CN114933988A - Pseudomonas with aerobic denitrification synchronous nitrogen and phosphorus removal performance - Google Patents
Pseudomonas with aerobic denitrification synchronous nitrogen and phosphorus removal performance Download PDFInfo
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Abstract
The invention discloses a Pseudomonas with aerobic denitrification synchronous nitrogen and phosphorus removal performance, which is named as Pseudomonas stutzeri JUST-1 and is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2022234. The bacterial strain obtained by screening and separating can realize the denitrification dephosphorization process under the aerobic condition, and has the performance of synchronously removing nitrogen and phosphorus in sewage: when TN and TP initial concentration are respectively 80mg/L and 90mg/L high-concentration nitrogen phosphorus synthesis sewage, the removal rate of TN and TP in 12 hours reaches 25.38 percent and 26.19 percent, and the removal rate of TN and TP in 24 hours reaches 77.11 percent and 73.57 percent; in actual municipal sewage with TN, TP and COD concentrations of 40.25mg/L, 3.89mg/L and 170mg/L respectively, after 20 hours of reaction, the removal rates of TN, TP and COD are 85.13%, 78.06% and 83.22%.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, relates to a biological method for municipal sewage treatment, and particularly relates to a pseudomonas strain with aerobic denitrification synchronous nitrogen and phosphorus removal performance.
Background
Municipal sewage treatment methods are mainly divided into physical, chemical and biological methods. The physical method is to remove the non-soluble substances in the sewage by adopting the modes of interception, filtration, precipitation and the like, and has simple structure but lower treatment degree. The chemical method is mainly used for removing soluble substances or colloids in sewage by adding medicaments to promote coagulation or generating redox reaction, has good treatment effect, but has higher cost and hidden danger of secondary pollution. The biological method achieves the purpose of purifying water quality by using microorganisms to absorb nutrient substances in sewage for metabolism, and has become a main method for removing nitrogen and phosphorus from municipal sewage due to higher treatment degree and lower cost.
Aiming at the current trend that municipal sewage discharge amount is sharply increased and low C/N ratio is shown, the traditional biological nitrogen and phosphorus removal technology has many problems of carbon source competition, sludge age contradiction, microbial mutual competition, substandard nitrogen and phosphorus discharge and the like. In order to solve the problems and the contradiction, on the basis of the traditional nitrogen and phosphorus removal principle, researchers develop a great deal of research aiming at improving a treatment device, controlling operation conditions, screening high-efficiency nitrogen and phosphorus removal strains and the like, and put forward a plurality of theories in the field of biological nitrogen and phosphorus removal.
In recent years, researchers put forward a theory of synchronously removing nitrogen and phosphorus by using aerobic denitrification, namely under an aerobic condition, aerobic denitrifying bacteria oxidize PHB by using nitrate or nitrite as an electron acceptor to obtain energy to promote the absorption of phosphorus. The microorganisms involved in the theory are aerobic denitrifying phosphorus removal bacteria, and are highly concerned by students due to the low requirement on carbon source, obvious nitrogen and phosphorus removal effect and easy control of reaction conditions. The screened aerobic denitrification phosphorus removal bacteria can enhance the effect of nitrogen and phosphorus removal, avoid competition among different functional strains, reduce the requirement of a carbon source, simplify a sewage treatment device, solve the key problem of the traditional nitrogen and phosphorus removal method to a certain extent, and is expected to provide a new idea for municipal sewage treatment.
The reported aerobic denitrifying phosphorus removal bacteria are few in types, mainly exist in pseudomonas, bacillus, ceramic bacterium, acinetobacter and other bacteria, are harsh in culture conditions, have low removal efficiency of nitrogen and phosphorus in sewage and are unstable in performance.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects of the prior art and simultaneously, efficiently and stably remove nitrogen and phosphorus in sewage in an aerobic tank, the invention provides pseudomonas with aerobic denitrification synchronous nitrogen and phosphorus removal performance.
The technical scheme is as follows: the Pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance is named as Pseudomonas stutzeri JUST-1 and is preserved in China center for type culture Collection with the address of China, Wuhan university, 430072; the preservation number is CCTCC NO: m2022234, date of deposit: 3, month and 9 days 2022. The strain is gram-negative bacteria, is short rod-shaped and has the length of 1-1.2 mu m. Bacterial colonies are milky yellow, translucent, irregular and flat in edges, smooth and viscous in surface and not easy to pick.
Preferably, the 16S rDNA sequence of the strain is shown in SEQ ID NO. 1:
AGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGAGTGGAGCTTGCTCCATGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTATGGCAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGGCTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCATTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTAAGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCTTCGGGGGGACGGTTACCACGGAGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCT。
the above sequence has been submitted to NCBI under accession number SRR 18120113. The 16S rDNA sequence is compared with a GenBank database by a BLAST analysis method, and the genetic relationship between the strain and Pseudomonas stutzeri is the closest, and the homology is as high as 99.99%.
The separation method of the pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance comprises the following steps:
s1, taking activated sludge in an aerobic pool of a sewage treatment plant, acclimatizing and culturing in an SBR reactor, taking fresh activated sludge at the last stage of an aerobic stage of the reactor, shake-culturing for 1-2 days according to the volume ratio of the sludge to the sterile denitrification dephosphorization culture medium of 1:9, transferring the cultured suspension into the fresh sterile denitrification dephosphorization culture medium according to the volume ratio of 1:9, and repeatedly transferring for 1 time every 2 days until the culture medium is uniform and has no sediment; wherein the activated sludge is sourced from an aerobic tank of a Zhenjiang Zhenzhou sewage treatment plant;
s2, taking the bacterial suspension subjected to selective enrichment of S1, performing gradient dilution by using sterile water, then coating the bacterial suspension on a denitrifying phosphorus removal agar culture medium plate, and performing inverted culture for 2 days;
s3, purifying the screened single bacterial colony by adopting a plate marking method, and repeatedly marking and purifying for 5-7 generations until a uniform single bacterial colony is formed and no abnormal bacterial colony appears;
s4, scraping a single thriving bacterium colony from the flat plate by using an inoculating ring, culturing the single thriving bacterium colony in a sterile denitrification dephosphorization culture medium for 24 hours by using a shaking table, taking the bacterium suspension in a centrifuge tube, centrifuging, pouring supernatant, washing by using PBS buffer solution, and re-suspending to prepare inoculation liquid.
Preferably, 1mL of the bacterial suspension in S2 is diluted 10 times, 100 times, 1000 times and 10000 times with sterile water.
Preferably, the temperature for culturing is 30-37 ℃.
Preferably, the denitrifying phosphorus removal culture medium comprises the following components: 1.0g/L NaCl, 0.5g/L MgCl 2 ，0.2g/L KH 2 PO 4 ，0.3g/L KCl，0.015g/L CaCl 2 ，80mg-N/L KNO 3 600mg-C/L lactic acid and 50mL Vickers salt solution, and the pH value is 7.2-7.4. The Vickers salt solution comprises the following components: 6.55g/L K 2 HPO 4 ·3H 2 O，2.5g/L MgSO 4 ·7H 2 O，2.5g/L NaCl，0.038g/L MnSO 4 ·H 2 O，0.05g/L FeSO 4 ·7H 2 And (O). The composition of the PBS buffer solution is as follows: 8.0g NaCl, 0.2g KCl、1.44g Na 2 HPO 4 、0.24g KH 2 PO 4 The pH was 7.4.
The pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance is applied to the sewage treatment process.
Preferably, the OD600 value of the inoculation liquid is 0.5A, the inoculation liquid is inoculated into the denitrification dephosphorization culture medium in a volume fraction of 1-5%, and the volume ratio of the inoculation liquid to the sewage is 1: 100-1: 20.
Preferably, when the initial concentration of TN and TP in the high-concentration nitrogen-phosphorus synthetic sewage is 80mg/L and 90mg/L respectively, the removal rate of the strain to TN and TP in 12 hours reaches 25.38% and 26.19%, and the removal rate of the strain to TN and TP in 24 hours reaches 77.11% and 73.57%.
Preferably, when the concentration of TN, TP and COD in the actual municipal sewage is 40.25mg/L, 3.89mg/L and 170mg/L respectively, the removal rate of TN, TP and COD in the sewage by the strain reaches 85.13%, 78.06% and 83.22% after 20 hours of reaction.
The principle of the strain for realizing the treatment of the high-concentration nitrogen and phosphorus sewage is as follows: under aerobic conditions, the denitrifying phosphorus removal bacteria oxidize PHB (poly-beta-hydroxybutyrate) in bacteria to obtain energy by taking nitrate or nitrite as an electron acceptor, so that inorganic phosphate in solution is absorbed to synthesize a phosphorus-containing polymer, and the aim of removing phosphorus in sewage is fulfilled.
Has the advantages that: the strain obtained by screening and separating can realize the performance of simultaneously removing nitrogen and phosphorus in sewage: when the initial concentration of TN and TP in the high-concentration nitrogen-phosphorus synthetic sewage is 80mg/L and 90mg/L respectively, the removal rate of TN and TP in 12 hours reaches 25.38 percent and 26.19 percent, and the removal rate of TN and TP in 24 hours reaches 77.11 percent and 73.57 percent; when the concentration of TN, TP and COD in the actual municipal sewage is respectively 40.25mg/L, 3.89mg/L and 170mg/L, the removal rate of TN, TP and COD after 20 hours of reaction reaches 85.13%, 78.06% and 83.22%.
Drawings
FIG. 1 is a gram stain chart of Pseudomonas stutzeri JUST-1 of the present invention.
FIG. 2 is a colony morphology map of Pseudomonas stutzeri JUST-1 of the present invention.
FIG. 3 is a morphological electron micrograph of Pseudomonas stutzeri JUST-1 of the present invention.
FIG. 4 is a tree diagram showing the development of the 16S rDNA sequence of Pseudomonas stutzeri JUST-1 and related species according to the present invention.
FIG. 5 is a diagram showing the denitrification and dephosphorization effect of Pseudomonas stutzeri JUST-1 on high-concentration nitrogen-phosphorus synthetic wastewater.
FIG. 6 is a graph showing the denitrification and dephosphorization effect of Pseudomonas stutzeri JUST-1 on the actual municipal sewage under aerobic conditions.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting thereof. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1: isolation, screening and characterization of strains
(I) Material preparation
1. Active sludge is obtained from an aerobic tank of a wastewater treatment plant of Zhenjiang province, China, and is put into an SBR reactor which runs stably in a laboratory for acclimatization culture, and fresh active sludge is collected at the terminal stage of an aerobic stage of the reactor to be used as a source for screening aerobic denitrifying phosphorus removal bacteria.
2. Culture medium
Denitrifying phosphorus removal culture medium: 1.0g/L NaCl, 0.5g/L MgCl 2 ，0.2g/L KH 2 PO 4 ，0.3g/L KCl，0.015g/L CaCl 2 ，80mg-N/L KNO 3 600mg-C/L lactic acid and 50mL Vickers salt solution, and the pH value is 7.2-7.4.
Vickers salt solution: 6.55g/L K 2 HPO 4 ·3H 2 O，2.5g/L MgSO 4 ·7H 2 O，2.5g/L NaCl，0.038g/L MnSO 4 ·H 2 O，0.05g/L FeSO 4 ·7H 2 O。
3. Laboratory apparatus and device
High-pressure steam sterilization pot, constant-temperature incubator, shaking table, ultraviolet-visible spectrophotometer, centrifugal machine and superclean bench.
(II) isolation and screening of the Strain
(1) 10mL of fresh activated sludge was transferred to a culture medium containing 90mL of sterile denitrifying phosphorus removal liquid and stirred to obtain a uniform suspension. After shaking culture at 32 ℃ and 120rpm for 1-2 days, 10mL of the suspension is transferred to 90mL of fresh sterile denitrifying phosphorus removal liquid culture medium. Transfer was repeated every 2 days until the medium was homogeneous and sediment-free. Taking 1mL of bacterial suspension which finishes the selective enrichment process, carrying out gradient dilution by using sterile water for 10, 100, 1000 and 10000 times, taking 200 mu L of diluent in each of the four gradients, coating the diluent on a denitrifying phosphorus removal agar culture medium plate, and placing the plate in a constant-temperature incubator at 32 ℃ for inverted culture for 2 days. And (3) purifying the screened single bacterial colony by adopting a flat plate scribing method, repeatedly scribing and purifying for 5-7 generations until an even single bacterial colony is formed, and preventing abnormal bacterial colonies from appearing. Scraping a thriving single bacterium from a flat plate by using an inoculating ring, dropping the thriving single bacterium into a sterilized denitrifying phosphorus removal culture medium, placing the culture medium into a shaker at 32 ℃ and 120rpm, culturing the bacterium suspension in the centrifuge tube after 24 hours, centrifuging the centrifuge tube for 5 minutes under the condition of 8000r/min, pouring supernatant, washing the supernatant for three times by using PBS buffer solution, and then carrying out resuspension to prepare the bacterium suspension serving as an inoculating liquid of a subsequent experiment, wherein the OD600 value of the bacterium suspension is about 0.5A. The inoculation liquid is inoculated into a 250mL conical flask filled with 100mL denitrifying phosphorus removal liquid culture medium in an inoculation amount of 2% (v/v), the conical flask is placed at 30 ℃ and is continuously cultured for 24h at 160rpm, samples are taken at 12h and 24h of the culture to determine the growth amount (OD600) of the strain and the concentration of TN and TP in a supernatant, and the TN and TP removal rate is calculated.
(2) After obtaining a stable single bacterial strain with the synchronous denitrification and dephosphorization function under the aerobic condition, taking the single bacterial strain to amplify in a denitrification and dephosphorization culture medium, washing the single bacterial strain for three times by using PBS buffer solution, then resuspending the single bacterial strain into bacterial suspension with OD600 of 0.5A, and storing the bacterial suspension at 4 ℃ for later use.
(III) characterization and identification of the Strain
1. Morphological characteristics and physiological and biochemical characteristics of bacterial colony
The obtained single colony with consistent size and shape is named as Pseudomonas stutzeri JUST-1, and the colony is milky yellow, translucent, irregular and flat in edge, smooth and viscous in surface and difficult to pick. The gram stain of the bacterium is negative (figure 1), and the bacterium is in the shape of a short rod and has the length of 1-1.2 mu m. The optimal growth temperature is 30-37 ℃, the optimal growth pH is 7-8, the colony morphology is shown in figure 2, and the bacteria morphology is shown in figure 3 of a scanning electron microscope.
2. Molecular identification of 16S rRNA of Pseudomonas stutzeri JUST-1
Adding 400 μ L Buffer SCL into the thallus to crack thallus, water bathing at 65 deg.C for about 1h, and mixing for 1 time every 10min until the mixture becomes clear. Then centrifuging and taking supernatant fluid for DNA extraction. The genome DNA was extracted by Ezup columnar bacteria genome DNA extraction kit (Shanghai Producer). Using the extracted DNA as a template, and carrying out PCR amplification by using a universal primer pair 27F/1492R aiming at the 16S rRNA gene of the bacteria, wherein the sequences of the primers are as follows: 27F (SEQ ID No.2), AGAGAGTTTGATCCTGGCTCAG; 1492R (SEQ ID NO.3), TACGGYTACCTTGTTACGACTT. And (3) delivering the PCR product to Shanghai workers for sequencing, comparing and analyzing a 16S rDNA sequence with a GenBank database by adopting a BLAST analysis method, finding that the genetic relationship of the strain and the Pseudomonas stutzeri is closest, the homology is as high as 99.99 percent, and integrating the physiological and biochemical and molecular identification results of the strain, wherein the strain is named as Pseudomonas stutzeri JUST-1. Sequences with higher homology were downloaded in GenBank and phylogenetic trees were constructed using the Neighbor-join algorithm in MEGA, see FIG. 4.
Example 2: application of Pseudomonas stutzeri JUST-1 in high-concentration nitrogen and phosphorus synthesis sewage treatment
The treatment capacity of the Pseudomonas stutzeri JUST-1 on high-concentration nitrogen and phosphorus synthetic sewage is investigated, and the degradation capacity of the Pseudomonas stutzeri JUST-1 on TN and TP in the synthetic sewage is studied. The synthetic sewage comprises the following components: 0.2g/LKH 2 PO 4 ，6.5g/L K 2 HPO 4 ·3H 2 O，0.6g/L KNO 3 0.563g/L lactic acid, pH 7.2, and autoclave at 121 ℃ for 20 minutes. Adding 100mL of synthetic sewage into a 250mL conical flask, adding 2mL of Pseudomonas stutzeri JUST-1 fixed bacterial suspension with OD600 value of about 0.5A, performing shake culture at 30 ℃ for 24h at 120 r/min, sampling every 6h, measuring the content of TN and TP in the solution, and calculating the removal rate of each index, as shown in FIG. 5. Results tableBright: the removal rates of the strain to TN and TP at 12h are respectively 25.38% and 26.19%, and the removal rates to TN and TP at 24h reach the maximum, respectively 77.11% and 73.57%.
Example 3: application of Pseudomonas stutzeri JUST-1 in actual municipal sewage treatment
The capability of Pseudomonas stutzeri JUST-1 in treating the actual municipal sewage is investigated, and the removal effect of the bacterium on TN, TP and COD of the actual municipal sewage is studied. In 3 groups of SBR reactors (the effective volume is 3L) which are operated in a laboratory simulation mode, the reactors are operated alternately in an anaerobic (8h) and aerobic (12h) mode, and only 100mL of Pseudomonas stutzeri JUST-1 bacterial suspension is added into an R1 reactor at the initial reaction; only 3500mg/L of activated sludge is added into the R2 reactor; the R3 reactor was charged with 3500mg/L activated sludge and 100mL of Pseudomonas stutzeri JUST-1 suspension. The content of municipal sewage in the SBR reactor is 2L, the sewage is taken from a Zhenzhou sewage treatment plant, the TN concentration is 40.25mg/L, the TP concentration is 3.89mg/L, and the COD concentration is 170 mg/L. Sampling once every 3 hours in the aerobic stage of the SBR reactor, and detecting the removal rate results of TN, TP and COD in the reactor as shown in figure 6, wherein after 20 hours of reactor operation, the removal rates of R1 to TN, TP and COD are 85.13%, 78.06% and 83.22% respectively; the removal rates of R2 to TN, TP and COD are respectively 62.98%, 57.84% and 67.94%; the removal rates of TN, TP and COD by R3 were 91.54%, 84.38% and 90.07%, respectively. The removal rate of TN, TP and COD in the R1 and R3 reactors added with Pseudomonas stutzeri JUST-1 is improved to a different extent than that in the R2 reactor.
Sequence listing
<110> university of Jiangsu science and technology
Zhenjiang microenvironment science and technology limited
<120> pseudomonas with aerobic denitrification synchronous nitrogen and phosphorus removal performance
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1525
<212> DNA
<213> Pseudomonas stutzeri
<400> 1
agagtttgat catggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60
ggatgagtgg agcttgctcc atgattcagc ggcggacggg tgagtaatgc ctaggaatct 120
gcctggtagt gggggacaac gtttcgaaag gaacgctaat accgcatacg tcctacggga 180
gaaagtgggg gatcttcgga cctcacgcta tcagatgagc ctaggtcgga ttagctagtt 240
ggtgaggtaa aggctcacca aggcgacgat ccgtaactgg tctgagagga tgatcagtca 300
cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga atattggaca 360
atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag 420
cactttaagt tgggaggaag ggcagtaagt taataccttg ctgttttgac gttaccaaca 480
gaataagcac cggctaactt cgtgccagca gccgcggtaa tacgaagggt gcaagcgtta 540
atcggaatta ctgggcgtaa agcgcgcgta ggtggttcgt taagttggat gtgaaagccc 600
cgggctcaac ctgggaactg catccaaaac tggcgagcta gagtatggca gagggtggtg 660
gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt ggcgaaggcg 720
accacctggg ctaatactga cactgaggtg cgaaagcgtg gggagcaaac aggattagat 780
accctggtag tccacgccgt aaacgatgtc gactagccgt tgggatcctt gagatcttag 840
tggcgcagct aacgcattaa gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca 900
aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960
aagaacctta ccaggccttg acatgcagag aactttccag agatggattg gtgccttcgg 1020
gaactctgac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttaaggtg ggcactctaa 1140
ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat catggccctt 1200
acggcctggg ctacacacgt gctacaatgg tcggtacaaa gggttgccaa gccgcgaggt 1260
ggagctaatc ccataaaacc gatcgtagtc cggatcgcag tctgcaactc gactgcgtga 1320
agtcggaatc gctagtaatc gtgaatcaga atgtcacggt gaatacgttc ccgggccttg 1380
tacacaccgc ccgtcacacc atgggagtgg gttgctccag aagtagctag tctaaccttc 1440
ggggggacgg ttaccacgga gtgattcatg actggggtga agtcgtaaca aggtagccgt 1500
aggggaacct gcggctggat cacct 1525
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tacggytacc ttgttacgac tt 22
Claims (10)
1. The Pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance is characterized in that the strain is named as Pseudomonas stutzeri JUST-1 and is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2022234.
2. The pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance according to claim 1, wherein the 16S rDNA sequence of the pseudomonas is shown as SEQ ID No. 1.
3. The separation method of the pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance as claimed in claim 1 or 2, which is characterized by comprising the following steps:
s1, taking activated sludge in an aerobic pool of a sewage treatment plant, acclimatizing and culturing in an SBR reactor, taking fresh activated sludge at the last stage of an aerobic stage of the reactor, shake-culturing for 1-2 days according to the volume ratio of the sludge to the sterile denitrification dephosphorization culture medium of 1:9, transferring the cultured suspension into the fresh sterile denitrification dephosphorization culture medium according to the volume ratio of 1:9, and repeatedly transferring for 1 time every 2 days until the culture medium is uniform and has no sediment;
s2, taking the bacterial suspension subjected to selective enrichment of S1, performing gradient dilution by using sterile water, then coating the bacterial suspension on a denitrifying phosphorus removal agar culture medium plate, and performing inverted culture for 2 days;
s3, purifying the screened single bacterial colony by adopting a plate marking method, and repeatedly marking and purifying for 5-7 generations until a uniform single bacterial colony is formed and no abnormal bacterial colony appears;
s4, scraping a single thriving bacterium colony from the flat plate by using an inoculating ring, culturing the single thriving bacterium colony in a sterile denitrification dephosphorization culture medium for 24 hours by using a shaking table, taking the bacterium suspension in a centrifuge tube, centrifuging, pouring supernatant, washing by using PBS buffer solution, and re-suspending to prepare inoculation liquid.
4. The method for separating the pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance according to claim 3, wherein 1mL of bacterial suspension is taken from S2, and the bacterial suspension is respectively diluted by 10 times, 100 times, 1000 times and 10000 times with sterile water.
5. The separation method of the pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance as claimed in claim 3, characterized in that the culture temperature is 30-37 ℃.
6. The method for separating the pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance according to claim 3, wherein a denitrification phosphorus removal culture medium comprises the following components: 1.0g/L NaCl, 0.5g/L MgCl 2 ，0.2g/L KH 2 PO 4 ，0.3g/L KCl，0.015g/L CaCl 2 ，80mg-N/L KNO 3 600mg-C/L lactic acid and 50mL Vickers salt solution, and the pH value is 7.2-7.4.
7. The application of the pseudomonas with the aerobic denitrification synchronous nitrogen and phosphorus removal performance in the sewage treatment process as claimed in claim 1 or 2.
8. The application of claim 7, wherein the OD600 value of the inoculation liquid is 0.5A, the inoculation liquid is inoculated into the denitrification dephosphorization culture medium in a volume fraction of 1-5%, and the volume ratio of the inoculation liquid to the sewage is 1: 100-1: 20.
9. The use of claim 7, wherein the strains achieve 25.38% and 26.19% removal rate of TN and TP in 12 hours, and 77.11% and 73.57% removal rate of TN and TP in 24 hours when the initial concentration of TN and TP in the high-concentration nitrogen-phosphorus synthetic sewage is 80mg/L and 90mg/L respectively.
10. The use of claim 7, wherein the strains achieve the removal rate of TN, TP and COD in the sewage of 85.13%, 78.06% and 83.22% after 20 hours of reaction when the concentration of TN, TP and COD in the actual municipal sewage is 40.25mg/L, 3.89mg/L and 170mg/L respectively.
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|---|---|---|---|---|
| CN103114062A (en) * | 2013-01-29 | 2013-05-22 | 山东大学 | Denitrifying phosphate-accumulating organism with nitrogen and phosphorus removal functions and applications thereof |
| CN106754570A (en) * | 2017-02-28 | 2017-05-31 | 重庆大学 | One plant of Pseudomonas stutzeri and its application |
| CN109943497A (en) * | 2019-01-25 | 2019-06-28 | 浙江省农业科学院 | One plant of Pseudomonas stutzeri and application thereof with aerobic denitrification function |
| CN113174345A (en) * | 2021-05-13 | 2021-07-27 | 安徽省农业科学院水产研究所 | Heterotrophic nitrification-aerobic denitrification strain for efficient denitrification and application thereof |
| CN113249254A (en) * | 2021-05-13 | 2021-08-13 | 安徽省农业科学院水产研究所 | Pseudomonas nitroreducens strain and application thereof |
| US20210269341A1 (en) * | 2019-01-15 | 2021-09-02 | Agro-environmental Protection Institute, Ministry Of Agriculture And Rural Affairs | Las-degrading and/or n-removing bacterium and application thereof |
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114062A (en) * | 2013-01-29 | 2013-05-22 | 山东大学 | Denitrifying phosphate-accumulating organism with nitrogen and phosphorus removal functions and applications thereof |
| CN106754570A (en) * | 2017-02-28 | 2017-05-31 | 重庆大学 | One plant of Pseudomonas stutzeri and its application |
| US20210269341A1 (en) * | 2019-01-15 | 2021-09-02 | Agro-environmental Protection Institute, Ministry Of Agriculture And Rural Affairs | Las-degrading and/or n-removing bacterium and application thereof |
| CN109943497A (en) * | 2019-01-25 | 2019-06-28 | 浙江省农业科学院 | One plant of Pseudomonas stutzeri and application thereof with aerobic denitrification function |
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