Pseudomonas stutzeri BBW831 and its applications

Abstract

The present invention discloses a Pseudomonas stutzeri BBW831 and its application, which relates to the field of biotechnology and is a marine aerobic denitrifying bacterium isolated and screened from the seabed sludge in the Beibu Gulf of China. It is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 2022938. The denitrification test results show that Pseudomonas stutzeri BBW831 has complete genes encoding all functional enzymes of the aerobic denitrification metabolic pathway, has the potential to denitrify nitrate to N2, and at the same time has excellent denitrification performance, showing good application potential for the treatment of high-salinity nitrogen-containing wastewater.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to Pseudomonas stutzeri BBW831 and applications thereof. Background Art

[0002] Excessive nitrogen emissions from industrial and agricultural activities are a major cause of eutrophication, leading to deteriorating water quality and reduced water function, affecting the survival of many aquatic organisms and even endangering human health. Inorganic nitrogen compounds, particularly nitrates, are the primary source of nitrogen pollutants in water, and their removal is a significant environmental concern worldwide.

[0003] Denitrification methods can be divided into physical, chemical and biological methods. Compared with physical and chemical denitrification methods, biological denitrification is a more cost-effective, efficient and eco-friendly method in wastewater treatment. It is usually achieved through nitrification and / or denitrification processes. First, ammonium is converted into nitrate through nitrification, and nitrate is gradually reduced to nitrite, nitric oxide (NO), nitrous oxide (N2O) and N2 through denitrification. The traditional view is that microorganisms with denitrification function are mainly anaerobic bacteria, and the denitrification process is initiated under completely anoxic conditions. However, since the first aerobic denitrifying bacterium Thiosphaera pantropha was reported in 1984, a variety of aerobic denitrifying microorganisms have been isolated from sludge in denitrification bioreactors, aquaculture ponds, wetlands and lakes. The discovery of aerobic denitrifying bacteria has opened up a new path for the development of biological denitrification technology. Practice has proved that aerobic denitrification process has the advantages of simple operation, energy saving, high efficiency and low operating cost compared with anaerobic denitrification process.

[0004] Mariculture has made a significant contribution to global protein supply and food security. However, the rapid development of mariculture has also brought about serious water pollution problems. In particular, the excessive production of inorganic nitrogen salts such as nitrate, nitrite, and ammonium has become a key constraint to the sustainable and healthy development of mariculture. my country has the world's largest mariculture industry and faces greater challenges in the treatment of mariculture effluent. Therefore, the screening and isolation of efficient denitrifying microorganisms for the treatment of high-salinity, nitrogen-containing mariculture wastewater is of great practical significance. Compared with terrestrial microorganisms, marine microorganisms are likely to develop unique metabolic and physiological capabilities due to their unique habitat. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a Pseudomonas stutzeri BBW831 and its application.

[0006] The technical solutions of the present invention are as follows:

[0007] A Pseudomonas stutzeri BBW831 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 2022938.

[0008] Preferably, it is obtained by separation and screening of seabed mud.

[0009] Preferably, the gene encoding the functional enzyme of the aerobic denitrification metabolic pathway is carried thereon.

[0010] Preferably, the functional enzymes include nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase.

[0011] Preferably, the genes include: narG, narH, narJ, narI, napB, napA, nirD, nirB, norD, norB, norC, nirS, norQ, nosZ.

[0012] The present invention also discloses a use of any of the above-described Pseudomonas stutzeri BBW831 under high salinity and / or high nitrite load conditions.

[0013] Furthermore, the high salinity is specifically 40-60 g / L NaCl solution.

[0014] Furthermore, the concentration of the nitrite is 100-150 mg / L.

[0015] The beneficial effects of the present invention are as follows: the Pseudomonas stutzeri BBW831 of the present invention is isolated from marine habitats and has complete genes encoding all functional enzymes of the aerobic denitrification pathway, so it has the potential to denitrify nitrate into N2 (NO3 - -N→NO2 - -N→NO→N₂O→N₂); the strain exhibited efficient and rapid denitrification performance and exhibited good tolerance and adaptability to stressful conditions such as high nitrite concentrations and NaCl salinity. The simple strategy of increasing the initial inoculum size effectively enhanced the denitrification capacity of Pseudomonas stutzeri BBW831 under these stressful conditions. These results demonstrate that Pseudomonas stutzeri BBW831 has excellent niche adaptability and holds great promise for application in wastewater treatment, particularly in the treatment of high-nitrogen and high-salinity marine aquaculture wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The colonies on the BTB agar plate are surrounded by a blue ring ( Figure 1 The middle area is blue) strain BBW831;

[0017] Figure 2The colony characteristics of strain BBW831 on LB agar plate (a) and its bacterial morphology under an optical microscope (10×100) (b);

[0018] Figure 3 is the phylogenetic tree of strain BBW831;

[0019] Figure 4 The genome map of Pseudomonas stutzeri BBW831 is shown;

[0020] Figure 5 Provide COG functional annotation for the Pseudomonas stutzeri BBW831 genome;

[0021] Figure 6 GO functional annotation for the Pseudomonas stutzeri BBW831 genome;

[0022] Figure 7 These are genes related to nitrogen metabolism in the genome of Pseudomonas stutzeri BBW831;

[0023] Figure 8 Graphs showing bacterial growth, pH, and nitrogen concentration changes of Pseudomonas stutzeri BBW831 in nitrate denitrification medium (8a), nitrite denitrification medium (8b), and mixed nitrogen source denitrification medium (8c);

[0024] Figure 9 Figure 9a shows the bacterial growth and nitrate denitrification rate of Pseudomonas stutzeri BBW831 at different NaCl concentrations, and the effect of different inoculum sizes on the nitrate denitrification rate of the bacteria under 50 g / L NaCl pressure (9b). DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below with reference to the applicant's specific experiments.

[0026] 1. Materials and Methods

[0027] 1.1 Strains

[0028] Pseudomonas stutzeri BBW831: isolated from marine mud in the Beibu Gulf waters of Zhanjiang City, Guangdong Province (20°33′08.42467″N, 109°36′51.68500″E), currently deposited in the China Center for Type Culture Collection on June 21, 2022 (deposit number: CCTCC NO: M 2022938), the deposit address is Wuhan University, Wuhan, China.

[0029] The specific separation and screening methods are as follows:

[0030] 1.1.1 Source of soil samples

[0031] The soil samples were collected from the seabed mud in the Beibu Gulf waters of Zhanjiang City, Guangdong Province (20°33′08.42467″N, 109°36′51.68500″E).

[0032] 1.1.2 Isolation and screening culture medium

[0033] Bromothymol blue (BTB) agar medium: KNO3 1.0 g / L, trisodium citrate 8.5 g / L, KH2PO4 1.0 g / L, FeSO4·7H2O 0.05 g / L, CaCl2 0.2 g / L, MgSO4·7H2O 1.0 g / L, 1% (g / v) bromothymol blue 1 mL / L, agar 20.0 g / L, pH 7.2, sterilization at 121°C for 20 min.

[0034] 1.1.3 Strain isolation

[0035] Weigh 5.0 g of seabed mud sample and add it to a triangular flask containing 50 mL of sterile water (with glass beads). Mix thoroughly. Pipette 5 mL of soil sample suspension and add it to 45 mL of LB liquid culture medium. Proliferate and culture at 30°C and 160 r / min for 48 hours. Pipette 1 mL of proliferation culture medium and dilute it to 10 -4 , absorb 10 -4 0.1 mL of the dilution solution was spread on a BTB agar plate, and a single colony with a blue ring was selected and streaked on an LB agar plate for purification three times. The purified single colony was stored for later use.

[0036] 1.1.4 Identification methods

[0037] The preserved strain was streaked onto LB agar plates and incubated at 30°C for 24 hours. The colony morphology was observed. A single colony was picked from the plate and Gram-stained before observation under an optical microscope.

[0038] Scrape the bacterial cells from the slant into a flask containing 20 mL of sterile water (containing glass beads). 1 mL of the culture medium was then transferred to LB liquid medium and incubated at 30°C and 160 rpm for 12 hours. A genomic DNA extraction kit was used to extract the bacterial DNA template. PCR amplification was performed using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-AAGTCGTAACAAGGTAACG-3′). The system consisted of 5 μL of template, 2 μL each of universal bacterial primers 27F and 1492R, 25 μL of 2× Taq PCR Master Mix, and 16 μL of sterile double-distilled water, for a total volume of 50 μL. Conditions included pre-denaturation at 95°C for 3 minutes, followed by 34 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 30 seconds. The amplification was then performed using a 5-minute extension at 72°C. The amplification was then stored at 4°C. The amplified fragments were detected by 1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA sequencing. The sequencing results were compared with the NCBI database, and the 16S rDNA sequences of the strains after alignment were selected to construct a phylogenetic tree using MEGA-X software.

[0039] The 16S rDNA gene sequence of strain BBW831 is as follows:

[0040]

[0041] 1.1.5 Isolation and identification of strain BBW831

[0042] After separation and screening on BTB agar plates, a single colony with a clear blue ring around it was obtained (e.g. Figure 1 After being cultured on LB agar plates, the colonies of strain BBW831 were light yellow, round, convex in the middle, translucent and moist ( Figure 2 a). Gram staining of the bacteria and observation under an optical microscope showed that the strain was rod-shaped and a Gram-negative bacterium ( Figure 2 b).

[0043] The 16S rDNA gene sequence of strain BBW831 is 1448 bp long (GenBank accession number: ON222741). The 16S rDNA sequence of strain BBW831 was compared with other deposited sequences in Genbank using the BLAST program in NCBI. The alignment results were used to construct a phylogenetic tree using the neighbor-joining method in MEGA X software. The results are shown in Figure 2. Figure 3 Strain BBW831 clustered with Pseudomonas stutzeri and had the highest homology with Pseudomonas stutzeri CCUG 11256. Strain BBW831 was identified as Pseudomonas stutzeri and named Pseudomonas stutzeri BBW831. It was deposited in the China Center for Type Culture Collection with accession number CCTCC NO: M 2022938.

[0044] 1.2 Culture medium

[0045] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0, sterilized at 121°C for 20 min. To prepare an LB slant, add 20 g / L agar to the medium.

[0046] Nitrate Denitrification Medium: 9.0 g / L sodium citrate, 1.0 g / L KNO₃, 4.0 g / L KH₂PO₄, 1.0 g / L K₂HPO₄, 0.2 g / L MgSO₄·7H₂O, 2 mL / L trace element solution, pH 7.5, sterilized at 121°C for 20 min. This medium is used to determine the nitrate denitrification performance of Pseudomonas stutzeri BBW831.

[0047] Nitrite Denitrification Medium: 9.0 g / L sodium citrate, 0.85 g / L NaNO₂, 4.0 g / L KH₂PO₄, 1.0 g / L K₂HPO₄, 0.2 g / L MgSO₄·7H₂O, 2 mL / L trace element solution, pH 7.5, sterilized at 121°C for 20 min. This medium is used to determine the nitrite denitrification performance of Pseudomonas stutzeri BBW831.

[0048] Mixed nitrogen source denitrification medium: 9.0 g / L sodium citrate, 0.333 g / L KNO₃, 0.335 g / L NaNO₂, 0.183 g / L NH₄Cl₃, 4.0 g / L KH₂PO₄, 1.0 g / L K₂HPO₄, 0.2 g / L MgSO₄·7H₂O, 2 mL / L trace element solution, pH 7.5, sterilized at 121°C for 20 min. This medium is used to determine the denitrification performance of Pseudomonas stutzeri BBW831 on mixed inorganic nitrogen sources.

[0049] The trace element solution added to the nitrate denitrification medium, nitrite denitrification medium and mixed nitrogen source denitrification medium consisted of the following components: ethylenediaminetetraacetic acid (EDTA) 50 g / L, ZnSO4·7H2O 3.92 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5.0 g / L, CoCl2·6H2O 1.61 g / L, CaCl2 5.5 g / L, (NH4)6Mo7O2·4H2O 1.1 g / L, and CuSO4·5H2O 1.57 g / L.

[0050] 1.3 Genome sequencing and functional annotation of Pseudomonas stutzeri BBW831

[0051] Pseudomonas stutzeri BBW831 was inoculated onto a 15×150 mm LB agar slant and incubated at 30°C for 48 hours. The fresh slant of Pseudomonas stutzeri BBW831 was washed with 10 ml of sterile water to remove bacterial cells. After shaking, 1 ml of the suspension was inoculated into a 250 ml Erlenmeyer flask containing 50 ml of LB medium. The suspension was incubated at 30°C and 160 rpm for 24 hours to prepare a seed culture. Then, 1 mL of the seed culture was inoculated into a 250 ml Erlenmeyer flask containing 50 ml of LB medium. The culture was incubated at 30°C and 160 rpm until the cells reached the logarithmic growth phase (approximately 20 hours). The resulting culture was centrifuged at 4°C and 10,000 rpm for 10 minutes. The cells were washed three times with distilled water and collected for genomic DNA extraction.

[0052] High-quality genomic DNA from Pseudomonas stutzeri BBW831 was extracted using a Qiagen genomic extraction kit. The concentration and purity of the extracted DNA were determined using an ultramicrospectrophotometer and a fluorescence quantifier. The whole genome of Pseudomonas stutzeri BBW831 was sequenced using the Illumina Novaseq 6000 sequencing platform, and the sequence assembly was completed using SPAdes v.3.9. Open reading frame (ORF) prediction was performed using Prodigal v2.60 software. All predicted protein sequences were compared with the NR (non-redundant protein database), Swiss-Prot database, COG (Clusters of orthologous groups) database, KEGG (Kyoto encyclopedia of genes and genomes) database, Interpro database, and Gene Ontology (GO) data to complete protein sequence functional annotation.

[0053] 1.4 Denitrification performance of Pseudomonas stutzeri BBW831

[0054] The denitrification performance of Pseudomonas stutzeri BBW831 was evaluated using nitrate denitrification medium, nitrite denitrification medium and mixed nitrogen source denitrification medium respectively: fresh cells of Pseudomonas stutzeri BBW831 were inoculated into LB medium (medium volume was 50 mL / 250 mL Erlenmeyer flask) and cultured at 30 ° C and 170 rpm for 24 h; the culture solution was centrifuged at 8000 rpm and 4 ° C for 10 minutes, and the collected bacterial cells were washed three times with deionized water; the collected bacterial cells were inoculated into nitrate denitrification medium, nitrite denitrification medium and mixed nitrogen source denitrification medium (medium volume was 200 mL / 250 mL Erlenmeyer flask), and the bacterial biomass OD after inoculation was 0. 600 The values ​​were all controlled within 0.05. After inoculation, the cells were cultured in a shaking incubator at 30°C and 200 rpm for 48 h, during which samples were taken every 12 h to determine the bacterial biomass (OD 600 ), pH and NO3 - -N, NO2 - -N and NH4 + -N concentration.

[0055] 1.5 Determination of nitrite denitrification performance of Pseudomonas stutzeri BBW831 at different inoculation rates

[0056] The effect of initial biomass on the nitrite denitrification performance of Pseudomonas stutzeri BBW831 was investigated by using four different inoculation sizes: fresh cells of Pseudomonas stutzeri BBW831 were inoculated into LB medium (medium volume was 50 mL / 250 mL Erlenmeyer flask) and cultured in a shaking incubator at 30°C and 170 rpm for 24 h; the culture solution was centrifuged at 8000 rpm and 4°C for 10 min, and the collected bacterial cells were washed three times with deionized water; the collected bacterial cells were inoculated into nitrite denitrification medium (medium volume was 200 mL / 250 mL Erlenmeyer flask), and the bacterial biomass OD after inoculation was 0. 600 The values ​​were controlled at 0.05, 0.6, 1.2 and 2.4 respectively; after inoculation, the cells were cultured in a shaking incubator at 30°C and 200 rpm for 24 h, and samples were taken every 8 h to determine the bacterial growth (OD 600 ) and NO2 - -Changes in N concentration.

[0057] 1.6 Determination of nitrate denitrification performance of Pseudomonas stutzeri BBW831 at different salinity levels

[0058] In order to evaluate the denitrification performance of Pseudomonas stutzeri BBW831 at different salinity levels, NaCl was added to the nitrate denitrification medium and set to six concentration gradients of 0, 10, 20, 30, 40 and 50 g / L. First, fresh cells of Pseudomonas stutzeri BBW831 were inoculated into LB medium (the medium volume was 50 mL / 250 mL Erlenmeyer flask) and cultured at 30 ° C and 170 rpm for 24 hours; the culture solution was centrifuged at 8000 rpm and 4 ° C for 10 minutes, and the collected bacterial cells were washed three times with deionized water; the collected bacterial cells were inoculated into nitrate denitrification medium with different NaCl gradients (the medium volume was 200 mL / 250 mL Erlenmeyer flask), and the bacterial biomass OD after inoculation was 0. 600 The values ​​were all controlled within 0.05. After inoculation, the cells were cultured in a shaking incubator at 30°C and 200 rpm for 12 h, and the bacterial growth (OD 600 ) and NO3 - -N concentration.

[0059] 1.7 Effect of different inoculum sizes on nitrate denitrification performance of Pseudomonas stutzeri BBW831 under high salinity

[0060] The effects of four different inoculum sizes on the nitrate denitrification performance of Pseudomonas stutzeri BBW831 at a NaCl concentration of 50 g / L were studied: fresh cells of Pseudomonas stutzeri BBW831 were inoculated into LB medium (medium volume was 50 mL / 250 mL Erlenmeyer flask) and cultured in a shaking incubator at 30°C and 170 rpm for 24 h; the culture solution was centrifuged at 8000 rpm and 4°C for 10 min, and the collected bacterial cells were washed three times with deionized water; the collected bacterial cells were inoculated into nitrate denitrification medium (medium volume was 200 mL / 250 mL Erlenmeyer flask) with a NaCl concentration of 50 g / L, and the bacterial biomass OD after inoculation was 0. 600 The values ​​were controlled at 0.05, 0.6, 1.2 and 2.4 respectively; after inoculation, the NO3 - -N concentration.

[0061] 1.8 Analytical methods

[0062] Bacterial growth (OD 600 ) determination: UV-5800PC spectrophotometer was used to measure the cell optical density (OD) of Pseudomonas stutzeri BBW831 culture solution at 600 nm. 600 ).

[0063] NO3 - -N, NO2 - -N and NH4 + Determination of -N concentration (mg / L): Pseudomonas stutzeri BBW831 was inoculated into the culture medium of nitrate denitrification medium, nitrite denitrification medium and mixed nitrogen source denitrification medium, and the supernatant was collected after centrifugation at 8000 rpm and 4°C; the supernatant was used to determine NO3 - -N, NO2 - -N and NH4 + -N concentration; NO3 - -N was determined by UV spectrophotometry, NO2 - -N was determined by N-(1-naphthyl)-1,2-diaminoethane dihydrochloride spectrophotometry, NH4 + -N was determined by Nessler's reagent spectrophotometer method.

[0064] NO3 - -N, NO2 - -N and NH4 + -N denitrification rate (%) and denitrification rate (mg·L -1 ·h -1 ) The calculation methods are respectively based on the public and Where C1 is the initial nitrogen concentration, C2 is the nitrogen concentration at culture time t, and t is the culture time of Pseudomonas stutzeri BBW831.

[0065] 2. Results and Analysis

[0066] 2.1 Analysis of the denitrification metabolic pathway of Pseudomonas stutzeri BBW831 based on genome sequencing

[0067] By sequencing the whole genome of Pseudomonas stutzeri BBW831 and annotating its functions, we analyzed its denitrification metabolic pathway at the gene level. The genome size of Pseudomonas stutzeri BBW831 is 4567965bp (ref. Figure 4 The COG, GO and KEGG databases were used to classify and annotate the Pseudomonas stutzeri BBW831 genome. Figure 5 ), Pseudomonas stutzeri BBW831 has a higher gene allocation in energy production and conversion (278 genes), amino acid transport and metabolism (285 genes), and inorganic ion transport and metabolism (274 genes), accounting for 7.21%, 7.39%, and 7.10% of the 3857 annotated genes, respectively. GO distribution shows (refer to Figure 6 ), and 1576, 2451, and 1566 genes were associated with biological processes, molecular functions, and cellular components, respectively.

[0068] According to the KEGG database, the functional annotation of genes involved in nitrogen metabolism in Pseudomonas stutzeri BBW831 was performed. A total of 44 genes related to nitrogen metabolism were found (refer to Figure 7 ), these genes are involved in denitrification, nitrate reduction and assimilation, amino acid metabolism and other nitrogen metabolism processes. Among them, a total of 14 genes (narG, narH, narJ, narI, napB, napA, nirD, nirB, norD, norB, norC, nirS, norQ, nosZ) are related to the denitrification pathway, which are responsible for encoding nitrate reductase (Nar and Nap), nitrite reductase, nitric oxide reductase and nitrous oxide reductase, as shown in Table 1. This shows that Pseudomonas stutzeri BBW831 has all the functional enzymes of the complete aerobic denitrification metabolic pathway and has the potential to gradually reduce nitrate to N2 (NO3 - -N→NO2 - -N→NO→N2O→N2).

[0069] Table 1 Functional genes related to the denitrification metabolic pathway in the genome of Pseudomonas stutzeri BBW831

[0070]

[0071]

[0072] In addition to nitrate denitrification genes, P. stutzeri BBW831 also contains the nasA and nirBD genes, encoding assimilative nitrate reductase and NADH nitrite reductase, respectively, suggesting that P. stutzeri BBW831 may have a metabolic pathway for reducing nitrate to ammonium. Furthermore, genes encoding glutamate dehydrogenase, glutamate synthetase, and glutamine synthetase, genes involved in amino acid metabolism, were also found in the genome of P. stutzeri BBW831.

[0073] 2.2 Denitrification performance of Pseudomonas stutzeri BBW831

[0074] The denitrification performance of Pseudomonas stutzeri BBW831 was studied using nitrate denitrification medium, nitrite denitrification medium and mixed nitrogen source denitrification medium. Figure 8 shown.

[0075] In the nitrate denitrification medium with nitrate as the only nitrogen source ( Figure 8 a), Pseudomonas stutzeri BBW831 grew rapidly within 0-12 hours, and its bacterial mass (OD 600 ) reached a maximum value of 0.884±0.050 at 12h; at the same time, NO3 - -N concentration decreased rapidly from the initial 166.10±3.75mg / L to 8.90±0.99mg / L within 0-12h, and the corresponding NO3 - -N denitrification rate reached 13.09 mg·L -1 ·h -1 .

[0076] In the nitrite denitrification medium with nitrite as the only nitrogen source ( Figure 8 b), bacterial count of Pseudomonas stutzeri BBW831 (OD 600 ) reached a peak of 0.725±0.006 at 24h, which was delayed by 12h compared with that in nitrate denitrification medium and was significantly lower than 0.884±0.050 in nitrate denitrification medium; NO2 - The -N concentration decreased from the initial 146.42 ± 14.17 mg / L to approximately 100 mg / L at 24 h, but did not decrease further thereafter.

[0077] Since nitrate, nitrite and ammonium often coexist in wastewater, the denitrification performance of Pseudomonas stutzeri BBW831 on mixed inorganic nitrogen sources was studied using a mixed nitrogen source denitrification medium. Compared with the denitrification medium with nitrate or nitrite as the only nitrogen source, the denitrification performance of Pseudomonas stutzeri BBW831 in the mixed nitrogen source denitrification medium ( Figure 8c) The bacterial growth was improved, and the bacterial volume OD at 12h was 600 The value reached 0.961±0.019; NO3 - -N, NO2 - -N and NH4 + -N denitrification rate within the first 12 h was close to 100%, and the denitrification rates were 5.13, 5.34 and 4.17 mg·L -1 ·h -1 .

[0078] The biological denitrification process (nitrate reduction to nitrite, nitrite reduction to NO, NO reduction to N2O, N2O reduction to N2) usually requires the participation of electrons, accompanied by OH - Therefore, in the absence of a buffer system, the pH of the denitrification process will increase. In nitrate denitrification medium, nitrite denitrification medium, and mixed nitrogen source denitrification medium, the pH value of Pseudomonas stutzeri BBW831 showed a clear upward trend throughout the denitrification process, which is consistent with the biochemical characteristics of the denitrification process.

[0079] It should be noted that Pseudomonas stutzeri BBW831 can denitrify 64.26±0.90 mg / L NO2 in the mixed nitrogen source medium. - -N was almost completely removed, however, NO2 in the nitrite denitrification medium - The denitrification rate of -N (concentration of 146.42±14.17 mg / L) was only about 30%. This phenomenon indicates that Pseudomonas stutzeri BBW831 has a certain adaptability and removal ability to nitrite, but excessive nitrite concentration may be toxic to the bacterial cells, thereby inhibiting the nitrite denitrification efficiency of the bacteria.

[0080] 2.3 Simple strategies to improve the denitrification capacity of Pseudomonas stutzeri BBW831 under high nitrite stress conditions

[0081] To mitigate the toxic effects of high nitrite concentrations, a simple strategy was developed to improve the nitrite denitrification efficiency of Pseudomonas stutzeri BBW831 by increasing the inoculum size. The results showed that the nitrite denitrification rate of Pseudomonas stutzeri BBW831 significantly increased with increasing initial bacterial biomass (see Table 2).

[0082] Table 2 Nitrite denitrification capacity of Pseudomonas stutzeri BBW831 at different inoculation rates

[0083]

[0084] a:RE meant the removal efficiency of NO2- -N

[0085] When the initial biomass (OD 600 ) were controlled at 0.05, 0.60, 1.20 and 2.40 respectively, after culturing for 8 hours, NO2 - -N denitrification rates were 1.19%, 44.73%, 79.02% and 99.46% respectively; the culture was continued for 8h (8-16h), the initial biomass (OD 600 ) under NO2 - -N concentrations further decreased to 2.85±0.44 and 0.71±0.17 mg / L, respectively, and the corresponding NO2 - -N denitrification rates reached 97.97% and 99.46% respectively; in sharp contrast to the high inoculation amount, at 0.05 initial biomass (OD 600 ) Under culture conditions, NO2 - -N denitrification rate was only about 35% until the end of the culture.

[0086] As can be seen from Table 3, the dynamic changes of bacterial growth show a similar trend to that of NO2 - -N removal efficiency was significantly different. 600 ) under culture conditions, although about 65% of NO2 - -N remains unused, but bacterial biomass (OD 600 ) increased from the initial 0.052±0.001 to 0.752±0.043 at the 24th hour, with a net increase of 0.700, which again showed that Pseudomonas stutzeri BBW831 showed good adaptability and tolerance to high concentrations of nitrite. It is particularly noteworthy that gradually increasing the initial bacterial biomass helps to improve the denitrification rate and denitrification rate of NO2-N, but the net increase of bacterial biomass shows a gradually decreasing trend. Specifically, at 0.60, 1.20 and 2.40 initial biomass (OD 600 ) under the culture conditions, the bacterial biomass (OD 600 ) were 0.518, 0.447 and 0.167, respectively, all lower than the initial biomass (OD 600 ) treatment. Based on the above results, it can be inferred that most of the NO2 - -N was removed by denitrification by Pseudomonas stutzeri BBW831, and only a small portion was used for cell growth.

[0087] Table 3 Changes in bacterial cell mass during nitrite removal by Pseudomonas stutzeri BBW831 at different inoculation sizes

[0088]

[0089] a:Net meant the net increase ofcell density(OD 600 )compare to the initial cell density(0h).

[0090] 1.4 Simple strategies to improve the denitrification capacity of Pseudomonas stutzeri BBW831 under high salinity stress conditions

[0091] Firstly, the nitrate removal performance of Pseudomonas stutzeri BBW831 at different salinity levels (0, 10, 20, 30, 40 and 50 g / L NaCl) was determined. Figure 9 a. The results showed that when the NaCl content in the nitrate denitrification medium was 0-20 g / L, the bacterial growth of Pseudomonas stutzeri BBW831 was significantly affected by NO3 - -N denitrification rates showed no significant difference, but when the NaCl concentration gradually increased to 30, 40 and 50 g / L, the bacterial growth and NO3 - -N denitrification rate showed a significant downward trend.

[0092] The above results indicate that the nitrate denitrification capacity of Pseudomonas stutzeri BBW831 at different salinity levels may be highly correlated with the bacterial growth. To verify this, 50 g / L NaCl was added to the nitrate denitrification medium, and the initial OD 600 The values ​​are controlled at 0.05, 0.6, 1.2 and 2.4 respectively. Figure 9 b. The results show that the increase in initial bacterial biomass can effectively increase NO3 - -N removal efficiency. When the initial biomass of Pseudomonas stutzeri BBW831 (OD 600 ) is controlled at 2.40, NO3 - -N denitrification rate reached 59.01%, compared with the initial biomass (OD 600 ) is 0.05, compared with the treatment with NO3 - -N denitrification rate increased by 1.75 times.

[0093] 3. Conclusion

[0094] The microbial aerobic denitrification process is driven by four nitrogen reductases: nitrate reductase (Nar and Nap), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide reductase (Nos). These enzymes sequentially reduce nitrate to nitrite, nitrite to NO, and NO to N2O or N2, achieving denitrification. Whole-genome sequencing and functional annotation revealed that Pseudomonas stutzeri BBW831 possesses complete genes encoding nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, indicating that the strain possesses the metabolic pathways required for complete denitrification of nitrate to N2. In Pseudomonas stutzeri BBW831, the narG, narH, narI, and narJ genes encode the α-, β-, and γ-subunits of the nitrate reductase Nar, and the molybdenum cofactor domain. napA and napB encode the napA and napB subunits of the nitrate reductase Nap, respectively. The nirD, nirB, and nirS genes encode the nitrite reductase Nir. Four nor genes (norD, norB, norC, and norQ) and one nosZ gene encode the nitric oxide reductase Nor and the nitrous oxide reductase Nos, respectively. Notably, many aerobic denitrifying microorganisms lack the nos gene encoding the nitrous oxide reductase Nos. Consequently, their denitrification end product is N2O rather than N2. Because N2O is a potent greenhouse gas, it can have a certain harmful effect on the atmospheric environment. This study showed that Pseudomonas stutzeri BBW831 has the nos gene encoding nitrous oxide reductase Nos, and has a metabolic pathway that completely reduces N2O to N2, which is of great significance for reducing N2O emissions during denitrification.

[0095] In terms of denitrification performance, Pseudomonas stutzeri BBW831 can denitrify NO3 in nitrate denitrification medium. - -N denitrification rate reached 13.09 mg·L -1 ·h -1 , which is significantly higher than the reported Pseudomonas stutzeri YZN-001 (11.46 mg·L -1 ·h -1 )[Zhang j,Wu P,Hao B,Yu Z(2011)Heterotrophic nitrification and aerobicdenitrification by the bacterium Pseudomonas stutzeri YZN-001.BioresourTechnol 102(21):9866–9869.], Pseudomonas stutzeri YG-24(7.33mg·L -1 ·h -1)[Li C, Yang J, Wang -1 ·h -1 )[Sun Y,Feng L,Li A,Zhang X,Yang J,Ma F(2017)Ammoniumassimilation:An important accessory during aerobic denitrification ofPseudomonas stutzeri T13.Bioresour Technol 234:264–272.] and Pseudomonas stutzeri ADP-19(3.03mg·L -1 ·h -1 )[Li B, Jing F, Wu D, Xiao B, Hu Z(2021)Simultaneous removal of nitrogen and phosphorus by a novel aerobic denitrifying phosphorus-accumulating bacterium, Pseudomonas stutzeri ADP-19. Bioresour Technol 321:124445.]. In addition, Pseudomonas stutzeri BBW831 can denitrify NO3 in mixed nitrogen source culture medium within 12 hours. - -N (62.28 ± 0.74 mg / L), NO2 - -N (64.26±0.90mg / L) and NH4 + -N (50.50±0.55 mg / L) was almost completely removed, showing good inorganic nitrogen denitrification ability.

[0096] It is reported that high concentrations of nitrite can produce toxic effects on microbial cells, thereby strongly inhibiting their denitrification activity. A nitrite concentration of 20 mg / L is generally considered to be the threshold for determining whether a microorganism has denitrification ability [Song Z, An J, Fu G, Yang X (2011) Isolation and characterization of an aerobic denitrifying Bacillus sp. YX-6 from shrimp culture ponds. Aquaculture 319 (1-2): 188–193.]. Pseudomonas stutzeri BBW831 was able to denitrify 146.42 ± 14.17 mg / L NO2 in nitrite denitrification medium. - The removal rate of -N is about 30%, and the strain can remove 64.26±0.90mg / L NO2 --N was almost completely removed, indicating that Pseudomonas stutzeri BBW831 has good tolerance and removal ability for nitrite. In addition, salinity is another key environmental factor affecting the microbial denitrification process. The increase in osmotic pressure caused by high salinity significantly inhibits the activity of microbial denitrifying enzymes [Uygur A, Kargi F (2004) Salt inhibition on biological nutrient removal from saline wastewater in asequencing batch reactor. Enzyme Microb Technol 34 (3-4): 313–318.]. The salt tolerance of Pseudomonas stutzeri BBW831 is similar to that of Pseudomonas balearica strain RAD-17 [Ruan Y, Taherzadeh MJ, Kong D, Lu H, Zhao H, Xu X, Liu Y, Cai L (2020) Nitrogen removal performance and metabolic pathways analysis of a novel aerobic denitrifying halotolerant Pseudomonas balearica strain RAD-17. Microorganisms 8:72.] and Pseudomonas sp. DN-23 [Li D, Liang X, Wu C (2020a) Characteristics of nitrogen removal and extracellular polymeric substances of a novel salt-tolerant denitrifying bacterium, Pseudomonas sp. DN-23. Front Microbiol 11:335.] is equivalent, that is, when the NaCl concentration is 30g / L and above, it will have a certain inhibitory effect on the denitrification performance of the strain. However, it is worth noting that when the simple strategy of increasing the initial inoculum size of Pseudomonas stutzeri BBW831 is adopted, the nitrite denitrification rate of the strain can reach as high as 99.46% within 8 hours, and under the condition of NaCl concentration as high as 50g / L, NO3 - -N denitrification rate can reach 59.01%.

[0097] In conclusion, Pseudomonas stutzeri BBW831 was isolated from a marine habitat and has complete genes encoding all functional enzymes of the aerobic denitrification pathway, thus having the potential to denitrify nitrate to N2 (NO3 - -N→NO2 - -N→NO→N₂O→N₂); the strain exhibited efficient and rapid denitrification performance and exhibited good tolerance and adaptability to stressful conditions such as high nitrite concentrations and NaCl salinity. The simple strategy of increasing the initial inoculum size effectively enhanced the denitrification capacity of Pseudomonas stutzeri BBW831 under these stressful conditions. These results demonstrate that Pseudomonas stutzeri BBW831 has excellent niche adaptability and holds great promise for application in wastewater treatment, particularly in the treatment of high-nitrogen and high-salinity marine aquaculture wastewater.

[0098] Under the premise that no conflict occurs, those skilled in the art may freely combine and superimpose the above-mentioned additional technical features.

[0099] The above descriptions are merely preferred embodiments of the present invention. Any technical solution that achieves the purpose of the present invention by substantially the same means shall fall within the scope of protection of the present invention.

Claims

1. A Pseudomonas stutzeri ( Pseudomonas stutzeri ), BBW831, characterized in that It is preserved in the China Center for Type Culture Collection, with the preservation number of CCTCC NO: M 2022938; it is obtained by separating and screening from submarine sludge; it has genes encoding functional enzymes for the aerobic denitrification metabolic pathway; the functional enzymes include nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase; This strain has an extremely efficient removal effect on the mixed N of NO3 − -N, NO2 − -N and NH4 + -N.

2. Application of Pseudomonas stutzeri Pseudomonas stutzeri BBW831 as described in claim 1 in denitrifying wastewater under conditions of high salinity and / or high nitrite load, characterized in that, Pseudomonas stutzeri The specific high salinity is a NaCl solution of 40 - 60 g / L, and the concentration of high nitrite is 100 - 150 mg / L.

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