A strain of denitrifying Zybelsella LJ1 with low-temperature resistant denitrification function and its application, product and method

Abstract

The present invention "a strain of denitrifying Zobellella denitrificans LJ1 with low-temperature resistant denitrification function and its application, product and method" belongs to the field of microbial technology. The present invention provides a strain of denitrifying Zobellella denitrificans LJ1 with low-temperature resistant denitrification function, and the preservation number is CGMCC NO: 24344. The present invention also provides the application of the strain LJ1 in complete denitrification, as well as a bacterial agent based on the strain LJ1 and a complete denitrification method. The LJ1 strain of the present invention has the ability of complete denitrification in a low-temperature environment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of denitrifying Zybelella LJ1 with low-temperature-resistant denitrification function and an application, product and method thereof. Background Art

[0002] With the development of social economy and agricultural production, a large amount of exogenous nitrogen has entered natural water bodies, causing serious nitrogen pollution and eutrophication problems in the water environment. At present, water resource shortage and low ecological quality of the water environment have become the main bottlenecks and outstanding shortcomings restricting my country's sustainable development and ecological civilization construction. - -N) is a very common form of nitrogen in nature and one of the important forms of nitrogen in the environment. Nitrate nitrogen pollution in the water environment mainly comes from agricultural irrigation processes, urban runoff, sanitation and irregular disposal of industrial waste. Nitrate nitrogen in rivers, lakes, etc. will cause serious eutrophication of water bodies. If there is a high concentration of nitrate nitrogen, i.e. nitrate, in drinking water, it will affect human health, such as methemoglobinemia in infants, bladder cancer, non-Hodgkin's lymphoma, ovarian cancer and digestive tract cancer in adults. Therefore, nitrate nitrogen in water bodies seriously affects human health, ecology and aquatic ecosystems, and the removal of nitrate nitrogen is crucial to the prevention and control of these problems. Constructed wetlands have been developed as a new type of sewage ecological treatment process since the 1970s and have been used to this day. It mainly uses plants, substrates, microorganisms and other methods to achieve efficient purification of sewage. Due to its high removal rate of total nitrogen, nitrate nitrogen and ammonium nitrogen, low operating costs and low clogging rate, it has been greatly promoted and utilized. It provides important technical support for the ecological management of natural wetlands such as rivers and lakes and the improvement of ecological quality.

[0003] Microorganisms are the main participants in wetland denitrification. The denitrification process involving microorganisms is the main way to reduce nitrate nitrogen and completely remove nitrogen in wetland treatment. Although there have been more and more studies on denitrification strains in recent years, the cultivation methods are different. As far as the current research situation is concerned, there are still factors such as strain activity and yield being limited by culture conditions. At the same time, the denitrification effect is also easily affected by low temperature environments and is poor. Greenhouse gas nitrous oxide is produced during the denitrification process, so it is currently very important to screen strains with complete denitrification functions and the ability to achieve efficient denitrification in low temperature environments, apply them to nitrogen-containing wastewater treatment, and reduce greenhouse gas emissions during denitrification.

[0004] Zobellella denitrificans is a microorganism of the genus Zobellella, and its main reported use so far is as a model strain.

[0005] There is no related report on denitrifying Zabela bacteria that is both low temperature resistant and has complete denitrification ability in the prior art of the present invention. Therefore, there is an urgent need to develop a denitrifying Zabela bacteria that is both low temperature resistant and has complete denitrification ability. Summary of the invention

[0006] Based on the above-mentioned gaps and needs in the prior art in the art, the present invention provides a denitrifying Zobellella denitrificans strain LJ1 which has complete denitrification ability and can still efficiently denitrify and denitrify under low temperature environment, and its applications, products and methods.

[0007] The technical solution of the present invention is as follows:

[0008] A denitrifying Zobellella denitrificans strain LJ1 with low-temperature resistant denitrification function is provided, wherein the deposit number thereof is CGMCC NO:24344.

[0009] The Zobellella denitrificans strain LJ1 with low-temperature-resistant denitrification function contains the gene encoding narG for membrane-bound nitrate reductase, the gene encoding napA for periplasmic nitrate reductase, the gene encoding nirK for Cu-type nitrite reductase, the gene encoding nirS for cytochrome cd1-type nitrite reductase, the gene encoding qnorB for nitric oxide reductase and the gene encoding nosZ for nitrous oxide reductase.

[0010] The complete denitrification comprises: the membrane-bound nitrate reductase and the periplasmic nitrate reductase napA of the strain LJ1 reduce nitrate to nitrite; the Cu-type nitrite reductase and the cytochrome cd1-type nitrite reductase of the strain LJ1 reduce nitrite to nitric oxide; the nitric oxide reductase of the strain LJ1 reduces nitric oxide to nitrous oxide; and the nitrous oxide reductase of the strain LJ1 reduces nitrous oxide to nitrogen gas.

[0011] Preferably, the low temperature resistance means that the strain LJ1 has the denitrification function in an environment below 15°C.

[0012] The invention discloses an application of a denitrifying Zobellella denitrificans strain LJ1 with a deposit number of CGMCC NO: 24344 in complete denitrification.

[0013] The complete denitrification comprises: the membrane-bound nitrate reductase and the periplasmic nitrate reductase napA of the strain LJ1 reduce nitrate to nitrite; the Cu-type nitrite reductase and the cytochrome cd1-type nitrite reductase of the strain LJ1 reduce nitrite to nitric oxide; the nitric oxide reductase of the strain LJ1 reduces nitric oxide to nitrous oxide; the nitrous oxide reductase of the strain LJ1 reduces nitrous oxide to nitrogen gas;

[0014] Preferably, the complete denitrification refers to: the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ1 sequentially perform the following reactions:

[0015] Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite;

[0016] Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide;

[0017] Nitric oxide reductase reduces nitric oxide to nitrous oxide;

[0018] Nitrous oxide reductase reduces nitrous oxide to nitrogen gas.

[0019] Preferably, the temperature of complete denitrification is 10-25°C

[0020] A bacterial agent, characterized in that it comprises: a denitrifying Zobellella denitrificans strain LJ1 with a preservation number of CGMCC NO: 24344 and having a low-temperature resistant denitrification function.

[0021] The bacterial agent also includes auxiliary materials.

[0022] A complete denitrification method, characterized in that a strain of Zobellella denitrificans LJ1 with a preservation number of CGMCC NO: 24344 and having a low-temperature resistant denitrification function is used to perform complete denitrification treatment on a sample to be treated.

[0023] The complete denitrification comprises: the membrane-bound nitrate reductase and the periplasmic nitrate reductase napA of the strain LJ1 reduce nitrate to nitrite; the Cu-type nitrite reductase and the cytochrome cd1-type nitrite reductase of the strain LJ1 reduce nitrite to nitric oxide; the nitric oxide reductase of the strain LJ1 reduces nitric oxide to nitrous oxide; the nitrous oxide reductase of the strain LJ1 reduces nitrous oxide to nitrogen gas;

[0024] Preferably, the complete denitrification refers to: the complete denitrification refers to: the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ1 sequentially perform the following reactions:

[0025] Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite;

[0026] Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide;

[0027] Nitric oxide reductase reduces nitric oxide to nitrous oxide;

[0028] Nitrous oxide reductase reduces nitrous oxide to nitrogen gas;

[0029] Preferably, the denitrification treatment time is 1-5 days and the temperature is 10-25°C.

[0030] The inoculation amount of the strain LJ1 in the sample to be treated is 10%;

[0031] Preferably, the sample to be processed is a nitrogen-containing sample;

[0032] Preferably, the nitrogen-containing refers to NO 3- .

[0033] The purpose of the present invention is to provide a high-efficiency denitrification bacterial strain and its application in sewage denitrification and river and lake purification.

[0034] The denitrifying strain of the invention is screened from the rhizosphere environment of the dominant plant cattail in the artificial wetland which has been in operation for more than ten years in the Beijing Wildlife Rescue Center, and the purified strain is obtained by enrichment culture, plate coating and streaking separation.

[0035] Cattail is the main plant used in wetland treatment. In the wetland treatment with long-term stable operation and good denitrification performance, a denitrifying bacterium LJ1 is isolated from the rhizosphere of cattail, and the strain belongs to Zobellella denitrificans in taxonomy. It has been confirmed by experiments that the LJ1 strain of the present invention contains the membrane-bound nitrate reductase encoding gene narG, the periplasmic nitrate reductase encoding gene napA, the Cu-type nitrite reductase encoding gene nirK, the cytochrome cd1-type nitrite reductase encoding gene nirS, the nitric oxide reductase encoding gene qnorB and the nitrous oxide reductase encoding gene nosZ, so that it has complete denitrification ability, that is, when the LJ1 strain of the present invention is used to denitrify nitrate-containing sewage, nitrate or nitrite can be completely converted into nitrogen. At the same time, the LJ1 strain of the present invention solves the problems of low activity and low yield of previously screened denitrifying bacteria, and the strain has potential application prospects in wetland treatment.

[0036] The application of the denitrifying strain LJ1 in artificial wetlands is characterized in that in the denitrifying medium, the denitrifying strain LJ1 3- The removal rates of -N and TN reached 89.21% and 90.17%, respectively.

[0037] The application method of the denitrifying strain LJ1 in artificial wetlands includes the following conditions: the inoculation amount is 0.1-2% of the volume of the artificial wetland water body; the implementation temperature is 10-25°C, the pH is weakly alkaline, and the DO value is 0-4.7 mg / L.

[0038] The benefits of the present invention are as follows: (1) The strain LJ1 obtained by the present invention has strong activity and high ability to remove nitrate nitrogen in water bodies, and has very important application value in artificial wetland aquaculture sewage, industrial wastewater and domestic sewage; (2) The strain LJ1 obtained by the present invention is easy to culture and has a large reproduction amount. (3) The strain LJ1 obtained by the present invention can carry out a complete denitrification process under low temperature environment, reduce greenhouse gas emissions in the intermediate process, and is suitable for denitrification, denitrification and sewage treatment under various temperature environments.

[0039] The deposit information of the Zobellella denitrificans strain LJ1 of the present invention is as follows:

[0040] Deposit number: CGMCC NO:24344;

[0041] Taxonomic nomenclature: Zobellella denitrificans;

[0042] Deposit date: January 19, 2022;

[0043] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0044] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the removal rate of nitrate and nitrite by the LJ1 strain in the fifth part of the experimental example of the present invention.

[0046] Figure 2 This is a line graph of the analysis of the gas component content in the complete denitrification process of the LJ1 strain in the fifth part of the experimental example of the present invention.

[0047] Figure 3 This is the growth curve of the LJ1 strain in Experimental Example 6 of the present invention under low temperature conditions.

[0048] Figure 4 This is the denitrification ability of the LJ1 strain in Experimental Example 7 of the present invention under different temperature conditions.

[0049] Figure 5 This is the amplification curve of the napA gene of the LJ1 strain in the fourth part of the experimental example of the present invention.

[0050] Figure 6 This is the amplification curve of the narG gene of the LJ1 strain in the fourth part of the experimental example of the present invention.

[0051] Figure 7 This is the amplification curve of the nirK gene of the LJ1 strain in the fourth part of the experimental example of the present invention.

[0052] Figure 8 The fourth part of the experimental example of the present invention is an amplification curve of the nirS gene of the LJ1 strain.

[0053] Fig. 9 This is the amplification curve of the qnorB gene of the LJ1 strain in the fourth part of the experimental example of the present invention.

[0054] Fig.10 This is the amplification curve of the nosZ gene of the LJ1 strain in the fourth part of the experimental example of the present invention.

[0055] Fig.11 Schematic diagram of the complete denitrification process of the LJ1 strain of the present invention. DETAILED DESCRIPTION

[0056] The content and technical effects of the present invention are further described in detail below in conjunction with specific embodiments and experimental examples, but the protection scope of the present invention is not limited thereto.

[0057] The first group of examples, strain LJ1 of the present invention

[0058] This group of embodiments provides a strain of Zobellella denitrificans LJ1 with low temperature resistant denitrification function. All the embodiments in this group have the following common features: the deposit number of the strain LJ1 is CGMCC NO:24344.

[0059] In a specific embodiment, the strain LJ1 simultaneously contains the gene encoding membrane-bound nitrate reductase narG, the gene encoding periplasmic nitrate reductase napA, the gene encoding Cu-type nitrite reductase nirK, the gene encoding cytochrome cd1-type nitrite reductase nirS, the gene encoding nitric oxide reductase qnorB and the gene encoding nitrous oxide reductase nosZ.

[0060] In some embodiments, the complete denitrification includes: the membrane-bound nitrate reductase and periplasmic nitrate reductase napA of strain LJ1 reduce nitrate to nitrite; the Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase of strain LJ1 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ1 reduces nitric oxide to nitrous oxide; and the nitrous oxide reductase of strain LJ1 reduces nitrous oxide to nitrogen gas.

[0061] Any act of utilizing, using, selling, promising to sell, producing, preparing, culturing, propagating, cloning, enriching, and fermenting the Zobellella denitrificans strain LJ1 with a preservation number of CGMCC NO: 24344 falls within the scope of protection of the present invention.

[0062] Based on the teachings and inspiration of the present invention, technicians in this field can select appropriate auxiliary materials for actual production needs in combination with commonly used technical means in the field of microbial technology, and prepare the denitrifying Zobellella denitrificans strain LJ1 with a preservation number of CGMCCNO: 24344 of the present invention into various dosage forms that meet various process production requirements, such as powders, tablets, liquids, etc.

[0063] Herein, the biocontrol bacteria, denitrifying Zobellella LJ1, LJ1, strain LJ1, LJ1 strain, and denitrifying Zobellella recorded in the content of the invention and the specific implementation methods all refer to: the denitrifying Zobellella (Zobellella denitrificans) strain LJ1 with the deposit number of CGMCCNO:24344 of the present invention.

[0064] In a preferred embodiment, the low temperature resistance means that the strain LJ1 has a denitrification function in an environment below 15°C.

[0065] The second group of examples, application of strain LJ1 of the present invention

[0066] This group of embodiments provides the application of a denitrifying Zobellella denitrificans strain LJ1 with a deposit number of CGMCC NO: 24344 and a low-temperature resistant denitrification function in complete denitrification.

[0067] In a preferred embodiment, the complete denitrification comprises: the membrane-bound nitrate reductase and the periplasmic nitrate reductase napA of strain LJ1 reduce nitrate to nitrite; the Cu-type nitrite reductase and the cytochrome cd1-type nitrite reductase of strain LJ1 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ1 reduces nitric oxide to nitrous oxide; the nitrous oxide reductase of strain LJ1 reduces nitrous oxide to nitrogen gas;

[0068] In a further embodiment, the complete denitrification refers to: the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ1 sequentially perform the following reactions:

[0069] Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite;

[0070] Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide;

[0071] Nitric oxide reductase reduces nitric oxide to nitrous oxide;

[0072] Nitrous oxide reductase reduces nitrous oxide to nitrogen gas.

[0073] In a specific embodiment, the complete denitrification refers to the sequential removal of nitrate, nitrite, nitric oxide, and nitrous oxide in the sample, and the final conversion into nitrogen gas.

[0074] In a preferred embodiment, the temperature of complete denitrification is 10-25°C.

[0075] The third group of embodiments, the bacterial agent of the present invention

[0076] This group of embodiments provides a bacterial agent. All embodiments of this group have the following common features: the bacterial agent comprises: a strain of Zobellella denitrificans LJ1 with a deposit number of CGMCC NO:24344.

[0077] In a further embodiment, the bacterial agent further includes: auxiliary materials.

[0078] In a more specific embodiment, the pharmaceutical excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0079] The dosage form of the bacterial agent of the present invention is not limited. According to the teachings and inspiration of the present invention, those skilled in the art can select suitable excipients for preparation based on actual production needs, combined with commonly used technical means in the field of microbial technology (for example, "Encyclopedia of Preparation Technology", "Pharmaceutical Preparation Technology", etc.), and prepare the denitrifying Zobellella denitrificans strain LJ1 with a deposit number of CGMCC NO: 24344 of the present invention into various other dosage forms that meet various process production requirements, such as powders, tablets, liquids, sprays, granules, capsules, etc.

[0080] The fourth group of embodiments, the complete denitrification method of the present invention

[0081] This group of embodiments provides a complete denitrification method. All embodiments in this group have the following common features: a strain of Zobellella denitrificans LJ1 with a preservation number of CGMCC NO: 24344 and having a low-temperature denitrification function is used to perform denitrification treatment on the sample to be treated.

[0082] In some embodiments, the complete denitrification comprises: the membrane-bound nitrate reductase and the periplasmic nitrate reductase napA of strain LJ1 reduce nitrate to nitrite; the Cu-type nitrite reductase and the cytochrome cd1-type nitrite reductase of strain LJ1 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ1 reduces nitric oxide to nitrous oxide; the nitrous oxide reductase of strain LJ1 reduces nitrous oxide to nitrogen gas;

[0083] Preferably, the complete denitrification refers to: Fig.11 As shown, the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ1 sequentially carry out the following reactions:

[0084] Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite;

[0085] Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide;

[0086] Nitric oxide reductase reduces nitric oxide to nitrous oxide;

[0087] Nitrous oxide reductase reduces nitrous oxide to nitrogen gas.

[0088] In some embodiments, the denitrification treatment time is 1-5 days, and the temperature is 10-25°C.

[0089] In other embodiments, the inoculation amount of the strain LJ1 in the sample to be treated is 10%;

[0090] Preferably, the sample to be processed is a nitrogen-containing sample;

[0091] Preferably, the nitrogen-containing refers to NO3 - .

[0092] Experimental example, acquisition and performance verification of the LJ1 strain of the present invention

[0093] 1. Screening of strains

[0094] Strain screening Rhizosphere soil of reed and cattail was collected from the treatment wetland that has been in operation for 13 years in Beijing Wildlife Rescue Center. The screening process is as follows: 10g rhizosphere soil and 20ml distilled water were mixed and inoculated into a 250ml conical flask containing denitrification liquid medium. The conical flask mouth was sealed with a sealing film to create an anaerobic environment, and placed in a 25℃ incubator for 7 days to enrich the culture medium. Among them, the denitrification liquid medium contains 1g K2HPO4, 0.8g NaNO3, 1gNaCO3, 0.03g CaCl2, 0.06g FeSO4·7H2O, 0.2g MgSO4·7H2O and 1000ml distilled water, and sterilized in an autoclave at 121℃ for 30min.

[0095] The enriched culture medium was diluted 10 -1 , 10 -2 , 10 -3 and 10 -4Finally, use a triangular coating rod to apply the diluted culture solution on the denitrification solid plate culture medium, and then pour the second layer of denitrification culture medium that has been cooled to below 40°C on the upper layer to form an anaerobic environment for culturing denitrifying microorganisms. After the second layer of culture medium solidifies, turn the plate upside down and place it in a 25°C incubator for cultivation. Observe the growth of microorganisms every day until obvious colonies are observed with the naked eye. Use an inoculation needle to select the strains grown in the culture medium, inoculate them on a new denitrification plate culture medium, streak them, and place them in a 15°C incubator for cultivation. Repeat this step until a single colony is formed.

[0096] Among them, the denitrification solid plate culture medium is made into two parts, one part is composed of 1g K2HPO4, 0.8g NaNO3, 1g NaCO3, 0.03g CaCl2, 0.06g FeSO4·7H2O, 0.2g MgSO4·7H2O and 500ml distilled water, and the other part is 20g agar dissolved in 500ml distilled water. Sterilize at 121℃ for 30min in an autoclave, and then mix the two parts in a clean bench.

[0097] 2. Verification of low temperature resistant denitrifying bacteria.

[0098] In order to further verify that the above-selected microorganisms have denitrification function, the sewage and sludge collected from the water inlet of the treatment wetland were first mixed in a ratio of 10:1 and then divided into different 250 ml conical bottles, and the contents of total nitrogen, nitrate nitrogen and nitrite nitrogen were determined. The single bacteria cultured in the previous step were picked and placed in different conical bottles, and the conical bottle mouths were sealed with sealing film. They were placed in a 15°C incubator and cultured for 5 days. The contents of nitrate nitrogen and nitrite nitrogen were then determined. When the removal rate was above 90%, it indicated that the strain was a denitrifying strain with high-efficiency denitrification ability in a low-temperature environment.

[0099] 3. Biological identification of denitrifying strain LJ1

[0100] The genomic DNA of strain LJ1 was extracted and used as a template to amplify the 16S rDNA of the strain using a pair of universal primers (27F / 1492R). The upstream primer was 27F (5'-AGAGTTTGATCCTGGCCA-3') (SEQ ID NO.1) and the downstream primer was 1492R (5'-GGTTACCTTGTTACGACTT-3') (SEQ ID NO.2).

[0101] The qPCR reaction system was (25 μL): 1 μL DNA, 12.5 μL RR340A Premix Ex Taq DNA polymerase, 9.5 μL ddH2O, and 1 μL each of upstream and downstream primers.

[0102] The PCR program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 30 cycles in total, extension at 72°C for 10 min, and finally storage at 4°C.

[0103] The PCR product was purified and sequenced by Paisono Biotechnology Co., Ltd. to obtain the 16S rDNA sequence of the strain. The 16S rDNA sequence obtained by sequencing was submitted to NCBI, and the homology sequence was compared with GenBank through software. The length of the 16S rDNA sequence of strain LJ1 was 1041bp, and the homology of the sequence with Zobellella denitrificans in the NCBI database (NCBI accession number: CP012621.1) reached 99.65%. Based on other biological characteristics of the strain, it was finally named Zobellella denitrificans LJ1 and sent for preservation. Its preservation information is as follows:

[0104] Deposit number: CGMCC NO:24344;

[0105] Taxonomic nomenclature: Zobellella denitrificans;

[0106] Deposit date: January 19, 2022;

[0107] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0108] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0109] 4. Denitrification functional gene test

[0110] The gene-specific amplification primers in Table 1 were used to obtain the ct values ​​of the denitrification functional genes narG, napA, nirK, nirS, qnorB, and nosZ in the strain by real-time fluorescence quantitative PCR, which were 21.04, 34.61, 24.08, 24.31, 26.56, and 24.39, respectively (see Table 1). Figure 5-Figure 10 ). This indicates that the strain has the membrane-bound nitrate reductase encoding gene narG involved in the denitrification process; the periplasmic nitrate reductase encoding gene napA; the nitrite reductase encoding genes nirK and nirS; the nitric oxide reductase encoding gene qnorB and the nitrous oxide reductase encoding gene nosZ, which can convert nitrate nitrogen into nitrite nitrogen, nitric oxide, nitrous oxide and nitrogen gas in sequence. Therefore, Zobellella denitrificans LJ1 is a denitrifying bacterium with complete denitrification.

[0111] Table 1

[0112]

[0113]

[0114] 5. Analysis of gas components produced by denitrification of strains

[0115] NO3 - -N as the nitrogen source was placed in a 100 mL serum bottle, sealed with a rubber stopper and an aluminum cap, and the air in the serum bottle was replaced with helium using an aeration cleaning system. 7 CFU / mL) were inoculated into serum bottles, and the contents of nitric oxide, nitrous oxide, and nitrogen in the system were measured every 4 hours at 25°C, and the removal rates of nitrate ions and nitrite ions were measured every 1 day. The results were as follows: Figure 1 and Figure 2 shown.

[0116] 6. Growth curve of denitrifying strain LJ1 under low temperature conditions

[0117] Add 100 ml of liquid culture medium to a 150 ml conical flask and inoculate 10 ml of bacterial solution (concentration 1×

[0118] 10 7 CFU / mL), set 10 replicates, cultured at 10℃ and 15℃ respectively, took out one replicate every 12h, measured using a spectrophotometer at a wavelength of 600nm, and drew a growth curve using the measured OD value. The results are shown in Figure 3 shown.

[0119] VII. Denitrification effect of denitrifying strain LJ1 on water bodies

[0120] The water was taken from the inlet of the artificial wetland of Beijing Wildlife Rescue Center, with a TN of 8.21 mg / L and a pH of 7.9. 3 In the reactor, the experimental group was inoculated with liquid bacterial agent of Zobellella denitrificans LJ1 (bacterial concentration was 1×10 7 CFU / mL), and the control group was inoculated with the same volume of liquid culture medium. The control conditions were 25℃, 15℃, and 10℃, DO value 0-2.1mg / L, and natural pH value. Samples were taken every 24h during the reaction to determine the TN content of the water. The results are as follows Figure 4As shown, after 5 days, at 25°C, the removal rate of TN by strain LJ1 reached 95.41%, at 15°C, the removal rate of TN by strain LJ1 reached 84.40%, and at 10°C, the removal rate of TN by strain LJ1 was 62.68%.

Claims

1. The denitrifying Zabela bacteria with the deposit number of CGMCC NO: 24344 ( Zobellella denitrificans ) Application of strain LJ1 in complete denitrification at 10℃.

2. A complete denitrification method, characterized in that: A strain of denitrifying bacteria with low temperature resistance and denitrification function (CGMCC NO: 24344) was used. Zobellella denitrificans ) strain LJ1 performs complete denitrification treatment on the sample to be treated; the complete denitrification treatment time is 1-5 days, the temperature is 10°C; the inoculation amount of the strain LJ1 in the sample to be treated is 10%; the sample to be treated contains NO3 - of samples.

Images