen was detected to first increase and then decrease, proving that this strain produces nitrate nitrogen through aerobic nitrification.
[0072] Example 7
[0073] The growth and denitrification / phosphorus removal characteristics of Klebsiella oxytoca TY when using ammonia nitrogen, nitrate nitrogen and nitrite nitrogen as a mixed nitrogen source.
[0074] Inoculate 1 mL of bacterial suspension into an Erlenmeyer flask containing 100 mL of simultaneous nitrification and denitrification medium III, and incubate at 30 °C and 120 rpm. Measure OD every 6 hours. 600 The NH4+ value was determined after centrifugation at 8000 rpm for 10 min, and the NH4+ of the supernatant was measured. + -N, NO3 - -N, NO2 - -N,PO4 3- -P and COD concentrations.
[0075] The results are as follows Figure 13 The results showed that the strain entered the logarithmic growth phase after 6 hours, while COD and NH4... + -N, NO3 - -N and PO4 3- -P begins to decrease, NO2 - -N initially increases and then decreases, indicating that this strain can achieve simultaneous nitrification and denitrification in a mixed nitrogen source of ammonia, nitrate, and nitrite. (COD, NH4) + -N, NO3 - -N, NO2 - -N and PO4 3- The maximum removal rates of -P were 79.388%, 89.668%, 99.083%, 97.547%, and 100%.
[0076] Example 8
[0077] Klebsiella oxytoca TY was investigated for its simultaneous denitrification and degradation of amoxicillin using organic carbon sources. 1 mL of bacterial suspension was inoculated into an Erlenmeyer flask containing 100 mL of antibiotic removal-denitrification medium. The culture conditions were 30 °C and 120 rpm. OD was measured every 6 hours. 600 The NH4+ value was determined after centrifugation at 8000 rpm for 10 min, and the NH4+ of the supernatant was measured. + -N, COD and amoxicillin concentrations.
[0078] Simultaneously, all bacterial suspensions were sterilized at 120℃ for 20 min and then inoculated into Erlenmeyer flasks containing 100 mL of antibiotic removal-denitrification medium. The culture conditions were 30℃ and 120 rpm. OD was measured every 6 h. 600 The NH4+ value was determined after centrifugation at 8000 rpm for 10 min, and the NH4+ of the supernatant was measured. + -N, COD and amoxicillin concentrations.
[0079] The results are as follows Figure 14 The results showed that the strain entered the logarithmic growth phase after 32 hours, during which COD, ammonia nitrogen, and amoxicillin began to degrade, and COD and NH4... +The maximum removal rates of -N and amoxicillin were 92.779%, 95.218%, and 92.208%, respectively, with maximum removal rates of 163.92 mgN / L / h, 4.97 mgN / L / h, and 0.77 mgN / L / h. Figure 15 This indicates that the strain's degradation of amoxicillin mainly relies on biodegradation and bioadsorption, and it can rapidly degrade amoxicillin using organic carbon sources. Its cometabolite system can maintain metabolic homeostasis by supplementing substrates, thereby enhancing its amoxicillin degradation capacity. Figure 16 As shown, the degradation products have low toxicity and can exist stably in the environment.
[0080] >16S rDNA sequence:
[0081]
Claims
1. A heterotrophic nitrifying-aerobic denitrifying strain that simultaneously degrades antibiotics, namely Klebsiella oxytoca TY, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 8, 2025, with accession number CGMCC No. 33884.
2. The heterotrophic nitrifying-aerobic denitrifying strain that simultaneously degrades antibiotics according to claim 1, characterized in that: The organic carbon sources used include sodium citrate, sodium succinate, sodium acetate, glucose, or sucrose; the inorganic nitrogen sources used include ammonia nitrogen, nitrate nitrogen, or nitrite nitrogen; the organic nitrogen sources used include alanine or glutamic acid; and the inorganic phosphorus source used is orthophosphate. Culture conditions: Sodium citrate as carbon source, C / N ratio of 20-40, initial ammonia nitrogen concentration of 80 mg / L, P / N ratio of 0.1, pH of 5.5-9.5, and temperature of 20-37℃.
3. The application of the heterotrophic nitrifying-aerobic denitrifying strain that simultaneously degrades antibiotics as described in claim 1, characterized in that, The optimal growth conditions are: carbon source = sodium citrate, C / N = 35, P / N = 0.1, initial ammonia nitrogen concentration = 80 mg / L, pH = 7.5, and temperature = 30℃.
4. The application of the heterotrophic nitrifying-aerobic denitrifying strain that simultaneously degrades antibiotics as described in claim 1, characterized in that: The bacteria are inoculated into synthetic wastewater with one or more of ammonia nitrogen, nitrate nitrogen, or nitrite nitrogen as the nitrogen source and orthophosphate as the phosphorus source, and carbon, nitrogen, and phosphorus are effectively removed under optimal culture conditions.
5. The application according to claim 4, characterized in that: The bacteria were inoculated into synthetic wastewater containing a mixture of organic carbon source and amoxicillin, and under optimal culture conditions, effective removal of carbon, nitrogen, and amoxicillin was achieved.