CN106244501A - One strain anti-antimony antibacterial NXH1 and application thereof - Google Patents
One strain anti-antimony antibacterial NXH1 and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of anti-antimony antibacterial acinetobacter calcoaceticus (AcinetobacterSp), preserving number is CGMCC No.12895.Bacterium high resistance antimony of the present invention, has anti-As simultaneously3+、Cd2+、Cr6+、Hg2Etc. various heavy, and the characteristic of promotion plant growing, the murder by poisoning to promotion plant strain growth, alleviation plant heavy metal is significant, has wide application potential in the biological restoration of the mining soil of heavy metal pollution.
Description
Technical field
The invention belongs to field of environmental biotechnology, be specifically related to a strain anti-antimony antibacterial NXH1 and application thereof.
Background technology
In recent years, along with antimony ore gathers the continuous expansion of scale, and the discharge of garbage in antimony industry, mining area week is caused
The soil on limit is by serious pollution.Have been found to be that a class has genotoxicity as environmental contaminants, antimony and antimonide
Material, and human body is had carcinogenesis (Huang et al., 1998; Takahashi et al., 2002;
Beyersmann et al., 2008).Additionally, antimony can also cause, injury of lung, hematuria, fibroblast be dead, sister dyes list
Body exchange, blood circulation diseases etc. (Huang et al., 1998).Antimony is by Environmental Protection Agency and council of the European Community
It is classified as preferential pollution administration thing, is also the pollutant closely monitored of the Japanese Environment Room.In recent years, the pollution situation of China's antimony more comes
The most serious, gradually by the extensive concern of various circles of society.Meanwhile, the smelting activity of adopting of antimony ore can cause and a huge sum of money for antimony association simultaneously
Belong to element such as arsenic, hydrargyrum etc. and enter mining area supergene environment, pollute farmland, mining area, not only affect crop growth, reduce crops
Quality.The soil in mining area be also result in the biggest destruction.How research repairs the difficult problem that mining soil has become urgently to be resolved hurrily.
Although antimony has the highest toxicity for the mankind, but some microorganisms can be in the environment of very high concentrations
Growth, it might even be possible to utilize this element as energy substance, or Sb (III) higher for toxicity is oxidized to toxicity relatively
Weak Sb (V), thus, the microorganism remediation of contaminated soil is theoretical and recovery technique just arises at the historic moment (rise and should wait, 2007).Micro-
Biological can be by thalline to the outer row of antimony, highly toxic Sb(III) be oxidized to hypotoxic Sb(V), microorganism is to antimony
Methylate, the mode such as thalline absorption reduces antimony to thalline and the toxicity of environment.Anti-antimony 1971, Lyalthova, reported first
1 strain antimony oxidizing bacteria, this bacterial strain can the antimony of trivalent be oxidized to the antimony of pentavalent for self provide energy (Lyalthova,
1971).Having the report on a small quantity about anti-antimony microorganism the most successively, in a word, up to the present oneself is through the anti-antimony microorganism of report
Kind the most less, therefore, it is to realize the important step that antimony pollution is repaired that screening has the microorganism of antimony resistance.
Few about the screening of anti-antimony microorganism and the correlational study report of application aspect at present, Liu Chengzuo (2012) screens
To a strain resistance to antimony microorganism, being identified as penicillium, its resistance to antimony concentration is 600mg/L.Strain in patent of the present invention is motionless
Bacillus (AcinetobacterSp.) the strain antibacterial in, its anti-antimony effect apparently higher than it has been reported that penicillium.And this is not
Lever bacterium has plant growth-promoting function, it is possible to produce IAA, siderophore and acc deaminase, has certain growth-promoting effect to plant
Really；Simultaneously to As3+、Cd2+、Cr6+、Hg2+Etc. various heavy, there is the strongest resistance, there is potential using value.
Summary of the invention
Present invention aim at the antibacterial providing a strain to have high resistance antimony, through 16S rDNA sequencing analysis, result form
Learn observe, be initially identified as acinetobacter calcoaceticus (AcinetobacterSp.), strain number is NXH1, and this bacterium has anti-As simultaneously3+、
Cd2+、Cr6+、Hg2Etc. various heavy, and promote the characteristic of plant growing.
The acinetobacter calcoaceticus of the present invention (AcinetobacterSp.) NXH1, is from Lengshuijiang, Hunan Xikuangshan Ore
The strain of separation screening in plant rhizosphere soil near smeltery of district, can enough tolerate the huge sum of money that antimony concentration is 2900mg/L
Genus is coerced.Bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 23rd, 2016, its
Referred to as CGMCC (unit address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postal
Political affairs encode: 100101), deposit number is CGMCC No.12895, survives after testing.
After cultivating 18h on LB solid medium, its colony characteristics is: bacterium colony is little, faint yellow, neat in edge, circular convex
Rise, smooth surface, moistening, non-wrinkled, opaque.Its somatic cells is characterized as: bacillus, and Gram’s staining is negative, without spore,
Growth is rapidly.
The 16S rDNA sequence of this bacterial strain being expanded by PCR, it is thus achieved that the amplified production of about 1400bp length, amplification is produced
Thing carries out sequencing through order-checking company, and by being checked order, row carry out BLAST comparison, result with the sequence in GenBank data base
Show, this bacterial strain and acinetobacter (AcinetobacterAcinetobacter calcoaceticus and addicted to oil acinetobacter calcoaceticus homology in)
Height, similarity is more than 98%.Combining form feature, cultural characteristic and 16S rDNA sequence analysis, this bacterial strain is defined as not lever
Bacterium (AcinetobacterSp.).
This bacterial strain is containing variable concentrations Sb(antimony potassium tartrate) CDM solid medium in cultivate certain time, observe
Its growing state, result shows, anti-Sb is very competent for this bacterium, and trivalent antimony toleration is reached 2900mg/L.
The method have the advantages that
The strain of the present invention is bolter the plant rhizosphere soil near smeltery of Lengshuijiang Xikuangshan Ore district of Hunan Province
A plant height anti-antimony (Sb) antibacterial selected, identified should for acinetobacter calcoaceticus (AcinetobacterSp.).Bacterial strain of the present invention except
There is the characteristic of the strongest preventing from heavy metal antimony, and this bacterial strain is to As3+、Cd2+、Cr6+、Hg2+Etc. heavy metal, there is the strongest resisting
Property.This bacterium also has plant growth-promoting function simultaneously, it is possible to produce IAA and siderophore both plant growth-promoting factors, and dirty to Sb
Plant growing in dye soil has facilitation, such as, have certain facilitation effect to growth of rape.The thinnest about anti-antimony
Research in terms of the multiple preventing from heavy metal ability of bacterium and growth-promoting effect at home and abroad yet there are no relevant report.Therefore, dirty from mining area
Contaminate the height anti-growth-promoting microorganism of ground screening heavy metal tolerance, to promoting local vegetation growth, the murder by poisoning of alleviation plant heavy metal
Significant.This bacterium has wide application potential in the biological restoration of the mining soil of heavy metal pollution.
Accompanying drawing explanation
Fig. 1 acinetobacter calcoaceticus of the present invention (AcinetobacterSp.) culture.
Fig. 2 acinetobacter calcoaceticus of the present invention (AcinetobacterSp.) 16S rDNA gene order.
Detailed description of the invention
It is further elucidated with the present invention below by the detailed description of detailed description of the invention, but is not the limit to the present invention
System, only illustrates.
The separation screening of embodiment 1 anti-antimony antibacterial and anti-Sb ability
Gather the plant rhizosphere soil sample near smeltery of Xikuangshan Ore district, Lengshuijiang, Hunan, weigh above-mentioned fresh soil
Earth sample 100g, adds appropriate antimony potassium tartrate ([C8H4K2O12Sb2·3(H2O)]), the ultimate density of antimony in soil sample is made to be
1000mg/kg, is put in 28 DEG C of constant incubator enrichment culture one week.Take above-mentioned soil sample 10g be put in equipped with 10 beades and
Fill in 90mL 0.85% NaCl sterile solution, 30 DEG C, 180 r min-1 shaking tables shake 30 min, makes sample fully scatter.
Take 0.1mL and be applied to (CDM culture medium prescription: MgSO in CDM culture medium4·7H2O 2.0 g, NH4Cl 1.0 g, Na2SO4 1.0
G, K2HPO40.013 g, CaCl2·2H2O 0.067 g, Na-lactate 5.0 g, agar 15.0 g, add distilled water extremely
1000mL, pH 7.2), wherein with antimony potassium tartrate as stress factors in CDM culture medium, make the concentration of antimony in final culture medium be
50mM, cultivates one week in 30 DEG C of constant incubators, observes its upgrowth situation every day.The bacterial strain above-mentioned initial gross separation gone out is carried out
The further purebred bacterial strain of isolated and purified acquisition, the strain after separating is received on inclined-plane, and 4 DEG C of preservations carry out subsequent experimental.
Inoculation (LB culture medium prescription: peptone 10g, yeast extract in LB culture medium by screening gained
5g, NaCl 10g, agar 15 g, add distilled water to 1000 mL, pH 7.2), activate 24 h, take 2 mL and proceed to equipped with 100
In 250 mL triangles of mL LB fluid medium, 30 DEG C, 150 r/min shaken cultivation, with nonvaccinated LB liquid culture
Base is comparison, selects the wavelength of 600nm, measures the optical density value of different incubation time bacterial suspension by microplate reader
(OD600).With incubation time as abscissa, OD600 is vertical coordinate, draws the growth curve of antibacterial, to understand the growth of bacterial strain
In the cycle, study the bacterial strain research suitable growth to toleration, resistance and the growth-promoting effect of the heavy metals such as Sb, As further for the later stage
The thalline in cycle.In CDM culture medium, add Sb storing solution, make the final concentration of the Sb in culture medium be respectively 1,2,4,6,8
With 10 mmol/L and concentrations above, improving heavy metal concentration successively according to experimental result, the anti-Sb bacterial strain preserved on inclined-plane is at LB
Culture fluid activates 24 h, on solid medium isolated and purified go out single bacterium colony, then be transferred to containing respective concentration Sb solid training
Support on base, observe strain growth situation, it is possible to the minimum inhibitory concentration that least concentration is this bacterial strain of suppression strain growth
(Minimum inhibitory concentration, MIC)。
By above-mentioned separation screening, it is thus achieved that some anti-Sb antibacterials, most of strain bacterium enter stable phase, bacterial strain logarithm at 48h
Phase is 6h-36h.Wherein, bacterial strain of the present invention (numbered NXH1) growth is very fast, is put into stable phase, follow-up reality after 24h
Test take cultivate 24h thalline study.By mensuration Sb to isolated strains minimum inhibitory concentration, find that bacterial strain NXH1 is to Sb's
Resistance is the highest, and minimum inhibitory concentration reaches 24mM, is converted into mass concentration substantially 2900mg/L.
The qualification of embodiment 2 NXH1 bacterial strain
This bacterial strain is carried out the sequence analysis of morphology, cultural characteristic and 16S rDNA.16S rDNA Molecular Identification is pressed
Carry out according to following steps: single colony inoculation of picking bacterium in liquid LB culture medium, 30 DEG C, 150r/min shaking table
Shaken cultivation, takes out culture fluid at 24h, and 5000r/min is centrifuged 1min and takes supernatant, tries according to bacterial genomes DNA extraction
Agent box (Tian Gen biochemical technology company limited provide), extracts bacterium colony DNA；Universal primer 27F and the 1492R antibacterial to extracting
DNA carries out PCR amplification；27F sequence is 5 '-AGA GTT TGA TCC TGG CTC AG-3 '；1492R sequence is 5 '-AAG
GAG GTG ATC CAG CCG CA-3 ' primer is synthesized by Beijing Bioisystech Co., Ltd of farsighted Boxing section；PCR product is entered
Row sequence, sequencing result BLAST in NCBI data base carries out sequence analysis, and carries out tetraploid rice.
The morphological features of NXH1 bacterial strain is as follows: bacterium colony is little, faint yellow, neat in edge, circular protrusions, smooth surface,
Moistening, non-wrinkled, opaque.Its somatic cells is characterized as: bacillus, and Gram’s staining is negative, and without spore, growth rapidly, is shown in
Fig. 1.These bacterium a length of 1415 bp of 16S rDNA gene order, are submitted to gene order on Genbank, carry out homology
Relatively, then use MEGA 6.0 Software on Drawing phylogenetic tree, so that it is determined that the kind of bacterial strain.Result show this sequence with
Acinetobacter calcoaceticus (AcinetobacterIn), the 16S rDNA gene order similarity of each bacterium is the highest, reaches more than 98%, ties simultaneously
Close colony morphology characteristic, thalline microscopic features determine NXH1 bacterial strain be acinetobacter calcoaceticus (AcinetobacterSp.), its 16S
RDNA gene order is as shown in Figure 2.It is general that this bacterium is deposited in China Committee for Culture Collection of Microorganisms on August 23rd, 2016
Logical microorganism center, it is referred to as CGMCC (unit address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology, postcode: 100101), deposit number is CGMCC No. 12895.
Embodiment 3: the acinetobacter calcoaceticus NXH1 toleration to various heavy
Preparation is containing variable concentrations As respectively3+、Cd2+、Cr6+、Hg2The CDM solid medium of heavy metal, Cd2+、Cr6+、Hg2With much money
Genus concentration, from 100mg/L, is followed successively by 100mg/L, 200mg/L, 400mg/L, 800mg/L, 1200mg/L, 1600mg/L, with
It is not added with the process of heavy metal as comparison.By acinetobacter calcoaceticus (AcinetobacterSp.) NXH1 inoculation is to without a huge sum of money
Activation culture 24h in the CDM culture medium belonged to, takes 0.1mL and is inoculated in respectively containing variety classes, the cultivation of variable concentrations heavy metal
In base, it is placed in 30 ° of C and cultivates 24h, observe thalline growing state, knot in the culture medium containing various types of, variable concentrations heavy metal
Fruit is shown in Table 1.As can be seen from the table, NXH1 bacterial strain is respectively provided with stronger toleration, to As to above-mentioned several heavy metal species3+、Cd2+、
Cr6+、Hg2Tolerable concentration be respectively 1600mg/L, 1200mg/L, 1200ml/L and 800mg/L.
Table 1 acinetobacter calcoaceticus NXH1 growing state under different heavy metal concentrations
Note: "+" represent well-grown；"-" represents and does not grows；" +/-" represents and can grow, but undergrowth
The plant growth-promoting characteristic of embodiment 4 acinetobacter calcoaceticus NXH1
There is the microorganism of Plant growth promotion often through secreting heteroauxing (Indole-3-acetic in environment
Acid, IAA), produce siderophore and acc deaminase, play the effect promoting plant growing.By following experimental implementation, permissible
Checking acinetobacter calcoaceticus NXH1 has plant growth-promoting characteristic.The Function Identification method producing IAA is as follows: specific in cultivating microorganism
Fluid medium adds 0. 5 g/L L-Trp (about 2. 5 mmol/L) high temperature sterilize afterwards, with toothpick strain bacterial strain chosen into
Fluid medium, in being placed on constant-temperature shaking incubator, 30 ° of C cultivate 24 hours, draw 2mL bacterium solution, add 2mL in 2mL bacterium solution
Salkowski reagent (12g FeCl3, 430mL 98% H2SO4, 570mL H2O) colour developing.The bacterium solution of pinkiness is then positive,
Illustrate that this bacterial strain produces IAA.The Function Identification method producing siderophore is as follows: preparation CAS detects culture medium, will divide in super-clean bench
Bacterial strain point from purification detects in culture medium at CAS, cultivates in the incubator of 30 DEG C；By observing bacterial secretory siderophore
The size of the orange haloing formed does qualitative screening, and orange haloing is the biggest, illustrates that bacterial strain produces siderophore ability the strongest.Acc deaminase
Authentication method is as follows: preparation DF culture medium (DF culture medium prescription: KH2PO44 g, Na2HPO4 6 g, MgSO4·7H2O 0.2 g,
FeSO4·7H2O 0.2 g, glucose 2g, gluconic acid 2 mL, citric acid 2 g, (NH4)2SO42 g, distilled water is settled to
1 L, pH 7.2), test strains is chosen in solid medium streak culture with clean toothpick, be placed in constant incubator, training
Support 24h, then test strains is chosen into being transferred to nitrogen-free and the ADF culture medium containing ACC (with 3 mmol/ with clean toothpick by bacterial strain
LACC replaces the (NH in DF culture medium4)2SO4For only nitrogen source) in, see after cultivating 24h in being placed on 30 DEG C of constant incubators
Examine result, ADF culture medium can grow the bacterial strain of bacterium colony, can primarily determine that there is acc deaminase activity.
CAS detection culture medium compound method is as follows:
Solution a: by 0.012gCAS(chrome azurol S) it is dissolved in 10ml distilled water, add containing 10mmol/LHCl 2ml's
1mmol/L FeCl3Solution；
Solution b: take 0.015gHDTMA(cetyl trimethylammonium bromide) it is dissolved in 8mL distilled water；
Dye solution c: be slowly added in solution b by solution a, rock gently so that the two mutual mix homogeneously of solution, obtains dye liquor c；
By 10 × MM9 saline solution: (Na2HPO430g, KH2HPO41.5g, NaCl 2.5 g, NH4Cl 5g, distilled water
500ml) in the clean triangular flask of the distilled water that 20mL and piperazine Diethanol sulfonic acid 6.04g there was added 150ml, with 50% after mixing
NaOH regulate pH to 6.8, and add agar powder 3.2g, and obtain culture medium d.
By dye solution c, culture medium d and 1mmol L-1CaCl2、1mmol·L-1 MgSO4·7H2O, the Fructus Vitis viniferae of 20%
Sugar is sterilized separately (115 DEG C, 20min), after the acid hydrolyzed casein filtration sterilization of 10%, is all placed in 50 DEG C of water-bath heat preservation for standby use.
Measure above-mentioned 0.2ml 1mmol/L CaCl respectively24ml, 1mmol/L MgSO4·7H2O 6ml, the cheese egg of 10%
Casamino acid and the glucose of 2ml 20%, add in culture medium d and add dye liquor c along bottle wall again, fully mix, and it is blue fixed to obtain
Property detection culture medium, be then poured in culture dish by every ware 30ml, be placed in aseptic operating platform stand-by.
By above-mentioned experiment, result shows: acinetobacter calcoaceticus NXH1 can produce IAA and siderophore, has potential plant
Growth-promoting function.
The embodiment 5 acinetobacter calcoaceticus NXH1 impact on growth of rape
Farmland with somewhere, Beijing is native as potting media.Soil crosses 20 mesh sieves after air-drying, and measures water content, to dry soil weight
20Kg weighs corresponding wind desiceted soil weight, the amount of the antimony that should add is configured to 250mL solution, fully mixes with above-mentioned soil, make soil
In earth containing antimony concentration be 20 mg/Kg, 50 mg/Kg, 100 mg/Kg, 250 mg/Kg, 500 mg/Kg, with field capacity 70%
Adding corresponding deionized water, balance 2 months under the conditions of being placed in lucifuge, be not added with antimony is right for the process that antimony concentration is 0mg/kg
According to, the soil dress basin after balance, standby.First choosing some full grains before experiment, Semen Brassicae campestris of uniform size is put in
10% hydrogen peroxide solution soaks 15min, then uses ddH2O rinses well, and the seed after sterilization is sprinkled upon uniformly and is covered with three layers
In the culture dish of gauze, in incubator cultivate 2d, choose when seed shows money or valuables one carries unintentionally the close planting seed of germinating energy in above-mentioned not
With in antimony pollution soil, every basin sows 30, and after planting every basin watering can accesses about 108Bacterium solution 10mL that CFU/g cultivates,
With the process that do not connects bacterium for comparison, each process sets 3 repetitions.Periodically watering, keep the illumination of h every day 8, plant is in greenhouse
Plant plant height, root length is measured after lower growth 14 d.As can be seen from Table 2: in the soil of different antimony concentration, add not lever
Each process Brassica campestris L plant height of bacterium NXH1 and root length are above not adding the process of bacterium, illustrate that acinetobacter calcoaceticus NXH1 is alleviating antimony to plant
Poison, promote that plant growing aspect has potential using value.
Table 2 acinetobacter calcoaceticus NXH1 is on Brassica campestris L plant height and the impact of root length
| Sb(mg/kg) | It is not added with bacterial strain high | Add bacterial strain high | It is not added with mycorhiza long | Add mycorhiza long |
| 0 | 12.43 | 13.34 | 3.43 | 6.45 |
| 20 | 13.23 | 14.96 | 4.38 | 7.56 |
| 50 | 13.77 | 15.42 | 4.15 | 7.22 |
| 100 | 13.17 | 14.05 | 3.68 | 6.10 |
| 250 | 12.76 | 13.42 | 3.77 | 5.27 |
| 500 | 11.68 | 12.39 | 3.38 | 6.16 |
Claims (4)
1. a strain anti-antimony antibacterial acinetobacter calcoaceticus (AcinetobacterSp.), preserving number is CGMCC No.12895.
2. acinetobacter calcoaceticus as claimed in claim 1 (AcinetobacterSp.) mining soil in heavy metal pollution is biological
Application in reparation.
Apply the most as claimed in claim 2, it is characterised in that: described heavy metal is Sb3+、As3+、Cd2+、Cr6+、Hg2+。
4. acinetobacter calcoaceticus as claimed in claim 1 (AcinetobacterSp.) application in plant growth-promoting.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220211A (en) * | 2018-04-02 | 2018-06-29 | 中国科学院微生物研究所 | One plant of thermophilic oil acinetobacter calcoaceticus NMB17 and its application in control of plant disease |
| CN111011159A (en) * | 2019-12-30 | 2020-04-17 | 北京林业大学 | Preparation method of ecological improvement matrix based on coal gangue and sludge |
| CN111961633A (en) * | 2020-09-09 | 2020-11-20 | 闽江学院 | Acinetobacter with hexavalent chromium removal and decoloration functions, separation and purification method and application |
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| CN111011159B (en) * | 2019-12-30 | 2021-11-05 | 北京林业大学 | Preparation method of ecological improvement matrix based on coal gangue and sludge |
| CN111961633A (en) * | 2020-09-09 | 2020-11-20 | 闽江学院 | Acinetobacter with hexavalent chromium removal and decoloration functions, separation and purification method and application |
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