CN115786180A - Pseudomonas stutzeri BBW831 and application thereof - Google Patents
Pseudomonas stutzeri BBW831 and application thereof Download PDFInfo
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Abstract
The invention discloses Pseudomonas stutzeri BBW831 and application thereof, relates to the technical field of biology, and relates to a marine aerobic denitrifying bacterium separated and screened from seabed sludge in northern gulf of China. It is preserved in China center for type culture Collection with a preservation number of CCTCC NO: m2022938. The denitrification test result shows that the Pseudomonas stutzeri BBW831 has complete genes for coding all functional enzymes in the aerobic denitrification metabolic pathway and has the function of denitrifying nitrate into N 2 Has excellent denitrification performance and shows good application potential suitable for treating high-salinity nitrogen-containing wastewater.
Description
Technical Field
The invention relates to the technical field of biology, in particular to pseudomonas stutzeri BBW831 and application thereof.
Background
Nitrogen element discharged by industrial and agricultural activities is one of main factors causing eutrophication of water body, thereby causing water quality deterioration and water body function reduction, further influencing the survival of a plurality of aquatic organisms and even harming human health. Among them, inorganic nitrogen compounds, especially nitrate, are the main source of nitrogen pollutants in water bodies, and the denitrification treatment thereof is also an environmental problem of high concern all over the world.
The denitrification method can be classified into a physical method, a chemical method and a biological method. Biological denitrification is a more cost-effective, efficient and eco-friendly method in wastewater treatment compared to physical and chemical denitrification methods, which are typically achieved by nitrification and/or denitrification processes. Firstly, ammonium is converted into nitrate through nitrification, and the nitrate is gradually reduced into nitrite, nitric Oxide (NO) and nitrous oxide (N) through denitrification 2 O) and N 2 . Traditionally, microorganisms with denitrification function are mainly anaerobes, and the denitrification process is started under the condition of complete oxygen deficiency. However, since the first aerobic denitrifying bacteria, thiophosphere pantoea pan (Thiosphaera panopha), 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 the aerobic denitrifying bacteria opens up a new way for the development of the biological denitrification technology, and practice proves that the aerobic denitrification process has the advantages of simple operation, energy conservation, high efficiency, low operation cost and the like compared with the anaerobic denitrification process.
Mariculture makes a significant contribution to the global protein supply and food safety. However, the rapid development of the mariculture also brings serious water pollution problems, and especially, the excessive generation of inorganic nitrogen salts such as nitrate, nitrite and ammonia salt becomes a key constraint factor for the sustainable and healthy development of the mariculture. China has the largest marine aquaculture industry in the world and faces greater challenges in the aspect of marine aquaculture tail water treatment. Therefore, the screening and separation of the high-efficiency denitrification microorganisms for treating the high-salinity nitrogen-containing mariculture wastewater have important practical significance. Marine microorganisms are highly likely to develop unique metabolic and physiological capabilities due to their specialized habitat as compared to terrestrial microorganisms.
Disclosure of Invention
The invention aims to solve at least one technical problem in the prior art and provides pseudomonas stutzeri BBW831 and application thereof.
The technical solution of the invention is as follows:
pseudomonas stutzeri BBW831, it is preserved in China center for type culture Collection, the collection number is CCTCC NO: m2022938.
Preferably, the method is obtained by separating and screening the seabed sludge.
Preferably, the gene encodes an aerobic denitrification metabolic pathway functional enzyme.
Preferably, the functional enzymes include nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase.
Preferably, the genes include: narG, narH, narJ, narI, napB, napA, nirD, nirB, norD, norB, norC, nirS, norQ, nosZ.
The invention also discloses application of the Pseudomonas stutzeri BBW831 in high salinity and/or high nitrite load condition.
Further, the high salinity is 40-60g/L NaCl solution.
Further, the concentration of the nitrite is 100-150mg/L.
The invention has the beneficial effects that: the Pseudomonas stutzeri BBW831 of the invention is separated from marine habitat, has complete genes for coding all functional enzymes in aerobic denitrification nitrogen removal path, and thus has the function of denitrifying and removing nitrogen from nitrate into N 2 Potential of (NO) 3 - -N→NO 2 - -N→NO→N 2 O→N 2 ) (ii) a The strain shows high-efficiency and rapid denitrification performance, and has good tolerance and adaptability to high-concentration nitrite, naCl salinity and other pressure conditions; the established simple strategy of increasing the initial inoculation amount effectively promotes SchneiderThe denitrification capability of pseudomonas BBW831 under the pressure conditions of high concentration nitrite and NaCl salinity and the like. The results show that the Pseudomonas stutzeri BBW831 has good ecological niche adaptability and has good application prospect in the field of wastewater treatment, particularly in the treatment of high-nitrogen and high-salt mariculture wastewater.
Drawings
FIG. 1 shows strain BBW831 with a blue ring (blue in the middle region of FIG. 1) around the colony on BTB agar medium plate;
FIG. 2 shows the colony characteristics (a) of the strain BBW831 on LB agar medium plate and the morphological characteristics (b) of its cells under an optical microscope (10X 100);
FIG. 3 is a phylogenetic tree of strain BBW831;
FIG. 4 is a genomic map of Pseudomonas stutzeri BBW831;
FIG. 5 is a COG functional annotation of the Pseudomonas stutzeri BBW831 genome;
FIG. 6 is a GO functional annotation of the Pseudomonas stutzeri BBW831 genome;
FIG. 7 shows the genes involved in nitrogen metabolism in the genome of Pseudomonas stutzeri BBW831;
FIG. 8 is a graph showing the cell growth, pH and nitrogen concentration trends of Pseudomonas stutzeri BBW831 in the nitrate denitrifying culture medium (8 a), nitrite denitrifying culture medium (8 b) and mixed nitrogen source denitrifying culture medium (8 c);
FIG. 9 shows the growth of the cells and the nitrate denitrification rate of Pseudomonas stutzeri BBW831 at different NaCl concentrations (9 a), and the effect of different inoculum sizes on the nitrate denitrification rate of the cells under 50g/LNaCl pressure conditions (9 b).
Detailed Description
The technical solution of the present invention will be further described below by the specific test of the applicant.
1. Materials and methods
1.1 strains
Pseudomonas stutzeri BBW831 (Pseudomonas stutzeri BBW 831): marine sludge (20 DEG 33 '08.42467' N,109 DEG 36 '51.68500' E) separated from northern gulf sea area of Zhanjiang city, guangdong province is currently preserved in China center for type culture Collection No. 6-21 in 2022 (the preservation number is CCTCC NO: M2022938) at the preservation address of Wuhan university, china.
The specific separation and screening method comprises the following steps:
1.1.1 sources of soil samples
The soil sample was taken from seabed sludge (20 ° 33.
1.1.2 isolation screening Medium
Bromothymol blue (bromothylol blue, BTB) agar medium: KNO 3 1.0g/L, trisodium citrate 8.5g/L, KH 2 PO 4 1.0g/L,FeSO 4 ·7H 2 O 0.05g/L,CaCl 2 0.2g/L,MgSO 4 ·7H 2 O1.0 g/L,1% (g/v) bromothymol blue 1mL/L, agar 20.0g/L, pH 7.2, and sterilization at 121 deg.C for 20min.
1.1.3 Strain isolation
Weighing 5.0g of seabed sludge sample, adding into a triangular flask filled with 50mL of sterile water (with glass beads), mixing well, sucking 5mL of soil sample suspension, adding into 45mL of LB liquid culture medium, and performing enrichment culture at 30 ℃ and 160r/min for 48 hours. Aspirate 1mL of multiplication medium and dilute it to 10 degrees in a gradient -4 Suction 10 -4 0.1mL of the dilution was spread on a BTB agar medium plate, and a single colony with a blue loop was selected and streaked on an LB agar medium plate and purified three times, and the purified single colony was stored for future use.
1.1.4 identification method
The preserved strain is streaked and inoculated on an LB agar medium plate, cultured for 24h at the temperature of 30 ℃, and the morphological characteristics of the colony are observed. Picking single colony in the plate, observing colony morphological structure in an optical microscope after gram staining.
The thalli on the strain inclined plane is scraped into a triangular flask filled with 20mL of sterile water (containing glass beads), inoculated into 1mL to LB liquid culture medium at 30 ℃ and 160R/min for 12h, a strain DNA template is extracted by using a genome DNA extraction kit, and PCR amplification is carried out by using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-AAGTCGTAACAAGGTAACG-3') respectively. The system is as follows: mu.L of template, 2. Mu.L of bacterial universal primers 27F and 1492R, 25. Mu.L of 2 XTaq PCR Master Mix, and 16. Mu.L of sterile double distilled water, wherein the total volume is 50. Mu.L. Conditions are as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 30s, and circulation for 34 times; after extension at 72 ℃ for 5min, the cells were stored at 4 ℃. The amplified fragment was detected by 1% agarose gel electrophoresis and sent to Biotechnology engineering (Shanghai) Co., ltd for 16S rDNA sequencing. And comparing the sequencing result with an NCBI database, selecting a 16S rDNA sequence of the compared strain, and drawing a phylogenetic tree by using MEGA-X software.
The 16S rDNA gene sequence of the strain BBW831 is as follows:
AACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGAGTGGAGCTTGCTCCATGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTATGGCAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGGCTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCATTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTAAGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCTTCGGGGGGACGGTTACCACGGAGTGATTCATGACT
1.1.5 isolation and identification of the Strain BBW831
Through the plate separation and screening of a BTB agar medium, a strain with an obvious blue ring (shown in figure 1) around a single colony is obtained, and the number of the strain is BBW831, which indicates that the strain possibly has the denitrification function. After cultivation on LB agar medium plates, colonies of the strain BBW831 were yellowish, round, convex in the middle, translucent and moist (FIG. 2 a). The bacterium was gram-stained, and the strain was rod-shaped and gram-negative when observed with an optical microscope (FIG. 2 b).
The 16S rDNA gene sequence of the strain BBW831 has the length of 1448bp (GenBank accession number: ON 222741). The 16S rDNA sequence of the strain BBW831 was compared with other deposited sequences in Genbank by BLAST in the online program NCBI, and phylogenetic trees were constructed using Neighbor-join method in MEGA X software, the results are shown in FIG. 3. The strain BBW831 is aggregated with Pseudomonas stutzeri to form a cluster, has the highest homology with Pseudomonas stutzeri CCUG 11256, is determined to be Pseudomonas stutzeri and is named as Pseudomonas stutzeri BBW831 (Pseudomonas stutzeri BBW 831) and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2022938.
1.2 culture Medium
LB culture medium: 10g/L tryptone, 5g/L, naCl g/L yeast extract, 7.0 pH, and 20min of sterilization at 121 ℃. In this, LB slants were prepared and 20g/L agar was added to the medium.
Nitrate denitrification culture medium: sodium citrate 9.0g/L, KNO 3 1.0g/L、KH 2 PO 4 4.0g/L、K 2 HPO 4 1.0g/L、MgSO 4 ·7H 2 0.2g/L of O, 2mL/L of trace element solution, pH 7.5 and sterilization at 121 ℃ for 20min. The method is used for measuring the denitrification performance of the Pseudomonas stutzeri BBW831 on nitrate.
Nitrite denitrification medium: sodium citrate 9.0g/L, naNO 2 0.85g/L、KH 2 PO 4 4.0g/L、K 2 HPO 4 1.0g/L、MgSO 4 ·7H 2 0.2g/L of O, 2mL/L of trace element solution, pH 7.5 and sterilization at 121 ℃ for 20min. By usingThe denitrification performance of the Pseudomonas stutzeri BBW831 on nitrite is measured.
Mixing a nitrogen source denitrification culture medium: sodium citrate 9.0g/L, KNO 3 0.333g/L、NaNO 2 0.335g/L、NH 4 Cl 0.183g/L、KH 2 PO 4 4.0g/L、K 2 HPO 4 1.0g/L、MgSO 4 ·7H 2 0.2g/L of O, 2mL/L of trace element solution, pH 7.5 and sterilization at 121 ℃ for 20min. Is used for measuring the denitrification performance of the pseudomonas stutzeri BBW831 on the mixed inorganic nitrogen source.
The microelement solution added into the nitrate denitrification culture medium, the nitrite denitrification culture medium and the mixed nitrogen source denitrification culture medium consists of the following components: ethylenediaminetetraacetic acid (EDTA) 50g/L, znSO 4 ·7H 2 O 3.92g/L、MnCl 2 ·4H 2 O 5.06g/L、FeSO 4 ·7H 2 O 5.0g/L、CoCl 2 ·6H 2 O 1.61g/L、CaCl 2 5.5g/L、(NH 4 ) 6 Mo 7 O 2 ·4H 2 O 1.1g/L、CuSO 4 ·5H 2 O1.57g/L。
1.3 genome sequencing and functional Annotation of Pseudomonas stutzeri BBW831
Pseudomonas stutzeri BBW831 was inoculated on LB agar slant (15X 150 mm) and cultured at 30 ℃ for 48 hours. The cultured fresh inclined plane of the pseudomonas stutzeri BBW831 is washed out of the somatic cells by 10ml of sterile water, 1ml of suspension liquid is inoculated into a 250ml triangular flask with 50ml of LB culture medium after shaking up, and shaking culture is carried out for 24h at 30 ℃ and 160rpm to prepare the seed culture solution. Then, 1mL of the seed culture was inoculated into a 250mL triangular flask containing 50mL of LB medium, shake-cultured at 30 ℃ and 160rpm until the cells were in the logarithmic phase (about 20 hours), the resulting culture was centrifuged at 4 ℃ and 10000r/min for 10 minutes, washed three times with distilled water, and then the cells were collected for extraction of genomic DNA.
High quality genomic DNA of Pseudomonas stutzeri BBW831 was extracted using Qiagen genome extraction kit, and the concentration and purity of the extracted DNA were determined using a ultraspectrophotometer and a fluorometer. The extracted pseudomonas stutzeri BBW831 adopts an Illumina Novaseq 6000 sequencing platform to complete whole genome sequencing and uses splicing software SPAdes v.3.9 to complete sequence splicing. Open Reading Frame (ORF) prediction was performed using ProdigalV2.60 software, and all predicted protein sequences were BLAST aligned with NR (non-redundant protein database), swiss-Prot database, COG (Clusters of orthologous groups, orthologic Cluster database), KEGG (Kyoto encyclopedia of genes and genes, kyoto encyclopedia database of genomes and genomes), interpro database, and Gene Ontology (GO) data to complete functional annotation of protein sequences.
1.4 measurement of Denitrification Performance of Pseudomonas stutzeri BBW831
The denitrification performance of pseudomonas stutzeri BBW831 was evaluated using a nitrate denitrification medium, a nitrite denitrification medium, and a mixed nitrogen source denitrification medium, respectively: inoculating fresh cells of Pseudomonas stutzeri BBW831 into LB culture medium (the culture medium is 50mL/250mL triangular flask), and shake-culturing at 30 ℃ and 170rpm for 24h; centrifuging the culture solution at 8000rpm and 4 deg.C for 10 min, and washing the collected thallus cells with deionized water for three times; inoculating the collected thallus cells into a nitrate denitrification culture medium, a nitrite denitrification culture medium and a mixed nitrogen source denitrification culture medium (culture medium loading is 200mL/250mL triangular flask), and inoculating thallus biomass OD 600 The values are all controlled to be 0.05; after inoculation, the cells were subjected to shake cultivation at 30 ℃ and 200rpm for 48 hours, during which a sample was taken every 12 hours for determining the biomass (OD) of the cells 600 ) pH and NO 3 - -N、NO 2 - -N and NH 4 + -the concentration of N.
1.5 determination of nitrite denitrification Performance of Pseudomonas stutzeri BBW831 under different inoculum sizes
The effect of initial biomass on nitrite denitrification performance of pseudomonas stutzeri BBW831 was examined by using four different inoculum sizes: inoculating fresh cells of Pseudomonas stutzeri BBW831 into LB culture medium (the culture medium is 50mL/250mL triangular flask), and shake-culturing at 30 ℃ and 170rpm for 24h; centrifuging the culture solution at 8000rpm and 4 deg.C for 10 min, and washing the collected thallus cells with deionized water for three times; inoculating the collected somatic cells into nitrite denitrification medium (medium loading 2)00mL/250mL triangular flask), OD of biomass of inoculated cells 600 The values are respectively controlled at 0.05, 0.6, 1.2 and 2.4; after inoculation, the cells were subjected to shake culture at 30 ℃ and 200rpm for 24 hours, during which time samples were taken every 8 hours for determination of the amount of cell growth (OD) 600 ) And NO 2 - -change in N concentration.
1.6 measurement of Denitrification Performance of Pseudomonas stutzeri BBW831 nitrate at different salinity levels
In order to evaluate denitrification performance of Pseudomonas stutzeri BBW831 at various salinity levels, naCl was added to the nitrate denitrification medium and set to six concentration gradients of 0, 10, 20, 30, 40, and 50g/L, respectively. Firstly, inoculating fresh cells of Pseudomonas stutzeri BBW831 into an LB culture medium (the culture medium loading is 50mL/250mL triangular flask), and carrying out shake culture at 30 ℃ and 170rpm for 24h; centrifuging the culture solution at 8000rpm and 4 deg.C for 10 min, and washing the collected thallus cells with deionized water for three times; the collected bacterial cells are respectively inoculated into nitrate denitrification culture media (culture medium loading is 200mL/250mL triangular flask) with different NaCl gradients, and the biomass OD of the inoculated bacterial cells 600 The values are all controlled to be 0.05; after inoculation, the cells were cultured by shaking at 30 ℃ and 200rpm for 12 hours, and the amount of cell growth (OD) was measured 600 ) And NO 3 - -N concentration.
1.7 Effect of different inoculum sizes on nitrate denitrification Performance of Pseudomonas stutzeri BBW831 at high salinity
The effect of four different inoculum sizes on the denitrification performance of the nitrate of Pseudomonas stutzeri BBW831 at a NaCl concentration of 50g/L was studied: inoculating fresh cells of Pseudomonas stutzeri BBW831 into LB culture medium (the culture medium is 50mL/250mL triangular flask), and shake-culturing at 30 ℃ and 170rpm for 24h; centrifuging the culture solution at 8000rpm and 4 deg.C for 10 min, and washing the collected thallus cells with deionized water for three times; the collected bacterial cells were inoculated into a nitrate-denitrifying medium (200 mL/250mL Erlenmeyer flask) having a NaCl concentration of 50g/L, and the microbial biomass OD after inoculation was determined 600 The values are respectively controlled at 0.05, 0.6, 1.2 and 2.4; after inoculation, NO was determined after 12h of shake cultivation at 30 ℃ and 200rpm 3 - -N concentration.
1.8 analytical methods
Cell growth amount (OD) 600 ) The determination of (1): the Optical Density (OD) of cells in the culture of Pseudomonas stutzeri BBW831 at 600nm was measured using a UV-5800PC spectrophotometer 600 )。
NO 3 - -N、NO 2 - -N and NH 4 + Determination of the N concentration (mg/L): inoculating pseudomonas stutzeri BBW831 to a culture solution of a nitrate denitrification culture medium, a nitrite denitrification culture medium and a mixed nitrogen source denitrification culture medium, centrifuging at 8000rpm and 4 ℃, and collecting a supernatant; supernatant for determination of NO 3 - -N、NO 2 - -N and NH 4 + -the concentration of N; NO (nitric oxide) 3 - -N is determined by UV spectrophotometry, NO 2 - -N is determined spectrophotometrically by N- (1-naphthyl) -1,2-diaminoethane dihydrochloride, NH 4 + -N is measured using a Nassner reagent spectrophotometer.
NO 3 - -N、NO 2 - -N and NH 4 + Denitrification Rate (%) and denitrification Rate (mg. L) of-N -1 ·h -1 ) The calculation methods respectively adopt public indications
And
wherein C1 is the initial nitrogen concentration, C2 is the nitrogen concentration at the time of culture t, and t is the culture time of Pseudomonas stutzeri BBW 831.
2. Results and analysis
2.1 genome sequencing-based analysis of Denitrification Metabolic pathway of Pseudomonas stutzeri BBW831
The denitrification metabolic pathway was analyzed from the gene level by whole genome sequencing and functional annotation of Pseudomonas stutzeri BBW 831. The genome size of Pseudomonas stutzeri BBW831 is 4567965bp (refer to FIG. 4). The pseudomonas stutzeri BBW831 genome was classified and functionally annotated using COG, GO and KEGG databases, respectively. COG classification showed (see fig. 5) that pseudomonas stutzeri BBW831 has higher gene allocation in functions of energy production and transformation (278 genes), amino acid transport and metabolism (285 genes), and inorganic ion transport and metabolism (274 genes), and the proportion of the three in 3857 annotated genes was 7.21%, 7.39%, and 7.10%, respectively. GO distribution shows (see fig. 6) 1576, 2451 and 1566 genes associated with biological processes, molecular functions and cellular components, respectively.
The genes involved in nitrogen metabolism in Pseudomonas stutzeri BBW831 were functionally annotated according to the KEGG database. There were found 44 genes associated with nitrogen metabolism (see FIG. 7) in common, and these genes were involved in denitrification, nitrate reductive assimilation, amino acid metabolism and other nitrogen metabolic processes. Among them, 14 genes (narG, narH, narJ, narI, napB, napA, nirD, nirB, norD, norB, norC, nirS, norQ, nosZ) in total are involved in the denitrification pathway and are responsible for encoding nitrate reductase (Nar and Nap), nitrite reductase, nitric oxide reductase and nitrous oxide reductase, as shown in Table 1. This indicates that Pseudomonas stutzeri BBW831 has all functional enzymes of the complete aerobic denitrification metabolic pathway and has the function of gradually reducing nitrate to N 2 Potential of (NO) 3 - -N→NO 2 - -N→NO→N 2 O→N 2 )。
TABLE 1 functional genes related to the Denitrification Metabolic pathway in the genome of Pseudomonas stutzeri BBW831
In addition to the nitrate denitrification gene, pseudomonas stutzeri BBW831 also contains the nasA and nirBD genes encoding assimilating nitrate reductase and NADH nitrite reductase, respectively, indicating that pseudomonas stutzeri BBW831 may have a metabolic pathway for the reduction of nitrate to ammonium. In addition, genes encoding glutamate dehydrogenase, glutamate synthase, and glutamine synthetase have also been found in the genome of Pseudomonas stutzeri BBW831, and these genes are involved in amino acid metabolism.
2.2 Denitrification Performance of Pseudomonas stutzeri BBW831
The denitrification performance of Pseudomonas stutzeri BBW831 was examined using a nitrate denitrification medium, a nitrite denitrification medium, and a mixed nitrogen source denitrification medium, respectively, and the results are shown in FIG. 8.
In nitrate-denitrifying medium with nitrate as sole nitrogen source (FIG. 8 a), pseudomonas stutzeri BBW831 rapidly grew in 0-12h, and its bacterial load (OD) 600 ) Reaches a maximum of 0.884 +/-0.050 at 12 h; at the same time, NO 3 - The N concentration is rapidly reduced within 0-12h, and is rapidly reduced to 8.90 +/-0.99 mg/L from the initial 166.10 +/-3.75 mg/L, and the corresponding NO 3 - The denitrification rate of-N reaches 13.09 mg.L -1 ·h -1 。
In nitrite denitrogenation Medium with nitrite as sole Nitrogen Source (FIG. 8 b), the biomass (OD) of Pseudomonas stutzeri BBW831 600 ) The peak value of 0.725 +/-0.006 is reached at 24h, the delay is 12 hours relative to that of the nitrate denitrification culture medium and is obviously lower than 0.884 +/-0.050 under the nitrate denitrification culture medium; NO 2 - The N concentration decreased from 146.42. + -. 14.17mg/L initially to about 100mg/L at 24h, but then did not decrease further.
Because nitrate, nitrite and ammonia salt in the wastewater frequently coexist, the denitrification performance of the pseudomonas stutzeri BBW831 on the mixed inorganic nitrogen source is researched by using the mixed nitrogen source denitrification culture medium. Compared with denitrification culture medium with nitrate or nitrite as only nitrogen source, the Pseudomonas stutzeri BBW831 in mixed nitrogen source denitrification culture medium (figure 8 c) has improved thallus growth, and thallus amount OD at 12h 600 The value reaches 0.961 plus or minus 0.019; NO 3 - -N、NO 2 - -N and NH 4 + The denitrogenation rate of N in the first 12h is close to 100 percent, and the denitrogenation rate reaches 5.13, 5.34 and 4.17 mg.L -1 ·h -1 。
Biological denitrification process (nitrate reduction to nitrite, nitrite reduction to NO, NO reduction to N 2 O，N 2 OtoIs originally N 2 ) Usually requires the participation of electrons accompanied by OH - Is generated. Thus, the pH of the denitrification process would increase if the buffer system were absent. In a nitrate denitrification culture medium, a nitrite denitrification culture medium and a mixed nitrogen source denitrification culture medium, the pH value of the whole denitrification process of the pseudomonas stutzeri BBW831 shows an obvious rising trend, which accords with the biochemical characteristics of the denitrification reaction process.
Note that Pseudomonas stutzeri BBW831 can denitrify the mixed nitrogen source to 64.26 + -0.90 mg/L of NO in the denitrification culture medium 2 - Almost complete removal of-N, however NO in nitrite denitrogenation medium 2 - The denitrogenation rate of-N (concentration of 146.42 +/-14.17 mg/L) is only about 30%. This phenomenon indicates that pseudomonas stutzeri BBW831 has a certain adaptability and removal capacity for nitrite, but an excessively high nitrite concentration may cause a certain toxicity to bacterial cells, thereby inhibiting the nitrite denitrification efficiency of the bacterial cells.
2.3 simple strategy for improving denitrogenation capability of Pseudomonas stutzeri BBW831 under high-concentration nitrite pressure
In order to alleviate the toxic effects of high concentrations of nitrite, a simple strategy was developed to increase the nitrite denitrification efficiency of pseudomonas stutzeri BBW831 inoculum size. The results show that the nitrite denitrification rate of Pseudomonas stutzeri BBW831 is significantly increased with the increase of the initial bacterial biomass (refer to Table 2).
TABLE 2 Pseudomonas stutzeri BBW831 nitrite denitrification capacity under different inoculum sizes
a：RE meant the removal efficiency of NO 2 - -N
When Pseudomonas stutzeri BBW831 is cultured in nitrite denitrification medium and when Pseudomonas stutzeri BBW831 is cultured in nitrite denitrification medium, the initial biomass (OD) 600 ) NO after 8h of culture at 0.05, 0.60, 1.20 and 2.40 respectively 2 - The nitrogen removal rates of-N were 1.19%, 44.73%, 79.02% and 99.46%, respectively; the cultivation was continued for 8h (8 th-16 th), with 0.60 and 1.20 primary biomass (OD) 600 ) NO of 2 - The N concentration is further reduced to 2.85. + -. 0.44 and 0.71. + -. 0.17mg/L, respectively, corresponding to NO 2 - The denitrogenation rate of N reaches 97.97% and 99.46% respectively; in sharp contrast to high inoculum sizes, at 0.05 initial biomass (OD) 600 ) Under culture conditions, NO 2 - the-N denitrification rate was only about 35% until the end of the culture.
As can be seen from Table 3, the dynamic change of the cell growth appeared together with NO 2 - The N removal efficiency is significantly different. Initial biomass (OD) at 0.05 600 ) Under culture conditions, although about 65% of NO is present 2 - -N remained unutilized, but cell biomass (OD) 600 ) Increasing from the initial 0.052. + -. 0.001 to 0.752. + -. 0.043 at 24h, the net increase in bacteria was 0.700, again indicating that Pseudomonas stutzeri BBW831 showed good adaptation and tolerance to high concentrations of nitrite. Of particular note, increasing the initial biomass of the bacteria gradually helped to increase NO 2 The denitrification rate and denitrification rate of-N, however, the net increase of biomass of the cells tended to decrease. Specifically, initial biomass (OD) at 0.60, 1.20 and 2.40 600 ) The bacterial biomass (OD) under the culture conditions of (1) 600 ) The maximum net gains were 0.518, 0.447 and 0.167, respectively, all below 0.05 initial biomass (OD) 600 ) Maximum net delta under treatment. From the above results, it can be inferred that most of NO 2 - N is removed by denitrification with Pseudomonas stutzeri BBW831, and only a small fraction is used for cell growth.
TABLE 3 variation of the amount of bacteria in the nitrite removal process of Pseudomonas stutzeri BBW831 under different inoculum sizes
a：Net meant the net increase ofcell density(OD 600 )compare to the initial cell density(0h).
1.4 simple strategy for improving denitrification capability of Pseudomonas stutzeri BBW831 under high salinity pressure condition
The nitrate denitrification performance of Pseudomonas stutzeri BBW831 was first determined at different salinity levels (0, 10, 20, 30, 40 and 50g/L NaCl, respectively). Referring to FIG. 9a, the results show that the cell growth and NO of Pseudomonas stutzeri BBW831 were observed when NaCl was 0-20g/L in the nitrate-denitrifying medium 3 - No significant difference was observed in the-N denitrification rates, but when the NaCl concentration was gradually increased to 30, 40 and 50g/L, the cell growth was observed to be different from that of NO 3 - the-N denitrification rate showed a significant decrease.
The above results indicate that the nitrate denitrification capacity of Pseudomonas stutzeri BBW831 at different salinity levels is likely to be highly correlated with the amount of bacterial growth. To verify this, 50g/L NaCl was added to the nitrate denitrification medium, and the initial OD after inoculation of Pseudomonas stutzeri BBW831 was added 600 The values were controlled at 0.05, 0.6, 1.2 and 2.4, respectively. Referring to FIG. 9b, the results show that the increase of initial biomass of bacteria can effectively increase NO 3 - -removal efficiency of N. When Pseudomonas stutzeri BBW831 initial biomass (OD) 600 ) Control at 2.40, NO 3 - The denitrification rate of-N reaches 59.01 percent and the initial biomass (OD) 600 ) Treatment ratio of 0.05, NO 3 - The nitrogen removal rate of-N is improved by 1.75 times.
3. Conclusion
The aerobic denitrification process of the microorganisms is respectively driven by four nitrogen reductases, namely nitrate reductase (Nar and Nap), nitrite reductase (Nir), nitric oxide reductase (Nor) and nitrous oxide reductase (Nos), in turn to reduce nitrate into nitrite, nitrite into NO and NO into N 2 O or N 2 Thereby achieving the denitrification effect. The whole genome sequencing and function annotation result shows that the Pseudomonas stutzeri BBW831 has complete genes for encoding nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, and the strain is proved to have the function of completely denitrifying and denitrifying the nitrate into N 2 The metabolic pathway of (1). In Pseudomonas stutzeri BBIn W831, the narG, narH, narI and narJ genes code the alpha-subunit, beta-subunit, gamma-subunit and molybdenum cofactor functional region in nitrate reductase Nar, and napA and napB code the napA and napB subunits in nitrate reductase Nap, respectively; the nirD, nirB and nirS genes are responsible for coding nitrite reductase Nir; four Nor (norD, norB, norC, norQ) and one nosZ gene are responsible for encoding nitric oxide reductase Nor and nitrous oxide reductase Nos, respectively. Notably, many aerobic denitrifying microorganisms lack the Nos gene encoding the no of nitrous oxide reductase, the end product of which is N 2 O instead of N 2 Due to N 2 O is a strong greenhouse gas and has certain harm to the atmospheric environment. This study showed that Pseudomonas stutzeri BBW831 has a Nos gene encoding a nitrous oxide reductase Nos, and has a structure in which N is substituted 2 Complete reduction of O to N 2 The pair reduces N during denitrification 2 The emission of O is of great significance.
Pseudomonas stutzeri BBW831 on NO in nitrate denitrogenation media in terms of denitrifying Denitrification Performance 3 - The denitrification rate of-N reaches 13.09 mg.L -1 ·h -1 Is obviously higher than that of 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 aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001.Bioresour Technol 102(21):9866–9869.]Pseudomonas stutzeri YG-24 (7.33 mg. L) -1 ·h -1 )[Li C,Yang J,Wang X,Wang E,Li B,He R,Yuan H(2015)Removal of nitrogen by heterotrophic nitrification-aerobic denitrification of a phosphate accumulating bacterium Pseudomonas stutzeri YG-24.Bioresour Technol 182:18–25.]Pseudomonas stutzeri T13 (7.09 mg. L) -1 ·h -1 )[Sun Y,Feng L,Li A,Zhang X,Yang J,Ma F(2017)Ammonium assimilation:An important accessory during aerobic denitrification of Pseudomonas stutzeri T13.Bioresour Technol 234:264–272.]And Pseudomonas stutzeri ADP-19 (3.03 mg. 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 NO in mixed nitrogen source denitrification medium within 12h 3 - -N(62.28±0.74mg/L)、NO 2 - -N (64.26. + -. 0.90 mg/L) and NH 4 + N (50.50 +/-0.55 mg/L) is almost completely removed, and good inorganic nitrogen denitrification capability is shown.
Nitrite is reported to exert toxic effects on microbial cells at high concentrations, thereby strongly inhibiting their denitrification activity, and nitrite concentrations of 20mg/L are also generally considered as thresholds for judging whether microorganisms have denitrification ability [ Song Z, an J, fu G, yang X (2011) Isolation and characterization of An aerobic denitrifying bacterium sp.YX-6from tank culture points.Aquaculture 319 (1-2): 188-193.]. Pseudomonas stutzeri BBW831 NO of 146.42 +/-14.17 mg/L in nitrite denitrification culture medium 2 - The removal rate of-N is about 30 percent, and the strain can remove 64.26 +/-0.90 mg/L NO in the mixed nitrogen source denitrification culture medium 2 - The almost complete removal of-N indicates that Pseudomonas stutzeri BBW831 has good tolerance and removal capability for nitrite. Furthermore, salinity is also another key environmental factor affecting the microbial denitrification process, and an 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 water heater in anaerobic batch reactor. Enzyme Microb technology 34 (3-4): 313-318.]. Salt tolerance of Pseudomonas stutzeri BBW831 salt tolerance of P.stutzeri BBW831 is consistent with the literature reports of P.barus Li Ali Pseudomonas RAD-17 (Pseudomonas balerarcian RAD-17) [ Ruan Y, taherzadeh MJ, kong D, lu H, ZHao H, xu X, liu Y, cai L (2020) Nitrogen removal performance and metabolic pathway analysis of a novel aerobic differentiation halogen strain RAD-17.Microorganisms 8.]And Pseudomonas DN-23 (Pseudomonas sp.DN-23) [ Li D, liang X, wu C (2020 a) Characteristics of nitrogen removal and extracellular polymeric substructures of a novel salt-degrading bacterium, pseudomonas sp.DN-23.FrontMicrobiol11:335.]That is, when the NaCl concentration is 30g/L or more, a certain inhibitory effect is exerted on the denitrification performance of the strain. However, it is worth noting that when a simple strategy of increasing the initial inoculation amount of Pseudomonas stutzeri BBW831 is adopted, the nitrite denitrification rate of the strain within 8h can reach 99.46%, and NO can reach 50g/L of NaCl 3 - The denitrification rate of-N can reach 59.01%.
In conclusion, pseudomonas stutzeri BBW831 isolated from marine habitat has complete genes encoding all functional enzymes of aerobic denitrification denitrogenation pathway, and thus has the function of denitrifying nitrate denitrogenation into N 2 Potential of (NO) 3 - -N→NO 2 - -N→NO→N 2 O→N 2 ) (ii) a The strain shows high-efficiency and rapid denitrification performance, and has good tolerance and adaptability to high-concentration nitrite, naCl salinity and other pressure conditions; the established simple strategy of increasing the initial inoculation amount effectively improves the denitrification capability of the pseudomonas stutzeri BBW831 under the pressure conditions of high-concentration nitrite, naCl salinity and the like. The results show that the Pseudomonas stutzeri BBW831 has good ecological niche adaptability and has good application prospect in the field of wastewater treatment, particularly in the treatment of high-nitrogen and high-salt mariculture wastewater.
The above additional technical features can be freely combined and used in superposition by those skilled in the art without conflict.
The above description is only a preferred embodiment of the present invention, and the technical solutions that achieve the objects of the present invention by substantially the same means are within the protection scope of the present invention.
Claims (8)
1. Pseudomonas stutzeri BBW831, characterized in that it is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2022938.
2. The Pseudomonas stutzeri BBW831 of claim 1, wherein the Bacillus stutzeri BBW831 is obtained by separation and screening of seabed sludge.
3. The Pseudomonas stutzeri BBW831 of claim 1, wherein it has a gene encoding an aerobic denitrification metabolic pathway-functional enzyme.
4. The Pseudomonas stutzeri BBW831 of claim 3, wherein the functional enzymes comprise nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase.
5. The Pseudomonas stutzeri BBW831 of claim 3, wherein said genes include: narG, narH, narJ, narI, napB, napA, nirD, nirB, norD, norB, norC, nirS, norQ, nosZ.
6. Use of the pseudomonas stutzeri BBW831 of any one of claims 1-5 in high salinity and/or high nitrite load conditions.
7. Use according to claim 6, wherein the high salinity is in particular a 40-60g/L NaCl solution.
8. Use according to claim 6, wherein the nitrite is present in a concentration of 100-150mg/L.
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| CN116508784A (en) * | 2023-05-11 | 2023-08-01 | 南京工业大学 | Application of palmitic acid in improving pseudomonas stutzeri in promoting plant growth and reducing nitrous oxide emission of farmland soil |
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| CN114703093A (en) * | 2022-03-18 | 2022-07-05 | 曲阜师范大学 | Facultative anaerobic complete denitrification aerogenic bacterium Y23 and application thereof |
| CN116508784A (en) * | 2023-05-11 | 2023-08-01 | 南京工业大学 | Application of palmitic acid in improving pseudomonas stutzeri in promoting plant growth and reducing nitrous oxide emission of farmland soil |
| CN116508784B (en) * | 2023-05-11 | 2024-01-19 | 南京工业大学 | Application of palmitic acid in improving pseudomonas stutzeri in promoting plant growth and reducing nitrous oxide emission of farmland soil |
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