CN113151068A - Shewanella denitrificans for degrading organic pollutants and application thereof - Google Patents
Shewanella denitrificans for degrading organic pollutants and application thereof Download PDFInfo
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- CN113151068A CN113151068A CN202110357164.XA CN202110357164A CN113151068A CN 113151068 A CN113151068 A CN 113151068A CN 202110357164 A CN202110357164 A CN 202110357164A CN 113151068 A CN113151068 A CN 113151068A
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/343—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of grease, fat, oil
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
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Abstract
The invention discloses a Shewanella denitrificans for degrading organic pollutants and application thereof. Shewanella sp.JM162201, deposited at the Guangdong province culture Collection (GDMCC) at 3/15 2021, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61585. the invention discloses a novel Shewanella sp JM162201 with the capability of degrading fat and protein and reducing nitrate and nitrite. Therefore, the Shewanella sp JM162201 disclosed by the invention has application value in the aspects of bioremediation, biodegradation, biological decontamination, water quality purification and the like.
Description
The technical field is as follows:
the invention belongs to the technical field of microorganisms, and particularly relates to a new bacterial species capable of degrading fat and protein and reducing nitrate and nitrite, namely Shewanella denitrificans, and application thereof in bioremediation, water quality purification and biodegradation.
Background art:
with the rapid development of industrial and agricultural industries and the improvement of social living standard in China, the discharge amount of industrial and agricultural sewage and domestic wastewater is increased rapidly, heavy burden is brought to the natural environment of China, and environmental safety and people health are threatened seriously. For example, sewage containing abundant organic pollutants and nitrogen is discharged into the environment, thereby aggravating eutrophication and pollution degree of rivers and lakes, causing mass propagation of phytoplankton and harmful microorganisms, consuming dissolved oxygen in water, leading fish, shrimp and the like to die in mass, further forming pollutants such as protein, fat, nitrate, nitrite and the like, finally leading the water to turn black and foul, and thoroughly collapsing an ecological system.
At present, the treatment technology for organic pollutants such as protein, fat and the like and inorganic pollutants such as nitrate, nitrite and the like in water mainly comprises chemical, physical and biological methods, wherein the biological treatment technology overcomes the defects of the traditional physical and chemical methods, has the advantages of simple operation, wide application range, good treatment effect, basically no secondary pollution and the like, and becomes a research hotspot in the field of environmental management in recent years. Therefore, it is of great significance to develop efficient water quality improving microorganisms.
Shewanella (Shewanella) is widely distributed in nature, has low temperature resistance and various respirators, can utilize various carbon sources, and can survive and propagate in environments with different salinity, temperature and the like. In addition, Shewanella can reduce metal, sodium thiosulfate and polycyclic aromatic hydrocarbon, petroleum and the like. The Shewanella has high application value in the aspects of bioremediation, biodegradation, biological decontamination, microbial fuel cells and the like.
The invention content is as follows:
the first purpose of the invention is to provide Shewanella sp JM162201 which can degrade fat and protein and reduce nitrate and nitrite.
Shewanella sp (Shewanella sp.) JM162201, which was deposited with Guangdong province culture Collection (GDMCC) at 3/15 2021, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61585.
the second purpose of the invention is to provide the application of the Shewanella sp JM162201 in bioremediation, water quality purification or biodegradation.
It is a third object of the present invention to provide a preparation for bioremediation, water purification or biodegradation, which contains Shewanella sp JM162201 as an active ingredient.
Preferably, the application of Shewanella sp JM162201 in degrading protein or fat is used.
Preferably, the application of Shewanella sp JM162201 in reducing nitrate and nitrite to realize denitrification is adopted.
Compared with the prior art, the invention has the following advantages:
the invention discloses a novel Shewanella sp JM162201 with the capability of degrading fat and protein and reducing nitrate and nitrite. Therefore, the Shewanella sp JM162201 disclosed by the invention has application value in the aspects of bioremediation, biodegradation, biological decontamination, water quality purification and the like.
Shewanella sp.JM162201, deposited at the Guangdong province culture Collection (GDMCC) at 3/15 2021, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61585.
drawings
FIG. 1 is a morphological diagram of a colony of Shewanella sp JM162201 cultured on an R2A plate for 48 hours.
FIG. 2 is a transmission electron microscope photograph of Shewanella sp JM162201 cell of the present invention.
FIG. 3 is a phylogenetic tree of Shewanella sp JM162201 and related strains according to the present invention; where the tree was built using the NJ method, only > 50% of the bootstrap coefficients are shown (1000 replicates).
FIG. 4 is a diagram of the polar lipid bilayer of Shewanella sp JM162201 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention; the parameters, ratios, etc. of the examples can be selected according to local circumstances without substantially affecting the results. Unless otherwise specified, the methods described in the examples are all conventional methods, and the reagents used are all conventional reagents or reagents formulated in a conventional manner.
Example 1 Shewanella sp JM162201 was isolated, purified and stored.
The culture water sample is collected from a certain culture farm in Taishan City in Jiangmen City. Adding 1mL of water sample into 9mL of sterile water, fully shaking and uniformly mixing, coating the diluent on an R2A flat plate by adopting a standard dilution gradient coating method, and culturing for 2 days at 28 ℃; colonies were then picked from the plate and streaked for purification to give strain JM 162201.
The colony morphology and cell transmission electron micrograph of the obtained strain JM162201 are shown in FIGS. 1 and 2.
The purified strain JM162201 was stored in the form of glycerol tubes (-80 ℃) and lyophilized tubes (4 ℃) respectively.
The strain JM162201 is generally cultured with R2A or 2216E at 28-30 ℃.
Example 2 identification of Shewanella sp JM 162201.
Molecular characterization of Shewanella sp JM162201
The genome of the strain JM162201 obtained in example 1 was extracted by the CTAB method. Its 16S rRNA gene was amplified using 27F and 1492R primers and sent to GenEWIZ, Kingzhi Biotech, Inc. for sequencing.
The length of the 16S rRNA gene sequence obtained after sequencing is 1466bp, and the nucleotide sequence is shown in SEQ ID NO. 1. The 16S rRNA gene sequence of JM162201 was most similar to Shewanella litorisemionis SMK1-12 (97.1%) in the EzbioCloud database. The phylogenetic tree of Shewanella sp JM162201 and related strains is shown in FIG. 3.
The genomic DNA of the strain JM162201 was transferred to Meiji biological medicine science and technology GmbH in the Shanghai, and genomic sequencing was performed by using Illumina Hiseq 4000 platform. After off-line, 8930862 double-ended original sequence reads with total base number of 1348560162bp are obtained. And assembling the genome by using SPAdes v3.11.1 and adopting a-careful mode and setting a k-mer value to be 127 to obtain a genome assembly file. Contigs with a length of less than 500bp and a coverage of less than 10 were removed. The genome was analyzed using QUAST on-line tool to obtain total length of 4436316bp, total 33 contigs, N50 of 513416bp, coverage of 303, and G + C content of 55.0%.
Collection of reference Strain Shewanella litoriseminis SMK1-12 from NCBI databaseTAnd Shewanella amazonensis SB2BTThe genome files (accession numbers GCA _016834455.1 and GCA _000015245.1, respectively). Using an in-line tool (https://www.ezbiocloud.net/tools/aniAnd http:// ggdc.dsmz.de) were calculated ANI and DDH, respectively.
The results showed that JM162201 and Shewanella litoriredinis SMK1-12TAnd Shewanella amazonensis SB2BTANI and DDH in between are respectively79.3%, 78.8%, 23.4% and 23.1%.
Second, Shewanella sp JM162201 morphology description
The biological characteristics were identified by culturing on R2A plates at 28 ℃ for 2 days as follows.
Colony morphology of Shewanella (Shewanella sp.) JM162201 was visually observed and photographed.
The cell morphology of Shewanella sp JM162201 was observed using an electron microscope (H7650, Hitachi).
Cells were stained using the cyclokay (HKM) gram stain kit.
Aerobic type of Shewanella (Shewanella sp.) JM162201 was measured by a puncture culture method and an anaerobic bag (Mitsubishi gas chemical, C-43).
The cell gliding motility of Shewanella (Shewanella sp.) JM162201 was examined by the method described by Bernardet (Bernardet JF & NakagawaY et al, 2002).
The cellular oxidase activity of Shewanella (Shewanella sp.) JM162201 was detected using a Kjeldahl oxidase kit (HKM).
Use of 3% H2O2Detecting the catalase activity of Shewanella (Shewanella sp.) JM162201 by using the solution;
detecting the physiological and biochemical characteristics of Shewanella (Shewanella sp.) JM162201 by using API ZYM, 20E and 20NE reagent strips;
identifying the fatty acid component of Shewanella (Shewanella sp.) JM162201 using gas chromatography and Sherlock microbiological Identification System (MIDI) System;
the polar lipid component was detected using the two-way spreading method (Tindall BJ & Sikorski J et al, 2007; Minnikin DE & O' donnell AG et al, 1984).
Quinoids were detected using HPLC (Collins MD & Pirouz T et al, 1977; Hiraishi A & UedaY et al, 1996).
The biological characteristics identified were as follows: shewanella sp JM162201 on R2A plate showed milky white, round, smooth surface and neat edges (FIG. 1); the cells are facultative anaerobic, motile, gram-negative, rod-shaped, about 1.4-2.3um long and about 0.4-0.6um wide (figure 2); can grow under the conditions of 10-40 deg.C (optimum temperature 28-30 deg.C), pH 4-10 (optimum pH 7-8), and NaCl (w/v) concentration 0-6% (optimum salt concentration 0-1%); can grow in various culture media such as Marine Agar culture medium, Marine Broth culture medium, 2216E culture medium, R2A culture medium and the like. Hydrolyzed Tween 20, Tween 40, Tween 80, casein, tyrosine and gelatin; starch and cellulose are not hydrolyzed; alkaline phosphatase, esterase (C4), lipoid esterase (C8), lipase-like enzyme (C14), arylamine leucine enzyme, arylamine valine enzyme, arylamine cystine enzyme, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α -glucosidase, N-acetyl-glucamine enzyme are positive; alpha-galactosidase, beta-uronidase, beta-glucosidase, alpha-mannosidase, beta-fucosidase are negative; can utilize D-glucose, D-mannose, N-acetyl-D-glucosamine, D-maltose, malic acid; l-arabinose, D-mannitol, potassium gluconate, capric acid, adipic acid, trisodium citrate, phenylacetic acid cannot be utilized; nitrate reduction and nitrite reduction were positive. As can be seen from FIG. 4, the polar lipids of Shewanella sp JM162201 mainly include PE and PG.
In conclusion, JM162201 is a new species of Shewanella, which is named: shewanella sp (Shewanella sp.) JM162201, which was deposited with Guangdong province culture Collection (GDMCC) at 3/15 2021, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61585.
appropriate changes and modifications to the embodiments described above will become apparent to those skilled in the art from the disclosure and teachings of the foregoing description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Sequence listing
<110> Guangdong Bowott Biotech Ltd
Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Microbiological Analysis and Testing Center)
GUANGZHOU WANGDAO BIOLOGICAL TECHNOLOGY Co.,Ltd.
<120> Shewanella denitrificans for degrading organic pollutants and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1543
<212> DNA
<213> Shewanella JM162201(Shewanella sp.)
<400> 1
taattgaaga gtttgatcat ggctcagatt gaacgctggc ggcaggccta acacatgcaa 60
gtcgagcggc agcacaaggg agtttactcc tgaggtggcg agcggcggac gggtgagtaa 120
tgcctgggaa cttgcccatt cgagggggat aacagttgga aacgactgct aataccgcat 180
acgccctacg ggggaaagag ggggatcttc ggacctctcg cgaatggata ggcccaggtg 240
ggattagcta gtaggtgagg taaaggctca cctaggcgac gatccctagc tgttctgaga 300
ggatggacag ccacactgga actgagacac ggtccagact cctacgggag gcagcagtgg 360
ggaatattgg acaatggggg aaaccctgat ccagccatgc cgcgtgtgtg aagaaggcct 420
tcgggttgta aagcactttc agtggggagg aaaggctgat ggttaatacc tatcagctgt 480
gacgttaccc acagaagaag caccggctaa ctccgtgcca gcagccgcgg taatacggag 540
ggtgcaagcg ttaatcggaa ttactgggcg taaagcgtgc gcaggcggtc tgttaagcga 600
gatgtgaaag ccccgggctc aacctgggaa ctgcatttcg aactggcaga ctagagtctt 660
gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc 720
ggtggcgaag gcggccccct ggacaaagac tgacgctcag gcacgaaagc gtggggagca 780
aacaggatta gataccctgg tagtccacgc cgtaaacgat gtctactcgg agtttggtgt 840
cttgaacact gggctctcaa gctaacgcat taagtagacc gcctggggag tacggccgca 900
aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 960
tcgatgcaac gcgaagaacc ttacctactc ttgacatcca gagaattcgg tagagatacc 1020
ttagtgcctt cgggaactct gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga 1080
aatgttgggt taagtcccgc aacgagcgca acccctatcc ttacttgcca gcgggtaatg 1140
ccgggaactt tagggagact gccggtgata aaccggagga aggtggggac gacgtcaagt 1200
catcatggcc cttacgagta gggctacaca cgtgctacaa tggtcggtac agagggttgc 1260
gaagccgcga ggtgaagcta atcccaaaaa gccggtcgta gtccggattg gagtctgcaa 1320
ctcgactcca tgaagtcgga atcgctagta atcgtggatc agaatgccac ggtgaatacg 1380
ttcccgggcc ttgtacacac cgcccgtcac accatgggag tgggctgcaa aagaagtggg 1440
tagcttaacc ttcgggaggg cgctcaccac tttgtggttc atgactgggg tgaagtcgta 1500
acaaggtagc cctaggggaa cctggggctg gatcacctcc tta 1543
Claims (5)
1. Shewanella sp JM162201 with deposit number: GDMCC No: 61585.
2. use of Shewanella sp JM162201 according to claim 1 for bioremediation, water purification or biodegradation.
3. The use according to claim 2, characterized in that it is the use of Shewanella sp JM162201 for the degradation of proteins or fats.
4. The use according to claim 2, characterized in that it is the use of Shewanella sp JM162201 for the reduction of nitrate, nitrite, effecting denitrification.
5. A preparation for bioremediation, water purification or biodegradation, which contains Shewanella sp JM162201 as an active ingredient.
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-
2021
- 2021-04-01 CN CN202110357164.XA patent/CN113151068A/en active Pending
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| WO2012061350A2 (en) * | 2010-11-01 | 2012-05-10 | E. I. Du Pont De Nemours And Company | Methods, strains, and compositions useful for microbially enhanced oil recovery: pseudomonas stutzeri |
| WO2016016677A1 (en) * | 2014-07-29 | 2016-02-04 | Total Sa | Bacterial method of treatment of oil reservoir and outcrop samples |
| CN109943503A (en) * | 2019-03-08 | 2019-06-28 | 同济大学 | The method of S.oneidensis MR-1 raising anaerobic denitrifying efficiency |
| CN110283739A (en) * | 2019-05-24 | 2019-09-27 | 中国科学院东北地理与农业生态研究所 | The denitrifying bacteria of one plant of salt tolerant and its application |
| CN111793573A (en) * | 2020-03-26 | 2020-10-20 | 大连理工大学 | Shewanella alga strain with functions of heterotrophic nitrate heterotrophic reduction and autotrophic nitrate heterotrophic reduction to ammonium, culture method and application thereof |
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