Comamonas testosteroni with alkali-resistant and low-temperature-resistant heterotrophic nitrification-aerobic denitrification characteristics and application thereof

Abstract

The present invention relates to a Comamonas testosteroni ( Comamonas testosterone )HR5, with the deposit number of CGMCC No.30691, has the ability of efficient heterotrophic nitrification-aerobic denitrification, and is resistant to low temperature, strong alkali and multiple antibiotics, and has good application prospects in the field of sewage denitrification treatment.

Description

Technical Field

[0001] The invention relates to Comamonas testosteroni with alkali-resistant and low-temperature-resistant heterotrophic nitrification-aerobic denitrification characteristics and application thereof. Background Art

[0002] Nitrogen-containing pollutants discharged into water bodies will cause eutrophication of the water bodies, affect the biodiversity of the aquatic environment, and disrupt the stability of the ecosystem. It is necessary to control the nitrogen concentration in the water bodies.

[0003] Biological denitrification has been a research hotspot in recent years. Traditional biological denitrification technology separates the two processes of autotrophic nitrification and anaerobic denitrification, and selects suitable nitrifying bacteria and denitrifying bacteria respectively. The whole process is complicated and costly.

[0004] Recently, the research on heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria has been gradually carried out. Heterotrophic nitrification-aerobic denitrification bacteria can not only use oxygen and nitrate or nitrite as electron acceptors to complete denitrification under aerobic conditions, but also simultaneously carry out heterotrophic nitrification to achieve ammonia nitrogen removal, showing efficient ammonia nitrogen and nitrate nitrogen removal capabilities. The HN-AD bacteria screened so far are mainly concentrated in the genus Acinetobacter ( Acinetobacter sp. ) and Pseudomonas ( Pseudomonas sp. ), which mainly achieves denitrification under medium temperature conditions (20~40℃), and cannot achieve the simultaneous removal of ammonia nitrogen and nitrate nitrogen under low temperature and strong alkaline conditions. Summary of the invention

[0005] The purpose of the present invention is to provide a Comamonas testosteroni HR5 which is resistant to low temperature and strong alkali and has efficient synchronous denitrification ability and application of the same in sewage denitrification.

[0006] The present invention adopts the following technical solution:

[0007] A testosterone bacteria Comamonas testosteroni )HR5, the deposit number is CGMCCNo.30691, deposited in the General Microbiology Center of China Culture Collection Administration, the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit date is May 20, 2024.

[0008] Furthermore, the Comamonas testosteroni HR5 can perform heterotrophic nitrification and denitrification.

[0009] Furthermore, the Comamonas testosteroni HR5 can perform aerobic denitrification.

[0010] Furthermore, the Comamonas testosteroni HR5 can simultaneously carry out heterotrophic nitrification and aerobic denitrification.

[0011] Furthermore, the Comamonas testosteroni HR5 can simultaneously perform nitrification-denitrification denitrification under low temperature and / or strong alkaline environment.

[0012] Furthermore, the Comamonas testosteroni HR5 is resistant to penicillin, oxacillin, ampicillin, cefazolin, cefuroxime sodium, amikacin, gentamicin, kanamycin, streptomycin, tetracycline, minocycline, erythromycin, azithromycin, norfloxacin, ciprofloxacin, lincomycin, chloramphenicol, clindamycin, chlorfenicol and metronidazole.

[0013] An application of the above-mentioned Comamonas testosteroni HR5 in sewage treatment.

[0014] An application of the above-mentioned Comamonas testosteroni HR5 in the treatment of nitrogen-containing alkaline sewage.

[0015] An application of the above-mentioned Comamonas testosteroni HR5 in the treatment of nitrogen-containing alkaline antibiotic wastewater.

[0016] A sewage treatment agent comprising the above-mentioned Comamonas testosteroni HR5.

[0017] The beneficial effects of the present invention are as follows: the Comamonas testosteroni HR5 strain of the present invention is resistant to low temperatures, adaptable to strongly alkaline environments, and has efficient simultaneous denitrification performance. It is also resistant to a variety of antibiotics, and may become a promising candidate for treating nitrogen-containing alkaline wastewater (especially nitrogen-containing alkaline wastewater containing antibiotics), and has good application prospects in the field of sewage denitrification treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the phylogenetic tree of strain HR5.

[0019] Figure 2 This is the growth curve of strain HR5.

[0020] Figure 3 This is the nitrogen removal effect of strain HR5 under different carbon sources.

[0021] Figure 4 This is the nitrogen removal effect of strain HR5 at 25℃.

[0022] Figure 5 This is the nitrogen removal effect of strain HR5 at 10℃.

[0023] Figure 6 This is the nitrogen removal effect of strain HR5 at 18℃.

[0024] Figure 7 This is the nitrogen removal effect of strain HR5 at pH = 10, 25℃.

[0025] Figure 8 This is the nitrogen removal effect of strain HR5 at pH = 10 and 10°C.

[0026] Fig. 9 These are the results of the drug resistance experiment for strain HR5. DETAILED DESCRIPTION

[0027] The present invention is described specifically and in detail below in conjunction with specific embodiments.

[0028] Example 1 Screening and Identification

[0029] 1. Screening of strains

[0030] Sludge samples were taken from the Shijiazhuang Wastewater Treatment Plant in Hebei Province, China. After mixing the fresh sludge sample, 15.0 ml was transferred to the enrichment medium, and cultured at 25°C and 90 r / min for 2-3 days. Then 10 ml of the supernatant was transferred to the fresh enrichment medium again. After culturing under the same conditions for 2-3 days, 10 ml of the supernatant was transferred to the fresh liquid acclimation medium, and cultured under the same conditions for 3 days. It was transferred to a new liquid acclimation medium every 3 days. The nitrate removal rate reached more than 98% for 3 consecutive times, and the strain was isolated.

[0031] Enrichment medium: NaCl 10g / L, tryptone 10g / L, yeast powder 5g / L.

[0032] Acclimation medium: KNO 3 0.1444 g / L, CH 3 COONa 0.469g / L, K 2 HPO 4 ·3H 2 O 0.75 g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, Nacl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O 0.05g / L, FeSO 4 7H 2 O 0.01 g / L. Solid medium is supplemented with 20 g / L agar.

[0033] Perform gradient dilutions and streak on solid acclimation medium, place the plate in a 25°C incubator for 2 to 3 days until clear single colonies grow on the plate.

[0034] Determination of denitrification performance: A single colony was inoculated into a liquid acclimation culture medium, cultured in a constant temperature incubator at 25°C for 2-3 days, 10 ml was extracted, and its nitrate-nitrogen removal effect was measured. The results showed that the strain labeled HR 5 had the highest nitrate-nitrogen removal rate (86%) and the best growth density.

[0035] 2. rDNA identification

[0036] The genomic DNA of strain HR5 was extracted and the PCR product was amplified using the universal primers 27F / 1492R for bacterial 16SrRNA gene amplification and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for sequence sequencing. The genus relationship of strain HR5 was identified by comparison analysis in the NCBI database. Comamonas testosterone The closest distance is 99.86% similarity. The results of phylogenetic tree homology analysis are as follows Figure 1 shown.

[0037] Based on the above analysis, strain HR5 was identified as Comamonas testosteroni ( Comamonas testosterone ) HR5. The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on May 20, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 30691.

[0038] Example 2 Growth curve of strain HR5

[0039] 2% HR5 bacterial solution was inoculated into fresh liquid acclimation medium (strain HR5 was first cultured in liquid acclimation medium to OD 600 The culture medium was placed in a constant temperature incubator at 90 r / min and 25°C. 2.5 ml of culture medium was taken every 6 hours. The OD of the culture medium was measured at a wavelength of 600 nm using the photoelectric turbidimetry method. 600 The growth curve of strain HR5 was obtained as follows Figure 2 shown.

[0040] The results showed that 0~12h is the lag phase, during which bacteria adapt to the new environment and store sufficient enzymes, energy and intermediate metabolites for cell volume increase, active metabolism and cell proliferation. 12~36h is the logarithmic growth phase, during which bacteria proliferate rapidly at geometric levels. 36~96h is the stable phase, during which the bacterial reproduction trend gradually decreases due to the consumption of nutrients in the culture medium and the accumulation of toxic metabolites. After 96h, it is the decay phase, during which the bacterial reproduction rate is lower than the rate of cell autolysis and death as the stable phase develops.

[0041] Example 3 Denitrification efficiency of strain HR5 with different carbon sources

[0042] Different carbon sources, sodium acetate, sodium succinate and sodium citrate, were prepared under the condition of C / N=9, and 5% of the HR5 bacterial liquid (same as in Example 2) was inoculated into the culture medium of different carbon sources, and cultured at 25°C and 90rpm. 10 ml of the culture medium was taken every 12 hours, and after filtering through a 0.45 μm filter membrane, the indicators such as nitrate nitrogen, ammonia nitrogen, nitrite nitrogen and dissolved total nitrogen were measured.

[0043] Denitrification medium containing sodium acetate is: KNO 3 0.1444 g / L, CH 3 COONa 0.615g / L, K 2 HPO 4 ·3H 2 O0.75g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O0.05g / L, FeSO 4 7H 2 O 0.01g / L.

[0044] The denitrification medium containing sodium succinate is: KNO 3 0.1444g / L, C 4 H 4 Na 2 O 4 6H 2 O 1.0125g / L, K 2 HPO 4 ·3H 2 O 0.75 g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O 0.05g / L, FeSO 4 7H 2 O 0.01g / L.

[0045] Denitrification medium containing sodium citrate is: KNO 3 0.1444g / L, C 6 H 5 Na 3 O 7 ·2H2 O 0.735g / L, K 2 HPO 4 ·3H 2 O 0.75 g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O 0.05g / L, FeSO 4 7H 2 O 0.01g / L.

[0046] The experiment showed that strain HR5 could utilize a variety of organic carbon sources for growth. The nitrogen removal effects of different carbon sources were as follows: Figure 3 As shown. Denitrification medium with sodium acetate as carbon source ( Figure 3 a), the initial nitrate nitrogen concentration was 19.39 mg / L and was removed to 1.21 mg / L after 24 hours, with a removal rate of 93.76%. The nitrite nitrogen accumulation reached the maximum at 18 hours, with a concentration of 8.13 mg / L, and the nitrite nitrogen concentration was 3.76 mg / L at 72 hours. Denitrification medium with sodium succinate as carbon source ( Figure 3 b), the initial nitrate nitrogen concentration was 19.34 mg / L and was removed to 1.14 mg / L after 36 hours, with a removal rate of 94.11%. The accumulation of nitrite nitrogen reached the maximum at 24 hours, with a concentration of 7.27 mg / L, and the nitrite nitrogen concentration reached 4.85 mg / L at 72 hours. In contrast, the denitrification medium with sodium acetate as the carbon source has a fast removal speed, is more suitable for the growth of bacteria, and has less nitrite nitrogen accumulation. The nitrate nitrogen with sodium citrate as the carbon source can remove about 70% ( Figure 3 c).

[0047] Example 4 Denitrification efficiency of strain HR5 at 25°C

[0048] 5% of the HR5 bacterial solution (same as in Example 2) was added to a denitrification medium, a nitrification medium and a simultaneous nitrification and denitrification medium with a carbon-nitrogen ratio of 9 and sodium acetate as a carbon source, and cultured at 25° C. and 90 rpm. 10 ml of the culture solution was taken every 12 h, filtered through a 0.45 μm filter membrane, and the nitrate nitrogen, ammonia nitrogen, nitrite nitrogen and dissolved total nitrogen and other indicators were measured.

[0049] Nitrification medium: (NH 4 ) 2 SO 4 0.0944g / L, CH 3 COONa 0.615g / L, K 2HPO 4 ·3H 2 O 0.75 g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O 0.05g / L, FeSO 4 7H 2 O 0.01g / L.

[0050] Denitrification medium: KNO 3 0.1444 g / L, CH 3 COONa 0.615g / L, K 2 HPO 4 ·3H 2 O 0.75 g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O 0.05g / L, FeSO 4 7H 2 O 0.01g / L.

[0051] The culture medium for simultaneous nitrification and denitrification is: (NH 4 ) 2 SO 4 0.04714g / L, KNO 3 0.0722g / L, K 2 HPO 4 ·3H 2 O0.75g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O0.05g / L, FeSO 4 7H 2 O 0.01g / L.

[0052] The nitrogen removal effect of C / N=9 is as follows Figure 4 The results showed that the denitrification medium ( Figure 4 a) The initial concentration of nitrate nitrogen was 19.31 mg / L, and the removal rate of denitrification medium reached 92.28% at 24 hours, when the concentration of nitrate nitrogen was 1.49 mg / L. The accumulation of nitrite nitrogen reached the maximum at 18 hours, with a concentration of 8.13 mg / L, and the concentration of nitrite nitrogen dropped to 3.76 mg / L at 72 hours. The removal rate of total dissolved nitrogen reached 87.1% at 72 hours. Nitrification medium ( Figure 4 b) The initial ammonia nitrogen concentration was 18.99 mg / L and was removed to 0.59 mg / L in 24 hours, with a removal rate of 96.89%, and no other nitrogen accumulation. The total nitrogen was removed from 16.66 mg / L to 0 mg / L in 36 hours. Figure 4 c) The initial concentration of nitrate nitrogen was 10.76 mg / L, which was reduced to 0.16 mg / L in 24 hours, and the removal rate reached 98.51%. The initial concentration of ammonia nitrogen was 9.29 mg / L, which was reduced to 0.17 mg / L in 12 hours, and the removal rate reached 98.17%. At 36 hours, the concentration of total nitrogen was 0.18 mg / L, and the removal rate reached 99.03%. This shows that strain HR5 can quickly remove nitrate nitrogen and accumulated nitrite nitrogen. It has good nitrification-denitrification efficiency at 25℃ and C / N=9.

[0053] Example 5 Denitrification efficiency of strain HR5 at 10°C

[0054] Denitrification medium, nitrification medium, and simultaneous nitrification and denitrification medium (same as in Example 4) were prepared with C / N=9 and sodium acetate as the carbon source. HR5 bacterial solution (same as in Example 2) was inoculated into different culture media at an inoculation rate of 5%. They were cultured in a constant temperature incubator at 10°C and 90 rpm. 10 ml of the culture solution was taken every 48 hours, and after being filtered through a 0.45 μm filter membrane, the nitrate nitrogen, ammonia nitrogen, nitrite nitrogen, and total dissolved nitrogen were measured.

[0055] The nitrogen removal effect of denitrification, nitrification, and simultaneous nitrification and denitrification culture medium at 10°C is as follows Figure 5 As shown. Denitrification medium within 168h ( Figure 5 a) can achieve 84.34% nitrate removal, from the initial nitrate concentration of 19.41 mg / L to 3.04 mg / L. At this time, the concentration of nitrite is 2.83 mg / L. Nitrification medium ( Figure 5 b) The initial ammonia nitrogen concentration was 17.89 mg / L and was removed to 0.55 mg / L in 168 hours, with a removal rate of 96.93%. There was no accumulation of other nitrogen, and the total nitrogen was removed from 15.88 mg / L to 0 mg / L. Figure 5c) The initial concentration of nitrate nitrogen was 10.61 mg / L, which was reduced to 0.70 mg / L after 168 hours, and the removal rate reached 93.4%. The initial concentration of ammonia nitrogen was 9.36 mg / L, which was reduced to 0.28 mg / L after 120 hours, and the removal rate reached 97.01%. At 168 hours, the concentration of dissolved total nitrogen was 1.81 mg / L, and the removal rate reached 90.72%.

[0056] Example 6 Denitrification efficiency of HR5 at 18°C

[0057] Denitrification medium, nitrification medium, and simultaneous nitrification and denitrification medium (same as in Example 4) were prepared with C / N=9 and sodium acetate as the carbon source. HR5 bacterial solution (same as in Example 2) was inoculated into different culture media at an inoculation rate of 5%, and cultured in a constant temperature incubator at 18°C ​​at 90 rpm. 10 ml of the culture solution was taken every 48 hours, filtered through a 0.45 μm filter membrane, and the nitrate nitrogen, ammonia nitrogen, nitrite nitrogen, and dissolved total nitrogen indicators were measured. The results are as follows: Figure 6 shown.

[0058] The results showed that within 168 h, the denitrification medium ( Figure 6 a) can achieve 84.34% nitrate removal, from the initial nitrate concentration of 19.41 mg / L to 3.04 mg / L. At this time, the concentration of nitrite is 2.83 mg / L. Nitrification medium ( Figure 6 b) The initial ammonia nitrogen concentration was 17.89 mg / L and was removed to 0.34 mg / L in 72 hours, with a removal rate of 98.1%. There was no accumulation of other nitrogen, and the total nitrogen was removed from 15.88 mg / L to 1.72 mg / L. Figure 6 c) The initial concentration of nitrate nitrogen was 10.61 mg / L, which was reduced to 0.88 mg / L after 72 hours, and the removal rate reached 91.71%. The initial concentration of ammonia nitrogen was 9.36 mg / L, which was reduced to 0.21 mg / L after 48 hours, and the removal rate reached 97.76%. At 120 hours, the concentration of total nitrogen was 1.20 mg / L, and the removal rate reached 93.85%.

[0059] Example 7 Denitrification efficiency of strain HR5 at high nitrogen concentration

[0060] Based on C / N=9 and sodium acetate as the carbon source, denitrification culture media with nitrogen concentrations of 50 mg / L and 100 mg / L were prepared. Each culture medium was inoculated with 5% HR5 bacterial solution (same as in Example 2) and cultured at 25°C and 90 rpm. 10 ml of the culture solution was taken every 24 hours, filtered through a 0.45 μm filter membrane, and the indicators such as nitrate nitrogen, ammonia nitrogen, nitrite nitrogen and dissolved total nitrogen were measured.

[0061] Denitrification medium (N=50mg / L) is: KNO3 0.361 g / L, CH 3 COONa 1.538g / L, K 2 HPO 4 ·3H 2 O0.75g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O 0.05g / L, FeSO 4 7H 2 O 0.01 g / L.

[0062] Denitrification medium (N=100 mg / L) is: KNO 3 0.722 g / L, CH 3 COONa 3.076g / L, K 2 HPO 4 ·3H 2 O0.75g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O0.05g / L, FeSO 4 7H 2 O 0.01g / L.

[0063] The test results are shown in Table 1.

[0064] Table 1 Removal effect of strain HR5 on nitrate nitrogen at high nitrogen concentration

[0065] .

[0066] In Table 1, the initial nitrate nitrogen concentration of the denitrification medium with N=50mg / L was 51.12mg / L, which decreased to 4.55mg / L after 96h, and the removal rate reached 91.1%; the dissolved total nitrogen decreased from the initial concentration of 56.59mg / L to 19.82mg / L after 96h, and the removal rate reached 64.98%. The initial nitrate nitrogen concentration of the denitrification medium with N=100mg / L was 95.60mg / L, which decreased to 21.38mg / L after 96h, and the removal rate reached 77.64%; the dissolved total nitrogen decreased from the initial concentration of 97.63mg / L to 78.55mg / L after 96h, and the removal rate reached 19.54%.

[0067] Example 8 Denitrification efficiency of strain HR5 under different pH environments

[0068] Based on C / N=9 and sodium acetate as the carbon source, the pH of the denitrification medium (same as in Example 4) was adjusted to 3, 5, 7, 10, and 12, respectively, and 5% of the HR5 bacterial solution (same as in Example 2) was inoculated, respectively, and cultured at 25°C and 90 rpm. 10 ml of the culture solution was taken every 12 hours, and after filtering through a 0.45 μm filter membrane, the nitrate nitrogen, ammonia nitrogen, nitrite nitrogen and dissolved total nitrogen and other indicators were measured.

[0069] The test results are shown in Table 2.

[0070] Table 2 Removal effect of nitrate nitrogen by strain HR5 at different pH values

[0071] .

[0072] As shown in Table 2, the initial concentration of nitrate nitrogen in the denitrification medium at pH = 7 was 19.66 mg / L, which was reduced to 2.19 mg / L after 24 hours, and the removal rate reached 88.86%. The initial concentration of nitrate nitrogen in the denitrification medium at pH = 10 was 19.66 mg / L, which was reduced to 1.66 mg / L after 24 hours, and the removal rate reached 91.56%. The initial concentration of dissolved total nitrogen was 19.54 mg / L, which was reduced to 4.21 mg / L after 24 hours, and the removal rate was 78.45%. The experiment showed that strain HR5 can still efficiently denitrify in a strong alkaline environment.

[0073] Example 9 Denitrification efficiency of strain HR5 at pH = 10, 25°C

[0074] Nitrification medium, synchronous nitrification and denitrification medium (same as Example 4), and short-range synchronous nitrification and denitrification medium with a pH of 10 were prepared with C / N=9 and sodium acetate as the carbon source. The HR5 bacterial solution (same as Example 2) was inoculated into different culture media at an inoculation rate of 5%. They were cultured in a constant temperature incubator at 25°C and 90rpm for 24h, 48h, 72h, 96h, 120h, 168h, and 192h, respectively. In addition, 10ml of the culture medium was taken from the nitrification medium at 216h and 240h, and after filtration through a 0.45μm filter membrane, the indicators such as nitrate nitrogen, ammonia nitrogen, nitrite nitrogen, and total dissolved nitrogen were measured. The results are as follows Figure 7 shown.

[0075] The medium for short-term simultaneous nitrification and denitrification is: (NH 4 ) 2 SO 4 0.04714 g / L, NaNO 2 0.04929g / L, K 2 HPO 4 ·3H 2 O 0.75 g / L, NaH 2 PO 4 ·2H 2 O 0.25g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, MgSO 4 7H 2 O0.05g / L, FeSO 4 7H 2 O 0.01g / L.

[0076] The results showed that the nitrification medium with ammonia nitrogen as the nitrogen source ( Figure 7 a), the initial ammonia nitrogen concentration was 15.35mg / L and the ammonia nitrogen concentration was 0.39mg / L at 192h, the removal rate reached 97.46%, and there was no accumulation of other nitrogen. Synchronous nitrification and denitrification medium with ammonia nitrogen and nitrate nitrogen as nitrogen sources ( Figure 7 b), the initial ammonia nitrogen concentration was 8.47 mg / L, and the initial nitrate nitrogen concentration was 9.76 mg / L, which decreased to 0.34 mg / L and 1.65 mg / L respectively after 72 hours, and the removal rates were 95.99% and 83.09% respectively. Short-range simultaneous nitrification and denitrification culture medium with ammonia nitrogen and nitrite nitrogen as nitrogen sources ( Figure 7 c) The initial ammonia nitrogen concentration of 8.6 mg / L was reduced to 0.34 mg / L in 72 hours, and the removal rate reached 96%. The initial nitrite nitrogen concentration of 9.58 mg / L was reduced to 3.98 mg / L in 96 hours, and the removal rate was 58.46%.

[0077] Example 10 Denitrification efficiency of strain HR5 at pH = 10, 10°C

[0078] Denitrification medium and simultaneous nitrification and denitrification medium (same as Example 4) with C / N=9 and sodium acetate as carbon source were prepared at pH=10. HR5 bacterial solution (same as Example 2) was inoculated into different culture media at an inoculation rate of 5%. They were cultured in a constant temperature incubator at 10°C. 10 ml of the culture solution was taken every 48 hours starting from the 5th day. After filtration through a 0.45 μm filter membrane, the nitrate nitrogen, ammonia nitrogen, nitrite nitrogen and total dissolved nitrogen were measured. The results are shown in Figure 8 shown.

[0079] The results showed that the denitrification medium with single nitrate as nitrogen source ( Figure 8 a), the initial nitrate nitrogen was 18.67 mg / L and then reduced to 1.70 mg / L in 360 hours, and the removal rate reached 90.89%. Figure 8 b) The initial ammonia nitrogen concentration of 8.47 mg / L was removed to 0.69 mg / L in 312 hours, and the removal rate reached 91.85%. The initial nitrate nitrogen concentration of 9.76 mg / L was removed to 1.36 mg / L in 408 hours, and the removal rate reached 86.07%. The initial total nitrogen of 16.19 mg / L was reduced to 5.92 mg / L in 408 hours, and the removal rate reached 63.43%. At the same time, under low temperature and alkaline conditions, strain HR5 can still survive and has a high efficiency in removing ammonia nitrogen and nitrate nitrogen.

[0080] Example 11 Antibiotic resistance test

[0081] The experimental methods, steps and evaluation criteria of the antibiotic resistance experiment (antibiotic paper strip susceptibility test) were based on the "M100 Antimicrobial Susceptibility Test Implementation Standard (13th Edition)" and "M02-A10 Antimicrobial Susceptibility Test Paper Strip Method Implementation Standard (Vol. 32 No. 1)".

[0082] The specific experimental process is as follows:

[0083] a) Prepare nutrient agar medium plates and adjust the pH to 7.2 ~ 7.4;

[0084] Nutrient agar medium: NaCl 10 g / L, tryptone 10 g / L, yeast powder 5 g / L, agar 20 g / L.

[0085] b) Use sterile blank drug sensitivity paper strips (Hunan Beekman Biological Reagent Co., Ltd.) to prepare paper strips with corresponding drug content.

[0086] c) Take HR5 bacterial solution (same as Example 2, OD 600 = 0.2), and diluted to the specified concentration with physiological saline before the experiment.

[0087] d) Use a disposable sterile cotton swab to dip into the diluted bacterial solution, squeeze out the water from the wall of the centrifuge tube, and then spread it evenly on the nutrient agar plate and dry it at room temperature for 5 minutes.

[0088] e) Use sterile tweezers to stick the paper containing antibiotics in the center of the nutrient agar plate. Set up 2 parallel replicates for each experiment, and set up 3 paper pieces containing sterile water in the center of the nutrient agar plate as a blank control.

[0089] f) Invert the plate within 15 minutes and incubate at 35°C for 18 to 24 hours.

[0090] g) Measure the diameter of the inhibition zone and observe the size of the inhibition zone.

[0091] The results are as follows Fig. 9 As shown in Table 3, in Table 3, the inhibition zone ≤14 mm was resistant (R), the inhibition zone ≥20 mm was sensitive (S), and the inhibition zone 15-19 mm was intermediate (I).

[0092] Table 3 Results of antibiotic resistance experiments of HR5 strain

[0093] .

[0094] The results showed that strain HR5 was resistant to penicillin, oxacillin, ampicillin and cefazolin among β-lactams, cefuroxime sodium among cephalosporins, amikacin, gentamicin, kanamycin and streptomycin among aminoglycosides, tetracycline and minocycline among tetracyclines, erythromycin and azithromycin among macrolides, norfloxacin and ciprofloxacin among quinolones, lincomycin among lincomycins, chloramphenicol among chloramphenicols, clindamycin among lincosamides, chlorfenicol among chloramphenicols, and metronidazole among nitroimidazoles.

[0095] It has intermediate effects on piperacillin and cephalexin of β-lactams, vancomycin and polymyxin B of polypeptides, levofloxacin of quinolones, and doxycycline of tetracyclines.

[0096] It is significantly sensitive to cephalosporins including ceftazidime, ceftriaxone, and cefoperazone, sulfonamides including cotrimoxazole, and carbapenems including imipenem.

[0097] Therefore, the HR5 strain has a certain resistance to multiple antibiotics and can be used to treat wastewater containing antibiotics, overcoming the effect of antibiotics on biological denitrification.

Claims

1. A Comamonas testosteroni ( Comamonas testosteroni ) HR5, characterized in that, The deposit number is CGMCC No.30691.

2. Use of Comamonas testosteroni HR5 as claimed in claim 1 in sewage treatment.

3. Use of Comamonas testosteroni HR5 as claimed in claim 1 in the treatment of nitrogen-containing alkaline sewage.

4. Use of Comamonas testosteroni HR5 as claimed in claim 1 in the treatment of nitrogen-containing alkaline antibiotic wastewater.

5. A sewage treatment agent comprising the Comamonas testosteroni HR5 as claimed in claim 1.

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