CN120699835B

CN120699835B - Listeria pulmonarius Y3 and application thereof

Info

Publication number: CN120699835B
Application number: CN202510930677.3A
Authority: CN
Inventors: 魏雨泉; 方雯瑄; 罗妍; 常素; 蒋正波; 李季; 许艇
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2025-07-07
Filing date: 2025-07-07
Publication date: 2025-12-05
Anticipated expiration: 2045-07-07
Also published as: CN120699835A

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Priestia* Y3 and its applications. *Priestia* Y3, classified as * Priestia sp.*, was deposited on March 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 34006. The *Priestia* Y3 provided by this invention has ammonia removal function, providing an effective biological deodorizing strain resource for efficient ammonia removal and deodorization in waste treatment processes.

Description

Listeria pulmonarius Y3 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a listeria monocytogenes Y3 and application thereof.

Background

Ammonia (NH ₃) is the main alkaline gas in the atmosphere and the major form of active nitrogen (Nr), producing approximately 2 million tons of NH ₃ per year worldwide, with agriculture (especially animal husbandry) being the largest source of NH ₃. Worldwide, agricultural NH ₃ emissions account for 90% of total emissions, with animal husbandry production accounting for about 60%. Meanwhile, industries such as combustion, vehicle transportation, chemical production, and waste management in urban areas also emit large amounts of NH ₃ into the atmosphere. As industry and agriculture expand and develop, the concentration of NH ₃ in the atmosphere increases, resulting in pollution of air, water and soil and loss of biodiversity, there is also a concern about NH ₃ emissions and treatment methods.

At present, the biological microbial agent technology is widely applied to ammonia removal, malodorous gas (NH ₃) is removed mainly through microbial metabolism, secondary pollution is not generated, the operation is simple, the investment and operation cost is low, a series of metabolic processes such as oxidation, nitrification and denitrification are carried out sequentially mainly depending on microorganisms, NH ₃ is used as nutrients, and finally stable-state compounds such as CO ₂、H₂ O and nitrate are generated, so that the preparation of the deodorant microbial agent by using the biological deodorization technology is a feasible method for removing NH ₃.

At present, microorganisms for removing nitrogen include photosynthetic bacteria, anaerobic ammonia oxidizing bacteria, aerobic denitrifying bacteria and the like, and the microorganisms have the defect of low ammonia nitrogen removal efficiency. Therefore, there is a lack of a microbial strain in the prior art that is capable of maintaining efficient and stable ammonia nitrogen removal under a variety of environmental conditions, particularly in the face of complex waste components.

Disclosure of Invention

In order to solve the defect of low ammonia nitrogen removal efficiency of microorganisms for removing nitrogen in the prior art, the invention develops a microorganism strain capable of still maintaining high-efficiency and stable ammonia nitrogen removal capability under various environmental conditions, particularly when facing complex waste components, and provides a listeria monocytogenes Y3 and application thereof. In order to achieve the above purpose, the present invention adopts the following technical scheme.

The invention provides a strain of Listeria Y3, wherein the Listeria Y3 is preserved in China general microbiological culture Collection center (CGMCC) No.34006 in the year 2025, month 3 and day 28, and the preservation unit address is the No. 1 West Song No.3 of the Korean region North Star of Beijing city.

The invention utilizes heterotrophic nitrification culture medium to screen and obtain a strain of bacteria with high ammonia nitrogen removal efficiency, and adds whole genome sequence analysis to more accurately determine strains based on the traditional strain identification technology, further optimizes the culture medium to accelerate the splitting and proliferation of thalli and activate the ammonia oxidase activity, improves the ammonia nitrogen removal effect, and provides technical support for the high ammonia nitrogen removal and deodorization in the waste treatment process. The Listeria monocytogenes Y3 provided by the invention is a microbial strain which can still maintain high-efficiency and stable ammonia nitrogen removal capability under various environmental conditions, especially in the face of complex waste components, has higher ammonia nitrogen removal rate, can further reduce the emission of ammonia in the waste treatment process, and solves the defect of low ammonia nitrogen removal efficiency of microorganisms for removing nitrogen in the prior art.

Preferably, the nucleotide sequence of 16S rRNA of the Listeria Y3 is shown as SEQ ID NO. 1.

The invention also provides a microbial agent comprising the listeria monocytogenes Y3.

The invention also provides application of the Listeria Y3 or the microbial agent in removing ammonia nitrogen in waste, wherein the waste comprises at least one of ammonia nitrogen-containing wastewater, landfill leachate and livestock and poultry manure.

Preferably, the listeria Y3 or the microbial agent is added to the wastewater containing ammonia nitrogen for reaction.

Preferably, the listeria Y3 or the microbial agent is used for metabolizing ammonia nitrogen in the ammonia nitrogen-containing wastewater by nitrification.

Preferably, the volume ratio of the listeria Y3 or the microbial agent is 1% -5% added to the ammonia nitrogen-containing wastewater.

Preferably, the OD ₆₀₀ value of the listeria monocytogenes Y3 or the microbial agent is 1.6-1.8.

Preferably, the reaction is carried out under the conditions of pH 7.8-8.2, temperature 34-36 ℃ and C/N of 12, wherein C/N refers to carbon-nitrogen ratio.

Preferably, the concentration of ammonia nitrogen in the ammonia nitrogen-containing wastewater is 200 mg/L-500 mg/L.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention provides a strain of Listeria monocytogenes Y3. The Listeria monocytogenes Y3 provided by the invention is a microbial strain which can still maintain high-efficiency and stable ammonia nitrogen removal capability under various environmental conditions, especially in the face of complex waste components, has higher ammonia nitrogen removal rate, can further reduce the emission of ammonia in the waste treatment process, and solves the defect of low ammonia nitrogen removal efficiency of microorganisms for removing nitrogen in the prior art.

2. Aiming at the health and environmental problems of residents brought by NH ₃, the invention aims to adopt a microbial deodorization technology to remove ammonia and provide strain resources. The bacterial strain with the ammonia removal effect is selected from the compost samples, the bacterial strain Y3 with the optimal ammonia removal capability is selected as an object, the bacterial strain is subjected to multiphase identification through 16S rRNA gene system development analysis, whole genome sequence analysis and morphological and physiological and biochemical characteristics, the classification status of the bacterial strain is determined, the optimal growth condition of the bacterial strain is determined through optimizing the culture conditions of environmental factors, pH, temperature, carbon source and C/N ratio of the bacterial strain, the ammonia nitrogen removal efficiency of the bacterial strain is improved, and effective biological deodorizing bacterial strain resources are provided for efficient ammonia removal and deodorization in the waste treatment process.

3. The invention screens out bacterial strain with high-efficiency ammonia removal effect from compost samples, takes the bacterial strain Listeria Y3 with optimal ammonia removal capability as an object, carries out multiphase identification on the bacterial strain through 16S rRNA gene system development analysis, whole genome sequence analysis, morphological characteristics and physiological and biochemical characteristics, and optimizes the culture condition of the bacterial strain to improve the ammonia nitrogen removal rate after definitely refining the classification status of the bacterial strain, thereby providing bacterial resource and potential functional products for high-efficiency biological ammonia removal and deodorization.

Drawings

FIG. 1 shows ammonia nitrogen removal rate of strain Y3 in the present invention.

FIG. 2 shows the growth curve of strain Y3 in the present invention.

FIG. 3 shows the colony and cell morphology of strain Y3 in the present invention, wherein FIG. 3A shows the colony morphology of strain Y3, and FIG. 3B shows the cell morphology of strain Y3.

FIG. 4 is a 16S rRNA gene evolutionary tree of strain Y3 of the present invention.

FIG. 5 is a genomic evolution tree of strain Y3 according to the present invention.

FIG. 6 shows ANI values for strain Y3 and a reference strain according to the invention, in which ：1：Y3;2：Priestia aryabhattaiJCM 13839;3：Priestia megateriumATCC 14581T;4：Metabacillus herbersteinensisCCM 7228;5：Bacillus basilensis403507-21;6：Robertmurraya yapensisXXST-01;7：Viridibacillus soliYIM B01967;8：Bacillus suaedaesalsaeRD4P76;9：Pseudoneobacillus rhizosphaeraeCIP 111885T;10：Bacillus pretiosusSAICEU11T;11：Bacillus proteolyticusMCCC 1A00365;12：Bacillus songklensisCCUG 61889;13：Bacillus nitratireducens4049;14：Metabacillus iocasaeDSM 104297T;15：Priestia flexaNBRC 15715T;16：Priestia veravalensisSGD-V-76T.

FIG. 7 shows the effect of pH on the growth of strain Y3 in the present invention.

FIG. 8 shows the effect of temperature on the growth of strain Y3 in the present invention.

FIG. 9 shows the effect of different carbon sources on the growth of strain Y3 according to the present invention.

FIG. 10 shows the effect of different carbon to nitrogen ratios on the growth of strain Y3 according to the present invention.

FIG. 11 shows ammonia nitrogen removal rate of strain Y3 in the present invention.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

Example 1 screening and identification of Strain Y3

1. Screening of Strain Y3

5G of compost samples (5 glass beads with the diameter of 3mm are put into the culture medium) are respectively added into 100mL of enrichment culture medium, the culture medium is placed on a shaking table with the temperature of 30 ℃ and the temperature of 180 r.min ^-1 for continuous constant-temperature culture until turbidity, 5mL of culture solution is added into fresh 100mL of enrichment culture medium, and the operation is the same as above, and the culture medium is continuously transferred four times, so that enriched bacterial liquid is obtained.

Wherein, the compost source is the North-great farm in Tangshan city.

The enrichment medium is prepared from sucrose 5.0g, ammonia water (concentration is 26%, filtering, sterilizing, adding after sterilizing) 5.0mL, sodium chloride 2.0g, potassium dihydrogen phosphate 2.0g, zinc sulfate 0.05g, peptone 2.0g, magnesium sulfate 0.5g, ferrous sulfate 0.04g and distilled water 1000mL.

Mixing 100 mu L of enriched bacterial liquid with sterile water according to the volume ratio of 1:9, mixing 100 mu L of mixed solution with sterile water according to the volume ratio of 1:9, repeating the steps, diluting the bacterial liquid to 10 ^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7 and 10 ^-8 times, respectively inoculating 0.1mL of bacterial liquid with dilution factors of 10 ^-5、10^-6、10^-7 and 10 ^-8 onto a double-sieve culture medium plate, repeating each dilution gradient for three times, and culturing in a 30 ℃ constant-temperature incubator in an inverted mode. And (3) picking single bacterial colony by using the sterilized toothpick, repeatedly streaking, separating and purifying until the bacteria are observed under a microscope, screening to obtain 10 strains, preserving the obtained pure strains by using glycerol with the volume percentage of 40%, and placing the pure strains in an ultralow temperature refrigerator at-80 ℃ for later testing.

Wherein 10 strains of bacteria are selected and obtained, and the strains are respectively named amycolatopsis (Amycolatopssp.), paramycolatopsis (Paraburkholderia sp.), pseudomonas (Pseudomonas sp.), bacillus subtilis (Bacillus sp.), lactobacillus (Lactiplantiella sp.), and Brevibacterium (Praestinopsis sp.) and Brevibacterium (Brevibacterium sp.).

10 Strains stored in glycerol are inoculated into 100mL of LB liquid medium for activation, the 10 strains are inoculated into 100mL of heterotrophic nitrification medium according to the inoculum size of 5% (v/v), the heterotrophic nitrification medium is cultivated at the constant temperature of 180 r.min ^-1 and 30 ℃, 5mL of bacterial liquid is respectively taken in an ultra-clean workbench during cultivation for 24h and 48h, the bacterial liquid is centrifuged for 2min in 12000 r.min ^-1, the content of ammonia nitrogen in the solution is measured according to the steps in the Nahner reagent spectrophotometry for measuring the environmental air and the ammonia of waste gas of HJ 533-2009, the content of the residual ammonia nitrogen in the solution is calculated according to a formula (11), then the ammonia nitrogen removal rate of the strains is calculated according to a formula (2), so that the ammonia oxidation capacity of each strain is compared, and the strain with better ammonia removal effect is selected from the bacterial liquid for subsequent experiments.

(1);

In the formula, A _{Bacteria (fungus)} is absorbance of the added bacterial liquid, A _{White color} is absorbance of a blank control group, a is intercept of a calibration curve, b is slope of the calibration curve, and V is sample volume in mL;

(2);

In the formula, the ammonia nitrogen content _CK is the ammonia nitrogen content in the heterotrophic nitrification medium with the same volume of non-inoculated bacteria, the unit is mg.L ^-1, and the ammonia nitrogen content _{Bacteria (fungus)} is the ammonia nitrogen content in the heterotrophic nitrification medium with the same volume of inoculated bacteria, the unit is mg.L ^-1.

Wherein, the heterotrophic nitrification culture medium is a liquid culture medium and is prepared from ：(NH₄)₂SO₄1.42 g·L^-1、MgSO₄0.06 g·L^-1、Na₃C₆H₅O₇12.25 g·L^-1、K₂HPO₄0.25 g·L^-1、NaCl 0.125 g·L^-1、Fe₂(SO₄)₃0.0025 g·L^-1 and MnSO ₄0.001 g·L^-1 with the following raw materials in final concentration.

The screened strain is further inoculated into 100mL of LB liquid medium, and the strain is subjected to shaking culture for 20h under the conditions of 30 ℃ and 180 r.min ^-1 until the liquid is turbid. Then 3mL of activated bacterial liquid is sucked into 100mL of heterotrophic nitrification culture medium, the culture medium is continuously cultured for 24h under the conditions of 180 r.min ^-1 and 30 ℃, each group is sampled every 2h, OD ₆₀₀ value is measured, 5mL of sterile liquid culture medium is sucked into a cuvette on an ultra-clean workbench, 600nm is selected, a BLANK button is pressed, the culture medium is discarded, 5mL of bacteria liquid to be tested is sucked, and the value is measured. Finally, the culture time of the strain is taken as an abscissa, the measured OD ₆₀₀ value is taken as an ordinate, and a growth curve is drawn. And screening out the strain with high growth rate and strong ammoxidation capacity by combining the growth rate of the strain and the ammonia nitrogen removal rate of the strain, and carrying out subsequent experiments.

The results are shown in FIGS. 1 and 2, wherein the ammonia removal efficiency of the strain Y3 is highest, the ammonia nitrogen removal rate is greater than 40% at 24h and 48h, and the strain growth rate is combined, so that the strain Y3 is selected for subsequent experiments.

2. Identification of Strain Y3

The strain Y3 is identified and described by adopting a multiphase identification mode such as molecular biology identification, phenotypic characteristic observation and the like, and the strain is proved to belong to a potential new species in the genus Listeria. The method comprises the following steps:

(1) Colony morphology characterization

Gram staining of the strain Y3 is negative, culturing is carried out on the strain Y3 in an LB culture medium at 30 ℃ for 24h ℃, the colony diameter of the strain Y3 is 4 mm-5 mm, the colony is yellow, round, semitransparent, wet in surface, glossy and neat in edge, the thallus is in a rod shape, the width of the thallus is 12-15 mu m, and the length of the thallus is 20-25 mu m (shown in figure 3).

(2) 16S rRNA sequence analysis:

Genomic DNA was extracted from the strain using a bacterial DNA extraction kit, and 16S rRNA gene was amplified using the genomic DNA of strain Y3 as a template and the universal primers 27F and 1492R of the 16S rRNA gene according to the PCR reaction systems and reaction procedures shown in tables 1 and 2. After splicing, similarity comparison with bacteria in the database was performed using NCBI online BLAST program, and phylogenetic tree of 16S rRNA gene was drawn using MEGA 11 software to determine phylogenetic relationship of strain Y3 and similar strain.

Wherein the bacterial DNA extraction kit is purchased from Beijing Bomaide Gene technology company.

The nucleotide sequence of the universal primer 27F is shown as SEQ ID NO.2, namely 5 '-AGAGTTTGATCCTGGGCTCAG-3'.

The nucleotide sequence of 1492R is shown as SEQ ID NO.3, namely 5 '-GGTTACCTTGTTACGACTT-3'.

TABLE 1 reaction system (50. Mu.L)

Table 2 reaction procedure

Note that "-" indicates that this step is not repeated.

As a result, strain Y3 was phylogenetically related to Listeria arvensis (P. aryabhattaiJCM 13839) as shown in FIG. 4. Thus, strain Y3 was identified as listeria monocytogenes Y3.

(3) Whole genome sequence analysis

The strain Y3 is sent to Beijing Aoweisen Gene technology Co., ltd, and the whole genome sequencing analysis is carried out on the established library by utilizing the second generation whole genome and third generation whole genome sequencing technology and combining a IIumina Hiseq sequencing platform and a PacBIO RS II real-time sequencing platform, and the on-line data are spliced and corrected by software. The Genome evolution tree can then be constructed using a typical Strain Genome Server TYGS (Type) Genome Server. To obtain more accurate classification status of the strains, the whole genome sequence of the strains was uploaded to an online platform JSpecies Web Server for comparison with all known strains in database Genbank, and the average nucleotide similarity between the two strains was calculated (Average Nucleotide Identity, ANI). DNA molecular hybridization values (DNA-DNA hybridization, dDDH) were obtained with known strains in the database using an online platform TYGS alignment, and a whole genome sequence phylogenetic tree was constructed.

The results are shown in FIG. 5. As shown in FIG. 6 and Table 3, the whole genome size of the strain Y3 was 9 Mb, the sequencing read length N50 of the genome completion map was 5202769bp, the mol% content of the genome G+C was 38.08%, and 5879 encoding genes were predicted. Phylogenetic tree divides the strain and Priestia aryabhattaiJCM 13839 into one branch, but the values of dDDH and ANIb between the strain Y3 and similar standard strains are 20.0% -67.9%, 66.8% -95.6%, and the values of dDDH and ANIb between the strain Y3 and the standard strain P. aryabhattaiJCM 13839 with the closest relativity are 67.9% and 95.6%, respectively, which are lower than the threshold value of new species division, thus proving that the strain Y3 is a new species in the genus of Listeria.

Wherein, the nucleotide sequence of the 16S rDNA of the Listeria Y3 is shown as SEQ ID NO. 1:

AAACGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAAAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCGCTTTTCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACGAGCAGTTACTCTTGTACTTGTTCTTCCCTAACAACAGAGTTTTACGACCCGAAAGCCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCACCGCGGGCCCATCTGTAAGTGATAGCCGAAACCATCTTTCAATCATCTCCCATGAAGGAGAAGATCCTATCCGGTATTAGCTTCGGTTTCCCGAAGTTATCCCAGTCTTACAGGCAGGTTGCCCACGTGTTACTCACCCGTCCGCCGCTAACGTCATAGAAGCAAGCTTCTAATCAGTTCGCTCGACTTGATGTATTAGGACGCGCACGATGTTATTAAAAA.

At present, the Listeria Y3 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation number of 34006 and a preservation unit address of North Star Xway No. 1,3 of the Korean area of Beijing city.

TABLE 3 dDDH values for Strain Y3 and reference Strain

(4) Physiological and biochemical characteristics

As a result of measuring the enzyme activity by API 20E, it was found from Table 4 that the strain Y3 was able to utilize o-nitrobenzene-galactoside, arginine, pyruvate, kohn gelatin, and further found that the strain Y3 was not able to utilize the same general carbohydrates as inositol, sorbitol, rhamnose, sucrose, melibiose, and plant-derived glycosides as amygdalin, arabinose, etc., as well as the inorganic salts as sodium thiosulfate, etc., and the nitrogen-containing heterocyclic compounds as urea, tryptophan, etc., as a result of measuring sugar alcohol substrates, and that the strain Y3 was not able to utilize the same general carbohydrates as lysine, ornithine, etc.

TABLE 4 physiological and biochemical index of strain Y3

Note that "+" indicates a positive reaction and "-" indicates a negative reaction.

EXAMPLE 2 culture conditions for Strain Y3

The invention provides a culture method suitable for the growth of a strain Y3 and high ammonia nitrogen removal rate. The optimization conditions comprise pH, temperature, carbon source and carbon nitrogen ratio, and the growth rate of the strain Y3 is used as a measurement index to determine the optimal culture medium.

(1) PH optimization

Preparing an LB liquid medium and a heterotrophic nitrification medium, sterilizing under high pressure, inoculating a strain Y3 into 100mL of the LB liquid medium for activation by an inoculating loop, performing shake culture at 30 ℃ and 180 r.min ^-1 h, inoculating into 100mL of the heterotrophic nitrification medium for culturing, setting the culture pH to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively, adjusting the pH of the heterotrophic nitrification medium by 0.1 mol.L ^-1 of dilute hydrochloric acid and 0.1 mol.L ^-1 of sodium hydroxide solution after the heterotrophic nitrification medium is sterilized, setting 3 repeats for each pH, performing shake culture at 30 ℃,180 r.min ^-1 for 24h, and measuring the optical density of the bacterial liquid by using the blank medium as calibration at the wavelength of 600nm every 2h by a spectrophotometer.

As shown in FIG. 7, strain Y3 grew in a pH range of 6.0 to 10.0, the optimum pH was 9.0, and the concentration of the obtained bacterial liquid reached the peak value and the OD value was 0.995 at the highest, by shaking culture for 8 hours.

(2) Temperature optimization

Preparing an LB liquid culture medium and a heterotrophic nitrification culture medium, sterilizing under high pressure, inoculating a strain Y3 into 100mL of the LB liquid culture medium by an inoculating loop, activating, shake culturing for 24 hours at 30 ℃,180 r min ^-1, inoculating into 100mL of the heterotrophic nitrification culture medium, culturing at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃ respectively, setting 3 repetitions of each temperature, shake culturing for 24 hours at 30 ℃,180 r min ^-1, and measuring the optical density of the bacterial liquid at the wavelength of 600nm by using a spectrophotometer at intervals of 2 hours by using a blank culture medium as a calibration.

As a result, as shown in FIG. 8, the strain Y3 was grown at a temperature ranging from 25℃to 40℃and at an optimum temperature of 40℃and the bacterial liquid concentration reached a peak value of 1.246 after cultivation of 6 h.

(3) Carbon source optimization

Preparing an LB liquid culture medium and a heterotrophic nitrification culture medium, sterilizing under high pressure, inoculating a strain Y3 into 100mL of the LB liquid culture medium for activation by an inoculating loop, performing shake culture for 24 hours at 30 ℃,180 r min ^-1, sucking 5% (v/v) of the bacterial liquid, inoculating the bacterial liquid into culture solutions respectively taking glucose, mannitol, sodium acetate, sodium succinate, sodium pyruvate and trisodium citrate as carbon sources, setting 3 repetitions of each culture solution, and measuring the optical density of the bacterial liquid by using a spectrophotometer at a wavelength of 600nm by taking a blank culture medium as a calibration at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h and 48h respectively.

As a result, as shown in FIG. 9, trisodium citrate was used as a carbon source, which was most advantageous for the growth of strain Y3.

(4) Carbon-nitrogen ratio optimization

Preparing an LB liquid culture medium and a heterotrophic nitrification culture medium, sterilizing under high pressure, inoculating a strain Y3 into 100mL of the LB liquid culture medium by an inoculating loop, activating, performing shake culture for 24h at 30 ℃ under 180 r min ^-1, fixing the nitrogen content in the culture medium after improvement to be 100 mg L ^-1, adding different amounts of optimal carbon sources into the culture medium with the C/N ratio of 4, 8, 12, 16, 20 and 24, inoculating 5% (v/v) bacterial liquid under aseptic conditions, and measuring the optical density of the bacterial liquid by using a spectrophotometer at the wavelength of 600nm by using a blank culture medium as a calibration at the time of 0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h and 48 h.

As shown in FIG. 10, the proper C/N ratio was favorable for the growth of the strain, the strain Y3 had an optimal C/N ratio of 8 and 12, too low a C/N ratio was unfavorable for the growth of the strain due to the shortage of the carbon source, and too high a C/N ratio was also suppressed to some extent due to the imbalance of the C/N ratio

Example 3 Ammonia nitrogen removal Rate for Strain Y3

And culturing the strain Y3 under the optimal culture conditions, inoculating 5% of bacterial liquid under the aseptic condition, setting 3 repetitions of each culture liquid, and measuring absorbance at the wavelength of 420nm at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h and 48h respectively to calculate the ammonia nitrogen removal rate.

Wherein, the optimal culture condition is that the pH is 8.0, the temperature is 40 ℃, trisodium citrate is used as a carbon source, and the carbon-nitrogen ratio is 12.

As a result, as shown in FIG. 11, the optimal ammonia nitrogen removal rate of the strain Y3 was 41.63% and 42.70% respectively after 48 hours of cultivation under the conditions that the C/N ratio was 8 and 12.

From the above results, it was found that among 10 strains selected from the soil and compost environment, strain Y3 was the strain having the highest ammoxidation rate and the highest growth rate. The genetic relationship with the strain P. aryabhattaiJCM 13839 is found to be nearest after the multiphase taxonomy identification, and the strain is a potential new strain. Strain Y3 had a higher removal rate at an optimized medium with pH 9.0, a temperature of 40℃and trisodium citrate as the carbon source and a carbon-nitrogen ratio of 8 or 12.

In conclusion, the invention screens out a strain with strong ammoxidation capability from the composting environment, and the ammonia nitrogen removal rate can reach 42.70% after the optimization of the culture medium.

It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and in order to prevent redundancy, the present invention describes a preferred embodiment.

While the preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts, all such variations and modifications are within the scope of the invention.

Claims

1. The strain of the listeria monocytogenes Y3 is characterized in that the listeria monocytogenes (Priestia sp.) Y3 is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 34006 in the 3 rd month of 2025.

2. The strain of listeria Y3 of claim 1, wherein the nucleotide sequence of 16S rRNA of listeria Y3 is set forth in SEQ ID No. 1.

3. A microbial agent comprising the listeria Y3 of claim 1.

4. Use of the listeria Y3 of claim 1 or the microbial agent of claim 3 for removing ammonia nitrogen from waste, wherein the waste comprises at least one of ammonia nitrogen-containing wastewater, landfill leachate and livestock manure.

5. The use according to claim 4, characterized in that the listeria Y3 or the microbial agent is added to the ammonia nitrogen-containing wastewater for the reaction.

6. The use according to claim 5, characterized in that said listeria Y3 or said microbial agent is used for metabolizing ammonia nitrogen in said ammonia nitrogen containing wastewater by nitrification.

7. The use according to claim 5, characterized in that the volume ratio of the listeria Y3 or the microbial agent is 1% -5% added to the ammonia nitrogen containing wastewater.

8. The use according to claim 7, wherein said listeria Y3 or said microbial agent has an OD ₆₀₀ value of 1.6 to 1.8.

9. The method according to claim 5, wherein the reaction is carried out at a pH of 7.8-8.2 and a temperature of 34-36 ℃ and a C/N of 12, wherein C/N means carbon nitrogen ratio.

10. The use according to claim 5, wherein the ammonia nitrogen concentration in the ammonia nitrogen containing wastewater is 200mg/L to 500mg/L.