CN109385388B

CN109385388B - Halophilic denitrifying bacteria YL5-2 and application thereof

Info

Publication number: CN109385388B
Application number: CN201811636221.2A
Authority: CN
Inventors: 徐军; 孙文妮; 张璐璐; 王开春; 田凤蓉; 李坤; 王强; 洪磊
Original assignee: Bluestar Lehigh Engineering Institute
Current assignee: Bluestar Lehigh Engineering Institute
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2021-09-14
Anticipated expiration: 2038-12-29
Also published as: WO2020134728A1; CN109385388A

Abstract

The invention discloses halophilic denitrifying bacteria YL5-2 and application thereof.A halophilic denitrifying bacterium YL5-2 is a new species of Halovibrio, is preserved in China general microbiological culture collection center with the preservation number of CGMCC NO.16315 and the preservation date of 2018, 8 and 20 days. The strain YL5-2 is a facultative or aerobic denitrifying bacterium, has salt tolerance of 3-32%, and can be treated with NO in salt concentration of 5-25%₃-N and NO₂Denitrification as electron acceptor. YL5-2 can be used for biological denitrification treatment of high-salinity wastewater with salt content more than 10%, and reduces the denitrification treatment cost of the wastewater.

Description

Halophilic denitrifying bacteria YL5-2 and application thereof

Technical Field

The invention relates to the field of environment-friendly microorganisms, in particular to halophilic denitrifying bacteria YL5-2 and application thereof.

Background

Nitrogen pollution is one of the important causes for eutrophication of water bodies. The denitrification treatment of the wastewater plays an important role in maintaining the water environment quality and preventing the water eutrophication. The nitrogen-containing wastewater discharged from the industries of petroleum, chemical industry, food processing, chemical fertilizer and the like has the characteristic of high salt. The salt content of some waste water produced in chemical industry and food processing is even higher than that of seawater. The traditional biological method has advantages of treating low-salinity nitrogen-containing wastewater, but can inhibit the metabolism of denitrifying microorganisms when the concentration of the wastewater is too high. The existence of the denitrifying bacteria with salt tolerance and halophilic provides theoretical possibility for biological denitrification of high-salinity wastewater.

The biological denitrifying bacteria comprise nitrifying bacteria and denitrifying bacteria. Biological denitrifying bacteria under the condition of high-salinity wastewater can also be divided into nitrifying bacteria and denitrifying bacteria. Denitrifying bacteria are a class of bacteria that are capable of reducing nitrate or nitrate to gaseous nitrogen compounds. Therefore, the screening, separation and cultivation of salt-tolerant and halophilic nitrifying bacteria and denitrifying bacteria from the environment become the key for solving the biological denitrification problem of high-salinity wastewater.

The method comprises the steps of separating and identifying moderately halophilic denitrifying bacteria from marine culture wastewater purification units (aquatic science and technology information, 2018, 45 (3): 149-154) to obtain the moderately halophilic denitrifying bacteria belonging to Halomonas, wherein the optimal metabolic growth conditions are that the temperature is 30 ℃, the salinity is 100g/L, the pH is 7.5-8.5, and the C/N ratio is 4: 1.

Separation identification and characteristics of a strain of mild halophilic denitrifying bacteria (Youyanli, Zhang Peiyu, and the like) at Qingdao university are separated from mature activated sludge for treating high-salt wastewater by 394-398 (application and environmental biology newspaper 2010,16 (3)) to obtain a strain of mild halophilic aerobic denitrifying bacteria YL-1, belonging to Dietzia sp, which can perform denitrification by using acetic acid, cane sugar, glucose, sodium citrate and sodium succinate at the concentration of 0-10% and the pH of 7.5-8.5.

The Nanjing university of industry calumniate Xiaoli (calumniate Xiaoli. separation and identification of halophilic denitrifying bacteria and research on denitrification characteristics of the halophilic denitrifying bacteria. 2013 Master graduation paper of Nanjing university of industry) uses sodium nitrate as a unique nitrogen source and a heterotrophic denitrification culture medium with salt concentration of 8%, and 6 halophilic denitrifying bacteria are obtained by enrichment, separation and screening from soil samples in a salt field of salt city: NY-1, NY-11 and NY-13 are all mycobacteria (Virgibacillus sp.), NY-8 and NY-10 are Halomonas sp, NY-4 is Marinobacter sp, the salinity growth range is 0-12%, and the optimal growth salt concentration is 3-8%. Wherein NY-4 has strongest denitrification capability, and NO is obtained when trisodium citrate is used as carbon source, salinity is 8%, C/N is 5, and pH is 8₃The removal rate of-N was 95%.

The prior art has few reports about halophilic denitrifying bacteria, particularly halophilic denitrifying bacteria under the condition that the salinity is more than 10%. The method for obtaining the halophilic denitrifying bacteria with the salt tolerance of more than 10 percent from the environment is the technical problem to be solved for biological denitrification of the wastewater.

Disclosure of Invention

The invention aims to solve the biological denitrification problem under the condition of high salt, in particular to the biological denitrification problem of high-salt wastewater under the condition that the salinity is more than 10 percent, and provides halophilic denitrifying bacteria which can use NO under the condition that the salinity is more than 10 percent₃-N as electron acceptor for denitrification.

Another purpose of the invention is to provide the application of the halophilic denitrifying bacteria.

The purpose of the invention can be realized by the following technical scheme:

the halophilic denitrifying bacteria YL5-2 disclosed by the invention is preserved in the China general microbiological culture collection center with the preservation number of CGMCC NO.16315 and the preservation date of 2018, 8 months and 20 days. Based on the differences of phylogenetic analysis of 16SrRNA, genome sequencing, DNA hybridization tests, fatty acid composition, respiratory quinone categories, physiological biochemistry and phenotypic characteristics and the like, the strain YL5-2 can be determined to be a new species of Halovibrio, and is named Halovibrio salipaludis sp.

The 16S rRNA sequence of the halophilic denitrifying bacteria YL5-2 is shown in SEQ ID NO. 1; GenBank/EMBL/DDBJ of the whole genome sequence has the accession number of NSKD 00000000.1.

The halophilic denitrifying bacteria YL5-2 disclosed by the invention are gram-negative, facultative aerobic, straight rod-shaped or vibrio parviticum characters of 0.5-0.8 mu m multiplied by 1.0-3.5 mu m, and bacterial colonies on a solid culture medium are smooth and light yellow through the movement of unipolar flagella.

The Vibrio salinophilus Halovibrio sp.YL5-2 can grow in the range of salt concentration of 3% -32%, pH6.5-11.0, and temperature of 15-45 ℃; the concentration of the most suitable growth salt is 10-25%, the most suitable growth pH is 7.5-8.0, and the most suitable growth temperature is 30-35 ℃.

The main respiratory quinone of Vibrio halophilic Halovibro sp.YL5-2 is Q-9, the main fatty acids are C18:1 omega 9C, C16:0, C19:0cyclo omega 8C and Summed Feature 8, and the main polar lipids are Diphosphatidylglycol (DPG), Phosphonomethylethanolamine (PE), Phosphonoglyycerol (PG) and Phosphonoglylcholine (PC).

The Halovibrio salina sp.YL5-2 can utilize bromine-succinic acid, propionic acid and acetic acid as unique carbon sources; but does not utilize D-maltose, D-fructose, D-galactose, D-cellobiose, stachyose, D-melibiose, N-acetyl-D-galactosamine, D-fucose, L-rhamnose, D-mannitol, D-galacturonic acid, D-aspartic acid, D-serine, D-glucuronic acid, L-lactic acid, quinic acid, mucic acid, D-malic acid, gamma-amino-butyric acid, formic acid, acetoacetic acid as the sole carbon source.

Has the advantages that:

the invention discovers a new species of Halovibrio, which is named Halovibrio salipaludis sp. The strain is a facultative or aerobic denitrifying bacterium, has a salt tolerance range of 3-32%, and can degrade various pollutants in wastewater within a salt concentration range of 5-25%. The halophilic denitrifying bacteria YL5-2 disclosed by the invention can be used for degradation, transformation and biological denitrification processes of pollutants under a high-salinity condition, and comprises high-salinity wastewater treatment, polluted seawater treatment, saline-alkali soil restoration, nitrogen nutrition consumption, algae excessive propagation inhibition, water body purification, substrate improvement and the like.

Description of the drawings:

FIG. 1 is a Transmission Electron Microscope (TEM) photograph (scale bar: 2 μm) of a Halovibrio sp.YL5-2 cell of Vibrio salina;

FIG. 2 is a polar lipid profile of Vibrio halophilus Halovibrio sp.YL5-2(a) and Halovibrio dentificans DSM15503(b), Halovibrio variabilis DSM 3050 (c). DPG: diphosphatidylglycerol; PG: phosphatidylglycol; AL: (ii) Aminolipid; PL: phospholipidd; PE: phospathylletholomine; PGNL: phosphoaminoglyolide; f: a first dimension; s: second dimension

FIG. 3 is a diagram of a 16S rRNA-based phylogenetic tree constructed from Halovibrio sp.YL5-2 and Halovibrio dentificans DSM15503 of Vibrio salina.

FIG. 4 is a phylogenetic tree diagram constructed by Halovibri sp.YL5-2 and Halovibri variabilis DSM 3050T of Vibrio salina and other strains based on the maximum simple Method (MP) of 16S rRNA.

FIG. 5 is a phylogenetic tree diagram constructed by Halovibrio sp.YL5-2 of Vibrio halophilus based on GGD matrix.

Biological material preservation information

YL5-2, classified and named as Halovibrio salipaludis, the preservation date is 20 days 8 months in 2018, the preservation unit is China general microbiological culture preservation management center, the preservation address is China academy of sciences microbial research institute No. 3, Xilu No.1, Beijing north morning district, the preservation number is: CGMCC NO. 16315.

Detailed Description

EXAMPLE 1 isolation and preservation of the halophilic denitrifying bacteria

The halophilic denitrifying bacteria YL5-2 was isolated from the soil deposited in Guermu sweat lake (36 deg.51 'N, 94 deg.95' E) in Qinghai province, China. The water of the salt lake of Kerr is at or close to the saturated salt concentration all the year round.

LB liquid culture medium with 20% NaCl concentration is prepared, 250mg/L of glycerol, 250mg/L of glucose and 50mg/L of methanol are added, and enrichment culture is carried out on the soil deposited in the salt lake of Carlsan at the temperature of 30 ℃ for 48 h. And (4) separating the strains in the enrichment culture solution by using YL solid culture medium. 1L of the culture medium contains the following components: glucose: 0.6g, trisodium citrate 0.5g, glycerol 2mL, yeast extract 0.8g, peptone 1.6g, dipotassium hydrogen phosphate 0.35g, potassium dihydrogen phosphate 0.1g, ammonium sulfate 0.25g, ammonium chloride 0.25g, MgSO₄ 0.5g，CaCl₂0.1g and NaCl 180 g; the trace element SL-410 mL, the pH value is 7.0-7.2; agar 2.5%.

The strain YL5-2 is preserved in China general microbiological culture collection center with the preservation number of CGMCC NO.16315 and the preservation date of 2018, 8 months and 20 days.

Example 2 sequence analysis and Whole Gene sequence analysis of halophilic denitrifying bacteria YL 5-216S rRNA

The Genomic DNA of the strain YL5-2 was extracted by using TaKaRa Kit (TaKaRa MiniBEST bacterial Genomic DNA Extraction 68Kit Ver.3.0).

The 16S rRNA was amplified using the universal bacterial primers 27F (5 '-AGAGTTTGATCMTGGCTCA G-3') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3'). PCR sequencing was performed by Shanghai Biotechnology Ltd. The complete 16S rRNA sequence of the strain YL5-2 is shown in SEQ ID NO. 1.

The whole genome sequencing of the strain YL5-2 adopts a high-throughput sequencing platform of Illumina MiSeq 2000, Shanghai Paixino Biotech Co., Ltd. Raw sequencing data were filtered and corrected using prinsoq (version number v 0.20.4) software, followed by base pairing of the genome using SOAP denovo software (version number v1.05) with default parameters, and then evaluation of genome integrity using CheckM software (version 1.03). Protein-encoded open reading frames were predicted using Glimmer software (version number 1.2). RNA prediction using RNAmmer software (version 1.2). The total genome sequence of the strain YL5-2 is 3,495,096bp, and the GenBank accession number is NSKD 00000000.1.

DNA-DNA hybridization experiments were carried out with strain YL5-2 and the most genetically developed strain of the Halovibrio model of the genus Vibrio. The method is provided by De Ley equal to 1970, and DNA hybridization value (dDDH) is obtained by one-to-one comparison of gene sequences by adopting a 2 nd mode (version number 2.0) of GGDC software. The results of DNA tests and analyses showed that the strain YL5-2 and Halovibro varilabilis DSM 3050^T、Halovibrio denitrificans DSM15503^TThe DNA-DNA hybridization values of (1) were 43.5% and 38.2%, respectively, well below the 70% threshold (a generally accepted threshold for speciation).

The nucleotide average identity value was obtained by 1000 repetitive topological tests using the base set of the whole genome sequence. The method is proposed by Goris equal to 2007, and the software used is MUMmer (version number 3.23) and Jspecies (version number 1.2.1). Based on the ANI threshold range (95-96%) of the species division proposed by Kim et al and Richter et al, ANI analysis was performed on the genome of strain YL5-2 and on its closely related genomes in GenBank (Table 1). The results showed that the Average Nucleotide Identity (ANI) value of strain YL5-2 with Tamilnaduibacter alinus Mi 7 was the highest and 88.5% (complementary Table S1), which is strain YL5-2^TA new species belonging to the genus Halovibrio provides a argumentation, named Halovibrio salipaludis sp.

TABLE 1 Strain YL5-2^TAverage Nucleotide Identity (ANI) and DDH values between closely related genomes in GenBank.

Example 3 phenotypic characterization and Biochemical characterization of the halophilic denitrifying bacteria YL5-2

Gram staining characteristics were tested using the BD gram staining kit.

Cell motility was determined using semi-MA medium (0.5% agar, w/v).

Cell morphology was examined by Transmission Electron Microscopy (TEM) analysis. That is, cells were picked from the exponentially growing culture solution, stained with 0.5% uranyl acetate, and photographed under a microscope (Tecnai Spirit, FEI, Hillsboro, OR, USA).

Oxidase activity Using oxidase kit (bioMerieux), by adding 3.0% H₂O₂The solution was poured into bacterial colonies and observed for bubble generation to determine catalase activity.

Temperature growth conditions were performed on YL liquid agar medium at 4, 10, 15, 20, 25, 30, 33, 37, 40, 45 and 50 ℃ respectively, with a constant pH of 7.5, comparing the strain YL5-2 at different temperatures^TThe growth rate determines its optimum growth temperature.

Salt tolerance was performed in YL agar and YL broth with 0.0-30.0% NaCl (w/v). Using buffer (Na)₂HPO₄/NaH₂PO₄(pH 5.0-7.0)，Na₂CO₃/NaHCO₃(pH 8.0-12.0)) the pH was adjusted to 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 (15.0% NaCl, 35 ℃) to determine the pH range suitable for growth.

Carbon source utilization capacity and enzyme activity assays API 20NE, API ZYM (bioMerieux) and Biolog GENIII microplates were used. All tests inoculated cells pre-grown on YL medium and diluted with relevant inoculation medium.

The phenotypic and biochemical characteristics of strain YL5-2 were identified as shown in Table 2:

TABLE 2 Halovibrio salina Halovibrio sp. YL5-2(a) and Halovibrio dentificas DSM15503^T(b)，Halovibrio variabilis DSM 3050^T(c) In the phenotypeComparison with physiological and biochemical characteristics

Description of the drawings: positive; negative.

Halophilic denitrifying bacteria YL5-2 was gram-negative, alkaline aerobic, straight rod-like or Vibrio mimicus shape of 0.5-0.8X1.0-3.5 μm and moved by unipolar flagella (FIG. 1).

The strain YL5-2 was grown aerobically with acetic acid and anaerobically with nitrate (API 20 NE).

Example 4 identification of fatty acids in Halovibrio sp.YL5-2 cells of Vibrio salina

Identification of cellular fatty acids YL5-2, Halovibro denitificans DSM15503 and Halovibro variabilis DSM 3050 cells were cultured on YL medium at 30 ℃ for 3 days. The method mainly comprises the following steps: scraping 100mg of cells from YL medium and saponifying with 50% methanol containing sodium hydroxide; the saponified cells were freeze-dried and then extracted for cellular fatty acids using chloroform/methanol/0.3% (w/v) NaCl aqueous solution at a ratio of 1:2:0.8 (v/v/v).

The total amount of cellular fatty acids was determined using the phosphomolybdic acid method.

Fatty acid qualitative and quantitative determinations were performed using a 6890N gas chromatograph (Agilent) and standard MIS library generation software in the Sherlock microbiological identification system (VERSION 6.0and Date 4, Microbial ID Inc., Newark, DE, USA) (Sasser, 1990).

Cell fatty acid-based identification of species was performed using DSMZ-related analytical tools in germany.

The results of cellular fatty acid identification are shown in FIG. 2.

The only respiratory quinone of YL5-2 was ubiquinone Q-9, identical to Halovibro variabilis DSM 3050.

The major cellular fatty acids of YL5-2 include C_18:1ω9c、C_16:0、C_19:0cyclo ω 8c and Summed feed 8 (table 3); the major polar lipids are Diphosphatidylglycerol (DPG), Phosphatidylcholine (PE), Phosphatedidylglycerol (PG), Phosphatidilcholine (PC) and two unidentified lipids (L). These are similar to the generic species Halovibrio varilabiliss and Halovibrio dentificas of YL 5-2.

TABLE 3 Halovibrio salina sp. YL5-2(a) and Halovibrio denitificans DSM15503^T(b)，Halovibrio variabilis DSM 3050^TComparison of cell fatty acid composition (%).

Description of the drawings:^*summed diets denote a mixture of two or three fatty acids that cannot be separated by GLC from the MIDI system. The Summed feature 3 includes C_16:1Omega 7C and/or C_16:1Omega 6C, Summed feature 8 includes C_18:1Omega 6C and/or C_18:1ω7c。

Example 5 fermentation culture test of Halovibrio sp.YL5-2 of Vibrio salina

(1) The components of the culture medium are 500mg/L of glycerol, 250mg/L of glucose, 500mg/L of methanol, 200mg/L of methylamine, 100-250 g/L of sodium chloride, 250mg/L of sodium acetate, 250mg/L of trisodium citrate, 100mg/L of yeast powder, 200mg/L of peptone and 200mg/L of beef extract, a small amount of trace elements and the pH value of 7.5-8.0. And sterilizing the liquid culture medium, and simultaneously controlling the water evaporation.

(2) After being inoculated in a 1L triangular flask, the culture is carried out for 48 hours at the temperature of 35 ℃, and the change of the salt concentration is influenced by the dissipation of the supplemented water in the culture process. After 48h, the OD600 values at 10%, 15%, 20% and 25% salt concentration were 1.72, 1.65, 1.62, 1.60 and 1.63, respectively. And performing two times of transfer culture, wherein the culture is performed for 48 hours after each transfer, and the OD600 is respectively 2.56, 2.68, 2.72, 2.68 and 2.52 after the acclimatization culture is completed.

(3)1L of the culture broth was inoculated into a 20L aerobic fermentor and fermentation culture was repeated with the medium components remaining unchanged. Still culturing for 48h at 35 deg.C under stirring speed of 100rpm with dissolved oxygen of 2.0-4.0 mg/L. After 48 hours, the OD600 of the bacterial liquid in the fermentation tank can reach 2.6-3.0.

(4) And (4) checking: sampling and observing every day by using a microscope in the culture process, and checking whether mixed bacteria grow; and meanwhile, the growth and morphological change conditions of YL5-2 are observed.

(5) As a result: test results show that the strain YL5-2 can be rapidly subjected to fermentation culture and amplification, which shows that YL5-2 has the potential of large-scale engineering application.

Example 6 salt tolerance test of Halovibrio sp.YL5-2 Vibrio salina

(1) The components of the culture medium are 50mg/L of glycerol, 25mg/L of glucose, 50mg/L of methanol, 20mg/L of methylamine, 250g/L of sodium chloride, 25mg/L of sodium acetate, 25mg/L of trisodium citrate, 10mg/L of yeast powder, 20mg/L of peptone, 20mg/L of beef extract and 20g/L of agar.

(2) Activating strains with fresh culture medium, and culturing for 3 days until thallus Porphyrae grows abundantly

(3) Salinity gradient setting: according to the enrichment screening conditions of YL5-2 strains, the salinity gradient of the culture medium is set to be 0%, 0.5%, 1%, 2%, 3%, 5%, 8%, 10%, 12%, 15%, 18%, 21%, 24%, 26%, 28%, 30%, 32% and 34%, respectively.

(4) Preparing a solid culture medium: the culture medium is prepared according to the set culture medium formula, the culture medium with higher salinity is sterilized after being melted by hot water, 5ml of evaporated water is added in each bottle, the volatile culture medium components are added after being sterilized and are evenly shaken, the plate is poured after being cooled to about 60 ℃, and the salinity is high and is easy to solidify, so that the pouring of the plate is fast (when the culture medium with 32% salinity is poured into the plate, a small amount of salt is precipitated after cooling and solidification, and a large amount of salt crystals are precipitated at 34% salinity).

(5) Inoculating and culturing: selecting a ring of fresh lawn under aseptic condition, inoculating to culture medium with various salinity, transferring from low salinity to high salinity in gradient manner, streaking with 34% salinity culture medium, separating out a large amount of salt crystals along with streaking track, sealing the culture dish with sealing film after inoculation, culturing at 35-37 deg.C for 3-7 days, and observing strain growth condition.

(6) And (3) test results: the salt tolerance range of YL5-2 strain is 3-32%. The YL5-2 strain can obviously observe newly grown lawn in 3-30% salinity culture medium within three days, but can grow to naked eyes in 32% saturated salinity culture medium for more than 7 days. The growth rate of YL5-2 is higher in the salt concentration range of 3% -30%.

TABLE 4 growth of YL5-2 on media with different salt concentrations

Example 7 salt-tolerant denitrifying capability test of halophilic denitrifying bacteria YL5-2

(1) Culture medium: acetic acid 2000mg/L, peptone 20mg/L, beef extract 20mg/L, NO₃the-N is 100mg/L, the NaCl concentration is 3% -30%, and the buffer solution is added to ensure that the pH value is 7.5-8.0.

(2) And (3) experimental design: the denitrification test was performed in 10 500mL flasks, each containing 300mL of medium, with 8 salinity gradients set at 3%, 6%, 10%, 12%, 15%, 20%, 25%, 30% salt content, and 2 replicates of the salt-free blank.

(3) Denitrification test: after sterilizing all samples, inoculating about 10mL of YL-5 culture solution, and culturing on a constant-temperature shaking table at the temperature of 30-35 ℃; the oscillation speeds are respectively 10 ppm; sampling and determining NO in the triangular flask after 24h, 48h and 72h respectively₃-the concentration of N. The test results are shown in the following table data:

TABLE 5 YL5-2 Denitrification for NO removal at different salt concentrations₃Test results of-N (unit: mg/L)

	0	0	3％	6％	10％	12％	15％	20％	25％	30％
											24h	99.6	99.5	91.3	76.2	68.8	67.5	66.4	67.6	72.6	89.6
48h	99.1	99.2	55.4	18.6	14.4	11.7	9.9	14.7	19.1	40.6
											72h	98.5	98.8	13.5	6.7	4.5	3.6	3.8	4.2	8.2	12.5

Example 8 salt-tolerant denitrifying capability test of halophilic denitrifying bacterium YL5-2

(1) Culture medium: acetic acid 2000mg/L, peptone 20mg/L, beef extract 20mg/L, NO₂the-N is 100mg/L, the NaCl concentration is 3% -30%, and the buffer solution is added to ensure that the pH value is 7.5-8.0.

(3) Denitrification test: after sterilizing all samples, inoculating about 10mL of YL-5 culture solution, and culturing on a constant-temperature shaking table at the temperature of 30-35 ℃; the oscillation speeds are respectively 10 ppm; sampling and determining NO in the triangular flask after 24h, 48h and 72h respectively₂-the concentration of N. The test results are shown in the following table data:

TABLE 6 YL5-2 Denitrification for NO removal at different salt concentrations₂Test results of-N (unit: mg/L)

	0	0	3％	6％	10％	12％	15％	20％	25％	30％
											24h	99.7	99.7	95.3	86.2	78.8	77.5	76.4	77.6	81.6	91.6
48h	99.3	99.1	62.4	20.3	16.5	14.8	11.3	15.7	19.1	30.6
											72h	99.0	98.6	15.8	9.7	6.6	4.2	3.8	7.3	8.5	15.3

Sequence listing

<110> Zhonglan Lianhai design research institute Co., Ltd

<120> halophilic denitrifying bacterium YL5-2 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1431

<212> DNA

<213> genus Halovibrio (Halovibrio sp.)

<400> 1

cccatggggg cagctacaca tgcagtcgag cggcagcagc tccttcggga ggctggcgag 60

cggcggacgg gtgagtaacg catgggaact tacccagtag tgggggatag cccggggaaa 120

cccggattaa taccgcatac gccctgaggg ggaaagcggg ctccggctcg cgctattgga 180

tgggcccatg tcggattagt tagttggtgg ggtaatggcc taccaaggcg acgatccgta 240

gctggtctga gaggatgatc agccacaccg ggactgagac acggcccgga ctcctacggg 300

aggcagcagt ggggaatatt ggacaatggg ggcaaccctg atccagccat gccgcgtgtg 360

tgaagaaggc cttagggttg taaagcactt tcagcaggga ggaaaagctg atcgttaata 420

ccggtcagtg ttgacgttac ctgcagaaga agcaccggct aactccgtgc cagcagccgc 480

ggtaatacgg agggtgcaag cgttaatcgg aattactggg cgtaaagggc gcgtaggcgg 540

tttggtaagc gagttgtgaa agccccgggc tcaacctggg aatggcaatt cgaactgcca 600

agctagaatg cagcagaggg cagtggaatt ccaggtgtag cggtgaaatg cgtagatatc 660

tggaggaaca ccagtggcga aggcgactgc ctgggctgac actgacgctg aggtgcgaaa 720

gcgtgggtag caaacaggat tagataccct ggtagtccac gctgtaaacg ctgagaacta 780

gtcgttgggg ctattagagc cttagtgacg cagctaacgc gataagttct ccgcctgggg 840

agtacggccg caaggttaaa actcaaatga attgacgggg gcccgcacaa gcggtggagc 900

atgtggttta attcgacgca acgcgaagaa ccttacctgg tcttgacatc ctgcgaactt 960

ggtagagata ccttggtgcc ttcgggagcg cagtgacagg tgctgcatgg ccgtcgtcag 1020

ctcgtgtcgt gagatgttgg gttaagtccc gtaacgagcg caacccttgt ccttagttgc 1080

cagcggtccg gccgggaact ctagggagac tgccggtgac aaaccggagg aaggtgggga 1140

tgacgtcagg tcatcatggc ccttacggcc agggctacac acgtgctaca atggggcgca 1200

cagagggcag caagcgcgcg agtgcaagcg aatcccttaa aacgcctcgt agtccggatc 1260

ggagtctgca actcgactcc gtgaagtcgg aatcgctagt aatcgcagat cagaatgctg 1320

cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggga gtggactgca 1380

ccagaagcgg ttagtctaac cttcgggagg acgatcgcca cggtgtctgt a 1431

Claims

1. Halophilic denitrifying bacteriumHalovibrio salipaludissp.nov YL5-2, which is characterized in that the preservation date is 2018, 8 months and 20 days, and the preservation number is CGMCC NO. 16315.

2. Use of the halophilic denitrifying bacteria YL5-2 of claim 1 in the treatment of high salinity wastewater with salinity of 3% -32%.

3. The use according to claim 2, characterized by the use of the halophilic denitrifying bacteria YL5-2 of claim 1 in the biological denitrification treatment of high salinity wastewater with salinity of 3% to 32%.

4. The use according to claim 3, wherein said halophilic denitrifying bacteria YL5-2 denitrify high salinity wastewater under anoxic or aerobic conditions.

5. The use according to claim 4, wherein said halophilic denitrifying bacteria YL5-2 denitrify high salinity wastewater with salinity of more than 10% under anoxic or aerobic conditions.

6. The use of the halophilic denitrifying bacterium YL5-2 of claim 1 in polluted seawater treatment and saline-alkali land remediation.

7. The use of the halophilic denitrifying bacterium YL5-2 of claim 1 in consuming nitrogen nutrition, inhibiting excessive algae reproduction, and purifying water.

8. Use of the halophilic denitrifying bacterium YL5-2 of claim 1 for modifying water body substrates.