Nitrite-resistant pseudomonas and use thereof

Abstract

The application provides a nitrite-tolerant Pseudomonas and application thereof, and the preservation number of the nitrite-tolerant Pseudomonas (Pseudomonas nitrititolerans) JSN170 strain is CGMCC No. 26623. The strain provided by the application is easy to breed, and has a fast growth speed and can reach a stable growth period in about 15 hours. The strain can still grow and breed under the condition of high-concentration nitrite and sodium chloride. The strain contains important nitrogen cycle key genes for reducing nitrite to nitrogen, has a complete denitrification path, and can quickly degrade nitrite in water (sea water or fresh water) by reducing the nitrite to nitrogen under aerobic conditions.

Description

Technical Field

[0001] This invention belongs to the field of microbial screening and application technology, specifically relating to a nitrite-resistant Pseudomonas aeruginosa and its application. Background Technology

[0002] With the increasing demand for aquatic products, my country's aquaculture industry has developed rapidly, making significant contributions to increasing the supply of high-quality animal protein, improving nutritional health, and ensuring food security for the people. However, the high concentrations of ammonia nitrogen and nitrite in aquaculture water caused by residual feed and the decomposition and metabolism of feces during the aquaculture process seriously endanger the health of farmed animals. How to quickly, efficiently, and safely reduce and control ammonia nitrogen, especially nitrite levels, in aquaculture water has always been a hot topic of concern.

[0003] Biological denitrification is considered one of the effective ways to remove ammonia nitrogen and nitrite from water bodies due to its safety, pollution-free nature, high efficiency, and economy. Biological denitrification refers to the process by which organic nitrogen and ammonia nitrogen in wastewater are converted into nitrogen gas through ammonification, nitrification, and denitrification under the combined action of microorganisms. Denitrification is the final step in biological denitrification. Traditional biological denitrification theory considers denitrification to be a strictly anaerobic process, where denitrifying bacteria reduce nitrate nitrogen and nitrite nitrogen to gaseous nitrogen (N2) under anaerobic conditions. Recent studies have found that some denitrifying bacteria can convert nitrate and nitrite into nitrogen gas in aerobic environments. Currently screened aerobic denitrifying bacteria are generally isolated from terrestrial environments such as lakes, aquaculture ponds, or sewage treatment plant sludge. They have slow growth rates and struggle to grow and reproduce in environments with high concentrations of nitrite and sodium chloride, making it difficult to degrade nitrite in water bodies within a short period. Summary of the Invention

[0004] The purpose of this invention is to provide a nitrite-tolerant Pseudomonas bacterium and its application. This strain can grow and reproduce under high concentrations of nitrite and sodium chloride; it can grow using nitrate, nitrite, and ammonium ions as the sole nitrogen source, thereby utilizing and reducing the ammonia nitrogen content in water; this strain has a complete denitrification metabolic pathway, reducing nitrite to nitrogen gas under aerobic conditions; it is suitable for efficiently degrading nitrite in aquaculture water (seawater or freshwater).

[0005] This invention first provides a highly efficient nitrite-degrading nitrite-tolerant Pseudomonas nitrititolerans strain JSN170, with accession number CGMCC No. 26623, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on February 21, 2023.

[0006] The 16S rRNA gene sequence of the aforementioned Pseudomonas nitrititolerans strain JSN170 is SEQ ID NO:1;

[0007] The nitrogen cycle-related genes of the *Pseudomonas nitrititolerans* strain JSN170 have the following nucleotide sequences: nirK gene (SEQ ID NO:2); nirS gene (SEQ ID NO:3); norB gene (SEQ ID NO:4); norC gene (SEQ ID NO:5); nosZ gene (SEQ ID NO:6); napA gene (SEQ ID NO:7); napB gene (SEQ ID NO:8); narG gene (SEQ ID NO:9); narH gene (SEQ ID NO:10); narI gene (SEQ ID NO:11); nirB gene (SEQ ID NO:12); and nirD gene (SEQ ID NO:13).

[0008] The housekeeping genes of the aforementioned Pseudomonas nitrititolerans JSN170 strain have the following nucleotide sequences: rpoB gene (SEQ ID NO:14), rpoD gene (SEQ ID NO:15), and gyrB gene (SEQ ID NO:16).

[0009] The nitrite-resistant Pseudomonas jSN170 provided by this invention is used for the efficient degradation of nitrite in aquaculture water.

[0010] The present invention also provides an article for degrading nitrite, the article comprising a live strain of the aforementioned nitrite-resistant Pseudomonas nitrititolerans JSN170.

[0011] One of the aforementioned products is a compound bacterial solution or a bacterial immobilization product.

[0012] In another aspect, the present invention provides a method for degrading nitrite in water, which uses the aforementioned nitrite-resistant Pseudomonas nitrititolerans JSN170 strain for treatment.

[0013] The strain provided by this invention has the following advantages:

[0014] 1) This strain is easy to reproduce and grows quickly, reaching a stable growth period in about 15 hours.

[0015] 2) This strain can tolerate high concentrations of nitrite and sodium chloride, and can still grow and reproduce under conditions of 150 mM nitrite and 6% sodium chloride.

[0016] 3) This strain contains a key gene for the nitrogen cycle, which reduces nitrite to nitrogen gas. It has a complete denitrification pathway and can rapidly degrade nitrite in water by reducing nitrite to nitrogen gas under aerobic conditions. Attached Figure Description

[0017] Figure 1 Electron micrograph of strain JSN170 cells;

[0018] Figure 2 Phylogenetic tree diagram of strain JSN170;

[0019] Figure 3 : Chromosomal genome diagram of strain JSN170;

[0020] Figure 4 Growth curve of strain JSN170;

[0021] Figure 5 : Growth of strain JSN170 under different high concentrations of nitrite;

[0022] Figure 6 Figures showing the growth of strain JSN170 using nitrate, nitrite, and ammonium ions as the sole nitrogen sources, respectively.

[0023] Figure 7 Sodium nitrite standard curve;

[0024] Figure 8 : Degradation curves of different concentrations of nitrite by strain JSN170;

[0025] Figure 9 : An experimental diagram of nitrite degradation in the water of a small-scale Litopenaeus vannamei shrimp farming pond;

[0026] Figure 10 : Experimental diagram of nitrite degradation in water of factory-scale Litopenaeus vannamei (SPAV) shrimp farming ponds Detailed Implementation

[0027] The strain of this invention was isolated from sediments in the sea area near Lanshan District, Rizhao City, Shandong Province. This strain grows rapidly and reproduces easily. It can grow and reproduce under high concentrations of nitrite and sodium chloride. It can grow using nitrate, nitrite, and ammonium ions as the sole nitrogen sources, thereby utilizing and reducing the ammonia nitrogen content in the water. This strain has a complete denitrification pathway, reducing nitrite to nitrogen gas under aerobic conditions. It is suitable for efficiently degrading nitrite in aquaculture water (seawater or freshwater).

[0028] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0029] Example 1: Isolation, classification and preservation of strain JSN170

[0030] 1) Sediment samples were collected from the sea area near Lanshan District, Rizhao City, Shandong Province (119°27.918′E, 35°09.050′N). The samples were transported to the laboratory under refrigeration. 10g of sediment sample was added to an Erlenmeyer flask containing 200 mL of sterile physiological saline and shaken for 2 hours. Using the dilution plating method, 100µL of the shaken water sample was spread onto 2216E medium plates (5g peptone, 1g yeast powder, 15g agar, 1L filtered seawater) for each dilution. The plates were incubated at 25℃ for 3-5 days. Single colonies were picked from the plates and streaked for purification. One of the strains was numbered JSN170.

[0031] Classification and identification of JSN170 strain:

[0032] 1) Morphological identification: Strawberry strain JSN170 was streaked onto LB agar plates (10 g peptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar, 1 L distilled water). After incubation at 25°C for 48 h, the colonies were approximately 1 mm in diameter, pale yellow, round, and with regular edges. It does not produce spores and is a Gram-negative bacterium. Microscopic examination showed that the cells were rod-shaped (0.4-0.5 × 1.5-2.2 µm) with polar flagella. Electron micrographs are shown below. Figure 1 As shown, motion.

[0033] 2) Phenotypic and chemical characteristics identification: Strain JSN170 is a facultative anaerobe, with a growth range of 0%-6% (w / v) in salinity. It is protease-negative, amylase-positive, and capable of degrading Tween-60. The main fatty acids include C. 18:1 ω7c, C 16:0 C 16:1 ω7c and C 12:0 The main respiratory quinone is ubiquinone 9 (Q-9).

[0034] 3) Molecular identification of 16S rRNA gene

[0035] Preparation of 16S rRNA gene template: A bacterial DNA extraction kit (TaKaRa Bio) was used. The specific procedure was as follows: 2 mL of fermentation broth from strain JSN170 was centrifuged at 10,000 rpm for 5 min. The supernatant was discarded, and the bacterial pellet was collected. The procedure was performed according to the kit instructions, ultimately yielding the DNA sample from JSN170 cells.

[0036] 16S rRNA gene cloning and sequencing. The sequence information of the primers used for PCR amplification is as follows:

[0037] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0038] 1541R: 5´-AAGGAGGTGATCCAGCCGCA-3′.

[0039] The PCR reaction system consisted of 60 μL: 30 μL of 2×Taq Mix, 3 μL each of primers 27F and 1492R, 3 μL of template, and sterile water to a final volume of 60 μL. PCR conditions were as follows: 95℃ for 5 min; 94℃ for 45 s; 57℃ for 1 min and 30 s; 72℃ for 1 min and 30 s, 30 cycles; and 72℃ for 10 min. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and its 16S rDNA nucleotide sequence was identified as SEQ ID NO:1. After comparison with the NCBI database, the 16S rDNA nucleotide sequence was found to belong to the genus *Pseudomonas*, with the closest related species being *Pseudomonas nitrititolerans*, showing 99.78% homology. Therefore, this bacterium was named *Nitrite-tolerant Pseudomonas* JSN170.

[0040] In the nucleotide sequence of the 16S rRNA gene of strain JSN170, the sequences before and after the fragment “GGTGCCTTCGGGA” are both “G”. However, for the type strain of Pseudomonas nitrititolerans and other species of Pseudomonas, the sequences before and after this fragment are not both “G”. Therefore, the nucleotide sequence “GGGTGCCTTCGGGAG” in the 16S rRNA gene of strain JSN170 is unique to this strain and distinguishes it from the type strain of Pseudomonas nitrititolerans and other species of Pseudomonas.

[0041] The 16S rRNA gene sequence of nitrite-resistant Pseudomonas jSN170 is SEQ ID NO:1.

[0042] The final selected strain of nitrite-resistant Pseudomonas JSN170 has the accession number CGMCC No. 26623. The depositary institution is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is February 21, 2023. The classification name is: Pseudomonas nitrititolerans JSN170.

[0043] Preservation of bacterial strains. Long-term preservation: Add the bacterial culture to a glycerol tube with a final glycerol concentration of 20% and store at -80°C for later use; Short-term preservation: Stir on an LB slant and store at 4°C.

[0044] Example 2: Phylogenetic analysis of nitrite-resistant Pseudomonas JSN170

[0045] Using MEGA 7.0 software, a phylogenetic tree was constructed using the neighbor-joining method. It was found that strain JSN170 and Pseudomonas nitrititolerans clustered on the same branch, indicating that strain JSN170 and Pseudomonas nitrititolerans are most closely related phylogenetically. Figure 2 ).

[0046] Example 3: Sequencing and Analysis of the Whole Chromosome Genome of Nitrite-Tolerant Pseudomonas JSN170

[0047] Step 1: Whole genome sequencing of nitrite-resistant Pseudomonas japonicus JSN170

[0048] Nucleic acid DNA was extracted using a bacterial DNA extraction kit (TaKaRa Bio), and the DNA samples were sent to Beijing Novogene Technology Co., Ltd. for whole-genome sequencing. The assembly analysis steps were as follows: Starting from the valid data after quality control of each sample, the genome was assembled using Unicycler software with second-generation and third-generation data, assembling the chromosome sequences into a circular genome, i.e., the final 0-gap complete sequence. The assembly results are as follows: the chromosome size is 4333546 bp (…). Figure 3 The whole genome sequencing data of nitrite-resistant Pseudomonas japonicus JSN170 are shown in Table 1.

[0049] Table 1: Statistical table of whole genome sequencing data of nitrite-resistant Pseudomonas jSN170

[0050] Genome size (bp) 4333546 G+C% 63.26 Gene number 4,071 Gene total length 3,824,721 Gene average length 940 Gene length / Genome (%) 88.26 Serial repeating sequence TR number 92 Total length (bp) of the tandem repeat sequence TR 5,267 Minisatellite DNA number 65 Minisatellite DNA total length (bp) 2,416 Microsatellite DNA number 6 Microsatellite DNA total length (bp) 270 Genomics Islands (GIs) number 9 GIs total length (bp) 117,200 Prophage number 7 Prophage total length 223727

[0051] Step 2: Key Genes of the Nitrogen Cycle. Based on the whole genome sequence analysis of strain JSN170, five important key genes for the reduction of nitrite to nitrogen were identified: nirK (encoding nitrite reductase), nirS (encoding nitrite reductase), norB (encoding nitric oxide reductase subunit B), norC (encoding nitric oxide reductase subunit C), and nosZ (encoding nitrous oxide reductase). This strain also contains key genes for the dissimilatory nitrate reduction pathway: napA, napB, narG, narH, narI, nirB, and nirD.

[0052] Their nucleotide sequences are as follows:

[0053] (1) The nucleotide sequence of the nirK gene is SEQ ID NO:2;

[0054] (2) The nucleotide sequence of the nirS gene is SEQ ID NO:3;

[0055] (3) The nucleotide sequence of the norB gene is SEQ ID NO:4;

[0056] (4) The nucleotide sequence of the norC gene is SEQ ID NO:5;

[0057] (5) The nucleotide sequence of the nosZ gene is SEQ ID NO:6;

[0058] (6) The nucleotide sequence of the napA gene is SEQ ID NO:7

[0059] (7) The nucleotide sequence of the napB gene is SEQ ID NO:8

[0060] (8) The nucleotide sequence of the narG gene is SEQ ID NO:9

[0061] (9) The nucleotide sequence of the narH gene is SEQ ID NO:10

[0062] (10) The nucleotide sequence of the narI gene is SEQ ID NO:11

[0063] (11) The nucleotide sequence of the nirB gene is SEQ ID NO:12

[0064] (12) The nucleotide sequence of the nirD gene is SEQ ID NO:13;

[0065] Step 3: Housekeeping genes. Based on the whole genome sequence analysis of *Pseudomonas nitrite-resistant* JSN170, the nucleotide sequences of the three housekeeping genes (rpoB, rpoD, and gyrB) of this strain are as follows:

[0066] (13) The nucleotide sequence of the rpoB gene is SEQ ID NO:14;

[0067] (14) The nucleotide sequence of the rpoD gene is SEQ ID NO:15;

[0068] (15) The nucleotide sequence of the gyrB gene is SEQ ID NO:16;

[0069] Step 4: Based on whole-genome data analysis, it was also found that in the 16S rRNA gene sequence, the sequences before and after the fragment "GGTGCCTTCGGGA" are both "G", consistent with the sequencing results of the 16S rRNA gene PCR product in Example 1. Therefore, the nucleotide sequence of the 16S rRNA gene of strain JSN170 is different from that of the type strain Pseudomonas nitrititolerans and other species of the genus Pseudomonas. "GGGTGCCTTCGGGAG" is unique to the nucleotide sequence of the 16S rRNA gene of strain JSN170.

[0070] Example 4: Growth curve of nitrite-resistant Pseudomonas jSN170

[0071] Step 1: Scrape the bacterial cells from the JSN170 slant and transfer them to 20 mL of LB seed culture medium. Incubate at 25°C with shaking for 24 hours.

[0072] Step 2: Transfer 5% of the inoculum to 100 mL of LB liquid medium and incubate at 30°C with shaking. Take samples every hour under aseptic conditions in a laminar flow hood, for a total of 24 hours.

[0073] Step 3: Using a UV spectrophotometer (PE, Lambda35), with a wavelength of 600 nm, and using uninoculated LB seed culture medium as a blank control, the OD value was measured (Table 2).

[0074] Step 4: Plot the JSN170 growth curve with time on the x-axis and OD value on the y-axis (Figure 4).

[0075] Table 2: Growth curve data of nitrite-resistant Pseudomonas japonicus JSN170

[0076] Time / h <![CDATA[OD 600nm ]]> 1 0.035 2 0.047 3 0.05 4 0.065 5 0.11 6 0.16 7 0.215 8 0.335 9 0.396 10 0.49 11 0.575 12 0.63 13 0.725 14 0.765 15 0.825 16 0.813 17 0.767 18 0.697 19 0.598 20 0.443 21 0.402 22 0.376 23 0.351 24 0.311

[0077] Example 5: Detection of the tolerance of strain JSN170 to high concentrations of nitrite

[0078] Step 1: Prepare test tubes containing liquid culture medium of different concentrations of sodium nitrite:

[0079] 1) Sodium nitrite 80mM (prepared with 0.552% NaNO2, 1% peptone, 0.5% yeast powder, and distilled water).

[0080] 2) Sodium nitrite 100mM (0.69% NaNO2, 1% peptone, 0.5% yeast powder, prepared with distilled water).

[0081] 3) Sodium nitrite 120mM (0.828% NaNO2, 1% peptone, 0.5% yeast powder, prepared with distilled water).

[0082] 4) Sodium nitrite 150mM (1.035% NaNO2, 1% peptone, 0.5% yeast powder, prepared with distilled water).

[0083] Step 2: Inoculate strain JSN170 into the above liquid test tube and incubate at 30°C for 7 days, then observe the growth of strain JSN170.

[0084] Step 3: Using a UV spectrophotometer (PE, Lambda 35), with a wavelength of 600 nm, and using uninoculated culture medium as a blank control, measure the OD value (Table 3 and...). Figure 5 ).

[0085] The results showed that the JSN170 strain had a strong tolerance to sodium nitrite and could still grow and reproduce at a high concentration of 150 mM sodium nitrite.

[0086] Table 3: Growth data of strain JSN170 tolerating high concentrations of nitrite

[0087] Different sodium nitrite concentrations <![CDATA[OD 600nm ]]> 80mM 0.532 100mM 0.421 120mM 0.238 150mM 0.169

[0088] Example 6: Growth experiment of strain JSN170 using nitrate, nitrite and ammonium ions as the sole nitrogen sources

[0089] Step 1: Prepare liquid test tube culture media with nitrate, nitrite, and ammonium ions as the sole nitrogen source respectively. (1) Liquid culture medium formula with nitrate as the sole nitrogen source: 0.2% KNO3, 0.02% MgSO4·7H2O, 0.08% K2HPO4·3H2O, 2% potassium sodium tartrate, 1% sodium chloride, 1 liter of distilled water. Sterilize at 121℃ for 20 minutes. (2) Liquid culture medium formula with nitrite as the sole nitrogen source: 0.2% NaNO2, 0.02% MgSO4·7H2O, 0.08% K2HPO4·3H2O, 2% potassium sodium tartrate, 1% sodium chloride, 1 liter of distilled water. Sterilize at 121℃ for 20 minutes. (3) Liquid culture medium formula with ammonium ions as the sole nitrogen source: 0.2% (NH4)2SO4, 0.02% MgSO4·7H2O, 0.08% K2HPO4·3H2O, 2% potassium sodium tartrate, 1% sodium chloride, and 1 liter of distilled water. The (NH4)2SO4 was filtered and sterilized. All other components were sterilized at 121°C for 20 minutes.

[0090] Step 2: Inoculate the JSN170 strain into the three different liquid test tubes mentioned above and incubate at 30°C for 7-10 days. Use the uninoculated control medium as a control, and perform three replicates for each experimental medium. Observe the growth of strain JSN170.

[0091] Step 3: Using a UV spectrophotometer (PE, Lambda 35), with a wavelength of 600 nm, and using uninoculated control liquid culture medium as a blank control, measure the OD value (Table 4 and...). Figure 6 ).

[0092] The results showed that the JSN170 strain can utilize nitrate, nitrite and ammonium ions as nitrogen sources for growth and reproduction, thereby reducing the ammonia nitrogen content in the water.

[0093] Table 4: Growth data of strain JSN170 using nitrate, nitrite, and ammonium ions as the sole nitrogen sources.

[0094]

[0095] Example 7: Detection of nitrate reducing capacity of strain JSN170

[0096] Preparation of reagents for nitrite content detection:

[0097] (1) Griess A reagent (10 g / L p-aminobenzenesulfonic acid solution): Accurately weigh 0.1 g sulfanilamide, dissolve it in 20% hydrochloric acid and make up to 100 mL, mix well, place in a brown bottle and store away from light.

[0098] (2) Griess B reagent (1 g / L naphthylethylenediamine hydrochloride solution): Accurately weigh 0.1 g of naphthylethylenediamine hydrochloride, dissolve it in distilled water and make up to 100 mL, mix well, place in a brown bottle and store away from light.

[0099] Referring to the *Handbook of Systematic Identification of Common Bacteria*, the liquid culture medium for nitrate reduction detection consisted of: 0.5% peptone, 0.1% yeast extract, 0.1% KNO3, and distilled water. Strain JSN170 was inoculated into this medium and cultured at 30°C for 3 days. Griess A and Griess B reagents were added sequentially to the fermentation broth. After standing for 2 minutes, the test sample was compared with the control group (nitrate reduction detection liquid culture medium without bacteria). Both the fermentation broth in the test sample tube and the control group remained colorless. After adding zinc powder, the fermentation broth in the test sample tube remained colorless, indicating that nitrate was no longer present. The control group, however, remained red. Referring to the *Handbook of Systematic Identification of Common Bacteria*, JSN170 was determined to have nitrate reduction capability.

[0100] Example 8: Experiment on the degradation of nitrite by nitrite-resistant Pseudomonas jSN170

[0101] Step 1: Prepare the nitrite standard curve

[0102] Prepare Griess A and Griess B reagents for nitrite content detection as in Example 7.

[0103] Accurately weigh 0.69 g of sodium nitrite, dissolve it in distilled water and bring the volume to 100 mL, mix well, and the nitrite concentration is 100 mM.

[0104] Take 2 mL of 100 mM sodium nitrite solution and dilute to 500 mL with distilled water. Mix well. The nitrite concentration is 400 μM.

[0105] Take 0, 1, 2.5, 5, 6, 7.5, 9 and 10 mL of sodium nitrite solution (400 μM) respectively, and dissolve it in 10, 9, 7.5, 5, 4, 2.5, 1 and 0 mL of distilled water to make final concentrations of 0, 40, 100, 200, 240, 300, 360 and 400 μM.

[0106] Each of the above 10 mL sodium nitrite solutions of different concentrations was treated with 50 μL of Griess A reagent, mixed, and allowed to react for 4 min; then 50 μL of Griess B reagent was added, mixed, and allowed to react in the dark for 30 min. The absorbance was measured at 540 nm using a UV spectrophotometer (PE, Lambda 35) (Table 5), and a nitrite standard curve was plotted. Figure 7).

[0107] Table 5: Data for the determination of nitrite standard curve

[0108] Nitrite concentration (μM) <![CDATA[A OD600nm ]]> 0 0.006 40 0.092 100 0.23 200 0.445 240 0.535 300 0.662 360 0.775 400 0.871

[0109] Step 2: Nitrite-resistant Pseudomonas jSN170 degrades nitrite at different concentrations

[0110] Preparation of 20 mL nitrite liquid culture medium with different concentrations:

[0111] (1) Weigh 0.5 g of peptone, 0.1 g of yeast powder and 1 g of sodium chloride, dissolve them in 100 mL of distilled water, and divide them into 5 Erlenmeyer flasks (100 mL) with volumes of 16 mL, 17 mL, 18 mL, 19 mL and 19.5 mL respectively.

[0112] (2) Weigh 1.038 g of sodium nitrite and dissolve it in 100 mL of distilled water until fully dissolved (nitrite concentration is 150 mM) to make sodium nitrite storage solution.

[0113] (3) Pipette 4 mL of the stock solution into a 16 mL LB Erlenmeyer flask, with a nitrite concentration of 30 mM;

[0114] Pipette 3 mL of the stock solution into a 17 mL LB Erlenmeyer flask; nitrite concentration 22.5 mM.

[0115] Pipette 2 mL of the stock solution into an 18 mL LB Erlenmeyer flask; nitrite concentration 15 mM.

[0116] Pipette 1 mL of the stock solution into a 19 mL LB Erlenmeyer flask; the nitrite concentration is 7.5 mM.

[0117] (4) Scrape the bacterial cells from the slant of nitrite-resistant Pseudomonas jSN170 into 20 mL of LB liquid medium and incubate at 25°C for 24 hours to obtain the seed culture. Transfer 5% of the inoculum to 20 mL Erlenmeyer flasks containing different concentrations of nitrite liquid medium and incubate at 30°C on a shaker (30 r / min). At 12, 24, 36, 72, 84, and 96 hours, transfer 1 mL of the bacterial culture to a sterile Eppendorf tube, centrifuge at 9000 rpm for 3 min, collect 100 μL of the supernatant, and add distilled water to a final volume of 10 mL (i.e., a 100-fold dilution). Add 50 μL of Griess A reagent, mix well, and react for 4 min; then add 50 μL of Griess B reagent, mix well, and react in the dark for 30 min. The blank control group consisted of 100 μL of uninoculated LB liquid medium, with distilled water added to a final volume of 10 mL (50 μL of Griess A reagent and Griess B reagent were added).

[0118] (5) The absorbance was measured at 540 nm using a UV spectrophotometer (PE, Lambda35). The corresponding nitrite amount was calculated based on the nitrite standard curve. The nitrite degradation curve of nitrite-resistant Pseudomonas japonicus JSN170 was plotted with time as the x-axis and nitrite amount as the y-axis (Table 6 and 10). Figure 8 )

[0119] Table 6: Data on nitrite degradation by nitrite-resistant Pseudomonas jSN170

[0120]

[0121] From Table 6 and Figure 8 It can be seen that the nitrite-tolerant Pseudomonas japonicus JSN170 can rapidly degrade high concentrations of nitrite. After 12 hours, the JSN170 strain degraded all the aforementioned high concentrations of nitrite, indicating that while obtaining nutrients (LB liquid medium) for growth and reproduction, the JSN170 strain can utilize sodium nitrite as a starting material to carry out dissimilatory nitrate reduction, especially denitrification. Under the above experimental conditions, when the nitrite content is less than 15 mM, the JSN170 strain can almost completely degrade nitrite within 24 hours; when the nitrite content is less than 30 mM, the JSN170 strain can almost completely degrade nitrite within 36 hours.

[0122] Example 9: Nitrite (NO2) levels in water used for small-scale culture of Litopenaeus vannamei - Degradation experiment

[0123] Step 1: A small-scale shrimp farming base for Litopenaeus vannamei was selected in Juzhen Town, Rudong County, Nantong City, Jiangsu Province. The experimental shrimp pond was 40 meters long, 8 meters wide, and 1 meter deep, with a water volume of approximately 320 cubic meters. The water temperature was 28℃, with bottom aeration and waterwheel oxygenation. 60,000 shrimp larvae were introduced per pond, and the shrimp were cultured for 45 days, reaching a length of 7-8 cm. The feed rate was 8 jin (4 catties) per meal, fed three times a day. 20% of the water was changed daily, with a salinity of 12‰ and dissolved oxygen levels of approximately 5-6 mg / L. Three experimental sheds were used. During the experiment, no disinfection or other microbial agents were added to the farming ponds.

[0124] Step 2: Determine the nitrite content in the water of the three groups of shrimp ponds before the experiment. Water samples were first tested on-site using a commercial colorimetric nitrite detection kit. The results showed that the nitrite content in all three groups was approximately 3.0 mg / L. Since the colorimetric kit can only detect a general range of nitrite levels, we froze the water samples and mailed them to the laboratory. The determination method was the same as in Example 8, and the nitrite standard curve was the same. Figure 7 The specific method is as follows: Add 50 μL of Griess A reagent to 10 mL of aquaculture water, mix well, and react for 4 min; then add 50 μL of Griess B reagent, mix well, and react in the dark for 30 min. Measure the absorbance at 540 nm using a UV spectrophotometer. Calculate the nitrite content according to the standard curve. The nitrite content in the three shrimp ponds was 60.8 µM-69.5 µM (approximately 2.8 mg / L-3.2 mg / L).

[0125] Step 3: Scrape bacteria from the nitrite-resistant Pseudomonas jSN170 slant and inoculate into six 200 mL sterile primary seed culture media (500 mL Erlenmeyer flasks). Incubate at 30°C with shaking for 24 hours. Primary seed culture medium formula: 10 g tryptone, 10 g glucose, 3.5 g yeast extract, 0.2 g concentrated amino acid stock solution, 0.2 g magnesium sulfate, 0.3 g potassium dihydrogen phosphate, 1 liter tap water. Adjust the pH to approximately 7.0 and sterilize.

[0126] Step 4: At an inoculum rate of 20%, transfer the above fermentation broth into three 2L secondary seed culture media (5L Erlenmeyer flasks), and incubate at 30°C with shaking for 24 hours. Secondary seed culture medium formula: 10g tryptone, 5g glucose, 5g molasses powder, 2g yeast extract, 0.2g concentrated amino acid stock solution, 0.2g magnesium sulfate, 0.3g potassium dihydrogen phosphate, 1L tap water, adjust pH to approximately 7.0, and sterilize.

[0127] Step 5: Transfer the secondary seed fermentation broth aseptically into a 100-liter fermenter. Rotate the fermenter at 150 rpm with an air volume of 3.5. The fermentation medium formula is: 1200 g tryptone, 500 g glucose, 1000 g molasses powder, 500 g yeast powder, 20 g concentrated amino acid stock solution, 20 g magnesium sulfate, 30 g potassium dihydrogen phosphate, and 80 liters tap water. Adjust the pH to approximately 7.0 and sterilize. Ferment for approximately 12 hours to obtain the JSN170 inoculum fermentation broth.

[0128] Step 6: Experiment on the degradation of nitrite in the aquaculture pond. The experimental period was eight days. Considering the high nitrite content in the pond water before the experiment, from the first to the fourth day, 960 ml of JSN170 fermentation liquid (3 ml of bacterial solution / per cubic meter of water) and 1600 g of molasses powder (5 g / per cubic meter of water) were evenly sprinkled into the aquaculture pond daily. This step was repeated on the sixth and seventh days, and no bacterial solution or molasses powder was added on the fifth day. The pond water was transported to the laboratory frozen each day. 10 mL of the pond water was taken, 50 μL of Griess A reagent was added, mixed well, and reacted for 4 min; then 50 μL of Griess B reagent was added, mixed well, and reacted in the dark for 30 min. The absorbance was measured at 540 nm. According to the nitrite standard curve, the nitrite (NO2) content in the aquaculture pond water was calculated. - ) content (Table 7 and Figure 9 ).

[0129] Table 7: Data on nitrite degradation by JSN170 bacterial strain in small-scale Litopenaeus vannamei shrimp farming ponds

[0130]

[0131] From Table 7 and Figure 9 It can be seen that the nitrite content in the water of the small greenhouse was high before the experiment. After the addition of nitrite-tolerant Pseudomonas nitrite-170 to the aquaculture pond, the nitrite content began to decrease continuously. After four consecutive days, the nitrite content decreased from a peak of about 69.5 µM (3.2 mg / L) to below 21.7 µM (1.0 mg / L). Even without the addition of any bacterial solution or molasses powder on the fifth day, the nitrite content remained at or below 10.8 µM (0.5 mg / L) from the sixth to the eighth day. The reason why the nitrite content could not be reduced to 0 was mainly due to the continuous generation of new nitrites from the daily feeding of the aquaculture pond. In summary, this indicates that nitrite-tolerant Pseudomonas nitrite-170 has grown and multiplied in the aquaculture pond water, becoming one of the dominant bacterial groups and playing a role in degrading nitrite.

[0132] In summary, the nitrite-tolerant Pseudomonas japonicus JSN170 of the present invention can effectively degrade and control the nitrite content in the water of small-scale culture ponds for Litopenaeus vannamei.

[0133] Example 10: Nitrite (NO2) levels in water used for factory-cultured Litopenaeus vannamei - Degradation experiment

[0134] Step 1: A factory-scale shrimp farming base for Litopenaeus vannamei was selected in Shouguang City, Shandong Province. Each farming pond contained approximately 40 cubic meters of water at a temperature of 28℃, with bottom aeration. Approximately 500 catties of live shrimp were kept per pond; the feed rate was 2 catties per meal, fed three times a day; approximately 30% of the water was changed daily, with a salinity of 5‰ and dissolved oxygen levels of approximately 5-6 mg / L. Three experimental groups were selected. During the experiment, no disinfection or other microbial agents were added to the farming ponds.

[0135] Step 2: Determine the nitrite content in the shrimp pond water of the three groups before the experiment. First, samples of the aquaculture water were tested on-site using a commercial colorimetric nitrite detection kit. The results showed that the nitrite content in the three groups of shrimp pond water was approximately 2.0 mg / L. Since the colorimetric kit can only detect a general range of nitrite levels in water, we froze the water samples and mailed them to the laboratory. The determination method was the same as in Example 8, and the nitrite standard curve was the same. Figure 7 The specific method is as follows: Add 50 μL of Griess A reagent to 10 mL of aquaculture pond water, mix well, and react for 4 min; then add 50 μL of Griess B reagent, mix well, and react in the dark for 30 min. Measure the absorbance at 540 nm using a UV spectrophotometer. Calculate the nitrite content according to the standard curve. The nitrite content in the three groups of shrimp pond water was 36.9 µM-43.5 µM (approximately 1.7 mg / L-2.0 mg / L).

[0136] Step 3: Scrape bacteria from the nitrite-resistant Pseudomonas jSN170 slant and inoculate into six 200 mL sterile primary seed culture media (500 mL Erlenmeyer flasks). Incubate at 30°C with shaking for 24 hours. Primary seed culture medium formula: 10 g tryptone, 10 g glucose, 3.5 g yeast extract, 0.2 g concentrated amino acid stock solution, 0.2 g magnesium sulfate, 0.3 g potassium dihydrogen phosphate, 1 liter tap water. Adjust the pH to approximately 7.0 and sterilize.

[0137] Step 4: At an inoculum rate of 20%, transfer the above fermentation broth into three 2L secondary seed culture media (5L Erlenmeyer flasks), and incubate at 30°C with shaking for 24 hours. Secondary seed culture medium formula: 10g tryptone, 5g glucose, 5g molasses powder, 2g yeast extract, 0.2g concentrated amino acid stock solution, 0.2g magnesium sulfate, 0.3g potassium dihydrogen phosphate, 1L tap water, adjust pH to approximately 7.0, and sterilize.

[0138] Step 5: Transfer the secondary seed fermentation broth aseptically into a 100-liter fermenter. Rotate the fermenter at 150 rpm with an air volume of 3.5. The fermentation medium formula is: 1200 g tryptone, 500 g glucose, 1000 g molasses powder, 500 g yeast powder, 20 g concentrated amino acid stock solution, 20 g magnesium sulfate, 30 g potassium dihydrogen phosphate, and 80 liters tap water. Adjust the pH to approximately 7.0 and sterilize. Ferment for approximately 10 hours to obtain the JSN170 inoculum fermentation broth.

[0139] Step 6: Experiment on the degradation of nitrite in a factory-scale aquaculture pond. On the first day, 200 ml of JSN170 fermentation broth (5 ml of bacterial solution per cubic meter of water) and 200 g of molasses powder (5 g per cubic meter of water) were evenly sprinkled into the aquaculture pond. The experimental period was eight days. This step was continued for the first three days. The step was repeated on the fifth and seventh days. No bacterial solution or molasses powder was added on the fourth and sixth days. The aquaculture pond water was transported to the laboratory frozen every day. 10 mL of the aquaculture pond water was taken, 50 μL of Griess A reagent was added, mixed well, and reacted for 4 min; then 50 μL of Griess B reagent was added, mixed well, and reacted in the dark for 30 min. The absorbance was measured at 540 nm. According to the nitrite standard curve, the nitrite content in the aquaculture pond water was calculated (Table 8 and...). Figure 10 ).

[0140] Table 8: Data on nitrite degradation by JSN170 bacterial strain in factory-scale Litopenaeus vannamei farming ponds

[0141]

[0142] From Table 8 and Figure 10 It can be seen that when nitrite-tolerant Pseudomonas japonicus JSN170 was added to a 40 cubic meter aquaculture pond, the nitrite content began to decrease continuously. After three consecutive days of use, the nitrite content decreased from a peak of 43.5 µM (2.0 mg / L) to below 21.7 µM (1.0 mg / L) on the fourth day. Even without the addition of any bacterial solution or molasses powder on the fourth and sixth days, the nitrite content remained at or below 21.7 µM (1.0 mg / L) from the fourth to the eighth day. The reason why the nitrite content could not be reduced to zero, as analyzed in Example 9, is mainly due to the continuous generation of new nitrites from daily feeding into the aquaculture pond. In summary, this indicates that nitrite-tolerant Pseudomonas japonicus JSN170 has grown and multiplied in the aquaculture pond water, becoming one of the dominant bacterial groups and playing a role in degrading nitrite.

[0143] In summary, the nitrite-tolerant Pseudomonas japonicus JSN170 of the present invention can effectively degrade and control the nitrite content in the water of factory aquaculture ponds.

Claims

1. A nitrite-resistant Pseudomonas aeruginosa, characterized in that, The aforementioned nitrite-resistant Pseudomonas aeruginosa ( Pseudomonas nitrititolerans The accession number of the object is CGMCC No. 26623.

2. The application of the nitrite-resistant Pseudomonas as described in claim 1 in the degradation of nitrite.

3. The application as described in claim 2, characterized in that, The application described is the degradation of nitrite in water.

4. A product for degrading nitrite, characterized in that, The product contains live bacteria of the nitrite-resistant Pseudomonas as described in claim 1.

5. The article of claim 4, characterized in that, The product is a bacterial solution or an immobilized bacterial product.

6. A method for degrading nitrite in water, characterized in that, The method described herein is to treat water bodies using the nitrite-tolerant Pseudomonas aeruginosa as described in claim 1.

7. The method as described in claim 6, characterized in that, The water body mentioned is the water body used for aquaculture of aquatic animals.

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