CN115181689A - Pseudosingle-cell shigella and application thereof - Google Patents
Pseudosingle-cell shigella and application thereof Download PDFInfo
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Abstract
The invention discloses a Pseudomonas stutzeri strain, which is named as Pseudomonas stutzeri LJ-1 in Latin science, has a preservation number of CCTCC NO: M2022353, and is preserved in China center for type culture Collection in Wuhan City, hubei province within 3 months and 31 days in 2022. The pseudomonas stutzeri strain LJ-1 can realize denitrification, is convenient to use and simple to operate, and reduces the sewage treatment cost; the strain LJ-1 can be prepared into a novel microecological bactericide and has good application prospect in composite wastewater treatment.
Description
Technical Field
The invention belongs to the field of microorganisms, and relates to a pseudounicellular shigella and application thereof.
Background
With the rapid development of economy, a great deal of industrial wastewater and urban sewage is increasingly discharged, and some untreated wastewater is discharged to rivers and lakes, so that the water body is seriously polluted, wherein the eutrophication of the water body is one of the most common problems, and the main reason of the eutrophication of the water body is that the nitrogen content in the water is too high, which can cause harm to some aquatic organisms and even human beings. Therefore, the denitrification of the sewage is urgent. Biological denitrification is a main way for removing nitrogen pollution in water. In the traditional denitrification process, the wastewater mainly passes through two processes of autoxidation and heterotrophic denitrification, and the two processes are respectively carried out in an aerobic reactor and an anoxic reactor, so that the traditional biological denitrification system is complex and high in cost.
The aerobic denitrification refers to a process of converting nitrate nitrogen into gaseous nitrogen under the action of a series of enzymes by aerobic denitrifying bacteria under aerobic conditions. The device can realize the nitrification and denitrification in the same reactor, thereby saving the floor area and the investment cost to a great extent. Although the enhanced application of the aerobic denitrifying bacteria can effectively increase the denitrification efficiency of the sewage treatment system in a short time, the loss of the bacteria is serious, and the bacteria cannot be stably maintained in the system for a long time. Microorganism aggregation is a key step for development and maintenance of a biological membrane, and the bacterial strain with high-efficiency membrane forming and denitrification capabilities can realize functional bacteria residence and high-efficiency denitrification.
Various aerobic denitrifying strains have been disclosed in the prior art, such as Acinetobacter (Acinetobacter) SYF26 [1] Achromobacter sp L16 [2] Klebsiella oxytoca strain YZ-12 [3] A63 (MN 093321), aeromonas caviae (Aeromonas caviae) RC-15 [4] . The above-listed Acinetobacter SYF26, achromobacter L16, klebsiella YZ-12 and Aeromonas caviae RC-15 all have high denitrificationThe efficiency can reach more than 95 percent, and the nitrogen in the water can be effectively removed; the Zubeniella A63 can not only remove nitrogen, but also can tolerate high-salinity wastewater, but can only complete denitrification in a neutral environment; acinetobacter SYF26, klebsiella YZ-12 and Aeromonas caviae RC-15 have high denitrification efficiency under low carbon nitrogen ratio, but can only adapt to neutral or weak alkaline environment, and cannot finish high denitrification efficiency under acidic environment.
The literature is presented:
[1]Su JF,Zhang K,Huang TL,Wen G,Guo L,Yang SF.Heterotrophic nitrification and aerobic denitrification at low nutrient conditions by a newly isolated bacterium,Acinetobacter sp.SYF26.Microbiology(Reading).2015Apr；161(Pt 4):829-837.
[2] the denitrification characteristics of the aerobic denitrifying bacteria Achromobacter sp.L16 [ J ] biotechnological report 2020, v.36; no.335 (06): 93-101.
[3] Leifeng, zhang Yan, sunyan Yan, liu \21855, the denitrification performance of heterotrophic nitrification-aerobic denitrifying bacteria YZ-12 and the treatment effect on the culture waste water [ J ]. Report of environmental engineering, 2022,16 (01): 301-310.
[4] Screening and identification of an aerobic denitrifying bacterium and denitrification characteristics [ J/OL ] of the aerobic denitrifying bacterium, namely Zhuhongxu, yanbenqin, high girl, dianthus hai and Yanqin, 2022.
Disclosure of Invention
In order to solve the problems, the invention provides a Pseudomonas stutzeri LJ-1 strain which has the function of aerobic denitrification and can directly convert nitrate nitrogen into a gas product; the device is applied to sewage treatment, is convenient to use and simple to operate, and reduces the sewage treatment cost.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
firstly, a Pseudomonas stutzeri strain is provided, the Pseudomonas stutzeri strain is Pseudomonas stutzeri strain LJ-1, the preservation number is M2022353, the Latin chemical name is Pseudomonas stutzeri LJ-1, the Pseudomonas stutzeri strain is preserved in China center for type culture preservation at 31 months 3 in 2022, and the preservation address is Wuhan city, hubei province in China.
Further, a preparation comprising said pseudomonas stutzeri strain is provided.
Further, the type of the preparation is any one of culture, culture extract, freeze-dried powder, fermentation liquor precipitate, fermentation liquor supernatant and fermentation liquor extract.
Furthermore, the freeze-dried powder comprises thallus of pseudomonas stutzeri LJ-1 and may also comprise a freeze-drying protective agent.
Further, the lyoprotectant can be one or more of polyhydroxy compounds, sugar, protein, polymers, amino acids, salts, amines and surfactants.
Further, a culture method of the pseudomonas stutzeri strain is provided, which comprises inoculating the pseudomonas stutzeri strain to a culture medium.
Further, the culture method is carried out under aerobic conditions with culture medium nitrogen concentration of 50-100mg/L, carbon-nitrogen ratio of 5-50, pH value of 5-11, and culture temperature of 25-35 deg.C.
Further, the culture method comprises the following steps: taking out the stored pure strain sample for later use; preparing a denitrification culture medium, and sterilizing at 121 ℃ for later use; inoculating a pure strain sample to a sterile denitrification culture medium; putting the strain into an incubator until the strain expansion culture is completed.
In some embodiments, the culturing method comprises the steps of: taking out the stored pure strain sample for later use; preparing a denitrification culture medium, and sterilizing at 121 ℃ for later use; inoculating a pure strain sample to a sterile denitrification culture medium in a clean bench; putting the strain into an incubator to wait for the completion of strain expansion culture.
Further, a microbial inoculum comprising said pseudomonas stutzeri strain is provided.
Preferably, the microbial inoculum can be prepared by culture or freeze-dried powder of pseudomonas stutzeri LJ-1.
Further, a sewage treatment agent comprising the pseudomonas stutzeri strain or the preparation is provided.
Preferably, the wastewater treatment agent comprises a culture of the pseudomonas stutzeri strain cultured to log phase.
Further, there is provided a method for treating wastewater, comprising adding said Pseudomonas stutzeri strain to wastewater.
Further, the pseudomonas stutzeri strain and/or the preparation are applied to sewage treatment.
Further, in the application, the strain is added into the sewage after the strain is subjected to amplification culture, or the strain is directly added into the sewage.
Further, the Pseudomonas stutzeri strain was cultured to a logarithmic phase, and the culture was added to sewage.
Further, the inoculation amount of the culture is 1% -5%.
Preferably, the inoculum size of the culture is 1% -3%, more preferably, the inoculum size of the culture is 1%.
Further, the sewage may be production sewage or domestic sewage, and the production sewage includes industrial sewage, agricultural sewage, and medical sewage.
Further, the sewage is sewage with high nitrogen content.
Furthermore, the sewage is wastewater generated by using a nitrate material as a raw material or an oxidant in the chemical industry, and wastewater generated by using nitrate or nitrite as an antioxidant in a livestock feed factory; the wastewater containing a large amount of ammonia nitrogen generated by oil refining, ferroalloy and the like is oxidized or nitrified to generate the high-concentration nitrate nitrogen wastewater.
Compared with the prior art, the invention has the following beneficial effects:
(1) The LJ-1 strain provided by the invention has an aerobic denitrification function, can directly convert nitrate nitrogen into a gas product, has less accumulation of nitrite nitrogen and ammonia nitrogen, and has NO within 20 hours when nitrate nitrogen is taken as a unique nitrogen source 3 - The concentration of N is reduced from the initial 100mg/L to 4.47mg/L, the removal rate is 4.83 mg/(L.h), and the removal rate reaches 96.53 percent, which is much higher than the removal rate of 78.4 percent of the monospora salina (Halomonas sp.) strain GJWA3 and the removal rate1.63 mg/(L.h) of the removal rate (from literature: su Mec, li \36191, pan Lu Qing, he Zi Yan, liu Liping, sinus Lei, zhang Meng.) A novel heterotrophic nitrification-aerobic denitrifying bacterium GJWA3 was used for the denitrification performance and quantitative determination [ J.sub.j.h ]]The university of china oceans journal (nature science edition), 2021, v.51; no.327 (10): 44.).
(2) The LJ-1 strain provided by the invention can have 90% of nitrogen removal capacity in a weak acid environment, which is obviously higher than that of the strain A63 (10% -20%) (from the literature: zhao Lin, duckweed, wujin Fang, pangweichen, mongolian and Congwang. The separation and identification of a salt-tolerant Zobenella sp and the nitrate reduction capacity [ J ]. Microbiological report 2020,47 (05): 1354-1365.).
(3) The pseudomonas stutzeri strain LJ-1 provided by the invention can realize denitrification, is convenient to use and simple to operate, and reduces the sewage treatment cost. The strain LJ-1 can be prepared into a novel microecological bactericide and has good application prospect in composite wastewater treatment.
(4) The Pseudomonas stutzeri strain LJ-1 provided by the invention has certain self-aggregation capability, and the self-aggregation and hydrophobicity indexes are 66% and 59% respectively. The self-aggregation ability was more significant compared to strain YL (self-aggregation ability 54.3%) studied by wanxia et al, strain LJ-1 (wanxia, denitrification and self-aggregation properties study of aerobic denitrifying bacteria Enterobacter sp.fl [ D.
Deposit description
The name of Chinese: pseudomonas stutzeri LJ-1;
latin learning name: pseudomonas stutzeri LJ-1;
the preservation number is as follows: CCTCC NO: M2022353;
the preservation organization: china center for type culture Collection;
the preservation address is as follows: wuhan city, hubei province, china;
preservation time: 3 months and 31 days 2022.
Drawings
FIG. 1 is a tree model of the evolution of strains;
FIG. 2 shows strain LJ-1 in NO 3 - Nitrogen removal capacity when N is a nitrogen source;
FIG. 3 shows strain LJ-1 at NH 4 + Nitrogen removal capacity when N is a nitrogen source;
FIG. 4 shows strain LJ-1 in NO 2 - Nitrogen removal capacity when N is a nitrogen source;
FIG. 5 is a graph showing the effect of different C/N on the nitrogen removal capacity of strain LJ-1;
FIG. 6 is a graph showing the effect of different pH on the nitrogen removal capacity of strain LJ-1;
FIG. 7 is the self-aggregation index of the strains at different incubation times;
FIG. 8 is a graph showing the hydrophobicity index of the strain at different incubation times.
Detailed Description
It should be noted that the raw materials used in the present invention are all common commercial products, and the sources thereof are not particularly limited.
Strain screening:
inoculating 5mL of activated sludge to a denitrification enrichment culture medium, culturing for three days, then inoculating 5mL of activated sludge to a fresh denitrification enrichment culture medium, repeating for 5 times, performing gradient dilution and coating on the culture solution of the last round to a denitrification identification culture medium, selecting a target colony, performing streak purification, and then verifying by using a denitrification test culture medium to finally obtain the pseudomonas stutzeri strain LJ-1.
Denitrification enrichment medium (amount of each component per liter of medium): KNO 3 5.0g, sodium succinate hexahydrate 16.67g, KH 2 PO 4 1.5g、Na 2 HPO 4 ·12H 2 O 10.55g、MgSO 4 0.1g、NH 4 Cl 0.3g, trace elements 2mL (trace elements composition (g. L) -1 )：FeCl 2 ·4H 2 O 1.8、CoCl 2 ·6H 2 O 0.25、NiCl 2 ·6H 2 O 0.01、CuCl 2 ·2H 2 O 0.01、MnCl 2 ·4H 2 O 0.70、ZnCl 2 0.1、H 3 BO 3 0.5、Na 2 MoO 4 ·2H 2 O 0.03、Na 2 SeO 3 ·5H 2 O 0.01)，pH 7.0。
EXAMPLE 1 morphological and molecular biological characterization of Strain LJ-1
The colony morphology of the pseudomonas stutzeri strain of the invention is as follows: yellowish, irregular edge, wrinkled surface, moist and translucent.
Molecular biological identification: using an amplification universal primer of a 16S rDNA gene, an upstream primer (27F) SEQ ID NO.1 and a downstream primer (1429R) SEQ ID NO.2, and taking LJ-1 bacterial liquid as a template to carry out PCR amplification to obtain a PCR product of 1449bp (SEQ ID NO. 3); the sequence was analyzed for sequence homology by the BLAST search program system of the National Center for Biotechnology Information (NCBI), and found to have 97% similarity to the 16S rDNA gene sequence of Pseudomonas stutzeri. FIG. 1 shows a phylogenetic tree constructed from the 16S rDNA gene of Pseudomonas stutzeri strain LJ-1.
According to the morphological characteristics, physiological and biochemical properties and molecular biological identification characteristics of the LJ-1 strain, the LJ-1 strain is identified to belong to Pseudomonas stutzeri and named as Pseudomonas stutzeri LJ-1 (Pseudomonas stutzeri LJ-1). Is preserved in China Center for Type Culture Collection (CCTCC) at 31/3/2022 with the preservation number of CCTCC NO: M2022353.
Example 2 Nitrogen removal of Strain LJ-1 under different Nitrogen sources
Culturing the strain LJ-1 to logarithmic phase, and inoculating to NH according to 1% of inoculum size 4 + -N、NO 3 - -N and NO 2 - Culturing in an-N culture medium at 30 ℃ and a table rotating speed of 160r/min for 60h, and sampling every 10h to determine the content change of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. The common components of the ammonia nitrogen, nitrate nitrogen and nitrite nitrogen test culture medium are as follows (the amount of each component in each liter of culture medium): sodium succinate hexahydrate 16.67g KH 2 PO 4 0.2g、MgSO 4 ·7H 2 0.2g of O, 2mL of trace elements (g/L) 2 ·4H 2 O 1.8，CoCl 2 ·6H 2 O 0.25，NiCl 2 ·6H 2 O 0.01，CuCl 2 ·2H 2 O 0.01，MnCl 2 ·4H 2 O 0.70，ZnCl 2 0.1，H 3 BO 3 0.5，Na 2 MoO 4 ·2H 2 O 0.03，NaSeO 3 ·5H 2 O0.01), and NH is used as nitrogen source 4 Cl、KNO 3 、Na 2 NO 2 Is the only nitrogenThe nitrogen concentration of the source preparation is 100mg/L, and the pH value is 7.0-7.3. As shown in FIG. 2, in the presence of NO 3 - NO within 20h when N is the sole nitrogen source 3 - The concentration of the-N is reduced to 4.47mg/L from the initial 100mg/L, the removal rate is 4.83 mg/(L.h), and the removal rate reaches 96.53 percent. When NH is generated as shown in FIGS. 3 and 4 4 + -N and NO 2 - NH when N is each the sole nitrogen source 4 + -N and NO 2 - the-N removal rates were 90.99% and 99.79%, respectively.
Example 3 Nitrogen removal of Strain LJ-1 in different C/N
The strain LJ-1 is cultured to logarithmic phase, and is inoculated into a nitrate nitrogen test culture medium (the components are the same as in example 2) with the C/N of 5, 10, 20, 30 and 50 according to the inoculation amount of 1 percent, and the culture is carried out for 30 hours at the temperature of 30 ℃ and the rotating speed of a shaking table of 160 r/min. The nitronium removal rate of the strain is higher than 80 percent when the C/N is in the range of 5-50. Particularly, the bacterial strain still has a high removal rate of 91.82 percent on nitrate nitrogen when the C/N is 5 (see figure 5).
Example 4 Nitrogen removal of Strain LJ-1 at different pH
The strain LJ-1 is cultured to a logarithmic phase and is inoculated into a culture medium with pH of 3, 5, 7, 9 and 11 according to the inoculation amount of 1 percent, and the main components (the amount of each component in each liter of the culture medium) are as follows: sodium succinate hexahydrate 16.67g, KH 2 PO 4 0.2g，MgSO 4 ·7H 2 O 0.2g，KNO 3 0.722g, 2mL of trace element (g/L): feCl 2 ·4H 2 O 1.8、CoCl 2 ·6H 2 O 0.25、NiCl 2 ·6H 2 O 0.01、CuCl 2 ·2H 2 O 0.01、MnCl 2 ·4H 2 O 0.70、ZnCl 2 0.1、H 3 BO 3 0.5、Na 2 MoO 4 ·2H 2 O 0.03、NaSeO 3 ·5H 2 O0.01), and culturing for 30 hours at the temperature of 30 ℃ and the rotating speed of a shaker of 160 r/min. The strain can grow at pH 5-11, preferably at pH 9, has nitrogen removing ability at pH 3-12, and has nitrogen removing ability of 90% at pH 5-11 (see FIG. 6)
Example 5 self-aggregation and hydrophobic Properties of Strain LJ-1
The strain was inoculated at 1% inoculum size into a 250mL Erlenmeyer flask containing 100mL of nitrate nitrogen test medium (same composition as in example 2), cultured at 30 ℃ for 60h at 160r/min, and the self-aggregation and hydrophobicity index was measured every 10h, indicating that the self-aggregation ability of the strain gradually increased within 10-40h and reached a maximum of 66% within 40h (see FIG. 7). The time-dependent change of the hydrophobicity of the strain (as shown in FIG. 8) is basically the same as the change trend of the self-aggregation capability of the strain, the hydrophobicity is gradually increased in the first 40h, and the maximum 59% is reached in 40 h.
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
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<120> Pseudosingle-cell Shigella and application thereof
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gcctaccgct gacgtataga catgcagtcg agcggatgag tggagcttgc tccatgattc 60
agcggcggac gggtgagtaa tgcctaggaa tctgcctggt agtgggggac aacgtttcga 120
aaggaacgct aataccgcat acgtcctacg ggagaaagtg ggggatcttc ggacctcacg 180
ctatcagatg agcctaggtc ggattagcta gttggtgagg taaaggctca ccaaggcgac 240
gatccgtaac tggtctgaga ggatgatcag tcacactgga actgagacac ggtccagact 300
cctacgggag gcagcagtgg ggaatattgg acaatgggcg aaagcctgat ccagccatgc 360
cgcgtgtgtg aagaaggtct tcggattgta aagcacttta agttgggagg aagggcagta 420
agttaatacc ttgctgtttt gacgttacca acagaataag caccggctaa cttcgtgcca 480
gcagccgcgg taatacgaag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcgc 540
gtaggtggtt cgttaagttg gatgtgaaag ccccgggctc aacctgggaa ctgcatccaa 600
aactggcgag ctagagtatg gcagagggtg gtggaatttc ctgtgtagcg gtgaaatgcg 660
tagatatagg aaggaacacc agtggcgaag gcgaccacct gggctaatac tgacactgag 720
gtgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat 780
gtcgactagc cgttgggatc cttgagatct tagtggcgca gctaacgcat taagtcgacc 840
gcctggggag tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc 900
ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggcc ttgacatgca 960
gagaactttc cagagatgga ttggtgcctt cgggaactct gacacaggtg ctgcatggct 1020
gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgt aacgagcgca acccttgtcc 1080
ttagttacca gcacgttaag gtgggcactc taaggagact gccggtgaca aaccggagga 1140
aggtggggat gacgtcaagt catcatggcc cttacggcct gggctacaca cgtgctacaa 1200
tggtcggtac aaagggttgc caagccgcga ggtggagcta atcccataaa accgatcgta 1260
gtccggatcg cagtctgcaa ctcgactgcg tgaagtcgga atcgctagta atcgtgaatc 1320
agaatgtcac ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgggag 1380
tgggattgct ctcagaagta gctagtctac accttcgggt gcacagtagc actagagatc 1440
attgctgga 1449
Claims (15)
1. The Pseudomonas stutzeri strain is characterized in that the preservation number is CCTCC NO: M2022353, the Latin chemical name is Pseudomonas stutzeri LJ-1, and the strain is preserved in China center for type culture Collection 3.31.2022.
2. A preparation comprising the pseudomonas stutzeri strain of claim 1.
3. The preparation according to claim 2, wherein the type of the preparation is any one of culture, culture extract, freeze-dried powder, fermentation broth precipitate, fermentation broth supernatant, and fermentation broth extract.
4. The method for culturing a Pseudomonas stutzeri strain according to claim 1, comprising inoculating said Pseudomonas stutzeri strain in a culture medium.
5. The culture method according to claim 4, wherein the culture is carried out under aerobic conditions at a nitrogen concentration of 50 to 100mg/L in the medium, a carbon-nitrogen ratio of 5 to 50, a pH of 5 to 11, and a culture temperature of 25 to 35 ℃.
6. The culture method according to any one of claims 4 to 5, comprising the steps of: taking out the stored pure strain sample for later use; preparing a denitrification culture medium, and sterilizing at 121 ℃ for later use; inoculating a pure strain sample to a sterile denitrification culture medium; putting the strain into an incubator until the strain expansion culture is finished.
7. A bacterial agent comprising the Pseudomonas stutzeri strain according to claim 1.
8. A wastewater treatment agent comprising the pseudomonas stutzeri strain of claim 1 or the preparation of any of claims 2-3.
9. The wastewater treatment agent according to claim 8, comprising a culture of the Pseudomonas stutzeri strain of claim 1 cultured to log phase.
10. A method for treating wastewater, which comprises adding the Pseudomonas stutzeri strain according to claim 1 to wastewater.
11. Use of a pseudomonas stutzeri strain according to claim 1 and/or a preparation according to any one of claims 2 to 3 in the treatment of wastewater.
12. The use according to claim 11, wherein the bacterial species is added to the wastewater after the culture for the amplification of bacterial species, or the bacterial species is added directly to the wastewater.
13. The use according to claim 12, wherein the pseudomonas stutzeri strain is cultured until after a logarithmic phase and the culture is added to the wastewater.
14. The use of claim 13, wherein the inoculum size of the culture is 1% to 5%.
15. The use of claim 14, wherein the inoculum size of the culture is 1% to 3%.
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| CN202210666724.4A CN115181689B (en) | 2022-06-13 | 2022-06-13 | Pseudomonas stutzeri and application thereof |
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| CN117384791A (en) * | 2023-10-18 | 2024-01-12 | 小菌主环境科技(武汉)有限公司 | Pseudomonas for promoting anaerobic iron ammoxidation process and application thereof in sewage treatment |
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| CN114591853A (en) * | 2022-02-24 | 2022-06-07 | 安徽师范大学 | Heavy metal-resistant aerobic denitrification strain with bisphenol A and nitrogen removal functions and application thereof |
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| Title |
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| LIANGFENG等: "Characterisation of Pseudomonas stutzeri T13 for aerobic denitrification: Stoichiometry and reaction kinetics", 《THE SCIENCE OF THE TOTAL ENVIRONMENT》 * |
| 问彩芳: "一株异养硝化—好氧反硝化菌株的分离鉴定及脱氮特性研究", 《中国优秀硕士学位论文全文数据库》 * |
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| CN117384791A (en) * | 2023-10-18 | 2024-01-12 | 小菌主环境科技(武汉)有限公司 | Pseudomonas for promoting anaerobic iron ammoxidation process and application thereof in sewage treatment |
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