CN111662847A - Enterobacter antimonoroxide and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an enterobacter antimonoroxide strain and application thereof. The enterobacter strain is obtained by separating and screening antimony-polluted soil from tin mine of Jiangjiang city, Hunan province, has high-efficiency antimony oxidation performance, can still survive in solution and soil with high antimony content, has good antimony tolerance, has high-efficiency antimony oxidation performance after being tested, and lays an important foundation for the development of a microbial adsorption antimony removal technology. At the same time, the strain binds Fe³⁺The modification effect is good, the oxidation efficiency can be improved, and the development and application potential is good.

Description

Enterobacter antimonoroxide and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to an enterobacter antimonoroxide strain and application thereof.

Background

Antimony (Sb) has chronic toxicity, carcinogenicity, and global mobility characteristics, and is a global pollutant and a priority control pollutant. Antimony (Sb) and compounds thereof can enter a human body through food chains, water and other ways, not only can the action of certain enzymes be influenced, but also diseases in the aspects of liver and cardiovascular systems can be caused, so that the world health organization, European Union and China all make strict limitation on the content of antimony in drinking water (<5 mu g/L), wherein the toxicity of trivalent antimony is ten times that of pentavalent antimony, and the oxidation of Sb (III) into Sb (V) is favorable for reducing the environmental hazard and risk of Sb (V). Some researches are made at home and abroad on how to oxidize Sb (III) into Sb (V), such as treatment technologies of adding chemical oxidants and the like. These techniques oxidize Sb (iii) to Sb (v), but have the disadvantages of low oxidation efficiency, high cost, complicated operation, easy generation of secondary pollution, etc. In recent years, scholars at home and abroad propose a microbial oxidation technology which is safe, economical, environment-friendly and high in oxidation efficiency. The screening and identification of efficient antimony-oxidizing microorganisms are one of the cornerstones of microbial antimony removal technology, but the research on the aspects is relatively less.

Disclosure of Invention

The invention provides an enterobacter antimonoroxide strain and application thereof, aiming at solving part of problems in the prior art or at least relieving part of problems in the prior art.

The invention is realized by that one strain of the antimony oxide enterobacter has a preservation number of CCTCC NO: M2020006. The Enterobacter (Enterobacter DHHN0903) is preserved in China center for type culture Collection, is named Enterobacter sp.AO-3, and the preservation unit is CCTCC for short, and the preservation date is 1 month and 3 days 2020.

Use of enterobacter antimonorxidans as described above for the oxidation of antimony.

Further, the antimony oxidation is specifically oxidation of Sb (iii) to Sb (v).

The application of the enterobacter antimonoroxide in the prevention and treatment of antimony pollution of soil or water body. The migration and transformation process control of Sb (III) and Sb (V) in soil or water is realized, and the toxicity and ecological hazard of antimony in the soil or water are reduced. The enterobacter strain DHHN0903 is used for converting Sb (III) in a water body into Sb (V) with lower toxicity by utilizing higher antimony oxidation degree and higher antimony oxidation speed, so that the detoxification effect is achieved, and the heavy metal Sb pollution is effectively prevented from the source.

Further, the application method comprises using Fe³⁺The ions promote the oxidation of antimony by enterobacter antimonoroxide. In particular to promote the oxidation of Sb (III) into Sb (V).

Further, the enterobacter antimonoroxide is used for treating a sample to be detected under the aerobic condition at the temperature of 30 ℃.

Further, said Fe³⁺With FeCl₃Is added to the reaction system.

Further, FeCl in the reaction system₃The concentration of (2) is 0.1 mol/L.

In summary, the advantages and positive effects of the invention are:

the enterobacter strain DHHN0903 is a gram-negative bacterium, can still survive in a solution with high antimony content and soil, has good antimony tolerance, and is convenient for bioremediation in an antimony-polluted environment. And through inspection, the antimony oxide has high-efficiency antimony oxidation, and lays an important foundation for the development of a microbial adsorption antimony removal technology. The strain has efficient oxidation performance on Sb (III) and has important significance on the development of a microbial oxidation antimony removal technology.

Meanwhile, the technology is environment-friendly; the strain culture conditions are simple, the preservation is easy, the industrial production is easy, and the development and application potential is good.

On the other hand, the modification effect of the strains combined with Fe is good, the oxidation efficiency can be improved, and the method has good development and application potentials.

Drawings

FIG. 1 is a photograph showing the colony morphology of Enterobacter antimonorum DHHN0903 on a plate;

FIG. 2 is a phylogenetic tree of Enterobacter bacteria of the present invention;

FIG. 3 is a graph of the oxidative performance of Enterobacter of the present invention;

FIG. 4 is a graph of the modified oxidation performance of Enterobacter of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The invention discloses an enterobacter antimonoroxide strain and application thereof, and the specific content is shown in the following examples.

Example 1 isolation, screening and characterization of high Performance Enterobacter antimonioxidans DHHN0903

The invention relates to screening from soil samples near a tailing dam of a Longwang pond of a tin mine in the city of the Cold Water Jiang of Hunan province, in particular to screening by flat plate scribing, and belongs to a conventional strain separation and screening experimental method in the field. The specific method and process are described as follows: weighing 10g of soil sample, and adding the soil sample into a sterilized 250ml conical flask; soaking in 90ml sterile water, shaking on a shaking table at 30 deg.C for 30min, and standing for 30 s; separating bacteria by serial dilution coating method, selecting single bacteria, streaking and purifying on solid culture medium, and performing pure culture of bacteria in 30 deg.C constant temperature incubator to obtain purified strain. The plate colony map is shown in FIG. 1. Physiological and biochemical characteristics and 16S rDNA sequence Analysis, and identification with reference to literature (Claesson MJ, O' Sullivan O, Wang Q, equivalent. comprehensive Analysis of Pyrosequencing and a Phylogenetic Microarray for expanding Microbial communication Structures in the Human Manual expression evaluation. AhmedN, ed.PLoS ONE.2009; 4(8): e 6669; Parks DH, Tyson, Hugenholtz P, Beiko RG.STAMP: static Analysis of taxonomic and functional profiles bioinformatics.2014; 30(21): 3123. 3124.) while the China center is entrusted to determine and analyze the gene sequence of the 16S rRNA deposited strain of microorganisms; and preliminarily determining the classification status of the microorganism strains to be detected according to the result, wherein the report number is CCTCC-JD-2019283. The gene test and the construction of the phylogenetic tree are shown in figure 2, and the result shows that the Enterobacter DHHN0903(Enterobacter DHHN0903) has the maximum similarity with the Enterobacter huaxiensis, and the similarity of the Enterobacter DHHN0903 and the Enterobacter huaxiensis reaches 99.34307%. Identified as Enterobacter, Enterobacter sp, designated DHHN 0903.

This example carried out a test analysis of Enterobacter DHHN0903 of the present invention by 16S rRNA sequence.

16S rRNA-related identification method:

carrying out PCR identification on the screened strains by using the universal primers of the bacteria: extracting template DNA according to the operation instruction of the bacterial DNA extraction kit. The 16S rRNA gene fragment of Enterobacter DHHN0903 was PCR amplified, cloned and sequenced using upstream primer 27F 5'-GAGTTTGATCCTGGCTCAG-3' and downstream primer 1492R 5'-TACGGTTACCTTGTTACGACTT-3' of the 16S rRNA conserved sequence.

Obtaining a 1433bp fragment after sequencing, obtaining the bacterium in enterobacter by sequencing under the accession number of MN720565 in GenBank, and the sequencing result of the amplified fragment is shown in SEQ ID No. 1.

The BLAST results for the 16S rRNA gene sequencing of enterobacter antimonorum DHHN0903 are given in the following table:

TABLE 1 BLAST comparison results

The physiological and biochemical properties of enterobacter DHHN0903 are as follows:

TABLE 2 physiological and biochemical characteristics of the strains-enzyme activity, carbon source assimilation

+: positive reaction; -: negative reaction; weak positive reaction

TABLE 3 physiological and biochemical characteristics of the strains-production of acid Using a carbon Source

The separation, purification and culture conditions and the detection and survival conditions of the strain are as follows: CDM Medium (MgSO)₄·7H₂O，2.0g；NH₄Cl，1.0g；Na₂SO₄，1.0g；K₂HPO₄，0.013g；CaCl₂·2H₂O, 0.067 g; na-lactate, 5.0 g; agar, 15.0 g; 1000mL of distilled water; pH 7.0); at 30 ℃.

The detection survivability conditions of the strain are as follows: CDM Medium (MgSO)₄·7H₂O，2.0g；NH₄Cl，1.0g；Na₂SO₄，1.0g；K₂HPO₄，0.013g；CaCl₂·2H₂O, 0.067 g; na-lactate, 5.0 g; agar, 15.0g (solid medium contains agar, liquid medium does not); 1000mL of distilled water; pH 7.0); at 30 ℃.

EXAMPLE 2 antimony resistance test (solid Medium) of high Performance antimony oxidizing bacterium DHHN0903

Scribing on CDM solid culture medium with different antimony concentrations by plate scribing, culturing in 30 deg.C constant temperature incubator, and measuring antimony resistance. The high-efficiency antimony oxidizing bacteria DHHN0903 can normally grow on a solid culture medium with the antimony concentration of 1-8000mg/L, and the growth of the antimony concentration of 9000mg/L is extremely slow and almost not.

Compared with other research reports, the resistance of enterobacter DHHN0903 to heavy metal antimony is higher than that of other reported microorganisms. The strain survives in a heavy metal pollution area for a long time, and the strong resistance to heavy metal antimony can be related to the complex living environment of the strain.

Example 3 Enterobacter DHHN0903 Oxidation Performance testing

Experiments preliminary studies were performed on the antimony oxidizing ability of enterobacter DHHN 0903. The medium used was CDM liquid medium to which Sb (III) was added at a concentration of 0.5 mmol/L. Wherein the sodium lactate is replaced by yeast extract. The yeast extract is used as a better comprehensive nutrient substance of a carbon source and a nitrogen source, and can promote the growth of the strain. Set up 3 parallel groups and 1 blank (no ligation)Inoculum), 250mL of culture solution is prepared for each group, high-temperature sterilization is carried out (121 ℃, 30min), 3% of DHHN0903 bacterial solution is inoculated after cooling to the appropriate temperature on an ultra-clean workbench, 4 groups of conical flasks are all put into a shaker at 30 ℃ and 150rpm for culture, and the experiment lasts for one week. The absorbance was measured every 4 hours, and a growth curve was plotted. The oxidation was measured simultaneously every 8 hours and an oxidation curve was plotted. Wherein the OD of the growth curve is measured by an ultraviolet spectrophotometer₆₉₀. In the oxidation curve measurement, the sample was centrifuged at 5000rpm for 5min, the supernatant was collected, and the concentrations of Sb (III) and Sb (V) in the supernatant were measured using an atomic fluorescence spectrometer (AFS-9700, Beijing Hai Shi instruments Co., Ltd.), and concentration curves were drawn, and the results are shown in FIG. 3. DHHN0903 was obtained with a maximum antimony oxidation rate of 21.6. mu. mol/L/h. The oxidation rate calculation formula is as follows:

wherein v is the reaction rate and S is the concentration of the substrate. Vmax represents the maximum speed, K_mIs the Michaelis-Menton constant. Wherein V_maxAnd K_mFrom (initial velocity)^-1And (initial substrate concentration)^-1The ratio of (a) to (b) is determined.

Example 4 Fe³⁺Ion-promoted oxidation rate of antimony by enterobacter DHHN0903

Enterobacter DHHN0903 was modified with iron salts and then tested for oxidation performance. The medium used was CDM liquid medium to which Sb (III) was added at a concentration of 0.5 mmol/L. Wherein the sodium lactate is replaced by yeast extract. Adding FeCl into the culture medium₃The concentration is 0.1mol/L, and the mixture is evenly mixed and shaken. 3 parallel groups and 1 blank control (no inoculation) are arranged, 250mL of culture solution is prepared for each group, high-temperature sterilization is carried out (121 ℃, 30min), after the culture solution is cooled to the appropriate temperature on an ultra-clean workbench, 3% of DHHN0903 bacterial solution is inoculated, 4 groups of conical flasks are all put into a shaker at 30 ℃ and 150rpm for cultivation, and the experiment lasts for one week. The oxidation was measured and an oxidation curve was plotted, as shown in FIG. 4.

According to the curve, Fe is not added³⁺Under ionic conditions, DHHN090The average oxidation rate of the bacterial liquid to Sb (III) is 2.10 mu mol/L/h; adding Fe³⁺Under the condition of ions, the average oxidation rate of the DHHN0903 bacterial liquid to Sb (III) is 2.85 mu mol/L/h. Description of Fe³⁺The ions promote the rate of oxidation of antimony by enterobacter DHHN 0903.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Hunan university of science and technology

<120> Enterobacter antimonicoxidans and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1433

<212>DNA

<213> Enterobacter (Enterobacter)

<400>1

gactggcggc gctaccatgc aagtcgagcg gtaacacagg gagcttgctc ctgggtgacg 60

agcggcggac gggtgagtaa tgtctgggaa actgcctgat ggagggggat aactactgga 120

aacggtagct aataccgcat aacgtcgcaa gaccaaagag ggggaccttc gggcctcttg 180

ccatcagatg tgcccagatg ggattagcta gtaggtgggg taacggctca cctaggcgac 240

gatccctagc tggtctgaga ggatgaccag ccacactgga actgagacac ggtccagact 300

cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat gcagccatgc 360

cgcgtgtatg aagaaggcct tcgggttgta aagtactttc agcggggagg aaggtgctga 420

ggttaataac ctcagcaatt gacgttaccc gcagaagaag caccggctaa ctccgtgcca 480

gcagccgcgg taatacggag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac 540

gcaggcggtc tgtcaagtcg gatgtgaaat ccccgggctc aacctgggaa ctgcattcga 600

aactggcagg ctagagtctt gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg 660

tagagatctg gaggaatacc ggtggcgaag gcggccccct ggacaaagac tgacgctcag 720

gtgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat 780

gtcgacttgg aggttgttcc cttgaggagt ggcttccgga gctaacgcgt taagtcgacc 840

gcctggggag tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc 900

ggtggagcat gtggtttaat tcgatgcaac gcgaagaacc ttacctactc ttgacatcca 960

gagaacttag cagagatgct ttggtgcctt cgggaactct gagacaggtg ctgcatggct 1020

gtcgtcagct cgtgttgtga aatgttgggt taagtcccgc aacgagcgca acccttatcc 1080

tttgttgcca gcggttaggc cgggaactca aaggagactg ccagtgataa actggaggaa 1140

ggtggggatg acgtcaagtc atcatggccc ttacgagtag ggctacacac gtgctacaat 1200

ggcatataca aagagaagcg acctcgcgag agcaagcgga cctcataaag tatgtcgtag 1260

tccggattgg agtctgcaac tcgactccat gaagtcggaa tcgctagtaa tcgtagatca 1320

gaatgctacg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt 1380

gggttgcaaa agaagtaggt agcttaacct tcgggagggc gctacccact tta 1433

Claims (8)

1. The preservation number of the enterobacter antimonoroxide is CCTCC NO: M2020006.

2. Use of enterobacter antimonorxidans as claimed in claim 1 for oxidation of antimony.

3. Use according to claim 2, characterized in that: the antimony oxidation is specifically the oxidation of Sb (iii) to Sb (v).

4. Use of enterobacter antimonorxidans as claimed in claim 1 for the prevention and treatment of antimony contamination of soil or water bodies.

5. Use according to any one of claims 2 to 4, characterized in that: the application method comprises the step of treating a sample to be tested by using the enterobacter antimonoroxide under the aerobic condition of 30 ℃.

6. Use according to claim 5, characterized in that: the application method also comprises the use of Fe³⁺The ions promote the oxidation of antimony by enterobacter antimonoroxide.

7. Use according to claim 6, characterized in that: said Fe³⁺With FeCl₃Is added to the reaction system.

8. Use according to claim 7, characterized in that: FeCl in the reaction System₃The concentration of (2) is 0.1 mol/L.

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